Introduction. Introduction. 1.1 Definition and epidemiology ofatherosclerosis. 1.2 Pathogenesis ofatherosclerosis

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1 1 1.1 Definition and epidemiology ofatherosclerosis 1.2 Pathogenesis ofatherosclerosis Classification ofatherosclerosis Hemodynamic forces Dyslipidemia Inflammation and Immunology Celturnover Matrix turnover and remodeling Neoangiogenesis Clinicalimplications 1.3 Plaque stability and acute thrombotic events Plaque erosion Calcified nodule Plaque rupture Pathogenesis of plaque rupture 1.4 Extracelular matrix homeostasis in advanced atherosclerosis Composition ofthe extracelular matrix Matricial constituents of the fibrous cap Cellular constituents of the fibrous cap Matrix degradation and its regulation Metalloproteinases Serine proteinases Cysteine proteinases 1.5 Thesis perspectives and outline 1.6 References 11

2 Chapter Definition and epidemiology of atherosclerosis One of the challenges in modern day medicine is the treatment of atherosclerosis related diseases like stroke and myocardial infarction. Clinical conditions that arise from acute ischemia are commonly caused by occlusive arterial thrombosis,mostly on atherosclerotic lesions. Atherosclerosis can be defined as progressive thickening of the arterial wall, which usually occurs at specific predilection sites throughout the entire arterial system such as bifurcations and branch points. 1, 2 A multi-factorial etiology underlies this disease and includes endothelium dysfunction and lipid accumulation in the arterial intima at the onset,and chronic inflammation and vessel remodeling at the more advanced stages of disease progression. 3-5 The resulting lesions,or plaques,cause progressive narrowing of the arteries and a dysfunctional regulation of vascular tone resulting in reduced flow capacity and eventually ischemia with clinical repercussions like angina pectoris and claudicatio intermittens. Advanced plaques may destabilize,leading to thrombus formation,acute vessel occlusion and infarction of downstream organs. 6,7 Although,contrary to common belief,the epidemiology of atherosclerosis is not entirely restricted to modern time or westernized societies 8-10,this disease has grown endemic in the industrialized world over the last century,and now is one of the major causes of morbidity and mortality in these regions. 11,12 Notwithstanding the fact that cardiovascular mortality decreased over the past thirty years (figure 1.1),in the Netherlands,cardiovascular disease was still the most important cause of death in Almost one third of these deaths could be related to ischemic heart disease and another 25% to stroke (figure 1.2). 80+ years years years <50 years Other 41,958 (30%) Cardiovascular disease Ischemic heart disease 15,973 (33%) Mortality per 100,000 (log scale) Respiratory disease 13,511 (9%) 48,799 (34%) Stroke 12,343 (25%) Other cardiovascular disaese 20,483 (42%) Cancer 38,087 (27%) Figure 1.1. Trend in cardiovascular mortality rate in various age groups over the past three decades in the Netherlands. The persistent decrease in mortality may be attributed to technological advances in invasive treatment and the introduction of improved pharmacotherapeutical agents. (Source:CBS) Figure 1.2. Distribution of the major causes of mortality in the Netherlands in Cardiovascular disease take up 24% of total mortality and approximately one third of these deaths can be related to ischemic heart disease. (Source:CBS) W hile many genetic predispositions,such as familial hypercholesterolemia and type I diabetes, have been shown to play a role in the susceptibility for atherosclerosis 14-17, environmental factors may be regarded as at least equally important. Risk factors,such as smoking and obesity,accelerate atherogenesis and increase the chance of acute ischemic events These life-style factors are often reversible and should be subject to aggressive prevention strategies by health 12

3 education programs. In the Netherlands, 30% of men and 20 % of women over 55 years old, smoke an average of 13 cigarettes per day. More than half the population over 55 may be regarded as overweight or obese. 13 Obesity not only increases atherosclerotic burden directly, but also increases risk of type 2 diabetes, another important cardiovascular risk factor affecting almost 15% of Dutch people over 65 in Other risk factors include physical inactivity, high blood pressure and dyslipidemia. These conditions act synergistically in increasing the risk for atherosclerosis and thrombosis. In 1999, more than 40% of the Dutch population had 2 and 15% had 3 or more risk factors. 13 Thus far, although prevention is regarded as the most favorable approach to reduce morbidity and mortality from atherosclerosis, it has been proven to be extremely difficult to modify these environmental risk factors and medical and surgical therapeutic strategies continue to be the mainstay in managing this disease. Over the past decades the effectiveness of pharmaceutical agents and more invasive therapies has progressed revolutionary. Agents such as HMG-CoA reductase inhibitors, or statins, as well as the evolution of percutaneous coronary intervention (PCI) from balloon angioplasty towards the introduction of drug eluting stents (DES) significantly contributed to the dramatic reduction in mortality from ischemic heart disease over the past three decades (figure 1.3). 13 Mortality per 100,000 (log scale) Ischemic heart disease Other cardiovascular disaese Cerebrovascular disaese Figure 1.3. Distribution of different categories of cardiovascular mortality in the Netherlands in Ischemic heart disease has been the primordial cause of cardiovascular death up to the nineties, but has seen a dramatic reduction over the past decades. Statin therapy, the evolution of percutaneous coronary intervention and, more recently, the introduction of drug eluting stents notably contributed to this reduction in mortality. (Source: CBS) Nevertheless, humans appear to be inherently prone to atherosclerosis. In future, further unraveling of pathophysiological mechanisms and predisposing genetic factors could lead to new therapeutic targets, early identification of high-risk patients and individually tailored prevention and treatment protocols with greater efficacy and less adverse effects. Progress in the elucidation of the molecular biology of atherosclerosis has accelerated exponentially in recent years through new screening techniques in genomics and proteomics. Advanced tools in molecular biology, for instance DNA microarray, RNA silencing and targeted gene transduction, and their ongoing development, keep contributing to disentangle the complex network of biochemical pathways that underlie the pathogenesis of atherosclerosis Pathogenesis of atherosclerosis The onset of atherosclerosis presumably takes place early in life. Lipid deposition and foam cell accumulation have been described as fatty streaks in the arterial intima in humans as young as 15 years of age. 22 As mentioned earlier, various etiologic factors, including dyslipidemia and endothelial dysfunction can 13

4 Chapter 1 promote permeability of the vascular endothelium and subsequent deposition of 23, 24 lipoproteins in the subendothelial matrix. Historically, atherosclerosis was regarded as a disease of vascular lipid accumulation and endothelium dysfunction was mainly left out of the picture. The response-to-injury hypothesis, introduced by Virchow in and later modified by Ross 26, postulates that noxious agents, such as shear stress, lipids and oxidation products, damage the endothelium, resulting in increased permeability and enhanced expression of cell adhesion molecules. The ensuing infiltration of inflammatory cells starts a complex process of persistent inflammation and vascular remodeling, which stretches over decades and leads to the development and progression of atheromatous plaques, aneurysms and 27, 28 arterial thrombosis (figure 1.4). Infiltrating leukocytes scavenge lipids and subsequently transform to foam cells, making up the bulk of early lesions. 29 The ensuing inflammation attracts more leukocytes and stimulates vascular smooth muscle cells (SMCs) to proliferate and migrate towards the intima. 30 The latter are the predominant source of extracellular matrix proteins, such as collagen and elastin, and importantly contribute to progressive plaque growth and arterial remodeling. 31, 32 During lesion progression, the ongoing inflammation and oxygen deprivation in the core of the plaque results in apoptotic and necrotic cell death This lipid-rich necrotic core is separated from the blood by a remaining fibrous cap, rich in SMCs and SMC-derived collagen. 36 Thrombosis can be caused by fracture of the fibrous cap, exposing the tissue factor rich gruel in the core of the lesion, or by endothelial erosion, exposing the subendothelial collagen, thereby starting platelet aggregation The resulting acute ischemia often is the first clinical presentation of atherosclerotic burden and by then atherosclerosis has already progressed to an advanced stage. 40 Figure 1.4. Atherosclerotic lesion development illustrated by the AHA/ACC classification of Early infiltration of leukocytes into the arterial intima is commenced by endothelial dysfunction, lipid accumulation and other adverse environmental cues. The resulting fatty streaks (type II lesions) can be found in humans as early as the second and third decade of life. Plaque growth eventually leads to apoptotic and necrotic cell death in the inner part of the lesion, releasing oxidatively modified lipid into the extracellular milieu. Type III lesions are characterized by the presence of several small lipid-rich pools, that can congregate to one large necrotic core in the center of the plaque (type IV, atheroma). Proliferation of smooth muscle cells and subsequent deposition of extracellular matrix components, such as elastin and collagen strengthen the fibrous cap, thus preventing the blood to come into contact with the tissue factor rich gruel of the necrotic core (type V). This protective fibrous cap, however, can be threatened by inflammatory processes that negatively affect matrix turnover, weakening the structural integrity of the cap, rendering it prone to rupture. This could either result in episodic plaque growth or in occluding arterial thrombosis (type VI). Source: Circulation. 1995;92:

5 Although biomedical research considerably increased our knowledge on the pathobiology of atherosclerosis, many facets and causal relations remain unexplained or unexplored. A better understanding of the many etiologic factors is important for the development of strategies that could identify atherosclerosis prone individuals and stabilize, or even regress, existing lesions, therewith preventing or treating atherosclerosis in general and acute ischemic events in particular Classification of atherosclerosis Both for research purposes and for risk assessment it is important to define the morphological features of various stages of atherogenesis. This enables us to standardize the description of atherosclerotic lesions and study the relation between plaque morphology and composition, pathophysiology and clinical repercussions. Presently, the 1995 AHA/ACC definitions form the most widely used classification system, defining six categories based on their histological features (figure 1.4 and 1.5). 28 The initial type I lesion merely contains isolated macrophage foam cells and exists already early in life. Type II lesion are macroscopically visible as small fatty streaks and present in over 65% of year old children. 29 Ongoing lipid accumulation and the presence of extracellular lipids form the intermediate type III plaque. In type IV lesions these small lipid deposits have coalesced into a larger central lipid pool and deposition of fibrous material characterizes the type V plaques. Migration of SMCs into the intima, their phenotypic shift towards a synthetic phenotype 41 and subsequent ECM accumulation give rise to fibroatheromatous or type Va lesions. In addition, type Vb lesions are partly calcified and type Vc lesions have a relatively small lipid component. Ultimately, plaques develop to complicated lesions, which consist of a large lipid-rich necrotic core with an overlying fibrous cap and contain cholesterol crystals and frequently also calcified material. The type VI lesions may give rise to plaque rupture (VIa), intraplaque hemorrhage (VIb) or thrombosis on a non-ruptured plaque (VIc); a sequence of events that eventually may lead to arterial occlusion and critical ischemia of end organs. Figure 1.5. AHA/ACC classification of atherosclerotic lesions and pathways of natural evolution and progression. From type I to type IV, changes in lesion morphology occur primarily due to increased accumulation of lipid components. The loop between types V and VI illustrates episodic lesion progression by thrombotic deposits on their surface. Source: Circulation. 1995;92:

6 Chapter 1 Alternatively, in 2000 Virmani and colleagues proposed a classification to describe advanced atherosclerotic lesions more adequately with regard to plaque 42, 43 stability. Fibrous lesions (type 1) and atheromatous plaques (type 2) are considered stable, while thin cap fibroatheroma (TCFA, type 3) with a relatively large necrotic core (>40%) and thin fibrous cap (<65 µm) are perceived as unstable. Healed cap ruptures (type 4) and acute plaque disruption or intraplaque hemorrhage (type 5) are distinct signs of plaque destabilization. In this classification, type 6 lesions are typified as plaque erosion which is defined as vascular thrombosis without any sign of fibrous cap discontinuity Hemodynamic forces Atherosclerotic lesions show a remarkably consistent distribution pattern throughout the arterial bed, being mostly confined to branch points of large and middle sized arteries. 2, 44 Typical sites of atherosclerotic burden include the carotid bifurcation, aortic arch, coronary arteries, the aorta near branch points of intercostal, renal and mesenteric arteries and the iliac bifurcation. These sites correspond to deviant hemodynamic conditions compared to the laminar flow pattern that is found in the greater part of the vasculature. 45, 46 The turbulence that occurs at these sites causes low and oscillatory shear forces on the endothelium and these biomechanical effects change both the geometry of endothelial cells and the gene expression profile. 47, 48 While the high shear stress of laminar flow promotes the expression of atheroprotective genes (e.g. TGF-, enos, PGI 2 thioredoxin) 49-51, low, oscillatory shear stress induces the expression of various cell adhesion molecules (e.g. VCAM- 1, ICAM-1) chemokines (MCP-1) 55, cytokines (TNF- ) 56, growth factors (PDGF- BB, Angiotensin-II) 57 and enzymes (NADH oxidase, MMP-9) 58, 59, that are involved in atherosclerotic plaque development and progression. In advanced lesions, circumferential stress from high arterial blood pressure not only affects gene expression, but also directly challenges the structural integrity of the protective fibrous cap that separates the blood from the plaques necrotic and highly thrombotic core. 60 The development of these types of lesions that are prone to rupture, so called vulnerable plaques, will be discussed in Dyslipidemia Elevated plasma lipid levels (total cholesterol > 5.0 mm) are a conditio sine qua non for atherogenesis. Circulating levels of LDL-cholesterol (LDL-C > 3.0 mm), triglycerides (TG > 1.7 mm) and lipoprotein a (Lp(a)) are significantly correlated to 61, 62 cardiovascular diseases. Conversely, levels of HDL particles are inversely correlated to cardiovascular disease because of their involvement in reverse cholesterol transport. 63 LDL has been identified to be the most important source of lipid accumulation within the arterial wall 23 and increased serum levels can either be caused by dietary intake or by genetic defects, such as LDL receptor deficiency in type I familial hypercholesterolemia. 14 These elevated serum lipid levels and endothelial dysfunction promote LDL influx and subendothelial retention by proteoglycans 24, 64 and the recently discovered SMC derived atherin. 65 Hydrolytic and oxidative modification of lipid components by infiltrated leukocytes facilitates LDL uptake by macrophages and contributes to the inflammatory response. Although macrophages only sparsely express specific LDL receptors, acetylated, oxidized and malon dialdehyde (MDA) LDL can be internalized through class A 16

7 (SRA) 69, 70 and B scavenger receptors (CD36, Cla-1) 71, macrosialin (CD68) 72, the macrophage receptor with collagenous structure (MARCO) 72 and the lectin-like oxldl receptor LOX Intracellular buildup of lipids give macrophages a foamy appearance and these foam cells are the major cellular component of early atherosclerotic lesions, fatty streaks. Lipoproteins are further oxidatively modified in the intracellular compartment 74 and excreted oxidized fatty acids and sterols can chemoattract and activate both of monocytes and vsmcs In addition, oxldl can upregulate the expression of various pro-atherogenic chemokines, cytokines and proteases, like MCP-1, IL-8 and MMP-9, by itself, enhancing monocyte transmigration into the intima, macrophage differentiation and inflammatory activation. 68, 78, 79 Finally, oxldl acts as a toxic agent for all cell types involved. Not only does oxldl facilitate apoptotic or necrotic cell death by direct oxidative stress and the release of lysosomal enzymes, but it impairs clearance of the apoptotic remnants as well, further enhancing inflammation 83 (see also 1.2.6) Inflammation and Immunology Although Virchow already described inflammation in fibroatheromatous lesions as early as the mid-19 th century 25, historically atherosclerosis has been mainly regarded as a lipid storage disorder, in which lipids accumulate in the arterial wall and thus lead to progressive stenosis and eventually completely obliterate the vessel lumen. The past decades it has become increasingly clear that inflammation plays a major role in the pathogenesis of atherosclerosis, both in fibrous and in lipidrich atheromatous lesions. 4, 27 In the response-to-injury hypothesis, Russell Ross postulates that endothelial damage by noxious agents and hemodynamic disturbances results in platelet aggregation and adhesion and release of growth factors, triggering SMC proliferation and a cascade of inflammatory responses. 26 Upregulation of adhesion molecules (e.g. VCAM-1, ICAM-1, P-Selectin) 52, 53, on the dysfunctional endothelium and release of a variety of chemokines (e.g. MCP-1, 67, 87, 88 IL-8, Fractalkine, GM-CSF) attracts leukocytes from the circulation and promotes their transmigration into the arterial wall. In contrast to classic inflammation, in which extravasation of leukocytes and plasma constituents into the surrounding tissue occurs, the thick arterial media is almost impermeable, keeping the infiltrated leukocytes, mainly monocytes and T-lymphocytes, confined to the subendothelial matrix. Recently, also mast cells have been described to be important actors in the pathophysiology of atherosclerosis by releasing a wide array of 89, 90 proteases that are involved in cell and matrix turnover. Monocytes are the most abundant of inflammatory cell types that infiltrate the subendothelium and differentiate into residential histiocytes or macrophages. The uptake and intracellular accumulation of modified LDL transforms the macrophages into foam cells and activates transcription factors in favor of an inflammatory gene expression profile. 91 Concurrently, cholesterol efflux can be impaired by the proteases cathepsin F, cathepsin S and chymase, degrading the cholesterol 92, 93 acceptors HDL and apoai, and by reduced expression of the ATP binding cassettes ABCA1 and ABCG1. 94, 95 The latter are regulated by the nuclear receptors PPAR- and 94, which also exert anti-inflammatory actions by interfering with the actions of NF B and activator protein-1 (AP-1) family members, thus inhibiting expression of IFN-, IL1, MMP-9, inos and CCR In normal physiology, inflammation protects the organism from infectious and noxious threats and mediates repair mechanisms after tissue damage. However, while an insufficient inflammatory response is hazardous to physiological integrity, an 17

8 Chapter 1 overactive and chronic reaction can be just as dangerous. In atherosclerosis, the primary inflammatory response, initiated by endothelial dysfunction, oxidative stress and hyperlipidemia, results in sustained chemoattraction, infiltration, retention and activation of leukocytes in which the balance between pro- and anti-inflammatory cytokines, the primary mediators of inflammation, favors chronic inflammation. Although many of these cytokines have been described elaborately regarding their basic immunologic functions, the exact role of many of these immune-modulators in atherosclerosis has yet to be unraveled. 100 While some cytokines promote proliferation and activation of macrophages (TNF-, IL-1, IL-18) and induce programmed cell death (TNF-, FasL, TRAIL) 35, , others attenuate ongoing inflammation and support tissue repair (IL-9, IL-10). 107, 108 Besides cytokines and chemokines, infiltrated leukocytes are an important source of growth factors, proteases and reactive oxygen species (ROS). The latter contributes to the chronicity of the inflammatory response by recruiting and activating inflammatory cells and by promoting cell death. 109 Growth factors induce SMC proliferation (b-fgf, TGF- ) 110, 111 and matrix metalloproteinases (MMPs) and cathepsins are pivotal for elastic lamina degradation and SMC migration (MMP-2, MMP-9, Cathepsin S) , facilitating plaque growth and matrix deposition. The implications of protease activity for cell and matrix turnover in the atherosclerotic plaque are discussed in 1.2.6, and more in detail in 1.4. Although monocytes and macrophages form the bulk of infiltrated leukocytes, lymphocytes importantly contribute to the regulation of both local and systemic inflammation. Classically, the immune response has been divided in an adaptive and an innate system in which the former enables the organism to quickly act against certain pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide (LPS), Hsp60 and oxldl. 116 The innate immunity mainly involves cells from the mononuclear lineage, i.e. macrophages and lymphocytes, and responds to foreign molecular patterns via scavenger and Toll-like receptors. 117 Ligation of the scavenger receptor induces endo- and phagocytosis 118, 119, while ligation of the Toll-like receptors activates the NF B and MAPK signaling pathways, resulting in the enhanced expression of genes involved in leukocyte recruitment, apoptosis and inflammation. 117, 120, 121 In addition, innate immunity can commence the activation of the cytotoxic complement system, which has also been implicated in atherogenesis. 122, 123 Taken together, the innate immunity constitutes a rapid first line of defense that can mobilize in minutes to hours, but persists in the context of atherosclerosis. Adaptive immunity, on the other hand, reacts against specific molecular structures and requires the generation of antigen receptors, i.e. T-cell receptors (TCRs) and immunoglobulins. 124 T-cells require strong stimulation by antigen presenting cells (APCs), initially dendritic cells (DCs), expressing a high amount of major histocompatibility complex type II (MHCII) on their surface, which enables them to present an antigen to a naïve T helper cell, and therewith instigating the adaptive immune response. Remaining memory T-cells show a lower activation threshold, making it possible for other APCs, such as macrophages, to reactivate a specific immune response in non-lymphoïd tissues. Regulatory T-cells modulate the process by secreting anti-inflammatory cytokines. 125 Deregulation of the immune response by a decreased activation threshold or molecular mimicry can result in autoimmune disease like rheumatoid arthritis and systemic lupus erythematosus. In atherosclerosis, antibodies have been found against oxldl and Hsp60, suggesting 126, 127 that (auto)-immunity does indeed play a role in this disease. 18

9 In addition to immune responses that are constricted to the arterial intima, circulating antigens and efflux of dentritic cells may result in a systemic immune activation by migrating to the lymphatic system and subsequently initiating a T-cell response. 128, 129 The correlation between elevated cytokine plasma levels (e.g. IL-12, IL-18 and IFN-γ) and acute coronary syndromes is suggestive for the contribution of 130, 131 systemic immune activation to the clinical manifestations of atherosclerosis. Summarizing, the initiation of atherosclerosis often represents a response of the innate immune system to the accumulation and modification of lipoproteins in the arterial intima. In addition, several studies suggest that microbial products, such as Hsp60 (Chlamidia pneumoniae) and CMV, are involved in the immune response in atherogenesis The subsequent rise in pro-inflammatory cytokines contributes to leukocyte adhesion (VCAM-1, ICAM-1), chemotaxis (MCP-1) and proliferation (GM-CSF). Infiltrated monocytes differentiate into macrophages that can internalize PAMPs via scavenger receptors. Antigen presentation by macrophages and dendritic cells to T-cells links innate to adaptive immunity in atherosclerosis. T-cells can be found in atherosclerotic lesion as early as monocytes. Most of these cells express CD4 and TCR +, a T-cell antigen receptor, indicating that they recognize molecular structures presented to them by macrophages or DCs. While in humans, these cells comprise two-third of lesional T-cells, in apoe-/- mice, 90% of the T-cell population is CD4 positive. 135, 136 Since many intimal cells secrete the Th1 activating cytokines IL- 12 and IL-18, the majority of CD4+ cells show Th1 properties by secreting IFN-, IL-2 and TNF-, therewith perpetuating inflammation By contrast, Th2 cytokines such as IL-10 are reported to have an anti-atherosclerotic effect. 140 However, human plaques express only little cytokines derived from the Th2 population, keeping the balance in favor of the pro-inflammatory Th1 related cytokines 141, while apoe deficient mice express Th2 associated cytokines only in extreme hypercholesterolemia Cell turnover Rather than just being a progressive accumulation of cells and extracellular matrix (ECM), the composition of atherosclerotic plaques is a dynamic balance of cell influx, proliferation and death and of both ECM deposition and degradation. Tangential wall stress, mechanical injury, oxidized lipids and activated inflammatory cells in the intima and various other adverse physical and biochemical stimuli all provoke VSMC proliferation and migration towards the arterial intima. 143, 144 Vital to this process is not only the mitogenic activation of cells, but also the degradation of the basal membrane and of elastic laminae, enabling SMCs to migrate into the lesion The concept that these lesional SMCs originate from the arterial media has been challenged several times. Benditt and Benditt suggested already in 1973 that many plaques appear to progress by monoclonal expansion of a subset of SMCs. 149 This could arise by selection of medial VSMCs that express a proliferative phenotype or expansion from invading circulating VSMC progenitors. 150 In addition, it has been suggested that bone marrow derived SMC-progenitors can infiltrate the plaque and that adventitial fibroblasts can transdifferentiate to a synthetic SMC phenotype The infiltration of inflammatory cells and the accumulation of intimal SMCs are counterbalanced by cell death mechanisms; i.e. necrosis and apoptosis. Necrosis is the most common form of cell death, arises from non-specific injury and includes 19

10 Chapter 1 cellular swelling and loss of membrane integrity. 155, 156 The subsequent release of cellular contents is accompanied by an inflammatory response and the chemoattraction of phagocytes. Conversely, apoptotic cell death is induced by specific stimuli, such as cytokines, ROS, free cholesterol or growth factor withdrawal, and involves regulation by several signal transduction pathways that converge in the activation of the so-called effector caspases 3, 6 and These activation pathways are complex and interdependent. Generally, two major categories of activation are distinguished: 1) ligand binding to receptors with an intracellular death domain (TNF, FasL) 158 and 2) release of cytochrome c from mitochondria by various adverse events (ROS, DNA damage, hypoxia, loss of cell-matrix interaction). 159 Morphologically, apoptosis is characterized by membrane blebbing, cytoplasm shrinkage, mitochondrial swelling and nuclear fragmentation. 156 Unlike with necrosis this form of programmed`cell death does not result in inflammation the apoptotic bodies can be phagocytosed by neighboring macrophages. The latter is essential for normal cell turnover and prevention of secondary necrosis with subsequent inflammatory response. 83, 160 Recently however, co-localization studies of apoptotic cells with macrophages in human endarterectomy sections revealed that phagocytosis of apoptotic bodies is impaired in atherosclerosis and that this is partly attributed to oxidative stress by oxldl and by oxidized red blood cells (RBCs) that may have entered the lesion through intraplaque hemorrhage (IPH). 83 Defective clearance of apoptotic cells can lead to necrotic core formation and persistent inflammation with augmented IL-6 and MCP-1 secretion. 161 In this manner, macrophage apoptosis contributes to necrotic core formation, while apoptosis of intimal SMCs can result in cell loss and increased inflammation within the overlying fibrous cap The latter may be illustrated by lesional p53 overexpression in murine carotid plaques resulting in fibrous cap thinning and an increased incidence of thrombotic events. 163 Bennett et al. demonstrated that VSMCs in advanced plaques display a higher level of apoptosis and slower rates of proliferation because of a defect in the phosphorylation of RB and a lower level of E2F transcriptional activity. 165 While in early plaques decreased proliferative activity and apoptosis of SMCs may impair plaque growth, in advanced plaques this is thought to compromise the production of ECM constituents rendering the fibrous cap more vulnerable to hemodynamic stress 166 as will be discussed in 1.3 and Matrix turnover and remodeling Smooth muscle cells are characterized by their immense phenotypic plasticity, enabling them to adapt to the continuously changing environmental circumstances. 167 Contractile SMCs are responsible for vascular tone, while the synthetic phenotype can fabricate a vast amount of ECM components including collagens, proteoglycans and elastin 168, 169, the properties of which are described in 1.4. This ECM production contributes to plaque growth in the initial stage of atherogenesis and reinforces the plaques resistance to disruption at advanced stages of lesion progression. 170 In turn, ECM constituents affect cellular differentiation and function. Collagen type I inhibits SMC proliferation and disruption of integrin-bound fibronectin may result in apoptosis Proteolytic enzymes, such as the matrix metalloproteinase (MMP) family and cathepsins, act in concert to 174, 175 degrade specific ECM components (see 1.4). Not only does this ECM degradation result in loss of structural proteins, but also in modulation of cellular function by altered cell-matrix interactions and by release of matrix bound bioactive molecules including TNF-, bfgf, TGF and VEGF Most important, the 20

11 dynamic balance between ECM synthesis and degradation contributes to the adaptive properties of the vessel wall. 181, 182 Arterial remodeling is a vital response to hemodynamic changes and to arterial injury. 183 In early stages of atherogenesis tissue repair mechanisms lead to constrictive remodeling, compromising arterial flow capacity. 184 Lumen patency can be preserved or restored by compensatory outward remodeling. However, while the degree of stenosis is hampered, plaque stability appears to be impaired in positively remodeled plaques. 185 In human coronaries, eccentric plaque growth is strongly correlated with calcification, macrophage infiltrates, a large lipid core and the risk of plaque disruption Neo-angiogenesis The normal arterial intima is devoid of capillaries and the intimal cells obtain oxygen and nutrients from the arterial lumen by diffusion. 155 However, when atherosclerotic plaques progress and the intima becomes thicker, neo-angiogenesis may develop in the deeper parts of the lesion in response to both hypoxia and oxidative stress. Oxygen deprivation and ROS cause upregulation of the transcription factor Hypoxia Inducible Factor (HIF)-1 effecting the expression of Vascular Endothelium Growth Factor (VEGF) which stimulates the proliferation and migration of endothelial cells. 186, 187 Besides hypoxia, inflammation is strongly associated with a vascular response as well. While in acute inflammation microvessels dilate and show increased permeability, chronic inflammation of the vessel wall is accompanied by proliferation of vasa vasorum and neo-angiogenesis, which in turn sustains the inflammatory process. Reactive oxygen species (ROS), IL-8 and mast cell derived b- FGF are but a few among the mediators that are involved in the development of 188, 189 lesional neovessels. Neo-angiogenesis promotes influx of leukocytes and lipids into the vessel wall contributing to the chronic inflammation. 190 In addition, extravasation of erythrocytes from disrupted neovessels initiate platelet and erythrocyte phagocytosis, leading to iron deposition, macrophage activation, ceroid production, foam cell formation and apoptosis. 191 Neo-angiogenesis can thus promote focal plaque expansion when microvessels become thrombotic or rupture prone. Moreover, intraplaque hemorrhage is associated with plaque destabilization. 192 Phagocytosed red blood cells increase intracellular free cholesterol promoting macrophage apoptosis and subsequent expansion of the necrotic core. 193, 194 Recent studies demonstrated that late-stage inhibition of neovascularization reduces macrophage accumulation and the progression of advanced plaques Clinical implications Summarizing, the pathogenesis of atherosclerosis involves a network of many different biochemical and physiological processes, including hemodynamics, lipid metabolism, inflammation, matrix biology and many more. These pathways are highly interdependent, greatly influenced by their environmental context and characterized by an immense redundancy, complicating the design of novel therapeutic strategies. Interestingly, pathogenic mechanisms that promote atherosclerotic plaque growth, including VSMC proliferation and migration and ECM production, are regarded protective in advanced stages of atherogenesis because of their stabilizing effect on the fibrous cap. Similarly, apoptotic stimuli may slow down lesion growth in early plaques, while limiting fibrous cap strength in advanced lesions. 196 This dual role of pathogenic actors also is apparent at a more molecular level. Whereas 21

12 Chapter 1 protease activity may facilitate SMC motility by degradation of the elastic lamina and basal membrane, thus improving plaque stability, these enzymes presumably are also involved in matrix degradation and subsequent fibrous cap thinning. 197 Hence, therapeutic interventions that are beneficial in early stages of atherogenesis may be detrimental for the solidity of larger complex lesions. Decreased plaque stability is a major cause of acute thrombotic events. 198 Mostly, thrombus formation is non-occlusive and contributes to episodic plaque growth. Occasionally, however, thrombosis results in vessel occlusion and acute ischemic events, such as myocardial infarction and stroke, involving tissue necrosis and remodeling, thus inflicting permanent damage and compromising organ function. Because unstable plaques are a key substrate for occlusive arterial thrombosis the following paragraphs describe the different mechanisms of plaque destabilization and focuses on the pathogenesis of plaque rupture. 1.3 Plaque stability and acute thrombotic events As mentioned before, acute ischemic events usually occur due to acute thrombosis at the site of an atherosclerotic plaque. In 1980, angiographic studies by DeWood et al revealed that an occlusive thrombus was responsible for most cases of acute myocardial infarction. 199 The establishment of coronary thrombosis as the most common cause of myocardial infarction led to the development and use of thrombolytic agents (e.g. streptokinase, urokinase). 200 Although this therapeutic modality is a very effective way of revascularization and is still important today, it is not possible to treat the actual culprit: the atherosclerotic lesion that is at risk to initiate acute thrombosis. As early as 1926, Benson postulated that coronary thrombi result from plaque disruption that exposes the lipid-rich core to the circulation. 201 In 1966, Constantinides was the first to show in autopsy studies that fracture of the fibrous cap in the atheroma was the immediate cause of coronary thrombosis. 202 Also, Davies demonstrated the importance of plaque fissuring and subsequent thrombosis in myocardial infarction, unstable angina, and sudden death due to ischemia. 203 Still, in the early eighties, the prevailing concept was that myocardial infarction resulted from occlusion at a site of high-grade stenosis. However, in 1988, Little showed that most of the myocardial infarctions were caused by coronary occlusion at sites with <50% stenosis. 204 The location of arterial occlusion could not be predicted by the severity of stenosis. Other studies later reported that coronary thrombosis resulting in myocardial infarction often occurred in coronary arteries with non-critical stenosis. 185, 205 These findings led to the introduction of the concept of the vulnerable plaque. 206 These unstable plaques do not necessarily lead to high-grade stenosis, but are at risk to initiate thrombosis, arterial occlusion, acute ischemia and eventually infarction. At present, three major mechanisms can be defined that may result in arterial thrombosis: plaque erosion, plaque rupture and eruption of a calcified nodule. While plaque rupture reflects fracture of the fibrous cap, exposing the tissue factor rich gruel in the necrotic core to the circulation, plaque erosion is more subtle and induces coagulation and platelet activation through endothelial desquamation, exposing the subendothelial matrix to the blood flow. Together with eruption of 22

13 calcified nodules, plaque erosion is classified as non-ruptured plaque. 205 Other forms of thrombosis in non-ruptured plaques may be described in the future. Figure 1.6 displays the morphological features of various types of vulnerable plaques. 207 In cases of non-thrombotic sudden cardiac death it is assumed that coronary spasm, emboli to the distal vasculature, or myocardial damage related to previous injury may 207, 208 account for a terminal arrhythmic episode. Figure 1.6. Different types of vulnerable plaques. Characteristically rupture-prone plaques (A) consist of a large (>40%) necrotic core and a thin (<65 µm) fibrous cap, that is at risk to disrupt (B). Another type of vulnerable plaque may entail a fibrocellular intima and a dysfunctional or even desquamated endothelium (C) that facilitates platelet aggregation and subsequent thrombosis (D). Intraplaque hemorrhage (E) can also be regarded as plaque destabilization. The accumulated erythrocytes in the intima promote inflammation and apoptosis and thus plaque progression and further destabilization. Thrombosis on an erupted calcified nodule is important expression of plaque vulnerability as well. Finally, the degree of stenosis (G) can be so critical that the ensuing flow reduction could facilitate vascular thrombosis. From: Circulation 2003;108: Plaque erosion An estimated 30-40% of culprit lesions in myocardial infarction could be 205, 209 appointed to non-ruptured plaques. Plaque erosion is the predominant mechanism in this category, but is not evenly distributed throughout the population. It is more common in women and young individuals <50 years of age and it is associated with smoking. 205 In the present classification of advanced atherosclerotic plaques as proposed by Virmani and co-workers plaque erosion is defined as arterial thrombosis on an atheroma without signs of plaque disruption or intraplaque hemorrhage (Figure 1.6C-D). 42 Indeed, in some cases a deep plaque injury cannot be identified and the thrombus appears to be superimposed on a de-endothelialized, but otherwise intact, plaque. Generally, lesions at risk for plaque erosion are less stenotic and are more fibrous than ruptured plaques. 205 The exposed intima consists of smooth muscle cells, proteoglycans and collagens, constituents that can activate platelets and therewith increase the risk of thrombosis

14 Chapter Calcified nodule Another, infrequent, mechanism of plaque destabilization is an eruptive calcified nodule from a thin-cap fibroatheroma (Figure 1.6F). The exact origin of this type of lesion is not known. Interestingly, they are commonly found in the right coronry artery, where torsion stress is maximal, suggesting that mechanical forces are responsible for eruption of a calcified nodule. 42 However, it has also been suggested that increased protease activity from the surrounding cellular infiltrate is important in the destabilization of these calcified lesions Plaque rupture Although rates of distribution of plaque destabilizing mechanisms vary in the numerous studies that have been conducted in this regard, plaque rupture is still perceived as the most common cause for acute occluding thrombosis, therewith accounting for ~60-70% of fatal acute myocardial infarctions and/or sudden coronary deaths. 42 In the face of this evidence it is crucial to realize that not all ruptured plaques result in occlusive arterial thrombosis and lead to fatal events. If a thrombus is non-occlusive it may be re-endothelialized, thus contributing to plaque progression. In fact, from the often layered appearance of coronary plaques it might be inferred that this is a common mechanism of advanced plaque growth in coronary arteries. 212 In recent years, it has become increasingly clear that acute thrombotic events are not only the result of destabilization of vulnerable plaques, but also result from vulnerable blood, a vulnerable myocardium and possibly other systemic factors. This insight led to the introduction of the term vulnerable patient, appreciating the importance of all mechanisms involved and their complex interdependency. 213 Despite these considerations, the rupture-prone plaque remains an important substrate for acute thrombotic events in vulnerable individuals. Consequently, plaque disruption is a critical target for clinical investigation and this thesis will principally concentrate on its complex pathobiology Pathogenesis of plaque rupture Rupture-prone plaques are morphologically well defined as plaques containing large lipid cores (>40%) and thin fibrous caps (<65 µm) (Figure 1.7). 38 Because of increased fluidity of these plaque and reduced tensile strength of the protective cap, structural integrity can be threatened by high circumferential hemodynamic stress. The fibrous cap is typically thin at the shoulder regions of the plaque and infiltrated with inflammatory cells. 39 The mechanisms of cap thinning are not well understood, but it is assumed that all pathophysiological processes described in 1.2, also participate in the pathophysiology of vulnerable plaque development and plaque rupture. Resilience of the fibrous cap is the resultant of a delicate and complex balance of extracellular matrix production and degradation, depending on 174, 214 cell turnover and proteolytic activity. Extensive apoptosis of both macrophages and smooth muscle cells have been reported in ruptured plaques, extending necrotic core volume and reducing 163, 164, 215, 216 thickness and strength of the fibrous cap, respectively. Virchow described "apoptosis"in the plaque over a century ago: "Thus we have here an active process which really produces new tissues, but then hurries on to destruction in 24

15 consequence of its own development." 217 In mouse models of advanced atherosclerosis it has been shown that downregulation of the anti-apoptotic Bax or upregulation of the pro-apoptotic p53 results in plaque progression, cap thinning and impaired plaque stability. 163, 218 Increased apoptotic activity, together with proliferative senescence, compromises SMC density in the fibrous cap and, being the predominant cell type that synthesizes ECM constituents, subsequently impairs deposition of structural matrix components, mainly type I and III collagens. The collagen synthesizing capacity of SMCs may also be downregulated directly, by proinflammatory cytokines such as IFN-, 219 or indirectly, by decreased expression of Hsp47, a chaperone protein ensuring the post-translational modification of procollagen -chains. 220 Besides affecting cell turnover and deposition of ECM constituents, mediators of inflammation, including TNF-, IL1, IL-18 and oxldl, also promote the expression, secretion and activation of proteolytic enzymes. 221 Although the exact role of the many different proteases that are expressed in the plaque are still under debate, it is widely assumed that the collagenolytic activity of the matrix metalloproteinases MMP-1 and -3 play an important role in fibrous cap thinning and plaque destabilization Conversely, proteases are pivotal for SMC mobility by breaking down the basal membrane surrounding the cell and enabling it to migrate towards the fibrous cap, thus favouring plaque stability. 174 Regarding the complexity of matrix biology in general and the effects of protease activity on plaque stability in particular, the following chapters will principally focus on the pathobiological effects of ECM modulating factors on advanced complex atherosclerotic plaques. Figure 1.7. Rupture-prone vulnerable plaques are characterized by large lipid-rich cores (>40%) and areas of fibrous cap thinning (<65 µm) without significant lumen narrowing. The caps tensile strength is compromised by loss of its structural proteins (i.e. collagen) both through impaired production and increased proteolytic degradation. (Source: Circulation. 2003;108:1664). 25

16 Chapter Extracellular matrix homeostasis in advanced atherosclerosis Because the structural integrity of an atheroma is dependent upon the fibrous cap overlying the liquefied necrotic core, thickness, elasticity and tensile strength are important characteristics in this regard and established by structural proteins of the extracellular matrix (ECM), including elastin and collagen. 226 VSMCs synthesize, secrete and assemble the bulk of these structural proteins as well as the ground matrix of proteoglycans in which they are embedded. 32, 168, 227 Driven by several inflammatory mediators in the intima, SMC phenotype shifts towards a synthetic state, activating genes that are involved in tissue repair and remodeling, 219, 228, i.e. ECM proteins, matrix degrading enzymes and growth factors. ECM production is determined by 1) the density of matrix synthesizing cells and 2) the rate of ECM synthesis, assembly and secretion by these cells. 231 Both determinants are regulated by inflammatory mediators, growth factors and oxidation products, which are highly abundant in the atherosclerotic plaque. Proteolytic enzymes, secreted by both inflammatory cells and SMCs, degrade ECM constituents and complete matrix turnover (figure 1.8). 174, 232 Not only does this dynamic balance of ECM assembly and degradation contribute to the adaptive character of tissues regarding repair and remodeling, but this continues breakdown and construction also 178, actively involves the ECM in cell function and turnover. Many functions of SMCs, such as adhesion, migration, proliferation, contraction, differentiation and apoptosis are determined by their pericellular context through surface adhesion receptors involved in cell-cell binding and cell-matrix interactions, such as integrins and Focal Adhesion Kinase (FAK) Disruption from fibronectin or dissociation of FAK results in accelerated SMCs apoptosis 239, 240, whilst binding of ECM degradation products to for instance V 3 integrin holds the same effect. 241 The strong interrelation between cells and their surrounding matrix is further exemplified by the notion that interstitial collagen impairs collagen synthesis in vitro and SMC migration is dependent upon newly formed collagen, providing a 242, 243 transcellular traction system for effective locomotion. Finally, numerous cytokines and growth factors show high affinity for matrix constituents, rendering the ECM to be a reservoir of bioactive molecules that can be released upon matrix degradation. Matrix metalloproteinase (MMP)-2 and -9 are able to cleave latent TGF- binding protein (LTBP)-1, therewith releasing TGF- from ECM-bound stores. 176 The same enzymes discharge the heparin-bound b-fgf, which in turn modulates collagen synthesis, SMC and EC proliferation and 244, 245 neoangiogenesis. Thus, the ECM is a dynamic structure that not only gives strength and stability to tissues, but also is actively involved in the coordination of cell turnover, recruitment and other (patho)physiological processes like inflammation and neovascularization Composition of the extracellular matrix Matricial constituents of the fibrous cap In general, the extracellular matrix could be described as a heterogeneous collection of both elastic and rigid polymeric proteins embedded in a ground matrix that consists of glycated proteins. These proteoglycans (PGs) are complex macromolecules consisting of glycoaminoglycans (GAG), such as heparin- and chondroitin sulphate, conjugated with a core protein and encompass 4 families 26

17 including a) chondroitin sulphate proteoglycans (CSPG), b) dermatan sulphate proteoglycans (DSPG), c) heparin sulphate proteoglycans (HSPG) and d) keratin sulphate proteoglycans (KSPG) of which the former 3 have been identified in the vessel wall. 232 They maintain viscoelastic properties of the ECM, organize structural proteins and affect cell function, differentiation, proliferation and migration as well as hemostasis, lipoprotein retention and lipolysis. 155, In atherosclerosis PGs have been reported to display altered characteristics such as increased affinity for 24, 249 apob100 containing lipoproteins (i.e. VLDL, LDL). One of the PG embedded polymeric fiber molecules that determines the elastic capacity of tissues is elastin. In the arterial wall, elastin fibers are principally synthesized by SMCs and fibroblasts and bundled to thick dense laminas that separate the intima from the media and the media from the adventitia forming a virtually impermeable barrier for proliferating and migrating cells and for macromolecules. 155 In conjunction with other layers of elastin in the arterial media these laminas grant the artery its elasticity which is important for both structural integrity and the dynamic conduction of blood flow. Disruption of the elastic laminas by proteolytic enzymes (e.g. MMP-2, MMP-9, cathepsins) enables extensive outward remodeling of the arterial wall, thus promoting aneurysm formation Not only does elastic lamina degradation result in improved SMC mobility, but elastin degradation products also actively stimulate SMC proliferation via binding to the elastin/laminin receptor, activating FAK and inducing Ca ++ influx. 253 ECM Homeostasis Production Degradation Cell Density Synthesis Active Proteases Protease Inhibitors TGF- b-fgf Angiotensin II TGF- PDGF-BB Stretch IL1, IL-18 TNF- oxldl Angiotensin II PDGF-BB TGF- TNF- MCP-1 IFN- b-fgf enos TGF- oxldl IL-8 Figure 1.8. Overview of ECM homeostasis and examples of several major determinants of matrix turnover. Cell density determines the amount of cells at a given site that is able to synthesize ECM components. It is the net result of proliferation, migration and apoptotic or necrotic cell death. ECM synthesis in an individual cell is affected by many environmental factors, including cytokines ad growth factors. Matrix degradation by is achieved by proteases. These enzymes are regulated by gene expression, secretion of the inactive zymogen, proteolytic activation and, finally, by specific physiological protease inhibitors. 27

18 Chapter 1 Another important and heterogenic group of ECM constituents produced by SMCs are the glycoproteins, which include fibronectin, vitronectin, laminin, thrombospondin and osteopontin. Fibronectin is an adhesive glycoprotein, binding to collagens, HSPG and proteases as well as to cells, influencing cell differentiation and survival. 172, 240, 254 Thrombospondin-1 and vitronectin promote SMC proliferation and migration. 255, 256 Osteopontin shows high affinity for collagen and stimulates migration of VSMCs. 237 It has been shown to accelerate lesion formation in a mouse model of atherogenesis. Moreover, osteopontin has been suggested as a regulator of 257, 258 calcification in human plaques. The most common family of ECM fiber proteins is collagen, which constitutes as much as 30% of dry human body weight. 155 Based on their different chemical build-up as much as 14 different members can be distinguished in this family. However, 95% of the collagens can be categorized to types I to IV (table 1.2). 155 Types I and III collagens form bundles of fibers and give structural integrity to the tissues, whereas type IV collagen forms thin amorphous membranes and is mainly found in basal membranes surrounding cells, giving them support, but also constraining cellular migration. 259 Type II collagens form very thin fibers, are strictly produced by chondrocytes and contribute to the strength of cartilaginous tissue. Taken together, type IV collagen is involved in the regulation of cell proliferation and migration and plays a selective role in the cells accessibility for small molecules through filtration, influencing cellular function. 145 Type I & III collagens, on the other hand, maintain tensile strength of tissues, form a substrate on which cells can migrate and can also directly influence cell function and behavior. Typically, type I collagen forms thick fibrils and is very rigid, whereas type III fibers are thinner and possess more elasticity. 260 The ratio of both types determines the tissues mechanical properties. 261, 262 Commonly, type I collagen is the predominant fiber protein in the extracellular space, but the type I/III ratio can be inverse in several diseases, such as osteogenesis imperfecta, rheumatoid arthritis and 226, 260 atherosclerosis. Moreover, variations in collagen organization affect its resistance to tangential forces. Burleigh et al. found that fibrous caps require higher collagen content than adjacent intima to withstand a given level of mechanical stress Collagen synthesis Basic building-blocks of collagens include the amino acids glycine (33.3%), proline (12%) and hydroxyproline (10%). 155 Especially hydroxyproline, post translational modified proline 263, is characteristic for collagen. Hence, the uptake of proline is an established method of collagen synthesis analysis. The synthesized polypeptide- -chains are translocated to the lumen of the endoplasmatic reticulum, where proline and lysine residues are hydroxylated L-ascorbic acid is an important enzyme co-factor in this process. The hydroxylysins can be glycosylated with galactose or glycosyl-galactose moieties by corresponding transferases. The heat shock protein (hsp)-47 is an important chaperone in this post-translational modification and its expression is regulated by growth factors and modified LDL. 220 Every type of -chain is synthesized with propeptides on both the N- and C- terminus, that control the correct sequence of assembly to a triple helix configuration and keep this newly formed procollagen soluble, preventing intracellular precipitation. Once secreted to the extracellular milieu, the C- and N-propeptides are proteolytically cleaved from the procollagen. The resulting tropocollagen is able to form long collagen fibrils, which are consolidated by cross-linking tropocollagen molecules via activity of the Cu ++ -dependent lysyl-oxidase. 267 In case of type I & III collagen the 28

19 fibrils are eventually assembled to larger fibers and bundles of fibers. The entire process is influenced by presence of proteoglycans and structural glycoproteins, that, like collagen, can be produced by SMCs as well. 172 Type Structural properties Source Interactions Function I Compact, thick fibrils with variable diameter forming fibers. Fibroblasts, osteoblasts, chondroblast, SMCs Dermatan sulphate Tensile strength II No fibers. Very thin fibrils. Chondroblast, chondrocyte Chondroitin sulphate Pressure resistance III Thin fibrils with a uniform diameter. SMC, fibroblast, adipocyte, Schwann cell Dermatan- & chondroitin sulphate Structure preservation with mechanical deformation IV No fibrils. Membraneous organization. Epithelium, endothelium, fibroblast, SMC, Heparan sulphate Support, attachment, filtration Table 1.2. Most common types of collagen in human and murine connective tissues Cellular constituents of the fibrous cap The key source of ECM components in the atherosclerotic plaque is the vascular smooth muscle cell that has phenotypically shifted from a contractile towards a synthetic state. 268 These intimal SMCs might have originated from medial SMCs, but, although still under debate, several reports suggest that in certain areas of the arterial vasculature infiltrated SMC progenitors make a significant contribution 150, 154 to the intimal cell population. In healthy mature vessels SMCs form a highly specialized cell population regulating vascular tone, blood pressure and blood flow distribution by their contractile functionality. These cells show extremely low proliferation rates, do not migrate and possess little synthetic activity. Reflecting their specialized function SMCs express a unique repertoire of gene products, including contractile proteins, 269, 270 ion channels and signaling molecules. Unlike skeletal and cardiac muscle cells, which are terminally differentiated, SMCs have retained a remarkable plasticity enabling them to undergo profound but 238, 271 reversible changes in phenotype in response to environmental circumstances. This can be observed during vascular development when SMCs show high proliferation rates and produce ECM products like elastin, collagen and proteoglycans that make up the bulk of the vessel wall. Analogous to vasculogenesis, SMCs play a critical role in vascular repair mechanisms by increased proliferation, migration and ECM deposition. SMC motility and survival is dependent upon the secretion of proteases (e.g. MMP-2, -9, Cathepsin S) to degrade the basal 115, 272 membrane enabling them to interact with the surrounding ECM. Besides playing a role in matrix turnover, SMCs also actively participate in the inflammatory response by producing cytokines, chemokines and adhesion molecules including IL- 273, 274 1, MCP-1 and VCAM-1. 29

20 Chapter 1 This functional adaptation to extracellular stimuli is transient, only lasts for the duration of these local environmental cues, and is an inherent property of differentiated SMCs. It is important to realize that SMCs can exibit a broad range of very different phenotypes of which the contractile and synthetic state can merely be regarded as extremes of the spectrum, but also includes SMC-derived foam cells. 275 The different phenotypes can in part be distinguished by the expression of specific markers. 276 However, many of these markers may also be expressed by other cell types, such as pericytes and fibroblasts, complicating identification and characterization of intimal cells in atherosclerotic lesions. The synthetic SMC is characterized by the loss and reorganization of cellular markers that are specific to contractile functionality, usually cytoskeleton proteins such as -SM-actin, SM22 and desmin. 277 By contrast, other cellular proteins are selectively upregulated in ECM 271, 276, 278 synthesizing cells including matrix Gla protein (MGP) and osteopontin. The abundance of growth factors and cytokines in atherosclerosis promotes proliferation, migration towards the intima and, subsequently, a synthetic behavior of SMCs leading to ECM accumulation and therewith inducing plaque growth at the early stages and plaque stabilization at the later stages of lesion development. Hence, while SMC accumulation can be regarded as detrimental in atherosclerosis because this contributes to lesion progression and arterial stenosis, the same process is essential for the stability of advanced plaques; without fully formed SMCrich fibrous caps lesions are prone to rupture. 170 Figure 1.8. Exceedingly simplified scheme of SMC phenotypic plasticity, with the dedifferentiated polygonal SMC on the left displaying its synthetic characteristics and the highly differentiated, spindle shaped contractile SMC on the right hand side of the spectrum. The phenotypic shifts are influenced by a large number of external factors that themselves are affected by one another, making the phenotypic regulation of these cells in the context of atherosclerosis extremely complex Matrix degradation and its regulation As discussed earlier, extracellular proteolysis is pivotal in many biological processes of the vascular wall, including vessel remodeling and wound healing, and plays a key role in cellular behaviour during atherogenesis. Extracellular proteolytic enzymes form a heterogenic family of which the metalloproteinases are probably the best described category. This group of zinc dependent proteases comprises the extensively investigated matrix metalloproteinases (MMPs), proteins with a disintegrin- and metalloproteinase domain (ADAMs) and the recently added pappalysins. 174, 232, 279 Other extracellular enzymes include the cysteine proteases (e.g. calpains, cathepsins), previously considered to be exclusive intracellular 30

21 enzymes, and the serine proteases, which form a rather mixed group of enzymes including tpa, neutrophilic elastase, chymase, tryptase and several fibrinolytic enzymes. 280 Metallo- and serine proteases show optimal activity at neutral ph and therefore are the most common enzymes to function extracellulaly. By contrast, cystein proteases work optimal in the acidic environment of lysosomes. 175 Their extracellular functions are not well understood, but it has recently become clear that several cathepsins can be secreted by atheroma associated cells and are highly expressed in human plaques 281, which emphasizes the need to investigate their function in the pathobiology of atherosclerosis Metalloproteinases In an attempt to elucidate the mechanisms by which a tadpole loses its tale, in 1962, Gross and Lapière first revealed the existence of a collagenase that cleaves the triple-helical collagen into a ¼ and a ¾ fragment, thus identifying the first member of the metalloproteinase family. 282 Belonging to the subfamily of metzincins, that contain Zn 2+ in their active site, MMPs (also known as matrixins) form the most prominent family of metalloproteinases and comprise at least 25 secreted or surfacebound proteases of which 14 have been described in vascular cells. 280 Based on structural homology and, partly overlapping, substrate specificity, the MMP family can be categorized to five subgroups: interstitial collagenases (MMP-1, -8 and -13), gelatinases (MMP-2 and -9), stromelysins/matrilysins (MMP-3, -7, -10 and -11), membrane-type MMPs (MT-MMPs) and others (table 1.4). Besides covering a wide variety of ECM constituents, MMPs also cleave a number of non-matrix substrates, such as TNF- and latent TGF-. 176, 283 The different, but overlapping, substrate specificities and the ability of various MMPs to activate other zymogens, suggest that individual MMPs need to act in concert with many other proteases to achieve matrix turnover. Typically, MMPs contain a signal sequence, a prodomain, a catalytic and a hemopexin-like domain. 279, 284 Latency is preserved by a conserved cystein residu in the prodomain sequence PRCGXPD, displacing the catalytic water from the active site, which holds the zinc-binding sequence HExGHxxGxxHS. Characteristically, in gelatinases this is interrupted by three repeats of a fibronectin type II-like sequence. 285, 286 The hemopexin domain anchors MMP-2 to the cell via integrin binding and may also mediate binding of MMP-9 to CD44, the hyaluron receptor. 287, 288 Furthermore this C-terminal domain is important for the recognition of large matrix molecule substrates and for the interaction with tissue inhibitors of metalloproteinases (TIMPs). 289 MMP activity is regulated at 4 levels: 1) induction or suppression of MMP gene expression, 2) trafficking of intracellular protease containing vesicles and subsequent secretion, 3) activation of the inactive zymogen and 4) inhibition of proteolytic activity by endogenous inhibitors. MMP expression is differentially regulated in the various cell types. In VSMCs, MMP-2 is constitutively expressed and can be upregulated by mechanical stretch. 290, 291 Together with MMP-9 it is rapidly induced after vascular injury and in response to inflammatory cytokines such as IL-1 and TNF These cytokines act in synergy with the growth factors PDGF and FGF-2 to promote MMP gene 228, 293 expression in VSMCs. In addition to soluble factors, interaction between VSMCs and T-cells induces MMP-1, -3, -8 and -9 via CD40 activation. 294 In 31

22 Chapter 1 macrophages, MMP-9 is the most abundant gelatinase and can be upregulated via TNF- and modified LDL. 148, 295, 296 The fibrogenic TGF- inhibits cytokine-mediated induction of MMP The expression of MMP-1 and -3 is not affected by cytokines in macrophages, but depends on intracellular lipid accumulation. 174 While simple exposure of macrophages to oxldl does not induce MMP-1 and -3 expression, it appears that the process of foam cell formation itself either promotes the secretion of an autocrine mediator or activates intrinsic transcription pathways leading to upregulation of MMP-1 and -3. Indeed, macrophage foam cells have been reported to secrete EMAP-II, an auto/paracrine factor with angiogenic properties 298 that promotes MMP-1 and -3 gene transcription (Newby et al., 2005, unpublished data). The promoter regions of many MMPs contain a TATA box and activator protein-1 (AP-1) binding site, while specific individual MMPs have several unique transcription factor binding sites, including a STAT-1 binding element (SBE) in MMP-1, stromelysin PDGF response element (SPRE) in MMP-3, and multiple NF B sites in MMP-1, -3 and Group MMPs Trivial names Principal known substrates Collagenases MMP-1 MMP-8 Interstitial Collagenase Neutrophil Collagenase Collagen type I, II, III, VII, VIII and X, gelatin, MMP-2 and -9 Collagen type I, II, III, VII, VIII and X, gelatin MMP-13 Collegenase-3 Collagen I, II, III, IV, gelatin, PAI-2, MMP-9 Gelatinases MMP-2 Gelatinase-A Collagen I, IV, V, VII, X, XI, XIV, elastin, laminin, fibronectin, MMP-13 MMP-9 Gelatinase-B Collagen IV, V, VII, X,, elastin, laminin, fibronectin Stromelysins MMP-3 Stromelysin-1 Collagen III, IV, IX, X, gelatin, MMP-1, -8, -9 and -13, MMP-1 MMP-10 Stromelysin-2 Collagen III, IV, V, gelatin, casein, MMP-1 and -8 MMP-7 Matrilysin Collagen IV and X, gelatin, fibronectin, versican MT-MMP MMP-14 MT1-MMP Collagen I, II, III, gelatin, MMP-2 and -13 MMP-15 MT2-MMP Gelatin, MMP-2 MMP-16 MT3-MMP MMP-2 MMP-17 MT4-MMP Others MMP-11 Stromelysin-3 MMP-12 Macrophage Metalloelastase Collagen IV, gelatin, elastin, fibronectin Table 1.4. Members of the MMP family that are expressed by atheroma-associated vascular cells, their presence in atherosclerotic plaques and the correlation of plasma levels with acute coronary syndromes (ACS). 32

23 Upon gene transcription, most MMPs are secreted as latent proforms that can be activated via cleavage of the pro-domain by other proteases, most commonly MMPs and plasmin 303, but also mast-cell derived serine proteases, such as trypsin and chymase 90, and the coagulation factors thrombin and factor Xa, linking thrombosis to ECM remodeling. 304, 305 Plasmin is an essential MMP activator in vivo and can be activated by urokinase-like plasminogen activator (upa) which is either secreted or associated with the membrane via binding to the upa receptor (upar). 306 In addition, plasminogen itself can bind to cells by interacting with annexin II, thus compartmentalizing MMP activity to the pericellular space. 307 Hence, increased MMP activity in vascular injury results from enhanced MMP expression and a complex cascade of catalytic activation counterbalanced by endogenous inhibitors (figure 1.10). Tissue inhibitors of metalloproteinases (TIMPs) form tight, but nonselective, complexes with MMPs, preventing their matrix degrading activity. 289 Four TIMP subtypes have been described thus far, each made up of two domains containing three disulfide bonds The N-terminal cysteine group from TIMP displaces a water molecule from the zinc ion in the active site and three C-terminal loops mediate complex formation with the hemopexin domain in gelatinases. TIMP-3 is unique in this family for its strong matrix binding capacity and its ability to prevent TNF receptor shedding by inhibiting ADAM-17, therewith promoting apoptosis. 312 TIMPs are constitutively expressed in atheroma associated cells, but in VSMCs, both TIMP-1 and -3 are also upregulated upon stimulation with the fibrogenic PDGF and TGF- 228, 313, whereas oxldl decreases TIMP-3 expression in macrophages. 314 Taken together, this suggests that inflammatory and immunogenic stimuli shift the MMP/TIMP balance towards proteolysis, while fibrogenic mediators appear to have the opposite effect. Cell membrane Towards proteolysis Enzymes Plasminogen upa receptor MMP-1 upa Plasmin MMP-3 MMP-9 Pro-MMP-13 MMP-13 MT-MMPs Inhibitors TIMP-1 TIMP-2 TIMP-3 PAIs 2-antiplasmin Matrix degradation Figure Highly simplified scheme of MMP interactions that result in a cascade of catalytic activation and matrix degradation, tightly controlled by tissue inhibitors of metalloproteinases. The variety of different activation pathways is extensive and interconnected on several levels leading to high redundancy. MT-MMPs TIMP-2 Progelatinase-A MMP-2 Activation from pro to active form Other inhibitors of MMP activity include plasminogen activator inhibitor-1 (PAI-1) 303 and the circulating 2-macroglobulin, a macromolecule that captures and completely encloses the MMP enzyme, preventing any interactions with its surrounding. Recently, tissue factor pathway inhibitor-2 (TFPI-2) has been recognized as a potent MMP inhibitor and its expression has been demonstrated in human atherosclerotic lesions. 315, 316 Though structurally similar to the anti-coagulant TFPI-1, the physiologic role of TFPI-2 in the coagulation cascade is relatively insignificant. 317 Conversely, it may both affect regulation of MMP activity and act as a mitogen for VSMCs

24 Chapter Serine proteinases The serine protease family is a heterogenic group of proteolytic enzymes that includes coagulation factors (thrombin, protein C, factor VII, IX, X and XII), and the fibrinolytic system. 319 Furthermore, neutrophils and mast cells secrete several serine proteases such as cathepsin G, chymase and tryptase and T lymphocytes contain granules with a variety of different serine proteases, termed granzymes. 280 Obviously, the fibrinolytic system plays an important role in cardiovascular disease and includes two important serine proteases that can convert plasminogen to the active serine protease plasmin: tissue-type plasminogen activator (tpa) and urokinase-like plasminogen activator (upa). While the former is soluble and shows high affinity for fibrin, resulting in clot dissolution, the latter is confined to the cell surface through a specific receptor (upar), compartmentalizing its proteolytic action to the pericellular space. As mentioned before, also plasminogen can be localized to the cell surface by annexin II, resulting in a contained plasmin activity that can influence MMP activity in its direct environment. 320 As with metalloproteases, serine protease activity can be inhibited by specific endogenous inhibitors. The most common group of these inhibitors is called the serpin family and includes plasminogen activator inhibitor-1 (PAI-1). The exact role of serpins in atherosclerosis still remains to be elucidated. While some described an atheroprotective role in early plaque development or accelerated atherogenesis in advanced lesions, other found no effect of PAI-1 on de novo atherogenesis The myxoma virus serpin Serp-1, protecting cells from viral infections, has recently been reported to inhibit de novo atherogenesis and promote plaque stability Cysteine proteinases Two major groups can be distinguished in the class of cysteine proteases: ICE related proteases and the papain superfamily. Most cathepsins belong to the papain family, including cathepsin B, H, L, S, C, K, O, F, V, X and W. 319 These lysosomal enzymes originally were thought to exclusively exert intracellular actions, such as degradation of unwanted proteins or invariant chain processing, important in antigen presentation. 326, 327 Hence, intracellular cathepsins prove to be key regulators of immunity. Not only does cathepsin S affect MCH-II function, but it is vital for NKT cell maturation as well. 328 However, recently it has become increasingly clear that cathepsins also are secreted into the surrounding matrix by macrophages, smooth muscle cells and endothelial cells to exhibit functions like ECM degradation and proteolytic modification of apolipoproteins and activation of other proteases. 175 Cathepsin K (CatK), for instance, is one of the most potent human collagenases and plays an important role in bone collagen metabolism. CatK deficiency significantly impairs bone growth in both humans and animals. 329 In turn, Cat D deficient mice display extensive intestinal necrosis secondary to a dramatic upregulation of inos in monocytic cells. 330 All of these intra- and extracellular actions regarding immunity, inflammation and cell and matrix turnover may have implications for atherosclerosis. Since Shi et al. first characterized human CatS in , cumulative evidence has suggested a role for this protease in atherogenesis. Indeed, both the elastolytic and collagenolytic cathepsin S (CatS) and K show markedly increased mrna levels in atherosclerotic plaques compared to the normal arterial wall. 281 Also, immunohistochemistry demonstrated high levels of both enzymes in atherosclerotic lesions, particularly in macrophages near the shoulder regions and in SMCs of the fibrous cap, suggesting that these cathepsins are involved in plaque stability. 332 Also, CatS and CatK protein expression has been found to be concentrated near areas of 34

25 elastic lamina fragmentation. Moreover, luminal and neovascular endothelial cells express CatS and CatS deficiency impairs microvessel growth, suggesting that this protease is involved in neo-angiogenesis as well, a process implicated in plaque growth and complication. 333 In vitro, cathepsins affect mechanisms that are known to be vital in the pathogenesis of atherosclerosis. CatS inhibits HDL3 induced cholesterol efflux from macrophage foam cells and various cathepsins (e.g. D,F,S,K) are able to modify apob100, inducing foam cell formation. 92 Furthermore, several studies show that lysosomal enzymes, including Cathepsin B, D and L, can directly induce apoptosis once released from the lysosomal compartment by processing procaspases. 81 As in humans, CatS was found to be elevated in mouse models of atherosclerosis as well, localizing mainly to intimal SMCs and macrophages and to medial SMCs. 332 Systemic CatS deficiency reduced initial plaque formation in LDL-/- mice 115, but no such effect could be observed in apoe-/- mice (Shi et al. 2004, personal communication). In LDLr-/- mice CatS deficiency led to reduced numbers of elastic lamina fragmentation and impaired transmigration of monocytes into the arterial intima, both elementary processes in the course of plaque development. 115 Conversely, deficiency of the endogenous cathepsin inhibitor cystatin C increased elastic lamina degradation and aortic dilatation in apoe-/- mice. 334 Taken together, the wide substrate specificity and the variety of different environmental contexts in which these proteases operate, point to a complex and pleiotropic role for cathepsins in the pathobiology of atherosclerosis (figure 1.11), which will be challenging to unravel. Figure Several pathogenic mechanisms in which cathepsins can be involved. Leukocyte transmigration could be facilitated by cathepsin dependent degradation of the endothelial basal membrane. Similarly, cathepsins are thought to play a role in neo-angiogenesis and BM degradation together with break-down of the elastic laminae by cathepsins are important for SMC migration towards the arterial intima. Finally, cathepsins plays a significant role in immunity and foam cell formation. (Source: Arterioscler Thromb Vasc Biol. 2004;24: ) 35