The effects of DPP-4 inhibitors on vascular endothelial...
Transcript of The effects of DPP-4 inhibitors on vascular endothelial...
The effects of DPP-4 inhibitors on
vascular endothelial function in
diabetes
A thesis submitted in fulfilment of the requirements for the degree of
Doctor of Philosophy
Salheen Madani T. Salheen, BSc, MSc
School of Medical Sciences
College of Science Engineering and Health
RMIT University
July 2015
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Declaration
I certify that except where due acknowledgement has been made, the work is that
of the author alone; the work has not been submitted previously, in whole or in part, to
qualify for any other academic award; the content of the thesis/project is the result of work
which has been carried out since the official commencement date of the approved research
program; any editorial work, paid or unpaid, carried out by a third party is acknowledged;
and, ethics procedures and guidelines have been followed.
Salheen Madani T. Salheen
Date: 28.07.2015
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Acknowledgement
A PhD project is a journey with ups and downs and also involves a lot of
hardwork, commitment, mental stress, frustrations and a sense of achievement on
successful completion. This acknowledgement is devoted to all those wonderful people
who were a part of this journey and who contributed in their own capacity to make it
happen and be presented in the present form.
First and foremost, I would like to express my sincerest and greatest gratitude to
my supervisor Professor Owen Woodman for offering me a wonderful opportunity to do
my doctoral research in his laboratory. His wide excellent knowledge and logical way of
thinking have been of great value to me. His understanding, encouraging and personal
guidance have provided a good basis for the present thesis. One simply could not wish for
a better or supportive supervisor. Thank you!
I am deeply grateful to my co-supervisor Dr Simon Potocnik for his help, support
and technical assistance which made my research simple and easy. We had fascinating
discussions and cheerful moments in the lab. It has been a privilege and a pleasure to work
with him.
I take this opportunity to thank all the academic, technical and the current (Kai and
Saher) staff in vascular research group at the Department of Cell Biology & Anatomy,
RMIT University, it has been a great pleasure working with each of you in one way or
another in the laboratory. To Kai, thank you for your technical assistance making the
laboratory an excellent environment to work in. I am also grateful to Saher for sharing and
providing animal tissue for experimentation and it has been a pleasure working with you
guys. I would also like to thank the Libyan Government represented by University of
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Tripoli and Ministry of Higher Education for awarding me scholarship to do my PhD
study.
This thesis is dedicated to my parents and in memory of them. Thank you both for
giving me strength to reach for the stars and chase my dreams. I would also like to express
my heartfelt thanks to my family to whom I owe a great deal. My brothers and sisters in
particular to my brothers Mussa Madani, Mohamed Madani, Dr Yousef Madani, and Fathi
Madani as well as my sisters Tibra Madani, Suaad Madani, Najia Madani, and Hanan
Madani for their morale support and encouragement.
Finally, my wonderful wife, Faten Laswad, deserve special mention who believed
that I can do this. Without your understanding, endless patience, support and
encouragement, it would have been impossible for me to finish this work. Thank you for
providing me with constant support, encouragement and understanding which is the pillar
behind my achievements. I owe every bit of my achievements to you.
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Publications
S.M. Salheen, U. Panchapakesan, C. Pollock, O.L. Woodman (2015). The DPP-4 inhibitor
linagliptin and the GLP-1 receptor agonist exendin-4 improve endothelium-dependent
relaxation of rat mesenteric arteries in the presence of high glucose. Pharmacol Res, 94,
26-33.
S.M. Salheen, U. Panchapakesan, C. Pollock, O.L. Woodman (2015). The Dipeptidyl
peptidase-4 inhibitor linagliptin preserves endothelial function in mesenteric arteries from
type 1 diabetic rats without decreasing plasma glucose. Manuscript submitted to Int J
Cardiol.
S.M. Salheen, J.CD. Nguyen, T.A. Jenkins, U. Panchapakesan, C. Pollock, O.L.
Woodman (2015). The DPP-4 inhibitor linagliptan restores endothelium-dependent
relaxation in mesenteric arteries from rats fed a western diet. Manuscript is in preparation.
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Conference abstracts
S.M. Salheen, U. Panchapakesan, C. Pollock, O.L. Woodman (2012) The DPP-4 inhibitor
linagliptan improves endothelium-dependent relaxation of rat mesenteric arteries in the
presence of high glucose. Proceeding of Australian Society of Clinical and Experimental
Pharmacologists and Toxicologists, Sydney, Australia, Poster 201, Abstract 428.
S.M. Salheen, U. Panchapakesan, C. Pollock, O.L. Woodman (2013) The DPP-4 inhibitor
linagliptan improves endothelium-dependent relaxation of rat mesenteric arteries in the
presence of high glucose and hyperglycaemia in STZ-induced diabetic rats. Proceeding of
Australian Society of Clinical and Experimental Pharmacologists and Toxicologists,
Melbourne, Australia, Poster 325, Abstract 130.
S.M. Salheen, U. Panchapakesan, C. Pollock, O.L. Woodman (2014) DPP-4 inhibitor
linagliptin restores endothelium-dependent relaxation in small mesenteric artery from type-
1 diabetic rats. Proceeding of Australian Society of Clinical and Experimental
Pharmacologists and Toxicologists, San Diego, USA, Poster 402, Abstract 1652.
S.M. Salheen, J.CD. Nguyen, T.A. Jenkins, U. Panchapakesan, C. Pollock, O.L.
Woodman (2014) The DPP-4 inhibitor linagliptan restores endothelial dysfunction of
mesenteric arteries in western diet fed rats. Proceeding of Australian Society of Clinical
and Experimental Pharmacologists and Toxicologists, Toronto, Canada, Poster 525,
Abstract 211.
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S.M. Salheen, U. Panchapakesan, C. Pollock, O.L. Woodman (2014) The DPP-4 inhibitor
linagliptin and the GLP-1 receptor agonist exendin-4 prevent high glucose-induced
impairment of endothelial function in rat mesenteric arteries. Proceeding of Australian
Society of Clinical and Experimental Pharmacologists and Toxicologists, Melbourne,
Australia, Poster 549, Abstract 323 and oral presentation.
Salheen M. Salheen, A. Mather, U. Panchapakesan, C. Pollock, O. L. Woodman (2014)
DPP-4 inhibitor linagliptin restores endothelium-dependent relaxation in small mesenteric
artery from type-1 diabetic rats. Proceeding of College of Science, Engineering and Health
Higher Degree by Research Student Conference, RMIT University, Melbourne, Australia,
Poster 104 and oral presentation.
S.M. Salheen, U. Panchapakesan, C. Pollock, O.L. Woodman (2014) The DPP-4 inhibitor
linagliptan and GLP-1 agonist exendin-4 improves endothelium-dependent relaxation of
rat mesenteric arteries in the presence of high glucose Proceeding of the International
Society of Cardiovascular Pharmacotherapy, Adelaide, Australia, Poster 549
Salheen S., Woodman O., Mather A., Panchapakesan U., Pollock C. (2014). DPP-4
inhibitor linagliptin restores endothelium-dependent relaxation in small mesenteric artery
from type-1 diabetic rats. The FASEB Journal, 28 (1 Supplement), 1051-4.
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Salheen, S. M., Nguyen, J. C., Jenkins, T. A., & Woodman, O. L. (2014). The DPP-4
Inhibitor Linagliptin Reverses Endothelial Dysfunction of Mesenteric Arteries From Rats
Fed a Western Diet. Arteriosclerosis, Thrombosis, and Vascular Biology, 34 (Suppl 1),
A211-A211.
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Table of contents
Declaration ...................................................................................................................................... i
Acknowledgement ....................................................................................................................... iii
Publications ................................................................................................................................... vi
Conference abstracts ................................................................................................................. vii
Table of contents ............................................................................................................................x
List of figures ................................................................................................................................xx
List of tables ............................................................................................................................... xxii
Summary .........................................................................................................................................1
Chapter 1 Literature review .......................................................................................................5
1.1 Introduction ............................................................................................................................5
1.2 Biological action of vascular endothelium ........................................................................7
1.2.1 Nitric oxide (NO) as EDRF ..........................................................................................9
1.2.2 Prostacyclin (PGI2) as EDRF .....................................................................................10
1.2.3 Endothelium-dependent hyperpolarization (EDH) as EDRF .................................12
1.2.3.1 Classical form of EDH response ........................................................................14
1.2.3.2 Non-classical form of EDH response ................................................................16
1.3 Generation of reactive oxygen and nitrogen species in diabetes ..................................18
1.3.1 Categories of reactive oxygen species and nitrogen species ..................................19
1.3.1.1 Superoxide anion ..................................................................................................20
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1.3.1.2 Hydroxyl radical ...................................................................................................20
1.3.1.3 Peroxynitrite ..........................................................................................................20
1.3.1.4 Hydrogen peroxide ...............................................................................................21
1.3.2 Diabetic sources of reactive oxygen and nitrogen species .....................................21
1.3.2.1 Mitochondria as a source of superoxide ............................................................22
1.3.2.2 Polyol pathway......................................................................................................26
1.3.2.3 Overproduction of Advanced glycation end-product (AGEs) ........................27
1.3.2.4 Activation of protein kinase C (PKC) ................................................................27
1.3.2.5 Increased hexosamine pathway activity ............................................................28
1.3.3 Sources of reactive oxygen and nitrogen species in vascular endothelial cells ...29
1.3.3.1 Xanthine oxidase ..................................................................................................29
1.3.3.2 NADPH oxidase ...................................................................................................29
1.3.3.3 Endothelial nitric oxide synthase ........................................................................30
1.3.4 Sources of endogenous antioxidants in diabetes .....................................................33
1.3.4.1 Catalase ..................................................................................................................33
1.3.4.2 Superoxide dismutase enzyme (SOD) ...............................................................33
1.3.4.3 Glutathione peroxidase (GPx).............................................................................34
1.3.4.4 Thioredoxin ...........................................................................................................34
1.4 Endothelial dysfunction in diabetes ..................................................................................35
1.4.1 Reduction of NO bioavailability ................................................................................36
1.4.1.1 Expression and phosphorylation of eNOS ........................................................37
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1.4.1.2 Direct reduction of NO bioactivity ..................................................................37
1.4.1.3 Augmented arginase activity ...............................................................................38
1.4.1.4 Amplified levels of ADMA.................................................................................38
1.4.1.5 Uncoupling of eNOS and BH4 ............................................................................39
1.4.3 Diminished EDH responses ........................................................................................41
1.4.3.1 Alteration in the expression or activity of endothelial KCa channels .............41
1.4.3.2 Transformed expression or activity of MEGJs .................................................43
1.5 The incretins and the dipeptidylpeptidase-4 inhibitors ..................................................44
1.5.1 Historical overview of the incretin concept .............................................................44
1.5.2 General description of incretin hormones and exendin-4 .......................................46
1.5.2.1 Glucose-dependent insulinotropic peptide (GIP) .............................................46
1.5.2.2 Glucagon-like peptide-1 (GLP-1).......................................................................47
1.5.2.3 The incretin mimetic exenatide (exendin-4) .....................................................48
1.5.3 Dipeptidylpeptidase and dipeptidylpeptidase inhibitors .........................................50
1.5.3.1 Dipeptidylpeptidase (DPP-4) ..............................................................................50
1.5.3.2 Dipeptidylpeptidase inhibitors ............................................................................51
1.5.4.2.1 The DPP-4 inhibitor linagliptin ...................................................................54
1.5.5 Biological actions of the gliptins as potential treatment for diabetic vascular
complications .........................................................................................................................55
1.5.5.1 Modulation of glucose homeostasis by the incretin system ............................55
1.5.5.2 Effects of DPP-4 inhibitors on vascular endothelial cells and NO ................57
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1.5.5.3 Effects of DPP-4 inhibitors on blood pressure .................................................58
1.5.5.5 Effects of DPP-4 inhibitors on inflammation and atherosclerosis .................59
1.5.5.4 Cardiovascular effects of DPP-4 inhibitors .......................................................60
1.6 Hypothesis ............................................................................................................................64
1.6.1 Specific aims of the project ........................................................................................64
Chapter 2 General Methods ......................................................................................................66
2.1 Animal experiments ............................................................................................................66
2.1.1 Induction of diabetes ...................................................................................................66
2.1.2 Blood glucose analysis ................................................................................................67
2.1.3 Estimation of glycated haemoglobin .........................................................................67
2.2 Isolation of vascular tissue .................................................................................................68
2.2.1 Preparation of mesenteric arteries .............................................................................68
2.3 Vascular functional experiments .......................................................................................71
2.3.1 Mesenteric arteries relaxation and contraction assays ............................................71
2.3.2 Estimation of basal NO level assay ...........................................................................74
2.4 Estimation of reactive oxygen species .............................................................................76
2.4.1 L-012 assay ...................................................................................................................76
2.4.2 Lucigenin assay ............................................................................................................76
2.5 Western Blot ........................................................................................................................77
2.5.1 Extraction of protein from tissue samples ................................................................77
2.5.2 Bradford protein assay ................................................................................................77
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2.5.3 Gel preparation for sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) ..........................................................................................................................78
2.5.4 Preparation of SDS-PAGE .........................................................................................79
2.5.5 Performance of low temperature SDS-PAGE ..........................................................80
2.5.6 Procedures of immunoblotting ...................................................................................80
2.6 Chemicals and Reagents ....................................................................................................81
2.7 Statistical analysis ...............................................................................................................81
Chapter 3 The DPP-4 inhibitor linagliptin and the GLP-1 receptor agonist exendin-4
improve endothelium-dependent relaxation of rat mesenteric arteries in the presence
of high glucose ..............................................................................................................................83
3.1 Introduction ..........................................................................................................................83
3.2 Methods ................................................................................................................................86
3.2.1 Isolation of mesenteric arteries ..................................................................................86
3.2.2 Vascular experiments ..................................................................................................86
3.2.3 Superoxide measurement in mesenteric artery ........................................................88
3.2.4 Reagents ........................................................................................................................88
3.2.5 Statistical analysis ........................................................................................................89
3.3 Results ..................................................................................................................................90
3.3.1 Effects of DPP-4 inhibitors and exendin-4 on vascular superoxide production ..90
3.3.2 Effect of high glucose, DPP-4 inhibitors, and exendin-4 on endothelium-
dependent relaxation .............................................................................................................92
3.3.3 Effect of pyrogallol on endothelium-dependent relaxation ....................................97
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3.3.4 Effect of linagliptin on NO-mediated relaxation ...................................................101
3.3.5 Effect of high glucose on NOS and Nox2 related protein expression ................101
3.3.6 Effects of linagliptin on EDH-mediated relaxation ...............................................104
3.4 Discussion ..........................................................................................................................107
3.4.1 Effects of DPP-4 inhibitors and exendin-4 on vascular superoxide generation 107
3.4.2 Effects of DPP-4 inhibitors and exendin-4 on vascular endothelial function ....108
3.4.3 Effect of linagliptin on NO-mediated relaxation ...................................................109
3.4.4 Effect of linagliptin on EDH-mediated relaxation ................................................110
3.4.5 Conclusion ..................................................................................................................110
Chapter 4 Acute treatment with the DPP-4 inhibitor linagliptan improves
endothelium-dependent relaxation of rat mesenteric arteries from STZ-induced type
1 diabetic rats .............................................................................................................................113
4.1 Introduction ........................................................................................................................113
4.2 Materials and Methods .....................................................................................................114
4.2.1 Experimental animals and induction of type 1 diabetes .......................................115
4.2.2 Vascular function assay ............................................................................................115
4.2.3 Assessment of ROS in mesenteric artery................................................................115
4.2.4 Reagents ......................................................................................................................116
4.2.5 Statistical analysis ......................................................................................................116
4.3 Results ................................................................................................................................116
4.3.1 Body weights and blood glucose .............................................................................116
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4.3.2 Effect of diabetes and linagliptin on superoxide production ...............................117
4.3.3 Effect of diabetes and linagliptin on vascular function ........................................120
4.3.4 Role of NO in normal and diabetic mesenteric arteries ........................................122
4.3.5 Role of EDH-type relaxation in normal and diabetic mesenteric arteries ..........122
4.3.5 Effect of linagliptin on NO-mediated relaxation ...................................................124
4.3.6 Effect of linagliptin on EDH-type relaxation .........................................................126
4.4 Discussion ..........................................................................................................................128
4.4.1 Effect of diabetes on superoxide and endothelium-dependent relaxation ..........128
4.4.2 Effect of diabetes on NO and EDH-type response ................................................129
4.4.1 Effect of linagliptin on vascular superoxide and relaxation .................................130
4.4.2 Effect of linagliptin on NO-mediated relaxation ...................................................130
4.4.3 Effect of linagliptin on EDH-mediated relaxation ................................................131
4.4.4 Conclusion. .................................................................................................................131
Chapter 5 The DPP-4 inhibitor linagliptan restores endothelial function of mesenteric
arteries from rats fed a western diet .....................................................................................134
5.1 introduction ........................................................................................................................134
5.2 Materials and Methods .....................................................................................................135
5.2.1 Experimental animals ................................................................................................135
5.2.2 Isolation of mesenteric arteries ................................................................................136
5.2.3 Vascular function experiments .................................................................................136
5.2.4 Measurement of superoxide generation in mesenteric arteries ............................137
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5.2.5 Western Blot ...............................................................................................................137
5.2.6 Reagents ......................................................................................................................137
5.2.7 Statistical analysis ......................................................................................................137
5.3 Results ................................................................................................................................138
5.3.1 Effect of WD on body weights ................................................................................138
5.3.2 Effect of WD and linagliptin on oxidative stress in mesenteric arteries ............138
5.3.3 Effect of linagliptin on endothelial function in mesenteric arteries from WD fed
rats .........................................................................................................................................138
5.3.4 Investigation of the contribution of NO and EDH-mediated relaxation in SD and
WD ........................................................................................................................................144
5.3.5 Effect of linagliptin on NO-mediated relaxation in WD ......................................144
5.3.6 Effect of WD on the expression of NOS and Nox2 ..............................................145
5.3.7 Effect of linagliptin on EDH-mediated relaxation in WD ....................................149
5.4 Discussion ..........................................................................................................................151
5.4.1 Effects of WD and linagliptin on oxidative stress .................................................151
5.4.2 Effect of WD diet on endothelial function .............................................................152
5.4.3 Effect of linagliptin on endothelial function in WD .............................................152
5.4.4 Effect of linagliptin on NO-mediated relaxation in WD fed rats ........................153
5.4.5 Effect of linagliptin on EDH-mediated relaxation in WD fed rats ......................153
5.5.5 Conclusion ..................................................................................................................154
Chapter 6 The DPP-4 inhibitor linagliptin preserves endothelial cell function in
mesenteric arteries from type-1 diabetic rats without decreasing plasma glucose ....156
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6.1 introduction ........................................................................................................................156
6.2 Materials and Methods .....................................................................................................159
6.2.1 Induction of type 1 diabetes .....................................................................................159
6.2.2 Linagliptin treatment .................................................................................................159
6.2.3 Isolation of mesenteric arteries ................................................................................160
6.2.4 Investigation of vascular function and reactivity ..................................................160
6.2.5 Estimation of basal release of NO in mesenteric arteries .....................................161
6.2.6 Western Blot ...............................................................................................................161
6.2.7 Measurement of superoxide release by mesenteric arteries .................................162
6.2.8 Materials......................................................................................................................163
6.2.9 Statistical analysis ......................................................................................................163
6.3 Results ................................................................................................................................164
6.3.1 Body weights and blood glucose .............................................................................164
6.3.2 Effect of linagliptin on vascular superoxide production .......................................164
6.3.3 Effect of diabetes and linagliptin on vascular function ........................................166
6.3.4 Determination of the relative contribution of NO and EDH to endothelium-
dependent relaxation ...........................................................................................................170
6.3.5 Effect of linagliptin on NO- and EDH-mediated relaxation in the mesenteric
arteries ...................................................................................................................................170
6.3.6 Effect of linagliptin on Nox2, total-eNOS and eNOS monomer/dimer .............175
6.4 Discussion ..........................................................................................................................177
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6.4.1. Effect of linagliptin on vascular endothelial function ..........................................177
6.4.2. Effects of linagliptin on NO-mediated relaxation in rats mesenteric arteries ...178
6.4.3 Effect of linagliptin on EDH-mediate relaxation in rats mesenteric arteries .....180
6.4.4 Conclusion ..................................................................................................................181
Chapter 7 General Discussion ................................................................................................183
7.1 Effects of diabetes and WD on vascular function.........................................................183
7.2 Protection of diabetes or WD-induced endothelial dysfunction by the DPP-4
inhibitors ...................................................................................................................................185
7.3 Future directions ................................................................................................................188
7.4 Conclusion .........................................................................................................................189
Chapter 8 References ................................................................................................................191
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List of figures
Figure 1.1 .......................................................................................................... 8
Figure 1.2 ........................................................................................................ 11
Figure 1.3. ....................................................................................................... 13
Figure 1.4 ........................................................................................................ 24
Figure 1.5 ........................................................................................................ 25
Figure 1.6 ........................................................................................................ 32
Figure 1.7. ....................................................................................................... 53
Figure 1.8. ....................................................................................................... 56
Figure 2.1 ........................................................................................................ 70
Figure 2.2 ........................................................................................................ 72
Figure 2.3 ........................................................................................................ 75
Figure 3.1 ........................................................................................................ 91
Figure 3.2 ........................................................................................................ 93
Figure 3.3 ........................................................................................................ 94
Figure 3.4 ........................................................................................................ 96
Figure 3.5 ........................................................................................................ 99
Figure 3.6 ...................................................................................................... 100
Figure 3.7 ...................................................................................................... 102
Figure 3.8 ...................................................................................................... 103
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Figure 3.9 ...................................................................................................... 106
Figure 4.1 ...................................................................................................... 119
Figure 4.2 ...................................................................................................... 121
Figure 4.3 ...................................................................................................... 125
Figure 4.4 ...................................................................................................... 127
Figure 5.1 ...................................................................................................... 140
Figure 5.2 ...................................................................................................... 141
Figure 5.3 ...................................................................................................... 142
Figure 5.4 ...................................................................................................... 143
Figure 5.5 ...................................................................................................... 147
Figure 5.6 ...................................................................................................... 148
Figure 5.7 ...................................................................................................... 150
Figure 6.1 ...................................................................................................... 167
Figure 6.2 ...................................................................................................... 168
Figure 6.3 ...................................................................................................... 169
Figure 6.4 ...................................................................................................... 173
Figure 6.5 ...................................................................................................... 174
Figure 6.6 ...................................................................................................... 176
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List of tables
Table 2.1 ......................................................................................................... 73
Table 3.1 ......................................................................................................... 95
Table 3.2 ......................................................................................................... 98
Table 3.3 ....................................................................................................... 105
Table 4.1 ....................................................................................................... 118
Table 4.2 ....................................................................................................... 123
Table 5.1 ....................................................................................................... 146
Table 6.1 ....................................................................................................... 165
Table 6.2 ....................................................................................................... 172
1
Summary
Diabetes mellitus is an independent risk factor for cardiovascular disease. It is well
known that hyperglycaemia causes overproduction of reactive oxygen species that leads to
vascular endothelial dysfunction. Endothelial cells, lining all blood vessels, modulate the
tone of the underlying smooth muscle by responding to mechanical forces and
neurohumoral mediators with the release of a variety of contracting and relaxing factors.
Endothelium-dependent relaxing factors include nitric oxide (NO), prostacyclin and an
unidentified cause of endothelium-dependent hyperpolarization (EDH). It is well
established that endothelial dysfunction plays a critical role in the initiation and
development of cardiovascular complications. The aim of this thesis was to investigate the
effects of a high concentration of glucose, hyperglycaemia in diabetes, and consumption of
a western, high fat, diet on the mechanism(s) of endothelium-dependent relaxation in rat
mesenteric artery. Further, the effect of acute and chronic treatment with dipeptidyl
peptidase-4 (DPP-4) inhibitor linagliptin was investigated on endothelium-dependent
relaxation in normal and diabetic rat mesenteric artery. As oxidative stress plays a critical
role in the initiation of endothelial dysfunction, it was hypothesised that drugs with
antioxidant activity might be helpful in maintaining normal vascular function in diabetes.
In the first study, the aim was to investigate the effects of DPP-4 inhibitors and a
glucagon-like peptide 1 receptor (GLP-1R) agonist, exendin-4, on the mechanism(s) of
endothelium-dependent relaxation in rat mesenteric arteries exposed to a high
concentration of glucose (40 mM). Organ bath techniques were employed to investigate
vascular endothelial function in rat mesenteric arteries in the presence of normal (11 mM)
or high (40 mM) glucose concentrations. Incubation of mesenteric artery rings with high
2
glucose for 2 h caused a significant increase in superoxide anion generation and a
significant impairment of endothelium-dependent relaxation. Exendin-4 and the DPP-4
inhibitor linagliptin, but not sitagliptin or vildagliptin, significantly reduced vascular
superoxide levels and improved endothelium-dependent relaxation in the presence of high
glucose. The beneficial actions of exendin-4, but not linagliptin, were attenuated by the
GLP-1R antagonist exendin fragment (9-39). Further experiments demonstrated that the
presence of high glucose impaired the contribution of both nitric oxide and endothelium-
dependent hyperpolarization to relaxation and that linagliptin improved both mechanisms
involved in endothelium-dependent relaxation. These findings demonstrate that high
glucose impaired endothelium-dependent relaxation can be improved by exendin-4 and
linagliptin, likely due to their antioxidant activity and independently of any glucose
lowering effect.
In the second study, the aim was to investigate the effect of the acute presence of
linagliptin, a DPP-4 inhibitor, in vitro on the mechanism(s) of endothelium-dependent
relaxation in rat mesenteric arteries isolated from streptozotocin (STZ)-induced diabetic
rats after 10 weeks of diabetes. The study demonstrated that endothelial dysfunction was
due to a decreased contribution of both NO- and EDH to endothelium-dependent
relaxation. Furthermore, endothelial dysfunction was associated with elevated oxidative
stress due the increased activity of NADPH-oxidase. This study demonstrated that acute
treatment with linagliptin reduced the levels of oxidative stress and improved endothelial
function by improving of the contribution of NO and EDH to endothelium-dependent
relaxation in mesenteric artery from the diabetic rats.
3
A high-fat ‘western’ diet (WD), a trigger for the development of type 2 diabetes,
may cause endothelial dysfunction as one of the earliest events in atherogenesis. In the
third study, the aim was to examine whether consumption of a WD affected endothelium-
dependent relaxation of rat mesenteric arteries and whether the DPP-4 inhibitor linagliptin
improves endothelium-dependent relaxation. Wistar Hooded rats were fed a standard diet
(SD, 7% total fat) or WD (21% total fat) for 17 weeks. Consumption of the WD
significantly increased superoxide release from mesenteric arteries assayed by lucigenin
chemiluminescence and linagliptin significantly reduced the vascular superoxide
production. WD significantly reduced the sensitivity to acetylcholine (ACh) and acute
treatment with linagliptin improved endothelial function. The contribution of EDH to
ACh-mediated relaxation was determined in the presence of L-NNA and ODQ to block
NOS and guanylate cyclase respectively. The study demonstrated that EDH-type
relaxation was improved by linagliptin. Furthermore, acute treatment with linagliptin also
significantly improved the contribution of NO (determined in the presence of TRAM-34 +
apamin to block IKCa and SKCa) to relaxation. The results demonstrated that acute
treatment with linagliptin in vitro significantly reduced vascular superoxide levels and
improved the contribution of both NO and EDH to preserve endothelium-dependent
relaxation in small mesenteric arteries isolated from rats fed a high fat diet.
In the fourth study, we hypothesized that long-term chronic treatment with DPP-4
inhibitor linagliptin in vivo (4 weeks, 2 mg/kg per day oral gavage), would improve
relaxation in mesenteric arteries from STZ-induced diabetic rats. The lucigenin-enhanced
chemiluminescence assay was used to measure superoxide production in mesenteric
arteries from normal and diabetic rats. The study demonstrated that chronic treatment with
linagliptin did not affect plasma glucose but markedly reduced the levels of superoxide
4
production and improved endothelium-dependent relaxation in mesenteric arteries from
type1 STZ-induced diabetic rats. Treatment with linagliptin selectively improved NO-
mediated relaxation in rats associated with an increased expression of eNOS, reduced
eNOS uncoupling and down regulation of the NADPH-oxidase subunit Nox2 expression.
In conclusion, this thesis extends our understanding of the pathological process
underlying endothelial dysfunction in the microvasculature caused by exposure to high
glucose concentrations in vitro, the consumption of a high fat diet, and sustained
hyperglycaemia. All of these factors caused oxidative stress via increased
activation/expression of NADPH-oxidase and eNOS uncoupling and subsequently
endothelial dysfunction. Long-term chronic treatment with linagliptin in STZ-induced
diabetic rats had no effect on plasma glucose levels, indicating that linagliptin exerted its
improvement in vascular function via a mechanism independent of glucose lowering. The
beneficial protective actions of linagliptin likely occurs via at least two mechanisms; a
GLP-1 receptor independent pathway in which the linagliptin is able to rapidly scavenge
ROS and inhibit NADPH oxidase as a source of superoxide production and reduced eNOS
uncoupling to preserve NO bioavailability or through a GLP-1 receptor dependent
pathway. These beneficial actions make linagliptin a potential adjunctive treatment for
diabetic vascular complications in patients with both type 1 and type 2 diabetes
importantly expanding the range of patients able to benefit from the use of this drug.
5
Chapter 1
Literature review
1.1 Introduction
Diabetes mellitus, a metabolic disorder characterized by hyperglycaemia, is a
disease that has been rapidly increasing worldwide. According to the fact sheet of the
World Health Organization (WHO), there are more than 346 million people worldwide
suffering from diabetes which can lead to complications such as atherosclerosis,
nephropathy, and retinopathy (WHO, 2011). In 2004, an estimated 3.4 million people died
from the consequences of high blood glucose (WHO, 2011). It has been reported that by
2030, about 438 million people worldwide are expected to have diabetes (Donald et al.,
2012). In 2010, the prevalence of diabetes mellitus in adults in the United Kingdom and
the United States was 7% and 11% respectively (Noble et al., 2011). In Australia, more
than 3.5 million Australians have diabetes. Diabetes mellitus in Australia is the cause of a
considerable portion of the total burden of diseases, and significant costs. For example,
Type 2 diabetes costs Australia $3 billion a year (Australia Diabetes, 2011).
Hyperglycaemia can result as a consequence of a deficiency in the release of
insulin or result from insulin resistance. Patients with diabetes are classified into two
groups; patients having type 1 (insulin-dependent) or type 2 (insulin-independent) diabetes
mellitus. Prolonged periods of type 1 or type 2 diabetes mellitus, and thus an extended
exposure to hyperglycaemia, can have severe effects on both the macro- and
microvasculature. This is as an outcome of disturbances in several metabolic and
6
haemodynamic mechanisms that occur as a result of abnormal blood glucose homeostasis.
Pathological conditions associated with these complications may develop in the nervous
system (neuropathy), the eye (retinopathy), kidney (nephropathy), and cardiovascular
system, all of which lead to the increased mortality and morbidity rates linked to advanced
cases of diabetes mellitus (Australia Diabetes, 2011).
The principle of current medicines used in the treatment of diabetes is based on the
idea of insulin replacement for type 1 diabetes, since insulin is not created by β-cells in the
pancreas, or to enhance the insulin sensitivity or secretion for patients who are suffering
from type 2 diabetes. These anti-hyperglycaemic treatments attempt to provide tight
control of glucose levels and decrease detrimental macro- and microvascular risks and
dysfunction caused by hyperglycaemia. But, large scale prospective studies for the two
types of diabetes 1 and 2 such as the Diabetes Control and Complications Trial
(DCCT/EDIC) and the UK Prospective Diabetes Study (UKPDS), demonstrated that tight
control and adjustment of glucose level may slow, but cannot prevent the development of
cardiovascular complications (David et al., 2005, Holman et al., 2008). Accumulating
evidence suggests that reactive oxygen species (ROS), provoked by hyperglycaemia is the
primary cause of the initiation and progression of diabetic cardiovascular complications
including impairment of the modulatory role of the vascular endothelium that could initiate
the development of these diabetic complications (Brownlee, 2001, De Vriese et al., 2000,
Pieper, 1998). As a consequence, it is desirable to obtain alternative therapies that directly
address the cardiovascular complications in order to minimize the high prevalence of
morbidity and mortality related to diabetes.
Incretin based therapies are a pharmacological class of glucose lowering agents
used as a new strategy in the treatment of patients with type 2 diabetes mellitus (Drucker,
2003). These compounds have less risk of hypoglycaemia and positively impact on the
7
obesity observed in this patient population (Amori et al., 2007). Dipeptidyl peptidase-4
(DPP-4) inhibitors, by inhibiting the action of the DPP-4 enzyme and increasing the
availability of glucagon-like peptide-1 (GLP-1) and/or increasing its receptor (GLP-1R)
stimulation, act to stimulate insulin secretion and inhibit glucagon secretion.
This review describes the critical role of high glucose, high fat diet and
hyperglycaemia in diabetes-induced ROS production and their detrimental effects on the
vascular endothelial cells. Furthermore, the evidence to suggest that DPP-4 inhibitors may
be a promising therapy for treating cardiovascular complications in diabetes independently
of their glucose lowering effect is described.
1.2 Biological action of vascular endothelium
The vascular endothelium, a simple monolayer of cells that covers the interior
surface of all blood vessels, has been recognized to have several important physiological
functions and is now considered as a purposefully placed, multifunctional organ (Epstein
et al., 1990). The endothelium is located as a barrier between the circulating blood and the
vascular smooth muscle and dynamically participates in the maintenance and control of
vascular function (Behrendt and Ganz, 2002). The vascular endothelium play an important
biological role in the regulation of vascular tone by producing endothelium-derived
relaxing factors (EDRF) which include nitric oxide (NO), prostacyclin (PGI2), and
endothelium-dependent hyperpolarisation (EDH) (Figure 1.1).
8
Figure 1.1 Diagram representing the endothelium-derived vasoactive substances. Nitric
oxide synthase (eNOS), stimulated by shear stress and/or acetylcholine, results in synthesis
and release of nitric oxide (NO) which exerts its action via relaxation of vascular smooth
muscle. The diagram also shows other endothelium-derived factors such as prostacyclin
(PGI2) and endothelium-dependent hyperpolarization (EDH). Adapted from (Vanhoutte et
al., 2009).
9
1.2.1 Nitric oxide (NO) as EDRF
In 1980 Furchgott and Zawadzki, reported that the endothelium was necessary to
mediate the ability of acetylcholine (ACh), and other agonists of muscarinic receptors, to
induce relaxation of rabbit isolated aorta (Furchgott and Zawadzki, 1980). This effect of
acetylcholine is mediated by an endothelium-derived relaxing factor that later was
identified as nitric oxide (NO) (Palmer et al., 1987, Moncada et al., 1991). NO is a small
molecule which readily diffuses to underlying smooth muscle and is generated, along
with L-citrulline, from the cationic amino acid L-arginine by the enzyme nitric oxide
synthase (eNOS) (Palmer et al., 1988). The activity of eNOS requires cofactors such as
nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide,
flavin dinucleotide (FAD), flavin mononucleotide (FMN), tetrahydrobiopterin (BH4) and
calmodulin (Bayraktutan, 2002, Hevel and Marletta, 1992) (Figure 1.2).
Besides the action of ACh, NO is synthesized and released from the endothelial
cells in response to a large variety of chemical, physical and humoral stimuli including
shear stress, platelet products (serotonin, adenosine diphosphate (ADP)), circulating
hormones (catecholamines), receptor-independent agonists (e.g. calcium ionophores) and
autocoids (histamine, bradykinin) which leads to stimulation of the eNOS enzyme and
release of NO.
NO is a very unstable molecule with a half-life of 4-50 seconds before it is
scavenged by oxygenated haemoglobin, superoxide anions or molecular oxygen (Rao,
1997, Nakagawa and Yokozawa, 2002). NO is recognized as a very strong vasodilator
which, in addition, possesses antiatherogenic properties such as reducing the adhesion of
platelets and leuckocytes to the endothelium, and inhibition of VSMC migration
(Radomski et al., 1987, Kubes et al., 1991). These actions are mainly mediated by the
activation of soluble guanylate cyclase (sGC) which increases formation of cyclic
10
guanosine monophosphate (cGMP) inside smooth muscle cells or platelets causing
relaxation and reduced aggregation respectively (Moncada et al., 1991, Ignarro et al.,
1981).
1.2.2 Prostacyclin (PGI2) as EDRF
Prostacyclin is thought to be one of the most important compounds in the
prostanoids, a family of bioactive lipid mediators that are produced by cyclooxygenase
(COX) from arachidonic acid (AA) present in the membrane of all cells of the body
(McAdam et al., 1999). Under normal physiological conditions, prostacyclin acts as an
effective vasodilator and contributes to the inhibition of platelet aggregation, adhesion of
leukocytes, and proliferation of vascular smooth muscle cells (VSMC) (Gryglewski et al.,
1976). These effects of PGI2 are controlled by the stimulation and activation of PGI2
receptors (IP) causing activation of adenylate cyclase and as a result, production of cyclic
adenosine monophosphate (cAMP) is increased (Narumiya et al., 1999).
11
Agonists
Figure 1.2 Shows the suggested mechanisms that regulate the creation of nitric oxide
(NO) in endothelial cells. Activation of receptors leads to an increase in the concentration
of Ca2+
. NO is synthesised by the conversion of L-arginine to L-citrulline through the
action of eNOS; endothelial nitric oxide synthase. Some cofactors are required to
synthesise NO such as BH4; tetrahydrobiopterin, calmodulin, NADPH; nicotinamide
adenine dinucleotide phosphate, FMN; flavin mononucleotide, and FAD; flavin adenine
dinucleotide. EC; endothelial cell. Adapted from (Vanhoutte et al., 2009).
GTP
[Ca2+]
Nitric oxide
L-arginine
L-citrulline
Nitric Oxide Synthase
(eNOS)
FAD Calmodulin
NADPH
BH4
FMN
EC
12
1.2.3 Endothelium-dependent hyperpolarization (EDH) as EDRF
EDH may be caused by an as yet chemically unidentified factor that is responsible
for causing hyperpolarization of the VSMC that is not due to the action of NO or PGI2
(Garland et al., 1995). EDH acts to regulate vasodilatation and mediates hyperpolarization
of vascular smooth muscles (McGuire et al., 2001, Luksha et al., 2009, Chen et al., 1988,
Edwards et al., 1998). The ability of EDH to contribute to endothelium- dependent
relaxation is mainly noted in resistance vessels (Shimokawa et al., 1996, Busse et al.,
2002) which are essential in maintaining blood pressure and regulating organ perfusion.
EDH induced relaxation can be classified into two general classes, that is, a classical form
and a non-classical form of EDH responses (Figure 1.3).
13
Figure 1.3 An illustration of the suggested EDH pathways associated with endothelial and
smooth muscle ion channel opening. EETs; epoxyeicosatrienoic acids, HNO; nitroxyl,
BKCa; large-conductance Ca2+
-activated K+ channel, SKCa; small-conductance Ca
2+-
activated K+ channel subtype 3, IKCa; intermediate-conductance Ca
2+-activated K-channel,
KIR; inwardly-rectifying K-channels, [Ca2+
]; intracellular calcium concentration, EDH;
endothelium-dependent hyperpolarization; MEGJ, myoendothelial gap-junction, EC;
endothelial cell, VSMC; vascular smooth muscle cell. Adapted from (Edwards et al.,
2010).
14
1.2.3.1 Classical form of EDH response
Endothelial cell activation by endothelium-dependent agonists such as ACh, leads
to increased intracellular Ca2+
which causes the beginning of the EDH response. Classical
EDH responses basically involve the opening of Ca2+
-activated K+
-channels (KCa) both
small conductance Ca2+–activated K
+ channels (SKCa) and intermediate conductance Ca
2+-
activated K+
channels (IKCa) (Edwards et al., 1998, Komori and Vanhoutte, 1990).
Application of a combination of apamin (selective SKCa blocker) and charybdotoxin (non-
selective inhibitor of large-conductance Ca2+
-activated K+
-channels (BKCa), and (IKCa)
abolished EDH responses, indicating the vital role of these channels since iberiotoxin, a
selective BKCa blocker, was unable to substitute for charybdotoxin (Waldron, 1994). The
SKCa and IKCa channels are located in the endothelium and are organized in endothelial
microdomains, especially within projections close to the adjacent smooth muscle cells and
nearby the inter-endothelial gap junctions. Hyperpolarization of the endothelial cells by the
stimulation and activation of either SKCa or IKCa channels leads to K+ efflux through them
which can act as a diffusible EDH which stimulates vascular smooth muscle Na+/K
+-
ATPase and inwardly-rectifying K-channels (KIR) respectively (Edwards et al., 1998,
Sandow et al., 2006, Absi et al., 2007), resulting in smooth muscle hyperpolarization and
relaxation.
The SKCa and IKCa channels are located in the endothelial cells in different
endothelial microdomains and they facilitate the hyperpolarization process in the vascular
endothelial cells through different downstream pathways (Sandow et al., 2006, Absi et al.,
2007, Dora et al., 2008). SKCa channels are located in caveolar partitions and preferentially
stimulate vascular smooth muscle cell KIR channels (Absi et al., 2007). This has been
supported by observations which demonstrated that the hyperpolarization of the VSMC,
because of the opening of endothelial SKCa channels by CyPPA, a selective opener of
15
SKCa, are specifically abolished not only by apamin, but also by a KIR selective blocker,
barium (Weston et al., 2010). In contrast, IKCa channels are non-caveolar (Weston et al.,
2008) and largely exist within the endothelial cell projections that cross the internal elastic
lamina. Hyperpolarization of VSMC, particularly by the activation of Na+/K
+-ATPase, is
due to the opening of IKCa channels (Sandow et al., 2009, Dora et al., 2008).
It is well recognized that K+
efflux is a mediator of some EDH responses
(Chrissobolis et al., 2000, Edwards et al., 1998, McNeish et al., 2005). The identification
of specialized microdomains that are located on both endothelial cells and vascular cells
has provided an enhanced understanding of how hyperpolarization can be enabled through
electrical coupling via projections for myoendothelial gap junctions (MEGJs), with the
probability of contribution from the changes in extracellular K+
which works as a chemical
mediator (Sandow et al., 2006). A number of experimental observations using rat
mesenteric artery demonstrate that there is a coupling between adjacent endothelial cells
by connexins such as CX37, CX40, and CX43 that are located in MEGJs which are
spatially close to zones where there are clusters of SKCa channels (Sandow et al., 2006).
On the other hand, densities of IKCa in the same blood vessels are established to be
spatially connected with CX37 and CX40 MEGJs. This is consistent with supporting
electrophysiological experiments which also suggested that IKCa were mainly expected to
be linked with microdomains that are close to MEGJs in the endothelial cells (Crane et al.,
2003). The significance of endothelial-VSMC communication and the presence of
specialised microdomains is also supported by data which demonstrate the possibility to
attenuate the electrical coupling of endothelial cells and VSMC through MEGJs by the
application of inhibitors to block cell-cell coupling, resulting in impairment of the
hyperpolarization and relaxation of VSMC by EDH (Edwards et al., 1999, Yamamoto et
al., 1999, Little et al., 1995, Mather et al., 2005). In general, K+ and myeoendothelial
16
electrical coupling as possible candidates for EDH seem equally attractive interpretations
of classical EDH responses, however perhaps not in all cases. Dora and colleagues, based
on data from small mesenteric artery of rat, suggested a model of EDH responses wherein
activation of endothelial cells by agonists such as ACh leads to increase Ca2+
in
endothelial cells, consequently activating SKCa with subsequent spread via MEGJs, and the
following activation of IKCa is dependent on the concentrations of Ca2+
in the area of
endothelial cell projections (Dora, 2010, Dora et al., 2008). By the activation of IKCa,
which seems to be limited to the endothelial cell projections, leads to hyperpolarization of
VSMC due to sufficient endothelial cell K+ efflux causing activation of VSMC Na
+/K
+-
ATPase and possibly KIR (Dora et al., 2008). Taken together, the mechanism of action of
these two classical EDH pathways may act in a separate, parallel or even synergistic way.
At any rate, activation of endothelial KCa channels as a starting point is essential in the
action of both pathways.
1.2.3.2 Non-classical form of EDH response
Whereas the classical EDH response works via the opening of endothelial KCa
channels, the non-classical EDH response causes hyperpolarization and relaxation of the
VSMC independently of channels on the endothelium. It is suggested that when the
endothelial cells are stimulated by shear stress or by local hormones such as bradykinin or
substance P, elements such as nitrogen oxides (NO and nitroxyl (HNO)), hydrogen
peroxide (H2O2), and epoxyeicosatrienoic acids (EETs) are released from the endothelial
cells and diffuse to VSMCs, causing hyperpolarization and relaxation (Bolotina et al.,
1994, Edwards et al., 2000, Andrews et al., 2009).
It is widely documented that NO is an endothelium-derived relaxing factor, causing
relaxation of the blood vessels through the activation of soluble guanylate cyclase (sGC)
17
(Palmer et al., 1987, Rees et al., 1989), but there is also evidence that NO causes VSMC
hyperpolarization through the opening of BKCa channels (Bolotina et al., 1994, Tare et al.,
1990). In addition, the one electron reduced and protonated form of NO, HNO is
synthesised endogenously and causes hyperpolarization and relaxation (Andrews et al.,
2009, Ellis et al., 2000, Wanstall et al., 2001). The hyperpolarizing effect of HNO can be
partially attenuated by iberiotoxin or 4-aminopyridine, demonstrating the involvement of
both BKCa and Kv channels (voltage-gated channels) respectively (Andrews et al., 2009,
Favaloro and Kemp-Harper, 2009, Yuill et al., 2011, Bullen et al., 2011).
Epoxyeicosatrienoic acids (EETs) are synthesised by the vascular endothelium as
cytochrome P450 metabolites of AA. EET production occurs in response to agonists such
as ACh and bradykinin or shear stress (Fisslthaler et al., 1999, Campbell et al., 1996) and
there is evidence to suggest that KCa channels on the VSMC and the endothelium are
activated by EETs producing hyperpolarization and relaxation (Baron et al., 1997, Hoebel
et al., 1997, Earley et al., 2005, Vriens et al., 2005, Edwards et al., 2000). Further
observations indicate that EETs are able to activate BKCa channels in the VSMC (Baron et
al., 1997, Edwards et al., 2000). It has been reported that bradykinin stimulates the
endothelial release of 11, 12 EET and 14, 15 EET which causes EDH responses in porcine
coronary arteries via activation of endothelial SKCa and IKCa, as well as maxi KCa-
dependent VSMC hyperpolarization. This effect was blocked by the EET antagonist 14,
15-EEZE in addition to iberiotoxin (Edwards et al., 2000, Weston et al., 2005).
Additionally, experimental results have shown the capacity of EETs to stimulate BKCa
channels in VSMC (Baron et al., 1997, Edwards et al., 2000). The mechanism of
activation of BKCa channels by EETs is complicated and isoform-dependent but the most
physiologically applicable pathway is through the activation of transient receptor potential
(TRP) channels in order to promote the influx of Ca2+
into the VSMC which then stimulate
18
the release of Ca2+
from ryanodine sensitive Ca2+
stores and subsequently, the local
increase of Ca2+
will activate BKCa which then leads to hyperpolarization and relaxation of
the VSMC (Earley et al., 2005, Vriens et al., 2005, Watanabe et al., 2003, Grgic et al.,
2009). In addition to activation of TRP channels in VSMC, TRP channels on the
endothelium can be activated by EETs to generate endothelial hyperpolarization and
consequently VSMC hyperpolarization (Earley et al., 2009, Fleming et al., 2007). Overall,
these different actions indicate that EETs display both classical and non-classical EDH
responses but, conclusions in regard to the mechanism of action of EETs differs depending
on the type of arteries examined (Baron et al., 1997, Earley et al., 2009, Edwards et al.,
2000, Fleming et al., 2007, Weston et al., 2005, Watanabe et al., 2003).
H2O2 is synthesized through the reduction of superoxide anions that occur either
spontaneously or by the enzyme Cu-Zn superoxide dismutase. Studies on the mice
mesenteric artery have demonstrated that H2O2 was able to cause relaxation and
hyperpolarization of VSMC via the opening of KCa channels and production of H2O2 is in
response to ACh stimulation (Fujiki et al., 2005, Shimokawa, 2010). This EDH-mediated
hyperpolarisation and relaxation in wild type and eNOS-/-
mice was inhibited by catalase,
which metabolises H2O2 (Fujiki et al., 2005, Shimokawa, 2010). In addition, there is
evidence that H2O2 -induced hyperpolarization is mediated, at least in part, by the opening
of BKCa (Barlow and White, 1998) and KATP channels (Wei et al., 1996).
1.3 Generation of reactive oxygen and nitrogen species in diabetes
Diabetes-induced hyperglycaemia is now recognized to result in oxidative stress
(Flammer and Luscher, 2010). “The term oxidative stress refers to a condition in which
cells are subjected to excessive levels of molecular oxygen or its chemical derivatives
19
called reactive oxygen species (ROS)”(Bayraktutan, 2002). Reactive oxygen species are
byproducts of normal cellular metabolism which can be valuable or detrimental to human
health. Oxidative stress describes a condition in which intracellular production of ROS
challenges the capability of cellular antioxidant systems to neutralise them, consequently
causing serious cellular damage and complications such as endothelial dysfunction (Cai
and Harrison, 2000). ROS can have either a useful or harmful effect to the body. The
beneficial effects of ROS are exerted at low physiological concentrations and are used in
host defence processes and also in many cellular signalling pathways such as responses to
growth factors and stress (Yoon et al., 2002). The detrimental effect of ROS occurs when
there is over production of ROS and/or impairment of endogenous antioxidant mechanisms
(Flammer and Luscher, 2010). Overproduction of ROS in diabetes may cause damage to
cellular DNA, lipids, and proteins through denaturation, thus impairing normal cellular
function. For example, during diabetes, overproduction of ROS in the vascular
endothelium may be the cause of endothelial dysfunction that could contribute to
cardiovascular complications. Cellular enzyme systems that act as important sources of
ROS include NADPH oxidase, endothelial nitric oxide synthase (eNOS), xanthine oxidase
and the mitochondrial respiratory chain. These systems are discussed in detail below.
1.3.1 Categories of reactive oxygen species and nitrogen species
Reactive oxygen and nitrogen species may be present either in the form of free
radicals or non-radicals. The free radicals are molecules that contain one or more unpaired
electrons and have a high level of reactivity. In mammalian cells, molecular oxygen acts as
a terminal electron acceptor in order to metabolize organic carbon and produce energy,
therefore, free radicals obtained from molecular oxygen are the most important type of
20
radical species. The main types of ROS are superoxide anion (O2.-), hydroxyl radicals
(˙OH), peroxynitrite (OONO-), and non-radicals such as H2O2.
1.3.1.1 Superoxide anion
Superoxide anions are a crucial source of ROS and have the ability to interact with
other molecules either directly or indirectly via enzymatic reactions or metal catalyzed
processes to increase the formation of secondary ROS. For example, in normal conditions,
superoxide is short-lived and either rapidly metabolized to hydrogen peroxide (H2O2)
through superoxide dismutase (SOD) or to peroxynitrite as a result of reaction with NO
(Reiter et al., 2000, Beckman, 2003).
1.3.1.2 Hydroxyl radical
The hydroxyl radical is considered as one of the most reactive free radicals.
Hydroxyl radicals have a very short half-life and have the ability to randomly oxidize
many of the close targets within cells such as DNA (mutation), lipids (peroxidation) and
proteins (oxidation) (Kehrer, 2000). The hydroxyl radical can be formed chemically either
through the reaction between superoxide and hydrogen peroxide (Haber–Weiss reaction;
H2O2 + O2˙-→O2 + OH
- + ˙OH) (Kehrer, 2000), or as a result of the reaction of hydrogen
peroxide in the presence of reduced metals (Fenton reaction; H2O2 + Fe2+
/Cu+→ Fe
3+/Cu
2+
+ OH- + ˙OH) (Thomas et al., 2009).
1.3.1.3 Peroxynitrite
The reaction between NO and superoxide produces a reactive oxidant form
peroxynitrite (Squadrito and Pryor, 1995, Szabo, 2003). Peroxynitrite is documented to act
21
as an influential oxidizing mediator causing nitration and hydroxylation of phenolic
compounds, particularly tyrosine residues (Reiter et al., 2000, Jourd'heuil et al., 2001). In
addition, hydroxyl radicals are generated by the protonation of peroxynitrite through the
following chemical reactions (Hogg et al., 1992):
NO. + O2
.- → ONOO
-
ONOO- + H
+ →
.OH +
.NO2
1.3.1.4 Hydrogen peroxide
Hydrogen peroxide is a small, stable, non-radical ROS that has the ability to readily
diffuse through both cellular and nuclear membranes. Physiologically, H2O2 is produced
by several pathways. For example, H2O2 can be produced either by peroxisomes from
molecular oxygen or as a consequence of superoxide anion dismutation by SOD. Within
the cells there are a number of enzymes that act to reduce H2O2 to H2O such as catalase,
glutathione peroxidase (GPX), and thioredoxin (Yeldandi et al., 2000, Carter et al., 1994).
1.3.2 Diabetic sources of reactive oxygen and nitrogen species
Under diabetic conditions, it is thought that high glucose levels (hypergylcaemia)
cause damage to tissues via four main pathways: (1) glucose and other sugars being highly
metabolised through the polyol pathway; (2) overproduction of advanced glycation
endproducts (AGEs) intracellularly and also increased coupling to its receptor; (3)
increased activity of the hexosamine pathway; and (4) activation of protein kinase C
(PKC) isoforms. The findings of clinical studies in which one of these pathways was
inhibited are unsatisfactory, as hyperglycaemia-induced tissue injury was not significantly
22
changed (Geraldes and King, 2010, Ramasamy and Goldberg, 2010), however, there is
evidence that all of these pathways are activated as a result of a single stimulus, that is
overproduction of ROS from mitochondria. Furthermore, by controlling mitochondrial
ROS, the tissue damage due to hyperglycaemia is minimized (Brownlee, 2001, Nishikawa
et al., 2000).
1.3.2.1 Mitochondria as a source of superoxide
Mitochondria play an important role in oxidative phosphorylation via the electron-
transport chain. In the mitochondria, the system involved in oxidative phosphorylation
consists of five major multi-enzyme complexes, called, complexes I, II, III, and IV and
ATP synthase and the mobile electron carrier ubiquinone (coenzyme Q), and cytochrome c
(Figure 1.4). Under normal conditions, when a molecule of glucose is taken up into the
cells, glycolysis causes production of pyruvate from glucose and the pyruvate then enters
the mitochondria. The pyruvate is then oxidized by the action of tricarboxylic acid (TCA)
cycle to produce nicotinamide adenine dinucleotide (NADH) together with flavin adenine
dinucleotide (FADH2). Transformation of electrons derived from oxidative reaction of
NADH and FADH2 activated through the electron transport chain involves complexes I-
IV to finally reach the electron acceptor, molecular oxygen. Throughout the course of
electron transfer through the chain, protons are driven across the mitochondrial membrane
yielding a voltage gradient. This voltage is then collapsed and finally yields ATP through
ATP synthase. During the electron transport procedure, and as a result of the escape of an
electron before it is added to an oxygen molecule, the mitochondria generate superoxide
radicals. The escape of electrons occurs at 2 prime locations ie complex I (NADH
dehydrogenase) and the interface between complex II and coenzyme Q (Turrens, 1980,
Turrens et al., 1985). Furthermore, the reduction of ROS occurs due to uncoupling protein-
23
1 (UCP-1) which leads to a collapse of the proton gradient across the mitochondrial
membrane. Under normal physiological settings these processes are firmly controlled
making this system an important source of ROS. It is estimated that around 1% of oxygen
is condensed to superoxide instead of entirely to water under normal physiological
conditions.
In diabetes, hyperglycaemia causes more pyruvate to be oxidized in the TCA cycle
and this acts to drive more electron donors (NADH and FADH2) into the electron
transport chain. This process increases the voltage gradient across the mitochondrial
membrane until a critical threshold is reached as a result of the increase of electron efflux
to the point at which the electron inside complex III is unsettled (Korshunov et al., 1997),
leading to generation of superoxide as a result of accumulation of electrons at coenzyme Q
which donates the electrons one at a time to molecular oxygen, thus producing superoxide
(Nishikawa et al., 2000). Overproduction of ROS by the mitochondria is considered as a
key initiating factor in the stimulation of four main fundamental pathways: (1) increased
flux of glucose and other sugars into the cells via the polyol pathway (Lee et al., 1995); (2)
increased production of intracellular glucose-derived advanced glycation endproducts
(AGEs) and binding to its receptor for AGEs (Brownlee, 1995); (3) stimulation of protein
kinase C (PKC) isoforms and (4) activation of hexosamine pathway (Koya and King,
1998) (Figure 1.5).
24
Figure 1.4 The generation of superoxide through the electron-transport chain in
mitochondria. Hyperglycaemia causes high mitochondrial membrane potential (ΔµH+) by
deriving electron donors from the TCA cycle (NADH and FADH2) and pumping protons
through the inner membrane of the mitochondria. Consequently this prevents electron
transport at complex III leading to an increase in the half-life of free radical intermediates
of coenzyme Q (ubiquinone) that produce superoxide from reduction of O2. Figure
obtained from (Brownlee, 2001).
25
Figure 1.5 A summary of signalling pathways causing diabetic tissue impairment due to
hyperglycaemia. Adapted from (Nishikawa et al., 2000).
26
1.3.2.2 Polyol pathway
The polyol pathway is essentially related to the family of aldo-keto enzymes that
are utilized as carbonyl substrates. NADPH acts to reduce the carbonyl compounds to their
particular sugar alcohols (polyol). The aldose-reductase enzyme acts to metabolise glucose
to sorbitol, thus, in diabetic states there is a high concentration of intracellular glucose
which is changed to sorbitol by aldose-reductase mediated by NADPH as a cofactor. It
seems that during hyperglycaemia, utilization of NADPH by this reaction prevents the
replacement of glutathione, which is needed to maintain glutathione peroxidase (GPx)
activity. This would eventually reduce cellular antioxidant activity and also because
NADPH is a cofactor required to reproduce reduced glutathione which is an important
scavenger of ROS, this could initiate or intensify intracellular oxidative stress (Ii et al.,
2004, Lee and Chung, 1999, Chung et al., 2003). It has been reported that in diabetic mice,
atherosclerosis is increased due to the overexpression of human aldose-reductase and
decreased gene expression that plays an important role in the regulation of glutathione
regeneration (Vikramadithyan et al., 2005).
Once the glucose is transformed to sorbitol, then the sorbitol is oxidized to fructose
in the presence of sorbitol deydrogenase. This process is accompanied by reduction of
NAD+ to NADH, providing more substrate to complex I of the mitochondrial respiratory
chain. As described above, in diabetes, the mitochondrial respiratory chain is recognized to
be a major supplier of ROS.
27
1.3.2.3 Overproduction of Advanced glycation end-product (AGEs)
In normal physiological situations, AGEs are produced from the non-enzymatic
reaction of glucose with other glycating compounds that are obtained from both glucose
and over-oxidation of fatty acids with proteins. In diabetic conditions, the production of
AGEs and its receptor is increased as a result of the constant action of hyperglycaemia and
oxidative stress (Fu et al., 1994, Thornalley et al., 1999). The binding of AGEs to its
receptors may lead to up-regulation of the transcription factor NF-κB via the activation of
cytokines which eventually causes an increase in the production of ROS. Therefore,
diabetes results in the damaging series of ROS formation, overproduction of AGE and its
receptor, activation of NF-κB, and cytokine production and excessive free radical
formation (Bayraktutan, 2002).
1.3.2.4 Activation of protein kinase C (PKC)
PKCs are a family of around eleven isoforms, with the ability to phosphorylate
various protein targets. Both Ca2+
ions and phosphatidyl serine are recognized as activators
of classical isoforms of PKC. The activity of these classic isoforms is also largely
enhanced by diacylglycerol (DAG). In addition, hyperglycaemia causes de novo synthesis
of DAG, leading to increased activity of mostly β- and δ- isoforms of PKC (Inoguchi et
al., 1992). This results in amplifying stress signaling pathways such as Jak/STAT,
p38MAPK, and NF-κB (Haneda et al., 1997). It is documented that activation of PKC has
been involved when the production of NO is suppressed (Derubertis and Craven, 1994),
and that the insulin-stimulated expression of eNOS in cultured endothelial cells has been
inhibited due to the activation of PKC (Kim et al., 2006). Furthermore, PKC activation
may enhance the activity of NADPH oxidase which acts as a ROS generating enzyme,
28
causing dysfunction of endothelial cells that can be ameliorated by using a PKC inhibitor
(Hink et al., 2001).
1.3.2.5 Increased hexosamine pathway activity
The hexosamine pathway has been demonstrated to be involved in the pathogenesis
of diabetic complications (Federici et al., 2002). In diabetes, hyperglycaemia has been
reported to reduce activity of glyceraldehyde-3-phosphate dehydrogenase in bovine aortic
endothelial cells through increased production of mitochondrial superoxide which in turn
causes increased activity of the hexosamine pathway (Du et al., 2000). In diabetes there is
high intracellular glucose and most of the glucose is metabolized firstly to glucose-6
phosphate via glycolysis and then subsequently to fructose-6 phosphate and then on
through the rest of the glycolytic pathway. However, some of fructose-6 phosphate is
diverted to act as a signalling pathway in which an enzyme GFAT (glutamine: fructose-6
phosphate amido-transferase) transforms fructose-6 phosphate into glucosamine-6
phosphate and finally forms UDP (uridine diphosphate) N-acetyl glucosamine. After that,
N-acetyl glucosamine uses serine and threonine amino acids residues as transcription
factors in a similar way to the normal phosphorylation process. The elevated modification
by this glucose amine causes pathological changes in gene expression (Sayeski and
Kudlow, 1996, Kolm-Litty et al., 1998, Wells and Hart, 2003). For example, elevated-
modification of the transcription factor Sp1 causes increased expression of transforming
growth factor-β1 and plasminogen activator inhibitor-1. Both have harmful effect on the
vasculature in diabetes (Du et al., 2000). Overall, increased glucose flux through the
hexosamine pathway has been documented as a factor in the pathogenesis of diabetic
complications and plays an important role both in hyperglycaemia-induced abnormalities
of glomerular cell gene expression (Kolm-Litty et al., 1998) and in cardiomyocyte
29
dysfunction in cell culture induced by hyperglycaemia (Clark et al., 2003). Furthermore, in
diabetes associated carotid artery plaque, modification of proteins in endothelial cells via
the hexosamine pathway is markedly increased (Federici et al., 2002).
1.3.3 Sources of reactive oxygen and nitrogen species in vascular endothelial cells
As discussed previously, vascular health is maintained by the appropriate function
of the endothelial cells. Mitochondria have been recognized as a major source of ROS
production in diabetes, but there are additional enzymes such as xanthine oxidase, NADPH
oxidase, and eNOS in the vascular endothelial cells that are thought to have a significant
role in increased ROS production.
1.3.3.1 Xanthine oxidase
In the endothelial cells, the enzyme xanthine oxidase acts to catalyze the oxidation
of hypoxanthine to urate by utilizing oxygen molecules as electron acceptors and liberating
ROS such as superoxide and hydrogen peroxide (Figure 1.6). Immunohistochemistry
studies demonstrated that xanthine oxidase is situated in endothelial cells (Jarasch et al.,
1981) and observations from experiments using electron spin resonance technologies
confirmed that xanthine oxidase is a crucial source of formation of vascular superoxide in
type I diabetes (Matsumoto et al., 2003).
1.3.3.2 NADPH oxidase
NADPH oxidase is a superoxide-producing enzyme primarily recognized as a
cytosolic enzyme complex in neutrophils where it contributes to the nonspecific defense
30
mechanism against pathogens via the formation of low quantities of superoxide by the
mechanism of electron transport (Babior et al., 2002). The NADPH oxidase complex
consists of five subunits involving a membrane-associated p22phox
and a gp91phox
subunit
and around four cytosolic subunits: p47phox
, p67phox
, p40phox
, and GTPase rac1 or rac2
(Babior, 2004). NADPH oxidase is also present at physiological levels in vascular tissues
(Valko et al., 2007). In addition to the control of vascular cell growth, proliferation,
activation, and migration, the NADPH oxidase-derived superoxide is also involved in
modulation of vascular tone (Feng et al., 2010). Conversely, in many of cardiovascular
diseases, including diabetes, NADPH activity and expression is up-regulated to form large
quantities of ROS causing oxidative stress (Figure 1.6) (Guzik et al., 2002, Takayama et
al., 2004, Ray and Shah, 2005, Drummond et al., 2011, Paravicini et al., 2004).
1.3.3.3 Endothelial nitric oxide synthase
The isoforms of NOS can be classified into three categories, namely, inducible
(iNOS), neural (nNOS), and endothelial (eNOS). The common action of all of these
isoforms is to convert L-arginine to L-citrulline and thus to liberate NO. This reaction is
catalyzed by a number of cofactors such as calmodulin, BH4, flavin mononucleotide, and
adenine dinucleotide. The second two isoforms produce low quantities of NO in a short
period of time and are mainly present in endothelial and neuronal tissue. In diabetic
conditions, where there is an increase of oxidative stress an unbalanced production of
nitric oxide and superoxide leads to further elevated formation of superoxide by the
NADPH oxidase, xanthine oxidase, and the mitochondria. The reaction between
superoxide and NO leads to the formation of a highly reactive intermediate called
peroxynitrite and lower NO bioavailability in the vascular regions, causing vascular
dysfunction (Figure 1.6). Peroxynitrite causes eNOS uncoupling, where the eNOS is
31
transformed from a dimer to a monomer to produce superoxide instead of NO. This also
occurs under conditions of diminishing levels of L-arginine and/or BH4 oxidation (Cai et
al., 2002, Hink et al., 2001, Ryoo et al., 2008, Münzel et al., 2010, Cai et al., 2005).
32
Figure 1.6 Scheme shows the suggested mechanism, sources, and pathways of the
production of ROS in endothelial cells. EC, endothelial cell; O2.-, superoxide anion; O2,
oxygen molecule; H2O2, hydrogen peroxide; NADPH, nicotinamide adenine dinucleotide
phosphate; NO, nitric oxide; OONO-
, peroxynitrite. Adapted from (Sugamura and
Keaney, 2011).
33
1.3.4 Sources of endogenous antioxidants in diabetes
Under diabetic conditions, the phenomenon of oxidative stress arises when the
cellular redox balance favours pro-oxidant production over endogenous antioxidant
defences (Rains and Jain, 2011). This is usually observed when there is an overproduction
of ROS (as described above) and/or in case of compromised function of endogenous
antioxidant systems. There are several sources of antioxidant systems involved
endogenously such as catalase, superoxide dismutase (SOD), glutathione peroxidase
(GPx), and thioredoxin.
1.3.4.1 Catalase
Catalase enhances the degradation of hydrogen peroxide to water and oxygen. In
diabetes, although there is increased expression of catalase, the activity was reduced due to
overproduction of hydroxyl radicals, thereby contributing to oxidative stress (Shi et al.,
2007, Heales, 2001, Góth et al., 2001, Góth, 2008).
1.3.4.2 Superoxide dismutase enzyme (SOD)
SOD catalyses the dismutation of superoxide producing oxygen and hydrogen
peroxide, having a vital role as an antioxidant agent to prevent cellular damage that may be
induced by superoxide radicals (Scandalios, 1993). Within the cells there are isoforms of
SOD situated in different compartments: (1) copper-zinc SOD (SOD 1) is located in the
cytoplasm, (2) manganese SOD (SOD 2) is positioned in the mitochondria, and also there
is extracellular copper-zinc SOD (SOD 3) (Faraci and Didion, 2004).
34
1.3.4.3 Glutathione peroxidase (GPx)
GPx reduces lipid hydroperoxides to lipid alcohol and reduces free hydrogen
peroxide to water (McCay et al., 1976). GPx1 is present in the cytoplasm of almost all
mammalian tissue and is the most abundant isoform of GPx. Under diabetic conditions, the
level of GPx expression has been reported to be diminished (Sindhu et al., 2004).
Additionally, studies show that GPx plays a role in the prevention of diabetes associated
atherosclerosis. For example, a study using ApoE knockout mouse demonstrated that the
extra deletion of GPx1 caused acceleration in the development of atherosclerosis by
upregulation of profibrotic and proinflammatory pathways (Lewis et al., 2007, Torzewski
et al., 2007). This accelerated progression of atherosclerosis was reduced by treatment of
knockout mice with ebselen, a mimetic of GPx1 (Chew et al., 2010).
1.3.4.4 Thioredoxin
The thioredoxin system is a fundamental system for preserving the balance of
cellular redox in endothelial cells and vascular smooth muscle (Yamawaki et al., 2003,
Yamawaki and Berk, 2005). The thioredoxin system includes; thioredoxin, thioredoxin
reductase, and NADPH that, through the interaction with redox-active centre of
thioredoxin (Cys, Gly, Pro, Cys), reduces the oxidized cystein groups on protein. This
forms a disulfide bond, which subsequently causes a reduction of thioredoxin reductase
and NADPH (Yamawaki et al., 2003). Thioredoxin peroxidase plays a role in the
antioxidant properties of thioredoxin by using SH groups as reducing equivalents. The
reduction of the oxidized form of thioredoxin peroxidase by the action of thioredoxin,
causes scavenging of ROS like hydrogen peroxide (Yamawaki et al., 2003). The
thiorodoxin-interacting protein, an endogenous inhibitor of thioredoxin, modulates the
antioxidant activity of thioredoxin. Under diabetic conditions, it has been demonstrated
35
that reduced activity of thioredoxin contributes to oxidative stress, where diabetes
increases the expression of thioredoxin-interacting protein (TXNIP) and reduces the
antioxidant activity of thioredoxin (Qi et al., 2009, Tan et al., 2011).
1.4 Endothelial dysfunction in diabetes
Endothelial dysfunction is defined as an impairment of the balance between
vasodilating and vasoconstricting substances that are produced by the endothelium (De
Vriese et al., 2000). It is considered an important contributor to the pathogenesis of
vascular diseases and the complications of diabetes mellitus (Hadi and Suwaidi, 2007).
Indeed, under physiological conditions there is a delicate balance of the release of
endothelium-derived relaxing factors and endothelium-derived contracting factors,
however, this sensitive balance is altered adversely in cardiovascular diseases, thus,
contributing to the detrimental effect on the vascular tissues and ending with damage to
organs (Tan et al., 2002). Endothelial dysfunction is recognized as an early and
independent predictor of the pathological cardiovascular outcomes of diabetes mellitus and
plays a major role in the initiation of cellular events and complications associated with
diabetic conditions (Hink et al., 2001). Endothelial dysfunction is associated with a
reduction in the relaxation of blood vessels in response to endothelium- dependent
vasodilators, such as ACh or bradykinin, or to flow-mediated vasodilatation. In humans,
studies to assess endothelial function by both invasive and non-invasive techniques have
been used in order to measure both agonist-induced and flow-mediated vasodilatation
(Barac et al., 2007, Buscemi et al., 2009). In addition to reduced production of
endothelium-derived relaxing factors such as NO, EDH, and PGI2, there are also changes
in the presence of proinflammatory molecules, the expression of adhesion molecules, and
36
enhanced production of vasoconstrictor molecules such as COX-derived metabolites of
arachidonic acid (AA), ROS, and endothelin-1 (ET1) that all have pathological
consequences. In diabetes, the impairment of endothelium-dependent relaxation is usually
associated with the production of ROS and increased expression of proinflammatory
molecules (Brownlee, 2001). Collectively, it is the combination of proinflammatory and
proliferative conditions and the impairment of endothelium-dependent responses to stimuli
that underlies endothelial dysfunction and offers the basis for the development of micro-
and macro-vascular disease. Although the aetiology of vascular endothelial dysfunction
under conditions of diabetes has been extensively examined in studies using humans or
animal models, the pathway which could be the primary mechanism to explain the
connection between diabetes and its associated vascular disease complications remains
unclear. Therefore, in the next section the possible mechanisms that play a role in the
initiation of endothelial dysfunction in diabetic vascular disease are described.
1.4.1 Reduction of NO bioavailability
Endothelium-derived NO bioavailability, as estimated by excess of NO over ROS
levels, is a one of the most important indices for determining vascular endothelial function.
Any condition in which there is reduced eNOS activity or where the generation of ROS is
elevated would lead to a reduction of NO bioavailability and consequent impairment of
endothelium-dependent relaxation. In diabetes there are many mechanisms involved in the
reduction of NO-mediated relaxation include: (1) over-generation of superoxide anions
leading to scavenging of NO and consequently increased levels of peroxynitrite; (2)
reduced synthesis of NO by eNOS enzyme due to eNOS uncoupling; (3) increased activity
of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of eNOS; (4) increased
37
uncoupling of eNOS and decreased bioavailability of BH4 which will be discussed in
detail in the following sectors.
1.4.1.1 Expression and phosphorylation of eNOS
In addition to inhibition of NO by superoxide, NO synthesis can be inhibited at an
enzymatic level. An alteration of the levels of eNOS expression may play a role in
endothelial dysfunction associated with diabetes. Using cultured endothelial cells and/or
isolated blood vessels from diabetic animals some studies have shown that the expression
of eNOS may be decreased (Okon et al., 2005, Srinivasan et al., 2004, Salt et al., 2003),
whereas there are other reports that the expression of eNOS is unchanged (Matsumoto et
al., 2006, Pannirselvam et al., 2006) or increased (Ding et al., 2007). It has been reported
that reduction of NO production may be attributed to the mechanism of phosphorylation of
eNOS at Ser1177 which activates the enzyme (Michell et al., 1999). Studies demonstrated
that in aortae from diabetic mice, phosphorylation of eNOS at Ser1177 was reduced
(Zhang et al., 2009, Zhong et al., 2007b, Leo et al., 2011b), as well as in renal arteries
(Zhong et al., 2007a), resulting in decreased synthesis of NO.
1.4.1.2 Direct reduction of NO bioactivity
In diabetes, the direct inactivation of NO has been attributed to the effect of
overproduction of ROS in the vasculature (Bitar et al., 2005). Indeed, several reports show
that diabetes causes imbalance of bioactive radicals by which the expression and/or
activity of pro-oxidant enzymes such as lipoxygenases increased, therefore, the generation
of superoxide anions increased and impairing endothelium-derived NO-mediated
38
relaxation (Woodman et al., 2008, Woodman, 2009, Malakul et al., 2008, Leo et al.,
2010).
1.4.1.3 Augmented arginase activity
One of the main roles of arginase is to catalyze the hydrolysis of L-arginine to
ornithine and urea, therefore, causing competitive inhibition of eNOS to utilize its
common substrate, L-arginine to synthesize NO. In diabetes, it has been reported that there
is an increase in the expression of arginase and as a consequent increase in its biological
activity causing impairment of endothelium-dependent relaxation which could be
ameliorated by acute application of an arginase inhibitor (Romero et al., 2008).
Furthermore, arginase activity is increased and NO synthesis is significantly reduced by
exposing endothelial cells to a medium containing a high glucose concentration (25
mmol/L, 24 hour) (Romero et al., 2008). The activity of arginase in cells exposed to high
glucose can be prevented by transfection of endothelial cells with arginase I small
interfering RNA which also normalizes NO production. Thus in diabetes, the activity of
arginase may have a role in reducing NO production (Romero et al., 2008, White et al.,
2006).
1.4.1.4 Amplified levels of ADMA
ADMA is an endogenous inhibitor of NOS. Under diabetic conditions, it has been
reported that the levels of ADMA increased causing impairment of the response to ACh in
endothelium-dependent relaxation in aortae (Xiong et al., 2003). In diabetes, the activity of
dimethylarginine dimethylaminohydrolase (DDAH), the enzyme that degrades ADMA, is
reduced causing accumulation of ADMA resulting in impairment of NOS activity (Xiong
39
et al., 2003). The levels of ADMA are significantly reduced by restoration of DDAH
activity through two mechanism, either by drug intervention or gene transfer which
increases the expression of DDAH and consequently increases NO synthesis and improves
endothelium-dependent relaxation in diabetic aortae (Lu et al., 2010, Xiao et al., 2009, Yin
and Xiong, 2005).
1.4.1.5 Uncoupling of eNOS and BH4
BH4 is a cofactor which plays an essential role in the modulation of eNOS activity.
BH4 is produced by guanosine 5-triphosphate cyclohydrolase I (GTPCH) (Li et al., 2010).
More than 60% of BH4 is expressed in the wall of blood vessels and associated with
endothelium and modulated by shear stress (Harrison et al., 2010, Tsutsui et al., 1996,
Widder et al., 2007, Li et al., 2010, De Bono and Channon, 2007). In order to be stabilized
as a dimer, eNOS requires appropriate levels of BH4, or the eNOS will act as an NADPH
oxidase if it is in a monomeric state, where the molecular oxygen becomes an electron
acceptor rather than L-arginine (Cai et al., 2005). In the monomeric state of eNOS,
superoxide anions are produced rather than NO (Cai et al., 2005). More important than the
level of BH4, it seems that the maintenance of optimal endothelial function depends
critically on the ratio of BH4 to oxidized biopterins. Indeed, it has been reported that the
levels of BH4 in small mesenteric arteries from db/db diabetic mice were not significantly
reduced in comparison to mesenteric arteries from normal animals, rather, the levels of
oxidized biopterin products such as 7,8-dihydrobiopterin (BH2) were increased, therefore,
the BH4/BH2 ratio was reduced (Pannirselvam et al., 2003). Furthermore, it has also been
reported that BH2 has the same affinity as BH4 to bind to eNOS, thus, BH2 competitively
displaces bound BH4 from its site in eNOS to impair NO production by forming an
uncoupled eNOS-BH2 complex, indicating the importance of BH4/BH2 ratio in order to
40
maintain the function of endothelial cells (Crabtree et al., 2008, Crabtree et al., 2009a).
Several studies showed that a reduced ratio of BH4/BH2 leads to a reduction of NO
bioavailability via uncoupling of eNOS causing endothelial dysfunction. This reduction of
ratio can be reversed by dietary provision of the BH4 precursors such as sepiapterin or
supplementation of BH4 (Cai et al., 2002, Cai et al., 2005, Hink et al., 2001). In addition to
overproduction of superoxide induced by hyperglycaemia and consequent decrease of the
ratio of BH4/BH2, it has also been demonstrated that the reduction of the BH4/ BH2 ratio is
modulated by the production of peroxynitrite. In diabetes which initiates the mechanism of
ubiquitination and 26S proteasome-dependent degradation of GTPCH which causes further
decrease in BH4/BH2 ratio (Wang et al., 2009, Wang et al., 2010, Xu et al., 2007). All
these effects induced by hyperglycaemia are produced by increased expression of SOD or
inhibition of eNOS or by the effects of inhibitors of 26S proteasome such as MG132 or
PR-11 (Xu et al., 2007). Similarly, the reduction of GTPCH and BH4 in STZ-induced
diabetic mice can be reversed by the SOD mimetic, tempol (Xu et al., 2007). In addition,
the BH4 biosynthetic pathway plays a role in maintenance of eNOS by BH4 recycling via
dihydrofolate reductase (DHFR) which has been identified as a critical modulatory factor
in the regeneration of BH4 from BH2 (Crabtree et al., 2011, Sugiyama et al., 2009,
Crabtree et al., 2009b). In diabetes, expression levels of DHFR could also participate in
the contribution to uncoupling of eNOS, but, the data in this regard are not consistent.
Several studies showed increases in the expression of DHFR in diabetes (Oak and Cai,
2007, Ren et al., 2008, Yamamoto et al., 2010). On the other hand, it has been reported
that in diabetic aorta the expression of DHFR was decreased (Wenzel et al., 2008). Despite
these inconsistencies of the reported data in regards to DHFR expression, all studies have
shown that in diabetes the ratio of BH4/BH2 was diminished and the endothelial
41
dysfunction was concomitant to uncoupling of eNOS (Oak and Cai, 2007, Ren et al., 2008,
Wenzel et al., 2008, Yamamoto et al., 2010, Channon, 2004).
1.4.3 Diminished EDH responses
The reports about the relative contribution of NO and EDH to endothelium-
dependent relaxation in mesenteric arteries from diabetic arteries are inconsistent. For
instance, it has been reported that the contribution of EDH to endothelium-dependent
relaxation is increased in diabetes to offer some compensation for the reduction of
bioavailability of NO (Shi et al., 2006). In contrast, another study showed that the action of
NO is not affected by diabetes and the contribution of EDH is selectively decreased (Wigg
et al., 2001). Generally, the EDH is complex in its nature and its contribution depends on
the vascular beds investigated. Several studies have endeavored to investigate the
impairment of EDH responses in arteries under pathological conditions such as diabetes
via investigating the underlying cellular mechanisms responsible for this impairment
(Burnham et al., 2006b, Young et al., 2008, Leo et al., 2011b). There are probable
pathways attributed to be responsible factors causes decrease in EDH responses in diabetes
include either modification of the expression or activity of KCa channel or impairment of
MEGJ expression or activity.
1.4.3.1 Alteration in the expression or activity of endothelial KCa channels
Changes in the expression or the activity of KCa channels could alter the
contribution of EDH to endothelium-dependent relaxation. In diabetes, the impairment of
EDH responses has been extensively investigated and its correlation to the expression level
of KCa channels, investigated but there is a lack of consistent data in this regard (Brondum
et al., 2010, Ding et al., 2005, Pannirselvam et al., 2006). For instance, it has been
42
reported that in STZ-induced diabetic ApoE-deficient mice, the expression of SKCa2 and
SKCa3 channels was reduced causing an impairment of EDH-mediated relaxation (Ding et
al., 2005). In contrast, another study showed that in insulin resistant Zucker fatty rats, even
though there is an impairment in EDH responses, the expression of SKCa3 channels was
augmented in small mesenteric arteries (Burnham et al., 2006a). Moreover, several other
reports showed that even where there is impaired EDH-mediated relaxation there is no
difference in the expression levels of KCa channels in diabetic arteries compared to normal
arteries (Pannirselvam et al., 2006, Brondum et al., 2010). It is important to mention that
the measurement of the expression levels of either protein or gene of the endothelial KCa
channels has the common problem that its commonly suggested that the channel function
reflected by the molecular weight of the native single channels reflects that of the
functional channel (Brondum et al., 2010, Hilgers and Webb, 2007). To some extent this is
not the case, as the functional channel in normal intact tissue has been reported to be
constituted by high molecular weight complexes of these channels (Chadha et al., 2010).
The deficiency of EDH-mediated relaxation response in mesenteric arteries from obese
animals is modulated by the decrease in the expression and/or activity of the KIR channels,
which is a downstream effect on SKCa-mediate the EDH responses (Haddock et al., 2011).
Furthermore, it has also been reported that the activity of KIR channel and/or the activity of
Na+-K
+-ATPase is diminished in diabetes (Makino et al., 2000). Overall, despite the lack
of consistent data in regard to the expression levels of KCa channels in the endothelial cells,
all the studies demonstrated that EDH-mediated relaxation is impaired in diabetic arteries,
indicating that the functional/bioactivity of the endothelial KCa channels.is a more essential
indicator of EDH type responses than endothelial KCa channel expression level.
43
1.4.3.2 Transformed expression or activity of MEGJs
Under diabetic conditions, myoendothelial cell communication can be directly
affected to reduce EDH-mediated relaxation via transforming the distribution and/or
expression of key vascular connexins of MEGJs (De Wit and Griffith, 2010). It has been
reported that the contribution of EDH to endothelium-dependent relaxation is reduced in
mesenteric arteries from db/db type 2 diabetic mice, although there is no change in mRNA
expression level for the key vascular connexins, Cx37, Cx40, Cx43, Cx45 (Pannirselvam
et al., 2006). On the other hand, a study in STZ-induced type 1 diabetic ApoE-deficient
mice showed that Cx37 expression was reduced in small mesenteric arteries (Ding et al.,
2005). Likewise, in aortic arteries from STZ-induced diabetic animals, the protein
expression of the connexins Cx37 and Cx40 was diminished (Hou et al., 2008) and also in
coronary arteries (Makino et al., 2008), suggesting that there is a connection between the
reduction in EDH-mediated relaxation in diabetes and the expression of connexins.
Furthermore, it has been reported that in small mesenteric arteries there is a general
reduction in mRNA and protein levels of Cx40 as well as EDH responses (Young et al.,
2008). The myoendothelial cell communication could be disturbed by the alteration of the
expression of the vascular connexin. A positive correlation has been demonstrated between
the levels of vascular connexin expression of Cx40 and Cx37 in the cremaster arterioles
from Cx40-deficient mice (De Wit, 2010). In Cx40-deficient mice, the conduction of
activated endothelium-dependent vasodilatation in cremaster arterioles is impaired due to a
decrease in the expression of Cx37 (Figueroa et al., 2003, De Wit, 2010). Therefore, in
diabetes, the impairment of EDH-mediated relaxation may be a result of the reduction of
Cx37 expression which also resulted from the reduction of the expression of Cx40.
Furthermore, it has been reported that in diabetes, the expression of Cx37, Cx40, and Cx43
can also be affected when high glucose levels inhibit the intercellular communications
44
through gap junction in VSMC in bovine aorta via PKC-mediated phosphorylation of
Cx43 (Kuroki et al., 1998). Similarly, Sato et al. (2002) reported that the activity of gap
junction communication in rat microvascular endothelial cells was reduced due to high
glucose and this reduction was concomitant with a reduction in Cx43 mRNA and protein
expression (Sato et al., 2002). Therefore, in diabetes, changes in the expression and/or
regulation of the connexins that make up MEGJs could play a critical role in the
endothelial dysfunction.
1.5 The incretins and the dipeptidylpeptidase-4 inhibitors
1.5.1 Historical overview of the incretin concept
The incretins are hormones that are secreted from enteroendocrine cells and
released from the gut into the blood stream within minutes of the ingestion of food (Baggio
and Drucker, 2007). The incretin hormones have a physiological role in the modulation of
the insulin secretory response to food nutrients (Baggio and Drucker, 2007). The “incretin
effect” is a term referring to the insulin secretory response due to the stimulation of
pancreatic β-cells by incretin hormones which have insulinotropic effect and account for at
least 50% of the total insulin secreted after oral glucose administration (Jose and Inzucchi,
2012). In 1902, Bayliss and Starling described the mechanism of pancreatic secretion,
where they found that the pancreas secretes juices via the pancreatic duct after acid
infusion into the digestive system, and this continued even when the innervation of the
intestine was inhibited (Bayliss and Starling, 1902). They conclude that the nature of the
signal that stimulates the pancreas was most likely a chemical, rather than nervous,
stimulus. After the interstinal wall had been stimulated by acid, they removed the extracts
and injected it into the blood stream and once again, they saw juices coming from
pancreatic duct of the injected animal, and they proved that the stimulation of the flow of
45
pancreatic juice was due to the stimulation of the pancreas by the extracts that must
contain a substance that was usually secreted from the intestinal wall into the blood stream
to stimulate the pancreas, and they called this substance “secretin” (Bayliss and Starling,
1902). Moor (1906) reported achieving some success in some experiments he applied on
young diabetic patients who received by mouth extracts of intestinal mucosa (Moore,
1906). This was the first trial based on incretin therapies in order to treat diabetes;
however, these experiments were essentially doomed, because the chemical nature of
peptides which would be degraded when given orally. In 1921, Banting and Best extracted
insulin from the pancreas to further investigate the probability of food entering the gut
causing release of an excitant into the blood stream to finally stimulate insulin secretion
and lower blood glucose (Banting et al., 1922). La Barre (1932) used the term “incretin” to
refer to an extract secreted in the upper part of the gut mucosa to cause hypoglycaemia (La
Barre and Ledrut, 1934). The concept of the incretin system progressed when
radioimmunoassays techniques for insulin become accessible. Between 1964 and 1976
there were at least three studies which independently showed that glucose administered
orally causes a greater insulin response when compared with intravenous glucose injection
although the glucose level obtained by intravenous injection was the same (Elrick et al.,
1964, McIntyre et al., 1964, Perley and Kipnis, 1967). Indeed, the three groups knew that
the oral intake of glucose leads to enhanced incretin release into the blood stream and
subsequently causes an increase in insulin secretion rather than did plasma glucose itself.
In 1971, Brown and Dryburgh isolated and identified the structure of amino acid from a
peptide that was obtained from intestinal mucosa (Brown and Dryburgh, 1971). It has been
reported that the exogenous administration of peptides causes inhibition of gastric acid
secretion in dogs and this peptide was called gastric inhibitory polypeptide (GIP) (Brown
and Dryburgh, 1971). Furthermore, Brown and colleagues identified that GIP had
46
insulinotropic properties (Dupre et al., 1973). GIP was deduced as the first incretin been
isolated and its properties identified. By the end of 1985, glucagon-like peptide-1 (GLP-1)
(7-36) was isolated as a peptide secreted in the gut and reported as a potent insulinotropic
factor (Schmidt et al., 1985). Subsequently clinical investigations demonstrated that GLP-
1 taken intravenously to increase plasma levels of GLP-1 to supraphysiological levels
could have the ability to increase insulin secretion and reduce glucose levels in patients
with type 2 diabetes mellitus. Thus incretin based therapy was proposed as a new approach
to treat type 2 diabetes mellitus.
1.5.2 General description of incretin hormones and exendin-4
1.5.2.1 Glucose-dependent insulinotropic peptide (GIP)
GIP, the first incretin hormone to be identified, consists of a single 42-amino acid
peptide chain derived from a 153-amino acid precursor (Takeda et al., 1987). It is a
member of the structurally related family of hormones including secretin, glucagon, and
vasoactive intestinal peptide. GIP was initially observed to inhibit gastric acid secretion as
well as gastrointestinal motility in dogs receiving supraphysiological dosages (Brown et
al., 1974). It has been reported that GIP at physiological plasma levels exerts glucose-
dependent stimulatory effects on the process of insulin secretion and enhances glucose
uptake by tissues via an insulin-mediated process (Dupre et al., 1973). Many reports
demonstrated that GIP production occurred in enterendocrine cells (called K-cells) which
are located in the proximal small intestine (duodenum and jejunum) and GIP is released as
a response to nutrients containing glucose or fat, and then subsequently released into the
blood-stream in an amount higher than the amount produced in the fasted state (Dupre et
al., 1973, Pederson et al., 1975, Elliott et al., 1993). It has been found in a study carried
out in fasting glucose concentration state that the effects of GIP strongly depends on the
47
glucose level in the blood, where administration of oral fat alone, such as oral corn oil,
without carbohydrate leads to stimulation of GIP release, however, this is not sufficient to
enhance insulin secretion, indicating that GIP-mediated insulin secretion is a glucose-
dependent process; i.e. it is necessary for glucose to be high in plasma in order to enhance
GIP to act on stimulation of insulin secretion (Ross and Dupre, 1978). The insulinotropic
effects of GIP can be achieved through the binding to its specific receptor (GIPR) causing
increased levels of intracellular cAMP and Ca2+
in pancreatic β-cells, and promoting cell
proliferation and cell survival in β-cells (Tru mper et al., 2001, Trumper et al., 2002). The
biological half-life of GIP is very short due to the rapid degradation of GIP by the action of
dipeptidyl peptidase 4 (DPP4). It has been reported that plasma GIP concentrations are
normal or increased in type 2 diabetes, however, the insulinotropic effects are lacking
(Ross et al., 1977, Vilsbøll et al., 2001). Although the mechanisms by which β-cells
respond to GIP remains unclear, some studies suggest that the decrease in physiological
response of β-cell to GIP was due to the effects of hyperglycaemia which causes down
regulation of GIPR expression/activity (Lynn et al., 2001, Zhou et al., 2007).
1.5.2.2 Glucagon-like peptide-1 (GLP-1)
GLP-1 was discovered through the cloning process and identification of the
proglucagon gene (Bell et al., 1983). GLP-1 is a product of post-translational cleavage of
the proglucagon gene via the prohormone convertase enzyme PC1/3 (Zhu et al., 2002,
Ugleholdt et al., 2004). GLP-1 has been considered as the second peptide that has incretin
activity and potentially stimulates insulin secretion from β-cells of the pancreas (Schmidt
et al., 1985). GLP-1 is primarily produced in enteroendocrine cells (called L-cells) that are
located within the enterocytes across the small bowel and ascending colon (Doyle and
Egan, 2007, Jang et al., 2007). There are multiple forms of GLP-1 hormone that have been
48
identified in humans and the COOH-terminally amidated form, GLP-1 (7-36) amide
represents the major circulating, biologically active form (at least 80%) with lesser amount
of the minor glucine extended form GLP-1 (7-37) (Ørskov et al., 1986). Similar to GIP,
GLP-1 is positively coupled to a specific receptor (GLP-1R) in order to achieve its
insulinotropic effects by increasing intracellular cAMP and Ca2+
levels in β-cells.
Furthermore, beside its insulinotropic effects, GLP-1 plays a role in inhibiting gastric
emptying and decreasing food intake (Willms et al., 1996), prevents secretion of glucagon
(Komatsu et al., 1989), thus minimizing the rate of the production of endogenous glucose
(Prigeon et al., 2003). Collectively, all of these actions should lower blood glucose levels
in type 2 diabetes. GLP-1 has also been reported to play a role in protecting β-cells from
apoptosis (Farilla et al., 2002) and to enhance the proliferation of β-cells via upregulation
of the transcription factor in β-cells, pancreatic duodenal homobox-1 protein (PDX-1)
(Perfetti et al., 2000, Stoffers et al., 2000), that recognizes increased transcription of the
insulin gene and up-regulates glucokinase and glucose transporter 2 (GLUT2) (Wang et
al., 1999). Several studies reported that the continuous treatment of type 2 diabetic patients
with GLP-1 can normalize blood glucose levels, enhance the function of β-cells, and
restore the first-phase of insulin secretion in β-cells (Holz et al., 1993, Zander et al., 2002),
hence, recently GLP-1/GLP-1R have become important therapeutic targets for treating
type 2 diabetes. GLP-1 has a short half-life (1.5-2 min) due to its degradation by the DPP-
4 enzyme.
1.5.2.3 The incretin mimetic exenatide (exendin-4)
Exenatide (exendin-4) is a 39–amino acid incretin mimetic, purified from the
salivary secretions of the Gila monster (Heloderma suspectum), which exerts its
glucoregulatory activities in a similar manner to mammalian incretin hormone glucagon
49
like peptide 1 (GLP-1) (Kolterman et al., 2003, Fineman et al., 2003, Nielsen and Baron,
2003, Nielsen et al., 2004). Exendin-4 causes glucose-dependent promotion of insulin
secretion and inhibition of high secretion of glucagon, and slows gastric emptying. The
process of glucose-dependent enhancement of insulin secretion by exendin-4 is mediated
by binding to the pancreatic GLP-1 receptor (Göke et al., 1993). In animal experimental
models of diabetes and in insulin secreting cell lines, both exendin-4 and GLP-1 improve
pancreatic β-cell function by increasing the expression of key genes responsible for insulin
biosynthesis and secretion (Nielsen et al., 2004). Several studies reported that exenatide
and GLP-1 are able to reduce food intake, enhance weight loss, and have an insulin-
sensitizing effect (Nielsen and Baron, 2003, Young et al., 1999, Nielsen et al., 2004,
Szayna et al., 2000, Gedulin et al., 2005). Investigators conclude that exendin-4 markedly
promotes the formation of new β-cells (Xu et al., 1999).
Besides the beneficial insulinotropic effects of exendin-4, it may have favorable
direct effects on endothelial cell function. It has been reported that exendin-4 reduces
macrophage adhesion to the endothelium, an early step in the formation of atherosclerotic
plaques and subsequent endothelial dysfunction (Arakawa et al., 2010). Findings in human
clinical trials showed that the acute administration of exendin-4 counteracts endothelial
dysfunction induced by ischemia and reperfusion in the heart (Ha et al., 2012) or after a
high fat meal (Koska et al., 2010). It has also been reported that the GLP-1 receptor
agonist liraglutide has a potential role in the inhibition of vascular disease progression via
the prevention of atherogenesis, stabilization of plaque and endothelial function together
with potential protection against major cardiovascular events in an ApoE−/−
mouse model
of atherosclerosis (Gaspari et al., 2013).
Both GLP-1 and exendin-4 have been shown to reduce the vulnerability of cells to
apoptotic signaling under conditions of oxidative stress (Chen et al., 2006, Tews et al.,
50
2009). Several mechanisms have been proposed to interpret this cytoprotective effect
including, counter-regulation in the reduction in proteins electron transport chain (Tews et
al., 2009), decrease the expression of pro-apoptotic thioredoxin-interacting protein
(TXNIP) implicated in cell glucose toxicity process (Chen et al., 2006), reduction of
endoplasmic reticulum stress (Tsunekawa et al., 2007), and down-regulation of NF-KB
which acts as a promoter to activate β-cells and stimulate NO synthase and NO pathway
(Liu et al., 2008).
1.5.3 Dipeptidylpeptidase and dipeptidylpeptidase inhibitors
1.5.3.1 Dipeptidylpeptidase (DPP-4)
DPP-4 is a complex enzyme consisting of 766 amino acids, expressed on the
surface of several cell types including vascular endothelial cells (Fadini and Avogaro,
2011, Augustyns et al., 1999). It is a serine aminopeptidase that cleaves GLP-1 and GIP
via dipeptide cleavages with alanine, proline, or hydroxyproline in the N-terminal amino
acids (Drucker, 2007). It has been reported that in vitro, DPP-4 cleaves many chemokines
and peptide hormones, however, in vivo, there are comparatively fewer peptides have been
identified as endogenous physiological substrates for DPP-4. Both GIP and GLP-1 are
endogenous physiological substrates for DPP-4. DPP-4 knockout mice studies showed
improved glucose tolerance concomitant with increased bioavailability of GIP and GLP-1
and enhanced secretion of insulin subsequent to oral glucose challenge. This was
accompanied by decreased accumulation of fat and diminished intake of food as well as
increased resistance to diet-induced obesity (Marguet et al., 2000, Conarello et al., 2003).
In vivo studies on mice which had their incretin receptors genetically inactivated showed
that the mechanisms transducing the glucoregulatory effects of DPP-4 inhibitors were
dominated by GIP and GLP-1 receptor-dependent pathways (Marguet et al., 2000). DPP-4
51
is expressed in the endothelial cells, particularly in the microvascular circulation
(Matheeussen et al., 2011), but it has been reported that the expression and the activity of
DPP-4 is only increased in microvascular endothelial cells in the presence of high glucose
(Pala et al., 2010). This suggests that DPP-4 inhibitors may reduce the detrimental effects
of hyperglycaemia on endothelial cells.
1.5.3.2 Dipeptidylpeptidase inhibitors
DPP-4 inhibitors are pharmacological glucose lowering compounds that have been
introduced as a new strategy for treatment of type 2 diabetes mellitus (Barnett, 2006,
Richter et al., 2008, Palalau et al., 2009, Hollander and Kushner, 2010, Scheen, 2011, Jose
and Inzucchi, 2012). In recent years, several structurally dissimilar DPP-4 inhibitors such
as (1) sitagliptin, (2) vildagliptin, (3) saxagliptin, (4) alogliptin, and (5) linagliptin (Figure
1.7), have been developed and are now being used clinically. The structural dissimilarities
of DPP-4 inhibitors could affect their beneficial pharmacological action. For example,
Kröller-Schön and colleagues (2012) reported that the DPP-4 inhibitor linagliptin
significantly inhibited the oxidative burst in human leucocytes but this was not shared with
other DPP-4 inhibitors such as sitagliptin, vildagliptin, alogliptin and saxagliptin (Kroller-
Schon et al., 2012). DPP-4 inhibitors act as incretin hormone enhancers which control
postprandial elevated levels of blood glucose by increasing insulin secretion and
decreasing glucagon secretion via glucose-dependent manner. Thus, in presence of high
plasma glucose levels, the incretin hormones signal to the liver to reduce glucose
production (Jose and Inzucchi, 2012). The DPP-4 inhibitors are able to reduce both fasting
plasma glucose (FPG) and post prandial glucose (PPG) levels (Aschner et al., 2006). It has
been reported that DPP-4 inhibitors do not reduce elevated blood glucose levels to a
greater extent than existing therapies (Fakhoury et al., 2009, Monami et al., 2010), but
52
they provide many potential benefits including the ability to induce a sustainable decrease
in glycated HbA1c that is accompanied by a low risk of hypoglycaemia and is not
associated with weight gain (Barnett, 2006).
53
Figure 1.7 DPP-4 inhibitors show dissimilarities in their chemical structure. Obtained
from (Lin et al., 2013).
54
1.5.4.2.1 The DPP-4 inhibitor linagliptin
Liangliptin (originally: BI 1356), developed by Boehringer Ingelheim
Pharmaceuticals (Ingelheim, Germany), inhibits the enzyme DPP-4 in a competitive and
reversible manner. It prevents the degradation of GLP-1 and GIP hormones and increases
insulin secretion from pancreatic β-cells (Gallwitz, 2012). Linagliptin has a unique chemical
structure based upon a xanthine scaffold structure (BI 1356; 8-(3-(R)-aminopiperidin-1-yl)-
7-but-2-ynyl-3-methyl-1-(4-methyl-quinazolin-2-ylmethyl)-3,7 dihydropurine-2, 6-Dione)
(Figure 1.7) (Deacon and Holst, 2010). Analysis of the X-ray crystal structure of linagliptin
in complex with human DPP-4 showed that the butynyl substituent of linagliptin occupies
the S1 hydrophilic pocket of the DPP-4 enzyme and the aminopiperidine substitute in the
xanthine scaffold occupies the S2 substitute and the primary amine of linagliptin interacts
with the key amino acid residues, (Deacon and Holst, 2010). Linagliptin is characterized by
its high potency and excellent selectivity for the DPP-4 enzyme (Thomas et al., 2008), with
a long duration of action which is advantageous for once-daily dosing (Thomas et al., 2008,
Eckhardt et al., 2007).
55
1.5.5 Biological actions of the gliptins as potential treatment for diabetic vascular
complications
1.5.5.1 Modulation of glucose homeostasis by the incretin system
In normal physiological conditions, the gut-derived incretin hormones GLP-1 and
GIP are released from the large and small intestine in response to food ingestion. Both of
these hormones stimulate insulin secretion and release from the pancreatic β-cells in
proportion to blood glucose levels (Jose and Inzucchi, 2012). In addition, GLP-1 decreases
glucagon secretion, slows gastric emptying, and increases satiety (Jose and Inzucchi, 2012,
Scheen, 2011, Drucker and Nauck, 2006). The actions of GLP-1 and GIP are rapidly
terminated by dipeptidyl peptidase-4 (DPP-4) (Figure 1.8). Thus, the compounds that
inhibit the biological action of DPP-4 enzyme such as DPP-4 inhibitors will prolong the
half-life and increase the bioavailability of the incretin hormones and consequently
improve their biological effect to maintain glucose homeostasis in individuals with type 2
diabetes.
56
Figure 1.8 The physiological effect of the incretin system. The DPP-4 enzyme is activated
after meal ingestion. As blood glucose levels increase in the blood, incretin hormones such
as GLP-1 and GIP are released from neuroendocrine cells in the intestinal tract. These
hormones cause insulin secretion from beta-cells in the pancreas. Additionally, in a
glucose dependent manner, GLP-1 decreases glucagon secretion from pancreatic alpha-
cells, increases the feeling of satiety, and slows gastric emptying. Through the action of
DPP-4, the incretin hormones (GLP-1 and GIP) are degraded rapidly into their inactive
forms. Blocking the action of DPP-4 increases the biological half-life of incretin hormones
and improves their physiological actions. Obtained from (Jose and Inzucchi, 2012).
57
1.5.5.2 Effects of DPP-4 inhibitors on vascular endothelial cells and NO
In diabetes, endothelial dysfunction plays an important role in the initiation and
development of vascular complications such as atherosclerosis, hypertension, and coronary
artery disease (Liu et al., 2012, Matsubara et al., 2012a). The risk of developing coronary
artery disease in type 2 diabetic patients is 2-4 fold higher compared to individuals without
diabetes (Grundy et al., 1999). In type 2 diabetes mellitus, treatment with gliptins showed
a potential vascular protective effect via GLP-1-dependent mechanisms (Hu et al., 2013).
Gliptin stimulate endothelium-dependent GLP-1 receptor (GLP-1R)-induced vasodilation
involving the cyclic adenosine monophosphate-dependent kinase A (PKA) pathway (Seino
et al., 2010). Involvement of PKA pathway sequentially activates several downstream
mediators which stimulate eNOS and increased production of NO (Liu et al., 2012). In
addition to engagement of GLP-1R-mediated signalling pathway, there is some gliptin
compounds that potentially exhibit improvement in endothelial function and acts against
pathological vascular developments via GLP-1R- independent signalling pathways (Shah
et al., 2011b, Kroller-Schon et al., 2012, Ding and Zhang, 2012). The gliptins vascular
protective effects are well documented in animal models. In study using mouse model of
hind limb ischemia, the DPP-4 inhibitor MK-0626 increased eNOS expression in vascular
tissue and showed potential improvement in blood circulation (Shih et al., 2014). Also, the
DPP-4 inhibitors sitagliptin and saxagliptin increased bioavailability of NO and improved
the endothelial function in spontaneously hypertensive rat models (Aroor et al., 2013, Liu
et al., 2012, Mason et al., 2012, Mason et al., 2011). The DPP-4 inhibitor linagliptin
improved endothelial function and reduced oxidative stress and inflammation in rat model
of lipopolysaccharide-induced septic shock (Kroller-Schon et al., 2012). It has also been
reported that linagliptin significantly improved the function of endothelial cells in a rat
model of renovascular hypertension (Chaykovska et al., 2013). Taken together, data from
58
these animal models under pathological challenges demonstrate that gliptins exhibit
enhancement in vasorelaxation and facilitate the increase in blood flow. Additionally,
gliptins also showed direct endothelium-dependent vasorelaxation in rat aortae ex vivo and
the DPP-4 inhibitor linagliptin was the most potent vasorelaxant and has antioxidant effect
which was not shared with other gliptins involved in the study such as alogliptin and
vildgaliptin (Kroller-Schon et al., 2012). In a study using human umbilical vein
endothelial cell culture, the acute presence of either alogliptin or its mediator, GLP-1,
caused eNOS upregulation and significant increases in activity and expression of eNOS
causing an increased in bioavailability of NO (Zhong et al., 2013, Ding and Zhang, 2012,
Shah et al., 2011b, Shah et al., 2011a). In several human clinical trials, gliptins exhibit
improvement in endothelial function with vasoprotective effects (Matsubara et al., 2012a,
Kubota et al., 2012, Van poppel et al., 2011, Noda et al., 2013, Suzuki et al., 2012, Fadini
et al., 2010). Potential vascular benefits have also been identified with gliptins such as
vildagliptin and alogliptin. These benefits may be due to different pathways eventually
causing an upregulation of NO-mediated relaxation (Van poppel et al., 2011, Noda et al.,
2013).
1.5.5.3 Effects of DPP-4 inhibitors on blood pressure
There is limited evidence that DPP-4 inhibitors affect blood pressure (BP). Recent
trials show that BP was significantly reduced with DPP-4 inhibition (Liu et al., 2012, Lee
et al., 2013, Chaykovska et al., 2013, Mistry et al., 2008, Ogawa et al., 2011). The BP in
Zucker diabetic fatty (ZDF) rats and SHRs was significantly reduced after less than 2-4
weeks of treatment with sitagliptin, saxagliptin, and linagliptin (Liu et al., 2012, Kroller-
Schon et al., 2012, Mason et al., 2012). Indeed, because GLP-1 is known to reduce salt
intake and increase urinary salt excretion, thus gliptins may show a reduction in BP in salt
59
sensitive hypertension (Ogawa et al., 2011). This beneficial effect was found in a trial in
patients having uncontrolled type 2 diabetes mellitus currently treated with
antihyperglycaemia and antihypertensive medications and the DPP-4 inhibitor sitagliptin
was introduced to their treatment programme (Ogawa et al., 2011). In contrast, other
studies report that the gliptins have little effect on BP (Jackson et al., 2008, Koren et al.,
2012, Van poppel et al., 2011).
1.5.5.5 Effects of DPP-4 inhibitors on inflammation and atherosclerosis
Atherosclerosis is an outcome of complex pathologic proinflammatory events in
the blood vessels and it is linked with activation of leukocytes with an increased
production of inflammatory cytokines (Hansson, 2005). The formation of atherosclerosis
plaques is due to the accumulation of inflammatory mediators in the blood vessels
(Barbieri et al., 2013, Ervinna et al., 2013). Hyperglycaemia is a major risk action for
atherosclerosis, due to increased oxidative stress and inflammation in addition to a
negative action on function of platelets (Barbieri et al., 2013). Taken together, all of these
risk factors promote the risk of atherosclerosis among type 2 diabetic patients.
Several animal studies demonstrated that gliptins have the capacity to delay the
development of atherosclerosis both in diabetic and nondiabetic conditions (Shah et al.,
2011a, Chaykovska et al., 2013, Ervinna et al., 2013, Terasaki et al., 2013, Ta et al.,
2011). Vildagliptin normalized the oxidative stress and inflammatory markers in the aortae
of Long-Evans Tokushima fatty rats (Matsui et al., 2011). Studies using apoE-null mouse
models showed that gliptins, such as alogliptin, anagliptin, linagliptin, and des-fluoro-
sitagliptin, markedly diminished the expression of proinflammatory cytokines and reduced
inflammation by inhibiting monocyte/macrophage activation and chemotaxis (Zhong et al.,
60
2013, Matsubara et al., 2012b). Furthermore, sitagliptin significantly decreased
postprandial plasma LDL and vLDL cholesterols and delayed the progression of
atherosclerosis in a hamster model of insulin-resistant fructose-induced hyperlipidaemia by
increasing incretin action and this was consistent with other findings of sitagliptin-treated
mice and also with apoE-deficient mice treated with alogliptin and anagliptin (Ervinna et
al., 2013, Ta et al., 2011, Hsieh et al., 2010). The beneficial effects of gliptins on
postprandial lipid profile may reduce the risk of cardiovascular pathology for individuals
with type 2 diabetes (Eliasson et al., 2012). Moreover, it has been reported that the DPP-4
enzyme itself may be inherently atherogenic, thus there is some speculation that may act as
a risk factor for atherosclerosis, because it stimulates cell proliferation in human coronary
smooth muscle and enhances monocyte migration, inflammatory reactions mediated by
macrophages, and tissue remodelling. Overall, it is still unclear whether the gliptins have
the ability to reduce hyperlipidaemia and cardiovascular consequences. Therefore new
investigations are needed to understand their potential to prevent the initiation and
development of atherosclerosis associated with diabetes.
1.5.5.4 Cardiovascular effects of DPP-4 inhibitors
Studies assessing the beneficial effects of DPP-4 inhibitors on actual
cardiovascular outcomes are limited, and their conclusions should be considered as highly
preliminary. Accumulating evidence suggests that the incretin-based therapy has
favourable cardiovascular outcomes, where the incretin has the potential to be used as
cardiovascular protective therapy in experimental models of myocardial infarction (MI)
and heart failure (Bose et al., 2005, Anagnostis et al., 2011, Jose and Inzucchi, 2012). In
human clinical trial, it has been reported that the DPP-4 inhibition by sitagliptin by
increasing the plasma concentrations of GLP-1 could protect the heart from ischaemic left
61
ventricular dysfunction during dobutamine stress echocardiography (DSE) in patients with
ischaemic heart disease (Khan et al., 2010).
In addition to the cardiac beneficial effects of DPP-4 inhibition which are possibly
mediated by GLP-1, DPP-4 inhibitors could also have direct effects on the risk factors
affecting the heart or vasculature, independently of the incretin system pathway. For
example, the mechanism which involves an enhanced signalling via the bone marrow
chemokine identified as stromal cell-derived factor (SDF)-1alpha. Once there is damage to
the blood vessels, local growth factors and cytokines are released and signal the bone
marrow to release endothelial progenitor cells directed to the injured places of the blood
vessels and differentiated into mature endothelial cells and contributes to the
reconstruction of the vasculature. Interestingly, one of the regulators of the endothelial
progenitor cells which enhance its mobilisation is SDF-1alpha which is known to be a
substrate for the DPP-4 enzyme. Thus the inhibition of DPP-4 will increase SDF-1alpha
concentrations to eventually promote the delivery of endothelial progenitor cells to the site
of injured blood vessels (Heissig et al., 2002). This beneficial effect of DPP-4 inhibition
on this chemokine was demonstrated by Fadini et al., who reported that after four weeks of
treatment of type 2 diabetic patients with sitagliptin, there was an increase in endothelial
progenitor cells, as well as an increase in SDF-1alpha levels, in addition to a reduction in
the proinflammatory chemokine monocyte chemotactic protein (Fadini et al., 2010).
Mistry and colleagues reported that non-diabetic patients with mild to moderate
hypertension treated with sitagliptin showed a small but significant reduction in their
systolic blood pressure (Mistry et al., 2008). In a clinical trial, treatment of individuals
with type 2 diabetes with sitagliptin, taken as a monotherapy was not associated with an
increased risk of major adverse cardiovascular events and was generally well tolerated in
clinical trials of up to 2 years in duration (Williams-Herman et al., 2010). Frederich et al.
62
evaluated the relative risk of cardiovascular events in trials assessing saxagliptin in
patients with type 2 diabetes mellitus and conclude that saxagliptin had a potential to
reduce cardiovascular events in diabetic patients (Frederich et al., 2010). A clinical trial
examined the relationship between linagliptin and cardiovascular outcomes in type 2
diabetic patients compared to other diabetic patients receiving a placebo with treatment
duration of at least 12 months. Linagliptin does not increase cardiovascular risk and, also,
to exhibit a potential reduction of adverse cardiovascular diabetic events such as
cardiovascular death, non-fatal myocardial infarction, non-fatal stroke, or hospitalisation
for unstable angina compared with placebo or other treatment (Johansen et al., 2011).
White et al. in a randomized clinical trial examined the effects of alogliptin on
cardiovascular end points such as cardiovascular death, non-fatal myocardial infarction
and non-fatal stroke in individuals with type 2 diabetes. The findings demonstrated that the
risk of cardiovascular events was lower in diabetic patients treated with alogliptin
compared with diabetic individuals receiving a placebo (White et al., 2010). Indeed,
several large-scale clinical trials planned specifically to evaluate the effects of DPP-4
inhibitors on cardiovascular events are currently proceeding including, TECOS
(Randomised, Placebo Controlled Clinical Trial to Evaluate Cardiovascular Outcomes
After Treatment With Sitagliptin in Patients With Type 2 Diabetes Mellitus and Inadequate
Glycaemic Control), EXAMINE (Cardiovascular outcomes Study of Alogliptin in
Subjects with Type 2 Diabetes and Acute Coronary Syndrome), SAVOR-TIMI 53
(Multicenter, Randomised, Doubleblinded, Placebo-Controlled Phase IV Trial to Evaluate
the Effect of Saxagliptin on the Incidence of Cardiovascular Death, Myocardial Infarction
or Ischaemic Stroke in Patients with Type 2 Diabetes) and CAROLINA (Cardiovascular
Outcome Study of Linagliptin Versus Glimepiride in Patients with Type 2 Diabetes).
Overall, small clinical studies suggesting that there are beneficial effects of DPP-4
63
inhibitors on diabetic cardiovascular risk. However, the relationship between the DPP-4
inhibitors and the actual cardiovascular outcomes remains poorly understood. To address
this doubt, several large scale clinical trials using different DPP-4 inhibitors are now
underway. Together, these large scale clinical trials could improve our understanding of
the potential effects of these glucose-lowering drugs on cardiovascular events in diabetes.
64
1.6 Hypothesis
The broad aim of the thesis is to investigate the pathological changes associated
with vascular dysfunction in the acute presence of high glucose or in small arteries from
diabetic rats or from high fat western diet (WD) fed rats. Based on our understanding of
the biological action and pharmacological activity of DPP-4 inhibitors, as discussed above,
we hypothesize that DPP-4 inhibitors may improve vascular endothelial function in
diabetes or western diet via GLP-1 dependent and/or independent pathways.
1.6.1 Specific aims of the project
(1) This project aims to investigate whether high glucose increases ROS production
and causes endothelial dysfunction after incubation of 2 hours. Further to investigate
whether the acute presence in vitro of DPP-4 inhibitors (linagliptin, sitagliptin,
vildagliptin) or the GLP-1R agonist, exendin-4 prevents the impairment of endothelial
function in small mesenteric arteries exposed to 40 mM glucose and to compare their
antioxidant effects and also to investigate the effects of linagliptin on the relative
contribution of NO-mediated and EDH-mediated relaxation to endothelium-dependent
responses in vessels exposed to high glucose.
(2) It is well documented that STZ-induced hyperglycaemia causes increases in
oxidative stress and impairment of endothelial function after a duration of 10 weeks. This
project aimed to determine the acute effects in vitro of the DPP-4 inhibitor (linagliptin) on
the mechanism(s) of endothelium-dependent relaxation in small mesenteric arteries from
STZ-induced type 1 diabetic and normal rats, where any benefit will occur independently
of any change in glucose levels where it is not possible for glucose concentration to
influence the action of linagliptin.
65
(3) It is well established that small arteries and arterioles are primarily involved in
determination of circulatory resistance and tissue perfusion. However, few if any studies
have explored the effect of WD on the change of microvascular function. This project
aimed to examine the effects of the acute presence of the DPP-4 inhibitor (linagliptin) in
vitro on the mechanism(s) of endothelium-dependent relaxation in small mesenteric
arteries from rats fed a high fat WD. Additionally, to explore the beneficial effects of
linagliptin on the contribution of NO and/or EDH-mediated relaxation in small resistance
arteries from WD fed rats.
(4) Based on the findings from the earlier studies, it was show that high glucose,
STZ-induced diabetes and western high fat diet causes endothelial dysfunction due to
impairment of the contribution of both NO-mediated and EDH-type relaxation, this was
associated with increased activity of NADPH-oxidase-derived superoxide production and
eNOS uncoupling in the mesenteric artery. Also based on the results from (1), (2), (3), it
was demonstrated that acute treatment with linagliptin in vitro reduced oxidative stress and
improved endothelial function in small mesenteric artery. The project aimed to investigate
the effects of chronic treatment in vivo with the DPP-4 inhibitor (linagliptin) on the
mechanism(s) of endothelium-dependent relaxation in small mesenteric arteries from STZ-
induced diabetic and normal rats, and if so, whether it acts via direct scavenging of ROS
and/or by inhibiting the sources of ROS production in the diabetic microvasculature, where
any benefit will occur independently of changes in glucose levels in STZ-induced type 1
diabetes. .
66
Chapter 2
General Methods
2.1 Animal experiments
Male Wistar rats were purchased from Animal Resource Centre (Perth, WA,
Australia). All the experimental animals were kept in the Research Animal Facility at
RMIT University. Animal welfare conditions were taken in account where all animal were
kept under controlled environments of illumination (12 h light/12 h darkness) and
temperature (20-25C). All animals received free access to food (standard pellet diet) and
water ad libitum. All the procedures were approved by the Animal Experimentation Ethics
committee of RMIT University and complied with the Australian National Health and
Medical Research Council code of practice for the care and use the animal for scientific
purposes (AEC approval numbers 1121 or 1309).
2.1.1 Induction of diabetes
Streptozotocin (STZ) which causes pancreatic islet cell destruction was used to
reduce insulin release thus generating an experimental model of type 1 diabetes. Sterile 0.1 M
citrate buffer at pH 4.5 was used to dissolve STZ and prepared to a stock concentration of 100
mg/ml. A single-dose of STZ was used to induce type 1 diabetes at 6-8 weeks of age (~200
g) after overnight fasting. The rats were temporarily exposed to heat lamps (~3-5 min) to
raise blood flow to the tail vein directly prior to the injection. To restrain the movement of
the rats, they were wrapped in a towel with the tail exposed for injection. Ethanol (70%)
was used to clean the tail to reduce the risk of infection and about 0.1 ml of STZ (50
67
mg/kg of body weight) was injected into the tail vein (26G/27G needle). Age-matched rats
used as a control group were injected with the same volume of vehicle (0.1 M citrate
buffer). After the injection of the STZ-treated and vehicle, the two groups were housed
separately and received free access to food and water.
2.1.2 Blood glucose analysis
One week after STZ injection, rats blood glucose levels were measured using a
glucometer (Accu-chek Advantage, Roche, U.S.A). Briefly, the rats were wrapped by
towel allowing access to the tail which was pre-heated under a lamp. Ethanol (70%) was
used to clean the tail to diminish the risk of infection. Applying a 26G needle, the tail vein
was lanced to let bleeding occur and one drop of blood was collected onto a blood glucose
strip fitted to the blood glucometer. The rats were considered diabetic if the blood
concentration was higher than 20 mM. The rats received a low dose of insulin (4-5 IU,
Protaphane, Novo Nordisk, NSW, Australia) to keep glucose <30 mM and to promote
weight gain and reduce mortality, without achieving euglycaemia. All high glucose
animals received insulin treatment on Mondays, Wednesday, and Fridays in the evening
(4-6 pm). The blood glucose for these rats was monitored weekly on Friday mornings (10-
11 am). At the time of tissue collection ie the end of experimental period, blood samples
were collected from the left ventricle using a heparinized 18G needle and 3 ml syringes,
and the glucose concentration was determined using the same procedure.
2.1.3 Estimation of glycated haemoglobin
Left ventricular blood was also transferred into vacuum blood collection tubes to
be used for subsequent analysis of glycated haemoglobin (HbA1c). The blood was diluted
68
1:1 with milliQ water in an eppendorf tube. HbA1C was measured using a Micromat
HbA1c analyser (Biorad, Sydney, NSW, Australia) following the manufacturer instruction.
2.2 Isolation of vascular tissue
At the time point of tissue collection with the end of experimental period, the rats
were killed by asphyxiation with CO2 followed by exsanguination. The mesenteric arteries
were immediately placed in ice-cold Krebs bicarbonate solution (4.7 mM KCl, 118 mM
NaCl, 1.18 mM MgSO4, 25 mM NaHCO3, 11.1 mM D-glucose and 1.6 mM CaCl2), which
were used for additional functional experiments and ROS measurement. The remaining
arteries were snap frozen in liquid nitrogen and stored at -80°C for subsequent western blot
analysis.
2.2.1 Preparation of mesenteric arteries
After the tissue collection, the entire tissue mass of mesenteric arteries was moved
onto a large sylgard coated Petri dish, having ice-cold Krebs biocarbonate solution. The
whole tissue of mesenteric arteries was pinned to the sylgard Petri dish using 26G needles
in order to expose the branches of mesenteric arteries. Small mesenteric arteries (the third
order branch of the superior mesenteric artery, internal diameter ~300 µm) were isolated,
cleared of fat and connective tissue using a dissection microscope and cut into small rings
of about 2 mm in length. Stainless steel wire (40-µm) in diameter was used and inserted
through the lumen of the mesenteric artery in a gentle approach and great care was taken in
inserting the wires to avoid any damage of the lumen of the artery (endothelium). The
rings of the mesenteric arteries after inserting of the wires were then mounted on a
Mulvany-Halpern myograph (model 610M, Danish Myo Technology, Aarhus, Denmark)
by fixing the wire onto the jaws of the myograph to the isometric force transducer side.
69
The first wire was used a guide to locate the lumen of the arteries, the second wire was
inserted in the same manner with great care and fixed to the jaws of the myography to the
micrometer side. The micrometer reading was increased thus that both the wires in the
lumen of the artery were slightly separated and were in close contact with the wall of the
artery (Figure 2.1). After the rings of the mesenteric arteries were mounted and fixed to the
myography, the vessels were allowed to stabilize at zero tension for around 15 min before
normalization. The standard optimization method was used to set the optimal value of
resting tension for each ring of the mesenteric arteries and was measured by applying the
resting tension-internal circumference relationship (Mulvany and Halpern, 1977;
McPherson, 1992). In summary, the rings of mesenteric arteries were stretched in slight
boosts (2 mN) with equilibration period was allowed between stretches. The passive
tension in the rings was recorded and the vessels stretched till the passive tension was
above 90 mm Hg (mean arterial blood pressure in rats). The rings of the mesenteric
arteries were then fixed to an internal circumference equal to 90% of the calculated
circumference at a passive transmural pressure of 13.3 kPa for the rest of experiment, the
maximum active wall tension is achieved as at this level of stretching is reached.
Therefore, the stretching of the vessels determine the passive tension-internal
circumference and achieves an internal circumference equal to 90% of that of the entire
blood vessels under transmural pressure of 100 mmHg. All the experiments were made in
equal criteria and at 37°C and bubbled with carbogen (95% O2 and 5% CO2). After the
normalization was performed, the mesenteric rings were fixed at the optimal tension for 30
minutes to stabilize and recorded on a chart recorder (model 3721; Yokogawa, Tokyo,
Japan).
70
Mulvany-Halpern myograph
Figure 2.1 Diagrammatic representation of the Mulvany-Halpern myography chamber
used to mount small mesenteric arteries. The ring segment of mesenteric artery was
mounted on jaws, one wire fixed to the force transducer and the other wire to the
micrometer in chamber of 5 ml volume containing Krebs buffer which continually aerated
with 5% CO2/ 95% O2.
71
2.3 Vascular functional experiments
After the process of stabilization was finished, the vessels were subjected to a
maximum contraction with an isotonic high K+-containing physiological saline solution
(KPSS), in which the Na+ ions were replaced by K
+ ions ([K
+]KPSS=123 mM). The tension
was resumed to basal tension, after a series of washouts with normal Krebs solution. In
order to evaluate the integrity of the endothelium, the arteries were precontracted with
phenylephrine (PE, 0.01-1 μM) to a level equal to (approximately 50%) of the KPSS
response and then relaxed with a high concentration of ACh (10 µM). If the relaxation
induced by ACh was larger than 80% of the precontraction, the endothelium was
considered intact and functionally fit to run the experiments. Mesenteric arteries with
endothelium that was not intact were excluded from the experimental studies.
2.3.1 Mesenteric arteries relaxation and contraction assays
After several washouts with normal Krebs, the mesenteric arteries were
precontracted again with PE (0.01-1 µM) to 50% of the KPSS response. Relaxant
responses to cumulative concentration additions of ACh (0.1 nM-10 µM) and sodium
nitroprusside (SNP, 0.01 nM-10 μM) were determined. All experiments were conducted in
the presence of indomethacin (10 µM) to inhibit the production of prostanoids and to block
any contribution of prostanoids to the vascular relaxation.Furthermore, cumulative
concentration-response curves to ACh and SNP were investigated subsequent to
incubation with pharmacological inhibitors (Table 2.1). The different combination of the
inhibitors is discussed in detail in each chapter. An example of a trace of the experimental
protocol is illustrated in figure 2.2.
72
Figure 2.2 A trace of the vascular relaxation protocol. Maximum contraction of the
mesenteric arteries was achieved with 123 mM KPSS. PE was used (black dots) to
precontract arteries and the endothelium was considered intact when the maximum
relaxation exceeded 80% relaxation. The cumulative concentration response curve to ACh
(10-10
-10-5
M) or SNP (10-11
-10-5
M) (red dots) were evaluated either in the absence or
presence of pharmacological inhibitors (Table 2.1).
73
Table 2.1 List of drugs used in vascular functional experiments
Drug Inhibitory activity Concentration
Indomethacin COX 10 µM
N-nitro-L-arginine (L-NNA) NOS 100 μM
1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one
(ODQ)
sGC 10 μM
1-[(2 chlorophenyl)(diphenyl)methyl]1H pyrazole
(TRAM-34)
IKCa 1 µM
Apamin SKCa 1 µM
Iberiotoxin (Ibtx) BKCa 0.1 µM
Tempol SOD mimetic 100 µM
Pyrogallol Superoxide
generator
30 µM
Linagliptin DPP-4 inhibitor 1 µM
Sitagliptin DPP-4 inhibitor 1 µM
Vildagliptin DPP-4 inhibitor 1 µM
Exendin-4 GLP-1R agonist 1nM-1µM
Exendin-Fragment (9-39) GLP-1R antagonist 1 µM
74
2.3.2 Estimation of basal NO level assay
After the endothelium was investigated and considered to be intact, the vessels
were precontracted again with PE (10-100 nM) to approximately 20% KPSS. The addition
of the NOS inhibitor, L-NNA (100 µM), would induce additional contraction to the
vessels. Due to removal of the basal level of NO that was acting against PE contraction.
The basal level of NO was assessed by the extent of L-NNA-induced contraction (Figure
2.3).
75
Figure 2.3 Pattern of the protocol to assess basal NO activity. The mesenteric arteries
reached maximum contraction induced by 123 mM K+. ACh (10
-5 M) was added to induce
relaxation and if the relaxation was greater than 80%, the endothelium was considered
intact. The addition of the NOS inhibitor, L-NNA (100 μM) to the arteries precontracted to
approximately 20% of KPSS, produced further contraction of the vessels and was used as
an indicator of the basal activity of NO.
76
2.4 Estimation of reactive oxygen species
The level of ROS was measured by the estimation of the superoxide production by
L-012 or lucigenin-enhanced chemiluminescence assays.
2.4.1 L-012 assay
L-012 is a chemiluminescent probe used to measure superoxide levels. L-012
reacts with superoxide, to produce photons which could be quantified by a photon counter.
The rings of rat mesenteric arteries were incubated in high glucose for 2 h and then
incubated at 37°C for 30 minutes in Krebs-HEPES buffer (composition (mM): NaCl 99.90,
KCl 4.7, KH2PO4 1.0, MgSO4·7H2O 1.2, D-glucose 11.0, NaHCO3 25.0, CaCl2·2H2O 2.5,
Na HEPES 20.0, pH 7.4) either alone, in the presence of tempol, a cell permeable SOD
mimetic (100 µM), linagliptin, DPP-4 inhibitor (1 µM), sitagliptin, DPP-4 inhibitor (1
µM), vildagliptin, DPP-4 inhibitor (1 µM), exendin-4, GLP-1 agonist (1 µM), and
exendin-fragment (9-39), (1 µM). 300 µL of Krebs-HEPES buffer, containing L-012 (100
µM, Wako Pure Chemicals, Osaka, Japan) and the appropriate treatments were placed into
a 96-well Optiplate, which was loaded into a Polarstar Optima photon counter (BMG
Labtech, Melbourne, VIC, Australia) to measure background photon emission at 37°C.
After background counting was completed, a single ring segment of mesenteric artery was
added to each well and photon emission was re-counted. Background counts were
subtracted from superoxide counts and normalized with dry tissue weight.
2.4.2 Lucigenin assay
The superior mesenteric artery was isolated, cleared of fat and connective tissue
and incubated in Krebs solution. The arteries were cut into small rings (2- to 3- mm-long
77
segments) in Krebs-HEPES buffer. The mesenteric arteries were incubated for 45 min at
37ºC in Krebs-HEPES buffer either alone or in the presence of NADPH (100 μM) as a
substrate for NADPH oxidase, diphenyliodonium (DPI, 5 µM), a flavoprotein inhibitor
that inhibits NADPH oxidase, and linagliptin (1 µM). 300 µl of Krebs-HEPES buffer,
containing lucigenin (5 µM) and the suitable treatment were placed into a 96-well
Optiplate. The Optiplate was loaded into a Polastar Optima photon counter (BMG
Labtech, Melbourne, VIC, Australia) to measure background photon emission at 37ºC.
After the background reading was recorded, a single ring section of mesenteric artery was
placed into each well and photon emission was recounted to measure the production of
NADPH oxidase- driven superoxide.
2.5 Western Blot
2.5.1 Extraction of protein from tissue samples
The mesenteric arteries from 2-3 rats of the same treatment groups were pooled,
snap frozen and considered as n=1. Using a 1 ml glass homogenizer, the mesenteric
arteries (~ 200 mg) were homogenized in lysis buffer solution consisting of (100 mM
NaCl, 10 mM Tris, 2 mM EDTA, 0.5% w/v sodium deoxycholate, 1% vol/vol triton X-
100, pH7.4, protease and phosphatase inhibitor cocktails (Roche, Sydney, NSW,Australia).
After 20 min of incubation with lysis buffer at 4ºC, the mesenteric arteries tissues were
homogenized and tissue samples were centrifuged for 10 min at 4ºC and 13,200 RPM. The
supernatant of tissue samples were collected and kept at -80ºC to be used when required.
2.5.2 Bradford protein assay
The Bradford assay was used to measure the protein concentration of the tissue
samples. Phosphate buffered saline (PBS) (100 μL) in a test tube was used to dilute the
78
tissue samples 1:100 and 100 μL of 0.2 M NaOH was added to the test tube. After an
incubation period of 15 minutes, milliQ water (600 μL) and 200 μL of red protein assay
reagent dye (Biorad, Sydney, NSW, Australia), were added to the test tube. The solution
which turned blue with the addition of protein was vortexed and 300 μL of the solution
was loaded to a 96-well plate and in a plate reader the absorbance (590 nM) of the samples
was measure. In addition, in the same 96-well plate, a bovine serum albumin (BSA)
standard curve was generated (0-20 μg/mL). Each of the standard and the protein samples
were applied in duplicate and if the absorbance of the tested samples were not compatible
with the range of absorbance in the standard curve, the unknown samples were further
diluted and the procedures were repeated as defined above. The standard curve was used to
calculate the protein concentration of the protein in the unknown samples. The amount of
protein in each sample was equal (30 µg) and the protein samples were aliquoted and PBS
was used to top up the samples so they contained identical amount of protein (30 μg ) in
the same total volume (10 μL or 15 μL) of 2X Sample buffer (20% w/v glycerol, 2% SDS,
62.5 mM Tris, 0.05% bromophenol blue and 5% -mercaptoethanol, pH 6.8) and was added
to each of the samples and stored at -80°C until required.
2.5.3 Gel preparation for sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE)
According to the manufacturers’ instructions, the plates were collected (Biorad,
Sydney, NSW, Australia). The 7.5%, 10%, 12% and 15% resolving gel (30% acrylamide,
distilled water, 1.5 M Tris, pH 8.8, 10% SDS, 10% ammonium persulfate and TEMED)
was arranged and moved into the glass plates by pipette; isopropyl alcohol (~100 μL) was
added to remove bubbles. Acrylamide-resolving gel was formed after 1-1.5 hours at room
temperature by the polymerization of the solution. The alcohol was removed once the
79
resolving gel had set by dabbing using filter paper and 4% stacking solution (30%
acrylamide, distilled water, 0.5 M Tris, pH 6.8, 10% SDS, 10% ammonium persulfate and
TEMED) was added on to the top of the resolving gel, the 15-well comb was inserted, and
the gel was left to polymerize at room temperature for 0.5-1 hour.
2.5.4 Preparation of SDS-PAGE
The method of SDS-PAGE was carried out by a mini-PROTEAN device (Biorad,
Sydney, NSW, Australia). Denaturation of protein samples (30 µg) in sample buffer was
completed by heating the samples at 95°C for 5 minutes. The prestained kaleidoscope
protein ladder (7 μL, Biorad, Sydney, NSW, Australia) and the denatured protein were
loaded into wells. The running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.3)
was used to perform the electrophoresis at 100 V until the separation was complete (2
hours). Subsequent to the process of electrophoresis the gels were carefully detached from
the gasket and equilibrated in ice-cold transfer buffer (25 mM Tris, 192 mM glycine, 20%
methanol, 0.037% SDS, pH 8.3) which works to eliminate additional salt and detergents
from the running buffer and leads to increase the conductivity of the transfer buffer and
reduce the amount of heat produced during the transfer. In the same transfer buffer, the
Hybond nitrocellulose membranes, filter papers and sponges were also equilibrated.
According to the manual for Biorad wet transfer cell, the sponges, filter papers,
nitrocellulose membranes and gels were assembled, and gels were transferred at 350 mA
for 2-3 hours. At the end of the transfer process, ponceau stain was used to confirm the
transfer of protein was successful.
80
2.5.5 Performance of low temperature SDS-PAGE
To investigate the expression of eNOS monomers and dimers, we used a low
temperature SDS-PAGE technique. As described in section 2.6.2, a 6% resolving gel was
prepared. In low temperature SDS-PAGE, an ice cold resolving and stacking gel were
used. Non-denatured protein sample (60 μg) and protein ladder (7 μL) were loaded into the
wells of 6% resolving gel and ice cold running buffer used to perform electrophoresis at 30
V overnight at 4°C. After the end of the electrophoresis stage, the proteins were transferred
from the gel to the nitrocellulose membrane at 30 V overnight at 4°C. Likewise, the
membrane was stained with ponceau stain to confirm the transferred of protein was
successfully performed.
2.5.6 Procedures of immunoblotting
To perform immunoblotting of eNOS, Nox2 and caveolin-1 protein levels (all BD
Transduction Laboratories, Lexington, KY, USA), calmodulin (Millipore, Billerica, MA,
USA), either by 5% w/v skim milk in Tris Buffered Saline plus 0.1% Tween-20 (TBST)
for 1 hour at room temperature or 5% BSA/TBST (phosphorylated proteins) for 1 hour at
4°C were used in order to block the nitro-cellulose membranes before the addition of the
primary antibodies (in 5 mL 3% BSA/TBST). Subsequently, the nitro-cellulose
membranes were incubated with primary antibodies (1:1000) overnight at 4°C. After that
the membranes were washed (3 x 10 minute washes with 10 mL TBST), and then
incubated with secondary antibody (sheep anti-mouse) (Millipore, Billerica, MA, USA) (in
5 mL, 5% skim milk/TBST) (1:2000) at room temperature for 1 hour. After the incubation,
the membranes were washed with 10 mL TBST (3 x 10 minute washes). The detection of
the secondary antibody which conjugated with horseradish peroxidase was performed after
the incubation of the membrane with either enhanced chemiluminescence reagents
81
(Amersham, GE Healthcare, Sydney, NSW, Australia) or Supersignal West Femto
(Thermo Scientific, Rockford, IL, USA) for 1 minute, and the detection of the
chemiluminescence signals on the membrane were achieved by digital image scanner
(Biorad Chemidoc). Densitometry used to quantify the detected protein bands. After
blocking non nonspecific binding with 5% w/v skim milk, nitro-cellulose membranes were
re-investigated with a loading control antibody (actin), diluted 1:1000 in 5 mL 1%
BSA/TBST. Followed by 3 x 10 minute washes of the membranes with 10 mL TBST
TBST, and once more explored with secondary antibody (Millipore, Billerica, MA, USA)
diluted 1:2000 in 5 mL 5% skim milk/TBST for 1 hour, and the bands were imagined and
qualified as described previously.
2.6 Chemicals and Reagents
All chemicals were purchased from Sigma-Aldrich (St Louis, MO, USA), except
for linagliptin (Boehringer Ingelheim, Germany), acetylcholine perchlorate (BDH
Chemicals, Poole, Dorset, UK), and ODQ (Cayman Chemical, Ann Arbor, MI, USA). All
chemicals were dissolved in distilled water, with the exception of L-NNA, which was
dissolved in 0.1 M sodium bicarbonate, indomethacin, which was dissolved in 0.1 M
sodium carbonate, ODQ, and TRAM-34 which was dissolved in dimethyl sulfoxide
(DMSO). Linagliptin, vildagliptin, sitagliptin, exendin-4, and exendin-fragment (9-39)
which were dissolved in distilled water.
2.7 Statistical analysis
All results are presented as the mean ± SEM, n indicates the number of
experiments. The concentration response data from rat isolated mesenteric arteries were
fitted to a sigmoidal curve using nonlinear regression (Prism version 6.0, GraphPad
82
Software, San Diego, CA, USA) to calculate the pEC50. The maximum relaxation (Rmax) to
ACh or SNP was determined as a percentage of the phenylpherine pre-contraction. pEC50
and Rmax values were compared and analysed using one way ANOVA or two way
ANOVA with post hoc analysis using Bonferroni’s test or Student’s unpaired t-test as
appropriate. p<0.05 was considered statistically significant.
83
Chapter 3
The DPP-4 inhibitor linagliptin and the GLP-
1 receptor agonist exendin-4 improve
endothelium-dependent relaxation of rat
mesenteric arteries in the presence of high
glucose
3.1 Introduction
Vascular disease is the main cause of morbidity and mortality in diabetes and
impairment of endothelial function is considered to be a principle factor in the
development of vascular disease (Grundy et al., 1999, De Vriese et al., 2000).
Hyperglycaemia is thought to be a critical cause of endothelial dysfunction resulting in
reduced NO release, generation of oxygen free radicals and impairment of endothelium-
dependent relaxation (Calles-Escandon and Cipolla, 2001, Rodriguez-Manas et al., 2003).
Acute exposure to high glucose reduces the sensitivity to acetylcholine-induced
endothelium-dependent relaxation (Guo et al., 2000) with a reduced NO release (Reyes-
Toso et al., 2002) associated with an increase in reactive oxygen species (Guo et al.,
2000).
Incretin-based therapies, including inhibitors of dipeptidyl peptidase-4 (DPP-4) and
glucagon-like peptide-1 (GLP-1) receptor (GLP-1R) agonists are effective in the treatment
of type 2 diabetes (T2DM) by increasing insulin secretion (Ussher and Drucker, 2014,
Drucker and Nauck, 2006). The incretin hormones, GLP-1 and glucose-dependent
insulinotropic polypeptide (GIP), are released by intestinal endocrine cells in response to a
84
meal (Kim and Egan, 2008). These hormones have a key role in the modulation of
pancreatic islet insulin release and, thus, of glucose homeostasis (Kim and Egan, 2008).
Drugs that inhibit the effect of DPP-4, which cleaves both GLP-1 and GIP, extend the half-
lives of these hormones and increase islet function and glycaemic regulation in T2DM
(Pratley and Salsali, 2007, Ahren et al., 2004).
In addition to their ability to lower glucose there is increasing evidence that DPP-4
inhibitors or GLP-1R agonists improve endothelial function in T2DM patients (J Motta et
al., 2012, Shah et al., 2011b) and in type 1 diabetic rats (Özyazgan et al., 2005). It has
been reported that GLP-1 improves endothelial function and exerts protective actions in
type-2 diabetic patients with coronary artery disease (Nystrom et al., 2004) and it has been
reported that linagliptin and sitagliptin improve endothelium-dependent relaxation in type
2 diabetic rats (Takai et al., 2014)
Diabetes is associated with overproduction of reactive oxygen species (ROS)
which is in turn associated with impaired NO release and/or activity causing endothelial
dysfunction (De Vriese et al., 2000, Leo et al., 2011b). There is growing evidence that
GLP-1R agonists and DPP-4 inhibitors may improve endothelial function. GLP-1R
agonists are reported to increase eNOS activity in isolated endothelial cells (Hattori et al.,
2010, Erdogdu et al., 2010) and to enhance endothelium-dependent dilatation in human
subjects (Basu et al., 2007). Similarly DPP-4 inhibitors increase eNOS activity (Shah et
al., 2011b) to increase eNOS expression in zucker obese rats (Aroor et al., 2013) and to
improve endothelium-dependent relaxation in spontaneously hypertensive rats (Liu et al.,
2012). By contrast, there is also a report that sitagliptin and alogliptin impair endothelium-
dependent dilatation in patients with type 2 diabetes (Ayaori et al., 2013). A contributing
factor to improved endothelial function may result from antioxidant effects of both GLP-
1R agonists and DPP-4 inhibitors (Kroller-Schon et al., 2012, Ávila et al., 2013, Erdogdu
85
et al., 2013, Shiraki et al., 2012, Ishibashi et al., 2010, Hendarto et al., 2012) which could
decrease NO inactivation by ROS.
As high glucose induced impairment of endothelium-dependent relaxation in
mesenteric arteries is associated with oxidative stress, thus, the aim of this study was to
investigate whether the acute presence of DPP-4 inhibitors (linagliptin, sitagliptin,
vildagliptin) or GLP-1R agonist, exendin-4 prevents the impairment of endothelial
function in small mesenteric arteries exposed to 40 mM glucose and compare the
antioxidant effects of linagliptin with those of sitagliptin, vildagiptin, and exendin-4 in
vitro. Furthermore, we sought to investigate the effects of linagliptin on the relative
contribution of NO-mediated and EDH-mediated relaxation to endothelium-dependent
responses in vessels exposed to high glucose. The interest in linagliptin was due to results
obtained in earlier part of study where linagliptin, but not the other DPP-4 inhibitors
reduced superoxide production in mesenteric arteries exposed to high glucose solution.
86
3.2 Methods
All procedures were approved by the Animal Experimentation Ethics committee of
RMIT University and complied with the Australian National Health and Medical Research
Council code of practice for the care and use of animals for scientific purposes (AEC
approval number 1121).
3.2.1 Isolation of mesenteric arteries
Male Wistar rats (~8-weeks of age, ~250g) were used. The mesenteric arcade was
isolated after rats were killed by asphyxiation with CO2 followed by exsanguination. The
mesenteric arteries were immediately placed in ice-cold Krebs bicarbonate solution (4.7
mM KCl, 118 mM NaCl, 1.18 mM MgSO4, 25 mM NaHCO3, 11.1 mM D-glucose and 1.6
mM CaCl2) containing the cyclooxygenase (COX) inhibitor indomethacin (10 µM), to
inhibit the production of prostanoids. The third order branch of the superior mesenteric
artery (internal diameter ~300 µm) was isolated, cleared of fat and connective tissue and
cut into small rings (~2 mm). The arteries were mounted on a Mulvany-Halpern myograph
(Danish Myo Technology, Aarhus, Denmark). The artery rings were stabilized at zero
tension for 15 min in Krebs solution bubbled with 95% O2/5% CO2 at 37ºC. Passive
tension of the blood vessels was identified through stretching to obtain an internal
circumference comparable to 90% of that in blood vessels under transmural pressure of
100 mmHg (Mulvany and Halpern, 1977).
3.2.2 Vascular experiments
The maximum contraction of the mesenteric arteries was assessed by addition of an
isotonic physiological saline solution containing a high concentration of K+
(123 mM,
87
KPSS). The basal tension was restored after 3-4 washes using Krebs solution. The
integrity of the endothelium was examined by precontraction of the mesenteric arteries
with phenylephrine (0.1-3 µM) to ~50% of the KPSS response and relaxed with a high
concentration of acetylcholine (ACh, 10 µM). ACh-induced relaxation of the precontracted
rings was more than 80% in all cases indicating that the endothelium was functionally
intact. The mesenteric arteries were incubated for 2 hours with 11 mM glucose or 40 mM
glucose and again precontracted with phenylephrine (0.1-3 µM) to ~50% of the KPSS
response. The high concentration of glucose (40 mM) was based on a previous reports
demonstrating that short incubations (4-6 h) with a similar concentration (44 mM)
impaired endothelial function in isolated blood vessels (Tesfamariam et al., 1991, Qian et
al., 2006). Cumulative concentration-response curves to the endothelium-dependent
relaxant, ACh (0.1 nM-10 µM) and the endothelium-independent relaxant, sodium
nitroprusside (SNP, 0.01 nM-10 µm) were determined after 20 min incubation with
linagliptin (1 µM), sitagliptin (1 µM), vildagliptin (1 µM) or exendin-4 (1 µM). The
concentration of linagliptin was based on the observations of Kroller-Schon et al. (Kroller-
Schon et al., 2012) that 1 µM was the lowest concentration to inhibit the respiratory burst
in polymorphonuclear leucocytes and in preliminary experiments we found that this
concentration had significant antioxidant activity and the same concentration was chosen
for other compounds. Exendin-4 was found to be equally effective to improve endothelial
function at a concentration of 1 µM (supplementary Figure 1). Relaxant responses were
also assessed in the presence of different combinations of N-nitro-L-arginine (L-NNA, 100
μM), an inhibitor of nitric oxide synthase (NOS), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-
1-one (ODQ, 10 µM), an inhibitor of soluble guanylate cyclase (sGC), apamin (1 µM), a
selective blocker of the small conductance calcium-activated K+
channel (SKCa), 1-[(2-
chlorophenyl) (diphenyl)methyl]-1H-pyrazole (TRAM-34, 1 µM), a blocker of
88
intermediate conductance calcium-activated K+
channel (IKCa), iberiotoxin (Ibtx, 100 nM),
a blocker of the large conductance calcium-activated K+
channel (BKCa), superoxide
dismutase (SOD, 50 U/mL) used to reduce superoxide, and pyrogallol (30 µM) used to
generate superoxide.
3.2.3 Superoxide measurement in mesenteric artery
Superoxide production in the mesenteric artery was measured by using L-012 and
lucigenin enhanced-chemiluminescence assays. Segments of mesenteric arteries were
incubated in 40 mM glucose for 2 h and then incubated at 37°C for 30 minutes in Krebs-
HEPES buffer (composition (mM): NaCl 99.90, KCl 4.7, KH2PO4 1.0, MgSO4.7H2O 1.2,
D-glucose 11.0, NaHCO3 25.0, CaCl2.2H2O 2.5, Na HEPES 20.0, pH 7.4) either alone or
in the presence of tempol, a SOD mimetic (100 µM) or linagliptin (1 µM). 300 µL of
Krebs-HEPES buffer, containing L-012 (100 µM, Wako Pure Chemicals, Osaka, Japan)
and the appropriate treatments were placed into a 96-well Optiplate, which was loaded into
a photon counter (Polarstar, BMG Labtech, Melbourne, VIC, Australia) to measure
background photon emission at 37°C. After background counting was completed, a single
ring segment of mesenteric artery was added to each well and photon emission was re-
counted. Background counts were subtracted from superoxide counts and normalized with
dry tissue weight. Artery-derived superoxide was also measured using lucigenin as
previously described (Leo et al., 2011b)
3.2.4 Reagents
All chemicals were purchased from Sigma-Aldrich (St Louis, MO, USA), except
for linagliptin (Boehringer Ingelheim, Germany), acetylcholine perchlorate (BDH
89
Chemicals, Poole, Dorset, UK), and ODQ (Cayman Chemical, Ann Arbor, MI, USA). All
chemicals were dissolved in distilled water with the exception of L-NNA, which was
dissolved in 0.1 M sodium bicarbonate, and indomethacin, which was dissolved in 0.1 M
sodium carbonate.
3.2.5 Statistical analysis
Results are presented as the mean±SEM, n indicates the number of experiments.
The concentration response data from isolated mesenteric arteries were fitted to a
sigmoidal curve using nonlinear regression (Prism version 6.0, GraphPad Software, San
Diego, CA, USA) to calculate the pEC50. The maximum relaxation (Rmax) to ACh or SNP
was determined as a percentage of the phenylephrine precontraction. pEC50 and Rmax
values were compared using one way ANOVA with post hoc analysis using Bonferroni’s
test. p<0.05 was considered statistically significant.
90
3.3 Results
3.3.1 Effects of DPP-4 inhibitors and exendin-4 on vascular superoxide production
Using L-012 and lucigenin chemiluminescence assays, the level of superoxide in
mesenteric arteries incubated in 40 mM glucose was demonstrated to be significantly
higher than arteries incubated in 11 mM glucose (Figure 3.1). Linagliptin (1 µM)
significantly reduced superoxide production in the presence of normal and high glucose
whereas exendin-4 (1 µM) only reduced ROS levels in the presence of high glucose
(Figure 3.1a, b). In contrast, sitagliptin (1 µM) and vildagliptin (1 µM) had no significant
effect on the superoxide levels in the mesenteric rings under either condition (Figure 3.1a).
The ability of linagliptin to reduce superoxide from arteries exposed to normal or high
glucose was also confirmed using lucigenin enhanced chemiluminescence (Figure 3.1b).
91
a)
b)
Figure 3.1 ROS measurement in mesenteric arteries using L-012 (a) and lucigenin (b) enhanced-
chemiluminesence assays. Superoxide levels were increased in mesenteric rings exposed to 40 mM
glucose. Superoxide levels detected in the presence of normal (11 mM) glucose were significantly
reduced by the SOD mimetic tempol (100 μM) or DPI (5 µM), a flavoprotein inhibitor that inhibits
NADPH oxidase and linagliptin (1 µM) but not by any of the other treatments. The elevation of
superoxide caused by high glucose was significantly attenuated by exendin-4 (1 µM) and
linagliptin but not by the other DPP-4 inhibitors. Results are shown as mean±SEM. n=6
experiments. *p<0.05 vs 11 mM glucose untreated, #
p<0.05 vs 40 mM glucose untreated,
Bonferroni’s test.
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3.3.2 Effect of high glucose, DPP-4 inhibitors, and exendin-4 on endothelium-
dependent relaxation
High glucose (40 mM) significantly reduced the sensitivity and maximum
relaxation to ACh, in the mesenteric arteries (Figure 3.2a, Table 3.3). The sensitivity and
maximum relaxation to ACh in mesenteric arteries exposed to mannitol (29 mM
mannitol+11 mM glucose) was similar to that determined in mesenteric arteries exposed to
11 mM glucose, indicating that the high glucose-induced impairment of the endothelium-
dependent relaxation was not due to a hyperosmotic effect (Figure 3.3). SNP responses
were unaffected by the high glucose (Figure 3.2b). Linagliptin (1 µM) significantly
increased the sensitivity to ACh and the maximum relaxation in arteries exposed to 40 mM
glucose (Figure 3.4, Table 3.1). The other DPP-4 inhibitors, sitagliptin (1 µM) and
vildagliptin (1 µM), showed a significant increase in the maximum relaxation, but no
change in the sensitivity to ACh-induced relaxation of mesenteric arteries exposed to 40
mM glucose whereas the GLP-1R agonist exendin-4 (1 µM), also caused a significant
increase in the maximum relaxation and the sensitivity to ACh in the presence of high
glucose (Figure 3.4, Table 3.1). To investigate the role of the GLP-1 receptor in the
beneficial effects of linagliptin and exendin-4, responses to ACh were tested during co-
incubation with the GLP-1 receptor antagonist exendin-fragment (9-39) (1 µM). The
presence of exendin-fragment (9-39) had no effect on ACh-induced relaxation of
mesenteric arteries exposed to 11 or 40 mM glucose (Table 3.2). In the presence of high
glucose exendin-fragment (9-39) prevented the ability of exendin-4 to improve ACh-
induced relaxation (Figure 3.5B) but did not affect the capacity of linagliptin to improve
the endothelium-dependent relaxation (Figure 3.5A, Table 3.2).
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Figure 3.2 Vascular function in mesenteric arteries. Cumulative concentration-response curves to
ACh (a) and SNP (b) in endothelium- undamaged mesenteric arteries. In each group of
experiments (a, b), mesenteric arteries were precontracted with PE to similar levels: (a) 11
mM glucose 65±3, 11 mM glucose+linagliptin 63±2, 40 mM glucose 61±3, 40 mM
glucose+linagliptin 60±1, (b) 11 mM glucose 60±2, 11 mM glucose+linagliptin 61±3, 40
mM glucose 62±3, 40 mM glucose+linagliptin 60±4 %KPSS, n=5-11 experiments. Results
are shown as mean±SEM. *pEC50 significantly different from 40 mM glucose
(Bonferroni’s test, p< 0.05).
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Figure 3.3 The addition of mannitol (29 mM) to glucose (11 mM) had no effect on the
endothelium-dependent relaxation indicating that the glucose-induced dysfunction was not due to
hyperosmosis. *pEC50 significantly different from 11 mM glucose and Mannitol (Bonferroni’s test,
p<0.05), #pEC50 signifiacantly different from 40 mM glucose (Bonferroni’s test, p<0.05), n=6-9.
Results are shown as mean±SEM.
95
Table 3.1 The effects of linagliptin, sitagliptin, vildagliptin and exendin-4, on endothelium-
dependent relaxation to ACh in mesenteric arteries in the presence of normal (11 mM) and high
(40 mM) glucose concentrations.
Glucose (11 mM) Glucose (40 mM)
ACh n pEC50 Rmax n pEC50 Rmax
Control 11 6.96±0.10 98±1 9 6.36±0.12* 91±2
*
Linagliptin 7 7.21±0.05 99±1 6 7.31±0.03# 97±1
#
Sitagliptin 8 6.59±0.05 98±1 9 6.31±0.05 98±1#
Vildagliptin 8 6.58±0.02 97±1 9 6.32±0.04 97±1#
Exendin-4 8 6.61±0.04 99±1 9 6.80±0.05b 97±1
#
All experiments were conducted in the presence of indomethacin (10 µM). n=the number
of experiments. *Significantly different to 11 mM glucose (p<0.05), ANOVA plus
Bonferroni’s multiple comparison test. #Significantly different to control within each
group (p<0.05), ANOVA plus Bonferroni’s multiple comparison test.
96
Figure 3.4 Cumulative concentration-response curves to ACh in endothelium-intact mesenteric
arteries exposed to 11 and 40 mM glucose. In each group of experiments, mesenteric arteries were
precontracted with phenylephrine to a similar level. All experiments were performed in the
presence of indomethacin. Results are shown as mean±SEM of 6-11 experiments. *pEC50
significantly different from 11 mM glucose (Bonferroni’s test, p<0.05). #pEC50 significantly
different from 40 mM glucose (Bonferroni’s test, p<0.05). See Table 3.1 for pEC50 and Rmax values
derived from these curves.
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3.3.3 Effect of pyrogallol on endothelium-dependent relaxation
The effect of the superoxide generator, pyrogallol on ACh-induced relaxation is
shown in (Figure 3.6). Pyrogallol (30 µM) significantly reduced the sensitivity to ACh
(pEC50 control 7.66±0.06, pyrogallol 7.07±0.06, p<0.05, Bonferroni’s multiple comparison
test) but the maximum relaxation to ACh was not affected (Rmax control 96±1%, pyrogallol
95±3%). The addition of superoxide dismutase (SOD, 50 U/mL) as a scavenger of
superoxide, improved the sensitivity to ACh in the presence of pyrogallol (7.64±0.07,
p<0.05, Bonferroni’s multiple comparison test) (Figure 3.6). The sensitivity to ACh in the
presence of pyrogallol was increased by linagliptin (7.64±0.14, p<0.05) or exendin-4
(7.17±0.12, p<0.05) demonstrating that linagliptin and exendin-4 effectively scavenged
superoxide ions produced by pyrogallol.
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Table 3.2 The effect of the GLP-1 receptor antagonist exendin-fragment (9-39) on the ability of
linagliptin and exendin-4 to preserve ACh-induced endothelium-dependent relaxation in
mesenteric arteries.
Glucose (11 mM) Glucose (40 mM)
ACh n pEC50 Rmax n pEC50 Rmax
Control 5 7.03±0.18 97±1 5 5.88±0.16* 86±3
Exendin-fragment (9-39) 5 6.51±0.06 94±2 5 6.37±0.06 88±2
Linagliptin+exendin-frag. ND ND 5 6.80±0.10# 97±2
Exendin-4+exendin-frag. ND ND 5 5.85±0.08 93±3
All experiments were conducted in the presence of indomethacin (10 µM).
n = the number of experiments. Results are shown as mean±SEM of 5 experiments
*Significantly different to 11 mM glucose (p<0.05), ANOVA plus Bonferroni’s multiple
comparison test. #Significantly different to 40 mM glucose (p<0.05), ANOVA plus Bonferroni’s multiple
comparison test, ND= Not determined.
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Figure 3.5 Demonstration of the beneficial effects of linagliptin (panel A) and exendin-4
(panel B), on responses to ACh in the presence of high glucose. The additional presence of
the GLP-1 receptor antagonist exendin fragment (9-39) did not affect the protective actions
of linagliptin but prevented the beneficial effect of exendin-4. In each group of
experiments, mesenteric arteries were precontracted with phenylephrine to a similar level.
All the experiments were conducted in the presence of indomethacin. Results are shown as
mean±SEM of 5 experiments. *pEC50 significantly different from 11 mM glucose
(Bonferroni’s test, p<0.05). #pEC50 significantly different from 40 mM glucose
(Bonferroni’s test, p<0.05). See Table 3.2 for pEC50 and Rmax values derived from these
curves.
100
Figure 3.6 Cumulative concentration-response curves to ACh in the absence (control) or presence
of pyrogallol (30 μM). Pyrogallol significantly decreased the sensitivity to ACh and this effect was
prevented by the presence of linagliptin or exendin-4. Mesenteric arteries were precontracted with
phenylephrine to a similar level. All the experiments were conducted in the presence of
indomethacin. Results are shown as mean±SEM of 5 experiments. *pEC50 significantly different
from control (Bonferroni’s test, p<0.05). The values for the pEC50 under different conditions are
given in the text.
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3.3.4 Effect of linagliptin on NO-mediated relaxation
To investigate the NO-mediated component of relaxation in mesenteric arteries
responses to ACh were assessed in the presence of the combination of TRAM-34 and
apamin. The addition of TRAM-34+apamin to vessel rings exposed to 11 mM glucose
reduced the sensitivity, but not the maximum relaxation, to ACh in comparison to the
control (Figure 3.7A, Table 3.3). By contrast, in arteries exposed to high glucose, the
addition of IKCa and SKCa blockers did not change the sensitivity to ACh, however, the
maximum relaxation was significantly diminished in comparison to the control (Figure
3.7B, Table 3.3). When responses to ACh were compared between arteries (Figure 3.5b)
high glucose reduced the maximum relaxation to ACh in the presence of TRAM-
34+apamin (Table 3.3), indicating that high glucose impaired the contribution of NO to
endothelium-dependent relaxation. The additional presence of linagliptin had no effect on
arteries exposed to 11 mM glucose but significantly increased the maximum relaxation
induced by ACh in arteries exposed to 40 mM glucose in the presence of TRAM-
34+apamin (Table 3.3) indicating that linagliptin enhanced the contribution of NO to
relaxation when the glucose level was elevated.
3.3.5 Effect of high glucose on NOS and Nox2 related protein expression
Incubation of the rings of mesenteric arteries in 11 mM glucose concentration did
not change the expression of total eNOS, whereas, 40 mM glucose concentration
significantly decreased the expression of total eNOS (Figure 3.8a). The expression of
Nox2 was significantly increased in the mesenteric arteries which incubated in 40 mM
glucose compared to 11 mM glucose (Figure 3.8b).
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Figure 3.7 Examination of the effect of high glucose on the contribution of NO to endothelium-
dependent relaxation in mesenteric arteries. Cumulative concentration-response curves to ACh in
the absence (control) or in the presence of TRAM-34+apamin in endothelium-intact mesenteric
arteries exposed to 11 mM (A) or 40 mM glucose (B). In each group of experiments, mesenteric
arteries were precontracted with phenylephrine to a similar level. All the experiments were
conducted in the presence of indomethacin. Results are shown as mean±SEM of 5-9 experiments.
*pEC50 (A) or Rmax (B) significantly different from control (Bonferroni’s test, p<0.05). #pEC50
significantly different from TRAM-34 + apamin (Bonferroni’s test, p<0.05). See Table 3.3 for
pEC50 and Rmax values.
103
Figure 3.8 Protein expression of eNOS (a) and Nox2 (b) in mesenteric arteries exposed to 11 mM
and 40 mM glucose determined by western blot analysis. High glucose (40 mM) significantly
reduced the expression of eNOS and increased the expression of Nox2. n=6 experiments.
Representative blots are shown on each the corresponding graphs. Results are shown as
mean±SEM. *Significantly different either from 11 mM glucose. (Student’s unpaired t-test,
p<0.05).
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3.3.6 Effects of linagliptin on EDH-mediated relaxation
To investigate the EDH-mediated relaxation, the contribution of NO was
eliminated by the presence of L-NNA+ODQ to inhibit eNOS and soluble guanylate
cyclase respectively. L-NNA+ODQ reduced the sensitivity to ACh in arteries exposed to
11 mM glucose (Figure 3.9A, Table 3.3). The sensitivity to ACh in arteries exposed to 40
mM glucose was also significantly reduced in comparison to arteries exposed to 11 mM
glucose in the presence of L-NNA+ODQ (Table 3.3), suggesting that high glucose
impaired the non-NO, non-prostanoid component of endothelium-dependent relaxation.
Linagliptin had no effect on EDH-mediated relaxation in mesenteric arteries exposed to 11
mM glucose (Figure 3.9A, Table 3.3) but improved the sensitivity to ACh-mediated EDH-
mediated relaxation in arteries exposed to high glucose (Figure 3.9B, Table 3.3). When the
contribution of NO was eliminated by L-NNA+ODQ, the maximum relaxation to ACh
was unimpaired in mesenteric arteries exposed to either normal or high glucose
concentration. The addition of TRAM-34 and apamin in the presence of L-NNA+ODQ
significantly reduced ACh-induced relaxation in arteries exposed to 40 mM glucose (Table
3.3). When Ibtx was added to the combination of L-NNA+ODQ+TRAM-34+apamin, the
relaxation to ACh in arteries exposed to 40 mM glucose was abolished (Table 3.3). In
artery rings exposed to 11 mM glucose, ACh continued to cause a maximum relaxation of
15% in the presence of combination of L-NNA+ODQ+TRAM-34+apamin and the
addition of Ibtx caused no further inhibition (Table 3.3). The presence of linagliptin had no
effect on the maximum relaxation induced by ACh in the presence of L-
NNA+ODQ+TRAM-34+apamin or with the addition of Ibtx in mesenteric arteries
exposed to 11 mM glucose (Table 3.3), in contrast, acute treatment with linagliptin
improved the ACh-induced relaxation in arteries exposed to 40 mM glucose in the
presence of L-NNA+ODQ+TRAM-34+apamin (Table 3.3).
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Table 3.3 Effect of L-NNA, ODQ and potassium channel blockers on ACh-induced relaxation of rat mesenteric arteries exposed to normal (11 mM) and high
(40 mM) glucose in the absence or presence of linagliptin (1 µM).
Glucose (11 mM) Glucose (11 mM)+linagliptin Glucose (40 mM) Glucose (40 mM)+linagliptin
ACh n pEC50 Rmax n pEC50 Rmax n pEC50 Rmax n pEC50 Rmax
Control 10 7.31±0.07 95±1 7 7.15±0.08 98±0 9 6.32±0.2# 91±2 6 7.25±0.06 97±1
TRAM34+apamin 6 6.55±0.0* 93±1 7 7.04±0.14 98±1 7 6.46±0.14 44±8
*,# 5 6.74±0.1
Ω 94±1
Ω
L-NNA+ODQ 9 6.64±0.12* 93±1 8 6.67±0.09
* 96±0 8 6.35±0.02 89±1 7 6.91±0.12
Ω 96±2
LNNA+ODQ+
TRAM-34+apamin
7 5.71±0.39* 15±2
* 5 5.52±0.2
* 32±10
* 9 ND 7±0
* 7 4.94±0.6
* 28±7
*
LNNA+ODQ+
TRAM-34
+apamin+Ibtx
4 ND 15±1* 5 ND 25±4
* 7 ND 1±0
* 6 ND 10±1
*
A comparison of the sensitivity (pEC50) and maximum relaxation (Rmax) to ACh in the absence (control), or the presence of TRAM-34 (1
µM)+apamin (1 µM), L-NNA (100 µM)+ODQ(10 µM), L-NNA (100 µM)+ODQ(10 µM)+TRAM-34 (1 µM)+apamin (1 µM) or L-NNA (100
µM)+ODQ(10 µM)+TRAM-34 (1 µM)+apamin (1 µM)+Ibtx (100 nM) in endothelium intact mesenteric arteries. All experiments were
conducted in the presence of indomethacin (10 µM).
n=the number of experiments.
*Significantly different to control within each group (p<0.05), Dunnet’s test, #Significantly different to 11 mM glucose within inhibitor group (p<0.05), Bonferroni’s test,
ΩSignificantly different to 40 mM glucose within inhibitor group (p<0.05), Bonferroni’s test,
ND, not determined.
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Figure 3.9 Examination of the effect of high glucose on the contribution of EDH to endothelium-
dependent relaxation in mesenteric arteries. Cumulative concentration-response curves to ACh in the
absence (control) or in the presence of L-NNA+ODQ in endothelium-intact mesenteric arteries
exposed to 11 mM (A) or 40 mM (B) glucose. In each group of experiments, mesenteric arteries were
precontracted with phenylephrine to a similar level. All the experiments were conducted in the
presence of indomethacin. Results are shown as mean±SEM of 7-9 experiments. *pEC50 significantly
different from control (Bonferroni’s test, p<0.05). #pEC50 significantly different from L-NNA+ODQ
(Bonferroni’s test, p<0.05). See Table 3.3 for pEC50 and Rmax values.
107
3.4 Discussion
This study demonstrated that impairment of endothelial function caused by high (40
mM) glucose in rat mesenteric arteries was prevented by the presence of the DPP-4 inhibitor
linagliptin or by exendin-4, a GLP-1R agonist. Both agents reduced the increase in vascular
ROS caused by the high glucose concentration whereas the other DPP-4 inhibitors sitagliptin
and vildagliptin failed to either reduce oxidative stress or to improve endothelial function.
Importantly, whereas the protective action of exendin-4 involved activation of the GLP-1R
the antagonist exendin fragment (9-39) had no effect on the beneficial action of linagliptin.
Thus linagliptin acted independently of GLP-1 as expected in this isolated tissue and rather
has direct antioxidant activity, perhaps by radical scavenging. High glucose impaired the
contribution of both NO and EDH to endothelium-dependent relaxation and linagliptin
improved the contribution of both of these relaxing factors.
3.4.1 Effects of DPP-4 inhibitors and exendin-4 on vascular superoxide generation
The findings in this study indicate that incubation of mesenteric arteries in high
glucose for 2 hours significantly increased generation of superoxide. This increase in
oxidative stress is consistent with other observations made in blood vessels isolated from rats
with either type 1 or type 2 diabetes (Leo et al., 2011b), which is considered a key cause of
endothelial dysfunction and consequent vascular disease (Fatehi-Hassanabad et al., 2010).
We investigated the ability of the DPP-4 inhibitors, linagliptin, sitagliptin, and vildagliptin as
well as the GLP-1R agonist, exendin-4 to reduce vascular superoxide levels and found that,
whereas linagliptin and exendin-4 showed a significant reduction of superoxide production,
the other DPP-4 inhibitors, sitagliptin and vildgliptin, had no significant effect. Takai et al.
(Takai et al., 2014) reported that linagliptin and sitagliptin improve endothelial function in
type 2 diabetic rats but it is likely that effect is, at least in part, secondary to the lowering of
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plasma glucose. Our observations are similar to that of Kroller-Schon et al. (Kroller-Schon et
al., 2012) who reported that whereas linagliptin significantly inhibited the oxidative burst in
human leucocytes sitagliptin, vildagliptin, alogliptin and saxogliptin were ineffective. The
difference in the antioxidant activity of the DPP-4 inhibitors may be explained by their
structural dissimilarities. Linagliptin has been reported to have antioxidant activity by
inhibiting xanthine oxidase (Yamagishi et al., 2014) but this seems unlikely to explain the
observations in isolated vascular tissue. Perhaps linagliptin acts as a radical scavenger in a
manner similar to polyphenols such as flavonoids (Woodman and Chan, 2004), supported by
its ability to prevent pyrogallol-induced impairment of endothelium-dependent relaxation.
There are reports that vildagliptin can reduce oxidative stress after chronic treatment in rats
with type 1 diabetes (Ávila et al., 2013) and after renal ischaemia and reperfusion (Glorie et
al., 2012) but the mechanism of action has not been established. Reports that exendin-4 has
antioxidant activity (Erdogdu et al., 2013, Padmasekar et al., 2013) and that GLP-1 decreased
ROS production from cardiac microvascular endothelial cells cultured in a high glucose
medium accompanied by a decreased expression of NADPH oxidase subunits (Wang et al.,
2013) indicate a role for activation of GLP-1R in vivo. Thus linigliptin, when administered in
vivo may have 2 mechanisms by which it acts as an antioxidant, direct radical scavenging and
increased activation of GLP-1R secondary to DPP-4 inhibition.
3.4.2 Effects of DPP-4 inhibitors and exendin-4 on vascular endothelial function
In this study, 40 mM glucose significantly reduced the sensitivity and maximum
response to the endothelium-dependent relaxant ACh without affecting endothelium-
independent relaxation as previously reported (Ceriello et al., 1996, De Vriese et al., 2000,
Cosentino et al., 1997, Cosentino et al., 2003). We confirmed that the impairment was not
due to hyperosmosis as the presence of mannitol (29 mM) in addition to normal glucose
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concentration had no effect on endothelium-dependent relaxation. To test whether DPP-4
inhibitors or activation of the GLP-1R improved endothelial function after exposure to high
glucose ACh responses were assessed in the presence of linagliptin, sitagliptin, vildagliptin
and exendin-4. Linagliptin and exendin-4 improved endothelium-dependent relaxation such
that the sensitivity and maximum response to ACh in the presence of high glucose was
restored to the control levels. By contrast sitagliptin and vildagliptin had no effect. The ability
to improve endothelial function parallels the capacity of linagliptin and exendin-4 to reduce
vascular superoxide in the presence of high glucose. To determine whether these beneficial
effects involved activation of the GLP-1R we repeated the experiments in the presence of the
antagonist exendin fragment (9-39) revealing that, whereas the protective actions of exendin-
4 were prevented by the antagonist, the actions of linagliptin were maintained demonstrating
that exendin-4 had GLP-1R-dependent actions but linagliptin did not. Data in this study is
consistent with reports showing that exendin-4 inhibited ROS-induced injury to endothelial
cells in a GLP-1R-dependent manner (Oeseburg et al., 2010, Erdogdu et al., 2013). Together
these observations suggest that when administered in vivo linagliptin has the capacity to exert
an antioxidant effect through 2 mechanisms, direct radical scavenging and by increasing the
activity of GLP-1 at its receptor.
3.4.3 Effect of linagliptin on NO-mediated relaxation
To further examine the mechanism of the improved response to ACh in the presence
of linagliptin we separately investigated the contribution of NO and EDH to relaxation. To
examine the role of NO-mediated relaxation, EDH-mediated relaxation was abolished by the
endothelial KCa channel blockers TRAM-34 and apamin. Under those conditions high
glucose reduced the maximum response to ACh indicating an impaired contribution of NO to
relaxation as has been reported previously (Cosentino et al., 1997) and as observed in animal
110
models of diabetes (Leo et al., 2011b). Interestingly, acute treatment with linagliptin in vitro
significantly increased the maximum relaxation to ACh in high glucose exposed arteries in
comparison to normal glucose. The restoration of NO activity by linagliptin treatment is
consistent with its antioxidant effect reducing vascular superoxide and increasing the
bioavailability of NO.
3.4.4 Effect of linagliptin on EDH-mediated relaxation
In order to evaluate the relative contribution of EDH to endothelium-dependent
relaxation, we examined ACh-induced relaxation in the presence of L-NNA and ODQ to
prevent the effect of NO synthesis and sGC activity respectively. The sensitivity to ACh in
mesenteric arteries exposed to high glucose was significantly reduced in the presence of L-
NNA and ODQ in comparison to normal glucose, indicating that high glucose impaired the
contribution of EDH to endothelium-dependent relaxation, as previously reported (Ozkan and
Uma, 2005, Liu and Gutterman, 2002, Clark and Fuchs, 1997). Acute treatment with
linagliptin of the mesenteric arteries exposed to high glucose showed enhanced EDH-
mediated relaxation which might be due to the reduction of superoxide levels by acute
linagliptin treatment.
3.4.5 Conclusion
Acute treatment of rat mesenteric arteries exposed to high glucose in vitro with the
DPP-4 inhibitor linagliptin or the GLP-1R agonist exendin-4 reduces superoxide generation
and preserves the contribution of both NO and EDH to endothelium-dependent relaxation.
Interestingly, whilst the protective actions of exendin-4 were GLP-1R dependent, linagliptin-
induced improvement of endothelium-dependent relaxation was not affected by the GLP-1R
111
antagonist consistent with a capacity to directly scavenge ROS. Linagliptin enhanced both
NO and EDH activity to improve endothelial function in the mesenteric arteries. Importantly,
under the conditions of these experiments, linagliptin and exendin-4 improved endothelial
function in the absence of any change in glucose levels. This suggests that it may be
worthwhile to explore the potential for these agents to improve vascular function in
pathologies beyond their current use in type 2 diabetes.
112
113
Chapter 4
Acute treatment with the DPP-4 inhibitor
linagliptan improves endothelium-
dependent relaxation of rat mesenteric
arteries from STZ-induced type 1 diabetic
rats
4.1 Introduction
The impairment of endothelium-dependent relaxation has been identified as a critical
factor in the initiation and development of diabetes-induced vascular complications (De
Vriese et al., 2000, Fatehi-Hassanabad et al., 2010). Diabetes mellitus type 2 is characterised
by insulin resistance and beta-cell dysfunction, causing hyperglycaemia and secondary
mirco-macrovascular complications (Bailey, 2008, Kimura et al., 2003). Hypoglycaemic
agents and insulin when used in the management of diabetes do not prevent the development
of parallel vascular complications (Brown et al., 2010, Holman et al., 2008). Under diabetic
conditions, generation of oxygen radicals, primarily superoxide anion as a consequence of
hyperglycaemia, has a key role in the pathogenesis of vascular complications (Brownlee,
2001, Forstermann, 2008, Li et al., 2013, Nishikawa et al., 2000). Potential targets for
pharmacological therapies include eNOS, NADPH-oxidase and mitochondria which all have
been reported as sources of ROS generation in diabetes (Fatehi-Hassanabad et al., 2010, Hink
et al., 2001).
Dipeptidyl peptidase-4 (DPP-4) inhibitors are compounds used in the management of
type 2 diabetes mellitus (Drucker and Nauck, 2006). We (Chapter 3) and other have
114
demonstrated that in addition to their glucose lowering action, the DPP-4 inhibitors might
exhibit a variety of other biological actions such as antioxidant and vasorelaxant effects.
(Kroller-Schon et al., 2012). Furthermore, a number of studies have revealed that DPP-4
inhibitors improve the activity of vascular eNOS (Shah et al., 2011b) or increased the
expression of eNOS, for example in Zucker obese rats (Aroor et al., 2013). In the previous
chapter, the acute antioxidant effect of the DPP-4 inhibitor linagliptin was demonstrated to
restore endothelial function in small mesenteric arteries exposed to a high glucose
concentration (Chapter 3). It was therefore hypothesised that acute treatment with linagliptin
would have the ability to reduce the oxidative stress and inhibit the source of ROS generation
in diabetic vasculature and consequently to improve endothelial function.
Endothelial dysfunction induced by hyperglycaemia in diabetes arises due to
impairment of both NO and/or EDH-mediated relaxation and vascular oxidative stress
(Heitzer et al., 2000, Mather et al., 2001, Antoniades et al., 2004, Leo et al., 2011b). Thus,
the aim of the present study was to explore whether acute treatment of small mesenteric
arteries from type 1 diabetic rats in vitro with the DPP-4 inhibitor linagliptin restores
microvascular endothelial function, and if so, whether the effects are through direct
scavenging of ROS. Additionally, we investigated whether linagliptin improved contribution
of NO and/or EDH to endothelium-dependent relaxation in diabetic arteries.
4.2 Materials and Methods
The experiments were approved by the Animal Experimentation Ethics Committee of
RMIT University (AEC approval number 1121) and complied with the Australian National
Health and Medical Research Council code of practice for the care and use of animals for
scientific purposes.
115
4.2.1 Experimental animals and induction of type 1 diabetes
Diabetes was induced according to the methods described in Chapter 2.1. Briefly,
male ~8 week old Wistar rats, weighing approximately 200 g, were randomly divided into
two groups: normal and diabetic. Type-1 diabetes was induced by a single injection of
streptozotocin (STZ, 50 mg/kg) into the tail vein after overnight fasting. The normal group
received an equivalent volume of the vehicle (0.1 M citrate buffer, pH 4.5). Blood glucose
was measured at the time of experiment for all groups of rats using ACCU-CHECK
Advantage II monitor (Roche, Mannheim, Germany). Rats with a blood glucose of >20
mmol/L were considered to be diabetic.
4.2.2 Vascular function assay
The vascular function experiments were performed according to methods described in
Chapter 2.3. Briefly, mesenteric arteries were precontracted to a similar level using PE (0.1-3
µM), and cumulative concentration-response curves to ACh (0.1 nM/L-10 µM) and SNP
(0.01 nM-10 µM) were investigated. In addition, responses to ACh and SNP were examined
after 20 minutes incubation with different combinations of L-NNA (100 µM), a non-selective
eNOS inhibitor, ODQ (10 µM), a sGC inhibitor, TRAM-34 (1 µM), a selective blocker of
IKCa, Ibtx (100 nM), a selective blocker of BKCa and apamin (1 µM), a SKCa inhibitor.
4.2.3 Assessment of ROS in mesenteric artery
ROS in the mesenteric artery was measured using either L-012 or lucigenin as
described in Chapter 2.4.1.
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4.2.4 Reagents
All chemicals were purchased from Sigma-Aldrich (St Louis, MO, USA), except for
ODQ (Cayman Chemical, Ann Arbor, MI, USA), and acetylcholine perchlorate (BDH
Chemicals, Poole, Dorset, UK). Linagliptin was a gift from (Boehringer Ingelheim,
Germany). All drugs were dissolved in distilled water with the exception of indomethacin,
which was dissolved in 0.1 M sodium carbonate, and L-NNA, which was dissolved in 0.1 M
sodium bicarbonate.
4.2.5 Statistical analysis
Data were expressed as mean ± SEM, n indicates the number of experiments. The
concentration response data from rat isolated mesenteric arteries were fitted to a sigmoidal
curve using nonlinear regression (Prism version 6.0, GraphPad Software, San Diego, CA,
USA) to calculate the pEC50. The maximum relaxation (Rmax) to ACh or SNP was determined
as a percentage of the phenylephrine precontraction. Differences between groups of pEC50
and Rmax values were compared and analysed using one way ANOVA with post hoc analysis
using Bonferroni’s test or Student’s unpaired t-test as appropriate. p<0.05 was considered
statistically significant.
4.3 Results
4.3.1 Body weights and blood glucose
Table 4.1 shows the levels of body weight gained, blood glucose, and HbA1c. Ten
weeks after treatment with STZ, the body weight gained was significantly higher in the
control than in diabetic group (Table 4.1). In diabetic rats, the blood glucose and HbA1c
levels were significantly greater compared to normal rats (Table 4.1).
117
4.3.2 Effect of diabetes and linagliptin on superoxide production
The superoxide level in mesenteric arteries was measured by L-012 and lucigenin
chemiluminescence assays. The level of superoxide generation in intact mesenteric arteries
from diabetic rats was significantly greater than in normal rats. Treatment with tempol, a cell
permeable SOD mimetic or DPI, a flavoprotein inhibitor that inhibits NADPH-oxidase,
reduced the production of superoxide anions to equivalent levels in normal and diabetic rats
(Figure 4.1a, b). The acute presence of linagliptin (1 µM) attenuated the generation of
superoxide anions in mesenteric arteries from normal and diabetic rats (Figure 4.1a, b).
118
Table 4.1 Mean body weight gained, blood glucose, and HbA1c levels at the end of experiment of
normal and diabetic rats.
n= the number of rats.
*Significantly different from normal group (Student’s unpaired t-test, p<0.05).
Results are shown as mean±SEM.
10 weeks after vehicle or STZ treatment
n Normal n Diabetic
Body weight (g) 10 524±17 9 372±16*
Blood glucose (mM) 8 5.3±0.2 9 28.5±1.0*
HbA1c (%) 9 5.2±0.1 9 13.0±0.2*
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Figure 4.1 Measurement of ROS in intact mesenteric arteries. In diabetic arteries, superoxide (a),
NADPH-oxidase activity (b) was increased and were decreased by acute presence of linagliptin or by
the presence of DPI, a flavoprotein inhibitor that inhibits NADPH oxidase, or tempol, a cell
permeable SOD mimetic. Results are shown as mean±SEM. n=5-9 experiments. *p<0.0001 vs
normal, #p<0.0001 vs diabetic.
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4.3.3 Effect of diabetes and linagliptin on vascular function
The mesenteric arteries from diabetic rats showed a significant impairment of their
relaxation response to ACh with a reduction in sensitivity, but not the maximum relaxation
(Figure 4.2a, Table 4.2). In contrast, the sensitivity (diabetic, 7.56±0 vs. normal, 7.47±0, n=
4-5, p<0.05) and maximum relaxation (diabetic, 97±16% vs. normal, 97±7%, n= 4-5,
p<0.05) to SNP were not affected (Figure 4.2b). The acute presence of linagliptin in arteries
from normal rats did not affect the sensitivity and response to ACh, but, in mesenteric arteries
from diabetic rats linagliptin, caused a significant increase in the sensitivity (Figure 4.2a,
Table 4.2). The presence of linagliptin did not affect the sensitivity or maximum response to
SNP in normal or diabetic rats (Figure 4.2).
121
Figure 4.2 Cumulative concentration-response curves to ACh (a), SNP (b) in endothelium-intact
mesenteric arteries. In each group of experiments (a, b), mesenteric arteries were precontracted with
PE to similar levels: (a) normal 60±1, normal+linagliptin 62±3, diabetic 59±1, diabetic+linagliptin
63±1, (b) normal 58±2, normal+linagliptin 57±3, diabetic 61±3, diabetic+linagliptin 59±4 %KPSS,
n=5-12 experiments. Results are shown as mean±s.e.m. *p<0.05 vs. diabetic. See Table 4.2 for pEC50
and Rmax values for ACh.
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4.3.4 Role of NO in normal and diabetic mesenteric arteries
The response to ACh-induced relaxation in mesenteric arteries from normal rats was
reduced by the presence of TRAM-34 and apamin to block the IKCa and SKCa channels
(Figure 4.3a, Table 4.2). In contrast, in mesenteric arteries from diabetic rats, the sensitivity
to ACh was unaffected by the presence of a combination of TRAM-34 and apamin (Figure
4.3b, Table 4.2), but the maximum relaxation was significantly reduced (Figure 4.3b, Table
4.2). The presence of a combination of TRAM-34, apamin and Ibtx inhibited the maximum
relaxation of vessels from both normal and diabetic rats (Table 4.2). This is indicating that
NO contributes to endothelium-dependent relaxation which is impaired due to diabetes.
4.3.5 Role of EDH-type relaxation in normal and diabetic mesenteric arteries
The sensitivity to ACh in both normal and diabetic mesenteric arteries were
significantly reduced by the presence of NO synthase inhibitor, L-NNA and a sGC inhibitor,
ODQ (Figure 4.4a, b, Table 4.2). When the contribution of NO was eliminated by L-
NNA+ODQ, the sensitivity to ACh was significantly lower in the diabetic compared to the
normal arteries (Table 4.2), indicating that diabetes impairs the contribution of a non-NO and
non-prostanoid factor to endothelium-dependent relaxation. The maximum relaxation to ACh
was unaffected by the presence of L-NNA+ODQ in normal or diabetic arteries.
123
Table 4.2 Effect of L-NNA, ODQ and potassium channel blockers on ACh-induced relaxation of mesenteric arteries from normal and diabetic rats in the
absence or presence of linagliptin (1 µM).
A comparison of the sensitivity (pEC50) and maximum relaxation (Rmax) to ACh in the absence (control), or the presence of TRAM-34 (1 µM)
+apamin (1 µM), L-NNA (100 µM)+ODQ (10 µM), L-NNA (100 µM)+ODQ (10 µM)+TRAM-34 (1 µM)+apamin (1 µM) or L-NNA (100 µM)
+ODQ (10 µM)+TRAM-34 (1 µM)+apamin (1 µM)+Ibtx (100 nM) in endothelium intact mesenteric arteries. All experiments were performed in
the presence of indomethacin (10 µM). n = the number of experiments.
*Significantly different to control within each group, p<0.05, Bonferroni’s test,
ФSignificantly different to normal within inhibitor group, p<0.05, Bonferroni’s test,
¥Significantly different to diabetic within inhibitor group, p<0.05, Bonferroni’s test. Results are shown as mean±SEM,
ND= not determined.
Normal Normal+linagliptin Diabetic Diabetic+linagliptin
ACh n pEC50 Rmax (%) n pEC50 Rmax (%) n pEC50 Rmax (%) n pEC50 Rmax (%)
Control 10 7.20±0.11 98±0 6 7.06±0.09 95±1 9 6.12±0.1Ф 96±1 8 7.03±0.09 96±1
TRAM34+apamin 6 6.62±0.12* 93±1 7 7.07±0.20 94±2 7 6.40±0.20 54±8
*Ф 6 6.71±0.3 93±2¥
L-NNA+ODQ 10 6.65±0.12* 92±2 10 6.49±0.20 96±2 8 5.66±0.06
*Ф 78±7 8 6.41±0.17¥ 91±7
LNNA+ODQ+
TRAM-34+apa+Ibtx
4 ND 6±1*
6 ND 6±0* 4 ND 4±0
* 4 ND 3±1
*
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4.3.5 Effect of linagliptin on NO-mediated relaxation
In order to investigate the role of the NO-mediated component of the relaxation,
responses to ACh were assessed in the presence of the KCa blockers, TRAM-34, apamin and
Iberiotoxin.. When the responses were compared between mesenteric arteries from normal
and diabetic rats, it is apparent that diabetes significantly reduced the maximum relaxation
induced by ACh in the presence of TRAM-34+apamin, indicating that NO-mediated
relaxation was impaired by diabetes. Acute treatment with linagliptin had no effect in normal
mesenteric arteries (Figure 4.3c, Table 4.2), but significantly increased the maximum
relaxation in mesenteric arteries from diabetic rats (Figure 4.3d, Table 4.2). A similar finding
was observed in linagliptin treated diabetic mesenteric arteries in the presence of combination
of TRAM-34+apamin+Ibtx, indicating that linagliptin was able to improve the contribution
of NO to endothelium-dependent relaxation in mesenteric arteries from diabetic rats (Table
4.2).
125
Figure 4.3 Contribution of NO to endothelium-dependent relaxation in mesenteric arteries.
Investigation of NO-mediated relaxation in mesenteric arteries isolated from normal (a), diabetic (b)
in the absence or presence of linagliptin, normal+linagliptin (c), diabetic+linagliptin (d) rats. In each
group of experiments, arteries were precontracted with PE to similar level. n=6-7 experiments.
Results are shown as mean±s.e.m. *p<0.05 vs diabetic. See Table 4.2 for values.
126
4.3.6 Effect of linagliptin on EDH-type relaxation
To identify the role of EDH-mediated relaxation, the action of NO-mediated
relaxation was abolished via the addition of L-NNA and ODQ. In the presence of L-
NNA+ODQ, the sensitivity but not the maximum response to ACh was significantly reduced
in mesenteric arteries from diabetic rats compared to normal rats, suggesting that diabetes
impaired the EDH type response. The presence of linagliptin in vitro had no significant effect
on ACh-induced EDH-mediated relaxation in mesenteric arteries from normal rats (Figure
4.4a, Table 4.2), but significantly increased the sensitivity to ACh in mesenteric arteries from
diabetic rats (Figure 4.4b, Table 4.2), indicating that linagliptin improved the contribution of
EDH-type relaxation in diabetic rats. The addition of the combination of L-
NNA+ODQ+TRAM-34+apamin+Ibtx, abolished ACh-induced relaxation in either untreated
diabetic or linagliptin-treated diabetic as well as untreated normal or linagliptin-treated
normal mesenteric arteries (Table 4.2).
127
Figure 4.4 Contribution of EDH to endothelium-dependent relaxation in mesenteric arteries.
Examination of EDH-type relaxation in mesenteric arteries isolated from normal and diabetic rats in
the absence or presence of linagliptin, (a), normal+linagliptin, (b) diabetic+linagliptin. In each group
of experiments, arteries were precontracted with PE to similar level. n=8-10 experiments. Results are
shown as mean±s.e.m. See Table 4.2 for values of pEC50 and Rmax derived from these data.
128
4.4 Discussion
This study demonstrated that, after 10 weeks of diabetes, endothelial dysfunction was
evident in the rat small mesenteric artery. This was due to a decreased contribution of both
NO-mediated and EDH-type relaxation to endothelium-dependent relaxation which
associated with increase in superoxide production. This study also demonstrated that acute
treatment of mesenteric arteries from type 1 STZ-induced diabetic rats attenuates the high
levels of oxidative stress caused by hyperglycaemia and ameliorates endothelial dysfunction
in mesenteric arteries from diabetic rats. Interestingly, linagliptin acted independently of
glucose lowering action and rather has direct antioxidant activity, possibly via radical
scavenging as demonstrated in (Chapter 3). Hyperglycaemia in type 1 STZ-induced diabetic
rats impaired the contribution of NO and EDH to endothelium-dependent relaxation and
acute treatment in vitro with linagliptin enhanced the contribution of both these mediated-
relaxation factors.
4.4.1 Effect of diabetes on superoxide and endothelium-dependent relaxation
In this study, the results indicate that type 1 STZ-induced diabetes augmented the
level of ROS production in mesenteric arteries which is consistent with previous reports (Leo
et al., 2010, Leo et al., 2011b, Inoguchi et al., 2000, Guzik et al., 2002). The increase in
superoxide was concomitant with a selective impairment of endothelium-dependent
relaxation in the mesenteric arteries. The level of superoxide production and the degree of
impairment of endothelial function is consistent with the opportunity that hyperglycaemia-
induced oxidative stress was responsible for the impairment of endothelium-dependent
relaxation in the mesenteric arteries. The overproduction of superoxide by the mechanism of
mitochondrial electron transport chain due to hyperglycaemia-induced oxidative stress
129
becomes an important interpretation for the pathogenesis of diabetic complications
(Brownlee, 2001). This mechanism is followed by the activation of four downstream
pathways: increased polyol pathway flux, increased formation of advanced glycosylation end
products, activation of protein kinase C, and increased hexosamine pathway flux (Nishikawa
et al., 2000).
4.4.2 Effect of diabetes on NO and EDH-type response
In the resistance vessels, such as the rat mesenteric artery, endothelium-dependent
relaxation is mediated by multiple factors including NO and EDH (Edwards et al., 2010).
High glucose concentration-induced oxidative stress has been associated with the impairment
of both NO- and EDH-mediated relaxation (Chapter 3) (De Vriese et al., 2000). In diabetes,
the increase in superoxide anions production can cause impairment in endothelium-dependent
relaxation via a number of mechanisms. Superoxide reacts with NO immediately, to form
peroxynitrite which simultaneously causes reduction in the availability of NO for relaxation
and increases peroxynitrite-induced toxicity. For example, peroxynitrite has been
demonstrated to uncouple endothelial NO synthase due to tetrahydrobiopterin (BH4)
oxidation, therefore additional stimulating the synthesis of superoxide (Münzel et al., 2010),
and leads to protein nitration of the calcium-activated potassium channels which are
responsible for EDH-type responses (Liu and Gutterman, 2002). Furthermore, it has been
reported that the peroxynitrite alone can cause impairment to EDH-type responses in the
mesenteric arteries, which might be improved by the existence of antioxidant treatment (Li et
al., 2008).
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4.4.1 Effect of linagliptin on vascular superoxide and relaxation
As we described early, in diabetes, increases in the generation of superoxide caused
an impairment in eNOS activity, increased NO inactivation, increased eNOS uncoupling via
oxidation of BH4, further increased activity of NADPH oxidase, and increased formation of
peroxynitrate which may promote the initiation of endothelial dysfunction. The presence of
linagliptin in vitro significantly reduced increased level of superoxide generation detected,
indicating that the linagliptin may reduce the oxidative stress, possibly by direct scavenging
of superoxide and improved endothelium-dependent relaxation in mesenteric arteries from
type 1 diabetic rats without affecting arteries from normal rats. Our findings in this study are
consistent with our findings in (Chapter 3) and other observations demonstrated that
treatment with linagliptin improves injury to cardiovascular tissue induced by salt-sensitive
hypertension which seem to be attributed to the reduction of oxidative (Koibuchi et al., 2014)
or enhancement of endothelial function and reduced vascular stress in aortic artery challenged
to experimental sepsis (Kroller-Schon et al., 2012).
4.4.2 Effect of linagliptin on NO-mediated relaxation
To investigate NO-mediated relaxation, ACh-induced relaxation was assessed in the
presence of endothelial KCa blockers to inhibit the EDH-mediated relaxation. This inhibition
of the EDH-type response, revealed significant decrease in the sensitivity to ACh and the
maximum relaxation in diabetic arteries compared to normal arteries. Acute treatment with
linagliptin significantly increased the maximum relaxation mediated by ACh in mesenteric
arteries from diabetic rats in comparison to untreated mesenteric arteries from diabetic rats,
indicating that linagliptin improved the contribution of NO to endothelium-dependent
relaxation in diabetic mesenteric arteries. The prevention of peroxynitrite formation and the
reduction of ROS activity in diabetic vasculature in rats might be at least in one part via rapid
131
free radical scavenging effect of linagliptin as we demonstrated in (Chapter 3), in order to
preserve the bioavailability of NO in vascular endothelial cells as we also observed in this
study. The response to SNP-induced endothelium-independent relaxation in this study was
comparable in both normal and diabetic rats, demonstrating that in both normal and diabetic
rats, the responses to exogenous NO-induced relaxation in smooth muscle cells was not
impaired.
4.4.3 Effect of linagliptin on EDH-mediated relaxation
In order to investigate the contribution of EDH-type relaxation to endothelium-
dependent relaxation, the NO-mediated relaxation was inhibited by the application of L-NNA
and ODQ to inhibit NO synthesis and sGC activity respectively. In the presence of L-
NNA+ODQ, the sensitivity to ACh was decreased in diabetic arteries when compared to
normal arteries, showing that diabetes impaired the contribution of EDH to endothelium-
dependent relaxation as previously reported (Leo et al., 2011b, Makino et al., 2000, Wigg et
al., 2001). It has been reported that superoxide generation by auto-oxidation of pyrogallol
might impair EDH type responses in rat mesenteric arteries (Ma et al., 2008). Furthermore, it
has been suggested that K+ as an one of EDH factor and the vasodilatation response to K
+ is
impaired in the mesenteric arteries in diabetic rats (Makino et al., 2000). Acute treatment
with linagliptin of mesenteric arteries from diabetic arteries improved EDH-mediated
relaxation which likely due to the antioxidant effect of linagliptin (Chapter 3).
4.4.4 Conclusion.
In the present study we have demonstrated that acute treatment of mesenteric arteries
from diabetic rats in vitro with the DPP-4 inhibitor linagliptin significantly reduces the
132
generation of superoxide and preserves the contribution of both NO and EDH to
endothelium-dependent relaxation. As we demonstrated in (Chapter 3), the attributed
beneficial protective effect of linagliptin in this study, at least in part, by rapidly scavenge
ROS and reduce oxidative stress to increase NO bioavailability and improve EDH-type
response in vascular endothelial cells. Interestingly, the beneficial effect of linagliptin to
protect NO and EDH activity observed in this study shows that it has potential as therapeutic
agent for use in the reduction and/or prevention of microvascular complications associated
with diabetes beyond its current use in type 2 diabetes.
133
134
Chapter 5
The DPP-4 inhibitor linagliptan restores
endothelial function of mesenteric arteries
from rats fed a western diet
5.1 introduction
Cardiovascular morbidity and mortality have been reported to be associated with
metabolic syndrome (Isomaa et al., 2001). Endothelial dysfunction is known to be linked to
obesity, atherogenesis, hypertension and insulin resistance that enhances the risk of
cardiovascular diseases (Caballero, 2003). It has been observed that obesity, particularly in
viscera, is correlated with the early stages of development of endothelial dysfunction (Arcaro
et al., 1999, Caballero, 2003). Excessive considerations have been devoted to high-fat content
of diet which is proposed to be a key event and responsible for the development of obesity
and subsequent vascular dysfunction, even before progress to type 2 diabetes mellitus
(Cuevas et al., 2000, Woo et al., 2004, Hajer et al., 2008) These observations demonstrate
that the consumption of high-fat diet is associated with the development of the impairment of
endothelium-dependent relaxation of large arteries due to transformed NO signaling
mechanisms (Molnar et al., 2005, Roberts et al., 2000).
It is well established that small arteries and arterioles are primarily involved in
determination of circulatory resistance and tissue perfusion. Nevertheless, few if any studies
have explored the effect of western diet (WD) on the change of microvascular function. Also,
it is of specific interest to investigate whether the DPP-4 inhibitor linagliptin which recently
we (Chapter 3 and 4) demonstrated was able to act as an antioxidant compound that can
135
ameliorate endothelial dysfunction in small mesenteric arteries isolated from STZ-treated
rats. Therefore, the aim of the studies presented in this chapter was to investigate whether
acute in vitro treatment with linagliptin reverses endothelial dysfunction in small resistance
mesenteric arteries from WD fed rat and, if so, whether it acts via direct scavenging of
superoxide anions and/or prevention of the sources of ROS generation in endothelial cells in
mesenteric arteries. Additionally, we sought to explore the beneficial effects of linagliptin on
the contribution of NO and/or EDH to endothelium-dependent relaxation in small resistant
arteries from WD fed rats.
5.2 Materials and Methods
All procedures were approved by the Animal Experimentation Ethics committee of
RMIT University and complied with the Australian National Health and Medical Research
Council code of practice for the care and use of animals for scientific purposes (AEC
approval number 1245).
5.2.1 Experimental animals
Male Wistar Hooded rats (Monash University, Australia) weighing 180–200 g at the
beginning of the study were used. At least one week acclimation was allowed prior to any
testing and during this period rats were handled daily to reduce stress. At the start of the
feeding regime, rats were housed in groups of four under a light/dark cycle (12 h/12 h), room
temperature (21±1°C) and humidity (30–70%) kept constant with water available ad libitum
in the home cage. Animals were randomly assigned to either a control (SD, Standard
AIN93G rodent diet, 7% total fat including 1.05% total saturated fatty acids; Specialty Feeds,
Perth, Australia) or western diet (WD SF00-219, 21% total fat including 1.80% total
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saturated fats and 0.15% cholesterol; Specialty Feeds, Perth, Australia). Animals were
allowed ad lib access to diets for 7 weeks after which they were placed on a food restricted
diet where animals received 70% of their normal daily food consumption for a period of 10
weeks. Food intake and body weight were measured twice weekly. The rats were killed 10
weeks after being placed on their restricted diet by asphyxiation by CO2 inhalation, followed
by decapitation, and their abdomen opened to isolate the mesenteric arteries. Blood samples
were obtained from the carotid arteries and the blood glucose levels were measured using a
one-touch glucometer (Roche, Sydney, New South Wales). Glycated haemoglobin (HbA1c)
was measured using the in2itTM
(II) analyser (Bio-Rad, Hercules, CA, USA).
5.2.2 Isolation of mesenteric arteries
After rats were killed, the mesenteric arteries were isolated and immediately kept in
ice cold Krebs bicarbonate solution. The third-order branch mesenteric arteries (internal
diameter ~200-300 µm) were isolated and prepared as described in Chapter 2.2.
5.2.3 Vascular function experiments
Vascular reactivity was evaluated as previously described (Chapter 2.3). Briefly, after
maximum contraction of the vessels and the assessment of the integrity of the endothelium,
the mesenteric arteries were precontracted with PE (0.1-3 µM) to attain comparable active
tension and cumulative concentration-response curves to ACh (0.1 nM – 10 µM) and SNP
(0.1 nM–10 µM) were obtained. In some experiments, cumulative concentration- responses
to ACh were assessed after 20 minutes incubation with different combinations of L-NNA
(100 μM), a non-selective NOS inhibitor, ODQ (10 μM), a sGC inhibitor, TRAM-34 (1 μM),
a selective blocker of IKCa and apamin (1 μM), a SKCa inhibitor.
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5.2.4 Measurement of superoxide generation in mesenteric arteries
Superoxide anion generation in the mesenteric arteries was measured using the
lucigenin-enhanced chemiluminescence assay as described in Chapter 2.5.1.
5.2.5 Western Blot
Tissue samples of first order, second order, and third order mesenteric arteries from 2
rats from the same treatment group were pooled and considered as n=1. Western blots were
accomplished as described in Chapter 2.7. To explore eNOS mono/dimer development in
tissue sample, a non-boiled sample was resolved by 6% SDS-PAGE at 4ºC overnight
(Chapter 2.7.5).
5.2.6 Reagents
All chemicals were purchased from Sigma-Aldrich (St Louis, MO, USA), except for
linagliptin (Boehringer Ingelheim, Germany), acetylcholine perchlorate (BDH Chemicals,
Poole, Dorset, UK), and ODQ (Cayman Chemical, Ann Arbor, MI, USA). All chemicals
were dissolved in distilled water with the exception of L-NNA, which was dissolved in 0.1 M
sodium bicarbonate, and indomethacin, which was dissolved in 0.1 M sodium carbonate.
5.2.7 Statistical analysis
Results are presented as the mean±SEM, n indicates the number of experiments. The
concentration response data from isolated mesenteric arteries were fitted to a sigmoidal curve
using nonlinear regression (Prism version 6.0, GraphPad Software, San Diego, CA, USA) to
calculate the pEC50. The maximum relaxation (Rmax) to ACh or SNP was determined as a
percentage of the phenylephrine precontraction. pEC50 and Rmax values were compared using
138
one way ANOVA with post hoc analysis using Bonferroni’s test or Student’s unpaired t-test
as appropriate. p<0.05 was considered statistically significant.
5.3 Results
5.3.1 Effect of WD on body weights
The body weights of SD and WD fed rats is shown in Figure 5.1. The final body
weight in WD fed rats was significantly higher than in SD fed rats.
5.3.2 Effect of WD and linagliptin on oxidative stress in mesenteric arteries
The level of superoxide generation in mesenteric arteries from SD or WD fed rats was
measured by using lucigenin-enhanced chemiluminesence. In WD fed rats, the level of
superoxide induced fluorescence was significantly higher than that in SD fed rats (Figure
5.2). Acute treatment with linagliptin (1 µM) significantly reduced the level of superoxide
generation detected in mesenteric arteries from WD fed rats (Figure 5.2).
5.3.3 Effect of linagliptin on endothelial function in mesenteric arteries from WD fed
rats
The level of contraction to the high K+
physiological saline solution (KPSS, 123
mmol/L) was similar in arteries from SD and WD rats (Figure 5.3). The endothelium-
dependent relaxation agonist ACh and the endothelium-independent relaxation agonist SNP
were used to assess the effect of linagliptin on the vascular function in mesenteric arteries
from SD and WD fed rats. In the mesenteric arteries from WD fed rats, the sensitivity to
ACh, but not the maximum relaxation was significantly decreased compared with SD fed rats
139
(Figure 5.4a), whereas, the sensitivity and the maximum relaxation to SNP were not altered
(Figure 5.4b). Acute treatment with linagliptin had no effect on the response of the
mesenteric arteries to ACh in SD fed rats, but in WD fed rats, linagliptin significantly
increased the sensitivity to ACh (Figure 5.4a).
140
Figure 5.1 Body weight gain over 10 wk of rats receiving a WD and SD. n=8-15. Results are shown
as mean±SEM. n=8-15 experiments. *p<0.0001 vs SD.
141
Figure 5.2 Superoxide levels in intact mesenteric arteries isolated from SD and WD fed rats. NADPH
activity was raised in WD mesenteric arteries which were attenuated with linagliptin treatment.
Results are demonstrated as mean±SEM. n=6-8 experiments. *p<0.05 vs SD #p<0.05 vs WD.
142
Figure 5.3 Mesenteric arteries isolated from SD and WD fed rats induced to maximum contraction by
KPSS (123 mM). KPSS caused a similar level of contraction in arteries from both groups of rats.
143
Figure 5.4 Vascular function in mesenteric arteries. Cumulative concentration-response curves to
ACh (a), SNP (b) in endothelium-intact mesenteric arteries. In each group (a, b), mesenteric arteries
were prcontracted with PE to a similar level: (a) SD 66±2, SD+linagliptin 64±1, WD 65±1,
WD+linagliptin 66±1, (b) SD 64±1, SD+linagliptin 64±1, WD 61±6, WD+linagliptin 66±1 %KPSS,
n=7-10 experiments. Results are shown as mean±SEM. *p<0.05 vs SD.
144
5.3.4 Investigation of the contribution of NO and EDH-mediated relaxation in SD and
WD
In mesenteric arteries from SD fed rats, the response to ACh was partially decreased
by either the combination of N-nitro-L-arginine (L-NNA), a NO synthase inhibitor and 1H-
[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a soluble guanylate cyclase inhibitor or
inhibitors of intermediate-conductance calcium-activated K+ channel (IKCa), small-
conductance calcium-activated K+ channel (SKCa) with 1-[(2
chlorophenyl)(diphenyl)methyl]1H pyrazole (TRAM-34), apamin and iberiotoxin (Ibtx)
respectively, indicating that both NO and EDH contribute to endothelium-dependent
relaxation. Moreover, the response of mesenteric arteries from WD fed rats to ACh-induced
relaxation was decreased in the presence of either NO inhibitors or EDH inhibitors compared
with SD fed rats suggesting that both NO and EDH-mediated relaxation were impaired by
consumption of WD by the rats.
5.3.5 Effect of linagliptin on NO-mediated relaxation in WD
In order to investigate the contribution of NO to endothelium-dependent relaxation, a
combination of IKCa and SKCa channel inhibitors (TRAM-34+apamin) were used. The
addition of TRAM-34+apamin to the mesenteric arteries from SD fed rats reduced the
sensitivity but not the maximum relaxation to ACh, compared to the control (Figure 5.5a,
Table 5.1). On the other hand, in mesenteric arteries from WD fed rats, the addition of IKCa
and SKCa inhibitors significantly reduced both the sensitivity and the maximum relaxation in
comparison to the control (Figure 5.5b, Table 5.1). In mesenteric arteries from WD fed rats,
the reduction in the responses to ACh (the sensitivity and the maximum relaxation) in the
presence of TRAM-34+apamin indicate that WD consumption impaired the contribution of
NO to endothelium-dependent relaxation. Acute treatment with linagliptin significantly
145
increased the response to ACh in the presence of TRAM-34+apamin by increasing the
sensitivity and the maximum relaxation in the arteries from WD fed rats (Table 5.1) indicating
that linagliptin has the ability to enhance the contribution of NO to endothelium-dependent
relaxation.
5.3.6 Effect of WD on the expression of NOS and Nox2
In mesenteric arteries from WD fed rats, the expression of eNOS significantly
decreased in comparison to mesenteric arteries from SD fed rats (Figure 5.6a). In WD fed
rats, the proportion of eNOS expressed as monomer was significantly higher than that in SD
fed rats (Figure 5.6b). Nox2 expression was significantly increased in mesenteric arteries
isolated from WD fed rats (Figure 5.6c).
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Table 5.1 Effect of L-NNA, ODQ and potassium channel blockers on ACh-induced relaxation of rat mesenteric arteries isolated from SD and WD fed rats in
the absence or presence of linagliptin (1 µM).
SD SD+linagliptin WD WD+lingaliptin
ACh n pEC50 Rmax n pEC50 Rmax n pEC50 Rmax n pEC50 Rmax
Control 8 7.56±0.08 Ω
94±2 8 7.44±0.09 Ω
94±0 8 6.97±0.06# 96±2 6 7.46±0.09
Ω 95±1
TRAM34+apamin 6 6.97±0.0* Ω
90±4 6 7.25±0.09 Ω
90±3 6 6.46±0.12*#
65±9 6 6.22±0.08 Ω
91±2
L-NNA+ODQ 5 6.71±0.10* Ω
97±1 5 6.88±0.09* Ω
96±0 5 6.24±0.06*#
95±1 5 6.86±0.05 Ω
93±0
LNNA+ODQ+
TRAM34+apamin+
Ibtx
5 ND 10±1* 5 ND 7±1
* 9 ND 6±1
* 7 ND 7±0
*
A comparison of the sensitivity (pEC50) and maximum relaxation (Rmax) to ACh in the absence (control), or the presence of TRAM-34 (1
µM)+apamin (1 µM), L-NNA (100 µM)+ODQ(10 µM), L-NNA (100 µM)+ODQ(10 µM)+TRAM-34 (1 µM)+apamin (1 µM) or L-NNA
(100 µM)+ODQ(10 µM)+ TRAM-34 (1 µM) +apamin (1 µM)+Ibtx (100 nM) in endothelium intact mesenteric arteries. All experiments
were conducted in the presence of indomethacin (10 µM).
n=the number of experiments.
*Significantly different to control within each group (p<0.05), Bonferroni’s test, #Significantly different to SD within inhibitor group (p<0.05), Bonferroni’s test,
ΩSignificantly different to WD within inhibitor group (p<0.05), Bonferroni’s test,
ND, not determined.
147
Figure 5.5 Contribution of NO to endothelium-dependent relaxation. Mesenteric arteries isolated
from both SD and WD fed rats to examine NO-mediated relaxation in the presence of TRAM-
34+apamin were isolated from SD (a), WD (b). In each group of experiments, arteries were
precontracted with PE to similar levels: 63±2 (a), 65±0.6 (b) %KPSS, n=8-9 experiments. Results are
shown as mean±SEM. #p<0.05 vs SD (control),*p<0.05 vs WD (control). See Table 5.1 for values.
148
Figure 5.6 Analysis of protein expression of eNOS (a, 130 kDa), eNOS dimer and monomer
measured in on representative WB (b, 260 kDa) and Nox2 (c, 58 kDa) in SD and WD mesenteric
arteries. In WD mesenteric arteries, the expression of eNOS significantly reduced and the proportion
of eNOS was expressed as the dimer decreased, and the expression of Nox2 increased. n=6
experiments. Results are shown as mean±SEM. *p<0.05 compared to SD.
149
5.3.7 Effect of linagliptin on EDH-mediated relaxation in WD
To investigate the contribution of EDH to endothelium-dependent relaxation, NO-
mediated relaxation was abolished by the addition of L-NNA+ODQ to block eNOS and
soluble guanylate cycalse respectively. The sensitivity to ACh-enhanced relaxation reduced in
the presence of L-NNA+ODQ in mesenteric arteries from SD fed rats (Figure 5.7a, Table
5.1). Also, the sensitivity to ACh, but not the maximum relaxation, in mesenteric arteries
from WD fed rats was significantly reduced in the presence of L-NNA+ODQ (Figure 5.7b,
Table 5.1), indicating that WD impaired the non-NO, non-prostanoid component in
endothelium-dependent relaxation. Acute treatment with linagliptin did not increase the
sensitivity to ACh and had no effect on EDH-mediated relaxation in arteries from SD fed rats
(Figure 5.7a, Table 5.1), however, linagliptin increased the sensitivity to ACh-induced EDH-
mediated relaxation in mesenteric arteries from WD fed rats (Figure 5.7b, Table 5.1). The
addition of TRAM-34, apamin, and Ibtx in the presence of L-NNA+ODQ significantly
reduced ACh-enhanced relaxation in mesenteric arteries from both SD and WD fed rats
(Table 5.1). Acute treatment with linagliptin did not improve the relaxation induced by ACh
in mesenteric arteries from SD or WD fed rats under those conditions.
150
Figure 5.7 Contribution of EDH to endothelium-dependent relaxation. Mesenteric arteries isolated
from both SD (a) and WD (b) fed rats to investigate EDH-mediated relaxation in the presence of L-
NNA+ODQ. In each group of experiments, arteries were precontracted with PE to similar levels:
62±0.7 (a), 65±0.8 (b) %KPSS, n=8-9 experiments. Results are shown as mean±SEM. See Table 1 for
values of pEC50 and Rmax.
151
5.4 Discussion
This study showed that endothelial dysfunction in mesenteric arteries caused by
consumption of WD (high fat diet) was prevented by acute treatment with the DPP-4 inhibitor
linagliptin. Linagliptin reduced the increased production in vascular ROS caused by WD
cosumption. Consumption of WD impaired the action of both NO and EDH and acute
treatment with linagliptin improved the contribution of both of these relaxing factors.
5.4.1 Effects of WD and linagliptin on oxidative stress
To investigate the effect of WD on oxidative stress, we measured the levels of
superoxide generation in mesenteric arteries from SD and WD fed rats. The findings in this
study indicate that the level of superoxide generation significantly increased in WD
mesenteric arteries compared to SD fed rats. These findings suggest that WD promotes the
development of oxidative stress and endothelial dysfunction primarily because of an
increased NADPH oxidase-derived superoxide anion production which may react with NO,
thereby enhancing the generation of the NO/superoxide reaction endproduct peroxynitrite
which in turn causes uncoupling of eNOS and inactivation of NO as noted in this study.
Furthermore, the findings also showed that the expression of Nox2 was increased in the
mesenteric arteries of WD fed rats, indicating that the impairment of endothelial-dependent
relaxation may be in part due to the overproduction of superoxide anions caused by increased
expression of the enzymatic source of superoxide generation. We inspected the ability of the
DPP-4 inhibitor linagliptin to decrease the levels of vascular superoxide and found that
linagliptin expressed a significant decline in superoxide generation in WD mesenteric
arteries by its ability to act as antioxidant compound as we have demonstrated in chapter 3.
152
5.4.2 Effect of WD diet on endothelial function
ACh was used to evaluate the effect of diet (SD and WD) on endothelium-dependent
relaxation. The findings of this study indicate that the consumption of WD causes impairment
of endothelial function in small resistant mesenteric arteries from these rats, and this is in
agreement with some reports which demonstrated that consumption of high fat diet induced
endothelial cell dysfunction in male and female rat aorta (Roberts et al., 2005, Reil et al.,
1999) or obesity-induced insulin resistance, hyperinsulinemia, and hypercholesterolemia that
have been anticipated to be involved in the development of endothelial dysfunction (Ohara et
al., 1993, Arcaro et al., 1999, Caballero, 2003, Busija et al., 2005). Thompson and colleagues
using coronary arteries and Woodman and colleagues using brachial arteries demonstrated
that the consumption of high fat diet caused impairment of endothelial cell function in male
and female pigs, respectively (Thompson et al., 2004, Woodman et al., 2003). The results in
this study demonstrated that the impairment of endothelial function may be attributed, in part,
to a reduced expression of eNOS, since WD consumption reduced the total protein content of
eNOS and reduced dimerization of eNOS in mesenteric arteries and this is consistent with the
report by Woodman et al, who noted that the consumption of high-fat diet induce a reduction
in brachial artery eNOS using immunohistochemistry (Woodman et al., 2003). Additionally,
SNP was used in order to investigate the effect of WD on vascular smooth muscle relaxation,
SNP-induced relaxation in this study was similar in both SD and WD fed rats, indicating that
the relaxation of smooth muscle in response to exogenous NO was not impaired in WD fed
rats.
5.4.3 Effect of linagliptin on endothelial function in WD
The findings in this study showed that rats fed a WD had a significant reduction in the
response to ACh-induced endothelium- dependent relaxation in comparison to SD fed rats.
153
Acute treatment with linagliptin in vitro improved endothelial function and increased
endothelium-dependent relaxation of mesenteric arteries isolated from WD fed rats.
Therefore we investigated whether linagliptin improved the endothelial function by
increasing the contribution of NO and/or EDH to the endothelium-dependent relaxation.
5.4.4 Effect of linagliptin on NO-mediated relaxation in WD fed rats
ACh-induced relaxation was used in order to investigate the contribution of NO to
endothelium-dependent relaxation in the presence of the combination of TRAM-34 and
apamin an IKCa and SKCa inhibitors respectively, in mesenteric arteries isolated from SD and
WD fed rats. Under the conditions of blocking the action of EDH-mediated relaxation, the
maximum relaxation response to ACh was significantly reduced in mesenteric arteries from
WD fed rats. Acute treatment with linagliptin in vitro, significantly improved the response to
ACh-induced relaxation and increased the maximum relaxation in WD mesenteric arteries in
comparison to the control. This is likely due to lingliptin reducing the oxidative stress by the
antioxidant effect which in turn increased the bioavailability of NO in mesenteric endothelial
cells by preventing the formation of peroxynitrite as a product of the reaction of superoxide
and NO via rapid free radical scavenging action of linagliptin (Chapter 3, 4). Overall, acute
treatment with linagliptin preserved the bioavailability of NO in mesenteric arteries of WD
fed rats.
5.4.5 Effect of linagliptin on EDH-mediated relaxation in WD fed rats
In addition to NO-mediated relaxation, the endothelium-dependent relaxation in rat
mesenteric arteries is mediated by both classical and non-classical EDH type responses
(Edwards et al., 2010, Leo et al., 2011b, Garland et al., 2011). To investigate the contribution
154
of EDH to endothelium-dependent relaxation in mesenteric arteries from SD and WD fed
rats, L-NNA+ODQ were added to prevent NO formation and sGC activity respectively. The
sensitivity to ACh-induced relaxation in WD mesenteric arteries was significantly decreased
in comparison to SD mesenteric arteries in the presence of L-NNA+ODQ, indicating that
feeding rats with WD for 10 weeks impaired the contribution of EDH to endothelium-
dependent relaxation. Acute treatment with linagliptin in vitro significantly improved the
sensitivity to ACh-induced relaxation in mesenteric arteries from WD fed rats, indicating that
linagliptin preserved the contribution of EDH to endothelium-dependent relaxation by
reducing the oxidative stress under the conditions of feeding animals with WD.
5.5.5 Conclusion
The findings in this study demonstrated that consumption of WD induced oxidative
stress, downregulation of vascular NOS, and reduced NO production to promote endothelial
dysfunction. The acute treatment in vitro with the DPP-4 inhibitor linagliptin improves the
endothelial function in mesenteric arteries developed in WD fed rats. This is probably via
conserving the beneficial effects of both NO and EDH-mediated relaxation. In this study we
showed that the DPP-4 inhibitor linagliptin has the ability to decrease the production of
superoxide anions which is perhaps, at least in part, via direct radical scavenging action. The
beneficial effect of DPP-4 inhibitor linagliptin by improving the vascular endothelial function
in small resistance mesenteric arteries in WD fed rats demonstrated in this study shows it
possesses the capability to act as a pharmacological intervention that may be anticipated to
improve the NO and EDH-mediated relaxation and could be valuable in the prevention of the
pathological role of WD in the development of endothelial dysfunction in the vasculature.
155
156
Chapter 6
The DPP-4 inhibitor linagliptin preserves
endothelial cell function in mesenteric
arteries from type-1 diabetic rats without
decreasing plasma glucose
6.1 introduction
Cardiovascular disease starts with risk factors such as hyperglycaemia in diabetes,
hypertension, and dyslipidaemia (Dzau et al., 2006). Endothelial dysfunction is considered a
critical factor in the initiation and development of vascular complications induced by diabetes
(De Vriese et al., 2000, Fatehi-Hassanabad et al., 2010). Macro- and microvascular
complications are presently the main causes of morbidity and mortality amongst diabetic
patients in both type 1 and type 2 diabetes mellitus (De Vriese et al., 2000). Endothelial cells
play an active role to regulate the basal vascular tone and reactivity of blood vessels in both
physiological and pathological conditions, by releasing a diversity of contracting and relaxing
factors in responding to stimulating factors such as mechanical forces and neurohumoral
mediators (Furchgott and Vanhoutte, 1989, Ferrara, 2002). The most important endothelium-
derived relaxing factors (EDRFs) are nitric oxide (NO), prostacyclin and endothelium-
dependent hyperpolarization (EDH) (Feletou and Vanhoutte, 1999, Woodman et al., 2000). It
has been demonstrated that the endothelial dyfunction caused by the impairment of eNOS
expression/activity and the reduction of NO production could be promoted by some risk
factors such as hypercholesterolemia and diabetes, thus, the endothelium-dependent
vasodilatation used as a parameter to investigate endothelial function in both physiological
and pathological circumstances (Arnal et al., 1999, Leo et al., 2011b).
157
Dipeptidyl peptidase-4 (DPP-4) is a glycoprotein peptidase broadly expressed in
various cell types which displays complex biological actions and has multifunctional
properties (Drucker, 2006, Drucker, 2007, Cordero et al., 2009, Yazbeck et al., 2009).
Inhibition of the DPP-4 system by DPP-4 inhibitors, which are class of blood glucose-
lowering drug, signifies a new approach in order to treat type 2 diabetes and take advantage
of their neutral effect on body weight and low risk of the occurrence of hypoglycaemia
(Panchapakesan et al., 2013, Zhong et al., 2013). DPP-4 inhibitors prolong the half-life of
incretins such as glucagon-like-peptide-1 (GLP-1) and glucagon induced peptide (GIP) by
inhibiting the degradation of these incretins and thus consequently lower blood glucose via
improved insulin secretion (Drucker, 2006). Interestingly, previous studies show beneficial
effects of GLP-1 in situations such as in the regulation of endothelial function and cardiac
remodeling (Zhao et al., 2006, Nikolaidis et al., 2004, Basu et al., 2007, Green et al., 2008).
The DPP-4 inhibitors have the ability to prevent the impairment of cardiac diastolic function
(Aroor et al., 2013), improve glomerulopathy (Nistala et al., 2014), ameliorate dysfunction in
rat aortic artery in experimental sepsis (Kroller-Schon et al., 2012) and reduce oxidative
stress in vascular endothelial cells (Ishibashi et al., 2013). We have recently found that acute
treatment with linagliptin ameliorate vascular dysfunction in mesenteric arteries exposed to
high concentration of glucose (40 mM) independently of glucose lowering effect.
We have previously demonstrated that in small arteries from STZ-treated rats with
diabetes-induced endothelial dysfunction results from the impairment of both NO-mediated
and EDH-mediated relaxation, associated with eNOS uncoupling and an increase in Nox2-
derived superoxide generation (Leo et al., 2011b). We have also shown that treatment with
3’,4’-dihydroxy flavonol reduces oxidant stress and improves endothelium-dependent
relaxation in type 1 diabetic rats (Leo et al., 2011a). Therefore, it is of particular interest to
examine whether linagliptin, a DPP-4 inhibitor which we have recently demonstrated is able
158
to also act as an antioxidant, can alleviate endothelial dysfunction in diabetic vasculature
independently of the glucose lowering properties. Thus, the aim of the present study was to
examine whether chronic in vivo treatment with linagliptin, a DPP-4 inhibitor with unique
xanthine-based structure, preserves endothelial function in small resistance mesenteric
arteries from type 1 STZ-induced diabetic rats and, if so, whether it acts via the mechanism of
direct scavenging of ROS and/or through the inhibition of the sources of ROS generation in
microvascular endothelial cells in diabetic mesenteric arteries independently of reducing
blood glucose effects.
159
6.2 Materials and Methods
Ethics statement: All the procedures were approved by the Animal Experimentation
Ethics committee of RMIT University (AEC approval number 1309) and complied with the
Australian National Health and Medical Research Council code of practice for the care and
use of animals for scientific purposes.
6.2.1 Induction of type 1 diabetes
Male Wistar rats (~ 8-weeks old and weighing approximately 200g) were randomly
divided into two groups: normal and diabetic. A single injection of streptozotocin (STZ, 50
mg/kg IV) after the rats were fasted overnight was used to induce type 1 diabetes, whereas,
rats in the control group received vehicle of a comparable volume (0.1 mol/L citrate buffer,
pH 4.5). Blood glucose was monitored and when greater than 25 mmol/L, rats were treated
with insulin (3-4 IU, s.c., 3 injections/ week, Protaphane, Novo Nordisk, NSW, Australia).
Blood glucose concentration was measured by a glucometer (Roche, Sydney, NSW,
Australia) and glycated haemoglobin (HbA1c) was measured using a Micromat HbA1c
analyser (Biorad, Sydney, NSW, Australia). Blood samples were collected from the left
ventricle at the end of the experimental period.
6.2.2 Linagliptin treatment
Six weeks after induction of diabetes, the two groups of rats were further divided into
4 subgroups (normal, normal+linagliptin, diabetic, diabetic+linagliptin) administering either
vehicle (1% carboxy methylcellulose, CMC) or linagliptin (2 mg/kg) by daily oral gavage for
a period of 4 weeks. The rats were administered the last dose of linagliptin at least 24 hours
prior to the collection of tissue for experimentation.
160
6.2.3 Isolation of mesenteric arteries
After ten weeks of STZ treatment, the rats were killed by asphyxiation with CO2
followed by exsanguination. The mesenteric arcade was isolated and collected and
immediately placed in ice-cold Krebs bicarbonate solution (4.7 mmol/L KCl, 118 mmol/L
NaCl, 1.18 mmol/L MgSO4, 25 mmol/L NaHCO3, 11.1 mmol/L D-glucose and 1.6 mmol/L
CaCl2) containing the non-selective cyclooxygenase (COX) inhibitor indomethacin (10
µmol/L), to inhibit the production of prostanoids. Small mesenteric arteries (third order
branch of the superior mesenteric artery, internal diameter ~300 µm) were isolated, cleared of
fat and connective tissue and dissected into rings of about 2 mm in length and then mounted
on a Mulvany-Halpern myograph (model 610M, Danish Myo Technology, Aarhus,
Denmark). After the rings were mounted, they were allowed to stabilize at zero tension for 15
min before normalization. The blood vessels were stretched to attain an internal pressure
comparable to 90% of that of the blood vessel under a transmural pressure of 100 mmHg
(McPherson, 1992, Mulvany and Halpern, 1977) to determine the passive tension-internal
circumference. All experiments were conducted at 37ºC in the Krebs solution bubbled with
carbogen (95% O2 and 5% CO2).
6.2.4 Investigation of vascular function and reactivity
After equilibration for 30 minutes, blood vessels were subjected to maximum
contraction by the addition of an isotonic physiological saline solution containing a high
concentration of K+
(123 mmol/L, KPSS). Basal tension of vessels was restored after several
washouts using Krebs solution. In order to investigate the integrity of the endothelium, the
vessels were precontracted with phenylephrine (0.1-3 µmol/L) to ~50% of the KPSS response
and relaxed with a high single dose of acetylcholine (ACh, 10 µmol/L). ACh-induced
relaxation of the precontracted rings was more than 80% in all cases indicating that the
161
endothelium was functionally intact. After several washouts, the mesenteric arteries were
precontracted again with phenylephrine (0.1-3 µmol/L) and cumulative concentration-
response curves to the endothelium-dependent relaxant agonist, ACh (0.1 nmol/L-10 µmol/L)
and the endothelium-independent relaxant agonist, sodium nitroprusside (SNP, 0.01 nmoL/l-
10 µmol/L) were examined. Furthermore, after 20 minutes, responses to ACh and SNP were
investigated with different combinations of N-nitro-L-arginine (L-NNA, 100 μmol/L), an
inhibitor of nitric oxide synthase (NOS), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one
(ODQ, 10 µmol/L), an inhibitor of soluble guanylate cyclase (sGC), apamin (1 µmol/L), a
selective blocker of the small calcium-activated K+
channel (SKCa), 1-[(2-chlorophenyl)
(diphenyl)methyl]-1H-pyrazole (TRAM-34, 1 µmol/L), a blocker of the intermediate
calcium-activated K+
channel (IKCa), and iberiotoxin (Ibtx, 100 nmol/L), a blocker of the
large calcium-activated K+
channel (BKCa).
6.2.5 Estimation of basal release of NO in mesenteric arteries
In order to investigate the effect of diabetes on the basal level of NO release,
endothelium- intact blood vessels were exposed to L-NNA (100 µmol/L) after precontraction
with phenylephrine (10-100 nmol/L) to ~ 20% KPSS. , A contractile response to L-NNA was
considered to indicate inhibition of basal release of NO (Leo et al., 2011b).
6.2.6 Western Blot
Western blots were performed by collecting the mesenteric arteries from normal and
diabetic rats. From the same treatment group, the endothelium-intact small mesenteric
arteries from 2 animals were pooled and considered as n =1. The same amount of protein
homogenate of the treatment animal groups were exposed to SDS-PAGE and analyzed
162
through western blot processes with mouse/rabbit primary antibodies (all 1:1000, overnight,
4oC) includes endothelial NO synthase (eNOS), Nox2 (all BD Transduction Laboratories,
Lexington, KY, USA). The membranes were reinvestigated with a loading control antibody
(actin), in order to normalize for the amount of protein. After 1 hour incubation at room
temperature with anti-mouse/rabbit secondary antibody (1:2000) (Millipore, Billerica, MA,
USA), all proteins were identified by enhanced chemiluminescence reagents (Amersham, GE
Healthcare, Sydney, NSW, Australia). Densitometry (Biorad Chemidoc, Sydney, NSW,
Australia) was used to quantify the protein bands expressed as a ratio of the loading control.
To examine tissue eNOS ratio of monomer/dimer formation, 6% SDS-PAGE at 4ºC used to
resolve a non-boiled sample and the membranes were examined and imaged as described
above.
6.2.7 Measurement of superoxide release by mesenteric arteries
To measure superoxide generation in mesenteric arteries, lucigenin-enhanced
chemiluminescence was used. The superior mesenteric artery was isolated, cleared of fat and
connective tissue and incubated for 45 minutes at 37ºC in Krebs- HEPES buffer solution
containing NADPH (100 µmol/L) as a substrate for NADPH-oxidase in the presence or
absence of diphenyliodonium (DPI, 5 µmol/L), a flavoprotein inhibitor that inhibits NADPH
oxidase. 300 µL of Krebs-HEPES buffer, containing lucigenin (5 µM) and the suitable
treatment were placed into a 96-well Optiplate. The Optiplate was loaded into a Polarstar
Optima photon counter (BMG Labtech, Melbourne, VIC, Australia) to measure background
photon emission at 37ºC. After the background reading was recorded, a single ring section of
mesenteric artery was placed into each well and photon emission was recounted. The
mesenteric arteries were placed in Krebs-HEPES buffer that contain either lucigenin (5 µM)
alone or with the presence of NADPH (100 μM) to measure the production of NADPH
163
oxidase- driven superoxide. The signal stimulated by NADPH was measured in the presence
or absence of DPI (5 µM). The background counts were subtracted from superoxide counts
and normalized with dry tissue weight.
6.2.8 Materials
Linagliptin was a gift from (Boehringer Ingelheim, Germany). All other chemicals
were purchased from Sigma-Aldrich (St Louis, MO, USA), except for ODQ (Cayman
Chemical, Ann Arbor, MI, USA), and acetylcholine perchlorate (BDH Chemicals, Poole,
Dorset, UK). All drugs were dissolved in distilled water with the exception of indomethacin,
which was dissolved in 0.1 M sodium carbonate, and L-NNA, which was dissolved in 0.1 M
sodium bicarbonate.
6.2.9 Statistical analysis
Data were expressed as mean ± SEM, n indicates the number of experiments. The
concentration response data from rat isolated mesenteric arteries were fitted to a sigmoidal
curve using nonlinear regression (Prism version 6.0, GraphPad Software, San Diego, CA,
USA) to calculate the pEC50. The maximum relaxation (Rmax) to ACh or SNP was determined
as a percentage of the phenylephrine precontraction. Differences between groups of pEC50
and Rmax values were compared and analysed using two way ANOVA with post hoc analysis
using Bonferroni’s test. p<0.05 was considered statistically significant.
164
6.3 Results
6.3.1 Body weights and blood glucose
The body weight, blood glucose and HbA1c levels of rats in the 4 groups are shown in
Table 1. Ten weeks after treatment with streptozotocin or vehicle, the final body weight in
normal rats was significantly higher than in diabetic rats (Table 6.1). Furthermore, in diabetic
rats, the blood glucose and HbA1c levels were significantly greater than in normal rats. Four
weeks treatment with linagliptin had no significant effect on body weight, HbA1c or blood
glucose levels in either normal or diabetic rats.
6.3.2 Effect of linagliptin on vascular superoxide production
The superoxide level in mesenteric arteries was measured by lucigenin-enhanced
chemiluminescence. In diabetic rats, the level of superoxide-induced fluorescence was
significantly higher than that in normal rats (Figure 6.1). After 4 weeks of treatment with
linagliptin, the level of NADPH oxidase-driven superoxide was significantly attenuated in
diabetic rats, but there was no effect in normal rats (Figure 6.1). In all groups,
diphenyliodonium was able to inhibit NADPH oxidase-driven superoxide production in the
mesenteric arteries (Figure 6.1).
165
Table 6.1 Mean body weight gained, blood glucose and HbA1c levels at the end of the experiment of
normal and diabetic rats with or without linagliptin (2 mg/kg oral gavage daily for 4 weeks) treatment.
10 weeks after vehicle or STZ treatment
n Normal n Normal+linagliptin n Diabetic n Diabetic+linagliptin
Body weight
(g)
8
498±18Ф
9
538±21Ф
11
346±11*¥
11
379±25*¥
Blood glucose
(mM)
8
5.1±0.3Ф
8
4.5±0.2Ф
11
26.4±2*¥
12
27.8±1.7*¥
HbA1c
(%)
7
6.2±0.2Ф
8
5.8±0.2Ф
10
14.8±0.6*¥
12
13.9±0.4*¥
n= the number of rats. *Significantly different from normal group (Bonferroni’s test, p<0.05), Ф
Significantly different to diabetic group, p<0.05, Bonferroni’s test. ¥
Significantly different to
Normal+linagliptin group, p<0.05, Bonferroni’s test. Results are shown as mean±SEM.
166
6.3.3 Effect of diabetes and linagliptin on vascular function
Both diabetes and linagliptin treatment had no effect on the level of contraction to the
high K+ physiological saline solution (KPSS, 123 mmol/L) (Figure 6.2). The sensitivity to
ACh but not the maximum relaxation was significantly reduced in mesenteric arteries from
diabetic rats (Figure 6.3a, Table 6.2), whereas the sensitivity and the maximum relaxation to
sodium nitroprusside (SNP) were not affected (Figure 6.3b). 4 weeks of linagliptin treatment
(2 mg/kg per daily, oral gavage) did not affect the response to ACh in mesenteric arteries
from normal rats, but in diabetic rats treated with linagliptin, the sensitivity to ACh was
significantly increased in comparison to the sensitivity and response to ACh in mesenteric
arteries from diabetic untreated rats (Figure 6.3a, Table 6.2). The treatment with linagliptin
had no effect on the response to SNP in normal rats or diabetic rats (Figure 6.3b).
The basal level of NO release was investigated by assessing the level of contraction
induced by L-NNA in mesenteric arteries precontracted with PE to 20% of the KPSS
response In normal mesenteric arteries the L-NNA- induced contraction was significantly
higher than that with diabetic mesenteric arteries (Figure 6.3c) showing that diabetes reduced
the basal release of NO. Treatment with linagliptin significantly increased the L-NNA-
induced contraction in diabetic mesenteric arteries compared to untreated diabetic arteries but
had no effect on L-NNA-induced contraction in normal mesenteric arteries (Figure 6.3c).
167
Figure 6.1 ROS measurement in intact mesenteric arteries. NADPH activity was raised in diabetic
mesenteric arteries which were reduced with linagliptin treatment. Results are shown as mean±SEM.
n=7-10 experiments. *p<0.05 vs normal, #p<0.05 vs diabetic.
168
Figure 6.2 Mesenteric arteries were induced to maximum contraction by KPSS. Exposure of
mesenteric arteries from both normal and diabetic rats with or without linagliptin (2 mg/kg per day
orally for 4 weeks) treatment to KPSS (123 mM). Diabetes or linagliptin treatment did not affect the
level of contraction of the mesenteric arteries.
169
Figure 6.3 Vascular function in mesenteric arteries. Cumulative concentration-response curves to
ACh (a), SNP (b), and basal NO release (c) in endothelium-intact mesenteric arteries. In each group
(a, b), mesenteric arteries were pre-contracted with PE to a similar level: (a) normal 66±2,
normal+linagliptin 64±1, diabetic 65±1, diabetic+linagliptin 66±1, (b) normal 64±1,
normal+linagliptin 64±1, diabetic 61±6, diabetic+linagliptin 66±1 %KPSS, n=7-10 experiments.
Results are shown as mean±SEM. **p<0.01, ***p<0.001. See Table 2 or results section for pEC50
and Rmax values.
170
6.3.4 Determination of the relative contribution of NO and EDH to endothelium-
dependent relaxation
In mesenteric arteries from normal rats, the ACh-induced relaxation was partially
reduced by either the combination of N-nitro-L-arginine (L-NNA), a NO synthase inhibitor
and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a soluble guanylate cyclase
inhibitor or inhibitors of intermediate-conductance calcium-activated K+ channel (IKCa),
small-conductance calcium-activated K+ channel (SKCa) with 1-[(2
chlorophenyl)(diphenyl)methyl]1H pyrazole (TRAM-34), apamin and iberiotoxin (Ibtx)
respectively, confirming that both NO and EDH were involved in endothelium-dependent
relaxation (Figure 6.4, Table 6.2). Further, the response to ACh-induced relaxation in
mesenteric arteries from diabetic rats was significantly further reduced in the presence of
either NO inhibitors or EDH inhibitors in comparison to normal rats, indicating that the
contribution of both NO and EDH to endothelium-dependent relaxation were impaired by
diabetes.
6.3.5 Effect of linagliptin on NO- and EDH-mediated relaxation in the mesenteric
arteries
The response to ACh was assessed in the presence of TRAM-34+apamin in order to
investigate the contribution of NO to endothelium- dependent relaxation. Treatment with
linagliptin for 4 weeks (2 mg/kg, daily oral gavage), significantly increased the maximum
relaxation to ACh in mesenteric arteries from diabetic rats but had no effect on responses in
normal rat arteries (Figure 6.4, Table 6.2) indicating that linagliptin could restore the
contribution of NO to endothelium-dependent relaxation in diabetes.
171
To investigate the contribution of EDH-mediated relaxation, the actions of NO were
inhibited by the addition of L-NNA and ODQ. Treatment of normal and diabetic rats for 4
weeks with linagliptin (2 mg/kg, daily oral gavage) significantly increased on ACh-induced
EDH mediated relaxation diabetic arteries but not in arteries from normal rats (Figure 6.5,
Table 6.2).
172
Table 6.2 Effect of L-NNA, ODQ and potassium channel blockers on ACh-induced relaxation of mesenteric arteries from normal and diabetic rats with or
without linagliptin (2 mg/kg oral gavage daily for 4 weeks) treatment in the presence of indomethacin.
Normal Normal+linagliptin Diabetic Diabetic+linagliptin
ACh
n
pEC50 Rmax (%)
n
pEC50 Rmax (%)
n
pEC50 Rmax (%)
n
pEC50 Rmax(%)
Control
8
7.26±0.13
99±0
9
7.02±0.12
94±3
10
6.14±0.11Ф¥
96±1
10
6.84±0.13¥
97±0
TRAM34+apamin
7
6.52±0.06*
91±2
7
6.29±0.10*
95±1
9
6.08±0.14
35±9*Ф¥
9
6.59±0.18
96±2¥£
L-NNA+ODQ
7
6.31±0.09*
95±0
7
6.45±0.09*
94±0
8
5.59±0.05* Ф¥
81±7
8
6.38±0.10¥£
95±0
LNNA+ODQ+
TRAM-34+apamin
7 5.62±0.29* 27±8
* 4 5.35±0.21
* 45±7
* 4 5.45±0.06
*
4±0* Ф¥
7 5.35±0.11* 2±1
* Ф
LNNA+ODQ+
TRAM-34+apa+Ibtx
4
5.27±0.17*
14±2*
4
5.31±0.07*
25±8*
ND
ND
A comparison of the sensitivity (pEC50) and maximum relaxation (Rmax) to ACh in the absence (control), or the presence of TRAM-34 (1
µM)+apamin (1 µM), L-NNA (100 µM)+ODQ(10 µM), L-NNA (100 µM)+ODQ(10 µM)+ TRAM-34 (1 µM) +apamin (1 µM) or L-NNA (100
µM)+ODQ(10 µM)+ TRAM-34 (1 µM) +apamin (1 µM)+Ibtx (100 nM) in endothelium intact mesenteric arteries. All experiments were performed
in the presence of indomethacin (10 µM). n = the number of experiments.
*Significantly different to control within each group (p<0.05), Bonferroni’s test, Ф
Significantly different to normal within inhibitor group, p<0.05, Bonferroni’s test.
¥Significantly different to normal+linagliptin within inhibitor group, p<0.05, Bonferroni’s test.
£Significantly different to diabetic within inhibitor group, p<0.05, Bonferroni’s test.
Results are shown as mean±SEM, ND= Not determined.
173
Figure 6.4 Relative contribution of NO to endothelium-dependent relaxation. Mesenteric arteries to
examine NO-mediated relaxation in the presence of TRAM-34+apamin were isolated from normal (a),
diabetic (b), normal+linagliptin (c), diabetic+linagliptin (d) rats. In each group of experiments, arteries
were precontracted with PE to similar levels: 63±2 (a), 65±0.6 (b), 65±1 (c), 63±1 (d) %KPSS, n=8-9
experiments. Results are shown as mean±SEM. See Table 6.2 for values.
174
Figure 6.5 Contribution of EDH to endothelium-dependent relaxation. Mesenteric arteries to examine
EDH-mediated relaxation in the presence of L-NNA+ODQ were isolated from normal (a), diabetic (b),
normal+linagliptin (c), diabetic+linagliptin (d) rats. In each group of experiments, arteries were
precontracted with PE to similar levels: 62±0.7 (a), 65±0.8 (b), 62±2 (c), 64±0.8 (d) %KPSS, n=8-9
experiments. Results are shown as mean±SEM. See Table 2 for values.
175
6.3.6 Effect of linagliptin on Nox2, total-eNOS and eNOS monomer/dimer
In diabetic rats, the expression of total eNOS in the mesenteric arteries was significantly
reduced as was the proportion of eNOS expressed as the dimer and the expression of Nox2 was
significantly increased (Figure 6.6). Treatment with linagliptin did not affect the expression or
dimerization of eNOS or the expression of Nox2 in normal rats. By contrast, in diabetic
mesenteric arteries after linagliptin treatment there was a significant increase in eNOS, both in
total and as a dimer, and a decrease in Nox2 expression
176
Figure 6.6 Western blot analysis of protein expression of eNOS (a, 130 kDa), eNOS dimers and
monomers (b, 260 kDa) and Nox2 (c, 58 kDa) in normal and diabetic mesenteric arteries with or without
linagliptin treatment. In diabetic mesenteric arteries, the expression of eNOS significantly reduced and the
proportion of eNOS expressed as the dimer reduced, and the expression of Nox2 increased. Treatment
with linagliptin increased the expression of eNOS significantly and reduced Nox2 expression and
increased the proportion of eNOS expressed as the dimer. Representative blots are shown on each of the
corresponding graphs. n=6 experiments. Results are shown as mean±s.e.m. *p<0.05, **p<0.01,
***p<0.001, ****p<0.0001.
177
6.4 Discussion
This study showed that treatment of type1 STZ-induced diabetic rats with the DPP-4
inhibitor linagliptin (2 mg/Kg, daily oral gavage) for 4 weeks decreases the levels of vascular
oxidative stress and improves endothelium-dependent relaxation in mesenteric arteries.
Importantly in this type 1 diabetes model the beneficial vascular effects of linagliptin were
achieved without affecting plasma concentrations of glucose or HbA1c demonstrating that
linagliptin has actions in addition to the capacity to lower plasma glucose. A similar dose of
linagliptin (1 mg/kg po) to that used in this study (2 mg/kg po) has been demonstrated to
effectively inhibit DPP-4 activity and to increase GLP-1 in Zucker diabetic fatty rats (Jones et
al., 2014) but in this type 1 model of diabetes where the beta pancreatic cells are destroyed by
necrosis it is not possible to elevate insulin secretion to decrease glucose. The dysfunction of
endothelial cells in the mesenteric arteries of diabetic rats is accompanied with an impairment of
the contribution of both NO and EDH-mediated relaxation to endothelium-dependent relaxation.
Treatment of diabetic rats with linagliptin improves the contribution of NO-mediated relaxation
to endothelium-dependent relaxation which associated with an increased expression of total
eNOS, decreased eNOS uncoupling and downregulation of Nox2 expression. In addition, the
contribution of EDH to endothelium-dependent relaxation in diabetic mesenteric arteries was
improved by the treatment with linagliptin.
6.4.1. Effect of linagliptin on vascular endothelial function
In the present study, Type 1 STZ-induced diabetes increased the level of vascular ROS
generation concomitant with a selective impairment of endothelium-dependent relaxation and an
increase in the expression of Nox2 in the mesenteric arteries consistent with previous reports (De
178
Vriese et al., 2000, Leo et al., 2011b, Ding et al., 2005). In diabetic rats, the in vivo treatment
with the DPP-4 inhibitor linagliptin (2 mg/kg, per day, oral gavage) for 4 weeks improved
endothelium-dependent relaxation in mesenteric arteries and decreased the generation levels of
oxidative stress associated with decreased Nox2 and improved coupling of eNOS in the
mesenteric arteries of diabetic rats. Our observations are similar to that of other observations that
have shown that treatment with the DPP-4 inhibitor linagliptin protects against cardiovascular
injury induced by salt-sensitive hypertension via a reduction of oxidative stress (Koibuchi et al.,
2014) or improves endothelial function and reduces vascular stress in experimental sepsis in
large conduit aortic artery (Kroller-Schon et al., 2012). Whilst there are various reports that the
DPP-4 inhibitor, linagliptin could ameliorate endothelial dysfunction induced by diabetes,
however, the mechanism of the vasoprotective effect of linagliptin remains ambiguous
particularly on the contribution of NO and EDH to endothelium-dependent relaxation in
diabetes.
6.4.2. Effects of linagliptin on NO-mediated relaxation in rats mesenteric arteries
In order to investigate NO-mediated relaxation, the EDH-mediated relaxation was
blocked by endothelial KCa channel blockers. This clearly showed impaired release of NO. Under
those conditions of blocking NO-mediate relaxation, the response to ACh and the maximum
relaxation was significantly reduced in the mesenteric arteries from diabetic rats in comparison
to mesenteric arteries from normal rats. Chronic treatment with linagliptin in vivo for 4 weeks
significantly increased the maximum relaxation to ACh in diabetic mesenteric arteries in
comparison to untreated diabetic mesenteric arteries. Furthermore, beside stimulated NO release,
basal NO release was also reduced in mesenteric arteries of diabetic rats, in response to NOS
179
inhibition the contraction was impaired and this was reversed in arteries from linagliptin treated
diabetic rats. Therefore, treatment with linagliptin was able to preserve the contribution of NO-
mediated relaxation to the endothelium-dependent relaxation in diabetic mesenteric arteries. In
our results, consistent with the impairment of NO mediated-relaxation we found that in the
diabetic mesenteric arteries the expression of eNOS was reduced and the proportion of eNOS
expressed as a dimer was decreased which indicated uncoupling of the total eNOS in the diabetic
mesenteric arteries. In addition, in linagliptin treated diabetic rats, the expression of eNOS was
comparable to the normal rats. Moreover, treatment with linagliptin stimulated the re-coupling of
eNOS, which was expressed as a dimer more than monomer compared to untreated mesenteric
arteries in diabetic rats and this normalization of eNOS expression and re-coupling of eNOS by
chronic treatment with linagliptin would elucidate the improvement of the endothelium-
dependent relaxation in mesenteric arteries of diabetic rats. A further factor to the repair of the
activity of NO by chronic treatment with liangliptin could be the decrease of vascular superoxide
generation in diabetic mesenteric arteries, and this could then increase the bioactivity of NO
through avoiding the forming of peroxynitrite (ONOO-) via the degradation of NO by superoxide
action. The reduction of ROS activity and prevention of peroxynitrite formation in diabetic
vasculature could be either due to the rapid free radical scavenging effect of linagliptin in order
to preserve the bioavailability of NO linagliptin through the inhibition of xanthine oxidase
(Yamagishi et al., 2014) or by reducing the expression and/or activity of the enzymatic source of
superoxide generation in the diabetic mesenteric arteries such as NAPDH oxidase subunit Nox2.
Taken together, chronic treatment with liangliptin reserved the beneficial activity of NO via
improving of eNOS expression, re-coupling of eNOS and reducing the expression of Nox2
which mediate superoxide generation in the mesenteric arteries of diabetic rats.
180
6.4.3 Effect of linagliptin on EDH-mediated relaxation in rat mesenteric arteries
The endothelium-dependent relaxation in rat mesenteric arteries is mediated by NO,
classical EDH and non-classical EDH pathways (Edwards et al., 2010). In order to evaluate the
contribution of EDH to endothelium-dependent relaxation in diabetes, we investigated the
endothelium-dependent relaxation in the presence of L-NNA and ODQ inhibitions to block NO
formation and sGC activity respectively. In addition to inhibit NOS, the guanylate cyclase
inhibitor was also used to confirm the inhibition of the action of NO derived from nitrosothiols, a
non-NOS sources of NO, which was earlier described to act as a source of NO in diabetic
conditions (Leo et al., 2010). The sensitivity to ACh in diabetic mesenteric arteries was
decreased significantly in comparison to normal mesenteric arteries in the presence of L-
NNA+ODQ, indicating that diabetes impaired the contribution of EDH to endothelium-
dependent relaxation, which is consistent with other reports (Leo et al., 2011b, Fukao et al.,
1997, Matsumoto et al., 2003, Matsumoto et al., 2005). Chronic in vivo treatment with
linagliptin improved EDH-mediated relaxation in mesenteric arteries of diabetic rats, indicating
that treatment with linagliptin preserved the contribution of EDH to endothelium-dependent
relaxation in diabetes. Although the main cause of the impairment of EDH in diabetic conditions
remains uncertain (Leo et al., 2011b, Matsumoto et al., 2003, Matsumoto et al., 2006, Makino et
al., 2000, Burnham et al., 2006a, Weston et al., 2008), however, is partly associated with over
generation of superoxide and oxidative stress. It is reported that auto-oxidation of pyrogallol
causes an increase in superoxide anions which could impair EDH-mediated relaxation in rat
mesenteric arteries (Ma et al., 2008). Our results in this study demonstrated that the impairment
of EDH-mediated relaxation in diabetes may be in part due to the overproduction of superoxide
181
and treatment of diabetic rats with lingaliptin improved the contribution of EDH to endothelium-
dependent relaxation in mesenteric arteries which could be as a result of the reduction of
superoxide levels by linagliptin treatment.
6.4.4 Conclusion
In this study we have demonstrated that the DPP-4 inhibitor linagliptin improves
endothelial function in mesenteric arteries in diabetic rats by preserving the activities of both NO
and EDH-mediated relaxation. In order to reduce inactivation of NO, linagliptin has protective
actions via at least two mechanisms. The DPP-4 inhibitor linagliptin was able to reduce the
generation of superoxide which may be due to direct radical scavenging action and/or attenuating
the enzymatic source for superoxide generation where the chronic treatment of diabetic rats with
liangliptin in vivo for 4 weeks prevents the uncoupling of eNOS, increases the expression of total
eNOS, and decreases Nox2, hence facilitates maintenance of endothelium-dependent relaxation.
Interestingly, the beneficial effects of linagliptin to preserve NO activity and improve EDH role
in relaxation demonstrated in this study displays that linagliptin has the ability to act as a
therapeutic compound independent of its glucose lowering action for use in the prevention of the
microvasculature complications of diabetes.
182
183
Chapter 7
General Discussion
7.1 Effects of diabetes and WD on vascular function
Diabetes mellitus is one of the fast growing diseases all over the world both in developed
and developing countries (Dunstan et al., 2002, Wild et al., 2004). Diabetes affects all the organs
in the body, however cardiovascular complications are a major cause of high prevalence of
morbidity and mortality in individuals with diabetes (Boyle et al., 2010). Vascular endothelial
function is identified as a vital independent risk factor for cardiovascular disease. In diabetes, the
endothelial dysfunction plays a critical role in the initiation and development of cardiovascular
complications (De Vriese et al., 2000, Halcox et al., 2002, Al Suwaidi et al., 2000). In this
thesis, we investigated the mechanisms underlying endothelial dysfunction over the course of
expose to high glucose concentration, STZ-induced type-1 diabetes, and western diet feeding
regime.
It is widely acknowledged that hyperglycaemia causes an increase in oxidative stress
which is known to cause endothelial dysfunction and is unfavourably related to an imbalance
between NO and superoxide production in vascular endothelial cells. In investigations of small
blood vessels (mesenteric arteries) exposed to high glucose, our findings (Chapter 3) indicate
that there was an endothelial dysfunction related to an increase in ROS production and
impairment in NO and EDH-mediated relaxation. Further investigation showed that the vascular
endothelial function improved by increased bioavailability of NO derived from eNOS pathway
184
and improved EDH-type response which leads to increasing the contribution of these mediated
relaxation factors to the endothelium-dependent relaxation in microvasculature. The results also
suggested that both NO and EDH-mediated relaxation are vital modulators of vascular health and
pharmacological compounds targeted to increase the bioavailability of NO and improve EDH-
type response could be used to maintain vascular endothelial function under the pathological
condition of diabetes.
In diabetes, prolonged hyperglycaemia causes overgeneration of ROS causing oxidative
stress which leads to an impairment of the action of NO and EDH-mediated relaxation and
subsequent endothelial dysfunction (De Vriese et al., 2000, Fatehi-Hassanabad et al., 2010)
(Chapters 4 and 6). During diabetes, hyperglycaemia results in oxidative stress via the increase in
NADPH oxidase expression/activity and uncoupling of eNOS that causes impairment of vascular
endothelial function. This impairment in endothelial function was concomitant with a reduction
in the contribution of both NO and EDH-mediated relaxation to the endothelium-dependent
relaxation. This indicates that the increase in the expression/activity of NADPH oxidase and
eNOS uncoupling are early contributors to hyperglycaemia-induced endothelial dysfunction. The
increase in expression/activity of endothelial NADPH-oxidase, suggests that the observed
impairment of EDH-type response may also be due to oxidative stress as a result of
overgeneration of superoxide in the microvasculature which cause KCa-channels (IKCa and SKCa)
alteration and interruption of downstream reactions of these channels (Leo et al., 2011b,
Burnham et al., 2006a) (Chapters 4 and 6). Thus, therapies targeting ROS derived from NADPH-
oxidase and eNOS uncoupling could have the potential to prevent microvascular complications
and endothelial dysfunction concomitant with diabetes.
185
Metabolic syndrome is generally defined as a combination of risk factors or abnormalities
strongly associated with obesity and insulin resistance which are noticeably associated with the
increase in the risk of development of cardiovascular disease and diabetes mellitus (Stein and
Colditz, 2004). ROS production is proposed to have an important role in the development of
insulin resistance (Furukawa et al., 2004, Lin et al., 2005, Urakawa et al., 2003, Diniz et al.,
2006). This study (Chapter 5) showed that the consumption of high fat diet (western diet) caused
endothelial dysfunction in the mesenteric microvasculature. The findings showed that the ROS
production in small blood vessels was significantly increased due to the consumption of western
diet. This increase in oxidative stress was through the increase in the expression/activity of
NADPH-oxidase and eNOS uncoupling that leading to vascular endothelial dysfunction. These
findings suggest that ROS production may be the early event promoting western diet-induced
pathologies and therefore may be an attractive therapeutic targeted for inhibiting insulin
resistance and obesity caused by consumption of high fat diet or western diet.
7.2 Protection of diabetes or WD-induced endothelial dysfunction by the DPP-4
inhibitors
Exposure of blood vessels to high glucose induced oxidative stress, that has been
demonstrated as a primary cause of premature vascular disease (Cosentino et al., 1997,
Cosentino et al., 2003, Tesfamariam and Cohen, 1992). A recent study demonstrated that the
DPP-4 inhibitor linagliptin, currently used as an antihyperglycaemic agent, was markedly more
potent than other DPP-4 inhibitors in its anitioxidant capacity (Kroller-Schon et al., 2012). In
this study (Chapter 3), the acute effects of the DPP-4 inhibitors linaglitin, sitagliptin and
vildagliptin and the GLP-1 receptor agonist exendin-4 were examined on the endothelial function
186
of blood vessels exposed to high glucose. The findings demonstrated that high glucose impaired
endothelium-dependent, but not endothelium-independent, relaxation in rat mesenteric arteries,
due to an increased production of superoxide and the acute treatment with linagliptin and
exendin-4 reduced vascular superoxide and improved endothelium-dependent relaxation and the
beneficial effect on endothelium-dependent relaxation of exendin-4, but not linagliptin, was
mediated by the GLP-1 receptor. Also, the NO and EDH-mediated relaxation were both impaired
in the presence of a high glucose concentration and the actions of both mediators were improved
by linagliptin. Taken together, the findings suggested that linagliptin and exendin-4 have
antioxidant effects that may not be shared with other DPP-4 inhibitors. This antioxidant action
may have additional benefit to the glucose lowering effects of linagliptin in the treatment of
diabetes.
In Chapter 4, the acute effect of the DPP-4 inhibitor linagliptin in vitro on microvascular
function from STZ-induced type 1 diabetic rats was investigated, demonstrating that acute
treatment with linagliptin markedly reduce the high level of superoxide and improved endothelial
function in diabetes. Endothelial dysfunction in the diabetic rats was associated with impairment
in the modulatory role of NO and EDH-mediated relaxation. Acute treatment with linagliptin in
vitro improved the contribution of both NO and EDH to the endothelium-dependent relaxation in
small blood vessels from the diabetic rats, indicating that the DPP-4 inhibitor has potential as
therapeutic agent for use in the reduction and/or prevention of microvascular complications
associated with diabetes beyond its current use in type 2 diabetes. In Chapter 5, the acute
treatment with DPP-4 inhibitor linagliptin in vitro on the endothelial function of small blood
vessels from western diet fed rats was investigated. The observed findings indicated that
superoxide production is increased in mesenteric arteries from western diet fed rats and in the
187
acute presence of linagliptin, the vascular superoxide production was significantly reduced.
Western diet caused endothelial dysfunction and both the NO and the EDH-mediated relaxation
was impaired in the microvasculature. The results showed that the acute presence of linagliptin
improved endothelium-dependent relaxation in the mesenteric arteries from western high fat diet
fed rats by selectively preserving the contribution of both NO and EDH-mediate relaxation. This
indicates that linagliptin could be in favour of the prevention of the pathological events caused
by western diet including the development of endothelial dysfunction in small arteries.
In Chapter 6, treatment with linagliptin in vivo in STZ-induced type 1 diabetic rats had no
effect on the plasma glucose level, indicating that the DPP-4 inhibitor improves the function in
blood vessels independently of a glucose lowering pathway. Furthermore, in normal animals,
treatment with linagliptin had no effect on vascular function. The beneficial action of linaglipin,
when administered in vivo may have at least two mechanisms of action. The linagliptin may
exhibiting its protective action via either as an antioxidant by rapidly directly scavenging ROS
and/or inhibit the enzymatic source for superoxide production (i.e. NADPH oxidase and eNOS
uncoupling) to preserve NO bioavailability or through a GLP-1 receptor dependent pathway
through increased activation of GLP-1R secondary to DPP-4 inhibition. The beneficial action of
linagliptin via its ability to reduce the oxidative stress in diabetic vasculature by inhibiting
superoxide-derived from NADPH oxidase and eNOS uncoupling makes the DPP-4 inhibitor
linagliptin a potential adjunctive treatment for diabetic vascular complications.
188
7.3 Future directions
Whilst the acute relaxant effects of the GLP-1 agonist exendin-4 are of interest, of great
significance is the investigation of the capacity of long-term chronic treatment with GLP-1
agonist exendin-4 to improve endothelial function in conditions of cardiovascular risk including
diabetes and western, high fat diet. This would help in better understanding of the effects of their
effect on endothelial function.
Acute treatment of small blood vessels from western high fat diet fed rats with the DPP-4
inhibitor linagliptin markedly improved vascular function may have been by inhibiting the
activity of NADPH oxidase derived ROS. Hence, future experiments would be useful to
investigate the effects of long-term chronic treatment with linagliptin on endothelial function in
conditions of western high fat diet.
The observations of preclinical studies have indicated a possible beneficial cardiovascular
action of DPP-4 inhibitors through both glucagon-like peptide 1 (GLP-1)-dependent and GLP-1-
independent pathways. Clinically, DPP-4 inhibitors improve several risk factors such as
improvement of blood glucose homeostasis, reduced blood pressure, improve postprandial
lipaemia, reduced inflammatory markers, reduced oxidative stress, and improved endothelial
function in individuals with type 2 diabetes. Some beneficial effect was also observed in patients
with ischaemic heart disease or congestive heart failure (Bose et al., 2005, Anagnostis et al.,
2011, Jose and Inzucchi, 2012). However, the actual relationship between the beneficial effects
of DPP-4 inhibitors such as linagliptin and cardiovascular events remains to be demonstrated,
thus investigation of the effect of DPP-4 inhibitor linagliptin on the ischemic/reperfusion cardiac
injury would help insight into the effects of gliptins such as linagliptin on cardiovascular
outcomes.
189
7.4 Conclusion
Taken together, the set of studies conducted in the present thesis have contributed to
better understanding of the beneficial action of the DPP-4 inhibitor linagliptin on endothelial
function independent of decreasing blood glucose levels. The data presented in the thesis
provides an insight into the effects of diabetic conditions and western high fat diet on the
mechanism(s) of endothelium-dependent relaxation. Also, data obtained in this thesis would help
in understanding the mechanism of action of the DPP-4 inhibitor linagliptin used in the treatment
of endothelial dysfunction in disorders such as diabetes and metabolic syndrome. Endothelial
dysfunction is the initial step of the commencement of cardiovascular complication such as
atherosclerosis. Conservation of endothelial function would delay and/or avoid complications of
cardiovascular risk factors and reduce the prevalence of the mortality and morbidity among
diabetic patients. As the DPP-4 inhibitor linagliptin treatment has demonstrated valuable effects
in improving endothelial function, it may be a useful adjunct therapy in the treatment of
endothelial dysfunction in diabetes independent of lowering blood glucose. This would make the
range of patients able to benefit from the use of this drug to be greatly expanded.
190
191
Chapter 8
References
Absi M, Burnham MP, Weston AH, Harno E, Rogers M, Edwards G (2007). Effects of methyl
beta-cyclodextrin on edhf responses in pig and rat arteries; association between skca
channels and caveolin-rich domains. Br J Pharmacol, 151, 332-340.
Ahren B, Landin-Olsson M, Jansson PA, Svensson M, Holmes D, Schweizer A (2004).
Inhibition of dipeptidyl peptidase-4 reduces glycemia, sustains insulin levels, and reduces
glucagon levels in type 2 diabetes. J Clin Endocrinol Metab, 89, 2078-2084.
Al Suwaidi J, Hamasaki S, Higano ST, Nishimura RA, Holmes DR, Lerman A (2000). Long-
term follow-up of patients with mild coronary artery disease and endothelial dysfunction.
Circulation, 101, 948-954.
Amori RE, Lau J, Pittas AG (2007). Efficacy and safety of incretin therapy in type 2 diabetes:
Systematic review and meta-analysis. JAMA, 298, 194-206.
Anagnostis P, Athyros VG, Adamidou F, Panagiotou A, Kita M, Karagiannis A, et al. (2011).
Glucagon-like peptide-1-based therapies and cardiovascular disease: Looking beyond
glycaemic control. Diabetes Obes Metab, 13, 302-312.
192
Andrews KL, Irvine JC, Tare M, Apostolopoulos J, Favaloro JL, Triggle CR, et al. (2009). A
role for nitroxyl (hno) as an endothelium‐derived relaxing and hyperpolarizing factor in
resistance arteries. Br J Pharmacol, 157, 540-550.
Antoniades C, Tousoulis D, Tountas C, Tentolouris C, Toutouza M, Vasiliadou C, et al. (2004).
Vascular endothelium and inflammatory process, in patients with combined type 2
diabetes mellitus and coronary atherosclerosis: The effects of vitamin c. Diabet Med, 21,
552-558.
Arakawa M, Mita T, Azuma K, Ebato C, Goto H, Nomiyama T, et al. (2010). Inhibition of
monocyte adhesion to endothelial cells and attenuation of atherosclerotic lesion by a
glucagon-like peptide-1 receptor agonist, exendin-4. Diabetes, 59, 1030-1037.
Arcaro G, Zamboni M, Rossi L, Turcato E, Covi G, Armellini F, et al. (1999). Body fat
distribution predicts the degree of endothelial dysfunction in uncomplicated obesity. Int J
Obes Relat Metab Disord, 23, 936-942.
Arnal JF, Dinh-Xuan AT, Pueyo M, Darblade B, Rami J (1999). Endothelium-derived nitric
oxide and vascular physiology and pathology. Cell Mol Life Sci, 55, 1078-1087.
Aroor AR, Sowers JR, Bender SB, Nistala R, Garro M, Mugerfeld I, et al. (2013).
Dipeptidylpeptidase inhibition is associated with improvement in blood pressure and
193
diastolic function in insulin-resistant male zucker obese rats. Endocrinology, 154, 2501-
2513.
Aschner P, Kipnes MS, Lunceford JK, Sanchez M, Mickel C, Williams-Herman DE (2006).
Effect of the dipeptidyl peptidase-4 inhibitor sitagliptin as monotherapy on glycemic
control in patients with type 2 diabetes. Diabetes care, 29, 2632-2637.
Augustyns K, Bal G, Thonus G, Belyaev A, Zhang X, Bollaert W, et al. (1999). The unique
properties of dipeptidyl-peptidase iv (dpp iv/cd26) and the therapeutic potential of dpp iv
inhibitors. Cur med chemist, 6, 311-328.
Australia Diabetes. 2011. Australia diabetes , the facts,
http://www.Diabetesnsw.Com.Au/australiandiabetescounil/media/pdfs/diabetes-fact-
sheet-2011.Pdf accessed on 07- 2011 [Online].
Ávila DDL, Araújo GRD, Silva M, Miranda PHDA, Diniz MF, Pedrosa ML, et al. (2013).
Vildagliptin ameliorates oxidative stress and pancreatic beta cell destruction in type 1
diabetic rats. Arch Med Res, 44, 194-202.
Ayaori M, Iwakami N, Uto‐Kondo H, Sato H, Sasaki M, Komatsu T, et al. (2013). Dipeptidyl
peptidase‐4 inhibitors attenuate endothelial function as evaluated by flow‐mediated
vasodilatation in type 2 diabetic patients. J Am Heart Assoc,, 2, e003277.
194
Babior BM (2004). Nadph oxidase. Curr Opin Immunol, 16, 42-47.
Babior BM, Lambeth JD, Nauseef W (2002). The neutrophil nadph oxidase. Arch Biochem
Biophys, 397, 342-344.
Baggio LL,Drucker DJ (2007). Biology of incretins: Glp-1 and gip. Gastroenterology, 132,
2131-2157.
Bailey CJ (2008). Metformin: Effects on micro and macrovascular complications in type 2
diabetes. Cardiovasc Drugs Ther, 22, 215-224.
Banting FG, Best CH, Collip JB, Campbell WR, Fletcher AA (1922). Pancreatic extracts in the
treatment of diabetes mellitus. Can Med Assoc J, 12, 141-146.
Barac A, Campia U, Panza JA (2007). Methods for evaluating endothelial function in humans.
Hypertension, 49, 748-760.
Barbieri M, Rizzo M, Marfella R, Boccardi V, Esposito A, Pansini A, et al. (2013). Decreased
carotid atherosclerotic process by control of daily acute glucose fluctuations in diabetic
patients treated by dpp-iv inhibitors. Atherosclerosis, 227, 349-354.
Barlow RS, White RE (1998). Hydrogen peroxide relaxes porcine coronary arteries by
stimulating bkcachannel activity. Am J Physiol Heart Circ Physiol, 275, 1283-1289.
195
Barnett A (2006). Dpp‐4 inhibitors and their potential role in the management of type 2 diabetes.
Int J Clin Pract, 60, 1454-1470.
Baron A, Frieden M, Bény JL (1997). Epoxyeicosatrienoic acids activate a high‐conductance,
ca2+‐dependent k
+ channel on pig coronary artery endothelial cells. J Physiol, 504, 537-
543.
Basu A, Charkoudian N, Schrage W, Rizza RA, Basu R, Joyner MJ (2007). Beneficial effects of
glp-1 on endothelial function in humans: Dampening by glyburide but not by glimepiride.
Am J Physiol Endocrinol Metab, 293, 1289-1295.
Bayliss WM, Starling EH (1902). The mechanism of pancreatic secretion. J Physiol, 28, 325-
353.
Bayraktutan U (2002). Free radicals, diabetes and endothelial dysfunction. Diabetes Obes Metab,
4, 224-238.
Beckman IS (2003). Nitric oxide, peroxynitrite and ageing. Critical Reviews of Oxidative Stress
and Aging: Advances in Basic Science, Diagno and Interven, 1, 54.
Behrendt D, Ganz P (2002). Endothelial function. From vascular biology to clinical applications.
Am J Cardiol, 90, 40-48.
196
Bell GI, Santerre RF, Mullenbach GT (1983). Hamster preproglucagon contains the sequence of
glucagon and two related peptides. Nature, 302, 716-718.
Bitar MS, Wahid S, Mustafa S, Al-Saleh E, Dhaunsi GS, Al-Mulla F (2005). Nitric oxide
dynamics and endothelial dysfunction in type ii model of genetic diabetes. Eur J
Pharmacol, 511, 53-64.
Bolotina VM, Najibi S, Palacino JJ, Pagano PJ, Cohen RA (1994). Nitric oxide directly activates
calcium-dependent potassium channels in vascular smooth muscle. Nature, 368, 850-853.
Bose AK, Mocanu MM, Carr RD, Brand CL, Yellon DM (2005). Glucagon-like peptide 1 can
directly protect the heart against ischemia/reperfusion injury. Diabetes, 54, 146-151.
Boyle JP, Thompson TJ, Gregg EW, Barker LE, Williamson DF (2010). Projection of the year
2050 burden of diabetes in the us adult population: Dynamic modeling of incidence,
mortality, and prediabetes prevalence. Popul Health Metr, 8, 29.
Brondum E, Kold-Petersen H, Simonsen U, Aalkjaer C (2010). Ns309 restores EDHF-type
relaxation in mesenteric small arteries from type 2 diabetic zdf rats. Br J Pharmacol, 159,
154-165.
Brown A, Reynolds LR, Bruemmer D (2010). Intensive glycemic control and cardiovascular
disease: An update. Nat Rev Cardiol, 7, 369-375.
197
Brown J, Dryburgh J, Ross S, Dupre J (1974). Identification and actions of gastric inhibitory
polypeptide. Recent Prog Horm Res, 31, 487-532.
Brown JC, Dryburgh JR (1971). A gastric inhibitory polypeptide. Ii. The complete amino acid
sequence. Can J Biochem, 49, 867-872.
Brownlee M (1995). Advanced protein glycosylation in diabetes and aging. Annu Rev Med, 46,
223-34.
Brownlee M (2001). Biochemistry and molecular cell biology of diabetic complications. Nature,
414, 813-820.
Bullen ML, Miller AA, Andrews KL, Irvine JC, Ritchie RH, Sobey CG, et al. (2011). Nitroxyl
(HNO) as a vasoprotective signaling molecule. Antioxid Redox Signa, 14, 1675-1686.
Burnham MP, Johnson IT, Weston AH (2006a). Impaired small-conductance ca2+
-activated k+
channel-dependent EDHF responses in type II diabetic zdf rats. Br J Pharmacol, 148,
434-441.
Burnham MP, Johnson IT, Weston AH (2006b). Reduced ca2+
-dependent activation of large-
conductance ca2+
-activated k+ channels from arteries of type 2 diabetic Zucker diabetic
fatty rats. Am J Physiol Heart Circ Physiol, 290, 1520-1527.
198
Buscemi S, Verga S, Tranchina MR, Cottone S, Cerasola G (2009). Effects of hypocaloric very-
low-carbohydrate diet vs. Mediterranean diet on endothelial function in obese women.
Eur J Clin Invest, 39, 339-347.
Busija DW, Miller AW, Katakam P, Erdos B (2005). Insulin resistance and associated
dysfunction of resistance vessels and arterial hypertension. Minerva Med, 96, 223-232.
Busse R, Edwards G, Feletou M, Fleming I, Vanhoutte PM, Weston AH (2002). EDHF:
Bringing the concepts together. Trends Pharmacol Sci, 23, 374-380.
Caballero AE (2003). Endothelial dysfunction in obesity and insulin resistance: A road to
diabetes and heart disease. Obes Res, 11, 1278-89.
Cai H, Harrison DG (2000). Endothelial dysfunction in cardiovascular diseases: The role of
oxidant stress. Circulation research, 87, 840-844.
Cai S, Alp NJ, Mcdonald D, Smith I, Kay J, Canevari L, et al. (2002). GTP cyclohydrolase i
gene transfer augments intracellular tetrahydrobiopterin in human endothelial cells:
Effects on nitric oxide synthase activity, protein levels and dimerisation. Cardiovasc Res,
55, 838-849.
199
Cai S, Khoo J, Mussa S, Alp N, Channon K (2005). Endothelial nitric oxide synthase
dysfunction in diabetic mice: Importance of tetrahydrobiopterin in eNOS dimerisation.
Diabetologia, 48, 1933-1940.
Calles-Escandon J, Cipolla M (2001). Diabetes and endothelial dysfunction: A clinical
perspective. Endocr Rev, 22, 36-52.
Campbell WB, Gebremedhin D, Pratt PF, Harder DR (1996). Identification of
epoxyeicosatrienoic acids as endothelium-derived hyperpolarizing factors. Circ Res, 78,
415-423.
Carter WO, Narayanan PK, Robinson JP (1994). Intracellular hydrogen peroxide and superoxide
anion detection in endothelial cells. J Leukoc Biol, 55, 253-258.
Ceriello A, Dello Russo P, Amstad P, Cerutti P (1996). High glucose induces antioxidant
enzymes in human endothelial cells in culture. Evidence linking hyperglycemia and
oxidative stress. Diabetes, 45, 471-477.
Chadha PS, Haddock RE, Howitt L, Morris MJ, Murphy TV, Grayson TH, et al. (2010). Obesity
up-regulates intermediate conductance calcium-activated potassium channels and
myoendothelial gap junctions to maintain endothelial vasodilator function. J Pharmacol
Exp Ther, 335, 284-293.
200
Channon KM (2004). Tetrahydrobiopterin: Regulator of endothelial nitric oxide synthase in
vascular disease. Trends Cardiovasc Med, 14, 323-327.
Chaykovska L, Alter ML, Von Websky K, Hohmann M, Tsuprykov O, Reichetzeder C, et al.
(2013). Effects of telmisartan and linagliptin when used in combination on blood pressure
and oxidative stress in rats with 2-kidney-1-clip hypertension. J Hypertens, 31, 2290-
2299.
Chen G, Suzuki H, Weston AH (1988). Acetylcholine releases endothelium-derived
hyperpolarizing factor and edrf from rat blood vessels. Br J Pharmacol, 95, 1165-1174.
Chen J, Couto FM, Minn AH, Shalev A (2006). Exenatide inhibits β-cell apoptosis by decreasing
thioredoxin-interacting protein. Biochem Biophys Res Commun, 346, 1067-1074.
Chew P, Yuen DY, Stefanovic N, Pete J, Coughlan MT, Jandeleit-Dahm KA, et al. (2010).
Antiatherosclerotic and renoprotective effects of ebselen in the diabetic apolipoprotein
e/gpx1-double knockout mouse. Diabetes, 59, 3198-3207.
Chrissobolis S, Ziogas J, Chu Y, Faraci FM, Sobey CG (2000). Role of inwardly rectifying k(+)
channels in k(+)
-induced cerebral vasodilatation in vivo. Am J Physiol Heart Circ Physiol,
279, 2704-2712.
201
Chung SS, Ho EC, Lam KS, Chung SK (2003). Contribution of polyol pathway to diabetes-
induced oxidative stress. J Am Soc Nephrol, 14, S233-5236.
Clark RJ, Mcdonough PM, Swanson E, Trost SU, Suzuki M, Fukuda M, et al. (2003). Diabetes
and the accompanying hyperglycemia impairs cardiomyocyte calcium cycling through
increased nuclear o-glcnacylation. J Biol Chem, 278, 44230-44237.
Clark SG, Fuchs LC (1997). Role of nitric oxide and ca++
-dependent k+ channels in mediating
heterogeneous microvascular responses to acetylcholine in different vascular beds. J
Pharmacol Exp Ther, 282, 1473-1479.
Conarello SL, Li Z, Ronan J, Roy RS, Zhu L, Jiang G, et al. (2003). Mice lacking dipeptidyl
peptidase iv are protected against obesity and insulin resistance. Proc Natl Acad Sci U S
A, 100, 6825-6830.
Cordero OJ, Salgado FJ, Nogueira M (2009). On the origin of serum CD26 and its altered
concentration in cancer patients. Cancer Immunol Immunother, 58, 1723-1747.
Cosentino F, Eto M, De Paolis P, Van Der Loo B, Bachschmid M, Ullrich V, et al. (2003). High
glucose causes upregulation of cyclooxygenase-2 and alters prostanoid profile in human
endothelial cells role of protein kinase c and reactive oxygen species. Circulation, 107,
1017-1023.
202
Cosentino F, Hishikawa K, Katusic ZS, Lüscher TF (1997). High glucose increases nitric oxide
synthase expression and superoxide anion generation in human aortic endothelial cells.
Circulation, 96, 25-28.
Crabtree MJ, Hale AB, Channon KM (2011). Dihydrofolate reductase protects endothelial nitric
oxide synthase from uncoupling in tetrahydrobiopterin deficiency. Free Radic Biol Med,
50, 1639-1646.
Crabtree MJ, Smith CL, Lam G, Goligorsky MS, Gross SS (2008). Ratio of 5, 6, 7, 8-
tetrahydrobiopterin to 7, 8-dihydrobiopterin in endothelial cells determines glucose-
elicited changes in no vs. Superoxide production by enos. Am J Physiol Heart Circ
Physiol, 294, 1530-1540.
Crabtree MJ, Tatham AL, Al-Wakeel Y, Warrick N, Hale AB, Cai S, et al. (2009a). Quantitative
regulation of intracellular endothelial nitric-oxide synthase (enos) coupling by both
tetrahydrobiopterin-enos stoichiometry and biopterin redox status insights from cells with
tet-regulated gtp cyclohydrolase i expression. J Biol Chem, 284, 1136-1144.
Crabtree MJ, Tatham AL, Hale AB, Alp NJ, Channon KM (2009b). Critical role for
tetrahydrobiopterin recycling by dihydrofolate reductase in regulation of endothelial
nitric-oxide synthase coupling: Relative importance of the de novo biopterin synthesis
versus salvage pathways. J Biol Chem, 284, 28128-28136.
203
Crane GJ, Gallagher N, Dora KA, Garland CJ (2003). Small- and intermediate-conductance
calcium-activated k+ channels provide different facets of endothelium-dependent
hyperpolarization in rat mesenteric artery. J Physiol, 553, 183-189.
Cuevas AM, Guasch V, Castillo O, Irribarra V, Mizon C, San Martin A, et al. (2000). A high-fat
diet induces and red wine counteracts endothelial dysfunction in human volunteers.
Lipids, 35, 143-148.
David M, Nathan M, Patricia A, Cleary M, Jye-Yu C, Backlund M, et al. (2005). Intensive
diabetes treatment and cardiovascular disease in patients with type 1 diabetes. N Engl J
Med, 353, 2643.
De Bono JP, Channon KM (2007). Endothelial cell tetrahydrobiopterin: Going with the flow.
Circ Res, 101, 752-754.
De Vriese AS, Verbeuren TJ, Van De Voorde J, Lameire NH, Vanhoutte PM (2000). Endothelial
dysfunction in diabetes. Br J Pharmacol, 130, 963-974.
De Wit C (2010). Different pathways with distinct properties conduct dilations in the
microcirculation in vivo. Cardiovasc Res, 85, 604-613.
De Wit C, Griffith TM (2010). Connexins and gap junctions in the edhf phenomenon and
conducted vasomotor responses. Pflugers Arch, 459, 897-914.
204
Deacon CF, Holst JJ (2010). Linagliptin, a xanthine-based dipeptidyl peptidase-4 inhibitor with
an unusual profile for the treatment of type 2 diabetes. Expert Opin Investig Drugs, 19,
133-140.
Derubertis F, Craven P (1994). Activation of protein kinase c in glomerular cells in diabetes.
Mechanisms and potential links to the pathogenesis of diabetic glomerulopathy. Diabetes,
43, 1-8.
Ding H, Aljofan M, Triggle CR (2007). Oxidative stress and increased enos and nadph oxidase
expression in mouse microvessel endothelial cells. J Cell Physiol, 212, 682-689.
Ding H, Hashem M, Wiehler WB, Lau W, Martin J, Reid J, et al. (2005). Endothelial
dysfunction in the streptozotocin-induced diabetic apoe-deficient mouse. Br J
Pharmacol, 146, 1110-1118.
Ding L, Zhang J (2012). Glucagon-like peptide-1 activates endothelial nitric oxide synthase in
human umbilical vein endothelial cells. Acta Pharmacol Sin, 33, 75-81.
Diniz YS, Rocha KK, Souza GA, Galhardi CM, Ebaid GM, Rodrigues HG, et al. (2006). Effects
of n-acetylcysteine on sucrose-rich diet-induced hyperglycaemia, dyslipidemia and
oxidative stress in rats. Eur J Pharmacol, 543, 151-157.
205
Donald M, Dower J, Ware R, Mukandi B, Parekh S, Bain C (2012). Living with diabetes:
Rationale, study design and baseline characteristics for an australian prospective cohort
study. BMC Public Health, 12, 8.
Dora KA (2010). Coordination of vasomotor responses by the endothelium. Circ J, 74, 226-232.
Dora KA, Gallagher NT, Mcneish A, Garland CJ (2008). Modulation of endothelial cell kca3.1
channels during endothelium-derived hyperpolarizing factor signaling in mesenteric
resistance arteries. Circ Res, 102, 1247-1255.
Doyle ME, Egan JM (2007). Mechanisms of action of glucagon-like peptide 1 in the pancreas.
Pharmacol Ther, 113, 546-593.
Drucker DJ (2003). Enhancing incretin action for the treatment of type 2 diabetes. Diabetes
Care, 26, 2929-2940.
Drucker DJ (2006). The biology of incretin hormones. Cell Metab, 3, 153-165.
Drucker DJ (2007). Dipeptidyl peptidase-4 inhibition and the treatment of type 2 diabetes:
Preclinical biology and mechanisms of action. Diabetes Care, 30, 1335-1343.
Drucker DJ, Nauck MA (2006). The incretin system: Glucagon-like peptide-1 receptor agonists
and dipeptidyl peptidase-4 inhibitors in type 2 diabetes. The Lancet, 368, 1696-1705.
206
Drummond GR, Selemidis S, Griendling KK, Sobey CG (2011). Combating oxidative stress in
vascular disease: Nadph oxidases as therapeutic targets. Nat Rev Drug Discov, 10, 453-
471.
Du XL, Edelstein D, Rossetti L, Fantus IG, Goldberg H, Ziyadeh F, et al. (2000).
Hyperglycemia-induced mitochondrial superoxide overproduction activates the
hexosamine pathway and induces plasminogen activator inhibitor-1 expression by
increasing sp1 glycosylation. Proc Natl Acad Sci U S A, 97, 12222-12226.
Dunstan DW, Zimmet PZ, Welborn TA, De Courten MP, Cameron AJ, Sicree RA, et al. (2002).
The rising prevalence of diabetes and impaired glucose tolerance: The australian diabetes,
obesity and lifestyle study. Diabetes Care, 25, 829-834.
Dupre J, Ross SA, Watson D, Brown JC (1973). Stimulation of insulin secretion by gastric
inhibitory polypeptide in man. J Clin Endocrinol Metab, 37, 826-828.
Dzau VJ, Antman EM, Black HR, Hayes DL, Manson JE, Plutzky J, et al. (2006). The
cardiovascular disease continuum validated: Clinical evidence of improved patient
outcomes part i: Pathophysiology and clinical trial evidence (risk factors through stable
coronary artery disease). Circulation, 114, 2850-2870.
Earley S, Heppner TJ, Nelson MT, Brayden JE (2005). Trpv4 forms a novel ca2+
signaling
complex with ryanodine receptors and bkca channels. Circ Res, 97, 1270-1279.
207
Earley S, Pauyo T, Drapp R, Tavares MJ, Liedtke W, Brayden JE (2009). Trpv4-dependent
dilation of peripheral resistance arteries influences arterial pressure. Am J Physiol Heart
Circ Physiol, 297, 1096-1102.
Eckhardt M, Langkopf E, Mark M, Tadayyon M, Thomas L, Nar H, et al. (2007). 8-(3-(r)-
aminopiperidin-1-yl)-7-but-2-ynyl-3-methyl-1-(4-methyl-quinazolin-2-ylmethyl)-3, 7-
dihydropurine-2, 6-dione (bi 1356), a highly potent, selective, long-acting, and orally
bioavailable dpp-4 inhibitor for the treatment of type 2 diabetes†. J Med Chem, 50, 6450-
6453.
Edwards G, Dora K, Gardener M, Garland C, Weston A (1998). K+ is an endothelium-
derived hyperpolarizing factor in rat arteries. Nature, 396, 269-272.
Edwards G, Feletou M, Gardener MJ, Thollon C, Vanhoutte PM, Weston AH (1999). Role of
gap junctions in the responses to edhf in rat and guinea-pig small arteries. Br J
Pharmacol, 128, 1788-1794.
Edwards G, Feletou M, Weston AH (2010). Endothelium-derived hyperpolarising factors and
associated pathways: A synopsis. Pflugers Arch, 459, 863-879.
Edwards G, Thollon C, Gardener MJ, Feletou M, Vilaine J, Vanhoutte PM, et al. (2000). Role of
gap junctions and eets in endothelium-dependent hyperpolarization of porcine coronary
artery. Br J Pharmacol, 129, 1145-1154.
208
Eliasson B, Möller-Goede D, Eeg-Olofsson K, Wilson C, Cederholm J, Fleck P, et al. (2012).
Lowering of postprandial lipids in individuals with type 2 diabetes treated with alogliptin
and/or pioglitazone: A randomised double-blind placebo-controlled study. Diabetologia,
55, 915-925.
Elliott RM, Morgan LM, Tredger JA, Deacon S, Wright J, Marks V (1993). Glucagon-like
peptide-1 (7-36)amide and glucose-dependent insulinotropic polypeptide secretion in
response to nutrient ingestion in man: Acute post-prandial and 24-h secretion patterns. J
Endocrinol, 138, 159-166.
Ellis A, Li CG, Rand MJ (2000). Differential actions of l-cysteine on responses to nitric oxide,
nitroxyl anions and edrf in the rat aorta. Br J Pharmacol, 129, 315-322.
Elrick H, Stimmler L, Hlad CJ, Jr., Arai Y (1964). Plasma insulin response to oral and
intravenous glucose administration. J Clin Endocrinol Metab, 24, 1076-1082.
Epstein FH, Vane JR, Änggård EE, Botting RM (1990). Regulatory functions of the vascular
endothelium. N Engl J Med, 323, 27-36.
Erdogdu O, Eriksson L, Xu H, Sjoholm A, Zhang Q, Nystrom T (2013). Exendin-4 protects
endothelial cells from lipoapoptosis by pka, pi3k, enos, p38 mapk, and jnk pathways. J
Mol Endocrinol, 50, 229-241.
209
Erdogdu Ö, Nathanson D, Sjöholm Å, Nyström T, Zhang Q (2010). Exendin-4 stimulates
proliferation of human coronary artery endothelial cells through eNOS, PKA-and
PI3K/Akt-dependent pathways and requires GLP-1 receptor. Mol Cell Endocrinol, 325,
26-35.
Ervinna N, Mita T, Yasunari E, Azuma K, Tanaka R, Fujimura S, et al. (2013). Anagliptin, a
DPP-4 inhibitor, suppresses proliferation of vascular smooth muscles and monocyte
inflammatory reaction and attenuates atherosclerosis in male apo e-deficient mice.
Endocrinology, 154, 1260-1270.
Fadini GP, Avogaro A (2011). Cardiovascular effects of DPP-4 inhibition: Beyond GLP-1.
Vascul Pharmacol, 55, 10-16.
Fadini GP, Boscaro E, Albiero M, Menegazzo L, Frison V, De Kreutzenberg S, et al. (2010).
The oral dipeptidyl peptidase-4 inhibitor sitagliptin increases circulating endothelial
progenitor cells in patients with type 2 diabetes: Possible role of stromal-derived factor-
1alpha. Diabetes Care, 33, 1607-1609.
Fakhoury W, Lereun C, Wright D (2009). A meta-analysis of placebo-controlled clinical trials
assessing the efficacy and safety of incretin-based medications in patients with type 2
diabetes. Pharmacology, 86, 44-57.
210
Faraci FM, Didion SP (2004). Vascular protection: Superoxide dismutase isoforms in the vessel
wall. Arterioscler Thromb Vasc Biol, 24, 1367-1373.
Farilla L, Hui H, Bertolotto C, Kang E, Bulotta A, Di Mario U, et al. (2002). Glucagon-like
peptide-1 promotes islet cell growth and inhibits apoptosis in zucker diabetic rats.
Endocrinology, 143, 4397-4408.
Fatehi-Hassanabad Z, Chan CB,Furman BL (2010). Reactive oxygen species and endothelial
function in diabetes. Eur J Pharmacol, 636, 8-17.
Favaloro JL, Kemp-Harper BK (2009). Redox variants of no (NO and HNO) elicit
vasorelaxation of resistance arteries via distinct mechanisms. Am J Physiol Heart Circ
Physiol, 296, 1274-1280.
Federici M, Menghini R, Mauriello A, Hribal ML, Ferrelli F, Lauro D, et al. (2002). Insulin-
dependent activation of endothelial nitric oxide synthase is impaired by o-linked
glycosylation modification of signaling proteins in human coronary endothelial cells.
Circulation, 106, 466-472.
Feletou M, Vanhoutte PM (1999). The alternative: EDHF. J Mol Cell Cardiol, 31, 15-22.
211
Feng J, Damrauer SM, Lee M, Sellke FW, Ferran C, Abid MR (2010). Endothelium-dependent
coronary vasodilatation requires NADPH oxidase-derived reactive oxygen species.
Arterioscler Thromb Vasc Biol, 30, 1703-1710.
Ferrara N (2002). Role of vascular endothelial growth factor in physiologic and pathologic
angiogenesis: Therapeutic implications. Semin Oncol, 29, 10-14.
Figueroa XF, Paul DL, Simon AM, Goodenough DA, Day KH, Damon DN, et al. (2003).
Central role of connexin 40 in the propagation of electrically activated vasodilation in
mouse cremasteric arterioles in vivo. Circ Res, 92, 793-800.
Fineman MS, Bicsak TA, Shen LZ, Taylor K, Gaines E, Varns A, et al. (2003). Effect on
glycemic control of exenatide (synthetic exendin-4) additive to existing metformin and/or
sulfonylurea treatment in patients with type 2 diabetes. Diabetes Care, 26, 2370-2377.
Fisslthaler B, Popp R, Kiss L, Potente M, Harder DR, Fleming I, et al. (1999). Cytochrome P450
2C is an EDHF synthase in coronary arteries. Nature, 401, 493-497.
Flammer AJ, Luscher TF (2010). Three decades of endothelium research: From the detection of
nitric oxide to the everyday implementation of endothelial function measurements in
cardiovascular diseases. Swiss medical weekly, 141, 3-8.
212
Fleming I, Rueben A, Popp R, Fisslthaler B, Schrodt S, Sander A, et al. (2007).
Epoxyeicosatrienoic acids regulate TRP channel dependent ca2+
signaling and
hyperpolarization in endothelial cells. Arterioscler Thromb Vasc Biol, 27, 2612-2618.
Forstermann U (2008). Oxidative stress in vascular disease: Causes, defense mechanisms and
potential therapies. Nat Clin Pract Cardiovasc Med, 5, 338-349.
Frederich R, Alexander JH, Fiedorek FT, Donovan M, Berglind N, Harris S, et al. (2010). A
systematic assessment of cardiovascular outcomes in the saxagliptin drug development
program for type 2 diabetes. Postgrad Med, 122, 16-27.
Fu MX, Wells-Knecht KJ, Blackledge JA, Lyons TJ, Thorpe SR, Baynes JW (1994). Glycation,
glycoxidation, and cross-linking of collagen by glucose. Kinetics, mechanisms, and
inhibition of late stages of the maillard reaction. Diabetes, 43, 676-683.
Fujiki T, Shimokawa H, Morikawa K, Kubota H, Hatanaka M, Talukder MA, et al. (2005).
Endothelium-derived hydrogen peroxide accounts for the enhancing effect of an
angiotensin-converting enzyme inhibitor on endothelium-derived hyperpolarizing factor-
mediated responses in mice. Arterioscler Thromb Vasc Biol, 25, 766-771.
Fukao M, Hattori Y, Kanno M, Sakuma I, Kitabatake A (1997). Alterations in endothelium-
dependent hyperpolarization and relaxation in mesenteric arteries from streptozotocin-
induced diabetic rats. Br J Pharmacol, 121, 1383-1391.
213
Furchgott RF, Vanhoutte PM (1989). Endothelium-derived relaxing and contracting factors.
FASEB J, 3, 2007-2018.
Furchgott RF, Zawadzki JV (1980). The obligatory role of endothelial cells in the relaxation of
arterial smooth muscle by acetylcholine. Nature, 288, 373-376.
Furukawa S, Fujita T, Shimabukuro M, Iwaki M, Yamada Y, Nakajima Y, et al. (2004).
Increased oxidative stress in obesity and its impact on metabolic syndrome. J Clin Invest,
114, 1752-1761.
Gallwitz B (2012). Linagliptin-a novel dipeptidyl peptidase inhibitor for type 2 diabetes therapy.
Clin Med Insights Endocrinol Diabetes, 5, 1-11.
Garland CJ, Hiley CR, Dora KA (2011). EDHF: Spreading the influence of the endothelium. Br
J Pharmacol, 164, 839-852.
Garland CJ, Plane F, Kemp BK, Cocks TM (1995). Endothelium-dependent hyperpolarization: A
role in the control of vascular tone. Trends Pharmacol Sci, 16, 23-30.
Gaspari T, Welungoda I, Widdop RE, Simpson RW, Dear AE (2013). The GLP-1 receptor
agonist liraglutide inhibits progression of vascular disease via effects on atherogenesis,
plaque stability and endothelial function in an apoe−/−
mouse model. Diab Vasc Dis Res,
10, 353-360.
214
Gedulin BR, Nikoulina SE, Smith PA, Gedulin G, Nielsen LL, Baron AD, et al. (2005).
Exenatide (exendin-4) improves insulin sensitivity and β-cell mass in insulin-resistant
obese fa/fa zucker rats independent of glycemia and body weight. Endocrinology, 146,
2069-2076.
Geraldes P, King GL (2010). Activation of protein kinase c isoforms and its impact on diabetic
complications. Circ Res, 106, 1319-1331.
Glorie LL, Verhulst A, Matheeussen V, Baerts L, Magielse J, Hermans N, et al. (2012). DPP4
inhibition improves functional outcome after renal ischemia-reperfusion injury. Am J
Physiol Renal Physiol, 303, 681-688.
Göke R, Fehmann H-C, Linn T, Schmidt H, Krause M, Eng J, et al. (1993). Exendin-4 is a high
potency agonist and truncated exendin-(9-39)-amide an antagonist at the glucagon-like
peptide 1-(7-36)-amide receptor of insulin-secreting beta-cells. J Biol Chem, 268, 19650-
19655.
Góth L (2008). Catalase deficiency and type 2 diabetes. Diabetes care, 31, e93.
Góth L, Lenkey Á, Bigler WN (2001). Blood catalase deficiency and diabetes in hungary.
Diabetes Care, 24, 1839-1840.
215
Green BD, Hand KV, Dougan JE, Mcdonnell BM, Cassidy RS, Grieve DJ (2008). GLP-1 and
related peptides cause concentration-dependent relaxation of rat aorta through a pathway
involving katp and camp. Arch Biochem Biophys, 478, 136-142.
Grgic I, Kaistha BP, Hoyer J, Köhler R (2009). Endothelial ca2+‐activated k
+ channels in normal
and impaired edhf–dilator responses–relevance to cardiovascular pathologies and drug
discovery. Br J Pharmacol, 157, 509-526.
Grundy SM, Benjamin IJ, Burke GL, Chait A, Eckel RH, Howard BV, et al. (1999). Diabetes
and cardiovascular disease: A statement for healthcare professionals from the american
heart association. Circulation, 100, 1134-1146.
Gryglewski RJ, Bunting S, Moncada S, Flower RJ, Vane JR (1976). Arterial walls are protected
against deposition of platelet thrombi by a substance (prostaglandin x) which they make
from prostaglandin endoperoxides. Prostaglandins, 12, 685-713.
Guo X, Liu WL, Chen LW, Guo ZG (2000). High glucose impairs endothelium-dependent
relaxation in rabbit aorta. Acta Pharmacol Sin, 21, 169-173.
Guzik TJ, Mussa S, Gastaldi D, Sadowski J, Ratnatunga C, Pillai R, et al. (2002). Mechanisms of
increased vascular superoxide production in human diabetes mellitus. Circulation, 105,
1656-1662.
216
Ha SJ, Kim W, Woo JS, Kim JB, Kim SJ, Kim WS, et al. (2012). Preventive effects of exenatide
on endothelial dysfunction induced by ischemia-reperfusion injury via KATP channels.
Arterioscler Thromb Vasc Biol, 32, 474-480.
Haddock RE, Grayson TH, Morris MJ, Howitt L, Chadha PS, Sandow SL (2011). Diet-induced
obesity impairs endothelium-derived hyperpolarization via altered potassium channel
signaling mechanisms. PLoS One, 6, e16423.
Hadi HA, Suwaidi JA (2007). Endothelial dysfunction in diabetes mellitus. Vasc Health Risk
Manag, 3, 853-76.
Hajer GR, Van Haeften TW, Visseren FL (2008). Adipose tissue dysfunction in obesity,
diabetes, and vascular diseases. Eur Heart J, 29, 2959-2971.
Halcox JP, Schenke WH, Zalos G, Mincemoyer R, Prasad A, Waclawiw MA, et al. (2002).
Prognostic value of coronary vascular endothelial dysfunction. Circulation, 106, 653-
658.
Haneda M, Araki S, Togawa M, Sugimoto T, Isono M, Kikkawa R (1997). Mitogen-activated
protein kinase cascade is activated in glomeruli of diabetic rats and glomerular mesangial
cells cultured under high glucose conditions. Diabetes, 46, 847-853.
217
Hansson GK (2005). Inflammation, atherosclerosis, and coronary artery disease. N Engl J Med,
352, 1685-1695.
Harrison DG, Chen W, Dikalov S, Li L (2010). Regulation of endothelial cell
tetrahydrobiopterin pathophysiological and therapeutic implications. Adv Pharmacol, 60,
107-32.
Hattori Y, Jojima T, Tomizawa A, Satoh H, Hattori S, Kasai K, et al. (2010). A glucagon-like
peptide-1 (glp-1) analogue, liraglutide, upregulates nitric oxide production and exerts
anti-inflammatory action in endothelial cells. Diabetologia, 53, 2256-2263.
Heales SJ (2001). Catalase deficiency, diabetes, and mitochondrial function. Lancet, 357, 314.
Heissig B, Hattori K, Dias S, Friedrich M, Ferris B, Hackett NR, et al. (2002). Recruitment of
stem and progenitor cells from the bone marrow niche requires MMP-9 mediated release
of kit-ligand. Cell, 109, 625-637.
Heitzer T, Krohn K, Albers S, Meinertz T (2000). Tetrahydrobiopterin improves endothelium-
dependent vasodilation by increasing nitric oxide activity in patients with type ii diabetes
mellitus. Diabetologia, 43, 1435-1438.
Hendarto H, Inoguchi T, Maeda Y, Ikeda N, Zheng J, Takei R, et al. (2012). GLP-1 analog
liraglutide protects against oxidative stress and albuminuria in streptozotocin-induced
218
diabetic rats via protein kinase a-mediated inhibition of renal NAD(P)H oxidases.
Metabolism, 61, 1422-1434.
Hevel JM, Marletta MA (1992). Macrophage nitric oxide synthase: Relationship between
enzyme-bound tetrahydrobiopterin and synthase activity. Biochemistry, 31, 7160-7165.
Hilgers RH, Webb RC (2007). Reduced expression of skca and ikca channel proteins in rat small
mesenteric arteries during angiotensin ii-induced hypertension. Am J Physiol Heart Circ
Physiol, 292, 2275-2284.
Hink U, Li H, Mollnau H, Oelze M, Matheis E, Hartmann M, et al. (2001). Mechanisms
underlying endothelial dysfunction in diabetes mellitus. Circ Res, 88, 14-22.
Hoebel BG, Kostner GM, Graier WF (1997). Activation of microsomal cytochrome P450 mono-
oxygenase by ca2+
store depletion and its contribution to ca2+
entry in porcine aortic
endothelial cells. Br J Pharmacol, 121, 1579-1588.
Hogg N, Darley-Usmar VM, Wilson MT, Moncada S (1992). Production of hydroxyl radicals
from the simultaneous generation of superoxide and nitric oxide. Biochem J, 2 419-424.
Hollander PA, Kushner P (2010). Type 2 diabetes comorbidities and treatment challenges:
Rationale for dpp-4 inhibitors. Postgrad Med, 122, 71-80.
219
Holman RR, Paul SK, Bethel MA, Matthews DR, Neil HA (2008). 10-year follow-up of
intensive glucose control in type 2 diabetes. N Engl J Med, 359, 1577-89.
Holz GG, Kühtreiber WM, Habener JF (1993). Pancreatic beta-cells are rendered glucose-
competent by the insulinotropic hormone glucagon-like peptide-1 (7-37). Nature, 361,
362-365.
Hou CJ, Tsai CH, Su CH, Wu YJ, Chen SJ, Chiu JJ, et al. (2008). Diabetes reduces aortic
endothelial gap junctions in ApoE -deficient mice: Simvastatin exacerbates the reduction.
J Histochem Cytochem, 56, 745-752.
Hsieh J, Longuet C, Baker C, Qin B, Federico L, Drucker D, et al. (2010). The glucagon-like
peptide 1 receptor is essential for postprandial lipoprotein synthesis and secretion in
hamsters and mice. Diabetologia, 53, 552-561.
Hu Y, Liu H, Simpson RW, Dear AE (2013). GLP-1-dependent and independent effects and
molecular mechanisms of a dipeptidyl peptidase 4 inhibitor in vascular endothelial cells.
Mol Biol Rep, 40, 2273-2279.
Ignarro LJ, Lippton H, Edwards JC, Baricos WH, Hyman AL, Kadowitz PJ, et al. (1981).
Mechanism of vascular smooth muscle relaxation by organic nitrates, nitrites,
nitroprusside and nitric oxide: Evidence for the involvement of s-nitrosothiols as active
intermediates. J Pharmacol Exp Ther, 218, 739-749.
220
Ii S, Ohta M, Kudo E, Yamaoka T, Tachikawa T, Moritani M, et al. (2004). Redox state-
dependent and sorbitol accumulation-independent diabetic albuminuria in mice with
transgene-derived human aldose reductase and sorbitol dehydrogenase deficiency.
Diabetologia, 47, 541-548.
Inoguchi T, Battan R, Handler E, Sportsman JR, Heath W,King GL (1992). Preferential
elevation of protein kinase c isoform beta ii and diacylglycerol levels in the aorta and
heart of diabetic rats: Differential reversibility to glycemic control by islet cell
transplantation. Proc Natl Acad Sci U S A, 89, 11059-11063.
Inoguchi T, Li P, Umeda F, Yu HY, Kakimoto M, Imamura M, et al. (2000). High glucose level
and free fatty acid stimulate reactive oxygen species production through protein kinase c-
-dependent activation of NAD(P)H oxidase in cultured vascular cells. Diabetes, 49, 1939-
1945.
Ishibashi Y, Matsui T, Maeda S, Higashimoto Y, Yamagishi S (2013). Advanced glycation end
products evoke endothelial cell damage by stimulating soluble dipeptidyl peptidase-4
production and its interaction with mannose 6-phosphate/insulin-like growth factor II
receptor. Cardiovasc Diabetol, 12, 125.
Ishibashi Y, Matsui T, Takeuchi M, Yamagishi S (2010). Glucagon-like peptide-1 (GLP-1)
inhibits advanced glycation end product (AGE)-induced up-regulation of VCAM-1
221
mRNA levels in endothelial cells by suppressing age receptor (RAGE) expression.
Biochem Biophys Res Commun, 391, 1405-1408.
Isomaa B, Almgren P, Tuomi T, Forsén B, Lahti K, Nissén M, et al. (2001). Cardiovascular
morbidity and mortality associated with the metabolic syndrome. Diabetes care, 24, 683-
689.
J Motta A, Koska J, Reaven P, Q Migrino R (2012). Vascular protective effects of diabetes
medications that mimic or increase glucagon-like peptide-1 activity. Recent Pat
Cardiovasc Drug Discov, 7, 2-9.
Jackson EK, Dubinion JH, Mi Z (2008). Effects of dipeptidyl peptidase iv inhibition on arterial
blood pressure. Clin Exp Pharmacol Physiol, 35, 29-34.
Jang H-J, Kokrashvili Z, Theodorakis MJ, Carlson OD, Kim B-J, Zhou J, et al. (2007). Gut-
expressed gustducin and taste receptors regulate secretion of glucagon-like peptide-1.
Proc Nat Acad Sci, 104, 15069-15074.
Jarasch ED, Grund C, Bruder G, Heid HW, Keenan TW, Franke WW (1981). Localization of
xanthine oxidase in mammary-gland epithelium and capillary endothelium. Cell, 25, 67.
Johansen O, Neubacher D, Von Eynatten M, Patel S, Woerle H (2011). Cardiovascular risk with
linagliptin in patients with type 2 diabetes: A pre-specified, prospective, and adjudicated
222
meta-analysis from a large phase iii program [abstract 0030-lb]. Proc 71st Annual Sci Ses
Am Diab Asso, 60, 24-28.
Jones RB, Vickers SP, Cheetham SC, Headland KR, Mark M,Klein T (2014). Effect of
linagliptin, alone and in combination with voglibose or exendin-4, on glucose control in
male zdf rats. Eur J Pharmacol, 729, 59-66.
Jose T, Inzucchi SE (2012). Cardiovascular effects of the DPP-4 inhibitors. Diab Vasc Dis Res,
9, 109-116.
Jourd'heuil D, Jourd'heuil FL, Kutchukian PS, Musah RA, Wink DA, Grisham MB (2001).
Reaction of superoxide and nitric oxide with peroxynitrite. Implications for peroxynitrite-
mediated oxidation reactions in vivo. J Biol Chem, 276, 28799-28805.
Kehrer JP (2000). The haber-weiss reaction and mechanisms of toxicity. Toxicology, 149, 43-50.
Khan FZ, Heck PM, Hoole SP, Dutka DP (2010). DPP-4 inhibition by sitagliptin improves the
myocardial response to dobutamine stress and mitigates stunning in a pilot study of
patients with coronary artery disease. Circ Cardiovasc Imaging, 3, 195-201.
Kim J, Montagnani M, Koh KK, Quon MJ (2006). Reciprocal relationships between insulin
resistance and endothelial dysfunction. Circulation, 113, 1888-1904.
223
Kim W,Egan JM (2008). The role of incretins in glucose homeostasis and diabetes treatment.
Pharmacol Rev, 60, 470-512.
Kimura F, Hasegawa G, Obayashi H, Adachi T, Hara H, Ohta M, et al. (2003). Serum
extracellular superoxide dismutase in patients with type 2 diabetes: Relationship to the
development of micro- and macrovascular complications. Diabetes Care, 26, 1246-1250.
Koibuchi N, Hasegawa Y, Katayama T, Toyama K, Uekawa K, Sueta D, et al. (2014). DPP-4
inhibitor linagliptin ameliorates cardiovascular injury in salt-sensitive hypertensive rats
independently of blood glucose and blood pressure. Cardiovasc Diabetol, 13, 157.
Kolm-Litty V, Sauer U, Nerlich A, Lehmann R, Schleicher E (1998). High glucose-induced
transforming growth factor beta1 production is mediated by the hexosamine pathway in
porcine glomerular mesangial cells. J Clin Invest, 101, 160.
Kolterman OG, Buse JB, Fineman MS, Gaines E, Heintz S, Bicsak TA, et al. (2003). Synthetic
exendin-4 (exenatide) significantly reduces postprandial and fasting plasma glucose in
subjects with type 2 diabetes. J Clin Transl Endocrinol, 88, 3082-3089.
Komatsu R, Matsuyama T, Namba M, Watanabe N, Itoh H, Kono N, et al. (1989).
Glucagonostatic and insulinotropic action of glucagonlike peptide i-(7–36)-amide.
Diabetes, 38, 902-905.
224
Komori K, Vanhoutte PM (1990). Endothelium-derived hyperpolarizing factor. Blood Vessels,
27, 238-245.
Koren S, Shemesh-Bar L, Tirosh A, Peleg RK, Berman S, Hamad RA, et al. (2012). The effect
of sitagliptin versus glibenclamide on arterial stiffness, blood pressure, lipids, and
inflammation in type 2 diabetes mellitus patients. Diabetes Technol Ther, 14, 561-567.
Korshunov SS, Skulachev VP, Starkov AA (1997). High protonic potential actuates a
mechanism of production of reactive oxygen species in mitochondria. FEBS Lett, 416,
15-18.
Koska J, Schwartz EA, Mullin MP, Schwenke DC, Reaven PD (2010). Improvement of
postprandial endothelial function after a single dose of exenatide in individuals with
impaired glucose tolerance and recent-onset type 2 diabetes. Diabetes care, 33, 1028-
1030.
Koya D,King GL (1998). Protein kinase c activation and the development of diabetic
complications. Diabetes, 47, 859-866.
Kroller-Schon S, Knorr M, Hausding M, Oelze M, Schuff A, Schell R, et al. (2012). Glucose-
independent improvement of vascular dysfunction in experimental sepsis by dipeptidyl-
peptidase 4 inhibition. Cardiovasc Res, 96, 140-149.
225
Kubes P, Suzuki M, Granger DN (1991). Nitric oxide: An endogenous modulator of leukocyte
adhesion. Proc Natl Acad Sci U S A, 88, 4651-5.
Kubota Y, Miyamoto M, Takagi G, Ikeda T, Kirinoki-Ichikawa S, Tanaka K, et al. (2012). The
dipeptidyl peptidase-4 inhibitor sitagliptin improves vascular endothelial function in type
2 diabetes. J Korean Med Sci, 27, 1364-1370.
Kuroki T, Inoguchi T, Umeda F, Ueda F, Nawata H (1998). High glucose induces alteration of
gap junction permeability and phosphorylation of connexin-43 in cultured aortic smooth
muscle cells. Diabetes, 47, 931-936.
La Barre J, Ledrut J (1934). Sur l'origine pancréatique et tissulaire de l'action hypoglycémiante
de l'incrétine préparée par hydrolyse pepsique. Arch Physiol Biochem, 40, 209-226.
Lee A, Chung SK, Chung S (1995). Demonstration that polyol accumulation is responsible for
diabetic cataract by the use of transgenic mice expressing the aldose reductase gene in the
lens. Proc Nat Acad Sci, 92, 2780-2784.
Lee AY, Chung SS (1999). Contributions of polyol pathway to oxidative stress in diabetic
cataract. FASEB J, 13, 23-30.
226
Lee TI, Kao YH, Chen YC, Huang JH, Hsu MI, Chen YJ (2013). The dipeptidyl peptidase-4
inhibitor-sitagliptin modulates calcium dysregulation, inflammation, and ppars in
hypertensive cardiomyocytes. Int J Cardiol, 168, 5390-5395.
Leo C-H, Hart JL, Woodman OL (2011a). 3′, 4′-dihydroxyflavonol reduces superoxide and
improves nitric oxide function in diabetic rat mesenteric arteries. PloS one, 6, e20813.
Leo C, Hart J, Woodman O (2011b). Impairment of both nitric oxide‐mediated and edhf‐type
relaxation in small mesenteric arteries from rats with streptozotocin‐induced diabetes. Br
J Pharmacol, 162, 365-377.
Leo CH, Joshi A, Woodman OL (2010). Short-term type 1 diabetes alters the mechanism of
endothelium-dependent relaxation in the rat carotid artery. Am J Physiol Heart Circ
Physiol, 299, 502-511.
Lewis P, Stefanovic N, Pete J, Calkin AC, Giunti S, Thallas-Bonke V, et al. (2007). Lack of the
antioxidant enzyme glutathione peroxidase-1 accelerates atherosclerosis in diabetic
apolipoprotein e–deficient mice. Circulation, 115, 2178-2187.
Li H, Horke S, Forstermann U (2013). Oxidative stress in vascular disease and its
pharmacological prevention. Trends Pharmacol Sci, 34, 313-319.
227
Li L, Rezvan A, Salerno JC, Husain A, Kwon K, Jo H, et al. (2010). GTP cyclohydrolase i
phosphorylation and interaction with GTP cyclohydrolase feedback regulatory protein
provide novel regulation of endothelial tetrahydrobiopterin and nitric oxide. Circ Res,
106, 328-336.
Li Y-F, Ru X-C, Wang H-P, Ma X, Jiang L-F, Xia Q (2008). Inhibition of superoxide anion-
mediated impairment of endothelium by treatment with luteolin in rat mesenteric artery.
FASEB J, 22, 1142-1144.
Lin K, Cai Z, Wang F, Zhang W, Zhou W (2013). Synthesis and biological evaluation of
xanthine derivatives on dipeptidyl peptidase 4. Chem Pharm Bull (Tokyo), 61, 477-482.
Lin Y, Berg AH, Iyengar P, Lam TK, Giacca A, Combs TP, et al. (2005). The hyperglycemia-
induced inflammatory response in adipocytes: The role of reactive oxygen species. J Biol
Chem, 280, 4617-4626.
Little TL, Beyer EC, Duling BR (1995). Connexin 43 and connexin 40 GAP junctional proteins
are present in arteriolar smooth muscle and endothelium in vivo. Am J Physiol, 268, 729-
739.
Liu L, Liu J, Wong WT, Tian XY, Lau CW, Wang Y-X, et al. (2012). Dipeptidyl peptidase 4
inhibitor sitagliptin protects endothelial function in hypertension through a glucagon–like
peptide 1–dependent mechanism. Hypertension, 60, 833-841.
228
Liu LB, Wang YP, Pan XD, Jiang SY, Chen Z (2008). Exendin-4 protected murine min6
pancreatic beta-cells from oxidative stress-induced apoptosis via down-regulation of NF-
kappab-iNOS-NO pathway. Yao Xue Xue Bao, 43, 690-694.
Liu Y,Gutterman DD (2002). Oxidative stress and potassium channel function. Clin Exp
Pharmacol Physiol, 29, 305-311.
Lu C-W, Guo Z, Feng M, Wu Z-Z, He Z-M, Xiong Y (2010). Ex vivo gene transferring of
human dimethylarginine dimethylaminohydrolase-2 improved endothelial dysfunction in
diabetic rat aortas and high glucose-treated endothelial cells. Atherosclerosis, 209, 66-73.
Luksha L, Agewall S, Kublickiene K (2009). Endothelium-derived hyperpolarizing factor in
vascular physiology and cardiovascular disease. Atherosclerosis, 202, 330-344.
Lynn FC, Pamir N, Ng EH, Mcintosh CH, Kieffer TJ, Pederson RA (2001). Defective glucose-
dependent insulinotropic polypeptide receptor expression in diabetic fatty Zucker rats.
Diabetes, 50, 1004-1011.
Ma X, Li Y-F, Gao Q, Ye Z-G, Lu X-J, Wang H-P, et al. (2008). Inhibition of superoxide anion-
mediated impairment of endothelium by treatment with luteolin and apigenin in rat
mesenteric artery. Life Sci, 83, 110-117.
229
Makino A, Ohuchi K, Kamata K (2000). Mechanisms underlying the attenuation of endothelium-
dependent vasodilatation in the mesenteric arterial bed of the streptozotocin-induced
diabetic rat. Br J Pharmacol, 130, 549-556.
Makino A, Platoshyn O, Suarez J, Yuan JX-J, Dillmann WH (2008). Downregulation of
connexin40 is associated with coronary endothelial cell dysfunction in streptozotocin-
induced diabetic mice. Am J Physiol Cell Physiol, 295, 221-230.
Malakul W, Thirawarapan S, Suvitayavat W, Woodman OL (2008). Type 1 diabetes and
hypercholesterolaemia reveal the contribution of endothelium-derived hyperpolarizing
factor to endothelium-dependent relaxation of the rat aorta. Clin Exp Pharmacol Physiol,
35, 192-200.
Marguet D, Baggio L, Kobayashi T, Bernard AM, Pierres M, Nielsen PF, et al. (2000).
Enhanced insulin secretion and improved glucose tolerance in mice lacking cd26. Proc
Natl Acad Sci U S A, 97, 6874-6879.
Mason RP, Jacob RF, Kubant R, Ciszewski A, Corbalan JJ, Malinski T (2012). Dipeptidyl
peptidase-4 inhibition with saxagliptin enhanced nitric oxide release and reduced blood
pressure and sicam-1 levels in hypertensive rats. J Cardiovasc Pharmacol, 60, 467-473.
230
Mason RP, Jacob RF, Kubant R, Walter MF, Bellamine A, Jacoby A, et al. (2011). Effect of
enhanced glycemic control with saxagliptin on endothelial nitric oxide release and cd40
levels in obese rats. J Atheroscler Thromb, 18, 774-783.
Matheeussen V, Baerts L, De Meyer G, De Keulenaer G, Van Der Veken P, Augustyns K, et al.
(2011). Expression and spatial heterogeneity of dipeptidyl peptidases in endothelial cells
of conduct vessels and capillaries. Biol Chem, 392, 189-198.
Mather KJ, Verma S, Anderson TJ (2001). Improved endothelial function with metformin in type
2 diabetes mellitus. J Am Coll Cardio, 37, 1344-1350.
Mather S, Dora KA, Sandow SL, Winter P, Garland CJ (2005). Rapid endothelial cell-selective
loading of connexin 40 antibody blocks endothelium-derived hyperpolarizing factor
dilation in rat small mesenteric arteries. Circ Res, 97, 399-407.
Matsubara J, Sugiyama S, Akiyama E, Iwashita S, Kurokawa H, Ohba K, et al. (2012a).
Dipeptidyl peptidase-4 inhibitor, sitagliptin, improves endothelial dysfunction in
association with its anti-inflammatory effects in patients with coronary artery disease and
uncontrolled diabetes. Circ J, 77, 1337-1344.
Matsubara J, Sugiyama S, Sugamura K, Nakamura T, Fujiwara Y, Akiyama E, et al. (2012b). A
dipeptidyl peptidase-4 inhibitor, des-fluoro-sitagliptin, improves endothelial function and
231
reduces atherosclerotic lesion formation in apolipoprotein e-deficient mice. J Am Coll
Cardiol, 59, 265-276.
Matsui T, Nishino Y, Takeuchi M, Yamagishi S (2011). Vildagliptin blocks vascular injury in
thoracic aorta of diabetic rats by suppressing advanced glycation end product-receptor
axis. Pharmacol Res, 63, 383-388.
Matsumoto T, Kobayashi T, Kamata K (2003). Alterations in edhf-type relaxation and
phosphodiesterase activity in mesenteric arteries from diabetic rats. Am J Physiol Heart
Circ Physiol, 285, 283-291.
Matsumoto T, Kobayashi T, Wakabayashi K, Kamata K (2005). Cilostazol improves
endothelium-derived hyperpolarizing factor-type relaxation in mesenteric arteries from
diabetic rats. Am J Physiol Heart Circ Physiol, 289, 1933-1940.
Matsumoto T, Miyamori K, Kobayashi T, Kamata K (2006). Specific impairment of
endothelium-derived hyperpolarizing factor-type relaxation in mesenteric arteries from
streptozotocin-induced diabetic mice. Vascul Pharmacol, 44, 450-460.
Mcadam BF, Catella-Lawson F, Mardini IA, Kapoor S, Lawson JA, Fitzgerald GA (1999).
Systemic biosynthesis of prostacyclin by cyclooxygenase (COX)-2: The human
pharmacology of a selective inhibitor of cox-2. Proc Natl Acad Sci U S A, 96, 272-277.
232
Mccay PB, Gibson DD, Kuo-Lan F, K Roger H (1976). Effect of glutathione peroxidase activity
on lipid peroxidation in biological membranes. Biochim Biophys Acta-Lipids and Lipid
Meta, 431, 459-468.
Mcguire JJ, Ding H, Triggle CR (2001). Endothelium-derived relaxing factors: A focus on
endothelium-derived hyperpolarizing factor (s). Cana J Physiol Pharmacol, 79, 443-470.
Mcintyre N, Holdsworth CD, Turner DS (1964). New interpretation of oral glucose tolerance.
Lancet, 2, 20-21.
Mcneish AJ, Dora KA, Garland CJ (2005). Possible role for k+ in endothelium-derived
hyperpolarizing factor–linked dilatation in rat middle cerebral artery. Stroke, 36, 1526-
1532.
Mcpherson GA (1992). Assessing vascular reactivity of arteries in the small vessel myograph.
Clin Exp Pharmacol Physiol, 19, 815-825.
Michell BJ, Griffiths JE, Mitchelhill KI, Rodriguez-Crespo I, Tiganis T, Bozinovski S, et al.
(1999). The akt kinase signals directly to endothelial nitric oxide synthase. Curr Biol, 9,
845-848.
233
Mistry GC, Maes AL, Lasseter KC, Davies MJ, Gottesdiener KM, Wagner JA, et al. (2008).
Effect of sitagliptin, a dipeptidyl peptidase-4 inhibitor, on blood pressure in nondiabetic
patients with mild to moderate hypertension. J Clin Pharmacol, 48, 592-598.
Molnar J, Yu S, Mzhavia N, Pau C, Chereshnev I, Dansky HM (2005). Diabetes induces
endothelial dysfunction but does not increase neointimal formation in high-fat diet fed
c57bl/6j mice. Circ Res, 96, 1178-1184.
Monami M, Iacomelli I, Marchionni N, Mannucci E (2010). Dipeptydil peptidase-4 inhibitors in
type 2 diabetes: A meta-analysis of randomized clinical trials. Nutr Metab Cardiovasc
Dis, 20, 224-235.
Moncada S, Palmer R, Higgs E (1991). Nitric oxide: Physiology, pathophysiology, and
pharmacology. Pharmacol Rev, 43, 109-142.
Moore B (1906). On the treatment of diabetus mellitus by acid extract of duodenal mucous
membrane. Biochem J, 1, 28-38.
Mulvany MJ, Halpern W (1977). Contractile properties of small arterial resistance vessels in
spontaneously hypertensive and normotensive rats. Circ Res, 41, 19-26.
Münzel T, Gori T, Bruno RM, Taddei S (2010). Is oxidative stress a therapeutic target in
cardiovascular disease? Eur Heart J, ehq396.
234
Nakagawa T, Yokozawa T (2002). Direct scavenging of nitric oxide and superoxide by green
tea. Food Chem Toxicol, 40, 1745-1750.
Narumiya S, Sugimoto Y, Ushikubi F (1999). Prostanoid receptors: Structures, properties, and
functions. Physiol Rev, 79, 1193-226.
Nielsen LL, Baron AD (2003). Pharmacology of exenatide (synthetic exendin-4) for the
treatment of type 2 diabetes. Curr Opin Investig Drugs, 4, 401-405.
Nielsen LL, Young AA, Parkes DG (2004). Pharmacology of exenatide (synthetic exendin-4): A
potential therapeutic for improved glycemic control of type 2 diabetes. Regul Pept, 117,
77-88.
Nikolaidis LA, Elahi D, Hentosz T, Doverspike A, Huerbin R, Zourelias L, et al. (2004).
Recombinant glucagon-like peptide-1 increases myocardial glucose uptake and improves
left ventricular performance in conscious dogs with pacing-induced dilated
cardiomyopathy. Circulation, 110, 955-961.
Nishikawa T, Edelstein D, Du XL, Yamagishi S-I, Matsumura T, Kaneda Y, et al. (2000).
Normalizing mitochondrial superoxide production blocks three pathways of
hyperglycaemic damage. Nature, 404, 787-790.
235
Nistala R, Habibi J, Aroor A, Sowers JR, Hayden MR, Meuth A, et al. (2014). DPP4 inhibition
attenuates filtration barrier injury and oxidant stress in the zucker obese rat. Obesity, 22,
2172-2179.
Noble D, Mathur R, Dent T, Meads C, Greenhalgh T (2011). Risk models and scores for type 2
diabetes: Systematic review. Bmj, 343, 7163-7163.
Noda Y, Miyoshi T, Oe H, Ohno Y, Nakamura K, Toh N, et al. (2013). Alogliptin ameliorates
postprandial lipemia and postprandial endothelial dysfunction in non-diabetic subjects: A
preliminary report. Cardiovasc Diabetol, 12, 8-12.
Nystrom T, Gutniak MK, Zhang Q, Zhang F, Holst JJ, Ahren B, et al. (2004). Effects of
glucagon-like peptide-1 on endothelial function in type 2 diabetes patients with stable
coronary artery disease. Am J Physiol Endocrinol Metab, 287, 1209-1215.
Oak J-H, Cai H (2007). Attenuation of angiotensin ii signaling recouples enos and inhibits
nonendothelial nox activity in diabetic mice. Diabetes, 56, 118-126.
Oeseburg H, De Boer RA, Buikema H, Van Der Harst P, Van Gilst WH, Silljé HH (2010).
Glucagon-like peptide 1 prevents reactive oxygen species–induced endothelial cell
senescence through the activation of protein kinase a. Arterioscler Thromb Vasc Biol, 30,
1407-1414.
236
Ogawa S, Ishiki M, Nako K, Okamura M, Senda M, Mori T, et al. (2011). Sitagliptin, a
dipeptidyl peptidase-4 inhibitor, decreases systolic blood pressure in japanese
hypertensive patients with type 2 diabetes. Tohoku J Exp Med, 223, 133-135.
Ohara Y, Peterson TE, Harrison DG (1993). Hypercholesterolemia increases endothelial
superoxide anion production. J Clin Invest, 91, 2546-2551.
Okon EB, Chung AW, Rauniyar P, Padilla E, Tejerina T, Mcmanus BM, et al. (2005).
Compromised arterial function in human type 2 diabetic patients. Diabetes, 54, 2415-
2423.
Ørskov C, Holst JJ, Knuhtsen S, Baldissera FG, Poulsen SS, Nielsen OV (1986). Glucagon-like
peptides glp-1 and glp-2, predicted products of the glucagon gene, are secreted separately
from pig small intestine but not pancreas*. Endocrinology, 119, 1467-1475.
Ozkan MH, Uma S (2005). Inhibition of acetylcholine-induced edhf response by elevated
glucose in rat mesenteric artery. Life Sci, 78, 14-21.
Özyazgan S, Kutluata N, Fer U, Afşar S, Özdaş S, Akkan AG, et al. (2005). Effect of glucagon-
like peptide-1 (7–36) and exendin-4 on the vascular reactivity in
streptozotocin/nicotinamide-induced diabetic rats. Pharmacology, 74, 119-126.
237
Padmasekar M, Lingwal N, Samikannu B, Chen C, Sauer H, Linn T (2013). Exendin-4 protects
hypoxic islets from oxidative stress and improves islet transplantation outcome.
Endocrinology, 154, 1424-1433.
Pala L, Pezzatini A, Dicembrini I, Ciani S, Gelmini S, Vannelli BG, et al. (2010). Different
modulation of dipeptidyl peptidase-4 activity between microvascular and macrovascular
human endothelial cells. Acta Diabetologica, 1-5.
Palalau AI, Tahrani AA, Piya MK, Barnett AH (2009). DPP-4 inhibitors in clinical practice.
Postgrad Med, 121, 70-100.
Palmer R, Ferrige A, Moncada S (1987). Nitric oxide release accounts for the biological activity
of endothelium-derived relaxing factor. Nature, 327, 524-526.
Palmer RM, Ashton D, Moncada S (1988). Vascular endothelial cells synthesize nitric oxide
from l-arginine. Nature, 333, 664-666.
Panchapakesan U, Mather A, Pollock C (2013). Role of GLP-1 and DPP-4 in diabetic
nephropathy and cardiovascular disease. Clin Sci (Lond), 124, 17-26.
Pannirselvam M, Ding H, Anderson TJ, Triggle CR (2006). Pharmacological characteristics of
endothelium-derived hyperpolarizing factor-mediated relaxation of small mesenteric
arteries from db/db mice. Eur J Pharmacol, 551, 98-107.
238
Pannirselvam M, Simon V, Verma S, Anderson T, Triggle CR (2003). Chronic oral
supplementation with sepiapterin prevents endothelial dysfunction and oxidative stress in
small mesenteric arteries from diabetic (db/db) mice. Br J Pharmacol, 140, 701-706.
Paravicini TM, Chrissobolis S, Drummond GR, Sobey CG (2004). Increased nadph-oxidase
activity and nox4 expression during chronic hypertension is associated with enhanced
cerebral vasodilatation to nadph in vivo. Stroke, 35, 584-589.
Pederson RA, Schubert HE, Brown JC (1975). Gastric inhibitory polypeptide: Its physiologic
release and insulinotropic action in the dog. Diabetes, 24, 1050-1056.
Perfetti R, Zhou J, Doyle ME, Egan JM (2000). Glucagon-like peptide-1 induces cell
proliferation and pancreatic-duodenum homeobox-1 expression and increases endocrine
cell mass in the pancreas of old, glucose-intolerant rats. Endocrinology, 141, 4600-4605.
Perley MJ, Kipnis DM (1967). Plasma insulin responses to oral and intravenous glucose: Studies
in normal and diabetic subjects. J Clin Invest, 46, 1954.
Pieper GM (1998). Review of alterations in endothelial nitric oxide production in diabetes:
Protective role of arginine on endothelial dysfunction. Hypertension, 31, 1047-1060.
Pratley RE, Salsali A (2007). Inhibition of dpp-4: A new therapeutic approach for the treatment
of type 2 diabetes. Curr Med Res Opin, 23, 919-931.
239
Prigeon RL, Quddusi S, Paty B, D'alessio DA (2003). Suppression of glucose production by glp-
1 independent of islet hormones: A novel extrapancreatic effect. Am J Physiol Endocrinol
Metab, 285, 701-707.
Qi W, Chen X, Holian J, Tan CY, Kelly DJ, Pollock CA (2009). Transcription factors krüppel-
like factor 6 and peroxisome proliferator-activated receptor-γ mediate high glucose-
induced thioredoxin-interacting protein. Am J Pathol, 175, 1858-1867.
Qian LB, Wang HP, Qiu WL, Huang H, Bruce IC, Xia Q (2006). Interleukin-2 protects against
endothelial dysfunction induced by high glucose levels in rats. Vascul Pharmacol, 45,
374-382.
Radomski MW, Palmer RM, Moncada S (1987). Endogenous nitric oxide inhibits human platelet
adhesion to vascular endothelium. Lancet, 2, 1057-1058.
Rains JL, Jain SK (2011). Oxidative stress, insulin signaling, and diabetes. Free Radic Biol Med,
50, 567-575.
Ramasamy R, Goldberg IJ (2010). Aldose reductase and cardiovascular diseases, creating
human-like diabetic complications in an experimental model. Circ Res, 106, 1449-1458.
Rao M (1997). Nitric oxide scavenging by curcuminoids. J Pharm Pharmacol, 49, 105-107.
240
Ray R, Shah AM (2005). Nadph oxidase and endothelial cell function. Clin Sci (Lond), 109, 217-
226.
Rees DD, Palmer RM, Moncada S (1989). Role of endothelium-derived nitric oxide in the
regulation of blood pressure. Proc Natl Acad Sci U S A, 86, 3375-3378.
Reil TD, Barnard RJ, Kashyap VS, Roberts CK, Gelabert HA (1999). Diet-induced changes in
endothelial-dependent relaxation of the rat aorta. J Surg Res, 85, 96-100.
Reiter CD, Teng RJ, Beckman JS (2000). Superoxide reacts with nitric oxide to nitrate tyrosine
at physiological ph via peroxynitrite. J Biol Chem, 275, 32460-32466.
Ren J, Duan J, Thomas DP, Yang X, Sreejayan N, Sowers JR, et al. (2008). IGF-I alleviates
diabetes-induced rhoa activation, enos uncoupling, and myocardial dysfunction. Am J
Physiol Regul Integr Comp Physiol, 294, 793-802.
Reyes-Toso CF, Roson MI, Albornoz LE, Damiano PF, Linares LM, Cardinali DP (2002).
Vascular reactivity in diabetic rats: Effect of melatonin. J Pineal Res, 33, 81-86.
Richter B, Bandeira-Echtler E, Bergerhoff K, Lerch CL (2008). Dipeptidyl peptidase-4 (DPP-4)
inhibitors for type 2 diabetes mellitus. Cochrane Database Syst Rev, 2, CD006739.
241
Roberts CK, Barnard RJ, Sindhu RK, Jurczak M, Ehdaie A, Vaziri ND (2005). A high-fat,
refined-carbohydrate diet induces endothelial dysfunction and oxidant/antioxidant
imbalance and depresses nos protein expression. J Appl Physiol (1985), 98, 203-210.
Roberts CK, Vaziri ND, Wang XQ, Barnard RJ (2000). Enhanced no inactivation and
hypertension induced by a high-fat, refined-carbohydrate diet. Hypertension, 36, 423-
429.
Rodriguez-Manas L, Angulo J, Vallejo S, Peiro C, Sanchez-Ferrer A, Cercas E, et al. (2003).
Early and intermediate amadori glycosylation adducts, oxidative stress, and endothelial
dysfunction in the streptozotocin-induced diabetic rats vasculature. Diabetologia, 46,
556-566.
Romero MJ, Platt DH, Tawfik HE, Labazi M, El-Remessy AB, Bartoli M, et al. (2008).
Diabetes-induced coronary vascular dysfunction involves increased arginase activity.
Circ Res, 102, 95-102.
Ross S, Brown J, Dupre J (1977). Hypersecretion of gastric inhibitory polypeptide following oral
glucose in diabetes mellitus. Diabetes, 26, 525-529.
Ross S, Dupre J (1978). Effects of ingestion of triglyceride or galactose on secretion of gastric
inhibitory polypeptide and on responses to intravenous glucose in normal and diabetic
subjects. Diabetes, 27, 327-333.
242
Ryoo S, Gupta G, Benjo A, Lim HK, Camara A, Sikka G, et al. (2008). Endothelial arginase ii:
A novel target for the treatment of atherosclerosis. Circ Res, 102, 923-932.
Salt IP, Morrow VA, Brandie FM, Connell JM, Petrie JR (2003). High glucose inhibits insulin-
stimulated nitric oxide production without reducing endothelial nitric-oxide synthase
ser1177 phosphorylation in human aortic endothelial cells. J Biol Chem, 278, 18791-
18797.
Sandow SL, Haddock RE, Hill CE, Chadha PS, Kerr PM, Welsh DG, et al. (2009). What's where
and why at a vascular myoendothelial microdomain signalling complex. Clin Exp
Pharmacol Physiol, 36, 67-76.
Sandow SL, Neylon CB, Chen MX, Garland CJ (2006). Spatial separation of endothelial small‐
and intermediate‐conductance calcium‐activated potassium channels (kCa) and connexins:
Possible relationship to vasodilator function? J Anat, 209, 689-698.
Sato T, Haimovici R, Kao R, Li A-F, Roy S (2002). Downregulation of connexin 43 expression
by high glucose reduces gap junction activity in microvascular endothelial cells.
Diabetes, 51, 1565-1571.
Sayeski PP, Kudlow JE (1996). Glucose metabolism to glucosamine is necessary for glucose
stimulation of transforming growth factor-α gene transcription. J Biol Chem, 271, 15237-
15243.
243
Scandalios JG (1993). Oxygen stress and superoxide dismutases. Plant Physiol, 101, 7-12.
Scheen AJ (2011). Linagliptin for the treatment of type 2 diabetes (pharmacokinetic evaluation).
Expert Opin Drug Metab Toxicol, 7, 1561-1576.
Schmidt W, Siegel E, Creutzfeldt W (1985). Glucagon-like peptide-1 but not glucagon-like
peptide-2 stimulates insulin release from isolated rat pancreatic islets. Diabetologia, 28,
704-707.
Seino Y, Fukushima M, Yabe D (2010). Gip and glp-1, the two incretin hormones: Similarities
and differences. J Diabetes Investig, 1, 8-23.
Shah Z, Kampfrath T, Deiuliis JA, Zhong J, Pineda C, Ying Z, et al. (2011a). Long-term
dipeptidyl-peptidase 4 inhibition reduces atherosclerosis and inflammation via effects on
monocyte recruitment and chemotaxis. Circulation, 124, 2338-2349.
Shah Z, Pineda C, Kampfrath T, Maiseyeu A, Ying Z, Racoma I, et al. (2011b). Acute dpp-4
inhibition modulates vascular tone through glp-1 independent pathways. Vasc
Pharmacol, 55, 2-9.
Shi Y, Ku DD, Man RY, Vanhoutte PM (2006). Augmented endothelium-derived
hyperpolarizing factor-mediated relaxations attenuate endothelial dysfunction in femoral
244
and mesenteric, but not in carotid arteries from type 1 diabetic rats. J Pharmacol Exp
Ther, 318, 276-281.
Shi Y, So KF, Man RY, Vanhoutte PM (2007). Oxygen-derived free radicals mediate
endothelium-dependent contractions in femoral arteries of rats with streptozotocin-
induced diabetes. Br J Pharmacol, 152, 1033-1041.
Shih C-M, Chen Y-H, Lin Y-W, Tsao N-W, Wu S-C, Kao Y-T, et al. (2014). Mk-0626, a
dipeptidyl peptidase-4 inhibitor, improves neovascularization by increasing both the
number of circulating endothelial progenitor cells and endothelial nitric oxide synthetase
expression. Curr Med Chem, 21, 2012-2022.
Shimokawa H (2010). Hydrogen peroxide as an endothelium-derived hyperpolarizing factor.
Pflügers Arch Eur J Physiol, 459, 915-922.
Shimokawa H, Yasutake H, Fujii K, Owada MK, Nakaike R, Fukumoto Y, et al. (1996). The
importance of the hyperpolarizing mechanism increases as the vessel size decreases in
endothelium-dependent relaxations in rat mesenteric circulation. J Cardiovasc
Pharmacol, 28, 703-711.
Shiraki A, Oyama J-I, Komoda H, Asaka M, Komatsu A, Sakuma M, et al. (2012). The
glucagon-like peptide 1 analog liraglutide reduces tnf-α-induced oxidative stress and
inflammation in endothelial cells. Atherosclerosis, 221, 375-382.
245
Sindhu RK, Koo JR, Roberts CK, Vaziri ND (2004). Dysregulation of hepatic superoxide
dismutase, catalase and glutathione peroxidase in diabetes: Response to insulin and
antioxidant therapies. Clin Exp Hypertens, 26, 43-53.
Squadrito GL, Pryor WA (1995). The formation of peroxynitrite in vivo from nitric oxide and
superoxide. Chem Biol Interact, 96, 203-206.
Srinivasan S, Hatley M, Bolick D, Palmer L, Edelstein D, Brownlee M, et al. (2004).
Hyperglycaemia-induced superoxide production decreases enos expression via ap-1
activation in aortic endothelial cells. Diabetologia, 47, 1727-1734.
Stein CJ,Colditz GA (2004). The epidemic of obesity. J Clin Endocrinol Metab, 89, 2522-2525.
Stoffers DA, Kieffer TJ, Hussain MA, Drucker DJ, Bonner-Weir S, Habener JF, et al. (2000).
Insulinotropic glucagon-like peptide 1 agonists stimulate expression of homeodomain
protein idx-1 and increase islet size in mouse pancreas. Diabetes, 49, 741-748.
Sugamura K, Keaney JF, Jr. (2011). Reactive oxygen species in cardiovascular disease. Free
Radic Biol Med, 51, 978-92.
Sugiyama T, Levy BD, Michel T (2009). Tetrahydrobiopterin recycling, a key determinant of
endothelial nitric-oxide synthase-dependent signaling pathways in cultured vascular
endothelial cells. J Biol Chem, 284, 12691-700.
246
Suzuki K, Watanabe K, Suzuki T, Ouchi M, Futami-Suda S, Igari Y, et al. (2012). Sitagliptin
improves vascular endothelial function in japanese type 2 diabetes patients without
cardiovascular disease. J Diabetes Mellitus, 2, 338.
Szabo C (2003). Multiple pathways of peroxynitrite cytotoxicity. Toxicol Lett, 140, 105-1012.
Szayna M, Doyle ME, Betkey JA, Holloway HW, Spencer RG, Greig NH, et al. (2000).
Exendin-4 decelerates food intake, weight gain, and fat deposition in zucker rats.
Endocrinology, 141, 1936-1941.
Ta NN, Schuyler CA, Li Y, Lopes-Virella MF, Huang Y (2011). DPP-4 (CD26) inhibitor
alogliptin inhibits atherosclerosis in diabetic apolipoprotein E-deficient mice. J
Cardiovasc Pharmacol, 58, 157-166.
Takai S, Sakonjo H, Jin D (2014). Significance of vascular dipeptidyl peptidase-4 inhibition on
vascular protection in zucker diabetic fatty rats. J Pharmacol Sci, 125, 386-393.
Takayama T, Wada A, Tsutamoto T, Ohnishi M, Fujii M, Isono T, et al. (2004). Contribution of
vascular nad(p)h oxidase to endothelial dysfunction in heart failure and the therapeutic
effects of hmg-coa reductase inhibitor. Circ J, 68, 1067-1075.
247
Takeda J, Seino Y, Tanaka K, Fukumoto H, Kayano T, Takahashi H, et al. (1987). Sequence of
an intestinal cDNA encoding human gastric inhibitory polypeptide precursor. Proc Natl
Acad Sci U S A, 84, 7005-7008.
Tan KC, Chow WS, Tam SC, Ai VH, Lam CH, Lam KS (2002). Atorvastatin lowers c-reactive
protein and improves endothelium-dependent vasodilation in type 2 diabetes mellitus. J
Clin Endocrinol Metab, 87, 563-568.
Tan SM, Zhang Y, Cox AJ, Kelly DJ, Qi W (2011). Tranilast attenuates the up-regulation of
thioredoxin-interacting protein and oxidative stress in an experimental model of diabetic
nephropathy. Nephrol Dial Transplant, 26, 100-10.
Tare M, Parkington H, Coleman H, Neild T, Dusting G (1990). Hyperpolarization and relaxation
of arterial smooth muscle caused by nitric oxide derived from the endothelium. nature,
346, 69-71.
Terasaki M, Nagashima M, Nohtomi K, Kohashi K, Tomoyasu M, Sinmura K, et al. (2013).
Preventive effect of dipeptidyl peptidase-4 inhibitor on atherosclerosis is mainly
attributable to incretin's actions in nondiabetic and diabetic apolipoprotein E-null mice.
PloS one, 8, e70933.
Tesfamariam B, Brown ML, Cohen RA (1991). Elevated glucose impairs endothelium-
dependent relaxation by activating protein kinase c. J Clin Invest, 87, 1643-1648.
248
Tesfamariam B, Cohen RA (1992). Free radicals mediate endothelial cell dysfunction caused by
elevated glucose. Am J Physiol Heart Circ Physiol, 263, 321-326.
Tews D, Lehr S, Hartwig S, Osmers A, Paslack W, Eckel J (2009). Anti-apoptotic action of
exendin-4 in ins-1 beta cells: Comparative protein pattern analysis of isolated
mitochondria. Horm Metab Res, 41, 294-301.
Thomas C, Mackey M, Diaz A, Cox D (2009). Hydroxyl radical is produced via the fenton
reaction in submitochondrial particles under oxidative stress: Implications for diseases
associated with iron accumulation. Redox Rep, 14, 102-108.
Thomas L, Eckhardt M, Langkopf E, Tadayyon M, Himmelsbach F, Mark M (2008). (R)-8-(3-
amino-piperidin-1-yl)-7-but-2-ynyl-3-methyl-1-(4-methyl-quinazolin-2-ylmethyl)-3, 7-
dihydro-purine-2, 6-dione (BI 1356), a novel xanthine-based dipeptidyl peptidase 4
inhibitor, has a superior potency and longer duration of action compared with other
dipeptidyl peptidase-4 inhibitors. J Pharmacol Exp Ther, 325, 175-182.
Thompson MA, Henderson KK, Woodman CR, Turk JR, Rush JW, Price E, et al. (2004).
Exercise preserves endothelium-dependent relaxation in coronary arteries of
hypercholesterolemic male pigs. J Appl Physiol (1985), 96, 1114-1126.
Thornalley PJ, Langborg A, Minhas HS (1999). Formation of glyoxal, methylglyoxal and 3-
deoxyglucosone in the glycation of proteins by glucose. Biochem J, 1, 109-116.
249
Torzewski M, Ochsenhirt V, Kleschyov AL, Oelze M, Daiber A, Li H, et al. (2007). Deficiency
of glutathione peroxidase-1 accelerates the progression of atherosclerosis in
apolipoprotein e-deficient mice. Arterioscler Thromb Vasc Biol, 27, 850-857.
Trumper A, Trumper K, Horsch D (2002). Mechanisms of mitogenic and anti-apoptotic
signaling by glucose-dependent insulinotropic polypeptide in beta(INS-1)-cells. J
Endocrinol, 174, 233-246.
Tru Mper A, Tru Mper K, Trusheim H, Arnold R, Go Ke B, Ho Rsch D (2001). Glucose-dependent
insulinotropic polypeptide is a growth factor for β (ins-1) cells by pleiotropic signaling.
Mol Endocrinol, 15, 1559-1570.
Tsunekawa S, Yamamoto N, Tsukamoto K, Itoh Y, Kaneko Y, Kimura T, et al. (2007).
Protection of pancreatic β-cells by exendin-4 may involve the reduction of endoplasmic
reticulum stress; in vivo and in vitro studies. J Endocrinol, 193, 65-74.
Tsutsui M, Milstien S, Katusic ZS (1996). Effect of tetrahydrobiopterin on endothelial function
in canine middle cerebral arteries. Circ Res, 79, 336-42.
Turrens J, Boveris, A (1980). Generation of superoxide anion by the nadh dehydrogenase of
bovine heart mitochondria. Biochem. J, 191, 421-427.
250
Turrens JF, Alexandre A, Lehninger AL (1985). Ubisemiquinone is the electron donor for
superoxide formation by complex iii of heart mitochondria. Arch Biochem Biophys, 237,
408-414.
Ugleholdt R, Zhu X, Deacon CF, Orskov C, Steiner DF, Holst JJ (2004). Impaired intestinal
proglucagon processing in mice lacking prohormone convertase 1. Endocrinology, 145,
1349-55.
Urakawa H, Katsuki A, Sumida Y, Gabazza EC, Murashima S, Morioka K, et al. (2003).
Oxidative stress is associated with adiposity and insulin resistance in men. J Clin
Endocrinol Metab, 88, 4673-4676.
Ussher JR, Drucker DJ (2014). Cardiovascular actions of incretin-based therapies. Circ Res, 114,
1788-1803.
Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M, Telser J (2007). Free radicals and
antioxidants in normal physiological functions and human disease. Int J Biochem Cell
Biol, 39, 44-84.
Van Poppel PC, Netea MG, Smits P, Tack CJ (2011). Vildagliptin improves endothelium-
dependent vasodilatation in type 2 diabetes. Diabetes care, 34, 2072-2077.
251
Vanhoutte PM, Shimokawa H, Tang EHC, Feletou M. (2009). Endothelial dysfunction and
vascular disease. Acta Physiologica, 196, 193-222.
Vikramadithyan RK, Hu Y, Noh HL, Liang CP, Hallam K, Tall AR, et al. (2005). Human aldose
reductase expression accelerates diabetic atherosclerosis in transgenic mice. J Clin Invest,
115, 2434-2443.
Vilsbøll T, Krarup T, Deacon CF,Madsbad S, Holst, Jens J (2001). Reduced postprandial
concentrations of intact biologically active glucagon-like peptide 1 in type 2 diabetic
patients. Diabetes, 50, 609-613.
Vriens J, Owsianik G, Fisslthaler B, Suzuki M, Janssens A, Voets T, et al. (2005). Modulation of
the ca2 permeable cation channel trpv4 by cytochrome p450 epoxygenases in vascular
endothelium. Circ Res, 97, 908-915.
Waldron G, Garland Cj (1994). Effect of potassium channel blockers on l-name insensitive
relaxations in rat small mesenteric artery. Can J Physiol Pharmacol, 72, 115.
Wang D, Luo P, Wang Y, Li W, Wang C, Sun D, et al. (2013). Glucagon-like peptide-1 protects
against cardiac microvascular injury in diabetes via a camp/pka/rho-dependent
mechanism. Diabetes, 62, 1697-1708.
252
Wang S, Xu J, Song P, Viollet B, Zou M-H (2009). In vivo activation of amp-activated protein
kinase attenuates diabetes-enhanced degradation of gtp cyclohydrolase i. Diabetes, 58,
1893-1901.
Wang S, Zhang M, Liang B, Xu J, Xie Z, Liu C, et al. (2010). AMPKα2 deletion causes aberrant
expression and activation of NAD(P)H oxidase and consequent endothelial dysfunction
in vivo role of 26S proteasomes. Circ Res, 106, 1117-1128.
Wang , Cahill CM, Pi Eyro MA, hou , Doyle M E, Egan JM (1999). Glucagon-like peptide-
1 regulates the beta cell transcription factor, PDX-1, in insulinoma cells. Endocrinology,
140, 4904-4907.
Wanstall JC, Jeffery TK, Gambino A, Lovren F, Triggle CR (2001). Vascular smooth muscle
relaxation mediated by nitric oxide donors: A comparison with acetylcholine, nitric oxide
andnitroxyl ion. Br J Pharmacol, 134, 463-472.
Watanabe H, Vriens J, Prenen J, Droogmans G, Voets T, Nilius B (2003). Anandamide and
arachidonic acid use epoxyeicosatrienoic acids to activate trpv4 channels. Nature, 424,
434-438.
Wei EP, Kontos HA, Beckman JS (1996). Mechanisms of cerebral vasodilation by superoxide,
hydrogen peroxide, and peroxynitrite. Am J Physiol, 271, 1262-1266.
253
Wells L, Hart GW (2003). O-glcnac turns twenty: Functional implications for post-translational
modification of nuclear and cytosolic proteins with a sugar. FEBS Lett, 546, 154-158.
Wenzel P, Daiber A, Oelze M, Brandt M, Closs E, Xu J, et al. (2008). Mechanisms underlying
recoupling of enos by hmg-coa reductase inhibition in a rat model of streptozotocin-
induced diabetes mellitus. Atherosclerosis, 198, 65-76.
Weston A, Porter E, Harno E, Edwards G (2010). Impairment of endothelial skca channels and
of downstream hyperpolarizing pathways in mesenteric arteries from spontaneously
hypertensive rats. Br J Pharmacol, 160, 836-843.
Weston AH, Absi M, Harno E, Geraghty AR, Ward DT, Ruat M, et al. (2008). The expression
and function of ca2+‐sensing receptors in rat mesenteric artery; comparative studies using
a model of type ii diabetes. Br J Pharmacol, 154, 652-662.
Weston AH, Félétou M, Vanhoutte PM, Falck JR, Campbell WB, Edwards G (2005).
Bradykinin‐induced, endothelium‐dependent responses in porcine coronary arteries:
Involvement of potassium channel activation and epoxyeicosatrienoic acids. Br J
Pharmacol, 145, 775-784.
White AR, Ryoo S, Li D, Champion HC, Steppan J, Wang D, et al. (2006). Knockdown of
arginase i restores no signaling in the vasculature of old rats. Hypertension, 47, 245-251.
254
White WB, Gorelick PB, Fleck P, Smith N, Wilson C, Pratley R (2010). Cardiovascular events
in patients receiving alogliptin: A pooled analysis of randomized clinical trials. Diabetes,
59, 105.
Who. 2011. World health organization. Diabetes fact sheet.
Http://www.Who.Int/mediacentre/factsheets/fs312/en/index.Html: Accessed on 06 05-
2015. [Online].
Widder JD, Chen W, Li L, Dikalov S, Thony B, Hatakeyama K, et al. (2007). Regulation of
tetrahydrobiopterin biosynthesis by shear stress. Circ Res, 101, 830-838.
Wigg SJ, Tare M, Tonta MA, O'brien RC, Meredith IT, Parkington HC (2001). Comparison of
effects of diabetes mellitus on an edhf-dependent and an edhf-independent artery. Am J
Physiol Heart Circ Physiol, 281, H232-40.
Wild S, Roglic G, Green A, Sicree R, King H (2004). Global prevalence of diabetes: Estimates
for the year 2000 and projections for 2030. Diabetes Care, 27, 1047-1053.
Williams-Herman D, Engel SS, Round E, Johnson J, Golm GT, Guo H, et al. (2010). Safety and
tolerability of sitagliptin in clinical studies: A pooled analysis of data from 10,246
patients with type 2 diabetes. BMC Endocr Disord, 10, 7.
255
Willms B, Werner J, Holst JJ, Orskov C, Creutzfeldt W, Nauck MA (1996). Gastric emptying,
glucose responses, and insulin secretion after a liquid test meal: Effects of exogenous
glucagon-like peptide-1 (glp-1)-(7-36) amide in type 2 (noninsulin-dependent) diabetic
patients. J Clin Endocrinol Metab, 81, 327-332.
Woo KS, Chook P, Chung WY, Sung RY, Qiao M, Leung SS, et al. (2004). Effects of diet and
exercise on obesity-related vascular dysfunction in children. Circulation, 109, 1981-
1986.
Woodman CR, Turk JR, Williams DP, Laughlin MH (2003). Exercise training preserves
endothelium-dependent relaxation in brachial arteries from hyperlipidemic pigs. J Appl
Physiol (1985), 94, 2017-2026.
Woodman OL, Chan E (2004). Vascular and anti-oxidant actions of flavonols and flavones. Clin
Exp Pharmacol Physiol, 31, 786-790.
Woodman OL, Malakul W, Cao AH, Xu Q, Ritchie RH (2008). Atrial natriuretic peptide
prevents diabetes-induced endothelial dysfunction. Life Sci, 82, 847-854.
Woodman OL, Malakul, Wachirawadee (2009). 3′, 4′-dihydroxyflavonol prevents diabetes-
induced endothelial dysfunction in rat aorta. Life Sci, 85, 54-59.
256
Woodman OL, Wongsawatkul O, Sobey CG (2000). Contribution of nitric oxide, cyclic gmp and
k+ channels to acetylcholine-induced dilatation of rat conduit and resistance arteries. Clin
Exp Pharmacol Physiol, 27, 34-40.
Xiao HB, Jun F, Lu XY, Chen XJ, Chao T, Sun ZL (2009). Protective effects of kaempferol
against endothelial damage by an improvement in nitric oxide production and a decrease
in asymmetric dimethylarginine level. Eur J Pharmacol, 616, 213-222.
Xiong Y, Fu YF, Fu SH, Zhou HH (2003). Elevated levels of the serum endogenous inhibitor of
nitric oxide synthase and metabolic control in rats with streptozotocin-induced diabetes. J
Cardiovasc Pharmacol, 42, 191-196.
Xu G, Stoffers DA, Habener JF, Bonner-Weir S (1999). Exendin-4 stimulates both beta-cell
replication and neogenesis, resulting in increased beta-cell mass and improved glucose
tolerance in diabetic rats. Diabetes, 48, 2270-2276.
Xu J, Wu Y, Song P, Zhang M, Wang S, Zou M-H (2007). Proteasome-dependent degradation of
guanosine 5′-triphosphate cyclohydrolase i causes tetrahydrobiopterin deficiency in
diabetes mellitus. Circulation, 116, 944-953.
Yamagishi S, Ishibashi Y, Ojima A, Sugiura T, Matsui T (2014). Linagliptin, a xanthine-based
dipeptidyl peptidase-4 inhibitor, decreases serum uric acid levels in type 2 diabetic
patients partly by suppressing xanthine oxidase activity. Int J Cardiol, 176, 550-552.
257
Yamamoto E, Nakamura T, Kataoka K, Tokutomi Y, Dong YF, Fukuda M, et al. (2010).
Nifedipine prevents vascular endothelial dysfunction in a mouse model of obesity and
type 2 diabetes, by improving enos dysfunction and dephosphorylation. Biochem Biophys
Res Commun, 403, 258-263.
Yamamoto Y, Imaeda K, Suzuki H (1999). Endothelium-dependent hyperpolarization and
intercellular electrical coupling in guinea-pig mesenteric arterioles. J Physiol, 514 505-
513.
Yamawaki H, Berk BC (2005). Thioredoxin: A multifunctional antioxidant enzyme in kidney,
heart and vessels. Current Opi Nephro Hyper, 14, 149-153.
Yamawaki H, Haendeler J, Berk BC (2003). Thioredoxin a key regulator of cardiovascular
homeostasis. Cir Res, 93, 1029-1033.
Yazbeck R, Howarth GS, Abbott CA (2009). Dipeptidyl peptidase inhibitors, an emerging drug
class for inflammatory disease? Trends Pharmacol Sci, 30, 600-607.
Yeldandi AV, Rao MS, Reddy JK (2000). Hydrogen peroxide generation in peroxisome
proliferator-induced oncogenesis. Mutat Res, 448, 159-177.
258
Yin QF, Xiong Y (2005). Pravastatin restores ddah activity and endothelium-dependent
relaxation of rat aorta after exposure to glycated protein. J Cardiovasc Pharmacol, 45,
525-532.
Yoon SO, Yun CH, Chung AS (2002). Dose effect of oxidative stress on signal transduction in
aging. Mech Ageing Dev, 123, 1597-1604.
Young AA, Gedulin BR, Bhavsar S, Bodkin N, Jodka C, Hansen B, et al. (1999). Glucose-
lowering and insulin-sensitizing actions of exendin-4: Studies in obese diabetic (ob/ob,
db/db) mice, diabetic fatty zucker rats, and diabetic rhesus monkeys (macaca mulatta).
Diabetes, 48, 1026-1034.
Young EJ, Hill MA, Wiehler WB, Triggle CR, Reid JJ (2008). Reduced edhf responses and
connexin activity in mesenteric arteries from the insulin-resistant obese Zucker rat.
Diabetologia, 51, 872-881.
Yuill KH, Yarova P, Kemp-Harper BK, Garland CJ, Dora KA (2011). A novel role for hno in
local and spreading vasodilatation in rat mesenteric resistance arteries. Antioxid Redox
Signal, 14, 1625-1635.
Zander M, Madsbad S, Madsen JL, Holst JJ (2002). Effect of 6-week course of glucagon-like
peptide 1 on glycaemic control, insulin sensitivity, and β-cell function in type 2 diabetes:
A parallel-group study. The Lancet, 359, 824-830.
259
Zhang H, Zhang J, Ungvari Z, Zhang C (2009). Resveratrol improves endothelial function role of
tnfα and vascular oxidative stress. Arterioscler Thromb Vasc Biol, 29, 1164-1171.
Zhao T, Parikh P, Bhashyam S, Bolukoglu H, Poornima I, Shen YT, et al. (2006). Direct effects
of glucagon-like peptide-1 on myocardial contractility and glucose uptake in normal and
postischemic isolated rat hearts. J Pharmacol Exp Ther, 317, 1106-1113.
Zhong J, Rao X, Rajagopalan S (2013). An emerging role of dipeptidyl peptidase 4 (DPP4)
beyond glucose control: Potential implications in cardiovascular disease. Atherosclerosis,
226, 305-314.
Zhong JC, Huang Y, Yung LM, Lau CW, Leung FP, Wong WT, et al. (2007a). The novel
peptide apelin regulates intrarenal artery tone in diabetic mice. Regul Pept, 144, 109-
1014.
Zhong JC, Yu XY, Huang Y, Yung LM, Lau CW, Lin SG (2007b). Apelin modulates aortic
vascular tone via endothelial nitric oxide synthase phosphorylation pathway in diabetic
mice. Cardiovasc Res, 74, 388-395.
Zhou J, Livak MF, Bernier M, Muller DC, Carlson OD, Elahi D, et al. (2007). Ubiquitination is
involved in glucose-mediated downregulation of gip receptors in islets. Am J Physiol
Endocrinol Metab, 293, 538-547.
260
Zhu X, Zhou A, Dey A, Norrbom C, Carroll R, Zhang C, et al. (2002). Disruption of pc1/3
expression in mice causes dwarfism and multiple neuroendocrine peptide processing
defects. Proc Natl Acad Sci U S A, 99, 10293-10298.