Parathyroid hormone related peptide is a naturally occurring, protein kinase A dependent angiogenesis inhibitor

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1 Prthyroi hormone relte peptie is nturlly ourring, protein kinse A epenent ngiogenesis inhiitor MANJIRI M. BAKRE 1, YUHONG ZHU 1, HONG YIN 1, DOUG W. BURTON 2, ROBERT TERKELTAUB 2, LEONARD J. DEFTOS 2 & JUDITH A. VARNER 1 22 Nture Pulishing Group 1 University of Cliforni, Sn Diego Comprehensive Cner Center, L Joll, Cliforni, USA 2 Deprtment of Meiine, University of Cliforni, n Veterns Affirs Meil Center, L Joll, Cliforni, USA Corresponene shoul e resse to J.A.V.; emil: jvrner@us.eu Angiogenesis is n importnt physiologil proess tht is exquisitely regulte y stimultory n inhiitory ftors 1 3. Sustine pthologil ngiogenesis results from n overlne of stimultory ftors n n insuffiieny of inhiitory ftors 1 3. An unerstning of the positive n negtive ftors tht influene vsulr prolifertion my enle the evelopment of new therpeutis to tret errnt ngiogenesis suh s tht ourring in ner, rthritis, linness n psorisis 1 3. Angiogenesis requires tivtion of quiesent enothelil ells y growth ftors; egrtion of sement memrne n isssoition of enothelil ells from the supporting vsulr smooth musle; prolifertion, survivl, migrtion n eventul ifferentition of these ells into lumen-ering strutures tht my lso e line with vsulr smooth-musle ells 1 3. Effetive inhiitors of ngiogenesis my lok one or more of these isrete proesses. In ft, severl nturlly ourring inhiitors of ngiogenesis hve een evelope s potentil therpeuti gents for ner 4 6. Prthyroi hormone relte peptie () n the losely relte prthyroi hormone (PTH) re peptie hormones tht regulte serum lium levels, vsulr tone n one formtion Nturlly ourring isoforms of exten from mino is 1 139, n 1 173; the lst is foun only in humns 8. Mie without ie shortly fter irth n hve skeletl normlities inluing shortene ones n inrese one vsulriztion Extrellulr PTH/ tivtes ell surfe G protein ouple reeptor (GPCR) tht then tivtes protein kinse A (PKA) n phospholipse C (refs. 13,14). This PTH1 reeptor is expresse on mny ells inluing honroytes, Pulishe online 19 August 22, oi:1.138/nm753 Angiogenesis is highly regulte proess tht results from the sequentil tions of nturlly ourring stimultors n inhiitors. Here, we show tht prthyroi hormone relte peptie, peptie hormone erive from norml n tumor ells tht regultes one metolism n vsulr tone, is nturlly ourring ngiogenesis inhiitor. Prthyroi hormone relte peptie or ten-mino-i peptie from its N terminus inhiits enothelil ell migrtion in vitro n ngiogenesis in vivo y tivting enothelil ell protein kinse A. Ativtion of protein kinse A inhiits ell migrtion n ngiogenesis y inhiiting the smll GTPse R. In ontrst, inhiition of protein kinse A reverses the nti-migrtory n nti-ngiogeni properties of prthyroi hormone relte peptie. These stuies show tht prthyroi hormone relte peptie is nturlly ourring ngiogenesis inhiitor tht funtions y tivtion of protein kinse A. vsulr smooth-musle ells n enothelil ells Mny of the physiologil funtions of re meite y tivtion of PKA (refs. 18 2). is lso proue y mny tumor ells; however, little is known out its effets on tumor vsulture. In ontrst, is known to use inrese serum lium in ner ptients (humorl hyperlemi of mlignny) n to promote lol one metstses 7,21,22. Humorl hyperlemi of mlignny results from stimultion of osteolsts to express RANKL, ftor tht inues osteolst ifferentition from mrophges, susequent egrtion of minerlize one n n eventul inrese in serum lium 23. Lol -meite one egrtion my ontriute to tumor ell oloniztion of one to form metstses 21,22. Reent stuies inite tht lso ts in n intrrine mnner to stimulte tumor ell prolifertion 24. As stuies inite tht my promote tumor progression ut lso my negtively regulte one vsulriztion, we sought to etermine if is involve in the physiologil regultion of ngiogenesis. Like other nturlly ourring ngiogenesis inhiitors suh s thromosponin-1 (ref. 1), enosttin 4, ngiosttin 5 n tumsttin 6, is proue in mesurle quntities y tumor ells. We foun tht inhiits growth ftor n tumor-inue ngiogenesis. The ngiostti effets of require tivtion of PKA y its G protein ouple reeptor. One tivte, PKA suppresses R tivtion, whih in turn inhiits enothelil ell migrtion n ngiogenesis. Our stuies show previously unknown signl trnsution pthwy tht negtively regultes ngiogenesis n inite tht lolize ministrtion of or tivtion of PKA my e useful strtegy to inhiit iseses ssoite with ngiogenesis. NATURE MEDICINE VOLUME 8 NUMBER 9 SEPTEMBER

2 Fluoresene intensity (perent of ontrol ) ARTICLES sline Cl CGRP 22 Nture Pulishing Group s sline 1 5 CGRP e f sline + + nti- IgG sline g 1 5 litonin h s Net tumor growth (mm 3 ) sline ontrol sline ontrol PTHRP+ nti- PTHRP+ lgg sline Fig. 1 inhiits ngiogenesis in vivo., CAMs stimulte with FGF or sline were trete with 1 173, litonin (Cl) or litonin gene relte peptie (CGRP)., ± s.e.m. ove kgroun. -, Cryosetions of CAMs immunostine with ntioy ginst α v β 3 (left n ) or ginst VWF (mile ) n then stine with hemtoxylin n eosin (H&E; right)., quntifition of immunoretive vessels per high-power fiel (HPF; mgnifition, 2)., FGF-stimulte CAMs trete with sline, or with -funtion-loking (nti-) or ontrol ntioies (IgG; ottom). Top, quntifition of loo vessel ± s.e.m. ove kgroun. e, Men fluoresene intensity in lystes of growth ftor eplete Mtrigel plugs ontining FGF n or FGF n litonin. f h, Mie ering -negtive DU145 tumors were trete with sline, or srmle ontrol peptie (ontrol). f, Net tumor volume fter tretment. Soli line, verge tumor volume. g, Cryosetions of tumors stine with H&E. h, Cryosetions immunostine for expression of vsulr ntigen CD31 (re) n DNA (lue). Top, quntifition of immunoretive vessels. Bottom, 2 mgnifition of ryosetions., P <.5 (Stuent s t-test). ontrol 996 NATURE MEDICINE VOLUME 8 NUMBER 9 SEPTEMBER 22

3 22 Nture Pulishing Group s FGF VEGF + FGF e Tumor weight (mg) s DU145 tumor suppresses ngiogenesis in vivo To evlute the funtion of in loo vessel evelopment, we teste the effets of on ngiogenesis in the hik horiollntoi memrne (CAM). We stimulte CAMs from 1-y-ol hik emryos with si firolst growth ftor (FGF) in the presene or sene of mino is ( ) or two other peptie hormones, litonin ( lium-regulting peptie hormone 25 ) n litonin gene relte peptie (CGRP, vsoiltory peptie hormone 25 ). Only inhiite ngiogenesis, whether nlyze y ounting loo vessel (Fig. 1; P <.1) or the ensity of vessels positive for integrin αvβ3 (ref. 26) (Fig. 1-; P <.1) or von Willern ftor (VWF) (Fig. 1-; P <.1). Anlysis of loo vessel morphology lso inite tht inhiite FGF-inue vsulr rnhing (Fig. 1). h no effet on the numer, size or omposition of pre-existing vessels, s etermine y mrosopi evlution of vessels in sline-stimulte CAMs or y exmintion of the numer n nture of αvβ3- n VWF-positive vessels in sline-stimulte CAMs (t not shown). These stuies inite tht seletively prevents the formtion of new mirovessels. The hlfmximl inhiitory ose of ws.1 µm (t not shown). This inhiition oul e loke y the ition of n ntioy loking funtion (8B12) irete ginst mino is 1 34 (ref. 27) (Fig. 1). lso loke FGF-stimulte ngiogenesis in the mouse (Fig. 1e; P <.1). These stuies inite tht funtions s n inhiitor of ngiogenesis. lso inhiits tumor ngiogenesis n growth. We trete nue mie ering DU145 prostte rinom tumors (tht i not express ) with ily intrvenous injetions of (finl serum onentrtion, 1 µm). suppresse tumor growth (Fig. 1f; P <.5), inue tumor nerosis (Fig. 1g) n inhiite tumor ngiogenesis (Fig. 1h; P <.1). Wheres slinetrete tumors oule in size uring the 1-y stuy, -trete tumors inrese in size y only 3%. These stuies inite tht inhiits tumor ngiogenesis s well s growth ftor inue ngiogenesis. Inhiition of ngiogenesis y gene elivery To etermine if gene elivery of might e useful strtegy for the therpeuti inhiition of Fluoresene intensity (perent of ontrol ) Fig. 2 Inhiition of ngiogenesis n tumor growth y gene elivery. n, Chiken emryos ering CAMs stimulte with FGF or VEGF were injete with enoviruses expressing or full-length., Bloo vessel ± s.e.m. ove kgroun (top) n ryosetions of trete CAMs immunostine with ntioies ginst (ottom). Arrow inites -positive loo vessel., Cryosetions of CAMs from immunostine with ntioies ginst α v β 3 : Immunoretive vessels per mirosopi fiel ( 2) were quntifie (top) n photogrphe (ottom)., Men fluoresene intensity in lystes of FGF-supplemente growth ftor eplete Mtrigel plugs ontining - or -expressing enoviruses. n e, CAMs ering DU145 prostte rinom tumors were injete with enoviruses expressing or full-length., Tumor weight (top) n pperne t 1 mgnifition (ottom) fter 7. e, Cryosetions of tumors from immunostine with ntioies ginst α v β 3 : Immunoretive vessels were quntifie per 2 mirosopi fiel (top) n were photogrphe (ottom)., P <.5 (Stuent s t-test). NATURE MEDICINE VOLUME 8 NUMBER 9 SEPTEMBER

4 22 Nture Pulishing Group ngiogenesis in vivo, we stimulte ngiogenesis in hik CAMs with FGF or vsulr enothelil growth ftor (VEGF). We then trnsue CAMs y injeting enoviruses expressing green fluoresent protein () or full-length (ref. 27) into the emryoni irultion. Virlly expresse, ut not, inhiite ngiogenesis stimulte y FGF (P <.1) or VEGF (P =.1), s shown y quntifition of loo vessel (Fig. 2, top) or integrin α v β 3 -immunoretive vessels (Fig. 2; P <.1). ws etete in loo vessels (rrows) in CAMs trnsue with using n ntioy irete ginst C-terminl mino is (ref. 28) (Fig. 2, ottom). Virlly expresse lso inhiite ngiogenesis in the ult mouse. Virl elivery of inhiite FGF-stimulte mouse ngiogenesis (Fig. 2; P <.5). Virlly inue expression of lso inhiite tumor growth on the CAM (Fig. 2; P <.3), initing tht lolly elivere n suppress tumor ngiogenesis. -trete tumors were smller, more oviously neroti n ssoite with fewer integrin α v β 3 -positive loo vessels thn -trete tumors (Fig. 2e, P <.1). Thus, virlly expresse n inhiit ngiogenesis n my serve s useful therpeuti ngiogenesis inhiitor. e Cells migrte Cells migrte meium nti- Cl CGRP α v β meium nti Cl α v β 3 sline litonin Cell tthment (Asorne 56 nm) Cell tthment (Asorne 56 nm) meium nti- Cl CGRP α v β meium nti- Cl α v β Fig. 3 The N-terminl 34 mino is of inhiit enothelil ell migrtion in vitro n ngiogenesis in vivo. n, Enothelil ell migrtion () n tthment () to vitronetin in the presene of meium, ntioy ginst v β 3 (nti- v β 3 ),, litonin (Cl) or CGRP. n, Enothelil ell migrtion () n tthment () to vitronetin in the presene of meium, ntioy ginst v β 3 (nti- v β 3 ), 1 141, 1 86, 1 34 or litonin (Cl). e, in FGF-stimulte CAMs trete with sline, 1 141, 1 86, 1 34, n litonin., P <.5 (Stuent s t-test). HPF, high-power fiel. inhiits enothelil ell migrtion To evlute the funtion of in enothelil funtions in vitro, we teste the effets of n other peptie hormones on enothelil ell migrtion on extrellulr mtrix sustrtes suh s vitronetin, ollgen n fironetin. signifintly inhiite ell migrtion on vitronetin (Fig. 3), ollgen n fironetin (t not shown), yet h no effet on tthment to these proteins (Fig. 3). Cell migrtion ws inhiite 5% y in the rnge of 1 1 µm. These stuies emonstrte tht inhiits enothelil ell migrtion, ut is not n inhiitor of integrin ligtion. Mpping of nti-ngiogeni tive sites of is ompose of severl omins with istint physiologil properties 29. To ientify the omin(s) of responsile for the nti-migrtory n nti-ngiogeni effets, we evlute the effets of vrious frgments of the peptie hormone in migrtion n ngiogenesis ssys. Like 1 173, frgments ontining the N terminus (1 141, 1 86 n 1 34) inhiite migrtion (Fig. 3), ut not tthment (Fig. 3), on vitronetin. Frgments lking the N terminus, suh s , were not le to inhiit ell migrtion (t not shown). In vivo, 1 141, 1 86 n 1 34 potently inhiite ngiogenesis (Fig. 3e). As the first 34 mino is ontin the ngiogenesis-inhiiting properties, we unertook further stuies to ientify the essentil resiues responsile for ngiogenesis inhiition. Stuies hve shown tht the first 6 mino is of PTH re require for tivtion of PTH/ reeptor signling, wheres the lst 15 re require for high-ffinity ining to the reeptor 29. Five of the first ten mino is in PTH n re ientil, n struture preitions inite these regions hve similr onformtions 3. Therefore, to further elinete the tive ngiogenesis- n ell migrtion-inhiition sites on, we ompre the tivities of frgments of extening from mino is 1 1, 1 34 n to tht of peptie onsisting of srmle version of 1 1. Wheres mino is 1 34 n 1 1 were similrly effetive in inhiiting ell migrtion on vitronetin, mino is n the srmle version of 1 1 i not inhiit ell migrtion (Fig. 4). The N-terminl frgment 1 1 inhiite ell migrtion in ose-epenent mnner, with mximum of 5% inhiition t onentrtion of 1 µm. 1 1 n 1 34 lso inhiite ngiogenesis in the CAM ssy, wheres the srmle 1 1 peptie i not (Fig. 4). 1 1 ws slightly less effetive t inhiiting ngiogenesis thn 1 34, however. 1 1 lso inhiite ngiogenesis in mouse moel of ngiogenesis, wheres the srmle 1 1 peptie i not (Fig. 4; P <.1). These stuies inite tht the first ten resiues re essentil for tivtion of the enothelil ell reeptor in vitro n in vivo. Angiogenesis n e inue y mny growth ftors. To etermine if is generl inhiitor of ngiogenesis, we evlute the effet of 998 NATURE MEDICINE VOLUME 8 NUMBER 9 SEPTEMBER 22

5 22 Nture Pulishing Group Cells migrte Fluoresene intensity (perent of ontrol) meium nti sr. Clitonin α v β sline srmle sline 1 34 on ngiogenesis stimulte y FGF, VEGF, interleukin (IL)-8 n tumor nerosis ftor (TNF)-α (Fig. 4). Angiogenesis inue y eh growth ftor ws inhiite y 1 34, with omplete inhiition ourring t 1 µm. The hlfmximl inhiitory onentrtion for eh growth ftor ws.1 µm or lower. Thus, is potent generl inhiitor of ngiogenesis. Mehnism of nti-ngiogeni effets To estlish the mehnism y whih inhiits ell migrtion in vitro n ngiogenesis in vivo, we evlute the effets of on enothelil ell signl trnsution. Like PTH, interts with the PTH1 reeptor, G protein ouple reeptor expresse on enothelil ells 17 tht tivtes PKA (refs. 13,14). 1 34, s well s ell-permele AMP (iutyryl AMP), rpily stimulte PKA tivity in enothelil ells (Fig. 5). PKA tivtion ws etetle in s few s 2 minutes, with mximum tivity 15 minutes fter stimultion with oth AMP n. To etermine if the nti-migrtory properties of result from signls trnsue through PKA, we evlute the effets of 1 34 on ell migrtion in the presene n sene of the PKA inhiitor N-(2-[p-romoinnmylmino]ethyl)-5-isoquinoline sulfonmie (H89) (Fig. 5). This PKA inhiitor loke the nti-migrtory properties of (P <.1). Furthermore, expression of muttionlly intive form of PKA (npka), whih loks PKA tivtion 31, suppresse the - n AMP-meite inhiition of enothelil ell migrtion (Fig. 5; P <.1). In ft, iret tivtion (perent inhiition) srmle Conentrtion (µm) Fig. 4 mino is 1 1 re suffiient to inhiit enothelil ell migrtion in vitro n ngiogenesis in vivo., Enothelil ell migrtion on vitronetin in the presene of meium, ntioy ginst α v β 3 (nti-α v β 3 ), 1 34, 1 1, 15 34, srmle version of 1 1 (sr. 1 1) or litonin., in FGF-stimulte CAMs trete with sline, 1 1, srmle version of 1 1 or 1 34., Men fluoresene intensity in lystes of growth ftor eplete Mtrigel plugs ontining purifie FGF n sline, 1 1 or srmle version of 1 1., CAMs stimulte y FGF (), VEGF (), IL-8 () or TNF-α () trete with sline or were ounte n perent inhiition of sline ontrol ws etermine., P <.5 (Stuent s t-test). of PKA y AMP (P <.1) or y trnsient trnsfetion with the PKA tlyti suunit (P <.1) lso inhiite enothelil ell migrtion (Fig. 5). We were le to etet expression of oth trnsgenes y western lot nlysis of lystes of trnsfete ells (Fig. 5e). These results inite tht inhiition of migrtion is PKA epenent n tht tivtion of PKA loks enothelil ell migrtion. The smll GTPse R is essentil in regulting ell motility y influening tin ssemly n lmellipoi extension 32. Cell hesion s well s growth-ftor stimultion upregulte R tivity in enothelil ells 33. Ativtion of R ws loke y, y expression of tivte PKA (Fig. 5f) n y AMP (not shown). Overexpression of muttionlly tive R (V12 R) overme the inhiition of ell motility meite y (P <.7), AMP (Fig. 5g; P <.3) or the PKA tlyti suunit (Fig. 5h; P <.6). These stuies inite tht tivtion of PKA inhiits ell migrtion y inhiiting R tivtion. Signl trnsution pthwys tht promote ell migrtion often lso promote ell survivl 34. As inhiits the migrtion of enothelil ells, it is possile tht it inhiits their survivl. In ft, inue poptosis of enothelil ells in ose-epenent mnner (Fig. 5i). As with the effets of on ell migrtion, -inue poptosis ws PKA epenent, s expression of ominnt negtive PKA suppresse this inution of ell eth (Fig. 5j), n suggeste tht iret tivtion of PKA my inue poptosis in enothelil ells. Enothelil ell expression of the tlyti suunit of PKA or exposure to iutyryl AMP inee inue poptosis in enothelil ells (Fig. 5k). These stuies inite tht not only inhiits enothelil ell migrtion, ut lso inues poptosis of enothelil ells in PKA-epenent mnner. inhiition of ngiogenesis is protein kinse A epenent Our stuies inite tht tivtion of PKA in vivo my inhiit ngiogenesis. We therefore evlute the involvement of PKA in the inhiition of ngiogenesis y. Either phrmologil or geneti inhiition of PKA reverse the inhiition of ngiogenesis. A seletive PKA inhiitor (H89) loke the suppression of ngiogenesis inue y 1 34 (Fig. 6; P <.2). Expression of muttionlly intive PKA (npka) lso reverse the inhiitory effets of (Fig. 6; P <.3). Furthermore, tivtion of PKA y AMP (Fig. 6; P <.3) or y expression of the tlyti suunit of PKA in the CAM (Fig. 6; P =.5) potently inhiite ngiogenesis. These stuies emonstrte tht inhiits ngiogenesis y tivting PKA. Reent stuies hve shown tht R tivity is require for ngiogenesis 35. Tken together, these stuies inite tht tivtion of PKA in enothelil ells in vivo uses R intivtion n proly inhiition of ell migrtion in vivo. In ition, or PKA my inhiit ngiogenesis y inuing poptosis of proliferting enothelil ells in vivo. In ft, NATURE MEDICINE VOLUME 8 NUMBER 9 SEPTEMBER

6 22 Nture Pulishing Group PKA tivity (% free C suunit) Cells migrte Cells migrte 2 AMP 1 meium Time (min) 2 1 npka me nti- PKA α v β 3 inh PKA inh meium srm. AMP peptie Cells migrte 1 5 V5 tg tive R totl R meium +AMP npka PKAt PKAt me PKAt Fig. 5 inhiits migrtion y loking R tivtion in PKA-epenent mnner., PKA tivity in enothelil ells in the presene of ulture meium (), () or iutyryl AMP (). Speifi tivity is expresse s perent of totl PKA tht n e iretly tivte y AMP in ell lystes., Enothelil ell migrtion on vitronetin in the presene of ulture meium, ntioy ginst αvβ3 (nti-α v β 3 ), 1 34, the protein kinse A inhiitor H89 (PKA inh) or omine with H89., The effet of muttionlly intive PKA (npka) on ell migrtion in the presene of, srmle ontrol peptie (srm. peptie) or AMP., The effet of AMP n trnsient trnsfetion with the PKA tlyti suunit (PKAt) on enothelil ell migrtion. e, Western lots of lystes of ells trnsfete with, npka or PKAt, inute with ntioies ginst V5 tg n. f, R tivity in enothelil ells trete with ulture meium or n in ells trnsfete with tivte PKA or. g, Migrtion of enothelil ells expressing muttionlly tive R (V12 R; +) or ( ) in the presene of ulture meium, or AMP. h, Migrtion on vitronetin of enothelil ells trnsfete with PKAt, V12 R plus PKAt, or V12 R plus. i, Quntifition of nnexin V positive enothelil ells ulture on vitronetin sustrtes in the presene of. j, Quntifition of nnexin V positive enothelil ells trnsfete with () or npka () n trete with ulture meium, or srmle ontrol peptie. k, Quntifition of nnexin V positive enothelil ells trnsfete with or PKAt n trete with ulture meium (me) or AMP., P <.5 (Stuent s t-test). e f g h i j k Cells migrte 1 5 V12 R Cells migrte me PThrP AMP V12 R PKAt Perent nnexinpositive ells Perent nnexinpositive ells Perent nnexinpositive ells Conentrtion (µm) 2 1 meium ontrol PKAt me AMP TUNEL stining of -trete CAMs showe tht this hormone inue poptosis in FGF- ut not sline-stimulte enothelil ells in vivo (Fig. 6e). Expression of the PKA tlyti suunit in FGF-stimulte CAMs or exposure of CAMs to AMP lso inue enothelil ell poptosis in vivo (Fig. 6f). These stuies inite tht inhiits ngiogenesis y iretly n speifilly inuing PKA-epenent enothelil ell poptosis n, furthermore, tht tivtion of PKA y hormonl, phrmologil or geneti mens is potent methos to inhiit ngiogenesis. Disussion is well-hrterize peptie hormone with physiologil effets on vsulr smooth musle, one n tumor ells We hve shown here tht n n N-terminl ten-minoi frgment inhiit enothelil ell migrtion n ngiogenesis, without ffeting quiesent loo vessels. lso inhiits tumor growth y inhiiting ngiogenesis. We hve lso seen similr effets with PTH (t not shown). tivtes PKA, therey suppressing tivtion of R n ell migrtion n inuing enothelil ell poptosis. Furthermore, inution of through gene therpy my e useful pproh to ngiogenesis inhiition. These re the first stuies to our knowlege to iretly exmine involvement of in ngiogenesis regultion. regultes vsulr tone y relxing vsulr smooth musle 7. Our stuies inite tht the vsoiltory effets of re istint from its effets on ngiogenesis, s tretment of tissues unergoing ngiogenesis with CGRP, nother vsoiltory hormone 25, hs no effet on the numer or qulity of vessels uring ngiogenesis. The effets of on ngiogenesis re speifi, s neither CGRP nor litonin, two peptie hormones with relte funtions tht re evolutionrily well onserve, inhiit ngiogenesis. Although hs no effet 1 NATURE MEDICINE VOLUME 8 NUMBER 9 SEPTEMBER 22

7 22 Nture Pulishing Group on the numer or size of pre-existing smooth musle or periytes (smooth musle like ells ssoite with pillries) in vivo, it oes inhiit FGF-inue inrese in esmin- n smooth musle tin positive vessels in vivo (t not shown). Like enothelil ells, esmin- n smooth musle tin positive periytes or smooth-musle ells proliferte in response to ngiogeni growth ftors 36. Unlike enothelil ells, however, oes not inue poptosis in smooth-musle ells or periytes (t not shown). These stuies suggest tht the min trget of uring ngiogenesis inhiition is the enothelil ell. The -meite inhiition of periyte prolifertion uring ngiogenesis my thus e seonry to its effet on enothelil ells. However, it is possile tht iretly ffets periytes uring ngiogenesis y n lterntive mehnism. my hve importnt funtions in the regultion of ngiogenesis uring emryoni evelopment, prtiulrly uring one formtion. Mie without ie shortly fter irth n hve skeletl normlities tht inlue shortene ones, elerte osteogenesis n inrese one vsulriztion promotes the prolifertion of honroytes, suppressing honroyte hypertrophy n ngiogenesis n therey mintining vsulr regions of rtilge During norml enohonrl one formtion, vsulr rtilge is grully reple y highly vsulrize n minerlize e Sl Sl PKAinh + PKA inh + FGF Sl + npka npka + + FGF AMP Sline FGF VEGF Sline PKAt + FGF one. Disruption of the refully regulte ptterns of vsulriztion n impir one formtion. For exmple, isruption of the pro-ngiogeni tivity of VEGF in one y systemi ministrtion of solule VEGF reeptor suppresses vsulr invsion of the hypertrophi rtilge n elys one minerliztion 37. Aitionlly, mie efiient in MMP-9, mtrix metlloproteinse tht is require for ngiogenesis, show elye osteogenesis, elongte ones n suppresse one vsulriztion 38. Together, these stuies inite tht, VEGF n MMP-9 funtion in opposing mnners to regulte one vsulriztion. Further etile stuies of eveloping tissues from -null mie my ientify itionl tissues in whih regultes vsulr evelopment. Thus, is proly essentil in the negtive regultion of ngiogenesis uring emryoni evelopment. my regulte the progression of soli tumors y inuing humorl hyperlemi of mlignny n lol one metstses 7, However, s with other nturlly ourring tumor-erive ngiogenesis inhiitors 39, relese of into the irultion y primry tumors my suppress the growth of istnt metstses. In ition, my iretly influene tumor ell prolifertion, s inite y its expression in prostte ner ells, where it seems to tivte nuler reeptors to promote tumor ell prolifertion 24. Intrellulr my f Fig. 6 meite inhiition of ngiogenesis is protein kinse A epenent., Sline- or FGF-stimulte CAMs trete with sline (Sl), 1 34, H89 (PKA inh) or plus H89. First sline refers to sline stimulte n sline trete CAMS. Seon sline refers to FGF stimulte n sline trete CAMS., FGF-stimulte CAMs trnsfete with pdna/v5-tgge DNA or pdna/v5-tgge muttionlly intive PKA (npka) DNA n trete with or sline., FGF-, VEGF- or slinestimulte CAMs trete with sline ( ) or iutyryl AMP (+)., FGF-stimulte CAMs trete with sline or trnsfete with pdna/v5-tgge PKA tlyti suunit (PKAt) or pdna/v5-tgge DNA., Dt represent quntifition of loo vessel., P <.5 (Stuent s t-test). e, Cryosetions of sline- or FGFstimulte CAMs trete with CGRP or (see Fig. 1 n ), immunostine to etet frgmente DNA s mrker of poptosis (green) n VWF (re). f, Cryosetions of FGF-stimulte CAMs trnsfete with PKAt DNA or trete with sline or AMP, immunostine to etet frgmente DNA s mrker of poptosis (green) n VWF (re). e n f, Digitlly merge imges show VWF-positive poptoti vessels (yellow), inite y rrows. NATURE MEDICINE VOLUME 8 NUMBER 9 SEPTEMBER 22 11

8 22 Nture Pulishing Group promote tumor ngiogenesis y inuing expression of IL-8 (ref. 24). In ft, previous stuy initing tht promotes ngiogenesis showe only tht onitione meium from trnsfete tumor ells n inue ngiogenesis 4. Our stuies with purifie n with -trnsfete tumor ells inite tht promotes ngiogenesis only when it ts in n intrrine mnner. As n nti-ngiogeni gent, my e useful therpeutilly if elivere t high onentrtions lolly. Our stuies using enovirlly expresse inite tht lol ministrtion of my e n effetive strtegy to inhiit ngiogenesis n tumor growth. tivtes G protein ouple trnsmemrne reeptors, therey stimulting enyl ylse n tivting protein kinse A (refs. 13,14). Mny of the physiologil funtions of, inluing vsulr smooth-musle relxtion, re PKA epenent Our urrent stuies emonstrte funtion for PKA in the inhiition of enothelil ell migrtion n ngiogenesis y. These stuies inite tht lol tivtion of PKA my lso e mens to inhiit ngiogenesis n iseses hrterize y ngiogenesis. Previous stuies hve shown tht tivtion of PKA y AMP n inhiit ell spreing, ytoskeleton formtion n intrellulr signling 41,42. Our t inite tht tivtion of PKA les to inhiition of the smll GTPse R, whih is require for ell migrtion in vitro 32,33 n for ngiogenesis in vivo 35. As R tivity is require for ngiogenesis in vivo, our stuies inite tht loks ngiogenesis y inhiiting R in PKAepenent mnner. Our t emonstrte tht is nturl ngiogenesis inhiitor n tht its lol elivery my e useful strtegy to inhiit pthologil ngiogenesis. More importntly, our stuies emonstrte tht tivtion of PKA y hormonl, geneti or phrmologil mens is n effetive strtegy for suppressing ngiogenesis. Methos Regents. N1 (green fluoresent protein) reporter n -expressing enovirus vetors were otine from D. Cheresh. Mouse PKA tlyti suunit n npka (RImut) DNA (from S. Tylor n S. MKnight) were sulone into topota pdna 3.1 V5/His y PCR-se TA loning oring to mnufturer s iretions (Invitrogen, Crls, Cliforni). My-tgge V12 R ws otine from M. Shwrtz. The sequene n orienttion of onstruts were verifie y DNA sequening. Cells (5 1 6 ) were trnsfete y eletroportion with totl of 3 µg DNA (2 µg expression vetors, 2 µg N1 n 8 µg pbluesript s rrier DNA) in 3 µl enothelil sl meium t 3 V n 45 µf. Expression levels of the trnsfete PKA suunits or were ssesse y western lot nlysis of ell lystes using ntioies ginst V5 or. Cells were ulture for 48 h efore eing use in experiments. Choriollntoi memrne ngiogenesis ssys. CAMs of 1-y-ol hiken emryos (MIntyre Poultry, Rmon, Cliforni) were stimulte with 3 ng FGF, VEGF, IL-8 or TNF-α (Genzyme,Cmrige, Msshusetts) or sline s esrie 43,44. CAMs were trete with 2 µl.1 1 µm peptie frgments 1 173, 1 141, 1 86, 1 34, 15 34, 1 1 or srmle 1 1 (HLQAHSVEDL), litonin, CGRP or sline 24 h fter the stimultion. Alterntively, 1 µm ws pplie together with 1 µg funtion-loking ntioy irete ginst (8B12) or 48 nm H89, seletive PKA inhiitor. Emryos stimulte with FGF or VEGF were lso injete intrvenously with plque-forming units of enoviruses expressing or (ref. 27). Aitionlly, FGF-stimulte CAMs were trnsfete y pplition of 4 µg N1, npka or PKA tlyti suunit plsmi DNA. In some experiments, 5-mg frgments of DU145 prostte rinom ell tumors were ple on CAMs. Emryos were injete intrvenously with plque-forming units of enoviruses expressing or, n tumors were exise fter 7. Ten emryos were use per group. Representtive CAMs were photogrphe t 1 mgnifition. were ounte in eh CAM t 3 mgnifition. Sttistil nlyses use the pire Stuent s t-test. Unfixe CAMs were flsh-frozen, ut into ryosetions n immunostine with ntioies ginst (9H7), αvβ3, smooth-musle tin, esmin n VWF. CAMs were lso stine y the terminl eoxinuleotiyltrnsferse-meite UTP-iotin nik en-leling (TUNEL) metho to etet frgmente DNA together with ntioies ginst VWF or esmin. All thin setions were photogrphe t 2 mgnifition. Experiments were one five to ten times. Mouse ngiogenesis n tumor ssys. Mouse ngiogenesis ssys were one s esrie 45 inorporting sline or 1 µg/ml FGF in growth ftor eplete Mtrigel. Pepties were inorporte in the Mtrigel t onentrtion of 1 µm n enoviruses t onentrtion of plque-forming units per 4 µl Mtrigel. Then, 3 fter injetion of Mtrigel, mie were injete intrvenously with FITC Griffoni (Bneire) simpliifoli letin. Plugs were then remove n fluoresene intensity ws mesure. Ten mie were use per group. Sttistil nlyses use the pire t-test. Nue mie were lso inoulte with negtive tumor ells. Then, 2 weeks lter, mie were trete for 1 with ily injetions of sline, or srmle ontrol peptie (1 µm finl onentrtion). Tumor volume ws mesure t ys n 1. Tumors were exise, ryopreserve, setione, immunostine for expression of CD31 n photogrphe t 2 mgnifition. Ten mie were use per group. Sttistil nlyses use the pire t-test. Institutionl pprovl ws otine for ll niml experimenttion. FITC nnexin V stining. Culture pltes were ote with 1 µg/ml vitronetin t 4 C for 16 h n loke with enture BSA. Humn umilil vein enothelil ells (HUVECs) were inute on pltes in the presene or sene of 1 µm or srmle peptie for 18 h, then inute with FITC nnexin V for 15 min in the rk. Wshe ells were fixe in 1% prformlehye, then ounterstine with DAPI (4,6-imiino-2-phenylinole). The perentge of nnexinpositive ells in five mirosopi fiels ws etermine t 2 mgnifition y fluoresene mirosopy. Cell ulture, migrtion n hesion ssys. HUVECs were grown in enothelil growth meium (ontining 2% fetl ovine serum, FGF n VEGF; Clonetis, Sn Diego, Cliforni). To rener HUVECs quiesent for R ssys, monolyers of enothelil ells were inute for 18 h in enothelil sl meium supplemente with.1% serum (Clonetis, Sn Diego, Cliforni). Cell migrtion n hesion ssys on ECM sustrtes were one s esrie efore 41,42. PKA ssys. PKA tivity ws mesure in HUVECs trete with 1 µm or 25 µm AMP essentilly s esrie 41. Culture meium ws remove from ell monolyers n ws reple with fresh ulture meium ontining 1 µm or 25 µm AMP. PKA tivity ws mesure 2 3 min lter. R ssys. R tivity ws mesure y PAK-ining-omin, PBD-ffinity preipittion s esrie 33 (Upstte Biotehnology, Syruse, New York). Enothelil ells were mintine in suspension for 2 h efore eing plte on vitronetin-ote pltes for 3 min in the presene or sene of 1 mm or AMP. Aknowlegments This work ws supporte y grnts from the Ntionl Institutes of Helth to J.A.V. (CA71619 n CA83133), to R.T. (AR47347) n to L.J.D. (DK6588). R.T. n L.J.D. re supporte y the Deprtment of Veterns Affirs. J.A.V. n L.J.D. re memers of the University of Cliforni, Sn Diego Cner Center. Competing interests sttement The uthors elre they hve no ompeting finnil interests. RECEIVED 27 JUNE; ACCEPTED 18 JULY NATURE MEDICINE VOLUME 8 NUMBER 9 SEPTEMBER 22

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