Associate Editor: K. E. SucklingCyclooxygenase and prostaglandin synthases in atherosclerosis: Recent insights and future perspectives
Introduction
Atherosclerosis is a complex disease and the precise molecular mechanisms contributing to its pathogenesis are not completely defined (Libby et al., 2002). While atherosclerosis was traditionally considered a mere lipid storage disorder, solid evidence in the last decades has led to the concept of atherosclerosis as a chronic inflammatory disease. In fact, inflammation characterizes all the phases of atherosclerosis progression, from the initial formation of the early lesions to the rupture of advanced plaques, both at systemic and local level. Indeed, because the systemic status of a patient has been associated with the incidence of plaque rupture, there has been much interest in potential circulating markers able to identify subjects at increased risk of plaque rupture. In this context, it has been shown that upregulation of inflammatory mediators such as C reactive protein (CRP), adhesion molecules [soluble intercellular adhesion molecule (sICAM)-1, soluble vascular cell adhesion molecule (sVCAM)-1, P-selectin], interleukin (IL)-6, IL-18, tumor necrosis factor (TNF)-α, and soluble CD40L predict future cardiovascular risk in a variety of clinical settings. From a local perspective, atherosclerotic plaques prone to rupture have histological characteristics that are different from stable plaques. Indeed, it has been widely shown that an inflammatory infiltrate, mainly composed of activated macrophages and T-lymphocytes, is present in all atherosclerotic plaques, but it is markedly more abundant in unstable than in stable plaques, and this strongly supports the crucial role of inflammation in plaque instability (Shah, 2003).
Several mechanisms linking inflammation to plaque instability have been proposed so far. In this context, it is recognized that important inflammatory enzymes and lipid mediators playing a crucial role in the pathophysiology of atherosclerosis progression derive from the cascade of arachidonic acid, a polyunsaturated fatty acid present in the phospholipids of cell membranes. This acid can be metabolized by different pathways originating a variety of biologically important compounds collectively known as prostanoids.
In recent years, we investigated some potential molecular mechanisms of atherosclerotic plaque instability (reviewed in Cipollone et al., 2005a, Cipollone et al., 2005b), all related to the expression of the inducible isoform of cyclooxygenase (COX), COX-2, the key enzyme in the conversion of arachidonic acid to prostanoids. The role of this protein in atherothrombosis appears to be quite complex, as it is an intermediate enzyme in the cascade of arachidonic acid and may generate not only proinflammatory but also anti-inflammatory compounds, according to the downstream synthase that is co-expressed with it.
In this article we review the role of cyclooxygenase-2 and downstream synthases in the vascular system and in atherothrombosis, summarizing our newest findings linking COX-2 to different plaque phenotypes.
The first steps of atherogenesis occur as a result of injury to endothelium by several factors, including elevated and modified low density lipoproteins (LDL), excessive blood pressure, free radicals, the products of glycoxidation associated with hyperglycemia, infectious agents, shear stress, or proinflammatory cytokines derived from excess adipose tissue. Following these stimuli, the endothelium undergoes rapid phenotypic changes, leading to activation of endothelial cells and impairment of their physiological functions, i.e. maintenance of blood circulation and fluidity, regulation of vascular tone, and inflammatory responses (Libby et al., 2002). Thus, homeostatic imbalance, impairment in vasorelaxation, oxidative stress and increased adhesiveness of the endothelium lining for circulating inflammatory cells become predominant in the injured vascular wall. The enhanced adhesion of blood leukocytes to the inner surface of the arterial wall is a key event in atherogenesis and is strongly promoted by the upregulation of the endothelial expression of cellular adhesion molecules, including ICAM-1, VCAM-1 and selectins. Once adherent to the endothelium, the blood leukocytes—mainly mononuclear phagocytes and T-lymphocytes—transmigrate into the tunica intima, a process promoted by chemoattractant mediators including monocyte chemotactic protein-1 (MCP-1). In the arterial wall, the blood-derived inflammatory cells promote and amplify a local inflammatory response that leads to generation of cytokines and foam cells, the lipid laden macrophages that characterize early atherosclerotic lesions. As this inflammatory process continues, the activated leukocytes and vascular cells release a variety of mediators, including growth factors that stimulate replication of smooth muscle cells (SMCs). These cells also are important players in atherosclerosis progression because of their consistent proliferation, migration from the tunica media into the intima, and synthesis of components of extracellular matrix, all of which contribute to plaque progression toward advanced lesions (Aikawa & Libby, 2004). Advanced plaques are mainly composed of connective tissue extracellular matrix (including collagen, proteoglycans, fibronectin elastic fibers), lipids (crystalline cholesterol, cholesteryl esters, phospholipids), calcium deposits, as well as inflammatory cells and SMCs (Davies, 1997). However, varying proportions of these components occur in different plaques, thus leading to a spectrum of lesion compositions. The extracellular lipids accumulated in the intima form the classic “lipid core”, a necrotic region containing oxidized lipids and prothrombotic factors, making the lipid highly thrombogenic when exposed to circulating blood. Under ill-defined circumstances, some plaques in some patients undergo physical disruption, and the subsequent formation of a superimposed platelet thrombus lead to the onset of the clinical manifestations of acute ischemic syndromes (Fuster et al., 2005).
The analysis of angiograms reveal that in about 75% of patients with acute ischemic syndromes, the culprit lesion site produced a < 70% diameter narrowing (Hackett et al., 1988). Thus plaques producing non-flow limiting stenoses account for more cases of plaque rupture and thrombosis than plaques producing a more severe stenosis. These observations support the hypothesis that plaque composition rather than plaque size is the real determinant of the plaque evolution toward rupture. Indeed, histological comparison of stable versus unstable plaques indicates that the latter have histomorphologic features quite different from intact plaques. Plaques prone to rupture are characterized by a large lipid core, often occupying > 40% plaque volume, as well as a thin fibrous cap, a structure that protects the deeper components of the plaque from contact with circulating blood. The biomechanical strength of the plaque's fibrous cap has considerable importance as a determinant of lesion stability, and thinning of the fibrous cap is generally considered to be a sign of vulnerability and a prelude to rupture. In this context, it is worth noting that fibrous caps from ruptured plaques typically contain less extracellular matrix (collagen and proteoglycans), a greater area of inflammatory cell infiltration and fewer SMCs than caps from intact plaques. Depletion of matrix components from the fibrous cap leading to cap thinning is caused, at least in part, by an imbalance between matrix synthesis and breakdown, leading to enhanced degradation and reduced synthesis of extracellular matrix. While depletion of matrix may be due to a reduction of the number or function of SMCs, enhanced matrix breakdown has been attributed to a family of matrix-degrading enzymes known as metalloproteinases (MMPs, in particular MMP-1, MMP-2, MMP-3, MMP-9) which are generated in atherosclerotic plaques mainly by inflammatory cells (macrophages, foam cells) and to a lesser extent by SMCs and endothelial cells (Jones et al., 2003). This family of enzymes plays a major role in both physiological and pathological vascular remodelling, entailing degradation and reorganization of the extracellular matrix (ECM) scaffold of the vessel wall. The activity of MMPs is tightly regulated at the level of gene transcription and is also regulated by their secretion in an inactive zymogen form that requires extracellular activation. Latent MMPs can be activated by plasmin, trypsin, and chymase (derived from degranulating mast cells), oxidized lipids, reactive oxygen species, chlamydial heat shock protein, CD40 ligation, inflammatory cytokines, and hemodynamic stress, all existing in atherosclerotic plaques. Finally, MMP generation and activity are also modulated by co-secretion of the tissue inhibitors of metalloproteinases (TIMPs). Thus, increased gene transcription, enhanced activation, and reduced activity of TIMPs can individually or together create a milieu for increased matrix proteolysis.
In the last few years, we focused our interest on two metalloproteinases, 92 kDa (MMP-9) and 72 kDa (MMP-2) gelatinases, specialized in the digestion of collagen fragments, that have been critically associated with acute ischemic events in humans (Cipollone et al., 2005a, Cipollone et al., 2005b). Firstly we characterized their expression pattern in carotid plaques from patients enlisted to undergo carotid endarterectomy for high-grade internal carotid artery stenosis. Patients were subdivided in symptomatic and asymptomatic according to NASCET classification (1991). The first group (symptomatic patients) included subjects who presented with clinical symptoms of atherothrombotic ischemic stroke or transient ischemic attack (TIA). Ischemic strokes were all well-defined atherothrombotic strokes clearly related to ulceration of a culprit plaque of the internal carotid artery and endarterectomy was performed within 120 days after the onset of symptoms in these patients. The second group (asymptomatic patients) included patients who had an asymptomatic carotid stenosis. We found that both of MMPs were expressed in the two groups of patients, but their expression and activity were markedly higher in plaques from symptomatic patients as compared to specimens from asymptomatic individuals, suggesting that MMPs play a crucial role in plaque instability. Thus the subsequent step of our research was to identify potential molecular mechanisms leading to MMP overexpression in this setting.
The starting point of our investigations was the observation that secretion of MMP-2 as well as MMP-9 by macrophages in human atherosclerotic plaques occurs through a mechanism dependent on prostaglandin (PG)E2, one of the compounds generated by the cyclooxygenase-mediated pathway (Corcoran et al., 1994). Thus, in the last years, we identified some of the mechanisms contributing to MMP overexpression in unstable plaques, all related to PGE2 biosynthetic pathway along the arachidonic acid cascade.
Section snippets
The cyclooxygenase-mediated pathway
Arachidonic acid metabolism plays an important role in the pathophysiology of acute ischemic syndromes, as compounds generated by this cascade, known as eicosanoids, are critical regulators of several pathophysiological responses, such as inflammation, blood clotting, wound healing, blood vessel tone, and immune responses. The first step in the formation of eicosanoids is the liberation of arachidonic acid from membrane-bound phospholipids, usually by the action of phospholipases, primarily
Prostaglandin E synthase
Prostaglandin E synthase catalyzes the conversion of the COX-product PGH2 to PGE2. This enzyme was characterized in 1999 by Jakobsson et al. (1999), who demonstrated for the first time that recombinant human microsomal glutathione-S-transferase (GST)-1-like 1 (MGST1-L1), a member of the MAPEG superfamily [for membrane-associated proteins involved in eicosanoid and glutathione (GSH) metabolism] was responsible for PGE2 biosynthesis. In the subsequent years, several isoforms of PGES have been
Prostacyclin synthase-mediated pathway in the vascular system
PGI2 is generated by the sequential action of COX and PGI2 synthase (PGIS), a member of the cytochrome P450 superfamily that specifically converts PGH2 to PGI2. PGIS was previously reported to be localized to plasma and nuclear membranes. In the last few years it was shown that PGIS, like its related cytochrome P450 proteins, is localized to endoplasmic reticulum but is also found at the perinuclear region including nuclear envelope (Helliwell et al., 2004). PGIS is constitutively expressed in
Prostaglandin F2α synthase
Prostaglandin F2 is synthesized via three pathways from PGE2, PGD2, or PGH2 by PGE2 9-ketoreductase, PGD2 11-ketoreductase, or PGH2 9,11-endoperoxide reductase, respectively (Watanabe, 2002). PGE2 9-ketoreductase has been demonstrated to be the key enzyme in the regulation of specific PGs in the endometrium during the perimplantation period. PGD2 11-ketoreductase exists as two isoforms: lung-type PGFS (or PGFS I) and liver-type PGFS (or PGFS II). PGFS-1 is expressed in lung and peripheral blood
Aspirin
Antithrombotic therapy is currently the cornerstone of the treatment of acute coronary syndromes. Numerous studies indicate the importance of inflammation in atherothrombosis and support therapeutic use of anti-inflammatory treatment. The use of aspirin has increased since it was shown to reduce the risk of myocardial infarction and stroke: because platelet aggregation is known to play a crucial role in thrombosis the administration of aspirin translates to clinical benefit in CAD, as
Coxibs and the message from the clinical trials
Selective inhibitors of COX-2 were developed on the basis of the assumption that COX-1 mediates the biosynthesis of physiological prostanoids that regulate vascular tone and mucosal integrity, whereas COX-2 is the inducible isoform responsible for the PG involved in inflammation. As predicted from biological experiments and animal models, specific blockade of the COX-2 system has a therapeutic anti-inflammatory effect associated with a lower risk of gastrointestinal (GI) complications,
Conclusions and future perspectives
The presence of different downstream synthases, the multiple and opposite biological effects of prostanoids, as well as the existence of different isoforms of prostanoid receptors make it difficult to define the role of COX-2 expression in atherosclerosis progression and in plaque rupture. The complexity surrounding the effects of COX-2 should be taken into consideration in the assessment of the potential benefits and risks of COX-2 inhibition. From a pharmacological standpoint, development of
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