Abstract
The homing of endothelial progenitor cells (EPC) to sites of ischemic injury is critical to successful neovascularization. We hypothesize that ultrasound-mediated (UM) transfection of stromal-cell derived factor-1 (SDF-1) would enhance engraftment of exogenously administered EPCs and improve neovascularization in a rat model of chronic hindlimb ischemia. Methods: Bone-marrow derived EPCs were isolated from donor F344 rats, cultured for 7 days, labelled with CMTMR and injected (1 x 10 6 ) into recipient animals via a jugular vein. Unilateral hindlimb ischemia was created by iliac artery ligation in 32 rats. At day 14 post ligation, microvascular blood volume (MBV) and blood flow (MBF) in the hindlimb muscles were assessed by contrast-enhanced ultrasound (CEU). Rats were assigned to 4 treatment groups: control untreated animals, EPCs alone, UM delivery of SDF-1 plasmid (500 μg DNA + 1x10 9 cationic microbubbles), and both UM delivery of SDF-1 + EPCs (n=8 per group). Perfusion was re-assessed at day 28. EPC engraftment was assessed using fluorescent microscopy. Circulating exogenous EPC numbers were determined by FACS at various time-points post injection. Results: Prior to EPC delivery, normalized MBV and MBF in ischemic muscle were similarly reduced in all groups. Control animals showed no changes in perfusion over time. By day 28, EPC treated animals had a significant increase in normalized MBV (1.04±0.24 vs 0.67±0.22, p<0.001), without significant improvements in MBF (0.44±0.13 vs 0.33±0.15, p=NS), while SDF-1/EPC treated animals had significant improvement in both normalized MBV (1.33±0.51 vs 0.67±0.22, p<0.001), and MBF (0.55±0.26 vs 0.33±0.15, p<0.001). Maximal exogenous circulating EPCs could be detected at 72 hours post injection. Fluorescent microscopy showed little to no CMTMR labelled cells in the non-ischemic hindlimb in all groups. In comparison, a rich population of cells was found in the ischemic hindlimb of both the SDF-1/EPC and EPC treated animals, with the SDF-1/EPC animals demonstrating the greatest density of EPCs. Conclusions: Ultrasound-mediated transfection of SDF-1 improves intravenous EPC targeting, enhancing EPC engraftment into chronically ischemic tissue and leading to greater neovascularization response.
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