doi:10.1152/ajpheart.00739.2011 302:H724-H732, 2012. First published 18 November 2011;Am J Physiol Heart Circ Physiol

D. McKinney, Frank Kuhr, Irena Levitan, Tohru Fukai and Masuko Ushio-FukaiJin Oshikawa, Seok-Jo Kim, Eiji Furuta, Cristiana Caliceti, Gin-Fu Chen, Ronaldsignaling and angiogenesis in endothelial cellsNovel role of p66Shc in ROS-dependent VEGF

You might find this additional info useful...

53 articles, 35 of which can be accessed free at:This article cites http://ajpheart.physiology.org/content/302/3/H724.full.html#ref-list-1

including high resolution figures, can be found at:Updated information and services http://ajpheart.physiology.org/content/302/3/H724.full.html

can be found at:AJP - Heart and Circulatory Physiologyabout Additional material and information http://www.the-aps.org/publications/ajpheart

This information is current as of April 30, 2012.

ISSN: 0363-6135, ESSN: 1522-1539. Visit our website at http://www.the-aps.org/.Physiological Society, 9650 Rockville Pike, Bethesda MD 20814-3991. Copyright © 2012 by the American Physiological Society. intact animal to the cellular, subcellular, and molecular levels. It is published 12 times a year (monthly) by the Americanlymphatics, including experimental and theoretical studies of cardiovascular function at all levels of organization ranging from the

publishes original investigations on the physiology of the heart, blood vessels, andAJP - Heart and Circulatory Physiology

on April 30, 2012ajpheart.physiology.org

Dow

nloaded from

Novel role of p66Shc in ROS-dependent VEGF signaling and angiogenesis inendothelial cells

Jin Oshikawa,1,3* Seok-Jo Kim,1,3* Eiji Furuta,1,3 Cristiana Caliceti,1,3 Gin-Fu Chen,2,3

Ronald D. McKinney,1,2,3 Frank Kuhr,4 Irena Levitan,4 Tohru Fukai,2,3 and Masuko Ushio-Fukai1,3

1Department of Pharmacology, Center for Lung and Vascular Biology, 2Departments of Medicine and Pharmacology, and3Center for Cardiovascular Research, and 4Pulmonary, Critical Care and Sleep Medicine, Department of Medicine,University of Illinois, Chicago, Illinois

Submitted 25 July 2011; accepted in final form 11 November 2011

Oshikawa J, Kim SJ, Furuta E, Caliceti C, Chen GF, McKin-ney RD, Kuhr F, Levitan I, Fukai T, Ushio-Fukai M. Novel role ofp66Shc in ROS-dependent VEGF signaling and angiogenesis in endothelialcells. Am J Physiol Heart Circ Physiol 302: H724–H732, 2012. Firstpublished November, 18, 2011; doi:10.1152/ajpheart.00739.2011.—p66Shc,a longevity adaptor protein, is demonstrated as a key regulator of reactiveoxygen species (ROS) metabolism involved in aging and cardiovasculardiseases. Vascular endothelial growth factor (VEGF) stimulates endothe-lial cell (EC) migration and proliferation primarily through the VEGFreceptor-2 (VEGFR2). We have shown that ROS derived from Rac1-dependent NADPH oxidase are involved in VEGFR2 autophosphor-ylation and angiogenic-related responses in ECs. However, a role ofp66Shc in VEGF signaling and physiological responses in ECs isunknown. Here we show that VEGF promotes p66Shc phosphoryla-tion at Ser36 through the JNK/ERK or PKC pathway as well as Rac1binding to a nonphosphorylated form of p66Shc in ECs. Depletion ofendogenous p66Shc with short interfering RNA inhibits VEGF-induced Rac1 activity and ROS production. Fractionation of caveolin-enriched lipid raft demonstrates that p66Shc plays a critical role inVEGFR2 phosphorylation in caveolae/lipid rafts as well as down-stream p38MAP kinase activation. This in turn stimulates VEGF-induced EC migration, proliferation, and capillary-like tube forma-tion. These studies uncover a novel role of p66Shc as a positiveregulator for ROS-dependent VEGFR2 signaling linked to angiogen-esis in ECs and suggest p66Shc as a potential therapeutic target forvarious angiogenesis-dependent diseases.

reactive oxygen species; vascular endothelial growth factor

ANGIOGENESIS IS INVOLVED in normal and postnatal developmentas well as reparative neovascularization that is associated withwound healing, ischemic heart and limb diseases, and chronicinflammatory diseases such as atherosclerosis. In endothelialcells (ECs), vascular endothelial growth factor (VEGF) in-duces angiogenesis by stimulating EC migration and prolifer-ation primarily through the autophosphorylation of VEGFreceptor type-2 (VEGFR2, KDR/Flk1; VEGFR2-pY), whichinitially occurs in part in caveolae/lipid rafts (19, 23, 32).Reactive oxygen species (ROS) function as key signalingmolecules to mediate various biological responses such as cellmigration, proliferation, and gene expression (11, 15, 37, 47).We and others (7, 48) reported that ROS derived from NADPHoxidase play an important role in VEGFR2 signaling andangiogenic responses in ECs, as well as ischemia-induced

neovascularization in vivo (9, 17, 42, 45). However, the un-derlying mechanisms are poorly understood.

The adaptor protein p66Shc is a life span determinant thatintegrates metabolic and longevity pathways and a key regu-lator for oxidative stress-mediated aging and cardiovasculardiseases (3, 8, 10, 13, 26, 28, 29, 36). The Shc protein isexpressed as three isoforms with relative molecular mass of 46,52, and 66 kDa (34). They consist of a phosphotyrosinebinding domain (PTB), a collagen homology domain (CH1),and a C-terminal Src homology 2 domain (SH2). In addition,p66Shc contains a unique N-terminal collagen homology do-main (CH2) that includes a Ser36. p66Shc phosphorylation atSer36 triggers a cascade of events leading to an increase inROS production (8, 14, 26, 35). Mice lacking p66Shc displayprolonged life span, reduced production of intracellular oxi-dants, and increased resistance to oxidative stress-inducedapoptosis (26). Accordingly, the majority of studies usingp66Shc!/! mice have defined the pathophysiological role ofp66Shc in cardiovascular disease where ROS represent a sub-stantial triggering component (3, 8, 10, 13, 28, 36). By con-trast, recent report (27) using isolated p66Shc!/! T cells showsthat p66Shc is required for the angiogenic response stimulatedby hypoxia.

Here we test the hypothesis that p66Shc may be involved inROS-dependent VEGF signaling linked to angiogenesis inECs. The present study demonstrates that VEGF stimulationincreases Ser36-phosphorylation of p66Shc in human umbili-cal vein ECs (HUVECs) at least in part through ERK, JNK, orPKC. We also found that VEGF promotes Rac1 associationwith a nonphosphorylated form of p66Shc and that p66Shc isinvolved in VEGF-induced Rac1 activation and ROS produc-tion, which promotes VEGFR2 autophosphorylation in cave-olae/lipid rafts and p38MAPK activation. This in turn contrib-utes to VEGF-induced angiogenic-related responses in ECs.These studies are the first to demonstrate the physiologicalfunction of p66Shc involved in VEGF-induced ROS produc-tion and VEGFR2-mediated redox signaling linked to angio-genesis in ECs.

METHODS

Materials. Antibodies to VEGFR2, phosphotyrosine (pY99), andpaxillin were from Santa Cruz Biotechnology. Antibody to phospho-Ser36-p66Shc was from Calbiochem. Antibodies to phospho-VEGFR2 (pY1175), phospho-p38MAPK, and phospho-ERK1/2 werefrom Cell Signaling. Anti-caveolin-1 and Shc antibodies were fromBD Biosciences. Human recombinant VEGF165 was from R&DSystems. Oligofectamine and Opti-MEMI reduced-serum mediumwere from Invitrogen. Other materials were purchased from Sigma.

* J. Oshikawa and S.-J. Kim contributed equally to this work.Address for reprint requests and other correspondence: M. Ushio-Fukai,

Dept. of Pharmacology Center for Lung and Vascular Biology, Center forCardiovascular Research, Univ. of Illinois at Chicago, 835 S. Wolcott,M/C868, E403 MSB, Chicago, IL 60612 (e-mail: mfukai@uic.edu).

Am J Physiol Heart Circ Physiol 302: H724–H732, 2012.First published November 18, 2011; doi:10.1152/ajpheart.00739.2011.

0363-6135/12 Copyright © 2012 the American Physiological Society http://www.ajpheart.orgH724

on April 30, 2012ajpheart.physiology.org

Dow

nloaded from

Cell culture. HUVECs were grown in Endo-Gro (Millipore) con-taining 5% FBS.

Immunoprecipitation and immunoblotting. Growth-arrested HUVECswere stimulated with VEGF (20 ng/ml), and cells were lysed in lysisbuffer at pH 7.4 (in mM: 50 HEPES, 5 EDTA, and 100 NaCl), 1%Triton X-100, protease inhibitors (10 "g/ml aprotinin, 1 mmol/lPMSF, and 10 "g/ml leupeptin), and phosphatase inhibitors (inmmol/l: 50 sodium fluoride, 1 sodium orthovanadate, and 10 sodiumpyrophosphate). Cell lysates were used for immunoprecipitation andimmunoblotting, as described previously (49).

H2O2 measurement. HUVECs grown on glass coverslips wereincubated with 20 "M 5-(and-6)-chloromethyl-2=,7=-dichlorodihydro-fluorescein diacetate, acetyl ester (CM-H2DCF-DA; Invitrogen) for 6min at 37°C and then Vectashield mounting medium with DAPI wasdropped on them. Cells were observed by confocal microscopy usingthe same exposure condition in each experiment. Relative DCF-DAfluorescence intensity with DAPI-positive cells was recorded andanalyzed using Image J.

Sucrose gradient fractionation. Caveolae/lipid rafts fractions wereseparated, as described previously (32, 38). Briefly, HUVECs (5.0 #107 cells) were homogenized in a solution containing 0.5 M sodiumcarbonate (pH 11), 1 mM sodium orthovanadate, and protease inhib-itors. The homogenates were adjusted to 45% sucrose by addition of90% sucrose in a buffer containing 25 mM MES (pH 6.5) and 0.15 MNaCl. A 5–35% discontinuous sucrose gradient was formed above andcentrifuged at 39,000 rpm for 16–20 h in a Beckman SW-40Ti rotor.From the top of the tube, 13 fractions were collected, and an equalvolume from each fraction was subjected to immunoblotting. Toquantify the protein expression levels in caveolae/lipid rafts andnoncaveolae/lipid rafts fractions, equal volume of fractions 4–5 werecombined for caveolae/lipid rafts and fractions 9–13 were combinedfor noncaveolae/lipid rafts.

Short interfering RNA transfection. HUVECs were grown to 40%confluence in 100-mm dishes and transfected with 10 nM controlshort intefering (si)RNA (from Ambion) and p66Shc siRNA (sense:5=-GAAUGAGUCUCUGUCAUCGCU-3= and antisense: 5=-CGAU-GACAGAGACUCAUUCCG-3= within the N;terminal CH2 domain,which is not found in p52 and p46 Shc) using Oligofectamine(Invitrogen), as described previously (52). Cells were used for exper-iments at 48 h after transfection.

Rac1 activity assay. Rac1 activation was measured using a G-LISARac activation assay kit (Cytoskeleton, Denver, CO) according to themanufacturer’s instruction. Briefly, serum-starved HUVECs werestimulated with VEGF (20 ng/ml) for 5 min. Cell lysates wereincubated on Rac-GTP affinity plates for 30 min at 4°C, and Rac1activation was measured by luminometry.

Modified Boyden chamber migration assay. Migration assays usinga modified Boyden chamber method were conducted in 24-wellTranswell chambers as described previously (49).

Wound scratch assay. HUVEC were grown to confluence in six-well plates, and a scratch was applied with a plastic pipette tip tomimic wound injury. After 24 h in a 37°C CO2 incubator in growtharrest medium containing 50 ng/ml VEGF, migrating cells wereassessed by the closure of the wound area, as described previously(21, 52).

Cell proliferation assay. HUVECs (105 cells) were seeded insixwell plates, and cell number with and without VEGF in 0.2% FBScontaining culture medium was determined by counting with a hemo-cytometer as described before (49, 52).

Capillary network formation assay. HUVECs transfected withcontrol siRNA or p66Shc siRNA were seeded on top of the thickgrowth factor-reduced Matrigel-coated wells (BD Biosciences) andincubated for 6 h at 37°C. Images were taken with a Nikon digitalcamera, and eight random fields per well were analyzed.

Fig. 1. VEGF increases Ser36 phosphorylation ofp66Shc through ERK, JNK, or PKC in endothelialcells (ECs). A: human umbilical vein ECs (HUVECs)were stimulated with VEGF (20 ng/ml) for indicatedminutes, and lysates were immunoprecipitated (IP)with anti-Shc antibody (Ab) or normal IgG (negativecontrol) and immunoblotted (IB) with anti-phospho-Ser36-p66Shc (pS36-p66Shc) Ab or Shc Ab. (n $ 3).Bottom: averaged data, expressed as fold change overbasal (means % SE). *P & 0.05 vs. control.B: HUVECs were pretreated with 20 "M LY294002(LY), 10 "M SB203580 (SB), 20 "M PD98059(PD), 20 "M SP600125 (SP), or 10 "M GF109203X(GF) for 30 min and stimulated with VEGF (20ng/ml) for 15 min. Lysates were measured for pS36-p66Shc (n $ 5). *P & 0.05 vs. vehicle 'VEGF.

H725ROLE OF p66Shc IN VEGF REDOX SIGNALING

AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00739.2011 • www.ajpheart.org

on April 30, 2012ajpheart.physiology.org

Dow

nloaded from

Statistical analysis. Results are expressed as means % SE. Statis-tical significance was assessed by Student’s paired two-tailed t-test orANOVA on untransformed data, followed by comparison of groupaverages by contrast analysis using the Super ANOVA statisticalprogram (Abacus Concepts, Berkeley, CA). A P value of &0.05 wasconsidered to be statistically significant.

RESULTS

VEGF increases Ser36 phosphorylation of p66Shc throughERK/JNK or PKC in ECs. To address the role of p66Shc inVEGF signaling, we examined the effect of VEGF on Ser36phosphorylation of p66Shc (pS36-p66Shc) in HUVECs. Figure 1Ashows that VEGF increased pS36-p66Shc within 5 min, whichpeaked at 15 min and remained above baseline for up to 60min. We examined the upstream pathways mediating pS36-p66Shc formation by VEGF. Figure 1B shows that the MEKinhibitor PD98059, the JNK inhibitor SP600125, or the proteinkinase C inhibitor GF109203X, but not the phosphatidylinosi-tol 3-kinase inhibitor LY294002 or the p38MAP kinase inhib-itor SB203580, significantly inhibited VEGF-induced p66Shcphosphorylation. These results suggest that VEGF increasespS36-p66Shc at least in part through ERK/JNK or PKC in ECs.

VEGF increases ROS production through p66Shc that as-sociates with Rac1 and regulates its activation in ECs. We nextexamined whether p66Shc is involved in VEGF-induced ROSproduction using DCF-DA that mainly detects intracellularH2O2 (32). Figure 2A shows that p66Shc siRNA selectivelyknocked down endogenous p66Shc protein expression withoutaffecting the expression of p52Shc or p46Shc protein in HUVECs.Under this condition, p66Shc siRNA significantly inhibitedVEGF-induced ROS production measured at 5 min (Fig. 2B) or30 min (not shown). We confirmed that pretreatment of ECswith polyethylene glycol-catalase before loading DCF-DAabolished the basal and VEGF-induced fluorescence signals, asreported previously (32).

Since we previously demonstrated that VEGF-induced ROSproduction is dependent on Rac1, a cytosolic component ofNADPH oxidase, we examined whether p66Shc binds to Rac1and/or involved in VEGF-induced Rac1 activation. Figure 3Ausing coimmunoprecipitation analysis shows that VEGF stim-ulation increased association of Rac1 with p66Shc within 5min, which continued above basal levels for !30 min. Rac1immunoprecipitates failed to bind to pS36-p66Shc, indicatingthat Rac1 binds to nonphosphorylated form of p66Shc. Figure3B shows that VEGF-induced Rac1 activation was signifi-cantly inhibited by p66Shc siRNA. These results suggest thatVEGF promotes p66Shc association with Rac1, which may berequired for activation of Rac1 and subsequent ROS produc-tion in ECs.

p66Shc is involved in VEGF-induced VEGFR2 autophos-phorylation in caveolae/lipid rafts and p38MAP kinase activa-tion in ECs. Since we previously demonstrated that ROSmediate VEGF-induced VEGFR2-pY, we next examined therole of p66Shc in VEGF signaling. Figure 4A shows thatknockdown of p66Shc with siRNA significantly inhibitedVEGF-induced VEGFR2-pY without affecting basal phos-phorylation or total VEGFR2 protein expression. We nextexamined the role of p66Shc in major VEGFR2 downstreamsignaling events such as p38MAPK and ERK1/2 activation,which are key mediators of VEGF-induced EC migration andproliferation, respectively (24, 40). Figure 4B shows that

p66Shc siRNA markedly inhibited VEGF-induced phosphory-lation of p38MAPK without affecting ERK1/2 phosphoryla-tion.

Since initial VEGFR2-pY increase occurs in part in cave-olae/lipid rafts (19, 23, 32) where a fraction of NADPHoxidase is activated (25, 46, 47, 53, 55), we next examined therole of p66Shc in this localized VEGFR2 activation. Sucrosegradient fractionation confirmed that VEGFR2 and Nox2 werelocalized in caveolin-1-enriched, caveolae/lipid raft fractions4–6 (Fig. 5A). Under this condition, VEGF stimulation rapidlyincreased VEGFR2-pY in caveolae/lipid rafts, which was sig-

Fig. 2. p66Shc is involved in VEGF-induced reactive oxygen species (ROS)production in ECs. A: Western blots analysis for p66Shc in HUVECs afterp66Shc short interfering (si)RNA treatment for 48 h. Lysates were immuno-blotted with anti-total Shc Ab, and the bands of p66Shc are shown at 66 kDa.B, top: dichlorofluorescein (DCF) fluorescence with DAPI staining was mea-sured by confocal microscopy in HUVECs transfected with control or p66ShcsiRNAs with or without 20 ng/ml VEGF simulation for 5 min. B, bottom:averaged data of DCF fluorescence intensity (fold change)/10 cells (n $ 3).*P & 0.05.

H726 ROLE OF p66Shc IN VEGF REDOX SIGNALING

AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00739.2011 • www.ajpheart.org

on April 30, 2012ajpheart.physiology.org

Dow

nloaded from

nificantly inhibited by p66Shc by siRNA (Fig. 5B). A parallelexperiment confirmed that p66Shc siRNA specifically knockeddown endogenous p66Shc protein expression in total lysates(not shown). These results suggest that p66Shc is involved inVEGFR2 autophosphorylation in caveolae/lipid rafts in ECs.

p66Shc is involved in VEGF-induced angiogenic relatedresponses in ECs. To determine the functional significance ofp66Shc in VEGF signaling, we examined the role of p66Shc inVEGF-induced EC migration and proliferation. Wound scratchassay (Fig. 6A) and modified Boyden chamber assay (Fig. 6B)show that depletion of p66Shc with siRNA significantly inhib-ited VEGF-induced directional EC migration. We also foundthat VEGF-induced EC proliferation was also significantlyinhibited by p66Shc siRNA (Fig. 6C). To determine the func-

tional consequence of p66Shc-mediated EC migration andproliferation, we examined the role of p66Shc in VEGF-induced capillary network formation on Matrigel. Figure 7Ashows that depletion of p66Shc significantly reduced the num-ber of capillary tube branches, branching sprouts as well astube length, suggesting that endogenous p66Shc plays animportant role in VEGF-induced angiogenic responses in ECs.

Fig. 3. VEGF stimulates p66Shc association with Rac1 and its activationthrough p66Shc in ECs. A: HUVECs were stimulated with VEGF (20 ng/ml)for indicated minutes. Cell lysates were IP with anti-Rac1 or Shc Abs ornormal IgG, and IB with anti-Shc or pS36-p66Shc Abs. Lysates without IP (NoIP) were IB with anti-Rac1 Ab. A, bottom: averaged data for fold change inp66Shc-Rac1 association, expressed as -fold change over basal. *P & 0.05.B: Rac1 activities were measured in HUVECs transfected with control orp66Shc siRNAs with or without 20 ng/ml VEGF stimulation for 5 min (n $ 3).*P & 0.05.

Fig. 4. p66Shc is involved in VEGF-induced VEGFR2 autophosphorylationand p38MAPK activation in ECs. HUVECs were transfected with control orp66Shc siRNAs. A: cells were stimulated with VEGF (20 ng/ml) for 5 min, andlysates were IP with anti-VEGFR2 Ab and IB with anti-phospho-tyrosine(pTyr) Ab. Same lysates were IB with anti-VEGFR2 or Shc Abs (n $ 4) *P &0.05. B: cells were stimulated with VEGF for indicated minutes and lysateswere IB with phospho-p38MAPK or phospho-ERK1/2 or p66Shc expression.*P & 0.05.

H727ROLE OF p66Shc IN VEGF REDOX SIGNALING

AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00739.2011 • www.ajpheart.org

on April 30, 2012ajpheart.physiology.org

Dow

nloaded from

DISCUSSION

Accumulating evidence reveals that p66Shc is a key regu-lator for various oxidative stress-dependent pathologies such asaging and cardiovascular diseases (3, 8, 10, 13, 26, 28, 29, 36).We (49) demonstrated that ROS derived from Rac1-dependentNADPH oxidase are involved in VEGFR2 autophosphoryla-tion and angiogenic-related responses in ECs. However, a roleof p66Shc in VEGF signaling and physiological responses inECs is unknown. The present study demonstrates that VEGFstimulation increases pS36-p66Shc at least in part throughERK/JNK or PKC pathway as well as p66Shc association withRac1, a component of NADPH oxidase in ECs. Moreover,depletion of p66Shc with siRNA blocks VEGF-induced Rac1activation and ROS production, thereby inhibiting VEGFR2-pYin caveolae/lipid rafts as well as p38MAPK activation. Func-tionally, p66Shc siRNA inhibits VEGF-induced EC migration,proliferation, and capillary tube formation. These findingssuggest that p66Shc functions as a positive regulator forROS-dependent VEGFR2 signaling linked to angiogenesis inECs (Fig. 7B).

p66Shc is shown to be phosphorylated at Ser36 by oxidativestress and insulin, EGF, endothelin-1 (5, 12, 20, 31), orangiotensin II (39) in various systems other than ECs. To ourknowledge, the present study provides the first evidence that akey angiogenesis growth factor, VEGF, stimulates Ser36 phos-phorylation of p66Shc. In response to stress factors, p66Shc isphosphorylated at Ser36 in N-terminal CH2 domain, which isnot found in p46Shc and p52Shc, leading to H2O2 generation(14, 35). The upstream kinases responsible for pS36-p66Shcformation have been reported in other systems, includingPKC( (14), PKC) (16), ERK (18), or JNK (1). Consistent with

previous reports, we found that inhibitors for MEK, JNK, orPKC, but not for phosphatidylinositol 3-kinase or p38MAPK,significantly reduce VEGF-induced pS36-p66Shc formation inECs. Since Ser36 in p66Shc lies in the MAPK consensusphosphorylation motif, ERK or JNK may be the kinases thatphosphorylate p66Shc. We confirmed that VEGF increasesERK and JNK phosphorylation within 5 min (unpublishedobservations). Thus our results suggest that VEGF increasespS36-p66Shc at least in part through ERK/JNK or PKC path-way in ECs. Detailed analysis of relationships among ERK,JNK, and PKC and identification of the subtypes of PKCinvolved in VEGF-induced pS36-p66Shc formation are thesubjects of future studies.

We and others (48) reported that VEGF stimulation ofECs rapidly increases ROS by activation of Rac1-dependentNADPH oxidase. In this study, we show that VEGF stimula-tion rapidly promotes Rac1 association with nonphosphory-lated form of p66Shc and that depletion of p66Shc with siRNAinhibits VEGF-induced Rac1 activation and ROS production.Given that Rac1 is a component of NADPH oxidase in ECs (2),these results suggest that p66Shc is at least involved in VEGF-induced ROS production via regulating Rac1-NADPH oxidasepathway. Consistent with our results, p66Shc is shown tomediate Rac1 activation through regulating a Rac-GEF (gua-nine nucleotide-exchange factor) such as son of sevenless 1(Sos1; Ref. 22) or (-Pix (5). Tomilov et al. (43) recentlydemonstrate that O2

! production by NADPH oxidase frommacrophage is decreased in p66Shc!/! mice. Of note, S36-p66Shc phosphorylation has been shown to be involved inmitochondrial ROS production (8, 14, 26, 35), and most recentstudy (51) shows that VEGF stimulation increases mitochon-

Fig. 5. p66Shc is involved in VEGF-induced VEGFR2autophosphorylation in caveolae/lipid rafts in ECs.A: sucrose gradient centrifugation was performed to iso-late caveolae/lipid rafts (C/LR), equal volume of eachfraction from top to bottom was IB with anti-VEGFR2,Nox2, caveolin-1, and paxillin (marker for non-C/LR)Abs. C/LR fraction were appeared in fractions 4–6. B,left: HUVECs were stimulated with or without VEGF (20ng/ml) for 5 min, and followed by C/LR fractionation.Equal amounts of proteins from C/LR fraction were IBwith anti-VEGFR2-pY1175, total VEGFR2, and caveo-lin-1 Abs (n $ 3). B, right: HUVECs transfected withcontrol or p66Shc siRNAs were stimulated with VEGF(20 ng/ml) for 5 min, and the C/LR fraction were IB withanti-VEGFR2-pY1175, total VEGFR2, and caveolin-1Abs. B, bottom: averaged data, expressed as VEGF-inducedfold change over basal in control siRNA-treated groups(means % SE). *P & 0.05.

H728 ROLE OF p66Shc IN VEGF REDOX SIGNALING

AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00739.2011 • www.ajpheart.org

on April 30, 2012ajpheart.physiology.org

Dow

nloaded from

drial ROS production in ECs. Thus one may speculate thatp66Shc binding to Rac1 is required for VEGF-inducedNADPH oxidase activation, while pS36-p66Shc is involvedin mitochondrial ROS production in ECs. This point iscurrently under investigation.

Since ROS are highly diffusible molecules, localized ROSsignal is important for efficient and specific activation of redoxsignaling events (6, 41, 47). VEGF-induced ROS are involvedin activation of VEGFR2 (48), which occurs in part at cave-olae/lipid rafts (19, 23, 32). In this study, we demonstrate thatp66Shc siRNA significantly inhibits VEGF-induced increasein VEGFR2-pY in total lysates and caveolin-enriched lipidrafts where a fraction of Nox2 is found. Given that NADPHoxidase is activated in part at lipid rafts in ECs (25, 47, 53, 55),our result suggests that thep66Shc-mediated increase in ROSthrough NADPH oxidase promotes VEGFR2-pY in these spe-cialized signaling domains. The mechanisms by which ROSmediate VEGFR2 phosphorylation remain unclear. Evidencesuggests that reversible oxidative inhibition of protein tyrosinephosphatases (PTPs) by ROS is an important mechanismthrough which ROS increase tyrosine phosphorylation-medi-ated growth factor signaling (11, 37, 44). Consistent with thisnotion, we (32) previously demonstrated that extracellular

H2O2 generated by extracellular superoxide dismutase lo-calized at caveolae/lipid rafts facilitates an increase inVEGFR2-pY via oxidative inactivation of PTPs DEP-1/PTP1B. Furthermore, Guo et al. (16) reported that the*1-adrenergic receptor promotes p66Shc-YY239/240 phos-phorylation in caveolae via a ROS-dependent manner incardiac myocytes. However, VEGF does not induce tyrosinephosphorylation of p66Shc in HUVECs (unpublished obser-vations). These results suggest that VEGF-stimulatedp66Shc/Rac1/ROS pathways may induce oxidative inacti-vation of PTPs localized at caveolae/lipid rafts, therebypromoting VEGFR2 activation.

We also examined the role of p66Shc in key VEGFR2downstream signaling kinases involved in angiogenesis includ-ing p38MAPK and ERK1/2, which mainly mediate EC migra-tion and proliferation, respectively. Knockdown of p66Shcsignificantly inhibits phosphorylation of p38MAPK but notERK1/2, by VEGF. This in turn reduces VEGF-induced ECmigration, proliferation, and capillary network formation, akey component of angiogenesis in ECs. Consistent with ourdata, a previous report (50) shows that p66Shc is involved inandrogen-induced prostate cancer cell proliferation. The lackof p66Shc siRNA effect on VEGF-induced ERK activation

Fig. 6. p66Shc is involved in VEGF-induced cell migration in ECs. HUVECs transfected with control or p66Shc siRNAs were used for measurement of ECmigration using modified Boyden chamber assay (A) or wound scratch assay (B) as well as EC proliferation (C). A: cell migration stimulated with VEGF for6 h was measured using modified Boyden chamber method. Bar graph represents averaged data, expressed as cell number counted per 10 fields (x 200) and foldchange over that in unstimulated cells (control). *P & 0.05. B: confluent monolayer of HUVECs were wound scratched in the presence of VEGF (20 ng/ml) tostimulate the EC migration toward the wound area. Images were captured immediately (0 h) and 24 h after the wound scratch. C: cells were cultured in 0.2%FBS containing medium with or without 20 ng/ml VEGF for 48 h and the cell number was measured. *P & 0.05, for the fold increase induced by VEGF vs.vehicle alone (n $ 3).

H729ROLE OF p66Shc IN VEGF REDOX SIGNALING

AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00739.2011 • www.ajpheart.org

on April 30, 2012ajpheart.physiology.org

Dow

nloaded from

suggests that other p66Shc downstream targets linked to ECproliferation than ERK1/2 may exist. Akt and/or forkheadrelated transcription factors FOXO3a/FKHRL1 (5, 16, 18, 30)or mammalian target of rapamycin-S6 kinase (33) will beadditional possible targets of p66Shc in VEGF signaling.

Previous studies (3, 4, 8, 10, 14, 26, 28, 35, 36) usingp66Shc knockout or knockdown approaches implicate a path-ological role of p66Shc in mediating apoptosis and endothelialdysfunction involved in oxidative stress-associated aging andcardiovascular diseases. Indeed, p66Shc activation has beenshown to induce oxidative stress and endothelial dysfunction,while p66Shc inhibition exhibits vasoprotective effects (3, 13).By contrast, our findings suggest a physiological function ofp66Shc in angiogenic signaling in ECs. Consistent with ourdata, Zaccagnini et al. (54) reported that p66Shc!/! micesubjected to ischemia/reperfusion show a more decreased cap-illary density in skeletal muscles than wild-type mice. Naldiniet al. (27) showed that hypoxia-induced angiogenic response isimpaired in p66Shc!/! T cells. Given that p66Shc!/! mice arenot embryonic lethal, these results suggest that p66Shc isinvolved in postnatal angiogenesis in response to injury but notdevelopmental angiogenesis. An opposite role of p66Shc maybe due to the fact that intracellular ROS act as a double-edgedsword depending on their concentration. In other words, a lowconcentration of ROS is required for biological responses suchas cell migration, while excess an amount of ROS produced inpathological conditions induce apoptosis or cytotoxic effects.Both cases may be mediated through p66Shc. Thus, under thecondition in which VEGF increases ROS that act as signalingmolecules in VEGFR2 signaling, p66Shc functions as a posi-tive regulator for angiogenic-related responses in ECs.

In summary, we demonstrate that VEGF stimulation pro-motes pS36-pp66Shc formation at least in part through theERK/JNK or PKC pathway as well as association of a non-phosphorylated form of p66Shc with Rac1 in ECs. We alsofound that p66Shc is involved in VEGF-induced Rac1 activa-tion and ROS production, thereby promoting VEGFR2 activa-tion in caveolae/lipid rafts, which in turn stimulates angiogenicresponses in ECs (Fig. 7B). These findings suggest that p66Shcis a key positive regulator of VEGF-induced ROS productionand VEGFR2-mediated redox signaling linked to angiogenesisin ECs. Our study should provide insight into p66Shc as apotential therapeutic target for various angiogenesis-dependentdiseases.

GRANTS

This research was supported by National Heart, Lung, and Blood InstituteGrants R01-HL-077524 and HL-077524-S1 (to M. Ushio-Fukai) and HL-070187 (to T. Fukai), American Heart Association National Council onRadiation Protection and Measurements Innovative Research Grant 0970336N(to M. Ushio-Fukai), and Uehara Memorial Foundation and Naito Foundation(to J. Oshikawa).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: J.O., S.-J.K., C.C., G.-F.C., T.F., and M.U.-F. con-ception and design of research; J.O., S.-J.K., E.F., C.C., G.-F.C., R.D.M., F.K.,and I.L. performed experiments; J.O., S.-J.K., E.F., C.C., G.-F.C., R.D.M.,F.K., and I.L. analyzed data; J.O., S.-J.K., C.C., G.-F.C., F.K., I.L., T.F., andM.U.-F. interpreted results of experiments; J.O., S.-J.K., E.F., G.-F.C., andM.U.-F. prepared figures; J.O. and M.U.-F. drafted manuscript; J.O., S.-J.K.,G.-F.C., T.F., and M.U.-F. edited and revised manuscript; J.O., S.-J.K., E.F.,C.C., G.-F.C., R.D.M., F.K., I.L., T.F., and M.U.-F. approved final version ofmanuscript.

Fig. 7. Role of p66Shc in capillary-like network formation in ECs andproposed model. A: HUVECs were seeded on Matrigel-coated plates in culturemedia containing VEGF for 6 h. Eight random fields per well were imaged,and representative pictures are shown (top). Averaged numbers of capillarytube branches, branching points, and tube length per field are shown (bottom;n $ 3). P & 0.05. B: proposed model for novel role of p66Shc in ROS-dependent VEGFR2 signaling and angiogenesis in ECs. VEGF stimulationpromotes pS36-pp66Shc formation through ERK/JNK/PKC as well as associ-ation of nonphosphorylated form of p66Shc with Rac1, a component of Nox2NADPH oxidase in ECs. p66Shc is involved in VEGF-induced Rac1 activationand ROS production, thereby promoting VEGFR2 activation in caveolae/lipidrafts, which in turn stimulates angiogenic responses in ECs.

H730 ROLE OF p66Shc IN VEGF REDOX SIGNALING

AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00739.2011 • www.ajpheart.org

on April 30, 2012ajpheart.physiology.org

Dow

nloaded from

REFERENCES

1. Arany I, Faisal A, Nagamine Y, Safirstein RL. p66Shc inhibits pro-survival epidermal growth factor receptor/ERK signaling during severeoxidative stress in mouse renal proximal tubule cells. J Biol Chem 283:6110–6117, 2008.

2. Babior BM. The NADPH oxidase of endothelial cells. IUBMB Life 50:267–269, 2000.

3. Camici GG, Schiavoni M, Francia P, Bachschmid M, Martin-PaduraI, Hersberger M, Tanner FC, Pelicci P, Volpe M, Anversa P, LuscherTF, Cosentino F. Genetic deletion of p66(Shc) adaptor protein preventshyperglycemia-induced endothelial dysfunction and oxidative stress. ProcNatl Acad Sci USA 104: 5217–5222, 2007.

4. Carpi A, Menabo R, Kaludercic N, Pelicci P, Di Lisa F, Giorgio M.The cardioprotective effects elicited by p66(Shc) ablation demonstrate thecrucial role of mitochondrial ROS formation in ischemia/reperfusioninjury. Biochim Biophys Acta 1787: 774–780, 2009.

5. Chahdi A, Sorokin A. Endothelin-1 couples betaPix to p66Shc: role ofbetaPix in cell proliferation through FOXO3a phosphorylation andp27kip1 down-regulation independently of Akt. Mol Biol Cell 19: 2609–2619, 2008.

6. Chen K, Craige SE, Keaney JF Jr. Downstream targets and intracellularcompartmentalization in Nox signaling. Antioxid Redox Signal 11: 2467–2480, 2009.

7. Colavitti R, Pani G, Bedogni B, Anzevino R, Borrello S, WaltenbergerJ, Galeotti T. Reactive oxygen species as downstream mediators ofangiogenic signaling by vascular endothelial growth factor receptor-2/KDR. J Biol Chem 277: 3101–3108, 2002.

8. Cosentino F, Francia P, Camici GG, Pelicci PG, Luscher TF, Volpe M.Final common molecular pathways of aging and cardiovascular disease:role of the p66Shc protein. Arterioscler Thromb Vasc Biol 28: 622–628,2008.

9. Ebrahimian TG, Heymes C, You D, Blanc-Brude O, Mees B, WaeckelL, Duriez M, Vilar J, Brandes RP, Levy BI, Shah AM, Silvestre JS.NADPH oxidase-derived overproduction of reactive oxygen species im-pairs postischemic neovascularization in mice with type 1 diabetes. Am JPathol 169: 719–728, 2006.

10. Fadini GP, Ceolotto G, Pagnin E, de Kreutzenberg S, Avogaro A. Atthe crossroads of longevity and metabolism: the metabolic syndrome andlife span determinant pathways. Aging Cell 10: 10–17.

11. Finkel T. Signal transduction by reactive oxygen species in non-phago-cytic cells. J Leukoc Biol 65: 337–340, 1999.

12. Foschi M, Franchi F, Han J, La Villa G, Sorokin A. Endothelin-1induces serine phosphorylation of the adaptor protein p66Shc and itsassociation with 14–3-3 protein in glomerular mesangial cells. J BiolChem 276: 26640–26647, 2001.

13. Francia P, delli Gatti C, Bachschmid M, Martin-Padura I, Savoia C,Migliaccio E, Pelicci PG, Schiavoni M, Luscher TF, Volpe M, Cosen-tino F. Deletion of p66shc gene protects against age-related endothelialdysfunction. Circulation 110: 2889–2895, 2004.

14. Giorgio M, Migliaccio E, Orsini F, Paolucci D, Moroni M, Contursi C,Pelliccia G, Luzi L, Minucci S, Marcaccio M, Pinton P, Rizzuto R,Bernardi P, Paolucci F, Pelicci PG. Electron transfer between cyto-chrome c and p66Shc generates reactive oxygen species that triggermitochondrial apoptosis. Cell 122: 221–233, 2005.

15. Griendling KK, Sorescu D, Ushio-Fukai M. NAD(P)H oxidase: role incardiovascular biology and disease. Circ Res 86: 494–501, 2000.

16. Guo J, Gertsberg Z, Ozgen N, Steinberg SF. p66Shc links alpha1-adrenergic receptors to a reactive oxygen species-dependent AKT-FOXO3A phosphorylation pathway in cardiomyocytes. Circ Res 104:660–669, 2009.

17. Haddad P, Dussault S, Groleau J, Turgeon J, Michaud SE, Menard C,Perez G, Maingrette F, Rivard A. Nox2-containing NADPH oxidasedeficiency confers protection from hindlimb ischemia in conditions ofincreased oxidative stress. Arterioscler Thromb Vasc Biol 29: 1522–1528,2009.

18. Hu Y, Wang X, Zeng L, Cai DY, Sabapathy K, Goff SP, Firpo EJ, LiB. ERK phosphorylates p66shcA on Ser36 and subsequently regulatesp27kip1 expression via the Akt-FOXO3a pathway: implication of p27kip1in cell response to oxidative stress. Mol Biol Cell 16: 3705–3718, 2005.

19. Ikeda S, Ushio-Fukai M, Zuo L, Tojo T, Dikalov S, Patrushev NA,Alexander RW. Novel role of ARF6 in vascular endothelial growthfactor-induced signaling and angiogenesis. Circ Res 96: 467–475, 2005.

20. Kao AW, Waters SB, Okada S, Pessin JE. Insulin stimulates thephosphorylation of the 66- and 52-kilodalton Shc isoforms by distinctpathways. Endocrinology 138: 2474–2480, 1997.

21. Kaplan N, Urao N, Furuta E, Kim SJ, Razvi M, Nakamura Y,McKinney RD, Poole LB, Fukai T, Ushio-Fukai M. Localized cysteinesulfenic acid formation by vascular endothelial growth factor: role inendothelial cell migration and angiogenesis. Free Radic Res 45: 1124–1135, 2011.

22. Khanday FA, Santhanam L, Kasuno K, Yamamori T, Naqvi A,Dericco J, Bugayenko A, Mattagajasingh I, Disanza A, Scita G, IraniK. Sos-mediated activation of rac1 by p66shc. J Cell Biol 172: 817–822,2006.

23. Labrecque L, Royal I, Surprenant DS, Patterson C, Gingras D,Beliveau R. Regulation of vascular endothelial growth factor receptor-2activity by caveolin-1 and plasma membrane cholesterol. Mol Biol Cell14: 334–347, 2003.

24. Lamalice L, Houle F, Jourdan G, Huot J. Phosphorylation of tyrosine1214 on VEGFR2 is required for VEGF-induced activation of Cdc42upstream of SAPK2/p38. Oncogene 23: 434–445, 2004.

25. Li PL, Gulbins E. Lipid rafts and redox signaling. Antioxid Redox Signal9: 1411–1415, 2007.

26. Migliaccio E, Giorgio M, Mele S, Pelicci G, Reboldi P, Pandolfi PP,Lanfrancone L, Pelicci PG. The p66shc adaptor protein controls oxida-tive stress response and life span in mammals. Nature 402: 309–313,1999.

27. Naldini A, Morena E, Pucci A, Pellegrini M, Baldari CT, Pelicci PG,Presta M, Ribatti D, Carraro F. The adaptor protein p66Shc is a positiveregulator in the angiogenic response induced by hypoxic T cells. J LeukocBiol 87: 365–369, 2010.

28. Napoli C, Martin-Padura I, de Nigris F, Giorgio M, Mansueto G,Somma P, Condorelli M, Sica G, De Rosa G, Pelicci P. Deletion of thep66Shc longevity gene reduces systemic and tissue oxidative stress,vascular cell apoptosis, and early atherogenesis in mice fed a high-fat diet.Proc Natl Acad Sci USA 100: 2112–2116, 2003.

29. Nemoto S, Combs CA, French S, Ahn BH, Fergusson MM, BalabanRS, Finkel T. The mammalian longevity-associated gene product p66shcregulates mitochondrial metabolism. J Biol Chem 281: 10555–10560,2006.

30. Nemoto S, Finkel T. Redox regulation of forkhead proteins through ap66shc-dependent signaling pathway. Science 295: 2450–2452, 2002.

31. Okada S, Kao AW, Ceresa BP, Blaikie P, Margolis B, Pessin JE. The66-kDa Shc isoform is a negative regulator of the epidermal growthfactor-stimulated mitogen-activated protein kinase pathway. J Biol Chem272: 28042–28049, 1997.

32. Oshikawa J, Urao N, Kim HW, Kaplan N, Razvi M, McKinney R,Poole LB, Fukai T, Ushio-Fukai M. Extracellular SOD-derived H2O2promotes VEGF signaling in caveolae/lipid rafts and postischemic angio-genesis in mice. PLos One 5: e10189, 2010.

33. Pani G. P66SHC and ageing: ROS and TOR? Aging (Albany, NY) 2:514–518, 2010.

34. Pelicci G, Lanfrancone L, Grignani F, McGlade J, Cavallo F, Forni G,Nicoletti I, Grignani F, Pawson T, Pelicci PG. A novel transformingprotein (SHC) with an SH2 domain is implicated in mitogenic signaltransduction. Cell 70: 93–104, 1992.

35. Pinton P, Rimessi A, Marchi S, Orsini F, Migliaccio E, Giorgio M,Contursi C, Minucci S, Mantovani F, Wieckowski MR, Del Sal G,Pelicci PG, Rizzuto R. Protein kinase C beta and prolyl isomerase 1regulate mitochondrial effects of the life-span determinant p66Shc. Sci-ence 315: 659–663, 2007.

36. Ranieri SC, Fusco S, Panieri E, Labate V, Mele M, Tesori V, FerraraAM, Maulucci G, De Spirito M, Martorana GE, Galeotti T, Pani G.Mammalian life-span determinant p66shcA mediates obesity-induced in-sulin resistance. Proc Natl Acad Sci USA 107: 13420–13425, 2010.

37. Rhee SG, Bae YS, Lee SR, Kwon J. Hydrogen peroxide: a key messen-ger that modulates protein phosphorylation through cysteine oxidation. SciSTKE 2000: PE1, 2000.

38. Song KS, Li S, Okamoto T, Quilliam LA, Sargiacomo M, Lisanti MP.Co-purification and direct interaction of Ras with caveolin, an integralmembrane protein of caveolae microdomains. Detergent-free purificationof caveolae microdomains. J Biol Chem 271: 9690–9697, 1996.

39. Sun L, Xiao L, Nie J, Liu FY, Ling GH, Zhu XJ, Tang WB, Chen WC,Xia YC, Zhan M, Ma MM, Peng YM, Liu H, Liu YH, Kanwar YS.p66Shc mediates high-glucose and angiotensin II-induced oxidative stress

H731ROLE OF p66Shc IN VEGF REDOX SIGNALING

AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00739.2011 • www.ajpheart.org

on April 30, 2012ajpheart.physiology.org

Dow

nloaded from

renal tubular injury via mitochondrial-dependent apoptotic pathway. Am JPhysiol Renal Physiol 299: F1014–F1025, 2010.

40. Takahashi T, Yamaguchi S, Chida K, Shibuya M. A single autophos-phorylation site on KDR/Flk-1 is essential for VEGF-A-dependent acti-vation of PLC-gamma and DNA synthesis in vascular endothelial cells.EMBO J 20: 2768–2778, 2001.

41. Terada LS. Specificity in reactive oxidant signaling: think globally, actlocally. J Cell Biol 174: 615–623, 2006.

42. Tojo T, Ushio-Fukai M, Yamaoka-Tojo M, Ikeda S, Patrushev NA,Alexander RW. Role of gp91phox (Nox2)-containing NAD(P)H oxidasein angiogenesis in response to hindlimb ischemia. Circulation 111: 2347–2355, 2005.

43. Tomilov AA, Bicocca V, Schoenfeld RA, Giorgio M, Migliaccio E,Ramsey JJ, Hagopian K, Pelicci PG, Cortopassi GA. Decreased super-oxide production in macrophages of long-lived p66Shc knock-out mice. JBiol Chem 285: 1153–1165.

44. Tonks NK. Redox redux: revisiting PTPs and the control of cell signaling.Cell 121: 667–670, 2005.

45. Urao N, Inomata H, Razvi M, Kim HW, Wary K, McKinney R, FukaiT,Ushio-Fukai M. Role of nox2-based NADPH oxidase in bone marrowand progenitor cell function involved in neovascularization induced byhindlimb ischemia. Circ Res 103: 212–220, 2008.

46. Ushio-Fukai M. Compartmentalization of redox signaling through NADPHoxidase-derived ROS. Antioxid Redox Signal 11: 1289–1299, 2009.

47. Ushio-Fukai M. Localizing NADPH oxidase-derived ROS. Sci STKE2006: re8, 2006.

48. Ushio-Fukai M. VEGF signaling through NADPH oxidase-derived ROS.Antioxid Redox Signal 9: 731–739, 2007.

49. Ushio-Fukai M, Tang Y, Fukai T, Dikalov SI, Ma Y, Fujimoto M,Quinn MT, Pagano PJ, Johnson C, Alexander RW. Novel role ofgp91(phox)-containing NAD(P)H oxidase in vascular endothelial growthfactor-induced signaling and angiogenesis. Circ Res 91: 1160–1167, 2002.

50. Veeramani S, Yuan TC, Lin FF, Lin MF. Mitochondrial redox signalingby p66Shc is involved in regulating androgenic growth stimulation ofhuman prostate cancer cells. Oncogene 27: 5057–5068, 2008.

51. Wang Y, Zang QS, Liu Z, Wu Q, Maass D, Dulan G, Shaul PW,Melito L, Frantz DE, Kilgore JA, Williams NS, Terada LS, NwariakuFE. Regulation of VEGF induced endothelial cell migration by mitochon-drial reactive oxygen species. Am J Physiol Cell Physiol 301: C695–C704,2011.

52. Yamaoka-Tojo M, Ushio-Fukai M, Hilenski L, Dikalov SI, Chen YE,Tojo T, Fukai T, Fujimoto M, Patrushev NA, Wang N, Kontos CD,Bloom GS, Alexander RW. IQGAP1, a novel vascular endothelialgrowth factor receptor binding protein, is involved in reactive oxygenspecies-dependent endothelial migration and proliferation. Circ Res 95:276–283, 2004.

53. Yang B, Oo TN, Rizzo V. Lipid rafts mediate H2O2 prosurvival effectsin cultured endothelial cells. FASEB J 20: 1501–1503, 2006.

54. Zaccagnini G, Martelli F, Fasanaro P, Magenta A, Gaetano C, DiCarlo A, Biglioli P, Giorgio M, Martin-Padura I, Pelicci PG, Capo-grossi MC. p66ShcA modulates tissue response to hindlimb ischemia.Circulation 109: 2917–2923, 2004.

55. Zhang AY, Yi F, Zhang G, Gulbins E, Li PL. Lipid raft clustering andredox signaling platform formation in coronary arterial endothelial cells.Hypertension 47: 74–80, 2006.

H732 ROLE OF p66Shc IN VEGF REDOX SIGNALING

AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00739.2011 • www.ajpheart.org

on April 30, 2012ajpheart.physiology.org

Dow

nloaded from