Maria Fazia

The Receptor RAGE as a Progression Factor Amplifying Arachidonate-Dependent Inflammatory and Proteolytic Response in Human Atherosclerotic Plaques Role of Glycemic Control

Background—RAGE (receptor for advanced glycation end products [AGEs]) plays a role in diabetic atherosclerosis. Recently, we have demonstrated enhanced expression of cyclooxygenase-2 and PGE synthase-1 (COX-2/mPGES-1) in human symptomatic plaques, and provided evidence that it is associated with metalloproteinase (MMP)-induced plaque rupture. However, the specific transmembrane signaling pathway(s) influencing plaque COX-2/mPGES-1 expression is unknown. The aim of this study was to characterize RAGE expression in human plaques and to correlate it with the inflammatory infiltration, COX-2/mPGES-1 and MMP expression, and with clinical evidence of diabetes. Methods and Results—Plaques obtained from 60 patients undergoing carotid endarterectomy were divided into diabetic and nondiabetic according to clinical evidence of type 2 diabetes. Plaques were subjected to analysis of RAGE, NF-&kgr;B, COX-2/mPGES-1, MMP-2 and MMP-9, lipid and oxidized LDL (oxLDL) content, and collagen content by immunohistochemistry and Western blot, whereas zymography was used to detect MMP activity. Immunohistochemistry was used to identify CD68+ macrophages, CD3+ T-lymphocytes, smooth muscle cells (SMCs), and HLA-DR+ inflammatory cells. Diabetic plaques had more (P <0.0001) macrophages, T-lymphocytes, and HLA-DR+ cells, more (P <0.0001) immunoreactivity for RAGE, activated NF-&kgr;B, COX-2/mPGES-1, and MMPs, increased (P <0.0001) gelatinolytic activity, reduced (P <0.0001) collagen content, and increased (P <0.0001) lipid and oxLDL content. Interestingly, RAGE, COX-2/mPGES-1, and MMP expression was linearly correlated with plasma level of HbA1c. Conclusions—In conclusion, this study demonstrates in humans that RAGE overexpression is associated with enhanced inflammatory reaction and COX-2/mPGES-1 expression in diabetic plaque macrophages, and this effect may contribute to plaque destabilization by inducing culprit metalloproteinase expression.

Association between enhanced soluble CD40L and prothrombotic state in hypercholesterolemia : Effects of statin therapy

Background—Hypercholesterolemia is associated with inflammation and the prothrombotic state. CD40-CD40 ligand (CD40L) interactions promote a prothrombotic response in nucleated cells. The aim of this study was to characterize the in vivo expression of soluble CD40L (sCD40L) in hypercholesterolemia, to correlate it with the extent of the prothrombotic state, and to investigate whether it may be modified by statins. Methods and Results—We studied 80 hypercholesterolemic patients and 80 matched healthy subjects. Hypercholesterolemic subjects had enhanced levels of sCD40L, factor VIIa (FVIIa), and prothrombin fragment 1+2 (F1+2) compared with healthy subjects. sCD40L correlated with total cholesterol and LDL cholesterol. Moreover, sCD40L was positively associated with in vivo platelet activation, as reflected by plasma P-selectin and urinary 11-dehydro-thromboxane B2, and with procoagulant state, as reflected by FVIIa and F1+2. Inhibition of cholesterol biosynthesis by pravastatin or cerivastatin was associated with comparable, significant reductions in sCD40L, FVIIa, and F1+2. Conclusions—This study suggests that sCD40L may represent the molecular link between hypercholesterolemia and the prothrombotic state and demonstrates that statin therapy may significantly reduce sCD40L and the prothrombotic state.

Suppression of the functionally coupled cyclooxygenase-2/prostaglandin E synthase as a basis of simvastatin-dependent plaque stabilization in humans.

Background—The clinical benefits of statins are attributed to changes in plaque composition that lead to reduced metalloproteinase (MMP) activity and plaque stabilization. However, the molecular mechanism of this effect is unclear. Recently, we demonstrated enhanced expression of isoforms of inducible cyclooxygenase (COX) and PGE synthase (COX-2/mPGES) in human symptomatic plaque and provided evidence that this is associated with MMP-induced plaque rupture. The aim of this study was to characterize the effect of simvastatin on inflammatory infiltration and the expression of COX-2/mPGES and MMPs in human carotid plaques. Methods and Results—Seventy patients with symptomatic carotid artery stenosis were randomized to the American Heart Association Step 1 diet plus simvastatin (40 mg/d) or the American Heart Association Step 1 diet alone for 4 months before endarterectomy. Plaques were subjected to analysis of COX-1, COX-2, mPGES, MMP-2 and MMP-9, lipid and oxidized LDL (oxLDL) content, and collagen content by immunocytochemistry, Western blot, and reverse transcription–polymerase chain reaction, whereas zymography was used to detect MMP activity. Immunocytochemistry was also used to identify CD68+ macrophages, CD3+ T-lymphocytes, smooth muscle cells (SMCs), and HLA-DR+ inflammatory cells. Plaques from the simvastatin group had fewer (P <0.0001) macrophages, T-lymphocytes, and HLA-DR+ cells; less (P <0.0001) immunoreactivity for COX-2/mPGES and MMPs; reduced (P <0.0001) gelatinolytic activity; increased (P <0.0001) collagen content; and reduced (P <0.0001) lipid and oxLDL content. Interestingly, COX-2/mPGES inhibition by simvastatin was completely reversed by mevalonate in vitro. Conclusions—This study demonstrates that simvastatin decreases inflammation and inhibits COX-2/mPGES expression in plaque macrophages, and this effect in turn may contribute to plaque stabilization by inhibition of MMP-induced plaque rupture.

Blockade of the Angiotensin II Type 1 Receptor Stabilizes Atherosclerotic Plaques in Humans by Inhibiting Prostaglandin E2–Dependent Matrix Metalloproteinase Activity

Background—Clinical trials have demonstrated that agents that inhibit the angiotensin II pathway confer benefit beyond the reduction of blood pressure alone. However, the molecular mechanism underlying this effect has yet to be investigated. Recently, we have demonstrated enhanced expression of inducible cyclooxygenase (COX) and prostaglandin (PG)E2-dependent synthase (COX-2/mPGES-1) in human symptomatic plaques and provided evidence that it is associated with metalloproteinase (MMP)-induced plaque rupture. Thus, the aim of this study was to characterize the effect of the angiotensin II type 1 (AT1) receptor antagonist irbesartan on the inflammatory infiltration and expression of COX-2/mPGES-1 and MMPs in human carotid plaques. Methods and Results—Seventy patients with symptomatic carotid artery stenosis were randomized to irbesartan (300 mg/d) or chlorthalidone (50 mg/d) for 4 months before endarterectomy. Plaques were subjected to analysis of COX-1, COX-2, mPGES-1, MMP-2, and MMP-9, angiotensin II, AT1, AT2, and collagen content by immunocytochemistry, Western blot, and reverse-transcriptase polymerase chain reaction, whereas zymography was used to detect MMP activity. Immunohistochemistry was also used to identify CD68+ macrophages, CD3+ T lymphocytes, smooth muscle cells (SMCs), and HLA-DR+ inflammatory cells. Plaques from the irbesartan group had fewer (P <0.0001) macrophages, T lymphocytes, and HLA-DR+ cells; less (P <0.0001) immunoreactivity for COX-2/mPGES-1 and MMPs; reduced (P <0.0001) gelatinolytic activity; and increased (P <0.0001) collagen content. It is worth noting that COX-2/mPGES-1 inhibition was observed after incubation in vitro with irbesartan but not with the selective AT2 blockade PD123,319. Conclusions—This study demonstrates that irbesartan decreases inflammation and inhibits COX-2/mPGES-1 expression in plaque macrophages, and this effect may in turn contribute to plaque stabilization by inhibition of MMP-induced plaque rupture.

Suppression of Rage as a Basis of Simvastatin-Dependent Plaque Stabilization in Type 2 Diabetes

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-&kgr;B, 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.

Balance Between PGD Synthase and PGE Synthase Is a Major Determinant of Atherosclerotic Plaque Instability in Humans

Objective—Inducible cyclooxygenase (COX-2) catalyzes the first step in prostanoid biosynthesis and is considered a proinflammatory enzyme. COX-2 and type 1 inducible PGE synthase (mPGES-1) have a role in metalloproteinase (MMP) release leading to plaque rupture. In contrast, lipocalin-type PGD synthase (L-PGDS) has been shown to exert antiinflammatory actions. Thus, in this study we investigated whether a shift from a PGDS-oriented to a PGES-oriented profile in arachidonate metabolism leads to inflammatory activation in rupture-prone plaque macrophages. Methods and Results—Atherosclerotic plaques were obtained from 60 patients who underwent carotid endarterectomy, symptomatic (n= 30) and asymptomatic (n= 30) according to evidence of recent transient ischemic attack or stroke. Plaques were analyzed for COX-2, mPGES-1, L-PGDS, PPARγ, IκB&agr;, NF-κB, and MMP-9 by immunocytochemistry, Western blot, reverse-transcriptase polymerase chain reaction, enzyme immunoassay, and zymography. Prostaglandin E2 (PGE2) pathway was significantly prevalent in symptomatic plaques, whereas PGD2 pathway was overexpressed in asymptomatic ones, associated with NF-κB inactivation and MMP-9 suppression. In vitro COX-2 inhibition in monocytes was associated with reduced MMP-9 release only when PGD2 pathway overcame PGE2 pathway. Conclusions—These results suggest that COX-2 may have proinflammatory and antiinflammatory properties as a function of expression of downstream PGH2 isomerases, and that the switch from L-PGDS to mPGES-1 in plaque macrophages is associated with cerebral ischemic syndromes, possibly through MMP-induced plaque rupture.

Preprocedural Level of Soluble CD40L Is Predictive of Enhanced Inflammatory Response and Restenosis After Coronary Angioplasty

Background—Inflammation plays a pathogenic role in the development of restenosis after percutaneous transluminal coronary angioplasty (PTCA). CD40–CD40L interaction is involved in the pathogenesis of atherosclerosis; however, its role in the pathophysiology of restenosis is still unclear. We tested the hypothesis that soluble CD40L (sCD40L) may be involved in the process of restenosis and that it exerts its effect by triggering a complex group of inflammatory reactions on endothelial and mononuclear cells. Methods and Results—We studied 70 patients who underwent PTCA and who had repeated angiograms at 6-month follow-up. Plasma sCD40L was measured before and 1, 5, 15, and 180 days after PTCA, whereas plasma soluble intercellular adhesion molecule-1, soluble vascular cell adhesion molecule-1, E-selectin, and monocyte chemoattractant protein (MCP)-1 were measured before and 24 hours after PTCA. Furthermore, the release of adhesion molecules and MCP-1 and the ability to repair an injury in endothelial cells, as well as the generation of O2− in monocytes, were analyzed in vitro after stimulation with serum from patients or healthy control subjects. Restenosis occurred in 18 patients (26%). Restenotic patients had preprocedural sCD40L significantly higher than patients with favorable outcomes (2.13±0.3 versus 0.87±0.12 ng/mL, P <0.0001). Elevated sCD40L at baseline was significantly correlated with adhesion molecules and MCP-1 generation after PTCA and with lumen loss at 6-month follow-up. Furthermore, high sCD40L was directly associated in vitro with adhesion molecules and MCP-1 generation and impaired migration in endothelial cells and with enhanced O2− generation in monocytes. Conclusions—We conclude that increased sCD40L is associated with late restenosis after PTCA. This may provide an important biochemical link between restenosis and aspirin-insensitive platelet activation. These results provide a rationale for studies with new antiplatelet treatments in patients who underwent PTCA.

Increased Expression of Transforming Growth Factor-β1 as a Stabilizing Factor in Human Atherosclerotic Plaques

Background and Purpose— Transforming growth factor-β (TGF-β) is a growth factor/cytokine involved in vascular remodeling and atherogenesis. Recent studies in apolipoprotein E-deficient mice have demonstrated a pivotal role of TGF-β in the maintenance of the balance between inflammation and fibrosis in atherosclerotic plaques. Furthermore, inhibition of TGF-β signaling has been shown to accelerate plaque formation and its progression toward an unstable phenotype in mice. However, if this mechanism is operative also in humans is still unknown. The aim of this study was to characterize the expression of TGF-β1 in human carotid plaque and to correlate it with the extent of inflammatory infiltration and collagen content with the clinical signs of plaque instability. Methods— Plaques were obtained from patients undergoing carotid endoarterectomy and divided into symptomatic and asymptomatic according to clinical evidence of recent transient ischemic attack or stroke. Plaques were analyzed for TGF-β1 expression by Immunocytochemistry, Western, and Northern blotting analysis. Immunocytochemistry was used to identify CD68+ macrophages, CD3 T lymphocytes, HLA-DR+ cells, and α-smooth muscle cells. Procollagen and interstitial collagen content were analyzed by immunohistochemistry and Sirius Red staining, respectively. Results— Plaque TGF-β1 mRNA was increased up to 3-fold in asymptomatic as compared with symptomatic plaques. Plaques from asymptomatic group had fewer (P<0.0001) macrophages and T lymphocytes compared with symptomatic plaques. TGF-β1 gene was transcriptionally active as demonstrated by increased (P<0.0001) TGF-β1 protein expression in asymptomatic plaques. Immunohistochemistry showed that TGF-β was mainly expressed in plaque shoulder and was associated with a comparable increase (P<0.0001) in plaque procollagen and collagen content. Conclusions— In conclusion, this study demonstrates the higher expression of TGF-β1 in human asymptomatic lesions and provides evidence that TGF-β1 may play an important role in the process of plaque stabilization.

Association Between Prostaglandin E Receptor Subtype EP4 Overexpression and Unstable Phenotype in Atherosclerotic Plaques in Human

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.

COX-2 and atherosclerosis.

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.