Wound Repair by P53 Silenced Human Endothelial Cell-Derived Conditioned Media, In Vitro

Introduction: Diabetes is associated with impaired wound healing. Studies have demonstrated that endothelial progenitor cells (EPCs) and mature human endothelial cells (EC) are susceptible to apoptosis in hyperglycemic environment and p53 gene silencing prevents cellular senescence and helps vascular regeneration. Our goal was to test whether cell conditioned media obtained from p53 silenced human cells can also improve cellular regeneration. Methods: Ad-human-P53 (TP53)-shRNA was used to silence P53 and Ad-scrambled-null-shRNA was used as control. To collect condition media, different human cells such as HEK, human CD14+ve cells and HUVEC (human umbilical vein endothelial cells) were cultured in exosome free FBS media for 5 days, following transduction with Ad-p53sh or Ad-null, as the case may be. After transduction, cells were grown in media containing 2% BSA (with no FBS and growth factors) for only 24 h and the collected supernatant was concentrated 10-fold to obtain the conditioned media (CM). Endothelial regeneration or wound healing was tested using endothelial wound healing assay kit (from Cell Biolabs # CBA-120-T). Results: We compared CM obtained from null and p53sh in HEK, CD14+ve and HUVEC cells on human endothelial cells. Proliferation capacity of the endothelial cells was greater with p53sh CM compared to null and non-transduced CM. The CM obtained from the untreated and null samples did not show increased proliferation in any of the 3 kinds of cells tested. The CM from p53sh HUVEC was more effective than CM from other cells. We are analyzing the CM (exosome and non-exosome fraction) for identification of specific secretory proteins. Conclusion: P53 silencing appears to improve HUVEC paracrine factors and this particular modality of cell free therapy may have therapeutic role in treating diabetes complications and even aging. Disclosure D. K. Rivera: None. S. Nandula: None. S. Sen: None. Funding American Heart Association