PROJECT SUMMARY This proposal presents a three-year research career development program focused on elucidating the molecular mechanisms and clinical significance of a pathologic macrophage population (Spp1+ macrophages) in peripheral arterial disease (PAD). The candidate is a currently an Assistant Professor of Surgery in the Division of Vascular & Endovascular Surgery at Duke University. He has clinical expertise in PAD and research experience in skeletal muscle biology (CTSA KL2). He has now chosen to focus on the role immune system, particularly macrophages, play in orchestrating skeletal muscle repair in PAD. Hence, he has sought additional training. The proposed experiments and didactic work will provide the candidate with a unique skillset that will enable him to transition to independence as a surgeon-scientist. Novel therapies for limb loss remain an unmet need. The long-term goal of my research program is to elucidate the molecular mechanisms of skeletal muscle regeneration in PAD and develop cellular therapies for limb preservation. Preliminary work in my lab has identified Spp1+ macrophages (Spp1+MΦ) as a pro-inflammatory macrophage population that is associated with failed muscle regeneration in PAD patients. The overall objectives of this proposal are to determine the mechanisms and clinical significance of Spp1+MΦ in PAD. The central hypothesis is that Spp1+MΦ inhibit muscle regeneration by inhibiting muscle stem cell (MuSC) and macrophage reparative programs. The rationale for this project is that determining the processes that regulate macrophage- mediated tissue repair and establishing a link between macrophage phenotype and clinical outcomes will provide a strong foundation for the development of immune-based therapies for PAD. The hypothesis will be tested by pursuing three specific aims: 1) Determine if Spp1+MΦ inhibit MuSC regeneration in the ischemic limb; 2) Determine if Spp1+MΦ inhibit macrophage polarization to a regenerative phenotype in the ischemic limb; and 3) Determine if Spp1+MΦ are positively correlated with myopathy, limb loss and mortality in human PAD patients. In Aims 1 and 2, we will perform hind limb ischemia on mice with conditional overexpression or ablation of Spp1 in myeloid cells. We will assess muscle regeneration and macrophage polarization via histologic, immunofluorescence (IF), and transcriptional analysis. In Aim 3, we will leverage our large institutional PAD tissue repository and use spatial transcriptomics and IF analysis to link Spp1+MΦ with dystrophic muscle and adverse clinical outcomes, such as major amputation and mortality. The research proposed in this application is innovative because it represents a dramatic paradigm shift in the scientific approach to PAD. This proposal places the investigative spotlight on the interplay between the immune system and skeletal muscle repair in PAD. The proposed research is significant because it is expected to provide a strong scientific justification f...