Project Summary In the United States, approximately 32% of all deaths every year are the result of cardiovascular disease. Of these cardiovascular diseases, peripheral artery disease (PAD) afflicts more than 8 million Americans. Most often caused by atherosclerosis, or the build up of plaque, the narrowed or occluded artery in a PAD patient's lower limbs results in reduced blood flow. This condition then causes pain during exercise (intermittent claudication) or potentially even pain at rest (critical limb ischemia). Current procedures, including balloon angioplasty, stenting, bypass grafting, or an atherectomy, treat more severe forms of PAD, but these methods often remain unsuccessful in the long term. In fact, many of these procedures result in restenosis, or recurrence of narrowing of the artery. Adding to the complexity of treating PAD, many patients also suffer from diabetes and hypercholesterolemia. These medical conditions further increase the risk of heart attack or stroke, and diabetic patients remain at a significantly higher risk of limb amputation. Therefore, a successful minimally invasive treatment is of great need for all populations of PAD patients An injectable hydrogel derived from decellularized skeletal muscle extracellular matrix (ECM) has shown promise in restoring tissue perfusion and encouraging repair of ischemic muscle. Our goal is to utilize this material in a more representative model of the PAD patient population. This biomaterial-alone approach has been tested with a single injection in healthy rats, but testing with multiple injections in a hypercholesterolemic mouse model will allow for a more accurate depiction of the health conditions of PAD patients. We hypothesize that multiple intramuscular injections of a decellularized skeletal muscle ECM will increase perfusion and encourage tissue regeneration in patients with PAD and hypercholesterolemia. We are proposing to accomplish the goal of testing a minimally invasive biomaterial delivery in disease models for PAD with the following specific aims: Aim 1: Demonstrate efficacy of decellularized skeletal muscle ECM in vivo to improve perfusion in a hypercholesterolemic mouse model of PAD. Aim 2: Examine effects of decellularized skeletal muscle ECM on tissue regeneration in a hypercholesterolemic mouse model of PAD. Aim 3: Elucidate mechanism of action of decellularized skeletal muscle ECM on perfusion restoration and tissue regeneration. By validating the minimally invasive delivery of a decellularized skeletal muscle ECM in a hypercholesterolemic mouse model of hindlimb ischemia, the results will provide further support of this biomaterial-alone approach as a method for treating PAD. The results from these studies will allow us to assess whether this material should be pursued for translation into PAD patients.