Abstract
Vascular endothelial growth factor (VEGF) stimulates endothelial cell (EC) migration and proliferation primarily through the VEGF receptor-2 (VEGFR2). We have shown that VEGF stimulates a Rac1-dependent NAD(P)H oxidase to produce reactive oxygen species (ROS) that are involved in VEGFR2 autophosphorylation and angiogenic-related responses in ECs. The small GTPase ARF6 is involved in membrane trafficking and cell motility; however, its roles in VEGF signaling and physiological responses in ECs are unknown. In this study, we show that overexpression of dominant-negative ARF6 [ARF6(T27N)] almost completely inhibits VEGF-induced Rac1 activation, ROS production, and VEGFR2 autophosphorylation in ECs. Fractionation of caveolae/lipid raft membranes demonstrates that ARF6, Rac1, and VEGFR2 are localized in caveolin-enriched fractions basally. VEGF stimulation results in the release of VEGFR2 from caveolae/lipid rafts and caveolin-1 without affecting localization of ARF6, Rac1, or caveolin-1 in these fractions. The egress of VEGFR2 from caveolae/lipid rafts is contemporaneous with the tyrosine phosphorylation of caveolin-1 (Tyr14) and VEGFR2 and with their association with each other. ARF6(T27N) significantly inhibits both VEGF-induced responses. Immunofluorescence studies show that activated VEGFR2 and phosphocaveolin colocalize at focal complexes/adhesions after VEGF stimulation. Both overexpression of ARF6(T27N) and mutant caveolin-1(Y14F), which cannot be phosphorylated, block VEGF-stimulated EC migration and proliferation. Moreover, ARF6 expression is markedly upregulated in association with an increase in capillary density in a mouse hindlimb ischemia model of angiogenesis. Thus, ARF6 is involved in the temporal-spatial organization of caveolae/lipid rafts- and ROS-dependent VEGF signaling in ECs as well as in angiogenesis in vivo.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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ADP-Ribosylation Factor 6
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ADP-Ribosylation Factors / biosynthesis
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ADP-Ribosylation Factors / genetics
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ADP-Ribosylation Factors / physiology*
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Amino Acid Substitution
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Animals
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Caveolae / drug effects
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Caveolae / metabolism*
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Caveolin 1
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Caveolins / genetics
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Caveolins / isolation & purification
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Caveolins / metabolism
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Cell Division / drug effects
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Cell Division / physiology
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Cell Fractionation
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Cell Movement / drug effects
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Cell Movement / physiology
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Endothelial Cells / cytology
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Endothelial Cells / enzymology
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Endothelium, Vascular / cytology
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Endothelium, Vascular / enzymology
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Enzyme Activation
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Female
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Focal Adhesions / drug effects
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Focal Adhesions / metabolism
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Hindlimb / blood supply
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Humans
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Ischemia / genetics
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Ischemia / metabolism
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Membrane Microdomains / drug effects
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Membrane Microdomains / metabolism*
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Mice
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Mice, Inbred C57BL
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Neovascularization, Physiologic / physiology*
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Phosphorylation / drug effects
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Point Mutation
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Protein Processing, Post-Translational / drug effects
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Protein-Tyrosine Kinases / metabolism
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Reactive Oxygen Species / metabolism
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Recombinant Fusion Proteins / physiology
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Signal Transduction / physiology*
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Superoxides / metabolism
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Umbilical Veins
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Vascular Endothelial Growth Factor A / pharmacology
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Vascular Endothelial Growth Factor A / physiology*
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Vascular Endothelial Growth Factor Receptor-2 / isolation & purification
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Vascular Endothelial Growth Factor Receptor-2 / metabolism
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rac1 GTP-Binding Protein / isolation & purification
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rac1 GTP-Binding Protein / metabolism
Substances
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ADP-Ribosylation Factor 6
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CAV1 protein, human
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Cav1 protein, mouse
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Caveolin 1
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Caveolins
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Reactive Oxygen Species
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Recombinant Fusion Proteins
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Vascular Endothelial Growth Factor A
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Superoxides
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Protein-Tyrosine Kinases
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Vascular Endothelial Growth Factor Receptor-2
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ADP-Ribosylation Factors
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ARF6 protein, human
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Arf6 protein, mouse
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rac1 GTP-Binding Protein