Effect of intensive atorvastatin therapy on prostaglandin E2 levels and metalloproteinase-9 activity in the plasma of patients with non-ST-elevation acute coronary syndrome.

Inflammation plays a pivotal role in the pathophysiology of non-ST elevation acute coronary syndromes (NSTEACS). Intensive statin therapy reduces the recurrence of cardiovascular events after acute coronary syndromes. The aim of this study was to examine nuclear factor-kappa B activity in peripheral blood mononuclear cells, prostaglandin E2 (PGE2) and leukotriene B4 levels, and matrix metalloproteinase-9 (MMP-9) activity in plasma from patients with NSTEACS (at 0 days, 4 days, 2 months, and 6 months), patients with stable coronary artery disease, and healthy controls. On day 4, patients with NSTEACS were randomized to receive atorvastatin 80 mg/day (n = 14) or standard treatment (n = 16) during 2 months to study its effect on these parameters. Nuclear factor-kappa B activity (by electrophoretic mobility shift assay), PGE2 levels (by enzyme-linked immunosorbent assay), and MMP-9 activity (by gelatin zymography) in the plasma of patients with NSTEACS were significantly increased compared with patients with coronary artery disease and healthy controls. At 6 months, MMP-9 activity was normalized, whereas nuclear factor-kappa B activity and PGE2 levels were still increased. Leukotriene B4 plasma levels (by enzyme-linked immunosorbent assay) were similar in patients with NSTEACS and those with coronary artery disease but were significantly higher than those of healthy subjects. There was a significant correlation between plasma PGE2 levels and MMP-9 activity in patients with NSTEACS (r = 0.754, p <0.01). Atorvastatin 80 mg/day reduced circulating PGE2 levels (median 222.4 [interquartile range 157.4 to 253.5] vs 550.8 [276.9 to 613.0] pg/ml, p = 0.006) and MMP-9 activity (0.0025 [0.0017 to 0.0035] vs 0.0280 [0.0057 to 0.0712] arbitrary units, p = 0.03). In conclusion, nuclear factor-kappa B activity in peripheral blood mononuclear cells, and plasma PGE2 levels and MMP-9 activity, increase during NSTEACS. Atorvastatin 80 mg/day normalizes PGE2 levels and MMP-9 activity, providing additional mechanisms by which intensive atorvastatin therapy may reduce the incidence of cardiovascular events.

Atorvastatin reduces the expression of prostaglandin E2 receptors in human carotid atherosclerotic plaques and monocytic cells: potential implications for plaque stabilization.

Prostaglandin E2 (PGE2), the product of cyclooxygenase-2 (COX-2) and prostaglandin E synthase-1 (mPGES-1), acts through its receptors (EPs) and induces matrix metalloproteinase (MMP) expression, which may favor the instability of atherosclerotic plaques. The effect of statins on EPs expression has not been previously studied. The aim of this study was to investigate the effect of atorvastatin (ATV, 80 mg/d, for one month) on EP expression in plaques and peripheral blood mononuclear cells (PBMC) of patients with carotid atherosclerosis. In addition, we studied the mechanisms by which statins could modulate EPs expression on cultured monocytic cells (THP-1) stimulated with proinflammatory cytokines (IL-1beta and TNF-alpha). Patients treated with atorvastatin showed reduced EP-1 (14 +/- 1.8% versus 26 +/- 2%; P < 0.01), EP-3 (10 +/- 1.5% versus 26 +/- 1.5%; P < 0.05), and EP-4 expression (10 +/- 4.1% versus 26.6 +/- 4.9%; P < 0.05) in atherosclerotic plaques (immunohistochemistry), and EP-3 and EP-4 mRNA expression in PBMC (real time PCR) in relation to non-treated patients. In cultured monocytic cells, atorvastatin (10 micromol/L) reduced EP-1/-3/-4 expression, along with COX-2, mPGES-1, MMP-9, and PGE2 levels elicited by IL-1beta and TNF-alpha. Similar results were noted with aspirin (100 micromol/L), dexamethasone (1 micromol/L), and the Rho kinase inhibitors Y-27632 and fasudil (10 micromol/L both). The effect of atorvastatin was reversed by mevalonate, farnesyl pyrophosphate, and geranylgeranyl pyrophosphate. On the whole, we have shown that atorvastatin reduces EPs expression in atherosclerotic plaques and blood mononuclear cells of patients with carotid stenosis and in cultured monocytic cells. The inhibition of EP receptors could explain, at least in part, some of the mechanisms by which statins could modulate the COX-2/mPGES-1 proinflammatory pathway and favor plaque stabilization in humans.

Overexpression of COX-2, Prostaglandin E synthase-1 and prostaglandin E receptors in blood mononuclear cells and plaque of patients with carotid atherosclerosis: regulation by nuclear factor-kappaB.

BACKGROUND AND OBJECTIVE: Prostaglandin E2 (PGE(2), a product of the cyclooxygenase 2 (COX-2) and membrane-associated Prostaglandin E Synthase (mPGES-1) pathway, has been implicated in the instability of atherosclerotic plaques. We have studied COX-2, mPGES-1 and PGE2 receptors (EPs) expression in peripheral blood mononuclear cells (PBMC) and atherosclerotic plaques of 29 patients with carotid stenosis as well as the effect of different nuclear factor-kappaB (NF-kappaB) inhibitors on COX-2, mPGES-1 and EPs expression in cultured monocytic cells (THP-1). METHODS: COX-2, mPGES-1 and EP expression was analyzed by RT-PCR (PBMC), immunohistochemistry (plaques) and Western blot (THP-1). PGE2 levels were determined by ELISA (plasma and cell supernatants). RESULTS: In relation to healthy controls, COX-2, mPGES-1 and EP-3/EP-4 mRNA expression was increased in PBMC from patients. In the inflammatory region of atherosclerotic plaques, an increase of COX-2, mPGES-1 and EPs expression was also observed. Activated NF-kappaB and COX-2, mPGES-1 and EPs proteins were colocalized in the plaque's cells. In cytokine-treated cultured THP-1, the NF-kappaB inhibitors parthenolide, Bay 11-7082 and PDTC reduced COX-2, mPGES-1 and EP-1/EP-3/EP-4 expression as well as PGE2 levels. By employing specific agonists and antagonists, we noted that the cytokine- and PGE2-induced metalloproteinase 9 (MMP-9) expression and activity occurs through EP-1/EP-3/EP-4, an effect downregulated by NF-kappaB inhibitors. CONCLUSIONS: Patients with carotid atherosclerosis depict an overexpression of COX-2, mPGES-1 and EPs simultaneously in the PBMC as well as in the vulnerable region of plaques. The studies in cultured monocytic cells suggest that NF-kappaB inhibitors and/or EPs antagonists could represent a novel therapeutic approach to the treatment of plaque instability and rupture.

HMG-CoA reductase inhibitors reduce I kappa B kinase activity induced by oxidative stress in monocytes and vascular smooth muscle cells.

Reactive oxygen species, such as superoxide anion (O2-) and hydrogen peroxide (H2O2), may act as second messengers of intracellular signaling and play a key role in the pathogenesis of atherosclerosis. The nuclear factor kappaB (NF-kappa B) is a redox-sensitive transcription factor that is involved in this process. The aim of the present study was to investigate the molecular mechanisms of action of statins on cultured vascular smooth muscle cells (VSMC) and monocytic cells (THP-1) under oxidative stress. In THP-1 and cultured VSMC, O2- caused an increase in NF-kappa B activation (P < 0.05) that was correlated with inhibitory I kappa B-alpha degradation. Atorvastatin or simvastatin decreased NF-kappa B activation induced by oxidative stress by around 50% in both cell types and was correlated with the I kappa B-alpha levels. In monocytes, O2- increased I kappa B kinase (IKK)-1 and IKK-2 activity (P < 0.05) and p38 and p42/44 activation and phosphorylation, which was reduced by statins. PD 98059 (p42/44 inhibitor) and SB20358 (p38 inhibitor) decreased NF-kappa B binding activity and prevented I kappa B-alpha degradation. However, we only observed a reduction in IKK-1 and IKK-2 activity with PD98059. Statins diminish NF-kappa B activation elicited by oxidative stress through the inhibition of IKK-1/-2, p38, and p42/44 activation. These data may help to further understand the molecular mechanisms of statins in cardiovascular disease.

Possible role of parathyroid hormone-related protein as a proinflammatory cytokine in atherosclerosis.

BACKGROUND AND PURPOSE: Parathyroid hormone-related protein (PTHrP) is a vasodilator peptide. In addition, PTHrP appears to affect vascular growth and to be a mediator of inflammation in rheumatic and brain disorders. We examined the possible role of PTHrP in the inflammatory process in atherosclerosis METHODS: We immunohistochemically analyzed the cellular localization of PTHrP, the type 1 PTH/PTHrP receptor (PTH1R), and monocyte chemoattractant protein-1 (MCP-1) in 26 human carotid atherosclerotic plaques. RESULTS: The inflammatory region of plaques was characterized by high PTHrP, PTH1R, and MCP-1 immunostaining in relation to the cap (0.75+/-0.1 versus 0.29+/-0.04, 0.5+/-0.1 versus 0.25+/-0.05, 0.72+/-0.2 versus 0.29+/-0.05, respectively; P<0.05). PTHrP and MCP-1 were colocalized in both resident and inflammatory cells in the plaque. Moreover, in cultured vascular smooth muscle cells (VSMC), PTHrP(1-36) increased MCP-1 mRNA (3-fold at 6 hours) and MCP-1 protein (2.5-fold at 24 hours). This effect was inhibited by either PTHrP(7-34) or various protein kinase A inhibitors and by the nuclear factor-kappaB (NF-kappaB) inhibitor parthenolide. Furthermore, PTHrP(1-36) elicited an increase in NF-kappaB activation in VSMC. The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor simvastatin inhibited the PTHrP(1-36) induction of both NF-kappaB activity and MCP-1 overexpression, and this was reversed by mevalonate. CONCLUSIONS: PTHrP appears to be a novel proinflammatory mediator in the atheroma lesion and may contribute to the instability of carotid atherosclerotic plaques. Our data provide a new rationale to understand the mechanisms involved in the beneficial effects of statins in atherosclerosis.

Simvastatin reduces NF-kappaB activity in peripheral mononuclear and in plaque cells of rabbit atheroma more markedly than lipid lowering diet.

OBJECTIVE: To study whether simvastatin reduces inflammation in atherosclerosis beyond its hypolipidemic effects. METHODS: Twenty-four rabbits with induced femoral injury and on an atherogenic diet were randomized to normolipidemic diet (n=9), or to continue the atherogenic diet while receiving simvastatin 5 mg/kg/day (n=9) or no treatment (n=6) for 4 weeks. RESULTS: As compared with no treatment, the normolipidemic diet significantly reduced lipid levels, while simvastatin produced nonsignificant reductions. In spite of this, NF-kappaB binding activity in peripheral mononuclear cells was reduced in the simvastatin group [2,958+/-5,123 arbitrary units (a.u.)] as compared with no treatment (49,267+/-20,084 a.u.; P<0.05) and normolipidemic groups (41,492+/-15,876 a.u.; P<0.05) (electrophoretic mobility shift assay). NF-kappaB activity in the atherosclerotic lesions was also reduced by simvastatin as compared to nontreated animals (4,108+/-3,264 vs. 8,696+/-2,305 nuclei/mm(2); P<0.05), while the normolipidemic diet induced only a nonsignificant diminution (P>0.05) (Southwestern histochemistry). Similarly, simvastatin decreased macrophage infiltration (4.6+/-12 vs. 19+/-12% of area staining positive; P<0.05) and the expression of interleukin-8 (24+/-12 vs. 63+/-21%; P<0.05) and metalloproteinase-3 (16+/-3 vs. 42+/-28%; P<0.05) (immunohistochemistry), while the reduction achieved by normolipidemic diet in all these parameters was again nonsignificant (P>0.05). CONCLUSIONS: These findings suggest that simvastatin reduces inflammation in atherosclerotic plaques and in blood mononuclear cells more than expected for the lipid reduction achieved.

3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors increase the binding activity and nuclear level of Oct-1 in mononuclear cells.

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) are drugs very effective to decrease low-density lipoprotein (LDL) cholesterol. In addition, a number of studies suggest that statins have other beneficial clinical effects beyond cholesterol lowering. We recently reported that statins decrease nuclear factor kappa B (NF-kappaB) binding activity in monocytes and vascular smooth muscle cells. We now explored the effect of two different statins, simvastatin and atorvastatin, in the activation of the octamer transcription factor Oct-1 on the monocytic cell line THP-1. Oct-1 is a nuclear factor that represses the transcription of proinflammatory genes such as interleukin-8, CD11c/CD18, vascular cell adhesion molecule-1 (VCAM-1) and platelet endothelial cell adhesion molecule-1 (PECAM-1). Low concentrations of both statins increased Oct-1 DNA binding activity (electrophoretic mobility shift assay) that was resolved into two specific bands. The upper one was supershifted by preincubation of nuclear extracts with anti-Oct-1 antibody. The lower one was supershifted by preincubation of nuclear extracts with an anti-Oct-2 antibody, also partially competed with 100 mol/l excess of cold activator protein-1 (AP-1) and attenuated by anti-c-Jun antibody. Both statins increased Oct-1 and Oct-2 nuclear protein levels (Western blot). In contrast, neither had any effect on PMA-differentiated cells, suggesting a distinct sensitivity between circulating monocytes and resident tissular macrophages. In addition, statins did not increase Oct-lipoprotein lipase binding activity that contains an Oct-1 binding element. The mRNA expression of interleukin-8, a chemokine containing Oct sites in its promoter, was diminished by statin pretreatment. Our results indicate that simvastatin and atorvastatin increase the activity of the transcriptional repressor Oct-1 in mononuclear cells, and could thus contribute to decrease the activation of these cells. These data suggest a possible novel mechanism supporting a certain anti-inflammatory effect of these two 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors.

3-Hydroxy-3-methyl-glutaryl coenzyme A reductase inhibitors, atorvastatin and simvastatin, induce apoptosis of vascular smooth muscle cells by downregulation of Bcl-2 expression and Rho A prenylation.

The mechanism by which 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) induce apoptosis in vascular smooth muscle cells (VSMCs) is unknown. In this work, we demonstrate that treatment of VSMCs with simvastatin and atorvastatin inhibited Bcl-2 expression in a time and dose-dependent manner, while Bax expression was not modified. This effect was reversed by mevalonate (100 micromol/l), farnesylpyrophosphate (5 micromol/l) or geranylgeranylpyrophosphate (5 micromol/l), suggesting the involvement of protein prenylation. The treatment of VSMCs with lipophilic statins was associated with decreased prenylation of p-21 Rho A and mevalonate, farnesyl pyrophosphate (F-PP) and geranylgeranyl pyrophosphate (G-PP) reversed prenylation to basal levels. In addition, overexpression of constitutively active Q63L Rho A prevented, at least in part, apoptosis induced by statins and downregulation of Bcl-2. We also investigated the participation of caspases (proteases) in the apoptosis induced by statins. The treatment of VSMCs with lipophilic statins induced activation of the caspase 9, the first caspase of the mitochondrial pathway. Coincubation of VSMCs with the caspase inhibitor ZVAD-fmk (100 micromol/l) significantly inhibited lipophilic statin-induced apoptosis. These findings indicate that the downregulation of Bcl-2 by Rho GTPases mediates statin-induced apoptosis and suggest a new potential mechanism of action for these drugs on the regulation of cell number in the atherosclerotic lesions.

Atorvastatin reduces the expression of cyclooxygenase-2 in a rabbit model of atherosclerosis and in cultured vascular smooth muscle cells.

Inflammation is involved in the genesis and rupture of atherosclerotic plaques. We assessed the effect of atorvastatin (ATV) on the expression of cyclooxygenase-2 (COX-2) and other proinflammatory molecules in a rabbit model of atherosclerosis. Fourteen animals underwent injury of femoral arteries and 2 weeks of atherogenic diet. Afterwards, they were randomized to receive either 5 mg/kg per day of ATV (n=8) or no treatment (NT, n=6) during 4 weeks, and were finally killed. ATV reduced lipid levels, neointimal size (0.13 (0.03-0.29) mm(2) vs 0.65 (0.14-1.81) mm(2), P=0.005) and the percentage of neointimal area positive for macrophages (1% (0-3) vs 19% (5-32), P=0.001), COX-2 (32% (23-39) vs 60% (37-81) P=0.019), interleukin-8 (IL-8) (23% (3-63) vs 63% (25-88) P=0.015), and metalloproteinase-3 (19% (12-34) vs 42% (27-93), P=0.010), without significant differences in COX-1 expression (immunohistochemistry). In situ hybridization confirmed a decreased expression of COX-2 mRNA (22% (5-40) vs 43% (34-59) P=0.038). The activity of nuclear factor-kappaB, which controls many proinflammatory genes including COX-2, was reduced in atherosclerotic lesions (3538 (2663-5094) vs 8696 (5429-11312)) positive nuclei per mm(2), P=0.001) and circulating mononuclear cells (2966 vs 17130 arbitrary units). In cultured vascular smooth muscle cells, ATV reduced the expression of COX-2 mRNA induced by IL-1beta and TNF-alpha without affecting COX-1 expression. In conclusion, ATV, besides decreasing a number of inflammatory mediators in the atherosclerotic lesion, significantly downregulates COX-2 both in vivo and in vitro. These anti-inflammatory actions could partially account for the reduction of acute coronary events achieved by statins.