Leukotriene B4 enhances the activity of nuclear factor-kappaB pathway through BLT1 and BLT2 receptors in atherosclerosis.

AIMS: Leukotriene B4 (LTB4) is a powerful chemoattractant and pro-inflammatory mediator in several inflammatory diseases, including atherosclerosis. It acts through its two membrane receptors, BLT1 and BLT2. The aim of this study was to determine the molecular mechanism involved in the proatherogenic effect of LTB4, BLT1 and BLT2 in atherosclerosis. Moreover, we characterized the expression of 5-lipoxygenase (5-LO) pathway and LTB4 receptors in blood and plaques from patients with carotid atherosclerosis. METHODS AND RESULTS: In cultured monocytic cells, LTB4 induced a rapid phosphorylation of mitogen-activated protein kinases (MAPKs ERK1/2 and JNK1/2) and PI3K/Akt via BLT1 and BLT2 in a pertussis toxin (PTX)-dependent mechanism (assessed via western blotting) and also increased nuclear factor-kappaB (NF-kappaB) DNA binding activity (assessed via EMSA) in a MAPK- and reactive oxygen species-dependent mechanism. Furthermore, LTB4 elicited interleukin-6, monocyte chemoattractant protein-1 and tumour necrosis factor-alpha mRNA overexpression also via BLT1 and BLT2 by a PTX- and NF-kB-dependent mechanism (assessed by real-time PCR), promoting an inflammatory environment. When compared with healthy subjects, patients with carotid atherosclerosis showed a significant increase in the expression of all the components of the 5-LO pathway and BLT1 and BLT2 mRNA (real-time PCR) in peripheral blood mononuclear cells and LTB4 plasma levels (ELISA). In these patients, an overexpression of 5-LO, leukotriene A-4 hydroxylase (LTA4-H) and BLT1 was noted in the inflammatory region of carotid plaques when compared with the fibrous cap (assessed by immunohistochemistry). CONCLUSION: The 5-LO pathway is enhanced in patients with carotid atherosclerosis. Furthermore, its product LTB4 phosphorylates MAPKs and stimulates NF-kappaB-dependent inflammation via BLT1 and BLT2 receptors in cultured monocytic cells. The blockade of this pathway could be a novel and potential therapeutic target in atherothrombosis.

Essential role of TGF-beta/Smad pathway on statin dependent vascular smooth muscle cell regulation.

BACKGROUND: The 3-hydroxy-3-methylglutaryl CoA reductase inhibitors (also called statins) exert proven beneficial effects on cardiovascular diseases. Recent data suggest a protective role for Transforming Growth Factor-beta (TGF-beta) in atherosclerosis by regulating the balance between inflammation and extracellular matrix accumulation. However, there are no studies about the effect of statins on TGF-beta/Smad pathway in atherosclerosis and vascular cells. METHODOLOGY: In cultured vascular smooth muscle cells (VSMCs) statins enhanced Smad pathway activation caused by TGF-beta. In addition, statins upregulated TGF-beta receptor type II (TRII), and increased TGF-beta synthesis and TGF-beta/Smad-dependent actions. In this sense, statins, through Smad activation, render VSMCs more susceptible to TGF-beta induced apoptosis and increased TGF-beta-mediated ECM production. It is well documented that high doses of statins induce apoptosis in cultured VSMC in the presence of serum; however the precise mechanism of this effect remains to be elucidated. We have found that statins-induced apoptosis was mediated by TGF-beta/Smad pathway. Finally, we have described that RhoA inhibition is a common intracellular mechanisms involved in statins effects. The in vivo relevance of these findings was assessed in an experimental model of atherosclerosis in apolipoprotein E deficient mice: Treatment with Atorvastatin increased Smad3 phosphorylation and TRII overexpression, associated to elevated ECM deposition in the VSMCs within atheroma plaques, while apoptosis was not detected. CONCLUSIONS: Statins enhance TGF-beta/Smad pathway, regulating ligand levels, receptor, main signaling pathway and cellular responses of VSMC, including apoptosis and ECM accumulation. Our findings show that TGF-beta/Smad pathway is essential for statins-dependent actions in VSMCs.

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.

Licofelone, a balanced inhibitor of cyclooxygenase and 5-lipoxygenase, reduces inflammation in a rabbit model of atherosclerosis.

Licofelone, a dual anti-inflammatory drug that inhibits 5-lipoxygenase (LOX) and cyclooxygenase (COX) enzymes, may have a better cardiovascular profile that cycloxygenase-2 inhibitors due to cycloxygenase-1 blockade-mediated antithrombotic effect and a better gastrointestinal tolerability. We examined the anti-inflammatory effect of licofelone on atherosclerotic lesions as well as in isolated neutrophils from whole blood of rabbits compared with a selective inhibitor of COX-2, rofecoxib. We also assessed the antithrombotic effect of licofelone in rabbit platelet-rich plasma. For this purpose, 30 rabbits underwent injury of femoral arteries, and they were randomized to receive 10 mg/kg/day licofelone or 5 mg/kg/day rofecoxib or no treatment during 4 weeks with atherogenic diet in all cases. Ten healthy rabbits were used as controls. Neutrophils and platelets were isolated from peripheral blood of rabbits for ex vivo studies. Licofelone reduced intima/media ratio in injured arteries, the macrophages infiltration in the neointimal area, monocyte chemoattractant protein-1 (MCP-1) gene expression, and the activation of nuclear factor-kappaB in rabbit atheroma. Moreover, licofelone inhibited COX-2 and 5-LOX protein expression in vascular lesions. Rofecoxib only diminished COX-2 protein expression and MCP-1 gene expression in vascular atheroma. Prostaglandin E(2) in rabbit plasma was attenuated by both drugs. Licofelone almost abolished 5-LOX activity by inhibiting leukotriene B4 generation in rabbit neutrophils and prevented platelet thromboxane B2 production from whole blood. Licofelone reduces neointimal formation and inflammation in an atherosclerotic rabbit model more markedly than rofecoxib. This effect, together with the antiplatelet activity of licofelone, suggests that this drug may have a favorable cardiovascular profile.

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.