Novel vascular endothelial growth factor D variants with increased biological activity.

Members of the vascular endothelial growth factor (VEGF) family play a pivotal role in angiogenesis and lymphangiogenesis. They are potential therapeutics to induce blood vessel formation in myocardium and skeletal muscle, when normal blood flow is compromised. Most members of the VEGF/platelet derived growth factor protein superfamily exist as covalently bound antiparallel dimers. However, the mature form of VEGF-D (VEGF-D(DeltaNDeltaC)) is predominantly a non-covalent dimer even though the cysteine residues (Cys-44 and Cys-53) forming the intersubunit disulfide bridges in the other members of the VEGF family are also conserved in VEGF-D. Moreover, VEGF-D bears an additional cysteine residue (Cys-25) at the subunit interface. Guided by our model of VEGF-D(DeltaNDeltaC), the cysteines at the subunit interface were mutated to study the effect of these residues on the structural and functional properties of VEGF-D(DeltaNDeltaC). The conserved cysteines Cys-44 and Cys-53 were found to be essential for the function of VEGF-D(DeltaNDeltaC). More importantly, the substitution of the Cys-25 at the dimer interface by various amino acids improved the activity of the recombinant VEGF-D(DeltaNDeltaC) and increased the dimer to monomer ratio. Specifically, substitutions to hydrophobic amino acids Ile, Leu, and Val, equivalent to those found in other VEGFs, most favorably affected the activity of the recombinant VEGF-D(DeltaNDeltaC). The increased activity of these mutants was mainly due to stabilization of the protein. This study enables us to better understand the structural determinants controlling the biological activity of VEGF-D. The novel variants of VEGF-D(DeltaNDeltaC) described here are potential agents for therapeutic applications, where induction of vascular formation is required.

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.

Blood flow remodels growing vasculature during vascular endothelial growth factor gene therapy and determines between capillary arterialization and sprouting angiogenesis.

BACKGROUND: For clinically relevant proangiogenic therapy, it would be essential that the growth of the whole vascular tree is promoted. Vascular endothelial growth factor (VEGF) is well known to induce angiogenesis, but its capability to promote growth of larger vessels is controversial. We hypothesized that blood flow remodels vascular growth during VEGF gene therapy and may contribute to the growth of large vessels. METHODS AND RESULTS: Adenoviral (Ad) VEGF or LacZ control gene transfer was performed in rabbit hindlimb semimembranous muscles with or without ligation of the profound femoral artery (PFA). Contrast-enhanced ultrasound and dynamic susceptibility contrast MRI demonstrated dramatic 23- to 27-fold increases in perfusion index and a strong decrease in peripheral resistance 6 days after AdVEGF gene transfer in normal muscles. Enlargement by 20-fold, increased pericyte coverage, and decreased alkaline phosphatase and dipeptidyl peptidase IV activities suggested the transformation of capillaries toward an arterial phenotype. Increase in muscle perfusion was attenuated, and blood vessel growth was more variable, showing more sprouting angiogenesis and formation of blood lacunae after AdVEGF gene transfer in muscles with ligated PFA than in normal muscles. Three-dimensional ultrasound reconstructions and histology showed that the whole vascular tree, including large arteries and veins, was enlarged manifold by AdVEGF. Blood flow was normalized and enlarged collaterals persisted in operated limbs 14 days after AdVEGF treatment. CONCLUSIONS: This study shows that (1) blood flow modulates vessel growth during VEGF gene therapy and (2) VEGF overexpression promotes growth of arteries and veins and induces capillary arterialization leading to supraphysiological blood flow in target muscles.

Growth factor-induced therapeutic angiogenesis and arteriogenesis in the heart--gene therapy.

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.

Gene therapy for ischemic cardiovascular diseases: some lessons learned from the first clinical trials.

Stimulation of angiogenesis, arteriogenesis, and lymphangiogenesis (i.e., therapeutic vascular growth) is a new concept for the treatment of ischemic cardiovascular diseases. A wealth of information is already available about the mechanisms and mediators of angiogenesis and arteriogenesis, which has led to the first randomized, controlled, phase II/III trials with recombinant growth factors or their genes. Even though end points predefined in the study protocols have been positive in several trials, it is still evident that the trials have not produced any clearly meaningful clinical benefits for the patients. This review addresses same questions and concepts related to the gene therapy-based applications of therapeutic vascular growth.

Gene transfer into rabbit arteries with adeno-associated virus and adenovirus vectors.

BACKGROUND: Gene transfer offers considerable potential for altering vessel wall physiology and intervention in vascular disease. Therefore, there is great interest in developing optimal strategies and vectors for efficient, targeted gene delivery into a vessel wall. METHODS: We studied adeno-associated viruses (AAV; 9 x 10(8) to 4 x 10(9) TU/ml) for their usefulness to transduce rabbit arteries in vivo in comparison with adenoviruses (Adv; 1 x 10(9) to 1 x 10(10) pfu/ml). 100 microl of viruses or placebo solution were injected intraluminally into transiently isolated carotid segments. RESULTS: In normal arteries AAV transduced mainly medial smooth muscle cells (SMC) while Adv transduced exclusively endothelial cells (EC). Mechanical injury to EC layer and internal elastic lamina enabled Adv to penetrate and transduce medial SMC. Transgene expression in EC after the AAV-mediated gene transfer was very low. The use of the EC-specific Tie-1 promoter did not lead to specific transgene expression in EC. Transgene expression in SMC persisted for at least 100 days after the AAV treatment whereas the Adv-mediated effect diminished in 14 days. AAV caused only a modest increase in EC VCAM-1 expression and proliferation rate of vascular cells as compared with the mock-treated arteries while Adv caused an extensive inflammatory cell infiltration, VCAM-1 expression, vascular cell proliferation and morphological damages. CONCLUSIONS: Significant differences were observed between the AAV and the Adv vectors in their patterns of arterial transduction and consequent inflammatory responses. These distinct properties may be utilized for different applications in vascular biology research and gene therapy for cardiovascular diseases.

Adenoviral catheter-mediated intramyocardial gene transfer using the mature form of vascular endothelial growth factor-D induces transmural angiogenesis in porcine heart.

BACKGROUND: It is unclear what is the most efficient vector and growth factor for induction of therapeutic vascular growth in the heart. Furthermore, the histological nature of angiogenesis and potential side effects caused by different vascular endothelial growth factors (VEGFs) in myocardium have not been documented. METHODS AND RESULTS: Adenoviruses (Ad) at 2 doses (2x10(11) and 2x10(12) viral particles) or naked plasmids (1 mg) encoding LacZ control, VEGF-A165, or the mature, soluble form of VEGF-D (VEGF-D(DeltaNDeltaC)) were injected intramyocardially with the NOGA catheter system into domestic pigs. AdVEGF-D(DeltaNDeltaC) gene transfer (GT) induced a dose-dependent myocardial protein production, as measured by ELISA, resulting in an efficient angiogenic effect 6 days after the injections. Also, AdVEGF-A165 produced high gene transfer efficacy, as demonstrated with immunohistochemistry, leading to prominent angiogenesis effects. Despite the catheter-mediated approach, angiogenesis induced by both AdVEGFs was transmural, with maximal effects in the epicardium. Histologically, strongly enlarged alpha-smooth muscle actin-positive microvessels involving abundant cell proliferation were found in the transduced regions, whereas microvessel density did not change. Myocardial contrast echocardiography and microspheres showed marked increases in perfusion in the transduced areas. VEGF-D(DeltaNDeltaC) but not matrix-bound VEGF-A165 was detected in plasma after adenoviral GT. A modified Miles assay demonstrated myocardial edema resulting in pericardial effusion with the higher AdVEGF doses. All effects returned to baseline by 3 weeks. Naked plasmid-mediated GT did not induce detectable protein production or vascular effects. CONCLUSIONS: Like AdVEGF-A165, AdVEGF-D(DeltaNDeltaC) GT using the NOGA system produces efficient transmural angiogenesis and increases myocardial perfusion. AdVEGF-D(DeltaNDeltaC) could be useful for the induction of therapeutic vascular growth in the heart.

VEGF-D is the strongest angiogenic and lymphangiogenic effector among VEGFs delivered into skeletal muscle via adenoviruses.

Optimal angiogenic and lymphangiogenic gene therapy requires knowledge of the best growth factors for each purpose. We studied the therapeutic potential of human vascular endothelial growth factor (VEGF) family members VEGF-A, VEGF-B, VEGF-C, and VEGF-D as well as a VEGFR-3-specific mutant (VEGF-C156S) using adenoviral gene transfer in rabbit hindlimb skeletal muscle. The significance of proteolytic processing of VEGF-D was explored using adenoviruses encoding either full-length or mature (DeltaNDeltaC) VEGF-D. Adenoviruses expressing potent VEGFR-2 ligands, VEGF-A and VEGF-DDeltaNDeltaC, induced the strongest angiogenesis and vascular permeability effects as assessed by capillary vessel and perfusion measurements, modified Miles assay, and MRI. The most significant feature of angiogenesis induced by both VEGF-A and VEGF-DDeltaNDeltaC was a remarkable enlargement of microvessels with efficient recruitment of pericytes suggesting formation of arterioles or venules. VEGF-A also moderately increased capillary density and created glomeruloid bodies, clusters of tortuous vessels, whereas VEGF-DDeltaNDeltaC-induced angiogenesis was more diffuse. Vascular smooth muscle cell proliferation occurred in regions with increased plasma protein extravasation, indicating that arteriogenesis may be promoted by VEGF-A and VEGF-DDeltaNDeltaC. Full-length VEGF-C and VEGF-D induced predominantly and the selective VEGFR-3 ligand VEGF-C156S exclusively lymphangiogenesis. Unlike angiogenesis, lymphangiogenesis was not dependent on nitric oxide. The VEGFR-1 ligand VEGF-B did not promote either angiogenesis or lymphangiogenesis. Finally, we found a positive correlation between capillary size and vascular permeability. This study compares, for the first time, angiogenesis and lymphangiogenesis induced by gene transfer of different human VEGFs, and shows that VEGF-D is the most potent member when delivered via an adenoviral vector into skeletal muscle.

Fibroblast growth factor 4 induces vascular permeability, angiogenesis and arteriogenesis in a rabbit hindlimb ischemia model.

Previous studies have shown that fibroblast growth factor (FGF)-1, FGF-2, and FGF-5 induce therapeutic angiogenesis. Here, we investigated the potential of FGF-4 for therapeutic neovascularization in comparison to vascular endothelial growth factor (VEGF), using adenoviral gene transfer in a novel rabbit hind limb ischemia model, with ischemia restricted to the calf. Magnetic resonance imaging and a modified Miles assay showed that both AdFGF-4 and AdVEGF given intramuscularly (i.m.) resulted in increases in vascular permeability and edema in transduced muscles 6 days after the gene transfer. In contrast, recombinant FGF-4 protein injected in the rabbit skin did not induce acute vascular permeability. Injections (i.m.) of AdFGF-4 and AdVEGF, but not intra-arterially administered AdVEGF, increased collateral growth, popliteal blood flow, and muscle perfusion compared with controls. The angiogenesis response consisted mainly of the enlargement of pre-existing vessels rather than an increase in capillary density. Adenoviral FGF-4 overexpression up-regulated endogenous VEGF, which may explain many of the effects thought to be specific for VEGF such as the increase in vascular permeability. This study demonstrates for the first time that FGF-4 induces vascular permeability, therapeutic angiogenesis, and arteriogenesis comparable to that of VEGF and could be useful for the treatment of peripheral vascular disease.

Expression of vascular endothelial growth factor and vascular endothelial growth factor receptor-2 (KDR/Flk-1) in ischemic skeletal muscle and its regeneration.

Vascular endothelial growth factor (VEGF) is a hypoxia-inducible endothelial cell mitogen and survival factor. Its receptor VEGFR-2 (KDR/Flk-1) mediates these effects. We studied the expression of VEGF and VEGFR-2 in ischemic human and rabbit skeletal muscle by immunohistochemistry and in situ hybridization. Human samples were obtained from eight lower limb amputations because of acute or chronic critical ischemia. In chronically ischemic human skeletal muscle VEGF and VEGFR-2 expression was restricted to atrophic and regenerating skeletal myocytes, whereas in acutely ischemic limbs VEGF and VEGFR-2 were expressed diffusely in the affected muscle. Hypoxia-inducible factor-1alpha was associated with VEGF and VEGFR-2 expression both in acute and chronic ischemia but not in regeneration. Hindlimb ischemia was induced in 20 New Zealand White rabbits by excising the femoral artery. Magnetic resonance imaging and histological sections revealed extensive ischemic damage in the thigh and leg muscles of ischemic rabbit hindlimbs with VEGF expression similar to acute human lower limb ischemia. After 1 and 3 weeks of ischemia VEGF expression was restricted to regenerating myotubes and by 6 weeks regeneration and expression of VEGF was diminished. VEGFR-2 expression was co-localized with VEGF expression in regenerating myotubes. Macrophages and an increased number of capillaries were associated with areas of ischemic muscle expressing VEGF and VEGFR-2. In conclusion, two patterns of VEGF and VEGFR-2 expression in human and rabbit ischemic skeletal muscle are demonstrated. In acute skeletal muscle ischemia VEGF and VEGFR-2 are expressed diffusely in the affected muscle. In chronic skeletal muscle ischemia and in skeletal muscle recovering from ischemia VEGF and VEGFR-2 expression are restricted to atrophic and regenerating muscle cells suggesting the operation of an autocrine pathway that may promote survival and regeneration of myocytes.