Direct inhibition by a statin of TNFalpha-induced leukocyte recruitment in rat pial venules - in vivo confocal microscopic study

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) have been shown to block leukocyte-endothelial interaction independently of their cholesterol-lowering properties. The effects of statins are generally attributed to a decrease in mevalonate caused by inhibition of HMG-CoA reductase, which results in an increase of nitric oxide (NO). However, a recent in vitro study demonstrated a novel effect which depended on the lipophilicity of statin and appeared to be unrelated to HMG-CoA reductase inhibition. The purpose of this study is to investigate whether the proposed mechanism actually operates in vivo. We examined the effects of simvastatin (lipophilic) and pravastatin (hydrophilic) on leukocyte behavior in a tumor necrosis factor alpha (TNFalpha)-induced leukocyte recruitment model. Leukocyte adhesion and rolling were examined in pial venules of rat brain by using confocal laser scanning microscopy after labeling leukocytes with rhodamine 6G. Experiments were conducted 4h after TNFalpha injection (0.5microg) in six groups: control, TNFalpha alone, TNFalpha + vehicle of simvastatin, TNFalpha + simvastatin (20mg/kg, 2ml/kg), TNFalpha + vehicle of pravastatin, and TNFalpha + pravastatin (40mg/kg, 2ml/kg). Statins and vehicles were injected subcutaneously for 3 days. TNFalpha caused a marked increase in rolling and adhered leukocytes. The number of adhered leukocytes in the simvastatin group was significantly less than in the vehicle group (276 +/- 38 cells/mm(2) versus 1155 +/- 89 cells/mm(2), P < 0.01), whereas pravastatin had little effect. Both simvastatin and pravastatin showed a tendency to decrease the number of rolling leukocytes, but there were no significant differences among TNFalpha-treated groups. Up-regulation of endothelial nitric oxide synthase (eNOS) mRNA or increased expression of P-selectin or intercellular adhesion molecule-1 (ICAM-1) was not observed, and therefore cannot account for the simvastatin-induced reduction of adhered leukocytes. Markedly different effect on leukocyte adhesion between simvastatin and pravastatin under comparable level of HMG-CoA reductase inhibitor was demonstrated in in vivo as was shown in in vitro study.