PROJECT SUMMARY This proposal describes the plan for Dr. Ulka Sachdev and her colleagues to investigate the regulation and expression of HMGB1, a nuclear protein and danger signal, in limb ischemia. These studies have relevance for a large portion of the population that suffers from peripheral arterial disease, or PAD. Patients with severe PAD are at risk for limb loss if procedures to improve blood flow can not be performed. These studies hope to elucidate a mechanism by which danger signals like HMGB1 can promote local regeneration of ischemic muscle. Her preliminary data has shown that HMGB1 is present in the nuclei of patients with PAD. In animal models, nuclear stores of HMGB1 can be mobilized by treating them with chloroquine, a commonly used drug. Mobilization of HMGB1 in this way occurs through activation of protein complexes called inflammasomes. While the interconnection of these pathways has been studied in inflammatory cells, it is not clear how they function in ischemic muscle. In muscle, these pathways may be protective. The first aim will characterize the role of inflammasome activation in HMGB1 release from myoblasts. Her preliminary data shows that myoblasts activate the inflammasome in response to ischemia. This aim will characterize inflammasome components that are present in myoblasts, determine which inflammasomes are activated after ischemia or nutrient depletion, and determine which inflammasome components are required for active release of HMGB1. The second aim will characterize how regulation of inflammasome-mediated HMGB1 release in muscle is regulated. The effects of chloroquine suggest that the process involves autophagy, a cell survival mechanism. This aim will characterize the cross-talk between autophagic, inflammasome pathways and HMGB1 expression in muscle. It is believed that inflammasome pathways involving caspase-1 will be protective, and will allow for regulated release of HMGB1. HMGB1 can then help promote muscle regeneration and possibly angiogenesis. In both specific aims 1 and 2, human muscle tissue from patients with peripheral arterial disease will be evaluated for inflammasome expression and function. Samples will be taken from patients with and without PAD who are undergoing operations for vascular disorders. Samples will also be taken from amputation specimens. These studies will provide important human correlates that may be important in evaluating significant pathways in PAD. Dr. Sachdev will be working with a team of experts in clinical management of PAD, imaging, muscle function and inflammasome signaling. The team may help define a new area of therapeutics for patients with arterial insufficiency.