Petra Korpisalo

Vascular Endothelial Growth Factor-B Induces Myocardium-Specific Angiogenesis and Arteriogenesis via Vascular Endothelial Growth Factor Receptor-1– and Neuropilin Receptor-1–Dependent Mechanisms

Background— New revascularization therapies are urgently needed for patients with severe coronary heart disease who lack conventional treatment options. Methods and Results— We describe a new proangiogenic approach for these no-option patients using adenoviral (Ad) intramyocardial vascular endothelial growth factor (VEGF)-B186 gene transfer, which induces myocardium-specific angiogenesis and arteriogenesis in pigs and rabbits. After acute infarction, AdVEGF-B186 increased blood vessel area, perfusion, ejection fraction, and collateral artery formation and induced changes toward an ischemia-resistant myocardial phenotype. Soluble VEGF receptor-1 and soluble neuropilin receptor-1 reduced the effects of AdVEGF-B186, whereas neither soluble VEGF receptor-2 nor inhibition of nitric oxide production had this result. The effects of AdVEGF-B186 involved activation of neuropilin receptor-1, which is highly expressed in the myocardium, via recruitment of G-protein-&agr; interacting protein, terminus C (GIPC) and upregulation of G-protein-&agr; interacting protein. AdVEGF-B186 also induced an antiapoptotic gene expression profile in cardiomyocytes and had metabolic effects by inducing expression of fatty acid transport protein-4 and lipid and glycogen accumulation in the myocardium. Conclusions— VEGF-B186 displayed strikingly distinct effects compared with other VEGFs. These effects may be mediated at least in part via a G-protein signaling pathway. Tissue-specificity, high efficiency in ischemic myocardium, and induction of arteriogenesis and antiapoptotic and metabolic effects make AdVEGF-B186 a promising candidate for the treatment of myocardial ischemia.

Stabilized HIF-1α is superior to VEGF for angiogenesis in skeletal muscle via adeno-associated virus gene transfer

Therapeutic angiogenesis provides a potential alternative for the treatment of cardiovascular ischemic diseases. Vascular endothelial growth factor (VEGF) is an important component of the angiogenic response to ischemia. Here we used adeno‐associated virus (AAV) gene delivery to skeletal muscle to examine the effects of VEGF vs. a stabilized form of hypoxia‐inducible factor‐1α (HIF‐1α). The recombinant AAVs were injected into mouse tibialis anterior muscle, and their effects were analyzed by immunohistochemistry and functional assays. These analyses showed that stabilized HIF‐1 α markedly increase capillary sprouting and proliferation, whereas VEGF164 or VEGF120 induced only proliferation of endothelial cells without formation of proper capillary structures. The Evans Blue permeability assay indicated that, unlike VEGF, HIF‐1 α overexpression did not increase vascular leakiness in the transduced muscle. Doppler ultrasound imaging showed that vascular perfusion in the HIF‐1 α treated muscles was significantly enhanced when compared to the controls and not further improved by co‐expression of the arteriogenic growth factors angiopoietin‐1 or platelet‐derived growth factor‐B. Our results show that AAV‐mediated transduction of a stabilized form of HIF‐1 α can circumvent the problems associated with overexpression of individual angiogenic growth factors. HIF‐1 α should thus offer a potent alternative for pro‐angiogenic gene therapy.

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.

Efficient Regulation of VEGF Expression by Promoter-Targeted Lentiviral shRNAs Based on Epigenetic Mechanism: A Novel Example of Epigenetherapy

Rationale: We studied a possibility that shRNAs can lead to transcriptional gene activation at the promoter level via epigenetic mechanism. Objective: The purpose of this study was to test the effects on vascular endothelial growth factor (VEGF-A) expression by promoter targeted small hairpin RNAs (shRNAs) in vitro and in experimental animals in vivo using stable local lentiviral gene transfer. Methods and Results: One shRNA was identified which strongly increased VEGF-A expression in C166 endothelial cells at mRNA and protein level whereas another shRNA decreased VEGF-A expression. Quantitative chromatin immunoprecipitation analysis revealed that the repressing shRNA caused epigenetic changes, which increased nucleosome density within the promoter and transcription start site and led to repression of VEGF-A expression. Epigenetic changes caused by the activating shRNA were opposite to those caused by the repressing shRNA. These results were confirmed in vivo in an ischemic mouse hindlimb model after local gene transfer where VEGF-A upregulation achieved by promoter-targeted shRNA increased vascularity and blood flow. Conclusions: We show that lentivirus-mediated delivery of shRNA molecules targeted to specific regions in the mVEGF-A promoter either induce or repress VEGF-A expression via epigenetic modulation. Thus, we describe a new approach of gene therapy, epigenetherapy, based on an epigenetic mechanism at the promoter level. Controlling transcription through manipulation of specific epigenetic marks provides a novel approach for the treatment of several diseases.

Vascular Endothelial Growth Factor-B Acts as a Coronary Growth Factor in Transgenic Rats Without Inducing Angiogenesis, Vascular Leak, or Inflammation

Background— Vascular endothelial growth factor-B (VEGF-B) binds to VEGF receptor-1 and neuropilin-1 and is abundantly expressed in the heart, skeletal muscle, and brown fat. The biological function of VEGF-B is incompletely understood. Methods and Results— Unlike placenta growth factor, which binds to the same receptors, adeno-associated viral delivery of VEGF-B to mouse skeletal or heart muscle induced very little angiogenesis, vascular permeability, or inflammation. As previously reported for the VEGF-B167 isoform, transgenic mice and rats expressing both isoforms of VEGF-B in the myocardium developed cardiac hypertrophy yet maintained systolic function. Deletion of the VEGF receptor-1 tyrosine kinase domain or the arterial endothelial Bmx tyrosine kinase inhibited hypertrophy, whereas loss of VEGF-B interaction with neuropilin-1 had no effect. Surprisingly, in rats, the heart-specific VEGF-B transgene induced impressive growth of the epicardial coronary vessels and their branches, with large arteries also seen deep inside the subendocardial myocardium. However, VEGF-B, unlike other VEGF family members, did not induce significant capillary angiogenesis, increased permeability, or inflammatory cell recruitment. Conclusions— VEGF-B appears to be a coronary growth factor in rats but not in mice. The signals for the VEGF-B–induced cardiac hypertrophy are mediated at least in part via the endothelium. Because cardiomyocyte damage in myocardial ischemia begins in the subendocardial myocardium, the VEGF-B–induced increased arterial supply to this area could have therapeutic potential in ischemic heart disease.

Activated Forms of VEGF-C and VEGF-D Provide Improved Vascular Function in Skeletal Muscle

The therapeutic potential of vascular endothelial growth factor (VEGF)-C and VEGF-D in skeletal muscle has been of considerable interest as these factors have both angiogenic and lymphangiogenic activities. Previous studies have mainly used adenoviral gene delivery for short-term expression of VEGF-C and VEGF-D in pig, rabbit, and mouse skeletal muscles. Here we have used the activated mature forms of VEGF-C and VEGF-D expressed via recombinant adeno-associated virus (rAAV), which provides stable, long-lasting transgene expression in various tissues including skeletal muscle. Mouse tibialis anterior muscle was transduced with rAAV encoding human or mouse VEGF-C or VEGF-D. Two weeks later, immunohistochemical analysis showed increased numbers of both blood and lymph vessels, and Doppler ultrasound analysis indicated increased blood vessel perfusion. The lymphatic vessels further increased at the 4-week time point were functional, as shown by FITC-lectin uptake and transport. Furthermore, receptor activation and arteriogenic activity were increased by an alanine substitution mutant of human VEGF-C (C137A) having an increased dimer stability and by a chimeric CAC growth factor that contained the VEGF receptor-binding domain flanked by VEGF-C propeptides, but only the latter promoted significantly more blood vessel perfusion when compared to the other growth factors studied. We conclude that long-term expression of VEGF-C and VEGF-D in skeletal muscle results in the generation of new functional blood and lymphatic vessels. The therapeutic value of intramuscular lymph vessels in draining tissue edema and lymphedema can now be evaluated using this model system.

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 &agr;-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.

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.

Promoting blood vessel growth in ischemic diseases: challenges in translating preclinical potential into clinical success

Angiogenic therapy, which involves the use of an exogenous stimulus to promote blood vessel growth, is an attractive approach for the treatment of ischemic diseases. It has been shown in animal models that the stimulation of blood vessel growth leads to the growth of the whole vascular tree, improvement of ischemic tissue perfusion and improved muscle aerobic energy metabolism. However, very few positive results have been gained from Phase 2 and 3 clinical angiogenesis trials. Many reasons have been given for the failures of clinical trials, including poor transgene expression (in gene-therapy trials) and instability of the vessels induced by therapy. In this Review, we discuss the selection of preclinical models as one of the main reasons why clinical translation has been unsuccessful thus far. This issue has received little attention, but could have had dramatic implications on the expectations of clinical trials. We highlight crucial differences between human patients and animal models with regards to blood flow and pressure, as well as issues concerning the chronic nature of ischemic diseases in humans. We use these as examples to demonstrate why the results from preclinical trials might have overestimated the efficacy of angiogenic therapies developed to date. We also suggest ways in which currently available animal models of ischemic disease could be improved to better mimic human disease conditions, and offer advice on how to work with existing models to avoid overestimating the efficacy of new angiogenic therapies.

Vascular Endothelial Growth Factor-Angiopoietin Chimera With Improved Properties for Therapeutic Angiogenesis

Background— There is an unmet need for proangiogenic therapeutic molecules for the treatment of tissue ischemia in cardiovascular diseases. However, major inducers of angiogenesis such as vascular endothelial growth factor (VEGF/VEGF-A) have side effects that limit their therapeutic utility in vivo, especially at high concentrations. Angiopoietin-1 has been considered to be a blood vessel stabilization factor that can inhibit the intrinsic property of VEGF to promote vessel leakiness. In this study, we have designed and tested the angiogenic properties of chimeric molecules consisting of receptor-binding parts of VEGF and angiopoietin-1. We aimed at combining the activities of both factors into 1 molecule for easy delivery and expression in target tissues. Methods and Results— The VEGF–angiopoietin-1 (VA1) chimeric protein bound to both VEGF receptor-2 and Tie2 and induced the activation of both receptors. Detailed analysis of VA1 versus VEGF revealed differences in the kinetics of VEGF receptor-2 activation and endocytosis, downstream kinase activation, and VE-cadherin internalization. The delivery of a VA1 transgene into mouse skeletal muscle led to increased blood flow and enhanced angiogenesis. VA1 was also very efficient in rescuing ischemic limb perfusion. However, VA1 induced less plasma protein leakage and myeloid inflammatory cell recruitment than VEGF. Furthermore, angioma-like structures associated with VEGF expression were not observed with VA1. Conclusions— The VEGF–angiopoietin-1 chimera is a potent angiogenic factor that triggers a novel mode of VEGF receptor-2 activation, promoting less vessel leakiness, less tissue inflammation, and better perfusion in ischemic muscle than VEGF. These properties of VA1 make it an attractive therapeutic tool.