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Aims: We evaluated for the first time the effects of angiogenic and lymphangiogenic AdVEGF-DΔNΔC gene therapy in patients with refractory angina. Methods and results: Thirty patients were randomized to AdVEGF-DΔNΔC (AdVEGF-D) or placebo (control) groups. Electromechanical NOGA mapping and radiowater PET were used to identify hibernating viable myocardium where treatment was targeted. Safety, severity of symptoms, quality of life, lipoprotein(a) [Lp(a)] and routine clinical chemistry were measured. Myocardial perfusion reserve (MPR) was assessed with radiowater PET at baseline and after 3- and 12-months follow-up. Treatment was well tolerated. Myocardial perfusion reserve increased significantly in the treated area in the AdVEGF-D group compared with baseline (1.00 ± 0.36) at 3 months (1.31 ± 0.46, P = 0.045) and 12 months (1.44 ± 0.48, P = 0.009) whereas MPR in the reference area tended to decrease (2.05 ± 0.69, 1.76 ± 0.62, and 1.87 ± 0.69; baseline, 3 and 12 months, respectively, P = 0.551). Myocardial perfusion reserve in the control group showed no significant change from baseline to 3 and 12 months (1.26 ± 0.37, 1.57 ± 0.55, and 1.48 ± 0.48; respectively, P = 0.690). No major changes were found in clinical chemistry but anti-adenovirus antibodies increased in 54% of the treated patients compared with baseline. AdVEGF-D patients in the highest Lp(a) tertile at baseline showed the best response to therapy (MPR 0.94 ± 0.32 and 1.76 ± 0.41 baseline and 12 months, respectively, P = 0.023). Conclusion AdVEGF-DΔNΔC gene therapy was safe, feasible, and well tolerated. Myocardial perfusion increased at 1 year in the treated areas with impaired MPR at baseline. Plasma Lp(a) may be a potential biomarker to identify patients that may have the greatest benefit with this therapy. [ABSTRACT FROM AUTHOR]

Highly increased blood flow and vascularity after angiogenic gene therapy have raised concerns of shunting and hemangioma-like blood pool formation that might decrease effective perfusion and ruin the beneficial effects of the therapy. Contrast enhanced ultrasound is a promising noninvasive tool for studying skeletal muscle perfusion. The objectives of the present study were to test bolus and infusion administrations of ultrasound microbubble contrast media in imaging vascular growth in skeletal muscle and assess the functionality of vessels grown with angiogenic gene therapy. Contrast enhanced ultrasound was used to study changes in skeletal muscle perfusion in normal and gene-transduced rabbit hindlimbs 6 days after gene transfer. Adenoviral gene transfer of VEGF (10e9-10e11 viral particles) or β-galactosidase control gene (10e11 viral particles) was done under anesthesia and induced up to 16-fold increases in relative tissue perfusion. Contrast intensity versus time curves were plotted and analyzed for contrast kinetics. Bolus administration of the contrast media was highly feasible in analyzing skeletal muscle blood flow and its kinetics. Maximal signal intensity of the bolus signal reflected relative changes in both blood flow and volume equally to the infusion method. Flow irregularities were detected after angiogenic gene therapy. In conclusion, bolus delivery of ultrasound contrast agent is highly feasible for the relative analysis of both quantity and quality of blood flow after angiogenic gene therapy. The kinetics of blood flow can and should be studied more extensively in both preclinical and clinical trials of angiogenic gene therapy since there is increasing evidence of flow irregularities in angiogenic vessels. [ABSTRACT FROM AUTHOR]

Background Cardiovascular patients suffer from reduced blood flow leading to ischaemia and impaired tissue metabolism. Unfortunately, an increasing group of elderly patients cannot be treated with current revascularization methods. Thus, new treatment strategies are urgently needed. Hypoxia-inducible factors ( HIFs) upregulate the expression of angiogenic mediators together with genes involved in energy metabolism and recovery of ischaemic tissues. Especially, HIF-2α is a novel factor, and only limited information is available about its therapeutic potential. Methods Gene transfers with adenoviral HIF-1α and HIF-2α were performed into the mouse heart and rabbit ischaemic hindlimbs. Angiogenesis was evaluated by histology. Left ventricle function was analysed with echocardiography. Perfusion in rabbit skeletal muscles and energy recovery after electrical stimulation-induced exercise were measured with ultrasound and 31 P-magnetic resonance spectroscopy (31 P- MRS), respectively. Results HIF-1α and HIF-2α gene transfers increased capillary size up to fivefold in myocardium and ischaemic skeletal muscles. Perfusion in skeletal muscles was increased by fourfold without oedema. Especially, Ad HIF-1α enhanced the recovery of ischaemic muscles from electrical stimulation-induced energy depletion. Special characteristic of HIF-2α gene transfer was a strong capillary growth in muscle connective tissue and that HIF-2α gene transfer maintained left ventricle function. Conclusions We conclude that both Ad HIF-1α and Ad HIF-2α gene transfers induced beneficial angiogenesis in vivo. Transient moderate increases in angiogenesis improved energy recovery after exercise in ischaemic muscles. This study shows for the first time that a moderate increase in angiogenesis is enough to improve tissue energy metabolism, which is potentially a very useful feature for cardiovascular gene therapy. [ABSTRACT FROM AUTHOR]

A large animal model of chronic myocardial ischemia and heart failure is crucial for the development of novel therapeutic approaches. In this study we developed a novel percutaneous one- and two-vessel model for chronic myocardial ischemia using a stent coated with a polytetrafluoroethylene tube formed in a bottleneck shape. The bottleneck stent was implanted in the proximal left anterior descending (LAD) or proximal circumflex artery (LCX), or in both proximal LCX and mid LAD 1 wk later (2-vessel model), and pigs were followed for 4-5 wk. Ejection fraction (EF), infarct size, collateral growth, and myocardial perfusion were assessed. Pigs were given antiarrhythmic medication to prevent sudden death. The occlusion time of the bottleneck stent and the timing of myocardial infarction could be modulated by the duration of antiplatelet medication. Fractional flow reserve measurements and positron emission tomography imaging showed severe ischemia after bottleneck stenting covering over 50% of the left ventricle in the proximal LAD model. Complete coronary occlusion was necessary for significant collateral growth, which mostly had occurred already during the first wk after the stent occlusion. Dynamic and competitive collateral growth patterns were observed. EF declined from 64 to 41% in the LCX model and to 44% in the LAD model 4 wk after stenting with 12 and 21% infarcted left ventricle in the LCX and LAD models, respectively. The mortality was 32 and 37% in the LCX and LAD models but very (71%) high in the two-vessel disease model. The implantation of a novel bottleneck stent in the proximal LAD or LCX is a novel porcine model of reversible myocardial ischemia (open stent) and ischemic heart failure (occluded stent) and is feasible for the development of new therapeutic approaches. [ABSTRACT FROM AUTHOR]

Aims Currently, it is still unclear which mechanisms drive metabolic benefits after angiogenic gene therapy. The side-effect profile of efficient angiogenic gene therapy is also currently incompletely understood. In this study, the effects of increasing doses of adenoviral (Ad) vascular endothelial growth factor-A (VEGF-A) were evaluated on vascular growth, metabolic benefits, and systemic side effects. Methods and results Adenoviral vascular endothelial growth factor-A or AdLacZ control was injected intramuscularly (109–1011 vp/mL) or intra-arterially (5 × 1011 vp/mL) into rabbit (n = 102) hindlimb muscles and examined 6 or 14 days later. Blood flow, tissue oedema, metabolic benefits, and the structure of angiogenic vessels were assessed using ultrasound imaging, modified Miles assay, arterial blood gas and metabolite analyses, and light and confocal microscopy, respectively. Safety analyses included cardiac ultrasound, electrocardiograms, and blood and tissue samples. Sprouting angiogenesis was already induced with low AdVEGF-A concentrations, whereas higher concentrations were needed to reach efficient capillary enlargement and increases in target muscle perfusion. Interestingly, metabolic benefits, such as improved aerobic energy metabolism and decreased metabolic acidosis during exercise, after AdVEGF-A administration were highly correlated to the level of capillary enlargement but not to sprouting angiogenesis. Several systemic dose-dependent side effects, including transient increases in liver, kidney, and pancreatic enzymes, and signs of cardiac effects were observed. Conclusion Efficient capillary enlargement leading to significant increases in tissue perfusion is needed to gain metabolic benefits after angiogenic gene therapy. However, the risk of systemic side effects can increase as the efficiency of angiogenic gene therapy is improved. Importantly, the unstable wall structure of the newly formed vessels seems not to compromise the metabolic benefits. [ABSTRACT FROM PUBLISHER]

Aims: Vascular endothelial growth factors (VEGFs) and their receptors (VEGF-Rs) are among the most powerful factors regulating vascular growth. However, it has remained unknown whether stimulation of VEGF-R1, VEGF-R2 or both of the receptors produces the best angiogenic responses in myocardium. The aim of this study was to compare the VEGF-R1-specific ligand VEGF-B186, VEGF-R2-specific ligand VEGF-E and VEGF-A165, which stimulates both receptors, regarding their effects on angiogenesis and left ventricular function in mice. Methods and results: High-resolution echocardiography was used to guide the closed-chest injections of adenoviral (Ad) vectors expressing VEGF-B186, VEGF-E, and VEGF-A165 into the anterior wall of the left ventricle in C57Bl/6J mice. Angiogenic and functional effects were analysed using histology, ultrasound and perfusion analyses 6 (D6) and 14 (D14) days after the Ad injection. AdVEGF-A165 induced a strong angiogenic response seen as an enlargement of myocardial capillaries whereas angiogenesis induced by AdVEGF-B186 and AdVEGF-E seemed more physiological. The increase in the capillary area was accompanied with an increase in myocardial perfusion at D6 after the gene injection. AdVEGF-A165 and AdVEGF-E induced endothelial-specific proliferation whereas AdVEGF-B186 mostly induced proliferation of cardiomyocytes. AdVEGF-A165 induced more pronounced tissue damage than AdVEGF-B186 and AdVEGF-E. Left ventricular function measured as ejection fraction did not change during the follow-up. AdVEGF-A165 increased both VEGF-R1 and VEGF-R2 protein expression whereas AdVEGF-B186 and AdVEGF-E did not affect endogenous receptor expression levels.Conclusion: AdVEGF-B186 and AdVEGF-E are equally potent in inducing therapeutic angiogenesis in mouse myocardium and produce less side effects than AdVEGF-A165. [ABSTRACT FROM PUBLISHER]

The article presents a discussion concerning a study on the vascular endothelial growth factor-A and platelet-derived growth factor-B combination gene therapy that prolongs the effects of angiogenic via recruitment of interstitial in the United States. The advantages of combination gene therapy are discussed. It also concludes that the combination of AdVEGF-A and AdPDGF-B intramuscular delivery causes prolonged angiogenic response.

Aims: We investigated the effects of angiogenic gene therapy with adenoviral placental growth factor131 (AdPlGF) on aerobic capacity and exercise tolerance in a rabbit hindlimb ischaemia model. We also assessed whether strong angiogenic changes such as capillary arterialization and formation of artery-venous shunts compromise oxygen transport to target tissues resulting in suboptimal therapeutic efficacy. [ABSTRACT FROM PUBLISHER]

Abstract: Myocardial ischemia is one of the most promising targets of gene therapy. Although several growth factors and delivery approaches have yielded positive results in preclinical studies, first clinical studies have shown little or no real clinical benefit to the patients. It is likely that less than optimal gene therapy approaches have been used so far, and more thorough preclinical studies are needed in order to establish safe, efficient pro-angiogenic therapy. Growth factor, gene transfer vector, delivery method and target microenvironment need to be chosen based on the therapeutic target. It has become apparent that induction of large collateral arteries in the myocardium may need a different approach than rapid growth of neovasculature around infarction scar. Large animal models are necessary in the determination of optimal therapeutic agent, dose and clinically relevant delivery strategy. [Copyright &y& Elsevier]

Critical lower limb ischemia is a common cause for amputation. To develop new therapeutic strategies, more information is needed about molecular mechanisms of tissue responses to ischemic stress and factors inducing angiogenesis. Using a DNA array of 8400 genes, gene expression patterns in human skeletal muscle samples collected from lower limbs amputated due to acute-on-chronic or chronic critical lower limb ischemia, were compared with the control samples collected from the same limb. The results were confirmed by RT-PCR and immunohistochemistry. In acute-on-chronic ischemia, 291 genes were significantly upregulated and 174 genes were downregulated (change in 5.5% of all genes) as compared to control samples. Significant induction of the hypoxia-inducible angiogenic pathway involving hypoxia-inducible factor-1α (HIF-1α), HIF-2α, vascular endothelial growth factor (VEGF) and its angiogenic receptor VEGFR-2, as well as tumor necrosis factor-α (TNF-α) with its downstream signaling machinery promoting inflammation and cell death, were found in acute-on-chronic ischemia. In chronic critical ischemia, gene expression changes were much less striking than in acute-on-chronic ischemia, with 74 genes significantly upregulated and 34 genes downregulated (change in 1.3% of all genes). In the chronic situation, the anabolic and survival factors, insulin-like growth factor-1 (IGF-1) and IGF-2, were upregulated in atrophic and regenerating myocytes together with attenuated HIF, VEGF, and VEGFR-2 expression in the same cells. In conclusion, acute-on-chronic and chronic human skeletal muscle ischemia result in distinct gene expression patterns. These findings may be of importance in the design of novel therapies, such as therapeutic vascular growth, for patients suffering from lower limb ischemia. [Copyright &y& Elsevier]