PROJECT SUMMARY/ABSTRACT Abdominal aortic aneurysm (AAA) is a permanent abdominal aorta expansion with high mortality but limited treatment options. Currently, there are no effective clinical medicines to prevent, delay, or reverse the growth or rupture of AAA, except an open or endovascular surgical repair for symptomatic aneurysms or aneurysms at high risk for rupture. Vascular smooth muscle cells (VSMC) are crucial in maintaining vascular wall integrity and function, while VSMC depletion is a characteristic feature of AAA. Ferroptosis is a type of programmed cell death dependent on iron and characterized by the accumulation of lipid peroxides. Smoking is a well- established AAA risk factor, and cigarette smoke extract induces VSMC ferroptosis. Glutathione peroxidase 4 (GPX4) has been identified as a critical inhibitor of ferroptosis and apoptosis. Human genetics and Gene Expression Omnibus database have demonstrated that decreased GPX4 expression is associated with an increased risk of AAA. Our preliminary studies demonstrate that GPX4 is significantly reduced in the aorta of AAA patients and animal models. Nicotine, a well-established AAA risk factor, reduced GPX4 and SMC contractile protein expression. We posit that AAA risk factors can result in VSMC death via an underappreciated pathway, i.e., ferroptosis in the artery resulting in AAA formation and rupture. We generated VSMC-specific Gpx4 knockout mice (Gpx4SMKO) by crossing Myh11-Cre with Gpx4flox/flox mice. When subjected to aneurysm induction by angiotensin II (Ang II) coupled with hypercholesterolemia, about 50% of Gpx4SMKO mice died due to AAA rupture. The incidence rate and maximal diameters in Gpx4SMKO mice were larger than in Gpx4flox/flox control mice. Specific Aim 1 will define the protective effects of VSMC-specific GPX4 in AAA using our novel VSMC-selective GPX4 transgenic mice. We will also examine the effects of VSMC-specific Gpx4 knockout using a different murine model with perivascular application of elastase, an AAA model that does not produce rupture. Aim 2 will determine the GPX4-dependent protective mechanisms in VSMC using gain- and loss-of-function strategies. We will examine the effects of GPX4 on AAA-relevant stimuli-induced cell death, VSMC phenotypic switch, and inflammatory responses in primary human and mouse abdominal aortic smooth muscle cells. Aim 3 will define that local activation of GPX4 attenuates AAA pathogenesis by enhancing VSMC capacity of resistance to pathologic factors. Further, we will examine whether the GPX4 activator protects against aneurysm development and rupture. Successful completion of the proposed studies will establish novel mechanisms regulating VSMC loss and vascular inflammation in AAA, which are likely to advance our understanding of the AAA formation and ultimately lead to novel strategies for treating AAA.