Vasculogenic Skin Reprogramming Requires TET-mediated Gene Demethylation in Fibroblasts for Rescuing Impaired Perfusion in Diabetes

Tissue nanotransfection (TNT) based topical electrophoretic delivery of Etv2, Foxc2, and Fli1 (EFF) plasmids achieves increased ischemic murine skin by inducing vasculogenic fibroblasts (VF). Human dermal fibroblasts respond to EFF nano electroporation with increased expression of numerous endothelial genes in vitro and this VF induction was associated with increased ten-eleven translocase 1/2/3 (TET) expression. Validation of VF induction using scRNAseq identified a transcript signature that was TET-dependent. TNTEFF also induced TET expression in fibroblasts in vivo and fibroblast-specific inducible overexpression of EFF-induced TET expression leading to VF transition in vivo. TET induction was associated with elevated 5-hmC abundance in VF. VF emergence required TET-dependent demethylation of fibroblast-borne endothelial genes in vivo and this effect augmented VF abundance and rescued perfusion in diabetic ischemic limbs. TNTEFF also rescued perfusion and closure of murine diabetic skin wounds and increased VF in cultured human skin explants. TET enzymes, suppressed in subjects with diabetes, play a pivotal role in promoting endogenous EFF-induced VF state change. TNT delivery of EFF transcription factors upregulates TET expression. This work is the first to recognize the significance of TET1/2/3 in the TNTEFF inducible formation of VF that form de novo blood vessels to rescue diabetic ischemic tissue.