Abstract Body: Introduction:
Diabetes is associated with impaired endothelial function. Studies have demonstrated that endothelial progenitor cells (EPCs) and mature endothelial cells (EC) are susceptible to apoptosis in hyperglycemic environment. Hyperglycemia leads to p53 gene over-expression and p53 gene silencing prior to hyperglycemia exposure prevents cellular senescence, improves mitochondrial function and helps vascular regeneration.. However, it is not possible to obtain sufficient quantities of secretome for therapeutic purposes from hematopoietic CD34+ve stem cells (HSCs). Our goal in this project was to test whether conditioned media (CM) obtained from p53 silenced human stem cells (other than CD34+ve cells) can also improve endothelial regeneration exposed to hyperglycemia. We compared CM obtained from adipose and bone marrow derived mesenchymal stem cells on endothelial regeneration Methods: Ad-human-P53 (TP53)-shRNA was used to silence P53 and Ad-scrambled-null-shRNA was used as control in bone marrow derived mesenchymal stromal cells. To collect conditioned media BM-MSCs 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% exosome free FBS for only 48h and the collected supernatant was concentrated 10-fold to obtain the CM. Endothelial regeneration was tested using endothelial wound healing assay kit ( from Cell Biolabs # CBA-120-T). We compared CM obtained from null and p53sh in adipose and bone marrow derived MSCs on human endothelial cells in normal and high glucose conditions Results:. We observed that CM from bone marrow derived MSCs increased the proliferation of endothelial cells in both normal and high glucose conditions, more so than adipose derived MSCs. We are currently studying the role of exosomes from this CM on cell proliferation and we are also analyzing the secretome (exosomal and non- exosomal fraction) by mass spectroscopy. Conclusion: P53 silenced BMMSC-CM appears to improve endothelial regeneration in hyperglycemia similar to the effect of p53-silenced HSCs. This maybe secondary to the fact that both these cells are derived from bone marrow and p53 silencing improves mitochondrial function. The CM obtained from BM-MSCs are in sufficient quantities and may have a prominent therapeutic role in treating diabetes vascular complications such as diabetic wound healing.