Sustained capillary enlargement induced by angiogenic gene therapy does not support post-ischemic muscle recovery of hyperlipidemic mice

Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Finish Academy (Clinical Researcher 324070 and Research Project 339560). Finnish Foundation for Cardiovascular Research Background Transient capillary enlargement has been shown to be essential for successful post-ischemic tissue recovery as it may facilitate the return of arterial driving pressure by supporting collateral maturation. It also leads to an intussusceptive increase in capillary density that supports efficient oxygen delivery and muscle regeneration. Hyperlipidemia has been shown to blunt post-ischemic capillary reactivity and hinder skeletal muscle recovery from ischemia. Therefore, it can be hypothesized that correction of capillary reactivity might improve post-ischemic muscle recovery under hyperlipidemia. Purpose The aim of this study was to test the ability of angiogenic VEGF gene therapy in improving post-ischemic muscle recovery under hyperlipidemia. Methods AdVEGF gene transfer was performed into the calf muscles of aged, hyperlipidemic LDLR-/-ApoB100/100 mice after acute ischemia induction. The effects of AdVEGF on capillary phenotype, tissue edema, restoration of blood flow parameters, microvascular hemoglobin oxygenation and tissue damage/regeneration were evaluated for up to 29 days post-procedure as compared to AdLacZ control using immuno- and histological analyses, magnetic resonance, contrast-enhanced ultrasound and photoacoustic imaging, respectively. Results AdVEGF gene therapy was able to promote capillary enlargement that led to recovery of arterial driving pressure in ischemic LDLR-/-ApoB100/100 muscles. However, capillary enlargement induced by AdVEGF appeared late, lasted up to 29 days and did not promote intussusception. Consequently, the negative effect of long-lasting enlargement, i.e., massive tissue edema, delayed blood flow recovery and aggravated ischemic tissue damage. Conclusion Proper time of induction and duration of capillary enlargement are crucial to yield a functional vascular response supporting post-ischemic tissue recovery.

Intramuscular vascular endothelial growth factor gene therapy: fact or fiction?

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. Adenoviral vascular endothelial growth factor-A or AdLacZ control was injected intramuscularly (10(9)-10(11) vp/mL) or intra-arterially (5 × 10(11) 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. 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.

Introduction: Angiogenic gene therapy has shown potential for treatment of peripheral ischemic diseases in animal models but unfortunately clinical trials have failed to show consistent improvements in patients. We hypothesized that the differential outcomes of animal and clinical trials could be explained by pathological differences between animal models and human disease. Methods: Chronic ischemia was developed by causing endothelial denutation via balloon catheters to limb arteries of New Zealand White rabbits (n=89) on a 1% cholesterol diet. Development of arterial narrowing and signs of ischemia were monitored weekly after the operation using ultrasound imaging, angiography and muscle force measurements. Adenoviral vascular endothelial growth factor (VEGF) or beeta-galactosidase control gene therapy was applied intramuscularly with the total dose of 3x10e11vp in 3ml of saline once muscles started showing signs of ischemia (at 2-6 weeks). The effects of gene therapy were followed 1, 2 and 4 weeks after gene transfers. Results: Atherosclerosis-resembling narrowing of the femoral artery was detected starting 2 weeks after denutation leading to chronically decreasing muscle function even in the presence of collateral formation and at least partial restoration of muscle blood flow. VEGF gene transfer induced capillary enlargement and improved muscle blood flow but at the same time increased muscle necrosis and worsened muscle function. Conclusions: Angiogenic gene therapy may not be the optimal treatment in chronic ischemia if muscle blood flow has already been restored by collateral circulation. The analysis of muscle capillary flow may help identify patients with chronic ischemia that could benefit from angiogenic therapies.

Bronchopulmonary dysplasia (BPD) and pulmonary emphysema, both significant global health problems, are characterized by a loss of alveoli. Vascular endothelial growth factor (VEGF) is a trophic factor required for endothelial cell survival and is abundantly expressed in the lung. We report that VEGF blockade decreases lung VEGF and VEGF receptor 2 (VEGFR-2) expression in newborn rats and impairs alveolar development, leading to alveolar simplification and loss of lung capillaries, mimicking BPD. In hyperoxia-induced BPD in newborn rats, air space enlargement and loss of lung capillaries are associated with decreased lung VEGF and VEGFR-2 expression. Postnatal intratracheal adenovirus-mediated VEGF gene therapy improves survival, promotes lung capillary formation, and preserves alveolar development in this model of irreversible lung injury. Combined VEGF and angiopoietin-1 gene transfer matures the new vasculature, reducing the vascular leakage seen in VEGF-induced capillaries. These findings underscore the importance of the vasculature in what is traditionally thought of as an airway disease and open new therapeutic avenues for lung diseases characterized by irreversible loss of alveoli through the modulation of angiogenic growth factors.

Vessel stabilization and the inhibition of side effects such as tissue edema are essential in angiogenic gene therapy. Thus, combination gene transfers stimulating both endothelial cell and pericyte proliferation have become of interest. However, there is currently little data to support combination gene transfer in large animal models. In this study, we evaluated the potential advantages of such a strategy by combining the transfer of adenoviral (Ad) vascular endothelial growth factor (VEGF)-A and platelet-derived growth factor (PDGF)-B into rabbit hindlimb skeletal muscle. AdLacZ alone or in combination with AdVEGF-A were used as controls. Contrast-enhanced ultrasound, modified Miles assay, and immunohistology were used to quantify perfusion, vascular permeability, and capillary size, respectively. Confocal microscopy was used in the assessment of pericyte-coverage. The transfer of AdPDGF-B alone and in combination with AdVEGF-A induced prominent proliferation of alpha-smooth muscle actin-, CD31-, RAM11-, HAM56-, and VEGF- positive cells. Although, pericyte recruitment to angiogenic vessels was not improved, combination gene transfer induced a longer-lasting increase in perfusion in both intact and ischemic muscles than AdVEGF-A gene transfer alone. In conclusion, intramuscular delivery of AdVEGF-A and AdPDGF-B, combined, resulted in a prolonged angiogenic response. However, the effects were most likely mediated via paracrine mechanisms rather than an increase in vascular pericyte coverage.

Post-translational regulation of a hypoxia-responsive VEGF plasmid for the treatment of myocardial ischemia

Approximately 15 million patients suffer from coronary and peripheral atherosclerotic diseases in the United States alone.1 The evolving development of medical and surgical therapies has significantly improved the physician’s ability to manage these patients, yet many continue to suffer debilitating symptoms from their disease and remain at risk for myocardial infarction, limb loss, and death. This clinical imperative, coupled with rapid advances in molecular biology, has led to the exploration of a plethora of therapeutic angiogenesis strategies. A method described in this issue of Circulation 2 offers yet another means by which angiogenesis may be achieved and thus potentially opens a new chapter in the ongoing search for novel approaches for the treatment of ischemia. In placing the novelty of the approach by Kanno et al into perspective, it is worth considering the important advances made during the last decade in the field of therapeutic angiogenesis. The investigators at the forefront of this field have not used a traditional pharmaceutical approach to search for potential angiogenic agents effective against vascular disease. Instead, they have chosen to arm themselves with the fruits of the molecular biology revolution by harnessing the power of endogenous human angiogenic factors. Vascular endothelial growth factor (VEGF) is the most potent and specific endogenous angiogenic factor yet identified, so it makes sense that it would have drawn the attention of cardiologists interested in angiogenic therapies. The demonstration that intra-arterial injection of recombinant VEGF could induce collateral formation in ischemic rabbit hindlimbs3 suggested that VEGF would be a useful angiogenic agent, and this has subsequently been confirmed by an impressive corpus of preclinical data. Phase I and II clinical trials are now under way to test the effects of VEGF in patients with ischemic vascular disease, and preliminary results from some of these trials have been …

Current Status of Cardiovascular Gene Therapy

Occluded arteries and ischemic tissues cannot always be treated by angioplasty, stenting or by-pass-surgery. Under such circumstances, viral gene therapy may be useful in inducing increased blood supply to ischemic area. There is evidence of improved blood flow in ischemic skeletal muscle and myocardium in both animal and human studies using adenoviral vascular endothelial growth factor (VEGF) gene therapy. However, the expression is transient and repeated gene transfers with the same vector are inefficient due to immune responses. Different baculoviral vectors pseudotyped with or without vesicular stomatitis virus glycoprotein (VSV-G) and/or carrying woodchuck hepatitis virus post-transcriptional regulatory element (Wpre) were tested both in vitro and in vivo. VEGF-D(ΔNΔC) was used as therapeutic transgene and lacZ as a control. In vivo efficacy was evaluated as capillary enlargement and transgene expression in New Zealand White (NZW) rabbit skeletal muscle. A statistically significant capillary enlargement was detected 6 days after gene transfer in transduced areas compared to the control gene transfers with baculovirus and adenovirus encoding β-galactosidase (lacZ). Substantially improved gene transfer efficiency was achieved with a modified baculovirus pseudotyped with VSV-G and carrying Wpre. Dose escalation experiments revealed that either too large volume or too many virus particles caused inflammation and necrosis in the target tissue, whereas 10(9) plaque forming units injected in multiple aliquots resulted in transgene expression with only mild immune reactions. We show the first evidence of biologically significant baculoviral gene transfer in skeletal muscle of NZW rabbits using VEGF-D(ΔNΔC) as a therapeutic transgene.

Objective To study the changes of apoptosis in brain after traumatic injury(TBI)treated with exogenous vascular endothelial growth factor(VEGF)gene therapy in oder to find out the role of exogenous VEGF gene in protectiog brain tissue.Method The injurea cerebral cortex from the rat models of brain with traumatic injury was injected with adenovirus(adenovirus,Ad)as the carrier of VEGF-165 gene(Ad-VEGF-165 Gene).RT-PCR and Western blot were used to detect the expression of VEGF mRNA and VEGF protein in the brain 6 h,24 h,3 d,7 d and 14 d after injury,and apoptosis in the injured location of brain was also detected by TUNEL successively after Ad-VECF-165 gene therapy.Results With exogenous Ad-VEGF-165 applied to the locally injured brain tissue after injury,VEGF mRNA and VEGF protein showed consistent expression and their expressions were significantly higher than those in trauma group and vehicle control group.Compared with the trauma group.the apoptosis in the gene therapy group 24 h,3 d and 7 d after injury presented a.significant reduction,and had close relationship with VEGF.(Control group:4.17±0.73;TBI group,24 h:47.18±6.01,3 d:79.44±11.23;TBI+VEGF group.24 h:28.72±5.31,3 d:54.18±7.66;P＜0.05).Conclusions The exogenous VEGF gene therapy administered to have protective effects on the local brain tissue in rats with traumatic injury in a certain time. Key words: Vascular endthelial growth factor(VEGF); Traumatic brain injury(TBI); Apoptosis; Gene therapy

Recent studies have emphasized the beneficial effects of the vascular endothelial growth factor (VEGF) on neurone survival and Schwann cell proliferation. VEGF is a potent angiogenic factor, and angiogenesis has long been recognized as an important and necessary step during tissue repair. Here, we investigated the effects of VEGF on sciatic nerve regeneration. Using light and electron microscopy, we evaluated sciatic nerve regeneration after transection and VEGF gene therapy. We examined the survival of the neurones in the dorsal root ganglia and in lumbar 4 segment of spinal cord. We also evaluated the functional recovery using the sciatic functional index and gastrocnemius muscle weight. In addition, we evaluated the VEGF expression by immunohistochemistry. Fluorescein isothiocyanate-dextran (FITC-dextran) fluorescence of nerves and muscles revealed intense staining in the VEGF-treated group. Quantitative analysis showed that the numbers of myelinated fibres and blood vessels were significantly higher in VEGF-treated animals. VEGF also increased the survival of neurone cell bodies in dorsal root ganglia and in spinal cord. The sciatic functional index and gastrocnemius muscle weight reached significantly higher values in VEGF-treated animals. We demonstrate a positive relationship between increased vascularization and enhanced nerve regeneration, indicating that VEGF administration can support and enhance the growth of regenerating nerve fibres, probably through a combination of angiogenic, neurotrophic and neuroprotective effects.

Introduction: Growth factors have shown potential for inducing angiogenesis and enhancing survival in the flap tissue. Targeted gene therapy has been shown to increase the bioavailability of various growth factors. In this study, we examined the effectiveness of preoperative vascular endothelial growth factor (VEGF) plasmid DNA transfection on the survival of the skin paddle in a rat pedicled TRAM flap model. Methods: Fifty-two rats were used. In part one, VEGF plasmid DNA was injected into the subcutaneous fascial layer of the upper abdominal walls of the rats. On postoperative day 4, biopsies were taken for histological review and VEGF protein quantification. In part two, the rats were divided into three groups. In the experimental group, the VEGF plasmid DNA was injected into the subcutaneous fascial layer in the area where the TRAM flap would be elevated. There were two control groups: one with plasmid containing no VEGF DNA and one with saline. The flaps were raised and examined 4 days after injection. Results: Flap tissue receiving VEGF plasmid DNA revealed angiogenesis. The VEGF level was significantly higher than of the tissue without receiving VEGF plasmid DNA injection. The mean viable area of the skin paddles receiving pre-operative VEGF plasmid DNA injection was significantly larger than that of flap receiving no VEGF plasmid DNA and saline injection. Conclusions: This study demonstrated that preoperative subcutaneous injection of VEGF plasmid DNA induced angiogenesis and improved the TRAM skin paddle survival.

Vascular endothelial growth factor gene therapy with intramuscular injections of plasmid DNA enhances the survival of random pattern flaps in a rat model

In this issue of Circulation , Baumgartner et al1 ). The plasmid DNA was injected directly into the muscle of ischemic limbs. Anatomic and functional efficacy was demonstrated by increased serum levels of VEGF, improved hemodynamic measurements and angiographic evaluation, reduced pain, increased healing of ischemic ulcers, limb salvage, and immunohistochemical evidence of proliferating endothelial cells in tissue specimens. The authors emphasize that this is the first medical therapy to achieve an increase in limb perfusion that is equivalent to or greater than successful surgical or percutaneous intervention. Direct intramuscular gene transfer of plasmid DNA appears to effectively stimulate collateral vessel growth, despite the lower transfection efficiency that is usually associated with gene therapy in the absence of a viral vector. This result has implications for other clinical trials of gene therapy that use intramuscular naked DNA. The fact that Isner’s group could prepare the plasmid for human use in a university medical center laboratory dedicated to this purpose reveals why gene therapy has moved so rapidly from the laboratory to the clinic, in contrast to protein therapy, which requires expensive manufacturing facilities and years of scale-up effort. Although the plasmid was injected into muscle of the ischemic limb, VEGF levels were apparently elevated in the whole circulation, as evidenced by transient peaks of VEGF in the serum and by edema of the ischemic limb and in some patients, in the opposite limb. The increased collateral vessels, however, are localized to the ischemic limb and do not develop in other areas of the body. This may reflect in part the short half-life of VEGF in the circulation (minutes) as well as the upregulation of receptors for VEGF in ischemic tissue. …