The statins are a class of HMG CoA-reductase inhibitors used clinically for their ability to lower serum cholesterol; however, not all of their clinical benefits, including enhanced endothelial cell (EC) barrier function, 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, including Rho and Rac, although we have previously reported the paradoxical activation of cytosolic Rac by simvastatin. While the inhibition of Rho attenuates actin stress fiber formation, promoting EC barrier function, the inhibition of Rac at the cell membrane prevents activation of NADPH oxidase and subsequent superoxide generation, known to be EC barrier disruptive. We sought to determine the relative regulatory effects of simvastatin on Rac and NADPH oxidase activities in the context of EC barrier protection. Human pulmonary artery ECs treated with simvastatin (5 μM, 16 hours) were found to have a significant decrease in membrane Rac (38% decrease), consistent with the inhibition of geranylgeranylation. Using a FITC-dextran transwell permeability assay, concomitant treatment of EC with xanthine (200 μM, 1 hour) and xanthine oxidase (30 mU/mL, 1 hour) to generate superoxide resulted in barrier disruption that was attenuated by simvastatin (5 μM, 16 hours, 49% decrease), consistent with the inhibition of NADPH oxidase. Moreover, LPS-induced (1 μg/mL) superoxide production measured by DHE fluorescence was also significantly reduced by simvastatin (50% decrease). Finally, compared with simvastatin treatment (5 μM, 16 hours), thrombin-induced permeability (1 U/mL, 5 minutes) was only modestly attenuated by the inhibition of Rac via siRNA (20% as effective as simvastatin), whereas the use of the Rho inhibitor Y-27632 (10 μM, 30 minutes) affected a more pronounced attenuation (70% as effective as simvastatin). These data suggest that EC barrier protection by simvastatin, although largely due to Rho inhibition, is also attributable to the inhibition of Rac at the cell membrane and the subsequent attenuation of superoxide generation by NADPH oxidase. Our findings contribute to defining mechanisms by which simvastatin modulates EC barrier properties, which may lead to new clinical applications.
Funding: HL077134-01, Parker B. Francis Fellowship.