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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2005;25:2295.)
© 2005 American Heart Association, Inc.

Vascular Biology

IQGAP1 Regulates Reactive Oxygen Species–Dependent Endothelial Cell Migration Through Interacting With Nox2

Satoshi Ikeda; Minako Yamaoka-Tojo; Lula Hilenski; Nikolay A. Patrushev; Ghulam M. Anwar; Mark T. Quinn; Masuko Ushio-Fukai

From Division of Cardiology (S.I., M.Y.-T., L.H., N.A.P., G.M.A., M.U.-F.), Department of Medicine, Emory University School of Medicine, Atlanta, Ga; and the Department of Veterinary Molecular Biology (M.T.Q.), Montana State University, Bozeman, Mt.

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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Objective— Endothelial cell (EC) migration is a key event for repair process after vascular injury and angiogenesis. EC migration is regulated by reorganization of the actin cytoskeleton at the leading edge and localized production of reactive oxygen species (ROS) at the site of injury. However, underlying mechanisms are unclear. We reported that IQGAP1, an actin binding scaffold protein, mediates VEGF-induced activation of gp91phox (Nox2)-dependent NAD(P)H oxidase and EC migration. We thus hypothesized that Nox2 and IQGAP1 may play important roles in ROS-dependent EC migration in response to injury.

Methods and Results— Using a monolayer scratch assay with confluent ECs, we show that ROS production is increased at the margin of scratch area and Nox2 translocates to the leading edge, where it colocalizes and associates with both actin and IQGAP1 in migrating ECs. Knockdown of IQGAP1 using siRNA and inhibition of the actin cytoskeleton blocked scratch injury-induced H₂O₂ production, Nox2 translocation and its interaction with actin, and EC migration toward the injured site.

Conclusions— These suggest that IQGAP1 may function to link Nox2 to actin at the leading edge, thereby facilitating ROS production at the site of injury, which may contribute to EC migration.

Using a monolayer scratch assay of confluent endothelial cells (ECs), we show that IQGAP1, an actin binding scaffold protein, functions to link Nox2 of NAD(P)H oxidase to actin at the leading edge, thereby facilitating ROS production at the site of injury, which may contribute to EC migration.

Key Words: actin cytoskeleton • endothelial cell migration • IQGAP1 • NAD(P)H oxidase • reactive oxygen species

	Introduction

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Endothelial migration is a key event during the repair of damaged vessels after vascular injury and angiogenesis, and this may contribute to limiting the development of atherogenesis.^1,2 Cell migration is regulated by the dynamic reorganization of the actin cytoskeleton, protrusion at the front of the cell, and retraction at the rear. It is a highly localized event, involving the generation of spatially and temporally restricted signaling molecules, including the small GTPase Rac1³ and phosphatidylinositol 3,4,5 trisphosphate [PI(3,4,5)P₃],⁴ the product of PI 3-kinase, at the site of the new leading edge. Although excess amounts of reactive oxygen species (ROS) are toxic, physiological levels of ROS serve as signaling molecules to regulate many growth and migratory responses.^5,6 ROS are also necessary for reparative angiogenesis in the ischemic heart⁷ and hindlimb⁸ as well as wound-healing in vivo.⁹ The PI 3kinase-Rac pathway is also involved in ROS production.¹⁰ In endothelial cells (ECs), endogenous H₂O₂ accumulates in actively migrating cells at the site of injury, which is required for cytoskeletal reorganization and cell migration.¹¹ However, underlying regulatory mechanisms are unclear.

In ECs, NAD(P)H oxidase is a major source of ROS.¹² ECs express NAD(P)H oxidase subunits that are identical to those found in phagocytes, including the membrane-bound gp91^phox (now known as Nox2) and p22^phox, the cytosolic components p47^phox and p67^phox, and Rac1.¹² On stimulation, cytosolic components translocate to the membrane to form a multimeric protein complex, leading to production of ROS.¹² Recently, 4 homologues of Nox2¹³ have been identified in nonphagocytic cells. Among them, Nox4 is abundantly expressed in ECs and involved in basal superoxide production.¹⁴ We reported that Nox2-derived ROS play an essential role in vascular endothelial growth factor (VEGF)-stimulated signaling linked to EC migration¹⁵ as well as neovascularization in vivo in response to VEGF¹⁵ and hindlimb ischemia.⁸

We have recently identified IQGAP1 as a novel VEGF receptor type2 (VEGFR2) binding protein¹⁶ and found that it is a critical regulator for VEGF-induced ROS production and EC migration. IQGAP1 is a scaffold protein that plays a pivotal role in regulating actin cytoskeleton, cell adhesion, and cell migration^17,18 by interacting directly with calmodulin, actin, active Rac1/Cdc42, ß-catenin, E-cadherin, the microtubule plus end-binding protein, CLIP-170, and a tumor suppressor protein, adenomatous polyposis coli (activated protein C [APC]).¹⁹ In actively migrating cells, IQGAP1 accumulates at the leading edge and cross-links actin filaments.^17,18 Recent evidence shows that IQGAP1 links active Rac1 to APC and CLIP170 to form a multi-molecular complex at the leading edge, thereby connecting actin cytoskeleton and microtubule dynamics during directional migration.¹⁹ We previously reported that IQGAP1 expression is dramatically increased in the luminal regenerating EC layers of balloon-injured carotid artery.¹⁶

Using a monolayer scratch assay with confluent HUVECs, we demonstrate here that H₂O₂ production is increased at the injured area and Nox2 translocates to the leading edge where it colocalizes and associates with both actin and IQGAP1 during active migration. Knockdown of IQGAP1 using siRNA and inhibition of actin cytoskeleton blocked scratch-induced H₂O₂ production, Nox2 translocation and its interaction with actin, and EC migration toward injured site. These results suggest that IQGAP1 may function as a scaffold to link Nox2 to actin at the leading edge, thereby facilitating ROS production at the site of injury, which may contribute to EC migration.

	Materials and Methods

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Mouse and rabbit anti-IQGAP1 antibodies were form BD Bioscience (for immunofluorescence) and Santa Cruz (for immunoprecipitation). Rabbit anti-Nox2 (gp91^phox) antibody is highly selective for human Nox2 protein, which has been demonstrated by peptide competition studies.²⁰ Other materials are in the online data supplement. Cell culture, measurements of intracellular H₂O₂ levels, a monolayer scratch assay, immunoprecipitation and immunoblotting, confocal immunofluorescence microscopy, synthetic siRNA and its transfection, and statical analysis are described in the Material and Methods section in the online data supplement (http://atvb.ahajournals.org).

	Results

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Nox2 Accumulates at the Leading Edge After Scratch Injury
We performed a monolayer scratch assay, which primarily measures migration toward the injured sites. Confluent monolayers of HUVECs were scratched, loaded with dichlorofluorescein diacetate (DCF-DA), and the change in dichlorofluorescein (DCF) fluorescence was monitored using immunofluorescence confocal microscopy. Scratch injury induced a significant increase in DCF fluorescence within 1 hour, peaked at 4 hours, which continued at least for 8 hours (Figure 1A) and gradually decreased to the basal levels within 12 hours (data not shown). We confirmed that DCF signal was abolished by preincubation with polyethylene glycol (PEG)-catalase (Figure IA, available online at http://atvb.ahajournals.org), suggesting that it mainly detects H₂O₂ production, as reported previously.¹⁶ At 4 hours after the scratch, cells at the margin of the scratched area produced significantly more H₂O₂ than did cells away from the injured sites in migrating ECs (Figure 1B).

View larger version (24K): [in this window] [in a new window]   Figure 1. ROS production and accumulation of Nox2 at the leading edge during EC migration after scratch injury. A, DCF fluorescence was measured after the scratch of confluent monolayers of HUVECs. Graphs represent averaged data, expressed as fold change over basal (set to 1). *P<0.05 vs control (0 hour) (n=7). B, DCF fluorescence (left) and subcellular localization of Nox2 at the scratched edge area (right) obtained with confocal microscopy at 4 hours after the injury. Small white arrows point to the leading edge and large arrows point to direction toward the scratched area. Images are representative of 4 independent experiments, and >80% cells show the identical staining patterns. C, Effects of antioxidants such as N-acetylcysteine (NAC, 10 mmol/L) for 1 hour or PEG-catalase (100 U/mL) for 24 hours on scratch-induced EC migration. Images are captured at immediately (0 hour) and 24 hours after the scratch, and are representative of 3 independent experiments.

To determine whether an increase in H₂O₂ production at the margin of scratched area was caused by the spatially restricted localization of Nox2, a critical component of endothelial NAD(P)H oxidase, we examined the subcellular localization of Nox2 in actively migrating ECs. Nox2 accumulated at the leading edge of the cell membrane in actively migrating ECs at the scratched area (Figure 1B; Figure IA). We confirmed the specificity of the Nox2 staining using Nox2 siRNA, which showed a dramatic decrease in Nox2-labeled immunofluorescence intensity at the leading edge of migrating ECs (Figure IB). Furthermore, nonimmune IgG (control) showed no staining (Figure IC). In contrast, in confluent HUVEC monolayers before scratch or in migrating ECs away from the scratched area, Nox2 was found predominantly in perinuclear and nuclear regions (Figure IB), which is consistent with the previous report.²¹ We then examined the functional role of ROS and Nox2 in EC migration induced by scratch injury. Pretreatment of HUVECs with antioxidants (N-acetylcysteine [NAC], a thiol antioxidant, and PEG-catalase) or Nox2 siRNA, which knockdowns Nox2 protein (Figure ID) markedly inhibited scratch-induced cell migration toward the injured sites (Figure 1C; Figure IIA, available online at http://atvb.ahajournals.org).

Nox2 Colocalizes and Associates With Actin After Scratch Injury
To determine whether accumulation of Nox2 to the leading edge depends on the actin cytoskeleton, we performed immunofluorescence and coimmunoprecipitation experiments to assess the possible interaction between Nox2 and actin. Nox2 colocalized with actin at the leading edge of the membrane protrusion and actin meshwork in migrating ECs, but did not colocalize with actin stress fibers in the cell body (Figure 2A). In nonscratched, confluent monolayer of ECs Nox2 partially colocalized with actin stress fibers (Figure IIB). Consistent with these results, Nox2 slightly coimmunoprecipitated with actin basally, and their association was further enhanced after scratch (Figure 2A).

View larger version (37K): [in this window] [in a new window]   Figure 2. Role of actin cytoskeleton in Nox2 localization at the leading edge after scratch injury. A, Upper: at 4 hours after the scratch, cells were double-labeled with rabbit anti-Nox2 antibody (left), followed by anti-rabbit fluoresceinisothiocyanate (FITC)-conjugated secondary antibody and Rhodamine Red X (RRX)-phalloidin (middle). Small white arrows point to the leading edge and large arrows point to direction toward the scratched area. Arrow heads in merged image indicate colocalization of Nox2 with F-actin at the leading edge. Images are representative of 4 independent experiments. A, Lower: Lysates from scratched HUVECs were immunoprecipitated with anti-Nox2 antibody, and immunoblotted with anti-actin or Nox2 antibody. Nox2 blots as 2 bands of molecular weight between 75 to 100 kDa protein, which is consistent with the variably glycosylated protein reported previously.33,34 Graph shows averaged data of Nox2-actin interaction, expressed as fold change over basal (0 hour). *P<0.05 vs control (0 hour) (n=4). B, Effects of latrunculin A (10 nmol/L for 1 hour) or jasplakinolide (50 nmol/L for 1 hour) on scratch-induced Nox2 localization at the leading edge. At 4 hours after the scratch, cells were double-labeled with anti-Nox2 antibody and RRX-phalloidin.

Role of Actin Cytoskeleton in Nox2 Localization at the Leading Edge and ROS Production After Scratch Injury
To determine the role of actin cytoskeleton in localization of Nox2 at the leading edge during active EC migration, we examined the effects of inhibition of actin depolymerization with latrunculin A or actin stabilization with jasplakinolide. Both latrunculin A and jasplakinolide inhibited the localization of Nox2 at the leading edge (Figure 2B) without significant effect on microtubule structure stained with -tubulin (Figure IIC). Furthermore, both inhibitors reduced the scratch-induced increase in DCF fluorescence without affecting basal levels (Figure IID).

IQGAP1 Colocalizes and Associates With Actin and Nox2 at the Leading Edge After Scratch Injury
IQGAP1 is an actin-binding scaffolding protein involved in regulating the actin cytoskeleton.^17,18 Thus, we examined whether IQGAP1 colocalizes and associates with Nox2 at the leading edge after the injury. In confluent monolayers of HUVECs immediately after the scratch, IQGAP1 was mainly localized at the cell margin (Figure 3A). However, in actively migrating ECs at 4 hours after scratch, IQGAP1 accumulated at the leading edge as well as the side opposite to the leading edge (Figure 3A). Of note, IQGAP1 predominantly colocalized with F-actin (Figure 3B) and Nox2 (Figure 4A) at the leading edge in migrating ECs. Moreover, coimmunoprecipitation analysis demonstrated that scratch injury promoted association of IQGAP1 with actin (Figure 3B) and Nox2 (Figure 4B), which correlated with their colocalization demonstrated by immunofluorescence study. Their association returned to basal levels when the ECs have completed migration (data not shown).

View larger version (59K): [in this window] [in a new window]   Figure 3. IQGAP1 colocalizes with actin at the leading edge after scratch injury. A, At 0 and 4 hours after the scratch, HUVECs were labeled with rabbit anti-IQGAP1 antibody, followed by anti-rabbit FITC-conjugated secondary antibody. Arrow heads indicate IQGAP1 localization at cell margin (0 hour) and leading edge (4 hours). More than 80% cells show the identical staining pattern. B, Upper: At 4 hours after the scratch, cells were double-labeled with rabbit anti-IQGAP1 antibody, followed by anti-rabbit FITC-secondary antibody (left) and RRX-phalloidin (middle). Small white arrows point to the leading edge and large arrows point toward the scratched area. Arrow heads indicate colocalization of IQGAP1 with F-actin at the leading edge. A, Lower: Lysates from scratched HUVECs were immunoprecipitated with anti-IQGAP1 antibody, and immunoblotted with anti-actin or IQGAP1 antibody. Graphs represent averaged data of IQGAP1-actin interaction, expressed as fold change over basal. *P<0.05 vs control (0 hour) (n=4).

View larger version (31K): [in this window] [in a new window]   Figure 4. IQGAP1 colocalizes with Nox2 at the leading edge after scratch injury. A, At 4 hours after the scratch, HUVECs were double-labeled with rabbit anti-Nox2 and mouse anti-IQGAP1 antibodies, followed by anti-rabbit FITC- and anti-mouse RRX-secondary antibody, respectively. NC indicates negative control, staining with nonimmune IgG. Small white arrows point to the leading edge and large arrows point toward the scratched area. Arrow heads in merged image indicate colocalization of Nox2 with IQGAP1 at the leading edge. B, Lysates from scratched HUVECs were immunoprecipitated with anti-Nox2 antibody, and immunoblotted with anti-IQGAP1 or Nox2 antibody. Graphs represent averaged data of IQGAP1-Nox2 interaction, expressed as fold change over basal. *P<0.05 vs control (0 hour) (n=4).

Role of IQGAP1 in Localization of Nox2 and Its Interaction With Actin at the Leading Edge After Scratch Injury
To determine the role of IQGAP1 in localization of Nox2 at the leading edge and association of Nox2 with actin, we examined the effects of knockdown of IQGAP1 using siRNA. IQGAP1 siRNA, but not scrambled siRNA, inhibited scratch-induced increase in F-actin at the lamellipodia of protruding cell membrane, but not in stress fibers, as well as accumulation of Nox2 at the leading edge (Figure 5A) and Nox2-actin interaction (Figure 5B). Quantification of Nox2-positive cells at the leading edge further confirmed scratch-induced translocation of Nox2 is significantly inhibited by IQGAP1 depletion (Figure IIIA, available online at http://atvb.ahajournals.org). Of note, IQGAP1 siRNA almost completely knocked down IQGAP1 protein without affecting the expression of Nox2, actin, and -tubulin (Figure 5B) or microtubule structure (Figure IIIB), confirming the specificity of IQGAP1 siRNA.

View larger version (35K): [in this window] [in a new window]   Figure 5. Role of IQGAP1 in localization of Nox2 and actin at the leading edge after scratch injury. HUVECs were transfected with scrambled (control) or IQGAP1 siRNAs, and scratched. A, After 4 hours, cells were labeled with RRX-phalloidin (top), or rabbit anti-Nox2 (middle) or anti-IQGAP1 (bottom) antibody, followed by anti-rabbit FITC-secondary antibody. Small white arrows point to the leading edge and large arrows point toward the scratched area. B, After 0 and 4 hours, lysates were immunoprecipitated with anti-Nox2 antibody, and immunoblotted with anti-actin or Nox2 antibody. Some lysates were immunoblotted with anti-IQGAP1, actin or -tubulin antibody. Blots are representative of >3 independent experiments. Graphs represent averaged data of Nox2-Actin interaction, expressed as fold change over basal. *P<0.05 vs scrambled siRNA-transfected cells (0 hour) (n=3).

Role of Actin Cytoskeleton and IQGAP1 in ROS Production and EC Migration After Scratch Injury
To determine the functional significance of interaction of IQGAP1 with Nox2 and actin at the leading edge, we examined the effects of IQGAP1 siRNA on H₂O₂ production and EC migration in response to injury. IQGAP1 siRNA significantly inhibited scratch-induced increase in DCF fluorescence without affecting basal levels (Figure IIIC), which is consistent with the results obtained with inhibition of actin cytoskeleton. Moreover, latrunculin A or jasplakinolide, as well as IQGAP1 siRNA, markedly inhibited scratch-induced cell migration (Figure 6A and 6B).

View larger version (32K): [in this window] [in a new window]   Figure 6. Role of IQGAP1 in actin cytoskeleton-dependent EC migration following scratch injury. HUVECs were pretreated with vehicle alone or latrunculin A (10 nmol/L for 1 hour) or jasplakinolide (50 nmol/L for 1 hour), or transfected with scrambled or IQGAP1 siRNAs, and scratched. Images were captured immediately (0 hour) and 24 hours after the scratch. A, The representative images of ECs treated with vehicle, latrunculin A, or jasplakinolide from 3 independent experiments are shown. B, Graphs represent averaged migrated cell number to the scratched area per field. *P<0.05 vs vehicle control (left) or scrambled siRNA-transfected cells (right) (n=3).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present study demonstrates that EC scratch injury stimulates accumulation of Nox2 at the leading edge of the cell, where it colocalizes and associates with both IQGAP1 and actin during active migration of ECs. This process correlates directly with the increase in H₂O₂ production at the margin of scratched area. We also found that knockdown of IQGAP1 using siRNA inhibits scratch-induced accumulation of Nox2 and actin at the leading edge, Nox2-actin interaction, H₂O₂ production, and EC migration toward the injured site. These results suggest that IQGAP1 may function as a scaffold to target/localize Nox2 at the leading edge and facilitate association with actin. As a result of these events, NAD(P)H oxidase activation at the sites of injury may contribute to EC migration.

Physiological concentrations of ROS function as signaling molecules and regulate reparative angiogenesis in the ischemic heart⁷ and hindlimb,⁸ as well as wound healing in vivo.⁹ We and others reported that Nox2 is a critical component of the endothelial NAD(P)H oxidase;^15,22 however, its role in EC migration after injury is unclear. Using a monolayer scratch assay with confluent HUVECs, we provide here the first evidence that Nox2 accumulates at the leading edge in actively migrating ECs. This response is associated with an increase in ROS production at the margin of scratched area. Our results are consistent with the notion that cell migration is a highly polarized event, which is dependent on the accumulation of active Rac1 and generation of PI(3,4,5)P₃ at the leading edge.^3,4 Of note, PI3-K-Rac1 pathway is an upstream mediator for activation of NAD(P)H oxidase.¹⁰ These suggest that scratch injury stimulates Nox2 accumulation to the leading edge where it promotes assembly of NAD(P)H oxidase, thereby increasing ROS production toward the injured area in migrating ECs. We also show that EC migration is significantly inhibited by NAC, PEG-catalase, or Nox2 siRNA, suggesting that Nox2-derived H₂O₂ plays an important role in EC migration. Of note, their inhibition is not complete, which may be attributable to the possibility that all the ROS involved in EC migration are not blocked completely by each intervention, or that ROS-independent pathways are also minimally involved.

We demonstrate that Nox2 colocalizes with actin at the leading edge, but not with stress fibers in the cell body, in actively migrating ECs. Coimmunoprecipitation of Nox2 and actin suggests that Nox2 and actin physically associates in vivo directly or indirectly through the intermediate proteins. The functional significance of interaction of Nox2 and actin is demonstrated by the observation that disruption of actin assembly with latrunculin A or disassembly with jasplakinolide inhibits scratch injury-stimulated accumulation of Nox2 at the leading edge, ROS production, and EC migration. Thus, an intact actin cytoskeleton may play an important role for proper localization of Nox2 at the leading edge, which is required for ROS production at the site of injury and EC migration. Molecular linkage between actin cytoskeleton and NAD(P)H oxidase has been demonstrated.^23–26 In ECs, Wu et al²⁷ reported that VEGF promotes p47^phox translocation to membrane ruffles via direct interaction with WAVE1, a promoter of the actin nucleation complex, which in turn activates NAD(P)H oxidase. Qian et al²⁸ showed that arsenic-induced NAD(P)H oxidase activation and EC migration are suppressed by cytochalasin D and jasplakinolide. In unstimulated ECs, Li et al²¹ reported that NAD(P)H oxidase(s) exist in a predominantly perinuclear location and are associated with actin filaments. Similarly, the present study shows that Nox2 is found at the perinuclear region and nucleus area, and partially colocalizes with actin stress fibers in nonscratched, confluent monolayer of ECs. This may explain at least in part the basal interaction of Nox2 and actin as demonstrated by coimmunoprecipitation. It is possible that actin filaments may provide a structural basis to stabilize the NAD(P)H oxidase in static cells. On scratching monolayer of ECs, the reorganized actin cytoskeleton, which is the main driving force for cell migration, may enable Nox2 to localize to the leading edge where actin polymerization and actin cross-linking are increased. Whether other NAD(P)H oxidase components are assembled together with Nox2 at this specific compartment during active EC migration is currently under investigation.

To gain insight into the molecular mechanisms of how Nox2 and actin interact at the leading edge, we examined the role of IQGAP1, an F-actin binding scaffold protein involved in regulating actin cytoskeleton.^17,18 It has been shown that IQGAP1 accumulates at the leading edge in actively migrating cells^16,29 and directly binds and cross-links actin filaments,^17,18,30 thereby regulating local actin assembly at the cell front. IQGAP1 also binds to active Rac1 through a GAP-related domain, thereby suppressing the intrinsic GTPase activity, which in turn increases active Rac1.^29,31 Thus IQGAP1 promotes cell migration and invasion in a Rac1-dependent manner.²⁹ We previously demonstrated that VEGF stimulation promotes recruitment of Rac1 to the IQGAP1 that associates with VEGFR2, which plays an essential role in VEGF-induced ROS production and EC migration.¹⁶ Here we show that IQGAP1 binds to and colocalizes with both Nox2 and actin at the leading edge during EC migration. This result suggests that IQGAP1 and Nox2 physically associate in vivo directly or indirectly through intermediate proteins such as F-actin which directly interacts with IQGAP1.³⁰ The functional significance of interaction of IQGAP1 with Nox2 and actin is demonstrated by the observation that IQGAP1 siRNA inhibits scratch-induced accumulation of Nox2 and actin at the leading edge, Nox2-actin interaction, H₂O₂ production, and EC migration. Although IQGAP1 has been shown to be involved in local control of microtubule stability through interacting with APC, active Rac1, and CLIP-170 at the leading edge in active migrating Vero cells,³² we found that IQGAP1 siRNA inhibits polarized actin assembly at the leading edge (Figure 5A) without significant effects on microtubule structure in HUVECs.

The present study suggests that IQGAP1 may function as an actin-binding scaffold protein to link/target Nox2 to actin cytoskeleton at the leading edge, thereby facilitating ROS production at the injured area, which may contribute to EC migration. Given that IQGAP1 expression is dramatically increased in the regenerating EC layers of balloon injured artery,¹⁶ it is tempting to speculate that IQGAP1 may contribute to the repair process of ECs through an increase in Nox2-derived ROS in vivo. These observations provide insight into a role of IQGAP1 in temporally and spatially organized ROS-dependent EC migration as well as a novel mechanism by which ROS are involved in endothelial migration.

	Acknowledgments

 
This work was supported by National Institutes of Health grants HL60728, and AR42426 (to M.T.Q), PO1 HL058000 and PO1 HL075209, an American Heart Association grant-in-aid 0555308B (to M.U.-F.), and an America Heart Association National Scientist Development Grant 0130175N (to M.U.-F.), and an American Heart Association Postdoctoral Fellowship 0425460B (to M.Y.-T.).

Received May 18, 2005; accepted September 14, 2005.

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