Therapeutic angiogenesis following intramuscular gene transfer of vascular endothelial growth factor 121 in a dog model of hindlimb ischemia

Vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen, has been shown to promote therapeutic angiogenesis in animal models of critical limb ischemia. Ischemic skeletal muscle is advantageous for taking up and expressing foreign genes transferred as naked plasmid DNA. Accordingly, we investigated the hypothesis that intramuscular administration of naked plasmid DNA encoding the 121-amino acid isoform of VEGF could augment collateral development and tissue perfusion in a dog hindlimb ischemia model. Unilateral hindlimb ischemia was surgically induced in Beagle dogs. Ten days later, animals received intramuscular injections of pVEGF121 plasmid directly in the ischemic muscles. Angiogenic effects were evaluated by angiography, calf blood pressure ratio and vasomotor reserve analyses. Thirty days after gene transfer, angiographically recognizable collateral vessels were increased in pVEGF121-treated animals compared with controls. Improvement in perfusion to the ischemic limb was documented by a significantly higher calf blood pressure ratio for pVEGF121 (0.79 \ub1 0.05) versus controls (0.56 \ub1 0.14, P<0.01). Vasomotor reserve assay suggested amelioration in blood availability at the microcirculation level in pVEGF121-treated animals. Hematological variables showed no significant modification due to the treatment. Our results suggest that intramuscular gene transfer of VEGF121 may promote therapeutic angiogenesis in critical limb vascular insufficiency

Spatiotemporal release of VEGF from biodegradable polylactic-co-glycolic acid microspheres induces angiogenesis in chick chorionic allantoic membrane assay

The file attached to this record is the author's final peer reviewed version. The Publisher's final version can be found by following the DOI link.While vascular endothelial growth factor (VEGF) is an acknowledged potent pro-angiogenic agent there is a need to deliver it at an appropriate concentration for several days to achieve angiogenesis. The aim of this study was to produce microspheres of biodegradable polylactic-co-glycolic acid (PLGA) tailored to achieve sustained release of VEGF at an appropriate concentration over seven days, avoiding excessive unregulated release of VEGF that has been associated with the formation of leaky blood vessels. Several formulations were examined to produce microspheres loaded with both human serum albumin (HSA) and VEGF to achieve release of VEGF between 3 and 10 ng per ml for seven days to match the therapeutic window desired for angiogenesis. In vitro experiments showed an increase in endothelial cell proliferation in response to microspheres bearing VEGF. Similarly, when microspheres containing VEGF were added to the chorionic membrane of fertilised chicken eggs, there was an increase in the development of blood vessels over seven days in response, which was significant for microspheres bearing VEGF and HSA, but not VEGF alone. There was an increase in both blood vessel density and branching – both signs of proangiogenic activity. Further, there was clearly migration of cells to the VEGF loaded microspheres. In summary, we describe the development of an injectable delivery vehicle to achieve spatiotemporal release of physiologically relevant levels of VEGF for several days and demonstrate the angiogenic response to this. We propose that such a treatment vehicle would be suitable for the treatment of ischemic tissue or wounds

Overexpression of hepatocyte growth factor/scatter factor promotes vascularization and granulation tissue formation in vivo

AbstractThe effect of hepatocyte growth factor/scatter factor (HGF/SF) during wound healing in the skin was investigated, using HGF/SF-overexpressing transgenic mouse model. Histological analysis of HGF/SF transgenic mouse excisional wound sites revealed increased granulation tissue with marked vascularization. Northern blot analysis demonstrated that, relative to control, vascular endothelial growth factor (VEGF) expression in transgenic skin was significantly higher at baseline and was robustly up-regulated during wound healing. Elevated levels of VEGF protein were detected immunohistochemically, predominantly in endothelial cells and fibroblasts within the granulation tissue of HGF/SF transgenic skin. Serum levels of VEGF were also elevated in HGF/SF transgenic mice. Thus, results from our study suggest that HGF/SF has a significant effect on vascularization and granulation tissue formation during wound healing in vivo, involving with induction of VEGF

Paradoxical Pro-angiogenic Effect of Low-Dose Ellipticine Identified by In Silico Drug Repurposing

Inadequate vessel maintenance or growth causes ischemia in diseases such as myocardial infarction, stroke, and neurodegenerative disorders. Therefore, developing an effective strategy to salvage ischemic tissues using a novel compound is urgent. Drug repurposing has become a widely used method that can make drug discovery more efficient and less expensive. Additionally, computational virtual screening tools make drug discovery faster and more accurate. This study found a novel drug candidate for pro-angiogenesis by in silico virtual screening. Using Gene Expression Omnibus (GEO) microarray datasets related to angiogenesis studies, differentially expressed genes were identified and characteristic direction signatures extracted from GEO2EnrichR were used as input data on L1000CDS2 to screen pro-angiogenic molecules. After a thorough review of the candidates, a list of compounds structurally similar to TWS-119 was generated using ChemMine Tools and its clustering toolbox. ChemMine Tools and ChemminR structural similarity search tools for small-molecule analysis and clustering were used for second screening. A molecular docking simulation was conducted using AutoDock v.4 to evaluate the physicochemical effect of secondary-screened chemicals. A cell viability or toxicity test was performed to determine the proper dose of the final candidate, ellipticine. As a result, we found ellipticine, which has pro-angiogenic effects, using virtual computational methods. The noncytotoxic concentration of ellipticine was 156.25 nM. The phosphorylation of glycogen synthase kinase-3β was decreased, whereas the β-catenin expression was increased in human endothelial cells treated with ellipticine. We concluded that ellipticine at sublethal dosage could be successfully repositioned as a pro-angiogenic substance by in silico virtual screening.ope

Endothelial cell plasma membrane biomechanics mediates effects of pro-inflammatory factors on endothelial mechanosensors: vicious circle formation in atherogenic inflammation

© 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).Chronic low-grade vascular inflammation and endothelial dysfunction significantly contribute to the pathogenesis of cardiovascular diseases. In endothelial cells (ECs), anti-inflammatory or pro-inflammatory signaling can be induced by different patterns of the fluid shear stress (SS) exerted by blood flow on ECs. Laminar blood flow with high magnitude is anti-inflammatory, while disturbed flow and laminar flow with low magnitude is pro-inflammatory. Endothelial mechanosensors are the key upstream signaling proteins in SS-induced pro- and anti-inflammatory responses. Being transmembrane proteins, mechanosensors, not only experience fluid SS but also become regulated by the biomechanical properties of the lipid bilayer and the cytoskeleton. We review the apparent effects of pro-inflammatory factors (hypoxia, oxidative stress, hypercholesterolemia, and cytokines) on the biomechanics of the lipid bilayer and the cytoskeleton. An analysis of the available data suggests that the formation of a vicious circle may occur, in which pro-inflammatory cytokines enhance and attenuate SS-induced pro-inflammatory and anti-inflammatory signaling, respectively.This research was funded by grant from “Fondo di Ateneo per la ricerca 2020”, University of Sassari, Italy.info:eu-repo/semantics/publishedVersio

Magnetic field dynamic strategies for the improved control of the angiogenic effect of mesenchymal stromal cells

project PTDC/EDM-EDM/30828/2017 SFRH/BD/114043/2015 co-financed by the ERDF under the PT2020 Partnership Agreement (POVI-01-0145-FEDER-007265), as well as from POR Lisboa 2020 grant PRECISE (Project N. 16394). Publisher Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland.This work shows the ability to remotely control the paracrine performance of mesenchymal stromal cells (MSCs) in producing an angiogenesis key molecule, vascular endothelial growth factor (VEGF-A), by modulation of an external magnetic field. This work compares for the first time the application of static and dynamic magnetic fields in angiogenesis in vitro model, exploring the effect of magnetic field intensity and dynamic regimes on the VEGF-A secretion potential of MSCs. Tissue scaffolds of gelatin doped with iron oxide nanoparticles (MNPs) were used as a platform for MSC proliferation. Dynamic magnetic field regimes were imposed by cyclic variation of the magnetic field intensity in different frequencies. The effect of the magnetic field intensity on cell behavior showed that higher intensity of 0.45 T was associated with increased cell death and a poor angiogenic effect. It was observed that static and dynamic magnetic stimulation with higher frequencies led to improved angiogenic performance on endothelial cells in comparison with a lower frequency regime. This work showed the possibility to control VEGF-A secretion by MSCs through modulation of the magnetic field, offering attractive perspectives of a non-invasive therapeutic option for several diseases by revascularizing damaged tissues or inhibiting metastasis formation during cancer progression.publishersversionpublishe

Modified polyethylene glycol hydrogels for growth factor delivery and controlled tissue invasion

The prevalence of cardiovascular disease and myocardial infarction-induced heart failure has risen significantly over recent years, emphasising the need for new, effective therapeutic strategies. A promising alternative approach is the cardiac delivery of potentially cardioprotective and regenerative growth factors from biomaterial scaffolds. One hydrogel system that has promise in this area is an injectable enzymatically degradable polyethylene glycol (PEG) hydrogel. Two modifications aimed at further optimising this system as a regenerative medicine scaffold were explored. Firstly, the covalent addition of heparin into the PEG backbone was assessed for its ability to stimulate angiogenesis by assessing the controlled release of basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) and placental growth factor 2 (PlGF-2), and also assaying endothelial cell sprouting in an in vitro 3D spheroid angiogenesis assay. The second modification involved overlaying an increasingly hydrolytic degradability on top of the enzymatically degradable background of the hydrogel. The potential of this modification to regulate the rate of hydrogel replacement by invading tissue was assessed in the 3D spheroid assay and a subcutaneous implant study in a rat model. The covalent coupling of heparin was found to substantially increase the rate of release of bFGF, VEGF and PlGF-2 over 20 days by 23%, 42% and 19%, respectively, relative to nonheparinised PEG hydrogels (p<0.01). A 3D spheroid-based angiogenesis assay was modified for use in quantifying endothelial cell sprouting in PEG hydrogels. bFGF and VEGF were shown to elicit a significant increase (2.3 – 2.4-fold increase) in average cumulative sprout lengths relative to that seen in the control spheroids (p<0.01). However, PlGF-2 did not stimulate a significant response (1.4-fold increase, p=NS). In follow up studies with heparinised hydrogels, it was found that the 3D angiogenesis was not rigorously established and ways forward are discussed. Enzymatically degradable PEG hydrogels that retained their enzymatic degradability with increasing levels of potential for hydrolysis were formed by increasing the proportion of PEGacrylate (PEG-Ac) and correspondingly decreasing the portion of PEG-vinyl sulfone (PEG-VS) monomers. PEG-Ac forms hydrolytically unstable bonds with the peptide crosslinker whilst 4 PEG-VS forms stable linkages. This approach was shown through swelling studies to be capable of generating a range of hydrolytic degradation rates. Sprouting of endothelial cells from PEG hydrogel embedded spheroids was shown to increase as the PEG-AC concentration increased. Importantly, the rate of tissue invasion in vivo was also shown to be positively correlated with the PEG-Ac concentration. The increased utility of these hydrogels to act as delivery vehicles for therapeutic agents, through covalent coupling of heparin, is promising for their use as regenerative medicine scaffolds. Additionally, so is the ability to finely tune tissue invasion by manipulating their hydrolytic degradability

Role of the Plasma Membrane Calcium ATPase as a Negative Regulator of Angiogenesis

Angiogenesis is the formation of new blood vessels from pre-existing ones. Unregulated angiogenesis is associated with several diseases such as diabetic retinopathy and tumour growth. Many signal transduction pathways have been implicated in the regulation of angiogenesis such as p38 mitogen-activated protein kinase (MAPK), phosphatidylinositol-3 kinase (PI3K), extracellular signal-related kinase 1/2 (Erk1/2) and of particular interest the calcineurin/nuclear factor of activated T-cell (NFAT) pathway. Inhibition of calcineurin activity by the drug cyclopsorin A (CsA) has been shown to inhibit processes required for successful angiogenesis such as in vitro cell migration, tube formation and additionally attenuates corneal angiogenesis in vivo. CsA is associated with severe side effects and therefore the identification of an endogenous regulator of this pathway would be beneficial. One possibility is the plasma membrane calcium ATPases (PMCAs). These high affinity calcium extrusion pumps have been shown to interact with calcineurin in mammalian cells and cardiomyocytes and down-regulate the calcineurin/NFAT pathway. This is hypothesised to be due to the interaction between the two proteins which maintains calcineurin in a low calcium micro-environment generated by the calcium removal function of the pump. Interestingly, PMCA4 has been shown to interact with calcineurin in endothelial cells. The aim of our study was to further our understanding of PMCA4s regulation of the calcineurin/NFAT pathway specifically in endothelial cells and establish if PMCA4 has a role in the regulation of angiogenesis. ‘Gain of function’ by adenoviral over-expression of PMCA4 and ‘loss of function’ by either si-RNA mediated knockdown of PMCA4 or isolation of PMCA4-/- MLEC were used as models. Over-expression of PMCA4 in HUVEC resulted in inhibition of the calcineurin/NFAT pathway with the opposite result occurring in the case of the knockout of PMCA4, identifying PMCA4 as a negative-regulator of the calcineurin/NFAT pathway in endothelial cells. Over-expression of PMCA4 significantly attenuated VEGF-induced protein and mRNA expression of the pro-angiogenic proteins RCAN1.4 and Cox-2, endothelial cell migration and in vitro and in vivo tube formation with the opposite result occurring in knockdown or knockout studies, confirming PMCA4 as a down-regulator of angiogenesis. Interestingly, over-expression or knockdown of PMCA4 had no effect on VEGF-induced HUVEC proliferation or Erk1/2 phopshorylation proposing PMCA4 may be a potential inhibitor of angiogenesis without compromising cell survival. Disruption of the interaction between PMCA4 and calcineurin by generation and ectopic expression of an adenovirus encoding the region of PMCA4 that interacts with calcineurin (428-651) (Ad-ID4) resulted in an increase in NFAT activity, RCAN1.4 protein expression and in vitro tube formation. These results identify the mechanism of PMCA4s inhibitory effect of the calcineurin/NFAT pathway and consequently angiogenesis is a result of the interaction between the two proteins. The novel findings of this study establish PMCA4 as a negative-regulator of the calcineurin/NFAT pathway in endothelial cells and angiogenesis. These results are far reaching and highlight a potential role for PMCA4 as a therapeutic target in a variety of diseases that are associated with pathological angiogenesis