Vascular Endothelial Growth Factor Plasmid Research Articles

Bone regeneration in rats with BMP-4 and VEGF plasmid DNA

Problem: Gene therapy offers a promising new method of bone regeneration. One technique uses bioresorbable polymer scaffolding at the bony defect site that contains plasmid DNA of bone growth factors. Previous studies have demonstrated bone growth using bone morphogenetic protein-4 (BMP-4) plasmid DNA. Research has suggested a synergistic effect of BMP-4 and vascular endothelial growth factor (VEGF) on bone regeneration. The present study aims to confirm bone regeneration with a polymer scaffold containing BMP-4 plasmid DNA in the rat calvarial defect model and to quantify a synergistic effect of BMP-4 and VEGF plasmid DNA in this model. Methods: Calvarial critical-sized defects were created in 100 male Sprague-Dawley rats. The animals were divided into 5 equal groups by treatment: none (negative control); VEGF plasmid DNA; BMP-4 plasmid DNA; VEGF and BMP-4 plasmid DNA; and autograft (positive control). The animals were euthanized at 4 and 8 weeks; calvaria were radiographed and histologically sectioned. Results: Radiomorphometry measured bone growth within the calvarial defects to quantify the bone regeneration effects of BMP-4 and VEGF plasmid DNA alone and in combination. Compared to control groups, significant bone growth was noted in the BMP-4 and BMP-4/VEGF groups, but no bone growth was noted in the VEGF group. No significant difference in bone growth was noted between the BMP-4 and BMP-4/VEGF groups. Microscopy of histologic sections (1 slide/specimen) confirmed the bone growth noted on radiomorphometry. Conclusion: BMP-4 plasmid DNA demonstrates significant bone growth in the rat calvarial defect model. VEGF plasmid DNA alone demonstrates no bone growth. When BMP-4 and VEGF plasmid DNA are combined, no synergistic effect is noted. Significance: Reconstruction of osseous craniofacial defects such as autografting for cleft palate and osteocutaneous free tissue transfer for mandibular defects often requires bone harvest with an associated donor site morbidity. Tissue engineering employing gene therapy for bone regeneration may offer a less morbid alternative. Support: None reported.

Therapeutic angiogenesis and vasculogenesis for ischemic disease: part II: cell-based therapies.

### Endothelial Progenitor Cells and Postnatal Vasculogenesis: Experimental Evidence The option of performing full-scale endothelial cell transplantation to optimize local neovascularization is daunting if even feasible. An alternative, attractive strategy is designed to exploit the conceptual notion that endothelial cells and hematopoietic stem cells were ultimately derived from a common precursor, the putative hemangioblast. Hematopoietic stem cells had been shown previously to be present in circulating blood, in quantities sufficient to permit their harvesting and readministration for autologous, in lieu of bone marrow, transplantation. The related descendants, endothelial progenitor cells, can be detected in the peripheral circulation.1,2 Initially, Flk-1 and a second antigen, CD34, shared by angioblasts and hematopoietic stem cells were used to isolate putative angioblasts from the leukocyte fraction of peripheral blood.1 Meanwhile, endothelial progenitor cells were isolated from human umbilical cord blood,3 bone marrow–derived mononuclear cells,4 and CD34+ or CD133+ hematopoietic stem cells1,5 and were successfully ex vivo expanded with the use of human peripheral blood mononuclear cells.6 These cells differentiated into endothelial cells, as shown by expression of various endothelial proteins (KDR, von Willebrand factor, endothelial nitric oxide synthase, VE-cadherin, CD146) and uptake of Dil-acetylated LDL and binding of lectin.1,7 In animal models of ischemia, heterologous, homologous, and autologous endothelial progenitor cells were shown to incorporate into sites of active neovascularization in ischemic and tumor tissue. Blood flow recovery and capillary density were markedly improved, and the rate of limb loss was significantly reduced after transplantation of human peripheral blood–derived endothelial progenitor cells8,9 or bone marrow mononuclear cells.10 Likewise, infusion of peripheral blood–derived endothelial progenitor cells,11 bone marrow mononuclear cells,12 or purified CD34+ cells13 improved neovascularization and myocardial function after infarction. Isolated CD34+ cells also increased impaired blood flow in diabetic mice.14 These findings provide evidence that exogenously …

Sp1-dependent regulation of the RTP801 promoter and its application to hypoxia-inducible VEGF plasmid for ischemic disease.

Gene therapy using vascular endothelial growth factor (VEGF) is a new potential treatment of ischemic disease. To be safe and effective, VEGF expression should be enhanced locally in ischemic tissue. In this study, we identified the cis-regulatory element for the hypoxia induction of the RTP801 promoter. In addition, pRTP801-VEGF was evaluated as a therapeutic plasmid in vitro. The cis-regulatory element for hypoxia induction was identified by deletion and mutation analyses. Antisense oligonucleotide co-transfection assay was performed to evaluate the role of Sp1. pRTP801-VEGF was constructed by the insertion of the RTP801 promoter into the VEGF plasmid. The hypoxia-inducible expression of VEGF was evaluated by in vitro transfection assay. In luciferase assay, the region between -495 and -446 was responsible for the hypoxia-induced transcription. The mutation of the Sp1 site in this region reduced hypoxia-induced transcription. In addition, co-transfection with antisense Sp1 oligonucleotide suggests that hypoxia induction of the RTP801 promoter is mediated by Sp1. In vitro transfection showed that pRTP801-VEGF had higher VEGF expression than pEpo-SV-VEGF. In addition, VEGF expression by pRTP801-VEGF was induced under hypoxia. With strong basal promoter activity and induction under hypoxia, pRTP801-VEGF may be useful for gene therapy for ischemic disease.

Cardiomyocyte hyperplasia after plasmid-mediated vascular endothelial growth factor gene transfer in pigs with chronic myocardial ischemia.

For over 40 years it has been proposed that cardiomyocyte hyperplasia may occur in hypertrophic human hearts. While this implies that heart myocytes can undergo cytokinesis, evidence of conventional cell division has been exceptionally reported. Recently, we found that gene transfer of vascular endothelial growth factor (VEGF) displays a mitogenic effect on adult cardiomyocytes. In the present study we searched for cardiomyocyte hyperplasia as evidence of VEGF-induced cardiomyocyte cytokinesis. Three weeks after implanting an Ameroid constrictor at the origin of the left circumflex artery, 16 pigs were randomized to receive 10 direct intramyocardial injections of 3.8 mg of plasmid encoding for VEGF (pVEGF) or empty plasmid. Five weeks later, hearts were weighed, myocyte diameter was measured in tissue sections, and myocyte length and nuclei number were studied in isolated myocytes. A resting echocardiogram was performed immediately before reoperation and before sacrifice to evaluate global and regional left ventricular function. Investigators were blinded to the study groups and nature of the injectate until the end of data analysis. No heart weight differences existed between groups. However, in the ischemic myocardium, pVEGF-treated hearts had 22% more cardiomyocytes per unit volume and exhibited significantly more oligonucleated (1 or 2 nuclei) cardiomyocytes than hearts receiving empty plasmid. In pigs with chronic myocardial ischemia, VEGF gene transfer induced cardiomyocyte cytokinesis, as revealed by cardiomyocyte hyperplasia. Our finding extends the previously reported mitogenic effect of VEGF on adult cardiomyocytes and supports the hypothesis that VEGF may have a therapeutic role in diseases characterized by myocardial cell loss.

Gene Transfer of Naked VEGF Plasmid Induces the Formation of Microvessels but not Mature Collaterals in Ischaemic Limb Muscles

Background: Several studies have demonstrated increased numbers of angiographically detectable collaterals after vascular endothelial growth factor (VEGF) gene transfer. However, VEGF appears to be insufficient for stimulating the growth of mature blood vessels. Therefore, we decided to reinvestigate in what way the VEGF gene transfer to rabbit ischaemic muscle can restore blood flow impaired by femoral artery excision.

Clinical trials of myocardial VEGF gene transfer

Preclinical studies have documented that gene transfer of plasmid DNA encoding for secreted angiogenic cytokines such as vascular endothelial growth factor (VEGF) can induce therapeutic angiogenesis in animal models. Based on these studies, we have conducted 4 Phase I clinical trials of direct intramyocardial injection of VEGF plasmid DNA in patients with otherwise inoperable myocardial ischemia. In the first 2 trials, VEGF-1 (30 patients) or VEGF-2 (30 patients) plasmid DNA was administered through a mini-thoracotomy incision in an open label nonrandomized fashion as sole therapy. The majority of patients in both study groups experienced a significant reduction in angina frequency and improvement in Canadian Cardiovascular Society (CCS) angina class associated with objective evidence of improved myocardial perfusion documented by myocardial perfusion imaging and left ventricular electromechanical mapping. The subsequent development of a catheter-based approach to myocardial gene transfer has allowed us to conduct 2 randomized blinded trials of VEGF-2 gene transfer in a total of 25 patients. While a placebo effect was evident early post-treatment, VEGF-treated patients demonstrated significantly better results in terms of improvement in angina class and objective evidence of improvement in myocardial perfusion. These early Phase I trials confirm that direct myocardial gene transfer with VEGF is safe (overall 1 year mortality 3.5%) and should thus permit larger scale, appropriately randomized trials to be performed in order to more definitively evaluate its therapeutic efficacy.

Cell-based gene transfer of vascular endothelial growth factor attenuates monocrotaline-induced pulmonary hypertension.

Pulmonary arterial hypertension is characterized by increased pulmonary vascular resistance secondary to a decrease in the caliber and number of pulmonary vascular channels. We hypothesized that the targeted overexpression of an angiogenic factor within the lung would potentially minimize the development and progression of pulmonary arterial hypertension by preventing the loss of existing vessels or by inducing the development of new blood vessels within the lung. We used a cell-based method of gene transfer to the pulmonary microvasculature by delivering syngeneic smooth muscle cells overexpressing vascular endothelial growth factor (VEGF)-A to inbred Fisher 344 rats in which pulmonary hypertension was induced with the pulmonary endothelial toxin monocrotaline. Four weeks after simultaneous endothelial injury and cell-based gene transfer, right ventricular (RV) hypertension and RV and vascular hypertrophy were significantly decreased in the VEGF-treated animals. Four weeks after gene transfer, the plasmid VEGF transcript was still detectable in the pulmonary tissue of animals injected with VEGF-transfected cells, demonstrating survival of the transfected cells and persistent transgene expression. In addition, delay of cell-based gene transfer until after the development of pulmonary hypertension also resulted in a significant decrease in the progression of RV hypertension and hypertrophy. These results indicate that cell-based VEGF gene transfer is an effective method of preventing the development and progression of pulmonary hypertension in the monocrotaline model and suggest a potential therapeutic role for angiogenic factors in the therapy of this devastating disease.

Effects of liposome-mediated gene transfer of VEGF in ischemic rat gracilis muscle.

The purpose of the current study was to determine the effects of vascular endothelial growth factor (VEGF) on muscle flap survival and vascularity in a rat gracilis ischemia-reperfusion model. A total of 12 adult male Wistar rats were divided into two groups (n = 6). The experimental group received the plasmid encoding VEGF(165) cDNA plus lipofectamine (cationic liposome) injected directly to the gracilis muscle following 4 h of ischemia. The control group received lipofectamine only. The viability and vascularity of the flaps were evaluated after 7 days of reperfusion. The data demonstrated that the VEGF plasmid- and lipofectamine-treated muscle flaps had significantly greater total survival and capillary count 7 days after reperfusion compared with the flaps treated only with lipofectamine. These results indicate that VEGF exerts a protective effect on ischemic skeletal muscle flaps.

Catheter-Mediated Vascular Endothelial Growth Factor Gene Transfer to Human Coronary Arteries after Angioplasty

Blood vessels are among the easiest targets for gene therapy. However, no data are available about the safety and feasibility of intracoronary gene transfer in humans. We studied the safety and efficacy of catheter-mediated vascular endothelial growth factor (VEGF) plasmid/liposome (P/L) gene transfer in human coronary arteries after percutaneous translumenal coronary angioplasty (PTCA) in a randomized, double-blinded, placebo-controlled study. The optimized angioplasty/gene delivery method was previously shown to lead to detectable VEGF gene expression in human peripheral arteries as analyzed from amputated leg samples. Gene transfer to coronary arteries was done with a perfusion-infusion catheter, using 1000 microg of VEGF or beta-galactosidase plasmid complexed with 1000 microl of DOTMA:DOPE liposomes. Ten patients received VEGF P/L, three patients received beta-galactosidase P/L, and two patients received Ringer lactate. Gene transfer to coronary arteries was feasible and well tolerated. Except for a slight increase in serum C-reative protein in all study groups, no adverse effects or abnormalities in laboratory parameters were detected. No VEGF plasmid or recombinant VEGF protein was present in the systemic circulation after the gene transfer. In control angiography 6 months later, no differences were detected in the degree of coronary stenosis between treatment and control groups. We conclude that catheter-mediated intracoronary gene transfer performed after angioplasty is safe and well tolerated and potentially applicable for the prevention of restenosis and myocardial ischemia.

Complete reversal of ischemic wall motion abnormalities by combined use of gene therapy with transmyocardial laser revascularization

Introduction:Transmyocardial laser revascularization is believed to induce an angiogenic response within ischemic myocardium. We evaluated transgene expression in the setting of transmyocardial laser revascularization and hypothesized that intramyocardial injection of plasmid DNA encoding the gene for vascular endothelial growth factor could enhance the revascularization achieved by transmyocardial laser revascularization, resulting in improved cardiac function. Methods: Ten Yorkshire pigs had carbon dioxide–transmyocardial laser revascularization or acupuncture sites with injections of an expression plasmid encoding the gene for β-galactosidase with or without HVJ-liposomes. Three days after transduction, transgene expression was determined by enzyme-linked immunosorbent assay. Six weeks after placement of a constrictor on the left circumflex artery, 29 pigs were randomized to ischemic controls (n = 5), transmyocardial laser revascularization (n = 4), transmyocardial laser revascularization with expression plasmid β-galactosidase injections (n = 5), expression plasmid-vascular endothelial growth factor injections (n = 4), or transmyocardial laser revascularization with expression plasmid-vascular endothelial growth factor (n = 5) and harvested 6 weeks after therapy. Six transmyocardial laser revascularization pigs had either expression plasmid β-galactosidase or expression plasmid-vascular endothelial growth factor injections and were harvested at 2 weeks. Normal pigs (n = 5) were included for comparison. Left ventricular free wall motion was assessed by a cardiologist in a blinded manner. Results: Transgene expression did not vary significantly with or without HVJ-liposomes in transfected transmyocardial laser revascularization myocardium. However, expression was detected in 56 of 60 (93%) transmyocardial laser revascularization–transfected sites, but in only 10 of 20 (50%) non-transmyocardial laser revascularization sites ( P < .001). All (5 of 5 hearts) of the transmyocardial laser revascularization–vascular endothelial growth factor treated hearts displayed no evidence of wall motion abnormalities. Only these hearts had a statistically significantly different rate of wall motion abnormality compared with ischemic controls ( P = .004). Conclusion: Transfection efficiency was improved with the use of transmyocardial laser revascularization. Wall motion abnormalities were completely reversed within 6 weeks after transmyocardial laser revascularization with the direct injection of an expression plasmid encoding vascular endothelial growth factor. (J Thorac Cardiovasc Surg 1998;116:763-9)