Wednesday, March 30, 2011

Angiopoiesis and bone regeneration via co-expression of the hVEGF and hBMP genes from an adeno-associated viral vector in vitro and in vivo

Angiopoiesis and bone regeneration via co-expression of the hVEGF and hBMP genes from an adeno-associated viral vector in vitro and in vivo

Chen Zhang1, Kun-zheng Wang1, Hui Qiang1, Yi-lun Tang1, Qian Li2, Miao Li2 and Xiao-qian Dang1
  1. 1Department of Orthopedic Surgery, the Second Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710004, China
  2. 2Department of Ultrasound, the Second Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710004, China
Correspondence: Xiao-qian Dang, E-mail dang_xiaoqian@sohu.com
Received 22 February 2010; Accepted 6 May 2010; Published online 28 June 2010.
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Abstract

Aim:

 
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-hVEGF165-internal ribosome entry site (IRES)-hBMP-7 (AAV-VEGF/BMP), rAAV-hVEGF165-GFP (AAV-VEGF), rAAV-hBMP-7-GFP (AAV-BMP), and rAAV-IRES-GFP (AAV-GFP). VEGF165 and BMP-7 gene expression was detected using RT-PCR. The VEGF165 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 VEGF165 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.

Conclusion:

 
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).

Keywords:

adeno-associated virus; vascular endothelial growth factor; bone morphogenetic protein (BMP); avascular necrosis of the femoral head (ANFH); gene therapy
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Introduction

Recent insight into the pathogenesis of avascular necrosis of the femoral head (ANFH) has not identified satisfactory methods to increase blood circulation in necrotic areas of the femoral head, to promote bone regeneration, or to prevent osteonecrosis. The rapid development of gene therapy technology is increasingly recognized as a new therapeutic option for the treatment of ANFH, especially through therapeutic neovascularization and bone formation. Among growth factors, vascular endothelial growth factor (VEGF) and bone morphogenetic protein (BMP) play important roles and have been extensively studied.
The VEGF family of growth factors is one of the most important cytokine families involved in angiogenesis. These factors promote the division of vascular endothelial cells and induce angiopoiesis. VEGF growth factors are essential for bone formation and repair during the bone regeneration process, which directly attracts endothelial cells and osteoclasts and enhances the differentiation of osteoblasts1, 2. BMP growth factors are the only signaling molecules that are individually sufficient for the induction of bone formation at orthotopic and heterotopic sites. They have defined roles in stimulating the proliferation and differentiation of mesenchymal and osteoprogenitor cells and have efficient bone induction activity3, 4. Because bone formation is a coordinated process involving the BMP and VEGF growth factors5, 6, orchestrating the timing with which these two factors are expressed may greatly enhance this process.
Choosing a safe and effective vector system to transfer and correctly express a target gene during gene therapy is important. Several different strategies have been examined for the delivery of genes of interest, including the use of naked DNA or an adenoviral vector. Treatment with naked DNA is simple and well tolerated by the recipient organism due to its low toxicity and weak induction of immune responses. However, the transduction efficiency is significantly lower when compared with other methods. The adenovirus has frequently been the vector of choice for gene transfer because it is able to transduce a variety of cells with high efciency. However, adenoviral vectors have major limitations, including a lack of sustained expression, the antigenicity of viral proteins that are targeted by both humoral immunity and cytotoxic T lymphocytes, and possible toxicity at high doses. However, there are many inherent features of the adeno-associated virus system that make it an attractive option as a human viral vector. AAV is a non-pathogenic, defective human parvovirus that requires the presence of a helper virus, such as adenovirus or herpes virus, for productive infection7, 8. Other advantages of this vector system include its low immunogenicity, its ability to transduce both dividing and non-dividing cells, the potential to integrate into specific sites, its ability to achieve long-term gene expression (even in vivo), and its broad tropism, allowing for the efficient transduction of diverse organs9. These features make AAV attractive and efficient for gene transfer in vitro and local injection in vivo.
To enhance neovascularization and bone regeneration during osteonecrosis therapy, we constructed adeno-associated viruses co-expressing hVEGF165 and hBMP-7 (rAAV-VEGF165-IRES-BMP-7) and detected their effect on gene expression and biological activity in vitro and in vivo. These data demonstrate the synergistic action of these two genes and may provide a new therapeutic option for ANFH.
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Materials and methods

Materials and reagents

The rAAV-hVEGF165-IRES-hBMP-7 (AAV-VEGF/BMP), rAAV-hVEGF165-GFP (AAV-VEGF), rAAV-hBMP-7-GFP (AAV-BMP), and rAAV-IRES-GFP (AAV-GFP) plasmids were constructed by Dr Xiang-hui HUANG. Human embryonic kidney cells-293 (HEK-293) and human umbilical vein endothelial cells (HUVECs) were obtained from the Department of Orthopedic Surgery in the Second Affiliated Hospital of Xi'an Jiaotong University. Male New Zealand rabbits (two months old, weighing 2.0–3.0 kg) were obtained from the experimental animal center of Xi'an Jiao Tong University. All animal protocols followed the recommendations and guidelines of the National Institutes of Health and were approved by the Xi'an Jiao Tong University Animal Care and Use Committee. The AAV helper-free system was obtained from Stratagene (La Jolla, CA, USA). A schematic representation of the structure of the plasmids in the AAV Helper Free System is provided in Figure 1A

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