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I I I I I I I I I I I I I I Ae+sPum Jo4rrul of Pmholoo. VbL 239, rb. 6s Datismt+n 1991 Cxg,.O.o."W%= An=WzWn CfPA6Dk8= WARNER-LAMBERT/PARKE-DAVIS AWARD LECTURE Viral Pathogenesis of Atherosclerosis Impact of Molecular Mimicry and Viral Genes David P. kajjar Prorrt dtt DE.pm-nea of BiocbernEspy and ParbohV, CrvxeH lfrriuastty Medtral Calkge, Nau York, New Yai Xurnan atibervgenaYs Lr a plafotsopic process uritb an wrdeflned catr.se. Several pathologic factors bave been lixkeid to tbe dLuase procesr, including asterial injury or activation of tbe endotbeliu"4 uticb may initiate proatberosclerotic eventa !ac Oe uessel u41L Atbeiosclerotic lesiosss aee rbaracterized in parS by tbe pracenoe of activated immune ul4 abnormal cell prollferardost, and alttreid cholesterpl metabo• ilsrn Tbese activated lmmunocompetent cells in Plaques produce vasoactive m.diators tbat can alter bomaostasic and maypromote the artsrlopotb}: Botb molecttlarand structtssrrl evidence irpserented thas berpesvirreses, by uaay of inductron of altened gene function and cellular cboGxterol metaboline, aou- pled wit#+ tbeir ability to activate coagulation and a monocyte r+eceptor on tbe infected eridotliellum, are inuolued in ma}orpatbogenic events assocYarrd rvr;tb atiKrosclerotis and d"rombaele Work from the au- tbor't taboratory, as unell as from otber researcb group; bavv sboum t6at avzan and buff&= bespetvi- rusas act rpectifiaally to induce alterations to the sur• ,Jizce and inner layers of the blood ueael u+a11 tfiat rnay pradispose to atbesarclerosis and its attendent dtnioal compii=tioss (AmJPadoi 1991. 13,9:1195- 1211) An important fsnure of the penesis of human atheroscle- rosis is its muttifacmrial nature. No single errtity has been tinked diroctty to its pathloQenesis in.,tMr animal models of tho disease or in the human art.riopaihy. Eviderlce is accurnulatirp that eady deveioprrwtW changes in spe- c'iflc vascular beds highly susceptlble to the disease are paramount to the laiter alterations of arterial metabolism that predisposes to subsequent atheromatous chanpas.'-'3 Utxloubtedly these changes are amplified by risk factors such as tlypercttiolesterolemia, hypenelr sion, smoiat~, and diabetes. For almost 20 years, interest in a viral cause of ath- erosderosis has continued, spurred by the oariy worlc of Bersditt and Bendrtf and Fabricant et a1.5° This perse- verance to define the vi4 cytopatfloJoyic mechanisms in4oived has certainly been enhanced by studies of an tulimal model ot viraily induced atherosclerosis under normocholesterolemic conditions,°"° and by the recog- nition of the association of this disease in patients who have underQore cardiac traalsplant surgery, butwho elso are infected with cyrprregalovirus,'a,' one of the seven herpesvin~ses now known to infect humans.12 Vascular injury purported to be invofved in early stages of the arteriopathy is also a corrrnon feature of acuta herpesvirus infection. Recent studies have identi- fied herpesvirus antigens and nucleic acids in the vas- cuiar vwaji"-'° after their passage through the endothe- lium. At the biochemical level, a variety of herpesvirus- induced paiflobiobgic changes have been documented within va,scular cells that impact on cellular lipid (choles- terol)° and connective tissue (proteoqlycans) metabo- lism.'° In fact, ttse harpesvirus-induced atteratiorls in lipid metabdism in vifro and in vivo paralW those patholcgic events rtininiscent of the artetiopatty found in the human disoase,cso4-2 Fi.rpecvinises induce an arterial lesion characteristic of leukocytodastic vasculibs. There is granuloeyte infiltra- tion in the artery, accompanied by ttuombin formadon AOCEQeO lor OubiQGm Au]ttQ ®.1491. AEeta=rsprirt r.4mrslo Dr. DariG P. M.pr. Prof®ordBbCtfar). iftry and P&p1?gy, COrT1d Unir.r.¢y MedP1! CObepe, 1300 YUk Aw- rue, nlea YoaK NY 10321. 1195 I

1196 HaiJar .SlpDet.nnbe+ 1.992. Vol. 139. No, 6 and fibrin deposition.2` Hence this veciar has the ability to influence not only atherosclerotic chatlges but also processes related to a thrombotic diathesis. Because the virus causes a fattyy-proliferative lesion in animals that oo- casionaly contains microthrombin,'such events support the hypothesis that surfaca expression of adhesion mof- ecules and intimal hyperpiasia may be initiated by a vi- relly induced event. This may trigQer cell proliferation by enhancement of growth factor or by cyrolune release from vascular cells or adherent macrophages, Or it alro may alter the balance between mitogen and growttt sup- pressor synthesis in the vascular well. Based on this background informaGon, both molecu- lar and structural evidence are highlighted to support the hypothesis that herpesviruses, by akering gene function and cellutar lipid metabolisrn, coupled with the abiliry to activate the coa8ulation system and a morxx,yte recep- tor on the endothefium, are invoived in pathogenic events associated with human atherosclerosis. Information about candidate virat or transforming genes potentialiy related to atherogenests a(so are presented. Epidemiologic Evidence Linking Herpesvirus and Atherosclerosis Herpesvirus infections are widespread in the general populatioon.2r-a' They are ubiquitous viruses found in most tissues of the body, even in blood ceils, By 10 years of age, more that15096 of children dertnnstrate antibod- ies to herpes simplex virus (H" type 1.25 The incidence of herpes simplex vinus, type 2. is more difficult to esti- mate, but two studies cite a prevalence of up to In the 35- to 44year-0id age group, substantially more than 1 t?96 demonstrate arrbbodies to HSV type 2,26-2' A more recent study demonstraied that the incidence of infection approaches 209'o in the population aged 15 to 74 years.27 It has now been documented that seven drf- ferent herpesvirus can infect hurnans.'2 Human atherosclerosis is not completeiy accounted for byall krown risk factors. Th;s suggests that tfle human risk factors may by synergistic with other, as of yet un- identi8ed, initiating factors.'•26 A direct seroepidernio- logic link between cytomegalovirus (CMV) infection and atherosclerosis has been suggosted.2° A cas¢.cantrof study was perforrrled wherein patients who underwent rardiovascular surQery were compared with a control group of subjects wittt similar cholesterol levels and epi- demiolog ic factors but who were not undergoing surgery. In approximatefy 160 pairs of patients, the prevalence of CMV antibodies was higher in the surgical group than in the control group (90% and 74%, respectively), and a greater percentage of surgical cases than controls had high trters of CMV antibodies (57% and 26%, respec- tively), There was no correlation between arrtibody titers and blood levels of cholesteroi or triplycerides. Interest ingly a recent Fremingham Heart Study failed to find an overall a5sociation between fevef blisters or cold sores, with a 6-year incidence of caronary heart disease in pa- tients 58 to 89 years of age.30 Previous work in this group of patienis had shown a strong correlation of a self- reported history with serologic evidence of previous herpes simplex virus type 1(I-iSV-1) infection. This study does not completely rule out tfze possibility of a relation- ship between HSV and atheroscierosis, particularty be- cause a subgroup ot women with recurrent cold sores had twice the risk of developing coronary heart disease. The strongest epidemiologic link between infection with herpesvin~ses and atherosclerosis in humans is in the heart transplant population. Several recent studies have demonstrated a strong correlation between CMV infecdon and accelerated atherosclerosis,'0" At Stan- ford University, 300 cardiac transplant pat;ents were treazed with immunosuppressan and were f0liowtd pro- spectrvely for the occurrence of CMV infection and the derelopment of atherosclerosis. Of those tested. 91 pa- tie„ts developed CMV infections, based on: 1) positive cultures for CMV. 2) demonstration of daacteristic CMV inclusion bodies in tissue samples, or 3) a fourfold rise in garrrra 3 imru.unoglobuiin (19G) CMV antibodies. After a 5-year folbvwup, the rate of graft bss due to acceserated aiherosderosis was 69% in CMV-infected patients, but only 37% in the non-CMV-infected group.10 Furthermore there was a 1t}fold greater incidence of patients who died with mare than 50% luminal obstruction of their cor- onary arteries. These individuals had CMV infections./0 Similar results wers reported from a sorne..what smaller study at the University of Minnesota." Thts study corn- pared rates of CMV infectiort and atherosclerosis in 102 immunosupprG;sed paisents who had received a car- diac transplant and survived for at least 1 year, At 2 years after transplant, 32% of the CMV-positive patients had corLxmiry artery disease, as opposed to 10°,6 of the CMV- negative patients. To date, these findings provide the strongest evidence in humans linking herpesvirus infeo- tion with atherosclerosis. Pathologic Evidence Linking Herpesviruses with Atherosclerosis After the publication in 1973 of a landmark study by Dr. Eari R Benditt on the possible rnonocknal nature of atf'~- erosclerotic Iesions.' his laboratory was one of the first groups to docvment the presence of herpesvirus nudeic acids in arterfal tissue removed during coronary bypass surgery. They used in sitU hybridizaibrt techniques." Approxirnaiel'y 10% of 160 tissue samples were positive in wit lea iiy ~ turt var I I I ticle da I Ing ~ sho I . DNI d em g. Cn diaiE c0uL Sug~ ataa senc mew Perh trof c thick F forrtx cal e VlCfin acid L-4 I I I I I I

Yra! Pathog.nesit of Ath.nosclerosis 1197 47 ORnels 1992, vcl 1,39, Na 6 I I I I r I a M I I I 1 F for herpeslirus. A second series of experiments exam- ined tissues that contained abnormally thickened intima Ffty percent of the samples reacted positively with an HSV•2 prabe. Postive staining occurred in cells bcated in discrete fod, characterized by increased cxllularity within or adjacent to the intima Melnick and his cot- lea9ues"•'s•'6 e)dended these studies, focusing primar- ily on CMV, which they were able to detect in cells cul- tured from arterial tissue derived from patiertis with ad- vanced (grade III) aiheroarteriosclerosis. In 1984, Gyorkey et al" reported the detection of vi- rians of the herpesvirus family in e(ec=m microscopic sections from aortae of 10 of 60 patients with atheroscie- rosis undergang cardiovascular surgery. These viral par- ticles in various stages of replication included empty nu- cleocapsids and virions with dense cores. They were present in smooth muscle and erxiothdial cells. Of 1360 grids that were examined, 35 show.d evidence of viral presence. Petrie et al,'s using in situ hybrid'¢aaion tech- niques, dernonstraied increased detection of CMV in cu4- tuned smooth muscle cells from arterial plaques by stain- ing for viral antigens, Most recentty, Hendridcs et al" showed that detection of CMV nuGeic acids by the poly- merase chain reaetien (PCR) was possible in 90% of samples obtained from patients with severe atheroscle- rosis as compared with only 50% in patients with minimal or no atherosclerosis. The presence of the complete viral genome was shown in these samples by botti dot blot DNA hybridization and PCR, using probes and primers derived from the immediate early and late genomic re- gions of CMV. Messenger RNA trarscribed from imme- diate-eariy CMV genes but not the late genomic regions could be demonstrated by in sltrj DNA hybridzation. This suggested that CMV exists in the vessel wall primarity in alatent state, wh.re ezpression of immediate-eary mes- senger RNA transcripts may occur withnn expression of messenger RNA coding for sttuctural capsid proteins." Perhaps this may lead to an alteration in factors that cor- trol growth in the vessel wall, predisposing to infimal thickening. Rnally, Yamashiroya and his colleagues' have per- formed both DNA hybrid'ization and immunohistochemk cal experiments on coronary vessels of young trauma victims to identify evidence of viral antigens or nucleic acid sequences at earlier stages of ttw art.riopattry, Ev- idence for HSV or CMV was detected in 8 of 20 speci- mens from coronary arteries. The viral DNA or ant;pens were found 'n celis of the irrtact luminal surface as well as in Jocal clusters of spindle-shaped or foam cells in the irtmal layer. Taken tngeti'w, this study and those rnen- tioned above support the hypodiesis that thase herpes- viruses may conm'bute to early events in ath.rogenesis, because the herp.svirus can be found in the vasculature at various stages of tr,e arteriopatrry. C3eariy herpesvin~ses can iMeci arterial calis. In faet. HSV types 1 and 2 can infect both bovine and human vascular endothelial cells" •`2 and vascular smooth mus- de cells.a Similarly hurnan CMV also can infect such cells.33" Infection of cells in vrbo with HSV resuhs in an inhibition of host cellular protein synthesis. This is de- picted in Figure 1. This 'shutoff' of host cell protein syn- thesis occurs in two stops. The first stage, occurring in the first few hours after infection. 'early shut4ff,' is medi- ated by a virion-associated protein and causes dissoG- ation of host celi mRfVAs from pofysomes. rendering the mRNA nonfunctional and enhancing mRNA degrada- tion.35m Kefalides and ZZaie" have demorutrated tttat this mechanism is relevant to the mRNAs encoding ex- tnscellular matrbc proteins in both human endothelial cells and human arterial smoottt muscle cells's infxted with HSV-1 fn vitfo. The delayed shut-off of host cell protein synthesis is thought to be mediated by a newly synthe- sized immediate eariy-viral gene product that subse- quently causes degradation of cellular messenger RNAs.9 Some of the viral genes responsible for the in- hibition of host cell protein synthesis have been mapped and are discussed elsewhere.'O 8iochemistry of Viral-induced Atherogenesis The concept that herpesviruses play a ma}or roie in the cause and pathogenesis of atherosderosis is supported by two observations:1) The findings of Paterson and Cot- tral, reported in 19b0.41 in wtuch res,rolymphomatosis and coronary scierosis induced by an infectious agent was linked to arteriosclerosis in chickens; and 2) intrecel- luiar and extracellular cholesterol accumulation was ob- served in cell cunures infected with feline herpesvfrus.s The work from this laboratory and those of our collab- ora¢ors have shown that atherosclerosis in chickens in- duced by an avian herpesvirus infection can provide con.siderable experimental evidence to support the car aept that herpesvfn1 ses are irnrolved in the pathogenesis of atherosclerosis." Pathogen-free normocholoster- olemic chickens were infected with 100 plaque-fomvnp units of an avian herpesvirus that causas Marek's dis- ease (MDV). Uninfecied pathogen-free chickens senved as a confrot. Mareks disease virus (MDV) is a herpesvi- rus that causes malignant lymphomas of T-cell origin in this animal moda!z Primarily nerve lesions and visceral tumors develop. An antigericafiy related herpesvirus to MDV, herpesvirus of turkeys (I-M'), has been used suo cessfully by the poultry industry as an effective vaccine against the neaplastic effects of MDV.`z Nonpathogenic for turkeys and chsckens, tttis naturaly oxurring herpes-

1198 Ha}lar VP Dea.+rtbe* 1991, Vo(. 1'J9, Na 6 I. Competition II. Dissociation nwinnlr tlt. Degradation I I Nost mRNA Namit trartslatian I I Djnooiatton of host a!l mRNA Irom poysomes Fgues 1. 1'flpadsttlCal seprnu of rfwpzbokgfc dotv+ga rlrs>ftg uadfj~trtdt~xf asbE•esdooSG~ 7bir srix+na desai6es dner srsps dw can l,ead m deviardproraie ry>v/xds in a'r manmaliart tell onat ClAafies'tgat uirb 6erpes v&uc PSm c'antpeaanas ocrras ubtra tha tsal n[RNit rut "eonyxte"aids ds6msndiNw in rfxearai nsRNApcnl in riie e.lldrav~ the rfmo~laeion.p~acrts yLt'srnesalhumr ojt~r'al btfcu1on wal mRNAS cros 6r dx p.edomistrW RNA e c4sQ{os t&a vr isantlrIIrd in rbe rnfrasd c.l; podua,g cswl P.ords+t !n addLrlon, dmwhaon of dw aas cetl mR1vAfm m mrpoly.+ernes em+ oms vt dx inf.ewd crllr. 3venupr>;,Q dse ne.mat aa,urarion of dw RN:, t+mcsaiprI fi,eat .br 3•3' dbw.Ytos on obupolyaome. WC rs dx ncaiarfnF codorr in eurQyvic celis jar vaulanon}. FbuUty, dagradarion of dw R1VA owuozpb b,v a va[o.i-+•rlaand o. nswlv syredis-ed uirally rnaMtd f=to•fs) (ddpiacd by -bw o+rsce>'tt moon fig:s) cat areoo 0169 baa a!{ prsdig,a" m dev.asuxi RNA rrisitstaao.: and b- poms'n rvndAset 7ba.O.r4ff ofP~owk OwuhesLc leads to aknurio.u bt a.rpta/Lelar maois and d.-4W+dprodr..arlo+t ef dcte eLMnW dw asr b¢oked ns ltptd aatabolrc acrativ. T!r cysopoaho/osio cl/eca indu& cyrtp4,wnlc lPid aaonulanort and aherariMec !n a'x eza+rwiluGb mrcri= Rep-trtmd rei& pembs,io.t" virus eliciLs an imrrtune response in chickens that prtt teCts aqainst subsequent tumor devetopment by MDY.`Z A mtsst interesting property of MDV is that it can cause atherosclerosis reproducibly in specific-pattlogen-free chickels"; and that imrnunizatlon of chickens with F VT can prevent MDV-indutxd atherosejerosis.a-' In tact, the distributionai, morphologic, and biochemical nature of the athernsclerotic lesions induced by MDV infection ciosely resembled human atherosclerosis' Coronary ar- tEries, the aorta, and its branches were affected, resulting in fatty or fatty-prvliferetivve-type lesions.' We showed that aortic tissue from the tlorrnocholes- terolernic, MDV-irtfected group had a significantiy higher content of free and esterffed chdesterd, triacyl9lycerots, and phosphdipids than did urUnfected controls.' Feed- inq ttle infected animal a high-cholesterol dfet produced a syrrergistic effect on Fipd accretion. Marek's disem virus infection also caused an abnormal rise in cho- 4oesteryl ester (CE) synthesis with a concomitant reduc- tion in CE hydrofysis in vi,ro, thus causing arterial lipid accumulatton.° Infecaon of cells with herpesviruses in vibv also produced profound effects on cellular eholes- tzrroi membolism2° 22 which paralleled our in vivo data Irtfection of avlan srrmooth musde cehs with MOV in vibe greatly incteased the accumuladon of choiesterol and CE20 This specific type of lipid accumulation, wf'tich atso oceurs durinq the human arteriopashy, was due to de- creased lysosomai and cytoplasmic CE hydrtalytie acov- ities. Detailed anatysis of enzyme actWaDOn showed that ttte CE cyc(e is aftered. nssufting in cytopiasmic CE ao- cumulariort"-~ (Figure 2). To defne some of the regulatory m®chanisms asso- ciated with the contrat of the CE cycle, including cyto- plasmic CE hydrolase, in herpesvirus-infected cells, the level of CE hydrolase activation in MDV-infected cells was examined in the presence of: 1) dibutyryl cyclic adenosine monophosphate (AMP), 2) dibutyryl cyclic AMP added together with protein kinase, or 3) agonists of adenyiate cyclase.1 Activation of cytoplasmic CE hydro- lase activity by cyclic AMP or protein kinase A was blocked in MDV-+nfeaed cells but not in uninfected celis or in cells infected with a control virus, turkey herpe.wirus (WT) 2i Furttnrnlore the rate of cholesterof efflux from arterial srmooth muscle cells challenged with dibutyryl cy- clic AMP was unchanged in MDV-infetrted cells as cort} pared with uninfected or HVT-infected ceils, in which ef- flux was actuaiy incteased.202' Hence it has been pro- posed that the reduced cytoplasmic CE hydroiase (NCEH) activity in lipid-laden, ht:rpesvirus-+nfected celis was due in part to the inability of the enzyme to be acti- vated by the cyrlic AMP-protein Idnase A mechanism.2' This may contribute to te patt+dogic changes seen in MOV-infected arteriai cells, such as intracellular CE ao- cunxtiatiotl. We e)dended these studies to human arterial snlooth muscle cells infected with herpes simplex virus (HSV) type 1: HSV also induced accumulation of saturated tri- acybycerois and CE in infected cells.22 The infected cvUs had roductions in the CE hydrolytic activities and in the enzyme itsetf, based on irtlmunopneciptrat+on data (Figure 3) and ac6vity assays because of decreased translaiion of the RNA that encodes the intracellular hy- W v c+i ~ t~.t ~ -,, W cn I I I I U c_ I I

Viral Patho9enesis of Atheroaclerosis 1199 yP Deeadr 1991, Vod 139. No. 6 I I I I I I I I Altered metabolic events Fi9ur. 2 Hnf+s ennapoxeaCan bva cdk 4adf~rg ro atre.ed mRaboL'c acraAty. T81r sclxrna depfca a byporberic aequenue by u6icb cvtulating berpc orracr nrars ceflr 6.vugli dx cav¢l wull m+d /wch to a(avsd merabollc ac Mry, 7ba isarYS cvadasrsrg ubw pnuams anekes b.v cauoV endodul;al uU vy~m, wee ebesr aelkecm be deJecadwirb be c:nsi Once dx s,t.ions me shed io cbe arrrsal ,vMcoat mrcV.L- ca14 bUo~btndtr{q ul1-0G• dsotertvyl es,cs (CF) rms mzs, .e.fib;a In deveatd CE bydrolase ~~ (ACEf1 and NYFFI). 7bis =m lead tb ino.aed CE a=w• vbn in OM e.d1 6s me forns of CEs.icb.ar 6jo1d rbvpkas uft* an a rbaacariuic fva. ~ o f~~ la~e~o cCe~ouc/am+t cdL Reg~•>y+ud droiases.?=' Because arachidonlc acid metabolitee such as prostacyclin (PGl2) and 124-IETE atter CE hydrolysis in arterial srrlooth musde cdts'. we rneesured these ei- casaraids in HSV-infected edls. We obsxrAd a reduc- tion in bolh spcnianeous t~basvline) and arachidanate- induced reiease of the cycbnxypenase producX PGI2 and a Iipnxypenase product, 12-HEfE': Hence we be- lieve that the herpesvirus-induced atherosckrosis re- sults, in parr, from attefations in metaboiic control of CE tmifficidng by eica~ in vasvJar cells. This concept is depicted in Figure 4. Role of Gytokines in Cholesterol Trafficking in HSV-infected Cells Recent studies have suggested that the immune system may participate in aMerosclerosis. because monocyres and T lymphocytes accumulate within the attlernsclerodc lesion.=Yral infectlon could induce mediator release from 'vrQrune and hernatopoietic cells, which may be respon- sibe for phenorypic changes in vascular cells. Of interest in this regard are recertt studies that show tttiat herpesri- ruses are capable of inducing messenger RNA expree- I

1200 NzJjar 4tP D.rmnbir 1991, voL 139, Na 6 I I I CTRL CTRL ' CELLS CELLS (IgG) CELLS Ad-2 ~ HSY A 6 C sion of marocyte-derived genes. For exarrtple, CMV in- fecacn in vibc induces macrophages to increase the ax- prestion of specific RNAs for intnrieLkin-1 0 (IL-1 P), tumor necrasis factor (1T4F), and colorry stimulatinq factor•43 The rde of TNF in the complex cytddne network in marn- maJian cells is not fuy understood, particularty regarding its andvirai ac&vity. Although TNF is an irxlucer of inter- feron messenger RNA transcripts, a well-documented cytoiane that has antiherpesviral activity, others have shown that TiVF's antiviral aetiviy is rxot abolished in the presence of anriserum to iraerferon," Wong and Go.d- deto have postulated that the antiviral activity of TlVF is attributab4e to lysis of viralty infected cells rather than to inductiort of interferon. Other poscibie mechanisnns to ex- plain the action of TNF irrvoh+e eitxosanaid metabdism, since TNF's y6c effects can be partialy abdistxd with cyck>exY9enase inhibaora Sudi studiea suggest that 0 Flqura 3. I e.f acid dbo- kst*yl ata' bydolase.4CFlifrorn bnprs rmc* ~a3eca ~ XSY-infectenf (mai = 0.7) SMC cYhr.ro. Bio synrbetic lat.JVrg of crll F.ot.ist uaas done uRm f35~ (40 uCi PW 5 Y 10•' Mllrl. Farh lane mnv.ifkd aiaDtu I mg of of! Praarn or a brnnogenan cwtslsrtng of 2 x 10e cells Gnnn.nobo6%t.e v.+re auhjeaed to SDyPAGE and autorad/oRfapbY uwa pw• fari/1Cd laae A, a cofool, m+mwtoColatt uas ohtained uub 8bc tcse of aormal rnosar 1RG. 7bis lovic siotta an arbarrm of .adtooc- tiurry at M, 3S•000 (arrour) (inolecu/ar reigi4a #aoeft at:e, x 10 "d), ubitb apprar- n.iarac ax molecutrr rscVbt of.tCES! Due to t6e Imge qumuiry ofprotein lonGed on dxs 8atx aestenW of thr soviceval p.ot.ft ,tueb a am (M, 43.0WA ndu1M msd purmcdlate JUa>rvma (M, 5?,lXJO-65,000.~ ond nryonn (1f. 200,000) pnripUared nonspecifttalty. lat.e a. bnmunoprec~ouatssf matera! +iro- taWfrwrs unrqf#=PA corraro! S+IGr wi.tg muGACEff nwrsoclolfal MmLaiy. (mSC) .t 6tsfd [s abott" by dx a•sotu carreparult»g to x, 55,000. lanu C, immursaprecipitared ACFFIfrvm adcna sur inf.cned cef(s fDM1 ui- rus aonrrol eroup.l larr D, rmmwiop•rcyo+- aand m-ial case~OrYlb~ to M, 55,000 fitr+c MV Infiiaed SMGs u crnuidnablp ltss imt tfrat fmtnd in Lmts B, l be lc.ls ef acsi+t antd wryatbi a8aort in latt R(and (ane C) K.+r b{q!a tlms ri5s trnotvsa atoun in laste 4 We aLso obsashd a prominsnt psotein hmid yop.~iar9 at b!, 150.000 in lanr D alty tr 6~6 mav Iz ojtx~P, w~m tspn. FI- W11y, we assayed AtM1 aah.try eo.ff ia.cnun nuprtUPttatlon m axocbe espeimant ta cDnfbrx that u.v ti+amsotoprecrpiixed ACEX• We fotaid tbai u'f could 6Mmfocpncfptrast about 60% of dm .1Lgf in dx all bttud on ow aaad,N,bw+d in ¢hr stpw,narasu mdpr/- 4Y Reprtnled unrrh pa•nutvon,2•1 cytdvnes, such as TNF, can promote synthesis of ei- cosanoid products that, in turn, can mediate specific bi- ofogic etfacts such as modulation of cholesterd merab- oiism, a concept originaly proposed by our laboratory.'b 8eaause HSV infectirn of vascular cells can produce a biochemical and cytopattabgic effect virwaly indistin- guishabie from atherosGerosis, we hypothesized that tfsese cytokines can prevent CE accretion in arterial srr*rth muscle cells that is associated wirh herpesvirus- induced atheroscierrsis `d Turnor neorosis factor and IL- 1, but not interferon, prevented CE accumularion in H5v- infected cells by induction of cyGic AMP-dependent CE hydrolysis. Tlvs effect was medisted through the arachi- donate 12-lipoxrygenase pattrway by 12-HETE, because prrtrP.Btrn€nt of aNis with a cocktail of lipoxygenase in- hibitors abotist,ed Me an6virai effect and 12-HETE pro- duction in the celi." 12-FiM is the rnajor lipoxygenase f- RVr dx 1 oni, met ove that turn hyd tirn kine virus Qaiiy tT1et caLL lipic ceU; I I I I I I I I Pot ~ anc AGr atte tt,ro assi I itiea I tein broz dicz acsc and dt2t I byr thro ever

Viral Pathogenesis of Ath.roscl.rosis 1201 NP Desonesr 1991. VoL 139. Na 6 I I 1.1 I I I I I I I Fgurs 4. Kvpa sara iRj.aion blaeiespb'r+d rrnrssduerion paebuavs in roeprioW onoot8 mu9cie cells 7hts modal sfiou4 6at bepcs tr• no• [n(OWon ieads to deae+zedlvsaeomad by. dro()k's f yF a. Fry ffie rtCfl•f ensVnu In a"- rion, lxfeaion leadr ro deoeasnd easn.iesion oj asartidonfc add X20.4,) to PGI, mnd 11- F= tttiio6 in rtan a9t /tad ro a reductd =knrron of adncylatr C•vclmr. Dwa.rzzrdc.v- a•!ir AMP prtxlutlion nuala in ds ull. Cws- drruing br ebis alicrvid mem6olie a.grstrwe of esmrs devxned ctietic &tfP nuf,.as lpss ac- mnarrae ofpvrebt lrtnrsv 14 atius na,ducs.ig rbe acruntiovt (rfsot.Rb pbopbo.Ylxxon) ol &e cyropfatmic CZ Lry+dnolan (Nffll) to Oe aicriee fo.m Jrom dx osnase fvm. 7laar meoatw/ic nv+wr Ot obr cyaoplasm, muplaL to dsaw rn dx+ hyaai-uK .aula bt r+uraeel/s<lm- CL• ae. ceria.i. CF101 ~C€ 4ACEN FA c2p:a ~ ~0,/ GH06+PA I P%.12•FETE +7a~ , AGT j M~s~ ~ I+Ca qe1a. AMP na~ PA~ metabdite found in arterial smoottz muscle celis-'° This overall conclusion is further supported by data that show tetTNFand 1L-1 enhance 12-E-IETE productlon. which in tum increases both intracellularcyclic AMP levels and CE hydroiysis." Coilectivdy Uiese findings identified for the first Ome a biochemical mechanism inwhved In a cyto- tdr~induced reduction of lipid accumulation in herpes- virus-infected ar[erial smooth muscle cells. This is pot&~ tially an important finding regarding control of chotesterol metabolism during virai-induced atherosclerosis, be- cause cytoldnes are importartt regulators of intreceliular lipid metabolism, not only in arterial smooth muscle ceHs,"g but also in monocyte-derived macrophages." Potential Links Between Atherosclerosis and Thrombosis A link between the coagufation system, fibrinotysis, and atherosclerosis has been proposed on the basis of thrombotc processes atthe level of the endothefum. The association between lesion progression, lipid abnom,al• ides invdving elevations of lipoproteins, including lipopro- tein (a) [Lp(a)], and the fibrin depasition is now being broadiy defired. Ear1y studes of Greenland Eakimos in- dicated that their diet enriched in pofyunsaturated fatty acids of fish oil origin, had an impact on p(ateiet function and eicosanoid production. It has been hypothesized that'rf the vessel wall is injured (or activated), for example, by herpesviruses, the endottielium can become pro- thrombotic, as evidenced by several cytopathologic events. These include enhanced thrombin generation and enhanced binding of platelets to endotheiium," de- lJCEt•tWML creased PG12 prvduction,`° enhanced tissue factor pro- duction, reduced thrombornodulin expression" and an inhibition of endothelial cell synthesis of heparan suifate proteogiycan," which is closely related structurally to heparin, a complex pofysaccharide that has anticoagu- lant activity. An example of molecular mimicry that relates throrn- bfltic processes to atherogenic events invotv@s the re- cent observadon of stnldng structurai homology benveen the apoprotein (a) component of human Lp(a) and plas- minogen,SO a major protein involved in fibrinoiysis.5' In- creased levels of Lp(a) have been associated with atti- enosclerasis in humans."L^-" Lipoprotein (a) as well as its apo(a) also have been shown to compete with plasrnino- gen for cellular binding sites,5t and Lp(a) has been as- sociated with enhanced functional, antigenic, and trarr script levels of plasminogen activator inhibitor (PAI) type I by the endothdium.S' Piasminogen activator inhibitor type 1 is the rapidiy acting physiologic inhibitor of both tissue-type and urokinaselike plasminogen act'rvators. The net effect of these events leads to down-fegulation of plasmin generation at endothelial cell surface.' Lipopro- tein (a) appears to function under some circumstances as an inhibitor of plasminogen activators, by competing vrith plasminogen for binding to streptokinase or by act- ing as a competitive inhibitor of tissue piasminogen acti- vator (t-PA) in the presence of fibnnogen. (KA Hajjar, per• sonal conmunication). Recendy we have observed that HSV infection may interfere with normal endothelial ceu flbrinoiysiss, as is the case with Lp(a), by inhibition of p41s- minogen binding and increasing PAl-1 activity, thereby preventing plasminogen activarion (unpublished obser- va>ions). The mechanisms of action of HSV infection and

1202 Hajjar 4fPLtsvnls 7997, vot 239, No, 6 virat protein production on processes related to fibnndy sis are currentty under investigation. Viral Acfivafi'on of the Coagulation Cascade An important event in the eary stages of atherosclerosis is the adhesion of bbod cells to altered endothetium. Cer- tainly both platelet (as well as neutrophil) and monocyte adhesion to the endothelium are processes attributable to the pathogenesis of thrombosis and atherosclerosis. Monocytes can migrate into ttie vessel media and begin accumuiating cholesterd, contributing to foam ceil for- mat;on? Herpes simplex virus-infected endothelium, as well as endothelium exposed to TNF orto IL•1, binds ptatelets,`6 granulocytas," and expresses tissue factor to a 9reater extent than it does to noninfected cells.`° Herpes simplex vinus-infected endothelium also expresses giyeoprvteins encoded by the HSV genome.s' These partiapate in mo- iecular mimicry because of their presumed furxaional role on the erxiothelium. For example, gtyzoprotein E (gE) can function as an Fc receptnr,55'5' and glycoprotein C (QC) can atso serve as a complement (C3b) receptor.s " A role for these proteins in the pathogenesis of endottie- ual injury is suggested by the observation that poryrr>or- phonuclear (PMN) celf adhesion to HSV-infected endot- he5um can be blocked by antiviral serum S8 Central to the pathogenesis of vascular injury is the localized activation of the coagulation cascade and the adhesion of circulating inflammatory cells to the exposed subendotheliaf vascular surface. Once adt*rent, tttiese cells can secrete growth factors, proteolytic enzymes, and cytoklnes that further activate the injured vessel sur• face.2-' For example, macrophage-derived cytokines such as TNF and IL-t induce endothelial expression of leukocyte adhesion molecules5° as well as tissue fac- tor.60 These events promote further leukocyte accumula- tion and locaJized tttrombin generation. Recently we demonstrated thar infection of endothe- lial cells prtxnotes enhanced monocyte adhssicn.c' En- hanced adhesion was blocked by rnonockmat arrtibod• fes to the viraliy encoded ceil surface giycoprotein g(C) but not by antibodies to g(D) or g(E). Adhesion nlsc was blocked by treating endothelial cells with specific throm- bin inhibitors or by growing ceus in prottirombin-depleted serum. This suggested that thrombin plays a role in the enhanced monocyte adhesion. Glycoprotein C bound and promoted activation of factor X on infected endothe- fal cells, thereby contributing to thrombin generation. To further support the hypothesis that g(C) was indeed irr volved in rnonocyte adhesion and factor X binding, we used cells (L cells) that do not normally express this gy coprotein but could be transfected with the gene for herpes-virus gtycoprotein C.6' We found ttiai factor X bound to transfected L cells thatwere induced to express g(C) by dezamethasone, a steroid horrnone that acti- vated the MMTV-LTR promoter region of an artificiat g(C) gene construct. Cross-linking and irnmunopreeipitation studies dernonstrated factor X-g(C) complex formation on the cell surface, suggesting that g(C}dependent thrombin generation by herpes-infected endothelium may be an important mediator of vascular pathology dur- ing viral infection. Thrombin itsstf can elicit a variety of cellular events. These include monocyte and neutrophil aGhesion, and cytokine refease and platelet activation on a damaged surface."-'61 Each one of these cytopatho• logic effects has been iinked to ft role of inflarnmaiion as it may present itself during the pathogenesis of athero- scterosis. Identificaf+on of a Monocyte Receptor on Herpesviral-infected Endob~elium Endottretial cells express several leukocyte receptors. in- duding GMP-140 (also known as PADGEM or CD 6Z), ELAM-1,ICAM-1 and 2, and VCAM-1 on their surfaces in response to cyrokine or exposure to other agonists.02 NorrnaltyGMP 140 is acytoptasmic protein found in rest- ing endothelial ceits found on the membrane of Weibel• Palade bodies ° After stimulation by thrombin, histamine or complement proteins. the Welbel-Pafade body is rap- idly franslocated, and its membrane becomes incorpo- rated into the plasma membrane, resulting in surface ez- pression of GMP 140.°0 This mechanism of new protein expression, ie, translocation from a preformed intracellu- lar membrane compartment to the cell surface, does not require de novo protein syrrttsP_sis. As described in the previous section, our recent data support tiie following model: HSV infection induces endothetial cell surface ex- pression of HSV g(C), which acts as a binding site for factor X. Concomitant generation of tissue factor converts bound factor X to an active prothrombinase, leading to generation of thrombin in tf* microenvironmerrt of the infection 6i Recenty we have exaended this hypothesis, because we now have evidence that thrombin can act in an autocrine manner to induce expression of the leuko- cyte receptor GMP1a0.a6' Our data indicate tftat mono- cyte adhesion induced by HSV infect;on is blocked by anti-GMP140. but not by anti-ELAM or antibodies to other adhesion molecules. This suggests that GMP-140 is a major receptor for monocytes on the HSV-infected endo- tltielium." These findings are summarized in Figure 5, which shows that HSV=utfected endotheiiat cells generate thrombin, which predisposes to increased rnonoc.yte ad- herence by ezpressing the monocyte receptor GMP-1a0. Thrombin generation by these cells is dependent on the 91 eXprf X bin As st, in ft (LFA ICAN cetis,' viralty spec Simii~ to stir syste bodiE I I I I I I I I I 1 TF HSV-; throrr weil i can n ~ platel tivatec may c injury, of ree ucts.` infecu endot Mc sites ii af fact, in9 to lappir gions ~ I I

Virat Pathogenesis of Ath.rosclerosis 1203 .VP Dasnlxr 1.9,91. VoL )-0, JJO, 6 I I I I I I I I I I I I I ...::::::.... HSV-infected EC SMC Fpun S. VbW aR reuwn of mr magulatlon ea=dr, HypodNffea! WttittW depiarng bau beprcs snPkr ivus fNSIV) ny4ztion G= lead m a p.or6rombon4 psarfi~rlwoeic ~rr o.r d*e,fdodwGar.a&faor by tnduang ihr ryr,mLes mid cpsaion o18lyKqo•ouv, (C) uticb ran ac+zr ar a 6indbig arrs)o• Faacr X a key pmvnrm•r of du encRularqn aataaxtr. 7bir tnoatfod birlQin,q ojFactor X and fa ruhseqirnt co>wasron jo dx aalxelvnS Facor \a, uar rtad ro dx aonarao3 ofpo&rowbvs to &onbrn rT,oao. l!a). Wben Facxor r1a it g-e.aed, d* = b+dtiaenwface eT.ezio.r of a nio+aocyrc mcep(or fGMP•14U ut~ frt non omfsomorr motacsYr ardbenorl ro dx andorb=m and +,uiu=~ af an isyrammeao.r atVo.iae by aaimrtns dw qyaoi- rirnevt Eace of d),,u cy,opzcorogrc fea- rm, psdipose ro a6ero- seL+osa ared dsombwiC expression of HSV g(C), which can act as a site for factor X binding and asscmblyoftfie protttrombinase complex as stated earlier, other receptors do not play a major role in this celt system. For ezample, the leukocyte integrirs (LFA-1. Mac-1, and p15o,95) mediate adhesion to ICAM-1 and iCAM-2 on cytokirtcstirnulated endotheliel cells,65 but do not appear to play a rore in adhesion to the virally infected celts, based on lack of inhibition seen with specific monocional antibodies or RGDS peptides.s` Simitarty VCAM-1, which mediates fympftocyte adhssion to stimulated endothelial cells,e6 is not functionai in this system, based on ladc of inhibbort with anti-VCAM anti- bodies." The significance of a procoagulant phenotype of HSV-infected cells is considerable: IocBi generation of thrombin at the site of infectdon may actt`rate platelets as weil as endothefial cells. Thcarnbin-stimulated platolets can adhere to monocytes, and ttterefore can recruit more platelets into ttv site of injury ?,6`-6' The presence of ao- tivated platelets and monocytes at the tocvs of infecrion ruy contribute further to the deYeiopmertt of vascular ;njury, chronic inflammation, and atherosckrosis by way of release of cytokines and lipoxygenase prod- ucts.","•6` Expression of adhesion motecules on HSV- infected cells thus may be an initiai step in v'rca}-rrxdiabsd endotheliaW injury and atherogen¢siz. Most recentiy, to identify the putative celMnteractlng sites in factor X, we have analyzed the stnucturat domains of factor X that are involved in the coordination of its bind- irsg to membrane receptors.6t A group of partially over- lapping synthetic peptides repre,sentatn+e of different re- gions of the factorX molemb Nwre used, Two unrelated surface membrane receptor regions that ceordinate this neaoqnition have been identified: CD11 bCD18 on mono- cytes and plycoprotein C(gC) on HSV-infected endothe- I'axn, ie, they recognize a comrnon strucaual motif in the catalytic domain of factor X.°° Each of the peptides we used blocked factot Xa-mediated monocyte procaagu• lant activity and suppressed monocyte adhesion, The stn.ctue of the catalytic domatn of factor X centaining the tfuee sites that are invofved in binding to herpesvirai g(C) and CD11 b/C018 was modelcd carnputationally. Factor X associates itself with herpesvirus-infected endoUheliaJ cell and monocyte rectptors using a similar mofecular reoognition motif. We were able to show that the vascular cell binding region of factor X is organ'ized into three dis- tinct interacting sites. The peptidyl anabgs prevent the consequences of vascular generation of tt,rombin such as PMN adhesion to endotheaial colls, chernotaxis, and monocyte-mediated deposition of insoluble 6brin. Our understanding of the cO biology of this system coupled with the molecuiar modeiing offers an unprecedented paradigm of the molecular compledty of this thrombotic process that can be linked to vira}-induced atherosclero- sis; it also directiy links the actinration of caagutariort pro- tens on vascular cells to typical irtAammato"rorrtbotic reactions. This was observed by corutituting three spa- tially distant surface Ioops that define a unique three- ddmensional celt-interacting network in the ligand, as shoNm in Figure 6. In summary, we observed that speciflc synthedc pep- tides inhibit facter X binding to maiocyrte CD11 b/CD18 and to g(C) expressed on herpesvirus-irdecteld endotfi-e iial cells. These peptides blocked rnonocyte generation I

1204 Hajjar AJP Dftv"rlas 1997, LbL 139. +No. 6 Fipure 6. Sss.a+sd n~orL! of dz Faaor x eardyKic do>+~ llomwlogy modd bLi4Gng tcdinlqua uwe usad ro eo.lsnT.ca a ntoder oJr6r crualyetr do.nairr oJJaaa-X frons a oyaal snArnor of aypcmogerz Snt.asirr[ly ebns&std re,qfont uWs vl.nrifstYt by etara! !n¢~e~.ziors of dhe=.ar-res of v3Ps>y; cGynroVyprr>R ekaroaie arrd ht1lbttnn, and rbr Faua' x myunrar uw aligrrard muraralh, uIrb dx sn7t+c.rcr of dw.e pomacec Avomc coa>dtNares fo. haaFbo.rr and eo,um,rdside drairu of fcaro• X awr amgsrrd daealy frri.n aVvcinoRen for dwe m~rwallv asuezie+d rMiom Gnordmata of noaroomxard arde dalnt uutn'rt sa r.cirsalty mnsmud rrgior~ ~ Od jor mininra= oe wlcrp ~ctib o~ osona Ato"ele aaadinaorr fEr ,~src7rsall,y c~scblr (loap) *Cgto.+s a~ere ra4en Jrvm a p.io.. ~rLdxd nec~dr.[ of Fvao. Xa rT. EaiqvsETOrs Lx a1.) 7be Eninal nsodrl sat.aure aar eneV mtnvniud tn sc{qaa fvst aUuuing all srde cf+alrzs to m-lca, w&d rben re(mnnq all IIde tl~evns togedxr uab rlar baek6unr atonrs of d t foop repsoru Baekhortt ef Facsur X uucih•ric dnmatm (rib8on rLicgrm++,d CaraA-r/c rnad btwr e.an ddr Wml mofaoe rcmuerj [r.yo p~ 1(G}DMFn) (67) r_PKlsofaeo (RYfuy Larip peprfde 3(IDRStDURG) (67), U'K riofau /eft cdcp peprlde 9 2YpnARr7w) (67J, ePIC sofao. (rop) ex•ardus rlsk ro Ilkrsr dxiva v+liow cPK (rou,ir orr+ar) rhr N•rn+.,isucs oJm. u4ui!}*c rfornain fs ltrwrd jur a6oco pepr/de 1, r6e o~bmwr bi+uiistg grmt r it apprpsi+nauly twrical and (o rbr nbht af dW. o-tarl and rbr Gu+mbuc: R ~(~~Id'L~ rderec oj Faaar x a,~rr.+~tes for mbic6 no sawsovJ enfor+rrarson ic atailable) c~ dr Garrrminta of~iri~L: 2. of thrombin and prevented monocyte adhesion to HSI- infected endokhelium, It is possib(etriai selective interrup- tion of coagulation by such symthetic analogs may impact positively to reduce vascular injury associated with morrocyte adhwrence and HSV infection of ehe endottie- (ium. Molecular Genetics of Atherosclerosis The monaclonal hypothesis of Benditt and Benditt` was based on the observation that 75% of ath.rosderotic plaques from human tissues removed at surgery or at autopsy derrronstrated a monocb)al pherotype of the gtucose-6 phosphate dehydrogenase (G-6-PD) enZyme, based on etecirophoretlc rrobiUty. This technique too+c advantage of the fact that the poiyrnorphic Eortns of this enzyme are expressed in an Xainked fashion. MediaJ cells from regions adjacent to atherosc{erotSc plaques frorn nonpiaque areas display equal arnounts of each pofymorphic form of the enzyme, as would be expected in an X-iinked gene, because one of the two X chromo- somes is inacvvated early in embryonic development, Smilar resuhs have been obtained by other laborato- ries °°'70 however the interpretation of these experiments in supporting the monoclonal hypothesis has been chai- lenged." The principal argument is that the G-frPD marker may be linked to sonle other gene that gives ttuse cells a selective growth advantage, ie, a'pheno- type-sefective advantage' wittw the proliferative lesion being trufy rna*ocfonel in origin. Tfw monoclonal hypottiesis purports that the srnooth muscle cell prel'rferacion seen in the ari,ernsclerotic lesion is similar to a benign tumor, for example, a uterine leio- n inft no I I in hu' rat ' _ (r1C - the nOr . D h ~ - nar Ma 3T; , forr re6 has pla I ~ 7, t: BLUt pla aic sior enc cer trar plac labc rYpE rec( with anti ang latc Thes gen anc cise unic f Iden rece I I I I I I , tervi mlh cedl: Cora taRz forrr I I

I I I I I I I I I I I I Il s I I . I rnyoma' A bgical extension of this hypothesis is the infererxx ttat afterations in the smooth muscle cell go-o- nome by chemical mutagens or vinuses may be involved in the smooth muscle cell proiiferation chardcteristic of human atherosGerosis. This tt*sis, in part, provided the ratiortale for testing the viral hypothesis in an animal modd that would produce an arteriopathy approxama6ng the human disease, Supporting such an idea is the ex- perimental observaqon of facal smooth cell profrferabon induced in the aortic intima of chickens treated with a chemical mutagen initiatien-pronwbon sequence." To extend these observations to the level of the ge- nome, Penn et al" reported the presence oftransforming DNA in samples derived from plaques of human coro- nary art.ries. Ptaque-derived DNA but not DNA from nor• mal human arteries had the capacity to transform NIH 3T3 ceils in virro. The transformed cell line was capable of forming turnors in nude mice. The oripinal observations regarding the presence of a transfomvng gene element have been eo¢endcd into an animal model of earty arterial plaques induced by administration of the carcinogen 7,12, dimethylbenz(a)enthracene to cockerels.'s Further studies on vascular smooth muscle derived from human plaques show enhanced expression of the proto- oncogene c~rryc.7° The concept that increased expres• s'brt of this transforrning gene is a generalized phenorrt- enon in atheros lerefc lesions has been challenged re- cently by Yew et al," who were unable to detect transforming activity in DNA isolated from atherosclerotic plaques of human carotid arteries. Nevettheless other laboraiories have detected a similar transformed phenio- type in cells derived from atherosclerotic lesions.7° This recent study reported the correlation of trartsformat;on with the absence of a 140-kd protein norrnaly secreted by these cells. Cther studdes have identified a 140-1cd antitumorigenic glycoprotein that acts as an anti- angiogenesis factor,7"O The loss ot this protein cerre- hdes with a transformed phenotype. Taken together, these Bndings raise the possibility that the transforming gene activity may represent ttrie loss of a normal anti- oncogene or a gene controlling cellular prolifen',DOn. Pr.- ase identity of the transforming 9sne or genes remains unidentihed. AlthouQh no transforming gene of viral origin has been identified to induce human atherosclerosis, the resutts of recent studies suggest tiat vascular ulls have the po- terttial to be transformed by viral genes. Natchtigat and colleeguess'.s" have shown thstt vascular smooth musGe cells can be transformed aftyrtransfection with a plasmid containirtg the BgIII N fragment (MTF9II) of HSV-2 Immor- tnlization is probaby ttsa most important attribute of trans- formation, because these cells normaly have a fmite life span in vr7ro.l' The transformed cells did not retain ttie viral DNA sequences, consistent with othsr models of Viral Pathog.n.zii of Atheroaclerosis 12fl5 Np Dewnyr Igg2, voL 739, Iva 6 HSV-induced trartsforrrtation. lnterestingy transfomtaiion of rat embryo cells by SV40 can result in marked accu- mulation of CE, apparently because of an alteration in the regulation of bw-density lipoprotein recepters.°' Thus these studies provide circumstantiat evidence that virally induced transformation can predispose to some of the characteris8cfsatures of atherogenesis. More studies are necessary to prove that the vessel wall actualy contains transformed cells that are part of the foam cell population in atherosclerodc plaque re9ions. In Figure 7, 1 have sum- marized three hypotheacal cytologic aherations induced by herpesvirus infection. The 2tthorogenic potential of vi- ruses in acute infection, chronic infection. with ongoing immunologic activation, and transfomsation of vascular smooth muscle ceUs is depicted. The mechanism of transformation by herpesvirus DNA fragmerrts has not been elucidated. Proposals have been made to irnpficate many factors, inctuding DNA stem Ioop structures, ribonucleotide reductase activity, mutagenesis, gene ampl'tfication, increased or altered expression of cellular genes, activation of endogenous viruses, protein kinase activity, and the possible interac- tjon between latency of the virus and transformatiort" No conclus+ve proof exists for any of these mechanisms. Rec.enty shuttle vectors have been empbyed to study sequence anaysis of the mutations induced in cellular DNA by HSV•1 `f Interestingly the maximal increase in mutatiort frequency was rtoted at 4 hours after HSV-1 infection, imp(cating an immediat"arly gene or early viral protein. :,,uence analysis of the chromosomal tragments may lead to information about putative cellular targets for the virally induced mutagenesis. Mutagenesis may be the explanation for a'hit and nn' mechanism of virally induced transforrnation.°6 Because vascular smooth muscle cells can be transformed by H51i-2,82 herpesvinus-induced transforrnation may be a contribu- tory mechanism to periods of uncontrolled growth in the athetosclerotic vessel wal1. This is an attractive proposal and needs to be explored experimentally. Herpesvirus Entry into Vascular Cells After s®veral decades of investigation, the pathway of herpesviral entry into cells shlt remains undefined, partly because of the cemplexity of the virion. Herpes simp4ex virus is a large DNA virus whose nucleocapsid is sur• rounded by a lipid envelope. Seven herpesvirus- encoded flfympmtdrs; (termed 98, gC, gD, gE, gG. gH, and gi) have been identified on the errvelope e7 Four of these, g(B), fl(C), g(D), and g(H), appear to play a role in infectivity. Three gycoproteins, g(Bj, g(D), and g(H), are essential for irtfectivity and appear to be invotved in the process of viral penetratian." The prvctss pamway by

1206 Hajjar .VP oa0sis 1991, VOL 139, hh s I I I I 2L nfironio , Yrf0otion I "1lwponr w Inpry': Immun. RAsponsr FUaAn9 gtomN fk0t0tSn00ytOkln• rfltlm .. - ,.. .1 •:... W.oi^• 5m rrottf.ntlon Figure ?. Tbnie ryrolagKalf,rraiiosu cuiuc.ri t ' by bslxs emus tnj.asrns A bjpor4eeie ModU Qyar.pulatsd 51fC pcorrWration depiaing wal inJertion of dx rxtal uxrll urbic6 ean rnd:am dree cytopadiolqgtc.eWa.• aclitz ISt»O[L 4$!d7 flOi lead !o a(1fffd o[l- !ulrs mo.fabology. /ipid axumular~on, al- tatd a>mdllwl~r nrvors: ClurvdJsosceioswl p.bp~ of cbe vr"ulz- ae!!s asid ucu+r is- Jlans,iaeion, wiib g•ou,d.,jaero•.and evroknit ntsase, pnaihfY naulting on .enoorb rnrc%rle oefl prolifewsian; chronic lnfealon, robicb mutd pravide ibe inu+tunolo,qic afrnuif fUr an ungontg erFl-"UdEu+.rt bsJfammaea.,v rr gorw uxb n4bequ" oaUuGr bftv%u*:n of t6o tV3sc! v4ll ttod+ng ao drsrku4.cs! ayroodb ANLYIQ cmll fsollfsaSYOn; d' tt.1t13(OfRU2ion, llfNd7 kadr ro II~ prOAfI/adtM sbKE a6e eelU (nu dw6 capaciry to dorenm8dau Rroutb aaid at!l dierisio~ approFirfarely. Re- P.•isreed widi Pa"sin'ia'r• which herpesviruses enter vascular cells to initiate an in- fecton, however, has not been completely cttiaracterized. We have become interested in the pathway of herpesvi- rus entry into vascular cells because the virus can induce arterial injury or activaGai associatad with lipid accretion and aff7erosclerosis. as discussed in previous sections. WuDunn and Spear'a have shown that the in'It;al at- tachmertt of virions to the cdl surface occurs when the virus binds to heparan sutiate. The virion envelope gy- coproteins involved in this attachment may inGude g(B) and g(D) or g(C). Recent studies in our laboratory,® and !n collaboration wittl ethers,°D suggest ihat after this ir+itial irrceraction. HSV-1 may use a basic fibroblast growth fac- tor (FGF) neceptor to enter some bovine and human en- dothelial and srnoottll musoie cells. Herpesviral infectiv+ty was partially blocked (<7t]9G) when the high-a}finity FGF receptor was blocked using FGF peptjdes that bind to tfe high-affinity FGF receptor. Not all vascular cells, such as the rat arterial smooth rnuscle cells, appear to use an FGF receptor complex as a major portal of entry for HS/ (preliminary observations). Tha reascns for this are un• ciear. We speculate that receptor density or receptor conforttsaf;on on the cell surface in specific animal spe- aes may alter the mode of viral penevatim In bovine ceUs, for example, we did observe thai the virus ap- p.ared to recognize the receptor, because our viral prepar-ations contained basic FGF. We believe that the virus uses FGF from the cell or matrix to penetrate other host cells (Figure 88). Of interest is the observation by Baird et aL90 that HSV preparations are capable of induc- ing phosphorylation of a 90-kd subsfrate that represents a specific intracellular response to basic FGF through activation of its receptor.80 M altemative hypothesis is that viral gfycoprote+ns interact directly with the receptor, perhaps because it may share some stnlcturat homolo- gies with basic FGF (Fgure 8A). One of the enveiope glycoproteins, g(D), is required for viral entry into cells, as evidenced by the following: 1) Mortoclonal antibodies that react specifically with g(D) inhibit virion uptake.°7 2) Mutant viruses lacking g(D) can adsorb to the cell sur- face, but carxrot penetrate °2 3) W-inactivated virions corttaining g(D) are capable of blocidng entry of HSV-1, whereas a similar quantity of inactivated virions lacking g(D) cannot inhibit virus entry.°2 The epitopes invoMed in g(D) function have been mapped with the use of de!etfon mutants and compie- mentation assays a'A` Specific mutations in HSV-1, g(D) have been stbwrt to correlate with neuroinvasiveness•w' Because g(D) is essential for virus penetration.92 this property may be due to the abi!'ity of the virus to penetrate cells of the peripheral nervous system and other organ syst.rns. Whether mutations in either the virion, cellular I I p9 t P'K ioa A4 Mse r6e~ via. dw t reo infe Cort OM use, cat cW er& atec I I I I I Gra , poR'

a I I I I I I I I I I I Viral Pathoflenstis of Atherosclerosis 1207 ~y/P pemnb.r Z491, VoL 139, Mn 6 } r F1pvn e. aorrbtr "xcbmrrtv oftYSVpvaevmro.t i,uo cssvlm arrts bv d~e basicJi7*v61aa grvuxh/aa~ (FCF) rtupra.. ru:o rncdecr miv prtyn+ad by uti~ ixrra sivrq~lae cvsa rHSSrI type 1, rnay piaim+ar~ t~cr.l~ aflx A: lnrac HSV Lisidr dbealy w die 6a+i'r fUFrrrepeo. owrrV ea t~+s p.~te.r eJ~ a~ n+~ slycoFsoret>as rpp) sue6 ar~prs~ ~CC„~ gFCVI. m~de.~rHJ o~e' od~Peio^ ro rbe avfoer of ea![r ay b.p~+ n~!/ait prxEOalyrvLt rHS Pe). SfofCCUlo~ mfmloy beru.+nvc t{ufe tou( g6CnpronYm am~dpasrions aJr6r fC'rF recepCVS>rap er.t4, 7bea I~PG nurv alfl ~u¢ ro nabil=e ~e HSV co~rP/ez on 86r isofcu of mr crilt by busncrlr,*g lcrurclly wkb tB.gA~vxbu: ffie v;ur tbo~ peneAa.s ~G. carn rt.ot.86 dmr t~sF .roepro.,, 8: r,raoQ NSV 'p~v4aGz" on fGF, dx +ma..al tf~d Jo. ~ FeP rn.pa; ajtrr ad"prion w dbe aN/ uia Hs•PG. FGF bis&dr to ia rrMor dbwly m+d ir 6uanaltwd buo dx c.K drP&ttng HSYaJon,q u+* a buo ebe eetC In bodr rnurins, orrr dae trts penevaaa Abe Gr!!, l+t tixn degrar ia sCpli=oss protrs Rep.inud uar6 pemissiortse receptors, or their expression will atter the susceptibilityr to infedion of individuals and their arteries are interesurx3 concepts, but they remain to be explored. Frortt a devei- opmerttal aspect, the concept that herpesviruses may use, in part, a growth factor or its receptor to penetrate cells in vivo is very intriguing bocaus® FGF levels in the celf have been liniced to processes related to cell differ- enbation, cell division, and aging that are often associ- ated witfl human atfteresclerosis. Summary Circumsrsntial evidence condrues to accumulate to sup- port the hypottasis that certain herpesviruses can cor~ tribute to specific paiho9enic events associated with ath- erosclerosis. This fncludes 1) evidence of widespread infection of herpes viruses in the fleneral population, wherein these viruses are found in the arterial wall, indud- ing lesiorrbcaring areas; 2) the recogni6on of the esso- aaiion between accelerated attwrosclerosis and CMV Infection in cardiac albgraft recipierus; 3) the demonstra- t;on of herpesvirus-+nduced art~.rosderosis occurring in animal models of the disease during normocholester- olemia; 4) profound mftects on chol8steror metabolism in human, bovine, and avian arterial smootr, muscle cdts infected with herpesvirusss, predisposing to free and es- teriTied cholesterol accumulation; 5) atterations in extra- cellular mztriz and enhancement of a procoaflulant milieu on vascular c.lls infected with herpesvirus in vitro; 6) in- duction of mortiocyte and PMN adhesion receptors on HSV-infected endotheliai cells; 7) transforming potentiat of herpesvuuses and the ability of vascular smaoih mus- cle cells to be transformed by such viruses: and finaliy, 8) the abiliry of herpesvirus to induce actnrati.on of specific cytokine genes that have been implicated in the growth regulation of the vessel wall during human atheragene- sis. Although the rea atior>ship of herpesvinuses and ait} erasclerosis wn humans is not unequivocalty direct, data are accumulzting triat indicate that specific viral vectors may ind@ed have a participatory role in the inducfion of arterial injury. This also includes activaDon of the coagu- la6on cascade with links to thrombotic processes and development of a complex aReriopattry, With the advent of the applicanon of new moiecuiar techniques to study transforming eu.ms in the arterial waN, undoubtedly new irtfortnation wili emerge regarding tte viral origin of atil- erosderosis. Acknowledgments The au>tar ttWks his fabws antl atwr cofleagues who particl- pamd in marry of the expernnents and Ciscussiorts about the irtterpretaDOns of thho worfc OaecriFed heroi+, TroesE indivibuals I

1208 Haj)ar NP De+-smbW 2991. YoC L39, Na 6 inchxie C. R'ctlerd Miniek. Cztherine Fabricant, Julius Fabricant. Domerlicic Falcdle, OrU Edngin. Ketlneth Porrerint2. Andrew Nichdsotl, RobeR Kaner, Timottly McCaNrey, and Roy S+7+~er- steirt Also the 8W1or wishes to Qratefully, acktlo+Aed9s Andrew Baird, Ctaud'a Basifico, Dario ARieti, Thcrtlas Edgingtan. Allm Man.xukfsani, Robert Fbrfdewic¢, end Hanroy Friedman for their coileborative effors. Fnaly many thanks ara earKled to Smn Smith, Aaron Marzus. Ralph Nachman, Michaef Gimbrale and RusseH Ross for the3r support and collegial advice everthe past 17 years. References 1. Schwars SM. Carnpbe0 GR. CampW JH: Repficadon of srrqctl~ muscle cdis in vaacular dlsaase. Cirt: Res 1886. 68:427-A4d 2. Rass R: Athernscbrosis; A probLem of the biofogy of arterial walt caUs and tfleir irlterIIcfiOns with bbod arnporlents. Ar- tetoscletnsis 1981, 1:293-,311 3. Ponwrantz KS. HaDar 0P: Etcasarbids in the regulation of arterial srnooth rruSSJe coll phenotype, prdiferative capac- iiy, and choie:,lrol metabcaism. Artenascterosis 19M, 9A 13- • d29 4. Benditi EP, Benditt JM: Eridence for a monocbnal origin of human asherosderodc plaques. Prno Nat Acad Sd U S A 1973, 70:1753-1756 5, Fabri= CG, Krook L, GiUecpie JH: Virus-induced chdes- terol crysrals. Science 1973, 181:566-567 fi Fabr'scant CG, Fabricant J, Lltrenta MM, Mhdc CR Virus- irtduced atneroeclLrosis. J Exp Med 1978. 148:33S.W 7, Minick CR, Fabricant CG, Fabricant J, Lttrenta MM: Aftroarterioscierosis induced by nkCtien v,if1 a nerpesti~- ruz. Am J Pafhd 1979, 96:673-fi6 8. Fabticarft CG. Fabrican[ J, Meicfc Cf3, Urenta MM: Herpes- virus indueed ameroscierasis in chitdcers. Fed Proc 1983, 422476-2479 9. Hajjar DP, Fabricant CG, MinirJc CR, Fabricant J; V~rug in- dueed asheroscferasis: Herpes virus infectian aRers aorilc chpJasterol metabolism and acwmulatiert. Am J Pathd 1986. 12262-70 10. Graiten MT. MorCnaCabrai CF, Stamee VA, Qyet PE, Sdrr, san EB, Shurrnerly NE Gymrreg;alovirus inte:.bon is assvci- ated with cardiac aliognsft rejection and atherosdeross. JAMA 1989. 261:3581 -3566 11. MacDalafd K Rector TS, Braunlan EA, Coubo SH, Oifvari MT: Assnciabort of ooronary artery disesse in prdfac vz n,s- plarrt recpidlta with cytorneqaloviru,s in(eolfon. Am J Patd 19®, 64:358~fi2 12. Frerrcal N. Schirtner EC, Wyan LS. Katso(am G, Raftrnan I, Darevsch RM, June CH- I3olalion of a new herpesvirus from human CD4T calls. Prx Nat Aced Sct U S A 1990, 87:748-752 13. Gyortcey F, Mehnic-h JL, Gulnn GA, Gyorfcey P, DeBakey ME Herpes viridae in t116 pndodlelial and srrtoom rnuccJe cclls of the proximal aorta of atheroscttrocis parients. Exp Mot Padhol 1 %4, 40:328-339 14. Bendtt EP, B2rrotrT, McCa:fgal JK: Yrtusas k+dhe Qpoiogy of alheroscWosis. Proc Nat Aced Sci US4 19w, 60:6386-- 638G 15. Melnick JL Petrie BL, Dreearrw GR. Burek J, McCdk~m Cii, Debakey ME: Cyton*9alcvirus antigen within human anarial $mooth rtnx.cle cel~. Lzncet 19B3, 2,644-6d7 16. Petrie BL Melhicfc JL Adarn E, Burek J, McColkm CH, De• 8akey ME Nudefc acid sequence of cytom®gafovirus in ealle cultured from human atterSal tissue. J Infecx Dis 1987, 155:1513-159 17. Hendricfcs MGR, Sal'vnens MMM. Vanbavetl CPA, Brupge- men CA; High prevaler+ae of latendy present cyUortegaiov`- rus in artenal walls of patierus suffering from prade III amernsderosis, Am J Pytiol 1Si90. 136,23-28 18. Yamashlrora HM, Ghosh L Yang A, f3obertson AL• Herpes viridae in the coronary arttries arxJ aorm of young traume vir,>mC Am J Paihd 1968, 130:71-79 19. Karler RJ, lotzo RV, Ztie Z, Kefalides NA; Inhibitidl of pra teogyoan synthesis in humfn endothelial oells aRer infncvon wittt f-fSV type-1, in vttro. Am J Respir Cef! Moi Bid t990. 2:d23.A31 20. Hgar DP, Fnfcone OJ, Fabricant CJ. Fabricant J: Attered cholestetd ester cycle is assoCiated vrith ppid accsmxuiiatian in h.rpssvirius irlfected avian arter'sal srnooWt muscle cells. J Bid Chem 1985, 260;6124-8128 21. Hafyar OP; Herpetviruc infection prevents activation af cycs plasrnie chd@yto~A oCtarase in arv~ial smooRt rrusc{a cells. J Biol Cttem 1986, 261:7611-7614 22. Hajjar DP, Pomerantz KB, Falcor+e DJ, Weksler B8, Grant Ak Herpes simppex virus irfttien in human arteriat cells: Impticatioru in ahcnsckrosie. J CGn Invest 1967, 80:1317- 1321 23. Hejar DP, N'dholron AC. HaDar KA, Sando GN. Summers SO: Deaeased rtmseriger RNA transia6on in harpesvirus- infeCted zrtnrial cells: EUcta on cholecteM ester hydolese, Prpc Nat Acad Sci U S A 1989, 86:3366-.'i370 24. Vertxlkrlri GM: ProinttaRr, natory and procoagulant effects of herpes simpiex infectbn on human endotfletum. 81ood Cells 1990, 16:209-216 25. Nahmies A, Jocey K Ep'rd~ of nerpes sirrtp{ex virus 1 an0 2. Viral lydectiorts d Humarx-Zpidermology arsd Conmoi. Edited by AS Evans. New York Plerxtm MediCat Book Company, 1978, pp 253-271 26. Rooney JP Epiderriology of herpes sirnplex, Herpes S"m- pkxYrus Infectitn: B'ioioQy, TreaDnent and Prev®ntion. Ann InOem Med 1993, 1ffl:404-419 27. Johrson RE Nahmias PJ, Magder (S. Lee FK, Sroolcs CA Srovden MA: A seroepidemlakglc survey of the prava- lerica of herpes simplex virue type 2 infecvon in the Unirad Sabc. N Engl J Med 1986. 321:7-12 28. t',orden T, Gartia?almieri MR, Kagon A, Kannel WB, ScMff- man J: Ofitererces in rmrcnary heart diseate in Fruning- ham, Hondulu and Puerto Rica J Chrfln pis 197a, 27:323- 3" 29. Adarn E, Meln'cc Jt, Probesfi.dd JL, Peo'ie BL Burtk J, Biilay KA, McCdlun CH, DeBakef+ ME Higtt letirels of cy- tarncpabvirus antibody in patiertts requiring vasaufv sur- gery for aftletoeGerosic lancat 1987. 2:291-293 30. Havlic RJ, Blecfcwekier WC, Kasbw R, Casielfi W: UnrktfY a 4 0 4 4 I I I I I I I I I I I 1 I C1 ' .+.W ~' . I

I I I I I I ! ms na ro- on 30, od r I t I I I 3 of )od nis ind I asaociation be~ clinicaliy eppareni herpas-+inri infocr don and cfxonary inddonoe at okier apes: The Frirringhzm Heart Study. ArtlrioxlGraCia 1489, 8:877-880 31. MacGrega RR, Priedrtlan HM, Macarak EJ, Kefalfdes Nlt Yrus infocdon of endott+elial cells irr.r.asas qranulocyte ad- h.ranw. J Chn Invesc 1960. fi5:1468-1a77 32, Frierirnan HM, Manrik EJ, MaaGregor RR, Wolfe J, KeFal- ides NA Vinis inF.ction of endothelial ee{La. J Irtfect Dis 1966, 143:26&-.279 33. Ho 00, Rota TR, Andraws CA Ffrr,dh MS: Replicalion of human cytomegnlovirus in .ndothaial cNls. J Infect Dis 1966, 150,956-.957 34. TumiloMcz JJ, Gawlik ME, PaAell BB• Tronron JJ: RepFica- Con of cytomegabvinu in human arteriaf smooth muede cells. J Vi rol 1985. 56:633-W 35. Sydiskia RJ, Roznan 8: Prfysornas and protein synthesis in calls infeGeC with a DNA virus. Scivnra 1966, 153:76-78 36. F.nwicJc ML Clark J, Earty and delayed shut oft of host prorein synchasit in eells infected with herpes sirrplWc virus. J G.n triroll fl83, 61:121-125 37. Kefelidee NA, Laie Z Hcrp.e =implsz vinu suppressan of humen endothetlal matr6c protain syntttlesis ia independerrt of viral protain syrtthesis. Lab Irw~t 1986, 55;328-136 38. Lonoon FS, Broycer JM, tiaie Z Kefatdee NA: Supprassion of hoat mRNA in human srrmth musch calls by a virion compatrxit faotor in herpes simplex virua type 1. Lab Invost 1990, 621 @r9-145 39, Nishiioka Y, 5ifversroein S: Degradntlon o/ celfular mRNA dur- ing infectian by herpes simplex vins. Proo Nat Acad Sc! USA 1977, 7423742374 40. KworxJ AD, Kruper A Frerkai N; Herpes simplez virus vi- rion hoat ahutoff fwCtion. J Vird 1988. 62912-921 41. Paterson JC, Cotiraf GE Erperirrpntal eoronary scieerosis: IIf. Lyrr+pfnmatasis as a causo of coronary scierosis in chickens. Atrh Pffittd 1950, 49:69p.•707 42. Gibbs CP, NaZer'san K, Velixr LF, Kung HJ: Fsaip'vo ho- mology exists b@tween {Narek disaase herposvirus and its • vaccine viruC, herpesvirua of turkeys. Proc Nat Arzd SrJ U S A 1984, 81:33E5-3,369 43. Dudding L Hashsf S. Clairk BD, Auron PE, Sporn S, HuanQ ES; Cytomegaiovirus infecdon stimulate5 expresion of monocyte associated mediazor ganas. J Irnmunol 19e9, 143:3343•3362 44. Kdtiase M, Horrifsen-DeStefano D, May LT. Vcak J, Sah- gai PB: Induction of grinl.rhron by tvrer necrosis facax. a homeostabc m.chanism in rhQ Contrd of cell prdiferatial. Cep 19A6. 45:656~ 45. Wortp GHW, Gaeddd DY: Tunor necrosis faotors n and ~ inhibit vitus replication and synerfliza with ~tprf.r~. Na- tuM 1986, $Z3:819-~ 46 EGr,Qin OR. Hajar DP: Evidalce for CytaWne rt9ulabon of chof.s[erd metabolism in hetpesvinia-infected arterial cells by the lipnxygonssp padlway, J Upid Res 1990. 31 c99- 306 47, Ishiba9tr S. IrwbaT. Shimarn H, HaradaK, Inoue l, Mofano H, Mori N, Gotoda T, Takaia F, Yamada N: l4bnocyte cok ony-cnmulatinQ faotar enhances uptake and degradation of acotytatod low density Iipoproteins and chofesterd astarifi- Yraf Pathogenesis of Ath.rosd.rosia 1209 .Slr Dere+.br L491, voL 139, M). 6 cetion in hunlan monocyte•deriv®d mnaapmapes. J Bioi Chem 19M, 265:1d109..14117 48. Visser MR. Tracy P8, Vercefbtti GM. Goodmsrt JL, White JG, .lxob HS; Enharxed thronlbin Qeneranon and platala birding on herpes simples virus infeUed endotlteliurn. Proc Nat Acad Sd U S A 1968. 85:8227-.8230 d9. Key NS, vercdlom GM, Wnkelmern JC. Motdow CF, Good- man JL Esmon NL, Evrt+on CT, Jacob HS: Infeclion of ves- oular endothdial cWfs wi0i herpes simplex virus arrsarres tissue fn= activiry and reduces thnxnbanodulin expres- sion. Proc Natl Acad Soi U S A 1990. 87:7095-7099 50. McLaan JW, Tomlinson JE. Kuang WJ, et a!: cONA se, quence of human apofipoprotsin (a) is homoiogus to piess- minagQn. Nsture 1987, 3C17:132-137 51. Hs.jjer K1i Gravish D, Breslow JL Nactlrtt$n RL• Upopro- tdn(a) modulebon of andattltfiai cetl surface fibrndysis and its potentlai rde i1 atherosdeross. Nature 19d9. 339:901- 3C6 52 Walton KW, Hitchens J, Meflnani HN. Khan M: A study ot mettnds of idonqficatiai and estination of Lp(a) lipoprote+n and of iis significanoe in heatttt, hyperfip•Kiemia and atMro- acierosis. Attwr~s 1974 20:323-346 53. Etingin OR, Hajar DP, Hapar KA Nachman RL Lipopror.in (a) rQgulate plasrninogen acavator inhibltor-1 eicpression in ended +elid xlls: a potandaf moohani6m in thrombogetlesis. J Bid Chem 1991, 266;245&-248,5 54. Friedman HM. Cohen GH, E'menExug Rj, Seidol CA Cines D& GTycnprcc.in C of herpes simpiex vinus 1 a= as a rectptor for the C3b cornplernent oornponcnt on infaaed cef+s. Nahxe 1984. 309:633-635 ii5. Cines D8. Lyss AP, 8ina M, Corkay R, KefaGdes NA, Fried- man HM: Fc and CS recepmrs indix,od by herpes simplex virus on ci; w^ed humen endarielial cells. J Cl'n Invest 1962, 69:123-128 56. Para MF, Baucke RB, Spear PG; GfywprIItein gE of herpes simplex vfrus type 1: Effo= of anti-gE on virion infecfnrity and on vintsrindxad Fc-I:inding reeeptors. J Yrd 1982, d1;129-136 57. Johnson DC, Frame MC, Ugas MW, Crnss AM, Stow ND: HSV IgG Fc r.captor acdvity depends on a complex of two viral ptlCOproteins gE and 91. J Vkol 1988, 62:1347-13b4 58. Vissx MR. Jafrb HS, Goodmen JL McCarthy JB, Furcht LT. Vercelacd GM: Granubcyte mediafed injury to herpes simplezvirus-i~ecmd humpn andoClernm. Lab Irnrest 19B9, 60:29Ca-3U4 59. Bevlacqua MP, Pober JS, MejeaL GR Fers W, Cotrar+ RS, Gimbnorw MA Reoombin8nt ttmtor necr= facax induceS pcnmagulant as.tivity in wltured txrt>an vascYdar endodle- liurn: CfwacTeriation and eamparaort with the aroons of ir><erffeuwfvt 1. Proc Natl Arad Sd U S A 1985, 83:4533-4537 6D. BerifscqLa MP, Pober JS, Mendrick DL Coirran RS. Gir- brnne MA: Iden 'hficabon of an inducible endothelial- leukocyte adhesion molecule. Proc Natf Acad Sci USA 1987. e4S238-9242 61. Eunpii OR, SUveirstdn RL Friedrnan HM, HaDar DP. Vfral acdvarion of the caagu6Tion cascade; Molecular intarac- tions at the surface of infecmd w,dott~el a! cells. Cell 1990, 61 fi57462

1210 Haljar NP Qt'rrRhe+ 1.992, Vo1. Ij9. No. 6 62. McEver RP: GMP•140: A reaptvr for re.rtrnphils and mono cyris on acamlzd platefets and etidoH>efum. J Cell Bio• ctom 1991,45:1-%-161 63, Hattori R. Harruhon KK, Fugara RD, McEver RP, Sims PJ: Stirrwlu.d secrzflon of endothdial von Willebrand factor is acr,ompanied by rapid redistribution to the cell surface of the inttxstlular granu4e membrane protein GMP•140. J Biot Ch.m 1989. 264:7768-7T71 64, Etingin OR, Silwrstein RL, HajFar DP: Idantificadon of a monooyte recaptor on herpesvinus-infectad endottwiia/ odlt. Pnx Natt Aoad Sci U S A 1991, 88:72Cb--72q3 65. Sanchez-Madrid F, Nagy JA, RobWns E, Slmon P, Springer TA: A twman leukocyt® d'rfforondatjon arwyen famiy with distinct a-subunits and a common p-subunft. J Exp Med 1983, 158:1785-1803 66. Osbom L Hession C, T¢ard R, vassallo C, Lutxw,tryj S, Ch1-Rcim G, Lobb R; Direct expression cbning of vascular oeU adhesion mdecule 1, a cytoklne-Inducod endotnalla1 procein that binCts to tyrr+phocytos. Call 1989. 59:1203-1211 67. Silverstein RL, Nachman RL: Thrombospondin binds to a~ and me661tos p~elet nionocYte adher.ion. J Cfh Invest 1987, 79:867-874 68, Altieri DC, Etingct OR, Fair DS, Brunck TK Galtosky JE, Hajjar DP, Edginpton T: Stn.tca.iral modf in Factor X modiates blnding to monocyte Ct)116/CD1 B and to gtympratein C on herp.zvinis-infoetad endothefiai ceus. Science, 1991, in priess 69. Peatson TA, Wang A. Sokz K Heptinstall RH: Ctonai char- acteristicc of fibrous plaques and fat.y strea}m from human aonas. Am J Pathol 1975, 81:379-355 70. Thomas WA. Reiner JM, Jana}ddevi K, Porerttin RA, Lee KT: Pop-0,don dyrtiamics of artprial calls during atherogenesis: X Study of montypism in athwasclerrAc lesiona of black wmien heterozYeous for gtucose•6 phosphate dehydroge- nase (G6PD). E>p Mol Peiftd 1979. 31:367-386 71. Thomas WA, Kim ON: Biolcgy of disease: Athenoaclervsis as a hyperplastic andror neoplastie prc,cess. Lab Invest 1983, 48:245-255 72 Linder 0, Garter SM. Glucose -6- pho:phato dehydrqe- rase rnosaicism: UGlizition as a ceu marker in trie study of leior~ Science 1g65, 150:67-8 73, Majesky MW, Reidy MA, Bendit< EP, Juchau MR: Foal sn1oou muzclo protifaratlan in the aortic indrta produced by an inttiation pronntion avquanco. Prize Natt Arad Sci U S A 198,5, 82;3450-34U 74, Penn A, Garu SJ, Warrat L Nasta D. Mindich B: Transforrn. Irg gem in human attunxderooc plaque DNA. Proc Naz Aoad SCI U S A 1986, 63:7951 795,5 75. Penn A, Snyder C: Arttriosderotic plaque dmdoprrxrn is pnxrcoed by potynuclear arometic hydrararbxm Carcno- flsnesls 1 g88, 9:2185-2189 76. Parkgs JL Cardetl Rfi, Hubbard FC, Hubbard D, M.ttz.r A, Penn A. Cu!tured human atherosclerotic plaque smooth nxxde ceUs retain rranstorrning pdential and dsplay en- hanced e)pression of the rnyo protoonccyene. Am J Pathd 1991, 138:76&-775 77. Yew PR. Ra{aNashfsth T8, Forresoar J, Barath P, Lustc AJ; NIH 3T3 transfonning gene is not a general feature of arh- eroacierotic plaque DNA. B'achen B'iophys Res Canmwn 1989, 165:1067-1071 78. Ahrrwd AJ, 0'Malley 8W, Yarsu FM: Presance of a putachro transfomiing geno in human arhefoscleroDc piaquas. Circu- hon 1990. III-35 79, RaStInejad F, Poiverini PJ. Bouck NP: Regulation of the ac- tivity of a nwv irYVbitor of angiogenesis by a cancer su¢ prossor gene. Call 1990, 5fx3 'W 80. Good DJ, Potverini PJ, Bouck NP. Reguation of the activity, of a ne+r<irY+ibitor of arp+ogenis is'smltulotogicalh and funo- tionally indisdngutshabla from a fragment of thrombxpon- din. Proc Nm1 Acad Sci USA 1990, 67:6fi24-6629 81. Nachtigal M, Legrand A Nagpal ML, Nachtigal SA Greertspan P: Trdr6formation of rabbit vascular srnooth musde celts by trarisfection with the eariy region of SVdO DNA. Am J Patho! 1990, 136;297-.306 82, Nachtigal M, Le9rand A, Greenspan P, Nachd9al SA, Nag- pal ML• ImrrnmJiz~on of rabNt v2scular smootn musGc calls afoer trarisfaction with a tragmertt of the BgLll N region of herpes simplpt virus tyA 2 DNA Irnennrology 1990, 31:166-174 81 Chen JK U L MdZure D8: Ahared kr++ density lipoprotein receptor mgulatiort is associated with choiesteryi ester ac• cumulation in simian virus 40 transtonmed rodent 6broblast cell Fr>GS. In Yrtro Cett Dev Biol 1968, 24:355-.9,5B 84. Maonab JCM: Herpes simplex vtn;s and hurnan cymrnag- aiovirus, their roles and morphological transfomtiatlon and genital earKw. J Gen Yrd 1987, 68:2525-2550 85. Hwanq CBC, Shinitae EJ: DNA seCuen:e ot mutations in- duced in cells by herpes simptex virus type-1, virotogy 1990, 178:180-188 86, Cameron IR, Park M, Dutfa BM, Ore A, Macnab JCM: HefpES Simptex vin,6 s@quenG@s invoNed in tfle inltiation of onoogenic morphobgical transfom-ation of rat cells are not required for mainterance of the transformed state. J Gen 1Arot 1965, 66:517-527 87. Herold BC, WuDum D, Sodys N, Spear PG: Gtycoprorein C of herpes simplez virus type 1 plays a principal nole in the absorption of virus to cells and in infecovity. J Nrot 1991, 65.1090-1098 68. WuD+m 0, Spear PG: Initial irtteracoai of herpes simplex virus with cdls is binding to heparan sutfatt. J Yrnl 1989, 63:52-58 89. Kaner RJ, Baird A, Marssukhatv A BasTca C, Sumrners BD, Roriciewicz RZ Hallar DP: Fbrobtast growth factor receptor is a portal ot cellular entry for herpes simplex virus type 1. Science 1990. 248:1410-1413 90. Baird IL Plorfcew;¢ RZ, Maher P, Kaner RJ, HaW 0P; As- saciation ot basic fibnoblast growth factor with herpos sim• pkx virus 1 mediares virion perietranon into vasc+Aar cells Nature 1990, 348341-346 91. Futler A0. Spear PG: Artti-gtycoprue+n 0 an6bodes that pemtit adsMtion but btcClc infection by herpos slmpt¢x vi• rtn 1 prtwnt virip-rto fusion at trw coU surfece. Proc Nat Acad Scl U S A 1867, 84:5454-6458 92. Johrsert DC, Ligas MW: Herpes simpbc vinkes tactdrV I cI 94. I I I I I I I 1 I I 1 I I I

Viral Pathogenesis of Ath.roscl.rosis 1211 eym Down6e. r931. VaL tJ9, Na 6 I I I I rau- ~ I29- ~. 1 I aan ao aaat I :9Y not , nC r C~E ! 1911 ~lex ~ I I ;t - ~Ils. /-S'/ hat vi- %J,p I h I gh'cprocein 0 are unable lo inhibit virus p.netraeon: Qt,ian- dtaDve e+ride+ice for viRus-spedfx ddl suftaoe recaptor6, J Yupl 1988, 624606-4612 33. Muggeridge Mi, Isoh VJ, Eiym RA, Tudaer TJ, Viruon AC, Gbrioso JC. Cahen GH, Eisenberg F{J. Antgenic arsalysis d a rnajor nesaryiiation site of herp.s sifipla virta gfympro- tain 0. usinp ddeSon mutants and monodonal annbody- rvastant murwts. J Vuoi 1988, 62:327a-3230 94. MuQQeridgv MI, Wfcox WC, Corien GH, Esenberg Fll: fdantlfication ot a aite on twrpes simpls5c virus type 1 grym protyin D ttnt fs ess.ndal for irtfectivity. J Virol 1990, 84:3617-3&26 95. Iz~ W Slewns JG: Molecular and biologiaJ cttaractar- izaaon of a hapEa simplWC virus ty;* 1(HSV-1) neuroinva- siverwrs gene. J Exp Med 1990, 172d87-496 96 KarMr R1, Haijar DP, Y4 gerxs and atheroQenesi.s. G.- nedc Factnrs in Atherosclorns+s--C'.andidate Genes and Processos. in Monogrdphs in Hunian Genetics. Editetl by aJ Lusis. JL Rdier, RS Sparks. New Torfc. KdrBer. 1991, in pro" I