Abstract 320: Apoptosis-Resistant CD34+ Cells Improve Collateral Circulation Post Femoral Artery Occlusion in Diabetic Mouse Model
Literature shows that EPCs contribute to increased collateral vessel formation following vaso-occlusion. However diabetes is associated with poor collateral vessel formation. We cultured human EPCs (defined as CD34+ cells) and exposed them to 5.5 mM (equivalent to 99 mg%, normal glucose, NG) and 20mM (equivalent to 360mg%, high glucose, HG) glucose, which resulted in significant EPC apoptosis within 48hrs. We noted HG exposure was associated with up-regulation of P53 and its downstream genes such as P21, PUMA and Caspase-3. We hypothesized that EPC apoptosis in HG is secondary to up-regulation of P53 and silencing or knocking out P53 may help neovascularization. We obtained mouse EPCs from P53 KO mice and observed that p53 null EPCs are more resistant to cell death in HG while retaining expression of essential endothelial genes. Next, we used Lenti and Adenovirus mediated siRNA methods to silence P53 in human EPCs (hEPCs) and P53 silenced hEPC showed better survival in HG. We developed unilateral femoral artery occlusion model to mimic peripheral vascular disease in streptozotocin induced type 1 diabetic or Leptin resistant type 2 diabetic mouse model. We delivered saline, P53 WT and P53 null mEPCs intra-muscularly around the femoral occlusion (n=10 in each group) and counted number of capillaries in a 20x microscopic field. We also quantified increased vessel formation by laser doppler assay of the ischemic and contralateral hind limbs upto 2 weeks, post EPC transplantation and conducted qRT-PCR and western blot of key endothelial genes such as eNOS, p-eNOS, VEGF-A, PECAM-1 and vWF from both quadriceps in all groups of mice.
Results: The group that received P53null EPCs had the most favorable outcome. Preliminary results from transplantation of lenti p53 silenced human CD34+ cells also indicate increased vessel formation compared to non p53 silenced hCD34+ cell in NOD-SCID diabetic mice. Summary: Transplantation of P53 null EPCs in presence of hyperglycemia help improve collateral vessel formation post femoral artery occlusion compared to WT-EPC transplanted group.
Conclusion: Our results indicate towards positive role of apoptosis resistant CD34+ cells in diabetic PVD by increasing collateral formation with significant clinical implications.
Author Disclosures: S. Sen: None. J. Jerry: None.
This research has received full or partial funding support from the American Heart Association.
- © 2014 by American Heart Association, Inc.