LETTER. Oxidative stress induces angiogenesis by activating TLR2 with novel endogenous ligands

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1 oi:.8/nture9 Oxitive stress inues ngiogenesis y tivting TLR with novel enogenous ligns Xioxi Z. West, *, Nikoly L. Mlinin *, Alon A. Merkulov, Mir Tishenko, Bethny A. Kerr, Ernest C. Boren, Eugene A. Porez, Roert G. Slomon & Ttin V. Byzov, Reiproity of inflmmtion, oxitive stress n neovsulriztion is emerging s n importnt mehnism unerlying numerous proesses from tissue heling n remoelling to ner progression,. Wheres the mehnism of hypoxi-riven ngiogenesis is well unerstoo,, the link etween inflmmtion-inue oxition n e novo loo vessel growth remins osure. Here we show tht the en prouts of lipi oxition, v-(-roxyethyl) pyrrole () n other relte pyrroles, re generte uring inflmmtion n woun heling n umulte t high levels in geing tissues in mie n in highly vsulrize tumours in oth murine n humn melnom. The moleulr ptterns of roxylkylpyrroles re reognize y Toll-like reeptor (TLR), ut not TLR or svenger reeptors on enothelil ells, leing to n ngiogeni response tht is inepenent of vsulr enothelil growth ftor. promote ngiogenesis in hinlim ishemi n woun heling moels through MyD88-epenent TLR signlling. Neutrliztion of enogenous roxylkylpyrroles impire woun heling n tissue revsulriztion n iminishe tumour ngiogenesis. Both TLR n MyD88 re require for -inue stimultion of R n enothelil migrtion. Tken together, these finings estlish new funtion of TLR s sensor of oxition-ssoite moleulr ptterns, proviing key link onneting inflmmtion, oxitive stress, innte immunity n ngiogenesis. Angiogenesis n either promote host efene n tissue repir or exerte orgn ysfuntion resulting in isese. In mny pthologies, ngiogenesis n inflmmtion re intimtely relte. Inflmmtory ells relese prongiogeni growth ftors, inluing vsulr enothelil growth ftor (), whih filitte neovsulriztion. Newly forme loo vessels enhne inflmmtory ell reruitment, therey promoting hroni inflmmtion. Leukoytes, in prtiulr myeloi ells, re guie y 8 n ontriute to 9 oxitive stress n the genertion of oxitive prouts, inluing hyroxy-v-oxolkenoi is n their esters (Supplementry Fig. ). When present in oxiize phospholipis, these moleules re reognize y the svenger reeptor CD n ontriute to therosleroti progression n pltelet hyper-retivity,. Hyrolysis followe y retion of the resulting unesterifie hyroxy-v-oxolkenoi is with proteins, or retion of the esterifie hyroxy-v-oxolkenoi is with proteins followe y hyrolysis, gives rise to fmily of roxylkylpyrrole protein uts (CAPs), mong them n similrly moifie ompouns (Supplementry Fig. ). These uts, present in oxiize low-ensity lipoprotein, umulte in therosleroti plques n re foun in the retin, where they promote horoil neovsulriztion n gerelte mulr egenertion,. These uts, in prtiulr, re trnsiently present uring woun heling, rehing mximum ys fter injury efore returning to originl levels when the woun hs hele (Fig., n Supplementry Fig. ). This inrese oinies with the reruitment of one-mrrow-erive ells (Supplementry Fig. ), whih generte itionl oxints 9. A sustntil proportion of (out % t ys, out % t ys) is present in F/8 mrophges (Fig. ) ut not in Gr- neutrophils (Supplementry Fig. ). High levels of oinie with intense woun vsulriztion, suggesting role for in woun ngiogenesis (Fig., ). In ontrst to wouns, in pthologil sttes levels were ontinuously elevte. In melnom, showing exessive vsulriztion n inflmmtion (ssesse y CD n CD8 stining, respetively), levels were elevte sixfol (Fig. ). Similrly, in murine melnom, levels were elevte ninefol (Supplementry Fig. ). In ontrst to woun n tumour tissues, in uninjure musle ws onfine to rteriolr smooth musle ells (Fig. e). umultion inrese in geing tissues (Fig. f). These t suggest role of in inflmmtionssoite vsulriztion. When teste on enothelil ells (ECs) from humn umilil vein, mouse lung or ort, h prongiogeni effet omprle to tht of, s evlute in vrious ssys (Fig. n Supplementry Figs n ). Similrly to, the effet of ws meite y integrins (Supplementry Fig. ). The prongiogeni effet ws epenent on the presene of pyrrole uts, n the protein moiety i not influene the effet of, euse uts ouple to mouse serum lumin, humn serum lumin or ipeptie were eqully effetive (Fig. n Supplementry Fig. ). However, in ontrst to, stimultion of ECs with i not result in phosphoryltion of reeptor (R) (Supplementry Fig. ). Moreover, -inue effets oth in vitro n in vivo were not erese y the R kinse inhiitor (ref. ) t onentrtion suffiient to lok -A effets (Fig., ); vehile lone h no effet (t not shown). In woun heling ssy, elerte vsulriztion n woun losure; this effet ws unimpee y, whih elye woun losure in ontrol nimls (Fig. ). Thus, tivtes the prongiogeni responses inepenently of /R signlling. Auts from the sme fmily of CAPs (Supplementry Fig. ), represente y roxypropylpyrrole (CPP), were lso prongiogeni (Fig. n Supplementry Figs, n ), initing tht the ECs respon to the moleulr pttern rther thn to prtiulr hemil moiety. To ientify reeptors meiting -inue ngiogenesis, we teste the role of CD n SR-BI svenger reeptors, s CD reognizes preursors of CAPs.BothnCPPutswereseffetiveon CD / n SR-BI / ECsstheywereonwil-typeells(Supplementry Fig. ), initing tht svenger reeptors re not involve in the reognition of these uts. Beuse ECs respon to the moleulr pttern of CAP, whih is hrteristi feture of oxitive stress, we propose the involvement of TLRs in -inue ngiogenesis. TLRs reognize severl mgessoite moleulr ptterns, inluing pthogen-ssoite moleulr ptterns (reviewe in refs, ) n ligns of host origin, Deprtment of Moleulr Criology, J. J. Jos Center for Thromosis n Vsulr Biology, NB, The Cleveln Clini Fountion, 9 Euli Avenue, Cleveln, Ohio 9, USA. Deprtment of Chemistry, Cse Western Reserve University, Cleveln, Ohio, USA. Tussig Cner Institute, The Cleveln Clini Fountion, 9 Euli Avenue, Cleveln, Ohio 9, USA. *These uthors ontriute eqully to this work. 9 NATURE VOL OCTOBER Mmilln Pulishers Limite. All rights reserve

2 RESEARCH ys 8 ys /CD CD CD8 Inrese over ontrol (%) CD e f Norml skin Melnom Norml skin Melnom CD Merge /CD8 /CD μm Fol inrese in FI μm Figure, n en prout of lipi oxition, is present in wouns, is elevte in melnom n umultes in geing tissues., n CD o-stining in norml n woune skin n 8 ys fter injury., Quntifie levels of n CD (n )., F/8 mrophge mrker n istriution in woun tissues n ys fter injury., n CD8 (top) or n CD (ottom) presene in humn skin n melnom. SMA 8 ys Merge μm ys ys /F/8 weeks /CD weeks μm Fol inrese in FI.... μm weeks weeks Right: quntifie levels of CD, CD8 n normlize to norml skin (n 8). FI, fluoresene intensity (ritrry units). e, Distriution of n CD (top) or n SMA (ottom) in murine skeletl musle. f, n CD o-stining in Vstus intermeius setions from n week ol mie. Right: quntifition of fluoresene normlize to -week-ol mie (n ). All vlues represent mens n s.e.m. Three sterisks, P,.. ontriuting to immune efene n to sterile inflmmtion, respetively. We fouse on TLR n TLR euse they re expresse on the enothelium, re implite in ngiogenesis n re known to reognize ro rnge of protein n lipi ligns. Anti-TLR, ut not ntioies ginst TLR, inhiite -inue, ut not -ipep e -ipep Chnge in tue length (%) g 8 TLR TLR IgG TLR +/+ -MSA CPP-HSA μm PmCSK f Numer of mirovessels -ipep μm Numer of mirovessels -ipep TLR / TLR +/+ TLR / 8 Fol inrese in tue length.... TLR +/+ TLR / PmCSK mm Fol erese in woun re * 8 -inue, tue formtion (Fig. e) n EC migrtion (Supplementry Fig. 8). To further ress the role of TLR in -riven ngiogenesis, ECs from TLR / mie were ompre with those from TLR / ontrols. TLR / ECs i not respon to tretment with or CPP in severl in vitro ssys; in ontrst, -triggere responses were not ffete y the lk of TLR (Fig. f n Supplementry Figs 8 n 9). To onfirm the role of TLR in prongiogeni responses in ECs, we teste the TLR syntheti lign PmCSK (ref. ). This lign inue roust sprouting of ECs from orti rings of TLR / mie ut not in TLR / mie (Fig. g). PmCSK ws s effetive s or in tuulogenesis n ell hesion ssys, n the effet of PmCSK ws eliminte y TLR ntioies (Supplementry Figs 9 n ). To investigte the reltive roles of n, we exmine the effet of R inhiitor,, or TLR eletion in woun heling. Inhiiting either pthwy h similr effet n in omintion they le to itive inhiition of woun losure (Supplementry Fig. ). Hving estlishe the role of TLR in the prongiogeni effets of on ECs, we speulte tht ministrtion might promote vsulriztion in ishemi or injure tissues. In hinlim ishemi moel, -ipeptie injetions resulte in -fol inrese of the pyrrole ut in musle tissue (Supplementry Fig. ), whih promote the revsulriztion of the ishemi lim (Fig. ) n restruturing of ollterl loo vessels ypssing the ligte femorl rtery Figure Prongiogeni effets of oxiize uts re epenent on pyrrole moiety n re meite y TLR ut not R signlling., Imges of mirovessels in orti ring ssy, trete with pyrrole uts s inite., Effet of on -ipeptie () n -stimulte prongiogeni response. Right: ssy quntifition, normlize to ontrol (n )., Growth of CD-positive loo vessels in Mtrigel plugs trete s inite. Right: quntifition of vsulr res (n )., Punh wouns trete with -ipeptie (-ipep) or on y. Right: quntifition of woun surfe res (top) n vsulr res (ottom) (n ). e, Quntifition of tuulogenesis y -ipeptie or in the presene of loking nti-tlr or nti-tlr ntioies, normlize to ontrol (n ). f, TLR / orti ring ell extrusion in response to -ipeptie or. Right, quntifition of mirovessels from TLR / n TLR / ultures (n ). g, Imges of TLR / n TLR / orti rings trete with the TLR lign PmCSK. Right: ssy quntifition (n ). All vlues represent mens n s.e.m. Asterisk, P,.; two sterisks, P,.; three sterisks, P,.., not signifint. OCTOBER VOL NATURE 9 Mmilln Pulishers Limite. All rights reserve

3 RESEARCH LETTER (Supplementry Fig. ). Exogenous ffete vsulriztion in TLR-epenent mnner (Fig. ). Consequently, injetion inrese loo flow in TLR / mie ut not in TLR / mie (Fig. ). In woun moel, exogenous elerte woun losure n vsulriztion in TLR / mie ut not in TLR / mie (Fig. ). Moreover, tumours implnte in TLR / mie showe mrkely erese vsulriztion n inrese res of nerosis (Supplementry Fig. ). To istinguish the effet of on ECs from tht on leukoytes, TLR / n TLR / mie were trnsplnte with TLR / one mrrow. In woun ssys, injetion of into TLR /. TLR / himers elerte woun losure, wheres TLR /. TLR / nimls hele more slowly n h no effet on woun losure (Fig. ). The ifferene ws ssoite with.-fol inrese in vsulture y tretment of TLR /. TLR / ut not TLR /. TLR / nimls with (Fig. e). Even in the sene of exogenous, vsulr re ws iminishe in wouns of TLR /. TLR / himers in omprison with TLR /.TLR / nimls (Fig. f). Moreover, melnom vsulriztion in TLR /. TLR / nimls ws reue reltive to tht in TLR /. TLR / or TLR /. TLR / himers (Fig. g). Thus, TLR on non-hemtopoieti ells meite vsulriztion inue y exogenous n it lso ontriute to woun n tumour ngiogenesis in the sene of n exogenous ut. Beuse is umulte t high levels uring woun heling (Fig. n Supplementry Fig. ) n in tumours (Fig. n Supplementry Fig. ), we resse the ontriution of enogenously generte uts to the proess of vsulriztion in these moels. Intrvenous ministrtion of neutrlizing ntioies ginst, ut not ontrol ntioies, resulte in more thn twofol erese in woun reovery (Fig. A). Vsulriztion of wouns ws lso inhiite y nti- ut not ontrol ntioies, emonstrting the signifine of enogenously generte in woun ngiogenesis (Fig. B n Supplementry Fig. ). Aministrtion of nti-, ut not ontrol ntioies, iminishe the progression n vsulriztion of melnom, n this effet ws itive to tht of the R inhiitor -ipeptie TLR +/+ TLR / TLR +/+ μm -ipeptie Numer of vessels per fiel mm TLR / TLR +/+ >TLR / e TLR +/+ > TLR +/+ g TLR +/+ > TLR / TLR +/+ > TLR +/+ TLR +/+ > TLR / /CD μm TLR +/+ TLR / TLR +/+ >TLR +/+ Fol inrese in vsulr re TLR / > TLR +/+ μm * TLR +/+ TLR /. TLR +/+ TLR / Perfusion rtio -ipeptie mm / >+/+ Figure -inue ngiogenesis in vivo is meite y TLR., Vsulriztion (SMA immunostining) in hinlims fter ishemi. Right: vsulr ensity quntifition. Fr right: vsulr re quntifition (n )., Left: perfusion rtio of hinlim loo flow fter tretment with (n 8). Right: Lser-Doppler imges efore, fter n ys fter hinlim injury. Pixel olouring is lirte in the sle unerneth. PU, ritrry perfusion units., Imges of woun on the inner skin flp on y fter injury., Wouns trete with -ipeptie in TLR / n TLR / hosts fter TLR / one mrrow trnsplnt. Right: quntifition of woun re (n ) Before Fol erese in woun re f 8 Before Dys fter surgery TLR +/+ > TLR +/+ TLR +/+ > TLR / μm 9 NATURE VOL OCTOBER Mmilln Pulishers Limite. All rights reserve + + TLR +/+ Before TLR / Bloo flow (PU), After < μm > μm ys fter e, Vsulriztion in -trete wouns fter one mrrow trnsplnt s inite. Right: quntifition of vsulr re, normlize to ontrol (n ). f, CD stining in woun grnultion tissue fter one mrrow trnsplnt. Arrowhes elinete the tissue ege. Right: vsulr res t grnultion tissue ege (less thn mm from the outer ege) n eeper res (more thn mm) (n ). g, CD-positive vsulture in melnom llogrfts, one mrrow trnsplnt s inite. Right: quntifition of vsulr re, n. All vlues represent mens n s.e.m. Asterisk, P,.; two sterisks, P,.; three sterisks, P,.., not signifint.

4 RESEARCH A IgG C D E -ma TLR TLR TLR mm IgG -ipeptie Fol erese in woun re IgG -ma Numer of mirovessels μm -ipeptie μm B IgG -ma mm μm IgG -ma (Fig. C). Thus, the /TLR xis rives -inepenent ngiogenesis in vriety of pthologil onitions. The key mehnism unerlying the ro lign speifiity of TLR (ref. ) is its heteroimeriztion with other memers of the TLR fmily, prtiulrly TLR n TLR (ref. ). In tue formtion ssy, nti-tlr n nti-tlr, ut not nti-tlr loking ntioies, Fol inrese in tue length * IgG ma +ma. TLR + + TLR + + TLR + + IgG + + Fol inrese in R GTP lo MyD88 +/+ MyD88 / F + + -MSA WT TLR / MyD88 / R GTP Totl R -MSA WT TLR / MyD88 / Figure Enogenous ontriutes to woun reovery n melnom vsulriztion; TLR-epenent responses to involve MyD88 n R. A, Wouns on y fter injury trete with ontrol IgG or nti- loking monolonl ntioy (ma). Right, quntifition of hnges in woun re (n ). B, CD-positive woun vsulture. Right: quntifition of vsulr re (n ). C, Melnom llogrft trete with ontrol IgG (), nti- ntioies, () n n nti- ntioies (). Left: immunostining of melnom CD. Right: tumour size on y. Fr right: quntifition of vsulr re (n ). D, -inue tuulogenesis in the presene of ontrol (IgG) or nti-tlr-, nti-tlr- or nti-tlr- loking ntioies. Right: quntifition of tue length, normlize to ontrol (n ). E, Imges of MyD88 / orti rings trete with -ipeptie. Right: mirovessel numers in MyD88 / n MyD88 / smples (n ). F, Ativtion of R in response to ; genotypes s inite; WT, wil type. Right: ssy ensitometry, normlize to ontrol (n ). All vlues represent mens n s.e.m. Asterisk, P,.; two sterisks, P,.; three sterisks, P,.., not signifint. iminishe the -inue ngiogeni response (Fig. D), initing the involvement of TLR/TLR heteroimers in reognition. It seems tht interts iretly with TLR, euse reominnt TLR oun the protein ut ut not the rrier protein lone (Supplementry Fig. ). Similrly to other TLR ligns, stimulte NF-kB in ell-se ssy (Supplementry Fig. ). Next, we evlute the role of MyD88, meitor of TLR signlling, in the prongiogeni tivity of. -inue EC sprouting is epenent on MyD88 euse MyD88 / ells i not respon to stimultion y (Fig. E). -inue ngiogenesis ws not ffete y the lk of MyD88 (Fig. E). Consiering tht inue ngiogenesis is integrin-epenent, we fouse on meitors of integrin n TLR signlling. R meites integrin-epenent migrtion in vsulr evelopment n is reporte to e tivte ownstrem of TLRs 8. Aoringly, we ssesse the loing of R with GTP in response to tretment with. GTP-oun R is inrese y tretment of TLR /, ut not TLR / or MyD88 / ells, with (Fig. F). Thus, lipi oxition prouts, represente y, promote the ngiogeni responses of ECs y tivting TLR signlling in MyD88-epenent mnner, resulting in R tivtion, whih in turn filittes integrin funtion. Tken together, our results estlish novel mehnism of ngiogenesis tht is inepenent of hypoxi-triggere expression. The prouts of lipi oxition re generte s onsequene of oxitive stress n re reognize y TLR, possily in omplex with TLR on ECs, n promote ngiogenesis in vivo, therey ontriuting to elerte woun heling n tissue reovery. If high levels of n its nlogues umulte in tissues, it my le to exessive vsulriztion, for exmple, in tumours. Contriution of the /TLR xis to ngiogenesis vries in ifferent physiologil settings, possily epening on the extent of oxitive stress. -riven ngiogenesis my e n ttrtive therpeuti trget, espeilly in ners resistnt to nti- therpy. Inflmmtion n oxition-riven ngiogenesis my our in other pthologies, for exmple theroslerosis, in whih rteril thikening n epen on its mirovsulture. In these settings there is n extensive genertion of oxitive prouts tht might promote therogenesis through TLR. Inee, it ws shown tht TLR / mie re protete from theroslerosis, n this effet oul e meite y ells other thn those erive from one mrrow 9. Thus, long with pthogen-ssoite n nger-ssoite moleulr ptterns, TLR reognizes n oxition-ssoite moleulr pttern. This funtion of TLR s sensor of oxitive stress revels the shortut link etween innte immunity, oxition n ngiogenesis. METHODS SUMMARY Immunostining ws omplete to ssess the presene of enogenous in vrious tissues. Tue formtion ssys, Mtrigel plug ssys, woun heling ssys, orti ring ssys, tumour implnttion moels, n hinlim ishemi moel evlute the ngiogeni effets of n TLR. TLR / n TLR / mie were use for the ove ssys n in one mrrow trnsplnttion stuies. MyD88 / mie n ommerilly ville R tivtion ssy were use to exmine the /TLR signlling pthwy. Full Methos n ny ssoite referenes re ville in the online version of the pper t Reeive Mrh; epte August. Pulishe online Otoer.. Jkson, J. R., See, M. P., Kirher, C. H., Willoughy, D. A. & Winkler, J. D. The oepenene of ngiogenesis n hroni inflmmtion. FASEB J., (99).. Coussens, L. M. & Wer, Z. Inflmmtion n ner. Nture, 8 8 ().. Mzzone, M. et l. Heterozygous efiieny of PHD restores tumor oxygention n inhiits metstsis vi enothelil normliztion. Cell, 89 8 (9).. Frisl, P., Mzzone, M., Shmit, T. & Crmeliet, P. Regultion of ngiogenesis y oxygen n metolism. Dev. Cell, 9 (9).. Cr, J. W. et l. Drusen proteome nlysis: n pproh to the etiology of gerelte mulr egenertion. Pro. Ntl A. Si. USA 99, 8 8 ().. Krin, M., Lwrene, T. & Nizet, V. Innte immunity gone wry: linking miroil infetions to hroni inflmmtion n ner. Cell, 8 8 (). OCTOBER VOL NATURE 9 Mmilln Pulishers Limite. All rights reserve

5 RESEARCH LETTER. Mrtin, P. Woun heling iming for perfet skin regenertion. Siene, 8 (99). 8. Niethmmer, P., Grher, C., Look, A. T. & Mithison, T. J. A tissue-sle grient of hyrogen peroxie meites rpi woun etetion in zerfish. Nture 9, (9). 9. Segl, A. W. How neutrophils kill miroes. Annu. Rev. Immunol., 9 ().. Porez, E. A. et l. Ientifition of novel fmily of oxiize phospholipis tht serve s ligns for the mrophge svenger reeptor CD. J. Biol. Chem., 8 8 ().. Porez, E. A. et l. Pltelet CD links hyperlipiemi, oxint stress n prothromoti phenotype. Nture Me., 8 9 ().. Gu, X. et l. Croxyethylpyrrole protein uts n utontioies, iomrkers for ge-relte mulr egenertion. J. Biol. Chem. 8, ().. Erhem, Q. et l. Croxyethylpyrrole oxitive protein moifitions stimulte neovsulriztion: implitions for ge-relte mulr egenertion. Pro. Ntl A. Si. USA, 8 8 ().. Mnley, P. W. et l. Anthrnili i mies: novel lss of ntingiogeni reeptor kinse inhiitors. J. Me. Chem., 8 9 ().. Plm, N. W. & Mezhitov, R. Pttern reognition reeptors n ontrol of ptive immunity. Immunol. Rev., (9).. Kwi, T. & Akir, S. Pthogen reognition with Toll-like reeptors. Curr. Opin. Immunol., 8 ().. Shi, H. et l. TLR links innte immunity n ftty i-inue insulin resistne. J. Clin. Invest., (). 8. Apetoh, L. et l. Toll-like reeptor -epenent ontriution of the immune system to ntiner hemotherpy n riotherpy. Nture Me., 9 (). 9. Kim, H. S. et l. Toll-like reeptor senses et-ell eth n ontriutes to the initition of utoimmune ietes. Immunity, ().. Vogl, T. et l. Mrp8 n Mrp re enogenous tivtors of Toll-like reeptor, promoting lethl, enotoxin-inue shok. Nture Me., 9 ().. Shefer, L. et l. The mtrix omponent iglyn is proinflmmtory n signls through Toll-like reeptors n in mrophges. J. Clin. Invest., ().. Jing, D. et l. Regultion of lung injury n repir y Toll-like reeptors n hyluronn. Nture Me., 9 ().. Grote, K. et l. Toll-like reeptor / stimultion promotes ngiogenesis vi GM- CSF s potentil strtegy for immune efense n tissue regenertion. Bloo, ().. Zhringer, U., Linner, B., Inmur, S., Heine, H. & Alexner, C. TLR promisuous or speifi? A ritil re-evlution of reeptor expressing pprent ro speifiity. Immunoiology, (8).. Aliprntis, A. O. et l. Cell tivtionnpoptosis y teril lipoproteinsthrough toll-like reeptor-. Siene 8, 9 (999).. Ozinsky, A. et l. The repertoire for pttern reognition of pthogens y the innte immune system is efine y oopertion etween toll-like reeptors. Pro. Ntl A. Si. USA 9, ().. Tn, W. et l. An essentil role for R in enothelil ell funtion n vsulr evelopment. FASEB J., (8). 8. Arie, L. et l. Toll-like reeptor -meite NF-kB tivtion requires R- epenent pthwy. Nture Immunol., (). 9. Mullik, A. E., Tois, P. S. & Curtiss, L. K. Moultion of theroslerosis in mie y Toll-like reeptor. J. Clin. Invest., 9 (). Supplementry Informtion is linke to the online version of the pper t Aknowlegements We thnk L. Hong for the help with synthesis of n CPP uts, Y. Cui for expressing n purifying nti- monolonl ntioies, n J. Cr for proviing nti- monolonl ntioy hyriom ells. This work ws supporte y NIH grnts HL, HL, CA8 to T.V.B., HL to E.A.P. n GM9 to R.G.S. n n Amerin Hert Assoition grnt (SDG) to N.L.M. Author Contriutions N.L.M., E.A.P. n T.V.B. esigne experiments. In vivo n ex vivo experiments were performe y X.Z.W. with help from A.A.M. n M.T. In vitro experiments were performe y N.L.M. with help from B.A.K. Melnom smples n their nlysis ws one y E.C.B. Synthesis of CAPs ws performe y R.G.S. The t were nlyse n plotte y X.Z.W. The mnusript ws written y N.L.M. n T.V.B. Author Informtion Reprints n permissions informtion is ville t The uthors elre no ompeting finnil interests. Reers re welome to omment on the online version of this rtile t Corresponene n requests for mterils shoul e resse to T.V.B. (yzovt@f.org). 9 NATURE VOL OCTOBER Mmilln Pulishers Limite. All rights reserve

6 RESEARCH METHODS Animl stuies. Wil-type CBL/ n DsRe trnsgeni mie were otine from Jkson Lortory, CD / mie from M. Ferio, SR-BI / mie from M. Krieger, n MyD88 / n TLR / mie from S. Akir n Jkson Lortory. All mie were mintine in the Biologil Resoures Unit of the Lerner Reserh Institute, reite y the Assoition for the Assessment n Areittion of Lortory Animl Cre Interntionl. All experimentl work ws onute in ompline with the Institutionl Animl Cre n Use Committee progrmme. Immunohistohemistry n imge nlysis. For immunofluoresene stining, we use rit polylonl nti- (esrie in ref. ), nti-cd (BioLegen), nti-sma (Rokln) n nti-cd8 (BD Biosienes) ntioies. We emee smples in OCT freezing meium n prepre tissue setions mm thik.setions were fixe in % prformlehye. After inution with primry ntioies, smples were wshe with PBS n expose to Alex Fluor-lelle seonry ntioy, nmely got nti-rit Alex Fluor88, nti-rt Alex Fluor8 or ntimouse Alex Fluor8 (Invitrogen). The slies were mounte with meium (DkoCytomtion) n imges were tken y either TCS-SP mirosope (Lei) or ZMZ mirosope (Nikon). For quntifition, the imges were nlyse with ImgePro softwre (Mei Cyernetis). Bone mrrow trnsplnt (BMT) n woun ssy. We performe BMT s esrie previously. In rief, we sujete two-month-ol mle wil-type TLR / or TLR / mie to irrition with totl ose of 9 Gy followe y one mrrow reonstitution y til vein injetion with one mrrow ells isolte from onor femurs. Eight weeks fter BMT, mie were use for woun heling ssys. A k punh woun heling moel ws use s esrie. Where inite, mie were given suutneous injetions (. mg per g oy weight) of -ipeptie (moleulr mss 8. D) t the time of injury n on ys, n. Seprtely, ontrol mie were sujete to k punh n injete suutneously with -ipeptie ( mg) n ( nmol) on lternte ys within m of the woun ege. Aorti ring ssy. The mouse orti ring ssy ws performe s esrie previously. Aorti rings were trete with -ipeptie (. mgml ), ( ng ml ) or PmCSK ( nm). Isoltion of enothelil ells. Mouse lungs were exise, mine, n igeste with ollgense-ispse regent (Rohe Dignosti). Digests were strine n the resulting ell suspension ws plte on flsks ote with mgml fironetin. Therefter, enothelil ells were isolte n hrterize s esrie previously. Tue formtion ssy. Humn umilil-vein enothelil ells or mouse lung enothelil ells were seee on Mtrigel-ote pltes (BD Biosiene). Meium ws supplemente with ( ng ml ) or protein uts (. mgml )s inite, n ells were further inute t uc for 8 h. Anti- loking ntioies (evelope y R.G.S.), nti-tlr n nti-tlr loking (BioLegen) n isotype ontrol ntioies (IgG; BioLegen) or nti-tlr n nti-tlr loking ntioies (InVivoGen) were e t mgml t the eginning of the experiment. Alterntively, we inute ells with nm (Enzo Life Sienes). Tue formtion ws oserve with phse-ontrst inverte mirosope, n photogrphs were tken of eh well. The t were quntifie y mesuring the length of tues with ImgePro softwre. Mtrigel plug ssy. Mtrigel ( ml) ontining ( nm) in omintion with ( ng) or -ipeptie ( mgml ) ws injete suutneously into CBL/ mie. Plugs were remove fter ys n setione. Hinlim ishemi moel. The femorl rtery ws ligte ner the ully rnhing eep femorl rtery, n seon ligtion ws ple in the proximity of the tiil rtery rnhing. The portion of the rtery n vein etween the ligtion points ws exise. -ipeptie ws ministere s solution in PBS (. mg per g oy weight) injete intrmusulrly istl to the ligtion site, with injetions every 8 h strting t the y of opertion. Intrmusulr ontent ws evlute y immunohistohemistry n quntifie with ImgePro softwre. Hinlim loo flow ws mesure y lser Doppler moorldi-ir ner-infrre lser Clss R (Moor Instruments) in ritrry perfusion units (PU). The results were plotte s rtio of the operte leg to the non-operte leg for eh niml to ount for vritions etween nimls. Tumour implnttion. B-F murine melnom ells (ATCC) were injete suutneously (. ells per injetion) in wil-type or BMT nimls s inite. Tumours were nlyse t y fter implnttion. Mie were injete intrperitonelly with ( mmol), nti- ntioy ( mg per g oy weight) or isotype ontrol (IgG, mg per g oy weight). R tivtion ssy. In eh ssy we use ells lyse in ml of uffer ( mm HEPES-KOH ph., mm NCl, % Noniet P, % glyerol, mm MgCl, mm EGTA). GST PAK PBD (Glutthione S-trnsferse p- tivte kinse p-ining omin) pre-sore on glutthione-grose es (Cytoskeleton) ws e s ml of % slurry followe y inution on rotry shker for min t uc. Bes were wshe four times with lysis uffer efore elution with SDS PAGE smple uffer. R ws etete y western lotting (lone ; BD Biosiene). Sttistil nlysis. All t re presente s mens n s.e.m. Proility vlues were se on pire t-test:, not signifint; sterisk, P,.; two sterisks, P,.; three sterisks, P,... Chen, J. et l. Akt regultes pthologil ngiogenesis, vsulr mturtion n permeility in vivo. Nture Me., 88 9 ().. Feng, W. et l. The ngiogeni response is itte y integrin on one mrrowerive ells. J. Cell Biol. 8, (8).. Mheleshwr, G. H., Somnth, P. R. & Byzov, T. V. Methos for isoltion of enothelil n smooth musle ells n in vitro prolifertion ssys. Methos Mol. Me. 9, 9 8 (). Mmilln Pulishers Limite. All rights reserve