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      Significance of Nitric Oxide and Peroxynitrite in Permeability Changes of the Retinal Microvascular Endothelial Cell Monolayer Induced by Vascular Endothelial Growth Factor


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          Reactive oxygen species (ROS) play an important role in signaling pathways stimulated by growth factors in vascular cells. We investigated whether vascular endothelial growth factor (VEGF), which is upregulated in diabetic retinopathy and atherosclerosis, is able to enhance production of ROS, and if so, whether ROS modulate endothelial permeability. ROS levels in bovine retinal microvascular endothelial cells (BMEC) were measured by the oxidation of 2′,7′-dichlorodihydrofluorescein (DCHF), and permeability was examined by monitoring the passage of albumin through BMEC monolayers. VEGF stimulated oxidation of DCHF in BMEC, an effect which was inhibited by superoxide dismutase (SOD) and the nitric oxide (NO) synthase inhibitor, N<sup>G</sup>-nitro- L-arginine methyl ester ( L-NAME), but not by D-NAME. Urate, a scavenger of peroxynitrite, attenuated the VEGF-induced oxidation of DCHF. VEGF elicited a significant increase in the macromolecule permeability of BMEC monolayers within 30 min. SOD did not modify the basal or the VEGF-stimulated hyperpermeability, but the combination of SOD and VEGF induced a transient reduction in permeability after 10 min. L-NAME, but not D-NAME, enhanced VEGF-induced hyperpermeability without affecting basal values. Urate did not modify the VEGF-induced changes in permeability. In conclusion, VEGF stimulates oxidation of DCHF, which most likely represents peroxynitrite formation, and induces an increase in permeability of BMEC monolayers. Activation of NO synthase seems to counteract this stimulatory effect of VEGF on endothelial permeability.

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          Vascular Endothelial Growth Factor Induces Endothelial Fenestrations In Vitro

          Abstract. Vascular endothelial growth factor (VEGF) is an important regulator of vasculogenesis, angiogenesis, and vascular permeability. In contrast to its transient expression during the formation of new blood vessels, VEGF and its receptors are continuously and highly expressed in some adult tissues, such as the kidney glomerulus and choroid plexus. This suggests that VEGF produced by the epithelial cells of these tissues might be involved in the induction or maintenance of fenestrations in adjacent endothelial cells expressing the VEGF receptors. Here we describe a defined in vitro culture system where fenestrae formation was induced in adrenal cortex capillary endothelial cells by VEGF, but not by fibroblast growth factor. A strong induction of endothelial fenestrations was observed in cocultures of endothelial cells with choroid plexus epithelial cells, or mammary epithelial cells stably transfected with cDNAs for VEGF 120 or 164, but not with untransfected cells. These results demonstrate that, in these cocultures, VEGF is sufficient to induce fenestrations in vitro. Identical results were achieved when the epithelial cells were replaced by an epithelial-derived basal lamina-type extracellular matrix, but not with collagen alone. In this defined system, VEGF-mediated induction of fenestrae was always accompanied by an increase in the number of fused diaphragmed caveolae-like vesicles. Caveolae, but not fenestrae, were labeled with a caveolin-1–specific antibody both in vivo and in vitro. VEGF stimulation led to VEGF receptor tyrosine phosphorylation, but no change in the distribution, phosphorylation, or protein level of caveolin-1 was observed. We conclude that VEGF in the presence of a basal lamina-type extracellular matrix specifically induces fenestrations in endothelial cells. This defined in vitro system will allow further study of the signaling mechanisms involved in fenestrae formation, modification of caveolae, and vascular permeability.
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            Superoxide generation by endothelial nitric oxide synthase: The influence of cofactors

            The mechanism of superoxide generation by endothelial nitric oxide synthase (eNOS) was investigated by the electron spin resonance spin-trapping technique using 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide. In the absence of calcium/calmodulin, eNOS produces low amounts of superoxide. Upon activating eNOS electron transfer reactions by calcium/calmodulin binding, superoxide formation is increased. Heme-iron ligands, cyanide, imidazole, and the phenyl(diazene)-derived radical inhibit superoxide generation. No inhibition is observed after addition of l -arginine, N G -hydroxy- l -arginine, l -thiocitrulline, and l - N G -monomethyl arginine to activated eNOS. These results demonstrate that superoxide is generated from the oxygenase domain by dissociation of the ferrous–dioxygen complex and that occupation of the l -arginine binding site does not inhibit this process. However, the concomitant addition of l -arginine and tetrahydrobiopterin (BH 4 ) abolishes superoxide generation by eNOS. Under these conditions, l -citrulline production is close to maximal. Our data indicate that BH 4 fully couples l -arginine oxidation to NADPH consumption and prevents dissociation of the ferrous–dioxygen complex. Under these conditions, eNOS does not generate superoxide. The presence of flavins, at concentrations commonly employed in NOS assay systems, enhances superoxide generation from the reductase domain. Our data indicate that modulation of BH 4 concentration may regulate the ratio of superoxide to nitric oxide generated by eNOS.
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              Vascular permeability factor/vascular endothelial cell growth factor-mediated permeability occurs through disorganization of endothelial junctional proteins.

              Vascular permeability factor/vascular endothelial growth factor stimulates endothelial proliferation, angiogenesis, and increased vascular permeability in vivo. We investigated mechanisms of vascular permeability factor-mediated endothelial monolayer permeability changes in vitro. [14C]Albumin flux across endothelial monolayers was measured following a 90-min exposure to vascular permeability factor (660 pM). Vascular permeability factor increased albumin flux to 3.4 times that of control albumin flux. Endothelial monolayers were also incubated for 90 min with vascular permeability factor plus Gö6976 (10 nM), staurosporine (1 microM), wortmannin (10 nM), AG126 (1 and 2.67 microM), and PD98059 (20 microM). Vascular permeability factor-mediated permeability was not blocked by Gö6976, an antagonist of "classical" protein kinase C, staurosporine, a pan-protein kinase C antagonist, nor wortmannin, a PI3-kinase blocker, but was blocked by incubation with AG126 or PD98059, inhibitors of mitogen-activated protein kinase activation. Immunofluorescent staining of the junctional proteins VE-cadherin and occludin showed a loss of these proteins from the endothelial junction that was prevented by co-incubation with AG126 or PD98059. These data demonstrate that vascular permeability factor increases albumin permeability across endothelial monolayers in vitro and suggests that permeability increases through rearrangement of endothelial junctional proteins involving the mitogen-activated protein kinase signal transduction pathway.

                Author and article information

                J Vasc Res
                Journal of Vascular Research
                S. Karger AG
                December 1999
                24 December 1999
                : 36
                : 6
                : 510-515
                aInstitut für Kardiovaskuläre Physiologie, Klinikum der Johann-Wolfgang-Goethe-Universität, Frankfurt, bPhysiologisches Institut, Justus-Liebig-Universität, Giessen, Germany; cInstitut de Recherches SERVIER, Suresnes, France
                25694 J Vasc Res 1999;36:510–515
                © 1999 S. Karger AG, Basel

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                Page count
                Figures: 4, References: 36, Pages: 6
                Research Paper


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