Abstract 239: Parthanatos is Involved in Hydrogen Peroxide Induced Vascular Smooth Muscle Cell Death

Abstract

Objectives: Oxidative stress underlies major vascular diseases including atherosclerosis and abdominal aortic aneurysm. Hydrogen peroxide (H2O2) is widely used to trigger oxidative stress in vitro for the study of apoptosis. However, we have previously shown that vascular smooth muscle cells (SMCs) respond to high concentrations (>1 mM) of H2O2 with necrosis. Traditionally regarded as incidental form of cell death, necrosis can occur through different mechanisms mediated by distinct intracellular signaling networks. The precise knowledge of death pathway is essential to the design of therapeutic strategy targeting cell death. The goal of the current study is to determine how H2O2 induces necrosis in SMCs. Methods: Mouse vascular aortic smooth muscle cell line, MOVAS, were treated with 3mM H2O2 for 2 hours, after which cell death was analyzed using flow cytometry and protein expression determined via western blot. Results: SMCs underwent apoptosis and necrosis in response to 0.3 and 3 mM H2O2, respectively. The 3mM H2O2 group died via a caspase-independent mechanism. Expression of common autophagy-associated proteins were unaffected. Additionally, different autophagy activators and inhibitors only moderately facilitated the pro-necrotic effect of H2O2. The H2O2-induced necrosis was not affected by necroptosis inhibitors including necrostatin-1s or by SiRNA silencing of necroptosis mediators RIP1, RIP3 and MLKL. Furthermore, ferroptosis and CypD inhibitors did not provide protection from necrosis induced by H2O2. In contrast, the necrotic response was attenuated by the PARP-1 inhibitor 3-aminobenzamide (37.10±13.72% vs 82.05±0.64%). Moreover, an PARP1 siRNA also reduced necrosis. PARP-1 is the central mediator of a necroptosis mechanism called parthanatos. Conclusions: Our data demonstrates that parthanatos constitutes a major mechanism underlying the necrotic response to high concentrations of H2O2. Current studies delineate the involvement of parthanatos in myocardial ischemia/reperfusion injury, cardiovascular ailments, and atherosclerosis. The present study may provide a new perspective on targeting PARP-1 for the protection of SMCs and likely cardiac myocytes against oxidative stress.