Macrophages and smooth muscle cells are important

Transcription

1 1363 Etpside Treatment Suppresses Athersclertic Plaque Develpment in Chlesterl-Fed Rabbits Margarita de la Llera-Mya, Gerge H. Rthblat, Jane M. Glide, and James M. England T study the mechanisms by which mncytes/macrphages and smth muscle cells cntribute t athersclertic lesins, we studied athersclertic plaque frmatin in chlesterl-fed rabbits treated with etpside, a drug that has been shwn t have several effects that culd interfere with the prpsed interactins between these tw cell types (M.W. Aarnudes et al, VirchwsArch B 1984;47: and M. Rzencweig et al, Cancer 1977;40: ). Our results shw that lng-term etpside treatment f New Zealand White rabbits maintained n a high-chlesterl diet decreases the extent f fatty streak frmatin in the artic intima. Mrever, the plaques frmed in the presence f etpside are thinner and at least fcally have less fibrus tissue and fewer smth muscle cell-derived fam cells than d plaques in cntrl rabbits. These effects are independent f the extent f the diet-induced hyperlipemia r an effect f etpside n bld cell cunt and may be related t the inhibitin f intimal cell prliferatin by etpside. (Arterisclersis and Thrmbsis 1992;12: ) KEY WORDS athersclersis plaque prgressin fam cells macrphages smth muscle cells hyperchlesterlemia Macrphages and smth muscle cells are imprtant cnstituents f the athersclertic plaque in naturally ccurring lesins 12 and in lesins btained frm chlesterl-fed experimental animals. 3-6 Recently, mnclnal antibdies specific fr macrphage r muscle cell antigens have been used t identify the phentype f lipid-laden fam cells within athersclertic plaques. 7 -" Based n antibdy reactivity, the majrity f cells in athersclertic lesins have been classified as either macrphage-derived (MFC) r smth muscle cell-derived (SMFC) fam cells. 12 Hwever, much remains t be learned regarding the rle f these tw cell types in the develpment f athersclersis. Current mdels f athersclertic plaque develpment prpse that the macrphage plays a crucial rle in the initiatin and prgressin f lesins. It is thught that bld mncytes adhere t and penetrate thrugh the vascular endthelium at sites prne t lesin develpment, 3813 which are als areas where mdified lipprteins can accumulate Macrphages can then avidly internalize these lipprteins via unregulated scavenger receptrs 16 t accumulate large intracellular stres f chlesteryl esters and becme fam cells in fatty streaks. By mechanisms that are nt understd but that may invlve the release f chemtactic and grwth factrs, 17 these lipid-laded macrphages can in turn trigger the migratin and prlifera- Frm the Departments f Physilgy and Bichemistry (M. de la L.-M, G.H.R., J.M.G.) and Pathlgy (J.M.E.), Medical Cllege f Pennsylvania, Philadelphia, Pa. Supprted by Natinal Institutes f Health grant HL and als by a generus grant frm the Jhnsn and Jhnsn Fcused Giving Prgram. Address fr crrespndence: Margarita de la Llera-Mya, PhD, Department f Bichemistry, Medical Cllege f Pennsylvania, 2900 Queen Lane, Philadelphia, PA Received December 3, 1991; revisin accepted July 20, tin f smth muscle cells int the intima, a hallmark f a prgressing athersclertic plaque > 19 The mechanism by which smth muscle cells becme fam cells in viv has nt been established, but it has recently been reprted that smth muscle cells in vitr can express unregulated cell-surface receptrs fr mdified lipprteins. 20 It has als been prpsed that lipid-rich macrphages can stimulate the frmatin f SMFCs by releasing chlesteryl ester-rich drplets that can then be internalized by smth muscle cells. 21 Thus, macrphages may prmte athersclersis by varius effects n smth muscle cells. T investigate the mechanisms by which smth muscle cells and mncytes/macrphages cntribute t the frmatin f athersclertic lesins, we studied the effects f the cytstatic drug etpside n athersclertic plaque develpment in chlesterl-fed rabbits. We chse t study the effects f etpside n artic lesins because this drug has been reprted t prduce mncytpenia in rabbits, 22 and as discussed abve, circulating mncytes have been implicated in the early events f fatty streak frmatin in several animal mdels f athersclersis Our results shw that lng-term treatment f chlesterl-fed New Zealand White (NZW) rabbits with etpside decreases the extent f plaque frmatin in the artic intima. The plaques frmed in etpside-treated rabbits are thinner, have decreased intimal accumulatin f fibrus tissue as visualized with histlgical stains, and have fewer SMFCs as measured immuncytchemically with the mnclnal antibdy HHF Althugh we bserved dramatic differences in lesin develpment in the drug-treated animals, these effects were fund t be independent f the extent f diet-induced hyperlipemia r changes in bld cell ppulatins. We prpse that etpside impairs plaque develpment by inhibiting intimal cell prliferatin.

2 1364 Arterisclersis and Thrmbsis Vl 12, N 11 Nvember 1992 Methds Rabbits Adult male NZW rabbits (average weight, 2.5 kg) were used in all experiments. The rabbits were individually caged in stainless steel cages and were hused in an animal care facility apprved by the American Assciatin fr Accreditatin f Labratry Animal Care and in accrdance with Natinal Institutes f Health guidelines. Cnventinal NZW rabbits were btained frm Ace Animals (Byertwn, Pa.). Specific pathgen-free (SPF) NZW rabbits were frm Hazletn Research Prducts (Denver, Pa.). NZW-SPF rabbits were used in sme experiments because they are mre inbred than the cnventinal rabbits and less prne t subclinical infectins. NZW-SPF rabbits were islated frm all ther rabbits and were shwn t be Pasteurella free n receipt and at the end f ur studies. All prcedures were reviewed and apprved by the Institutinal Animal Care and Use Cmmittee. Etpside Treatment and Mnitring Rabbits were treated with the cytstatic drug etpside (VP16-213, VePesid, Bristl-Myers Onclgy Divisin, Evansville, Ind.), a ptent inhibitr f mncytic murine leukemia 23 that has been shwn by thers t prduce mncytpenia in Chinchilla rabbits. 22 We btained etpside as a gift frm Dr. William Bradner (Bristl-Myers) and used a ttal weekly dse f 40 mg/kg bdy wt injected subcutaneusly as tw 20 mg/kg dses. Etpside was diluted with sterile phsphatebuffered saline t the apprpriate dse just befre injectin. Since etpside is frmulated in a cmplex vehicle that maintains the drug in slutin, we injected cntrl rabbits with vlumes and cncentratins f vehicle equivalent t thse received by the drug-treated rabbits. The vehicle was prepared accrding t the frmula in the drug package insert and cntained citric acid, benzyl alchl, Tween 80, plyethylene glycl 300, and ethanl. All rabbits were bled nce a week frm the marginal ear vein. These bld samples were used fr Culter cunts (cunter mdel ZM, Culter Electrnics, Hialeah, Fla.) f red (RBC) and white (WBC) bld cells and platelets, as well as differential cunts n smears stained with Wright-Giemsa (Diff-Quick Stain, American Scientific Prducts, Kankakee, 111.). Hematcrits were measured using standard techniques. These parameters were used t mnitr drug effects. Rabbits were treated with etpside ne week befre the start f the high-chlesterl diet, and treatment was cntinued thrughut the time the rabbits were kept n the athergenic diet. Occasinally, lng-term etpside treatment has been fund t cause prfund and rapid drps in bth hematcrit and RBC cunts. Therefre, if hematcrit levels drpped by 30% r mre, the etpside treatment was either suspended r decreased until the hematcrit stabilized. By this criterin NZW-SPF rabbits appeared t have a lwer tlerance t etpside than did cnventinal rabbits and, n the average, received less f the drug (average ttal dse f etpside given: NZW-SPF, 460±45 mg per rabbit; NZW, 585±153 mg per rabbit). Liver functin tests were dne by the labratries f the veterinary schl f The University f Pennsylvania n bld samples frm sme f the rabbits used in each experiment. Serum levels f glutamine pyruvate transaminase and bilirubin were elevated in etpside-treated rabbits n the high-chlesterl diet, but similar elevatins were als seen with the diet alne, and the average values fr these parameters were nt significantly different between these tw grups (/>=0.69 fr glutamine pyruvate transaminase and /?=0.18 fr bilirubin). Histlgical evaluatin f liver and spleen samples frm rabbits that had been treated with etpside fr several weeks shwed n abnrmalities. Diets Athersclertic lesins were induced by feeding the rabbits a 2% chlesterl-6% peanut il diet fr 8 weeks. 24 This athergenic diet was prepared by disslving the chlesterl (USP grade, anhydrus, Sigma Chemical C., St Luis, M.) in the peanut il (Planters) and then using this mixture t cat pellets f the standard chw (Agway Pr Lab Diet, Ithaca, N.Y.). The diet was prepared every 2 weeks and was stred in sealed cntainers at 4 C. All rabbits n the chlesterl diet were initially fed g/day, and the amunt eaten was mnitred. If necessary, the amunt f diet given each animal was adjusted t ensure that bth etpside-treated and untreated rabbits cnsumed the same amunt f chlesterl. Ttal chlesterl was measured n pstprandial plasma samples, btained nce a week frm the ear vein, using a standard enzymatic assay (Sigma prcedure N. 352, Sigma), and these weekly measurements were pltted against time. The area under each curve was integrated by weighing the paper cntaining the traces f each curve t calculate the average daily plasma chlesterl level fr each rabbit. In the experiments with NZW-SPF rabbits, after 4 weeks the 2% chlesterl diet was cut with equal amunts f standard chw t lwer the chlesterl cntent t 1% and t maintain the serum chlesterl levels belw 2,000 mg/dl. Ttal triglyceride was als measured enzymatically (triglyceride-gpo, prcedure N. 339, Sigma) in sme samples. In additin, agarse gel electrphresis (Paragn Lip Electrphresis System, Beckman Instruments Inc., Brea, Calif.) was dne n plasma samples btained frm all rabbits at varius intervals during chlesterl feeding and at the time the animals were killed. Electrphresis was dne t ensure that bth etpside-treated and untreated rabbits develped the beta-migrating very lw density lipprtein (j3-vldl) that has been described in the literature. 25 Tissue Preparatin Rabbits were given a lethal dse f sdium pentbarbital (Butler C., Clumbus, Ohi). Immediately after death, bld was cllected by heart puncture; serum was prepared and stred at 4 C until use. The arta was dissected frm the arch t the bifurcatin, gently rinsed with nrmal saline, and pened alng the psterir wall. After remving as much f the periartic tissue as pssible, the pened artas were flattened n strips f paper, intima side up. The vessels adhered t the paper strips and were fixed face dwn with 10% buffered frmalin (Fisher Scientific, Pittsburgh, Pa.) vernight at rm temperature.

3 de la Llera-Mya et al Etpside Suppresses Athersclersis 1365 Analysis f Lesins Fixed artas were stained with Sudan IV t visualize areas f athersclertic plaque. Staining was dne by flating the vessels face dwn fr 25 minutes at rm temperature in a slutin f 0.5% Sudan IV (Fisher Scientific C., Fair Lawn, N.J.) in freshly prepared 70% isprpanl. 26 The stain was differentiated with several rinses in 70% isprpanl. Templates f the stained vessels were drawn n clear acrylic sheets, and the extent f grss lesin was quantified with a dt-cunting methd by superimpsing each template ver a dt grid with a 2-mm 2 grid size (Letratne LT-914, Letraset, England) and cunting the ttal number f dts cvered by each vessel and the number cvered qnly by lesins. 27 Tissue fr histlgical analysis (3 mm thick) was taken frm the macrscpically mst apparent plaques. At least tw plaques were sampled frm each arta; ne plaque was always frm the artic arch, and the ther sample f plaque was usually taken frm the thracic arta, althugh in sme animals prminent plaques in the abdminal arta were sampled. Adjacent histlgical sectins were stained with hematxylin-esin, Verheff-van Giesn's elastin, and Massn's trichrme stains. T analyze the cellular rigin f fam cells fund in the artic lesins, immuncytchemical staining was dne using the well-characterized mnclnal antibdies HHF-35 and RAM-ll. HHF-35 was btained frm Enz Bichemicals Inc. (New Yrk), and RAM-11 was a kind gift frm Dr. Allen M. Gwn. Staining with these antibdies was dne with standard prcedures using an avidin-bitin cmplex/perxidase technique fr visualizatin (Vectastain ABC kit, Vectr Crp., Burlingame, Calif.). T determine if there were significant quantitative histlgical differences in plaques btained frm either etpside r cntrl rabbits, histlgical sectins f plaques frm the artic arch were stained with Massn's trichrme stain and HHF-35, cded, and randmized; these sectins were then reviewed by fur independent bservers wh assigned each sectin a scre frm 1 (lw reactivity) t 4 (high reactivity). Scres frm all bservers were averaged fr each experimental grup, and the statistical significance f the results was analyzed by using Student's / test. Measurement f lesin thickness was dne by using a micrscpe eyepiece micrmeter. The mean intimal thickness was calculated frm measurements f the distance frm the luminal surface t the internal elastic lamina, determined at ju.m intervals thrughut the entire length f the plaque sectin, and Student's t test was als used t determine statistical significance. Althugh histlgical assessment was dne in bth experiments, quantitative histlgical analyses were dne nly n slides prepared frm tissue btained in experiment 2. Chemical Analysis f Tissues Frmalin-fixed vessels frm all rabbits in experiment 2 were rinsed with 70% isprpanl fllwed by nrmal saline. The vessels were bltted gently, and wet weights were determined. Entire individual artas were minced, except fr tw 3-mm-lng sectins taken frm each vessel fr histlgical analysis. The minced tissues were resuspended in 2.5 ml water fr each 0.5 g wet weight and were hmgenized with a Plytrn. Aliquts (0.5 ml) f hmgenates were transferred t tared test tubes t determine wet weights. Ttal lipids were extracted frm equivalent aliquts accrding t the methd f Flch et al. 28 Chlesterl was measured by gas-liquid chrmatgraphy with chlesterl methyl ether (Sigma) as an internal standard. 29 Ttal chlesterl was measured after hydrlysis by fllwing the methd f Ishikawa et al, 30 and the chlesterl ester cntent was calculated by difference. Althugh all extracts cntained a residual amunt f Sudan IV, it was determined that it did nt interfere with the quantificatin f either chlesterl r its esters. Experiments With Cells in Culture ill A macrphages were seeded in 35-mm wells (Cell Wells, Cstar, Crning, N.Y.) and grwn t cnfluency in RPMI-1640 supplemented with 10% heat-inactivated fetal bvine serum. Test media fr cell incubatins cntained 10% f each serum t be tested in RPMI supplemented with gentamicin (50 /ug/ml, Tri Bi Labratries, State Cllege, Pa.). Incubatins with test media were started by replacing grwth media with the test media after gently rinsing the cells with RPMI Cells were incubated with test media fr 24 hurs at 37 C. Cell incubatins were dne in triplicate. At the end f the incubatins test media were remved, and the cell mnlayers were rinsed with warm phsphatebuffered saline. Cell lipids were extracted by incubating the mnlayers with isprpanl vernight as previusly described. 29 Cell chlesterl and chlesteryl ester cntent was quantified by gas-liquid chrmatgraphy as described abve. Cell prtein was quantified by the methd f Markwell et al 31 after the mnlayers were extracted with isprpanl. 29 Statistical Analysis Statistical cmparisns between grups were made by using analysis f cvariance, t test, Mann-Whitney nnparametric tests, and linear crrelatin. Differences were cnsidered significant at a prbability value f 0.05 r less. The quantitative data are reprted as mean±sd. The analysis f cvariance was dne by ur statistician, Dr. Edward Graceley. Other statistical tests were dne by using a cmputerized prgram (Graph PAD In Stat, Graph PAD Sftware). Results Plasma Chlesterl and Lipprteins Plasma chlesterl in bth etpside-treated and cntrl rabbits increased prgressively frm a nrmal level f mg/dl (NZW, 36±12.5 mg/dl; NZW-SPF, 50±8.5 mg/dl) t ver 1,500 mg/dl after 4 weeks n the high-fat diet. The average daily plasma chlesterl level was calculated fr each rabbit by integrating the areas under the chlesterl-versus-time curves btained frm the weekly plasma chlesterl measurements. There were equivalent increases in the average daily plasma chlesterl levels and bdy weights fr bth etpsidetreated and cntrl rabbits (Table 1), and there were n significant differences in the average amunt f diet eaten by each grup. Hwever, the amunt f diet eaten by each rabbit was nt significantly crrelated t either the daily plasma chlesterl level r the plasma chlesterl level measured at the time the animals were killed.

4 1366 Arterisclersis and Thrmbsis Vl 12, N 11 Nvember 1992 TABLE 1. Average Weights and Plasma Chlesterl Levels fr Cntrl and Etpside-Treated Rabbits Experiment NZW rabbits Cntrl («= 12) Etpside (TI = 11) NZW-SPF rabbits Weight (kg)* 3.1 ± ±0.2 Plasma chlesterl (mg/dl)t l,906±513 l,470±498 Cntrl (n=9) 2.9±0.2 1,614±479 Etpside (n = ll) 2.8±0.1 1,474±391 NZW, New Zealand White; SPF, specific pathgen free. *Mean±SD f all weekly weight determinatins fr each grup f animals. tmean±sd f average daily plasma chlesterl values fr each grup f animals. Plasma triglyceride levels increased slightly frm a basal level f mg/dl (NZW, 58±18 mg/dl; NZW-SPF, mg/dl) with the high-fat diet and seemed t be accentuated when plasma chlesterl levels were ver 1,000 mg/dl (NZW, 181 ±93 mg/dl; NZW-SPF, 97±57 mg/dl). Agarse gel electrphresis f serum samples btained at varius intervals during chlesterl feeding demnstrated that the hyperchlesterlemia seen in all f the chlesterl-fed rabbits was accmpanied by the appearance f a characteristic /3-VLDL particle, which is cnsidered t be athergenic. 25 T determine if lipprteins frm the etpsidetreated rabbits culd be internalized by macrphagelike cells t prduce fam cells, J774 macrphages were incubated with nrmlipemic rabbit serum and with hyperlipemic serum btained frm untreated and etpside-treated rabbits that had been n the athergenic diet fr 4 weeks. As shwn in Table 2, when added at equivalent chlesterl cncentratins, hyperlipemic sera frm either drug-treated r cntrl rabbits caused a similar increase in cellular chlesterl. As expected, the excess chlesterl was stred as chlesteryl ester in drplets that were evident in the lipid-laded cells when these were examined by phase micrscpy. 29 Effects f Etpside n Artic Athersclersis Since a high-chlesterl diet induces lipid-rich athersclertic lesins in the rabbit arta, Sudan IV was used t quantify the extent f intimal area cvered by 90 BO A EXP * 20 O 10 SE n 90 B ' " «t* 1 fl 1 EXP. 2 1 BO O O O 10 O O * 0 1 1* _!_ 1 I PLASMA CHOLESTEROL (mg/dl) FIGURE 1. Scatterplts shwing effect f etpside n lesin frmatin. Extent f Sudan IV staining as a functin f the average daily plasma chlesterl cncentratin was calculated by integrating the chlesterl-versus-time curves fr each rabbit (see "Methds"). Experiment 1 (panel A) was dne with cnventinal New Zealand White (NZW) rabbits, and experiment 2 (panel B) was dne with specific pathgen-free NZW. Open circles represent individual rabbits fed the highchlesterl diet as described in "Methds." Clsed circles represent individual rabbits fed the high-chlesterl diet and treated with etpside as described in "Methds." plaque Pled data btained frm bth cnventinal and NZW-SPF rabbits demnstrated a significant difference in the percentage f the intimal surface that was stained with Sudan IV in the etpside-treated grup cmpared with the cntrl grup (cntrl, 37±25% versus etpside, 13±15%,/?<0.001). Figure 1 shws the extent f Sudan IV staining in each vessel as TABLE 2. Chlesterl Accumulatin in J774 Cells Expsed t Serum Frm Cntrl and Etpside-Treated Rabbits Cell chlesterl (/ig/mg cell prtein) Treatment 1. 10% FBS (0.034 mg/ml chlesterl) 2. 10% rabbit serum+etpside (0.030 mg/ml chlesterl) 3. 10% HRS (2.25 mg/ml chlesterl) 4. 10% HRS+etpside (2.24 mg/ml chlesterl) Ttal ± Unesterified 29± ±8 Esterified ± ±22 FBS, fetal bvine serum; HRS, hyperlipemic rabbit serum. Cnfluent mnlayers f J774 cells were incubated with the indicated sera fr 24 hurs, after which the chlesterl cntent was determined as described in "Methds." Nrmal rabbit serum plus etpside was pled frm three rabbits. Hyperlipemic rabbit serum was btained frm single rabbits. Three different hyperlipemic sera frm etpside-treated rabbits were tested. Data shwn are frm a typical experiment. Values are mean+sd f three determinatins.

5 de la Llera-Mya et al Etpside Suppresses Athersclersis 1367 a functin f the average daily plasma chlesterl cncentratin. The data are presented separately fr each grup f rabbits. As shwn in Figure 1A (cnventinal NZW rabbits) and Figure IB (NZW-SPF rabbits), etpside-treated animals develped fewer Sudan IV-psitive lesins at all levels f hyperlipemia, and an analysis f cvariance f the extent f lesins cmpared with the area under the chlesterl-versus-time curve fr each rabbit demnstrated that this difference was significant in each experiment (significance f F is fr experiment 1 and fr experiment 2). We measured the chlesterl cntent f the artas btained in the secnd experiment. A cmparisn f the extent f intimal area cvered by Sudan IV-psitive lesins and the chlesterl cntent f the tissue demnstrated a significant crrelatin fr bth etpside-treated animals (r=0.74) and the untreated cntrl animals (r=0.84), and the average chlesterl cntent f the artas in the cntrl grup shwed a trend tward an elevated value cmpared with the drug-treated grup by the Mann-Whitney tw-sample test (p=0.063). Despite quantitative differences in the extent f plaque, as expected the lesins were mst frequently fund in the artic arch and at the intercstal stia in the thracic arta f bth cntrl and etpside-treated animals in bth experiments. 17 Effect f Etpside n the Mlcranatmy f Athersclertic Plaques The thickness and cellular cmpsitin f chlesterl-induced athersclertic plaques in etpside-treated rabbits differed frm thse f cntrls. The mean thickness f plaques in the etpside-treated grup was 88.5 ±16.9 fim cmpared with ±16.0 /xm in the cntrl grup (p=0.0243). The plaques in cntrl animals shwed cnsiderable mrphlgical hetergeneity in that the central areas f the plaques were predminantly cellular, with nly fcal accumulatin f fibrus (trichrme- r elastin-stain-psitive) tissue (Figures 2A and 2E), whereas peripheral areas f plaques were ften less cellular and cntained substantial accumulatins f fibrus tissue (Figures 2C and 2G). In cntrast, athersclertic plaques in etpside-treated animals appeared t be mre hmgeneus and cntained sparser accumulatins f fibrus tissue than did plaques in cntrl animals (Figures 2B, 2D, 2F, and 2H). Mnclnal antibdies reactive fr either a macrphage-specific antigen (RAM-11) r muscle-specific actin (HHF-35) were used in immunhistchemical assays t analyze the cellular cnstituents f athersclertic plaques. As expected, plaques in cntrl animals cntained large, glbular fam cells that were RAM-11 psitive and smaller, spindle-shaped fam cells that were HHF-35 psitive; these are designated macrphage-derived fam cells (MFCs) r smth muscle cell-derived fam cells (SMFCs), respectively. In cntrl animals, MFCs and SMFCs were nt unifrmly distributed within plaques. MFCs were relatively mre abundant in regins in which extracellular tissue was sparse (Figure 21). Cnversely, SMFCs were relatively mre abundant in regins rich in fibrus tissue (Figure 2O). In cntrast t the cntrl animals, fewer SMFCs appeared t be present within plaques f etpsidetreated animals (Figures 2J, 2L, 2M, and 2P). T determine if these histlgical impressins f quantitative differences in plaque cmpnents between the tw grups f rabbits were significant, slides stained with either Massn's trichrme r HHF-35 mnclnal antibdy were scred in a blinded analysis by fur independent bservers as described in "Methds." Average scres fr trichrme-psitive cllagen material were significantly different by a ne-tailed t test (/?=0.0046). Average scres fr the HHF-35 stain fr SMFCs were als significant by ne-tailed t test (p=0.0498). Thus, the plaque that frmed in the cntrl animals was enriched in fibrus tissue and shwed a trend tward a higher cntent f SMFCs. Effects f Etpside Treatment n Bld Cell Ppulatins Since an initial aim f this investigatin was t emply etpside t reduce the number f circulating mncytes and t determine if such a reductin influenced plaque develpment, careful attentin was given t mnitring mncyte levels thrughut the entire treatment perid. Culter cunts f RBCs and WBCs, as well as differential cunts n bld smears, were perfrmed fr all animals n a weekly basis. Platelet cunts were dne every 3-4 weeks. In all rabbits fed chlesterl, there was a significant decrease in the RBC cunt and hematcrit when cmpared with rabbits fed a standard diet; hwever, n decreases in the ttal WBC cunt were seen with either the high-chlesterl diet alne r the high-chlesterl diet and etpside (Table 3). The platelet cunt was the same in bth drug-treated and untreated chlesterlfed rabbits (data nt shwn). Etpside treatment did nt change the average lymphcyte cunt but did cause a slight but significant increase in the number f granulcytes in NZW rabbits (Table 3). Since bth the percentage f the WBC ppulatin represented by mncytes as well as the abslute number f WBCs can vary widely in nrmal rabbits, there was cnsiderable verlap in the abslute mncyte cunts f bth cntrl and etpside-treated grups thrughut the experiment. Thus, it was difficult t assess whether the etpside-treated rabbits were cnsistently different in their mncyte cunts thrughut their 8 weeks n the high-chlesterl diet, and a stringent statistical analysis f all the data pints using analysis f cvariance culd nt demnstrate statistical differences between etpside-treated and cntrl animals with either cnventinal r NZW-SPF rabbits (data nt presented). Mrever, the average mncyte cunt during treatment culd nt be statistically crrelated with the extent f plaque measured with Sudan IV staining (r=0.216). Discussin Our experimental results demnstrate that treatment f chlesterl-fed rabbits with the drug etpside causes a statistically significant decrease in the extent f plaque frmed in the artic intima. The decrease in plaque frmatin is independent f the extent f dietinduced hyperlipemia (Figure 1). In bth cntrl and treated animals, plaque was mst frequently fund in the artic arch and at the intercstal stia in the thracic arta. The lesins frmed in the absence f etpside resembled early fibrus plaques, which have been described in rabbits 7-8 and nnhuman pri-

6 1368 Arterisclersis and Thrmbsis Vl 12, N 11 Nvember 1992 Central Cntrl Peripheral Etpside Cntrl Etpside ib& N t t FIGURE 2. Phtmicrgraphs shwing the effect fetpside n the micranatmy f athersclertic plaques. Panels A-D shw sectins stained with the Verheff-van Giesn technique fr elastin in which elastin fibers stain black (arrw indicates fcal elastin accumulatin in panel A). Panels E-H shw sectins stained with the Massn 's trichrme technique, in which cllagen fibers stain blue (arrw indicates fcal cllagen accumulatin in panel E). Panels I-P are immunperxidase assays, in which tissue sectins were reacted with either mnclnal antibdy RAM-II, directed against a rabbit macrphage-specific antigen (panels I-L) r mnclnal antibdy HHF-35, directed against muscle cell specific actin (panels M-P; arrws indicate scattered HHF-35psitive fam cells in panel N). Panels I-P have been cunterstained with hematxylin. Bar represents 100 fim.

7 de la Llera-Mya et al Etpside Suppresses Athersclersis 1369 TABLE 3. Experiment NZW rabbits Cntrl (n = 12) Etpside (n = ll) NZW-SPF rabbits Cntrl (n=9) Etpside (n = ll) Published values Bld Cell Cunts f Cntrl and Etpside-Treated Rabbits RBCs (cunts/1 xlo~ 12 ) WBCs (cunts/1 xlo~ 9 ) Befre After Befre After 5.4± ± ± ( ) 3.9±0.3* 3.4±0.7* 4.1±0.5* 3.5±0.5* 6.8± ± ± ± ( ) 6.7± ± ± ±1.4 Lymphcytes (cunts/1 xlo" 9 ) Befre After 4.3 ± ± ± ± ± ± ± ( ) Granulcytes (cunts/1 xlo" 9 ) Befre After 2.0± ± * 2.0± ± ± ± ( ) RBCs, red bld cells; WBCs, white bld cells; NZW, New Zealand White; SPF, specific pathgen free. Values represent mean±sd f all measurements (see "Methds") befre (average fr all rabbits) and after (average fr rabbits in each grup) the treatment phase f the experiment. Published values are frm Reference 32. *p<0.05. mates, 1119 and which represent a develpmental transitin between the fatty streak and mre advanced lesins. 7 ' In cntrast, the athersclertic lesins in chlesterl-fed rabbits that had been treated with the cytstatic drug etpside were less extensive and thinner and, at least fcally, cntained less fibrus tissue and SMFCs when cmpared with the mre fibrus lesins seen in the untreated cntrl animals. Since numerus studies in animal and human 11 arterial tissues suggest that with time sme fatty streaks develp int fibrus plaques, we prpse that etpside treatment suppresses this cnversin and therefre reduces the prgressin f early athersclertic lesins t mreadvanced plaques. The mechanism by which etpside suppresses plaque prgressin and develpment is nt established, but several bservatins indicate that its actin in this study is nt slely due t a systemic effect f the drug. First, there were n significant differences in ttal plasma chlesterl cncentratins between drugtreated and the cntrl rabbits, and it was shwn that all chlesterl-fed rabbits had circulating )3-VLDL. Mrever, hyperlipemic serum frm drug-treated animals was shwn t induce macrphage fam cell frmatin in vitr. Thus, etpside treatment did nt interfere with the intestinal absrptin f chlesterl r the frmatin f abnrmal, remnant-like lipprteins. Secnd, althugh etpside has been shwn t cause mncytpenia in shrt-term experiments with Chinchilla rabbits, 22 n cnsistent, significant difference in the average abslute mncyte number culd be demnstrated between etpside-treated and untreated rabbits during the 8 weeks the rabbits were n a high-chlesterl diet. Likewise, there was n difference in the ttal RBC, WBC, r platelet cunt between these tw grups. Thus, the reductin in athersclertic plaque develpment seen with etpside treatment des nt appear t be due t a significant drug-induced reductin in the number f circulating mncytes r platelets, cells that have been implicated in athergenesis. 18 Third, the prminent accumulatin f MFCs in the athersclertic lesins f the etpside-treated animals suggests that the drug des nt interfere with either the recruitment f mncytes/macrphages int areas f the vessel wall prne t lesin develpment r the intracellular accumulatin f lipid that leads t the appearance f fam cells. Hwever, the bservatin that bth the extent and thickness f lesins are diminished in etpside-treated animals des suggest that drug treatment results in a quantitative defect in verall plaque frmatin. Mrever, the fatty streaks that frmed in the etpsidetreated animals did nt seem t readily mature t mre advanced fibrus plaques. The results presented here raise the pssibility that etpside affects prcesses that are intrinsic t the plaque. It is thught that the prgressin f athersclertic plaques frm fatty streaks t mre advanced fibrus lesins is clsely related t the cellular cmpnents f these lesins and that the appearance and prliferatin f SMFCs in the vascular intima heralds this transitin Our results indicate that the accumulatin f SMFCs and fibrelastic tissue in plaques is nt slely related t the duratin f expsure f the vessel wall t elevated levels f plasma chlesterl, since bth etpside-treated and cntrl rabbits became hyperlipemic shrtly after the start f the athergenic diet. Hwever, we did find that in cntrl rabbits there was a spatial crrelatin between the accumulatin f SMFCs in plaques and the presence f fibrelastic tissue. This was particularly evident at the periphery f plaques but culd als be seen in ther areas f plaque that cntained accumulatins f SMFCs. Since the ppulatin f SMFCs may influence the cmpsitin f the extracellular matrix in the athersclertic plaque, 2 agents that disrupt the accumulatin f SMFCs culd suppress fibrelastic plaque develpment. In additin, since the appearance f SMFCs in plaques is at least partially dependent n the intimal prliferatin f smth muscle cells 34 and etpside has been shwn t be cytstatic fr a variety f cell types, 23 we speculate that the etpside-mediated suppressin f fibrelastic plaque develpment may be at least partially explained by the inhibitin f smth muscle cell prliferatin in the intima. The cytstatic effect f etpside may als explain the decreased extent f fatty streak frmatin in the treated rabbits, since experimental evidence suggests that areas f active endthelial cell prliferatin are mre permeable t lipprtein and ther bld cmpnents 35 and the accumulatin f these cnstituents is thught t be a precursr f plaque frmatin. Therefre, it is pssible that the frequency f such high-permeability areas is reduced because f the cytstatic effects f etpside. Finally, there is evidence that macrphage prliferatin ccurs at sites f lesins 33 and

8 1370 Arterisclersis and Thrmbsis Vl 12, N 11 Nvember 1992 that this step may be als impaired in etpside-treated rabbits, thereby diminishing the extent f athersclertic lesins. Our results d nt eliminate the pssibility that etpside interferes with athersclertic plaque develpment by mechanisms ther than cytstatic effects. Fr example, there is much evidence t suggest that grwth and chemtactic factrs may be lcally released frm varius cells t act in a paracrine fashin at sites f plaque develpment. 17 It is pssible that etpside culd interfere with these putative mechanisms by inhibiting the maturatin r activatin f mncytes r the activatin f tissue macrphages, s that the mncyte-derived fam cells fund in lesins f drug-treated rabbits lack sme as-yet-undefined paracrine functin critical t the prgressin f plaques. Althugh future studies are needed t identify the mechanism by which etpside inhibits athersclertic lesins, it is clear that this animal mdel ffers an pprtunity t investigate factrs that prmte the initiatin and prgressin f the macrphage-rich fatty streak and t develp therapeutic interventins that retard plaque develpment. Acknwledgments Mr. Vinh Van Nguyen prvided excellent technical assistance. We thank Dr. William Bradner fr prviding etpside, Dr. Allen Gwn fr prviding the RAM-11 antibdy, and Dr. Page Mrahan fr helpful discussins. References 1. Stary HC: Macrphages, macrphage fam cells and eccentric intimal thickening in the crnary arteries f yung children. Athersclersis 1987;64: Rss R, Wight TN, Strandness E, Thule B: Human athersclersis: I. Cell cnstitutin and characteristics f advanced lesins f the superficial femral artery. Am J Pathl 1984;114: Gerrity RG: The rle f mncytes in athergenesis: I. Transitin f bld-brne mncytes int fam cells in fatty lesins. Am J Pathl 1981;103: Jris I, Zand T, Nunnari JJ, Krlikwski FJ, Magn G: Studies n the pathgenesis f athersclersis: I. Adhesin and emigratin f mnnuclear cells in the arta f hyperchlesterlemic rats. Am J Pathl 1983;113: Fwler SD, Mazer EP, Greenspan P: Fam cells and athergenesis. Ann N YAcad Sci 1985;454: Parker F, Odland GF: A crrelative histchemical, bichemical and electrnmicrscpic study f experimental athersclersis in the rabbit arta with special reference t the my-intimal cell./4m 7ftjtfi/1966;48: Rsenfeld ME, Tsukada T, Chait A, Bierman EL, Gwn AM, Rss R: Fatty streak expansin and maturatin in Watanabe heritable hyperlipemic and cmparably hyperchlesterlemic fat-fed rabbits. Arterisclersis 1987;7: Rsenfeld ME, Tsukada T, Gwn AM, Rss R: Fatty streak initiatin in Watanabe heritable hyperlipemic and cmparably hyperchlesterlemic fat-fed rabbits. Arterisclersis 1987;7: Klurfeld DM: Identificatin f fam cells in human athersclertic lesins as macrphages using mnclnal antibdies. Arch Pathl Lab Med 1985;109: Masuda J, Rss R: Athergenesis during lw level hyperchlesterlemia in the nnhuman primate: 1. Fatty streak frmatin. Arterisclersis 1990;10: Gwn AM, Tsukada T, Rss R: Human athersclersis: II. Immuncytchemical analysis f the cellular cmpsitin f human athersclertic lesins. Am J Pathl 1986;125: Tsukada T, Rsenfeld ME, Rss R, Gwn AM: Immuncytchemical analysis f cellular cmpnents in athersclertic lesins. Arterisclersis 1986;6: Watanabe T, Hirata N, Yshikawa Y, Nagafuchi Y, Tyshima H: Rle f macrphages in athersclersis. Lab Invest 1985;53: Siminescu N, Vasile E, Lapu F, Ppescu G, Siminescu M: Prelesinal events in athergenesis. Am J Pathl 1986;123: Schwenke DC, Carew TE: Initiatin f athersclertic lesins in chlesterl-fed rabbits: I. Lcal increases in arterial LDL cncentratin precede develpment f fatty streak lesins. Arterisclersis 1989;9: Brwn MS, Gldstein JL: Lipprtein metablism in the macrphage: Implicatins fr chlesterl depsitin in athersclersis. Annu Rev Bichem 1983;52: Hanssn GK, Jnassn L, Seifert PS, Stemme S: Immune mechanisms in athersclersis. Arterisclersis 1989;9: Rss R: The pathgenesis f athersclersis An update. N Engl J Med 1986;314: Masuda J, Rss R: Athergenesis during lw level hyperchlesterlemia in the nn-human primate: II. Fatty streak cnversin t fibrus plaque. Arterisclersis 1990;10: Pitas RE: Expressin f the acetyl lw density lipprtein receptr in rabbit fibrblasts and smth muscle cells. / Bil Chem 1990; 265: Wlfbauer G, Glick JM, Minr LK, Rthblat GH: Develpment f the smth muscle cell: Uptake f macrphage lipid inclusins. Prc NatlAcad Sci U S A 1986;83: Aarnudse MW, Lamberts HB, Dijk F, Vs J, de Vries AJ: Mncytes and radiatin-induced athermatsis in rabbits. Virchws Arch B 1984;47: Rsencweig M, VnHff DD, Henney JE, Muggia FM: VM26 and VP16-213: A cmparative analysis. Cancer 1977;40: Kritchevsky D, Tepper SA: Chlesterl vehicle in experimental athersclersis: Part II. Peanut il. Athersclersis 1971;14: Mahley RW, Innerarity TL, Brwn MS, H YK, Gldstein JL: Chlesteryl ester synthesis in macrphages: Stimulatin by /3-very lw density lipprteins frm chlesterl-fed animals f several species. J Lipid Res 1980;21: Hlman R, McGill HC, Strng JP, Geer JC: Technics fr studying athersclertic lesins. Lab Invest 1958;7: Mitchell JRA, Cranstn WI: A simple methd fr the quantitative assessment f artic disease. J Atherscler Res 1965;5: Flch JM, Lees M, Slane-Stanley GHA: A simple methd fr the islatin and purificatin f ttal lipids frm animal tissues. J Bil Chem 1957;226: McClskey HM, Rthblat GH, Glick JM: Incubatin f acetylated lw-density lipprtein with chlesterl-rich dispersins enhances chlesterl uptake by macrphages. Bichim Biphys Acta 1987; 921: Ishikawa TT, MacGee J, Mrrisn JA, Glueck CJ: Quantitative analysis f chlesterl in 5 t 20 /a.1 f plasma. J Lipid Res 1974;15: Markwell MAK, Haas SM, Bieber LL, Tlbert NE: A mdificatin f the Lwry prcedure t simplify prtein determinatin in membrane and lipprtein samples. Anal Bichem 1978;87: Sandersn JH, Phillips CE: An Atlas f Labratry Animal Hematlgy, New Yrk, Oxfrd University Press, 1981, pp Lewis JC, Taylr RG, Jerme WG: Fam cell characteristics in crnary arteries and artas f White Carneau pigens with mderate hyperchlesterlemia. Ann N YAcad Sci 1985;454: Rsenfeld ME, Rss R: Macrphages and smth muscle cell prliferatin in athersclertic lesins f WHHL and cmparably hyperchlesterlemic fat-fed rabbits. Arterisclersis 1990; 10: Chuang PT, Cheng H-J, Lin S-J, Jan K-M, Lee MML, Chien S: Macrmlecular transprt acrss arterial and venus endthelium in rats. Arterisclersis 1990; 10: