PROJECT SUMMARY Chronic wounds present a costly social and medical dilemma, particularly in patients with type II diabetes, but there are no effective treatments. These wounds are characterized by chronic inflammation, severe microvascular complications, and therefore lack expansion of the granulation tissue and proliferation necessary to heal the wound. Recently, exosome administration has emerged as a potent therapy for promoting wound healing but the mechanisms underlying the therapeutic effect are mostly unknown. Exosomes are secreted membranous nanovesicles that can be isolated from cell culture of multipotent stromal cells. In a preclinical type II diabetic model, a single local administration of exosomes was very effective and reduced time to closure to nearly that of wild type mice. We found extensive neovascularization in granulation tissue of exosome-treated wounds and presence of large numbers of macrophages immunoreactive for arginase1, typically indicative of a pro-healing phenotype. Exosomes from multipotent stromal cells carry plasminogen activator inhibitor-1 (PAI-1) among other angiogenesis-associated proteins. When we applied a PAI-1 inhibitor simultaneously with exosomes, the beneficial effect of exosome treatment was partially mitigated. Exosomes can have multiple signaling pathway targets, and PAI-1 is a major component. Our long term objective is to understand the molecular mechanisms we need to address to alter the chronic wound state, and actually promote wound closure. An approach like this is necessary to understand the rationale and safety of exosome treatment. In this study, we are investigating the hypothesis that PAI-1 in bone marrow MPSC exosomes mediates adaptive effects on macrophages and ECs in chronic wound beds, thereby promoting expansion of granulation tissue with effective changes in wound healing trajectory. In Aim 1, we will determine whether overexpression or loss of PAI-1 in exosomes affects the typically delayed diabetic wound healing. We will assess spatiotemporal distribution of macrophages and endothelial cells. In Aim 2, we will analyze the changes in wound bed macrophages and endothelial cells at a single cell resolution, using an integrated epitope and transcriptomics approach. Additionally, in in vitro culture with inflammatory conditions, we will determine molecular changes in macrophages and microvascular endothelial cells, downstream of exosome uptake. The results will demonstrate the mechanisms orchestrating the efficacy of exosome treatment in normalizing inflammation and promoting diabetic wound closure. We will perform the study under the mentorship of Dr. Bruce Cronstein, Dr. Ann Marie Schmidt, and Dr. Thorsten Kirsch, and gain expertise in modeling chronic diseases in preclinical study designs, hyper-inflammatory disease states, macrophage assays, and exosome-mediated mechanisms and cellular communication in a chronic cutaneous wound bed. The collaborative and scientific learning enviro...