Autologous skeletal myoblasts transduced with a new adenoviral bicistronic vector for treatment of hind limb ischemia

Abstract

Objective: We aimed to achieve angiogenic synergism between human vascular endothelial growth factor165 (VEGF165) and angiopoietin-1 (Ang-1) using a new adenoviral bicistronic vector concurrently with cell therapy to repair an ischemically damaged hind limb in a rabbit model. Methods: Rabbit autologous primary skeletal myoblasts were isolated and labeled with retrovirally transduced LacZreporter gene, 4,6-diamidino-2-phenylindole (DAPI), and 5-bromo-2′-deoxyuridine (BrdU). Hind limb ischemia was created in 48 female New Zealand White rabbits by means of femoral artery ligation at 8 different places, and was assessed at angiography. Animals were randomized to receive intramuscular injection of either Dulbeco's Modified Eagle Medium (DMEM;group 1, n = 8), nontransduced myoblasts (group 2, n = 10), or myoblasts transduced with Ad-Null (group 3, n = 10), Ad-VEGF (group 4, n = 10), or Ad-Bicis (group 5, n = 8). Six weeks after treatment neovascularization in the limb was assessed at angiography. The animals were euthanized, and tissue was harvested for histologic study. Results: Extensive transplanted myoblast survival was observed in all cell-transplanted groups, as visualized with DAPI, BrdU, and LacZ staining. Angiographic blood vessel count revealed enhanced neovascularization in group 5 (25.14 ± 5.14) compared with group 4 (13.62 ± 4.52), group 3 (6.09 ± 0.09), group 2 (4.67 ± 3.49), and group 1 (3.18 ± 7.76). Immunostaining for von Willebrand factor confirmed significantly increased capillary density (P <. 01) at high-power microscopic field in group 5 (19.04 1.59) compared with group 4 (15.31 ± 1.55), group 3 (6.53 ± 0.97), group 2 (5.69 ± 0.51), and group 1 (3.03 ± 0.20). Conclusion: Simultaneous expression of VEGF and Ang-1 from bicistronic vector transduced skeletal myoblasts potently stimulated enhanced functional neovascularization in a rabbit model of limb ischemia. Clinical Relevance: Angiogenesis requires interplay between various anti-angiogenic and pro-angiogenic growth factors, their receptors, and effector cells. Hence more recent studies have focused on the use of a combination of factors or their genes to develop functionally stable blood vessels. Combining angiogenic synergy between vascular endothelial growth factor165 (VEGF165) and angiopoietin-1 (Ang-1) using a bicistronic vector simultaneously expressing the genes of these 2 growth factors, together with myoblast therapy, would have the additional advantage of inducing myogenesis concurrent with angiogenesis in the treatment of ischemically injured limbs. Of note, synergy between VEGF 165 and Ang-1 to produce functional and leak-resistant blood vessels and myoblast-based cell therapy may be a promising and safe option for therapeutic angiogenesis in the clinical perspective.