Jeffrey M. Isner

Clinical trials of cardiovascular gene therapy, whether using viral (53%) or nonviral (47%) vectors, have thus far disclosed no evidence indicative of inflammatory or other complications, including death, directly attributable to the vector used. Indeed, despite the fact that initial trials of cardiovascular gene therapy targeted patients with end-stage vascular disease, including critical limb ischemia and refractory myocardial ischemia, the mortality for patients enrolled in clinical trials of cardiovascular gene therapy reported to date compares favorably with mortality for similar groups of patients in contemporary controlled studies of medical or interventional therapies. The most common morbidity reported after cardiovascular gene transfer is lower extremity edema; in contrast to data involving genetically engineered mice, however, evidence of life- or limb-threatening edema has not been described in any patients, including patients after gene transfer for myocardial ischemia. Concerns regarding the potential for angiogenic cytokines to promote the progression of atherosclerosis are not supported by angiographic follow-up of patients with coronary or peripheral vascular disease. The levels and duration of gene expression investigated for therapeutic angiogenesis transfer have been unassociated with hemangioma formation. Likewise, there is little evidence from either preclinical or clinical studies to support the notion that the administration of angiogenic growth factors, per se, is sufficient to stimulate the growth of neoplasms. Patients enrolled in clinical studies of angiogenic cytokines, including patients with diabetes and a previous history of retinopathy, have disclosed no evidence to suggest that ocular pathology is a risk of angiogenic growth factor gene transfer.

The pathogenetic basis for diabetic neuropathy has been enigmatic. Using two different animal models of diabetes, we have investigated the hypothesis that experimental diabetic neuropathy results from destruction of the vasa nervorum and can be reversed by administration of an angiogenic growth factor. Nerve blood flow, as measured by laser Doppler imaging or direct detection of a locally administered fluorescent lectin analogue, was markedly attenuated in rats with streptozotocin-induced diabetes, consistent with a profound reduction in the number of vessels observed. A severe peripheral neuropathy developed in parallel, characterized by significant slowing of motor and sensory nerve conduction velocities, compared with nondiabetic control animals. In contrast, 4 weeks after intramuscular gene transfer of plasmid DNA encoding VEGF-1 or VEGF-2, vascularity and blood flow in the nerves of treated animals were similar to those of nondiabetic control rats; constitutive overexpression of both transgenes resulted in restoration of large and small fiber peripheral nerve function. Similar experiments performed in a rabbit model of alloxan-induced diabetes produced comparable results. These findings support the notion that diabetic neuropathy results from microvascular ischemia involving the vasa nervorum and suggest the feasibility of a novel treatment strategy for patients in whom peripheral neuropathy constitutes a secondary complication of diabetes.

Background —We investigated the therapeutic potential of ex vivo expanded endothelial progenitor cells (EPCs) for myocardial neovascularization. Methods and Results —Peripheral blood mononuclear cells obtained from healthy human adults were cultured in EPC medium and harvested 7 days later. Myocardial ischemia was induced by ligating the left anterior descending coronary artery in male Hsd:RH-rnu (athymic nude) rats. A total of 10 ⁶ EPCs labeled with 1,1′-dioctadecyl-1 to 3,3,3′,3′-tetramethylindocarbocyanine perchlorate were injected intravenously 3 hours after the induction of myocardial ischemia. Seven days later, fluorescence-conjugated Bandeiraea simplicifolia lectin I was administered intravenously, and the rats were immediately killed. Fluorescence microscopy revealed that transplanted EPCs accumulated in the ischemic area and incorporated into foci of myocardial neovascularization. To determine the impact on left ventricular function, 5 rats (EPC group) were injected intravenously with 10 ⁶ EPCs 3 hours after ischemia; 5 other rats (control group) received culture media. Echocardiography, performed just before and 28 days after ischemia, disclosed ventricular dimensions that were significantly smaller and fractional shortening that was significantly greater in the EPC group than in the control group by day 28. Regional wall motion was better preserved in the EPC group. After euthanization on day 28, necropsy examination disclosed that capillary density was significantly greater in the EPC group than in the control group. Moreover, the extent of left ventricular scarring was significantly less in rats receiving EPCs than in controls. Immunohistochemistry revealed capillaries that were positive for human-specific endothelial cells. Conclusions —Ex vivo expanded EPCs incorporate into foci of myocardial neovascularization and have a favorable impact on the preservation of left ventricular function.

Background The impact of hyperlipidemia on collateral vessel development in vivo remains enigmatic. We sought to determine the anatomic extent and functional capacity of the collateral bed that develops in response to limb ischemia in a well characterized animal model of spontaneous hypercholesterolemia, the Watanabe heritable hyperlipidemic (WHHL) rabbit. We further characterized the impact of exogenous angiogenic cytokine administration on collateral vessel development and function in the same animal model. Methods and Results Weight-matched 6-month-old male homozygous WHHL (n=9) and normal New Zealand White (NZW) (n=9) rabbits underwent surgical resection of one femoral artery. Ten days later, the ischemic hindlimb was evaluated for collateral vessel formation, blood flow, and tissue damage. Collateral vasculature was less extensive among WHHL than NZW, as indicated by a significant reduction in angiographic score (0.19±0.02 versus 0.35±0.03, P <.001) and capillary density (46.4±4.1 versus 78.9±4.6/mm ² , P <.0002). This was associated with a reduction in calf blood pressure index (9.5±3.5% versus 32.8±2.8%, P <.0001), arterial blood flow (7.5±0.6 versus 13.6±0.7 mL/min, P <.0001), and muscle perfusion index (40.1±3.2% versus 65.9±2.0%, P <.0001) and an increase in muscle necrosis (48.16±5.41% versus 25.90±3.83% negative 2,3,5-triphenyltetrazolium chloride staining, P <.004). Treatment of WHHL rabbits (n=9) with recombinant human vascular endothelial growth factor produced a statistically significant improvement in all functional as well as anatomic indices of collateral development. Conclusions Collateral vessel development associated with hindlimb ischemia in vivo is severely attenuated in an animal model of spontaneous hypercholesterolemia but nevertheless may be augmented by administration of angiogenic cytokines.