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Circulation Research. 2005;96:467-475
Published online before print February 3, 2005, doi: 10.1161/01.RES.0000158286.51045.16
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(Circulation Research. 2005;96:467.)
© 2005 American Heart Association, Inc.

Molecular Medicine

Novel Role of ARF6 in Vascular Endothelial Growth Factor–Induced Signaling and Angiogenesis

Satoshi Ikeda*, Masuko Ushio-Fukai*, Lian Zuo, Taiki Tojo, Sergey Dikalov, Nikolay A. Patrushev, R. Wayne Alexander

From the Division of Cardiology, Department of Medicine, Emory University School of Medicine, Atlanta, Ga.

Correspondence to Masuko Ushio-Fukai, PhD, Division of Cardiology, Emory University School of Medicine, 1639 Pierce Dr, Rm 319, Atlanta, GA 30322. E-mail mfukai{at}emory.edu


*    Abstract
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 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
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.


Key Words: ARF6 • caveolin • vascular endothelial growth factor • endothelial cell • angiogenesis


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Vascular endothelial growth factor (VEGF) stimulates endothelial cell (EC) migration and proliferation primarily through the VEGF type 2 receptor (VEGFR2, KDR/Flk-1),1 thereby contributing to angiogenesis in vivo. In ECs, VEGF binding initiates autophosphorylation of VEGFR2, which is followed by activation of key signaling enzymes, including MAP kinases and Akt.1 We and others showed that VEGF activates the small GTPase Rac1 that is important for VEGFR2-mediated signaling linked to formation of lamellipodia associated with focal complexes, cell migration, and proliferation2–4 as well as activation of NAD(P)H oxidase, a major source of reactive oxygen species (ROS) in ECs.3 Recently, Labrecque et al5 reported that VEGFR2 is localized within caveolae/lipid rafts in the basal state. Caveolae are flask-shaped cell membrane invaginations containing the major structural protein caveolin and are subsets of lipid rafts that compartmentalize various signaling molecules including Rac1, receptors, G-protein, and enzymes.6,7 Caveolin-1 binds to VEGFR2 and negatively regulates receptor activity and VEGF stimulates tyrosine phosphorylation of caveolin.5 The mechanisms by which these responses are mediated are incompletely understood.

ADP-ribosylation factor 6 (ARF6) is a member of the Ras superfamily of small GTPases and is involved in membrane trafficking, Rac1-mediated membrane ruffling, cortical actin remodeling, cell motility, and activation of phospholipase D.8 ARF6 also colocalizes with Rac1 in endosomes, and the two are simultaneously transported to the plasma membrane during motility.9 Both Rac1 and ARF6 have nucleotide-dependent interactions with the Arfaptin and Arfophilin proteins,10 which may play a role in their localization and transport linkage. The localization of ARF6 is guanine nucleotide dependent; in its GDP-bound form, it localizes to the cytosol and endosomal compartments, and when bound to GTP, it translocates to the plasma membrane11 with ARNO, its specific nucleotide exchange factor.12 ARF6 is activated by various growth factors such as hepatocyte growth factor13 and colony-stimulating factor-114 as well as by G protein–coupled receptor agonists.15 We hypothesized that ARF6 may be involved in Rac1-related, VEGF-mediated signaling and angiogenesis-related responses in ECs.

We demonstrate that ARF6 colocalizes with Rac1 and VEGFR2 in caveolae/lipid rafts and is necessary for VEGFR2 autophosphorylation and EC migration and proliferation. These mechanisms are related to Rac1 activation, ROS production, and trafficking of VEGFR2 away from caveolae/lipid rafts. Activated VEGFR2 colocalizes with tyrosine-phosphorylated caveolin at focal complexes/adhesions, which may be associated with VEGF-induced EC migration and proliferation. Moreover, ARF6 expression is increased in association with an increase in capillary density in a mouse hindlimb ischemia model of angiogenesis. These results suggest that ARF6 plays an important role in caveolae/lipid rafts–dependent VEGF signaling linked to angiogenesis.


*    Materials and Methods
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 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Materials
Anti-ARF6 antibody was from Chemicon or Santa Cruz Biotechnology. Antibodies to Rac1, caveolin-1, and mouse phosphocaveolin-1 (pY14) were from BD Bioscience. Anti–phospho-VEGFR2 (pY1054) antibody was obtained from Biosource (see the Expanded Materials and Methods section in the online data supplement available at http://circres.ahajournals.org).

Immunoprecipitation and Immunoblotting
Human umbilical vein endothelial cells (HUVECs) were stimulated with agonists and cells were lysed. Cell lysates were used for immunoprecipitation and immunoblotting as described previously (see the online data supplement).

Adenovirus Transfection
The adenoviruses expressing dominant-negative, GTP-binding defective mutant ARF6 [Ad.ARF6(T27N)] and wild-type ARF6 (Ad.ARF6 WT) were kindly provided by Dr Michael Czech at the University of Massachusetts Medical Center, and mutant caveolin-1(Y14F) in which tyrosine 14 is mutated to phenylalanine [Ad.Cav-1(Y14F)] was kindly provided by Dr Cynthia Mastick at the University of Nevada. HUVECs were incubated with various multiples of infection (MOI) of either Ad.ARF6(T27N), Ad.ARF6 WT, Ad.Cav-1 (Y14F), or Ad.LacZ (control) and successfully infected cells were used for experiments as described previously3 (see the online data supplement).

Rac Activity Assay
Rac activity was assessed with an assay kit (Upstate Biotechnology) using a GST-conjugated PAK-1 protein-binding domain peptide, which binds only to Rac-GTP, as described previously.3

Superoxide Measurement by High-Performance Liquid Chromatography
Superoxide measurement using dihydroethidium (DHE) with an superoxide measurement by high-performance liquid chromatography (HPLC)–based assay was performed with minor modification as described previously (see the online data supplement).

Modified Boyden Chamber Migration Assay
Migration assays using a Modified Boyden Chamber method were conducted in 24-well transwell chambers as described previously.3,16

In Vitro Proliferation Assay
HUVECs were cultured in 0.2% FBS containing medium with or without stimulants for 48 hours, and cell number was counted with a hemocytometer as described previously.3,16 In some experiments, cells were extracted for immunoblotting.

Purification of Caveolae Fractions
Caveolae/lipid raft membranes were isolated as described previously (see the online data supplement).

Confocal Immunofluorescence Microscopy
HUVECs on glass coverslips were processed as previously described16 (see the online data supplement).

Mouse Ischemic Hindlimb Model
Study protocols were approved by the Animal Care and Use Committee of Emory University School of Medicine. Female C57BL/6J mice (8 to 9 weeks of age, The Jackson Laboratory; Bar Harbor, Me) were subjected to unilateral hindlimb surgery and hindlimb blood flow was measured by a laser Doppler blood flow (LDBF) analyzer as we reported previously17 (see the online data supplement).

Histological Analysis
Paraffin-embedded tissue slices (7 µm) were used for hematoxylin and eosin (H&E), ARF6, and Griffonia simplicifolia lectin staining (see the online data supplement).

Cell culture and statistical analysis are available in online data supplement.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
Role of ARF6 in VEGF-Induced Autophosphorylation of VEGFR, Rac1 Activation, and ROS Production
To determine the role of ARF6 in VEGF signaling, we first examined whether ARF6 is involved in VEGF-stimulated VEGFR2 autophosphorylation in HUVECs. As shown in Figure 1A, infection of cells with Ad.ARF6(T27N) at 10 MOI significantly inhibited VEGF-induced VEGFR2 tyrosine phosphorylation without affecting VEGFR2 expression. In contrast, either Ad.LacZ or Ad.ARF6 WT at 10 MOI had no effect, suggesting that inhibitory effects of ARF6(T27N) on VEGFR2 autophosphorylation are not due to the nonspecific overexpression of ARF6 protein but to the specific blockade of ARF6 activation.



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  		Figure 1. ARF6- and VEGF-induced VEGFR2 autophosphorylation, Rac1 activation, and ROS production. HUVECs were infected with Ad.LacZ (control), Ad.ARF6 WT, or Ad.ARF6(T27N) and stimulated with 20 ng/mL VEGF. A, After VEGF stimulation for 3 minutes, lysates were immunoprecipitated with anti-VEGFR2 antibody, followed by immunoblotting with anti-phosphotyrosine (pTyr) antibody. B, After VEGF stimulation for 3 minutes, lysates bound to PAK-1 agarose were immunoblotted with anti-Rac antibody to measure Rac1 activity. C, Lysates were immunoprecipitated with anti-ARF6 antibody, followed by immunoblotting with anti-Rac1 or anti-ARF6 antibody. Bottom panel shows averaged data, expressed as fold change over basal (the ratio in untreated cells was set to 1). *P<0.05 for the change induced by VEGF. D, Cells stimulated VEGF were used for HPLC-based DHE fluorescence analysis and graphs represent averaged data, expressed as fold change in the relative increase in DHE fluorescence intensity over that in unstimulated cells. *P<0.05 for the changes induced by VEGF vs vehicle alone. Values were calculated from more than 3 independent experiments.

Because we have previously shown that Rac1-dependent increases in ROS are involved in autophosphorylation of theVEGFR2,3 we examined the role of ARF6 in VEGF-stimulated Rac1 activation and ROS production. As shown in Figure 1B, overexpression of ARF6(T27N) significantly inhibited the VEGF-stimulated Rac1 activation, as measured at 3 minutes by a pulldown assay. To gain further insight into the relationship between ARF6 and Rac1, we examined whether VEGF stimulation promotes interaction between ARF6 and Rac1 in association with the increase in Rac1 activity. As shown in Figure 1C, VEGF stimulation promoted ARF6 coimmunoprecipitation with Rac1 within 2 minutes, which was contemporaneous with VEGF-induced Rac1 activation (Figure 1B). Furthermore, Figure 1D shows that overexpression of ARF6(T27N) significantly inhibited the VEGF-stimulated increase in ROS production, as measured by HPLC-based DHE fluorescence assays. These results suggest that ARF6 is an upstream mediator for Rac1, with which it interacts, and which may contribute, at least in part, to ROS production that mediates VEGFR2 autophosphorylation in ECs.

ARF6 Cofractionates With VEGFR2 in Caveolin-Enriched Fractions and Regulates VEGFR2 Localization
VEGFR2 is present in endothelial caveolae and associates with caveolin-1 in the basal state.5 Dissociation of VEGFR2 from caveolae/caveolin-1 seems to be essential for VEGFR2 tyrosine phosphorylation and downstream signaling.5 Thus, we examined whether ARF6 also localizes in caveolae-like microdomains using detergent-free OptiPrep gradient cell fractionation as described.18 As shown in Figure 2A, Western analysis of sequential fractions from the gradient showed that in unstimulated ECs, ARF6 was mainly found in buoyant, lower-density fractions containing VEGFR2, Rac1, and caveolin-1 without paxillin, a marker protein for focal complexes/adhesions. VEGF stimulation for 20 minutes did not affect the distribution of VEGFR2, ARF6, Rac1, or caveolin-1 in the gradient fractions (Figure 2A). The amount of VEGFR2 in the caveolin-enriched fraction 3, however, was significantly reduced (Figure 2B), but not that of ARF6 or Rac1 or caveolin-1 (data not shown). We examined whether ARF6 is involved in VEGF-stimulated egress of VEGFR2 from caveolae/lipid rafts in HUVECs. As shown in Figure 2B, overexpression of ARF6(T27N) significantly inhibited VEGF-induced reduction of VEGFR2 in the caveolin-enriched fractions. These data suggest that ARF6 is involved in regulating VEGFR2 movement out of caveolae/lipid rafts, which is an essential step for full activation of VEGFR2-mediated signaling.5



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  		Figure 2. Localization of VEGFR2, ARF6, and Rac1 in caveolae fractions and ARF6 mediates VEGF-stimulated VEGFR2 moving out from these fractions. A, HUVECs were stimulated with 20 ng/mL VEGF for 20 minutes. Caveolae fractions were immunoblotted for VEGFR2, ARF6, Rac1, caveolin-1, or paxillin. The blots are representative of three separate experiments. PC represents positive control (HUVEC lysates). B, Equal amounts of caveolin-enriched fraction 3 were immunoblotted with anti-VEGFR2 or caveolin-1 antibodies in HUVECs infected with Ad.LacZ (control) or Ad.ARF6(T27N). Bottom panel shows averaged data, expressed as fold change of VEGFR2 level over basal. Values are the mean±SE for 3 independent experiments. *P<0.05 for changes induced by VEGF vs vehicle alone.

ARF6 Is Involved in VEGF-Induced Tyrosine Phosphorylation of Caveolin-1
VEGF stimulates tyrosine phosphorylation of caveolin-1 in association with VEGFR2 activation.5 Thus, we tested whether ARF6 is involved in VEGF-induced caveolin-1 phosphorylation in HUVECs. VEGF stimulation increased tyrosine phosphorylation of caveolin-1 within 2 minutes and peaked at 5 minutes, as detected by a monoclonal caveolin antibody specifically recognizing tyrosine 14-phosphorylated caveolin-1 (pY14-caveolin-1) (Figure 3A). The VEGF-induced tyrosine phosphorylation of caveolin-1 was significantly inhibited by overexpression of ARF6(T27N) (Figure 3B). We also found that ARF6(T27N) had no effect on ERK1/2 phosphorylation by VEGF (data not shown), providing evidence that the effect of ARF6(T27N) on phosphorylation of caveolin-1 is specific. After VEGF stimulation, pY14-caveolin-1 also inducibly associated with VEGFR2 (Figure 3C). The time course of association of pY14-caveolin-1 with VEGFR2 was contemporaneous with the dissociation of VEGFR2 from nonphosphorylated caveolin-1 (Figure 3C). Only a fraction of total caveolin is associated with VEGFR2 in the basal state ({approx}13%) and a similar portion is tyrosine phosphorylated in VEGF-stimulated HUVECs. These data are consistent with a model in which ARF6 is involved in the egress of VEGFR2 from the caveolae/lipid rafts as it dissociates from caveolin-1. ARF6 is also required for the contemporaneous VEGF-induced phosphorylation of caveolin-1 (pY14) and the association of pY14-caveolin-1 with VEGFR2.



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  		Figure 3. ARF6- and VEGF-induced tyrosine phosphorylation of caveolin-1 (Cav-1) and its association with VEGFR2. A, HUVECs were stimulated with 20 ng/mL VEGF, and lysates were immunoblotted with anti–phospho-Cav-1 (pY14) or Cav-1 antibody. B, HUVECs infected with Ad.LacZ (control) or Ad.ARF6(T27N) were stimulated with VEGF for 5 minutes. *P<0.05 for increase induced by VEGF vs vehicle alone. Values were calculated from more than 3 independent experiments. C, HUVECs lysates were immunoprecipitated with anti-VEGFR2 antibody, followed by immunoblotting with anti–phospho-Cav1 (pY14) or Cav-1 antibody. Blots are representative of 3 separate experiments.

Colocalization of Autophosphorylated VEGFR2 and Tyrosine Phosphorylated Caveolin-1 at Focal Complexes/Adhesions
To determine the spatial relationship between activated VEGFR2 and pY14-caveolin-1, we examined their subcellular localization using confocal microscopy. As shown in Figure 4A, on stimulation with VEGF, autophosphorylated VEGFR2 colocalized with vinculin, an integral protein of focal complexes/adhesions19 as well as with pY14-caveolin-1 (Figure 4B). The staining of these proteins shows small dot-like structures at the edge of lamellipodia, which is consistent with focal complexes,20,21 and is reproducible; more than 90% of cells show the identical patterns. Thus, activated VEGFR2 interacts with pY14-caveolin-1 at focal complexes contemporaneously on release from caveolin-1/caveolae after VEGF stimulation. In contrast, immunofluorescence staining with anti–caveolin-1 and VEGFR2 antibodies show that the majority of nonphosphorylated caveolin and VEGFR2 are not localized in focal complexes/adhesions, suggesting that only a minor fraction of total caveolin-1 and VEGFR2 is tyrosine phosphorylated and concentrated in these specialized membrane domains (data not shown).



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  		Figure 4. Colocalization of autophosphoryalted VEGFR2 and tyrosine phosphorylated caveolin-1 in focal complexes/adhesions. HUVECs were stimulated with 50 ng/mL VEGF for 10 minutes, and double-labeled with rabbit anti–phospho-VEGFR (pY1054) antibody and mouse anti-vinculin (A) or anti-phosphocaveolin (pY14) (B) antibody, followed by anti-rabbit FITC-conjugated and anti-mouse Rhodamine Red X-conjugated secondary antibodies, respectively. Scale bar=20 µm. All fluorescence images were taken at 5 different fields/well, and the single cell images are representative of more than 3 different experiments.

Role of ARF6 and Tyrosine Phosphorylated Caveolin-1 in Localization of Autophosphorylated VEGFR2 at Focal Complexes/Adhesions
To determine the role of ARF6 and pY14-caveolin-1 in translocation of autophosphorylated VEGFR2 from caveolae/lipid rafts to focal complexes/adhesions, we examined the effects of overexpression of ARF6(T27N) and mutant caveolin-1 [Cav-1(Y14F)] on localization of phospho-VEGFR2 and pY14-caveolin-1 in HUVECs. As shown in Figure 5, ARF6(T27N) markedly reduced the presence of phospho-VEGFR2, basally and after VEGF stimulation, in focal complexes/adhesions, an effect of which is consistent with the results obtained by biochemical analysis (Figure 1A). In contrast, overexpression of Cav-1(Y14F) reduced the localization of activated VEGFR2 at focal complex-like structures and increased its staining at larger structures that have the appearance of focal adhesions (Figure 5). The overall level of VEGF-stimulated VEGFR2 phosphorylation was slightly reduced. We also confirmed that staining of pY14-caveolin-1 is almost completely abolished in ARF6(T27N) and Cav-1(Y14F)-overexpressing ECs (data not shown), supporting the specificity of anti–phospho-caveolin antibody. These immunofluorescence data are consistent with the possibility that ARF6 regulates the level of VEGFR2 autophosphorylation, at least in part, through acting on Rac1-ROS pathways, whereas its effect downstream on pY14-caveolin-1 plays an important role in proper localization of activated VEGFR2 at focal complexes.



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  		Figure 5. Involvement of ARF6 and phosphorylated caveolin in VEGF-induced localization of VEGFR2. HUVECs infected with Ad.LacZ (control), Ad.ARF6(T27N), or Ad.Cav-1(Y14F) were stimulated with 50 ng/mL VEGF for 10 minutes. Cells were labeled with rabbit anti–phospho-VEGFR (pY1054) antibody, followed by anti-rabbit Rhodamine Red X-conjugated secondary antibody. Scale bar=20 µm. All fluorescence images were taken at 5 different fields/well, and the single cell images are representative of more than 3 different experiments.

Involvement of ARF6 and Tyrosine Phosphorylated Caveolin-1 in VEGF-Stimulated Cell Migration and Proliferation
We examined the functional role of ARF6 and pY14-caveolin-1 in VEGF-stimulated cell migration in ECs using modified Boyden chamber methods.3,16 Overexpression of ARF6(T27N) and Cav-1(Y14F), but not LacZ (control) or ARF6 WT, inhibited VEGF-stimulated cell migration without affecting basal responses (Figure 6A through 6C). Neither ARF6(T27N) nor Cav-1(Y14F) affected migration induced by the angiogenic factor, sphingosine 1-phosphate (S1P), the effect of which is not mediated through ROS,3 confirming the specificity for VEGFR2-mediated responses. Similarly, ARF6(T27N) and Cav-1(Y14F), but not of LacZ or ARF6 WT, blocked VEGF-stimulated cell proliferation (Figure 6D). These results strongly suggest that both ARF6 and pY14-caveolin-1 are involved in VEGF-stimulated EC migration and proliferation. Furthermore, ARF6 protein expression is significantly increased in association with the increase in cell number during VEGF-stimulated EC proliferation (Figure 7).



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  		Figure 6. ARF6 and phosphocaveolin-1 in VEGF-induced cell migration and proliferation. HUVECs infected with Ad.LacZ (control), Ad.ARF6 WT, Ad.ARF6(T27N), or Ad.Cav-1(Y14F) were used for measurement of EC migration (A through C) and proliferation (D). A, B, and C, Infected cells were stimulated with 50 ng/mL VEGF or 10 µmol/L S1P for 6 hours, and cell migration was measured by the modified Boyden chamber method. Bar graph represents averaged data, expressed as cell number counted per 10 fields (x200) and fold change in cell number over that in unstimulated LacZ-infected cells (control). *P<0.05 for the change induced by stimulants vs vehicle alone. D, Infected cells were cultured in 0.2% FBS containing medium with or without 20 ng/mL VEGF for 48 hours, and the cell number was measured. *P<0.05 for the increase induced by VEGF vs vehicle alone. Values are the mean±SE for 3 independent experiments.



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  		Figure 7. ARF6 protein expression during VEGF-induced EC proliferation. HUVECs were cultured in 0.2% FBS containing medium with 20 ng/mL VEGF for 48 hours, and lysates and cells were used for immunoblotting with anti-ARF6 antibody and measurement of cell number, respectively. Values are the mean±SE for 3 independent experiments. *P<0.05 for the increase induced by VEGF vs vehicle alone.

Induction of ARF6 Protein Expression in Mouse Ischemic Hindlimb Model of Angiogenesis
EC migration and proliferation are key events for angiogenesis, which involves new capillary formation from preexisting vessels. To assess the role of ARF6 in in vivo angiogenesis, we examined the expression of ARF6 in a mouse hindlimb ischemia model in which angiogenesis is dependent on VEGF.22 Figure 8A shows blood flow recovery using LDBF analysis in ischemic and nonischemic limbs after femoral artery ligation and demonstrates that hindlimb blood flow was markedly decreased immediately after surgery (day 0) and recovered to the level of that of the nonischemic limb by day 7. As shown in Figure 8B and 8C, H&E staining of ischemic- and nonischemic hindlimb sections obtained at 7 days after operation showed an increase in small capillary-like structures in ischemic tissues. Immunohistochemical analysis of serial sections demonstrates that ARF6 protein expression was dramatically increased in the ischemic hindlimbs compared with nonischemic ones, which was accompanied by an increase in density of lectin-positive capillary ECs. Western analysis also shows that ARF6 protein expression was markedly increased in the ischemic hindlimb tissues at 7 days after operation in association with the increase in VEGF expression (Figure 8D). Given the functional role of ARF6 in EC migration and proliferation, these data suggest that ARF6 may be involved also in the process by which new blood vessels are formed in vivo.



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  		Figure 8. Induction of ARF6 protein expression in mouse ischemic hindlimb model of angiogenesis. Hindlimb ischemia was induced by the right femoral artery ligation as described in Materials and Methods. A, Laser Doppler blood flow analysis: Arrows indicate a low perfusion signal (dark blue) detected at immediately after operation (day 0) and a high perfusion signal (yellow to red) detected on day 7 in the ischemic hindlimbs. B, H&E staining (arrows indicate capillary-like structures) and immunostaining of nonischemic and ischemic tissues with anti-ARF6 antibody (green) or lectin which stains ECs of capillaries (red) at 7 days after ischemia as visualized with a Zeiss fluorescence microscope. NC indicates staining without primary antibody (negative control). C, Number of ARF6-positive cells and lectin-positive capillaries in nonischemic and ischemic tissues at 7 days after operation were counted in 5 randomly selected high power fields (x400, per mm2). *P<0.05 vs nonischemic tissue. D, ARF6 (left) and VEGF (right) protein expression in nonischemic and ischemic tissues at 0 and 7 days after operation. Bottom panel shows averaged data, expressed as fold change over basal. Values are the mean±SE for 3 independent experiments. *P<0.05 for the changes between day 0 and 7 as well as nonischemic and ischemic tissues.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
The molecular mechanisms involved in VEGFR2-mediated signaling linked to angiogenic responses in ECs are incompletely understood. The present study demonstrates that ARF6 plays a critical role in VEGFR2 autophosphorylation, EC migration, and proliferation, at least in part through regulating Rac1 activity, ROS production, and VEGFR2 egress from caveolae/lipid rafts. We also found that activated VEGFR2 localizes at focal complexes/adhesions in association with tyrosine phosphorylated caveolin-1, a putative positive regulator for VEGF signaling in ECs. Furthermore, ARF6 expression is markedly increased in the mouse hindlimb ischemia model of angiogenesis.

We demonstrated previously that VEGF rapidly activates Rac1 and promotes Rac1 translocation to the plasma membrane, both of which are involved in autophosphorylation of VEGFR2 in ECs.3 The dominant-negative, GTP-binding defective ARF6(T27N) mutant, but not ARF6 WT, significantly inhibits VEGFR2 autophosphorylation in HUVECs (Figure 1A), suggesting that the effect of ARF6(T27N) is due to blockade of ARF6 activation specifically. The inhibition of VEGF-induced Rac1 activation by overexpression of ARF6(T27N) supports this notion (Figure 1B). Thus, ARF6 is an upstream mediator of Rac1-GTP loading, and this activation likely reflects its association with Rac1 (Figure 1C). Relationships between ARF6 and Rac1 in other systems have been reported. Boshans et al9 showed that the G protein–coupled receptor agonist bombesin-induced Rac1 activation is ARF6-dependent in CHO cells transfected with Rac1. Overexpression of ARNO, a guanine nucleotide exchange factor for ARF6, promotes cell migration, in part through activation of Rac1.23 Moreover, ARF6 has been shown to be involved in Rac-induced membrane ruffling through regulating Rac1 translocation to the plasma membrane9,24 and via interacting with the Rac1 binding protein POR1.25 Thus, ARF6 and Rac1 regulate various biological responses in a coordinated manner. Although the mechanism by which ARF6 modulates Rac1 activity remains to be determined, it is plausible that ARF6 may activate Rac1 through regulating a Rac-GEF (guanine nucleotide-exchange factor) such as Tiam1 or Sos-1.26

Rac1 is a critical component of Nox2-based NAD(P)H oxidase, a major source of ROS, in ECs,27 and we demonstrated that the Rac1-dependent increase in ROS is important for VEGF-induced VEGFR2 autophosphorylation.3 ARF6(T27N) significantly inhibits VEGF-stimulated increase in O2– production in ECs (Figure 1D). Theses results suggest that the ARF6/Rac1/ROS pathways are involved in VEGF-induced VEGFR2 autophosphorylation. The mechanisms by which ROS mediate VEGFR2 phosphorylation remain unclear. Accumulating evidence suggest that protein tyrosine phosphatases (PTPs) are direct targets of ROS and that reversible oxidative inhibition of PTPs by ROS is an important mechanism through which ROS increase tyrosine phosphorylation events in growth factor signaling.28,29 Indeed, various PTPs including HCPTPA, SHP-1, and SHP-21 have been shown to bind to VEGFR2. Thus, it is possible that ARF6(T27N) may inhibit Rac1-dependent ROS production, thereby enhancing PTPs activity, which in turn downregulates VEGFR2 autophosphorylation. The precise underlying mechanism requires further investigation.

A variety of receptors and signaling proteins such as VEGFR2, EGF receptor (EGF-R), PDGF receptor, Rac1, Ras, eNOS, and cSrc are concentrated within caveolae-like microdomains via interacting with caveolin-1.7 The present study demonstrates in HUVECs that ARF6 is localized basally in caveolin-enriched fractions, which also contain VEGFR2 and Rac1. VEGF stimulation resulted in releasing VEGFR2 from caveolae/lipid rafts without affecting localization of ARF6, Rac1, or caveolin-1 in these fractions (Figure 2). Consistent with our results, Labrecque et al5 have shown that caveolin-1 associates with inactive VEGFR2 basally and undergoes rapid dissociation from the receptor after VEGF stimulation in bovine aortic ECs. Similarly, our group reported that EGF-R transactivation by angiotensin II is associated with release of EGF-R from caveolin-1 in vascular smooth muscle cells (VSMCs).30 ARF6(T27N) significantly inhibits VEGF-induced egress of VEGFR2 from caveolae/lipid rafts (Figure 2B), suggesting that ARF6 is involved in this process. Although the underlying mechanisms are unknown, it is possible that ARF6 may act at the level of VEGF receptor, because the dominant-negative ARF6 mutant inhibits its tyrosine phosphorylation and activation. EGF-stimulated migration of EGF-R from caveolae is dependent on the presence of the active kinase and the regulatory domains of the receptor.31 Given that caveolin-1 binding to VEGFR2 inhibits VEGFR2 activity,5 ARF6 may act a positive regulator for VEGFR2 function by contributing to the dissociation of caveolin-1 from VEGFR2 in caveolae/lipid rafts, thereby facilitating release of active VEGFR2 out of these microdomains.

Coimmunoprecipitation (Figure 3C) and immunofluorescence (Figure 4) studies show that activated VEGFR2, contemporaneously on their VEGF-stimulated release from caveolin-1/caveolae, interact with pY14-caveolin-1 at focal complexes, appearing as small dot-like structures at the edge of lamellipodia.32 Formation of focal complexes is induced by Rac1,20 and VEGF-induced Rac1 activation (Figure 1B) and tyrosine phosphorylation of caveolin-1 are inhibited by ARF6(T27N) (Figure 3B). Thus, the ARF6-Rac1 pathway may play an important role in the formation of focal complexes to which activated VEGFR2 and phosphocaveolin-1 are recruited from caveolae/lipid rafts. The reduction by both ARF6(T27N) and Cav-1(Y14F) of VEGF-induced localization of phospho-VEGFR2 at focal complexes/adhesions supports this notion (Figure 5). Thus, these results suggest that ARF6 regulates the level of VEGFR2 autophosphorylation through acting on Rac1-ROS pathways, whereas downstream phosphorylation of caveolin-1 is required, at least in part, for proper compartmentalization of activated VEGFR2 at focal complexes. Caveolin-1 is tyrosine phosphorylated by various growth factors including insulin-like growth factor,33 EGF,34 and VEGF.5 Phospho-caveolin is localized at focal adhesions after stimulation with angiotensin II and EGF in VSMC30 and A431 cells,35 respectively. Furthermore, phospho-caveolin interacts with Grb7,35 low molecular weight protein tyrosine phosphatase,36 and Csk, a negative regulator for Src.37 Thus, phospho-caveolin-1 may function as a critical scaffolding protein for growth factor–mediated signaling by serving as a docking site for phosphotyrosine-binding molecules at focal complexes/adhesions.

Functional roles of both ARF6 and phospho-caveolin in VEGF signaling is demonstrated by the observations that overexpression of ARF6(T27N) and Cav1(Y14F), but not ARF6 WT, significantly inhibit VEGF-stimulated cell migration and proliferation without affecting basal responses in HUVECs (Figure 6). These results suggest that ARF6 activation and its downstream phosphorylation of caveolin-1 play an important role in VEGF-induced, angiogenic-related responses in ECs. In contrast, S1P-induced EC migration is not affected by either ARF6(T27N) or Cav-1(Y14F), suggesting a specific role of ARF6 and pY14-caveolin in EC migration stimulated by VEGF. Liu et al38 demonstrated that S1P-, but not VEGF-, stimulated cell migration is mediated through Rho associated kinase in ECs. We showed that an Rac1-dependent increase in ROS is involved in VEGF-, but not S1P-, stimulated EC migration and proliferation.3 Given that Rac1 activity and ROS generation are regulated by ARF6 (Figure 1), these results indicate that the ARF6/Rac1/ROS pathway plays an essential role in VEGFR2-mediated signaling linked to angiogenic responses in ECs. Our results are consistent with, and perhaps provide some unifying mechanistic context for, previous reports that ARF6-dependent migration is mediated through Rac1 in epithelial cells,23 phospho-caveolin is involved in EGF-stimulated cell migration in 293T cells,35 and EC migration is associated with dissociation of caveolin-1 from caveolae and relocalization of pY14-caveolin to the cell front.39 Understanding the precise relationships between Rac1 and phospho-caveolin in VEGF signaling requires further investigation.

VEGF is a potent endogenous regulator for angiogenesis in response to ischemia,22 and EC migration and proliferation are key events for angiogenesis in vivo. Immunohistochemical and Western analysis demonstrate that expression of ARF6 is markedly increased in hindlimb tissues after ischemia, as reflected in an increase of lectin-positive, newly-formed capillaries (Figure 8). Our in vivo data are consistent with in vitro evidence that ARF6 expression in cultured ECs is increased in association with the increase in cell numbers during VEGF stimulation (Figure 7), suggesting that ARF6 expression reflects the growth state of ECs. Because ARF6 is involved in VEGF-stimulated EC migration and proliferation, the functional consequence of upregulation of ARF6 in the neovasculature is likely to be consistent with an important role of ARF6 in postnatal angiogenesis, which is dependent on NAD(P)H oxidase–derived ROS.17

In summary, ARF6 plays an important role in VEGFR2 autophosphorylation and in EC migration and proliferation through regulating Rac1 activity and ROS production. Phospho-caveolin appears to be a positive regulator for VEGF signaling and is associated with VEGFR2 movement from caveolae/lipid rafts to focal complexes/adhesions. ARF6 expression is upregulated in an animal model of angiogenesis. These results provide insight into a role of ARF6 in the temporal-spatial organization of caveolae/lipid rafts–dependent angiogenic-related VEGF signaling responses in ECs as well as in angiogenesis in vivo.


*    Acknowledgments
 
This work was supported by NIH grant HL60728 (to R.W.A. and M.U.-F.) and an AHA National Scientist Development Grant 0130175N (to M.U.-F.). Experiments/data analysis/presentation were performed in part through the use of the Internal Medicine Imaging Core, supported by NIH grants PO1 HL058000 and PO1 HL075209. We thank Drs Minako Yamaoka-Tojo and Lula Hilenski for technical assistance and helpful discussion.


*    Footnotes
 
*Both authors contributed equally to this study. Back

Original received October 6, 2004; revision received January 6, 2005; accepted January 25, 2005.


*    References
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