The statins are a class of HMG CoA-reductase inhibitors used clinically for their ability to reduce serum cholesterol levels via inhibition of the prenylation pathway. However, not all of their clinical benefits, including both vascular barrier protection and reduced superoxide generation, can be attributed to their lipid-lowering properties. One potential mechanism of these effects is via inhibition of geranylgeranylation, a covalent modification that allows translocation to the cell membrane and activation of the small GTPases Rho and Rac, mediators of cytoskeletal rearrangement. While statins inhibit Rho, we previously reported the paradoxical activation of Rac (Rac-GTP) in endothelial cell (EC) after prolonged treatment with simvastatin (5 μM, 16 hours). In this study, upon membrane fractionation and subsequent Western blotting, we report a 37% reduction in Rac translocation to the EC membrane by simvastatin (5 μM, 16 hours) relative to untreated control cells, consistent with the inhibition of geranylgeranylation and evidence of a separate mechanism of simvastatin-mediated Rac activation. In addition, as Rac is required for activation of the NADPH oxidase complex and subsequent superoxide anion generation, we examined the effect of simvastatin on the peripheral translocation of P47phox, an NADPH oxidase component. Simvastatin affected a 47% reduction in P47phox at the EC membrane relative to control cells. Finally, we measured transendothelial electrical resistance and quantified the effects of simvastatin pretreatment on sphingosine 1-phosphate-induced barrier enhancement, an event dependent on Rac. S1P-induced (1 μM) EC barrier function was increased by simvastatin (5 μM, 16 hours) 70% at peak effect relative to S1P-treated control cells. These data are consistent with a functional role for Rac in simvastatin-mediated EC barrier protection and further define the mechanism by which simvastatin is able to directly modulate EC.
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