AAV-mediated VEGF gene transfer into skeletal muscle stimulates angiogenesis and improves blood flow in a rat hindlimb ischemia model.
OBJECTIVE
Clinical trials on therapeutic angiogenesis using vascular endothelial growth factor (VEGF) are ongoing, however the benefits of these therapies are still controversial. To establish a more efficient gene transfer method for ischemic diseases, we investigated the therapeutic potential of adeno-associated virus (AAV)-mediated VEGF gene transfer.


METHODS
We produced VEGF(165)-expressing AAV vectors (AAV-VEGF). HEK-293 cells were transduced with AAV-VEGF in vitro and VEGF expression and secretion were examined. We used a rat ischemic hindlimb model and AAV-VEGF was administered intramuscularly into the ischemic limb. Gene expression was evaluated by RT-PCR and ELISA. Six weeks after gene transfer, we measured the blood flow of limb vessels and the skin temperature of limbs. Histochemical examination was performed to illustrate capillary growth.


RESULTS
Western blotting and ELISA revealed VEGF protein expression and secretion from AAV-VEGF-transduced HEK-293 cells. VEGF mRNA and protein expression was consistently observed in the injected muscle at least 10 weeks after the injection, while no VEGF mRNA could be detected at remote organs. The mean blood flow in AAV-VEGF-transduced ischemic limbs was significantly higher than in AAV-LacZ-transduced limbs. Capillary density was significantly higher in AAV-VEGF-injected tissues than in AAV-LacZ-injected tissues.


CONCLUSIONS
This study demonstrates that (1) AAV-mediated VEGF gene transfer into rat skeletal muscles is efficient and stable without ectopic expression, and (2) AAV-mediated VEGF gene transfer stimulates angiogenesis and thereby improves blood flow in a rat hindlimb ischemia model. These findings suggest that AAV-mediated VEGF gene transfer may be useful for treatment of ischemic diseases.
Shimpo
Masahisa
M
Division of Cardiovascular Medicine, Jichi Medical School, Minamikawachi-Machi, Tochigi 329-0498, Japan.
Ikeda
Uichi
U
Maeda
Yoshikazu
Y
Takahashi
Masafumi
M
Miyashita
Hiroshi
H
Mizukami
Hiroaki
H
Urabe
Masashi
M
Kume
Akihiro
A
Takizawa
Toshihiro
T
Shibuya
Masabumi
M
Ozawa
Keiya
K
Shimada
Kazuyuki
K
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D002478:Cells, Cultured; D003097:Collateral Circulation; D004195:Disease Models, Animal; D016228:Endothelial Growth Factors / Q000235:genetics / Q000502:physiology*; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D006439:Hemodynamics; D006614:Hindlimb / Q000098:blood supply; D007511:Ischemia / Q000503:physiopathology / Q000628:therapy*; D008222:Lymphokines / Q000235:genetics / Q000502:physiology*; D008297:Male; D018482:Muscle, Skeletal / Q000098:blood supply*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012039:Regional Blood Flow; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D016228:Endothelial Growth Factors; D008222:Lymphokines; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors