BM-derived endothelial progenitor cells (EPCs) have a favorable impact on tissue perfusion in ischemic heart and skeletal muscle, and represent novel therapeutic targets for angiogenesis. However, EPCs isolated from patients with cardiovascular (CV) risk factors show functional impairment and in preclinical studies do not posses the same pro-angiogenic potency as compared to those isolated from healthy control patients. EPC dysfunction may be related to impaired secretion of angiogenic factors, including reduced expression of eNOS, and may be improved by gene transfection. We investigated the efficacy of lentiviral versus jetPEI transfection of eNOS plasmid of EPCs from CV patients, comparing their angiogenic effects in a mouse model of hindlimb ischemia. EPCs were isolated from the blood of human subjects with CV risk factors (Framingham risk score >15%) and transduced them using lentivirus vectors (MOI of 3) containing either eNOS or GFP or with the chemical transfection agent jetPEI. Unilateral hindlimb ischemia was created in athymic nude mice (n=41) (day 0). On day 3, 5x105 human EPCs (GFP- and eNOS-transfected (n=12 for each)) or control (saline (n=5)) were injected directly into the medial thigh and gastrocnemius muscles of the ischemic limb (total volume of 0.1 mL). Immediately following surgery (day 0) and on day 28, hindlimb perfusion measurements were made using laser Doppler perfusion imaging (LDPI, Moor Instruments). After femoral artery ligation and excision, perfusion to the ischemic leg was reduced by laser Doppler imaging to ∼20% of the contralateral control limb. Over the next 28 days, endogenous angiogenesis resulted in an increase in blood flow to the ischemic leg to ∼40% in control, GFP-transfected and saline-treated animals. With administration of both viral and jetPEI eNOS transfected EPCs, perfusion increased to (59.7 +/−10.1%, (viral), 57.08+/−3.8% (jetPEI)(n=6 patients, p<0.05 versus controls). There was no significant difference between the viral and jetPEI transfection methods in regards to perfusion and no significant difference in mRNA expression by in vitro testing. eNOS transfection improves the angiogenic potency of EPCs derived from patients with CV risk factors, resulting in increased perfusion after acute hindlimb ischemia. JetPEI is a safe and feasible chemical transfection method, which is comparable to viral transfection and may be more attractive for clinical trials due to regulatory concerns over viral transfection.

Background: Endothelial progenitor cells (EPCs) from patients with coronary arterydisease (CAD) or CAD risk factors exhibit greatly reduced regenerativecapacity, which likely contributes to the relatively modest nature of the benefit seen in recent clinical trials of autologous cell therapy postmyocardial infarction. We hypothesized that eNOS overexpression will improve the functional capacity of EPCs from these patients. Methods and Results: EPCs were isolated from the peripheral blood ofpatients with high Framingham risk scores (FRS > 15%) and were transducedusing lentiviral vectors containing either eNOS or GFP (sham). We observed that eNOS-transduction significantly improved migration toward chemotactic factors(VEGF and SDF-1) compared to sham-transduced cells. EPCs were co-cultured witha mature endothelial cell (EC) line on Matrigel to measure their pro-angiogenicfunction in vitro. eNOS-transduced EPCs induced longer angiogenic tubes withmore branch points compared to sham-transduced cells, and exhibited higherassociation with EC tubes. We did not observe a significant difference in the adhesion of EPCs to an EC layer pre-activated with TNF-?, suggesting that the association to angiogenictubes is likely through a different mechanism. In immunodeficient mice, eNOS-transduced EPCs resulted in significant improvement in ischemic hindlimbperfusion compared to sham-transduced cells. PCR arrays revealed changes in angiogenic and pro-survivalgene expression in response to eNOS overexpression, providing preliminaryinsight into the mechanisms underlying its beneficial actions. Conclusions: The present data show that reduced regenerative activity of EPCsisolated from CAD patients can be significantly improved by the overexpressionof eNOS. The combination of cell and gene therapy may improve the efficacy ofautologous cell therapies for cardiovascular disease.