Abstract 621: Increased Recruitment of Embryonic EPCs in a Chronic Ischemic Hindlimb Model by Transient p65-Overexpresssion
Therapeutic neovascularization can be achieved by vascular growth factors or by mobilisation or exogenous application of endothelial progenitor cells. Murine embryonal EPCs (tie-2+, c-kit+, Sca-1+, CD34-, flk-1 low) are capable of inducing therapeutic angiogenesis in a chronic hindlimb model (rabbit). In the present study, we aimed at modulation of eEPC recruitment to the ischemic tissue by overexpression of p65, an NF κB subunit. Transient p65 overexpresssion induced E-selectin, ICAM-1 and VCAM-1. In contrast, stable p65-transfected eEPCs displayed no effect on ICAM-1- and VCAM-1-expression and only a marginal increase in E-selectin expression.
Methods: 7 days after femoral artery excision, 5x106 eEPCs (wild type=wt, p65 transient=p65t, p65 stable=p65s, n=5/group) were retroinfused into the anterior tibial vein. Recruitment of diI-labeled eEPCs in the ischemic gastrocnemic muscle was investigated 2 days after retroinfusion of wt-, p65t- and p65s-eEPCs. At d7 and 35 angiography of both hindlimbs was performed for collateral quantification (% of d7 level) and frame count score (cinedensitometry, % of d7 level). Capillary/muscle fiber ratio was assessed at d35.
Results: eEPC recruitment at d2 was elevated after transient p65-transfection (116±23 cells/field vs. 45±7, wt). Whereas no difference was found in capillary density of p65-t-eEPC-and wt-eEPC-treated ischemic muscles (1,44±0.10 vs. 1.41±0.09, respectively), collateral growth (wt 129±5 vs. 107±7% in controls) was increased by p65t-eEPCs (146±4%). The wt-eEPC-induced gain of perfusion (151±5% vs. 109±10% in controls) was further increased by p65t-eEPC transfusion (205±24%). Interestingly, p65s-eEPCs had no effect on collateral growth (131±7%) and perfusion-improvement (172±18%), compared to wt-eEPCs.
We conclude that transient p65-transfection in embryonic EPCs increases recruitment to ischemic muscle tissue via induction of an inflammatory phenotype, resulting in a further gain of perfusion. The lack of a functional effect of stable p65-transfection indicates a short therapeutic window of p65 transfection, most likely limited by an intracellular feed back mechanism counteracting the activity of overexpressed p65 within days.