Vascular Endothelial Growth Factor cDNA Research Articles

A Biosafety Study of Human Umbilical Cord Blood Mononuclear Cells Transduced with Adenoviral Vector Carrying Human Vascular Endothelial Growth Factor cDNA In Vitro.

The biosafety of gene therapy remains a crucial issue for both the direct and cell-mediated delivery of recombinant cDNA encoding biologically active molecules for the pathogenetic correction of congenital or acquired disorders. The diversity of vector systems and cell carriers for the delivery of therapeutic genes revealed the difficulty of developing and implementing a safe and effective drug containing artificial genetic material for the treatment of human diseases in practical medicine. Therefore, in this study we assessed changes in the transcriptome and secretome of umbilical cord blood mononuclear cells (UCB-MCs) genetically modified using adenoviral vector (Ad5) carrying cDNA encoding human vascular endothelial growth factor (VEGF165) or reporter green fluorescent protein (GFP). A preliminary analysis of UCB-MCs transduced with Ad5-VEGF165 and Ad5-GFP with MOI of 10 showed efficient transgene expression in gene-modified UCB-MCs at mRNA and protein levels. The whole transcriptome sequencing of native UCB-MCs, UCB-MC+Ad5-VEGF165, and UCB-MC+Ad5-GFP demonstrated individual sample variability rather than the effect of Ad5 or the expression of recombinant vegf165 on UCB-MC transcriptomes. A multiplex secretome analysis indicated that neither the transduction of UCB-MCs with Ad5-GFP nor with Ad5-VEGF165 affects the secretion of the studied cytokines, chemokines, and growth factors by gene-modified cells. Here, we show that UCB-MCs transduced with Ad5 carrying cDNA encoding human VEGF165 efficiently express transgenes and preserve transcriptome and secretome patterns. This data demonstrates the biosafety of using UCB-MCs as cell carriers of therapeutic genes.

Modified Rat Hepatocellular Carcinoma Models Overexpressing Vascular Endothelial Growth Factor

PurposeTo compare tumor vascularity in 4 types of rat hepatocellular carcinoma (HCC) models: N1S1, vascular endothelial growth factor (VEGF)-transfected N1S1 (VEGF-N1S1), McA-RH7777, and VEGF-transfected McA-RH7777 (VEGF-McA-RH777) tumors. Materials and MethodsThe N1S1 and McA-RH7777 cell lines were transfected with expression vectors containing cDNA for rat VEGF. Eighty-eight male Sprague–Dawley rats (weight range, 400–450 g) were randomly divided into 4 groups (ie, 22 rats per model), and 4 types of tumor models were created by using the N1S1, VEGF-N1S1, McA-RH7777, and VEGF-McA-RH777 cell lines. Tumor vascularity was evaluated by perfusion computed tomography (CT), enzyme-linked immunosorbent assay of VEGF, CD34 staining, angiography, and Lipiodol transarterial embolization. Intergroup discrepancies were evaluated by Kruskal–Wallis test. ResultsArterial perfusion (P < .001), portal perfusion (P = .015), total perfusion (P < .001), tumor VEGF level (P = .002), and microvessel density (MVD; P = .007) were significantly different among groups. VEGF-McA-RH7777 tumors showed the greatest arterial perfusion (46.7 mL/min/100 mL ± 15.5), total perfusion (60.7 mL/min/100 mL ± 21.8), tumor VEGF level (3,376.7 pg/mL ± 145.8), and MVD (34.5‰ ± 7.5). Whereas most tumors in the N1S1, VEGF-N1S1, and McA-RH7777 groups showed hypovascular staining on angiography and minimal Lipiodol uptake after embolization, 5 of 6 VEGF-McA-RH7777 tumors (83.3%) presented hypervascular tumor staining and moderate to compact Lipiodol uptake. ConclusionsMcA-RH7777 tumors were more hypervascular than N1S1 tumors, and tumor vascularity was enhanced further by VEGF transfection. Therefore, the VEGF-McA-RH7777 tumor is recommended to mimic hypervascular human HCC in rats.

Inhibition of intimal hyperplasia via local delivery of vascular endothelial growth factor cDNA nanoparticles in a rabbit model of restenosis induced by abdominal aorta balloon injury.

In-stent restenosis (ISR) is one of the major factors affecting long-term outcomes of percutaneous coronary interventions. Vascular endothelial growth factor (VEGF) has been hypothesized to have a positive role in preventing ISR, however, this remains controversial. The aim of the present study was to assess whether nanoparticles can be used to deliver VEGF to injured arteries and whether this is beneficial in preventing restenosis. New Zealand White rabbits were randomly divided into a control group, an empty nanoparticles group and a VEGF nanoparticles group (n=6 in each group). Polylactic-polyglycolic acid VEGF nanoparticles were prepared using a phacoemulsification method. A rabbit model of restenosis was established following abdominal aorta balloon injury, and VEGF gene nanoparticles, empty nanoparticles or normal saline were delivered locally at the site of injury via a GENIE Catheter™ perfusion balloon. Intimal proliferation determination and immunohistochemistry analysis were performed at day 28 following arterial injury. Compared with the control and empty nanoparticle groups, the neointima area (0.49±0.09, 0.48±0.08 and 0.19±0.11 mm2, respectively; P<0.001) and proliferation index (0.32±0.03, 0.32±0.05 and 0.13±0.06, respectively; P<0.001) were significantly lower in the VEGF nanoparticles group. In addition, in the VEGF nanoparticles group, the immunoreactivity of α-actin and proliferating cell nuclear antigen were significantly lower (P≤0.001), while the immunoreactivity of VEGF was higher (P=0.01). Therefore, the results revealed that local delivery of VEGF gene nanoparticles reduced intimal thickening and cell proliferation following abdominal aorta balloon injury in a rabbit model, demonstrating the efficacy of this therapy against restenosis.

Modified Model of VX2 Tumor Overexpressing Vascular Endothelial Growth Factor

PurposeTo determine whether upregulated expression of vascular endothelial growth factor (VEGF) in VX2 cells can increase vessel density (VD) and reduce tumor necrosis. Materials and MethodsThe VX2 cell line was transfected with expression vectors containing cDNA for rabbit VEGF. Stable clones producing rabbit VEGF (VEGF-VX2) were selected. VEGF-VX2 cells (n = 5 rabbits) or nontransfected VX2 cells (controls; n = 5 rabbits) were implanted into leg muscle of 10 rabbits. The animals were sacrificed at day 21. Tumor volume, percentage of necrosis, VD, and VEGF concentration in tumor protein extract were quantified. ResultsOverexpression of VEGF by VX2 cells augmented tumor implantation efficiency 100% and favored cyst formation. The tumor volume was significantly larger for VEGF-VX2 transfected tumors versus controls (P = .0143). Overexpression of VEGF in VX2 cells significantly increased the VD of the tumors (P = .0138). The percentage of necrosis was reduced in VEGF-VX2 tumors versus controls (19.5% vs 38.5 %; P = .002). VEGF concentration in VEGF-VX2 tumors was significantly higher than in control tumors (P = .041) and was correlated with tumor volume (ρ = .883, P = .012). ConclusionsThe overexpression of VEGF increased tumor growth and vascularization, favored cyst formation, and reduced tumor necrosis. This new phenotype of the VX2 tumor may offer some advantages over classic models of VX2 tumor for evaluating anticancer therapies.

MRNA in situ hybridization analysis of VEGF in chronic lymphocytic leukemia

Background: Several factors render chronic lymphocytic leukemia (CLL) an interesting subject for study by researchers. These include marked progress in understanding the molecular biology of normal and neoplastic lymphocytes and recent advances in molecular genetics techniques. Among molecular markers, vascular endothelial growth factor (VEGF), have been widely studied.Objective: The aim of the study is to evaluate the role of VEGF in the pathogenesis of CLL and its role in disease progression.Patients, materials and methods: A retrospective cross-sectional study was done on 60 patients with chronic lymphocytic leukemia (45 males &amp; 15 females) compared with 20 controls (anemic patients), all recruited at the Medical City Hospital/ Teaching Laboratories/ Baghdad from January 2004 to December 2007. The bone marrow biopsy of each was re-examined histologically. In situ hybridization was performed utilizing biotin labeled VEGF cDNA probes.Results: The frequency of VEGF positivity was 95% (57 of 60 cases). A statistically significant inverse correlation was found between increasing VEGF scores and lower packed cell volume level and lower platelet count. A statistically significant association was found between advanced modified Rai stage and patients with high score. In addition, there was a statistically significant positive correlation between increasing scores of VEGF and advancing clinical stage. Conclusions: The score of VEGF is high among patients with advanced clinical stage of the disease.

VEGF and BMP-6 enhance bone formation mediated by cloned mouse osteoprogenitor cells

New strategies such as combined utilization of growth factors may provide a better treatment for difficult fractures. We have demonstrated enhanced angiogenesis and osteogenesis through the actions of vascular endothelial growth factor (VEGF) and bone morphogenetic protein-6 (BMP-6) on the osteogenic differentiation of a cloned mouse osteoprogenitor cell in vitro and ectopic bone formation in vivo. Human VEGF and BMP-6 genes expressed together produced a significant increase in alkaline phosphatase activity, expression of the RunX2 and osteocalcin genes and mineralization. Microcomputed tomographic analysis of subcutaneous implants consisting of cells transfected with VEGF and BMP-6 cDNA and delivered on a 3D poly (lactic-co-glycolic acid) scaffold confirmed the additive effects between VEGF and BMP-6. Ectopic bone formation in the VEGF plus BMP-6 group was greatest compared to that in either VEGF or BMP-6 alone. This is the first study that demonstrates osteogenesis in vitro and in vivo through the additive effects of VEGF and BMP-6.

Effects of Increased Renal Tubular Vascular Endothelial Growth Factor (VEGF) on Fibrosis, Cyst Formation, and Glomerular Disease

The role of vascular endothelial growth factor (VEGF) in renal fibrosis, tubular cyst formation, and glomerular diseases is incompletely understood. We studied a new conditional transgenic mouse system [Pax8-rtTA/(tetO)(7)VEGF], which allows increased tubular VEGF production in adult mice. The following pathology was observed. The interstitial changes consisted of a ubiquitous proliferation of peritubular capillaries and fibroblasts, followed by deposition of matrix leading to a unique kind of fibrosis, ie, healthy tubules amid a capillary-rich dense fibrotic tissue. In tubular segments with high expression of VEGF, cysts developed that were surrounded by a dense network of peritubular capillaries. The glomerular effects consisted of a proliferative enlargement of glomerular capillaries, followed by mesangial proliferation. This resulted in enlarged glomeruli with loss of the characteristic lobular structure. Capillaries became randomly embedded into mesangial nodules, losing their filtration surface. Serum VEGF levels were increased, whereas endogenous VEGF production by podocytes was down-regulated. Taken together, this study shows that systemic VEGF interferes with the intraglomerular cross-talk between podocytes and the endocapillary compartment. It suppresses VEGF secretion by podocytes but cannot compensate for the deficit. VEGF from podocytes induces a directional effect, attracting the capillaries to the lobular surface, a relevant mechanism to optimize filtration surface. Systemic VEGF lacks this effect, leading to severe deterioration in glomerular architecture, similar to that seen in diabetic nephropathy.

Bipartite vectors for co‐expression of a growth factor cDNA and short hairpin RNA against an apoptotic gene

Although human islet transplantation is a promising approach for treating type I diabetes, its success is limited as a result of the poor survival rate of transplanted islets. Expression of a growth factor gene to promote revascularization and silencing of pro-apoptotic genes before transplantation may improve the outcome of islet transplantation. In the present study, we constructed bipartite plasmid vectors to co-express a vascular endothelial growth factor (VEGF) cDNA and short hairpin (sh)RNA targeting inducible NO synthase (iNOS) gene. First, we screened shRNA sequences against human iNOS by transfecting plasmids encoding shRNA targeting different start sites of human iNOS. Then, the effect of different promoters [such as H1, U6 and cytomegalovirus (CMV)] and micro RNA backbones on gene silencing was determined. No statistical difference in iNOS gene silencing was observed for the shRNA with H1, U6 and CMV promoters. In addition, a conventional shRNA showed better silencing of the iNOS gene compared to shRNA containing mir375 and mir30 backbones. A bipartite plasmid was also constructed with mir30-shRNA and a VEGF cDNA controlled by a single CMV promoter. This plasmid showed a better silencing effect compared to plasmid without VEGF cDNA. In the present study, we have successfully constructed bipartite vectors co-expressing a VEGF cDNA and a shRNA against the iNOS gene. These vectors could be attractive candidates for improving the survival of transplanted islets.

Local Gene Transfer and Expression Following Intramuscular Administration of FGF-1 Plasmid DNA in Patients With Critical Limb Ischemia

NV1FGF is an expression plasmid encoding sp.FGF-1(21-154) currently under investigation for therapeutic angiogenesis in clinical trials. NV1FGF plasmid distribution and transgene expression following intramuscular (IM) injection in patients is unknown. The study involved six patients with chronic critical limb ischemia (CLI) planned to undergo amputation. A total dose of 0.5, 2, or 4 mg NV1FGF was administered as eight IM injections (0.006, 0.25, or 0.5 mg per injection) 3-5 days before amputation. Injected sites (30 cm(3)) were divided into equally sized smaller pieces to assess spatial distribution of NV1FGF sequences (PCR), NV1FGF mRNA (reverse transcriptase-PCR), and fibroblast growth factor-1 (FGF-1)-expressing cells (immunohistochemistry). Data indicated gene expression at all doses. The distribution area was within 5-12 cm for NV1FGF sequences containing the expression cassette, up to 5 cm for NV1FGF mRNA, and up to 3 cm for FGF-1-expressing myofibers. All FGF receptors were detected indicating robust potential for bioactivity after NV1FGF gene transfer. Circulating levels of NV1FGF sequences were shown to decrease within days after injection. Data support demonstration of plasmid-mediated gene transfer and expression in muscles from patients with CLI. FGF-1 expression was shown to be limited to injection sites, which supports the concept of multiple-site injection for therapeutic use.

062 Application of gene therapy to tendon healing: modification of tenocytes with exogeneous pdgf and vegf genes to promote collagen production

Purpose: Gene therapy provides a useful method to enhance wound healing, but it has not been attempted in healing of wounds in flexor tendons in the hands, which inherently lack sufficient intrinsic healing capacity. To explore the potential of application of gene therapy to tendon wound healing, we genetically modified tenocytes with the platelet-derived growth factor-B (PDGF-B) and the vascular endothelial growth factor (VEGF) gene and investigated the expression of the genes for collagen production in an in vitro model of the proliferating tenocytes. Method: Tenocytes were obtained from cultures of rat intrasynovial tendons and randomly distributed to 34 dishes. The tenocytes in dishes in two experimental groups (n = 9, each group) were treated for 12 hours with the plasmid containing the PDGF-B cDNA or the VEGF cDNA and were then cultured for 5 days; tenocytes in the other eight dished received sham vector and the tenocytes in the control dishes (n = 8) did not receive the exogenous gene. Efficiency of the gene transfer was evaluated by detection of the presence of the transgene in the tenocytes by reverse transcription polymerase chain reactions (RT-PCR). Levels of expression of type I and III collagen, and TGF-β genes were determined by quantitative analysis of the products of RT-PCR. Results: Expression of the type I collagen gene was significantly increased by transfer of the exogenous PFDGF gene to the tenocytes (p < 0.001). Transfer of the PDGF-B gene did not affect the expression of TGF-β and the type III collagen genes significantly. Expression of TGF-β gene increased significantly in the cells treated with exogenous VEGF cDNA (p = 0.039). Expression of type I and III collagen genes by tenocytes was minimally affected by transfer of the VEGF gene to the tenocytes and was significantly weaker than that stimulated by PDGF-B gene therapy (p = 0.001). Efficient gene transfer was confirmed by the presence of the PDGF-B gene or the VEGF gene in the tenocytes receiving the transferred genes. Conclusions: Transfer of exogenous PDGF-B gene significantly enhances expression of the type I collagen gene of the tenocytes. Transfer of exogenous VEGF gene has very limited effects on promotion of collagen production in the proliferating tenocytes. In contrast, transfer of the VEGF gene significantly increases expression of the TGF-β gene. This study suggests that transfer of the PDGF-B gene may offer a novel way of effectively promoting healing of intrasynovial flexor tendons. VEGF gene therapy is not as beneficial as PDGF-B gene therapy to tendon healing and may increase activities of TGF-β that are associated with adhesion formations.

Establishment of a Sandwich ELISA Method for Detection of Vascular Endothelial Growth Factor in Serum Samples of Hepatocellular Carcinoma Patients

To establish a sandwich ELISA method for detecting vascular endothelial growth factor (VEGF) in sera of population and the patients with hepatocellular carcinoma (HCC). Full length and two truncated human VEGF cDNA sequences were amplified from a commercial plasmid pBLAST49-hVEGF by PCR and inserted into the prokaryotic-expression plasmid pET-32a or pGEX-2T. Various VEGF proteins were expressed and purified from E. coli in His-Trx or GST fusion forms. The specific VEGF antibodies were elicited in experimental rabbits and mice by immunization of the full length VEGF fusion protein His-Trx-VEGF1-165. After purification of antibodies with chromatograph of Protein G, a sandwich ELISA technique was established. Serum VEGF levels were evaluated in 229 adults and 291 HCC patients. SDS-PAGE displayed that the molecular weights of the expressed full length (His-Trx-VEGF1-165), N-terminal (His-Trx-VEGF1-100) and C-terminal (GST-VEGF100-165) human VEGF fusion proteins were about 38KD, 31KD, and 33KD, respectively. Western blots confirmed that the prepared antisera were able to recognize both prokaryoticly and eukaryoticly expressed recombinant VEGF proteins. Assays of serially diluted His-Trx-VEGF1-100 by the established sandwich ELISA method showed that the linear range of the standard curve was 0.625-320 ng/mL, with the squared correlation coefficient R2 = 0.991. Screening of a serum panel containing 291 serum samples of HCC patients and 229 health adults revealed that the average VEGF level in HCC patients was higher than that in healthy controls, with a statically significant difference. The established sandwich ELISA reflects the level of serum VEGF and provide scientific basis for screening metastasis and recurrence of HCC using serum VEGF as an index.

Vascular Endothelial Growth Factor Mediates Intracrine Survival in Human Breast Carcinoma Cells through Internally Expressed VEGFR1/FLT1

BackgroundWhile vascular endothelial growth factor (VEGF) expression in breast tumors has been correlated with a poor outcome in the pathogenesis of breast cancer, the expression, localization, and function of VEGF receptors VEGFR1 (also known as FLT1) and VEGFR2 (also known as KDR or FLK1), as well as neuropilin 1 (NRP1), in breast cancer are controversial.Methods and FindingsWe investigated the expression and function of VEGF and VEGF receptors in breast cancer cells. We observed that VEGFR1 expression was abundant, VEGFR2 expression was low, and NRP1 expression was variable. MDA-MB-231 and MCF-7 breast cancer cells, transfected with antisense VEGF cDNA or with siVEGF (VEGF-targeted small interfering RNA), showed a significant reduction in VEGF expression and increased apoptosis as compared to the control cells. Additionally, specifically targeted knockdown of VEGFR1 expression by siRNA (siVEGFR1) significantly decreased the survival of breast cancer cells through down-regulation of protein kinase B (AKT) phosphorylation, while targeted knockdown of VEGFR2 or NRP1 expression had no effect on the survival of these cancer cells. Since a VEGFR1-specific ligand, placenta growth factor (PGF), did not, as expected, inhibit the breast cancer cell apoptosis induced by siVEGF, and since VEGFR1 antibody also had no effects on the survival of these cells, we examined VEGFR1 localization. VEGFR1 was predominantly expressed internally in MDA-MB-231 and MCF-7 breast cancer cells. Specifically, VEGFR1 was found to be colocalized with lamin A/C and was expressed mainly in the nuclear envelope in breast cancer cell lines and primary breast cancer tumors. Breast cancer cells treated with siVEGFR1 showed significantly decreased VEGFR1 expression levels and a lack of VEGFR1 expression in the nuclear envelope.ConclusionsThis study provides, to our knowledge for the first time, evidence of a unique survival system in breast cancer cells by which VEGF can act as an internal autocrine (intracrine) survival factor through its binding to VEGFR1. These results may lead to an improved strategy for tumor therapy based on the inhibition of angiogenesis.

Expression Level, Tissue Distribution Pattern and Prognostic Impact of Vascular Endothelial Growth Factor (VEGF) and Its Receptors Flt-1 and KDR in Different Subtypes of Non-Hodgkin Lymphoma (NHL).

Background: VEGF is the main regulator of tumour angiogenesis via its receptors, Flt-1 and KDR. It is expressed by tumour cells and normal stroma cells, and, beyond angiogenesis, it can stimulate tumour growth via autocrine and paracrine pathways.Material and methods: Pre-therapeutic lymph-node biopsies from 152 NHL cases were investigated. These included 62 cases of follicular lymphoma (FL), 20 of which had histologically verified transformation to aggressive lymphoma, 46 cases of diffuse large B-cell lymphoma (DLBCL) and 44 cases of peripheral T-cell lymphoma (PTCL). VEGF, Flt-1 and KDR were investigated at protein level by immunohistochemistry, and at mRNA level by non-isotopic' in situ' hybridisation. For the latter, a fluorescein-conjugated probe was obtained from pGEM3Zf plasmid containing 1–581 bp of human VEGF cDNA. The staining intensity was scored semiquantitatively (0–3) and subdivided in ‘focal'or ‘diffuse'depending on morphological evaluation. Expression of VEGF and its receptors was correlated to the morphologically estimated microvascular density (Chalkley method).Results: VEGF protein expression was detected in 88 % FL, 91% DLBCL and 95% PTCL. As expected, PTCL had a stronger and more diffuse staining pattern than FL and DLBCL. Flt-1 protein expression was detected in 95 % FL, 96% DLBCL and in virtually all cases of PTCL. Lymphoma cells expressing KDR were also seen in all biopsies. A diffuse staining pattern was more frequent in FL. VEGF mRNA was expressed in all subtypes at similar levels although DLBCL displayed a diffuse staining pattern more frequently than other histologies. Higher VEGF protein expression in lymphoma cells was correlated with higher microvascular density (p=0.005), while a negative correlation was observed between receptor expression (Flt-1 and KDR) and Chalkley count (p=0.01 and 0.003, respectively). Patients with FL, who had diffuse VEGF staining of lymphoma cells, had poorer overall survival than those with focal staining (p=0.04). Diffuse KDR staining was correlated with a higher risk of transformation (p=0.05). In fact, a remarkably high microvessel density was found in interfollicular as opposed intrafollicular areas and high interfollicular Chalkley count was correlated with histologic transformation to DLBCL (p=0.01). In ‘de novo' DLBCL, high KDR expression was correlated with poor treatment response (p=0.03) and a grade 3 KDR intensity in lymphoma cells had a significant adverse impact on overall survival (p<0.001). In PTCL, a diffuse tissue distribution of VEGF mRNA correlated to poorer overall survival (p=0.0015). Flt-1 expression had no influence on outcome in the investigated lymphoma subtypesConclusion: VEGF and its receptors are expressed by lymphoma cells in all investigated NHL subtypes. The staining pattern and intensity of VEGF and KDR differed in the three subtypes, while Flt-1 distribution was similar. High VEGF and KDR expression had adverse prognostic impact in DLBCL and PTCL.

Endogenous production of nitric oxide contributes to proliferation effect of vascular endothelial growth factor-induced malignant melanoma cell.

The objectives of this study were to observe the effect of overexpression of vascular endothelial growth factor (VEGF) on the proliferation of the malignant melanoma (MM) cell line A375, and to study the role of nitric oxide (NO) in this process and the mechanism of VEGF induced-A375 cell proliferation. The VEGF(165) cDNA was transfected into A375 cells by electroporation. VEGF mRNA and protein in A375 cells were detected by RT-PCR and ELISA. The proliferation of A375 cells was assessed by cell counting and MTT assay. Protein expression of iNOS, eNOS and nNOS was detected by Western blotting. NO production in A375 cell supernatant was measured by the nitrate reductase method. VEGF mRNA in A375 cells was significantly increased 72 h and 96 h after transfection of VEGF(165) cDNA, as were VEGF protein, NO and iNOS levels. However, protein expression of eNOS and nNOS was not detected in either transfected or untransfected cells. Proliferation of A375 cells transfected with VEGF(165) cDNA was enhanced. The nitric oxide synthase inhibitor l-NAME could dose-dependently inhibit the proliferation of A375 cells evoked by VEGF. These results indicate that VEGF enhances the expression of iNOS in A375 cells and results in an increase in NO formation, which may be important in the process of VEGF-induced proliferation of A375 cells.

680. Application of Gene Therapy to Tendon Healing: Modification of Tenocytes with Exogenous PDGF and VEGF Genes to Promote Collagen Production

Top of pageAbstract Purpose: Gene therapy provides a useful method to enhance wound healing, but it has not been attempted in healing of wounds in flexor tendons in the hands, which inherently lack sufficient intrinsic healing capacity. To explore the potential of application of gene therapy to tendon wound healing, we genetically modified tenocytes with the platelet derived growth factor-B (PDGF-B) and the vascular endothelial growth factor (VEGF) gene and investigated the expression of the genes for collagen production in an in vitro model of the proliferating tenocytes. Method: Tenocytes were obtained from cultures of rat intrasynovial tendons and randomly distributed to 34 dishes. The tenocytes in dishes in 2 experimental groups (n=9, each group) were treated for 12 hours with the plasmid containing the PDGF-B cDNA or the VEGF cDNA and were then cultured for 5 days; tenocytes in the other 8 dished received sham vector and the tenocytes in the control dishes (n=8) did not receive the vector and the gene. Efficiency of the gene transfer was evaluated by detection of the presence of the transgene in the tenocytes by reverse transcription polymerase chain reactions (RT-PCR). Levels of expression of type I and III collagen, and TGF-beta genes were determined by quantitative analysis of the products of RT-PCR and were statistically analyzed. Results: Expression of the type I collagen gene was significantly increased by transfer of the exogenous PFDGF gene to the tenocytes (p<0.001). Transfer of the PDGF-B gene did not affect the expression of TGF-beta and the type III collagen genes significantly. Expression of TGF-beta gene increased significantly in the cells treated with exogenous VEGF cDNA (p=0.039). Expression of type I and III collagen genes by tenocytes was minimally affected by transfer of the VEGF gene to the tenocytes and was significantly weaker than that stimulated by PDGF-B gene therapy (p=0.001). Efficient gene transfer was confirmed by the presence of the PDGF-B gene or the VEGF gene in the tenocytes receiving the transferred genes. Conclusions: Transfer of exogenous PDGF-B gene significantly enhances expression of the type I collagen gene of the tenocytes. Transfer of exogenous VEGF gene has very limited effects on promotion of collagen production in the proliferating tenocytes. In contrast, transfer of the VEGF gene significantly increases expression of the TGF-beta gene. This study suggests that transfer of the PDGF-B gene may offer a novel way of effectively promoting healing of intrasynovial flexor tendons. VEGF gene therapy is not as beneficial as PDGF-B gene therapy to tendon healing and may increase activities of TGF-beta that are associated with adhesion formations.

Enhancement of Ischemic Flap Survival by Prefabrication with Transfer of Exogenous PDGF Gene

Treatment of skin flaps by means of gene therapy has been introduced recently as a novel approach to enhance viability of ischemic skin flaps. Transfer of the platelet-derived growth factor (PDGF) to enhance survival of the ischemic skin flap has not been explored. In this study, the authors investigated the effect of the transfer of the PDGF cDNA on survival and vascularity of the ischemic random flap in a rat model, and compared the effects of PDGF gene therapy to those of vascular endothelial growth factor (VEGF) gene therapy. A total of 45 adult Sprague-Dawley rats were randomly divided into four groups. The PDGF gene therapy group (n=10) received the plasmid containing the PDGF cDNA with liposome injected to the dermis of the flap. A saline control group (n=10) received physiologic saline only, and the vector control group (n=10) received liposome plus vector without the PDGF gene segment. In the fourth group (n=15), the VEGF gene was transferred to the flap. Seven days later, a dorsal random flap including the injection area was raised. One rat each from the saline and vector control groups died during the study period and were excluded. The viability of the flap and vascularity within the flaps were assessed 7 days after flap elevation. The PDGF plasmid-treated flaps had significantly greater survival area (60.8+/-7.8 percent) compared with the flaps treated with saline (52.3+/-5.0 percent) and those treated with liposome and vector (50.7+/-5.9 percent). PDGF gene therapy had effects on survival of the flap similar to VEGF gene therapy (57.6+/-5.2 percent, after transfer of VEGF cDNA). Neovascularization with the flap tissues was confirmed by immunohistochemical staining of von Willebrand factor, a marker specific for angiogenesis. The number of newly-formed blood vessels in the transgenic flaps was significantly greater than that of the vessels in the flaps receiving the saline. The findings of this study indicate that transfer of the PDGF cDNA effectively enhances neovascularization of the ischemic skin flap and increases the viability of the flap, and transfer of the PDGF gene is as efficient as transfer of the VEGF gene in improving viability of the skin flap. This study suggests that PDGF gene therapy may be a novel strategy for the treatment of ischemic skin flaps.

Tendon healing in vitro: Modification of tenocytes with exogenous vascular endothelial growth factor gene increases expression of transforming growth factor β but minimally affects expression of collagen genes

It is not clear how the transfer of exogenous growth factor genes to tenocytes affects collagen production. An increase in collagen production enhances the repair but an increase in growth factors that stimulate tissue fibrosis may cause adhesion. Gene therapy is a new way to regulate tendon healing but it has been explored rarely. We genetically modified tenocytes with the vascular endothelial growth factor (VEGF) gene and investigated the expression of the genes for collagen production in an in vitro model of the proliferating tenocytes. Tenocytes were obtained from cultures of rat intrasynovial tendons and distributed randomly to 25 dishes. The tenocytes in the experimental dishes (n = 9) were treated for 12 hours with plasmid containing the VEGF complementary deoxyribonucleic acid and then were cultured for 5 days; the tenocytes in the control dishes (n = 8) did not receive the exogenous gene. Tenocytes in the other dishes received exogenous platelet-derived growth factor (PDGF) gene for comparison of the effects of VEGF gene therapy. Efficiency of the gene transfer was evaluated by presence of the transgene in the tenocytes which was detected by reverse transcription polymerase chain reactions. Levels of expression of types I and III collagen and transforming growth factor (TGF)-beta genes were determined by quantitative analysis of the products of reverse transcription polymerase chain reactions. Expression of the TGF-beta gene increased significantly in the cells treated with exogenous VEGF cDNA. Expression of type I and III collagen genes by tenocytes was affected minimally by transfer of the VEGF gene to the tenocytes and was significantly weaker than that stimulated by PDGF gene therapy. Efficient gene transfer was confirmed by the presence of the VEGF complementary deoxyribonucleic acid in the tenocytes receiving the transferred gene. Transfer of exogenous VEGF gene has very limited effects on the promotion of collagen production in the proliferating tenocytes. This study suggests that VEGF gene therapy is not as beneficial as PDGF gene therapy to tendon healing and may increase the activities of TGF-beta that are associated with adhesion formations.

The use of transformed Escherichia coli for experimental angiogenesis induced by regulated in situ production of vascular endothelial growth factor – an alternative gene therapy

Defects in angiogenesis (blood vessel formation) are responsible for two most important causes of death in developed countries (ischemic heart disease and cancer). Vascular endothelial growth factor (VEGF) plays a pivotal role in physiological and pathological regulation of angiogenesis. In the last years several studies have indicated the possibilities of VEGF in the therapy of ischemic heart disease. However, especially VEGF gene therapy (naked DNA, plasmids and adenovirus mediated) is associated with adverse side effects regarding the expression regulation. To prepare bacterial strains producing VEGF using plasmids containing the VEGF cDNA for the use in experimental angiogenesis. Escherichia coli strain BL21(DE3) was transformed with Bluescript vector containing the inserts with cDNA sequences coding VEGF-A isoforms (VEGF121, VEGF164, VEGF189). Selection of recombinants was achieved by cultivating E. coli cells on ampicillin-added medium. The expression of target genes in the T7 expression system was induced by isopropyl-beta-D-thiogalactoside (IPTG). Polyacrylamide gel electrophoresis of the cell lysates showed the presence of polypeptides of molecular weight corresponding with known values of VEGF isoforms. Blood vessel formation induced by bacterial VEGF production was proved in vivo in mice seven days after intraperitoneal injection of transformed bacteria by light microscopy. CONCLUSION AND HYPOTHESIS: In summary, E. coli strain expressing VEGF was prepared and its biological effect confirmed. Bacteria, which produce angiogenic factors, provide a new modality for experimental angiogenesis and may be also suitable for clinical use. The in situ production of therapeutic proteins using optimalized prokaryotic expression systems can represent a useful tool for treatment based on molecular biomedicine. The main advantage of the described approach lies in the enhanced regulation control--bacterial expression can be regulated positively (induction by exogenous low molecular weight agents) and negatively (application of antibiotics). The hypothesis of alternative gene therapy should be proved in further studies.

AdVEGF165 gene transfer increases survival in overdimensioned skin flaps

Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis. VEGF A also plays an important role in wound healing of the skin by promoting angiogenesis and by stimulating blood vessel growth. Therefore we tested the hypothesis that flap survival could be increased by the preoperative injection of AdVEGF(165). We studied the effect of AdVEGF(165) in an overdimensioned ischemic random-pattern-flap model in the rat (n = 50) with a length-to-width ratio of 4 : 1. VEGF cDNA was administered in two concentrations of 5 x 10(8) plaque-forming units (pfU) and 1 x 10(9) pfU using a recombinant adenoviral vector. Recombinant virus was injected subdermally 7, 3 or 0 days prior to flap harvest for the lower concentration and 7 days prior for the higher concentration. Flap survival and necrosis were observed at day 7, the day the animals were sacrificed. Adenoviral gene transfer with VEGF(165) 3 and 7 days before flap harvest showed a significantly increased flap survival of 50% together with a significantly reduced necrosis (p < 0.01). Injection using a titer of 1 x 10(9) pfU 7 days prior to surgery increased flap survival even more, though failing to reach statistical significance compared to the lower concentration. VEGF protein concentration in the injected skin was significantly higher than in controls (p < 0.01). Flap perfusion was increased as well, demonstrated by indocyanine green (ICG) fluoroscopy (p < 0.001). Our results confirm the important role of VEGF(165) on angiogenesis in ischemic flaps. Indeed by injecting VEGF(165) at 3 to 7 days preoperatively in a concentration of 1 x 10(9) pfU our data show that length-to-width ratio for random-pattern-flaps could be increased from 2 : 1 to 3 : 1 and therefore may allow a wider range of applications of this simple flap technique.

Effect of antisense VEGF cDNA transfection on the growth of chronic myeloid leukemia K562 cells in vitro and in nude mice

To further elucidate the role of vascular endothelial growth factor (VEGF) in the pathogenesis of chronic myeloid leukemia (CML), we transfected K562 cells with a VEGF 121cDNA sense vector (S), an antisense (AS) vector or vector (V) alone. The growth of transfected cells was investigated by MTT and colony-formation assays, and apoptosis was measured by flow cytometry (FCM) of Annexin-V-FITC/PI dual labeled cells. Transfected cells were subcutaneously transplanted into nude mice and the microvessel density (MVD) of tumor masses was determined by vWF immunohistochemistry staining. We tested the supernatant of different transfected K562 cells against human bone marrow endothelial cells (BMECs), and examined the synergic effects of antisense VEGF 121cDNA and IFNα or STI571 on the proliferation and apoptosis of K562 cells. We found that K562/AS transfectants exhibited a 49% reduction in VEGF secretion, whereas K562/S transfectants exhibited a 3-fold increase in VEGF secretion, all in comparison to the vector controls. K562 cells transfected with antisense VEGF 121cDNA showed growth retardation in vitro. In transplanted nude mice in vivo, transfection of implanted cells with antisense VEGF 121cDNA resulted in decreased tumor MVD, and increased apoptosis in the presence of IFNα. Taken together, these results suggest that VEGF may be involved in the pathogenesis of CML through autocrine and paracrine mechanisms, and that anti-VEGF therapy alone or in combination with conventional treatment may be beneficial for CML patients.