P708Homocysteine induces apoptosis of arterial smooth muscle cells via NOX4-produced reactive oxygen

Introduction: Elevated levels of homocysteine (Hcy) form a vascular risk factor and can induce apoptosis of endothelial cells and vascular smooth muscle cells. Recent studies have shown that NADPH oxidase (NOX)-mediated reactive oxygen species (ROS) play a role in Hcy-induced apoptosis of endothelial cells. In this study we have analyzed the effect of Hcy on the different NOX isoforms and their role in human arterial smooth muscle cells (SMCs). Methods: Human arterial SMCs isolated from the human umbilical cord were incubated with 100 μM Hcy for 24 hours and were analyzed for cell viability by determining phosphatidylserine exposure via Flow cytometry and caspase 3 activity measurements. With use of digital imaging microscopy, expression of NOX1, 2 and 4 and production of ROS were studied. NOX4 small interference RNA (siRNA) were used to specify the role of NOX4 in Hcy-induced apoptosis. Results: Hcy induced a significant increase in phosphatidylserine exposure and caspase 3 activity in arterial SMC. This was accompanied by a significant translocation of NOX4 from the cytoplasm to (peri)nuclear regions, coinciding with a significant increase in local ROS production. Hcy did not affect expression levels, nor subcellular localization of NOX1 and NOX2. Knock-down of NOX4 using siRNA significantly reduced Hcy-induced (peri)nuclear NOX4 expression, concomitant ROS production and apoptosis in isolated arterial SMCs. Conclusion: Pathophysiological concentrations of Hcy induce apoptosis of human arterial SMCs via NOX4 mediated ROS production.