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