Ex vivo gene delivery of ephrin-B2 induces development of functional collateral vessels in a rabbit model of hind limb ischemia

ObjectiveIn this study, we delivered ephrin-B2 to the ischemic hind limb of rabbits using an ex vivo method of gene transfer and evaluated whether the in vivo application of ephrin-B2 contributed to the development of functional collateral vessels. Ephrin-B2 is a transmembrane ligand of several Eph receptors and bidirectional signaling between ephrin-B2 and Eph-B4 is considered to be essential in angiogenesis and the development of arteries and veins.MethodThe left femoral artery of male Japanese White rabbits was excised to induce limb ischemia, and a primary culture of autofibroblasts was obtained from a skin section. Nineteen days later, the gene expressing ephrin-B2 (ephrin group) or β-galactosidase gene (control group) was adenovirally transfected to the cultured auto-fibroblasts (5 × 106 cells); then 48 hours later, the gene-transduced cells were injected through the left internal iliac artery of the same rabbit. At 28 days after injection, the development of collateral vessels and their function were assessed (control group, n = 12; ephrin group, n = 10).ResultsThe gene expressing ephrin-B2 was successfully transferred to the rabbit autofibroblasts, and ephrin-B2, expressed on the cell membrane, possessed binding ability with its receptor, Eph-B4. Calf blood pressure ratio (control group: 0.523 ± 0.047 vs ephrin group: 0.658 ± 0.049, P < .0001), angiographic score (0.344 ± 0.091 vs 0.525 ± 0.109, P = .0006), in vivo blood flow of the left internal iliac artery (rest: 11.963 ± 2.806 vs 17.202 ± 3.622 mL/min, P = .0014; maximum: 27.652 ± 10.377 vs 43.400 ± 7.108 mL/min, P = .0007), collateral conductance (32.740 ± 7.408 vs 54.489 ± 18.809 mL/min/100 mm Hg, P = .0097), and capillary density of the left thigh muscle (118.517 ± 18.669 vs 167.400 ± 31.271, P = .0002) showed significant improvement in the ephrin-B2 group compared with controls.ConclusionThese findings suggest that auto-fibroblasts expressing ephrin-B2 potentially promote arteriogenesis as well as angiogenesis in the adult vasculature, resulting in the development of functional collateral vessels to an ischemic lesion.Clinical RelevanceTherapeutic enhancement of collateral vessel development is a promising strategy for the treatment of vascular occlusive disease. We previously presented a new method for gene transfer of basic fibroblast growth factor and showed excellent collateral development in animal models of limb ischemia and myocardial ischemia. In the present study, we delivered ephrin-B2 by using our therapeutic strategy and determined whether in vivo gene transfection with ephrin-B2 induces collateral development in animal model. It would suggest that ephrin-B2 provides potential for therapeutic angiogenesis