Vascular regeneration with adenovirally reprogrammed endothelial cells and their long-term neovascularization effects

Reprogrammed endothelial cells (rECs) have been generated directly from human fibroblasts for cell therapy by overexpression of a transcription factor, ETV2, through lentiviral vector. However, due to the nature of lentiviral vector, rECs induced with this vector system are less desirable. By using adenoviral vector, we sought to generate rECs with vascularizing and therapeutic potential in clinically more compatible manner. Furthermore, we aimed to enhance cell engraftment and neovascularization potential by encapsulating rECs with a peptide amphiphile (PA) nanomatrix gel. We employed adenoviral vector harboring ETV2 to reprogram human fibroblasts into ECs and examined characteristics and therapeutic effects of rECs induced with adenoviral ETV2 (Ad-ETV2). We found that Ad-ETV2 transduction into fibroblasts induced the expression of EC genes and repressed the expression of fibroblast-related genes. KDR expressing cells at D6 (Ad-rECs) displayed the EC functional phenotypes in vitro assays and expressed proangiogenic factors at high level. Transcriptomic analysis of Ad-rECs by RNA-seq confirmed the conversion into ECs by demonstrating that fibroblast-related genes were down-regulated whereas vascular development and angiogenesis-related genes were up-regulated in Ad-rECs. ETV2 transduction suppressed fibroblast characteristics and induced endothelial characteristics in fibroblasts during the reprogramming process. Transplantation of these Ad-rECs into ischemic hindlimb enhanced the recovery of blood flow and augmented capillary density. These Ad-rECs clearly contributed to new vessel formation through direct vascular incorporation and possibly vessel guidance over 12 months. Also, encapsulation of rECs into PA-RGDS enhanced Ad-rEC engraftment in the ischemic tissue and vessel incorporation. Here we demonstrate that, using clinically suitable adenoviral vector system transducing ETV2, human somatic cells can be directly reprogrammed to functional and therapeutically potent ECs. Ad-rECs were able to efficiently recover impaired blood flow and induce repair of ischemic tissue. Moreover, PA-RGDS nanomatrix gel improved engraftment and, consequently induced long-term, neovascularization effects or Ad-rECs. This PA-RGDS encapsulated Ad-rECs can serve as an efficient therapeutic platform for vascular regeneration.