PTP1b is a physiologic regulator of vascular endothelial growth factor signaling in endothelial cells.

PTP1b is a Physiologic Regulator of VEGF Signaling in Endothelial Cells Anthony A. Lanahan, Diana Lech, Alexandre Dubrac, Jiasheng Zhang, Zhen W. Zhuang, Anne Eichmann and Michael Simons Circulation. published online June 30, 2014; Circulation is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 2014 American Heart Association, Inc. All rights reserved. Print ISSN: 0009-7322. Online ISSN: 1524-4539

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DOI: 10.1161/CIRCULATIONAHA.114.009683

PTP1b is a Physiologic Regulator of VEGF Signaling in Endothelial Cells

Running title: Lanahan et al.; PTP1b regulation of VEGF signaling

Anthony A. Lanahan, PhD1; Diana Lech, BA1; Alexandre Dubrac, PhD1; Jiasheng Zhang, PhD1; D1,2 Zhen W. Zhuang, MD1; Anne Eichmann, PhD1; Michael Simons, MD

1

Yale Ya lee Cardiovascular Carrdi d ovvas ascu cular Research Center, Section Sectio on of o Cardiovascularr Medicine, Med edicine, Dept of Internal Medicine; Medici ine ne;; 2De Dept D ptt of of Cell Cell Biology, Bio iolo logy lo gy,, Yale gy Y lee University Ya Uni niveersityy School Scho Sc hool ho o of ol of Medicine, Medi Medi dici cine ci n , New New Haven, H ve Ha ven, n, C CT T

Address Addr Ad dres dr esss for es for Correspondence: Corr Co rres rr espo es pond po nden nd ence en ce:: ce Michael Mich Mi chae aell Simons, Simo Si mons ns, MD Yale Cardiovascular Research Center 300 George St. New Haven, CT 06520 Tel: 203-785-7000 Fax 203-785-5144 E-mail: [email protected]

Journal Subject Codes: Vascular biology:[96] Mechanism of atherosclerosis/growth factors, Vascular biology:[97] Other vascular biology

1 Downloaded from http://circ.ahajournals.org/ at Universite de Sherbrooke on July 1, 2014


Abstract

Background—Regulation of vascular endothelial growth factor receptor-2 (VEGFR2) signaling is a control point that determines the extent of vascular tree formation. Recent studies demonstrated an important role played by VEGFR2 endothelial trafficking in control of its activity and suggested the involvement of a phosphotyrosine phosphatase 1b (PTP1b) in this process. This study was designed to define the role of PTP1b in endothelial VEGFR2 signaling and its role in regulation of angiogenesis and arteriogenesis. Methods and Results—We generated mice carrying an endothelial-specific deletion of PTP1b and examined the effect of this knockout on VEGF signaling, angiogenesis and arte aarteriogenesis rteeriog ogen og enes en esis es is in vitro and in vivo. PTP1b knockout endothelial cells had increased VEGF-dependent activation of ERK proliferation ER RK signaling, sign si gnnal alin ing, ssprouting, in proouting, migration and prolifer pr erration compared to to controls. con onntr trol o s. Endothelial PTP1b mice increased retinal and Matrigel implant angiogenesis, accelerated wound healing nnull ulll m ice had in incr c ea cr ease seed re reti tina ti nall an na nd Ma Matr rigell im mpllan nt an ngiog giog genessis is,, ac acc celleraate tedd wo ounnd he eal alin ingg in pointing enhanced arteriogenesis was poin po inti in ting ti ng tto o en enha hanc nced nc ed aangiogenesis. nggio ioge geeneesi sis. s. IIncreased nccreeas aseed ar arte teeriiog o en enes esiis es is w ass ddemonstrated emoonsstra em strate t d by observations te obseervvati vatioons ons recovery arterial large numbers arterioles of faster reco ove very ry ooff ar arte teriial bblood te lo ood fflow l w an lo andd la larg rgee nu rg numb mb ber e s of o nnewly ewly ew ly fformed orme or medd ar me rteeri riol o es ol e in the hindlimb ischemia mouse model. PTP1b endothelial knockout also rescued impaired blood flow recovery after common femoral artery ligation in synectin null mice. Conclusions—PTP1b is a key regulator of endothelial VEGFR2 signaling and plays an important role in regulation of the extent of vascular tree formation.

Key words: angiogenesis, endothelial cell, vascular endothelial growth factor receptor, vascular endothelial growth factor, arteriogenesis



Introduction Recent studies involving signal transduction by vascular endothelial growth factor (VEGF)-A in endothelial cells demonstrated the existence of an important regulatory mechanism involving intracellular trafficking of VEGF receptor-2 (VEGFR2). 1 The presumed role of this event is movement of a VEGFR2-containing endosome from a phosphatase-rich environment to the part of the cytoplasm where VEGFR2 is not being actively dephosphorylated. In particular, this mechanism plays a critical role in VEGF-induced ERK-1/2 activation that has been linked to the development of arterial circulation (arteriogenesis).2-5 VEGF-induced ERK activation involves ligand-driven dimerization of thee rreceptor ecep ec ep pto torr leading eading to trans- phosphorylation of C-loop activation loop tyrosines 1054 and 11059. 0559. T This, hiss, iin hi n turn, urn, leads to phosp phosphorylation sphorylation of a number off cyt cytoplasmic y opplasmic domain tyrosines including the Y1175 1175 75 ite,, ph phosph phor oryl or ylat attio i n of w h ch iiss re hi rrequired quir qu ired ed ffor or E RK K activ iv vat atio ion. io n 6, 7 O Once ncee phosphorylated, nc p ossph ph phor oryl or y at yl ated ed,, Y117 site, phosphorylation which ERK activation.

serves erv ves e as a bi bind binding n ing sit site te ffor or P PLCȖ LCȖ and LCȖ nd ssubsequent ubseqquent qu ac activation cti tiva vati va tion ti on ooff the th he Raf1/MEK/ERK Raf11/M MEK EK/E /ERK K ccascade. asscaade.. unusual aspect this process requirement VEGFR2 Onee un unusua ual as aspe p ct ooff th his pro roce c ss is thee re requ quir irem ementt fo forr VE VEGF G R2 pphosphorylation hosppho ho h ry ryla lati tion aand n nd trafficking. raf affi fick ckin ingg IIn n th thee ab abse absence senc ncee of V VEGFR2 EGFR EG FR22 en endo endocytosis, docy cyto tosi siss ER ERK K ac acti activation tiva vati tion on iiss pr prac practically acti ticcal ally ly aabsent bsen bs entt as sseen eenn ee following knockouts of EphrinB2, aPKC or PAR3. A complete, or nearly complete, lack of ERK activation leads to a total failure of vascular development as seen in VEGFR2 knockouts, mice carrying a Y1175F knock-in mutation or mice with knockouts of EphrinB2, aPKCs or PAR3. 8-10 Once endocytosed, VEGRF2 undergoes an important trafficking step that is essential for full activation of the ERK signaling cascade. Delayed trafficking of the receptor from clathrincoated pits to EEA1 early endosomes reduces the extent of ERK activation due to partial dephosphorylation of its Y1175 site. The trafficking event requires formation of a neuropilin-1



(Nrp1, another VEGF-A receptor)-synectin-myosin-VI complex. A knockout of any of these genes or a knock-in of Nrp1 missing its PDZ domain (Nrp1cyto) that is required for its binding to the PDZ scaffold protein synectin (Gipc1), results in reduced Y1175 phosphorylation and decreased ERK activation. In vivo this manifests itself as impaired embryonic and adult arteriogenesis including reduced arterial vessel density, branching and decreased lumen diameter. 4, 5 This trafficking-dependent phosphorylation of VEGFR2 suggests the existence of a phosphatase that dephosphorylates the Y1175 site. Studies using in vitro siRNA knockouts of various protein tyrosine phosphatase in endothelial cells with impaired VEGFR2 trafficking (synectin synectin or myosin-VI null knockouts) demonstrate that only phosphotyrosine ph phosphatase hos osph ph hat atas asee 1b as (PTP1b) PTP1b) knockdown rescues VEGF-induced ERK activation. 5 In agreement with this is the ob bse serv rv vat atio ion io n th thatt ooverexpression ve verexpression of PTP1b in norm rmaal endothelial ccells rm e ls rreduces, el educ ed u es, while knockdown observation normal increases, ncrrea e ses, VEG VEGF-induced EG GFF indu duceed ER ERK K ac activation ctiivat vatioon 111 while whilee iinhibition nhhib ibit itio io on of P PTP1b TP1b aactivity TP1b cti tivvit vity ty uusing s ng a cchemical si hemi he m cal mi inhibitor nhi hibi bito bi torr re to rescues esc scue uess bblood loo oodd flow flow rrecovery eccov over eryy an er andd ar arte arteriogenesis eri riog ogeenessiss iin og n th the he hind hhindlimb ind ndli l mb iischemia li sche sc hemi he mam mi model oddell in Gipc1-/-/ mice. e.5 F Finally, in nal ally ly,, VEGFR2 ly VEGF VE GFR2 GF R2 can can be be immunoprecipitated immu im muno mu nopr no prec pr ecip ec ipit itat it ated at ed w with ithh PT it PTP1 PTP1b P1bb 11 aand P1 nd tthe he ttwo w proteinss wo have been visualized together on Rab5 endosomes using structured illumination microscopy (SIM) super-resolution imaging.12 These data point to PTP1b as the likely phosphatase regulating VEGF-induced ERK activation. However, whether PTP1b actually normally regulates VEGFR2 signaling in vivo in endothelial cell-specific manner and plays a role in its various biological activities remains unknown. In this study we set out to examine this question by selectively deleting the phosphatase in mouse endothelial cells. We find that endothelial-specific PTP1b (PtpN1ECKO) deletion is associated with increased ERK signaling and increased angiogenesis and



arteriogenesis. These results demonstrate that PTP1b is an important endogenous endothelial cell regulator of VEGF-A signaling.

Materials and Methods Reagents and Antibodies Human fibronectin (BD Biosciences#356008), Matrigel Basement Membrane Matrix Growth Factor Reduced (BD#354230), IGF-1(Sigma), VEGF-A165 (#293-VE R&D systems), Antibodies: anti-CD31 (BD#55370)anti-VE-cadherin (Santa Cruz#SC6458), anti-phospho p44/42 MAP kinase (phospho-ERK, Cell Signaling#9106), anti-p44/42 MAP kinase (total ERK, Cell Signaling#9102), anti-PTP1b (Santa Cruz#sc-1718), anti phospho IGF-1 Re Rece cept p or pt Receptor Beta(Tyr1131) (CS#3921), anti IGF-1 Receptor Beta (CS#3027). We used appropriate secondary antibodies an ntiibo bodi dies di ess tthat hat were ha werre we re conjugated to horseradish pperoxidase e oxidase (Vecto er (Vector or Lab Laboratories) ab bor oraatories) or fluorescently labeled abeele l d (Life Te Tech Technologies). chno ch nolo l gi lo gies ess). ). IIsolectinB4 soleect sole ctin i B4 wa in was as ppurchased urch chaseed ch ed ffrom ro om M Molecular olecu cula l r Pr la Prob Probes obes ob es ((Invitrogen). Invi In vitr vi trrog gen en). ) Mouse Mous Mo usee ge us generation gene nerrati ne ration on n oxP/ P/lloxP P/ P To obtain mi mice ice hhomozygous omoz om ozzyg y ou us fo fforr a co cond conditional nd dit i io iona nall al na aallele lele le l ooff PT le PTP1 PTP1b(Ptpn1) P1b( P1 b(Pt b( (Pt Ptpn pn n1) Ptpn1 Ptp tpn1 n1lloxP/loxP mice m mi ce13 were

crossed with Cdh5-CreERT2 mice (gift of R. Adams)10. Cre activity was induced in 3 week old mice by 5 consecutive daily intraperitoneal injections of tamoxifen (0.1ml of a 20mg/ml solution in corn oil). The phenotypes of mutant mice were analyzed at 6-10 weeks of age and littermate animals were used as controls. All animal experiments were performed under a protocol approved by the Institutional Animal Care and Use Committee of Yale University. Isolation of murine endothelial cells Primary arterial endothelial cells were isolated from adult mouse dorsal aorta as previously described. 14 Arteries from 4 mice were harvested, pooled, minced with scissors and then



digested in 25ml collagenase 0.2% (w/v) at 37oC for 45 min. The crude cell preparation was pelleted and resuspended in DPBS. The cell suspension was incubated with PECAM-1-coated beads (IgG Dynal beads from Dynal Corp., Great Neck, NY) at room temperature for 10min. with end-over-end rotation. Using a magnetic separator, the bead-bound cells were recovered, washed andresuspended in complete culture medium[DMEM containing 20% fetal calf serum, supplemented with 100ug/ml heparin, 100ug/ml ECGF growth supplement (ECGF: Biomedical Technologies, Stoughton, MA), and nonessential amino acids, sodium pyruvate, L-glutamine, and antibiotics at standard concentrations], and then plated in a 0.1% gelatin-coated 10cm tissue culture dish. Migration and proliferation of endothelial cells The xCELLigence RTCA DP analyzer was used to measure migration, adhesion and pr rollif ifer erat atio tio ion n ooff w ildt il d ype and PTP1b knockout ccells el s in response too 0.5% ells 5% % FBS, FBS, 20% FBS, VEGFproliferation wildtype A (1100ng/ml) 10 andd FGF2(100ng/ml). an FGF2 FG F (1 F2 (100 00ng 00 ng/m ng /ml) l).. For l) Forr migration mig ig gratioon the thee CIM C IM-Pl M-Pl P atte 16 was was uused, sed, sed d, tthe he bbottom he ottto om A(100ng/ml) CIM-Plate ch ham ambe berr co be ont ntai ainnedd th thee gr grow owth t ffactor th acto ac torr and to and 40 40,0 000 0 ce cel llss we were re aadded ddeed dd ed tto o th he to topp chamber ch ham mbe ber and and the th he chamber contained growth 40,000 cells the plate monito oreed ev ver eryy 5 mi inu nute tess fo fforr 24 4 hhours. ours ou rs.. F rs or pr prol olif ifer if erat er atio at io on an aand d ad adhe hesi he sion si o tthe on he E -P Plate 16 wass monitored every minutes For proliferation adhesion E-Plate used, the plates were coated with 0.1% gelatin and 5000 cells per well were added to media containing the growth factor. For proliferation the plate was monitored every 15 minutes for 80 hours and for adhesion the plate was monitored every 5 minutes for 4 hours. For each condition at least 4 replicate wells were used. siRNA Transfection siRNAs (FlexiTube siRNA) were purchased from QIAGEN. HUVECs were transfected with 25 pmol siRNA per 6-well with 2.5 ml RNAiMax (Invitrogen) according to the instructions of the manufacturer. Cells were used for experiments 48 hr after transfection.



In Vitro EC Cord Formation Cells were starved overnight in 0.5% FBS, detached with trypsin and seeded at a density of 60,000 cells per well in a 24 well plate containing 0.5ml per well of reduced-growth factor matrigel (BD Biosciences). Cells were in media containing 0.5% FBS, VEGF-A(100ng/ml) or FGF2(100ng/ml); 1ml per well. After 8 hours cells were fixed with 4% paraformaldehyde and 5 random images were taken of each well (10X objective) and the total length of cord formation was quantified for each field. The mean of 10 total lengths per well represent an experimental point. Cord length was assessed using the NeuronJ plug-in of ImageJ. In Vitro Sprouting Assay resuspended After siRNA transfection, HUVECs (250,000 cells/well in 6-well plates) were re esu susp s en ende dedd in de fibrinogen solution (2.5 mg/ml fibrinogen [Sigma-Aldrich] in EBM-2 [Lonza] supplemented with wi th h 2% 2% FBS FBS and an nd 50 mg/ml aprotinin [Sigma-Aldrich]), [Sigma-Al Aldr Al d ich]), and plated dr platted e onn top top of a precoated fibrin layer (fibrinogen with [Sigma-Aldrich] ayeer (fibrinoge genn ssolution olu luti t on on cclotted lo ott tted ed w i h 1 U tthrombin it hromb binn [S Sigm Sigm gmaa-Al Alddriich] ffor orr 220 0 min min at a 337_C). 7_C) 7_ C). Th C) Thee second was clotted Wi-38 cells/well), EBM-2 eco cond nd llayer ay yer ooff fib ffibrin ibrin brin w a clo as lo ott tted ed ffor or 1 hhrr at 337_C. or 7_C 7_ C. W i-38 ccells ellls ((250,000 25 50, 0 00 00 ce cell lls/ ll s//we welll) ll), ), inn EBM E BM-22 supplemented upplemented ed d with witth 2% FBS FBS and and d 25 25 ng/ml n /m ng /mll VEGF, VEGF VE GF,, were GF were then the henn plated plat pl ated at ed on on top top of of the the fibrin fib bri rinn layers. Cultures were incubated at 37C, 5% CO2. After 4–6 days, cultures were labeled with 4 mg/ml Calcein AM for 1 hr, and imaged by fluorescence using a standard FITC filter. Hindlimb Ischemia Model As previously described, 14 the femoral artery was ligated at 2 positions spaced 5 mm apart and the arterial segment between the ligatures was excised. Tissue perfusion was assessed preoperatively, immediately post-operatively, and 3, 7, and 14 days after surgical intervention. Flow images of the foot were acquired using a Moor Infrared Laser Doppler Imager (LDI; Moor Instruments Ltd) at 37.0°C to 38.0°C under ketamine/xylazine (80/5 mg/kg) anesthesia. Data



were analyzed with Moor LDI image processing software V3.09 and reported as the ratio of flow in the right/left (R/L) hindlimb after background subtraction. Micro-CT Angiography 2D mCT scans were acquired with a GE eXplore MS Micro-CT System, using a 400 cone-beam with angular increment of 0.5 degrees and 8-27 micron slice thickness at a voltage of 163.2 mAs, 80; kVp. mCT data were transferred to a Dell Dimension computer with 3D volume rendering software (version 3.1, Vital Images, Inc. Plymouth, MN) and microview software (version 1.15, GE medical system). NIH ImageJ (National Institutes of Health, Bethesda, MD) and Image Pro Plus (Media Cybernatics) software were used to analyze vessel number, diameter, area, volume, and arterial density. Wound Healing Assay and Analysis Wo oun undd healing heal he alin ing studies stuudies were provided by the microsurgery st mic icro rosurgery core at Yalee C ardiovascular Research Wound Cardiovascular C ennter. Mice wer eree ana aest sthe heti tize zedd bby y in ntraaperritoneaal inje ectio on off a kketamine etam min ne (1 (10 00m mg/k /kg) g)/x xylaz azin ine Center. were anaesthetized intraperitoneal injection (100mg/kg)/xylazine 10m 0mg/ g/kg kg)) solution. solutiionn. Wounds so Woun Wo u dss were wer eree created creaateed with with th a sterile stter e il ile 6-mm 6-mm biopsy bio iops psyy punch pu h in the he back bacck skin n (10mg/kg) Miltex Inc,, PA) PA) without wit itho hout u injuring inj njur urin ng the the underlying unde un derl rlyi ying n muscle. ng mus uscle. e. Wound Wou ound nd regions reg egiion onss were were photographed pho hoto togr g aphed (Miltex using a Leica M125 microscope with an HC80 HD camera (Leica, Germany) on days 0, 1, 3, 5 and 7. Wound area was calculated using NIH ImageJ software. Wound sizes at different time points were expressed as percentage of the wound area on day 0. In vivo Matrigel Assay and Analysis Growth factor-depleted Matrigel plugs (0.5ml) containing either FGF2(100ng/ml) or heparin (10U) with VEGF-A165 (100 ng/ml) were injected subcutaneously on both sides of C57BL/6 mice . Seven days later, Matrigel plugs were excised and stained with a rat anti-mouse monoclonal CD31 antibody. CD31-positive vessels were counted in randomly acquired images



using ImageJ. Immunohistochemistry of Whole-mount Retinas P5 pups were sacrificed and the eyes removed and prefixed in 4% PFA for 20 minutes at room temperature. The retinas were dissected out and blocked overnight at 4°C in blocking buffer (0.1 M Tris-HCl, 150 mM NaCl 1% Blocking Reagent (PerkinElmer), 0.5% Triton X100). After washing with Pblec (1 mM MgCl2, 1 mM CaCl2, 0.1 mM MnCl2, 1% Triton X-100 in PBS), the retinas were incubated with IsolectinB4 in Pblec overnight followed by incubation with the corresponding secondary antibody for 2 hr at room temperature. Then the retinas were mounted in fluorescent mounting medium (DAKO, Carpinteria, CA, USA). Images were acquired using a Perkin Elmer UltraVIEW VoX spinning disc confocal microscope. microsco ope pe.. Quantification was performed on wildtype and PTP1b ECKO neonates; 20-25 images per group were aacquired were cqui cqui uire r d an re nd Bi B ological CMM Analyzer so oft f ware16 was use seed to o qquantify ua uantify vascular area and and Biological software16 used nnumber um mb of vess mber vessel ssel e bbranch el raanchh po poin points in nts pper er iimage. er magge. Stat St Statistical atis at isti is t ca ti call An Anal Analysis alys ysis ys is All data are sshown hown ho wn aass me m mean an n ± st standard tan anda dard da rdd eerror rror rr or ooff th the he mean m an me n (SEM) (SE SEM) M) or standard sta tand ndar nd ardd deviation ar deevi viat atio at ionn (SD) as io bars in the histograms. Differences were considered statistically significant if p 0.05 by Student’s t test in case of two group comparison or by Mann-Whitney U, Kruskal-Wallis and two-way ANOVA followed by Bonferonni analysis as appropriate.

Results In vitro effects of endothelial cell PTP1b deletion. To test the effect of PTP1b deletion on the response of endothelial cells to various growth factors, primary mouse endothelial cells (EC) from PtpN1fl/fl mice were treated with an



adenovirus carrying the Cre gene (Ad-Cre) or control virus (Ad-CMV), growth arrested and then stimulated with IGF-1, VEGF-A165 or FGF2. As expected, PTP1b deletion resulted in enhanced activation of IGF1 receptor (IGF1Rȕ) and ERK signaling (Fig. 1A,B). Consistent with the hypothesis that PTP1b downregulates VEGF signaling, ERK activation by VEGF was also increased in knockout compared to wild type EC (Fig. 1C,D). On the other hand, stimulation with FGF2 detected no differences in ERK activation suggesting that PTP1b is not involved in FGF signaling (Fig. 1E,F). In agreement with increased ERK activation by VEGF, VEGFR2 phosphorylation was also increased in PTP1b-/- compared to control endothelial cells (Suppl. Fig. 1A,C) despite a decrease in total VEGFR2 levels (Suppl. Fig.1B). Furthermore, there were no differences in Akt or p38MAPK activation in response to VEGF in PTP1b-/- vvs. s. control con ntr trol ol endothelial cells (Suppl. Fig. 1). In agreement In agr greeme mennt with these data, PTP1b-/- eendothelial me ndothelial cells demonstrated ndo dem e onnst strrated faster proliferation (Fig. Figg. g. 2A-C) an andd m migration igr graatio ionn ((Fig. io Fig. Fi g. 22D-F) D-F) iinn re D-F response espponsse to V VEGF EG GF but but not not FG F FGF2 F2 or or fe feta fetal taal bo bov bovine vine ne sserum eruum er stimulation. tim imul ulat ul a io at ion. n. Furthermore, Fur urtthe thermo ermore ree, cord cord r formation for orma mati tiionn in in the th he inn vitro vitrro ro Matrigel Matri atriige g l assay assa as s y was sa was also a so al s accelerated acc cceleeraated ated byy VEGF-A, bu but ut no nott FG FGF2 F2 orr fe feta fetal t l bo bbovine vine vi nee sserum erum er um ((Fig. Fiig. 22G-I). G-II).. GIn vivo effects of endothelial cell PTP1b deletion: angiogenesis. Endothelial-specific deletion of PTP1b was achieved by crossing PtpN1fl/fl mice with Cdh5CreERT2 line as described in the Methods section. The effectiveness of deletion in the resulting PtpN1ECKO mice was assessed by isolating endothelial cells from the heart, lungs and aorta of Ptpn1ECKO and control littermates mice and testing them for PTP1b expression. As previously reported for this Cre line, all endothelial cells tested demonstrated nearly complete target gene deletion15 (Suppl Fig 2). To test the role of PTP1b in angiogenesis, we first employed an in vitro sprouting assay.



HUVEC that were exposed to either control or PTP1b siRNA were treated with VEGF-A or saline and the extent of sprouting was determined as outlined in the Methods. PTP1b knockdown was associated with a highly significant increase in sprouting both in the absence or presence of VEGF-A (Fig. 3A, B). To check the validity of this result in vivo, we analyzed vascular sprouting in the retinas of P5 PtpN1ECKO and control littermates pups (Fig. 3 C-E). As in the case of the in vitro assay, endothelial PTP1b knockout was associated with a significantly increased sprouting as demonstrated by increased number of branch points and vessel density. At the same time, there was no change in the extent of vascular coverage of the retinas (Fig. 3F). Finally, Ptpn1ECKO mice had larger numbers of tip cells along the vascular front as assessed by the number of filopodia per vessel length (Fig. 3G,H). healing We next studied the effect of this knockoutt in the skin wound hea ling response that is he heavily, eav avil ilyy, il y, bbut ut nnot ot eexclusively, xc xclusively, angiogenesis-depen angiogenesis-dependent. nde d nt. In agreeme agreement ent n wi with ith iin n vitro results showing increased PtpN1 ncrrea e sed proliferation prollifferaation onn and and migration mig igra rati tioon ti on ooff PtpN pN N1-/- eendothelial ndottheli liaal ccells, ellls,, wo el woun wounds unds ds closed clo lose sedd significantly siign g if ific ican ic an ntl ty ECKO CKO O faster fa ast ster er iinn Pt PtpN1 tpN pN11ECK ccompared ompa om p reed to llittermate pa ittterm it term mat atee co ccontrol ntro nt ro ol mi mice ce ((Fig. Fig. 4A,B) Fig. 4A,B A,B)

To more mor oree specifically sppec ecif ifficcallly explore expplo lore ree the he effect eff ffec ectt of this ec th his deletion del elet etio et ionn on VEGF-driven io VEG EGFF dr Fdriv i en angiogenesis, iv ang ngio iogenesis, wee io employed an in vivo Matrigel model. PtpN1ECKO and littermate control mice were implanted with Matrigel pellets containing VEGF-A or FGF2. Quantification of capillary ingrowth seven days later demonstrated increased angiogenesis in implants containing VEGF-A but not FGF2 in PtpN1ECKO mice (Fig. 4C,D). In vivo effects of endothelial cell PTP1b deletion: arteriogenesis. We next evaluated if PTP1b deletion has an effect on arteriogenesis in adult tissues. To this end, hindlimb ischemia was induced in PtpN1ECKO and littermate control mice and the kinetics, extent of blood flow recovery and anatomical extent of arteriogenesis were evaluated. PTP1b knockout



was associated with accelerated blood flow recovery as early as day 3 but the final extent was the same as in controls (Fig. 5A,B). Micro-CT analysis of the hindlimb vasculature demonstrated a significantly larger number of small arteries in PtpN1ECKO compared to control mice both above and below the knee (Fig. 5C-E). There were no differences in arterial vessel density at 14 days when the extent of blood flow recovery was the same in both groups (data now shown). These results suggest that PTP1b affects the rate but not the extent of new artery formation. We finally studied whether PTP1b deletion would restore blood flow in synectin knockout mice since impaired VEGF signaling in this strain is thought to be due to the reduced rate of VEGFR2 trafficking leading to increased PTP1b-driven receptor dephosphorylation. To his end, PtpN1ECKO mice were crossed onto the previously described Synectin null nuulll ((Gipc1 Gipc Gi pc11-/--) pc this strain train14. The hindlimb ischemia model was then usedd to study arteriogenesis in double knockout -/ mice ((PtpN1 mice PtpN Pt pN11-/pN /Gipc1 /Gi Gippc1 Gi pc -/--) vs. littermate controls (both (bo both bo h single knockouts knocko koouts and and wild type animals).

In agreement agree eeme mentt with me witth previous prev pr evio ev ious us results, ressul ults, PTP1b PT b deletion deleetiion was was associated assoc ocia iateed with ia witth wi th accelerated acc c el eler erratted ed bblood lo lood flow and increased while processes synectin fl low w rrecovery e ovver ec eryy an nd in inc creaase sedd ar aarteriogenesis teri te riog ri ogen en nessis w hile hi le bboth othh pr roc oces essse sess we were r rreduced re ed ducced iin n sy syne ectiin in knockouts ((Fig. Fiig. 6A,B). 6A, A,B B). ) P PTP1b TP1b TP 1bb kknockout noock kou outt on tthe h bbackground he acckg kgro roun ro undd of ssynectin un yn nec ecti tinn de ti dele deletion leti t on ffully ti ully ul l restored arteriogenesis, returning it to levels seen in control animals (Fig. 6A,B).

Discussion Taken together, the data in this study point to a critical physiological role of PTP1b in the regulation of VEGFR2 signaling. Endothelial-specific deletion of this phosphatase resulted in a VEGF-driven increase in ERK activation that is central to arterial blood vessel growth and formation6, and an increase in VEGF-driven cell proliferation and migration. In vivo this translated into increased angiogenesis and arteriogenesis.



VEGF signaling is one of the most tightly regulated processes in endothelial biology. Several tyrosine phosphatases have been reported to be associated with VEGFR2 including CD148/Dep1 16-18, VE-PTP 19-22 and PTP1b, 4, 11 the subject of the current study. As with all phosphatases, these molecules have a broad spectrum of activity and are capable of dephosphorylating a number of targets. 23 However, their precise role in the vasculature has not been defined. Mice with an early embryonic inactivation of VE-PTP demonstrate normal vasculogenesis but defective yolk sac angiogenesis leading to mid-embryonic lethality.20 A knockout of CD148/DEP1 is also lethal (at E10.5) with the mice demonstrating enlarged aberrantly developed vasculature and impaired pericyte investment.18 In contrast, mice with global deletion of PTP1b appear grossly normal and a no vascular defects have been beeen reported rep por orte tedd to te date. 24, 25 Neve Ne v rtheele ve less ss, recent studies suggested tha hatt P ha TP1b shouldd play playy an important role in VEGF Nevertheless, that PTP1b signaling. ign nal a ing. In particular, parrtiicu cula lar,, we la we previously prev pr evio io ously usly y reported reporrteed that th hat PTP1b PTP TP1b TP 1b b associates ass ssoociiate tess with with early early arly endosomes end ndos osom omes om ess containing co ont n ai aini n ngg V ni VEGFR2 EGFR EGFR FR22 an aand ndd th that at V VEGFR2-dependent EGF EG FR22-d dep epen en nde d nt aactivation cttiv ivat atio at ionn of io o E ERK RK iiss iimpaired mpai mp aire ai r d in re n Sy Synectin ynec ynec e ti tinn nul nnull u t (Gipc1 Gipc1-/-/ ) or Nr Nrp1 p cyto p1 kknock-in n ck no k-iin mi m mice. ce..44,, 5 Th ce This is iiss th thought hou ough g t to bbee du gh duee too prolonged prol pr olon ol onge on g d exposure ge expo ex posu po sure of su

VEGFR2-containing endosomes to PTP1b leading to a partial dephosphorylation of the VEGFR2 Y1175 site that is crucial for ERK activation. In vitro overexpression of PTP1b decreases VEGF-induced endothelial ERK activation11 while impaired VEGF-induced ERK activation in synectin null endothelial cells can be restored by a knockdown of PTP1b expression. Finally, treatment of Gipc1-/- mice with a PTP1b inhibitor restored impaired arteriogenesis.5 These results suggest that PTP1b regulates the extent of VEGFR2 Y1175 site phosphorylation thereby regulating the magnitude and duration of ERK activation. However, there is no direct genetic demonstration of the role of PTP1b in endothelial VEGF signaling and



it is not known how its deletion would affect embryonic vascular development or angiogenesis. The current study was designed to address this gap. Endothelial cells derived from PtpN1ECKO mice demonstrated increased Y1175 VEGFR2 phosphorylation and enhanced ERK activation in response to VEGF-A165, consistent with the proposed role of PTP1b as a phosphatase controlling this site’s phosphorylation. At the same time, we observed no change in VEGF-driven Akt and p38-MAPK activation. The mechanism of VEGF-A-dependent activation of the former is not well established, but it is not dependent on Y1175 phosphorylation. The activation of the latter is dependent on Y1241 VEGFR2 phosphorylation,6 the site not controlled by PTP1b activity.4, 5 Several assays, in vitro and in vivo, demonstrating increased endotheliall sprouting, sprrou o tiing ng,, la larg larger rger numbers of tip cells, faster wound healing and more extensive neovascularization in Matrigel ECKO E EC O implants mpl plan an nts iin n Pt PtpN1 P pN N1ECK mice, are consistent withh enhanced enhanced d angiogenic angioge genicc response. ge ressponse. Accelerated re ECKO ECK O blood bloood flow rec recovery covverry and and in incr increased crrea ease sedd arte aarteriolar r eriolarr ddensity enssitty inn PtpN P PtpN1 tpN pN11EC mice m ice in tthe ic he hhindlimb in ndl dlim imbb is im isch ischemia chem h mia

mo model ode dell an andd th thee re rescue esccue ooff im impa impaired paair ired ed bblood lood fflow loo low lo w rrecovery e ov ec oveeryy in n ssynectin ynec yn ecti ec tiin nu null lll m mice icee by eendothelial ic ndoothheli nd heliial PTP1b deletion deletiion demonstrate dem emon onnst s raate PTP1b’s PTP TP1b 1b’ss role 1b rol o e in in regulation regu re gula gu lati la tion ti on of of arteriogenesis. arte ar teeri riog o en og enes esis es is.. is PTP1b activity is not specific to VEGFR2. Indeed, we observed increased IGF-induced ERK activation as well, the result consistent with the known role of this phosphatase in IGF receptor activation.24, 25 At same time FGF signaling was not affected. One important difference between VEGF and IGF vs. FGF signaling pathways is that while VEGFR2 undergoes clathrindependent endocytosis, 26 FGFR1, the principle FGF receptor in endothelial cells, undergoes macropinocytic uptake.27 It is conceivable that differences in early trafficking of these receptors determine their susceptibility to PTP1b-induced dephosphorylation. Another important consideration is that this study employed an endothelial specific



deletion of PtpN1 since systemic inhibition of it activity may have affected the function of blood-derived macrophages, the cell type that is crucial to postnatal arteriogenesis. Finally, this study further supports the critical role played by endothelial cells in arteriogenesis,28 as selective manipulation of PTP1b expression in these cells had a profound effect on this process. Overall, these studies illustrate that PTP1b-driven dephosphorylation of VEGFR2 is an important physiological regulator of vascular growth and emphasize the critical role endothelial VEGF signaling plays in angiogenesis and arteriogenesis.

Conflict of Interest Disclosures: None.

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Figure Legends:

Figure 1. Increased IGF-1 and VEGF-A signaling in PTP1bECKO cells relative to WT. (A, C, E) IGF1, FGF2 and VEGF-A signaling in wildtype and PTP1b null endothelial cells. Western blotting of total cell lysates isolated from floxed PTP1b cells treated with control CMV adenovirus or CRE adenovirus for three days and then starved overnight. Confluent, serumstarved cells were stimulated for the times indicated with 50ng/ml of IGF-1, FGF2 or VEGF-A. (A) Phosphorylation of the tyrosine residue 1131 of the IGF1 receptor and p44/p42 MAP kinase is increased in PTP1bECKO cells relative to WT following IGF-1 treatment. (C) Phosphorylation of p44/p42 MAP kinase is increased in PTP1bECKO cells relative to wildtype foll following low owin i g VE in VEGF VEGF-A GF--A GF treatment. reatment. (E) Phosphorylation of p44/p42 MAP kinase is not changed in PTP1bECKO cells elaati tive ve tto o wi wil ldty ty ype following FGF2 treatment. (B, (B, D, F) Quantif iffic i attio on of o relative pERK relative wildtype Quantification ECKO O induction ndu uct c ion by IIGF1, GF1, F GF FGF2 GF22 an GF andd VE VEGF-A EGF GF-A A iin nw wildtype illdtype pe and annd nd PTP1b PTP1b TP1bECK ccells e lss ((n=3), el n=3), n=3 3) (S (SD, D, * P P

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