Abstract 12118: Regenerative Potential Of P53 Silenced Human Endothelial Progenitor Stem Cells (hEPCs) In Diabetes
Literature shows that peripheral blood derived EPCs can be matured to adult EC, however senescence of EPCs in high glucose (HG) is also known to occur, which may reduce the number of EPCs leading to poor wound healing in diabetes. This senescence may be secondary to p53 activation. We cultured hEPCs and exposed them to 5.5 mM (equivalent to 99mg%) and 20mM (equivalent to 360mg%) glucose and assessed cell survival by FACS analysis using propidium iodide (PI) stain. There was significant cell death noted in HG within 48hrs, compared to much less cell death of commercially obtained human umbilical vein endothelial cells (HUVEC), which are mature endothelial cells (EC). As senescence of EPCs in HG has been linked to p53 activation, we obtained mouse peripheral blood derived EPCs from mouse p53 KO and WT animals and observed that p53 KO EPCs are more resistant to death compared to p53 WT in HG. We cultured EPCs from p53KO mouse peripheral blood which evolved into mature mouse EC (MEC) without senescence. MEC retained all endothelial properties such as cobblestone appearance, tube-formation on matrigel and several EC gene expression such as pecam-1 (also known as CD-31), vwf (von-Willebrand's factor), kdr (also known as vascular endothelial growth factor receptor 2, VEGF-2), e-nos (endothelial nitric oxide synthase) were maintained at similar level to WT EPC. We subsequently used Lenti-shRNA to silence p53 in human EPCs and noted better survival and maturity towards adult human EC with no loss of function compared to HUVEC for over 4wks. These findings illustrate that EPCs are more susceptible to death in HG than mature EC which may explain long-term vascular complications and poor healing in diabetes. EPC from p53KO are resistant to HG injury compared to EPC from p53WT. It is possible to culture stable MEC and human EC lines from EPC of p53KO and p53 silenced hEPCs from mouse and human blood, respectively. These cell lines maintained all usual endothelial cell characters without loss of function for more than 1 month. P53 silencing to prevent EPC senescence in high glucose may help in vital tissue repair and regeneration in diabetes.
- © 2010 by American Heart Association, Inc.