Johanna E. Markkanen

VEGF-D Is the Strongest Angiogenic and Lymphangiogenic Effector Among VEGFs Delivered Into Skeletal Muscle via Adenoviruses

Abstract— 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 (&Dgr;N&Dgr;C) VEGF-D. Adenoviruses expressing potent VEGFR-2 ligands, VEGF-A and VEGF-D&Dgr;N&Dgr;C, 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-D&Dgr;N&Dgr;C 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-D&Dgr;N&Dgr;C–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-D&Dgr;N&Dgr;C. 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.

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 (2×1011 and 2×1012 viral particles) or naked plasmids (1 mg) encoding Lac Z control, VEGF-A165, or the mature, soluble form of VEGF-D (VEGF-D&Dgr;N&Dgr;C) were injected intramyocardially with the NOGA catheter system into domestic pigs. AdVEGF-D&Dgr;N&Dgr;C 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 &agr;-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&Dgr;N&Dgr;C 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&Dgr;N&Dgr;C GT using the NOGA system produces efficient transmural angiogenesis and increases myocardial perfusion. AdVEGF-D&Dgr;N&Dgr;C could be useful for the induction of therapeutic vascular growth in the heart.

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.

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.

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.

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

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.

15-Lipoxygenase-1 Prevents Vascular Endothelial Growth Factor A– and Placental Growth Factor–Induced Angiogenic Effects in Rabbit Skeletal Muscles via Reduction in Growth Factor mRNA Levels, NO Bioactivity, and Downregulation of VEGF Receptor 2 Expression

Human 15-lipoxygenase-1 (15-LO-1) is an oxidizing enzyme capable of producing reactive lipid hydroperoxides. 15-LO-1 and its products have been suggested to be involved in many pathological conditions, such as inflammation, atherogenesis, and carcinogenesis. We used adenovirus-mediated gene transfers to study the effects of 15-LO-1 on vascular endothelial growth factor (VEGF)-A165– and placental growth factor (PlGF)-induced angiogenesis in rabbit skeletal muscles. 15-LO-1 significantly decreased all angiogenic effects induced by these growth factors, including capillary perfusion, vascular permeability, vasodilatation, and an increase in capillary number. The effects are attributable to the reduction in the amount of VEGF-A165 and PlGF transcripts by 15-LO-1, resulting in reduced protein expression. The most likely mediator of the VEGF family–induced capillary vasodilatation is nitric oxide (NO), which is produced by NO synthases. Endothelial NO synthase protein expression and NO synthase activity were significantly induced by VEGF-A165, and these inductions were reduced by 15-LO-1. VEGF-A165 induces its angiogenic effects primarily via vascular endothelial growth factor receptor (VEGFR)2, and also PlGF mediates angiogenic signaling via VEGFR2, even though it binds to VEGFR1. VEGFR2 expression is induced by peroxisome proliferator-activating receptor γ. We showed by quantitative RT-PCR and immunohistochemistry that expression of endogenous rabbit peroxisome proliferator-activating receptor γ and VEGFR2 were significantly increased in the growth factor–transduced muscles, but these inductions were efficiently prevented by 15-LO-1. In conclusion, the results suggest that expression of 15-LO-1 has an efficient antiangiogenic effect in vivo via reduction in growth factor mRNA levels, NO bioactivity, and VEGFR2 expression.

Distinct Architecture of Lymphatic Vessels Induced by Chimeric Vascular Endothelial Growth Factor-C/Vascular Endothelial Growth Factor Heparin-Binding Domain Fusion Proteins

Vascular endothelial growth factor (VEGF)-C and VEGF-D are composed of the receptor-binding VEGF homology domain and a carboxy-terminal silk homology domain that requires proteolytic cleavage for growth factor activation. Here, we explored whether the C-terminal heparin-binding domain of the VEGF165 or VEGF189 isoform also containing neuropilin-binding sequences could substitute for the silk homology domain of VEGF-C. Such VEGF-C/VEGF–heparin-binding domain chimeras were produced and shown to activate VEGF-C receptors, and, when expressed in tissues via adenovirus or adeno-associated virus vectors, stimulated lymphangiogenesis in vivo. However, both chimeras induced a distinctly different pattern of lymphatic vessels when compared with VEGF-C. Whereas VEGF-C–induced vessels were initially a dense network of small diameter vessels, the lymphatic vessels induced by the chimeric growth factors tended to form directly along tissue borders, along basement membranes that are rich in heparan sulfate. For example, in skeletal muscle, the chimeras induced formation of lumenized lymphatic vessels more efficiently than wild-type VEGF-C. We conclude that the matrix-binding domain of VEGF can target VEGF-C activity to heparin-rich basement membrane structures. These properties may prove useful for tissue engineering and attempts to regenerate lymphatic vessels in lymphedema patients.

Gene Therapy for Restenosis Current Status

Atherosclerosis is a major cause of morbidity and mortality in Western world. Vascular occlusion caused by atherosclerosis usually requires invasive treatment, such as surgical bypass or angioplasty However, bypass graft failure and restenosis limit the usefulness of these procedures, with 20% of patients needing a new revascularisation procedure within 6 months of angioplasty. Numerous pharmacological agents have been investigated for the prevention of restenosis but none has shown undisputed efficacy in clinical medicine.Gene transfer offers a novel approach to the treatment of restenosis because of easy accessibility of vessels and already existing gene delivery methods. It can be used to overexpress therapeutically important proteins locally without high systemic toxicity, and the therapeutic effect can be targeted to a particular pathophysiological event. Promising results have been obtained from many pre-clinical experiments using therapeutic genes or oligonucleotides to prevent restenosis. Early clinical trials have shown that plasmid- and adenovirus-mediated vascular gene transfers can be conducted safely and are well tolerated. Ex vivo gene therapy with E2F-decoy succeeded in reducing graft occlusion rate after surgical bypass in a randomised, double-blind clinical trial.In the future, further development of gene delivery methods and vectors is needed to improve the efficacy and safety of gene therapy. Also, better knowledge of vascular biology at the molecular level is needed to find optimal strategies and gene combinations to treat restenosis. Provided that these difficulties can be solved, gene therapy offers an enormous potential for clinical medicine in the future.

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-DΔNΔC) 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-DΔNΔC, the cysteines at the subunit interface were mutated to study the effect of these residues on the structural and functional properties of VEGF-DΔNΔC. The conserved cysteines Cys-44 and Cys-53 were found to be essential for the function of VEGF-DΔNΔC. More importantly, the substitution of the Cys-25 at the dimer interface by various amino acids improved the activity of the recombinant VEGF-DΔNΔC 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-DΔNΔC. 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-DΔNΔC described here are potential agents for therapeutic applications, where induction of vascular formation is required.