Lipid Composition and Metabolism of Thromboatherosclerotic Lesions Produced by Continued Endothelial Damage in Normal Rabbits

Transcription

1 Lipid Cmpsitin and Metablism f Thrmbathersclertic Lesins Prduced by Cntinued Endthelial Damage in Rabbits By Allan J. Day, Frank P. Bell, Sean Mre, and Rbert Friedman ABSTRACT Thrmbathersclertic and fibrus lesins were prduced by endthelial damage with plyethylene catheters inserted int the artas in rabbits n a nrmal diet. Tw weeks after insertin f the catheters, the cncentratin f bth free chlesterl and chlesteryl ester in the thrmbathersclertic lesins was significantly greater than that in the adjacent nrmal intima. A further increase in the cncentratin f free chlesterl and particularly f chlesteryl ester ccurred during the remainder f the 4-mnth study perid. Gas-liquid chrmatgraphy indicated that the raised thrmbathersclertic lesins cntained mre chlesteryl leate and less chlesteryl linleate than did either the nrmal intima r the fibrus lesins. The incrpratin f [l- 14 C]leic acid int cmbined lipid in the artas incubated in vitr shwed that, by 2 weeks, tw t three times mre leic acid had been incrprated int chlesteryl ester in the thrmbathersclertic raised lesins than in the nrmal intima. A similar increase was demnstrated at 2 and 4 mnths. When [ 32 P] phsphate was used as a precursr, incrpratin int lecithin was higher and incrpratin int phsphatidyl insitl was lwer in the raised lesins than they were in the nrmal intima. Fibrus lesins did nt differ significantly frm the adjacent nrmal intima in their incrpratin f either f these precursrs int lipid. Therefre, the accumulatin f chlesteryl ester in the thrmbathersclertic lesins resulted in part frm synthesis in the arterial wall, which was stimulated by the prminent platelet cmpnents f the lesins. KEY WORDS athergenesis arterial metablism phsphlipid chlesterl arterial injury platelet thrmbi chlesteryl ester The rle f elevated serum lipids as an etilgical factr in the develpment f athersclertic vascular disease has been studied extensively. In cmparisn, the rles f injury r mural platelet thrmbi as agents primarily respnsible fr lipid accumulatin in the early athersclertic lesin have received less attentin. Mst f the experimental techniques in which injury r thrmbembli are used t prduce experimental athersclersis cause fibrus lesins (1-5), and elevatin f serum lipid levels is nrmally necessary t unmask the effect f injury r thrmbembli n lipid accumulatin in the lesin (3, 6-9). Hand and Chandler (10) have demnstrated, hwever, that autlgus platelet thrmbi injected int Frm the Department f Pathlgy, McMaster University, Hamiltn, Ontari, Canada. This study was supprted by Grants MA 2168 and MT 3067 frm the Medical Research Cuncil f Canada. Dr. Day was a Visiting Prfessr, McMaster University, n study leave during 1972 frm the Department f Physilgy, University f Melburne, Australia. Dr. Bell is a Research Schlar f the Canadian Heart Fundatin. Received Octber 9, Accepted fr publicatin January 10, Circulatin Retearch, Vl. XXXIV. April 1974 nrmchlesterlemic rabbits give rise t lipidcntaining lesins. The suggestin that platelet lipid cntributes directly t the lipid accumulatin f the early athersclertic lesins, hwever, is untenable, because the lipid cmpsitin f bth platelets and rganized platelet thrmbi differs markedly frm that f the athersclertic lesin (11, 12). Mre prbably, platelet uptake by arterial wall cells (macrphages and smth muscle cells) is assciated with stimulated lipid synthesis in the resulting lesin. Lipid-cntaining athersclertic lesins have recently been prduced in rabbits and rats n nrmal diets fllwing acute transverse injury t the artic endthelium (13,14). Thrmbtic lesins prduced in situ by chrnic endthelial damage with plyethylene catheters implanted in the arta f rabbits n nrmal diets have als been shwn t develp int lesins resembling fatty streak (15) and fibrfatty (15, 16) lesins in man. The mrphlgy f these lesins has recently been reprted (15), and histchemical evidence has been btained that agrees with the cnclusin that stimulatin f lipid synthesis by smth muscle cells and macrphages accunts fr sme f the accumulated lipid. The 467

2 468 DAY, BELL, MOORE, FRIEDMAN suggestin that the lipid accumulatin in these lesins prduced by catheter-induced injury is assciated with the stimulatin, pssibly by the platelet thrmbi which are frmed, f lipid synthesis in the smth muscle cells was investigated in the present paper. The lipid cmpsitin and the lipid synthesis f different lesins evlving frm platelet thrmbi r small fibrus lesins prduced by cntinued endthelial damage in rabbits n nrmal diets are als presented. Methds New Zealand white rabbits ( kg) were fed Purina rabbit chw thrughut the experiment. Plyethylene catheters (1.2 mm,.d.) were inserted int the arta under sdium pentbarbital anesthesia thrugh an incisin in the femral artery. The catheters were passed int the arta t apprximately the midthracic regin and sutured in place by a stay stitch arund the femral artery. After 2 weeks, 2 mnths, r 4 mnths, the rabbits were killed with sdium pentbarbital, and the whle arta frm the heart t the iliac bifurcatin was remved with the catheter still in situ. Adventitial cnnective tissue was remved, and the arta was pened lngitudinally t display the lesins prduced. Tw types f lesins (fibrus and raised) were present. The fibrus lesins were flat and pearly in appearance crrespnding t similar lesins described by Mre (15). These lesins were present at all f the time intervals studied, althugh at 2 weeks their presence was limited. Raised lesins were pendulus and, in sme cases, appeared as large rganized mural thrmbi n the endthelium. In ther cases, they were invasive and enclsed the catheter. These raised lesins crrespnded t the raised and tunnel lesins described by Mre (15). Macrscpically nrmal intima between the lesins and its subjacent media were used fr cmparisn. The tw types f lesins and the nrmal intima were separately dissected, tgether with the underlying media] tissue in each case. Careful dissectin with cilia frceps permitted the separatin f the intima-media segments crrespnding t the tw types f lesins and the nrmal intima-media. Full details f the mrphlgy f the tw types f lesins studied have been reprted previusly (15). The fibrus lesins did nt stain fr lipid; they were nrmally cvered with endthelium and cntained appreciable numbers f smth muscle cells. The raised lesins cntained platelet fibrin thrmbi and shwed lipid staining as early as 2 days after insertin f the catheters. Mst f this lipid was present intracellularly in macrphages and fam cells. Sme f these lipphage cells shwed smth muscle cell characteristics by electrn micrscpy; fat was prminent in medial smth muscle cells at 2 weeks. At later time intervals, extracellular lipid became mre prminent with fam cells surrunding the lipid pl. CHEMICAL ANALYTICAL STUDIES Because the tissues cntained relatively small amunts f lipid, it was necessary t pl material frm 3-5 rabbits fr examinatin in batches. Twelve rabbits (three batches) were examined at 2 weeks, 19 r, fr sme analyses, 21 rabbits (fur r six batches) were studied at 2 mnths, and 6 rabbits (tw batches) were studied at 4 mnths. The cmbined nrmal intima, fibrus lesins, r raised lesins in the batches at each time interval were hmgenized and extracted with a chlrfrm-methanl slutin (2:1 v/v), and the resulting lipid extract was washed as described by Flch et al. (17). The prtein precipitate present after extractin with the chlrfrm-methanl slutin was separated fr determinatin f dry defatted weight and DNA cntent (18). The lipid extract was reserved fr determinatin f its free chlesterl, chlesteryl ester, and lipid phsphrus cntent. Because f the small quantity f chlesteryl ester present in the extracts, the fllwing prcedure, which is essentially the prcedure described by Day and Wahlqvist (19), was adpted. An internal standard cntaining a knwn amunt f chlesteryl heptadecanate and a knwn tracer amunt f 14 C-labeled chlesterl was added t the lipid extract. The chlesteryl heptadecanate prvided a basis fr quantificatin f the chlesteryl esters which were measured as methyl esters by gas-liquid chrmatgraphy fllwing initial separatin f the chlesteryl esters by thin-layer chrmatgraphy. Recvery f free chlesterl was mnitred by the labeled chlesterl. N internal standard fr phsphlipid was added. After the additin f the internal standard, the chlesterl, phsphlipid, and chlesteryl esters in the lipid extract were separated by thin-layer chrmatgraphy n silica gel G using a N-hexane-diethyl ether-acetic acid slvent system (146:50:4 v/v/v). Spts were visualized by spraying with 0.2% dichlrflurescein. Chlesteryl ester was scraped directly int ampules and methyl-esterified with 5% H 2 SO 4 in methanl; its fatty acid cmpsitin and cntent were determined by gasliquid chrmatgraphy as described in the fllwing sectin. Free chlesterl was eluted with chlrfrm; a sample was cunted t mnitr recvery, and anther sample was assayed by gas-liquid chrmatgraphy. Phsphlipid was eluted by the methd f Arvidsn (20); a sample f the eluate was used fr determinatin f lipid phsphrus (21) and anther sample was methyl-esterified with 5% H 2 SO 4 in methanl fr determinatin f its fatty acid cmpsitin by gas-liquid chrmatgraphy. GAS-LIOUID CHROMATOQRAPHY Methyl esters were separated n 15% diethyleneglycl-succinate clumns using a gas chrmatgraph (Hewlett Packard 402) at a clumn temperature f 170 C. Separatin f standards KA and KD (Applied Science Labratries) shwed a percent cmpsitin cmparisn with an errr f less than 10% fr minr cmp- Circulatin Research, Vl. XXXIV, April 1974

3 AORTIC LIPID ACCUMULATION IN THROMBOATHEROSCLEROSIS 469 nents (less than 5% f the mixture) and an errr f less than 5% fr majr cmpnents (mre than 10% f the mixture). Calculatin f the mass f individual fatty acids was carried ut using retentin time by peak height as a basis. METABOLIC STUDIES At each time interval (2 weeks, 2 mnths, and 4 mnths), fur r five rabbits were used fr metablic study. They were identical in all respects t the rabbits used fr the crrespnding chemical study except that data were btained with single rabbits rather than by pling. After the artas had been remved and the adventitial cnnective tissue dissected away, the artas were incubated fr 4 hurs at 37 C in 10 ml f incubatin medium cntaining a slutin f Media 199 and nrmal rabbit serum t which had been added apprximately 5 /ic f [l- 14 C]leic (59 mc/mm (New England Nuclear) and apprximately 100 /tc f [ 32 P] phsphate (Carrier Free New England Nuclear). Streptmycin and penicillin (50 //.g and 50 units/ml, respectively) were als added t the incubatin medium. After incubatin, the artas were remved and washed thrughly in 0.9% sdium chlride slutin; the nrmal intima and the fibrus and raised lesins were separated and extracted separately with a chlrfrm-methanl slutin as previusly described. Extracts were washed as described by Flch et al. (17), and the ttal chlesterl cntent and the cunts/min f bth 14 C and 32 P were determined. A sample f the lipid extract was then separated int phsphlipid, chlesterl, fatty acid, triglyceride, and chlesteryl ester by thin-layer chrmatgraphy n silica gel G using a N-hexane-diethyl etheracetic acid slvent system (146:50:4 v/v/v). Individual lipid spts were identified by spraying with 0.2% dichlrflurescein and cunted directly fllwing scraping int cunting vials using the dixane-water scintillatr described by Snyder (22). The incrpratin f bth [l- 14 C]leic acid and [ 32 P]phsphate int individual phshlipids was fllwed by separatin f the lipid extract int individual phsphlipids by thin-layer chrmatgraphy using the methd f Skipski et al. (23). Phsphlipid spts were identified with 0.2% dichlrflurescein and cunted directly with the dixane-water scintillatr (22) after they had been scraped int cunting vials. Cunting was dne with a liquid scintillatin cunter and duble-labeled techniques t separate 14 C and 32 P. Quenching and 32 P decay were mnitred and crrected fr where necessary. All data were expressed as cunts per minute f the respective lipid I4 C r 3Z P incrprated per micrgram f DNA in the respective prtin f the artery nrmal intima, fibrus lesin, r raised lesin. CHEMICAL ANALYSIS Lipid phsphrus was determined by the methd f Bartlett (21). Chlesterl was determined in samples f the lipid extracts r in the eluates by the autmated pr- Circulatin Reiearch, Vl. XXXIV, April 1974 cedure f Blck et al. (24) using an autanalyzer (Technicn). DNA was determined by the methd f Dische (18). STATISTICAL ANALYSIS Analyses f variance were carried ut n bth analytical and metablic data frm the three grupsnrmal intima, fibrus lesins, and raised lesins at each time interval. Statistical significance was calculated by Student's t-test. Results The cncentratin f lipids in the nrmal arta and in the fibrus and raised lesins at the three time intervals studied is shwn in Table 1. The free chlesterl cncentratin f the fibrus lesin was nly marginally increased ver that f the nrmal intima. The free chlesterl cntent f the raised lesin, hwever, was cnsiderably increased ver bth the crrespnding nrmal intima and the crrespnding fibrus lesin (P < 0.01 at bth 2 weeks and 2 mnths). Chlesteryl ester shwed the mst marked increase f all the lipids in the raised lesins. At 2 weeks, 114 /u,g chlesterl ester/mg DNA was present cmpared with 17 fig/mg DNA in the adjacent nrmal intima. The chlesteryl ester cncentratin f the raised lesins rse t 538 ig/mg DNA and 597 fig/mg DNA at 2 mnths and 4 mnths, respectively. These values were significantly different cmpared with the chlesteryl ester cncentratin f either nrmal intima r fibrus lesins (P<0.01. The marked rise in the chlesteryl ester cntent in the raised lesins accunted fr an elevatin in the percent f chlesteryl ester in the arta frm 2.8% in the nrmal intima t 8.3, 24.5, and 28.6% in the raised lesin at 2 weeks, 2 mnths, and 4 mnths, respectively. Changes in the chlesteryl ester cntent f the fibrus lesins were much smaller; the levels did nt differ significantly frm thse in the adjacent nrmal intima. The increased amunt f chlesteryl ester present in the nrmal intima at 2 mnths (64.5 (ig/mg DNA) requires sme explanatin. The amunt f chlesteryl ester present in the nrmal artery is extremely small; therefre, small amunts f chlesteryl ester culd accunt fr marked changes in chlesteryl ester cncentratin. It is pssible that, althugh the apparently nrmal artery studied was macrscpically free f fatty depsits, micrscpic fat-cntaining lesins culd have been present. Mre (15) has reprted that flat fatty lesins are seen micrscpically in similar material cnsidered nrmal by grss examinatin. Despite

4 470 DAY, BELL, MOORE, FRIEDMAN TABLE 1 Lipid Cntent f Arta and Fibrus and Raised Lesins at Varius Perids after Insertin f Plyethylene Catheters 2 Weeks 2 Mnths 4i Mnths Fibrus Raised Fibrus Raised FlbrusRalsd Free chlesterl (/ig/mg DNA) Chlesteryl ester Wmg DNA) % Ester Lipid phsphrus (Mg/mg DNA) 577±31 C 17-b 2.8* b 78' 773 ±118 b 31 ± 8 d 3.8± 0.8 d 73 ± ±28 c>b 114 ±13 W 8.3 ± 0.9 w 56 ± ±91 b 64.5±23.1 C 7.8± 2.2 -" 112 ± 3.5* 973 ±193 d 201 ± 71 b 16.2± 2.3* 114 ± 3.0* 1664 ±108 w 538 ± 53 c - b 24.5± 1.2 c -» 91 ± 8.7*- e All values are means ± SE. The data were btained after 2 weeks frm three batches (12 rabbits), after 2 mnths frm fur batches (19 rabbits), and after 4 mnths frm tw batches (6 rabbits). Numbers in the same rw with the same superscripts are significantly different at the fllwing levels: a-a P< 0.05, b-b P<0.01, c-c P< 0.001, d-d P< 0.01, and e-e P<0.05. The significance f difference was determined by Student's t-test 'Data were btained frm tw batches (eight rabbits). tdata were btained frm three batches. this relatively high cntent f chlesteryl ester in the macrscpically nrmal intima at 2 mnths, the raised lesins frm the same artas shwed cnsiderably higher levels f chlesteryl ester. The lipid phsphrus cncentratin f the raised lesin was slightly less than that f the crrespnding nrmal intima r fibrus lesin, but this difference was nly statistically significant at 2 mnths (P<0.05). The fatty acid cmpsitin f the chlesteryl esters present in the nrmal intima and in the fibrus and raised lesins is shwn in Table 2. In view f the small amunt f material present, data was nt available fr the fibrus lesins at 2 weeks. The prprtin f chlesteryl leate had increased in the raised lesin at 2 weeks cmpared with that in the nrmal intima, and this effect became marked by 2 mnths when chlesteryl leate had increased and chlesteryl linleate had decreased in relatin t their values in the nrmal intima. Hwever, the chlesteryl ester fatty acid cmpsitin f the fibrus lesins at 2 mnths was almst identical with that f the nrmal intima. This change frm chlesteryl linleate t chlesteryl leate in the raised lesin is demnstrated by the 18:1/18:2 rati at 2 mnths which changed frm 1.57 and 1.56 in the nrmal intima and the fibrus lesins, respectively, t 2.51 in the raised lesin. Only tw batches were studied at 4 mnths and an even further elevatin in the prprtin f TABLE 2 Per cent Distributin f Chlesteryl Etter Fatty Acids f Arta and Fibrus and Raised lesins Fatty acid 16:0 (Palmitic) 16:1 (Palmitlic) 18:0 (Stearic) 18:1 (Oleic) 18:2 (Linleic) 20:4 (Arachidnic) 18:1/18:2 * Weeks Raised t 16.8 ± ± ± ± ± ± ±0.3 * ±0.9 ± 1.5* ± 0.2 ± 1.1 ± 1.9* ± 1.8 ±0.09 c 21Mnths Fibrus* ±0.5 ±0.2 b ±0.5 ±0.4 d ±2.0 e ±1.8 i ±0.11 b Raised 15.9 ± ± 0.1*- b 5.6 ± ± LI 0 -" ± 0.9" 6.7 ± ± 0.16*^ Fibrus Mnths Raised Numbers in the same rw with the same superscripts are significantly different at the fllwing levels: a-a P< 0.05, b-b P< 0.01, c-( P< 0.001, d-d P< 0.001, and e-e P<0.05. 'Data are means f tw batches (eight rabbits). fdata are means ± SE f three batches (12 rabbits). tdata are means ± SE f fur batches (19 rabbits). Data are means ± SEf six batches (21 rabbits). I Data are means f tw batches (six rabbits). Curuliin RatanK Vl. XXXIV, April 1974

5 AORTIC LIPID ACCUMULATION IN THROMBOATHEROSCLEROSIS 471 TABLE 3 Percent Distributin f Phsphlipid Fatty Acids f Arta and Fibrus and Raised Lesins Fatty add " 2 Weeks Raisedt 2 Mnths* Fibrus Raised 4 Mnths' Fibrus Raised 16:0(Palmitic) :0(Stearic) :l(Oleic) :2(Linleic) :4(Arachidnic) ± ± ± ± ± ± 0.3" 19.8 ± ± ± ± 0.2^ 19.2 ± ' 19.8 ± ± ± ± 0.9 d 33.9 ± 4.4 ab 23.4 ± ± ± ± 3.0 cd Numbers, in the same rw with the same superscripts are significantly different at the fllwing levels: a-a P< 0.01, b-b P< 0.01, c-c P< 0.001, and d-d P< "Data are means f tw batches (eight rabbits). fdata are means ± SEf three batches (12 rabbits). tdata are means ± SE f fur batches (19 rabbits). chlesteryl leate and a further reductin in chlesteryl linleate were present; therefre, the 18:1/18:2 rati had altered frm 1.71 in the nrmal intima (similar t that at 2 mnths) t 3.81 in the raised lesins. The phsphlipid fatty acid cmpsitin f the nrmal arta and f the tw types f lesins is shwn in Table 3. Again, data fr the fibrus lesin was nt available at 2 weeks because f the paucity f material at this time. The phsphlipid fatty acid cmpsitin f the fibrus lesin at the tw time intervals studied was almst identical t that f the nrmal intima. The phsphlipid fatty acid cmpsitin f the raised lesin, hwever, markedly cntrasted with that f the nrmal intima; elevated palmitic (16:0) and reduced arachidnic (20:4) acid were bserved at all time intervals. This difference was clearly established in the rabbits studied after 2 weeks and persisted essentially unchanged t 4 mnths. METABOLIC EXPERIMENTS The incrpratin f [l- 14 C]leic acid int ttal lipid, phsphlipid, triglyceride, and chlesteryl ester in the nrmal arta and in the fibrus (2 and 4 mnths) and raised lesins is shwn in Table 4. Since different incubatin media were used at different time intervals, cmparisn between similar grups frm different time intervals cannt be made. Cmparisn within each time interval, hwever, is valid, since identical incubatin media were used fr each f the artas, and, f curse, the tw lesins and the nrmal intima were incubated in the same incubatin media prir t their dissectin. The figures fr the raised lesin at 2 weeks include Circulatin Research, Vl. XXXIV, April 1974 data frm ne aberrant rabbit that shwed very high incrpratin figures in relatin t the cntent f DNA. The variatin indicated by the standard errrs fr these figures was extremely high, and the crrespnding means and standard errrs fr the three remaining rabbits are given in parentheses tgether with the ttal figures. The marked elevatin in chlesterl cncentratin f the raised lesins which was demnstrated in the analytical studies is als shwn in the metablic series. The ttal chlesterl cncentratin f the raised lesins rse t 2.95 fig/fig DNA and 2.57 fig/fig DNA at 2 mnths and 4 mnths, respectively. The incrpratin f leic acid int lipid was lwer in the raised lesins than it was in the crrespnding nrmal lesins at bth 2 weeks and 2 mnths (Table 4). This finding may reflect the larger amunt f inert thrmbus present in the raised lesins. Incrpratin int lipid in the nrmal intima and the fibrus and raised lesins was similar, hwever, by 4 mnths. The relative incrpratin f [l- M C]leic acid int phsphlipid, triglyceride, and chlesteryl ester differed, hwever, in the raised and the fibrus lesins cmpared with the nrmal arta. At each f the time intervals (2 weeks, 2 mnths, and 4 mnths), there was appreciable diversin f label t chlesteryl ester in the raised lesin. This feature can be seen by bserving the incrpratin f leic acid int chlesteryl ester in relatin t incrpratin int phsphlipid (Table 4). The rati f chlesteryl ester t phsphlipid incrpratin was significantly higher fr the raised lesin cmpared with that fr the crrespnding nrmal intima at 2 weeks (P< 0.001), at 2 mnths (P<0.0l), and at 4

6 2 Weeks 2 Mnths 4 Mnths ± ± ± ± ±0.035" 2.57±0.61 ac Raised Fibrus Raised Fibrus Raised > m > a m Z m TABLE 4 Incrpratin f [l - I4 C] Oleic Acid int Cmbined Lipid in Arta and Fibrus and Raised Lesins (cunts/min incrprated!ng DNA) M t 2 Weeks Raised 2 Mnths 4 Mnths Fibrus Raised Fibrus Raised Ttal lipid Phsphlipid Triglyceride Chlesteryl ester Rati f chlesteryl ester t phsphlipid Ttal chlesterl cncentratin DNA) 89.3 ± ±5.3 C 28.5 ± ± ±0.005 e 89.3 ± ± ± ± ±0.01 l e ± ± (0.952) (56.1 ±11.7) (16.5±4.4) c (16.7 ±2.4) (4.4 ±1.3) 228 ± ±13.4" 69.2± ± ±0.030 c 287 ± ±28.4 c 100± ± ±0.026 d 140 ± ±6.0 ac 43.2 ± ± ±0.008 cd 89.0 ± ± ± ± ±0.009 a 103.5± ± ± ± ± ±0.128 e 1.34±0.184 f 2.95±0.329 ef 0.873±0.184 a 0.796±0.049 c The data btained after 2 weeks and 4 mnths were frm fur rabbits (means ± SE). Numbers in parentheses are means ± SE fr three rabbits. The data btained after 2 mnths were frm five rabbits. Numbers in the same rw with the same superscripts are significantly different at the fllwing levels: a-a P< 0.05, c-c P < 0.01, d-d P < 0.01, e-e P < 0.001, and f-fp< TABLE 5 Incrpratin f[l - I4 C] Oleic Acid int Individual Phsphlipids in Arta and Fibrus and Raised Lesins (cunts/min incrprated/tig DNA Sphingmyelin Lecithin Phsphatidyl insitl Phsphatidyl ethanlamine 0.29 ± 0.59 a 0.18 ± 0.08 (0.10 ± 0.01) a 2.06 ± 0.59 cd 0.44 ± ± 0.06 d 31.7 ± 3.50" 23.1 ± 10.9 (12.5 ± 3.5)" 79.6 ± 9.3 a 90.6 ± 18.2 b 32.5 ± 4.1 ab 8.3 ±0.9 c 3.6 ± 1.75 (1.86 ± 0.37) c 13.1 ± 2.3 a 21.7 ± 6.3 b 5.14 ± 0.75 al> 7.8 ±0.92 c 3.7 ± 1.8 (2.0 ± 0.5) c 16.8 ± 2.1 a 21.1 ± ± 1.19 a - c ± ± ± ± ± ± ± ± ± ± ± ± 0.60 The data btained after 2 weeks and 4 mnths were frm fur rabbits (means ± SE). The numbers in parentheses are means ± SE f three rabbits. The data btained after 2 mnths were frm five rabbits. Numbers in the same rw with the same superscripts are significantly different at the fllwing levels: a-a P< 0.05, b-b P< 0.05, c-c P< 0.01, and d-d P< 0.01.

7 AORTIC LIPID ACCUMULATION IN THROMBOATHEROSCLEROSIS 473 I60 I c <x KJ M q N t ' O5 <D O M -H -H M -H H d h Ol h d w _: eri f-. IS CM CO QQ n uj O h c d i -H -H -H -H -H 00 <N O5 F- 00 ai * d H ^ t~- CO T (N t- <M t c «-i 1 -! CM O I 00 -H -H -H -H -H +i O 00 O5 -H O ri ^ ID 6! > H n m «y ««cj in -^ c r-: «? -w «OONHH +H +1 -H -H -H e»» OON c in d ^ 2 i CO N a "^* c c d -H -H -H -H -H ^! O en CO IO H 1^ lo c -! in t^ "? -* H -H (N I H -H -H -H -H CSI * t *** CO n K: in in-; looi'mo -H -H -H -H -H OS «J CO O s O> -H i c c c -H -H +t -H +1 (D O» 1/5 tp i i 66 i CD -H OS 00 ss CO u in T "* i-: -! CM a> < -H -H -H -H -H -H t ^* c h<>i in i in i 73 'a 5 c Si) -D - g d H O5 IO d ḏ H s p ḏ H 10 d t 00 O5 d -H <D O5 O H s 0 CSI g O T JZ IK = a ^ '" a a» -a a ȯ6 ' 0 a C - «O 0) > E.5. t j M?! «2 S-8 S 5 g S -S -Hi a 2.SP mnths (P<0.05). There was n significant difference, hwever, in the rati f chlesteryl ester t phsphlipid between the fibrus lesin and the nrmal intima at the tw time intervals studied. Sme cmment is necessary regarding the difference between the incrpratin int chlesteryl ester and phsphlipid in the nrmal intima at 2 mnths cmpared with that at 2 weeks and 4 mnths. As was indicated fr the crrespnding analytical data, the amunt f chlesteryl ester present in the nrmal intima at 2 mnths was higher than that present at either 2 weeks r 4 mnths. This higher amunt was reflected in the higher relative incrpratin int chlesteryl ester in the nrmal intima at 2 mnths cmpared with that at 2 weeks and 4 mnths and might have been due t sme micrscpic lesin present in the macrscpically nrmal intima at this stage. Hwever, the increased relative incrpratin int chlesteryl ester in the raised lesin was still marked at each f the time intervals. The incrpratin f [l- 14 C]leic acid int individual phsphlipids in the three prtins f the artas is shwn in Table 5. When ne takes int accunt the reduced amunt incrprated int ttal phsphlipid in the raised lesins at 2 weeks and 2 mnths (Table 4), it is apparent that there was n marked change in the relative incrpratin f leic acid int the individual phsphlipids in either the raised r the fibrus lesins frm that incrprated in the nrmal arta. The pssible exceptin is the incrpratin int sphingmyelin: a marked reductin in [l- 14 C]leic acid incrpratin was a feature f the raised lesin at all time intervals. Hwever, incrpratin int sphingmyelin was very lw in all f the areas studied and at each time interval. The incrpratin f [ 32 P] phsphate int phsphlipids by the different prtins f the arta is shwn in Table 6. The reduced incrpratin int phsphlipid in the raised lesin frm the rabbits studied at 2 weeks and 2 mnths seen in the [l- 14 C]leic acid incrpratin studies was als demnstrated fr the 32 P incrpratin. In the nrmal intima the tw majr phsphlipids were lecithin and phsphatidyl insitl, and, at bth 2 weeks and 4 mnths, incrpratin int phsphatidyl insitl exceeded that f lecithin; therefre, the rati f phsphatidyl insitl t lecithin incrpratin was 2.21 and 1.58 at 2 weeks and 4 mnths, respectively. At 2 mnths, the majr phsphlipids labeled in the Circulatin Research, VL XXXIV. April 1974

8 474 DAY, BELL, MOORE, FRIEDMAN macrscpically nrmal intima were phsphatidyl insitl and lecithin, but they were present in apprximately equal amunts with a rati f phsphatidyl insitl t lecithin incrpratin f Data fr the fibrus lesin at 2 mnths and 4 mnths were essentially the same as thse fr the nrmal intima with marked incrpratin int phsphatidyl insitl and with ratis f phsphatidyl insitl t lecithin incrpratin similar t thse fr the crrespnding nrmal intima. The incrpratin f [ 32 P] phsphate int phsphatidyl insitl in the raised lesin, hwever, was appreciably less than that fr the crrespnding nrmal intima r fibrus lesin. Althugh labeling f this cmpnent was appreciable, that f lecithin was much higher and accunted fr abut tw-thirds f the [ 32 P] phsphate incrprated int phsphlipid in the raised lesin. The reduced incrpratin int phsphatidyl insitl is indicated by the ratis f phsphatidyl insitl t lecithin incrpratin given in Table 6. At each time interval, this rati was significantly less than that fr the crrespnding nrmal intima. Discussin The cncentratin f bth free chlesterl and chlesteryl ester increased appreciably in the raised lesin cmpared with that in the crrespnding nrmal intima at each f the time intervals studied. The relative increase in the cncentratin f chlesteryl ester, hwever, far exceeded that f free chlesterl. Tw weeks after insertin f the catheters, chlesteryl ester cncentratin had increased t six t seven times that f the crrespnding nrmal intima, and by 4 mnths it had risen t ver thirty times that f the nrmal intima; at this time the chlesteryl ester accunted fr almst 29% f the ttal chlesterl present. The chlesteryl esters that accumulated in the raised lesins cntained mre chlesteryl leate and less chlesteryl linleate than did thse in the crrespnding nrmal intima. Bndjers and Bjrkerud (25) have als reprted the accumulatin f chlesteryl leate in lesins develped in rabbits n a nrmal diet fllwing mechanical trauma. The accumulatn f chlesteryl leate within lipphage cells in human fatty steak lesins is als well recgnized (26). In the lesins prduced in this study, the prgressive elevatin f chlesteryl leate suggests that preferential esterificatin f chlesterl with leic acid ccurs within the lipid-cntaining cells f the raised lesin. The different cmpsitin f the small amunt f chlesteryl esters in-the nrmal intima cmpared with that f chlesteryl esters accumulating in the raised lesin supprts this pssibility. Entry and retentin f chlesteryl esters frm the plasma cannt, therefre, explain the cmpsitinal changes. In the nrmal intima, little chlesterl esterificatin ccurs, and levels f chlesterl-esterifying enzymes are lw (27, 28). Hwever, chlesterl-esterifying activity is stimulated in the athersclertic lesin (27-29). In chlesterl-fed animals, this stimulatin is related t increased entry f chlesterl frm the bld. Other etilgical agents, hwever, may als stimulate chlesterl-esterifying enzymes. In the present experiments, serum lipid levels were lw, but ther etilgical factrs, namely endthelial injury and platelet mural thrmbi were present. The metablic studies perfrmed in the present experiments suggest that these agents are assciated with the stimulatin f chlesterl esterificatin in the arterial wall. The raised lesins had a higher capacity t incrprate fatty acid int chlesteryl ester than did the crrespnding nrmal intima. Direct cnfirmatin f increased chlesterl-esterifying enzyme activity in cell-free preparatins f raised lesins is precluded, hwever, by the relatively small amunt f material available. N infrmatin abut the rigin f the free chlesterl r f the chlesterl cmpnent f the chlesteryl ester in the lesins studied is available frm the present wrk. It is pssible that this chlesterl came frm the plasma in the rabbits n nrmal diets, althugh their plasma chlesterl levels were arund 50 mg/100 ml. The rigin f this chlesterl, hwever, needs t be investigated further. Mrphlgical details f the lesins prduced in the present study have been reprted previusly (15). At the early stage f develpment f the raised lesin, platelet and fibrin thrmbi were prminent and much f the lipid was present intracellularly in the macrphages and fam cells. By 2 weeks, significant numbers f smth muscle cells had als appeared. By 2 mnths and 4 mnths, a certain amunt f the lipid was extracellular but, even at these time intervals, cellular infiltratin was marked; fam cells and smth muscle cells were prminent. There is abundant evidence fr the active invlvement f fam cells and smth muscle cells in lipid synthesis in the develping athersclertic lesin (28, 30-32); it appears likely that, in the lesins described in the present paper, these cells with their cntained chlesterlesterifying enzymes (33) are the direct surce fr Circulatin Retearch, Vl. XXXIV, April 1974

9 AORTIC LIPID ACCUMULATION IN THROMBOATHEROSCLEROSIS 475 the chlesteryl ester which accumulates in the lesin. Direct evidence is nt available at present t suggest that the develpment f these cells r their chlesterl-esterifying enzyme activity is directly stimulated by injury r by platelet thrmbi, but the evidence frm the present wrk makes such a cnclusin prbable. The ttal phsphlipid cncentratin in the raised lesin was similar t that in the nrmal intima. The fatty acid cmpsitin f the phsphlipid, hwever, was markedly different in the raised lesin frm that in the nrmal intima because the palmitic acid cntent was higher and the arachidnic acid cntent was lwer than they were in the crrespnding nrmal intima at each f the time intervals studied. The incrpratin f [ 32 P] phsphate int phsphlipid by the raised lesin als differed frm that f the nrmal intima. Relatively less [ 32 P] phsphate was incrprated int phsphatidyl insitl and mre was incrprated int phsphatidyl chline in the raised lesin than in the nrmal intima. The high relative incrpratin f [ 32 P] phsphate int phsphatidyl insitl in the nrmal intima has been previusly bserved in bth rabbit and prcine artas (34, 35). Als, with the develpment f the athersclertic lesin in the chlesterl-fed rabbit, a shift frm phsphatidyl insitl t phsphatidyl chline synthesis ccurs (34), and this shift persists in cells prepared in tissue culture frm artas f chlesterlfed rabbits (36). The significance f the changing pattern f phsphlipid synthesis in the develping athersclertic lesin is nt clear. Hwever, the similarity f the raised lesins described in the present paper t this characteristic is striking and, therefre, this raised lesin resembles metablically that f the athersclertic lesin in the chlesterl-fed rabbit. The marked changes in chemical cmpsitin and in metablism f the raised lesin cntrasted with the cmpsitin and metablism f the fibrus lesins. The fibrus lesins did, hwever, shw sme chlesteryl ester accumulatin after 2 mnths. This bservatin agrees with the mrphlgical findings previusly described (15). As previusly mentined, the apparently nrmal intima at 2 mnths als shwed an elevated chlesteryl ester cncentratin cmpared with that f the nrmal intima at 2 weeks and 4 mnths. Mre (15) has indicated that at 2 mnths micrscpic lesins similar t thse present in the fibrus lesins may be present in macrscpically nrmal intima, and their presence may well ac- Circulatin Reuarch, VL XXXIV, April 1974 cunt fr sme f the anmalus findings reprted fr the nrmal intima at 2 mnths with respect t its cmpsitin and metablism. Althugh there was evidence fr sme accumulatin f chlesteryl ester in the fibrus lesins, its fatty acid cmpsitin was essentially the same as that f the crrespnding nrmal intima, and the incrpratin f [l- l4 C]leic acid int chlesteryl ester and f [ 32 P] phsphate int phsphlipid was similar at 2 mnths when maximal chlesteryl ester accumulatin ccurred in this lesin. N evidence fr the accumulatin f this lipid by metablism in the fibrus lesin is frthcming. Acknwledgment The technical assistance f Mr. Brian Blmfield, Mrs. Bnnie Wegerich, and Mrs. Gwen Cuture is gratefully acknwledged. We are als indebted t Mr. Timthy Day fr assistance with cmputer prgraming in calculatin f data. References 1. HARRISON, C.V.: Experimental pulmnary arterisclersis. J Pathl 60: , HEARD. B.E.: Experimental study f thickening f the pulmnary arteries f rabbits prduced by the rganizatin ffibrin.j Pathl 64:13-19, HEPTINSTALL, R.H.: Effects f high bld chlesterl n the pulmnary arterial changes prduced in rabbits by the injectin f bld clt. Br J Exp Pathl 38: , J0RCENSEN, L., ROWSELL, H.C., HOVlG, T., AND MUSTARD, J.F.: Reslutin and rganizatin f platelet-rich thrmbi in cartid arteries f swine. Am J Pathl 51: , WOOLF, N., BRADLEY, J.W.P., CRAWFORD. T., AND CARSTAIRS, K.C.: Experimental mural thrmbi in the pig arta: Early natural histry. Br J Exp Pathl 49: , THOMAS. W.A., O'NEAL, R.M., AND LEE, K.T.: Thrmbemblism, pulmnary arterisclersis, and fatty meals. Arch Pathl 61: , FRIEDMAN. M., AND BYERS, S.O.: Experimental thrmbathersclersis. J Clin Invest 40: , BYERS. S.O., AND FRIEDMAN, M.: Lipidizatin induced by bne and glass fragments within an arterial plaque. Br J Exp Pathl 47: , PRIOR, J. T., AND HARTMANN, W.H.: Effect f hyperchlesterlemia upn intimal repair f the arta f the rabbit fllwing experimental trauma. Am J Pathl 32: , HAND. R.A., AND CHANDLER, A.B.: Athersclerticmetamrphsis f autlgus pulmnary thrmbembli in the rabbit. Am J Pathl 40: , SMITH. E.B.: Quantitative and qualitative cmparisn f the lipids in platelets, artic intima, and mural thrmbi. Cardivasc Res 1: ,1967.

10 476 DAY, BELL, MOORE, FRIEDMAN 12. CRAIC, I.H., BELL, F.P., AND SCHWARTZ, C.J.: Thrmbsis 25. and athersclersis: Organizatin f pulmnary thrmbembli in the pig: Individual phsphlipids, fatty acid cmpsitin f lecithin, sphingmyelin, esterified chlesterl and 3 H-chlesterl specific activity. Exp Ml Pathl 18: , BJORKERUD, S.: Athersclersis initiated by mechanical trauma in nrmlipidemic rabbits. J Athersclersis Res 9: , BONDJERS, G., AND BJORNHEDEN, T: Experimental 27. athersclersis induced by mechanical trauma in rats. Athersclersis 12: , MOORE, S.: Thrmb-athersclersis in nrmlipemic rab- 28. bits: Result f cntinued endthelial damage. Lab Invest 29: , SUMIYOSHI, A., MORE, R.H. AND WEIGENSBERG, B.I.: Artic 29. fibrfatty type athersclersis frm thrmbus in nrmlipidemic rabbits. Athersclersis 18:43-57, FOLCH, J., LEES, M., AND SLOANE-STANLEY, G.H.: Simple 30. methd fr the islatin and purificatin f ttal lipids frm animal tissues. J Bil Chem 226: , DISCHE, Z.: Clr reactins f nucleic acid cmpnents. In 31. Nucleic Acids, vl. 1, edited by E. Chargaff and I. N. Davidsn. New Yrk, Academic Press, 1955, pp DAY, A.J., AND WAHLQVIST, M.L.: Chlesterl ester and 32. phsphlipid cmpsitin f nrmal artas and f athersclertic lesins in children. Exp Ml Pathl 13: , ARVIDSON, G.A.E.: Reversed-phase partitin f thin-layer chrmatgraphy f rat liver lecithins t yield eight simple phsphatidyl chlines. J Lipid Res 8: , BARTLETT.G.R.: Phsphrus assay in clumn chrmatgra- 34. phy. J Bil Chem 234: , SNYDER, F.: Radiassay f thin layer chrmatgrams: Highreslutin znal scraper fr quantitative 14 C and 3 H 35. scanning f thin layer chrmatgrams. Anal Bichem 9: , SKIPSKI, V.P., PETERSON, R.F., AND BARCLAY, M.: Quantitative analysis f phsphlipids by thin-layer chrma- 36. tgraphy. Bichem J 90: , BLOCK, W.D., JARRETT, K.J., AND LEVINE, J.B.: Imprved autmated determinatin f serum ttal chlesterl with a single clr reagent. Clin Chem 12: , BONDJERS. C, AND BJORKERUD, S.: Arterial repair and athersclersis after mechanical injury: IV. Uptake and cmpsitin f chlesteryl ester in mrphlgically defined regins f athersclertic lesins. Athersclersis 15: , SMITH, E.B., EVANS, P.H., AND DOWNHAM, M.D.: Lipid in the artic intima: Crrelatins f mrphlgical and chemical characteristics. J Athersclersis Res 7: , LOFLAND, H.B., ANDCLARKSON.T.B.: Certain metablic patterns f athermatus pigen artas. Arch Pathl 80: ,1965. DAY, A.J., AND WAHLQVIST, M.L.: Uptake and metablism f 14 C-labeled leic acid by athersclertic lesins in rabbit arta. Circ Res 23: , PROUDLOCK, J.W., AND DAY, A.J.: Chlesterl esterifying enzymes f athersclertic rabbit intima. Bichim Biphys Acta 260: ,1972. DAY, A.J., NEWMAN, H.A.I, AND ZILVERSMIT, D.B.: Synthesis f phsphlipid by fam cells islated frm rabbit athersclertic lesins. Circ Res 19: ,1966. DAY, A.J., AND TUME, R.K.: In vitr incrpratin f 14 C- labelled leic acid int cmbined lipid by fam cells islated frm rabbit athermatus lesins. J Athersclersis Res 9: , WAHLQVIST. M.L., DAY, A.J., AND TUME. R.K.: Incrpratin f leic acid int lipid by fam cells in human athersclertic lesins. Circ Res 24: ,1969. PROUDLOCK. J.W., DAY, A.J., AND TUME. R.K.: Chlesterl esterifying enzymes f fam cells islated frm athersclertic rabbit arta. Athersclersis 18: ,1973. NEWMAN, H.A.I., DAY. A.J., AND ZILVERSMIT, D.B.: In vitr phsphlipid synthesis in nrmal and athermatus rabbit artas. Circ Res 19: ,1966. ZILVERSMIT, D.B.: Metablism f arterial lipids. In Athersclersis, Prc 2d Int Sympsium n Athersclersis, edited by R. J. Jnes. New Yrk, Springer-Verlag, 1970, pp DAY, A.J.: Lipid metablism by rabbit artic intimal and medial cells in tissue culture. Virchws Arch [Path Anat], in press. Circulatin Retearch, VL XXXIV, April 1974