Elsevier

Atherosclerosis

Volume 193, Issue 2, August 2007, Pages 438-444
Atherosclerosis

Anti-inflammatory effects of atorvastatin: Modulation by the T-786C polymorphism in the endothelial nitric oxide synthase gene

https://doi.org/10.1016/j.atherosclerosis.2006.07.020Get rights and content

Abstract

Statins produce cholesterol-independent, anti-inflammatory effects, which result at least in part from increased endothelial nitric oxide production. These effects may be modulated by polymorphisms in the endothelial nitric oxide synthase (eNOS) gene. Here, we examined whether the T-786C polymorphism of eNOS gene affects the concentrations of markers of atherosclerosis and inflammation (sCD40L, sVCAM-1, sICAM-1, sP-selectin, MCP-1, high sensitivity (hs)-CRP, MMP-2, MMP-9, and TIMP-1). We also studied whether atorvastatin-induced anti-inflammatory effects are modulated by this polymorphism. Healthy male volunteers (N = 200), Caucasians, non-smokers, were genotyped for the T-786C polymorphism by restriction fragment length polymorphism. Subjects with TT or CC genotype received placebo for 14 days followed by 14 days of treatment with atorvastatin, 10 mg/day p.o. The concentrations of inflammatory markers were measured with ELISA kits or by gelatin zymography. Serum cholesterol and LDL-cholesterol were significantly reduced after atorvastatin treatment in both genotype groups (P < 0.05). No significant differences between genotype groups were found in the concentrations of the inflammatory markers after placebo. However, atorvastatin significantly reduced the concentrations of sCD40L, sVCAM-1, sP-selectin and MMP-9 in subjects with CC (but not TT) genotype (P < 0.05). While atorvastatin decreased hs-CRP levels in both genotype groups (P < 0.05), no significant effects were found on the concentrations of sICAM-1, MCP-1, pro-MMP-9, pro-MMP-2 and TIMP-1. These results suggest no effects for the T-786C polymorphism on the concentrations of inflammatory markers. However, this polymorphism modulates the anti-inflammatory effects of atorvastatin. These findings may be relevant for the primary prevention of cardiovascular events in subjects with CC genotype, who may be at increased cardiovascular risk and could benefit from treatment with statins.

Introduction

Vascular endothelial cells produce nitric oxide (NO), which is a major contributor to vasodilatation and to the anti-inflammatory and anti-thrombotic properties of the vascular wall [1]. Endothelial nitric oxide synthase (eNOS) is the enzyme responsible for the majority of NO production in the cardiovascular system. This enzyme is encoded by a gene presenting a clinically relevant polymorphism in the promoter region (T-786C), which has been associated with impaired NO production and cardiovascular diseases [2], [3], [4]. This polymorphism was suggested to reduce eNOS gene promoter activity by approximately 50% [2], thereby lending experimental support to a physiologic role for this polymorphism. Importantly, this polymorphism increases the susceptibility to endothelial dysfunction and coronary artery disease [2], [5], [6], [7]. However, while many studies report significant associations between eNOS gene polymorphisms or haplotypes and cardiovascular diseases [4], [8], [9], [10], the therapeutic implications of such allelic variations remain to be determined [11].

Statins inhibit cholesterol synthesis in the liver by blocking the conversion of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) to mevalonate [12]. However, in addition to their cholesterol lowering properties, statins produce additional, cholesterol-independent, beneficial effects [12] that are encountered early in the course of lipid lowering therapy [13]. These so-called pleiotropic effects include anti-inflammatory effects on a number of tissues and cell types [14]. For example, statins may reduce the formation of pro-inflammatory mediators such as C-reactive protein (CRP) by hepatocytes [15]. Moreover, statins may alter gene expression in key cells involved in atherogenesis. For example, statins may reduce the expression of P-selectin [12], [16], vascular adhesion molecule (VCAM)-1 and intercellular adhesion molecule (ICAM)-1 [12], [14], which are cellular adhesion molecules involved in the early steps of leucocyte recruitment to the vessel wall [16]. In addition, statins may attenuate inflammatory cell infiltration by targeting monocyte chemoattractant protein (MCP)-1, which is a chemokine regulating leukocyte recruitment into sub-endothelial space [14]. Statins also play anti-inflammatory and immunoregulatory effects by modulating matrix metalloproteinase (MMP)-9/tissue inhibitor of metalloproteinase (TIMP)-1 [17], [18], [19] or CD40 ligand (CD40L) [12], [19]. Of particular importance, altered expression or activity of MMP-2 and MMP-9 have been reported to play a role in a variety of pathological conditions affecting the cardiovascular system [20], [21], [22], [23], [24], [25], [26]. Indeed, the circulating level of MMP-9 has been recently suggested to be a blood biomarker helping in the diagnosis of important cardiovascular diseases [20], [21]. These recent findings are consistent with the notion that plasma MMP-9 and MMP-2 may have diagnostic and prognostic value.

While at least part of the anti-inflammatory effects produced by statins result from increased endothelial NO production [12], [14], no previous study has examined whether these effects are modulated by clinically relevant eNOS gene polymorphisms. Indeed, this suggestion is supported by recent findings demonstrating that fluvastatin produces stronger increases in the transcriptional activity of eNOS gene associated with the CC genotype for the T-786C polymorphism of eNOS gene [27]. Moreover, whereas there is one study showing an association between the G894T polymorphism of eNOS gene and inflammatory markers [28], no previous study has examined whether the T-786C polymorphism of eNOS gene is associated with inflammatory markers. In the present study, we examined whether this polymorphism affects the circulating concentrations of soluble markers of atherosclerosis and inflammation (sCD40L, sVCAM-1, sICAM-1, sP-selectin, MCP-1, high sensitivity (hs)-CRP, MMP-2, MMP-9 and TIMP-1). We also studied whether atorvastatin-induced anti-inflammatory effects are modulated by the T-786C polymorphism of eNOS gene.

Section snippets

Subjects and study design

Approval for use of human subjects was obtained from the Institutional Review Board at the Faculty of Medicine of Ribeirao Preto, University of Sao Paulo, Brazil, and each subject provided written informed consent. The present work was carried out in accordance with the ethics standards of the Helsinki Declaration. Healthy male volunteers (N = 200; age range: 18–56 years), Caucasians, non-smokers and not taking any medications, were recruited from local population and genotyped for the T-786C

Results

The TT, TC and CC genotype frequencies in the 200 healthy subjects included in the present study were 36, 51 and 13%, respectively. Here, we have not studied heterozygotes because the CC and TT genotype groups would provide much more reliable information regarding the possible effects associated with the T-786C polymorphism. Table 1 summarizes the clinical and laboratorial characteristics of the 30 subjects enrolled in the present study. All subjects were healthy male, Caucasians, non-smokers

Discussion

The main novel findings reported here are: (1) the T-786C polymorphism in the eNOS gene does not significantly affect the circulating concentrations of inflammatory markers; (2) atorvastatin produced significant anti-inflammatory effects in healthy subjects with CC genotype, but not in subjects with TT genotype. These findings suggest that the T-786C polymorphism modulates the anti-inflammatory effects of atorvastatin. Although our findings do not provide a mechanistic insight into disease

Acknowledgments

Financial support: Fundação de Aparo a Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). We thank Laboratorios Pfizer Ltda. for providing atorvastatin, and Juliana A. Uzuelli for technical assistance. The sponsors had no involvement in data analyses and writing of the manuscript.

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