Nooijer, Ramon de
Citation
Nooijer, R. de. (2005, December 12). Modulation of the Extracellular Matrix in Advanced
Atherosclerosis. Retrieved from https://hdl.handle.net/1887/3751
Version:
Corrected Publisher’s Version
License:
Licence agreement concerning inclusion of doctoral thesis in the
Institutional Repository of the University of Leiden
1.1 Definition and epidem iology of atherosclerosis
1.2 Pathogenesis of atherosclerosis
1.2.1 Classification of atherosclerosis
1.2.2 Hem odynam ic forces
1.2.3 Dyslipidem ia
1.2.4 Inflam m ation and Im m unology
1.2.5 Cell turnover
1.2.6 Matrix turnover and rem odeling
1.2.7 Neoangiogenesis
1.2.8 Clinical im plications
1.3 Plaque stability and acute throm botic events
1.3.1 Plaque erosion
1.3.2 Calcified nodule
1.3.3 Plaque rupture
1.3.3.1 Pathogenesis of plaque rupture
1.4 Extracellular m atrix hom eostasis in advanced atherosclerosis
1.4.1 Com position of the extracellular m atrix
1.4.1.1 Matricial constituents of the fibrous cap
1.4.1.2 Cellular constituents of the fibrous cap
1.4.2 Matrix degradation and its regulation
1.4.2.1 Metalloproteinases
1.4.2.2 Serine proteinases
1.4.2.3 Cysteine proteinases
1.5 Thesis perspectives and outline
1.6 References
1.1 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 2002.13 Almost one third of these deaths could be related to ischemic heart
disease and another 25% to stroke (figure 1.2).
W hile many genetic predispositions, such as familial hypercholesterolemia and type I diabetes, have been shown to play a role in the susceptibility for
atherosclerosis14-17, 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.18-20 These “life-style” factors are often
reversible and should be subject to aggressive prevention strategies by health
Cancer 38,087 (27%) Cardiovascular disease 48,799 (34% ) Other 41,958 (30% ) Respiratory disease 13,511 (9% ) Ischemic heart disease 15,973 (33% ) Stroke 12,343 (25%) Other cardiovascular disaese 20,483 (42% ) Cancer 38,087 (27%) Cardiovascular disease 48,799 (34% ) Other 41,958 (30% ) Respiratory disease 13,511 (9% ) Ischemic heart disease 15,973 (33% ) Stroke 12,343 (25%) Other cardiovascular disaese 20,483 (42% ) 80+ years 65-79 years 50-64 years <50 years M o rt a lit y p e r 1 0 0 ,0 0 0 ( lo g s c a le ) 80+ years 65-79 years 50-64 years <50 years M o rt a lit y p e r 1 0 0 ,0 0 0 ( lo g s c a le )
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 2002.
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
1999.13 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
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.21
1.2 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
M o rt a lit y p e r 1 0 0 ,0 0 0 ( lo g s c a le )
Ischemic heart disease
Cerebrovascular disaese Other cardiovascular disaese
M o rt a lit y p e r 1 0 0 ,0 0 0 ( lo g s c a le )
Ischemic heart disease
Cerebrovascular disaese Other cardiovascular disaese
M o rt a lit y p e r 1 0 0 ,0 0 0 ( lo g s c a le )
Ischemic heart disease
Cerebrovascular disaese Other cardiovascular disaese
promote permeability of the vascular endothelium and subsequent deposition of
lipoproteins in the subendothelial matrix.23, 24 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 1856 25 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
arterial thrombosis (figure 1.4).27, 28
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.33-35 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.37-39 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
1995.
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).
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.
1.2.1 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 12-14 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’
phenotype41 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
Alternatively, in 2000 Virmani and colleagues proposed a classification to describe advanced atherosclerotic lesions more adequately with regard to plaque
stability.42, 43 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.
1.2.2 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-ȕ, eNOS, PGI2 thioredoxin)49-51, low, oscillatory
shear stress induces the expression of various cell adhesion molecules (e.g.
VCAM-1, ICAM-1)52-54 chemokines (MCP-1)55, cytokines (TNF-Į)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 §1.3.
1.2.3 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
cardiovascular diseases.61, 62 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
proteoglycans24, 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.66 67 68
(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-1.73 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 compartment74 and excreted oxidized fatty acids and sterols can
chemoattract and activate both of monocytes and vSMCs.75-77 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,80-82 but it impairs clearance of the apoptotic
remnants as well, further enhancing inflammation83 (see also §1.2.6).
1.2.4 Inflammation and Immunology
Although Virchow already described inflammation in fibroatheromatous
lesions as early as the mid-19th century25, 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
lipid-rich 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, 84-86 on
the dysfunctional endothelium and release of a variety of chemokines (e.g. MCP-1,
IL-8, Fractalkine, GM-CSF)67, 87, 88 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
proteases that are involved in cell and matrix turnover.89, 90
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
acceptors HDL and apoAI,92, 93 and by reduced expression of the ATP binding
cassettes ABCA1 and ABCG1.94, 95 The latter are regulated by the nuclear receptors
PPAR-Į and –Ȗ94, which also exert anti-inflammatory actions by interfering with the
actions of NFțB and activator protein-1 (AP-1) family members, thus inhibiting
expression of IFN-Ȗ, IL1ȕ, MMP-9, iNOS and CCR2.96-99
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-Į, IL-1ȕ, IL-18)101-103 and induce
programmed cell death (TNF-Į, FasL, TRAIL)35, 104-106, 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-ȕ)110,
111
and matrix metalloproteinases (MMPs) and cathepsins are pivotal for elastic
lamina degradation and SMC migration (MMP-2, MMP-9, Cathepsin S)112-115,
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, §1.2.7 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 phagocytosis118, 119, while
ligation of the Toll-like receptors activates the NFțB 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
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
systemic immune activation to the clinical manifestations of atherosclerosis.130, 131
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.132-134 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 TCRĮȕ+, 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-Ȗ, IL-2
and TNF-Į, therewith perpetuating inflammation.137-139 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 cytokines141, while apoE
deficient mice express Th2 associated cytokines only in extreme
hypercholesterolemia.142
1.2.5 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.145-148 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. 151-154
cellular swelling and loss of membrane integrity.155, 156 The subsequent release of cellular contents is accompanied by an inflammatory response and the chemo-attraction 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 7.157 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
(TNFĮ, 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.162-164 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 stress166 as will be discussed in §1.3 and §1.4.
1.2.6 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 elastin168, 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.171-173 Proteolytic enzymes, such
as the matrix metalloproteinase (MMP) family and cathepsins, act in concert to
degrade specific ECM components (see §1.4).174, 175 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
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.43
1.2.7 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)-1Į 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
lesional neovessels.188, 189
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.195
1.2.8 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
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,
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
account for a terminal arrhythmic episode.207, 208
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;108:1664.
1.3.1 Plaque erosion
An estimated 30-40% of culprit lesions in myocardial infarction could be
appointed to non-ruptured plaques.205, 209 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
1.3.2 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.211
1.3.3 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.
1.3.3.1 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
cell turnover and proteolytic activity.174, 214
Extensive apoptosis of both macrophages and smooth muscle cells have been reported in ruptured plaques, extending necrotic core volume and reducing
thickness and strength of the fibrous cap, respectively.163, 164, 215, 216 Virchow
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
pro-inflammatory cytokines such as IFN-Ȗ,219 or indirectly, by decreased expression of
Hsp47, a chaperone protein ensuring the post-translational modification of
procollagen Į-chains.220 Besides affecting cell turnover and deposition of ECM
constituents, mediators of inflammation, including TNF-Į, IL1ȕ, 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.222-225 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.
1.4 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,
i.e. ECM proteins, matrix degrading enzymes and growth factors.219, 228, 229 230
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
actively involves the ECM in cell function and turnover.178, 233-235
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).236-238 Disruption
from fibronectin or dissociation of FAK results in accelerated SMCs apoptosis239, 240,
whilst binding of ECM degradation products to for instance ĮVȕ3 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
transcellular traction system for effective locomotion.242, 243
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-ȕ binding protein (LTBP)-1, therewith releasing TGF-ȕ 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
neoangiogenesis.244, 245
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.
1.4.1 Composition of the extracellular matrix
1.4.1.1 Matricial constituents of the fibrous cap
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, 246-248 In atherosclerosis PGs have
been reported to display altered characteristics such as increased affinity for
apoB100 containing lipoproteins (i.e. VLDL, LDL).24, 249
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.250-252 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
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.
ECM Homeostasis
Production Degradation
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
calcification in human plaques.257, 258
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
atherosclerosis.226, 260 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.226
1.4.1.1.1 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 proline263, is characteristic for collagen. Hence, the uptake of
proline is an established method of collagen synthesis analysis. The synthesized polypeptide-Į-chains are translocated to the lumen of the endoplasmatic reticulum,
where proline and lysine residues are hydroxylated.264-266 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 Į-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
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.
1.4.1.2 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
to the intimal cell population.150, 154
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,
ion channels and signaling molecules.269, 270
Unlike skeletal and cardiac muscle cells, which are terminally differentiated, SMCs have retained a remarkable plasticity enabling them to undergo profound but
reversible changes in phenotype in response to environmental circumstances.238, 271
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
membrane enabling them to interact with the surrounding ECM.115, 272 Besides
playing a role in matrix turnover, SMCs also actively participate in the inflammatory response by producing cytokines, chemokines and adhesion molecules including
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 Į-SM-actin, SM22Į
and desmin.277 By contrast, other cellular proteins are selectively upregulated in ECM
synthesizing cells including matrix Gla protein (MGP) and osteopontin.271, 276, 278
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
SMC-rich fibrous caps lesions are prone to rupture.170
1.4.2 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
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
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 plaques281, which emphasizes the need to investigate their
function in the pathobiology of atherosclerosis.
1.4.2.1 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 Zn2+ in their active site, MMPs (also known as matrixins) form the most
prominent family of metalloproteinases and comprise at least 25 secreted or
surface-bound 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-Į and latent TGF-ȕ.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-Į.292 These cytokines act
in synergy with the growth factors PDGF and FGF-2 to promote MMP gene
expression in VSMCs.228, 293 In addition to soluble factors, interaction between
macrophages, MMP-9 is the most abundant gelatinase and can be upregulated via
TNF-Į and modified LDL.148, 295, 296 The fibrogenic TGF-ȕ inhibits cytokine-mediated
induction of MMP-12.297 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 NFțB sites in
MMP-1, -3 and -9.299-302
Group MMPs Trivial names Principal known substrates
Collagenases MMP-1 Interstitial
Collagenase
Collagen type I, II, III, VII, VIII and X, gelatin, MMP-2 and -9
MMP-8 Neutrophil
Collagenase 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
