Pravastatin suppresses the interleukin‐8 production induced by thrombin in human aortic endothelial cells cultured with high glucose by inhibiting the p44/42 mitogen activated protein kinase

3-Hydroxy-3-methylglutaryl co-enzyme A reductase inhibitors (statins) prevent the progression of atherosclerosis by lowering cholesterol. However, the effect of statins on the synthesis of pro-inflammatory cytokines from endothelial cells has not yet been fully investigated. Here, we examined the effect of pravastatin, one of the statins, on IL-8 synthesis induced by thrombin in human aortic endothelial cells (AoEC) cultured with high glucose concentrations. Pravastatin significantly decreased the IL-8 synthesis induced by thrombin. Pravastatin inhibited the p44/42 MAP kinase activity induced by thrombin, but did not inhibit the p38 MAP kinase activity. Translocation of ras protein from the cytosol to plasma membrane was inhibited by pravastatin. Pravastatin inhibit the activator protein-1 activity, but did not inhibit the activation of IκB-α. Dominant negative ras inhibited the p44/42 MAP kinase activity induced by PMA. Our results suggest that pravastatin inhibits IL-8 synthesis by blocking the ras-MAP (p44/42) kinase pathway rather than nuclear factor-κB. Pravastatin may prevent atherosclerosis not only by lowering cholesterol levels, but also by suppressing IL-8 synthesis in AoEC through the inhibition of p44/42 MAP kinase, and this may be more beneficial in diabetic patients than in non-diabetics. Keywords: Pravastatin, interleukin-8, endothelium, signal transduction, thrombin, MAP kinase, diabetic macroangiopathy Introduction The development of atherosclerosis is a complicated process starting from endothelial dysfunction and leading to migration of smooth muscle cells into the intima. Several causes of endothelial dysfunction include lipid disorder, diabetes mellitus, hypertension, and cigarette smoking. Following endothelial dysfunction, the migration of leukocytes into the subendothelial space and migration of medial smooth muscle cells into intimal layer of arteries occur (Ross, 1999). Such cellular responses are regulated by growth factors and cytokines such as fibroblast growth factor, platelet derived growth factor, tumour necrosis factor (TNF)-α, interleukin (IL)-1, IL-6, IL-8 and monocyte chemoattractant protein (MCP)-1. We previously reported that in aortic endothelial cells (AoEC) high concentrations of glucose stimulated the IL-8 production, but did not stimulate IL-1, IL-6 and TNF-α production (Urakaze et al., 1996; Temaru et al., 1997). Therefore, we postulated that IL-8 plays some role in the pathogenesis of diabetic angiopathy. Furthermore, IL-8 has been implicated in the pathogenesis of atherosclerosis because the migration of aortic smooth muscle cells is induced by IL-8 (Yue et al., 1993, 1994) and also IL-8 is expressed in human atheroma (Koch et al., 1993; Wang et al., 1996; Rus et al., 1996). Recently, Boisvert et al. (1998) have reported that mice lacking IL-8 receptors are less susceptible to atherosclerosis. These data suggest that IL-8 may have an important role in the formation of human atherosclerotic lesions by inducing the accumulation of both leukocytes and smooth muscle cells. On the other hand, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors are generally used as therapeutic agents for the treatment of hypercholesterolaemia. These drugs have an inhibitory effect on cholesterol synthesis mainly in the liver by inhibiting the conversion of HMG-CoA to mevalonate, the rate-limiting step of in vivo cholesterol synthesis (Goldstein & Brown, 1990). As a result, the plasma levels of low density lipoprotein (LDL) cholesterol is decreased, which leads to a longer survival rate with less incidence of the vascular events due to atherosclerosis (Scandinavian Simvastatin Survival Study Group, 1994; Shepherd et al., 1995; Sacks et al., 1996; The Long-Term Intervention with Pravastatin in Ischaemic Disease (LIPID) Study Group, 1998; Plehn et al., 1999). However, in the West of Scotland Coronary Prevention Study pravastatin, an HMG-CoA reductase inhibitor, showed greater benefits with a lower risk of cardiovascular events, even although the pravastatin treated patients showed a similar level of LDL cholesterol compared with the placebo subjects (West of Scotland Coronary Prevention Study Group, 1998). These data suggest that lipid changes may not fully account for the clinical benefits of pravastatin, and that it may have another beneficial effects. Indeed, other reports showed that HMG-CoA reductase inhibitors improved the endothelial dysfunction present in atherosclerosis, enhanced endothelial nitric oxide synthase (eNOS) mRNA expression (Hernandez-Perera et al., 1998; Endres et al., 1998), suppressed plasminogen activator inhibitor-1 mRNA expression in endothelial cells (Essig et al., 1998), and inhibited the proliferation of smooth muscle cells (Munro et al., 1994). However, it has not yet been fully investigated as to whether pravastatin may affect the production of pro-inflammatory cytokines especially IL-8 in vascular endothelial cells. In this paper, we attempted to clarify the effect of pravastatin on the regulation of IL-8 synthesis induced by thrombin in human aortic endothelial cells cultured with high concentrations of glucose. We report here that pravastatin significantly inhibits the synthesis of IL-8 induced by thrombin at both the protein and mRNA levels via the inhibition of ras-raf-MAP (p44/42) kinase and transcription factor, activating protein (AP)-1. Our results suggest that pravastatin may prevent atherosclerosis by suppressing IL-8 synthesis in AoEC through the inhibition of p44/42 MAP kinase. Therefore, pravastatin may not only represent a cholesterol-lowering agent, but it could also contribute to improve the endothelial dysfunction present in diabetic macroangiopathy.