Autologous skeletal myoblasts transduced with a new adenoviral bicistronic vector for treatment of hind limb ischemia.
OBJECTIVE
We aimed to achieve angiogenic synergism between human vascular endothelial growth factor 165 (VEGF 165 ) 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).


CONCLUSIONS
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.
Department of Surgery, National University of Singapore.
Muhammad I
MI
Niagara
Husnain Kh
HKh
Haider
Lei
L
Ye
Valerie S W
VS
Koh
Yean T
YT
Lim
Kian K
KK
Poh
Ruowen
R
Ge
Eugene K W
EK
Sim
D000256:Adenoviridae; D042683:Angiopoietin-1 / Q000235:genetics*; D000818:Animals; D005260:Female; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D006614:Hindlimb / Q000098:blood supply*; D007511:Ischemia / Q000628:therapy*; D023421:Models, Animal; D018482:Muscle, Skeletal / Q000502:physiology; D032446:Myoblasts / Q000502:physiology; D018919:Neovascularization, Physiologic / Q000235:genetics; D011817:Rabbits; D014161:Transduction, Genetic / Q000379:methods; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics*
D042683:Angiopoietin-1; D042461:Vascular Endothelial Growth Factor A