Project Summary Hematopoietic stem cells (HSCs) possess the remarkable capacity to self-renew and sustain the entirety of the blood and immune system throughout the lifetime of an individual. HSC maintenance depends upon signals and paracrine factors produced by the bone marrow (BM) microenvironment or “niche.” HSC regeneration following myelotoxicity requires regeneration of vascular ECs within the niche. However, the precise mechanisms through which BM ECs regulate HSC regeneration remain incompletely understood. Elucidation of the mechanisms that govern HSC regeneration could have significant medical impact since a substantial percentage of cancer patients receive HSC-depleting high dose chemotherapy, radiation therapy or myeloablative hematopoietic cell transplantation in the treatment of their disease. Hematopoietic toxicities from such regimens commonly cause hospitalizations, infectious complications and delays in curative therapy. We have identified an autocrine mechanism in which BM ECs secrete a protein, semaphorin 3A (SEMA3A), in response to myelotoxic irradiation, and this protein binds to an EC receptor, NRP1, causing increased BM EC death and delayed BM vascular regeneration. We hypothesize that targeted inhibition of SEMA3A or its receptor, NRP1, on BM ECs will block SEMA3A-NRP1 signaling in BM ECs following injury, thereby facilitating the regeneration of BM ECs and the BM vascular niche wherein HSCs reside. In so doing, we propose that early restoration of the BM vasculature will promote HSC regeneration and early hematopoietic reconstitution after myelosuppression. Indeed, our preliminary results suggest that antibody-mediated inhibition of NRP1 decreases BM apoptosis following irradiation and accelerates BM vascular regeneration in irradiated mice. Importantly, systemic anti-NRP1 treatment also promotes the early recovery of white blood cells, neutrophils, myeloid progenitor cells, HSCs with long-term repopulating capacity, and increases survival of irradiated mice. We propose to utilize pharmacologic and cell-specific genetic models to characterize the functions of SEMA3A and NRP1 in regulating BM vascular regeneration and hematopoietic regeneration. We will also interrogate the role of integrin function in mediating SEMA3A effects on BM EC function and will evaluate the effects of modulation of SEMA3A-NRP1 signaling on human BM EC function and human hematopoietic regeneration. Our broad, long-term objective is to define the role of the SEMA3A-NRP1 pathway in regulating BM vascular regeneration as a novel platform for therapeutic human hematopoietic regeneration.