Elsevier

Atherosclerosis

Volume 174, Issue 2, June 2004, Pages 305-313
Atherosclerosis

Simvastatin potenciates PGI2 release induced by HDL in human VSMC: effect on Cox-2 up-regulation and MAPK signalling pathways activated by HDL

https://doi.org/10.1016/j.atherosclerosis.2004.01.037Get rights and content

Abstract

High density lipoproteins (HDL) induce prostacyclin (PGI2) release in vascular smooth muscle cells (VSMC) by up-regulation of cyclooxygenase-2 (Cox-2). Our goal was to analyse the mechanisms underlying this effect, and its potential modulation by HMG-CoA reductase inhibition in human VSMC. The contribution of mitogen-activated protein kinase (MAPK) signalling pathways was assessed by Western blot analysis and using specific inhibitors [PD098059 for p42/44 MAPK kinase (MEK); SB203580 for p38 MAPK or L-JNKI1 for c-Jun N-terminal kinase-1 (JNK-1)]. HDL-induced PGI2 release was inhibited by rofecoxib (a specific Cox-2 inhibitor, 5 μM). HDL induced the early activation of p42 MAPK, p38 MAPK and JNK-1. p42/44 MAPK was the major pathway involved in both Cox-2 up-regulation and PGI2 synthesis; p38 MAPK was also involved in both processes while JNK inhibition only affected PGI2 synthesis. Pertussis toxin (an inhibitor of Gαi/Gαo proteins) prevented MAPK activation and inhibited both Cox-2 up-regulation and PGI2 release. Genistein (a tyrosine kinase inhibitor) inhibited PGI2 release without affecting MAPK activation or Cox-2 up-regulation. Simvastatin (0.1–1 μM) increased HDL-induced PGI2 release (≈45% at 1 μM) but did not significantly modify early MAPK activation or Cox-2 expression. Simvastatin alone did not significantly affect PGI2 release. Our results suggest that mechanisms associated with G protein-coupled receptor activation, trigger Cox-2 up-regulation and PGI2 release via multiple MAPK signalling pathways in VSMC. The mechanism is independent of tyrosine kinase receptors, although cytosolic tyrosine kinases could activate Cox-2 post-translationally. The potential contribution of HDL to vascular homeostasis, via increases in PGI2 synthesis, could be enhanced by HMG-CoA reductase inhibitors.

Introduction

The protective effect of high density lipoproteins (HDL) against coronary artery disease has been extensively evaluated through experimental and epidemiological studies [1], [2]. Reverse cholesterol transport is the most widely accepted mechanism for this protective effect; however, HDL elicits additional effects on vascular cells, among them the synthesis of endogenous antiatherogenic molecules such as nitric oxide [3] and prostacyclin (PGI2) [4]. PGI2, the major eicosanoid synthesised in the mammalian vasculature, exerts a wide spectrum of vasoprotective functions [5]; in fact, PGI2 analogues are currently being assayed in clinical trials for the treatment of some vascular diseases. Therefore, the atheroprotective effects of HDL could be, at least in part, dependent on the modulation of vascular PGI2 production.

The rate-limiting enzyme in the biosynthesis of PGI2 is prostaglandin endoperoxide H synthase (cyclooxygenase, Cox) which catalyses the conversion of arachidonic acid to PGH2, the first committed step in the biosynthesis of a wide range of eicosanoids. The two Cox isoforms [Cox-1 (constitutive) and Cox-2 (inducible)] have been differentially involved in vascular functions [6], [7]. Cox-2 has currently been associated with pro-inflammatory/pro-atherogenic stages, due to its up-regulation in monocyte-derived macrophages present in atherosclerotic lesions [8]. However, Cox-2 may contribute to vascular PGI2 formation in healthy humans [9], and data from both genetically modified mice [10] and wild type animal models [11], indicate that Cox-2-derived PGI2 prevents both local thrombosis and neo-intima formation. In addition, Cox-2 also contributes to the innate defensive mechanisms of the myocardium [12].

HDL induce PGI2 release through Cox-2-dependent mechanisms in vascular smooth muscle cells (VSMC) [13], [14] and endothelial cells [15]. The aim of the present study was to determine the signalling pathways involved in Cox-2 up-regulation and PGI2 release induced by HDL in human VSMC, and to assess its modulation by HMG-CoA reductase inhibitors (statins), drugs which are able to modulate cell signalling and vascular function [16], [17]. Our study shows that in human VSMC, HDL increase Cox-2 expression and PGI2 release via multiple MAPK signalling pathways [p42/44 MAPK (ERK), p38 MAPK and JNK-1] induced by mechanisms dependent on G protein-coupled receptor (GPCR) activation. In addition, we show that clinical doses of simvastatin increase PGI2 release induced by HDL. Nevertheless, simvastatin did not significantly change early MAPK phosphorylation or Cox-2 up-regulation.

Section snippets

Cell culture

Human VSMC were obtained from non-atherosclerotic aortic segments of explanted hearts from transplant operations performed at the Hospital de Sant Pau (Barcelona, Spain) as described [16]. Cells were incubated at 37 °C in a humidified atmosphere of 5% CO2 in medium 199 (M199) supplemented with 20% foetal calf serum (FCS), 2% human serum, antibiotics (100 u/ml penicillin and 0.1 mg/ml streptomycin) and l-glutamine (2 mM). Cells used in the experiments were between the third and fifth passage.

VSMC

Rofecoxib inhibits HDL-induced PGI2 release

HDL induced Cox-2 protein levels and PGI2 release in human VSMC in a time- (Fig. 1) and dose-dependent manner [PGI2 levels ranging from 2.9±0.4 to 11.6±1.2 ng/ml at 7.5 and 60 mg/dl, respectively]. The effect of HDL was greater than that produced by interleukin-1β (IL-1β) (4.4±0.46 ng/ml at 10 ng/ml), a well-known Cox-2 inducer. Rofecoxib abolished HDL-induced PGI2 synthesis without affecting Cox-2 protein levels (Fig. 1).

HDL increase Cox-2 transcription through the activation of MAPK pathways

HDL (30 mg/dl) promoted the early up-regulation of Cox-2 mRNA levels (Fig. 2A

Discussion

In previous studies we reported that HDL induce PGI2 release in VSMC through a mechanism requiring Cox-2 up-regulation [13], [14] and cPLA2 activation [14]. In the present study we show that in VSMC HDL promote Cox-2 expression and PGI2 release through multiple MAPK [p42 MAPK, p38 MAPK and JNK-1] pathways activated via mechanisms triggered by GPCR. The pathways involved in Cox-2 expression/PGI2 release induced by HDL are summarised in Fig. 9. In addition, low concentrations (in the therapeutic

Acknowledgements

This work has been funded in part by grants from FIS-PI020392, PN 2000-0174 and Fundación de Investigación Cardiovascular (FIC)-Catalana Occidente. Itziar Escudero was the recipient of a research fellowship from Ministerio de Educación y Ciencia of Spain. We thank Olga Bell and Silvia Aguiló for their technical assistance.

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    Both authors contributed equally to this work.

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