Vascular endothelial growth factor (VEGF165) is a highly specific vascular endothelial growth factor that can be used to treat many cardiovascular diseases. The development of anti-tumor drugs and disease detection reagents requires highly pure VEGF165 (at least 95% purity). To date, the methods for heterologous expression and purification of VEGF165 require multiple purification steps, but the product purity remains to be low. In this study, we optimized the codons of the human VEGF165 gene (vegf165) according to the yeast codon preference. Based on the Pichia pastoris BBPB vector, we used the Biobrick method to construct a five-copy rhVEGF165 recombinant expression vector using Pgap as the promoter. In addition, a histidine tag was added to the vector. Facilitated by the His tag and the heparin-binding domain of VEGF165, we were able to obtain highly pure rhVEGF165 (purity > 98%) protein using two-step affinity chromatography. The purified rhVEGF165 was biologically active, and reached a concentration of 0.45 mg/mL. The new design of the expression vector enables production of active and highly pure rhVEGF165 ) in a simplified purification process, the purity of the biologically active natural VEGF165 reached the highest reported to date.
Codon/genetics* ; Humans ; Pichia/genetics* ; Recombinant Proteins/genetics* ; Saccharomycetales ; Vascular Endothelial Growth Factor A/genetics* ; Vascular Endothelial Growth Factors

OBJECTIVEGene therapy has been becoming one of the most attractive medical areas. But the using of gene therapy in plastic surgery is relatively scarce. Our purpose was to investigate the effect of naked plasmid encoding Vascular Endothelial Growth Factor on the viability of the random skin flap by directly injected subcutaneously.

METHODS30 female Sprague-Dawley rats randomly divided into three groups. A random dorsal skin flap of 3 cm x 9 cm was elevated in each of the rats. And 1 ml double-distilled water solution was injected subcutaneously, which was only water in group 1 during the operation, 200 micrograms VEGF cDNA plasmid in group 2 during the operation, 200 micrograms pcDNA3.1/zeo(+)--VEGF in group 3, 24 hours before the operation, respectively. 7 days after the operation, all the animals were sacrificed by overdose anesthetic. The survival tissue was measured with planimetry. Two samples were harvested from each group for pathological check and immunohistochemical test.

RESULTSImmunohistochemical staining demonstrated that there was human VEGF deposited around the capillary in the flaps treated with VEGF gene. The flaps treated with VEGF gene had a larger percentage of survival skin (group 1 = 47% +/- 5.4%, group 2 = 65.4% +/- 6.3%, group 3 = 72.3% +/- 8.5%, P < 0.05).

CONCLUSIONVEGF gene directly injected into subcutaneous can express VEGF. It makes the gene therapy simple and practical and will be promising future in the tissue transplantation.

Animals ; Endothelial Growth Factors ; genetics ; Female ; Genetic Therapy ; Injections, Subcutaneous ; Lymphokines ; genetics ; Rats ; Rats, Sprague-Dawley ; Surgical Flaps ; physiology ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors

OBJECTIVETo study the expression of vascular endothelial growth factor (VEGF) and its receptors, the fms-like tyrosine-1 (flt-1) and kinase insert domain-containing receptor (KDR) in endometrial carcinoma and investigate the functions of VEGF and its receptors for endometrial carcinoma angiogenesis and its relation to the grade of tumor.

METHODSImmunocytochemistry and in situ hybridization technique were used to measure the level of VEGF, flt-1, KDR protein and mRNA in endometrial carcinoma tissue from 23 patients and endometrial samples from 6 normal menopausal women. A few endometrial carcinoma samples were homogenized for Western blot analysis. The blood vessel density was estimated by counting blood vessels stained with endothelial marker VIII factor.

RESULTSThe VEGF and its receptors were widely expressed in the cytoplasm of endothelial cells and tumor cells of endometrial carcinoma. The level of VEGF protein in endothelial cells and endometrial cancer cells of grade II and III tumor tissues was higher than that in grade I and normal menopausal endometrium (P < 0.05). VEGF mRNA did not show higher expression with the increase of tumor grade but its expression in normal tissue was lower than that in cancer (P < 0.05). The expression of flt-1 protein and mRNA in endothelial cells got higher in III than in grade II and I (P < 0.05), but invariable in cancer cells (P > 0.05), flt-1 expression in cancer was higher than that in normal menopausal endometrium either in endothelial cells or in cancer cells (P < 0.05). The expression of KDR protein in endothelial and cancer cell was high but did not alter with the increase of tumor grade (P > 0.05), the level of its mRNA was higher in cancer than that in normal tissue (P < 0.05). The microvascular density in grade III (48 +/- 12) was higher than that in grade II (26 +/- 16), grade I (27 +/- 14) and normal menopausal tissue (26 +/- 11, P < 0.05).

CONCLUSIONSThe expression pattern of VEGF, flt-1 and KDR protein and mRNA increased with the increase of tumor grade in endometrial carcinoma indicates that VEGF and its receptors contribute to the neovascularization of tumors and is one of the factors that relate to rapid tumor growth of endometrial carcinoma.

Endometrial Neoplasms ; metabolism ; physiopathology ; Endothelial Growth Factors ; genetics ; metabolism ; Extracellular Matrix Proteins ; metabolism ; Female ; Gene Expression ; Humans ; Intercellular Signaling Peptides and Proteins ; genetics ; metabolism ; Lymphokines ; genetics ; metabolism ; Neovascularization, Pathologic ; RNA, Messenger ; metabolism ; Receptors, Vascular Endothelial Growth Factor ; genetics ; metabolism ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factor Receptor-1 ; Vascular Endothelial Growth Factor Receptor-2 ; metabolism ; Vascular Endothelial Growth Factors

OBJECTIVETo construct a retroviral vector carrying human vascular endothelial growth factor (hVEGF (121)) cDNA for evaluation of the possibility of VEGF gene therapy in ischemic bone disease.

METHODShVEGF(121) cDNA was obtained from the plasmid pCDI/VEGF(121) and cloned into retroviral plasmid pLXSN. Recombinant plasmid was transferred to the retro virus packaging cell, PT-67, by lipofectamine mediated gene transfer. Mouse bone marrow stromal cells (MSCs) were transfected by the retrovirus. The integration of the hVEGF(121) cDNA into MSC genomic DNA and expression of the VEGF gene was detected. Proliferation assays of human umbilical vein endothelial cells (HUVECs) by VEGF(121) in culture medium were performed.

RESULTSRecombinant pLXSN/VEGF(121) was correctly constructed and confirmed by restriction endonuclease analysis and DNA sequencing analysis. hVEGF(121) gene was integrated into MSC genomic DNA after transfection, and the VEGF(121) protein was expressed. Proliferation assays showed VEGF(121) in culture medium was a biologically active protein and had a mitogenic effect on HUVEC.

CONCLUSIONSRecombinant retroviral vector carrying hVEGF(121) cDNA was successfully constructed. VEGF (121) protein expressed by MSCs had mitogenic effect biologically. This provides a further foundation for VEGF gene therapy for bone ischemic disease and bone tissue engineering.

Animals ; Bone Marrow Cells ; metabolism ; Cell Division ; DNA, Complementary ; genetics ; Endothelial Growth Factors ; genetics ; Endothelium, Vascular ; cytology ; Genetic Therapy ; Humans ; Lymphokines ; genetics ; Mice ; Plasmids ; Retroviridae ; genetics ; Stromal Cells ; metabolism ; Transgenes ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors ; Virus Assembly

To investigate the effect of vascular endothelial growth factor (VEGF) on beta1 integrin (VLA-4 and VLA-5) activation ability in K562 cells transfected with antisense VEGF121 cDNA, K562 cells were transfected with antisense (As), sense (S) and vector (V, pcDNA(3)). Flow cytometry was used to evaluate the expression rate of VLA-4 (CD49d/CD29) and VLA-5 (CD49e/CD29) and beta1 integrin activation ability in the transfected K562 cells. The results showed that the expression rates of VLA-4 and VLA-5 were more than 92% in the transfected K562 cells and there was no difference among the K562/V, K562/S and K562/As cells. However, beta1 integrin activated 9EG7 expression rate in K562/As cells was higher than that in K562/V cells [(75.6 +/- 10.5)% vs (41.2 +/- 2.1)%, P < 0.01)] after activation with beta1 integrin activator 8A2. It is concluded that function of beta1 integrin to be activated is restored in K562 cells transfected with antisense VEGF121 cDNA.
DNA ; genetics ; DNA, Antisense ; genetics ; Endothelial Growth Factors ; genetics ; metabolism ; Flow Cytometry ; Humans ; Integrin alpha4beta1 ; biosynthesis ; Integrin alpha5beta1 ; biosynthesis ; Intercellular Signaling Peptides and Proteins ; genetics ; metabolism ; K562 Cells ; Lymphokines ; genetics ; metabolism ; Transfection ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors

OBJECTIVETo construct the adenoviral vector bringing hVEGF(121) cDNA for evaluation of the possibility of VEGF gene therapy in ischemic bone disease.

METHODSHuman vascular endothelial growth factor (hVEGF(121)) cDNA obtained from the plasmid pCDI/VEGF(121) was cloned into plasmid pshuttle and further cloned to Adeno-X Viral DNA. The recombinant adenoviral plasmid was identified and then transferred to the adenoviral packaging cell HEK293 by lipofectamine mediated gene transfer method to pack the virus. After titilating the virus, the mouse bone marrow stromal cells (MSC) were transfected by the adenovirus and the expression of VEGF gene was detected.

RESULTSThe recombinant Adeno-VEGF(121) was correctly constructed and confirmed by restriction endonuclease analysis and DNA sequencing analysis. After MSCs were tranfected by the virus, RT-PCR showed that hVEGF(121) mRNA was transcripted from the hVEGF(121) gene. Western blot and immune histochemistry showed VEGF(121) protein was expressed in transgene MSCs.

CONCLUSIONThe recombinant adenoviral vector bringing hVEGF(121) cDNA was successfully constructed and the transgene MSC expressed hVEGF gene in vitro, it provided the further foundation of VEGF gene therapy for bone ischemic diseases.

Adenoviridae ; genetics ; Blotting, Western ; Cells, Cultured ; DNA, Complementary ; genetics ; Endothelial Growth Factors ; genetics ; metabolism ; Gene Expression ; Gene Transfer Techniques ; Genetic Vectors ; genetics ; Humans ; Immunohistochemistry ; Lymphokines ; genetics ; metabolism ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors

OBJECTIVE: To assess the association of single nucleotide polymorphisms (SNPs) of vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor 1 (VEGFR1) pathways-related genes and the risk of pre-eclampsia. METHODS: In total 178 pregnant women with pre-eclampsia (case group) and 100 healthy pregnant women (control group) during the third trimester were enrolled. The SNPs of VEGF rs3025039, rs2010963 and VEGFR1 rs3812867, rs55875014 and rs722503 loci were determined by PCR-restriction fragment length polymorphism (PCR-RFLP) assay. The levels of serum VEGF and sVEGFR1 were also determined. And their association with pre-eclampsia was analyzed. RESULTS: The systolic blood pressure, diastolic blood pressure and sVEGFR1 of the case group were significantly higher than those of the control group, while the VEGF level was significantly lower than that in the control group (P<0.05). Allelic frequencies of the VEGF (rs3025039, rs2010963) and VEGFR1 (rs3812867, rs55875014, rs722503) have fit the Hardy-Weinberg equilibrium (P>0.05). The frequency of T allele of VEGF at rs3025039 locus in the case group was higher than that in the control group (P<0.05). There were significant differences in VEGF at rs3025039 locus under dominant and co-dominant models in case group (P<0.05). Compared with those with CC, the risk was higher in patients with CT or TT genotypes (P<0.05). The systolic and diastolic blood pressure and sVEGFR1 in pre-eclampsia pregnant women with CT or TT genotypes were significantly higher than those with the CC genotype, while their VEGF level was significantly lower (P<0.05). No significant difference was found in allelic frequencies of other four loci between the two groups (P>0.05). CONCLUSION Polymorphisms of rs3025039 locus of VEGF gene is associated with the occurrence of pre-eclampsia. The variant at this locus may affect the activity of VEGF and influence the development of pre-eclampsia.
Case-Control Studies ; Female ; Genetic Predisposition to Disease ; Genotype ; Humans ; Polymorphism, Single Nucleotide ; Pre-Eclampsia/genetics* ; Pregnancy ; Vascular Endothelial Growth Factor A/genetics* ; Vascular Endothelial Growth Factor Receptor-1/genetics* ; Vascular Endothelial Growth Factors/genetics*

OBJECTIVETo investigate the feasibility of endothelialization of bioprosthesis by transfer of vascular endothelial growth factor (VEGF) gene.

METHODSBovine pericardium treated with glutaraldehyde and L-glutamic acid was positioned into the pig right atrium. pcD(2)/hVEGF(121) gene (1 mg) was transferred into the right ventricular myocardium using surgical sutures Reverse transcri ption polymerase chain reaction (RT PCR) was employed to evaluate the expression of myocardial VEGF mRNA. The determination of concentrations of VEGF protein in blood from both the right atrium and peripheral vein, and histological and ultrastructural analysis of implanted bovine pericardium were completed simultaneously.

RESULTSThe concentration of VEGF derived from the right atrium in pcD(2)/hVEGF(121) group was significantly higher than that in the pcD(2) group 10 days after VEGF gene transfer (P < 0.01). The expression of myocardial VEGF mRNA in pcD(2)/hVEGF(121) group was much higher in comparison with that in the pcD(2) group. The morphological analysis demonstrated that the coverage rate of host endothelium in the pcD(2)/hVEGF(121) group was 2.6 times as fast as that in the pcD(2) group at 16 days after VEGF(121) gene transfer (P < 0.01). Entire endothelialization occurred at 30 days after VEGF gene transfer. In addition, higher expression of myocardial VEGF mRNA was still available.

CONCLUSIONSVEGF gene transfer by surgical suture can remarkably accelerate endothelialization of bioprosthesis, which may provide a new approach for inhibiting biological valve calcification and improve biocompatibility and long-term durability of the bioprosthesis.

Animals ; Bioprosthesis ; Endothelial Growth Factors ; analysis ; genetics ; Endothelium, Vascular ; physiology ; Female ; Gene Transfer Techniques ; Heart Valve Prosthesis ; Humans ; Lymphokines ; analysis ; genetics ; Male ; RNA, Messenger ; analysis ; Swine ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors

OBJECTIVETo evaluate the effect of local application of vascular endothelial growth factor (VEGF) via adenovirus-mediated gene transfer on survival of full thickness flaps selected randomly in rats.

METHODSThirty Sprague-Dawley rats weighing 480-520 g were used in this study. A dorsal flap (8 cm x 2 cm) in full thickness with the pedicle located at the level of the iliac crest was designed. Then the rats received 1,012 pfu replication-deficient recombinant adenovirus carrying VEGF (AdCMV-VEGF group, n=10), 1,012 pfu recombinant beta-galactosidase adenovirus (AdCMV-Gal group, n=10) and 1 ml saline (saline group, n=10), respectively, in the distal two thirds of the proposed flap by means of subdermal injection at 8 different locations. Three days after treatment, the flaps were elevated as originally designed and sutured back in situ. The survival rate of the flaps was evaluated on day 7 after operation.

RESULTSThe survival rate of the flaps in the AdCMV-VEGF group increased significantly as compared with those of the AdCMV-Gal group (P<0.01) and the saline group (P<0.01). Immunohistochemical staining showed that VEGF was expressed in the survival flaps injected with AdCMV-VEGF. Histological analysis showed that more granulation tissues and angiogenesis were observed in the AdCMV-VEGF group than those in the AdCMV-Gal and the saline groups.

CONCLUSIONSLocal application of adenovirus-mediated VEGF165 cDNA may efficiently improve the survival of ischemic skin flaps.

Adenoviridae ; genetics ; Animals ; Endothelial Growth Factors ; genetics ; Genetic Therapy ; Intercellular Signaling Peptides and Proteins ; genetics ; Lymphokines ; genetics ; Male ; Neovascularization, Physiologic ; Rats ; Rats, Sprague-Dawley ; Surgical Flaps ; Transfection ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors

OBJECTIVETo investigate the effect of phVEGF165 transfer on vascular remodelling after balloon injury and its possible mechanisms.

METHODS90 New Zealand white rabbits were divided randomly into 3 groups: group I (balloon injury group), group II (pAdtrackCMV group) and group III (pAdtrackCMV-VEGF165 group). All animals were given hypercholesterol diet for 7 days before experiment and continued to receive hypercholesterol diet until being killed. Each group was further divided into five subgroups according to the sacrifice time (3 days, 1, 2, 4 and 8 weeks after transfection). Blood samples and arteries were harvested for further analysis.

RESULTSAt the end of 2 weeks, areas of neointima plus media of group III were smaller than those of group I and II (P < 0.05). The areas under external elastic membrane were larger in group III at 4 weeks and lumen stenosis rates were significantly lower than group I and II (P < 0.05 or 0.01). In group III, VEGF165, metalloproteinases (MMPs) -1, -2, -9 and their tissue inhibitors (TIMPs) 1, 2 could be detected from 3 days after gene transfer and reached the highest level at 2 weeks time and could not be detected by 8 weeks time. In groups I and II, MMP-2 and TIMP-1, -2 could be detected during the whole procedure and the value of TIMP1/MMP1 was significantly higher than in group III (P < 0.01).

CONCLUSIONRemodelling is the main reason for restenosis (RS) after vascular balloon injury. Local pAdtrackCMV-VEGF165 transfer can specifically change the expression of MMPs and facilitate the positive remodelling process, hence, inhibiting restenosis.

Angioplasty, Balloon ; adverse effects ; Animals ; Coronary Restenosis ; etiology ; pathology ; Endothelial Growth Factors ; genetics ; physiology ; Intercellular Signaling Peptides and Proteins ; genetics ; physiology ; Lymphokines ; genetics ; physiology ; Male ; Matrix Metalloproteinases ; metabolism ; Rabbits ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors