Baculovirus-mediated vascular endothelial growth factor-D(ΔNΔC) gene transfer induces angiogenesis in rabbit skeletal muscle.

BACKGROUND: 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. METHODS: 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. RESULTS: 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 ß-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. CONCLUSIONS: 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.

Capillary enlargement, not sprouting angiogenesis, determines beneficial therapeutic effects and side effects of angiogenic gene therapy.

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 (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. 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.

Vascular endothelial growth factor-A and platelet-derived growth factor-B combination gene therapy prolongs angiogenic effects via recruitment of interstitial mononuclear cells and paracrine effects rather than improved pericyte coverage of angiogenic vessels.

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.

Therapeutic angiogenesis with placental growth factor improves exercise tolerance of ischaemic rabbit hindlimbs.

AIMS: We investigated the effects of angiogenic gene therapy with adenoviral placental growth factor(131) (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. METHODS AND RESULTS: Hindlimb ischaemia was surgically induced in New Zealand White rabbits (n = 20) that a day later received intramuscular (i.m.) AdPlGF or AdLacZ (3 x 10(11)vp) gene transfer (GT). Corresponding GTs were also done in healthy non-ischaemic rabbits (n = 10). Muscle energy metabolism and skeletal muscle perfusion were studied non-invasively before GT and at 6 and 28 days using (31)P-magnetic resonance spectroscopy and contrast pulse sequence ultrasound, respectively. Oedema was quantified using modified Miles assay at sacrifice. AdPlGF increased perfusion 7.8-fold and improved aerobic capacity of ischaemic limbs 45% compared with AdLacZ controls (P < 0.05) at 6 days. In non-ischaemic limbs, strong angiogenic response to GT, including capillary arterialization and acute oedema, did not impair muscle energy metabolism. CONCLUSION: This study shows that proangiogenic gene therapy can significantly improve performance of ischaemic limbs and supports the concept of therapeutic angiogenesis for the treatment of patients with ischaemia.

Gamma-secretase inhibitor treatment promotes VEGF-A-driven blood vessel growth and vascular leakage but disrupts neovascular perfusion.

The Notch signaling pathway is essential for normal development due to its role in control of cell differentiation, proliferation and survival. It is also critically involved in tumorigenesis and cancer progression. A key enzyme in the activation of Notch signaling is the gamma-secretase protein complex and therefore, gamma-secretase inhibitors (GSIs)--originally developed for Alzheimer's disease--are now being evaluated in clinical trials for human malignancies. It is also clear that Notch plays an important role in angiogenesis driven by Vascular Endothelial Growth Factor A (VEGF-A)--a process instrumental for tumor growth and metastasis. The effect of GSIs on tumor vasculature has not been conclusively determined. Here we report that Compound X (CX), a GSI previously reported to potently inhibit Notch signaling in vitro and in vivo, promotes angiogenic sprouting in vitro and during developmental angiogenesis in mice. Furthermore, CX treatment suppresses tumor growth in a mouse model of renal carcinoma, leads to the formation of abnormal vessels and an increased tumor vascular density. Using a rabbit model of VEGF-A-driven angiogenesis in skeletal muscle, we demonstrate that CX treatment promotes abnormal blood vessel growth characterized by vessel occlusion, disrupted blood flow, and increased vascular leakage. Based on these findings, we propose a model for how GSIs and other Notch inhibitors disrupt tumor blood vessel perfusion, which might be useful for understanding this new class of anti-cancer agents.

New aspects in vascular gene therapy.

Advances in clinical gene therapy have been modest although significant progress has been made during the past few years. New viruses have been introduced and new results have been collected from preclinical and clinical studies. This review will focus on cardiovascular and especially proangiogenic gene therapy. Recent results from preclinical developments and clinical trials will be discussed.

The effects of VEGF-R1 and VEGF-R2 ligands on angiogenic responses and left ventricular function in mice.

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-B(186), VEGF-R2-specific ligand VEGF-E and VEGF-A(165,) 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-B(186,) VEGF-E, and VEGF-A(165) 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-A(165) induced a strong angiogenic response seen as an enlargement of myocardial capillaries whereas angiogenesis induced by AdVEGF-B(186) 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-A(165) and AdVEGF-E induced endothelial-specific proliferation whereas AdVEGF-B(186) mostly induced proliferation of cardiomyocytes. AdVEGF-A(165) induced more pronounced tissue damage than AdVEGF-B(186) and AdVEGF-E. Left ventricular function measured as ejection fraction did not change during the follow-up. AdVEGF-A(165) increased both VEGF-R1 and VEGF-R2 protein expression whereas AdVEGF-B(186) and AdVEGF-E did not affect endogenous receptor expression levels. CONCLUSION: AdVEGF-B(186) and AdVEGF-E are equally potent in inducing therapeutic angiogenesis in mouse myocardium and produce less side effects than AdVEGF-A(165).

High-resolution ultrasound perfusion imaging of therapeutic angiogenesis.

OBJECTIVES: The purpose of this study was to test the feasibility of contrast pulse sequence (CPS) ultrasound imaging for high-resolution perfusion imaging after gene transfer (GT) for therapeutic angiogenesis. BACKGROUND: Imaging modalities capable of accurate and feasible perfusion measurement are essential for the preclinical and clinical development of therapeutic angiogenesis. However, current methods suffer from compromises between spatial and temporal resolution and sensitivity. Contrast pulse sequence ultrasound is a recently developed real-time perfusion imaging method that generates high-contrast agent-to-tissue specificity and spatial resolution. METHODS: Contrast pulse sequence ultrasound was used to noninvasively assess parameters of blood flow 6 days after adenoviral vascular endothelial growth factor (AdVEGF) GT in rabbit and mouse hind limbs with bolus intravenous injection of a microbubble contrast medium. Blood volume, mean transit time, perfusion, and time to the arrival of the contrast bolus were calculated with the gamma variate function. Contrast-enhanced power Doppler ultrasound (CEU), dynamic contrast-enhanced (DCE) magnetic resonance imaging (MRI), and histological capillary measurements were used as reference methods. RESULTS: Blood volume and perfusion increased over 40- and 20-fold, respectively, 6 days after AdVEGF GT in rabbit skeletal muscles. Perfusion values measured with CPS correlated well with those obtained with CEU (r = 0.975) and DCE-MRI (r = 0.854). However, CPS provided superior spatial and temporal resolution showing blood flow in vessels of only 10 to 20 mum in diameter. Contrast pulse sequence ultrasound was also feasible for imaging of therapeutic angiogenesis in mouse hind limbs both at the arterial and capillary levels. The CPS ultrasound revealed that AdVEGF mainly induces angiogenesis in adipose tissue rather than in the skeletal muscle of mouse hind limbs. CONCLUSIONS: Contrast pulse sequence ultrasound is an efficient and accurate noninvasive real-time perfusion imaging modality in small laboratory animals and also offers a means for the assessment of muscle perfusion in future clinical trials of therapeutic angiogenesis.