hVEGF165 Gene Research Articles

Construction of Tissue-engineered Cartilage In Vivo from Microtia Chondrocytes After Transfection with Human VEGF165 Genes Mediated by a Recombinant Adeno-Associated Viral Vector.

To evaluate the transfection efficiency of cultured chondrocytes from individuals with microtia (microtia chondrocytes) with the recombinant adeno-associated virus vector rAAV2-hVEGF165-IRES-EGFP and hVEGF165 in vitro. To test whether VEGF165 gene-modified microtia chondrocytes can enhance the survival and quality of tissue-engineered cartilage. The recombinant plasmid rAAV2-hVEGF165-IRES-EGFP was inserted into rAAV2 virus vectors to construct rAAV2-hVEGF165-IRES-EGFP using the AATMaxTM system. The second-passage microtia chondrocytes were divided into 3 groups in vitro: the Ctr group (without transfection), Exp1 group (transfected with rAAV2-IRES-EGFP), and Exp2 group (transfected with rAAV2-hVEGF165-IRES-EGFP). At 24 h, 48 h, 72 h and 7 d after transfection, cell viability was measured by MTT staining. Transfection efficiency was determined by the rate of fluorescence-positive cells. The mRNA expression of hVEG165 was detected by RT-PCR (reverse transcription PCR) and agarose gel electrophoresis, and the VEGF165 protein levels in the supernatant fluids were measured by ELISAs. The second passage microtia chondrocytes with (Exp) and without (Ctr) transfection of VEGF165 genes were mixed with 0.5 ml 30% Pluronic F-127 at 4°C and then injected subcutaneously into the opposing side of the back of nude mice. Eight weeks after injection, the cartilage-like tissues of nude mice were harvested for morphological and histologic examination. Chondrocyte viability increased in a time-dependent manner but did not differ among the 3 groups at the same time point. The mRNA and protein levels of VEGF increased in a time-dependent manner in the 3 groups. The mRNA and protein levels of VEGF165 were much higher in the Exp 2 group than in the Ctr and Exp 1 groups at the same time point, but the levels were not significantly different between the Exp 1 and Exp 2 groups. Both the Ctr group and the Exp1 group formed mature cartilage with mature lacunar structures, metachromatic matrices, collagen, and elastic fibers, and the structure of neonatal cartilage was not significantly different between the 2 groups. However, the wet weight of the neonatal cartilage was much larger in the Exp group (127.4±12.4 mg) than in the Ctr group (58.5±12.2 mg, p < 0.05). VEGF protein staining also showed a higher level in the Exp group. The HVEGF165 gene was transfected efficiently into microtia chondrocytes using the recombinant adeno-associated virus vector rAAV2-hVEGF165-IRES-EGFP. After transfection, the mRNA and protein levels of hVEGF165 increased in a time-dependent manner. VEGF165 gene-modified microtia chondrocytes showed enhanced survival in vivo but did not improve the texture of tissue-engineered cartilage. This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description ofthese Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Construction and identification of recombinant adenovirus vector and observation of its transfecting rabbit bone marrow mesenchymal stem cells

To construct and identify adenovirus vector co-expressing hBMP2 and hVEGF165 fusion protein which labeled with green fluorescence protein, and laying the foundtion of the effect of hBMP2 and hVEGF165 gene inducing BMMSCs differentiation to osteoblast and bone defect repaired in the body. BMP2 and VEGF165 gene was amplified from cDNA library by PCR and inserted to the polyclonal site of adenovirus shuttle plasmid pAd-MCMV-GFP. Ad-BMP2- VEGF165 was recombinated and propagated in HEK293 cells by co-transfecting with the constructed recombinant shuttle plasmid pAd-MCMV-BMP2-VEGF165 and adenovirus helper plasmid pBHGloxΔ E1, 3Cre. The recombinant adenovirus was purified and virustiter was determined, and then to research GFP expression and to calculate the adenovirus transfection rate in rabbit BMMSCs. The recombinant adenovirus vector Ad-BMP2-VEGF165 was successfully constructed by the methods of gene analyzing, colony PCR, Western blotting and observing GFP expression, and the titer of the adenovirus was 1×1010 PFU /ml. Recombinant adenovirus vector containing hBMP2 and hVEGF165 gene was successfully constructed and its high titer was obtained.

Osteogenic and Angiogenic Potency of VEGF165-Transfected Canine Bone Marrow Mesenchymal Cells Combined with Coral Hydroxyapatite in Vitro.

To explore the osteogenic and angiogenic potential of human vascular endothelial growth factor 165 (hVEGF165) gene-transfected canine bone marrow mesenchymal stem cells (BMSCs) combined with coral hydroxyapatite (CHA) scaffold. We constructed a lentiviral vector and transfected canine BMSCs with the best multiplicity of infection. Osteogenesis was induced in the transfected groups (GFP-BMSCs group and hVEGF-BMSCs group) and non-transfected group (BMSCs group), followed by the evaluation of alkaline phosphatase (ALP) activity and alizarin red S staining. Cells from the three groups were co-cultured with CHA granules, respectively to obtain the tissue-engineered bone. MTT assay and fluorescence microscopy were employed to assess cell proliferation and adhesion. The expression of osteogenic and angiogenic related genes and proteins were evaluated at 7, 14, 21, and 28days post osteoinduction in cell culture alone and cell co-culture with CHA, respectively using RT-PCR and ELISA. The hVEGF165 gene was transfected into BMSCs successfully. Higher ALP activity and more calcified nodules were found in the hVEGF-BMSCs group than in the control groups (p < 0.001). Cells attached and proliferated in CHA particles. Both cells cultured alone and cells co-culture with CHA expressed more osteogenic and angiogenic related genes and proteins in the hVEGF-BMSCs group compared to the GFP-BMSCs and BMSCs groups (p < 0.05). High expression of hVEGF165 in BMSCs potentially promote the osteogenic potential of BMSCs, and synergically drive the expression of other osteogenic and angiogenic factors. hVEGF-BMSCs co-cultured with CHA expressed more osteogenic and angiogenic related factors, creating a favorable microenvironment for osteogenesis and angiogenesis. Also, the findings have allowed for the construction of a CHA-hVEGF-BMSCs tissue-engineered bone.

Endothelial cells modified by adenovirus vector containing nine copies hypoxia response elements and human vascular endothelial growth factor as the novel seed cells for bone tissue engineering.

Vascularization is one of the hotspots during the development of new therapeutic strategies for bone tissue engineering, which can alleviate hypoxic circumstance and prevent transplant failure. Vascular endothelial growth factor (VEGF) gene transfection using recombinant adenovirus (Ad) vector can effectively promote angiogenesis, but uncontrolled long-term continuous expression of VEGF brings safety concern. Here we constructed a recombinant Ad vector containing nine copies of HRE promoter and the hVEGF165 gene, which conserved the oxygen sensitivity of hypoxia-inducible factor-1/hypoxia response elements (HIF-1/HRE). After transfection into human umbilical vein endothelial cells (HUVEC), the hVEGF165 mRNA and protein levels were much higher in response to hypoxia, as revealed by RT-PCR and ELISA, respectively. Furthermore, Ad-9HRE-hVEGF165 vector effectively promoted proliferation, migration and tube formation of HUVEC under hypoxic conditions. Thus we believe that the Ad-9HRE-hVEGF165 vector can contribute to the regulation of vascularization, which may provide a new approach for a better control of the expression of hVEGF165 during bone tissue engineering.

Repair of rabbit radius defect by using recombinant of hBMP2/hVEGF165 modified BMSCs with porous nano-hydroxyapatite/polyamide 66 composite

Objective To assess the effect of bone defect repair using the recombinant of adenovirus-mediated hBMP2 and hVEGF165 genes transfer of BMSCs with porous nano-hydroxyapatite/polyamide 66 (n-HA/PA66). Methods Sixty male adult New Zealand rabbits were assigned to groups A, B and C according the completely random design, with 20 rabbits per group. Bone defect of 15 mm in length was made in the middle segment of bilateral radii in rabbits. In Group A, the defects were filled with nothing on the left side in blank controls (Group A1) and with n-HA/PA66 material alone on the right side (Group A2). In Group B, the defects were filled with hVEGF165/BMSCs/n-HA/PA66 on the left side (Group B1) and hBMP2/ BMSCs/n-HA/PA66 on the right side (Group B2). In Group C, the defects were filled with BMSCs/n-HA/PA66 on the left side (Group C1) and hBMP2/hVEGF165/BMSCs/n-HA/PA66 on the right side (Group C2). Radiological analysis, HE staining, and Masson coloration were performed 2, 4, 8 and 12 weeks after operation. Results Radiographs, HE staining and Masson staining taken 8 weeks after cell transplantation showed large amount of new cartilage grown into the defect area and massive bony tissue formation around the margin in Group C2. At postoperative 12 weeks, Group C2 showed transplants were surrounded by outer bone tissues with superior bone repair effect to other groups (P 0.05). Conclusion hBMP2/hVEGF165 genes transferred BMSCs seeded on porous n-HA/PA66 can contribute to osteogenesis during the repair of rabbit radius defect. Key words: Bone morphogenetic proteins; Vascular endothelial growth factors; Bone defect

Sequential, timely and controlled expression of hVEGF165 and Ang-1 effectively improves functional angiogenesis and cardiac function in vivo

We aimed to control the gene expression of vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1) in the ischemic heart to explore the feasibility of sequential, timely and controlled multigene expression as a means of improving therapeutic angiogenesis in vivo. Adult rabbit myocardial infarction models were surgically established (n=120). Hypoxia-inducible factor-1α-hypoxic response element (HIF1α-HRE) and Tet (tetracycline)-On advanced gene control systems were reconstructed for controlled expression of the human VEGF165 (hVEGF165) and Ang-1 genes, respectively. Recombinant adeno-associated viruses (rAAV)-9HRE-hVEGF165 and rAAV-TRE-Tight-Ang-1 were delivered into the ischemic myocardium for 12 weeks. Reverse transcription-polymerase chain reaction, western blotting and immunofluorescence staining were used to detect gene and protein expression. Vessel functionality, vascular permeability and animal cardiac function were also evaluated. Under the control of the HIF1α-HRE and Tet-On gene control systems, the expression of the exogenous hVEGF165 and Ang-1 genes was consistent in the ischemia control. In the sequential group, we found that the number of functional vessels with a larger diameter and more vascular branches was increased, and vascular permeability was significantly reduced. In addition, animal heart function was significantly improved compared with the non-sequential and hVEGF165- or Ang-1-only groups (P<0.05, P<0.05, respectively). Sequential, timely and controlled expression of the hVEGF165 and Ang-1 genes in vivo is a new therapeutic angiogenesis strategy that can effectively promote functional vessel regeneration and can improve cardiac function in ischemic heart disease.

An in vitro study of pcDNA 3.0-hVEGF165 gene transfection in endothelial progenitor cells derived from peripheral blood of rabbits

The present study investigated the effect of the VEGF165 gene on adhesion, migration, and proliferation of endothelial progenitor cells (EPCs) derived from peripheral blood of rabbits. Peripheral blood mononuclear cells were isolated from rabbits by density gradient centrifugation with Ficoll-Plaque Plus. EPCs were characterized by immunofluorescence and immunostaining. A pcDNA 3.0-hVEGF165 expression vector was constructed and EPCs were transfected with the pcDNA 3.0-hVEGF165 gene. The EPCs derived from peripheral blood of rabbits were successfully transfected with pcDNA 3.0-hVEGF165. ELISA showed that the expression of VEGF165 increased significantly in the EPCs transfected with the hVEGF165 gene compared to control cells. Compared to control EPCs, EPCs transfected with the hVEGF165 gene had significantly enhanced adhesion, migration, and proliferative ability in vitro.

Targeted Delivery of Human VEGF Gene via Complexes of Magnetic Nanoparticle-Adenoviral Vectors Enhanced Cardiac Regeneration

This study assessed the concept of whether delivery of magnetic nanobeads (MNBs)/adenoviral vectors (Ad)–encoded hVEGF gene (AdhVEGF) could regenerate ischaemically damaged hearts in a rat acute myocardial infarction model under the control of an external magnetic field. Adenoviral vectors were conjugated to MNBs with the Sulfo-NHS-LC-Biotin linker. In vitro transduction efficacy of MNBs/Ad–encoded luciferase gene (Adluc) was compared with Adluc alone in human umbilical vein endothelial cells (HUVECs) under magnetic field stimulation. In vivo, in a rat acute myocardial infarction (AMI) model, MNBs/AdhVEGF complexes were injected intravenously and an epicardial magnet was employed to attract the circulating MNBs/AdhVEGF complexes. In vitro, compared with Adluc alone, MNBs/Adluc complexes had a 50-fold higher transduction efficiency under the magnetic field. In vivo, epicardial magnet effectively attracted MNBs/AdhVEGF complexes and resulted in strong therapeutic gene expression in the ischemic zone of the infarcted heart. When compared to other MI-treated groups, the MI-M+/AdhVEGF group significantly improved left ventricular function (p<0.05) assessed by pressure-volume loops after 4 weeks. Also the MI-M+/AdhVEGF group exhibited higher capillary and arteriole density and lower collagen deposition than other MI-treated groups (p<0.05). Magnetic targeting enhances transduction efficiency and improves heart function. This novel method to improve gene therapy outcomes in AMI treatment offers the potential into clinical applications.

Expression of vascular endothelial growth factor gene mediated by lipofectamin in canine bone marrow

Objective To investigate the vascular endothelial growth factor (VEGF) secretion potential of canine bone marrow mesenchymal stem cells (BMSCs) that were transfected with hVEGF165 gene,and to provide scientific basis for stem cell treatment in avasctlar bone necrosis. Methods Marrow stromal cells were obtained from the tibia of adult dogs.The cells were isolated and differential cultured in vitro to obtain BMSCs.An hVEGF165 gene transfection group and a control group were set up.In the gene transfection group after passage the recombinant pcDNA32hVEGF165 plasmid was transfected into the BMSCs with lipofectamin and the transfection efficiency was observed under fluorescent microscope.For both groups VEGF165 expression levels in these BMSCs were quantified by ELISA at 1,3,5,7,9,11and 13 days.MTT method was applied to detect cell proliferation and draw the growth curve. Results Canine BMSCs were successfully transfected with hVEGF165 via lipofectamin and secreted VEGF proteins.VEGF secretion in culture media of hVEGF165 transfection group was higher than that of the control group ( P ＜ 0.05).ELISA detected increased VEGF secretion in 48 hours of canine BMSCs culture which peaked in 1week in the hVEGF165 transfection group.VEGF concentration was around 300 ng/L,being 10 folds of that of the control group.The difference was significant ( P ＜ 0.05).BMSCs growth curve of the transfection group and control group was not significantly different ( P ＞ 0.05 ).Conclusion hVEGF165 can be transferred into canine BMSCs using gene transfection technique.The transfected BMSCs express VEGF protein of bidogical activity.Cell proliferation is not affected. Key words: Vascular endothelial growth factor; Bone mesenchymal stem cells; Lipofectamin; Plasmids; VEGF165

Novel hyperbranched polyamidoamine nanoparticle based gene delivery: Transfection, cytotoxicity and in vitro evaluation

In this study, hyperbranched polyamidoamine (hPAMAM) was developed as a novel non-viral gene vector for the first time. The hPAMAM was synthesized using a modified “one-pot” method. DNA was then bound to hPAMAM at different weight ratios (whPAMAM/wDNA). The higher weight ratio could bring larger particle size and higher zeta potential of hPAMAM–DNA complexes. The encapsulated DNA was protected by hPAMAM from degradation for over 3h. Under the optimal condition, high gene transfection efficiency could be achieved in COS7 (47.47±1.42%) and HEK293 (40.8±0.98%) cell lines. And hPAMAM showed rather minor cytotoxicity in vitro (cell viability=91.38±0.46% in COS7 and 92.38±0.61% in HEK293). The hPAMAM mediated human vascular endothelial growth factor 165 (hVEGF165) gene transfected cells could express hVEGF165 stably for 14 days, with the peak expression at day 2. In conclusion, hPAMAM based gene delivery was economical, effective and biocompatible, and may serve as a promising non-viral vehicle for gene therapy.

English

The experiment adopting reverse transcription polymerase chain reaction (RT-PCR) technology, amplified hVEGF165 gene fragments from human leukemia cells HL-60. hVEGF165 gene was reconstructed in pIRES2-EGFP and transferred into the human placenta-derived mesenchymal stem cells (HPMSCs) by liposome-mediated method successfully. The mRNA and protein of hVEGF165 in the transferred cells was detected by RT-PCR and Western blot, and the results showed that hVEGF165mRNA and the protein expressed by HPMSCs transfected with pIRES2-EGFP-hVEGF165 was significantly more than HPMSCs transfected with pIRES2-EGFP. EGFP expression was observed under fluorescence microscope, which proved that the report gene was successfully transferred to the target cells. hVEGF biology activity and cell proliferation activity of HPMSCStransfected with pIRES2-EGFP-hVEGF165 was detected by MTT array, which showed that hVEGF165 can promote the proliferation of HPMSCS; however, hVEGF165 biology activity of HPMSCS&nbsp;transfected with pIRES2-EGFP-hVEGF165 was significantly more than HPMSCs transfected with pIRES2-EGFP. Identification of multipotentiality showed that HPMSCS&nbsp;transfected with pIRES2-EGFP-VEGF165 still maintained multipotentiality. &nbsp; Key words: Transfect, human placenta-derived mesenchymal stem cells, hVEGF165.

Hypoxic response elements and Tet-On advanced double-controlled systems regulate hVEGF165 and angiopoietin-1 gene expression in vitro

Angiogenesis in ischemic tissue is a complex and multi-gene event. In the study, we constructed hypoxic response elements (HRE) and the Tet-On advanced double-controlled systems and investigated their effects on the expression of hVEGF 165 and angiopoietin-1 ( Ang-1) genes in rat cardiomyocytes exposed to hypoxia and pharmacologic induction. We infected neonatal rat cardiomyocytes with recombinant rAAV-rtTA-Rs-M2/rAAV-TRE-Tight-Ang-1 and rAAV-9HRE-hVEGF 165. Our results indicated that the viral titer was 1×10 12 vg/mL and the viral purity exceeded 98%. hVEGF 165 expression was induced by hypoxia, but not by normoxia ( P < 0.001). Ang-1 expression was evident under doxycycline induction, but undetectable without doxycycline induction ( P < 0.001). Immunofluorescence staining showed that positively stained hVEGF 165 and Ang-1 protein appeared only under both hypoxia and doxycycline induction. We demonstrate here that HRE and the recombinant Tet-On advanced double gene-controlled systems sensitively regulate the expression of hVEGF 165 and Ang-1 genes in an altered oxygen environment and under pharmacological induction in vitro.

Gene expression promoted by the SV40 DNA targeting sequence and the hypoxia-responsive element under normoxia and hypoxia

The main objective of the present study was to find suitable DNA-targeting sequences (DTS) for the construction of plasmid vectors to be used to treat ischemic diseases. The well-known Simian virus 40 nuclear DTS (SV40-DTS) and hypoxia-responsive element (HRE) sequences were used to construct plasmid vectors to express the human vascular endothelial growth factor gene (hVEGF). The rate of plasmid nuclear transport and consequent gene expression under normoxia (20% O2) and hypoxia (less than 5% O2) were determined. Plasmids containing the SV40-DTS or HRE sequences were constructed and used to transfect the A293T cell line (a human embryonic kidney cell line) in vitro and mouse skeletal muscle cells in vivo. Plasmid transport to the nucleus was monitored by real-time PCR, and the expression level of the hVEGF gene was measured by ELISA. The in vitro nuclear transport efficiency of the SV40-DTS plasmid was about 50% lower under hypoxia, while the HRE plasmid was about 50% higher under hypoxia. Quantitation of reporter gene expression in vitro and in vivo, under hypoxia and normoxia, confirmed that the SV40-DTS plasmid functioned better under normoxia, while the HRE plasmid was superior under hypoxia. These results indicate that the efficiency of gene expression by plasmids containing DNA binding sequences is affected by the concentration of oxygen in the medium.

Influence on the Osteogenic Activity of the Human Bone Marrow Mesenchymal Stem Cells Transfected by Liposome-Mediated Recombinant Plasmid pIRES-hBMP2-hVEGF165 In Vitro

Bone marrow mesenchymal stem cells (BMSCs) is an attractive option for use as seed cells in tissue engineering strategies. Bone morphogenetic proteins (BMPs) to be involved in the formation of various tissue types including bone, cartilage, tendon, and ligament. Vascular endothelial growth factor (VEGF) is a promising reagent for inducing angiogenesis. Constructed a coexpressing vector of human bone morphogenetic protein 2 (hBMP-2) and vascular endothelial growth factor 165 (hVEGF165). The vectors were transfected into proliferated BMSCs isolated from healthy adult bone marrow. The expression of hBMP-2 and hVEGF165 genes of BMSCs were assayed by Western-blot, and the level of alkaline phosphatase activities of BMSCs was determined by RT-PCR analysis of osteocalcin mRNA expression. The levels of collagen I by immunohistochemical staining were also determined.BMSCs transfected with reconstructed plasmid pIRES-hBMP-2-VEGF165 could secret a high level of BMP-2 and hVEGF165. The production of type I collagen, the activity of alkaline phosphatase, and the expression of osteocalcin mRNA were also significantly improved in the transfected BMSCs, compared with the control group.The exogenous hBMP-2 and hVEGF165 genes can be expressed constitutively in the transfected BMSCs, and the lineage-committed differentiation abilities of BMSCs containing combination of genes BMP-2 and VEGF165 are enhanced.

Deproteinized bone with VEGF gene transfer to facilitate the repair of early avascular necrosis of femoral head of rabbit

To explore a new method for early avascular necrosis of femoral head (AVNFH) therapy. Sixty-nine AVNFH New Zealand adult rabbits were randomly divided into three groups with equal number. In Group A, deproteinized bone (DPB) that absorbed with recombinant plasmid pcDNA3.1-hVEGF165 was implanted into the drilled tunnel of necrotic femoral head. In Group B, only DPB was implanted. In Group C, only tunnel was drilled without DPB or plasmid implanted. Femoral head specimens were obtained at postoperative 1, 2, 4, 8, 16 weeks. The expression of VEGF165 and collagen I was detected by immunohistochemistry. Bone formation was detected generally by X-ray. Angiogenesis and the repair of the femoral head were observed histologically. The expression of VEGF 165 could be detected 2 weeks after implantation in Group A, but it was not observed in other groups. The result of collagen I expression had a significantly difference 2, 4 and 8 weeks after operation in Group A from those in other groups (P < 0.01). X-ray results indicated that there was more bone formation in Group A than in other groups. The regenerated capillary vessels staining result of necrotic femoral head in Group A was significantly different from those in other groups at postoperative 2 and 4 weeks (P < 0.01). Transfection of hVEGF165 gene enhances local angiogenesis and DPB-VEGF compound improves the repair of necrotic femoral head. Deproteinized bone grafting with VEGF gene transfer provides a potential method for the treatment of osteonecrosis.

Treatment for osteonecrosis of femoral head by hVEGF-165 gene modified marrow stromal stem cells under arthroscope

To understand the biological characteristics and osteogenic potential of hVEGF-165 gene modified marrow stromal stem cells and investigate the effect and value of treatment for osteonecrosis of femoral head by hVEGF-165 gene modified marrow stromal stem cells under arthroscope. rAAV-2-hVEGF-165 plasmids were extracted and transfected into rabbit marrow stromal stem cells. hVEGF-165 mediated by adeno-associated virus (AAV) was used to transfect rMSCs. The transfection efficiency was detected with enhanced green fluorescent protein under fluorescence microscope. hVEGF-165 mediated by adeno-associated virus (AAV) was used to transfect rMSCs. Virus transfection stayed overnight after 90% cell converged. MOI was 105. The transcription and expression of hVEGF-165 protein expression were detected by RT-PCR and Western blot. The necrotic bone was emptied and then MSCs were implanted under arthroscope. The histology of femoral head was inspected at postoperative 2 - 8 weeks. The expression of hVEGF-165 gene could be found distinctly in the transfected rabbit MSCs and hVEGF-165 protein in the supernatants of transfected cell cultures. The transfection efficiency of adeno-associated virus (AAV) transfected rMSCs was 70%. And rAAV-2-hVEGF-165 transfected rMSCs achieved an effective expression by RT-PCR and Western blot. hVEGF-165 could be found after a 48-hour transfection and peaked at Day 10. Immunohistochemical detection showed that the implanted rMSCs was positive at Week 2 and strong positive at Week 8. The compressive strength of the hVEGF-165 gene group approached that of normal control. hVEGF-165 gene transfected rabbit MSCs can express hVEGF-165 with highly biological activity. It provides provided a basis for employing hVEGF-165 gene and MSCs based gene therapy for ONFH repairing and regeneration. rAAV-2-hVEGF-165/MSCs may be implanted accurately under arthroscope. Implantation of human BMP-2 gene transfected BMSCs can repair early-stage experimental femoral head necrosis.

Genetic engineering of human stem cells for enhanced angiogenesis using biodegradable polymeric nanoparticles

Stem cells hold great potential as cell-based therapies to promote vascularization and tissue regeneration. However, the use of stem cells alone to promote angiogenesis remains limited because of insufficient expression of angiogenic factors and low cell viability after transplantation. Here, we have developed vascular endothelial growth factor (VEGF) high-expressing, transiently modified stem cells for the purposes of promoting angiogenesis. Nonviral, biodegradable polymeric nanoparticles were developed to deliver hVEGF gene to human mesenchymal stem cells (hMSCs) and human embryonic stem cell-derived cells (hESdCs). Treated stem cells demonstrated markedly enhanced hVEGF production, cell viability, and engraftment into target tissues. S.c. implantation of scaffolds seeded with VEGF-expressing stem cells (hMSCs and hESdCs) led to 2- to 4-fold-higher vessel densities 2 weeks after implantation, compared with control cells or cells transfected with VEGF by using Lipofectamine 2000, a leading commercial reagent. Four weeks after intramuscular injection into mouse ischemic hindlimbs, genetically modified hMSCs substantially enhanced angiogenesis and limb salvage while reducing muscle degeneration and tissue fibrosis. These results indicate that stem cells engineered with biodegradable polymer nanoparticles may be therapeutic tools for vascularizing tissue constructs and treating ischemic disease.

Vascular endothelial growth factor promotes cardiac stem cell migration via the PI3K/Akt pathway

VEGF is a major inducer of angiogenesis. However, the homing role of VEGF for cardiac stem cells (CSCs) is unclear. In in vitro experiments, CSCs were isolated from the rat hearts, and a cellular migration assay was performed using a 24-well transwell system. VEGF induced CSC migration in a concentration-dependent manner, and SU5416 blocked this. Western blot analysis showed that the phosphorylated Akt was markedly increased in the VEGF-treated CSCs and that inhibition of pAkt activity significantly attenuated the VEGF-induced the migration of CSCs. In in vivo experiments, rat heart myocardial infarction (MI) was induced by left coronary artery ligation. One week after MI, the adenoviral vector expressing hVEGF165 and LacZ genes were injected separately into the infarcted myocardium at four sites before endomyocardial transplantation of 2 × 10 5 PKH26 labeled CSCs (50 μL) at atrioventricular groove. One week after CSC transplantation, RT–PCR, immunohistochemical staining, Western blot, and ELISA analysis were performed to detect the hVEGF mRNA and protein. The expression of hVEGF mRNA and protein was significantly increased in the infarcted and hVEGF165 transfected rat hearts, accompanied by an enhanced PI3K/Ak activity, a greater accumulation of CSCs in the infarcted region, and an improvement in cardiac function. The CSC accumulation was inhibited by either the VEGF receptor blocker SU5416 or the PI3K/Ak inhibitor wortmannin. VEGF signaling may mediate the migration of CSCs via activation of PI3K/Akt.

Transplantation of Nanoparticle Transfected Skeletal Myoblasts Overexpressing Vascular Endothelial Growth Factor-165 for Cardiac Repair

We investigated the feasibility and efficacy of polyethylenimine (PEI) based human vascular endothelial growth factor-165 (hVEGF165) gene transfer into human skeletal myoblasts (HSM) for cell based delivery to the infarcted myocardium. Based on optimized transfection procedure using enhanced green fluorescent protein (pEGFP), HSM were transfected with plasmid-hVEGF165 (phVEGF165) carried by PEI (PEI-phVEGF165) nanoparticles. The transfected HSM were characterized for transfection and expression of hVEGF165 in vitro and transplanted into rat heart model of acute myocardial infarction (AMI): group-1=DMEM injection, group-2= HSM transplantation, group-3= PEI-phVEGF165-transfected HSM (PEI-phVEGF165 myoblast) transplantation. A total of 48 rats received cyclosporine injection from 3 days before and until 4 weeks after cell transplantation. Echocardiography was performed to assess the heart function. Animals were sacrificed for molecular and histological studies on the heart tissue at 4 weeks after treatment. Based on optimized transfection conditions, transfected HSM expressed hVEGF165 for 18 days with >90% cell viability in vitro. Apoptotic index was reduced in group-2 and group-3 as compared with group-1. Blood vessel density (x400) by immunostaining for PECAM-1 in group-3 was significantly higher (P=0.043 for both) as compared with group-1 and group-2 at 4 weeks. Regional blood flow (ml/min/g) in the left ventricular anterior wall was higher in group-3 (P=0.043 for both) as compared with group-1 and group-2. Improved ejection fraction was achieved in group-3 (58.44+/-4.92%) as compared with group-1 (P=0.004). PEI nanoparticle mediated hVEGF165 gene transfer into HSM is feasible and safe. It may serve as a novel and efficient alternative for angiomyogenesis in cardiac repair.

In vivo h-VEGF165 gene transfer improves early endothelialisation and patency in synthetic vascular grafts☆

Small-diameter synthetic vascular graft performance is inferior to autologous vein grafts. This study tested the hypotheses that local in vivo administration of plasmids encoding for human vascular endothelial growth factor (VEGF), or co-administration of plasmids encoding for human vascular endothelial growth factor/plasmids encoding for fibroblast growth factor-2 in the tissues surrounding a porous synthetic vascular graft would enhance graft endothelialisation and, consecutively, graft patency. First, optimal gene for small-diameter synthetic graft endothelialisation was studied in rat abdominal aorta model (n=132): plasmids encoding for human vascular endothelial growth factor; co-administration of plasmids encoding for human vascular endothelial growth factor/plasmids encoding for fibroblast growth factor-2; or control plasmids were injected around 60 microm ePTFE graft. Second, optimal small-diameter synthetic graft design for endothelialisation was explored in rabbit abdominal aorta model (n=90). Various ePTFE grafts or pre-clotted polyester grafts were used with/without plasmids encoding for human vascular endothelial growth factor. Third, clinically used medium-size synthetic grafts were investigated with/without plasmids encoding for human vascular endothelial growth factor in dog carotid (n=20) and femoral arteries (n=15). Endothelialisation was assessed in midgraft area with scanning electron microscopy. In rats, plasmids encoding for human vascular endothelial growth factor enhanced endothelialisation; whereas co-administration of plasmids encoding for human vascular endothelial growth factor/plasmids encoding for fibroblast growth factor-2 had worst outcome at 1 week (NS), 2 weeks (P=0.01) and 4 weeks (P=0.02). In rabbits, pre-clotted polyester grafts had a trend for faster endothelialisation than ePTFE grafts (P=0.08); whereas plasmids encoding for human vascular endothelial growth factor enhanced endothelialisation compared to controls at 2 weeks (P=0.06), however, the effect reversed at 4 weeks (P=0.03). In dogs, synthetic graft patency was improved by plasmids encoding for human vascular endothelial growth factor in femoral position (P=0.103); whereas all carotid grafts were patent at 6 weeks. Thus, these data suggested that endothelialisation was fastest in pre-clotted polyester grafts; and that local application of plasmids encoding for human vascular endothelial growth factor had a potential to improve early endothelialisation and patency in synthetic vascular grafts.