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EXPERIMENTALTHRONIBO-ATHEROSCLEROSIS*

By MEYERFRIEDMANAND SANFORD0. BYERS

(From The Harold Brunn Institute, Mount Zion Hospital and Medical Center,San Francisco, Calif.)

(Submitted for publication December 27, 1960; accepted March 3, 1961)

Thrombosis long has been known as an oftenoccurring sequent of coronary atherosclerosis, butuntil quite recently, very few investigators seri-ously considered that thrombosis of itself incitedatherosclerosis. This possibility, initially pro-posed by von Rokitansky (1), was apparently re-futed by Virchow (2) and this seeming refutation,together with the discovery by Anitschkow (3) ofthe atheroma-inducing propensities of a diet richin cholesterol, discouraged further considerationof atherosclerosis as of thrombogenic origin.

However, Duguid (4-6) has revived the throm-bogenic etiology of atherosclerosis. He stressedthe detection of fibrin in typical coronary andaortic atherosclerotic plaques as a probable se-quent of an earlier thrombosis. He called atten-tion to the presence of fibrous-lined lamellae inplaques, which suggested to him that the patho-genetic process might be intermittent rather thanchronically progressive. He believed that he couldobserve evolutionary stages in the transformationand organization of mural thrombi into lipid-richatherosclerotic plaques. Finally he emphasized,as other pathologists also have done (2, 7-10),that the lipid deposit in the human atheroscleroticplaque (see Figure 1, A) is usually deeply situ-ated, whereas it is superficially accumulated in theatherosclerotic plaque observed in the rabbit con-tinuously fed cholesterol. It is important topoint out, however, that if the hypercholestero-lemic-atherosclerotic rabbit is allowed to returnto its usual normocholesterolemic level, eventuallythe lipid in its plaque also may become deeply em-bedded (11).

The observations of Duguid have been con-firmed by some pathologists (12-15) but his con-clusions have not been accepted either by all

* Aided by grants from the National Institutes ofHealth, the Life Insurance Medical Research Fund, theAmerican Heart Association, the San Francisco HeartAssociation, the Sacramento Heart Association and theContra Costa County Heart Association.

pathologists (7, 9) or by the majority of thoseinvestigators who have oriented their thoughts andactions to disturbances in lipid dynamics (eitherwithin the arterial wall or in the blood stream).These latter particularly have continued to be pro-foundly influenced by the ease with which experi-mentally induced hypercholesterolemia leads toatherosclerosis and have generally discounted thefact that the site of deposition of lipid in the athero-sclerotic animal continuously receiving cholesterolis quite different from that usually observed inhuman coronary atherosclerosis (compare A andH in Figure 1).

Obviously, as McLetchie (9) observed, thereis need to evaluate Duguid's contention by ex-perimental means. In short, beginning with athrombus arising in situ, can the morphologicalfacsimile of human coronary atherosclerosis bereproduced?

Recently we have been able to induce thrombusformation in the arterial system by either the intro-duction of a specially treated magnesium alloy.coil (16) or polyethylene tubing (17). It there-fore appeared important to us to study the evolu-tion of such thrombi in both normocholesterolemicand hypercholesterolemic rabbits. The resultsobtained indicated that the production of a throm-bus invariably led to the formation of a cellularplaque. In the presence of hypercholesterolemicplasma this experimental plaque accumulatedlipid in sites which simulated exactly the distribu-tion of lipid observed in the human atheroscleroticplaque.

METHODS

Both acute and chronic experiments were done. Inthe acute experimental study, 10 hypercholesterolemicrabbits that had been given a WN7ayne rabbit chow dietwith added cholesterol (2 per cent) and cottonseed oil(2 per cent) for 3 months had a 1.5 cm long magnesiumalloy coil inserted into the abdominal aorta below the siteof exit of the renal arteries; 8 similar rabbits receiveda 0.5 inch hemicylindrical segment cut from polyethylenetubing (ID 0.045, OD 0.062 inch) in the same portion

1139

MEYERFRIEDMANAND SANFORD0. BYERS

of their aortas. The method of preparation and insertionof both the coil (16) and polyethylene segment (17)have been described previously. For control purposes,8 normocholesterolemic rabbits received the coil and 8,the tubing; 72 hours later, after blood serum sampleswere obtained, the animals were sacrificed and thethrombi removed. The serum samples and the totalthrombi, or plaques, were analyzed for cholesterol by ap-propriate methods (18). Previous to analysis, the poly-

there remained only a residuum of fragmented metallicsalts. The thrombi produced by the coil method alsowere analyzed for triglyceride content (19). In addi-tion, sections of each thrombus were stained with Mal-lory's phosphotungstic acid hemotoxylin stain (PTAH)and Sudan IV.

Several types of chronic studies were done. The firststudy used 68 rabbits in whose aorta a magnesium al-loy coil was inserted; 32 of these rabbits also were

ethylene was removed from its associated thrombus, or placed on a cholesterol (2 per cent) cottonseed oil (2 perplaque. The wire could not be similarly removed since cent) enriched diet, and the remaining 36 animals (serv-

FIG. 1

A. LOW-POWERVIEW OF A SECTION OF ANTERIORDESCEND-ING BRANCHOF THE LEFT CORONARYARTERYOF A 57 YEAROLD MANWHOHADDIED OF AN ACUTECORONARYTHROMBO-SIS OF THE CIRCUMFLEXBRANCHOF THE SAMEARTERY(SU-DAN IV STAIN IN ALL COLOR SLIDES). This is the typicalhistological picture of a moderately diseased coronaryartery and is presented for purposes of comparison withthe succeeding slides. Note the eccentric position of theplaque, the deeply situated lipid separated by fibrous-like tissue free of stainable lipid, both from the lumenand from the partially thinned and fragmented media.The essential dissimilarity of this histological structurewith that found in the spontaneously developing athero-sclerosis of the cholesterol-oil fed rabbit can be seen bycomparing this illustration with H.

B. RABBIT MW-4: LOW-POWERVIEW OF THE THROMBOTICPROCESSIN A CHOLESTEROL-OIL FED RABBIT 21 DAYS AFTERINTRA-AORTIC COIL INSERTION. The cellular envelopmentand penetration of the original thrombus and the frag-mentation of the subjacent media are clearly visible.Sudanophilia of the basal lateral areas and the subjacentmedial remnants also is depicted. Note the absence ofsudanophilia in the more peripheral portions of the en-veloping and penetrating newly formed tissue. Frag-ments of metallic salts also are shown.

C. RABBIT MG22-3: ANOTHERLOW-POWERVIEW OF THETHROMBOTICPROCESSIN A CHOLESTEROL-OIL FED RABBIT 21DAYS AFTER INTRA-AORTIC COIL INSERTION. Here thesudanophilia in the basal central area of the newly de-veloping tissue just adjacent to the media is clearlyshown. Note the partial disorganization and sudanophiliaof the media underlying the thrombotic process and theirabsence in those portions of the media not in juxtapositionwith the process. In this section large masses of metal-lic salts persist despite penetration of hyperplastic intimaltissue which also contains some extracellular pools oflipid.

D. RABBIT RR-25: LOW-POWERVIEW OF PLAQUE, 84DAYS AFTER INSERTION OF COIL IN A CHOLESTEROL-OIL FEDRABBIT. The sudanophilia deeply situated in the basaland lateral areas of the plaque is clearly shown. Frag-mentation and sudanophilic infiltration of the media sub-jacent to the plaque also can be seen. Some metallicsalts still remain in the plaque. The freedom fromatherosclerotic involvement of the more distant portionsof the aorta was a usual finding. The essential similar-

ity of this plaque to that found in the human coronaryartery and its dissimilarity to the atherosclerotic processdeveloping spontaneously in the cholesterol-oil fed rab-bit can be seen by comparing the above photographwith A and H, respectively.

E. RABBIT RR-32: LOW-POWERVIEW OF PLAQUE, 84DAYS AFTER INSERTION OF COIL IN A CONTROLRABBIT FEDTHE STOCK DIET. Here dense fibrous tissue containingresidual metallic salts at its base has completely replacedthe original thrombus. Note the complete absence ofsudanophilia. In other respects, however, its structuralcharacteristics are similar to those of plaque shown in D.

F. RABBIT R3-12: LOW-POWERVIEW OF PLAQUE, 84DAYS AFTER INSERTION OF HEMICYLINDRICAL SEGMENTOFPOLYETHYLENETUBING IN A CHOLESTEROL-OIL FED RABBIT.The cleft in the center of the plaque was the area oc-cupied by the tubing whose trough and its containedthrombus had faced the lumen. A portion of this throm-bus, staining a brownish color and encircled by a thinrim of new intimal tissue, can still be seen in the cleftarea. Note the absence of sudanophilia in this throm-bus remnant and its dense accumulation in the basal cen-tral and lateral areas of the hyperplastic intima. Ac-tually, the wall of the tubing itself elicited the total in-timal reaction, since it had isolated the thrombus in itstrough from the intimal surface of the aorta. Never-theless, this plaque, and also that shown in D, bears astrong likeness in its essential structural details to thehuman plaque depicted in A.

G. RABBIT Z-3 NO. 10: LOW-POWERVIEW OF PLAQUE,168 DAYS AFTER INSERTION OF COIL IN A RABBIT FED CHO-LESTEROL-OIL DIET DURING THE LAST HALF OF THE PERIOD.This dense, relatively acellular plaque with only the slight-est peripheral tint of sudanophilia resembles the 84-dayold plaque of the stock-fed rabbit shown in E despite thecholesterol and oil feeding. The fragmentation of themedia is well shown.

H. RABBIT Z-3 NO. 10: LOW-POWERVIEW OF THE SPON-TANEOUSLYDEVELOPEDATHEROSCLEROSISIN THE ARCH OFTHE AORTA OF THE SAME RABBIT WHOSETHROMBUS-IN-DUCEDPLAQUEIS ILLUSTRATED IN G. The superficial siteof lipid deposition in the hyperplastic intima contrastsstrongly with the sites of lipid accumulation shown in thehuman plaque A and in the thrombus-induced plaque ofthe rabbit fed cholesterol-oil concomitantly with intra-aortic coil or tubing insertion (D and F).

1140

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ing as controls) were given ordinary Purina rabbitchow. Serum samples for cholesterol analyses were ob-tained from all rabbits every 3 weeks, excepting thoserabbits sacrificed at 7, 14 and 21 days; from these ani-mals a single terminal serum sample was obtained. Attime of sacrifice, the entire aorta was inspected andgraded for gross atherosclerosis (18). The thromboticremnant was then identified and, after a small sectionwas obtained for histological study, the total process in-cluding the new intimal overgrowth (when this had oc-curred) was removed and analyzed for its cholesterolcontent. In addition, for control purposes a 5 cm sec-tion of abdominal aorta directly cephalad to the throm-botic process was removed and also analyzed for its cho-lesterol content. This chronic study continued for 84days.

The second study used 20 rabbits. Polyethylene tub-ing was inserted into each abdominal aorta; 11 of the rab-bits were then placed immediately on the cholesterol-cottonseed oil enriched diet. The other 9 rabbits werefed the stock diet. Eight rabbits (4 on the stock diet and4 on the cholesterol-oil enriched diet) were sacrificed 21days, and the remaining 12 rabbits were sacrificed 84 days,after insertion of tubing. Gross and histological studiesof the thrombotic process of all rabbits were done. Inaddition, similar serum and tissue analyses and histo-logical examinations were carried out on the 12 rabbitsallowed to live for 84 days after the insertion of tubing.

The third study used 26 rabbits; all of these also re-ceived the magnesium alloy coil, but they were at firstgiven the stock diet. Twenty-one days after the coilhad been inserted, 8 of these rabbits were placed on thecholesterol-cottonseed oil enriched diet and continuedthereon for an additional 84 days; another 14 were atfirst given the stock diet for 84 days after the coil hadbeen inserted and then placed on the cholesterol-cotton-seed oil enriched diet for an additional 84 days. The re-maining 4 rabbits were continued on the stock diet forthe entire 168 day period. The analytic and histologicalstudies were also done on these rabbits.

RESULTS

A. Characteristics of the newly formed throm-bus in the normal and hypercholesterolemic rab-bit. The thrombi removed 3 days after the inser-tion of either the coil or tubing were found to befreshly suspended-still unattached to the intimaof the aorta (16) in both the normo- and hyper-cholesterolemic rabbits. It was thus obvious thatmaterial arising from the blood stream itself wasunder study.

Both types of induced thrombi appeared pinkishwhite. Those produced by the coil were con-siderably larger than those formed in the tubingtrough and still contained a plethora of metallicsalts as previously described (16). It is of in-

terest, however, that the average weight of eithertype of thrombus was not any greater in the hy-per- than in the normocholesterolemic animals(Table I). Upon histological study, strands offibrin were observed in both types of thrombi;both were relatively devoid of cellular elements,but rich in platelets. None of the thrombi of eithertype in either the normo- or hypercholesterolemicanimals exhibited a positive staining reaction forlipid.

The analytical results were of considerable in-terest. The coil-induced thrombi in the hypercho-lesterolemic rabbits contained an average of 840mg ofcholesterol and 657 mg of triglyceride per100 g of dry tissue (dried over silica-gel) ascompared with an average of 418 mg of cholesteroland 464 mg of triglyceride per 100 g of dry tissuefound in the thrombi of the normocholesterolemicrabbits. When these figures were used to calcu-late the amount of cholesterol in the total thrombi,it was found that these thrombi contained lessthan 0.2 mg of total cholesterol. Moreover, thosein the hypercholesterolemic animals contained anabsolute amount of only about 0.1 mg more totalcholesterol than was found in the thrombi of thenormocholesterolemic rabbits, although, of course,the percentage of cholesterol was greater inthrombi from hypercholesterolemic rabbits.

The cholesterol analyses of the tubing-inducedthrombi in both normo- and hypercholesterolemicrabbits also indicated that a minimal amount ofcholesterol was deposited in the early thrombus.Thus (Table I), although the average cholesterolcontent of the thrombi in the hypercholesterolemicrabbits was greater than that in the normocho-lesterolemic rabbits, neither type of rabbit hadthrombi that contained more than 0.04 mg of totalcholesterol, and the average difference in the totalcholesterol content of the thrombi of the two seriesof rabbits was approximately 0.01 mg-a mostinsignificant quantity.

B. The chronic changes occurring in thrombi inrabbits fed excess cholesterol immediately afterinsertion of the coil. Seven days after coil in-sertion, the aortic thrombus was observed in boththe hypercholesterolemic and control rabbits tobe lightly adherent to the underlying intima.Upon histological study, newly invading tissuecomposed of cells containing large nuclei and palecytoplasm coming directly from the intima be-

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EXPERIMENTALTHROMBO-ATHEROSCLEROSIS 1143

TABLE I

The effect of immediate cholesterol-oil feeding upon cholesterol content of coil- and tubing-induced thrombi and plaques

Coil Aorta Thrombotic process Av. ratioor tubing Av. serum air-dried TP/A

Rabbits Av. wt inserted cholesterol cholesterol Av. wt (dry) Cholesterol cholesterol*

no. g no. days mg % mg/100 g mg mg/100 gI.

Seriest 10 2,930Range:SE of mean

Series A 3 3,020

Rabbits fed cholesterol-oil diet immediately after insertion of coil3 644t

(208-1,383)±101.0

7 115t(41-226)

B 3 2,960 14 2211:(86-313)

C 4 3,032 21 348t(178-612)

D 8 3,104 42. 311§(118-804)

±69.0E 14 3,438 84 724§

(630-948)±66.0

1,095(820-1,400)

±42.0735

(578-844)

871(804-962)

1,206(748-1,660)

832(613-1,030)

±49.01,063

(730-1,480)4:87.0

II. Rabbits fed stock diet after insertion of coilSeries 0 8 2,398 3 54

(42-78)-i-2.4

Series A 3 3,125 7 33t(27-36)

B 3 2,860 14 25t(15-36)

C 6 2,980 21 39t(22-42)±3.1

D 24 3,438 84 51 §(30-71)

±1.4

508(501-604)

±35.0631

(518-768)654

(565-707)730

(520-710)±62.0532

(294-973)4116.0

29 418(15-40) (329-500)

±3.1 ±18025 693

(14-33) (629-757)24 960

(19-28) (854-1,100)14 1,274

(8-24) (468-1,810)±3.3 ±188.0

8 1,016(8-9) (459-2,095)±0.3 ±83.0

0.83

1.10

1.47

1.74

1.90

III. Rabbits fed cholesterol-oil diet immediately after insertion of tubingSeries 0 t 8 2,978 3 320t

(108-435)±40.0

Series A 7 3,482 84 1,506§(1,091-2,180)

±62.0

1,090(788-1,262)

±195.01,974

(778-5,550)±89.0

1.9(0.8-6.5)

±0.95.1

(1.4-7.7)±=0.8

IV. Rabbits fed stock diet after insertion of tubingSeries 0 8 2,864 3 70

(37-102)±4.8

Series A 5 3,346 84 35§(27-45)

±2.6

770 1.5(619-892)

±49.0437

(418-474)±32

(0.4-4.0)±0.35

1.8(0.8-3.0)

±0.37

1,898(562-2,790)

±294.017,189

(4,250-29,100)±3,700.0

1,508(620-2,750)

±2081,991

(1,305-2,980)±212

1.73

8.77

1.96

4.55

* TP/A cholesterol ratio equals cholesterol concentration of thrombotic processcholesterol concentration of adjacent aortic segment'

t These rabbits were hypercholesterolemic at time of coil or tubing insertion, having been fed the cholesterol-oilenriched diet for the preceding 3 months. The cholesterol values given represent average of 3 monthly values.

Represents serum cholesterol value on day of sacrifice.§ Represents average of all serum cholesterol values obtained every 3 weeks.

25(17-33)

±3.235

(29-40)25

(20-33)16

(12-20)17

(11-22)±1.413

(9-24)41.3

840(575-1,148)

+48939

(850-988)1,432

(1,170-1,680)2,774

(1,690-3,820)4,359

(1,965-8,230)±7406,240

(2,520-12,560)±890.0

0.77

1.28

1.65

2.30

5.25

5.85

MEYERFRIEDMANAND SANFORDO. BYERS

neath the central basal portion of the thrombuswas observed in the three normocholesterolemicand the three already slightly hypercholesterolemicanimals. A few capillaries could be detected ac-companying these cells. The media beneath thisarea of hyperplasia appeared somewhat altered inthat the elastic lamellae were distorted and fre-quently fragmented. No evidence of acute inflam-mation was observed. Finally, despite the moder-ate elevation of the serum cholesterol in the threerabbits ingesting the cholesterol-oil enriched diet,(Table I) no evidence of sudanophilia was ob-served in any of the specimens.

Cholesterol analyses of the aortic segment andthe thrombotic process (Table I) suggested thata greater amount of cholesterol was present inthe thrombotic masses of the hypercholesterolemicrabbits, but the ratio of the cholesterol content ofthe thrombotic process to that of the adjacentaorta (TP/A cholesterol ratio) was approxi-mately the same (Table I) in both groups ofanimals.

Thrombi observed 14 days after their insertioninto three normocholesterolemic and three hyper-cholesterolemic rabbits exhibited a more tenaciousadherence to the vessel well. Extension of thehyperplastic intimal tissue up along the sides ofthe thrombi could be detected grossly.

Upon histological study it could be seen thatthe intimal new growth had invaded the centralbasal area of the thrombus even farther and hadascended along its sides. At this time, very smallamounts of lipid were first observed but only inthe specimens obtained from the three rabbits fedthe cholesterol-oil enriched diet; moreover, thislipid was confined chiefly to the basal areas ofthe newly formed tissue either beneath the throm-bus or ascending the latter's sides. This lipid wasapparently both intra- and extracellular, but notrue foam cells were seen. The media lying be-neath the thrombus showed even more disorgani-zation.

The average cholesterol content of the threethrombotic processes was considerably greater inthe hyper- than in the normocholesterolemic rab-bits (Table I). The TP/A cholesterol ratio inboth groups of rabbits also had increased.

All of the aortic thrombi of the 10 rabbits sacri-ficed 21 days after insertion of the coil now werecovered b)y newly formed intimal tissue (Figure

FIG. 2. RABBIT MGR-4: TYPICAL APPEARANCEOF THE

THIROMBOTICPROCESS21 DAYS AFTER INSERTION OF COIL INTO

THE AORTA. Although three segments are seen, they ac-tually formed a continuous spiral mass prior to incisionof the aorta. At this time the original thrombus is en-tirely covered by newly formed intimal tissue. The whitespots observed in the lowest mass are residual remnantsof metallic salts lying just beneath the newly formed tis-sue 'envelope.

2). Such thrombi both in the normal and hyper-cholesterolemic rabbits were still grayish-whitein color, but they could be stripped from the aortaonly with difficulty. At this time, the averageweight of the thrombotic process was approxi-mately the same in the two groups of rabbits.Spontaneous atherosclerosis involving any areaof the aorta was not observed grossly in the hy-percholesterolemic rabbits.

There was a very marked difference in the his-tological appearance of the thrombotic processesof the two groups of rabbits. Although all thrombi(still containing large amounts of metallic salts)now were covered completely as well as invadedby newly formed intimal cells, only the thromboticprocesses of the hypercholesterolemic rabbitsshowed well marked deposits of lipid. This lipid,chiefly exfracellular, was still found in those por-

1144

EXPERIMENTALTHROMBO-ATHEROSCLEROSIS

tions of newly formed intimal tissue at the baseand along the lower sides of the original thrombusand also in the inner third of the now completelydisorganized media immediately subjacent to theoriginal thrombus (Figure 1, B and C). It wasthus apparent that the major portion of the excesslipid seen either arose in situ or came from an in-tramural rather than a transintimal source. Itwas of interest too that no lipid was ever ob-served at this time in the aortic intima not oc-cupied by the thrombotic area (Figure 1, C).

The average cholesterol content of the totalthrombotic process (including of course the basaland lateral intimal hyperplastic tissue) in the hy-percholesterolemic rabbits was over twice thatfound in the same process in the normal rabbits(Table I). Also the TP/A cholesterol ratio hadincreased to 2.30. The average cholesterol con-centration of the thrombotic processes of the normalrabbits had increased also but to a lesser extent(Table I).

Eight cholesterol-oil fed rabbits were sacrificed42 days after coil insertion. No significant spon-taneous atherosclerosis was observed in any por-tion of the aorta of these eight rabbits. At thistime, however, the original site of coil insertionwas marked by a serpiginous, pinkish plaque, wellcovered with intimal tissue, approximately 1.5 cmlong (i.e., the same length as the coil that had beeninserted) and 3 to 5 mmwide. Along the lateralbase of this plaque newly formed blood vesselswere quite prominent. Five of these eight plaqueswhen incised showed a central core of yellowish,semiliquefied waxy material.

Upon histological examination, the initial throm-bus appeared to be almost completely replaced bythe intimal tissue that had grown in from the baseand along the sides. Already the most peripheralportions of intimal tissue forming the plaque hadbegun to resemble dense fibrous tissue containingscattered elastic fibers. Only some residual metal-lic salts remained to indicate that the plaque hadbeen induced by a thrombus. The central core ofthe plaque, however, in most instances exhibitednecrosis. The lipid deposit, which had increasedgreatly, was almost totally extracellular and ac-cumulated in lake-like masses at the basal areas ofthe plaque. The more peripheral areas of mostplaques appeared relatively free of lipid.

The average cholesterol content of these eight

plaques was 4,359 mg per 100 g of dry tissue.At this time there remained no doubt that the proc-ess was accumulating cholesterol far more avidlythan was the adjacent aortic segment that hadbeen analyzed (TP/A cholesterol ratio: 5.25).

Fourteen hypercholesterolemic and 24 normalrabbits were sacrificed 84 days after insertion ofthe coil. Twelve of the 14 hypercholesterolemicrabbits exhibited slight to moderate spontaneousatherosclerosis (grade 1-3) in the arch and thor-acic portions of the aorta. Only 4 of these 14 rab-bits, however, exhibited any gross spontaneousatherosclerosis in that portion of the abdominalaorta immediately above or below the plaque.

The gross appearance of the plaques in the twogroups of animals differed markedly (Figure 3);whereas in the normal rabbits the plaques usuallyhad shrunk to a dense, hard, rather flat, whitish-gray plaque, those of the 14 hypercholesterolemicanimals appeared larger, frequently exhibiting awaxy yellowish color. In general too, the aortaadjacent to these latter plaques appeared more dis-torted. Eight of .the 14 plaques in the hyper-cholesterolemic rabbits exhibited a necrotic corefrom which yellow, waxy, thick fluid could be ex-pressed. Similar central necrosis of the plaquesin the normal rabbits was never observed.

At histological examination, the plaque of thehypercholesterolemic rabbits exhibited a struc-ture that resembled rather markedly that seen inthe diseased human coronary artery (Figure 1, D;compare with A). Thus the eccentric position ofthe thrombo-atherosclerotic plaque, the dense lipiddeposit situated quite deep in the plaque, thefibrous cap, and the relative immunity of theaortic intima not subjacent to the process arewell illustrated. In two of the plaques small areasof calcification also were observed. These fibrousplaques with deeply accumulated lipid at the baseof the plaque and in the subjacent portion of themedia were never seen in the spontaneous athero-sclerosis occurring in the rabbit during the firstfew months of cholesterol feeding (see H, Figure1).

The plaque occurring in the normal rabbits 84days after coil insertion was composed (see E,Figure 1) almost entirely of dense, fibrous-liketissue interspersed with occasional elastic fibersand containing some residual metallic salts at thebase of the plaque. Thinning and distortion of

1145


served in either the cholesterol-fed or the normalrabbits. The polyethylene tubing in every caseenclosed a greyish-white mass which adhered tothe intima of the aorta (Figure 4). Close inspec-tion revealed that this mass was covered with in-timal tissue growing over it and separating it fromthe tubing wall. In addition to this, the outerwall of the tubing itself also was partially coveredby a bilateral extension of living intimal tissue.

Histological examination of these plaques re-vealed that, in most of them, the original rela-tively acellular thrombus was almost completelyreplaced by intimal tissue invading and encirclingthe original thrombus in much the same fashionas that observed in the coil-induced thrombi.Again, as in the coil-induced process after 21 days,

FIG. 3. RABBITS MGS-18 AND MGU-31: THEPLAQUES

FORMING84 DAYS AFTER INTRA-AORTIC COIL INSERTION INTO

THE CHOLESTEROL-OIL FED RABBIT (MG S-18) AND INTOTHE CONTROLRABBIT FED THE STOCK DIET (MG U-31).Fully developed plaques have now replaced the initialthrombus mass. The plaque in each animal was actuallya spiral continuous process but was cut into three sec-tions on incision of the aorta. Note the greater thicknessand the lipid infiltration of the plaque segments of therabbit fed the cholesterol-oil enriched diet (Mg S-18).Also note the distortion of the aorta adjacent to theplaque. The plaque segments of the control rabbit(Mg U-31) are flatter, thinner and translucent and ex-hibit pinpoint areas composed of small blood vessels.

the media subjacent to the process almost alwayswas observed.

C. The chronic changes occurring in thrombi inrabbits fed excess cholesterol immediately afterinsertion of tubing. As previously described (17),the trough of the polyethylene tubing was occu-pied by a relatively cell-free, fibrin- and platelet-rich thrombus within 72 hours after its insertion.When the aorta of the eight rabbits sacrificed21 days after introduction of the tubing wasopened, no spontaneous atherosclerosis was ob-

FIG. 4. RABBIT W2-272: THE THROMBOTICPROCESS21DAYS AFTER INSERTION OF A SEGMENTOF POLYETHYLENETUBING INTO THE AORTA. The polyethylene has beenlifted and pulled to the side. The whitish-grey thrombusis attached to the intima from which newly formed tis-sue has arisen to begin to cover the thrombus and alsothe outer wall of the tubing.

1146


R3 #4 IFIG. 5. RABBITS R3-4 AND R3-1: THE PLAQUES FORM-

ING 84 DAYS AFTER INTRA-AORTIC POLYETHYLENETUBING

SEGMENTINSERTION INTO THE CHOLESTEROL-OIL FEI) RAB-

BIT (R3-4) AND INTO THE CONTROLRABBIT (R3-1) FED

THE STOCK DIET. Fully developed plaques covering thetubing (removed prior to photography) and its containedthrombus have now developed. The thick, lipid-infil-trated walls of this plaque in the cholesterol-oil fed rab-hit (R3-4) contrast strongly with the poorly visualized,flat, translucent plaque of the control animal (R3-1).The two lateral dark lines running lengthwise representthe space occupied by the walls of the tubing. Thenew intimal growth had grown within the trough of thetubing and also had ascended the latter's outer walls.The silk stay sutures can still be seen.

the basal portions of the intimal tissue invadingand encircling the thrombotic remnants in thecholesterol-fed rabbits exhibited lipid chiefly inextracellular aggregations. Thus, except for theabsence of metallic salt fragments, the histologicalappearance of the plaques arising after tubing in-

sertion was essentially similar to that of the coil-induced plaques.

All of the seven cholesterol-fed rabbits sacrificed

84 days after insertion of the tubing showed ex-tensive confluent atherosclerosis in the arch andthoracic portions of the aorta (grade 3-4), butthe abdominal aorta of four of them was free ofvisible atherosclerosis, and in the remaining threethere were only scattered small plaques. In eachof these seven rabbits- the polyethylene tubingin the aorta was totally covered by a thick, ob-viously lipid-containing tissue (Figure 5). Thetrough of two of the tubings was found to facethe lumen rather than the wall of the aorta. Instrong contrast, the plaques present in the aortaof the control normocholesterolemic rabbits weresmaller, flatter, more translucent, and devoid ofgrossly detectable lipid. The plaques in bothgroups of rabbits were relatively tough and onehad the impression of cutting through scar tissuewhen transverse incisions were made in eithertype of plaque. The interior of the plaque in sixhypercholesterolemic animals was occupied by thesame type of yellowish, waxy, thick fluid ob-served in some of the coil-induced plaques.

Similar to the histological structure observedin the coil-induced plaques, the tubing-inducedplaque in the cholesterol-fed rabbit showed inevery case dense, extracellular accumulation oflipid in the basal central and lateral areas (Figure1, F). Usually the media immediately beneaththe plaque was thinned, slightly disorganized, andoccasionally infiltrated with lipid situated bothintra- and extracellularly. In the aorta of thetwo animals whose tubing trough had faced thelumen of the aorta, the original clot therein hadbeen invaded by new intimal tissue which, as-cending the lateral walls of the tubing, then haddipped into the trough to begin to replace the ini-tial thrombus. This is shown in Figure 1, F.which illustrates several very interesting points.The section shows quite well that the lipid depo-sition is in the basal intimal tissue that was sepa-rated right from the beginning from any contactwith the thrombus by the impermeable walls ofthe polyethylene tubing. This, of course, cogentlysuggests that the intimal lipid could not have beenderived from the original thrombus. Confirmingthis probability, the residual portion of the throm-bus itself showed no lipid. The section also indi-cates that the hyperplastic intimal tissue reaction,with its rich deposition of chiefly extracellularlipid, has been a response not to the thrombus but

1147


to the wall of the tubing itself. Finally, the sec-tion bears a much closer resemblance to the typeof atherosclerosis observed in the human coronaryartery than does that observed in our rabbit fedexcess cholesterol (Figure 1, compare F with Aand H).

The plaques of the five control rabbits in gen-eral showed the same type of cellular structureobserved in the plaques of the cholesterol-oil fedrabbits; however, the total plaque was consider-ably smaller and completely devoid of sudanophilicmaterial except for one, in which a few sudano-philic droplets were seen in the basal central area.As observed in the experimental animals, theplaques of these control rabbits exhibited whatwe considered to be fibrous transformation of theinitial intimal extension.

The plaques of both the experimental and controlrabbits contained far more cholesterol than did theimmediately adjacent aorta. Thus (Table I),the average cholesterol content of the controlplaques was more than four times that found intheir aortas. Even more striking was the findingthat the average cholesterol concentration of theplaques of the cholesterol-oil fed rabbits was morethan eight times that found in either their ownaortas or in the plaques of the control rabbits.This greater concentration of cholesterol in thetubing-induced plaques, as compared with thatfound in the coil-induced plaques, was probablydue not only to the greater degree of hypercho-lesterolemia achieved in the rabbits bearing the

tubing but also to the absence of metallic frag-ments.

D. The chronic changes occurring in thrombiof rabbits not fed cholesterol immediately afterinsertion of the coil. The plaques of the eight rab-bits fed the cholesterol-oil diet for 84 days, begin-ning 21 days after coil insertion, were examinedboth histologically and chemically. They ap-peared to present essentially the same cellularstructure and about the same amount of sudano-philia both in the basal central and basal lateralportions of the plaque as did plaques from rabbitsfed cholesterol-oil immediately. Moreover, theiraverage cholesterol content was as great (TableII) as that found in the 84-day old plaques of therabbits fed the cholesterol-oil diet immediately af-ter coil insertion.

On the other hand, when the 14 rabbits, fedthe cholesterol-oil enriched diet for 84 days be-ginning 84 days after coil insertion, were sacri-ficed, a most interesting phenomenon was ob-served. Twelve of these 14 rabbits exhibitedmoderate atherosclerosis of the ascending portionof their aortas (grade 1-3) but all of their coil-induced plaques appeared shrunken and grosslydevoid of fat deposition and markedly resembledthe type of plaque observed in the four controlrabbits fed the stock diet over the same period(Figure 6).

Histologically too, only 1 of the 14 hypercho-lesterolemic rabbits had a coil-induced plaque thatcontained sudanophilic material. Even in this

TABLE II

The effect of delayed cholesterol-oil feeding upon coil-induced plaques

Thrombotic process Av. ratioCoil Av. serum Aorta TP/A

Rabbits Av. wt inserted cholesterol* cholesterol Av. wt (dry) Cholesterol cholesterol

no. g no. days mg % mg/1OOg mg mg/1OOg

8RangeSE of mean

I. Rabbits fed cholesterol-oil diet 21 days after coil inserted2,998 105 876 1,242 14

(720-1,035) (730-1,830) (8-24)±42.0 ±t156.0 4±1.6

II. Rabbits fed cholesterol-oil diet 84 days after coil inserted14 3,553 168 741 1,419 8

(309-1,101) (510-4,290) (5-12)±62.0 ±405.0 ±0.65

III; Control rabbits fed stock diet4 3,284 168 57

(38-64)583

(391-937)8

(8-9)

8,083(3,130-11,700)

± 950.0

2,097(880-4,020)

±260.0

560(488-600)

6.50

1.47

0.96

* Average of all serum cholesterol determinations obtained every 3 weeks.

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FIG. 6. RABBITS 445 AND 461: THE PLAQUES FORMING

168 DAYS AFTER INTRA-AORTIC COIL INSERTION. AlthoughRabbit 445 had been fed the cholesterol-oil enriched dietfor the last half of the total period, its plaque segmentsin general resemble those of the control rabbit 461 fedthe stock diet. Compare these plaque segments of Rab-bit 445 with those of Rabbit Mg S-18 of Figure 3.

one plaque (Figure 1, G), the degree of sudano-philia was insignificant compared with that foundin the same rabbit's ascending aorta (Figure 1,H). In short, a firm, relatively acellular, fibrousplaque, essentially similar to that found in thestock-fed rabbits (compare E and G in Figure 1),was observed in these rabbits despite their pro-

longed cholesterol-oil feeding and accompanyinghypercholesterolemia.

The chemical analyses also indicated that a plaquealready 84 days old possessed a relative immunityto experimentally induced dietary hypercholes-terolemia not shared by the plaque only 21 daysold or less. Thus (Table II), the average cho-lesterol of these plaques was 2.097 mg per 100 ml,a value only 1.47 times that of the adjacent ab-dominal aorta. It should be noted, however, thatthe average cholesterol content of the plaques ofthe four control rabbits allowed to survive for a

similar period of 168 (lays was considerably lessthan that of the plaques of other rabbits fed thestock diet and allowed to survive for only 84 daysafter coil insertion.

DISCUSSION

Weare well aware of the probability that someof the results of this present study will be con-sidered by some investigators as important prin-cip)ally because they furnish direct experimentalproof of the ability of a thrombus to elicit the for-mation of a plaque bearing a striking chemical aswell as architectural resemblance to the plaquefound in the human artery. Certainly it cannotbe denied that starting with a thrombus, we in-variably induced a plaque. Nor can it be deniedthat if the cholesterol concentration of the rabbit'sblood was elevated to that of man or higher, lipidandl cholesterol accumulation quickly ensued inthese plaques and that they, similar to humanplaques, frequently exhibited internal degenera-tion, necrosis and liquefaction.

But these same experiments also appeared tous to demonstrate that the formation of a plaqueneed not rely solely upon a thrombus for its ini-tiation, growth or maturation. Certainly in ourown experiments with hypercholesterolemic rab-bits, an atherosclerotic plaque resulted whetherthe initial injury or stimulus to the vessel wallwas caused by the fulminant sort of thrombus in-duced by metal coil, by the bland type resultingfrom introduction of the polyethylene tubing,or even by mere contact with the tubing itself.Moreover, the type of plaque and its evolution in-duced by our thrombi was essentially similar tothat induced by needle scraping of the aortic in-tima (10, 20), by cauterization of the adventitia(21), by explantation of the carotid arteries (22),and by freezing of the aorta (8, 23). Indeed, it isprobable that the fragmentation of the internalelastic membrane observed in the arteries of in-fants (24), and the intramural hemorrhage sooften observed in the arteries of adults (25) con-ceivably could induce the same type of athero-sclerotic plaque that has been produced ex-perimentally.

Despite the variety and multiplicity of theabove agents and events capable of injuring oraltering the arterial wall and hence inducing

1149


atherosclerotic reactions, all of them are followed,as is well known, by intimal proliferation or hy-perplasia. Weconsider it quite probable that theessential sameness of all human atheroscleroticplaques may be due, not to the sameness of initiat-ing agent, but to the sameness of intimal hyper-plasia and its peculiar properties which thesevarious agents elicit.

It is of course possible that the initial tissue in-jury itself, by giving rise to a state of inflamma-tion, as Boyd (26), Winternitz, Thomas andLeCompte (27) and Waters (28-31) believe,invariably elicits the intimal hyperplasia and per-haps the lipid and cholesterol accumulation oc-curring therein. It is difficult to refute this pos-sibility if only because all of the above agentsseemingly induce tissue injury or change, and thelatter might be expected to elicit, if not all, atleast several of the classic phenomena associatedwith inflammatory processes. In our own experi-ments however we, as did Taylor, Balwin andHass (23), failed to observe the presence of asignificant number of inflammatory cells in ourlesions at any stage. Moreover, the lipid andcholesterol accumulation observed in the plaquesof rabbits that were not made hypercholesterolemicuntil 21 days after clot induction was no less thanthat observed in the plaques of rabbits made hy-percholesterolemic immediately after thrombusformation. Whereas these facts make us reason-ably certain that acute inflammation could not beresponsible for the lipid and cholesterol accumu-lation, they do not rule out the possible operationof a chronic inflammatory process. Indeed, themere presence of a rapidly growing tissue in ahigh pressure system such as is present in the ar-terial lumen might lead to a new series of in-juries or inflammatory reactions, or both.

The present studies indicated quite clearly, webelieve, that in the early stages of intimal hyper-plasia, stainable lipid was first deposited chieflyin the basal and lateral areas of the formingplaque. Whether such lipid represented an in-creased synthesis in situ or an exudate (31) is aquestion that remains to be answered. Certainlyit was clearly shown that it could not have beenderived from any lipid initially present in thethrombus as Curran, Thomas and O'Neal (32)believed. It was of great interest to us, too, toobserve that, although the newly developing

plaque exhibited tremendous proneness to lipidand cholesterol accumulation, the mature plaque,as Ssolowjew (21, 22) also observed in his ex-periments, exhibited a striking immunity to suchaccumulation. The basal deposition of lipid inthe induced plaque of hypercholesterolemic rab-bits contrasted strikingly with the superficialsubendothelial lipid accumulation occurring inthe spontaneous atherosclerosis developing in theaortic arch of these animals. In view of the simi-larity of the human plaque to the induced one andits well known dissimilarity to the spontaneousatherosclerotic area developing in the rabbit main-tained continuously hypercholesterolemic (2, 7),it seems possible that the human atheroscleroticplaque may also quite often be the sequent of adiscontinuous insult rather than of a continuoustransintimal penetration of lipid and cholesterol.Such an insult could be a thrombotic one, but itcould just as well be hemorrhagic, physical orchemical.

Central necrosis with occasional ulcer forma-tion is frequently observed in the human arterialplaque. Its cause has not been determined, al-though it has been postulated (2, 12, 33) that itoccurs as a result of central ischemia. This, how-ever, does not appear to be the responsible agentbecause, in our experiments, none of the plaquesof the normocholesterolemic animals ever ex-hibited central disintegration or necrosis. Onthe other hand, it was found in almost all of thelipid-laden plaques of the hypercholesterolemicanimals. Thus, it is our belief that it is thehuge depots of lipid and cholesterol themselvesthat effected the necrosis we observed so often.These may also be associated with the pathogene-sis of the central necrosis occurring in humanarterial plaques.

It was rather disconcerting to observe that theplaques induced in the control normocholesterole-mic rabbits, while considerably smaller than thosefound in the hypercholesterolemic rabbits, werenot insignificant in size, despite their absence ofstainable lipid and relatively low cholesterol con-tent. Similar relatively lipid-free plaques arealso found in human arteries (2, 12, 34, 35). In-deed, from the studies of Moon (24) and Enos,Beyer and Holmes (33) it seems extremely doubt-ful that such plaques could have been caused byan initial deposit of lipid or cholesterol. Rather,

1150


it would appear that the plaque served as a sitfor later accumulation of these substances. Iiour.studies, in which the total process could beobserved at all stages, there could be no doubthat a plaque's origin did not depend exclusivelyupon a prior deposit of lipid or cholesterol; tothe contrary, it invariably depended upon a preceding intimal hyperplasia. This latter phe-nomenon in turn did not at all stem always froman initial infiltration or accumulation of lipidor cholesterol. However, the later accumulationof such substances almost always intensified,hence aggrandized, the intimal overgrowth.

SUMMARY

The experimental induction of intra-arterialthrombi by two different techniques led, in thehypercholesterolemic rabbit, to a plaque exhibitinga striking gross and histological resemblance tothat observed in human atherosclerosis. Theseinduced thrombo-atherosclerotic plaques con-sisted of dense, relatively acellular fibrous tissuehaving deeply embedded lipid and were frequentlysubject to central fatty degeneration and lique-faction.

The propensity of these plaques to accumulatethe excessive amounts of lipid and cholesterol ac-tually observed was found to be due to the intimalhyperplastic tissue that the thrombi evoked. Theavidity of this living intimal tissue for these sub-stances, however, was confined to the earlystages of the tissue's growth. After final fibroustransformation of such tissue, actual protectionagainst the accumulation of lipid and cholesterolwas observed.

The possible relevance of these experimentalstudies to the general pathogenesis of athero-sclerosis was discussed.

ACKNOWLEDGMENT

The authors wish to express their deep thanks to C.Omoto, W. Hayashi and Elizabeth Wang for theirtechnical assistance. The costs of publishing the colorplate in this article were defrayed by a contribution fromthe Wm. S. Merrell Company. Their assistance isgratefully acknowledged.

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