Angiopoiesis and bone regeneration via co-expression of the hVEGF and hBMP genes from an adeno-associated viral vector in vitro and in vivo.
OBJECTIVE
To investigate the therapeutic potential of adeno-associated virus (AAV)-mediated expression of vascular endothelial growth factor (VEGF) and bone morphogenetic protein (BMP).


METHODS
Four experimental groups were administered the following AAV vector constructs: rAAV-hVEGF(165)-internal ribosome entry site (IRES)-hBMP-7 (AAV-VEGF/BMP), rAAV-hVEGF(165)-GFP (AAV-VEGF), rAAV-hBMP-7-GFP (AAV-BMP), and rAAV-IRES-GFP (AAV-GFP). VEGF(165) and BMP-7 gene expression was detected using RT-PCR. The VEGF(165) and BMP-7 protein expression was determined by Western blotting and ELISA. The rabbit ischemic hind limb model was adopted and rAAV was administered intramuscularly into the ischemic limb.


RESULTS
Rabbit bone marrow-derived mesenchymal stem cells (BMSCs) were cultured and infected with the four viral vectors. The expression of GFP increased from the 7th day of infection and could be detected on the 28th day post-infection. In the AAV-VEGF/BMP group, the levels of VEGF165 and BMP-7 increased with prolonged infection time. The VEGF(165) and BMP-7 secreted from BMSCs in the AAV-VEGF/BMP group enhanced HUVEC tube formation and resulted in a stronger osteogenic ability, respectively. In rabbit ischemic hind limb model, GFP expression increased from the 4th week and could be detected at 8 weeks post-injection. The rAAV vector had superior gene expressing activity. Eight weeks after gene transfer, the mean blood flow was significantly higher in the AAV-VEGF/BMP group. Orthotopic ossification was radiographically evident, and capillary growth and calcium deposits were obvious in this group.


CONCLUSIONS
AAV-mediated VEGF and BMP gene transfer stimulates angiogenesis and bone regeneration and may be a new therapeutic technique for the treatment of avascular necrosis of the femoral head (ANFH).
Zhang
Chen
C
Department of Orthopedic Surgery, the Second Affiliated Hospital of Xi'an Jiaotong University, China.
Wang
Kun-zheng
KZ
Qiang
Hui
H
Tang
Yi-lun
YL
Li
Qian
Q
Li
Miao
M
Dang
Xiao-qian
XQ
D000818:Animals; D015153:Blotting, Western; D001854:Bone Marrow Cells / Q000378:metabolism; D019485:Bone Morphogenetic Proteins / Q000235:genetics*; D001861:Bone Regeneration / Q000235:genetics*; D000229:Dependovirus / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D005271:Femur Head Necrosis / Q000503:physiopathology / Q000628:therapy; D005786:Gene Expression Regulation; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006614:Hindlimb; D006801:Humans; D007511:Ischemia / Q000503:physiopathology / Q000628:therapy; D008297:Male; D059630:Mesenchymal Stem Cells / Q000378:metabolism; D018919:Neovascularization, Physiologic / Q000235:genetics*; D011817:Rabbits; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics*
D019485:Bone Morphogenetic Proteins; D042461:Vascular Endothelial Growth Factor A