Endothelial Growth Factor Gene Transfer Research Articles

Vascular endothelial growth factor gene transfer therapy for coronary artery disease: A systematic review and meta-analysis.

It is not clear whether treatment by vascular endothelial growth factor (VEGF) gene transfer can improve myocardial ischemia through a proangiogenesis mechanism and is effective against coronary artery disease (CAD). We aimed to perform a systematic review and meta-analysis of randomized controlled trials (RCTs) that compared VEGF gene therapy and standard treatments in CAD. We systematically searched the PubMed, Embase, and Cochrane databases and relevant references for RCTs (published up to May 2018; no language restrictions) and performed meta-analysis using both fixed and random effects models. Our primary outcome measures were mortality and serious cardiac events. The secondary outcome measures were follow-up left ventricular ejection fraction (LVEF), change in LVEF (ΔLVEF), and angina outcomes. The registration number is CRD42017058430. Of 524 identified studies, 14 were eligible and were included in our analysis. At a mean follow-up of 6months, VEGF gene therapy demonstrated a decreased risk of serious cardiac events (11.7% vs 21.2%, relative risk: 0.56; 95% confidence interval (CI): 0.37, 0.84; P=0.005) and a slight improvement in follow-up LVEF (weighted mean difference: 1.95; 95%CI: 1.28, 2.62). Furthermore, VEGF gene therapy using adenoviral vectors showed more potential benefit in terms of the risk of serious cardiac events, ΔLVEF, and Canadian Cardiovascular Society angina class. Nevertheless, mortality and angina frequency scores were not different. Vascular endothelial growth factor gene therapy appears to be safe and effective regarding serious cardiac events, with greater benefit when using adenoviral vectors. This meta-analysis highlights the need for further exploration in these areas.

Effect of vascular endothelial growth factor gene transfer on infarct size, left ventricular function and myocardial perfusion in sheep after 2 months of coronary artery occlusion

In large mammalian models of acute myocardial infarction (AMI), plasmid-mediated vascular endothelial growth factor (pVEGF) gene transfer has been shown to induce angio-arteriogenesis, proliferation of myocyte precursors and adult cardiomyocyte mitosis, reducing infarct size at 15 days after coronary artery occlusion. However, it is unknown whether these effects persist at longer follow-up times, nor how they affect cardiac performance. We thus assessed infarct size, left ventricular (LV) function and perfusion in 2-month-old ovine AMI. Adult sheep with coronary artery occlusion were randomized to blindly receive ten intramyocardial injections of 3.8 mg of pVEGF or empty plasmid distributed at the infarct border. Three and 60 days later, LV perfusion (single-photon emission computed tomography) and function (stress echocardiography) were assessed. Finally, hemodynamics (LV catheterization), scar size and peri-infarct histology were studied. Infarct size was 30% smaller in pVEGF-treated sheep (23.6 ± 1.9% versus 32.7 ± 2.7% of the LV; p < 0.02). Percentage fractional shortening and wall thickening at the infarct border improved after pVEGF, as did myocardial perfusion and LV wall motion under pharmacological stress. Global LV function did not differ between groups, although the force-frequency response was preserved in pVEGF group and lost in placebo animals. These effects were associated with angio-arteriogenesis and proliferation of cardiomyocyte precursors. In sheep with AMI, pVEGF gene transfer affords long-term infarct size reduction, yielding regional LV function and perfusion improvement and reducing remodeling progression. These results suggest the potential usefulness of this approach in the clinical setting.

Combined VEGF gene transfer and erythropoietin in ovine reperfused myocardial infarction

BackgroundIn reperfused acute myocardial infarction (RAMI), cardioprotective treatments may enhance myocardial salvage and hence reduce the area of necrosis. Based on studies showing that plasmid-mediated vascular endothelial growth factor (pVEGF) gene transfer reduces infarct size by combining angio-arteriogenic and cardiomyogenic effects and that erythropoietin (EPO) exerts anti-apoptotic actions in animal models of AMI, we aimed to assess if their association would reduce infarct size to a larger extent than any of them individually in a large mammalian model of RAMI. MethodsAdult sheep subjected to 90-minute coronary artery occlusion received upon reperfusion intramyocardial pVEGF 3.8mg plus intravenous EPO 1000IU/kg (n=8), pVEGF (n=8), EPO (n=8) or placebo (n=8). ResultsFifteen days after treatment, infarct size was smaller in the 3 treatment groups (pVEGF+EPO: 8±1%; pVEGF: 16±5%; EPO: 13±4%) compared to placebo (25±7%, p<0.001). However, in the EPO+VEGF group infarct size was significantly smaller than in the groups receiving EPO or VEGF individually (p<0.05). DNA fragmentation, a hallmark of late apoptosis, was significantly lower in sheep receiving EPO. The combined treatment, while not affecting global left ventricular performance, improved regional peri-infarct function and prevented over-time expansion of the post-infarct perfusion defect. ConclusionsCombined pVEGF and EPO treatment might be clinically useful to enhance the benefits of early revascularization in patients with acute myocardial infarction.

Effects of Blood-Activating and Stasis-Removing Drugs Combined with VEGF Gene Transfer on Angiogenesis in Ischemic Necrosis of the Femoral Head

To observe the promoting effects of blood-activating and stasis-removing Chinese drugs combined with vascular endothelial growth factor (VEGF) gene transfer on angiogenesis in ischemic necrosis of the femoral head. Forty Japanese giant-ear rabbits were randomly divided into a control group, a model group, a Chinese drug group, a gene group, and a combined group. After 8 weeks of treatment, the rate of VEGF positive cell expression in the synovium of the femoral head was measured using the immunohistochemical method, and the number of blood vessels in the femoral head was measured by digital subtraction angiography. The rate of VEGF positive cell expression in the model group was significantly lower than that in the Chinese drug group (P < 0.05) and very significantly lower than those in other groups (P < 0.01); but in the combined group it was significantly higher than in the Chinese drug group (P < 0.05). The differences in the number of blood vessels in area A between the model group and other groups were not statistically significant. However, in area B, the number of blood vessels significantly increased in the control group, the gene group and the combined group as compared with the model group (P < 0.05), and in the combined group the number of blood vessels was significantly more than in the gene group (P < 0.05); but in the Chinese drug group it was not significantly different than the model group (P > 0.05). Either the blood-activating and stasis-removing Chinese drugs or VEGF gene transfer can promote the angiogenesis and building of collateral circulation for femoral head ischemic necrosis, and the combined therapy with Chinese drugs or VEGF gene transfer may show a better therapeutic effect. The present study provides an experimental basis for clinical application of the combined therapy with the blood-activating and stasis-removing Chinese drugs and VEGF gene transfer.

Low‐dose simultaneous delivery of adenovirus encoding hepatocyte growth factor and vascular endothelial growth factor in dogs enhances liver proliferation without systemic growth factor elevation

Hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF) gene transfer proved to enhance liver regeneration. However, elevation of their plasma levels may induce potentially serious distant effects such as tumorigenesis or proliferative retinopathy. This study was performed to examine whether simultaneous administration of low-dose adenovirus encoding HGF and VEGF genes in dogs will stimulate liver proliferation but without inducing liver toxicity or systemic elevation of HGF and VEGF levels. Adult dogs received an intravenous injection of low-dose adenoviral vectors encoding human HGF and VEGF (HGF/VEGF), beta-galactosidase (lacZ) or phosphate-buffered saline (PBS). Liver proliferation was measured using the proliferating cell nuclear antigen (PCNA) immunostaining labelling index. HGF and VEGF plasma concentrations and transaminases were repeatedly measured. Transgene expression was evaluated using reverse-transcription polymerase chain reaction. Human HGF and VEGF expressions were detected only in the liver of HGF/VEGF dogs at day 2 after injection but declined at sacrifice (day 7). No expression was detected in the liver of the lacZ or PBS groups. Plasma levels of HGF and VEGF were not statistically different from those in the lacZ group (P=0.81, P=0.22 respectively). The PCNA labelling index was five-fold higher in the HGF/VEGF group compared with the lacZ group (P<0.01). No immunostaining was detected in the PBS group. Transaminases were only elevated (P<0.01) in the lacZ group compared with the other groups. We showed that simultaneous administration of low-dose adenoviral vectors encoding human HGF and VEGF genes can induce transgene expression and liver proliferation without liver toxicity or systemic growth factor elevation.

Vascular endothelial growth factor gene transfer for diabetic polyneuropathy: A randomized, double‐blinded trial

Randomized, blinded trial of intramuscular gene transfer using plasmid vascular endothelial growth factor (VEGF) to treat diabetic polyneuropathy. Diabetic patients with polyneuropathy were randomized to receive a VEGF-to-placebo ratio of 3:1. Three sets of injections were given at eight standardized sites adjacent to the sciatic, peroneal, and tibial nerves of one leg. Primary outcomes were change in symptom score at 6 months and a prespecified overall clinical and electrophysiological improvement score. Secondary outcomes were differences in symptoms, examination scores, visual analog pain scale, nerve conduction, and quantitative sensory testing. Thirty-nine patients received plasmid VEGF and 11 received placebo. Mean symptom score improved in both legs at 6 months, favoring VEGF over placebo (-1.2 +/- 0.5 vs -0.9 +/- 0.5; p < 0.01 after adjustment for change in the untreated leg) and compared with the untreated leg (-0.7 +/- 0.5; p = 0.02). The region of sensory loss and visual analog pain scale improved in the treated group (-1.5 vs -0.5; p = 0.01). Twelve of 39 VEGF versus 2 of 11 placebo patients met criterion for overall improvement. Other measures including nerve conduction potentials did not improve. There were 84 adverse events in VEGF patients, and 22 were serious; there were 51 events in placebo patients, and 2 were serious. Intramuscular plasmid VEGF gene transfer improved diabetic neuropathic symptoms, meeting primary end-point criteria for efficacy but not affecting most secondary measures. Treatment was associated with more serious adverse events that did not reach statistical significance. These results are not conclusive but may justify further clinical study.

Delivery of hypoxia-inducible VEGF gene to rat islets using polyethylenimine

Islet transplantation is a promising strategy for treatment of diabetes. However, islets are exposed to hypoxia in the process of isolation and transplantation and prone to apoptosis. Vascular endothelial growth factor (VEGF) gene transfer is one of the promising strategies to address this problem. However, VEGF expression in the cells under normoxia is undesirable since it may induce pathological angiogenesis. Therefore, VEGF expression should be regulated to avoid this problem. In this study, hypoxia-inducible VEGF gene was transferred to islets using a non-viral carrier. Rat islets were transfected with high molecular weight PEI (25 kDa, PEI25K), low molecular weight PEI (2 kDa, PEI2K), and polyamidoamine dendrimer (PAMAM). PEI25K had higher transfection efficiency to rat islets than PAMAM or PEI2K. The hypoxia-inducible gene expression vector, pRTP801-Luc or pRTP801-VEGF was transferred to rat islets using PEI25K. Transfection assay with pRTP801-Luc showed that luciferase expression was induced in rat islets under hypoxia. In addition, transfer of pRTP801-VEGF showed that VEGF gene expression was higher under hypoxia than normoxia in rat islets. In conclusion, delivery of pRTP801-VEGF using PEI25K induces VEGF level specifically under hypoxia and may be useful for the development of anti-apoptotic strategies for islet transplantation.

Percutaneous Transendocardial Delivery of Self-complementary Adeno-associated Virus 6 Achieves Global Cardiac Gene Transfer in Canines

Achieving efficient cardiac gene transfer in a large animal model has proven to be technically challenging. Previous strategies have used cardiopulmonary bypass or dual catheterization with the aid of vasodilators to deliver vectors, such as adenovirus, adeno-associated virus (AAV), or plasmid DNA. Although single-stranded AAV (ssAAV) vectors have shown the greatest promise, they suffer from delayed expression, which might be circumvented using self-complementary vectors. We sought to optimize cardiac gene transfer using a percutaneous transendocardial injection catheter to deliver adeno-associated viral vectors to the canine myocardium. Four vectors were evaluated--ssAAV9, self-complementary AAV9 (scAAV9), scAAV8, scAAV6--so that comparison could be made between single-stranded and self-complementary vectors as well as among serotypes 9, 8, and 6. We demonstrate that scAAV is superior to ssAAV and that AAV 6 is superior to the other serotypes evaluated. Biodistribution studies revealed that vector genome copies were 15-4,000 times more abundant in the heart than in any other organ for scAAV6. Percutaneous transendocardial injection of scAAV6 is a safe, effective method to achieve efficient cardiac gene transfer.

Increased tissue perfusion promotes capillary dysplasia in the ALK1-deficient mouse brain following VEGF stimulation

Loss-of-function activin receptor-like kinase 1 gene mutation (ALK1+/-) is associated with brain arteriovenous malformations (AVM) in hereditary hemorrhagic telangiectasia type 2. Other determinants of the lesional phenotype are unknown. In the present study, we investigated the influence of high vascular flow rates on ALK1+/- mice by manipulating cerebral blood flow (CBF) using vasodilators. Adult male ALK1+/- mice underwent adeno-associated viral-mediated vascular endothelial growth factor (AAVVEGF) or lacZ (AAVlacZ as a control) gene transfer into the brain. Two weeks after vector injection, hydralazine or nicardipine was infused intraventricularly for another 14 days. CBF was measured to evaluate relative tissue perfusion. We analyzed the number and morphology of capillaries. Results demonstrated that hydralazine or nicardipine infusion increased focal brain perfusion in all mice. It was noted that focal CBF increased most in AAVVEGF-injected ALK1+/- mice following hydralazine or nicardipine infusion (145+/-23% or 150+/-11%; P<0.05). There were more detectable dilated and dysplastic capillaries (2.4+/-0.3 or 2.0+/-0.4 dysplasia index; P<0.01) in the brains of ALK1+/- mice treated with AAVVEGF and hydralazine or nicardipine compared with the mice treated with them individually. We concluded that increased focal tissue perfusion and angiogenic factor VEGF stimulation could have a synergistic effect to promote capillary dysplasia in a genetic deficit animal model, which may have relevance to further studies of AVMs.

A promising strategy for the treatment of ischemic heart disease: Mesenchymal stem cell-mediated vascular endothelial growth factor gene transfer in rats

Treatment of ischemic heart disease (IHD) remains a worldwide problem. Gene therapy, and recently, cell transplantation, have made desirable progress. A combination of appropriate stem cells and angiogenic genes appears promising in treating IHD. To study the results of angiogenesis and myogenesis induced by transplantation of the adenovirus carrying human vascular endothelial growth factor 165 (Ad-hVEGF(165))-transfected mesenchymal stem cells (MSCs) in IHD compared with direct MSC transplantation or Ad-hVEGF165 delivery. Cultured MSCs were transfected by Ad-hVEGF(165), and secreted VEGF was measured by ELISA in vitro. Ad-hVEGF(165)-transfected MSCs (MSC/VEGF group), MSCs (MSC group), Ad-hVEGF(165) (VEGF group) or a serum-free medium (control group) was injected into syngeneic Wistar rats immediately after left coronary artery occlusion. All cells were marked with CM-DiI (Molecular Probes, USA) before transplantation. One week after treatment, messenger RNA expression of hVEGF(165) in the MSC/VEGF group was found to be significantly higher than in other groups by reverse transcriptase-polymerase chain reaction analysis. One month after cell transplantation, left ventricular (LV) ejection fraction, capillary density of the infarcted region, infarct size and hemodynamic parameters (including LV end-diastolic pressure, LV+dP/dt and LV-dP/dt) were measured and immunohistochemical analysis was performed. A high level of VEGF was expressed by Ad-hVEGF(165)-transfected MSCs. LV ejection fraction, mean capillary density of the infarcted region and hemodynamic parameters were significantly improved in the MSC/VEGF group compared with the MSC group, the VEGF group and the control group (P<0.001 for all). Partly transplanted MSCs showed the cardiomyocyte phenotype, expressed desmin and cardiac troponin T, and resulted in angiogenesis in the ischemic myocardium. However, a few transplanted MSCs incorporated into the vascular structure and most of the new vascular components were host-derived. The combined strategy of MSC transplantation and VEGF gene therapy can produce effective myogenesis and host-derived angiogenesis, resulting in the prevention of progressive heart dysfunction after myocardial infarction.

Hypoxic response elements control expression of human vascular endothelial growth factor165 genes transferred to ischemia myocardium in vivo and in vitro

Vascular endothelial growth factor (VEGF) gene transfer with recombinant adeno-associated viral (rAAV) vector for ischemia heart disease therapy is being increasingly studied. However, uncontrolled long-term expression of VEGF may cause some side effects. Therefore, an attempt to develop an effective gene control system for safeguarding against such side effects should be made. Pathphysiologically, an ideal control system for VEGF gene expression is letting it respond to hypoxia. We used nine copies of hypoxic response element (HRE) to regulate expression of hVEGF(165) in the myocardium, and tried to elucidate the feasibility and safety of the application of the HIF-1-HRE system. Cardiomyocytes of neonatal Sprague Dawley rats were cultured and incubated with rAAV-9HRE-hVEGF(165), and pig ischemic heart models were established and rAAV-9HRE-hVEGF(165) was injected into ischemia myocardium. RT-PCR, Western blot, ELISA, and immunohistochemistry were used to determine hVEGF(165) expressions of cultured cardiomyocytes and myocardium under hypoxic and reoxygenation conditions. The results of RT-PCR and ELISA determinations revealed that, in cultured cardiomyocytes, expressions of hVEGF(165)mRNA and protein were up-regulated under hypoxic conditions. After 4 h of reoxygenation, hVEGF(165)mNRA expression was decreased, and disappeared following 8 to 12 h of reoxygenation (P < 0.01). RT-PCR and Western blot also showed that, under myocardial ischemia, hVEGF(165) expression was increased significantly (P < 0.01). Following myocardial reperfusion, both hVEGF(165)mRNA and protein expressions were inhibited (P < 0.01). The new vessels in the reperfusion condition were decreased. This study suggested that 9HRE can effectively control hVEGF(165) gene expression in vivo and in vitro. It has feasibility for using the HIF-1-HRE system for regulation of angiogenic factor expression in ischemia heart.

Adenovirus mediated vascular endothelial growth factor gene transfer protects dopaminergic neurons in Parkinson's disease: experiment with rats

To evaluate the protective effect of adenovirus mediated vascular endothelial growth factor 165 (VEGF165) gene transfer on dopaminergic neurons in Parkinson disease (PD). Adenovirus vector coding VEGF165 (Ad-VEGF165) was injected into the striate bodies of 16 SD rats, and adenovirus Ad-LacZ was injected into 25 rats and phosphate-buffered saline (PBS) was injected into 16 rats as controls. Then 6-hydroxydopamine (6-OHDA) was injected to establish PD model. X-gal staining was used to detect the expression of the report gene LacZ in the brain of the Ad-LacZ group 3 d, 2 w, and 6 w after injection, 3 rats in each time point. RT-PCR was used to detect the VEGF165 mRNA expression in the striate body of the rats of the 3 groups 2 weeks after injection, 3 rats for each group. Western blotting was performed to check the protein expression of VEGF165 in the striate bodies of the rats of the 3 groups 2 weeks after injection, 3 rats for each group. A certain numbers of rats in each group underwent rotational behavior analysis 1, 2, and 6 weeks after the 6-OHDA injection. Immunohistochemistry was used to examine the number of tyrosine hydroxylase (TH) positive neuron, density of TH-positive fiber in striate body and black substance, laminin-positive vessel density, and glial fibrillary acidic protein (GFAP) positive glial cells. High performance liquid chromatography-electric-chemical discharge (HPLC-ECD) was performed to detect the contents of dopamine (DA) and its metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the striatum. Beta-gal was expressed in the striate bodies of all Ad-LacZ transfected rats, showing the successful transfection of LacZ gene. The mRNA expression and protein expression of VEGF165 in the striate body were significantly higher in the Ad-VEGF165 group than in the other 2 groups. The apomorphine-induced rotation number in the Ad-VEGF165 group was 8.3 turns/min +/- 8.7 turns/min 1 week after the transfection, then gradually decreased, and became 5.0 turns/min +/- 4.4 turns/min 6 weeks after. The rotation numbers of the Ad-LacZ and PBS group were 14.7 turns/min +/- 11.2 turns/min and 13.9 turns/min +/- 8.3 turns/min respectively 1 week after the injection, then increased gradually, and became 20.2 turns/min +/- 13.6 turns/min and 21.8 turns/min +/- 11.8 turns/min respectively 6 weeks later, all significantly higher than those of the Ad-LacZ group (all P < 0.01). The ratios of TH-positive cells in the black substance in the transfected side was 0.42 +/- 0.11, and the density of fibers in the striate body of the transfected side to that of the contralateral side was 0.56 +/- 0.10 in the Ad-VEGF165 group, both significantly higher than those of the Ad-LacZ group (0.20 +/- 0.10 and 0.28 +/- 0.09) and PBS group (0.22 +/- 0.13 and 0.24 +/- 0.08), (all P < 0.01). The ratio of laminin-positive vessel density of the transfected side to that of the contralateral side in the Ad-VEGF165 group was 2.09 +/- 0.42, and the ratio of GFAP-positive glial cells of the striate body of the transfected side to that of the contralateral side was 2.77 +/- 1.21 in the Ad-VEGF165 group, both significantly higher than those in the Ad-LacZ group (1.01 +/- 0.16 and 1.64 +/- 0.28) and the PBS group (1.04 +/- 0.09 and 1.56 +/- 0.62) (P < 0.01 and 0.05). HPLC-ECD showed that the contents of DA, HAV, and DOPAC of the striate body at the destroyed side in the Ad-VEGF165 group were all significantly higher than those in the other 2 groups (all P < 0.01). The ratios to the DA, DOPAC, and HVA of the destroyed side striate body to those of the contralateral side in the Ad-VEGF165 group was 0.35 +/- 0.11, 0.46 +/- 0.09, and 0.38 +/- 0.09 respectively, all significantly higher than those in the Ad-LacZ group (0.17 +/- 0.15, 0.21 +/- 0.07, and 0.16 +/- 0.05) and PBS group (0.19 +/- 0.06, 0.20 +/- 0.09, 0.14 +/- 0.03) (all P < 0.01). Gene transfer of Ad-VEGF165 has a protective effect on the dopaminergic neurons of PD. The proliferation of vessels and glial cells induced by VEGF may involve in the process of neuroprotection to the dopaminergic neurons of PD.

Antisense vascular endothelial growth factor suppressed corneal neovascularization in rats

To explore whether recombinant adeno-associated virus mediated antisense vascular endothelial growth factor (rAAV-aVEGF) gene transfer inhibits the development of corneal neovascularization (CNV) in a rat model. rAAV-aVEGF(165) and recombinant adeno-associated virus mediated Lacz (rAAV-Lacz) were constructed by three-plasmid cotransfection methods. Forty-eight Sprague-Dawley (SD) rats were divided into two groups randomly. CNV were induced in vivo by alkaline cauterization of the central cornea. Twenty-four rats were injected with rAAV-aVEGF(165) (10(10) pfu/ml) into conjunctiva; another rats were injected with rAAV-Lacz (10(10) pfu/ml) into conjunctiva as controls. The Lacz gene expression was evaluated by 5-bromo-4-chloro-3-indolyl-beta-D-galactoside (X-gal) immunohistochemical staining. 1 to 30 days post-cautery, CNV was evaluated by morphometric analysis, and expression of VEGF was evaluated by immunohistochemistry. Two days later, 21.36% +/- 1.07% Lacz gene expression was detected in conjunctival sac in control group. 30 d later, 28.02% +/- 1.16% Lacz gene remained expression. Morphometric analysis and immunohistochemistry demonstrated rAAV-mediated antisense VEGF(165) significantly inhibited CNV. The VEGF(165) protein was decreased post-cautery compared to control group injected with. rAAV-Lacz (F = 1639.22, F = 2187.16, F = 719.17, P < 0.01). This study suggests that rAAV-aVEGF(165) can sufficiently inhibit the cautery-induced CNV, and the effect is associated with the inhibition of VEGF production.

Combined Angiopoietin-1 and vascular endothelial growth factor gene transfer restores cavernous angiogenesis and erectile function in a rat model of hypercholesterolemia

Hypercholesterolemia-related endothelial cell dysfunction and decreased endothelium-derived nitric oxide formation may account for impaired angiogenesis and subsequent erectile dysfunction. Angiopoietin-1 (Ang1) is a critical angiogenic factor for vascular maturation and enhances vascular endothelial growth factor (VEGF)-induced angiogenesis in a complementary manner. We hypothesized that combined adenovirus-delivered human Ang1 (ad-Ang1) and VEGF165 (ad-VEGF165) gene transfer might promote angiogenesis cooperatively in a rat model of hypercholesterolemic erectile dysfunction and result in a recovery of erectile function. Ad-Ang1 and ad-VEGF165 were injected either alone or in combination into the corpus cavernosum of the penis. Combined gene transfer of both ad-Ang1 and ad-VEGF165 significantly increased cavernous angiogenesis, eNOS phosphorylation, and cGMP expression compared with that in the groups treated with either therapy alone. Erectile function, as evaluated by electrical stimulation of the cavernous nerve 2 and 8 weeks after treatment, was completely restored in the combined treatment group, whereas intracavernous injection of either ad-Ang1 or ad-VEGF165 alone elicited partial improvement. The results indicate that combined application of angiogenic factors may enhance cavernous angiogenesis cooperatively by reinforcing the endothelium both structurally and functionally, which results in an additive effect on erectile function in hypercholesterolemic rats.

Adventitial VEGF 165 gene transfer prevents lumen loss through induction of positive arterial remodeling after PTCA in porcine coronary arteries

Negative arterial remodeling still plays an important role in the pathogenesis of coronary restenosis even in the era of interventional stenting (e.g. arterial narrowing occurs proximal and distal of a stented segment). Previous studies suggest that increased angiogenesis and inhibited regression of injury-induced adventitial microvessels prevents negative remodeling. We have examined the effect of local vascular endothelial growth factor (VEGF 165) gene transfer on adventitial microvessel angiogenesis/regression and arterial remodeling after coronary angioplasty. Twenty pigs underwent angioplasty, each one in two major coronary arteries, followed by plasmid liposome gene transfer with either VEGF 165 or control gene LacZ (50 μg DNA with 50 μg of Lipofectine) into the (peri)adventitial space using a needle injection catheter. Arteries were examined at days 1, 7, 14, and 28. Local delivery of VEGF 165 gene into the outer compartments of balloon-injured porcine coronary arteries reduced lumen area loss due to distinct positive remodeling (arterial enlargement). Prevention of adventitial microvessel regression, enhanced adventitial elastin accumulation, reduced adventitial myofibroblast numbers, and a pronounced adventitial inflammatory response considered as a part of arterial healing seem to be the main VEGF-mediated mechanisms indicating the therapeutic potential of VEGF for restenosis prevention.

Effect of VEGF gene transfer on the bleomycin-induced pulmonary hypertension in immature rabbits

To investigate the effect of vascular endothelial growth factor (VEGF) gene transfer on the bleomycin(BLM)-induced pulmonary hypertension in immature rabbits. Immature rabbits were divided into 4 groups; control, BLM, liposome and trans-gene groups. The systolic, diastolic and mean pulmonary artery pressure (PASP, PADP, MPAP) were measured by micro-catheter, the pathological changes and the expression of VEGFmRNA and eNOSmRNA of endothelial cells in pulmonary arteries were evaluated by HE stain and in situ hybridization. (1) The PAP of BLM and liposome groups was higher than that of control and trans-gene groups. The PASP was 16.5+/-2.9, 25.2+/-7.0, 24.4+/-6.0 and 18.3+/-2.7 mmHg; the PADP was 8.8+/-4.2, 13.1+/-3.8, 13.7+/-4.6 and 10.2+/-2.6 mmHg; the MPAP was 12.1+/-4.0, 18.4+/-4.7, 18.4+/-5.1 and 14.1+/-2.5 mmHg in control, BLM, liposome and trans-gene groups respectively. (2) The thickness of wall increased and the cavity became narrow, and the thickness index (TI) and area index (AI) increased in middle and small pulmonary arteries of BLM and liposome groups. The TI was 0.52+/-0.16, 0.65+/-0.16, 0.63+/-0.11 and 0.55+/-0.13; and the AI was 0.74+/-0.17, 0.84+/-0.14, 0.85+/-0.08 and 0.79+/-0.12 in control, BLM, liposome and trans-gene groups,respectively. (3) The level of VEGFmRNA and eNOSmRNA expression in pulmonary arterial endothelial cells decreased in BLM and liposome groups. The level of VEGFmRNA and eNOSmRNA expression in trans-gene group was higher than that in BLM and liposome groups, but lower than that in control group. VEGFmRNA was 0.83+/-0.09, 0.45+/-0.11, 0.45+/-0.13 and 0.65+/-0.18; eNOSmRNA was 0.79+/-0.12, 0.45+/-0.12, 0.50+/-0.14 and 0.56+/-0.08 in control, BLM, liposome and trans-gene groups, respectively. VEGF gene transfer in immature rabbits with BLM-induced pulmonary hypertension could attenuate the increasing of PAP and wall thickness in middle and small pulmonary arteries, and increase the level of VEGFmRNA and eNOSmRNA expression in pulmonary arterial endothelial cells.

Improved Survival of Ischemic Cutaneous and Musculocutaneous Flaps after Vascular Endothelial Growth Factor Gene Transfer Using Adeno-Associated Virus Vectors

A major challenge in reconstructive surgery is flap ischemia, which might benefit from induction of therapeutic angiogenesis. Here we demonstrate the effect of an adeno-associated virus (AAV) vector delivering vascular endothelial growth factor (VEGF)165 in two widely recognized in vivo flap models. For the epigastric flap model, animals were injected subcutaneously with 1.5 x 10(11) particles of AAV-VEGF at day 0, 7, or 14 before flap dissection. In the transverse rectus abdominis musculocutaneous flap model, AAV-VEGF was injected intramuscularly. The delivery of AAV-VEGF significantly improved flap survival in both models, reducing necrosis in all treatment groups compared to controls. The most notable results were obtained by administering the vector 14 days before flap dissection. In the transverse rectus abdominis musculocutaneous flap model, AAV-VEGF reduced the necrotic area by >50% at 1 week after surgery, with a highly significant improvement in the healing process throughout the following 2 weeks. The therapeutic effect of AAV-VEGF on flap survival was confirmed by histological evidence of neoangiogenesis in the formation of large numbers of CD31-positive capillaries and alpha-smooth muscle actin-positive arteriolae, particularly evident at the border between viable and necrotic tissue. These results underscore the efficacy of VEGF-induced neovascularization for the prevention of tissue ischemia and the improvement of flap survival in reconstructive surgery.

Superior neovascularization and muscle regeneration in ischemic skeletal muscles following VEGF gene transfer by rAAV1 pseudotyped vectors

Recombinant adeno-associated virus serotype 2 (rAAV2) vector has been widely employed for gene therapy. Recent progress suggests that the new serotypes of AAV showed a better performance than did AAV2 in normal tissues. Here, we evaluate the potential role of human vascular endothelial growth factor (VEGF) gene transfer using rAAV vector pseudotyped with serotype 1 capsid proteins (rAAV1) in the treatment of muscle ischemia. In ischemic skeletal muscles, the rAAV1-LacZ vector allowed higher level, broader distribution, and long-lasting gene expression compared with the rAAV2-LacZ vector. Muscle VEGF165 production following the rAAV1-VEGF165 vector injection was 5–10 times higher than that following the rAAV2-VEGF165 vector injection. VEGF165 production mediated by the rAAV1-VEGF165 vector stimulated a large set of neovascularization with relatively mature vascular structures and enhanced muscle regeneration in the ischemic skeletal muscles. Thus, the rAAV1-VEGF165 vector mediated gene transfer may be a therapeutic approach to peripheral vascular diseases.

A Regenerative Role for Bone Marrow Following Experimental Colitis: Contribution to Neovasculogenesis and Myofibroblasts

Bone marrow (BM) cells form differentiated adult lineages within nonhematopoietic tissues, with a heightened propensity with increasing regenerative pressure dictated by disease. We have previously shown that BM cells engraft into the gut and contribute substantially to the subepithelial intestinal myofibroblast population in the lamina propria. To investigate the reparative capacity of BM in inflammatory bowel disease (IBD), a well-established model of experimental colitis was used. Lethally irradiated female mice were rescued by a BM transplant from male donors. Colitis was induced 6 weeks posttransplantation by injection of trinitrobenzene sulfonic acid (TNBS), and tissues were analyzed 1-14 days later. Donor-derived cells were detected by in situ hybridization using a Y chromosome-specific probe, and their phenotype was determined by immunohistochemistry. TNBS-induced colitis was manifest as patchy lesions that increased in severity between days 1 and 8, and the mucosa gradually regenerated between days 8 and 14. The contribution of BM to intestinal myofibroblasts was significantly increased in regions of colitis compared with noninflamed regions. Furthermore, BM-derived endothelial cells, pericytes, and vascular smooth muscle cells were frequently interspersed throughout blood vessels, suggesting that these cells facilitate angiogenesis in tissue repair, substantiated by a significant increase in the incidence of BM-derived vascular smooth muscle cells in colitic compared with noninflamed regions. Blood vessels formed entirely from BM-derived cells were also seen, suggesting a role for BM in neovasculogenesis. Our data show that BM contributes to multiple intestinal cell lineages in colitis, with an important function in tissue regeneration and vasculogenesis after injury.

Vascular endothelial growth factor (VEGF) gene transfer enhances surgical revascularization of necrotic bone

Avascular necrosis of bone is a relatively common clinical condition caused by inflammatory conditions, steroid or other drug use, and trauma that affect many different sites in man. Revascularization of the necrotic bone is slow to occur, often resulting in bone resorption and eventual collapse of the involved bone. Rapid revascularization and subsequent bone remodeling may lead to improved outcomes. Surgical revascularization with arterovenous bundles (AV bundles) or vascularized bone grafts results in neoangiogenesis and bone remodeling. Gene transfer of an angiogenic factor to the vessel wall may be an additional strategy to further accelerate this process. In this study, we examined the effectiveness of vascular endothelial growth factor (VEGF) gene transfer to augment surgical revascularization of necrotic bone. An adenoviral vector, either with the VEGF gene (VEGF-A) or identical virus without the cDNA VEGF insert (ADV-ΔE1) was used to transduce endothelial cells in rabbit saphenous arteries. The artery was then placed with its venae comitantes as an AV bundle into necrotic iliac crest bone in vivo. Angiogenesis in the necrotic bone was quantified by bone blood flow measurement and assessment of vessel density following microangiography. The extent of neoangiogenesis was significantly greater in the VEGF group than the control group at 1 week postoperatively.