Abstract We0056: Therapeutic role of P53 silencing along with mitochondrial superoxide dismutase in hyperglycemic endothelial cells.

Introduction: Diabetes is associated with endothelial dysfunction and increased reactive oxygen species (ROS). Studies have demonstrated that endothelial progenitor cells (EPCs) and mature endothelial cells (EC) both are susceptible to apoptosis in hyperglycemia (HG) with increased ROS. p53 gene silencing (p53sh) prevents cellular senescence and helps vascular regeneration in HG. Previously we have shown that p53 silenced EPC and even associated conditioned media (CM) helped EC regeneration in HG. Our goal was to test whether CM obtained from p53 silenced and SOD2 antioxidant overexpressed human bone marrow derived mesenchymal stromal cells (BM-MSCs) can improve EC function in HG. Methods: Ad-human-P53 (TP53)-shRNA is used to silence P53 and Ad-humanSOD2 was used to over-express SOD2. Ad-scrambled-null-shRNA was used as control in BM-MSCs. To collect CM, BM-MSCs were cultured in exosome free media for 5days, following transduction with Ad-p53sh and Ad-SOD2 simultaneously or with Ad-null constructs, as the case may be. The collected supernatant was concentrated 10-fold to obtain the conditioned media (CM). Endothelial regeneration in HG was tested using endothelial wound healing assay kit ( from Cell Biolabs # CBA-120-T). Results: We compared CM obtained from null and p53sh-SOD2 in BM-MSCs on human endothelial cells in HG conditions. Number of cells in the scratch area increased significantly in the p53sh-SOD2 CM compared to null and non-transduced CM. Post secretome therapy hyperglycemia exposed endothelial cells showed increase in mt-SOD (SOD2) . Conclusion: P53 silencing followed by SOD2 over-expression further improved the number and function of human-EC cells in HG and possibly more than p53-sh-BMMSC CM. This CM may have therapeutic role in treating EC-dysfunction related complications in diabetes.