VEGF-E activates endothelial nitric oxide synthase to induce angiogenesis via cGMP and PKG-independent pathways

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Abstract

Vascular endothelial growth factor-A (VEGF), which binds to both VEGF receptor-1 (Flt1) and VEGFR-2 (KDR/Flk-1), requires nitric oxide (NO) to induce angiogenesis in a cGMP-dependent manner. Here we show that VEGF-E, a VEGFR-2-selective ligand stimulates NO release and tube formation in human umbilical vein endothelial cells (HUVEC). Inhibition of phospholipase Cγ (PLCγ) with U73122 abrogated VEGF-E induced endothelial cell migration, tube formation and NO release. Inhibition of endothelial nitric oxide synthase (eNOS) using l-NNA blocked VEGF-E-induced NO release and angiogenesis. Pre-incubation of HUVEC with the soluble guanylate cyclase inhibitor, ODQ, or the protein kinase G (PKG) inhibitor, KT-5823, had no effect on angiogenesis suggesting that the action of VEGF-E is cGMP-independent. Our data provide the first demonstration that VEGFR-2-mediated NO signaling and subsequent angiogenesis is through a mechanism that is dependent on PLCγ but independent of cGMP and PKG.

Section snippets

Materials and methods

Reagents. Recombinant VEGF-E (orf virus strain D-1701) and VEGF-A were purchased from RELIATech (Brauschweig, Germany). Growth factor-reduced Matrigel was obtained from BD Biosciences (Cowley, UK). l-NNA (NG-NO2-l-arginine), ODQ (1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one), KT-5823 and U73122 were obtained from Calbiochem (Nottingham, UK). In vitro Angio kit was purchased from TCS Biologicals (Buckingham, UK). All other cell culture reagents and chemicals were obtained from Sigma Chemical

VEGF-E induces an in vitro angiogenic response

Immunoprecipitation and Western blot analysis were used to demonstrate that VEGF-E induces autophosphorylation of VEGFR-2 in PAEVEGFR-2 cells and HUVEC (Fig. 1A), but not in PAEVEGFR-1 cells (data not shown). VEGF-E was shown previously to induce the migration of PAEVEGFR-2, but not of PAEVEGFR-1[21]. In primary cultures, VEGF-E also increased endothelial cell migration (Fig. 1B). To determine whether selective VEGFR-2 stimulation alone can induce in vitro angiogenesis, the ability of VEGF-E to

Discussion

VEGF-E is likely to be a superior candidate for therapeutic angiogenesis as VEGF-A is associated with edema, hemorrhage and leukocyte recruitment [24]. This inflammatory response is not seen with VEGF-E overexpression [25]. This study provides the first evidence that activation of VEGFR-2 by VEGF-E induces phosphorylation of eNOS, stimulates the release of NO to promote angiogenesis in a cGMP- and PKG-independent manner. VEGF-E was identified as a VEGFR-2 specific viral homologue of VEGF-A

Acknowledgments

This work was supported by grants from the British Heart Foundation, the Wellcome Trust and the European Vascular Genomics Network, a network of excellence supported by the European Community’s Sixth Framework Programme for Research Priority 1 ‘Life Sciences, Genomics and Biotechnology for Health’ (contract LSHM-CT-2003-503254).

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