Project Summary The ability to restore the microvasculature and improve perfusion, by expanding the population of endothelial cells in vivo, would be a major advancement in cardiovascular medicine and create a novel therapeutic approach to cardiovascular disease. The proposed studies, aimed at identifying the role of O-GlcNAcylation in transdifferentiation and epigenetic plasticity, may reveal a novel mechanism of neovascularization and uncover new avenues for therapeutic angiogenesis and vasculogenesis (Carmeliet, 2005, Isner 1999). Activation of inflammatory signaling pathways is required to drive fibroblasts to undergo angiogenic transdifferentiation and become endothelial cells in vitro (Sayed, 2015). This process requires cell autonomous innate immune signaling, which triggers global changes in expression and activity of epigenetic modifiers (Lee, 2012). The term “transflammation” describes the process by which innate immune signaling promotes epigenetic plasticity and phenotypic fluidity. Recently, it has been shown that a glycolytic shift contributes to transdifferentiation by increased nuclear acetyl-CoA for histone acetylation (Lai, 2019). Although this glycolytic switch links metabolism to epigenetic modelling, the contribution of other metabolites to epigenetic plasticity in neovascularization remains unexplored. Preliminary data indicate that O-GlcNAcylation, and nutrient driven post-translational modification (PTM), is significantly elevated in angiogenic transdifferentiation and neovascularization following hindlimb ischemia. This observation raises the exciting possibility that O-GlcNAcylation links cell fate plasticity to metabolism in vascular transdifferentiation, and that targeting this PTM may be a new therapeutic avenue in regenerative medicine. The overarching hypothesis is that O-GlcNAcylation enhances neovascularization in recovery from ischemic injury by facilitating cell fate plasticity, and that perturbations to O-GlcNAcylation through genetic and pharmacologic manipulation will attenuate transdifferentiation and diminish neovascularization. The immediate goal will be to determine if O-GlcNAcylation is required for angiogenic transdifferentiation and to identify targets of O-GlcNAcylation contributing to epigenetic plasticity and cell fate fluidity in vascular transdifferentiation and neovascularization. The proposed studies on O-GlcNAcylation in transdifferentiation will identify an epigenetic mechanism for cellular plasticity and a novel therapeutic target to enhance endogenous neovascularization for treatment of cardiovascular disease. The long term goal is to evaluate the therapeutic efficacy of targeting O-GlcNAcylation to enhance vascular recovery in non-ischemic and ischemic models of cardiovascular disease, such as heart failure and myocardial infarction. Aim 1: Determine the role of O-GlcNAcylation in transdifferentiation and DNA accessibility in vitro. Aim 2: Determine the role of O-GlcNAcylation in transdiffe...