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Cyclooxygenase (COX) is the rate limiting enzyme catalyzing the conversion of arachidonic acid into prostanoids, lipid mediators critically implicated in a variety of physiological and pathophysiological processes, including inflammation, vascular and renal homeostasis, and immune responses. Since the early 1990s it has been appreciated that two isoforms of COX exist, referred to as COX-1 and COX-2. Although structurally homologous, COX-1 and COX-2 are regulated by two independent and quite different systems and have different functional roles. In the setting of acute ischemic syndromes it has been recognized that COX pathway plays an important role; however, whereas the function of platelet COX-1 in acute ischemic diseases is firmly established, the role of COX-2 in atherothrombosis remains controversial. The complex role of COX-2 in this setting is also confirmed by the unexpected cardiovascular side effects of long-term treatment with COX-2 inhibitors. In this article, we review the pattern of expression of COX-2 in the cellular players of atherothrombosis, its role as a determinant of plaque vulnerability, the effects of the variable expression of upstream and downstream enzymes in the prostanoid biosynthesis on COX-2 expression and inhibition.

Objective: Receptor for advanced glycation end products (AGEs) (RAGE) plays a central role in the process of plaque rupture in diabetic patients. Recently, it has been reported that RAGE may be downregulated by improving glycemic control. In contrast, despite being well known that RAGE may be induced in human vessels in a glucose-independent fashion, also by myeloperoxidase (MPO)-dependent AGE generation, no data exist regarding the possibility of a pharmacological modulation of glucose-independent RAGE generation. Thus, the aim of this study was to characterize the effect of simvastatin on the expression of RAGE and RAGE-dependent plaque-destabilizing genes in human atherosclerotic plaques., Methods and Results: Seventy type 2 diabetic patients with asymptomatic carotid artery stenosis (>70%) were randomized to American Heart Association (AHA) step 1 diet plus simvastatin (40 mg/d) or AHA step 1 diet alone for 4 months before endarterectomy. Plaque expression of MPO, AGEs, RAGE, NF-kappaB, COX-2, mPGES-1, matrix metalloproteinase (MMP)-2 and MMP-9, lipid and oxidized LDL (oxLDL) content, procollagen 1, and interstitial collagen was analyzed by immunohistochemistry and Western blot; zymography was used to detect MMP activity. Plaques from the simvastatin group had less (P<0.0001) immunoreactivity for MPO, AGEs, RAGE, p65, COX-2, mPGES-1, MMP-2, and MMP-9, lipids and oxLDL; reduced (P<0.0001) gelatinolytic activity; increased (P<0.0001) procollagen 1 and collagen content; and fewer (P<0.0001) macrophages, T-lymphocytes, and HLA-DR+ cells. Of interest, RAGE inhibition by simvastatin, observed not only in plaque sections but also in plaque-derived macrophages, was reverted by addition of AGEs in vitro., Conclusions: This study supports the hypothesis that simvastatin inhibits plaque RAGE expression by decreasing MPO-dependent AGE generation. This effect in turn might contribute to plaque stabilization by inhibiting the biosynthesis of PGE2-dependent MMPs, responsible for plaque rupture.

The inducible isoform of cyclooxygenase-2 (COX-2) plays a role in pathophysiologic processes like inflammation and pain but is also constitutively expressed in tissues such as the kidney or vascular endothelium, where it exerts important physiologic functions. Although much evidence exists that implicates COX-2 in atherosclerosis, its role in this setting remains substantially uncertain. This observation is also confirmed by the results of clinical trials of selective COX-2 inhibitors. Treatment with these drugs, developed with the assumption that they would be as effective as nonselective COX inhibitors but without their gastrointestinal side effects, has been reported to be associated with an increased cardiovascular risk. In this article, we review the pattern of expression of COX-2 in the cellular players of atherothrombosis, its role as a determinant of plaque vulnerability, and the vascular effects on prostanoid inhibition by COX-2 inhibitors.

Several clinical trials have shown that agents blocking the renin-angiotensin system reduce the incidence of acute ischaemic events. This effect was independent from blood pressure reduction and was presumably related to plaque stabilisation. With the aim of investigating potential mechanisms underlying this effect, carotid plaques were analysed in a recent study from patients randomised to treatment with the angiotensin receptor blocker irbesartan, or the diuretic chlorthalidone for 4 months before carotid endarterectomy. It was found that irbesartan decreased inflammatory infiltration, increased collagen content and downregulated prostaglandin E2-dependent metalloproteases as a consequence of suppression of inducible COX-2/prostaglandin E synthase. This article reviews the results of this study and the most recent evidence that supports the possibility that angiotensin II receptor blockers represent a novel therapy for plaque stabilisation.

Inflammation plays a central role in the development of atherosclerotic disease, from the early phases of lesion formation to plaque disruption, the main underlying cause of acute ischemic syndromes. Arachidonic acid metabolism is implicated in the pathophysiology of ischemic syndromes affecting the coronary or cerebrovascular territory, as demonstrated by biochemical measurements of eicosanoid biosynthesis and the results of inhibitor trials in these settings. In particular, much attention has been focused on the pathway catalyzed by cyclooxygenase (COX), which leads to the generation of a variety of lipid mediators known as prostanoids. Two COX isozymes have been characterized, COX-1 and COX-2, that differ in terms of regulatory mechanisms of expression, tissue distribution, substrate specificity, and preferential coupling to upstream and downstream enzymes. Whereas the role of platelet COX-1 in acute ischemic diseases is established, the role of COX-2 in atherothrombosis remains unclear. In this article, we summarize the findings from our group suggesting a crucial role for COX-2 in modulating atherosclerotic plaque stability or instability, according to the variable expression of upstream and downstream enzymes in the prostanoid biosynthesis.

Microalbuminuria is the earliest clinical evidence of diabetic nephropathy, but the mechanisms linking hyperglycemia and kidney complications are not clear. The aim of this study was to evaluate whether enhanced oxidative stress in patients with microalbuminuria can contribute to diabetic nephropathy development through downregulation of the antiapoptotic gene Bcl-2 that promotes in turn a pro-inflammatory status. We studied 30 patients with type 1 diabetes (15 with and 15 without microalbuminuria) compared to 15 matched healthy controls. Plasma oxidant status, and expression of Bcl-2, activated NF-kB, inducible Nitric Oxide synthase (iNOS), and monocyte chemoattractant protein (MCP)-1 in circulating monocytes were evaluated at baseline and after 8-week oral vitamin E treatment (600 mg b.i.d.). Bcl-2 expression was significantly reduced in microalbuminuric diabetic patients as a consequence of increased oxidant burden secondary to persistent hyperglycemia. Bcl-2 down-regulation was associated with enhanced expression of NF-kB, iNOS and MCP-1, and showed a strong correlation with the albumin excretion rate. Low Bcl-2 expression and high inflammatory status were normalized by vitamin E both in vivo and in vitro. Our study showed that Bcl-2 down-regulation in diabetic patients with poor glycemic control results in the activation of the NF-kB pathway leading to the development of nephropathy. Vitamin E might provide a novel form of therapy for prevention of nephropathy in diabetic patients in which an acceptable glycemic control is difficult to achieve despite insulin therapy.

Objective: We recently demonstrated that inducible cyclooxygenase/PGE synthase-1 (COX-2/mPGES-1) are overexpressed in symptomatic plaques in association with PGE2-dependent metalloproteinase (matrix metalloproteinase [MMP]) biosynthesis and plaque rupture. However, it is not known which of the 4 PGE2 receptors (EP1-4) mediates macrophage metalloproteinase generation. The aim of this study was to characterize EP1-4 expression in plaques from symptomatic and asymptomatic patients undergoing carotid endarterectomy and correlate it with the extent of inflammatory infiltration, COX-2/mPGES-1 and MMP expression and clinical features of patients' presentation., Methods and Results: Plaques were analyzed for COX-2, mPGES-1, EP1-4, MMP-2, and MMP-9 by immunohistochemistry, reverse-transcription polymerase chain reaction and Western blot; zymography was used to detect MMP activity. We observed strong EP4 immunoreactivity, only very weak staining for EP2, and no expression of EP1 and EP3 in atherosclerotic plaques. EP4 was more abundant in MMP-rich symptomatic lesions, whereas EP2 was no different between symptomatic and asymptomatic plaques. Finally, MMP induction by PGE2 in vitro was inhibited by the EP4 antagonist L-161 982, but not by its inactive analog L-161 983 or by the EP2 antagonist AH6809., Conclusions: This study shows that EP4 overexpression is associated with enhanced inflammatory reaction in atherosclerotic plaques. This effect might contribute to plaque destabilization by inducing culprit metalloproteinase expression.

Objective: The participation of 5-lipoxygenase (5-LO) in the development of atherosclerosis has been suggested by recent studies. However, a role for 5-LO as a modulator of atherosclerotic plaque instability has not been previously reported in humans. Thus, the aims of this study was to analyze the expression of 5-LO in human carotid plaques and to investigate the mechanism by which this enzyme could lead to plaque instability and rupture., Methods and Results: We obtained atherosclerotic plaques from 60 patients undergoing carotid endarterectomy. We divided the plaques into symptomatic and symptomatic according to clinical evidence of plaque instability. Clinical evidence of plaque instability was provided by the assessment of recent ischemic symptoms attributable to the stenosis and by the presence of ipsilateral cerebral lesion(s) determined by computed tomography. Plaques were analyzed for CD68+ macrophages, CD3+ T cells, alpha-actin+ smooth muscle cells, 5-LO, cyclooxygenase 2, matrix metalloproteinase (MMP)-2, and MMP-9 by immunohistochemical, immunoblotting, and densitometric analyses. MMP activity was assessed by zymography. Leukotriene (LT) B4 and collagen were quantified by ELISA and Sirius red polarization, respectively. The percentage of macrophage-rich and T-cell-rich areas was larger in symptomatic compared with asymptomatic patients (25+/-6% versus 8+/-4%, P<0.0001, and 74+/-17 versus 18+/-4 cell/mm2, P<0.003). 5-LO expression was higher in symptomatic compared with asymptomatic plaques (24+/-4% versus 6+/-3%, P<0.0001) and was associated with increased MMP-2 and MMP-9 expression (27+/-4% versus 7+/-3%, P<0.0001, and 29+/-5% versus 8+/-2%, P<0.0001) and activity and with decreased collagen content (6.9+/-2.4% versus 17.8+/-3.1%, P<0.01). Immunofluorescence showed that 5-LO and MMPs colocalize in activated macrophages. Notably, higher 5-LO in symptomatic plaques correlated with increased LTB4 production (18.15+/-3.56 versus 11.27+/-3.04 ng/g tissue, P<0.0001)., Conclusions: The expression of 5-LO is elevated in symptomatic compared with asymptomatic plaques and is associated with acute ischemic syndromes, possibly through the generation of LTB4, subsequent MMP biosynthesis, and plaque rupture.