British Journal of Pharmacology (2005) 145, & 2005 Nature Publishing Group All rights reserved /05 $30.00

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1 British Journl of Phrmcology (25) 145, & 25 Nture Pulishing Group All rights reserved /5 $3. Endothelin-1 enhnces oxidtive stress, cell prolifertion nd reduces poptosis in humn umilicl vein endothelil cells: role of ET B receptor, NADPH oxidse nd cveolin-1 1,4 Feng Dong, 1,4 Xiochun Zhng, 2 Loren E. Wold, 3 Qun Ren, 3 Zhojie Zhng &,1,2 Jun Ren 1 Division of Phrmceuticl Sciences & Center for Crdiovsculr Reserch nd Alterntive Medicine, University of Wyoming, Lrmie, WY , U.S.A.; 2 Deprtment of Phrmcology, Physiology nd Therpeutics, University of North Dkot, Grnd Forks, ND 5823, U.S.A. nd 3 Deprtment of Zoology nd Physiology, University of Wyoming, Lrmie, WY , U.S.A. Keywords: Arevitions: 1 Endothelin-1 (ET-1), n endothelium-derived vsoctive peptide, prticiptes in the regultion of endothelil function through mechnisms tht re not fully elucidted. This study exmined the impct of ET-1 on oxidtive stress, poptosis nd cell prolifertion in humn umilicl vein endothelil cells (HUVEC). HUVECs were chllenged for 24 h with ET-1 (1 pm 1 nm) in the sence or presence of the ET B receptor ntgonist BQ788 (1 mm) or the NADPH oxidse inhiitor pocynin (1 mm). Rective oxygen species (ROS) were detected using chloromethyl-2,7 -dichlorodihydrofluorescein dicette. Apoptosis ws evluted with 4,6 -dimidino-2 -phenylindoldihydrochloride stining nd y the cspse-3 ssy. Cell prolifertion ws mesured y colorimetric ssy. Expression of NADPH oxidse, Akt, pakt, Bcl-2, Bx, IkB, cveolin-1 nd enos ws evluted y Western lot nlysis. 2 ET-1 significntly enhnced ROS genertion nd cell prolifertion following 24-h incution, oth of which were prevented y BQ788 or pocynin, consistent with the ility of ET-1 to directly upregulte NADPH oxidse. ET-1 itself did not ffect poptosis ut ttenuted homocysteineinduced poptosis through n ET B receptor-medited mechnism. Western lot nlysis indicted tht ET-1 llevited homocysteine (Hcy)-induced poptosis, likely cting y ntgonizing the Hcy-induced decreses in Akt, pakt, pakt-to-akt, Bcl-2-to-Bx rtios nd increses in Bx nd cveolin-1 expression. Furthermore, ET-1 downregulted expression of cveolin-1 nd enos, which ws ttenuted y BQ788 or pocynin. 3 In summry, our results suggest tht ET-1 ffects oxidtive stress, prolifertion nd poptosis possily through ET B, NADPH oxidse, Akt, Bx nd cveolin-1-medited mechnisms. British Journl of Phrmcology (25) 145, doi:1.138/sj.jp Pulished online 14 Mrch 25 Endothelin-1; oxidtive stress; poptosis; prolifertion; NADPH oxidse; cveolin-1 Ac-DEVD-pNA, N-cetyl-Asp-Glu-Vl-Asp p-nitronilide; CM-H 2 DCFDA, 5-(6)-chloromethyl-2,7 -dichlorodihydrofluorescein dicette; DAPI, 4,6 -dimidino-2 -phenylindoldihydrochloride; ECGS, endothelil cell growth supplement; ERK, extrcellulr signl-regulted kinse; ET-1, endothelin-1; Hcy, homocysteine; HUVEC, humn umilicl vein endothelil cells; JNK, c-jun NH 2 -terminl kinse; MAP, mitogen-ctivted protein; MAP kinse: mitogen-ctivted protein kinse NADPH, nicotinmide denine dinucleotide phosphte; NFkB, nucler trnscription fctor kb; NO, nitric oxide; pakt, phosphorylted Akt; PKB, protein kinse B; ROS, rective oxygen species Introduction Author for correspondence t: Center for Crdiovsculr Reserch nd Alterntive Medicine & Division of Phrmceuticl Sciences, University of Wyoming, Lrmie, WY , U.S.A.; E-mil: jren@uwyo.edu 4 These uthors contriuted eqully to this work. Pulished online 14 Mrch 25 Endothelin-1 (ET-1), 21-mino-cid polypeptide produced y vsculr endothelil cells, possesses potent vsoctive ctivity nd hs een implicted in the pthophysiology of mny crdiovsculr diseses such s hypertension, therosclerosis nd hypercholesterolemi (Brton et l., 1998; Best et l., 1999; Schiffrin, 21). ET-1 hs een shown to exert its iologicl effects through inding to specific G proteincoupled memrne receptors, nmely ET A nd ET B sutypes. ET A receptor is locted minly on vsculr smooth muscle cells nd trnsmits the signl for vsoconstriction nd prolifertion, wheres the ET B receptor is present predominntly on endothelil cells nd medites vsorelxtion through nitric oxide (NO) nd prostcyclin (Schiffrin, 21). The lnce etween vsoconstriction nd vsorelxtion or ET A nd ET B receptors is the most importnt fctor in determining the regionl lood flow nd lood pressure regultory effects of ET-1 (Schiffrin, 21). Production of ET-1 hs een found to e elevted under numer of hypertensive sttes such s slt-sensitive hypertension (Schiffrin, 21). In ddition to its vsoctive effects, ET-1 lso ffects cell prolifertion, tissue remodeling nd cell survivl

2 324 F. Dong et l NADPH oxidse, cveolin-1 nd endothelin-1 in vrious cell types including vsculr smooth muscle nd endothelil cells (Ziche et l., 1995; Slni et l., 2; Schiffrin, 21). ET-1 hs een demonstrted to enhnce vsculr superoxide (O 2 ) production nd to promote cell prolifertion through induction of rective oxygen species (ROS) (Wedgwood et l., 21; Li et l., 23, ). However, the precise role of ET-1 s modultor of poptosis, the geneticlly progrmmed cell deth process, remins controversil. ET-1 hs een reported to e n ntipoptotic fctor in vrious cell types including endothelil nd vsculr smooth muscle cells (Shichiri et l., 1997; 2; Del Buflo et l., 22). On the contrry, the propoptotic effect of ET-1 hs een documented in humn melnom cells (Okzw et l., 1998). Nevertheless, the signling pthwys responsile for the nti- vs propoptotic properties of ET-1 hve not een elucidted. Signling components of the ET-1 cscde such s the ET-1 receptor, extrcellulr signl-regulted kinse (ERK) nd mitogen-ctivted protein (MAP) kinse molecules hve een found to coloclize with cveolin-1 in cveole (Chun et l., 1994; Peiro et l., 2; Hu et l., 23). Cveolin-1 is the mjor component of cveole, the flskshped memrne invgintions prticipting in multiple cellulr processes such s vesiculr trnsport, cholesterol homeostsis nd perhps, most importntly, signl trnsduction (Okmoto et l., 1998). Cveolin-1 is known to suppress growth nd cell prolifertion (Engelmn et l., 1997; Kifor et l., 23). Incresed cveolin-1 expression is ssocited with oth mcrophge poptosis (Grglovic & Dory, 23) nd ntipoptosis in certin prostte cncer-derived cell types (Timme et l., 2). It is postulted tht regultion of prolifertion or cell deth y cveolin-1 my e cell specific (Grglovic & Dory, 23). However, whether cveolin-1 is involved in ET-1-induced cell prolifertion nd ntipoptosis processes remins unknown. The im of the present study ws to exmine the cusl link mong ET-1-induced responses on ROS genertion, poptosis nd cell prolifertion in humn umilicl vein endothelil cells (HUVEC). We lso exmined expression of protein kinse B (PKB)/Akt, phosphorylted Akt (pakt), nti- nd propoptotic enzymes Bcl-2 nd Bx, respectively, the nucler trnscription fctor kb (NFkB) inhiitory suunit IkB, cveolin-1 nd its downstrem signling molecule enos following 24-h tretment of ET-1. Methods HUVEC cell culture HUVECs were purchsed from the Americn Type Culture Collection (ATCC, Mnsss, VA, U.S.A.) nd cultured in Kight s F12K medium with 2 mm L-glutmine, 1.5 g l 1 sodium icronte,.1 mg ml 1 heprin,.3.5 mg ml 1 endothelil cell growth supplement (ECGS), 1% fetl ovine serum, 1 mgml 1 streptomycin nd 1 mlml 1 penicillin (ATCC, Mnsss, VA, U.S.A.) t 371C with 5% CO 2. HUVECs (1 5 cells ml 1 ) were cultured for, 8, 16 nd 24 h with ET-1 (1 pm 1 nm, endogenous ET-1 ¼ 2 1 pm) for the time-dependent response of ET-1. For susequent studies, confluent HUVECs were treted with ET-1 (1 pm 1 nm) for 24 h in the sence or presence of the nicotinmide denine dinucleotide phosphte (NADPH) oxidse inhiitor pocynin (1 mm), ET B ntgonist BQ788 (1 mm) or homocysteine (Hcy; 1mM) serving s propoptosis control. Hcy or hyperhomocysteinemi is n integrl component of mny crdiovsculr, neurodegenertive nd lcoholic diseses. Hcy my unlesh inflmmtory meditors including NFkB, IL-1 nd IL-6, promote genertion of superoxide nion (O 2 ) leding to oxidtive stress nd endoplsmic reticulum stress en route to ultimte cell injury such s poptosis nd inflmmtion (Ji & Kplowitz, 24). Intrcellulr fluorescence mesurement of ROS The memrne-permele proe 5-(6)-chloromethyl-2,7 -dichlorodihydrofluorescein dicette (CM-H 2 DCFDA) (Moleculr Proes, Eugene, OR, U.S.A.) enters the cells nd produces fluorescent signl fter intrcellulr oxidtion y ROS such s H 2 O 2. Intrcellulr oxidnt stress ws monitored y chnges in fluorescence intensity resulting from intrcellulr proe oxidtion ccording to our previously descried method (Privrtsky et l., 23). Following 24-h ET-1 (1 pm 1 nm) tretment with or without pocynin or BQ788, HUVECs were loded with 1 mm CM-H 2 DCFDA for 3 min t 371C nd wshed with PBS uffer. Cells were smpled rndomly using n Olympus BX-51 microscope equipped with n Olympus MgnFiret SP digitl cmer nd ImgePro imge nlysis softwre (Medi Cyernetics, Silver Spring, MD, U.S.A.). Fluorescence ws clirted with InSpeck microspheres (Moleculr Proes). More thn 5 cells per tretment group were evluted using the grid-crossing method for cell selection in more thn five visul fields per experiment (4 6 experiments). Apoptosis with 4,6 -dimidino-2 - phenylindoldihydrochloride (DAPI) stining We used DNA-specific fluorochrome stining technique to ssess the effect of ET-1 on poptosis (Li et l., 24). Typicl poptotic chnges comprise condenstion of chromtin, its presence long the periphery of the nucleus nd segmenttion of the nucleus. The rte of poptosis ws determined s the percentge of poptotic nuclei (nuclei with condensed nd/or segmented chromtin were counted s poptotic nuclei) per visul field. In rief, HUVECs growing on glss coverslips were rinsed in PBS nd fixed with 4% prformldehyde for 15 min t room temperture, then wshed with PBS. DAPI (1 mgml 1 ) ws dded to the fixed cells efore eing exmined y fluorescence microscopy. More thn 5 cells per tretment group were evluted rndomly in more thn five visul fields per experiment (4 6 experiments). Apoptosis with cspse-3 ssy Cspse-3 is n enzyme ctivted during induction of poptosis. The cspse-3 ctivity ws determined ccording to the pulished method (Li et l., 24). Briefly, 1 ml of PBS ws dded to flsk contining ET-1 (1 pm 1 nm)- treted HUVECs (with or without 1 mm pocynin, 1 mm BQ788 or 1 mm Hcy), monolyer HUVECs were scrped nd collected in microfuge tue. Cells were pelleted y centrifugtion t 1, g t 41C for 1 min. The superntnt ws discrded, nd the cells were lysed in 1 ml of

3 F. Dong et l NADPH oxidse, cveolin-1 nd endothelin ice-cold cell lysis uffer (5 mm HEPES, ph 7.4,.1% CHAPS, 1 mm dithiothreitol (DTT),.1 mm EDTA,.1% NP4). The ssy for cspse-3 ctivity ws crried out in 96-well plte. Ech well contined 2 ml of cell lyste, 7 ml of ssy uffer (5 mm HEPES, ph 7.4,.1% CHAPS, 1 mm NCl, 1 mm DTT nd 1 mm EDTA) nd 1 ml of cspse-3 colorimetric sustrte N-cetyl-Asp-Glu-Vl-Asp p-nitronilide (Ac-DEVD-pNA) (Sigm Chemicls, St Louis, MO, U.S.A.). The 96-well plte ws incuted t 371C for 2 h, during which time cspse in the smple ws llowed to cleve the chromophore pna from the sustrte molecule. Asornce redings were otined t 45 nm with the cspse-3 ctivity eing directly proportionl to the colorimetric rection. Protein content ws determined using the Brdford (1976) method. Cell prolifertion ssy Cell prolifertion ws ssessed with colorimetric cell prolifertion kit (Oncogene, Sn Diego, CA, U.S.A.), which mesures the incresed ctivity of cellulr mitochondril dehydrogenses tht cn cleve the tetrzolium dye WST-1 to formzn. The formzn formtion is then quntified y mesuring the chnge in sornce t 45 nm in microplte reder. The ctivity of mitochondril dehydrogenses is proportionl to cell numer. In rief, HUVECs were seeded onto 96-well pltes nd incuted in 15 ml culture medium t 371C inco 2 tissue culture incutor for 24 h contining ET- 1(1nM) with or without pocynin (1 mm) or BQ788 (1 mm). After 24 h incution, 1 ml of the WST-1 leling mixture ws dded nd further incuted t 371C for 2.5 h. The sornce ws red t 45 nm using Spectr Mx 19 Microplte Spectrophotometer (Moleculr Devices, Sunnyvle, CA, U.S.A.) (Li et l., 24). Western lot nlysis of NADPH oxidse p47 phox, Akt, pakt, Bcl-2, Bx, cveolin-1 nd enos HUVECs were collected nd sonicted in lysis uffer contining 2 mm Tris (ph 7.4), 15 mm NCl, 1 mm EDTA, 1mM EGTA, 1% Triton,.1% SDS nd protese inhiitor cocktil. Equl mounts of protein lystes (5 mg per lne) were seprted on 15% (cveolin-1, p47 phox, Bcl-2, Bx, -ctin, IkB nd phospho-ikb) or 7% (Akt, pakt nd enos) SDS polycrylmide gels in minigel pprtus (Mini- PROTEAN II, Bio-Rd) nd trnsferred to nitrocellulose memrnes (.2 mm). The memrnes were locked in 5% (w v 1 ) nonft milk in TBS-T uffer, nd then incuted with nti-cv-1 (1 : 1), nti-p47 phox (1 : 1), nti-bx (1 : 5), nti-bcl-2 (1 : 5), nti-akt (1 : 1), nti-phospho-akt (1 : 1), nti-ikb (1 : 1), nti-phospho-ikb (1 : 1), nti-enos (1 : 1) nd -ctin (1 : 5) ntiodies t 41C overnight. Anti-cveolin-1 polyclonl ntiody ws purchsed from Sigm. Anti-p47 phox monoclonl ntiody ws kindly provided y Dr Mrk T. Quinn from Montn Stte University (Bozemn, MT, U.S.A.). All other ntiodies were purchsed from Cell Signling Technology (Beverly, MA, U.S.A.). After incution with the primry ntiody, lots were incuted with either nti-mouse or ntirit IgG HRP-linked ntiodies t dilution of 1 : 5 for 12 min t room temperture. Immunorective nds were detected using the Super Signl West Dur Extended Durtion Sustrte (Pierce, Milwukee, WI, U.S.A.). The intensity of nds ws mesured with scnning densitometer (model GS-8; Bio-Rd) coupled with Bio-Rd PC nlysis softwre (Privrtsky et l., 23). For ll Western lot nlysis experiments, -ctin ws used s n internl loding control. Dt nlysis For ech experimentl series, dt re presented s men7 s.e.m. Sttisticl significnce (Po.5) for ech vrile ws estimted y two-wy nlysis of vrince (ANOVA) or t-test, where pproprite. A Dunnett s test ws used for post hoc nlysis when required. Results Effect of ET-1 on oxidtive stress: involvement of ET B receptor nd NADPH oxidse Figure 1 demonstrted the time-dependent response of ET-1 (1 pm 1 nm) on ROS genertion. While ET-1 filed to elicit ny effect on ROS genertion within 18 h of incution, ET-1 (1 pm 1 nm) significntly promoted ROS genertion fter 24 h of incution. Longer incution time (up to 48 h) of ET-1 did not elicit ny further enhncement of ROS genertion (dt not shown). Therefore, 24 h ws used for ll susequent ET-1 incution experiments. Figure 2 depicted tht 24 h incution of ET-1-induced enhncement of ROS reched plteu phse etween 1 pm nd 1 nm. Interestingly, oth the ET B receptor ntgonist BQ788 (1 mm) nd the NADPH oxidse inhiitor pocynin (1 mm) completely prevented ET-1-induced elevtion in ROS genertion (Figure 2), suggesting likely involvement of ET B receptor nd NADPH oxidse in ET-1-induced ROS genertion. Neither pocynin nor BQ788 ffected ROS genertion itself (Figure 2). The ET A receptor ntgonist BQ123 (1 mm) did not ffect ET-1-induced ROS genertion (dt not shown), supporting the notion tht the ET A receptor sutype does not exist in HUVECs (Duerrschmidt et l., 2). DCF Fluorescent Intensity pM ET-1 1pM ET-1 1nM ET Incution Time (hr) Figure 1 Time-dependent response of ET-1 (1 pm 1 nm) on intrcellulr ROS genertion in HUVECs. ROS genertion ws detected using CM-H 2 DCFDA. Men7s.e.m., n ¼ 4, Po.5 vs respective control group. 24

4 326 F. Dong et l NADPH oxidse, cveolin-1 nd endothelin-1 1 pm ET-1 1 pm ET-1 c d 1 nm ET-1 1 nm ET-1 1 nm ET-1 e f g.6.5 DCF Fluorescent Intensity pM ET-1 1pM ET-1 1nM ET-1 Apocynin Figure 2 Effect of ET B receptor ntgonism nd NADPH oxidse inhiition on ET-1-induced ROS genertion. HUVECs were incuted with ET-1 (1 pm 1 nm) for 24 h in the sence or presence of the ET B receptor ntgonist BQ788 or the NADPH oxidse inhiitor pocynin efore ROS ws detected using CM-H 2 DCFDA. () ; () 1 pm ET-1; (c) 1 pm ET-1; (d) 1 nm ET-1; (e) 1 nm ET-1 þ pocynin (1 mm); (f) 1 nm ET-1 þ BQ788 (1 mm). (g) Summry of four independent experiments. Men7s.e.m., n ¼ 4 6 experiments, Po.5 vs control, Po.5 vs 1 nm ET-1. BQ788 Effect of ET-1 on poptosis in HUVEC Our results indicted tht ET-1 lone (1 pm 1 nm) did not significntly ffect poptosis in HUVECs following 24-h tretment (Figure 3j nd k). As expected, Hcy (Hcy, 1 mm) elicited overt poptotic cell deth mesured with DAPI stining nd in the cspse-3 ctivity ssy. While lower concentrtions of ET-1 (1 nd 1 pm) did not ffect Hcyinduced poptosis, higher levels of ET-1 (1 nd 1 nm) significntly ttenuted Hcy-induced poptosis in HUVECs. The ET-1-induced ntipoptotic effect ginst Hcy ws olished y the ET B receptor ntgonist BQ788 (1 mm) (Figure 3 i). BQ788 itself did not ffect Hcy-induced poptosis (dt not shown). Effect of ET-1 on cell prolifertion in HUVEC vi n ET B -NADPH oxidse-dependent pthwy Figure 4 shows tht ET-1 (1 nm) significntly promoted cell prolifertion of HUVECs fter 24-h incution, which ws completely prevented y the NADPH oxidse inhiitor pocynin (1 mm) nd the ET B receptor ntgonist BQ788 (1 mm), suggesting tht ET B receptor nd/or NADPH oxidse likely medite the prolifertive response of ET-1. Neither pocynin nor BQ788 lone ffected cell prolifertion (Figure 4). To confirm the involvement of NADPH oxidse in ET-1-induced prolifertion nd ROS genertion in HUVECs, Western lot nlysis ws performed nd the results indicted tht ET-1 (1 nm) significntly

5 F. Dong et l NADPH oxidse, cveolin-1 nd endothelin Hcy (1 mm) c 1 pm ET-1+Hcy 1 pm ET-1+Hcy 1 nm ET-1+Hcy 1 nm ET-1+Hcy +Hcy d e f g h Apoptosis (%) DAPI stining 1pMET-1 1pMET-1 1nM ET i Cspse-3 Activity (% of ) Hcy j 1 DAPI Stining k 1. Apoptosis (%) Cspse-3 Activity (% of ) pMET-1 1pMET-1 1nM ET-1 1pMET-1 1pMET-1 1nM ET-1 Figure 3 Effect of ET-1 on sl nd Hcy-induced poptosis. HUVECs were incuted with 1 mm Hcy for 24 h in the sence or presence of ET-1 (1 pm 1 nm) or BQ788 (1 mm). () ; () Hcy; (c) Hcy þ 1 pm ET-1; (d) Hcy þ 1 pm ET-1; (e) Hcy þ ; (f) Hcy þ 1 nm ET-1; (g) Hcy þ þ BQ788. (h) Summry of 4 6 independent DAPI poptotic ssys. (i) Effect of ET-1 on Hcy (1 mm)-induced chnge in cspse-3 ctivity. (j) Effect of ET-1 on sl poptosis (DAPI stining); (k) Effect of ET-1 on cspse-3 ctivity. Men7s.e.m., n ¼ 4 6 experiments, Po.5 vs control; Po.5 vs 1 mm Hcy; þ Po.5 vs Hcy þ. upregulted protein expression of the p47 phox suunit of NADPH oxidse fter 24-h incution. Protein expression of -ctin confirmed equl loding etween ET-1-treted nd untreted groups (Figure 4). This result vlidted direct ctivtion of NADPH oxidse y ET-1, which ws consistent with the inhiitory effect of the NADPH oxidse inhiitor pocynin on ET-1-induced ROS genertion nd cell prolifertion.

6 328 F. Dong et l NADPH oxidse, cveolin-1 nd endothelin-1 p47 phox Aritrry opticl density Cell prolifertion p47 phox β-ctin Apocynin BQ788 Effect of ET-1 on Akt, Bcl-2, Bx, IkB, cveolin-1 nd enos protein expression 44Kd To exmine the potentil signling pthwys involved in the ET-1-induced ntipoptotic nd prolifertive response, severl relevnt nti-/propoptotic signls were exmined in Hcytreted HUVECs using Western lot nlysis following 24-h ET-1 incution. Figure 5 reveled tht Hcy (1 mm) significntly depressed protein levels of totl Akt, pakt nd the rtio of pakt-to-totl Akt, ll of which my e ntgonized y coincution with ET-1 (1 nm). ET-1 itself significntly enhnced pakt nd pakt-to-totl rtio. Neither Hcy nor ET-1 significntly ffected the Bcl-2 expression following 24-h incution. However, Hcy (1 mm) significntly enhnced expression of the propoptotic gene Bx nd reduced the Bcl-2-to-Bx rtio, oth of which were reversed y ET-1 (1 nm) (Figure 6). ET-1 itself did not significntly ffect expression of 42KD Figure 4 () Effect of ET-1 on prolifertion in HUVEC. HUVECs were incuted with ET-1 (1 nm) for 24 h with or without pocynin nd BQ788. () Effect of ET-1 (1 nm) on the p47 phox suunit of NADPH oxidse in HUVECs fter 24 h incution using immunolot technique. Inset shows representtive lot of immunostining using nti-p47 phox nd nti--ctin (s loding control) ntiodies. Men7s.e.m., n ¼ 4 6 experiments, Po.5 vs control; Po.5 vs 1 nm ET-1. AKT Phospho-AKT c d AKT levels (Aritrry opticl density) p-akt levels (Aritrry opticl density) p-akt : AKT rtio Hcy + 6Kd 6Kd Bcl-2 or Bx (dt not shown). While neither Hcy (1 mm) nor ET-1 (1 nm) ffected totl protein expression of the NFkB inhiitory suunit IkB, they oth stimulted phosphoryltion of IkB (which removes the inhiition of IkB on NFkB). ET-1 +Hcy +Hcy Figure 5 Effect of ET-1 (1 nm) on Hcy-induced response of Akt nd pakt in HUVEC. HUVECs were incuted with Hcy (1 mm) in the sence or presence of ET-1 (1 nm) for 24 h. () Representtive lots showing immunostining with nti-akt or nti-pakt ntiodies in HUVECs; (, c) Akt nd pakt protein expression fter 24- h incution Hcy (1 mm) with or without ET-1 (1 nm); (d) pakt-to- Akt rtio. Men7s.e.m., n ¼ 4 6 experiments, Po.5 vs control, Po.5 vs Hcy (1 mm).

7 F. Dong et l NADPH oxidse, cveolin-1 nd endothelin did not ffect Hcy-induced phosphoryltion of IkB (Figure 7). Hcy (1 mm) significntly enhnced the protein expression of cveolin-1, which my e ntgonized y ET-1 (1 nm). ET-1 induced downregultion of cveolin-1 expression, which ws olished y pocynin or BQ788, suggesting tht oth the ET B receptor nd NADPH oxidse re likely involved in ET-1- induced regultion of cveolin-1 (Figure 8). On the other hnd, oth Hcy (1 mm) nd ET-1 (1 nm) significntly reduced enos protein expression. Hcy-induced decrese in enos ws further mplified with the presence of ET-1. ET-1-induced decrese in enos expression ws ttenuted y pocynin nd BQ788, suggesting tht oth the ET B receptor nd NADPH oxidse re likely involved, t lest in prt, in ET-1-induced regultion of enos (Figure 9). Protein expression of -ctin ws exmined in ll Western lot nlyses to ensure equl protein loding (dt not shown). Discussion Our results indicted tht ET-1 enhnced oxidtive stress in HUVECs following 24-h incution. The oservtion of enhnced ROS genertion in response to ET-1 exposure is consistent with the finding tht ET-1 is cple of promoting O 2 genertion, however, through n ET A receptor-nadph oxidse-medited pthwy in the vsculture (Li et l., 23). Recent evidence suggested tht ROS, such s O 2 nd H 2 O 2, my ply role in the prolifertion of vsculr smooth muscle cells (Wedgwood et l., 21) nd firolsts (Cheng et l., 23). ROS my elicit wide vriety of iologicl/pthologicl responses, depending upon the cell type, the mgnitude nd the durtion of exposure. It ws suggested tht low doses of ROS re mitogenic nd promote cell prolifertion, wheres intermedite doses my result in temporry or permnent growth rrest. Severe oxidtive stress due to high ROS environment usully leds to cell deth vi either poptotic or necrotic mechnisms (Mrtindle & Holrook, 22). ET-1 hs een shown to rescue cells from poptosis induced y vrious poptotic stimuli including pclitxel, NO, serum deprivtion nd c-myc (Shichiri et l., 1998; 2; Del Buflo et l., 22). Consistently, our results showed tht ET-1 possesses n ntipoptotic effect ginst Hcy through n ET B - medited mechnism. Our study reveled tht the ntipoptotic mechnisms of ET-1 ginst Hcy-induced poptosis my e medited through its ntgonism ginst Hcy-induced decrese in pakt : Akt rtio, increse in Bx expression nd decrese in Bcl-2 : Bx rtio. These propoptotic (Bx) nd ntipoptotic (Akt, Bcl-2) molecules pper to disply tissue nd cell specificity in response to ET-1 exposure. For exmple, ET-1 ws shown to induce expression of Bcl-2 in crdic myocytes vi cyclosporin A-dependent mnner without ffecting Bx expression (Kkit et l., 21). No effect ws oserved for ET-1 on Bcl-2 or Bx expression in vsculr smooth muscle cells (Diep et l., 2). ET-1 hs een reported to stimulte Akt phosphoryltion, which ws olished y the ET B receptor ntgonist BQ788 (Del Buflo et l., 22). Akt phosphoryltion ws demonstrted to occur within minutes of ET-1 receptor inding, peked t 3 min nd susequently declined, consistent with the notion of rpid signling nd prolonged desensitiztion of ET-1 signl pthwys (Liu et l., 23). Interestingly, our study reveled significntly elevted Akt phosphoryltion following 24-h incution of ET-1, Bcl2 Bx c Bcl2(Aritrry opticl density) Bx(Aritrry opticl density) d Bcl2 : Bx rtio _ + + Hcy + Hcy + Hcy 26Kd 23Kd Figure 6 Effect of ET-1 (1 nm) on Hcy-induced response of Bcl-2 nd Bx in HUVEC. HUVECs were incuted with Hcy (1 mm) in the sence or presence of ET-1 (1 nm) for 24 h. () Representtive lots showing immunostining with nti-bcl-2 or nti-bx ntiodies in HUVECs; (, c) Bcl-2 nd Bx protein expression fter 24-h incution Hcy (1 mm) with or without ET-1 (1 nm); (d) Bcl-2- to-bx rtio. Men7s.e.m., n ¼ 4 6 experiments, Po.5 vs control, Po.5 vs Hcy (1 mm).

8 33 F. Dong et l NADPH oxidse, cveolin-1 nd endothelin IkB 41KD Cv-1 22KD Phospho-IkB IkB levels (Aritrry opiticl density) KD Cv-1levels (Aritrry opiticl density) Cv KD c Phospho-IkB levels (Aritrry opiticl density) Cv-1levels (Aritrry opiticl density) d Phospho-IkB : IkB rtio Figure 7 Effect of ET-1 (1 nm) on Hcy-induced response of totl nd phosphorylted IkB in HUVEC. HUVECs were incuted with Hcy (1 mm) in the sence or presence of ET-1 (1 nm) for 24 h. () Representtive lots showing immunostining with nti-ikb or ntiphospho-ikb ntiodies in HUVECs; (, c) IkB nd phospho-ikb protein expression fter 24-h incution Hcy (1 mm) with or without ET-1 (1 nm); (d) phospho-ikb-to-ikb rtio. Men7s.e.m., n ¼ 4 6 experiments, Po.5 vs control. Figure 8 () Effect of ET-1 (1 nm) on Hcy-induced response of cveolin-1 (Cv-1) expression in HUVEC. HUVECs were incuted with Hcy (1 mm) in the sence or presence of ET-1 (1 nm) for 24 h. () Effect of NADPH oxidse nd ET B receptor inhiition on ET-1- induced decrese in Cv-1 protein expression in HUVEC. HUVECs were incuted with ET-1 (1 nm) for 24 h with or without pocynin (1 mm) nd BQ788 (1 mm). Insets show representtive immunostining using nti-cv-1 ntiody. Men7s.e.m., n ¼ 4 6 experiments, Po.5 vs control; Po.5 vs ET-1 (1 nm). suggesting tht 24 h incution of ET-1 my not led to desensitized Akt phosphoryltion nd susequently insulin resistnce, which occurs for chronic ET-1 tretment in vivo (Wilkes et l., 23). Although NFkB hs een implicted in ET A receptor ctivtion-induced cell prolifertion nd inhiition of poptosis (Mngelus et l., 21), dt from our present study do not fvor role of NFkB in ET-1-induced ntipoptotic effect ginst Hcy since ET-1 nd Hcy oth triggered phosphoryltion of the NFkB inhiitory suunit IkB (Figure 7). ET-1 hs lso een shown to ctivte cscde of signling molecules including ERK1/2, p38 MAP kinse nd c-jun N-terminl kinse (JNK) (Cheng et l., 23), which my ply role in ET-1-medited protection ginst poptosis (Mrtindle & Holrook, 22). Further study is wrrnted to

9 F. Dong et l NADPH oxidse, cveolin-1 nd endothelin enos enos levels (Aritrry opiticl density) enos enos levels (Aritrry opiticl density) Kd elucidte the role of these signling pthwys in ET-1-elicited ntipoptotic effect. One interesting finding is tht ET-1-elicited ntipoptotic effect ginst Hcy is mirrored to some extent y ltertions in the levels of cveolin-1 (Figure 8). Cveolin-1 orgnizes proteins in cveole, thus enling finely tuned regultion of physiologicl responses (Okmoto et l., 1998). As suppressor of growth nd cell prolifertion (Engelmn et l., 1997; Kifor et l., 23), cveolin-1 level is ssocited with poptosis (Grglovic & Dory, 23). Overexpression of cveolin-1 my sensitize cells to poptotic stimuli, possily vi inhiition of PI-3 kinse nd/or ctivtion of cspse-3 (Zundel et l., 2; Liu et l., 21). This is consistent with our finding of similr chnges of cspse-3 ctivity nd cveolin-1 expression in response to ET-1 exposure. It is suggested tht ET B receptor forms complex with cveolin-1 in cells in which these two 14KD Figure 9 () Effect of ET-1 (1 nm) on Hcy-induced response of enos expression in HUVEC. HUVECs were incuted with Hcy (1 mm) in the sence or presence of ET-1 (1 nm) for 24 h. () Effect of NADPH oxidse nd ET B receptor inhiition on ET-1-induced decrese in enos protein expression in HUVEC. HUVECs were incuted with ET-1 (1 nm) for 24 h with or without pocynin (1 mm) nd BQ788 (1 mm). Insets show representtive immunostining using nti-enos ntiody. Men7s.e.m., n ¼ 4 6 experiments, Po.5 vs control; Po.5 vs ET-1 (1 nm). proteins were co-expressed. The ET B /cveolin-1 complex ws formed efficiently only when ET B ws unoccupied or ound to n ntgonist. ET-1 my dissocite this complex. In contrst, ET A (lthough not present in HUVECs; Duerrschmidt et l., 2) cn ind to cveolin-1 regrdless of lignd-inding sttus. Cveolin-1 utilizes its scffolding domin (residues 82 11) nd the C-terminl domin (residues ) to ind to ET B receptor (Ymguchi et l., 23). We speculte tht ET-1 my compete with cveolin-1 for the ET B receptor, nd therefore disrupt the ET B /cveolin-1 complex nd ssocited poptosis. Nevertheless, the precise reltionship etween cveolin-1 nd poptosis my e complicted since metsttic prostte cncer cells exhiit incresed expression of cveolin-1 nd reduced poptosis (Nsu et l., 1998; Li et l., 21). NO prticiptes in the regultion of ROS genertion nd poptosis y oth inducing nd suppressing ROS genertion nd poptosis depending upon the species or cell types. NO ws shown to inhiit prolifertion, induce ROS genertion nd poptosis (Gordon et l., 21; Del Buflo et l., 22). Results from our current study displyed tht 24-h tretment of Hcy or ET-1 reduced enos expression, indicting tht the protection of ET-1 ginst Hcy-induced poptosis is unlikely relted to ltertion of enos protein expression. However, ET-1 downregulted enos expression ws medited through n ET B -NADPH oxidse-dependent mechnism, similr to its effect on ROS genertion. Reduced enos expression in response to ET-1 exposure is likely to ply role in ET-1- induced ROS genertion. ET-1 further decresed Hcy-induced enos protein expression (Figure 9). This my e ttriuted to ET-1-induced ROS ccumultion since ROS itself cn reduce enos expression nd the ssocition of enos with cveolin-1 (Peterson et l., 1999). Diminished enos protein expression nd NOS ctivity hve een reported following ET-1 nd Hcy tretment (Li et l., 22; Tognetti et l., 23), suggesting the existence of functionl reltionship etween ET-1 nd enos leding to ROS genertion. It should e noted tht ET-1 through ET B my lso stimulte NO production in sinusoidl endothelil cells s result of Akt ctivtion nd susequently enos phosphoryltion (Del Buflo et l., 22). enos is n importnt sustrte for Akt nd my e ctivted y pakt (Liu et l., 23). The discrepnt responses of ET-1 on enos nd Akt phosphoryltion re not fully understood, ut my e relted to the durtion of ET-1 tretment. Experimentl limittions Although our study indicted tht ET-1 is implicted in the regultion of ROS, poptosis nd cell prolifertion likely through n ET B receptor/nadph oxidse-medited pthwy(s) in HUVEC, these results did not fully ddress if enhnced cell prolifertion my e responsile for discrepnt responses of ROS genertion nd ntipoptosis ginst Hcy upon ET-1 exposure. An mple mount of evidence suggests tht incresed cell prolifertion is usully ccompnied with reduced poptosis in response to extrcellulr stimuli including ET A receptor ctivtion (Mngelus et l., 21). However, inhiition of cell prolifertion is often ssocited with enhnced poptosis (de Ruijter et l., 24). Nevertheless, these complex events mong ROS genertion, cell prolifertion nd poptosis in response to ET-1 exposure re not yet understood nd wrrnt further work. In ddition, we do not know if the cells eing studied were in the quiescent or

10 332 F. Dong et l NADPH oxidse, cveolin-1 nd endothelin-1 prolifertive stte. Position of the cell cycle my e n importnt fctor for poptosis. Since ET-1 my ctivte the ERK1/2, p38 MAP kinse nd JNK signling molecules (Cheng et l., 23), potentil prticiption of these signling pthwys in ET-1-induced cell prolifertion should not e ruled out t this time. Lstly, the Western lot technique used for signling mechnisms of ET-1 in our study is somewht semiquntittive nd the degree of chnge in these proteins ws reltively modest. It should not e considered the sole evidence of mechnistic link, without confirmtory functionl s well s specific gene overexpression or deletion dt. In summry, our results suggested tht ET-1 my induce oxidtive stress, prolifertion nd protect ginst Hcy-induced poptosis in HUVEC through ET B receptor/nadph oxidsedependent pthwys. Our study indicted tht ntgonism ginst Hcy-induced decrese in Akt phosphoryltion nd Bcl-2/Bx rtio s well s downregulted cveolin-1 protein expression involving n ET B /NADPH oxidse-dependent pthwy my ply role in ET-1-medited cellulr effects (such s ntipoptosis) in HUVECs. Further study is underwy to estlish the definitive connection mong ROS genertion, cell prolifertion nd poptosis, s well s role of cveolin-1 nd enos in ET-1-induced regultion of cell survivl. This work ws supported in prt y grnts from Ntionl Center for Reserch Resources (P2 RR1564) to University of Wyoming (R.O. Kelley, PI), NIH R3 AG nd Americn Hert Assocition Pcific Mountin Affilite (355521Z) to J.R. We pprecite helpful discussion from Dr Alex F. Chen (Michign Stte University, Est Lnsing, Michign). References BARTON, M., HAUDENSCHILD, C.C., D USCIO, L.V., SHAW, S., MUNTER, K. & LUSCHER, T.F. (1998). 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