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

Original Article

Acta Pharmacologica Sinica (2010) 31: 821–830; doi: 10.1038/aps.2010.67; published online 28 June 2010

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 osteoblasts^{1, 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 activity^{3, 4}. Because bone formation is a coordinated process involving the BMP and VEGF growth factors^{5, 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 efficiency. 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 infection^{7, 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.

Figure 1.

(A) Schematic representation of the structure of plasmids in AAV Helper Free System. (B) Conceptual diagram of construction of pAAV-hVEGF₁₆₅-IRES-hBMP-7. hVEGF₁₆₅ gene (600 bp) and hBMP-7 gene (1300 bp) were respectively inserted into upstream MCS and downstream MCS located on either side of IRES sequence (631 bp). The length of the bicistronic frame is 2.5 kb.
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rAAV vector production

The construction of 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) vectors was carried out as previously described¹⁰. The structure of the pAAV-hVEGF₁₆₅-IRES-hBMP-7 vector is shown in Figure 1B. IRES sequences were incorporated into the pAAV MCS to construct a bicistronic vector with two multiple cloning sites. Then, the hVEGF₁₆₅ (Pubmed NM-003376) and hBMP-7 (Pubmed NM-001719) genes were inserted into the upstream and downstream MCS, respectively. The length of the bicistronic frame is 2.5 kb, which is within the capacity of the vector. The AAV helper-free system was used to generate recombinant AAV. HEK-293 cells were cultured in H-DMEM supplemented with 10% fetal bovine serum containing 20 mg/mL penicillin-streptomycin and incubated with 5% CO₂ at 37 ^oC. The AAV vector was co-transfected with the pAAV-helper and pAAV-RC vectors into HEK 293 cells by a calcium phosphate method according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). A primary virus stock was collected 72 h after transfection and further concentrated and purified by chloroform/PEG8000 protocols¹¹. The recombinant adeno-associated virus had a titer of 5.5×10¹¹ vp/mL.

Rabbit bone marrow-derived mesenchymal stem cells (BMSC) culture and rAAV infection in vitro

Male New Zealand rabbits were used to obtain rabbit BMSCs. The cells were harvested by gently flushing the tibiae and femora with L-DMEM. Density gradient centrifugation and adherent screening methods were used to isolate BMSCs as previously described¹². The cells were cultured in L-DMEM supplemented with 10% fetal bovine serum containing 20 mg/mL penicillin-streptomycin and incubated with 5% CO₂ at 37 ^oC. Following the 3rd passage, BMSCs (5×10⁴ cells/well) were seeded onto 24-well plates 24 h before rAAV infection. By taking into account the cytopathogenic effect, infection efficiency, and cost of recombinant virus, we determined that the best multiplication of infection (MOI) for infecting rabbit BMSCs with rAAV was 5×10⁴ vp/cell. The four rAAV virus variants were introduced into BMSCs using this MOI. Cells were incubated as above and were swirled gently at 30-min intervals. One hour later, the medium was replaced with L-DMEM supplemented with 10% fetal bovine serum. Medium was then completely replaced every three days.

Rabbit hind limb ischemia model and rAAV infection in vivo

Male New Zealand rabbits were kept under specific pathogen free conditions and supplied with sterile food and acidified water. The hind limb ischemia model was developed as described previously¹³. Rabbits were anesthetized with an intraperitoneal injection of sodium pentobarbital (50 mg/kg). Under a surgical microscope, a vertical longitudinal incision was made in the right hind limb. The right femoral arteries were separated from the origin of the external iliac artery, ligated, and completely excised. Immediately after ligation of the femoral artery, the four rAAV virus variants were each injected into five different sites¹⁴ on the three major thigh muscles of each rabbit (5.5×10¹¹ vp/20 μL per site), including the adductor (two sites), the quadriceps (two sites), and the semimembranous (one site) muscles. Subsequently, the skin was sutured. After surgery, all animals were housed under standard conditions (temperature: 21±1 °C; humidity: 55%–60%) with food and water continuously available. The hind limbs were mobilized without any fixation. To prevent infection, animals received prophylactic injections of gentamicin (0.03 mg·kg⁻¹·d⁻¹, im) within 3 days after surgery.

Rabbits were sacrificed at various time points post-injection to characterize gene expression efficiency and the effects on angiopoiesis and bone regeneration in vivo. Each group contained 30 rabbits and was divided into four experimental subgroups: group A (n=6) was examined at week 2 for GFP expression (n=3) and immunoblotting (n=3), group B (n=6) at week 4 for GFP expression (n=3) and immunoblotting (n=3), group C (n=9) at week 6 for GFP expression (n=3), immunoblotting (n=3), and ELISA (n=3), and group D (n=9) at week 8 for GFP expression (n=3) and for blood flow measurement, X-ray radiography, and immunohistochemistry (n=6).

Reporter gene (GFP) expression in vitro and in vivo

Following 3, 7, 14, and 28 days of infection with AAV-GFP virus in vitro, the expression of GFP protein was observed by inverted fluorescence microscopy. At 2, 4, 6, and 8 weeks post-injection in vivo, the muscles injected with the AAV-GFP virus were sliced by the frozen section method and the expression of the GFP protein was observed as above. Each assay was performed in triplicate.

Preparation of culture medium and assessment of VEGF₁₆₅ and BMP-7 gene expression

Total cellular RNA was isolated at 1, 2, 3, 7, 14, 21, and 28 days following infection with the AAV-GFP, AAV-VEGF, AAV-BMP or AAV-VEGF/BMP viruses using TRIzol Reagent (Invitrogen). Extracted RNA was treated with DNase I (Takara, Tokyo, Japan) to eliminate DNA contamination, and first-strand cDNA was synthesized with random hexamer primers using the reverse first-strand cDNA synthesis kit from MBI Fermentas (Glen Burnie, MD, USA). PCR was performed to amplify humanVEGF₁₆₅ (forward primer 5′-CCATCGATATGAACTTTCTGCTGTCTTG-3′; reverse primer 5′-CGGAATTCTCACCGCCTCGGCTTGTC-3′) and BMP-7 (forward primer 5′-GGCCGGATCCATGCACGTGCGCTCACTGCG-3′; reverse primer 5′-GGCCGTCGACCTAGTGGCAGCCACAG-3′). β-actin (forward primer 5′-GAGGGAAATCGTGCGTGAC-3′; reverse primer 5′-TAGGAGCCAGGGCAGTAATCT-3′) was detected by RT-PCR as an internal control. PCR was performed using the following program: 94 °C for 3 min for one cycle and 35 cycles at 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 45 s. The PCR products were electrophoresed on ethidium bromide-stained 2.0% agarose gels. Each assay was performed in triplicate.

Muscle extract preparation and assessment of VEGF₁₆₅ and BMP-7 gene expression

At 2, 4, and 6 weeks following injection with the AAV-GFP, AAV-VEGF, AAV-BMP, or AAV-VEGF/BMP viruses, the frozen muscles were pulverized in liquid nitrogen and homogenized in 3 mL of ice-cold lysis buffer (1% Nonidet P-40; 50 mmol/L Tris-HCl, pH 7.4; 150 mmol/L NaCl; 200 U/mL aprotinin; 1 mmol/L phenylmethylsulfonyl fluoride, PMSF). The tissue lysates (50 mg of protein) were separated by 12% polyacrylamide gel electrophoresis and blotted onto polyvinylidene difluoride membranes. Immunoblotting was performed with anti-human VEGF₁₆₅ and BMP-7 antibodies and the specific binding of the antibody was visualized with an ECL detection system. At 6 weeks post-injection, muscle extracts were measured with an enzyme-linked immunosorbent assay (ELISA) kit using the Biotrak ELISA system (R&D, Minneapolis, MN, USA) according to the manufacturer's instructions. Each assay was performed in triplicate.

Angiogenic and osteogenic in vitro assays

Tube formation assay

 

HUVECs were cultured as previously described¹⁵. Basement membrane matrigel matrix (BD, Bedford, MA, USA) was diluted by serum-free medium, added to a 24-well plate, and incubated at 37 °C for 30 min to allow solidification to occur. HUVECs (5×10⁴ cells/well) were seeded on the matrigel and fresh L-DMEM medium supplemented with 10% FBS was added. Next, 1 mL of culture supernatant was harvested from the AAV-GFP, AAV-VEGF, AAV-BMP, or AAV-VEGF/BMP groups 14 days post-infection and added to the 24-well plate. The plate was then incubated at 37 ^oC with 5% CO₂ for 12 h. The images of tube formation were captured under a light microscope from three random fields, and quantification of the tubes was analyzed by image processing software (Media Cybernetics, USA) to assess the biological activity of VEGF in vitro.

Mineralization assay

 

BMSCs were infected with the four virus groups above. The cells were then cultured in L-DMEM supplemented with 10% fetal bovine serum containing 20 mg/mL penicillin-streptomycin with 5% CO₂ at 37 °C (the culture medium did not contain osteogenic induction factors, such as ascorbic acid, β-glycerophosphate, or dexamethasone). Mineralization effects were detected by von Kossa and alizarin red (AZR) staining¹⁶ for calcium deposits 4 weeks post-infection and observed using an inverted phase contrast microscope. The images of mineral nodules were captured under a light microscope from three random fields, and quantification of the mineral nodules was analyzed by image processing software to assess the biological activity of BMP in vitro.

Blood flow measurement and orthotopic bone formation in vivo

Eight weeks after injection, rabbits in the four groups were anesthetized with an intraperitoneal injection of sodium pentobarbital (50 mg/kg). Blood flow in the anterior tibial artery of ischemic and normal hind limbs was measured at rest with an Aspen Advanced Doppler ultrasound device from Acuson (Siemens Medical Solutions, Mountain View, CA, USA) using a perivascular flow probe and calculated by the inlay automatic processing software. The data were expressed as a percentage of the contralateral limbs. Three separate measurements were performed for each rabbit at every time point and the results were averaged. In addition, rabbits in the four groups were subjected to X-ray radiography to assess orthotopic bone formation.

Histological assessment

Eight weeks after injection, thigh muscle tissue sections of ischemic limbs from the four groups were harvested and fixed in 10% neutral-buffered formalin. To identify the proliferation of capillary endothelial cells, tissue sections were immunostained for CD34. The monoclonal antibody against CD34 was applied at a 1:500 dilution after blocking with 1% normal bovine serum. Subsequent incubation with biotinylated horse anti-mouse IgG and an ABC Elite kit (Santa Cruz) was performed. The number of CD34-positive vessels was counted at a magnification of 200×, and twenty fields from each typical slide were counted (mean number of capillaries per square millimeter). To assess orthotopic bone formation, the slides were stained by von Kossa staining to detect mineralization.

Statistical analysis

The results are reported as means±standard deviation. The normality of the data distribution was assessed with the Shapiro-Wilk (W) test. ANOVA followed by the Fisher's test was conducted to assess differences among treatment groups. Statistical significance was set at a P-value less than or equal to 0.05. The SPSS mathematical statistics software used for this analysis was purchased from SPSS Inc (version 8; SPSS Inc, Chicago, IL, USA).

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Results

Animal condition after rAAV infection

There were no symptoms of local or systemic toxicity after rAAV infection. In the region of the injection sites, no inflammatory reaction, such as rubeosis, engorgement, or abscessus, was observed. The activities of all animals were normal. There was no systemic toxicity, such as nutation, instability of gait, anhelation, retardation, cyanosis, or convulsion. No animals died before the end of the experiments.

GFP gene expression

In vitro: GFP protein expression could be detected on the third day post-infection. However, the efficiency and density of infection were unstable. The expression of GFP protein increased from the 7th day and could be detected at 28 days post-infection (Figures 2A, 2B). In vivo: With prolonged infection time, GFP protein expression increased from the 4th week and could be detected at 8 weeks post-infection (Figures 2C, 2D).

Figure 2.

Representative images of GFP protein expression. (A–B) on the 3rd, 7th, 14th, and 28th days after rAAV-IRES-GFP virus transfection in vitro. (A) Magnification×100; (B) Magnification×200; (C–D) on the 2nd, 4th, 6th, and 8th weeks after rAAV-IRES-GFP virus injection in vivo. (C) Magnification×100; (D) magnification×200.
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Efficient genes expression of hVEGF₁₆₅ and hBMP-7

To confirm hVEGF₁₆₅ and hBMP-7 gene expression in vitro, RT-PCR assays were performed. As shown in Figure 3A–3D, the sizes of the PCR products for VEGF₁₆₅, BMP-7, and β-actin were 600 bp, 1300 bp, and 340 bp, respectively. With prolonged infection time, the intensity of the VEGF₁₆₅ and BMP-7 bands increased in the AAV-VEGF/BMP group. Together, these data demonstrate that VEGF₁₆₅ was expressed in the AAV-VEGF and AAV-VEGF/BMP groups but not in the AAV-BMP and AAV-GFP groups and that BMP-7 was expressed in the AAV-BMP and AAV-VEGF/BMP groups but not in the AAV-VEGF and AAV-GFP groups. Protein expression of 2, 4, and 6 weeks following injection with AAV-VEGF/BMPin vivo is shown in Figure 3E–3H. Expression of the VEGF₁₆₅ and BMP-7 proteins was visualized by Western blot analysis. Strong staining at the expected molecular weights of 23 kDa (hVEGF₁₆₅), 55 kDa (hBMP-7), and 43 kDa (β-actin) was observed. With prolonged infection time, the intensity of the VEGF₁₆₅ and BMP-7 bands increased. These data demonstrate that VEGF₁₆₅ was expressed in the AAV-VEGF and AAV-VEGF/BMP groups but not in the AAV-BMP and AAV-GFP groups and that BMP-7 was expressed in the AAV-BMP and AAV-VEGF/BMP groups but not in the AAV-VEGF and AAV-GFP groups. As shown in Figure 3I, 3J, the production of hVEGF₁₆₅ and hBMP-7 was quantified in relevant muscle extracts 6 weeks post-injection. The average amounts of hVEGF₁₆₅ protein in the AAV-VEGF/BMP and AAV-VEGF groups were significantly higher than those in the AAV-GFP and AAV-BMP groups (P<0.05, n=30). The average amounts of hBMP-7 protein in the AAV-VEGF/BMP and AAV-BMP groups were significantly higher than those in the AAV-GFP and AAV-VEGF groups (P<0.05, n=30).

Figure 3.

Expression of hVEGF₁₆₅ and hBMP-7. (A–D) Representative images of RT-PCR assay of AAV-GFP group (A), AAV-VEGF group (B), AAV-BMP group (C) and AAV-VEGF/BMP group (D). The size of the PCR products for hVEGF₁₆₅, hBMP-7, and β-actin were 600 bp, 1300 bp, and 340 bp, respectively. With prolonged infection time, the brightness of the VEGF₁₆₅ or BMP-7 bands increased in AAV-VEGF/BMP group. No hVEGF₁₆₅ or hBMP-7 band could be detected in AAV-GFP group. Track 1–7 stands for the 1st, 2nd, 3rd, 7th, 14th, 21st, and 28th days post-transfection. (E–H) Representative images of Western blotting assay of AAV-GFP group (E), AAV-VEGF group (F), AAV-BMP group (G) and AAV-VEGF/BMP group (H). The molecular weights of hVEGF₁₆₅, hBMP-7, and β-actin were 23 kDa, 55 kDa and 43 kDa respectively. Strong staining with the expected molecular weight was observed in AAV-VEGF/BMP group, and no hVEGF₁₆₅ or hBMP-7 band was observed in AAV-GFP group. (I) ELISA assay for VEGF protein expression. The data is expressed as the mean±SD from three independent experiments. ^bP<0.05 vs AAV-GFP group, ^eP<0.05 vs AAV-BMP group. (J) ELISA assay for BMP protein expression. The data is expressed as the mean±SD from three independent experiments. ^bP<0.05 vs AAV-GFP group. ^eP<0.05 vs AAV-VEGF group.
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Biological activity of hVEGF₁₆₅ and hBMP-7 in vitro

As shown in Figure 4A, hVEGF₁₆₅ secreted from BMSCs in the AAV-VEGF/BMP group enhanced HUVEC migration, proliferation, and tube formation in comparison with the other three groups. The number of tubes in the AAV-VEGF/BMP group was significantly higher than that in the AAV-GFP and AAV-BMP groups. However, there was no statistical difference between the AAV-VEGF/BMP group and the AAV-VEGF group (Figure 4B). In addition, the mineralization effect of hBMP-7 was detected by von Kossa (Figure 5A) and alizarin red staining (Figure 5B). The AAV-VEGF/BMP group displayed stronger osteogenic activity than all the other groups. The number of mineralized nodules in the AAV-VEGF/BMP group was significantly higher than that in the AAV-GFP and AAV-VEGF groups. However, there was no statistical difference between the AAV-VEGF/BMP group and the AAV-BMP group (Figure 5C).