Differential endothelial hydrogen peroxide signaling via Nox isoforms: Critical roles for Rac1 and modulation by...

Statins have manifold protective effects on the cardiovascular system. In addition to lowering LDL cholesterol levels, statins also have antioxidant effects on cardiovascular tissues involving intracellular redox pathways that are incompletely understood. Inhibition of HMG-CoA reductase by statins not only modulates cholesterol synthesis, but also blocks the synthesis of lipids necessary for the post-translational modification of signaling proteins, including the GTPase Rac1. Here we studied the mechanisms whereby Rac1 and statins modulate the intracellular oxidant hydrogen peroxide (H₂O₂) via NADPH oxidase (Nox) isoforms. In live-cell imaging experiments using the H₂O₂ biosensor HyPer7, we observed robust H₂O₂ generation in human umbilical vein endothelial cells (HUVEC) following activation of cell surface receptors for histamine or vascular endothelial growth factor (VEGF). Both VEGF- and histamine-stimulated H₂O₂ responses were abrogated by siRNA-mediated knockdown of Rac1. VEGF responses required the Nox isoforms Nox2 and Nox4, while histamine-stimulated H₂O₂ signals are independent of Nox4 but still required Nox2. Endothelial H₂O₂ responses to both histamine and VEGF were completely inhibited by simvastatin. In resting endothelial cells, Rac1 is targeted to the cell membrane and cytoplasm, but simvastatin treatment promotes translocation of Rac1 to the cell nucleus. The effects of simvastatin both on receptor-dependent H₂O₂ production and Rac1 translocation are rescued by treatment of cells with mevalonic acid, which is the enzymatic product of the HMG-CoA reductase that is inhibited by statins. Taken together, these studies establish that receptor-modulated H₂O₂ responses to histamine and VEGF involve distinct Nox isoforms, both of which are completely dependent on Rac1 prenylation.