ABSTRACT The prevalence of diabetes, obesity, and vascular disease in an aging population is fueling a surge in chronic skin wounds, which affect >8 million Medicare beneficiaries at a cost of >$96 billion per year. An estimated 2.5% of US population is affected by chronic wounds. Diabetic ulcers (DU), complicated by infection/ischemia/high reactive oxygen species, often suffer from poor perfusion. Reliance on VEGF therapy to improve perfusion makes logical sense, yet clinical study outcomes fall far short of expectations. Possible factors limiting VEGF therapy outcomes include insufficient local concentration of VEGF signaling partners and regression of immature vessels. It is critically important to troubleshoot barriers and to establish a therapeutic regimen that delivers desirable functional outcomes. To circumvent the low-efficiency hurdle of VEGF single-gene “monotherapy”, development of “combined gene therapy” was initiated to deliver of two or more angiogenic molecules to substantially enhance efficacy. Current literature presents compelling evidence recognizing a critical role of active lipid mediators such as eicosanoids in enabling VEGF-dependent angiogenesis. However, the implementation of VEGF therapy has not tested the significance of these lipid mediators as necessary adjuvant. Recently we have reported that in diabetic ischemic skin endothelial cells, PLCγ2 levels remain low accounting for diminished efficiency of VEGF therapy. However, the underlying mechanisms of such downregulation remain unknown. Emerging evidence demonstrate that hyperglycemia leads to DNA methylation and epigenetic silencing in the complex interplay between genes and the environment in DU subjects. In addition to endothelium, PLCγ2 enables macrophage function via TREM2 signaling. TREM2+ macrophages participate in tissue angiogenesis post-injury. The central hypothesis is that hyperglycemia dependent loss of vascular PLCγ2– myeloid TREM2 crosstalk compromises VEGF-induced increase in diabetic wound-edge vascularization. Gene targeted DNA demethylation has potential to rescue PLCγ2 gene expression. The proposed line of work is the first to study CRISPR/dCas9-based in vivo targeted epigenetic editing of dermal endothelial cells as a therapeutic intervention for enabling VEGF therapy. Three aims are proposed: Aim 1. Determine the epigenetic mechanisms underlying PLCγ2 regulation of VEGF signaling under hyperglycemic conditions. Aim 2. Elucidate the molecular mechanisms by which PLCγ2 regulates wound tissue vascularization. Aim 3. Evaluate the functional significance of endothelial-targeted PLCγ2 augmentation in vascular-myeloid crosstalk as it relates to diabetic wound vascularization. The proposed research will unveil the critical role of PLCγ2 and the impact of this important element in VEGF signaling to help improve the efficiency and robustness of vasculogenic VEGF therapy in diabetic wound healing.

PUBLIC HEALTH RELEVANCE STATEMENT Clinical trials on VEGF therapy to rescue diabetic vasculopathy has produced limited to no benefits. Troubleshooting VEGF therapy in a way that would help maximize clinical outcomes would fill a major void in the current healthcare needs. The proposed research will unveil the critical role of PLCγ2 in VEGF signaling to help improve the efficiency and robustness of vasculogenic VEGF therapy in wound healing in diabetic subjects.