Microenvironment Changes (in pH) Affect VEGF Alternative Splicing

Vascular endothelial growth factor-A (VEGF-A) has several isoforms, which differ in their capacity to bind extracellular matrix proteins and also in their affinity for VEGF receptors. Although the relative contribution of the VEGF isoforms has been studied in tumor angiogenesis, little is known about the mechanisms that regulate the alternative splicing process. Here, we tested microenvironment cues that might regulate VEGF alternative splicing. To test this, we used endometrial cancer cells that produce all VEGF isoforms as a model, and exposed them to varying pH levels, hormones, glucose and CoCl2 (to mimic hypoxia). Low pH had the most consistent effects in inducing variations in VEGF splicing pattern (VEGF121 increased significantly, p < 0.001, when compared to VEGF145, 165 or 189). This was accompanied by activation of the p38 stress pathway and SR proteins (splicing factors) expression and phosphorylation. SF2/ASF, SRp20 and SRp40 down-regulation by siRNA impaired the effects of pH stimulation, blocking the shift in VEGF isoforms production. Taken together, we show for the first time that acidosis (low pH) regulates VEGF-A alternative splicing, may be through p38 activation and suggest the possible SR proteins involved in this process

Soluble perlecan domain i enhances vascular endothelial growth factor-165 activity and receptor phosphorylation in human bone marrow endothelial cells

<p>Abstract</p> <p>Background</p> <p>Immobilized recombinant perlecan domain I (PlnDI) binds and modulates the activity of heparin-binding growth factors, <it>in vitro</it>. However, activities for PlnDI, in solution, have not been reported. In this study, we assessed the ability of soluble forms to modulate vascular endothelial growth factor-165 (VEGF₁₆₅) enhanced capillary tube-like formation, and VEGF receptor-2 phosphorylation of human bone marrow endothelial cells, <it>in vitro</it>.</p> <p>Results</p> <p>In solution, PlnDI binds VEGF₁₆₅in a heparan sulfate and pH dependent manner. Capillary tube-like formation is enhanced by exogenous PlnDI; however, PlnDI/VEGF₁₆₅mixtures combine to enhance formation beyond that stimulated by either PlnDI or VEGF₁₆₅alone. PlnDI also stimulates VEGF receptor-2 phosphorylation, and mixtures of PlnDI/VEGF₁₆₅reduce the time required for peak VEGF receptor-2 phosphorylation (Tyr-951), and increase Akt phosphorylation. PlnDI binds both immobilized neuropilin-1 and VEGF receptor-2, but has a greater affinity for neuropilin-1. PlnDI binding to neuropilin-1, but not to VEGF receptor-2 is dependent upon the heparan sulfate chains adorning PlnDI. Interestingly, the presence of VEGF₁₆₅but not VEGF₁₂₁significantly enhances PlnDI binding to Neuropilin-1 and VEGF receptor-2.</p> <p>Conclusions</p> <p>Our observations suggest soluble forms of PlnDI are biologically active. Moreover, PlnDI heparan sulfate chains alone or together with VEGF₁₆₅can enhance VEGFR-2 signaling and angiogenic events, <it>in vitro</it>. We propose PlnDI liberated during basement membrane or extracellular matrix turnover may have similar activities, <it>in vivo</it>.</p

Sweet cues: How heparan sulfate modification of fibronectin enables growth factor guided migration of embryonic cells

Growth factors regulate a diverse array of cellular functions including proliferation, survival and movement, and the ability to do this often involves interactions with the extracellular matrix (ECM) and particularly heparan sulfate proteoglycans (HSPGs). HSPGs have been shown to sequester growth factors and to act as growth factor co-receptors or receptors themselves. Recent studies, however, have revealed a new role for HSPGs in mediating the interactions of growth factors with the ECM. Specifically, heparan sulfate has been shown to modulate fibronectin structure to reveal previously masked growth factor binding sites. In vivo, this mechanism appears to control the guidance of migrating cells during embryonic development as HSPG-modification of fibronectin enables direct platelet derived growth factor-fibronectin interactions necessary for this process. A model based on this observation is discussed here as well as the possibility that other growth factors/morphogens utilize similar mechanisms involving fibronectin or additional ECM proteins

Acidic preconditioning improves the proangiogenic responses of endothelial colony forming cells

Objective: Acidosis is present in several pathological conditions where vasculogenesis takes place including ischemia, tumor growth and wound healing. We have previously demonstrated that acidosis induces human CD34+ cell apoptosis. Considering that endothelial colony-forming cells (ECFC) are a subpopulation of CD34+ cells and key players in vasculogenesis, in the present study we investigated the effect of acidosis on the survival and functionality of ECFC. Approach and results: Endothelial colony-forming cells obtained by differentiation of human cord blood CD34+ cells in endothelial growth medium-2 for 14–21 days were exposed at pH 7.4, 7.0 or 6.6. We found that acidosis failed to induce ECFC apoptosis and, although an early reduction in proliferation, chemotaxis, wound healing and capillary-like tubule formation was observed, once the medium pH was restored to 7.4, ECFC proliferation and tubulogenesis were augmented. Stromal cell derived factor-1 (SDF1)-driven migration and chemokine receptor type 4 surface expression were also increased. The maximal proangiogenic effect exerted by acidic preconditioning was observed after 6 h at pH 6.6. Furthermore, preconditioned ECFC showed an increased ability to promote tissue revascularization in a murine model of hind limb ischemia. Immunoblotting assays showed that acidosis activated AKT and ERK1/2 and inhibited p38 pathways. Proliferation rises triggered by acidic preconditioning were no longer observed after AKT or ERK1/2 inhibition, whereas p38 suppression not only mimicked but also potentiated the effect of acidosis on ECFC tubule formation abilities. Conclusions: These results demonstrate that acidic preconditioning greatly increases ECFC-mediated angiogenesis in vitro including ECFC proliferation, tubulogenesis and SDF1-driven chemotaxis and is a positive regulator of microvessel formation in vivo.Fil: Mena, Hebe Agustina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Lokajczyk, Anna. Université Paris Descartes; Francia. Inserm; FranciaFil: Dizier, Blandine. Inserm; FranciaFil: Strier, Sergio E.. Ciudad Autónoma de Buenos Aires. Hospital "Bernardino Rivadavia"; ArgentinaFil: Voto, Liliana S.. Ciudad Autónoma de Buenos Aires. Hospital "Juan A. Fernández"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Boisson Vidal, Catherine. Université Paris Descartes; Francia. Inserm; FranciaFil: Schattner, Mirta Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Negrotto, Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentin