Regulation of Connective Tissue Growth Factor (CTGF) in

hypertension-induced end organ damage

Piet Finckenberg

Institute of Biomedicine

Pharmacology

University of Helsinki

Academic Dissertation

To be presented, with the permission of the Faculty of Medicine, University of Helsinki, for public

examination in the auditorium “Richard Faltin” of the Fourth Department of Surgery, Helsinki

University Central Hospital, Helsinki, on 12 December, 2003, at 12 noon.

Supervisors

Docent Eero Mervaala Professor Leena Lindgren

Institute of Biomedicine, Pharmacology Department of Anaesthesia

University of Helsinki Tampere University Hospital

Helsinki, Finland Tampere, Finland

Reviewers

Professor (emer.) Eero Saksela Docent Ilkka Pörsti

Department of Pathology Department of Internal Medicine

University of Helsinki Tampere University Hospital

Helsinki, Finland Tampere, Finland

Opponent

Professor Gerald F. DiBona

Department of Internal Medicine

University of Iowa College of Medicine

and Veterans Administration Medical Center

Iowa City, Iowa, USA

Cover: Myocardial Expression of CTGF mRNA in dTGR rat

ISBN 952-91-6656-7 (paperback) ISBN 952-10-1498-9 (PDF, http://ethesis.helsinki.fi)

Helsinki, 2003, Yliopistopaino

TABLE OF CONTENTS

LIST OF ORIGINAL PUBLICATIONS 7

ABBREVIATIONS 8

ABSTRACT 10

1 INTRODUCTION 12

2 REVIEW OF THE LITERATURE 14

2.1 CONNECTIVE TISSUE GROWTH FACTOR (CTGF) 14

2.1.1 Structure and physiological functions 14

2.1.2 Regulation of CTGF expression 17

2.1.3 Role of CTGF in pathological states 18

2.1.4 Therapeutic approaches to prevent CTGF overexpression 20

2.2 HYPERTENSION-INDUCED END ORGAN DAMAGE 20

2.2.1 Lesions of the heart, vasculature and kidney 20

2.2.1.1 Sympathetic nervous system in the pathogenesis of hypertension-induced

end organ damage 21

2.2.1.2 Involvement of the mechanical factors 22

2.2.1.3 Involvement of the hormonal factors and inflammation 23

2.2.1.3.1 Angiotensin II 26

2.3 ANIMAL MODELS USED IN THE PRESENT STUDY 30

2.3.1 Spontaneously hypertensive rat (SHR) (Study I) 30

2.3.2 Double transgenic rat harboring human renin and angiotensinogen genes (dTGR)

(Study II, III, IV) 30

2.4 COMPOUNDS USED IN THE PRESENT STUDY 31

2.4.1 Enalapril (study I) 31

2.4.2 Valsartan (study I, II) 31

2.4.3 -Lipoic acid (study III) 32

2.4.4 Cyclosporine A (CsA) (study I, II) 32

2.4.5 Tacrolimus (FK 506) (study IV) 33

2.4.6 Everolimus (SDZ RAD) (study II) 33

2.4.7 Magnesium (study IV) 34

3 AIMS OF THE STUDY 35

4 MATERIALS AND METHODS 36

4.1 EXPERIMENTAL ANIMALS 36

4.2 EXPERIMENTAL DIETS AND DRUG TREATMENTS 36

4.3 MEASUREMENT OF BLOOD PRESSURE 37

4.4 METABOLIC STUDIES 37

4.5 COLLECTION OF THE SAMPLES 37

4.6 HISTOLOGY 37

4.6.1 Sample preparation 37

4.6.2 Tissue morphology and scoring of the samples 38

4.7 DETECTION OF mRNA FOR CTGF, COLLAGENS I AND III, AND TGF ββββ1 38

4.7.1 In situ hybridization 38

4.7.2 Northern blot 39

4.7.3 Reverse transcriptase-polymerase chain reaction (RT-PCR) assay for CTGF mRNA 40

4.8 IMMUNOHISTOCHEMISTRY 40

4.9 BIOCHEMICAL DETERMINATIONS 41

4.10 STATISTICAL ANALYSIS 41

4.11 ETHICS OF THE STUDY 41

5 RESULTS 42

5.1 BRIEF SUMMARY OF THE MAIN RESULTS 42

5.2 BLOOD PRESSURE AND CARDIAC HYPERTROPHY 43

5.3 URINARY PROTEIN EXCRETION 43

5.4 HISTOPATHOLOGY 44

5.4.1 Myocardial lesions 44

5.4.2 Renal lesions 45

5.5 EXPRESSION OF CTGF 46

5.5.1 Myocardial expression of CTGF 46

5.5.2 Renal expression of CTGF 47

5.5.3 Time- and blood pressure-dependent expression of CTGF 47

6 DISCUSSION 49

6.1 METHODOLOGICAL ASPECTS 49

6.1.1 Increased RAS activity in SHR and dTGR 49

6.1.2 Blood pressure measurements 50

6.2 EFFECT OF BLOOD PRESSURE ON CTGF EXPRESSION 51

6.3 EFFECT OF ANG II AND INFLAMMATORY RESPONSE

ON CTGF EXPRESSION 52

6.4 POSSIBLE CLINICAL IMPLICATIONS 55

7 SUMMARY AND CONCLUSIONS 56

ACKNOWLEDGEMENTS 57

REFERENCES 60

ORIGINAL PUBLICATIONS 75

7

LIST OF ORIGINAL PUBLICATIONS

This thesis is based on the following original publications, referred to in the text by the Roman

numerals I-IV, and some unpublished data:

I Finckenberg P, Lassila M, Inkinen K, Pere AK, Krogerus L, Lindgren L, Mervaala E, Vapaatalo H, Nurminen ML, Ahonen J. Cyclosporine induces myocardial connective tissue growth factor in spontaneously hypertensive rats on high-sodium diet. Transplantation 2001, 71:951-958

II Finckenberg P, Inkinen K, Ahonen J, Merasto S, Louhelainen M, Vapaatalo H, Müller D, Ganten D, Luft F, Mervaala E. Angiotensin II induces connective tissue growth factor gene expression via calcineurin-dependent pathways. Am J Pathol 2003, 163:355-366

III Mervaala E, Finckenberg P, Lapatto R, Muller DN, Park JK, Dechend R, Ganten D, Vapaatalo H, Luft FC. Lipoic acid supplementation prevents angiotensin II-induced renal injury.

Kidney Int 2003, 64:501-508 (P. F. sharing the first authorship with E. M.)

IV Finckenberg P, Merasto S, Lindgren L, Vapaatalo H, Müller D, Luft F, Mervaala E. Magnesium supplementation prevents angiotensin II-induced myocardial damage and CTGF overexpression.

(Submitted)

The original publications are reprinted with the permission of the copyright holders.

8

ABBREVIATIONS

ACE angiotensin converting enzyme

Ang angiotensin

ANOVA analysis of variance

AP-1 activator protein-1

AT1 angiotensin II type 1 receptor

AT2 angiotensin II type 2 receptor

ATP adenosine triphosphate

BCE-1 basal control element-1

cAMP cyclic adenosine monophosphate

CD4 co-receptor expressed on helper T cells

CD8 co-receptor expressed on cytotoxic T cells

CHF congestive heart failure

Col I collagen type I

Col III collagen type III

CsA cyclosporine A

CTGF connective tissue growth factor

DCM dilated cardiomyopathy

DNA deoxyribonucleic acid

dTGR double transgenic rat harboring human renin and angiotensinogen genes

ECM extracellular matrix

ED1 antibody recognizing rat monocytes and macrophages

ED2 antibody recognizing rat monocytes

EMSA electrophoretic mobility shift assay

FGF fibroblast growth factor

FKBP FK506 binding protein

GAPDH glyceraldehyde-3-phosphate dehydrogenase

G-CSF granulocyte colony-stimulating factor

GM-CSF granulocyte-macrophage colony-stimulating factor

hAOGEN human angiotensinogen

HPLC high-pressure liquid chromatography

hREN human renin

9

ICAM-1 intercellular adhesion molecule-1

IFN interferon

IGF insulin-like growth factor

IL interleukin

LVH left ventricular hypertrophy

MAP(K) mitogen-activated protein (kinase)

mTOR mammalian target of rapamycin

mRNA messenger ribonucleic acid

NADPH nicotinamide adenine dinucleotide phosphate

NFAT nuclear factor of activated T cells

NF-κB nuclear factor kappa B

PCNA proliferating cell nuclear antigen

PDGF platelet-derived growth factor

PKC protein kinase C

PRA plasma renin activity

RAS renin-angiotensin system

RDA recommended daily allowance

RT-PCR reverse transcriptase-polymerase chain reaction

ROS reactive oxygen species

SBP systolic blood pressure

SD Sprague-Dawley rat

SHR spontaneously hypertensive rat

TβRE transforming growth factor beta-response element

TGFβ transforming growth factor beta

TNF tumor necrosis factor alpha

TOR target of rapamycin

TSP-1 thrombospondin-1

VCAM-1 vascular cell adhesion molecule-1

VSMC vascular smooth muscle cell

WKY Wistar Kyoto rat

10

ABSTRACT

Hypertension is a major risk factor for the development of cardiovascular complications such as

atherosclerosis, myocardial infarction, stroke and renal failure. In common to these adverse events

is the accumulation of connective tissue in the involved structures. The exact mechanisms

contributing to the regulation of connective tissue homeostasis in hypertension are still obscure.

Recently an important regulator of the connective tissue synthesis was found, and named

“connective tissue growth factor” (CTGF). During the recent years accumulating evidence has

connected CTGF overexpression with various inflammatory and fibrotic diseases. The present

series of studies were conducted to investigate the role of the CTGF in hypertension-induced end

organ damage. Special attention was focused on angiotensin II (Ang II), an effector molecule of the

renin-angiotensin system (RAS), as a regulator of the CTGF gene expression.

Cyclosporine (CsA)-treated spontaneously hypertensive rats (SHR) on high sodium diet (7-9 weeks

old) and double transgenic rats harboring human renin and angiotensinogen genes (dTGR) (3-4

weeks old) were used as hypertensive animal models with increased RAS activity. The roles of the

blood pressure and Ang II as regulators of the CTGF gene expression were studied. To further

elucidate the regulatory mechanisms of the CTGF gene, intervention studies with antihypertensive

drugs (ACE inhibitor enalapril, AT1 receptor antagonist valsartan), immunosuppressives (CsA,

tacrolimus, everolimus), antioxidant (-lipoic acid) as well as with magnesium were conducted.

Blood pressure was recorded, the relative organ weights measured, mediators of inflammation were

detected with immunohistochemistry and electrophoretic mobility shift assay. Tissue morphology

was examined and the expression of myocardial and renal CTGF mRNA was determined with in

situ hybridization, Northern blot and RT-PCR.

Arterial hypertension induced the expression of the myocardial CTGF mRNA in CsA-treated SHRs.

The expression of the CTGF gene correlated closely with blood pressure, and was prevented with

concomitant antihypertensive treatment by RAS antagonists enalapril and valsartan.

In dTGRs, Ang II induced the expression of the CTGF in a time- and blood pressure-dependent

manner. This Ang II-induced expression of the CTGF co-located with marked perivascular

inflammatory response, cellular proliferation, vascular damage, and was associated with the

activation of the redox-sensitive transcription factors NF-κB and AP-1.

11

The pathological expression of the CTGF in dTGRs was prevented by AT1 receptor antagonist

valsartan. Suppression of the Ang II-mediated perivascular inflammatory response by an

antioxidant -lipoic acid or calcineurin inhibitors CsA and tacrolimus protected against the

overexpression of the CTGF in the dTGR. Dietary magnesium supplementation protected against

the induction of the CTGF gene in the dTGR. Additive therapeutic effects were found with

magnesium supplementation and tacrolimus treatment.

Taken together, these findings provide evidence that (1) high blood pressure induces the myocardial

expression of the CTGF gene (2) Ang II induces CTGF gene expression via AT1 receptor in a time-

and blood pressure-dependent manner (3) oxidative stress induces CTGF expression (4) systemic

magnesium balance regulates the expression of the CTGF.

12

1 INTRODUCTION

According to the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of

High Blood Pressure (JNC), high blood pressure is defined as a blood pressure of >140/90 Hg

(Chobanian et al., 2003). In a great majority of adult patients no identifiable cause for hypertension

can be found. High blood pressure affects every fourth person in the industrialized countries. The

correlation between elevated blood pressure and cardiovascular complications has been clearly

demonstrated (for review, see Staessen et al., 2003).

Hypertension is among the most important risk factors for the development of several

cardiovascular diseases (atherosclerosis, coronary artery disease, myocardial infarction, stroke and

renal failure) (Chobanian et al., 2003) which necessitate a huge burden to medical care and

expences. The exact mechanisms contributing to the hypertension-induced end organ (blood

vessels, heart, kidney) damage remain obscure. The prevalence of morbid cardiovascular episodes

among the treated hypertensive patients remains higher when compared to the normal population

(for review, see Schiffrin, 2002a). This casts the question about the nature of hypertension-induced

end organ damage.

The pathological changes in the heart, blood vessels and kidneys of the hypertensive patients

include vascular remodeling, inflammation and vast connective tissue accumulation leading to

scarring of the involved organs (Mezzano et al., 2001; Dostal, 2001; reviewed by Williams, 2001).

Scarring of the heart or kidneys leads to the deterioration and ultimately to the failure of the organ

function. The accumulation of connective tissue is controlled by various growth factors. Much

attention has previously been focused on transforming growth factorβ1 (TGFβ1), a regulator of

collagen synthesis (Lijnen et al., 2000). Recently, however, a new growth factor, termed

“connective tissue growth factor” (CTGF), acting downstream from TGFβ1 was found (for review,

see Moussad and Brigstock, 2000). The expression of the connective tissue growth factor (CTGF),

which mediates the TGFβ1-induced collagen synthesis, has been noted to increase in the

atherosclerotic lesions, myocardial infarctions and renal fibrosis (Oemar et al., 1997; Ito et al.,

1998; Ohnishi et al., 1998). The regulation of the fibrosis-promoting CTGF gene is probably under

various vasoactive substances, growth factors and locally acting cytokines.

13

Currently, much attention is focused on angiotensin II (Ang II), the effector molecule of the renin-

angiotensin system (RAS). Ang II has an important role in the regulation of blood pressure,

vascular tone and body fluid balance (Weir and Dzau, 1999). The results of the recent Heart

Outcomes Prevention Evaluation (HOPE) study and others have shown that antagonizing the action

of Ang II has important protective effects on cardiovascular system, even independent of blood

pressure reduction (Yusuf et al., 2000; Schiffrin, 2002a). In addition to its role as a blood pressure

regulator, Ang II acts also as a growth factor contributing to cardiac hypertrophy and synthesis of

connective tissue in the heart, kidneys and blood vessels (Dostal, 2001). Ang II has been reported to

cause local inflammatory response by generating reactive oxygen species (ROS) and promote the

accumulation of connective tissue via the activation of TGFβ1 (Fakhouri et al., 2001; Sorescu and

Griendling, 2002).

The present study was designed to explore the role of the CTGF in the pathogenesis of

hypertension-induced end organ damage. In particular the influence of blood pressure and Ang II on

CTGF gene regulation was examined. As Ang II is a known promoter of hypertension and

cardiovascular complications, we used two hypertensive animal models with increased activity of

the renin-angiotensin system; namely, cyclosporine-treated spontaneously hypertensive rats (SHR)

on high sodium diet (Mervaala et al., 1997), and double transgenic rats harboring human renin and

angiotensinogen genes (dTGR) (Ganten et al., 1992). To further clarify the regulatory mediators of

the CTGF gene expression; we conducted intervention studies with antihypertensive drugs,

antioxidant, magnesium supplementation, as well as with anti-inflammatory immunosuppressive

drugs.

14

2 REVIEW OF THE LITERATURE

2.1 CONNECTIVE TISSUE GROWTH FACTOR (CTGF)

2.1.1 Structure and physiological functions

The term “connective tissue growth factor” (CTGF) was first introduced in 1991 by Bradham et al.

to describe a novel growth factor that was secreted by human endothelial cells in culture.

The CTGF gene belongs to the CCN (acronym of CYR61/CEF-10, CTGF/Fisp-12 and Nov) family

of growth factors (Moussad and Brigstock, 2000). The gene for human CTGF located in

chromosome 6q23.1 comprises five exons and four introns and has several regulatory elements such

as activator protein-1 (AP-1), nuclear factor kappa B (NF-κB), cyclic adenosine monophosphate

(cAMP), SMAD, and a unique transforming growth factor beta (TGF)-response element (TβRE)

in its promoter region (Figure 1) (Martinerie et al., 1992; Grotendorst et al., 1996; for review, see

Brigstock, 1999; Moussad and Brigstock, 2000). This element is different from the other reported

TGF -responsive elements and is not present in other genes regulated by TGF (Grotendorst et al.,

1996). Prior translation the CTGF mRNA is a target for posttranscriptional regulation by various

factors (Figure 2). The CTGF is a cysteine-rich peptide with four domains containing 349 amino

acids and a secretory signal peptide at the N terminus (reviewed in Gupta et al., 2000) (Figure 3).

The CTGF was first isolated from the endothelial cell culture (Bradham et al., 1991). Subsequently,

it has been detected in fibroblasts, cartilagenous cells and chondrocytes, cancer cell lines, smooth

muscle cells, renal podocytes and mesangial cells, hepatic sinusoidal cells, myofibroblasts,

pancreatic acinar and ductile cells, bronchoalveolar ductile cells, as well as in serum, tear fluid and

urine (Tamatani et al., 1998; Gupta et al., 2000; Sato et al., 2001; for review, see Blom et al., 2002;

Riser et al., 2003; van Setten et al., 2003).

15

Figure 1. Genomic structure of the CTGF with hypothesized regulatory elements in the promoter region (modified from Blom et al., 2002).

Ang II = angiotensin II AP-1 = activator protein-1

ROS = reactive oxygen species

cAMP = cyclic adenosine monophosphate TGF1 = transforming growth factor beta1

NF-κB = nuclear factor kappa B

T RE = transforming growth factor beta-response element

BCE-1 = basal control element-1

NO = nitric oxide

PGE2 = prostaglandin E2

IGF = insulin-like growth factor

promoter

intron

exon

I II III IV V

Genomic CTGF DNA signal sequence: secretion

IGF binding protein: IGF binding

von Willebrand factor: oligomerization

thrombospondin type I repeat: cell attachment

cysteine knot: receptor binding, dimerization

5’ 3’

Regulatory elements and hypothesized regulators of the promoter region

SMAD binding

AP-1 binding

Stretch responsive element

BCE-1 (T RE)

NF-κB binding site

SMADs

NF-κB

TGF 1

AP-1

Stretch

ROS

Thrombin

cAMP binding

Ang II

cAMP

NF-κB TGF 1

PGE2

NO

+

− + +

+ ++

+

−

+++

++

+

High glucose

+

16

Figure 2. Organization of the CTGF mRNA with hypothesized regulatory elements in the untranslated 3’ region (modified from Gupta et al., 2000).

Figure 3. Modular structure of the CTGF peptide (modified from Gupta et al., 2000).

Structure of translated CTGF peptide

Domain I Domain IVDomain IIIDomain II

Signal peptide

IGF binding

von Willebrand factor type C repeat

Hinge region

Thrombospondin type I repeat

C-terminal domain

N-terminal C-terminal

CTGF mRNA

3’ untranslated region

5’ 3’

Regulatory elements and hypothesized regulators of the untranslated 3’ region

RNA destabilizer

5’ 3’Signal peptide Domain I Domain II Domain III Domain IV

CYP1A1 silencer

IFN silencer

TNF silencer

TNF

−−IFN

−Shear stress

(If present, predisposes mRNA for degradation)

IFN = gamma interferon CYP1A1 = cytochrome P450 1A1 tumor necrosis factor alpha = TNF

17

The CTGF participates in several physiological processes including embryonic development and

differentiation, endochondral ossification, female reproductive tract function in the uterus and

ovary, wound healing, and angiogenesis (Moussad and Brigstock, 2000; Ivkovic et al., 2003).

The bioactivity of the CTGF is still under intense research. Depending on the cell types, CTGF has

been noted to promote mitosis, chemotaxis, stimulate apoptosis, angiogenesis, synthesis of

collagens, fibronectin and 5-integrin (Frazier et al., 1996; Gupta et al., 2000).

The mechanisms by which CTGF exerts its effects on target cells remain more or less unsolved.

Previous studies have demonstrated that adhesion of the endothelial cells, platelets, and fibroblasts

to CTGF is mediated through different integrin receptors. In the study by Babic et al. (1999),

murine CTGF was noted to modulate endothelial cell adhesion, migration, survival and

angiogenesis via 5β3 integrin receptor-dependent pathways. In addition, Segarini et al. (2001)

identified the low density lipoprotein receptor-related protein/2-macroglobulin receptor (LRP2) as

a receptor for CTGF in fibroblasts (Schober et al., 2002). By signaling through the integrins, a

mechanistic interpretation is provided for the chemotactic and mitogenic properties of the CTGF, as

well as for its functions in extracellular matrix (ECM) remodeling during angiogenesis and wound

healing (Blom et al., 2002).

After binding to its receptors, CTGF is presumably internalized into the endosomes, translocated to

the cytosol, phosphorylated by protein kinase C (PKC) and transported to the nucleus where it

stimulates the production of RNAs (Wahab et al., 2001).

2.1.2 Regulation of CTGF expression

Both in endothelial cells and smooth muscle cells the expression of the CTGF is induced by TGF1

(Hishikawa et al., 1999). Although several factors are capable of regulating CTGF gene expression,

it is generally believed that CTGF is transcriptionally activated primarily through TGFβ1. In

addition to TGFβ1, CTGF gene expression is also regulated by high glucose, tumor necrosis factor

α (TNFα), vascular endothelial growth factor (VEGF), cortisol, cAMP, coagulation protease

thrombin, prostaglandin E2 (PGE2), drugs such as iloprost and statins, as well as with

cytomegalovirus infection (Duncan et al., 1999; Murphy et al., 1999; Ricupero et al., 1999;

Abraham et al., 2000; Pereira et al., 2000; Suzuma et al., 2000; Howell et al., 2001; Stratton et al.,

2001; Eberlein et al., 2001; Inkinen et al., 2001). Furthermore, it has been recently shown that other

growth factors such as the platelet-derived growth factor (PDGF), epidermal growth factor (EGF),

and fibroblast growth factor (FGF) also activate CTGF gene expression at a transcriptional level

18

(Brigstock, 1999; Moussad and Brigstock, 2000). The intracellular pathways regulating CTGF are

still not clear. Very recently, Leask et al. (2003) reported that SMAD, PKC and Ras/MEK/ERK

(kinase) pathways are necessary for the TGF1-mediated induction of the CTGF promoter.

Induction of the CTGF promoter is antagonized by c-Jun or by MEKK1 (kinases), suggesting that a

proper balance between the Ras/MEK/ERK and JNK MAPK cascades is necessary for the induction

of the CTGF. Other important regulators of the CTGF might also be NF-κB, AP-1 and reactive

oxygen species (ROS), which are known mediators of inflammation. ROS stimulates the expression

of the CTGF through the activation of Janus kinases and AP-1 (Park et al., 2001).The role of the

NF-κB is more complex as, depending on the cell type, its activation has been shown to induce or

reduce the expression of CTGF (Blom et al., 2002). Interestingly, it has been previously noted that

the activity of the CTGF can be induced by varying the amount of tension (cyclic stretch and static

pressure) imposed on cells (Hishikawa et al., 2001; McCormick et al., 2001). This effect could be

explained by the presence of a stretch-responsive element in the CTGF promoter, which might

regulate the activity of the CTGF under conditions of mechanical stress (Blom et al., 2002).

2.1.3 Role of CTGF in pathological states

As a mediator of fibrotic processes, the expression of the CTGF has been noted to increase in

several pathological conditions involving inflammation and connective tissue accumulation

(Table 1). The expression of the CTGF has been reported to be practically absent in normal human

arteries, but highly enhanced in the intimal smooth muscle cells of atherosclerotic lesions and in the

myocardial infarcts (Oemar et al., 1997; Ohnishi et al., 1998). In the human kidney, the expression

of the CTGF has been found to be markedly increased in the glomerular and interstitial

inflammatory lesions with cellular proliferation and matrix accumulation e.g. IgA nephropathy,

chronic transplant rejection, crescentic glomerulonephritis, focal glomerulosclerosis, lupus nephritis

and membranoproliferative glomerulonephritis (Ito et al., 1998). Furthermore, the up-regulation of

the CTGF has been noted in numerous other pathological states and lesions (scleroderma, keloids,

dermatofibroma, malignant melanoma and other malignancies, chronic pancreatitis, hepatitis and

liver cirrhosis, Crohn’s disease and ulcerative colitis, pulmonary fibrosis and sarcoidosis) (Gupta et

al., 2000).

19

The role of the CTGF in inflammation-wound healing is evident. CTGF mediates the normal scar

formation in the lesion. Physiologically the activity of TGFβ1 and CTGF diminishes as the wound

heals and an adequate scar-formation is achieved. However, in pathologic conditions where the

inflammatory component remains abundant, the expression of the CTGF and hence the production

of collagens stay elevated. The chronic induction of the CTGF expression results in a pathologic

fibrosis of the involved organ and development of improper scars, like keloids (Igarashi et al.,

1996).

Table 1. Enhanced expression of CTGF in different diseases.

Scleroderma, eosinophilic fascitis, Dupuytren's contracture Igarashi et al., 1996

Desmoplastic malignant melanoma Kubo et al., 1998 Mammary carcinoma Frazier and Grotendorst, 1997

Chronic pancreatitis di Mola et al., 1999

Pancreatic cancer Wenger et al., 1999

Liver fibrosis, hepatitis C Paradis et al., 1999 Crohn’s disease, ulcerative colitis Dammeier et al., 1998

Idiopathic pulmonary fibrosis, sarcoidosis, asthma Allen et al., 1999; Burgess et al., 2003

Atherosclerosis Oemar et al., 1997

IgA nephropathy, crescentic glomerulonephritis, focal glomerulosclerosis, lupus nephritis, membranoproliferative glomerulonephritis and diabetic nephropathy

Ito et al., 1998; Wahab et al., 2001

Glioblastoma Pan et al., 2002

Ischaemic heart disease Chen et al., 2000

Cartilaginous tumors Shakunaga et al., 2000

Cerebral infarction Schwab et al., 2000

Disease Reference

Malignancies

Renal diseases

Cardiovascular diseases

Pulmonary diseases

Gastro-intestinal diseases

Dermatological diseases

Riser et al., 2003

2.1.4 Therapeutic approaches to prevent CTGF overexpression

During the last couple of years, accumulating evidence has postulated CTGF as an attractive target

for antifibrotic therapy. Although anti-TGFβ1 therapy has been shown to suppress the expression of

the CTGF, it is also pro-carcinogenic and pro-inflammatory in experimental models (Murphy et al.,

1999). Therefore the use of anti-CTGF therapy without dampening the growth-regulating effect of

TGFβ1 could be appropriate.

Recently, a neutralizing antibody and an antisense oligonucleotide to CTGF have been developed.

These molecules have been shown to block the TGF-stimulated collagen synthesis in various

preclinical models (Blalock et al., 2003). In clinical use, there are no specific drugs to prevent the

overexpression of the CTGF. The down-regulation of the CTGF has been noted with drugs having

diverse actions. Previously, Stratton et al. (2002) have shown that prostacyclin derivatives (iloprost)

are able to prevent the induction of the CTGF in fibroblasts and in patients with scleroderma

through the activation of PKA and blockade of the Ras/MEK/ERK signaling. Also, the cholesterol-

lowering drugs 3-hydroxy-3-methylglutaryl-coenzyme A (HMG CoA) reductase inhibitors (statins)

have been shown to suppress the expression of the CTGF via inhibition of RhoA (Eberlein et al.,

2001). Moreover, Candido et al. (2002) have demonstrated that angiotensin convertase enzyme

(ACE) inhibitor therapy inhibits the CTGF overexpression in the aorta of the diabetic

apolipoprotein E-deficient mice.

2.2 HYPERTENSION-INDUCED END ORGAN DAMAGE

2.2.1 Lesions of the heart, vasculature and kidney

Cardiovascular and renal complications associated with hypertension have become one of the major

risk factors for disability and premature death in the industrialized countries. The hypertensive heart

diseases (left ventricular hypertrophy (LVH) and dilated cardiomyopathy (DCM)) resulting from

the hypertrophy of cardiac muscle fibers and/or deposition of extracellular matrix (fibrosis) can lead

to deterioration of cardiac function and ultimately to morbid and lethal cardiac events. Vascular

diseases (atherosclerosis and arteriosclerosis), originating from the thickening and stiffening of the

arteries, lead to hypoperfusion of different organs and can cause sudden death by occlusion of the

arteries (myocardial infarction, stroke) (for review, see Escobar, 2002). Strokes are strongly

associated with hypertension as hypertension-associated atheromatous plaques narrow brain

arteries, predisposing to local clot formation and ischaemia (for review, see Dickinson, 2003).

20

21

The renal damage, termed benign nephrosclerosis, can be characterized by a decline in arterial,

tubular and glomerular function (Bohle and Ratschek, 1982). The hypertension-associated renal

damage can lead to renal dysfunction and worsening of the hypertension. Non-insulin dependent

diabetes mellitus is the leading cause of end-stage renal disease requiring dialysis or kidney

transplantation in industrialized countries (Gerth et al., 2002; Keane et al., 2003). Diabetic

nephropathy with its resultant hypertension has exponentially increased the need and expences of

medical care in industrialized countries (Gerth et al., 2002; Williams et al., 2002).

2.2.1.1 Sympathetic nervous system in the pathogenesis of hypertension-induced end organ

damage

Sympathetic nervous system has a central role in controlling blood pressure and electrolyte balance.

Besides regulating adrenalin secretion from the adrenals, sympathetic nerves control renal blood

flow, sodium excretion and renin secretion (for review, see DiBona, 2000). Although the

importance of sympathetic nervous activation in the pathogenesis of essential hypertension is well

documented, the exact pathophysiology of the sympathetic nervous dysfunction present remains to

be elucidated. In addition to the increased rate of sympathetic nerve firing there are several

explanations for the increased spillover of noradrenaline to plasma, providing a neurogenic basis of

hypertension (reviewed by Esler et al., 2001). Sympathetic nervous system overactivity is present in

a proportion of patients with essential hypertension, principally in younger ones with hypertension

in its developmental phase (Esler et al., 1989). Sympathetic stimuli regulate the volume of

myocytes, proliferation of smooth muscle cells and ECM production, and thereby contribute to

cardiac and vascular remodeling (Hu et al., 1999; O’Gallaghan and Williams, 2002; Schlaich et al.,

2003). Previously Aggarwal et al. (2002) have demonstrated an elevated noradrenergic activity in

human congestive heart failure (CHF) and a positive correlation between this and the level of

whole-body noradrenaline spillover. Moreover, LVH has been shown to associate with increased

sympathetic activity largely confined to the heart, suggesting that increased cardiac noradrenaline

release is related to the development of LVH (Schlaich et al., 2003). As enhanced levels of

sympathetic activity promote hypertension, cardiac arrhytmias and increased activity of RAS,

treatment with prazosin, clonidine, ganglionic blockers, reserpine and -adrenoceptor blockers have

been widely used in the treatment of cardiovascular diseases.

22

2.2.1.2 Involvement of the mechanical factors

Heart and vasculature

In the heart, arterial hypertension causes myocardial hypertrophy. When enhanced hemodynamic

load is imposed on the left ventricle for a prolonged time the physical stress results in hypertrophic

responses such as the activation of the phosphorylation cascades of many protein kinases,

expression of specific genes and an increase in protein synthesis ultimately leading to the

impairment of the cardiac function and malignant enlargement of the myocardium (pathological

hypertrophy) (Wikman-Coffelt et al., 1979). During this pathological process, the production of

vasoactive peptides such as Ang II, is increased which plays a critical role in the induction of

cardiohypertrophic responses (for review, see Zou et al., 2002).

Along with the enlargement of cardiomyocytes, there are alterations in the ECM which provides the

supportive structure for the myocytes and capillaries (for review, see Weber, 2001). In the process

of cardiac hypertrophy, fibroblasts enhance their synthesis of fibrillar collagens, which leads to the

accumulation of collagens in the interstitium, and around the myocardial arteries. Subsequently this

leads to the stiffening of the myocardium and deterioration of cardiac contractile function (Weber,

2001). Although it is difficult to separate the effects of mechanical and hormonal stimuli, in vitro

studies support a role for mechanical stress in regulating the cardiac hypertrophy. It has been

previously shown that the stretching of the muscle fibre leads to enhanced synthesis of contractile

proteins (Wikman-Coffelt et al., 1979). Even the stretch directed towards a single cardiac cell

evokes the activation of hypertrophy-associated genes (c-fos, c-jun, c-myc, -actin, β-myosin)

(Sadoshima et al., 1992). Besides the genes associated with cardiomyocyte hypertrophy, the

synthesis of cardiac collagens and eventually cardiac fibrosis is also regulated by mechanical

stimuli (Carver et al., 1991).

Increased wall tension due to pressure overload serves as a growth promoting stimulus for smooth

muscle cells (VSMC) within the vessel wall. This leads to the proliferation and hypertrophy of the

VSMC. In addition, activated smooth muscle cells secrete components of the ECM (collagens)

(Sumpio et al., 1988). Together with the concomitant endothelial dysfunction this leads to the

stiffening of the arterial structure, increased peripheral resistance and weakened responsiveness to

vasorelaxing factors (arteriosclerotic changes) (Shepherd, 1990; reviewed by Jeffery and Morrell,

2002).

23

Kidney

The importance of the kidneys in regulating blood pressure was comprehensively reviewed in 1972

by Guyton et al.. Kidneys regulate blood pressure and systemic sodium and fluid balance via the

action of renin, Ang II, aldosterone and vasopressin. A high intake of sodium can promote “salt-

sensitive” hypertension and it has been previously shown that a high sodium intake predicts

mortality and risk of coronary heart disease, independent of other cardiovascular risk factors,

including blood pressure (Tuomilehto et al., 2001). As the kidneys have a sophisticated auto-

regulatory system for the adjustment of the intrarenal blood flow, it seems probable that the renal

lesions accompanying hypertension are not a direct consequence from the pressure overload.

Instead the increased renal vascular resistance seems to be present already at the early stages of

hypertension (reviewed by Ruilope et al., 1994). Therefore, it seems possible that in humans,

arterial hypertension may sometimes be a consequence from the alterations in the renal circulation

or nephron number (Keller et al., 2003). Support to this notion comes from the transplantation

studies, where the “hypertensive kidney” has been transplanted into normotensive animals which

thereafter become hypertensive (Dahl and Heine, 1975).

2.2.1.3 Involvement of the hormonal factors and inflammation

The prevalence of pressure-overload cannot solely explain the occurrence of organ lesions in the

hypertensive patients, as the frequency of morbid cardiovascular episodes among the treated

hypertensive patients remains higher when compared to the normal population. Recently, much

attention has been focused on the hormonal mechanisms in the development of the end organ

damage. With ever-increasing data from numerous studies, hormonal factors and inflammatory

reactions are today considered as the key effectors in contributing to the development of

myocardial, vascular and renal lesions associated with hypertension (Figure 4) (Ridker et al., 1997;

for review, see Lagrand et al., 1999). Very recently, in the Framingham study, it was shown that

increases in the serum inflammatory markers, particularly elevated interleukin-6, were associated

with increased risk of congestive heart failure in a patient population without prior myocardial

infarction (Vasan et al., 2003).

24

Figure 4. Simplified scheme of events leading from vascular injury to fibrosis (modified from Franklin, 1997).

Heart and vasculature

The most common injury to the heart is the myocardial infarction. During the first weeks after a

massive infarction, the myocardium is susceptible to rupture. The function of the repair process is to

maintain the structural integrity. The replacement of the contractile tissue by scar leads to impaired

myocardial function by decreasing the myocardial contractility. In hypertensive heart disease,

resistance arteries (including coronary vessels) undergo remodeling processes. Coronary arteries

develop accumulation of collagen in the adventitial layer (perivascular fibrosis) where bundles of

collagen extend also into the interstitial areas of the myocardium (interstitial fibrosis) (Silver et al.,

1990). The atherosclerotic lesions develop within several years and are characterized by smooth

Vascular injury

diabetes traumahypertension myocardial

infarction

Activation of the inflammatory cells

Release of cytokines, growth factors and matrix proteins

Activation of the mesenchyme-derived cells

Proliferation, synthesis of extracellular matrix

Vascular fibrosis

25

muscle cell proliferation, chronic inflammatory reaction and deposition of lipid and matrix in the

subintimal space.

Kidney

Depending on the nature of the lesion, the healing process in the kidney results in fibrosis of the

affected glomeruli and interstitium. The hypertension-associated lesions usually comprise damage

to the afferent arteries with the thickening of the medial layer with connective tissue. In more severe

cases the afferent arteries have marked concentric hypertrophy and/or fibrinoid necrosis. In

association with arterial damage there is often glomerular (glomerular ischaemia,

glomerulosclerosis) and tubular (tubulo-interstitial fibrosis) lesions (Bohle and Ratschek, 1982).

The inflammation-healing process

The repair process of the affected organ begins immediately after injury and thus overlaps with the

inflammatory response. It is through continuum of inflammatory reactions and deposition of ECM

that the injuries are healed. The connection between inflammation and fibrosis can be seen in the

early phases of embryonic development when there is no inflammatory response to an injuring

stimulus. The healing occurs by proliferation of the cells surrounding the lesion and no fibrosis

occurs.

Whether the injuring stimulus has been a myocardial infarction following a coronary artery

occlusion or an effect by an endogenous hormone, the healing process eventuates in fibrous tissue

formation (Figure 4). On the first stage of the inflammatory phase, platelet-derived cytokines

attract cells from the leukocyte axis and polymorphonuclear neutrophilic leukocytes (PMN)

infiltrate the lesion. These cells appear within hours from the injury and their function is to remove

the cellular debris from the wound. The infiltration of PMN is soon followed by the accumulation

of monocytes/macrophages which remove debris from the lesion and release pro-inflammatory

cytokines and interleukins; colony-stimulating factors (GM-CSF), growth factors (TGFβ1,

thrombospondin-1 (TSP-1), PDGF, FGF) and ROS. Lymphocytes possess the ability for antigen-

specific recognition and they control the function of other leukocytes. B lymphocytes participate in

the humoral response by secreting various antibodies and T lymphocytes, in turn, regulate the

cellular inflammatory response. In addition to the cells specific to the injured tissue, the stromal

cells (fibroblasts, smooth muscle cells, endothelial cells and inflammatory cells) have a major role

in repair. In response to growth factors released by other cells, fibroblasts begin to proliferate and

secrete components of the ECM. The fibroblast-derived collagens assemble to form the fibrillar

network that eventually becomes the scar of the damaged area.

26

2.2.1.3.1 Angiotensin II

From the large number of growth factors involved in the regulation of inflammation and

regeneration, much attention has recently been paid on Ang II.

Ang II is the active effector of the RAS. Ang II regulates blood pressure, plasma volume and the

activity of the sympathetic nervous system. It has also a fundamental role as a growth factor in

myocardial, vascular and renal remodeling (for review, see Touyz and Schiffrin, 2000). Originally

described as a systemic humoral system, Ang II is currently known to be produced also in several

tissues by the activation of local RAS (for review, see Bader et al., 2001). Under conditions of salt

or volume depletion or sympathetic activation, the protease renin, derived from the macula densa of

the juxtaglomerular apparatus, cleaves the liver-derived peptide angiotensinogen to form

angiotensin I (Ang I). The main effector of the RAS, Ang II, is then formed by the ACE. ACE

exists usually in an endothelium-bound form with particularly high concentrations in the lung

(Bader et al., 2001). In addition to ACE, Ang II can be formed by other enzymes. Generation of

Ang II has been noted to occur via the activation of chymase, cathepsin G and tonin (reviewed by

Dzau et al., 1993). Local (tissue) RAS is activated under pathophysiological conditions where the

synthesized Ang II appears to contribute to altered tissue function and morphology (Hirsch et al.,

1990). Furthermore, in some tissues, locally formed Ang II may exceed the importance of

circulating Ang II (Navar et al., 1996). Ang II exerts its effects via binding to its receptors located

in the cell surface. Two main subtypes of Ang II receptors have been cloned, described and

classified, namely the G-protein-coupled AT1 and AT2 receptors (Timmermans et al., 1993). Two

other subtypes of Ang II receptors have also been described but are not yet included in the

definitive classification of the mammalian AT receptors. To date, AT1 receptors have been shown to

mediate most of the physiological actions of Ang II, and this subtype is predominant in the control

of Ang II-induced vascular functions (Touyz and Schiffrin, 2000). In contrast to the AT1 receptor,

the physiological role of the AT2 receptor remains elusive. It has been suggested that the AT2

receptor counteracts the Ang II-induced effects of the AT1 receptor (Nakajima et al., 1995;

reviewed by Siragy and Carey, 2001). Although the main function of smooth muscle cells is

vasoconstriction, it has also become evident that the Ang II-induced stimulation of the AT1

receptors has a major role in the proliferation of VSMC and fibroblasts, deposition of ECM and

inflammation (Dostal, 2001; Mezzano et al., 2001).

27

Signaling events generated by Ang II

Upon binding to its receptor, Ang II causes an increase in the intracellular free Ca2+, Na+ and an

efflux of Mg2+ (Touyz and Schiffrin, 2000). Ang II activates growth promoting signal pathways

(Janus kinases, MAP-kinases, calcineurin and p70S6-kinase) and mediators of cell migration (focal

adhesion kinase) and regulators of ROS and inflammation (nicotinamide adenine dinucleotide

phosphate (NADPH) oxidases, NF-κB, AP-1) (Schmelzle and Hall, 2000; Molkentin and Dorn,

2001; Liu et al., 2003). The Ang II-mediated activation of intracellular kinases leads to the

induction of early growth-response genes and regulation of the vascular and cardiac growth,

remodeling and repair (reviewed by Berk and Corson, 1997).

The calcium-calmodulin, Ser/Thr protein phosphatase calcineurin and its downstream

transcriptional effector nuclear factor of activated T cells (NFAT) play pivotal roles in the immune

response. Activation of calcineurin leads to dephosphorylation and nuclear translocation of NFAT

and regulation of several inducible genes in T cells. Recently, a role for calcineurin-dependent

pathways in the signal transduction of cardiac growth has been postulated. Stimulation of

calcineurin is capable of driving cardiac hypertrophy, heart failure, and death in experimental

animal models (Molkentin et al., 1998; reviewed by Molkentin, 2001). Ang II stimulates the

hypertrophy and proliferation of cardiomyocytes and cardiofibroblasts through the induction of

calcineurin thus suggesting a role for calcineurin-dependent pathways in the pathogenesis of Ang

II-induced cellular hypertrophy and proliferation (Fu et al., 1999).

Previous studies have provided compelling evidence that Ang II controls the process of cardiac and

vascular hypertrophy also via AT1 receptor-mediated activation of p70S6-kinase which regulates

the cellular response to nutrients, organization of the actin cytoskeleton, membrane traffic, protein

degradation, PKC signaling, ribosome synthesis and possibly, mechanisms of cancer (reviewed in,

Schmelzle and Hall, 2000). In addition, p70S6-kinase has a central role in inflammatory reactions

(Giasson and Meloche, 1995; Takano et al., 1996; Lehman et al., 2003).

Ang II and inflammation

A link between the RAS and inflammation-wound healing has been previously noted. Ang II

stimulates the proliferation of lymphocytes and regulates their activation through AT1 receptor-

mediated induction of calcineurin phosphatase (Nataraj et al., 1999). Chronic activation of RAS,

resulting in a permanent elevation in plasma and tissue Ang II, leads to perivascular inflammation

and fibrosis, vascular remodeling, myocyte necrosis and subsequent scar formation, even in the

absence of hypertension (Figure 5) (Ratajska et al., 1994; Mervaala et al., 1999; Weber, 2001).

Ang II stimulates NADPH oxidase to produce ROS such as superoxide anions and hydrogen

28

peroxide, which participate in the regulation of VSMC growth, apoptosis of cardiomyocytes,

induction of vascular inflammatory response, impairment of endothelial-dependent vascular

relaxation, atherosclerosis and pathogenesis of mechanical stress-induced cardiac hypertrophy

(Figure 5) (Aikawa et al., 2001; Aikawa et al., 2002, reviewed by Griendling et al., 2000). Our

group has previously shown that Ang II-mediated inflammatory reaction in the heart, kidney and

vasculature, characterized by leukocyte infiltration and vascular adhesion molecule overexpression

are due to Ang II-induced ROS formation and activation of the redox-sensitive transcription factors

NF-κB and AP-1 (Müller et al., 2000a). Moreover, we have shown that Ang II controls the

migration of mature dendritic cells even by blood pressure-independent mechanisms and activates

monocytes/macrophages even in the uninjured areas (Mervaala et al., 1999; Müller et al., 2002).

Figure 5. Different mechanisms of Ang II-induced vascular inflammation (modified from Ruiz-Ortega et al., 2001)

Secretory products Reactive oxygen species

O2-, H2O2, OH-

Cytokines and growth factors Interleukins, TNF , MCP-1, TGF , PDGF, FGF, G-CSF, GM-CSF, CTGF

Matrix proteins Fibronectin, thrombospondin

Enzymes Proteases, collagenases, elastase, ACE

Activated monocyteAdhesion and transmigration

Secreting monocytes

Ang II (Promotes chemotaxis, activation, adhesion and transmigration)

Ang II

Endothelial cells

Smooth muscle cells

Ang II = angiotensin II ACE = angiotensin converting enzyme

FGF = fibroblast growth factor

GM-CSF = granulocyte-macrophage colony-stimulating factor

G-CSF = granulocyte colony-stimulating factor

CTGF = connective tissue growth factor MCP-1 = monocyte chemoattractant protein-1 PDGF = platelet-derived growth factor

TNF = tumor necrosis factor alpha TGF = transforming growth factor beta

Circulating monocyte

Secretory products

29

Ang II-mediated induction of growth factors

Ang II has a major role in regulating the cardiac and renal growth and structure. In addition to the

pressure-induced mechanical factors, hypertrophy of the heart is also governed by the growth-

promoting action of Ang II. Similarly, a number of kidney diseases, and their progression to the

end-stage renal disease, are driven, in part, by the effects of Ang II. Ang II is a powerful mitogen

and an ECM promoting factor for many cell types. It increases DNA synthesis, promotes cell-cycle

progression and up-regulates the expression of growth factors such as endothelin-1, TGFβ1, PDGF,

and FGF which induce cellular hypertrophy, hyperplasia and production of the ECM (Stirling et al.,

1990; Gibbons et al., 1992; Nakahara et al., 1992; Kaiura et al., 2000). The Ang II mediated

production of collagen and fibronectin in the heart and kidney is primarily mediated by TGF1

(Kagami et al., 1994; Everett et al., 1994; Funck et al., 1997).

TGFβ is one of the most potent profibrotic stimuli to fibroblasts. TGF participates in the normal

development, wound repair, and several pathological processes including cardiac, renal and liver

fibrosis, development of atherosclerosis, and arteriosclerosis (for review, see Border and Noble,

1994; Ihn, 2002). At the site of tissue injury, TGF (released from infiltrating lymphocytes,

platelets, activated macrophages, injured myocytes and fibroblasts) is first secreted in a latent form.

The latent form is then activated by other peptides, such as TSP-1. Activation of the TGF1

increases the expression of collagen types I, III, VI, VII and X and fibronectin (for reviews, see

Massague, 1990; Govinden and Bhoola, 2003). The production of the ECM is further enhanced by

the inhibitory effect of the TGF on matrix degradation by decreasing synthesis of proteases and

increasing levels of protease inhibitors (Massague, 1990; Govinden and Bhoola, 2003). The

functional importance of the TGF in wound healing can be seen after myocardial infarction.

Weeks after infarction the TGF receptor density is markedly increased in both the infarcted and

noninfarcted myocardium and it is anatomically coincident with sites of fibrosis (Sun et al., 1998).

Besides participating in the reparative wound healing response, TGF also controls the fibrosis

caused by myocardial pressure-overload. As Ang II is known to cause mechanical stress

(hypertension), fibrosis via the activation of the TGF1, perivascular inflammation, inflammatory

end organ damage characterized by an abundance of leukocytes and activation of the inflammatory

mediators (NF-κB, AP-1), the involvement of Ang II in regulating the expression of the CTGF

deserves further notice.

30

2.3 ANIMAL MODELS USED IN THE PRESENT STUDY

2.3.1 Spontaneously hypertensive rat (SHR) (Study I)

The SHR is the most widely used animal model for human hypertension. In the SHR the arterial

hypertension and essential human hypertension share many similarities in the pathogenic

mechanisms (Trippodo and Frohlich, 1981). The SHR strain was developed in 1963 by Okamoto

and Aoki. The SHR has been shown to possess a genetic susceptibility to (salt-sensitive)

hypertension (St. Lezin et al., 1997). Blood pressure in the SHR rapidly increases during the first 3-

4 postnatal months after which it stabilizes at an elevated level. The SHR has an enhanced

sympatho-adrenal drive and develop functional and structural vascular lesions (impaired

vasodilatation, vascular remodeling) (Trippodo and Frohlich, 1981; Koller and Huang, 1994). To

examine the pathogenesis of the CsA-induced hypertension and end organ damage, we have

developed an animal model, i.e. the CsA-treated SHR on high sodium diet in which magnesium

deficiency as well as increased RAS activity is involved in the pathogenesis of the CsA toxicity

(Mervaala et al., 1997; Lassila et al., 2000).

2.3.2 Double transgenic rat harboring human renin and angiotensinogen genes (dTGR)

(Study II, III, IV)

The transgenic rat-strain carrying the human angiotensinogen TGR-(hAOGEN) and human renin

TGR-(hREN) genes was originally developed for the study of human renin inhibitors which

ordinarily do not function in rats. The human components of the RAS in the dTGR do not interact

with the endogenous rat RAS (Ganten et al., 1992; Bohlender et al., 1997). Hypertension, vascular

inflammation and end organ damage in the dTGR are due to increased Ang II synthesis by the

human components of the RAS. The development of the myocardial, renal, and vascular damage in

the dTGR is age-dependent. In fact, immediately after weaning at the age of 3-4 weeks, myocardial

and renal tissue morphology, assessed by light microscopy, are indistinguishable between the dTGR

and the normotensive SD rat. In contrast, marked inflammatory cell recruitment, mainly consisting

of monocytes and macrophages, as well as an up-regulation of the intercellular adhesion molecule-1

(ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and the exctracellular matrix component

fibronectin, are already present at an early age of the dTGR (Mervaala et al., 1999; Müller et al.,

2000b). These findings have been interpreted as follows: Ang II triggers inflammatory response and

31

exerts direct pro-inflammatory effects by blood pressure-independent mechanisms related to the

activation of the redox-sensitive transcription factors NF-κB and AP-1 as well as oxidative stress

(Müller et al., 2002).

2.4 COMPOUNDS USED IN THE PRESENT STUDY

2.4.1 Enalapril (Study I)

ACE inhibitors are nowadays widely used in the treatment of essential and renovascular

hypertension. Numerous large clinical studies have shown long-term reductions in the incidence of

the myocardial infarction, stroke, cardiac arrest, heart failure, and atherosclerosis in patients treated

with ACE inhibitors (Sleight, 2002).

ACE inhibitors prevent the ACE-mediated conversion of angiotensin I to angiotensin II. Thus,

inhibition of the ACE diminishes the formation of Ang II. This in turn, leads to vasodilatation and a

decrease in blood pressure. In addition, the blockade of the ACE promotes the bioavailability of

vasodilatating substance bradykinin. As Ang II is an important regulator of renal blood flow,

sodium homeostasis and mineralocorticoid aldosterone, the ACE inhibition promotes natriuresis by

increasing renal blood flow, sodium excretion and by preventing the action of aldosterone. In

addition to the antihypertensive effect, ACE inhibitors have been shown to diminish the

inflammatory response associated with the end organ damage. Therefore ACE inhibitors are also

used to prevent the progressive cardiovascular and renal damage in diabetes mellitus. The most

common adverse effects of the ACE inhibitors: dizziness, headache and cough are usually well

tolerated (Gradman et al., 1995).

2.4.2 Valsartan (Study I, II)

Valsartan belongs to the family of the Ang II receptor AT1-antagonists. AT1 receptor antagonists

are currently used in the treatment of essential hypertension. In clinical trials, valsartan has been

shown to reduce symptoms, enhance the quality of life and lessen morbidity and mortality in

patients (for review, see Manohar and Pina, 2003). In patients who cannot tolerate ACE inhibitors

because of angioedema or severe cough, an AT1 receptor antagonist can be succesfully used as an

alternative. Recently, valsartan has been approved as a treatment of heart failure in patients who

cannot be maintained on an ACE inhibitor therapy (Manohar and Pina, 2003).

32

The antihypertensive and anti-inflammatory effects of valsartan resemble closely the ones seen with

the ACE inhibitor enalapril. By specifically blocking the AT1 receptor, valsartan does not directly

affect the synthesis of Ang II. In contrast, it prevents the action of Ang II at the receptor level. As it

has been hypothesized that the AT2 receptor-mediated action of Ang II could have some beneficial

effects, including anti-fibrotic action, the blockade of only one subtype (AT1) of the Ang II

receptors might have additional therapeutic effects. The most common adverse effects associated

with valsartan therapy include: headache, dizziness and tiredness. As the RAS plays a crucial role in

the normal renal development neither the ACE inhibitors nor the AT1 receptor blockers can be used

during gestation (for review, see Guron and Friberg, 2000).

2.4.3 -Lipoic acid (Study III)

In humans, lipoic acid is currently used to improve the glucose effectiveness, and to prevent the

progression of diabetic neuropathy. Its efficacy has also been examined in the treatment of various

neurological disorders (for review, see Ziegler et al., 1999). Lipoic acid is a sulfur-containing

coenzyme required for the mitochondrial dehydrogenase reactions leading to the ATP formation.

Lipoic acid and the reduced form, dihydrolipoate, are potent natural antioxidants. Lipoic acid reacts

with superoxide, hydroxyl radicals, hypochlorous acid, peroxyl radicals, and singlet oxygen

(Carreau, 1979; Packer et al., 1995; Moini et al., 2002). It has been previously reported that dietary

lipoic acid supplementation decreases oxidative stress, ameliorates hypertension and protects

against renal and vascular injuries in various animal models (Kocak et al., 2000; Suh et al., 2001).

Treatment with lipoic acid is usually well tolerated without any major adverse effects (Evans et al.,

2002).

2.4.4 Cyclosporine A (CsA) (Study I, II)

The calcineurin inhibitor CsA is a fungal cyclic polypeptide, which has been widely used to prevent

rejection of organ transplants. After its introduction, CsA has increased graft survival and decreased

patient mortality significantly compared to more conventional immunosuppressives (Merion et al.,

1984). Moreover, the use of CsA to treat several autoimmune diseases has increased during the

recent years.

CsA was the first T lymphocyte selective drug to inhibit lymphocyte function without concomitant

myelosuppression. CsA binds to cyclophilin to inhibit the phosphatase calcineurin, and thereby

prevents the dephosphorylation of NFAT and production of inflammatory cytokines, particularly

33

interleukin-2 (IL-2), which is critical for T lymphocyte proliferation and maturation, and interferon

γ (IFN γ) which is critical for macrophage activation (Liu et al., 1992).

Despite its beneficial effect on the long-term graft survival, the mortality due to cardiovascular

diseases and renal failure is greater among the CsA-treated graft recipients than among the normal

population. This is probably a consequence of the severe and common adverse effects of the CsA,

hypertension and nephrotoxicity which could be mediated by the CsA-induced activation of RAS

(Julien et al., 1993; for review, see Textor et al., 1994).

2.4.5 Tacrolimus (FK506) (Study IV)

The calcineurin inhibitor tacrolimus is a macrolide which effectively prevents rejection of

organ transplants with a concomitant decrease in patient morbidity and mortality (Margreiter,

2002).

Similarly to CsA, tacrolimus also inhibits the activity of calcineurin and NFAT and hinders the

production of inflammatory cytokines IL-2 and IFN γ. Although this occurs via different pathways

(CsA binds to cyclophilin and tacrolimus binds to FK506 binding protein (FKBP)), the inhibition of

calcineurin produces similar pharmacologic effects with both drugs. Clinical studies suggest similar

graft and patient survival with either CsA or tacrolimus. The relative frequency of chronic rejection

in patients treated with tacrolimus versus CsA has not been shown to differ. The side-effect profile

of CsA and tacrolimus are more or less similar. However, certain side-effects, such as gingival

hyperplasia, hirsutism, hyperlipidemia and hypertension are more commonly noted with CsA,

whereas tremor and glucose intolerance are associated more often with tacrolimus (for review, see

Morales et al., 2001; Hood, 2002).

2.4.6 Everolimus (SDZ RAD) (Study II)

The mammalian target of rapamycin (mTOR) inhibitor everolimus is a macrolide

immunosuppressant used to prevent the inflammatory graft-rejection among the organ transplant

recipients. It has been used alone and, more commonly, with calcineurin inhibitors. The synergistic

effect of the everolimus and calcineurin inhibitors have shown low rates of acute rejection,

improved rates of patient and graft survival, and better renal function in organ transplant recipients

(for review, see Nashan, 2002a).

34

Everolimus is a proliferation signal inhibitor that blocks growth factor-driven proliferation of the T

cells, B cells and VSMC by inhibiting the activation of the p70S6-kinase. Everolimus has been

shown to reduce acute rejection, limit the CsA-induced nephrotoxicity, protect against

cytomegalovirus infections, and inhibit vascular remodeling (for review, see Nashan, 2001; Nashan,

2002b). Previous studies have provided strong evidence that the p70S6-kinase plays an important

role also in Ang II-induced protein synthesis, vascular and cardiac hypertrophy, and possibly,

inflammatory response (Giasson and Meloche, 1995; Takano et al., 1996). The most common

adverse effects of the everolimus are thrombocytopenia and hyperlipidemia but not hypertension.

2.4.7 Magnesium (Study IV)

In epidemiological studies, altered magnesium balance has been linked to the prevalence of

hypertension and hypertensive heart diseases. Some subgroups of hypertensive patients (e.g.

African-Americans, the elderly, patients with insulin resistance or pre-eclampsia) have a high

incidence of altered magnesium metabolism and pre-eclamptic and eclamptic patients are

consistently treated with magnesium (Touyz et al., 1987; Touyz et al., 1992; Touyz, 2003). In

addition, a relationship has been noted between Ang II, magnesium and blood pressure (Touyz and

Schiffrin, 2000). Magnesium has a multitude of functions in the cardiovascular system. Magnesium

regulates contractile proteins, transport of other cations (calcium, sodium and potassium), DNA and

protein synthesis, cell replication, and oxidative-phosphorylation (Touyz, 2003). Serum magnesium

is regulated within a range of 0.7-1.1 mmol/l, and the current recommended daily allowance (RDA)

for magnesium is 400mg/d (National Public Health Institute, Finland, 1998).

In experimental models of hypertension, hypomagnesemia causes elevation in blood pressure and

increased reactivity to vasoconstrictors (Miyagawa et al., 2000). Thus, it may be possible that Ang

II-dependent hypertension is associated with cardiovascular effects of hypomagnesemia. It has been

recently demonstrated that Ang II promotes magnesium efflux and decreases intracellular and

serum magnesium possibly by stimulating the Na+/Mg2+ exchanger in VSMC. In addition,

imipramine and quinidine, inhibitors of Na+/Mg2+ exchanger have been shown to attenuate the Ang

II–induced hypertension in rats (Touyz and Yao, 2003).

35

3 AIMS OF THE STUDY

Since its discovery in 1991, CTGF has received growing attention as a mediator of fibrotic

processes. Intense research by various groups has provided much information about the regulation

and nature of the CTGF. The enhanced expression of the CTGF has been linked to several diseases

associated with inflammation and connective tissue accumulation. However, the role of the CTGF

in the pathogenesis of the hypertension-induced end organ damage and its connections to RAS has

not yet been established.

By using CsA-treated SHR on high sodium diet and dTGR as experimental animal models,

the specific aims of the study were:

• To investigate whether the arterial hypertension promotes myocardial

CTGF gene expression.

• To examine whether Ang II regulates CTGF gene expression.

• To evaluate whether the antagonists of the RAS protect against the induction of the CTGF.

• To investigate the effects of immunosuppression and antioxidant therapy on the CTGF gene

expression.

• To examine whether magnesium supplementation ameliorates Ang II-induced myocardial

damage and CTGF overexpression.

36

4 MATERIALS AND METHODS

4.1 EXPERIMENTAL ANIMALS

In Study I, 48 male spontaneously hypertensive rats (SHR) and 8 Wistar-Kyoto (WKY) rats (Harlan

Sprague Dawley, Indianapolis, IN, USA) aged 7-9 weeks at the beginning of the experiment were

used.

Studies II, III, and IV included 120 male 3-4 week-old double transgenic rats harboring human

renin and angiotensinogen genes (dTGR) (RCC, Füllinsdorf, Switzerland) and 40 age-matched

normotensive Sprague Dawley rats (SD) (M&B, Ejby, Denmark).

The rats were housed in a standard laboratory facility (temperature 22-24°C, a 12-hour light-dark

cycle). The rats had free access to tap water and feed during the experiment.

4.2 EXPERIMENTAL DIETS AND DRUG TREATMENTS

In Study I, rats were placed on high-sodium diet during the treatment (sodium 2.6%, magnesium

0.2%, potassium 0.8%, calcium 1.0%, phosphorous 0.75%) The food was prepared by adding

sodium chloride to the commercially available rat chow (R36, Finnewos Aqua, Helsinki, Finland).

In Studies I-IV, enalapril (30 mg/kg/d. Leiras Ltd, Turku, Finland), valsartan (3 and 30 mg/kg/d.

Novartis Ltd, Basel, Switzerland), -lipoic acid (350 mg/kg/d. Sigma Aldrich Finland, Helsinki,

Finland), and magnesium (0.6 % of the dry weight of the feed) were mixed in the standard food to

produce the planned daily doses. Normal rat chow served as the control diet. Drug concentration in

the chow was calculated based on the fact that food consumption of an adult rat is approximately

20-25 g/day. The actual intake of drugs was verified in the metabolic studies.

Cyclosporine (2 and 5 mg/kg/d. Novartis Ltd, Basel, Switzerland) and tacrolimus (1 mg/kg/d.

Fujisawa Europe, Munich, Germany) were administered subcutaneously (I, II, IV). Everolimus (2.5

mg/kg/d. Novartis Ltd, Basel, Switzerland) was administered by gavage (II).

37

4.3 MEASUREMENT OF BLOOD PRESSURE

Systolic blood pressure and heart rate from the pretrained rats were measured by using a tail-cuff

blood pressure analyzer (Apollo-2AB Blood Pressure Analyzer, Model 179-2AB, IITC Life

Science, Woodland Hills. CA., USA). Before the measurement, the rats were kept at +30°C for 30

min to enable the pulsations of the tail artery to be detected. Systolic blood pressure was calculated

from the arithmetic mean of three consecutive results without disturbances in the signal.

4.4 METABOLIC STUDIES

During the last week of the experiments (I, III, IV) (weekly in Study II), the rats were housed

individually in metabolic cages. The consumption of food and water was measured, and the

estimated intake of drugs was calculated. Urine was collected over a 24-hour period, urine volumes

were measured and samples stored at -80°C until the biochemical determinations were performed.

4.5 COLLECTION OF THE SAMPLES

At the end of the experiments, the rats were made unconscious with CO2/O2 (70%/30%; AGA,

Riihimäki, Finland) and decapitated. In Study II, an additional group of SD rats was sacrificed at

the age of 4 weeks, and dTGR rats at the age of 4, and 5.5 weeks.

Blood samples were taken into chilled tubes with or without an anticoagulant (EDTA 4.5 mM).

Plasma and serum were stored at -80°C until the biochemical determinations were performed. The

heart and kidneys (I-IV) and pulmonary artery (II) were excised, washed with ice-cold saline,

blotted dry and weighed. Tissue samples for mRNA detection were snap-frozen in liquid nitrogen

and samples for immunohistochemistry in isopentane (-35°C), then all were stored at -80°C until

assayed. Samples for conventional morphology, immunohistochemistry and in situ hybridization

were fixed with 10 % formaline and processed in paraffin with routine techniques.

4.6 HISTOLOGY

4.6.1 Sample preparation

The samples for histology were fixed with 10 % formaline for 24-48 hours and processed in

paraffin through ascending ethanol concentrations and xylene. Sections (3-5 m) were cut with

38

a microtome, rehydrated through xylene and descending ethanol concentrations. The sections were

then stained with Masson’s trichrome and examined under a conventional light microscope without

knowledge of the sample identity.

4.6.2 Tissue morphology and scoring of the samples

In the heart, the arteries and ventricular fibrous tissue formation were evaluated. Each sample was

scored from 0 to 3 (I, III) or from 0 to 4 (II, IV) according to the morphological changes.

Furthermore, in the kidneys, interstitial, tubular and glomerular changes were evaluated. The

samples were scored from 0 to 4 (II-IV) according to the morphological changes. Thereafter, the

damage score was calculated from the arithmetic mean of each group (n=4-10).

4.7 DETECTION OF mRNA FOR CTGF, COLLAGENS I AND III, AND TGF ββββ1

4.7.1 In situ hybridization

cDNA clones specific for human CTGF mRNA (1.1kb in pRc/CMV, provided kindly by G.

Grotendorst, University of Miami, USA) (Study I, II, IV), rat proα1 I collagen mRNA (α1 R2) and

proα1 III collagen mRNA (pRGR5) (Study I-II); rat TGFβ1 mRNA (Study II) were used for the

hybridization. The plasmids containing the inserted probe were linearized with restriction enzymes

to allow transcription of digoxigenin-labeled sense and antisense RNA probes according to the

Riboprobe Synthesis Kit method (Boehringer Mannheim, Mannheim, Germany). The paraffin

sections (5 µm) of the heart (Study I-IV), kidney (Study II) and pulmonary artery (Study II) were

placed onto Superfrost (Menzel-Gläser, Braunschweig, Germany) slides. The sections were

deparaffinized and hydrated through descending ethanol concentrations. Pretreatment included

incubation with 10 µg/ml proteinase K and 4 % paraformaldehyde post-fixation. The sections were

hybridized with labeled RNA-probe in the concentration of 1ng/µl at +50oC for 18 h. The slides

were washed under stringent conditions including treatment with RNAse A. Digoxigenin-labeled

probes were detected following the methods from the DIG-detection kit (Boehringer Mannheim,

antibody dilution 1:2000). After a 16-hour colour substrate incubation the slides were

counterstained with hematoxylin and coverslipped with aqueous-based mounting medium. Staining

was accepted positive when seen with the antisense probe only.

39

4.7.2 Northern blot

RNA extraction

Total RNA was extracted from the heart (Study I, II, IV) and kidney (Study II) by using the method

of Chomczynski and Sacchi (1987) and the RNA content was determined spectrophotometrically at

wavelengths 260 nm and 280 nm. Aliquots of the total RNA were size-separated by electrophoresis

on agarose gels. After the integrity and uniform loading of the gels were confirmed with ethium

bromide staining, the RNA was transferred from the gels to nylon membranes by blotting

(MagnaGraph Nylon Transfer Membrane; Micron Separations Inc., Westborough, MA, USA.

PosiBlot, Stratagene, La Jolla, CA, USA). The membranes were placed on filter papers, dried, UV-

crosslinked (Spectrolinker, Spectronics Corporation, Westbury, NY, USA) and stored at +4°C until

hybridization.

Labeling of the probe

The PCR amplified cDNA clones for human CTGF mRNA 1.1kb in pRc/CMV (Study I, II, IV), rat

proα1 I collagen mRNA (α1 R2) and rat proα1 III collagen mRNA (pRGR5) (Study I, II), rat TGFβ1

mRNA (Study II), and rat GAPDH mRNA (I, II, IV) as a reference probe were labeled with (32P)dCTP

by random priming (High Prime DNA labeling kit, Boehringer Mannheim). The unincorporated

label was separated with elution (NICK Column, Amersham Pharmacia Biotech AB, Uppsala,

Sweden).

Hybridization

The hybridization was performed at +42ºC for 18 h in hybridization solution (50% formamide, 1M

NaCl, 1% SDS, 5x Denhardt’s solution, 10% dextran sulphate and 100µg/ml herring sperm DNA).

The filters were washed three times with 0.1% SDS in 2x SSC at +22ºC and twice with 0.1% SDS

in 0.2 SSC at +55ºC. The bound probe was detected by autoradiography at -70ºC using Kodak X-

omat films (Eastman Kodak Company, NY, NY, USA). The films were developed and scanned.

The band intensities were measured with computer densitometry (Bio Image Intelligent Quantifier,

B. I. Systems Corp. Ann Arbor, MI, USA). The amount of the detected mRNA for CTGF, proα1 I

collagen, proα1 III collagen and TGFβ1 were calculated in relation to the expression of GAPDH

mRNA of the corresponding sample.

40

4.7.3 Reverse transcriptase-polymerase chain reaction (RT-PCR) assay for CTGF mRNA

The RNA was extracted from the whole hearts (II, III) and right ventricle (II) with Trizol reagent

(Gibco Life Technologies, Invitrogen Corporation, Paisley, UK), according to the instructions of the

manufacturer. After extraction, a 1 µg aliquot of the total RNA was treated with DNAase 1

(Deoxyribonuclease 1, Sigma Chemical Co., USA) and reverse-transcribed to cDNA by incubation

for 50 minutes at +45ºC in the presence of reverse transcriptase (Enhanced Avian HS RT-PCR kit,

Sigma Chemical Co., USA). 2 µl of cDNA was subjected to a quantitative real time polymerase

chain reaction (PCR) by Lightcycler Instrument (Roche Diagnostics, Neuilly sur Seine, France) for

the detection of the CTGF mRNA and GAPDH mRNA. The samples were amplified using

FastStart DNA Master SYBR Green 1 (Roche Diagnostics) in the presence of forward and reverse

primers (Sigma Genosys, London, UK). After amplification the quality of the PCR products was

analyzed and the quantities of the CTGF and GAPDH PCR products were quantified with an

external standard curve amplified from a purified PCR product.

4.8 IMMUNOHISTOCHEMISTRY

Cryosections (5 µm) were treated with acetone (10 min at -20°C), chloroform (30 min at room temp

(RT)) and air-dried. Primary antibodies (mouse anti-rat ED1 (Study II, III) ED2 (Study II), CD4

(Study II, III), CD8 (Study II, III) (Serotec Ltd, Oslo, Norway), anti-rat CTGF (for paraffin

sections) (Study II, IV) (Abcam, Cambridge, UK) and anti-mouse PCNA (Study II) (Stressgen

Biotechnologies Corp. Victoria, BC Canada), were applicated for 30 min at RT. The slides were

then treated with secondary antibody (peroxidase-conjugated rabbit anti-mouse immunoglobulins;

DAKO A/S, Glostrup, Denmark) for 30 min at RT. After treatment with the third antibody (HRP-

F(ab´)2 goat anti-rabbit (Zymed, SF, CA, USA) for 30 min at RT, the reaction was made visible by

an AEC (3-amino-9-ethyl carbazole) solution containing hydrogen peroxidase. Finally, the samples

were counterstained with hematoxyline and mounted. From the processed cardiac samples, the

amount of the CTGF positive label was scored from 0 to 4 by using a light microscope (IV). From

the renal samples, the whole cortical and medullary area was photographed and the relative amount

of primary antibody positive label per sample was determined with computerized densitometry

(Leica IM500 and Leica QWIN software; Leica Microsystems AG, Heerbrugg, Switzerland).

In situ detection of DNA fragmentation (Study II) was made by the TUNEL method according to

the procedures in Apoptosis Detection Kit (Apoptag Peroxidase in Situ Apoptosis Detection Kit,

Intergen Company, Oxford, UK). In brief, the cardiac and renal sections (5µm) were deparaffinized,

41

hydrated and treated with proteinase K. Endogenous peroxidase was quenched with 3% hydrogen

peroxide. Terminal deoxynucleotidyl transferase (TdT) enzyme was applied on the specimens and

incubated at +37°C for one hour. The sections were labeled with an anti-digoxigenin conjugate and

developed with a peroxidase substrate. Finally, the slides were counterstained with hematoxyline,

dehydrated and mounted. The degree of apoptosis was measured by counting the number of the

apoptotic cells (excluding the leukocytes) in 10 high-powered fields for each slide.

4.9 BIOCHEMICAL DETERMINATIONS

Urinary protein concentration (I) was measured by the Lowry method (Lowry et al., 1951).

Urinary albumin concentration (II-IV) was measured by ELISA, using rat albumin as a standard

(Celltrend GmbH, Luckenwalde, Germany).

Serum lactate dehydrogenase (s-LDH) activity (II) was determined by UV-kinetic analysis (Hitachi

912, United Laboratories, Helsinki, Finland).

Serum ionized magnesium [S-Mg2+] (IV) was measured with ion-selective electrode (NOVA CRT

8+ electrolyte analyzer (Nova Biomedical, Waltham, NJ, USA).

4.10 STATISTICAL ANALYSIS

Statistical analysis was carried out by one-way analysis of variance (ANOVA) supported by the

Tukey’s test (I) or Fisher’s Least-Significant-Difference test (LSD) (II-IV) for pairwise

comparisons. The two-way ANOVA for repeated measurements was applied for data consisting of

repeated observations. The correlation between the SBP and CTGF expression (I) was calculated

with Pearson’s correlation matrix followed by the two-tailed t-test. The level of statistical

significance was regarded as P<0.05. The results are expressed as mean ± S.E. mean. The data was

analyzed by using SYSTAT statistical software (SYSTAT Inc, Evanston, IL, USA).

4.11 ETHICS OF THE STUDY

The study-protocols were approved by the Animal Experimentation Committee of the Institute of

Biomedicine, University of Helsinki, Finland.

42

5 RESULTS

5.1 BRIEF SUMMARY OF THE MAIN RESULTS

Study I

The calcineurin inhibitor cyclosporine induced a marked hypertension, myocardial damage and

CTGF overexpression in the SHRs on high-sodium diet. The expression of the CTGF correlated

with blood pressure and was effectively prevented with an ACE inhibitor enalapril and an AT1

receptor antagonist valsartan.

Study II

Ang II induced marked inflammatory end organ damage and CTGF overexpression in the dTGRs.

Myocardial expression of the CTGF increased in a time- and blood pressure-dependent manner and

located in the myocardial structures affected by increased inflammatory response and pressure-load.

Treatment with an immunosuppressant cyclosporine and an AT1 receptor antagonist valsartan

protected against the Ang II-induced inflammatory response and CTGF overexpression thus

showing the importance of the Ang II and calcineurin signaling in the regulation of the CTGF gene.

Study III

Ang II induced severe inflammatory end organ damage, activation of the redox-sensitive

transcription factors (NF-κB, AP-1) and CTGF overexpression in the dTGRs. The Ang II-induced

infiltration of inflammatory cells, activation of inflammatory mediators NF-κB and AP-1 as well as

the overexpression of the CTGF was effectively prevented by suppression of the oxidative stress

with the antioxidant -lipoic acid.

Study IV

The Ang II-induced inflammatory end organ damage and CTGF overexpression in the dTGRs was

prevented with concomitant dietary magnesium supplementation. A calcineurin inhibitor tacrolimus

produced similar beneficial effects as cyclosporine in the Study II. Magnesium supplementation also

improved the therapeutic effects of the tacrolimus, which produced marked hypomagnesemia when

given as monotherapy.

43

5.2 BLOOD PRESSURE AND CARDIAC HYPERTROPHY

In the SHRs on high-sodium diet (I), CsA (5 mg/kg/d) produced a marked hypertensive effect

during the six-week treatment period. Concomitant enalapril and valsartan treatment antagonized

the hypertensive effect of the CsA. CsA tended to further promote LVH but the difference did not

reach statistical significance (P=0.098). Enalapril and valsartan protected against the development

of LVH.

During the three-week experimental periods, untreated dTGRs developed a marked hypertension

and cardiac hypertrophy compared to the age-matched normotensive SD rats (II-IV). Valsartan

treatment (II) normalized blood pressure and cardiac hypertrophy in the dTGRs. Calcineurin

inhibitors CsA (2 mg/kg/d) and tacrolimus (II, IV) produced a modest decrease in blood pressure

and cardiac hypertrophy. Dietary magnesium had a slight antihypertensive effect (IV) with a

decrease in the cardiac weight-to-body weight ratio. In contrast, combination treatment with

tacrolimus and magnesium (IV) normalized blood pressure and ameliorated cardiac hypertrophy.

Treatment with lipoic acid (III) had a mild antihypertensive effect without a significant reduction in

cardiac hypertrophy. Mammalian TOR inhibition by everolimus (II) had no effect on blood pressure

or cardiac hypertrophy in the dTGRs.

5.3 URINARY PROTEIN EXCRETION

Urinary protein (I) or albumin (II-IV) was measured as an index of the renal damage.

In the SHRs on high sodium diet, CsA caused a marked proteinuria which was 3.5-fold higher than

in the control group. In addition, CsA treatment resulted in an elevated level of serum creatinine and

a decline in a creatinine clearance. Antagonism of the RAS with enalapril or valsartan prevented the

increase in the urinary protein excretion. Nevertheless, despite the treatments, the urinary protein

excretion in the SHRs remained at a higher level than usually seen in their normotensive counter

parts, Wistar Kyoto rats.

The dTGRs developed a marked albuminuria, which was from 20 to 150-fold higher than in the SD

rats (II-IV). The AT1-receptor antagonist valsartan, as well as CsA, tacrolimus (alone or combined

with magnesium) and lipoic acid (II-IV) protected against the development of albuminuria

effectively, whereas everolimus (II) had a less marked effect. In contrast, magnesium treatment did

not have an effect on the urinary albumin excretion.

44

5.4 HISTOPATHOLOGY

5.4.1 Myocardial lesions

In the SHRs receiving high sodium diet (I), every ventricular sample had pathological alterations.

The majority of the epicardial and intramuscular arteries showed adventitial scarring, medial

thickening and smooth muscle cell vacuolization. The ventricular muscle had patches of thin

fibrotic scars and signs of recent infarcts. The lesions in the CsA (5 mg/kg/d) group were more

severe than in the control group. The epicardial and intramuscular arteries manifested marked

adventitial fibrosis, varying degrees of medial thickening and smooth muscle cell vacuolization.

Also the number and area of the observed myocardial scars exceeded those seen in the control

group. The cardiac damage index value did not differ significantly between the control and CsA

groups. In contrast, the cardiac damage index value was markedly lower with the higher dose of

valsartan when compared to the CsA group.

Treatment with the ACE inhibitor enalapril or the lower dose of the angiotensin AT1 receptor

antagonist valsartan prevented the development of the CsA-induced pathological alterations,

whereas the higher dose of valsartan seemed to be even more beneficial.

The hearts of the untreated dTGRs (II-IV) showed severe pathological alterations. The epicardial

and intramuscular arteries had perivascular inflammation, adventitial scarring, medial thickening

and sometimes fibrinoid necrosis. The intramuscular arteries showed marked medial/intimal

hyperplasia (concentric hypertrophy) leading to vessel occlusion. The ventricular muscle had

patches of fibrotic scars and signs of recent infarcts with inflammation. Cell proliferation as well as

apoptosis was increased in the hearts of the untreated dTGRs. Treatment with the AT1 receptor

antagonist valsartan (II) prevented the Ang II-induced morphological damage in the heart.

Calcineurin inhibition with CsA (2 mg/kg/d) (II) had no major effect on the cardiac morphology.

Although CsA protected against the Ang II-induced inflammation, cell proliferation, apoptosis and

myocardial infarcts, arteries were still notably affected. Interestingly, calcineurin inhibitor

tacrolimus (IV) had an evident cardioprotective effect. Lipoic acid (III) effectively prevented Ang

II-induced vascular lesions and myocardial infarcts. Treatment with magnesium alone (IV) had a

slight cardioprotective effect, whereas combination treatment with tacrolimus provided a very

45

effective inhibition against the Ang II-induced myocardial lesions. Interestingly, myocardial

damage tended to worsen by everolimus (II). Everolimus treatment caused numerous infarcts with a

marked accumulation of monocytes/macrophages. Intramuscular and epicardial arteries were

notably thickened and had areas of fibrinoid necrosis and perivascular inflammation.

5.4.2 Renal lesions

The description of the renal histopathology in the Study I has been previously published (Lassila et

al., 2000). In the SHRs on high sodium diet (I), there were minor pathological alterations involving

a small portion (4%) of the glomeruli. Neither tubular nor interstitial changes were detected. In

contrast, in the CsA group one-third of the glomeruli were apparently damaged. Within the affected

glomeruli, the pathological changes varied from a slight mesangial matrix expansion to a severe

necrosis and capillary collapse. Interstitial fibrosis of striped pattern was also found in the cortical

areas of the CsA-treated SHRs. Tubular atrophy and dilatation located in the tubuli surrounded by

fibrotic tissue. The large renal arteries in the CsA group were mostly normal, only a slight

thickening of the adventitia was found, whereas smaller arteries had medial changes ranging from a

minor smooth muscle cell vacuolization to a massive smooth muscle and endothelial cell

proliferation. All the CsA-induced pathological changes were completely prevented by blocking the

RAS with enalapril or valsartan (Lassila et al., 2000).

The kidneys of the untreated dTGRs (II-IV) had severe histopathological changes as indicated by

the increased renal damage index. The damage in the glomeruli ranged from a slight mesangial

expansion to a capillary collapse, glomerular sclerosis or necrosis. The intrarenal arteries had

varying degrees of medial/intimal hyperplasia and fibrinoid necrosis of the vessel wall. There was

marked perivascular and periglomerular inflammation characterized by an abundance of ED1, ED2,

CD4 and CD8 immunopositive cells. In addition, the redox-sensitive intracellular mediators of

inflammation, NF-κB and AP-1 were activated in the dTGRs (II). The tubules were affected to a

minor extent. Some tubuli were atrophied or dilated when surrounded by interstitial fibrotic tissue.

Part of the tubuli were necrotic or had peritubular fibrosis with inflammation. The Ang II-induced

cellular apoptosis located primarily in the epithelium of the proximal tubules. Valsartan, CsA,

tacrolimus and lipoic acid (II-IV) prevented the Ang II-induced renal damage efficiently, whereas

magnesium (IV) had lesser effect. The combination treatment with magnesium and tacrolimus

produced a very effective protection against the Ang II-induced renal damage. Although everolimus

partially prevented the development of albuminuria in the dTGRs (II), it still increased the amount

and severity of the renal damage in the dTGR. Lesions of the intrarenal arteries were similar but

46

more severe than in the dTGR group. Proximal tubules had necrosis and apoptosis. Patches of

interstitial fibrosis and ED1/ED2 positive inflammatory cells were seen throughout the sample.

However, everolimus partially attenuated the T cell-mediated inflammatory response, and cell

proliferation in the kidneys.

5.5 EXPRESSION OF CTGF

5.5.1 Myocardial expression of CTGF

In the SHRs (I), in situ hybridization showed a minor CTGF mRNA positive label in the epicardial

artery media where it was expressed by a small number of smooth muscle cells. There was a faint

CTGF expression in the ventricular fibrotic scars. CTGF mRNA expression was markedly

upregulated in the arterial media of the CsA group, where the positively labeled cells had the

phenotype of a smooth muscle. Equally to the control group, the CTGF positive label was detected

mostly in the vascular media, and in the extensive ventricular scars. Concomitant treatment with

enalapril and the lower dose of valsartan prevented the increment in CTGF mRNA expression,

whereas treatment with the higher dose of valsartan reduced CTGF expression by 75% compared to

the control group, and by 85% compared to the CsA-treated SHR on high sodium diet. When the

expression of the myocardial CTGF mRNA was compared between the 14-week-old hypertensive

SHR rats and the age-matched normotensive WKY rats on low-sodium diet, we noticed a strain-

specific induction of the CTGF mRNA in the SHRs.

In the untreated dTGRs (II-IV), as detected with in situ hybridization, immunohistochemistry and

Northern blot, CTGF mRNA and protein expression was markedly up-regulated especially in the

arterial media. A strong CTGF mRNA expression was also detected in the inflamed areas of the

vasculature and muscle. The fibrotic scars and infarcts of the ventricle had a strong CTGF mRNA

positive label in the outer boundaries (fresh infarct) or throughout the area (scar) of the lesion. In

the SD group, a faint CTGF mRNA expression was detected in the media of epicardial and

intramuscular arteries. In the ventricular muscle only a few positively labeled cells were noted.

Valsartan, CsA, tacrolimus, lipoic acid (0.047±0.018 vs. 0.2±0.049; P=0.002; n=6) and magnesium

(II-IV) prevented the Ang II-induced CTGF gene expression completely, whereas everolimus

treatment (II) had no effect on the CTGF expression.

47

5.5.2 Renal expression of CTGF

In the kidneys of the dTGRs (II), CTGF mRNA and peptide expression located mainly in the renal

corpuscles, arteries and in the interstitium. There was a marked up-regulation of the CTGF mRNA

expression in the media of the arcuate, interlobular and afferent arteries. Also, an enhanced

expression was detected in the interstitial and glomerular areas with inflammation, necrosis or

fibrosis. The SD group had a slight CTGF mRNA positive signal mainly in the same areas as in the

dTGR rats. However, there was no detectable signal in the tubules or interstitium, whereas a diffuse

signal was expressed in the vascular media. Treatment with valsartan and CsA protected effectively

against the Ang II-induced increase in CTGF mRNA. Treatment with everolimus (II) resulted in an

opposite effect. It further increased the expression of the renal CTGF mRNA.

5.5.3 Time- and blood pressure-dependent expression of CTGF

In the SHRs (I), we noticed a close correlation between the systolic blood pressure and myocardial

CTGF expression.

The expression of the myocardial CTGF mRNA was similar in the juvenile (3-4 week-old) dTGRs

and SD rats (II). Both in the heart and kidneys of the dTGRs, the temporal expression of the CTGF

mRNA revealed a strong up-regulation in the gene expression at the onset of hypertension between

the postnatal weeks 4 and 5.5. The difference in CTGF mRNA expression between the weeks 5.5

and 7 was less pronounced. At the age of 7 weeks, CTGF mRNA expression in the heart and

kidneys of the untreated dTGRs was 3.5 and 1.65 -fold higher as compared to the normotensive SD

rats, respectively.

To further elucidate the role of blood pressure as a regulator of the CTGF expression, also low-

pressure cardiovascular structures (right ventricle, pulmonary artery) were examined. The

expression of the CTGF mRNA in the right ventricle of the 7-week-old SD and dTGRs indicated a

noticeable but not significant increase in the amount of the CTGF mRNA in dTGR. In the

pulmonary arteries of the SD and dTGRs the CTGF mRNA positive signal was almost absent,

indicating no differences between these groups.

The main findings and effects of treatment with different compounds in corresponding animals are

summarized in Table 2.

Tab

le 2

. Mai

n fin

ding

s in

the

rats

and

effe

cts

of tr

eatm

ent w

ith d

iffer

ent c

ompo

unds

in c

orre

spon

ding

ani

mal

s (S

tudy

I−IV

).

Stu

dy

Str

ain

Tre

atm

ent

SB

P

Car

diac

hy

pert

roph

y M

yoca

rdia

l C

TG

F m

RN

A

I S

HR

I I I I S

HR

S

HR

S

HR

SH

R

HS

HS

+C

sA

HS

+C

sA+

Ena

30

HS

+C

sA+

Val

3

HS

+C

sA+

Val

30

−−

−

↑(↑

)

Car

diac

dam

age

− ↑↑ ↓

↓↓↓

↓↓

↓↓

↓↓

↓↓

↓ ↓↓

(↓)

↓ ↓↓

dT

GR

=

d

oub

le t

ran

sgen

ic r

at h

arb

orin

g hu

man

ren

in a

nd

angi

ote

nsi

no

gen

gen

es

SH

R

=

spo

nta

neo

usl

y h

yper

ten

sive

rat

CsA

=

cy

clo

spo

rine

(5m

g/kg

/d s

.c.

in S

tud

y I;

2m

g/kg

/d s

.c.

in S

tud

y II)

HS

=

h

igh

sod

ium

die

t (s

odiu

m 2

.6%

of t

he

cho

w d

ry w

eigh

t)

En

a 30

=

en

alap

ril 3

0m

g/kg

/d p

.o.

Val

3

=

vals

arta

n 3

mg/

kg/d

p.o

. V

al 3

0

=

vals

arta

n 3

0m

g/kg

/d p

.o.

Ren

al d

amag

e

− ↑↑

↓↓

↓↓

↓↓

Mg

=

Tac

ro

=

tacr

olim

us

1m

g/kg

/d s

.c.

-lip

oic

aci

d

=

-lip

oic

aci

d 3

50m

g/kg

/d p

.o.

SD

=

S

pra

gue

Daw

ley

rat

Eve

rolim

us

=

(SD

Z R

AD

) 2

.5m

g/kg

/d p

.o.

mag

nes

ium

(0

.6%

of t

he

cho

w d

ry w

eigh

t) a

aa

aa

bb

bb

b

bb

bb

b

bb

bb

b

SB

P

=

syst

olic

blo

od

pre

ssu

re

↑↑

II

dTG

R

Veh

icle

↓↓

↓↓

↓↓

↓↓

II S

D

Veh

icle

II

dTG

R

Val

30

↑↑

↑

↑

− c

ontr

ol; (

↑) s

light

ly in

crea

sed;

↑ in

cre

ased

; ↑↑

sub

stan

tially

incr

ease

d;

↔ n

o si

gnifi

cant

cha

nge

; (↓) s

light

ly d

ecr

eas

ed; ↓

decr

eas

ed; ↓

↓ s

ubst

antia

lly d

ecre

ased

; a

com

pare

d to

SH

R+

HS

(S

tud

y I)

; b

com

pare

d to

SH

R+

HS

+C

sA (

I); c co

mpa

red

to S

D

rats

(II-

IV);

d c

ompa

red

to u

ntre

ated

dT

GR

s (I

I-IV

).

II ↓

↓

↓

↓(↓

) dT

GR

C

sA

II ↓

↔↓

↑

↑dT

GR

E

vero

limus

III

↓

↓

↓↓

dTG

R

-Lip

oic

aci

d

IV

↓

↔

↓

dTG

R

Mg

IV

↓↓

↓

↓↓

dTG

R

Tac

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48

6 DISCUSSION

The hypertension-induced end organ damage predisposes the hypertensive patients to numerous

cardiovascular complications leading to premature disability and mortality. The deterioration of the

cardiovascular and renal function results partly from the pathologic inflammatory reaction and

connective tissue accumulation occurring also by blood pressure-independent mechanisms.

Although the research has today focused to explore the role of locally acting growth factors and

mediators of connective tissue formation, the exact mechanisms contributing to the hypertension–

induced inflammatory end organ damage remain still more or less obscure.

This study investigated the role of the CTGF in the pathogenesis of hypertension-induced end organ

damage in two hypertensive animal models with increased RAS activity: CsA-treated SHRs on high

sodium diet and dTGRs. As regulators of the CTGF expression, the participation of the increased

blood pressure and Ang II were studied.

6.1 METHODOLOGICAL ASPECTS

6.1.1 Increased RAS activity in SHR and dTGR

The SHR is the most widely used animal model for human hypertension. The SHR is an inbred

strain which develops hypertension and its complications with increasing age. Although criticized

as being an inappropriate model for human hypertension, the arterial hypertension in the SHR and

essential human hypertension share many similarities in the pathogenic mechanisms (Trippodo and

Frohlich, 1981). To examine the pathogenesis of the CsA-induced hypertension and end organ

damage, we have developed an animal model for CsA toxicity, i.e. the CsA-treated SHR on high

sodium diet (Mervaala et al., 1997). This animal model expresses increased RAS activity and

develops hypertension and renal lesions similar to CsA-treated humans (Mervaala et al., 1997;

Lassila et al., 2000). Importantly, the development of hypertension and nephrotoxicity is achieved

by using an adequate drug dosage (CsA 5mg/kg/d) producing therapeutically high, but relevant

blood concentration (750 ng/ml) (Mervaala et al., 1997).

Previously, majority of the experimental studies focusing on the pathogenesis of CsA toxicity were

conducted in normotensive rats receiving either normal chow with sodium concentration

approximately 0.25-0.3 % w/w, or sodium-depleted diet (Burdmann et al., 1995; Shihab et al.,

1999). In these animal models a fairly high CsA dosage (usually 15 mg/kg/d producing

49

50

blood CsA concentrations of 5000-6000 ng/ml) causes renal damage similar to that found in the

CsA-treated patients (Shihab et al., 1999). However, even though CsA induces pronounced renal

damage in these animal models, the rats usually remain normotensive. Therefore these animal

models are not very suitable for studying the mechanisms of CsA-induced hypertension.

In experimental rat models CsA increases PRA regardless of the sodium content of the diet

(Burdmann et al., 1995; Mervaala et al., 1997). Although the administration of CsA in humans is

usually associated with volume-expanded, low renin form of hypertension, CsA has also been

reported to elevate PRA in cardiac and liver transplant patients and plasma Ang II levels in patients

with psoriasis (Julien et al., 1993; Edwards et al., 1994). Since the clinical CsA treatment in

humans is partly limited by the drug-induced hypertension and nephrotoxicity, and the CsA-induced

hypertension has been shown to depend on the level of dietary salt (Curtis et al., 1988; Textor et al.,

1994), we believe that our animal model provides valuable information when studying the CsA-

induced hypertension and its associated complications.

The dTGR was originally developed for studying human renin inhibitors which ordinarily do not

function in rats. The human components of RAS in the dTGR do not interact with the endogenous

rat RAS (Ganten et al., 1992; Bohlender et al., 1997). Hypertension, vascular inflammation and end

organ damage in the dTGR are due to increased Ang II synthesis by the human components of the

RAS. Ang II concentrations in the circulation and in the tissues are about 3 to 5-fold higher in the

dTGR compared to non-transgenic SD controls (Mervaala et al., 2000b). Therefore, the dTGR can

be considered as an appropriate model for studying the Ang II-induced pathologic effects on blood

pressure and end organ structure/function.

6.1.2 Blood pressure measurements

In the present study, blood pressure was measured indirectly by the tail-cuff method. When a study

design requires monitoring of a large number of animals, the non-invasive tail-cuff method is

commonly chosen. The advantage of this method is its relative simplicity and low cost compared to

e.g. the radiotelemetric method. Tail-cuff recording correlates well with direct blood pressure

recordings where intra-arterial catheters are used (Bunag and Butterfield, 1982). However, tail-cuff

recording may overestimate the real blood pressure levels possibly affected by ambient temperature,

restrainers, smells, noise, and the tail-cuff itself. To minimize the stress-inducing stimuli, the blood

pressure measurements in the present study were performed on pretrained rats by the same person

in a quiet and peaceful environment.

51

6.2 EFFECT OF BLOOD PRESSURE ON CTGF EXPRESSION

In the present study, we noticed a close correlation between the systolic blood pressure and

myocardial CTGF expression in the SHRs. The CsA-treatment (5 mg/kg/d) produced a malignant

hypertension with marked end organ damage without an inflammatory component. In addition to

elevated mechanical strain, the induction of the CTGF in CsA-treated SHRs could have also been a

consequence from other, yet unresolved events, like hypoxia or production of the ROS (Nishiyama

et al., 2003).

Next, the effect of blood pressure on the CTGF mRNA expression was examined more closely by

using the dTGRs. Although the expression of the myocardial CTGF mRNA was similar in 3-4

week-old SD and dTGR rats, a marked up-regulation of the myocardial and renal CTGF gene

expression in the dTGRs occurred between the postnatal weeks 4 and 5.5, and to a lesser extent

between the weeks 5.5 and 7. We noticed that the up-regulation of the CTGF gene occurred at the

onset of Ang II-induced hypertension before an overt myocardial and renal damage was observed.

For a further evaluation of the blood pressure-dependent expression of the CTGF, cardiac structures

not affected by the systolic blood pressure (right ventricle, pulmonary artery) were examined. The

CTGF mRNA level in the right ventricle of the 7-week old dTGRs was noticeably but not

significantly higher than in the SD rats.

Furthermore, when we examined the expression of the CTGF mRNA in the pulmonary arteries of

the dTGRs and SDs, there was no notable difference between these two rat strains. The CTGF

mRNA positive signal was almost completely absent from all the samples studied, which is in

contrast to the marked difference we have noted in the CTGF mRNA expression between the

myocardial arteries in the dTGR and SD.

Although it is difficult to separate the effects of hemodynamic and humoral factors in an in vivo

model, there are several reports which propose increased mechanical stress as an inducer of the

CTGF and connective tissue synthesis. In vitro studies have revealed that static and cyclic stress

enhances the expression of the CTGF (Riser et al., 2000; Hishikawa et al., 2001). The pathways by

which the induction of the CTGF is regulated in the conditions above remain obscure. The recently

discovered stretch-responsive element in the CTGF promoter region might be involved in the

regulation of the CTGF under mechanical stimuli. Previously, a mechanical strain-responsive

element has been found in the rat collagen I gene and the induction of collagen III gene has been

noted in association of cyclic mechanical stretch (Carver et al., 1991; Lindahl et al., 2002).

Furthermore, the studies by van Wamel et al. (2002) have shown that TGFβ1 mRNA expression in

stationary cardiomyocytes is increased by the release of Ang II from stretched cardiomyocytes.

52

6.3 EFFECT OF ANG II AND INFLAMMATORY RESPONSE ON CTGF EXPRESSION

Wound heals through inflammation and connective tissue formation. An exception can be seen in

the early phases of embryonic development when there is no inflammatory response to an injuring

stimulus. The healing occurs by the proliferation of the cells surrounding the lesion and no fibrosis

occurs. Interestingly, in the early embryonic stage CTGF participates in the organogenesis, and

even injected CTGF mRNA does not promote fibrosis, but instead it results in an abnormal

differentiation of embryonic structures (Abreu et al., 2002).

In several studies, the up-regulation of the CTGF expression has been noted in conjunction with

inflammatory lesions. Induction of the CTGF has been shown in the inflammatory diseases of

kidneys, lungs, liver, pancreas, skin, cardiovascular system, and colon (Table 1). Besides having an

inflammatory component, characteristic of these diseases is also an abnormal homeostasis of

connective tissue. The difficulty in auto-immune diseases is the prolonged state of inflammation

and activity of fibrosis-promoting growth factors, which does not seem to serve any physiological

function. This “inflammation-wound healing” occurs without previous traumatic episode and reacts

to the organisms own molecules as hostile invaders.

The participation of the RAS and Ang II in the processes of inflammation-connective tissue

formation has been clearly demonstrated. Besides acting as a circulating humoral system, RAS

operates also locally. The local RAS is particularly active in atherosclerotic arteries in which

increased expression of the ACE and AT1 receptor can be seen in the inflammatory cells of

atherosclerotic plaques (Fukuhara et al., 2000; Gross et al., 2002). In numerous experimental

studies, it has been noted that hindering the activity of RAS, either with ACE inhibitors or AT1

receptor antagonists, protects against the Ang II-induced NF-κB-mediated neointimal inflammation

in atherosclerosis, NF-κB-mediated inflammation of the renal cortex, inflammatory experimental

glomerulonephritis and diabetic nephropathy (Morrissey and Klahr, 1997; Hernandez-Presa et al.,

1998; Ruiz-Ortega et al., 1998; for review, see Schiffrin, 2002b).

Hypertension, vascular inflammation and end organ damage in the dTGR are due to increased Ang

II synthesis by the human components of the RAS (Mervaala et al., 2000b). In the dTGRs, marked

inflammatory cell recruitment, mainly consisting of monocytes and macrophages, as well as the up-

regulation of the adhesion molecules VCAM-1, ICAM-1, and the extracellular matrix component

fibronectin, are already present immediately after weaning (Mervaala et al., 1999; Müller et al.,

2000b). The interpretation made from these findings is that Ang II triggers inflammatory response

53

and exerts direct pro-inflammatory effects even by blood pressure-independent mechanisms related

to the activation of the redox-sensitive transcription factors NF-κB and AP-1, as well as oxidative

stress.

In the present study, we showed that in the dTGRs, the expression of the myocardial CTGF mRNA

was markedly up-regulated in the vascular media and in the inflamed and infarcted areas. Induction

of the renal CTGF mRNA was located mainly in the medial layer of the arteries, in the interstitium

and in the necrotic glomeruli. Both in the heart and kidneys, the expression of the CTGF co-located

very closely with infiltrated leukocytes suggesting an important role for inflammatory response in

the CTGF gene regulation. Furthermore, we noticed that the induction of the CTGF in the dTGRs

was associated with the concomitant activation of inflammatory mediators NF-κB and AP-1. In

contrast, in the CsA-treated (5 mg/kg/d) SHRs on high sodium diet, we did not notice any signs of

myocardial inflammation. Despite the lack of the visible inflammation in this model, there were

numerous scars in the myocardium indicating previous phases of reparative fibrosis and presence of

leukocytic cells. In addition, the contribution of the NF-κB and AP-1 in regulating the gene

expression of the CTGF in the CsA-treated SHR can be proposed on the basis of the recent findings

by Asai et al. (2003), who showed that chronic CsA-treatment in rats promotes the activity of the

AP-1 and NF-κB. The 50-fold increment in the observed plasma renin activity of the CsA group

compared to the group receiving vehicle, proposes the involvement of the RAS in regulating the

expression of the CTGF. It has been previously reported that CsA stimulates RAS and up-regulates

Ang II receptors in human vascular smooth muscle cells (Avdonin et al., 1999; for review, see

Lassila, 2002). Furthermore, high sodium diet has also been shown to promote cardiac ACE activity

in rats (Kreutz et al., 1995). The role of the RAS in regulating CTGF can be further proposed by the

presence of the NF-κB, AP-1 and TGFβ1 regulatory elements in the CTGF gene promoter region.

Besides stimulating the activity of the NF-κB and AP-1, Ang II induces fibrosis in the heart and

kidneys through TGFβ1 (Everett et al., 1994; Dostal, 2001). In the dTGRs, by using in situ

hybridization CTGF mRNA was noticed to co-locate very closely with TGFβ1 mRNA. This

suggests also the involment of the TGFβ1 as a regulator of the CTGF in this model. However, the

induction of the TGFβ1 occurred only in the kidneys of the dTGRs. Therefore, further studies would

be required to determine the role of the TGFβ1 as a regulator of the CTGF gene expression in this

model.

54

In the present study, the regulation of the CTGF expression seemed to involve the following Ang II

dependent pathways:

1. Ang II signaling through the AT1 receptor appeared to be involved as the expression of the CTGF

was markedly decreased by valsartan in the dTGRs and CsA-treated SHRs. In the SHRs, treatment

with valsartan resulted in a lower CTGF expression than could have been presumed from its

antihypertensive effect. This supports the findings by Candido et al. (2002) who demonstrated a

marked vascular CTGF overexpression and ACE activity in diabetic apolipoprotein-E knock-out

mice. ACE inhibitor therapy inhibited the development of atherosclerotic lesions and diabetes-

induced ACE and CTGF overexpressions in the aorta.

2. In the dTGRs, ROS seemed to be involved in the regulation of the CTGF gene, as the treatment

with antioxidant lipoic acid hindered the activity of the redox-sensitive NF-κB and AP-1, and

prevented the induction of the CTGF gene. Ang II stimulates NADPH oxidase to produce ROS,

which participate in the regulation of the VSMC growth, induction of vascular inflammatory

response, impairment of endothelial-dependent vascular relaxation, pathogenesis of cardiac

hypertrophy and stimulation of the CTGF expression (Griendling et al., 2000; Park et al., 2001).

3. In the present study, the Ang II-stimulated activation of the T cell calcineurin phosphatase

seemed to be involved in the Ang II-induced expression of the CTGF in dTGRs, as the blockade of

the calcineurin activity with CsA or tacrolimus protected against the inflammatory response and up-

regulation of the CTGF mRNA. Previously, it has been shown that human T cells synthesize CTGF

and Ang II promotes the proliferation and function of T cells via calcineurin dependent pathway

(Nataraj et al., 1999; Workalemahu et al., 2003). Nataraj et al. (1999) also found that calcineurin

inhibition completely blocked the ability of the Ang II to induce proliferation of the cultured splenic

lymphocytes, demonstrating that calcineurin is required for the stimulation of the lymphocyte

proliferation by AT1 receptors. Increased activity of the calcineurin has been connected to other

adverse events e.g. cardiac hypertrophy, heart failure, overexpression of the TGFβ1, activation of

the NF-κB, and organ transplant rejection (Molkentin et al., 1998; Molkentin and Dorn, 2001). In

our previous studies it was noted that, in dTGRs, Ang II induces inflammation even by blood

pressure-independent mechanisms and the inflammation with the subsequent end organ damage can

be prevented with calcineurin inhibition. (Mervaala et al., 2000a; Mervaala et al., 2000b).

55

4. The redox-sensitive mediators of inflammation, NF-κB and AP-1 probably mediated the final,

DNA-binding step in the CsA-treated SHRs and in the dTGRs. This could be proposed by the

presence of the NF-κB and AP-1-binding elements in the CTGF gene promoter region. To support

this speculation, a marked activation of the NF-κB and AP-1 was noticed in dTGRs. Treatment with

antioxidant lipoic acid resulted in a diminished activity of the NF-κB, AP-1, and suppression of the

CTGF gene expression. Furthermore, in the dTGRs, magnesium supplementation, which has been

shown to diminish the activation of the AP-1 and NF-κB, protected against the induction of the

CTGF gene (Asai et al., 2003). Although controversial data exist, NF-κB and AP-1 are today

considered as positive regulators of the CTGF expression (Blom et al., 2002).

6.4 POSSIBLE CLINICAL IMPLICATIONS

Currently, ACE inhibitors, AT1 receptor antagonists, and statins are widely used to prevent the

cardiovascular end organ damage in arterial hypertension. Besides having anti-hypertensive or

LDL-lowering effects, these drugs have been shown to diminish the accumulation of adverse

connective/scar tissue in the heart, kidney and vasculature. Despite the various beneficial effects of

the aforementioned drugs, the antifibrotic therapy could also be targeted on a single molecule.

Previous expectations focused on TGFβ1, which has a central role in connective tissue synthesis.

Unexpectedly, studies with anti-TGFβ1 antibody and TGFβ1-knockout mice have revealed the

importance of the TGFβ1 as a controller of growth and inflammation. In numerous experimental

studies, inhibition of the TGFβ1-signaling has resulted in hyperinflammation, atherosclerosis, and

malignancies (Murphy et al., 1999; Mallat et al., 2001; Engle et al., 2002). CTGF is induced by

various components of injuring conditions (high glucose, mechanical strain and inflammation) and

the presence of the CTGF in tissue samples and body fluids has been shown to correlate with the

degree of diabetic nephropathy and other fibrotic processes (Tamatani et al., 1998; Sato et al., 2001;

Riser et al., 2003). Whether CTGF mediates the immunomodulatory or anti-carcinogenic effects of

the TGFβ1 is not yet known. Acting as a downstream mediator of the TGFβ1-induced collagen

production, CTGF may provide a more selective drug target in anti-fibrotic therapy. The blockade

of the CTGF may thus avert the possible dangers associated with the antagonism of the TGFβ1.

On the basis of recent investigations CTGF has a potential of serving as a suitable drug target or an

early marker of various inflammatory/fibrotic disease states and a reliable indicator of response to

therapy.

56

7 SUMMARY AND CONCLUSIONS

This study investigated the expression of the CTGF in the pathogenesis of the hypertension-induced

end organ damage by using two hypertensive animal models with increased RAS activity: the CsA-

treated SHR on high sodium diet and the dTGR.

The main findings and conclusions are as follows:

• Arterial hypertension induced myocardial CTGF mRNA expression.

• In the dTGRs, Ang II induced CTGF expression in a time- and blood pressure-dependent

manner primarily via AT1 receptor stimulation. Ang II-induced CTGF overexpression was

linked to increased oxidative stress and inflammatory response of the vascular wall.

• Blockade of the RAS activity either with the ACE inhibitor or the AT1 receptor antagonist

prevented the overexpression of the CTGF gene.

• Calcineurin inhibitors CsA and tacrolimus as well as the antioxidant lipoic acid effectively

protected against the induction of the CTGF gene in the dTGR.

• Magnesium supplementation reduced the Ang II-induced myocardial damage and CTGF

overexpression. Additive therapeutic effects were found withmagnesium supplementation

andcalcineurin inhibitor.

57

ACKNOWLEDGEMENTS

This study was carried out at the Institute of Biomedicine, Pharmacology, of the University of

Helsinki during 1999-2003.

First, I wish to express my warm gratitude to my supervisor, Docent Eero Mervaala. His humoristic

and “workaholic” attitude kept me busy during these years. Working with him gave me loads of

knowledge, research funds ;-) and hopefully, some professional attitude in the field of

cardiovascular research.

My warm thanks also to my other supervisor, Professor Leena Lindgren, whose benevolently fierce

character is matched only by her professional skills.

I owe my respectful thanks to Professor Heikki Vapaatalo, the former head of the Department of

Pharmacology. He is a professor of the “good-old times” whose ideas and knowledge are of the

highest value.

My heartfelt thanks go to late Professor Juhani Ahonen, the father of this study. The kindness,

friendship and ideas of this “fly-fisherman” were unique. I would like to dedicate this work to him.

Professor Eero Saksela and Docent Ilkka Pörsti, the reviewers appointed by the Medical Faculty of

the University of Helsinki, are gratefully acknowledged for their valuable criticism and comments.

I would also like to thank Professors Esa Korpi, Heikki Karppanen, Ilari Paakkari and Ismo

Virtanen for the various valuable conversations we have had during these years.

I would like to express my sincere gratitude to Professor Krister Höckerstedt for providing the

laboratory at the Surgical Hospital at my disposal.

I am grateful to Docent Marja-Leena Nurminen, who kindly welcomed me in the cardiovascular

research group. She gave me (in her own peculiar way) the principles of the cardiovascular

pharmacology and research work. Our group had pleasant evenings with good wine and food

provided by Marja-Leena, and her husband Manu.

58

Respectful acknowledgements go to Marika Collin, whose coordination skills in various

assessments provided the needed feasible experimental methods. I am still laughing when I am

thinking our “smartass”-theories about everything.

I am grateful to my friend Markus Lassila, for his cheerful attitude in everyday life. Our research

work shared many topics which was beneficial for both of us. Thanks also go to Riikka Nevala and

Timo Vaskonen. We shared many delightful moments in the work and leisure time.

Cheerful gratitude goes to Saara Merasto and Marjut Louhelainen who provided the much needed

assistance during this research work. Their companionship and attitude gave me many laughs (and gray

hair).

I would also like to thank my friends and colleagues Kaija Inkinen, Docent Riitta Korpela, Docent

Irmeli Lautenschlager, Docent Ilkka Tikkanen, Docent Pekka Rauhala, Zhong Jian Cheng, Antti

Ohvanainen, Juha Ketonen, Anna-Stina Lax, Ilkka Reenilä, Petri “Pirtu” Vainio, Anna-Kaisa Pere,

Antti Väänänen, Teemu Helkamaa, Timi Martelius, Anneli von Behr, Toini Siiskonen, Sari

Laakkonen, Tarja Markov, Stephen Venn, Jesus Santisteban, Erkka Solatunturi, Eeva Harju,

Kristiina Haasio, Tuula Lähteenmäki and others.

I was fortunate to work with highly-skilled collaborators. My sincere thanks go to Professors

Friedrich Luft and Detlev Ganten; Docents Dominik Müller, Leena Krogerus and Risto Lapatto.

I would like to thank the following people: R.H. Böeckel, Andrew Hilary, Brendan Mulachy, Harri

Mauriainen, Henri Pulla, Marusha, Raymond Bourque and others for the various activities,

happenings and leisure time.

I am grateful to my relatives for their mental, professional (and financial) support and company I

received during these years.

My warmest thanks to my chequered, 18-year-old, four-footed, always demanding friend, Enni.

Thanks to Matilda “Mato Matala”, for keeping me up on my toes (and giving me more gray hair).

59

Finally, I owe my special gratitude to my very special soul mate Maija for giving me the necessary

kicks in everyday life. Without her, I think my greatest accomplishments would have been in the

areas of barbecue or kitty-litter-cleaning business.

This work was financially supported by the Academy of Finland, the Sigrid Jusélius Foundation,

the Kidney Foundation, the Research and Science Foundation of Farmos, and the Aarne Koskelo

Foundation.

Helsinki, October 2003

Piet Finckenberg

# 9

60

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