Matrix Metalloproteinase-7 Degradation of Fetuin Blocks Fetuin-Mediated Inhibition of Mineralization

by

Reyhaneh Rezaei

A thesis submitted in conformity with the requirements for the degree of Master of Science

Discipline of Periodontology, Faculty of Dentistry University of Toronto

© Copyright by Reyhaneh Rezaei 2013

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Matrix Metalloproteinase-7 Degradation of Fetuin Blocks Fetuin-Mediated Inhibition of Mineralization

Reyhaneh Rezaei

Master of Science

Discipline of Periodontology, Faculty of Dentistry University of Toronto

2013 Abstract

Mechanisms underlying the association between atherosclerosis and periodontitis are not

defined. Matrix metalloproteinases (MMPs) are increased in untreated periodontitis.

Fetuin, a serum protein, inhibits vascular mineralization in humans. I hypothesized that

MMP-7 affects fetuin function. In vitro mineralization was conducted based on methods

developed by Hunter and colleagues. Mineralization was quantified using absorbance

measurements (at 540 nm) of alizarin red bound to nascent crystals. Mineralization assays

included intact MMP-3 and MMP-7-treated human and bovine fetuin (0-2 µM).The

inhibition of mineralization mediated by fetuin (2 µM) was maximal at 3.5-4 hours.

Transmission electron microscopy and electron diffraction analysis of crystals formed

revealed the presence of hydroxyapatite. SDS-PAGE demonstrated that MMP-7 is more

effective in digesting fetuin than MMP-3. The inhibitory effect of fetuin on

mineralization was more reduced by MMP-7 digestion compared to MMP-3. Fetuin

inhibits mineralization in vitro and this effect is reduced by MMP-7 degradation.

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Dedication

I would like to dedicate my thesis to my beloved husband, Alireza.

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Acknowledgements

I thank Christopher McCulloch and Howard Tenenbaum for their guidance in the conduct

of my master’s research project. I thank Craig Simmons who was an invaluable member

of my advisory committee and who provided useful input. I thank Doug Holmyard (Mt.

Sinai Hospital, Dept. of Pathology, Toronto, ON) for advice and help with the assessment

of mineralization by electron microscopy and by electron diffraction analysis. I am

grateful to Graeme Hunter and Yinyin Liao (University of Western Ontario) for

providing me with their protocol for assessing hydroxyapatite growth in vitro. I thank

Kerry D'Costa, Wilson Lee, Carol Laschinger and Ryan Schure who contributed to the

various figures in this thesis. The research was supported financially by grants from the

Canadian Institutes of Health Research (MOP36332) and the Ontario Heart and Stroke

Foundation (T7085) to CAM who is also supported by a Canada Research Chair (Tier 1).

The authors report no commercial relationships to the funding sources for this study or no

known conflicts of interest related to this study.

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Table of Contents

Abstract……………………………………………………………….……………….…………………..ii

Dedication………………………………………………………………………………..iii

Acknowledgments.……………………………………………………………………....……………..iv

Table of Contents…………………………………………………...…………………………………..v

List of Tables and Figures…………………………………………………………………………..vii

List of Abbreviations………………………………………………………………….……………..viii

Literature Review……………………………………………...………………………………………..1

1.Periodontal Diseases…………………………….………………………………………………...…1

1.1. Definition and Classification……………………………………………………………1

1.2.Etiology…………………………………………..…………………………………………..1

1.3.Pathogenesis of Periodontitis……………………………………..……………………..3

1.4.Enzymes Present in gingival crevicular fluid……………………………...…………4

1.5.Matrix Metalloproteinases…………..……………………………………………………6

2.Cardiovascular Diseases……………………………………………………………………………8

2.1.Definition and Classification………………………………………….…………………8

2.2.Blood Vessels……………………………………………………………….………………9

2.3.Atherosclerosis……………………………………………………………………………10

2.4.Atherosclerosis and Periodontitis……………………………………………………..12

2.5.Pathogenesis of Atherosclerosis……………………………………………………….15

2.6.Mechanism of Vascular Calcification………………………………………………..16

2.7.Regulation of Vascular Calcification/Ossification…………………………….…..16

3.Fetuin/AHSG……………………………….………………………………………………………...18

3.1.Discovery and Measurement in Blood Proteins……………………………………18

3.2 Structure of Human AHSG/Bovine Fetuin………………………………………….19

3.3.Biological Activities……………………………………………………………………..19

3.4.Degradation of Fetuin……………………………………………………………………21

4.Study of Mineralization In vitro……………………………….……………………………….22

4.1.In vitro Mineralization Assays………………………………………………………...22

4.2.Assessments of Mineralization In vitro……………………………………………...23

5.Statement of the Problem……………………………….………………………………………..26

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Objectives……………………………….……………………………………………………………….26

Hypothesis……………………………….……………………………………………………………...26

6.Material and Methods……………………………….……………………………………………27

6.1. Cell-free In vitro Mineralization……………………………………………….….…27

6.2.Fetuin-mediated Inhibition of Mineralization In vitro……………….……….…..27

6.3.Crystal Species Formed in Mineralization Assays………………………………...28

6.4.Effect of MMP Cleavage of Fetuin on its Ability to Inhibit Mineralization....29

6.5.Statistical Analyses……………………………………………………………………....29

7.Results……………………………….…………………………………………………………………30

7.1.Cell Free In vitro Mineralization……………………………….……………………..30

7.2. Fetuin Inhibition of Mineralization In vitro………………………………………..30

7.3. Crystal Species Formed in Mineralization Assays…………………………....…..31

7.4.Effect of MMP Cleavage of Fetuin Inhibition of Mineralization………………31

8.Discussion……………………………….…………………………………………………………….33

Conclusions……………………………….…………………………………………………………….36

Tables and Figures……………………………….…………………………………………………...37

Copyright Acknowledgments……………………………….……………………………………..52

Bibliography……………………………….…………………………………………………………...53

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List of Tables and Figures

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TABLE 1: Definition of sensitivity, specificity, relative risk, biomarker

FIGURE 1: MMP domain structure

FIGURE 2: Human AHSG domain structure

FIGURE 3: Fetuin interaction with BCP

FIGURE 4A: Bragg’s formula

FIGURE 5A: Alizarin red staining of minerals and cetylpyridinum extraction for

absorbance measurement

FIGURE 5B: Mineralization time course over 4 hours

FIGURE 6A: Mineralization time course over 4 hours with fetuin at 2µM

FIGURE 6B: Mineralization assessment at 3.5 hours in the presence of fetuin at various

concentrations

FIGURE 7A: TEM analysis of crystal species in the presence and absence of fetuin

FIGURE 7B: Electron diffraction analysis of crystal species

FIGURE 8A: SDS-PAGE analysis of bovine fetuin in the presence of MMP-7 and

MMP-3

FIGURE 8B: SDS-PAGE analysis of human AHSG in the presence of MMP-7 and

MMP-3

FIGURE 8C: Mineralization assessment in the presence of intact and MMP-7 and

MMP-3 treated bovine fetuin

FIGURE 8D: Mineralization assessment in the presence of intact and MMP-7 and

MMP-3 treated human AHSG

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List of Abbreviations

AHSG Alpha-2-Heremans Schmid glycoprotein

AP Alkaline phosphatase

AST Aspartate aminotransferase

BMP Bone morphogenic protein

Cbfa Core-binding factor subunit alpha

CCDC Cambridge Crystallographic Data Centre

CRP C-reactive protein

CVD Cardiovascular disease

ECM Extracellular matrix

EDA Electron diffraction analysis

ESRD End-stage renal disease

GCF Gingival crevicular fluid

HA Hydroxyapatite

ICSD Inorganic Crystal-Structure Database

IL Interleukin

LDL Low-density lipoprotein

M-CSF Macrophage colony-stimulating factor

MCP Monocyte chemoattractant protein

MMP Matrix metalloproteinase

MT-MMP Membrane-type matrix metalloproteinase

RANKL Receptor Activator of Nuclear Factor

Runx Runt-related transcription factor 2

TEM Transmission electron microscopy

TIMP Tissue inhibitor of metalloproteinases

TGF Transforming growth factor

TNF Tumour necrosis factor

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VCAM Vascular cell adhesion molecule

VSMC Vascular smooth muscle cell

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Literature Review

1. Periodontal Diseases

1.1. Definition and Classification

Periodontitis is a destructive disease of the periodontium, which comprises the supporting

structure of the teeth including the alveolar process, periodontal ligament and root

cementum of the teeth. Periodontal diseases have been classified extensively; one widely

used classification emerged from the 1999 International Workshop for Classification of

Periodontal Diseases1. The most commonly employed diagnostic tools for assessment of

periodontitis are the clinical examination and radiographic assessment, which together are

used for evaluating the presence and the severity of periodontal diseases1.

Periodontitis, which is one of the periodontal diseases considered in the 1999

workshop, affects 10-15% of humans in a severe form2. The prevalence of moderate

periodontitis is greater than severe periodontitis, however a wide range of prevalence of

periodontitis has been reported due in part to the lack of consistent applications of

rigorous definitions and clinical measurements of these diseases3.

1.2. Etiology

Periodontitis is an infectious disease driven by tooth-born, bacterial biofilms, which

initiates cascades of destructive, inflammatory processes within the periodontium4.

Destruction of the periodontium arises as a consequence of interactions of the destructive

actions of periodontopathogenic bacteria and host immune responses. Currently, there is

great interest in the discovery of the biological impact of risk factors such as smoking and

diabetes that are associated with more severe periodontitis.

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The microbial populations that are associated with periodontal destruction4 have

been investigated using a large number of methods including DNA probe hybridization

and checkerboard analyses5. Five major complexes of microbial population in the oral

cavity have been identified among which Bacteroides forsythus, Porphyromonas

gingivalis and Treponema denticola have been strongly associated with progressive

periodontal destruction and bleeding on probing5. Control of these virulent organisms is

one of the primary goals of periodontal therapy4. According to Löe and co-workers,

clinical gingivitis can be treated with effective control of microbial biofilms 6. Lindhe and

co-workers suggest that control of microbial biofilms is effective in clinical management

of periodontal diseases 7.

As alluded to above, the host immune response to biofilms and their byproducts

contributes to periodontal destruction. The host response is manifested by an increase in

serum antibody to causative organisms 8,9 and is responsible for mediating host protective

systems. These protective responses prevent the apical spread of biofilms down the root

surface and into the underlying connective tissues, but at the same time activate a wide

variety of immune-mediated processes that lead to destruction of bone, cementum and the

soft connective tissues of the periodontium. Further, periodontitis is thought to be a multi-

factorial disease; risk factors such as uncontrolled systemic diseases (e.g. diabetes),

smoking and genetic predisposition can contribute to the progression of periodontal

destruction 10-12.

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1.3. Pathogenesis of Periodontitis !

Periodontitis, as introduced above, results from interactions between microbial pathogens

and immune regulators expressed by the susceptible host 13. Important insights that

inform our current understanding of the pathogenesis of periodontitis have arisen from

several different types of investigations. These include epidemiological studies that have

focused on the influence of risk factors in modulating the host response, and from twin

and cohort studies that have sought to identify the role of genetically-determined and

etiological factors 14,15.

Periodontitis is considered to be a chronic inflammatory condition; the disease-

induced changes at the cellular level are, in large part, a reflection of host responses to

bacterial metabolites and possibly bacterial invasion16. Periodontal lesions are

accompanied by increased vascularity, capillary vasodilation, capillary permeability and

gingival crevicular fluid (GCF) flow 17-19. Increased capillary permeability may facilitate

the migration of inflammatory cells into marginal periodontal lesions but may also lead to

increased release of inflammatory and anti-inflammatory cytokines by immune cells into

local tissues. Alternatively, the capacity for bacteria from adherent biofilms to enter the

systemic circulation and lymphatic drainage system is also enhanced by increased

vascular permeability 20. As a result of the flow of inflammatory interstitial fluid through

the gingival pocket, a large number of inflammatory markers and enzymes have been

identified in GCF. Indeed, GCF has been explored as a diagnostic tool to help

complement traditional clinical assessments of periodontal diseases including

measurements of probing depth, bleeding on probing, assessment of biofilms, loss of

attachment and radiographic assessment showing loss of bone 1,21-23. In periodontitis, the

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enzymes released as a consequence of the immune response seen in this disease might

contribute to the destruction of periodontal tissues. Despite the injurious nature of these

factors it has also been recognized that these, and in particular host-derived enzymes in

GCF, might provide non-invasive diagnostic tools (i.e. biomarkers) that could be used for

monitoring the clinical course of periodontitis in patients who have received a diagnosis

of or are undergoing treatment 24.

1.4. Enzymes Present in GCF

Several host-derived enzymes are present in the GCF in abundance; their levels and/or

activities may provide insights into pathogenesis and progression of periodontitis, and

could be of diagnostic value. Some of the prominent GCF enzymes are considered below.

Alkaline phosphatase (AP) is a glycosylated, membrane-bound hydrolytic

enzyme, which increases the local concentration of phosphate and has been implicated in

the turnover of periodontal ligament, cementum and maintenance of bone homeostasis 24.

At sites affected by periodontitis, alkaline phosphatase is secreted primarily by

neutrophils in GCF alkaline phosphatase may originate from bacteria in biofilms 24. The

levels of alkaline phosphatase in GCF may serve as an indicator of the progression of the

disease and could be used to assess treatment effects 25 and the presence of inflammation

26.

ß-glucuronidase is a neutrophil-derived enzyme that is released from lysosomes; it

hydrolyzes interstitial connective tissue matrices, particularly proteoglycans 21,24,27.

Increased levels of ß-glucuronidase may indicate progressive periodontitis 28. In a

longitudinal study, ß-glucuronidase levels were found to be higher in periodontal sites

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with progressive attachment loss compared to stable controls 23. The levels of ß-

glucuronidase are reduced after periodontal therapy 21. The risk of progressive

periodontitis is increased 6-14 fold in patients with elevated ß-glucuronidase levels 27.

Measurement of ß-glucuronidase levels in GCF has been used for its evaluation as a

diagnostic test and these data indicate reasonably high levels of sensitivity and

specificity29. Notably, ß-glucuronidase is a marker for the presence of macrophages30,

which are present in the gingival tissues of patients with periodontitis31.

Elastase is a proteolytic enzyme stored in lysosomes 27 of neutrophils that is

involved in digestion of phagocytosed bacteria. When released into the extracellular

matrix, elastase can degrade elastin, fibronectin and collagen24. Elevated levels and

activity of elastase are found in patients with periodontitis, which correlate with increased

probing depths 32,33,34. Elastase levels may be predictive of future periodontal attachment

loss 35 and are reduced after treatment of experimental gingivitis 36.

Aspartate aminotransferase (AST) is an enzyme found mostly in red blood cells,

liver, heart and muscle; its presence in interstitial fluid is associated with tissue damage

and necrosis 27. Aspartate aminotransferase levels are elevated at sites with clinical

attachment loss compared to control sites with no attachment loss, which has been

demonstrated in experimental periodontitis induced in beagle dogs37.

Cathepsins are a family of proteolytic enzymes that are elevated in the GCF of

patients with periodontitis compared to gingivitis 24. Cathepsin B is a cysteine protease

that is released from macrophages 27. Cathepsins B and D are present in GCF of patients

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with periodontitis and have been assessed as diagnostic tools for estimating the severity

of periodontitis and response to periodontal therapy 21,24,27.

Matrix metalloproteinases are thought to be important enzymes that regulate the

initiation and progression of periodontitis; they could also be important in the putative

association between periodontitis and disorders such as cardiovascular diseases (see

below; see sections 2.4&2.5). Therefore, these enzymes will be discussed in more detail.

1.5. Matrix Metalloproteinases

Matrix metalloproteinases (MMPs) are zinc-dependent endoproteinases that degrade

extracellular matrix components including various collagens and basement membrane

proteins 38,39. MMPs are involved in several biological processes such as embryogenesis,

tissue remodeling, wound healing, angiogenesis, and in pathological disorders such as

cancer, arthritis and periodontitis 39,40. The human genome for MMPs encodes 24

homologous proteinases (MMP 1-3 and 7-28) 38.

MMPs consist in general of a signal peptide domain, a pro-domain, a zinc-dependent

catalytic domain and a hemopexin domain39 (Figure 1). The signal peptide participates in

cellular trafficking and helps guide the zymogen for secretion 41. The pro-domain is

typically an 80-residue polypeptide that extends from the catalytic domain to the N-

terminal of the protein and contains the conserved sequence PRCGXPD 38,41. The

catalytic domain is required for MMPs to provide cleavage of specific substrates. Subtle

differences in the sequence of this domain account for some of the variations of preferred

substrate cleavage by different MMPs. The catalytic domain consists of a five-stranded β-

sheet with three α-helices. Helix B, forms the base of the active-site cleft and contains a

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Zn2+ ion binding motif (3 histidine residues) 41. The catalytic domain is stabilized by one

Zn2+ ion on the upper surface in the ‘S-loop’ and by two or three Ca2+ ions, depending on

the specific MMP38,42. The hemopexin domain is present in all MMPs except MMP-7 and

MMP-26 and plays an important role in determining substrate specificity. It consists of 4

hemopexin modules and is stabilized by an intra-domain disulfide bridge and central

structural Ca2+ions43. The hemopexin C domain plays an essential role in triple helicase

activity, which is important in the initial degradation of fibrillar collagen 38.

MMPs are categorized as collagenases, gelatinases, stromelysins, matrilysins,

membrane type and a miscellaneous group. Collagenases consist of MMP-1, 8 and 13;

they can cleave interstitial collagens I, II and III 38,39. The gelatinases MMP-2 and 9

degrade gelatin and denatured collagens 39. MMP-9 can also digest basement membrane

collagen (i.e. type IV). Elevated levels of MMP-9 were found in the GCF of patients with

gingivitis and periodontitis 38,44. MMP-2 cleaves collagen type I, II and III proteins and

this enzyme has been implicated in a large number of biological processes including

osteogenesis39. Gelatinases bind to gelatin, collagen and laminin as a result of their

fibronectin-like domain.

The stromelysins (MMP-3, 10) exhibit similar substrate specificity. Importantly,

MMP-3 can activate other MMPs 39. The matrilysins MMP-7 and 26, are capable of

degrading a broad array of different matrix proteins. Notably, MMP-7 is also associated

with the degradation of cell-surface molecules including pro-α-defensin, Fas-ligand, pro-

tumor necrosis factor (TNF)-α and E-cadherin39.

Membrane-type MMPs include MMP-14, 15, 16 and 24 (trans-membrane type I) and

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MMP-17 and 25 (glycosylphosphatidylinositol-bound MMPs) 39. The MMPs in this

category can digest matrix proteins including collagen, and contribute to angiogenesis

and to activation of MMP-2 from its ‘pro’ form 45.

A group of MMPs consisting of MMP-12, 19, 20, 22, 23 and 28 are in the

miscellaneous category. MMP-12 is expressed by macrophages and is necessary for

macrophage migration 46. MMP-19 is implicated in rheumatoid arthritis 47. MMP-20 is

found in newly formed enamel and digests amelogenin39. MMP-28 is found in

reproductive tissues and is activated in the Golgi apparatus and consequently, it is

secreted into the extracellular matrix as an active enzyme 48.

Tissue inhibitors of MMPs (TIMP-1, 2, 3 and 4) are proteins that inhibit the catalytic

activity of MMPs. TIMPs are implicated in the inhibition of active MMPs, the activation

of pro-MMP-2, promotion of cell growth and proliferation, matrix binding, inhibition of

angiogenesis and the induction of apoptosis 49. The C-terminal and N-terminal domains

of TIMPs bind to the catalytic domains of MMPs at a molar ratio of 1:1 39,49. TIMP-2 and

TIMP-3 are effective inhibitors of the membrane-type MMPs while TIMP-1 cannot

inhibit MT-MMP. TIMP-3 is an inhibitor of tumor necrosis factor-α converting enzyme

and of MMP-1, 2, 3, 9 and 13. TIMP-4 can inhibit MMP-1, 3, 7 and 9 38,39,49. Since

TIMPs are major regulators of the activities of MMPs their application for treatment of

cardiovascular diseases and cancer is under intense investigation 50.

2. Cardiovascular Diseases

2.1. Definition and Classification

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Cardiovascular diseases refer to diseases affecting the heart and the vascular system and

in particular those of lung, brain, kidney and peripheral arteries and veins; these diseases

account for 29% of deaths world-wide 11. It is currently thought that the most common

causes of cardiovascular diseases are hypertension and atherosclerosis. According to the

American Heart Association, hypertension refers to systolic blood pressures of >140 mm

Hg and diastolic blood pressures of >90 mm Hg. Atherosclerosis refers to the stiffening

and narrowing of blood vessels, which compromises blood flow to end organs while also

increasing hypertension due to the loss of vascular elasticity.

Cardiovascular diseases affecting the coronary arteries can cause ischemia in the

heart muscle and eventually tissue necrosis and cardiac arrest. Abnormalities in

pulmonary artery function, such as pulmonary hypertension, cause right side heart failure

and increased pressure in the atria. Cerebrovascular diseases affect blood flow to the

brain; rapid loss of blood to the brain is defined as stroke and can be either ischemic

(blockage of flow to an area of brain) or hemorrhagic (leakage and accumulation of blood

in an area of brain following the development of a ‘burst’ cerebral vessel).

Cerebrovascular accidents (i.e. stroke) cause focal necrosis of brain and affect brain

function corresponding to the affected area 51. A better understanding of blood vessel

anatomy, physiology and the causes of disturbed function could lead to improved

treatments for cardiovascular diseases. For a more detailed discussion of these issues

please see Nabel 200352; further discussion here is beyond the scope of this thesis.

2.2. Blood vessels

Blood vessels comprise arteries, veins, venules and capillaries, a nomenclature that is

based on vessel function and anatomy. Arteries carry oxygenated and deoxygenated

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blood from the heart to the systemic organs or the lungs respectively. Arteries are

comprised of three layers: tunica intima, tunica media and tunica adventitia. The tunica

intima is the inner layer and is lined by simple squamous endothelial cells. The tunica

media is the middle layer and comprises smooth muscle cells, elastic fibers and a large

variety of extracellular matrix proteins. The tunica adventitia is the outermost layer and is

composed of well-organized matrix proteins that provide structural strength to the vessel.

Large vessels with a substantial elastic component such as the aorta contain substantial

amounts of the protein elastin, which maintains the elasticity of vessels during systole

and diastole.

2.3. Atherosclerosis

Atherosclerosis refers to the development of plaques within the intimal layers of the

affected artery. It is thought that the accumulation of these pathological structures might

explain the development of atheromas and are probably the major factors relating to the

development of increased arterial stiffness, left ventricular dysfunction and heart

failure53.The formation of atheromas arises from lipid accumulation and endothelial

injury within the intimal layer, which are the precursors of an inflammatory process that

leads to the formation of arterial plaques, which can subsequently calcify. Calcification

of atheromas may not be an ectopic mineralization phenomenon but could involve

osteodifferentiation and the development of osteoblasts within the intima. These cells can

produce ectopic mineralized bone, which may be seen as vascular calcification.

The major risk factors for atherosclerosis are hypercholesteremia, obesity, diabetes,

smoking and hypertension. Inflammatory diseases such as periodontitis are associated

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with atherosclerosis and cerebrovascular accidents but the underlying mechanisms that

mediate these possible associations are not defined 54.

Hypercholesteremia, which manifests as chronically high levels of cholesterol in

serum, contributes to plaque formation and narrowing of the arterial lumen 55.

Hypercholesterolemia may be a manifestation of a genetic abnormality (e.g. familial

hypercholesterolemia) but may be influenced also by environmental factors including

high dietary fat or obesity 56,57. Indeed, obesity is one of the most important risk factors

for atherosclerosis and coronary artery disease; it also increases risks of myocardial

infarction and stroke. A healthy diet and maintenance of a healthy body weight

significantly reduce low-density lipoprotein and the risk of cardiovascular diseases. Some

data indicate that loss of body weight in adults is associated with reductions of low-

density lipoprotein 58.

Diabetes is a major risk factor for cardiovascular diseases. The chronic

hyperglycemic state and insulin resistance may disturb the function of endothelial cells,

vascular smooth muscle cells and platelets 59. Diabetes is associated with decreased nitric

oxide formation and increased endothelin production, which promotes inflammation and

the production of vascular smooth muscle59.

Smoking generates a very large array of metabolic abnormalities and is a major

environmental risk factor for cardiovascular diseases. For example, smoking increases

oxidation of low-density lipoprotein, which contributes to progression of

atherosclerosis60. Nicotine in serum is associated with low levels of nitric oxide, which is

a physiological regulatory factor that prevents thrombosis and platelet activation61.

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Smoking also increases the levels of markers of systemic inflammation including C-

reactive protein, IL-6 and TNF-α; all of these molecules may drive the process of

atherosclerosis60.

Hypertension is an important risk factor for cardiovascular diseases; it causes

damage to endothelial and vascular smooth muscle cells. Hypertension increases vascular

smooth muscle hypertrophy and causes oxidative stress because of reduced perfusion and

increased free radical formation. These conditions contribute to disturbances of

endothelial function that in turn promote critical inflammatory processes that lead to

atherosclerosis 62.

2.4. Atherosclerosis and Periodontitis

According to the NHANES III cohort study, patients with severe periodontal diseases are

at greater risk (~4 times greater risk) for the development of myocardial infarction

compared to patients without periodontitis 63. Several prospective, cohort studies have

demonstrated 1.5-2.5 higher risk of complications of atherosclerosis in adult patients with

periodontal diseases 64. In a retrospective study, a statistically significant correlation was

found between the extent of carotid artery calcification (as measured in panoramic

radiographs) and alveolar bone loss 65. Specifically, the study showed that individuals

with ~25% alveolar bone loss exhibited unilateral or bilateral carotid calcification while

subjects with ~10% bone loss did not show carotid calcification.

Other studies have provided epidemiological evidence suggesting an association

between cardiovascular diseases and periodontitis 66,67. Notably, several risk factors (e.g.

smoking, hypertension, male gender, low socioeconomic status and aging 68) are

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associated with both periodontitis and cardiovascular diseases in human populations, but

it is difficult to correct for poorly understood but potentially common underlying causal

factors that impact both disease processes. Therefore it is not obvious whether

periodontitis has any impact upon the pathogenesis of cardiovascular diseases. The

relationships that have been demonstrated epidemiologically might be related to similar

underlying pathophysiological processes for these diseases and similar etiologic factors

(e.g. smoking).

There are biologically plausible data that could explain, at least in part, the

association between periodontitis and cardiovascular diseases. For example, periodontal

pathogens have been shown in atherosclerotic plaques. Other data have shown

improvement in the levels of systemic markers of inflammation and improved endothelial

function after treatment for periodontal disease. There is still no solid explanation for

these findings, which have demonstrated a statistically significant correlation between

cardiovascular diseases and periodontitis. A brief outline of some of the biologically

plausible explanations will be outlined below 54.

Periodontal pathogens can be introduced into the systemic circulation (i.e.

bacteremia) on a daily basis because of physical disturbances to periodontal tissues (e.g.

tooth brushing)69,70. Bacteremias arising from chronic periodontal infections could

damage endothelial cells and vascular smooth muscle cells, a process that may contribute

to atherosclerosis 71. Conceivably, bacteremias associated with the oral microflora could

also promote platelet aggregation and thrombus formation72. Porphyromonas gingivalis,

which is prominent in the subgingival flora of patients with periodontitis, can be isolated

from the serum after dental scaling73. Actinobacillus actinomycetemcomitans, an

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abundant pathogen in subgingival biofilms of patients with periodontitis, has also been

isolated from atherosclerotic plaques 70. Collectively, these findings suggest the

possibility that periodontal pathogens may enter the blood stream through the periodontal

pocket wall and contribute to atherosclerotic plaque formation, an important underlying

cause of cardiovascular diseases 71. However, the presence of these organisms within

atherosclerotic plaques does not mean a priori that these micro-organisms cause vascular

damage or exacerbation of the formation of atheromas.

In addition to data showing periodontal pathogens in atherosclerotic plaques,

other studies have shown that treatment of periodontitis can lead to improvements in the

levels of systemic markers of inflammation including reductions of serum lipid levels and

overall improvements of endothelial cell function 74,75. The numbers of leukocytes and

plasma cells, and the levels of C-reactive protein, are higher in patients with periodontitis

compared with periodontally healthy patients 76-78. Data from retrospective cohort studies

indicate that levels of systemic inflammatory markers (e.g. C-reactive protein, IL-6)

decrease six months after initial periodontal therapy 79,80. High serum cholesterol levels

and increased low-density lipoproteins have been associated with the presence of deeper

periodontal pockets 81. In a case-control study, levels of low-density lipoprotein and

triglycerides were respectively, 13% and 39% higher in patients with moderate

periodontitis compared to healthy controls 82.

The endothelial cell lining of the tunica intima of blood vessels plays a major role in

maintaining circulatory homeostasis. Patients with advanced periodontitis exhibit

endothelial dysfunction, which increases risk of developing cardiovascular diseases 78.

While non-surgical periodontal therapy causes transient bacteremias 54 and increases

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systemic inflammation, endothelial cell function is improved six months after treatment

83. This finding is notable as endothelial dysfunction is an important component in the

pathogenesis of atherosclerosis.

2.5. Pathogenesis of Atherosclerosis

Large and medium vessels, such as the aorta and coronary arteries, are affected

commonly by atherosclerosis56, but whole vessels do not seem to calcify and so the

location of calcified atherosclerotic plaques throughout the human vasculature is highly

variable. Calcified lesions are mainly restricted to the tunica intima and typically grow

larger as the atheroma expands 84.

Calcification in the tunica media is seen in Monckeberg’s medial sclerosis, which

consists of a mineralized extracellular matrix 85 and manifests as a linear deposition of

calcified particles along elastic laminae and with circumferential calcified rings in more

advanced stages of the disease 85.

The development of atheromas is associated with a large number of metabolic

disturbances, including interference of systemic lipid balances and damage to endothelial

cells, which in some cases is associated with turbulent blood flow adjacent to vessel

walls56. Injured endothelial cells express adhesion surface molecules such as VCAM-186;

injury also leads to increased expression of chemokines and recruitment of monocytes

and T-cells87. Inflammatory cells release cytokines, which promote smooth muscle cell

aggregation, proliferation and local oxidation of low-density lipoproteins 56. Leukocytes

migrate between endothelial cell junctions and into the tunica media, a process that is

facilitated by MMPs. Smooth muscle cells involved in the formation of atheromatous

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!16!

plaques, release pro-inflammatory factors such as IL-1β, TNF-α and β, IL-6, M-CSF,

MCP-1, IL-18 and CD-40L. These signals promote inflammatory cell proliferation,

aggregation and the formation of lipid-loaded foam cells (derived from monocytes and

macrophages) in the atheroma. The accumulation of foam cells, smooth muscle cells and

inflammatory cells in the atheroma leads to narrowing of the blood vessel lumen and

when advanced, can compromise end-organ blood flow and contribute to the

development of hypertension 56. Calcification within atheromatous plaques is thought to

be mediated by tightly regulated processes, which are considered next.

2.6. Mechanism of Vascular Calcification

Vascular calcification and arterial hardening increase the risk of cardiovascular events

and mortality, particularly in subjects with end-stage renal disease and diabetes88.

Elevated calcium and phosphate levels, along with cell-mediated calcification processes,

can promote vascular calcification.

While the mechanisms underlying vascular calcification are not well-

defined, it is thought that one possible contributory system may involve the conversion of

vascular smooth muscle cells into cells that are phenotypically similar to osteoblasts 89.

Elevated extracellular phosphate levels up-regulate phosphate transport through

sodium/phosphate exchangers and channels 89. Elevated intracellular phosphate levels

may increase the expression of Runx2/Cbfa-1, which are osteogenic transcription factors

84,89. Elevated extracellular calcium may also enhance phosphate-dependent, open

probability of the Pit-1 channel 89. Growth factors such as TGF-β and BMP-2 can further

enhance osteogenic differentiation by vascular smooth muscle cells further84,89.

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!17!

Elevated levels of extracellular phosphate ion concentration may be able to

promote the formation by vascular smooth muscle cells and matrix vesicles of apoptotic

bodies, which act as nucleating sites for mineralization84. Elevated extracellular calcium

ion concentration can also promote transport of calcium ions into matrix vesicles through

calcium-permeable channels89.

2.7. Regulation of Vascular Calcification/Ossification

The loss of inhibition of vascular calcification may promote osteogenic cell

differentiation in atherosclerosis. Some of the prominent molecular determinants of

vascular mineralization and ossification include osteopontin, osteoprotegrin, matrix Gla

protein and fetuin 85.

Osteopontin inhibits mineralization by preventing the nucleation and aggregation

of calcium oxalate crystals, as shown in in vitro studies 90. In vivo studies have reported

the development of calcium oxalate crystals in kidney stones in mice that are null for

osteopontin 91.

Osteoprotegrin, an important regulator of osteogenesis, has complex effects on

mineralization. Osteoprotegrin inhibits RANKL signaling and osteoclast formation 92.

The inhibitory effect of osteoprotegrin on arterial calcification has been examined in

osteoprotegrin null mice 93,94. In one clinical study, high levels of osteoprotegrin were

associated with increased risk for coronary artery disease93.

Matrix Gla protein is a major inhibitor of calcification and is present at high levels

in cartilage and smooth muscle cells 85. There is extensive intra-vascular calcification in

matrix Gla protein null mice 95. Matrix Gla protein can bind calcium and decrease serum

!

!18!

calcium concentration 96; it also binds elastin and prevents the formation of crystal

nucleation complexes 97. Matrix Gla protein can inhibit the signaling activity of BMP-2

and the osteogenic pathway of vascular calcification. This pathway may involve

osteogenic phenotypic changes of vascular smooth muscle cells that arise in part from

elevated extracellular phosphate and calcium levels 94.

Fetuin/Ahsg may inhibit calcification of atheromas by a variety of mechanisms (see

below), which may include inhibition of osteoblast-like cell differentiation and direct

inhibition of mineral crystal formation. Its effects on osteodifferentiation are thought to

be related to a region in the Ahsg molecule that acts as a decoy receptor. Ahsg can bind to

and sequester osteoinductive factors including the BMPs (i.e. inhibition of a cell-

mediated mineralization pathway), while other domains of the protein bind calcium

phosphate, thereby acting as a ‘crystal poison’ which interferes with nucleation and

growth of mineral crystals 98. Notably, there are low serum levels of Ahsg in

hemodialysis patients and these individuals are at increased risk for developing vascular

calcification 99.

3. Fetuin/AHSG

3.1. Discovery and Measurement in Blood Proteins

Fetuin, a prominent serum protein, was first isolated from bovine serum in 1944 by

Pederson 89,100. Fetuin has also been isolated from the sera of sheep, pigs, mice and

humans and is present at higher levels in fetal compared to adult sera 101. Fetuin belongs

to the cysteine super-family of cysteine protease inhibitors 88. The human homolog of

fetuin is alpha-2-Heremans Schmidt-glycoprotein (AHSG), which is named after the two

!

!19!

discoverers of this hepatic protein 89. Fetuin is produced by multiple organs in utero but is

produced only by the liver in adults; in both mice and humans fetuin is the product of a

single gene. The concentration of Ahsg in the serum of children is higher than that

measured in adults 102; the concentration in the latter is ~10µM 88.

3.2. Structure of Human AHSG/Bovine Fetuin

Human AHSG is a heterodimeric, three-domain protein, composed of cystatin domain 1,

cystatin domain 2 and domain 3103 (Figure 2).

Bovine fetuin is composed of two light chains (1c-1 and 1c-2)101,104. The glycoprotein N-

terminal domain contains 2 N-linked and 3 O-linked polysaccharides that terminate with

sialic acid residues; these residues have high affinity for divalent cations (e.g. calcium) as

well as a number of anti-inflammatory molecules 100. Human and bovine fetuin have

~70% similarity in their amino acid sequence and exhibit the same protease-sensitive

sites and conserved cysteine residues 101. As noted above, a single gene codes for bovine

and human fetuin101. The D1 and D2 domains of fetuin (see figure 2) inhibit

proteolysis105.

3.3. Biological Activities

Fetuin/Ahsg has been implicated in multiple biological activities. For example, Ahsg

inhibits insulin signal transduction based on its partial phosphorylation and structural

similarities with the insulin receptor, which is a tyrosine kinase 100,103. Fetuin/Ahsg are

one of the major non-collagenous proteins of bone and teeth, which suggests a potential

role in regulation of calcification in those structures106-108.

!

!20!

One of the important but poorly described functions of fetuin is its inhibitory role in

vascular mineralization. Serum fetuin levels are reduced in end-stage renal disease, which

is associated with elevated phosphate and calcium levels 88. Based on studies of rat

calvarial cell cultures, fetuin inhibits the ability of basic calcium phosphate particles to

mediate precipitation and modulation of apatite formation 98. Fetuin-null mice exhibit

extensive soft tissue calcification in heart, skin, kidney and the vasculature 88,109,110.

Fetuin can exert its inhibitory effects on mineralization through cell-mediated

mechanisms and also by its ability to block hydroxyapatite (HA) crystal formation and

regulation of the binding properties of mineral carriers. Fetuin inhibits spontaneous basic

calcium phosphate particle precipitation by 50-60% in cell-free systems; this inhibition is

increased to 90% in the presence of vascular smooth muscle cells 88. The involvement of

fetuin in cell-mediated regulation of mineralization may involve apoptosis, vesicle

calcification or phagocytosis of apoptotic cells 88. Fetuin inhibits vascular smooth muscle

cell apoptosis by reducing caspase 3,8,9-mediated cleavage of cytoplasmic proteins,

which disturbs cell-signaling pathways that are important for apoptosis. Fetuin also

prevents vesicle-mediated nucleation of mineralization by sequestration of vesicles

released by apoptotic vascular smooth muscle cells 88. Fetuin can inhibit the pro-

inflammatory activities of neutrophils by binding to calcium phosphate crystals, which in

turn can induce cytokine secretion by other inflammatory cells (e.g. macrophages) and

vascular smooth muscle cells 89. Finally, fetuin may regulate mineralization by binding to

TGF-β family proteins (including BMPs), thereby inhibiting BMP-mediated induction of

osteoblast cell differentiation and indirectly, the formation of a mineralized bone

matrix111,112.

!

!21!

Fetuin inhibits mineralization in the absence of cells by binding to basic calcium

phosphate, thereby preventing precipitation 88. Fetuin binds to aggregates of calcium and

phosphate to form pre-nucleation, cluster-laden fetuin monomers89. This process

stabilizes supersaturated and “metastable” mineral solutions, which are transiently stable

colloids that are normally cleared through their passage in the circulation 89 (Figure 3).

The binding of fetuin is attributed to the amino acid sequence of the D1 domain, which

consists of repeating aspartic and glutamic acids that enable fetuin to act as a “mineral

chaperone”. In this process, mineral species are transported from the extracellular space

to the blood89,113. Fetuin prevents growth of mineral crystals by forming a dense

monolayer protein around the mineral core that acts to shield the crystal 89,114. Fetuin

monomers can bind to calcium ions, which contributes to small ion buffering capacity 114.

Fetuin affects mainly calcium-phosphate crystal growth rather than the nucleation in the

context of regulation of mineralization 89,115.

3.4. Degradation of Fetuin

The biological activity of fetuin may be disturbed because of its degradation by enzymes

that are found in circulating blood. Fetuin is cleaved by MMP-7 and MMP-3 104 but the

functional significance of this degradation is not understood. MMP-7 cleaves fetuin at

Pro277-Ser278 and Arg294-His295, which liberates the 1c-1 and 1c-2 chains. MMP-3

cleavage of fetuin also liberates 1c-1 and 1c-2, a cleavage that generates two lighter

chains 104. Limited digestion of human Ahsg by MMP-2, MMP-9 and MMP-7 has been

reported 40,116. Complete digestion of fetuin occurs after 12 hours of incubation with

MMP-2 and MMP-9 but a 10-fold molar excess of these enzymes is required for

complete fetuin digestion 40, a concentration which seems unlikely to occur in vivo.

!

!22!

4. Study of Mineralization In vitro

4.1. In vitro Mineralization Assays

Controlled mineralization in tissues such as bone, teeth and calcified cartilage

(biomineralization) is the result of complex interactions involving calcium and phosphate

ions, inhibitors, nucleating proteins and the substrate to be mineralized 107,117. Despite the

supersaturated nature of fluids such as blood, saliva, urine and interstitial fluid, they do

not normally exhibit spontaneous calcification because of the balance provided by critical

regulatory factors that govern the mineralization process 118,119. Studies of mineralization

in vivo have helped guide the development of in vitro assays that are used to define the

roles played by different extracellular proteins in this process.

Hunter and coworkers conducted experiments using cell-free, steady-state agarose

gels for studying the growth of HA crystals120. The gels are comprised of 2% agrose in a

buffer containing 150 mM NaCl, 0.01% NaN3 and 10 mM Tris HCl (pH 7.4, 37°C), and

are incubated for 5 days. Phosphate and calcium solutions are introduced at opposite ends

of the gels at various concentrations of supra-threshold (7.5 mM) and sub-threshold (5.5

or 6.0 mM) conditions. The effect of inhibitory proteins such as osteopontin was studied

under supra-threshold conditions at various concentrations120. Nucleation of HA was

studied using the same system with different concentrations of agrose, NaCl, HEPES and

NaN3 at sub-threshold concentrations of calcium and phosphate107. Sub-threshold

conditions have been used for studying nucleating agents in concentrations above the

!

!23!

spontaneous threshold for mineralization, HA formation might not be increased in the

presence of the naturally occurring nucleating agents107.

O’Young and coworkers developed an in vitro assay in which ‘seed crystals’ of HA

were added to a super-saturated solution of HA at 37°C, pH=7.4 along with constantly

maintained concentrations of Ca2+(0.5 mM), phosphate (0.3 mM) and NaCl (150 mM)118.!

If!the!concentration!of!ions is not maintained during the assay, the mineralization

potential is lost, without the effects of any exogenous protein. The growth rate of HA

formation was measured in the presence of different protein concentrations to study the

effect of the protein of interest on the growth of HA crystals. Azzopardi and coworkers

used a similar assay with 0.25 mg/ml HA as HA seed crystals117.

4.2. Assessment of Mineralization In vitro

Simple, qualitative tests for mineralization can be conducted using Von Kossa or alizarin

red staining. These stains indicate whether any mineral species are present in the sample,

but the nature of the mineral species can only be identified using electron or x-ray

diffraction analyses. The relative abundance of mineralization in vitro can be quantified

by absorbance spectrophotometry at 540 nm of mineral-bound alizarin red 121.

Von Kossa staining is used for low-resolution quantification of mineral species in

a sample. It estimates the relative abundance of phosphate ions, which produce a black

stain when bound by Von Kossa’s reagent (10% neutral-buffered formalin, 5% sodium

carbonate, 2.5% silver nitrate). The silver nitrate binds to phosphate ions in the mineral

crystals; sodium carbonate enhances the color of the mineral-bound silver nitrate.

Although Von Kossa staining is an effective method for quantifying mineralization, it is

!

!24!

not sufficient alone to define the nature of the mineralization. Other methods, such as x-

ray diffraction or electron microscopy, are needed to define the nature of the crystalline

species that are deposited122.

Alizarin red binds to calcium ions in crystals and is an effective staining method

for identifying the relative abundance of mineralized species in a sample. Cetylpyridinum

chloride extraction of the alizarin red stain from the sample is then used to quantify the

relative amount of mineralization using colorimetric measurements 123.

As described above, while Von Kossa or alizarin red staining can be used to

estimate the relative abundance of mineralized crystal species in a sample, electron or x-

ray diffraction analyses are needed to characterize the nature of the crystal species. In this

context Aron Klug received the Nobel prize for development of crystallographic electron

microscopy methods, which use electron scattering data to study the 3-dimensional

structure and orientation of atoms within crystals 124. The spatial distribution and

diffraction patterns derived from electron diffraction analyses are related to the lattice

type of the crystal; the intensity of the diffraction patterns is related to the arrangement of

the atoms within the crystal cell. For example, symmetrical diffraction patterns reflect the

symmetry of the crystalline structure. Single crystals produce a spot diffraction image

while polycrystalline samples produce a ring that comprises several spot scatter

patterns144,145,146.

The pattern of the deflected beam from a crystal is described by Bragg’s formula,

2d sinθ=nλ, where λ is the wavelength of the incident wave, d is the spacing between the

!

!25!

planes in the lattice, and θ is the angle between the incident beam and the scattering

planes (Figure 4)147.

The d-spacing measurements are compared to gold standard values, which are

derived from either the ICSD or CCDC databases and which contain respectively,

100,000 or 500,000 data entries that correspond to various types of crystal species 144,145.

To obtain precise estimates of the nature of crystal species found in biological samples,

transmission electron microscopy or scanning electron microscopy are used143. Electron

microscopes use collimated electron beams to obtain an image of mineral crystals that

can then be used to visualize the shapes of the species more precisely and thereby type

the crystals that are formed 143,144,145,146.

Another method for visualizing the 3-dimensional structure of crystals employs x-ray

diffraction analysis, which is similar to electron diffraction but uses an x-ray beam as the

incident beam instead of electrons. X-ray diffraction provides information on the

structure, physical and chemical properties of the crystal. The x-ray diffraction pattern of

a crystalline substance is unique, analogous to the electron diffraction pattern. The

difference between electron diffraction and x-ray diffraction analyses is that the

wavelength of the electron beam is shorter and hence more reflections occur with larger

diffraction angles and scattering148.

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!26!

5. Statement of the Problem

In cross-sectional studies, severe periodontitis has been associated with cardiovascular

diseases including patients that exhibit calcifying atheromas. The underlying mechanisms

that may link these two diseases are not defined. The loss of function of proteins that

might inhibit calcification, particularly in the vascular system such as fetuin, could lead to

increased vascular calcification. Endoproteinases associated with inflammation such as

matrix metalloproteinases could disturb the inhibitory effects of fetuin. As MMPs are

released into the circulatory system as a result of inflammation of the periodontium, I

considered that MMP-mediated degradation of fetuin and disturbance of the regulation of

calcification could explain the possible association of periodontal diseases with

calcification of atheromas. Although I have reviewed two possible pathways by which

fetuin could regulate mineralization (osteoblast cell differentiation, inhibition of

mineralization), I chose to focus here on fetuin/AHSG regulation of mineralization.

Objectives:

1. Optimize conditions for cell-free in vitro mineralization

2. Optimize the concentration of fetuin that inhibits mineralization in vitro

3. Identify the type(s) of crystal species formed in mineralization assays

4. Examine whether fetuin inhibition of mineralization is affected by MMP-7 or

MMP-3-induced cleavage of fetuin

Hypothesis:

Matrix metalloproteinase degradation of fetuin inhibits fetuin-mediated inhibition of

mineralization.

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!27!

6. Material and Methods

6.1. Cell-free In vitro Mineralization

The assay for measurement of hydroxyapatite crystals formed in vitro was based on an

original paper by Chen and co-workers 125 and modified by Graeme Hunter (University of

Western Ontario, London, ON). In 96-well, non-tissue culture-treated plates (total

volume of 100 µl per well) the following solutions were added sequentially: 10 µl

phosphate solution (KH2PO4, 10 mM; Na2HPO4, 20 mM; NaCl, 150 mM; pH 7.4), 10 µl

calcium solution (CaCl2, 50 mM; Hepes, 50 mM; NaCl, 150 mM; pH 7.4) and 80 µl of

buffer solution (HEPES, 50 mM; NaCl, 150 mM; pH 7.4). The control groups were

arranged into columns in the plates. Control groups included phosphate solution only,

calcium solution only or buffer solution only. The control groups were analyzed at the

same time as test groups (phosphate, calcium and buffer solutions). After incubations (up

to 4 hours at room temperature), the supernatant was removed, leaving the nascent

hydroxyapatite crystals bound to the bottom of the well. Electron microscopy (see below)

was used to examine the crystalline structures that were formed at the bottom of the

wells.

Presumptive crystals of HA (and with this type of analysis, apatite) were stained with

75 µl alizarin Red S (0.5% alizarin Red S; pH 4.2; 5 minutes of staining). The unbound

alizarin red solution was removed by pipetting. To dissolve the crystal-bound alizarin red,

cetylpyridinium chloride solution (100 µl; 100 mM) was added to each well and

incubated at room temperature for 30 minutes. The relative abundance of hydroxyapatite

crystals formed in each well was estimated from the amount of Alizarin Red S stain

!

!28!

remaining in the cetylpyridinium chloride solution, which was measured by absorbance at

540 nm with a spectrophotometer. These experiments, each with 8 replicates, were

repeated three times and the mean values were used for analyses.

6.2. Fetuin-mediated Inhibition of Mineralization In vitro

Various concentrations of bovine fetuin (0.5, 1.0, 1.4, 2.0 μM) were added to the

mineralization solutions to assess the inhibitory effect of fetuin on mineralization and to

determine whether MMP degradation of fetuin would affect mineralization in vitro. To

study the kinetics of mineralization in the presence or absence of fetuin, mineralization

was quantified at 30 minutes time intervals. The experiment was repeated with 8

replicates during each trial; mean values were used to plot time-courses over 4 hours.

6.3. Crystal Species Formed in Mineralization Assays

The nature of the crystals formed in the assays was assessed by embedding crystalline

material removed from the bottom of dishes and examination by electron microscopy.

Briefly, the dried mineral was scraped from the bottom of culture wells, placed into

electron microscopy block molds and embedded in Quetol-Spurr resin. Sections (100 nm

thick) were cut on an RMC MT6000 ultramicrotome, placed on formvar coated-grids,

imaged with a FEI Tecnai 20 transmission electron microscope and the images were

stored electronically.

To examine the type(s) of crystal species formed in the presence or absence of fetuin,

electron diffraction analysis was performed. The d-spacing values of the samples were

compared with those of authentic hydroxyapatite crystal standards.

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!29!

6.4. Effect of MMP Cleavage of Fetuin on its Ability to Inhibit

Mineralization

Bovine and human fetuin were treated with activated human MMP-7 or activated mouse

MMP-3 at an enzyme to substrate ratio of 1:60. Incubations of bovine and human fetuin

with MMPs were conducted overnight to ensure optimal digestion of fetuin by the MMP

under test. MMP-mediated fetuin degradation fragments were assessed by SDS-PAGE;

gels were stained with Coomassie blue to identify protein bands. MMP-treated bovine or

human fetuin at 0 or 2 μM was incubated with the mineralizing solutions and

mineralization quantified as described above.

6.5. Statistical Analyses

For all data shown, experiments were repeated 2-3 times and were conducted on different

days. For each individual experiment, at least 5 replicates were analyzed. For continuous

variables, means and standard deviations were computed. For the binding experiments,

means, standard errors were computed. Comparisons between groups were assessed with

analysis of variance and individual group differences were analyzed post hoc with

Tukey’s test. The type I error threshold for estimation of statistical significance was set at

p<0.05.

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!30!

7. Results

7.1. Cell-free In vitro Mineralization

The calcium phosphate and buffer control groups demonstrated absorbance

measurements of 0.5 and 0.4 absorbance units respectively at the end of 4 hour

incubations. The test groups in which all ions were included in the test solutions showed

~2.5 absorbance units (Figure 5A), suggesting that mineralization had occurred under

these experimental conditions.

In the test group in which all components of the assay were added sequentially, there

was measureable mineralization. In preliminary trials of the test group, assessment of

mineralization by spectrophotometric absorbance (540 nm) of mineral-bound alizarin red

showed that there was a time-dependent (0-4 hours) increase of absorbance from ~0.5 to

2.5 absorbance units, which peaked at about 3.5 hours (Figure 5B).

7.2. Fetuin Inhibition of Mineralization In vitro

The inhibitory effects of fetuin on HA mineralization in vitro were assessed. When the

mineralization assays were conducted with fetuin added to the incubation medium, fetuin

exerted a concentration-dependent inhibitory effect on mineralization (0-2 µΜ). When

the mineralization assays were conducted with bovine fetuin (2 μM), absorbance values

were reduced ~5-fold (p<0.001) in comparison to the same assay conducted in the

absence of fetuin (4 hours incubations; Figure 6A).

The mineralization assays were conducted at various concentrations of fetuin (0.5,

1.0, 1.4, 2.0 µM) and absorbance was measured after 3.5 hours. This time point was

!

!31!

identified as the time at which maximal mineralization occurred under control conditions.

In brief, the data demonstrate that fetuin has a concentration-dependent inhibitory effect

on mineralization (Figure 6B).

7.3. Crystal Species Formed in Mineralization Assays

Transmission electron microscopic imaging of mineralized samples conducted in the

presence or absence of fetuin at various concentrations showed needle-shaped or rod-

shaped crystal species, which are consistent with the appearance of HA crystals 126,127

(Figure 7A).

Electron diffraction analysis of the hydroxyapatite mineralization showed that the d-

spacing and intensities of the pattern of the HA formed in the in vitro assay matched the

published standards (Figure 7B). These data showed that even fetuin inhibited

mineralization but there was no evidence for total inhibition. Further, in the presence of

fetuin, the mineral that was formed was HA.

7.4. Effect of MMP Cleavage on Fetuin Inhibition of Mineralization

Bovine and human fetuin were treated with MMP-7 and MMP-3 and the digestion

products examined by SDS-PAGE. After 24 hours of MMP-7 digestion of human or

bovine fetuin, small fetuin fragments (~17 kDa) were observed (Figure 8A&B).

When mineralization assays were conducted with human or bovine fetuin that had

been degraded by MMP-3 (24 hours at E:S=1:60), the measurements of mineralization

were not substantially different compared with intact fetuin (p>0.2). In contrast, for

human fetuin that was degraded by MMP-7, its ability to inhibit mineralization was

!

!32!

reduced by at least 3.4-fold more compared to intact fetuin (p<0.001). When bovine

fetuin was degraded by MMP-7, there was also a 4-fold increase of mineralization

compared to intact bovine fetuin (p<0.001). Notably, the overall impact of MMP-7 on the

ability of fetuin to inhibit mineralization was more marked with bovine fetuin than with

human fetuin. These data also show that the smaller fetuin digestion fragments (~17kDa)

generated by MMP-7 do not have the capacity to inhibit mineral formation unlike the

native molecule (Figure 8C&D).

!

!33!

8. Discussion

In this study, a simple mineralization assay was used based on the spontaneous and rapid

formation of HA crystals in vitro, an approach that enabled study of the effect of intact

and degraded fetuin on mineralization. The data show that fetuin reduces mineralization

by inhibiting nucleation processes; since it was added simultaneously with the

mineralizing solutions that contained supersaturated concentrations of calcium and

phosphate ions. The rationale for this experimental approach is that fetuin physiologically

inhibits calcification by preventing nucleation of crystal formation107. This inhibitory

effect cannot be assessed after the peak of mineralization (3.5 hrs.) since there is a natural

and significant reduction of mineral formation after this duration of incubation, possibly

due to loss of mineral ions. The reduction of mineralization after 3.5 hours could also be

due to a decrease in pH after consumption of the OH- as a result of formation of HA

crystals128. One of the challenges with the conduct of the mineralization assays in this

study was maintaining a constant pH for the duration of the mineralization assay. Despite

these pitfalls, the mineralization assay data showed increased mineralization for the

duration of the assay (3.5 hours) in the absence of fetuin. This trend was the same in

assays that included fetuin and which also exhibited a decrease in the absorbance

measurements.

Although mineralization was reduced in the presence of fetuin, the nature of the

crystals that were formed did not apparently change, indicating that fetuin inhibited

precipitation of apatite crystals, which is in accord with current ideas on fetuin regulation

of mineralization and with other data showing that in biological systems, apatite is the

!

!34!

first crystal to emerge during mineralization, despite the possibility that other minerals

might form129.

Notably, the in vitro calcification experiments presented in this thesis are an

approximation of what occurs in vivo, which involves the interaction of other regulatory

molecules119. The interaction of fetuin with other proteins and enzymes in serum needs to

be considered to study its effect on calcification of atheromas and a possible association

with periodontitis. Further, only one type of mineralization assay was conducted here;

other types of assays may provide additional insights107.

My main findings are that while intact bovine and human fetuin inhibit HA

formation in vitro, after digestion of fetuin by MMP-7, the inhibitory effect on

mineralization was greatly reduced. In contrast, MMP-3-digested fetuin inhibited

mineralization similar to that of native fetuin. Therefore, MMP-7, a prominent enzyme in

periodontal lesions130 may contribute to the inhibition of vascular mineralization that is

seen in patients with periodontitis131,132.

Periodontitis and atherosclerotic vascular disease are important pathological

disorders due to their high prevalence54. Further , periodontitis and calcifying atheromas

have been associated in epidemiological studies 133,134, suggesting the possibility of a

common underlying mechanism that link these two diseases. The endothelial dysfunction

observed in patients with periodontitis is comparable to that seen in patients with

hypertension78,135. While there is some evidence for the involvement of periodontal

pathogens in the formation of atheromas and damage to endothelial cells 136, the

underlying mechanisms that link periodontitis and the formation of calcifying atheromas

are not defined 54. Notably, enzymes released into the circulation such as MMP-7 may

!

!35!

contribute to the pathogenesis of atherosclerosis 137 but currently we are uncertain

whether there are marked increases of MMP-7 in serum of patients with periodontitis. In

this context, MMP-3 and MMP-7 levels are increased in serum of patients with gastric

cancer 138 and MMP-9 levels are increased in the serum of patients with periodontitis 139.

Based on the results obtained here, MMP-7 can digest fetuin and disturb its

inhibitory effects on mineralization in vitro. If serum levels of MMP-7 are found to be

higher in patients with periodontitis and can cleave fetuin in vivo, this notion could

explain in part the association between periodontitis and calcified atheroma formation.

Further, measurement of serum levels of MMP-7 could be considered as a potentially

useful tool to assess the risk of developing calcifying atheroma in patients with

periodontitis.

! !

!

!36!

Conclusions

My data support the notion that fetuin, which protects against vascular calcification,

can be degraded by proteolytic enzymes that are increased in periodontitis and other

inflammatory diseases. If this main finding can be confirmed in other, more in-depth

studies, it may explain the epidemiological association between periodontitis and

vascular calcification.

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!37!

Table

Table 1-Definition of sensitivity, specificity, relative risk, biomarker.

Term Definition

Sensitivity The proportion of people with disease who will have a positive test result140

Specificity The proportion of people without the disease who will have a negative test result140

Relative Risk A measure of association between exposure to a particular factor and risk of a certain outcome. It is defined as the ratio of risk in the exposed and unexposed groups141.

Biomarker A molecule that is measured objectively and evaluated as an indicator of normal biologic processes, pathogenic processes, and pharmacologic responses to a therapeutic intervention142

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!38!

Figures

Figure 1-MMP domain structure39-S: signal peptide, Pro: pro-domain, Cat: catalytic domain, Zn: zinc ion, Hpx: hemopexin domain, Fn: fibronectin domain, G: GPI anchor, Cp: cytoplasmic domain, Ca: cysteine array region, Ig: IgG-like domain, V:!vitronectin!insert,!I:!type!I!transmembrane!domain,!II:!type!II!transmembrane!domain.

amino acid sequence of the catalytic domain is related toMMP-1. MMPs generally consist of a prodomain, a catalyticdomain, a hinge region, and a hemopexin domain (see Figure1). They are either secreted from the cell or anchored to theplasma membrane. On the basis of substrate specificity,sequence similarity, and domain organization, vertebrateMMPs can be divided into six groups (see Figure 1 and Table1), as described below. An extended version of Table 1,including MMP substrates, is available in the online datasupplement (available at http://www.circresaha.org).

CollagenasesMMP-1, MMP-8, MMP-13, and MMP-18 (Xenopus) are inthis group. The key feature of these enzymes is their ability tocleave interstitial collagens I, II, and III at a specific sitethree-fourths from the N-terminus. Collagenases can alsodigest a number of other ECM and non-ECM molecules.

GelatinasesGelatinase A (MMP-2) and gelatinase B (MMP-9) belong tothis group. They readily digest the denatured collagens,gelatins. These enzymes have three repeats of a type IIfibronectin domain inserted in the catalytic domain, whichbind to gelatin, collagens, and laminin.12 MMP-2, but notMMP-9, digests type I, II, and III collagens.13,14 AlthoughMMP-2 null mice develop without any apparent abnormali-ty,15 mutations in human MMP-2 resulting in the absence ofactive enzyme are linked with an autosomal recessive form ofmulticentric osteolysis, a rare genetic disorder that causesdestruction and resorption of the affected bones.16 Thissuggests that MMP-2 in humans is important forosteogenesis.16

StromelysinsStromelysin 1 (MMP-3) and stromelysin 2 (MMP-10) bothhave similar substrate specificities, but MMP-3 has a proteo-lytic efficiency higher than that of MMP-10 in general.Besides digesting ECM components, MMP-3 activates anumber of proMMPs, and its action on a partially processedproMMP-1 is critical for the generation of fully activeMMP-1.17 MMP-11 is called stromelysin 3, but it is usually

grouped with “other MMPs” because the sequence andsubstrate specificity diverge from those of MMP-3.

MatrilysinsThe matrilysins are characterized by the lack of a hemopexindomain. Matrilysin 1 (MMP-7) and matrilysin 2 (MMP-26),18also called endometase,19 are in this group. Besides ECMcomponents, MMP-7 processes cell surface molecules suchas pro–!-defensin, Fas-ligand, pro–tumor necrosis factor(TNF)-!, and E-cadherin. Matrilysin 2 (MMP-26) also di-gests a number of ECM components.

Membrane-Type MMPsThere are six membrane-type MMPs (MT-MMPs): four aretype I transmembrane proteins (MMP-14, MMP-15, MMP-16, and MMP-24), and two are glycosylphosphatidylinositol(GPI) anchored proteins (MMP-17 and MMP-25). With theexception of MT4-MMP, they are all capable of activatingproMMP-2. These enzymes can also digest a number of ECMmolecules, and MT1-MMP has collagenolytic activity ontype I, II, and III collagens.20 MT1-MMP null mice exhibitskeletal abnormalities during postnatal development that aremost likely due to lack of collagenolytic activity.21 MT1-MMP also plays an important role in angiogenesis.22 MT5-MMP is brain specific and is mainly expressed in thecerebellum.23 MT6-MMP (MMP-25) is expressed almostexclusively in peripheral blood leukocytes and in anaplasticastrocytomas and glioblastomas but not in meningiomas.24,25

Other MMPsSeven MMPs are not classified in the above categories.Metalloelastase (MMP-12) is mainly expressed in macro-phages26 and is essential for macrophage migration.27 Besideselastin, it digests a number of other proteins.MMP-19 was identified by cDNA cloning from liver28 and

as a T-cell–derived autoantigen from patients with rheuma-toid arthritis (RASI).29Enamelysin (MMP-20), which digests amelogenin, is pri-

marily located within newly formed tooth enamel. Ameloge-nin imperfecta, a genetic disorder caused by defective enamelformation, is due to mutations at MMP-20 cleavage sites.30

Figure 1. Domain structure of MMPs. Thedomain organization of MMPs is as indicated: S,signal peptide; Pro, propeptide; Cat, catalyticdomain; Zn, active-site zinc; Hpx, hemopexindomain; Fn, fibronectin domain; V, vitronectininsert; I, type I transmembrane domain; II, type IItransmembrane domain; G, GPI anchor; Cp,cytoplasmic domain; Ca, cysteine array region;and Ig, IgG-like domain. A furin cleavage site isdepicted as a black band between propeptideand catalytic domain.

828 Circulation Research May 2, 2003

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Figure 2-Human Ahsg domain stucture89–Human Ahsg consists of 3 domains; cystatin domain1, cystatin domain2 and domain3

share a function with fatty acid binding proteins, a family ofabundantly expressed 14-kDa to 15-kDa proteins. Like fetuin,fatty acid binding proteins reversibly bind hydrophobic li-gands, including saturated and unsaturated long-chain fattyacids, and other lipids with high affinity.40

Because of their rich complex glycosylation pattern, fe-tuins serve as model substances for lectin and glycoproteinresearch. Lectin binding should always be seriously consid-ered when fetuin binding to cells and to the extracellularmatrix is studied. On a practical note, the strong binding ofpertussis toxin to terminal sialic acid residues in fetuin formthe basis of a Food and Drug Administration-approvedpertussis toxin test.41,42 Fetuin-A sequestration of lectinsproved a major complication in experimental cytotoxic ther-apy using cancer cell–specific antibodies coupled to theRicinus communis agglutinin ricin.43 The immunotoxins wererapidly cleared by the asialoglycoprotein receptor and causedliver toxicity.44

In a search for natural transforming growth factor-beta(TGF-!) receptor antagonists, a sequence homology wasfound between TGF-! receptor type II and fetuin-A.45 TheTGF-! receptor II homology 1 domain from fetuin boundpreferentially to bone morphogenetic protein (BMP)-2. Full-length fetuin-A bound directly to TGF-!1 and TGF-!2 andwith greater affinity to the TGF-!–related BMP-2, BMP-4,and BMP-6. Finally, and likely impinging on fetuin’s role inmineralization biology, fetuin or neutralizing anti-TGF-!antibodies blocked osteogenesis and deposition of calcium-containing matrix in mineralizing cell cultures.46 An alteredbone phenotype in fetuin-A–deficient mice (Ahsg!/!) wasexplained accordingly in terms of failure to block TGF-!–dependent signaling in osteoblastic cells.47 Tumorigenesisexperiments using Ahsg!/! mice further supported the hy-pothesis that fetuin-A is an antagonist of TGF-! in vivo, inthat it inhibited intestinal tumor progression.48

Antiinflammatory Role of Fetuin-AFetuin-A, one of the most abundant fetal plasma proteins, wasfound to be essential for the inhibition of the proinflammatorycytokine tumor necrosis factor production by spermine and itssynthetic analogues.49,50 Accordingly, the strong fetal expres-sion of fetuin and spermine have been associated with thetolerance of the fetus, “nature’s transplant,” by mothers.51

The strong antiinflammatory effects of fetuin were verified invivo using several models of inflammation, includinglipopolysaccharide-induced miscarriage in rats,52 carrageenaninjection,53 cerebral ischemic injury in rodents,54 and cecalligation and puncture in mice.55 In all cases fetuin-A wasassociated with reduced inflammatory response and increasedsurvival, and administering additional fetuin generally im-proved outcome.

Thus fetuin-A generally may be regarded as antiinflam-matory.56 The antiinflammatory property of serum "2-HSglycoprotein/human fetuin-A was further supported by thedemonstration that fetuin-A is a potent and specificcrystal-bound inhibitor of neutrophil stimulation by hy-droxyapatite crystals.57 Calcium phosphate crystals induceproinflammatory cytokine secretion through the NLRP3inflammasome in monocytes/macrophages,58 – 60 cell deathin human vascular smooth muscle cells,61 and cell activa-tion in chondrocytes.62– 64 Antiapoptotic activity offetuin-A has been observed in smooth muscle cells65 anddampening of the cell-specific responses would generallybe expected to alleviate the detrimental consequences oflocal inflammation, cell death, and cartilage degradation.The proven protective function of fetuin-A in many animalmodels of inflammation,52–55 the inhibition of proinflam-matory compounds,50,51,66 – 68 and the inhibition of crystal-induced neutrophil activation57 collectively suggest thatfetuin-A may generally protect during pathological miner-alization as well.69

AALA A F N A

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AHSG1 AHSG1/AHSG2 AHSG2T230, T238 T230M, T238S551±38mg/L 542±31 494±23 mg/L

*

**

Cystatin domain 2

Domain 3

Cystatin domain 1

Figure 2. Cartoon of human fetuin-A/"2-HS glycoprotein showing cystatin-likedomains 1 and 2 and a third unrelateddomain in green, yellow, and blue shad-ing, respectively. The cotranslational andposttranslational modifications disulfidebridges (C-C in yellow) Ser-phosphoryla-tion sites (S in red), protease-sensitivesites (R-K, dibasic tryptic cleavage site;L-L, chymotryptic cleavage site; R-T,furin-sensitive cleavage site; all in green),allelic variants (T230/T238; M230/S238)depicted as orange asterisks, and Asn-linked complex N-glycosylation sites, andSer/Thr O-glycosylation sites depicted asblue symbols, respectively. Data takenfrom.6,7,13,96

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Figure 3- Fetuin interaction with BCP. This figure illustrates the interaction of fetuin with mineral particles: the green-colored structure is the cystatin-like D1 domain; the yellow structure is the D2 domain; the blue structure is the D3 domain; the red structure refers to basic calcium phosphate 89.

respectively, all accelerate the particle ripening process,demonstrating that calciprotein particle maturation followsthe Arrhenius law. Secondary calciprotein particles are sta-bilized by a compact outer fetuin-A monolayer against furthergrowth (Figure 4E) for up to 30 hours at body temperature,which is ample time for clearing of calciprotein particles fromcirculation.

The formation and maturation of calciprotein particles canbe followed by optical monitoring110 of mineralizing solu-tions. Supplemental Figure I (available online at http://www.circresaha.org) shows dynamic light scatter diagramsillustrating that the transformation from primary to secondarycalciprotein particles is fetuin-A concentration-dependent.Higher fetuin-A concentrations cause prolonged stability ofboth calciprotein monomers and calciprotein particles, andthus a right-shift in the transformation curves. A clinical testcould use the kinetic parameters of the precipitation reaction,thus measuring the overall calcification risk in biologicalfluids, eg, in the blood of dialysis patients.

Protein–mineral complexes are soluble precursors of phys-iological mineralization of bones and teeth as well. Recentresearch has shown that biomineralization starts with amor-phous mineral–protein complexes containing fetuin-A.123–126

Price et al127–129 have shown that fetuin-A is criticallyrequired to direct mineralization to the interior of syntheticmatrices that have size exclusion characteristics similar tothose of collagen. Fetuin-A does so by selectively inhibitingmineral growth outside of these matrices. This mineralizationby inhibitor exclusion is likely redundant, because fetuin-A–deficient mouse bone is mineralized perfectly well. The micenevertheless show a bone phenotype of stunted femur length,indicating premature growth plate mineralization and dis-turbed osteogenic signaling.47

Nanobacteria: A Red Herring From MarsNanobacteria initially described nanoscopic life forms de-tected by electron microscopy in rock sediments.130 Similarentities were discovered in meterorites from Mars131 and,finally, in cell culture.132 Nanobacteria attracted a lot ofscientific and economical attention as a causal agent of majordiseases, including vascular calcification, kidney stones, andcancer.132–136 In the year 2000, Cisar et al137 determined thatsimple mixtures of phospholipids and calcium phosphatecrystals closely resembled nanobacteria in that they showedlife-like growth and replication. Nucleic acid sequencespreviously thought to be diagnostic markers of nanobacteriawere in fact diagnostic of common laboratory contami-nants.137 Two groups of researchers finally solved the riddleof pathological mineralizing nanobacteria.120,138 A series ofexperiments on the origin of putative nanobacteria showedthat calcium phosphate together with proteins and furthernonmineral compounds formed nanoparticles that resemblednanobacteria in shape and behavior.139 Minerals containingnanoparticles had a high binding capacity for charged mole-cules, including ions, carbohydrates, lipids, and nucleic acids.Depending on the exact composition, mixtures of mineral andnonmineral compounds sustained either crystallization ofhydroxyapatite mineral or the formation of complex protein–mineral complexes.122,140 It was found that the main proteincomponent of the nanobacteria was fetuin-A.120 Besidesfetuin-A, serum albumin and apolipoproteins were also iden-tified;121 hence, the term nanobacteria was exchanged for themore apt term calcifying nanoparticles, virtually identicalwith the protein–mineral complexes, calciprotein particles, orfetuin–mineral complexes described before.

Calciprotein Particles Metabolism and ClearingFigure 5 illustrates the putative metabolism of calciproteinparticles, soluble protein–mineral complexes, which are now

Figure 4. Formation of primary and sec-ondary calciprotein particles (CPP) fromfetuin-A monomer and amorphous mineralprecursors. A, Hydroxyapatite precursorviewed along the [001] axis. The dottedlines confine the hexagonal unit cell. Thecircle represents a Ca9(PO4)6 Posner clus-ter as a building block of the apatite struc-ture. B, A three-dimensional view of fivePosner clusters juxtaposed to the acidic!-sheet of the computer-modeled ami-noterminal fetuin-A domain D1. Thedomain surface is plotted semitransparentwith negative charge indicated in red andpositive charge indicated in blue. C, Acomputer-generated homology modelstructure illustrating the fetuin-A–mineralinterface. The fetuin-A protein structurewas modeled after published structures forcystatin-like domains 1 (green) and 2 (yel-low).176 Domain 3 (blue) was modeled afterthe Escherichia coli protein malonyl-CoA:acyl carrier protein transacylase(1MLA).177 Fetuin-A does not influence the

formation of mineral nuclei. However, fetuin-A does prevent the rapid growth and aggregation of nuclei and, thus, mineral precipita-tion.112 D, Primary CPP are spherical and rather unstructured agglomerates of mineral (clusters) and fetuin-A, whereas (E) secondaryCPP consist of a crystalline mineral core covered by fetuin-A. In conclusion, fetuin-A effectively shields the mineral phase, leading tostable CPP that can be transported and cleared, as shown in Figure 5. The computer graphics were generated using the softwarepackages VESTA,178 VMD,179 and APBS.180

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Figure 4-Bragg’s formula147- 2d sinθ=nλ. λ: wavelength of the incident wave, d: spacing between the planes in the lattice, θ: the angle between the incident beam and the scattering planes

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Figure 5A-Alizarin red staining of minerals and cetylpyridinum extraction for absorbance measurement. Non-tissue culture treated well plates were used to add the ions sequentially. The alizarin red was added to stain the mineral particles. Alizarin red was extracted using cetylpyridinium chloride. Absorbance measurements were done at 540 nm after 30 minutes. (24 well plate dish was used here for illustration purposes)

Phosphate Calcium Buffer Buffer All All

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Figure 5B-Mineralization time course over 4 hours. Absorbance was measured at 540 nm every 30 minutes. The mean values+SEM were plotted versus time to obtain the time course of mineralization in the absence of fetuin. The peak value was obtained at 3.5 hours.

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Figure 6A-Mineralization time course over 4 hours with fetuin at 2 µM. Absorbance was measured at 540 nm every 30 minutes in the presence of fetuin at 2µM concentration. The mean values+SEM were plotted versus time to obtain the time course mineralization in the presence of fetuin and compare to the results from the control group of no fetuin. Same trend was noted with significantly smaller values in the presence of fetuin

!!

Fetuin Inhibition of Mineralization

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Figure 6B-Mineralization assessment at 3.5 hours in the presence of fetuin at various concentrations. In vitro mineralization assay was conducted in the presence of bovine fetuin at 0.5, 1.0, 1.4, 2.0 µM concentration. The mean+SEM values at 3.5 hours were used to plot the bar graph. As fetuin concentration increased, the absorbance measurement values decreased (dose-dependent).

Inhibition of Mineralization by Fetuin (3.5 hr incubation)

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Figure 7A-TEM analysis of crystal species in the presence and absence of fetuin. TEM imaging was used to analyze the crystal species formed in the absence (control) and presence of fetuin at various concentrations. The needle shape of the mineral species suggests the hydroxyapatite nature of the crystal species

.

TEM Analysis Demonstrating Effects of Fetuin on Formation of Apatite Crystals

in Mineralization Assay

Control Fetuin- 0.5 µM Fetuin- 1.0 µM

Fetuin- 1.4 µM

Fetuin- 2.0 µM

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Figure 7B-Electron diffraction analysis of crystal species. Electron diffraction analysis was also used to further confirm the presence of mineral species and their characteristics. The d-spacing values of the samples were compared to the gold standard database. The result confirmed the presence of hydroxyapatite formation Electron Diffraction Analysis Demonstrating Formation of

Apatite Crystals in Mineralization Assay System

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Figure 8A-SDS-PAGE analysis of bovine fetuin in the presence of MMP-7 and MMP-3. Bovine fetuin was treated with MMP-7 and MMP-3 at a substrate to enzyme ration of 1:60 and was incubated overnight. The digestion of fetuin with MMP-7 seemed more pronounced than the digestion with MMP-3. MMP-7 and MMP-3 Cleavage of Fetuin

Native Fetuin MMP-7 MMP-3 95

MMP-Treated Bovine Fetuin

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Figure 8B-SDS-PAGE analysis of human AHSG in the presence of MMP-7 and MMP-3Human AHSG was incubated with MMP-7 and MMP-3 1:60 ratio. MMP-7-mediated cleavage of human AHSG was largely complete after 24 hours but was less advanced with MMP-3.

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Figure 8C-Mineralization in the presence of intact fetuin or MMP-7 and MMP-3 treated bovine fetuin. In vitro mineralization assays were conducted in the presence of no fetuin, 2 µM fetuin, MMP-3 degraded or MMP-7-degraded fetuin. The mineralization was significantly inhibited in the presence of intact fetuin and MMP-3-degraded fetuin, which is consistent with the incomplete digestion of fetuin by MMP-3.

Impact of MMP-treated Bovine Fetuin on Mineralization (2 µM; 4 hours)

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Figure 8D-Mineralization in the presence of intact fetuin, MMP-7 and MMP-3- treated human fetuin. In vitro mineralization assays were conducted in the presence of no human fetuin, 2 µM fetuin, MMP-3-degraded fetuin and MMP-7-degraded fetuin. Mineralization was inhibited in the presence of intact fetuin and MMP-3-treated AHSG, and to a lesser extent by MMP-7-degraded fetuin.

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Copyright Acknowledgements

Portions of this work is in press in the Journal of Periodontal Research in 2012.

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