Abstract 361: Cyclophilin A Mediates Angiotensin II--Stimulated NADPH Oxidase Activation in Vascular Smooth Muscle Cells

Abstract

Objective: Cyclophilin A (CyPA) is a ubiquitously expressed cytosolic protein that possesses PPIase activity and scaffold function. CyPA regulates Angiotensin II (Ang II) induced reactive oxygen species (ROS) production in vascular smooth muscle cells. However, the mechanism of this CyPA regulation remains unclear. We hypothesized that CyPA regulates plasma membrane translocation of NADPH oxidase cytosolic subunit, p47phox, which is required for NADPH oxidase structural organization and activity. Methods and results: Immunofluorescence studies in rat aortic smooth muscle cells revealed that CyPA translocated from the cytosol to the plasma membrane in response to Ang II in a time dependent manner with a peak at 10min (46.4±5.4 fold increase). Mouse Aortic Smooth Muscle Cells (MASM) were isolated from mice lacking CyPA (CyPA-/-) and wild type controls (WT), treated with Ang II (100nM) and immunofluorescence analysis was performed. Ang II induced p47phox plasma membrane translocation at 10min in WT mice. However, p47 phox translocation was significantly inhibited in CyPA -/- MASM. CyPA and p47phox colocalized at the plasma membrane in response to Ang II. Further analysis using subcellular fractionation studies confirmed that Ang II induced p47phox plasma membrane translocation was inhibited in CyPA -/- MASM compared to WT (1.2±2.7 vs 4.3±3.4 fold increase). Coimmunoprecipitation analyses confirmed that Ang II increased CyPA association with p47phox in a time dependent manner (2.5±3.4 fold increase at 10min). Finally, pretreatment with the PPIase activity inhibitor, cyclosporine A (1uM), could not inhibit CyPA association with p47phox and CyPA mediated p47phox translocation to the plasma membrane. Conclusion: These data suggest that Ang II promotes an association between CyPA and p47phox that enhances plasma membrane translocation of p47phox. This is proposed to increase the NADPH oxidase activity thereby increasing cellular ROS production. This process is independent of the PPIase activity of CyPA. Therefore, inhibition of the CyPA and p47phox association could be a future therapeutic target for Ang II induced ROS regulated cardiovascular diseases such as atherosclerosis and abdominal aortic aneurysm formation.