IMMUNOLOGIC ARTERIAL INJURY IN AT H EROG ENESIS* C. Richard Minick Department of Pathology The New York Hospital-Cornell Medical Center New York. New York 10021

INTRODUCTION Injury to the arterial wall is probably a primary causative factor in arteriosclerosis. Arterial injury, the resulting necrosis, inflammation, intimal hyperplasia, and other features of repair may favor the deposition of blood-borne lipid a t the site of injury and thereby lead to atherosclerosis. Therefore, it is essential to our understanding of human arteriosclerosis to determine those causes of arterial injury and the nature of the local reactive changes that may be important in the genesis of arterial disease in man and other animals. There is considerable evidence to suggest that inflammation that involves the arterial wall, and particularly that which results from immunologic injury, may be important in the development of arteriosclerosis in man.’5-21 In this regard, the wellknown association of syphilitic aortitis and atherosclerosis comes to mind. Further, rheumatic injury to cardiac valves, aortas, and coronary arteries may lead to arterio~-~~~~~ sclerosis, which, in some instances, may evolve as a t h e r o ~ c l e r o s i s . ~Premature coronary atherosclerosis occurs in young men in association with healed coronary ~~~ injury to arteritis, like that seen in rheumatic heart d i ~ e a s e . ”Immunologic coronary arteries in lupus erythematosus may lead to the premature development of coronary a t h e r o s c l e r ~ s i s .Moreover, ~ ~ ~ ~ ~ rapidly progressive occlusive atherosclerosis is known to occur in hornografts, in particular in cardiac homo graft^.^^*^^ Data obtained from clinical observations and from experiments indicates that immunologic injury plays an important role in the pathogenesis of arterial lesions in lupus erythematosus and graft rejection and in the pathogenesis of rheumatic cardiovascular disease.’6,2’ Immunologic injury may also be important in the pathogenesis of syphilitic cardiovascular disease.2R The concept that arteritis and immunologic arterial injury may be important in the genesis of atherosclerosis is intellectually appealing, because atherosclerosis and arteritis share major pathogenic features. In atherosclerosis, macromolecules, such as lipoproteins, are deposited and retained in the arterial wall in excess quantity. This increased endothelial transport of lipoprotein is thought to be essential to both the intimal hyperplasia and the fat deposition. Increased endothelial permeability is a cardinal feature of inflammation. Circulating mediators of inflammation increase endothelial permeability and may permit access of large quantities of serum protein and fluid to the arterial wall and adjacent tissue. In the instance of immunologic arterial injury due to antigen-antibody complexes, it has been proposed that the release of vasoactive substances from platelets and leukocytes alters endothelial permeability and permits entry of macromolecules, including immune complexes, into the arterial ‘Supported by Research Grant HL-01803 from the National Heart Institute, by grants from The Cross Foundation and The John Polachek Foundation for Medical Research, and by Grant 5T IGM 78 from the Division of General Medical Sciences, United States Public Health

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wall, where they may interact with complement and leukocytes and lead to arterial n e c r o s i ~ . ~Similar ~ * ~ ~ immunopharmacologic *~~ mechanisms may be important in increasing endothelial permeability in atopy. Moreover, changes in the structural integrity of the endothelium are thought to be an early feature of arterial changes in some stages of graft rejection. In the presence of hypercholesterolemia, changes in endothelial integrity that result from these immunologic phenomena would permit large quantities of lipoprotein to enter the arterial wall. Thus, immunologic responses to many environmental antigens, including those in infecting microorganisms, vaccines, antibiotics and other drugs, food, and tobacco, and to antigens in one's own tissue, may be an important causative factor of an even greater amount of arterial disease in man, some of which may evolve as atherosclerosis. For several years, we have investigated the possibility that arterial injury may be a primary causative factor in athero-arterioclerosis. Specifically, we have investigated the role that immunologic arterial injury and the subsequent reparative The results of our changes may play in the production of athero~clerosis.~~~'-~~*~~-~~ experiments demonstrate that the synergy of immunologic injury to arteries and hypercholesterolemia can lead to atherosclerosis in rabbits. In many instances, the arterial lesions bear close resemblance to atherosclerosis in man. This paper will report the results of these experiments. A N D METHODS MATERIALS

Immunologic injury to arteries was induced in rabbits by repeated intravenous injections of foreign serum protein or by graft rejection induced in heterotopically placed cardiac homografts. Hypercholesterolemia was induced by feeding cholesterol-supplemented, or semisynthetic lipid-rich, cholesterol-poor Total serum cholesterols, and, in some instances, serum phospholipids and triglycerides, were estimated as described previously." Aortas, pulmonary arteries, and cardiac valves were examined grossly at the time of autopsy, and visible atherosclerotic lesions were quantitated. Hearts were cut into approximately 12 blocks, and sections of coronary arteries in each block were examined microscopically. In some experiments, sections of splenic, gastric, mesenteric, femoral, subclavian, hepatic, renal, carotid, cerebral, and pulmonary arteries and aortas were also examined microscopically. Arterial lesions were tabulated as to size of artery involved and histologic character of lesions. Selected blocks of tissue were prepared for transmission electron microscopy by standard techniques. Portions of arteries were also prepared for scanning electron microscopy. In preparation for scanning microscopy, rabbits were perfused with 1% glutaraldehyde in Sorenson's phosphate buffer at 90 mm Hg for 40 min. Blocks of tissue were then fixed in 1% glutaraldehyde for 1 hr and washed in phosphatebuffered saline and postfixed in I % osmium in Millonig's phosphate buffer. After washing in phosphate buffer and distilled water, they were dehydrated sequentially first in a graded series of alcohols and then in a graded series of amyl acetate solutions in alcohol. Blocks of tissue were then critical point dried in carbon dioxide at a pressure of 1300 1 b / h 2and a temperature of 39-41°C.

RESULTS AND COMMENT In this report, we will discuss changes in the coronary arteries and aortas, although similar lesions were seen in other arteries. The changes in the coronary arteries will be emphasized because of the predilection of immunologic injury to in-

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FIGURE1. Fatty-proliferative change induced in main coronary artery of a rabbit that received repeated injections of foreign serum protein and was fed a cholesterol-supplemented diet for 82 days; elastic stain. x 145. (From Minick et By permission of The Journal of Experimental Medicine.)

volve coronary arteries and the special importance of coronary atherosclerosis as a cause of morbidity and mortality in man. Results of our initial experimentss indicated that rabbits of group I l l , which were fed cholesterol-supplemented diets and repeatedly injected with horse serum, four injections of 10 cm3/kg at intervals of 16 days, developed fatty-proliferative lesions of large, medium, and small coronary arteries that bore close resemblance to some stages of human atherosclerosis (FIGURESI & 2). In contrast, control rabbits of group I, which were fed cholesterol-supplemented diets but not injected with horse serum, developed predominately fatty lesions, which occurred primarily in small 3). coronary arteries, that bore little resemblance to human atherosclerosis (FIGURE Rabbits of group 11, which received repeated intravenous injections of horse serum and lipid-poor diets, developed proliferative fibromuscular intimal thickening in 4). Many of these lesions resemlarge, medium, and small coronary arteries (FIGURE bled fibromuscular intimal thickening in man, which is called diffuse or progressive intimal thickening.

FIGURE 2. Fatty-proliferative change in main coronary artery of a 35-year-old man with aortic stenosis and marked coronary atherosclerosis. Note similarity to change in rabbit artery illustrated in FIGURE I; hematoxylin and eosin. x 114. (From Minick e f a/.* By permission of The Journal of Experimental Medicine.)

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FIGURE3. Fatty, nearly acellular change in the wall of a small intramyocardial artery. Change is characteristic of those induced in rabbits by cholesterol-supplemented diet or semisynthetic lipid-rich cholesterol-poor diet; hematoxylin and eosin. x 200. (From Minick & Murphy." By permission of The American Journal of Pathology.)

No grossly visible changes were seen in the linings of the aortas of rabbits of group 11, which were injected with foreign serum protein and fed lipid-poor diets. Rabbits of groups I and 111 developed grossly visible yellow-white fatty streaks and plaques in the aortas. There was a significantly greater amount of fatty change in aortas of rabbits of group 111, which received the dietary supplement of cholesterol

FIGURE4. Musculoelastic intimal thickening of large coronary artery. Change is characteristic of those induced in rabbits that received repeated injections of foreign serum protein over many months and diets low in lipid. Similar lesions were also seen in rabbits that were injected with foreign serum protein and fed cholesterol-supplemented semisynthetic lipid-rich diets; hematoxylin and eosin. x100. (From Minick & Murphy." By permission of The American Journalof Pathology.)

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and concomitant injections of foreign serum than in rabbits of group I, which received cholesterol supplement and no injections of foreign serum protein. Thus, the degree of aortic fatty change was greater in rabbits that were fed cholesterol-supplemented diets and repeatedly injected with foreign serum protein. Moreover, both with respect to their distribution in the coronary arterial tree and their proliferative character, the coronary arterial lesions of rabbits of group 111 were unlike those in group I but similar to those of rabbits in group 11. Because rabbits in both groups I1 and 111 were similarly injected with horse serum, it is reasonable to infer that in group 11, in the absence of a large amount of lipid in the blood, proliferative lesions without fatty change developed a t sites of immunologic injury and that in rabbits of group 111, in the presence of increased blood lipid, fatty-proliferative lesions developed at these sites. The inference is supported by the observation that in group I , the elevation of serum lipid similar to that in group 111 induced in the absence of immunologic arterial injury arterial lesions that were only fatty and limited almost solely to small arteries, never large arteries. During the past several years, unexpectedly severe and apparently rapidly developing atherosclerotic changes in coronary arteries have been found to occur in some human cardiac homo graft^.^',^^ It is noteworthy that these arterial changes bore striking resemblance to the coronary atherosclerosis induced in the rabbits in our previous experiments by the synergy of immunologic injury and hypercholesterolemia. Therefore, we performed experiments designed to test the hypothesis that the synergy of immunologic injury to coronary arteries, due to cardiac homograft rejection and hypercholesterolemia, can result in coronary a t h e r o s c l e r ~ s i sHetero.~~ topic cardiac homotransplants were performed in the necks of 60 rabbits. Rabbits were divided into four experimental groups. Rabbits of groups I and I1 were fed a diet supplemented with 0.4% cholesterol by weight, and rabbits of groups 111 and IV were fed a lipid-poor diet. In addition, rabbits of groups I1 and IV were injected with immunosuppressive drugs, methyl prednisolone, and azathioprine. Hearts and aortas of recipient rabbits served as controls. Most rabbits fed lipid-poor diets had no grossly visible lesions in the transplanted segment of the aorta. Rabbits fed cholesterol-supplemented diets had grossly visible fatty changes in the transplanted segment of the ascending aorta, and in some aortas, almost the entire surface was so affected. Invariably, this arteriosclerotic change stopped abruptly at the anastomosis of the transplanted ascending aorta with the recipient’s carotid artery. Either no fatty change or only slight fatty change was present in aortas of recipients. In transplanted hearts, coronary arterial lesions developed that were different in quality, quantity, and distribution from those found in recipient hearts. In rabbits fed a lipid-poor diet, arterial lesions were mainly proliferative and mostly without fatty change, and arteries of all sizes were affected. In rabbits fed a cholesterol supplement, some lesions likewise were proliferative without fatty change, but the majority were fatty-proliferative. Some fatty-proliferative lesions bore close resemblance to human coronary atherosclerosis. In contrast, in hearts of recipient rabbits fed a lipidpoor diet, no arterial lesions developed; in hearts of the recipient rabbits fed cholesterol-rich diets, only occasional fatty lesions developed in small intramyocardial arteries. In the two experiments summarized above, rabbits were fed a cholesterol supplement that resulted in an average serum cholesterol of between 500 and 700 mg%, and their arteries were subjected to intense immunologic injury induced over a short period of time. In man, the amount of hypercholesterolemia is rarely this great; also, as compared with the arterial lesions induced in those rabbits, arterial injuries in man are likely to be more protracted or recurrent at longer intervals over a much longer

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period of time. Accordingly, it appeared reasonable to suggest that atherosclerosis that even more closely resembles that which occurs in man could be induced in rabbits by more chronic arterial injury in combination with a semisynthetic lipid-rich cholesterol-poor diet that results in an amount of blood cholesterol of the same magnitude as that in man. In long-term experiments, chronic atherosclerosis, which bore marked similarity to that in man, was induced in rabbits by the combined action of foreign protein injected repeatedly at intervals of 4-8 weeks and semisynthetic lipid-rich cholesterolpoor diets fed concomitantly for as long as 17 months." Feeding these semisynthetic lipid-rich diets to rabbits of groups I and 111 resulted in an increase of blood cholesterol from the normal range of 50 to 80 mg%, seen in most rabbits of group I1 fed diets low in lipid, to values that averaged between 200 and 250 mg%.

FIGURE5. Coronary atherosclerosis in the same man referred to in FIGURE2. Note lipid pools deep in the lesion with overlying fibrornuscular cap; Weigerthematoxylin and eosin. x30. (From Minick & Murphy." By permission of The American Journalof Pathology.)

As in the previous experiments, rabbits of group 111, which were repeatedly injected with foreign serum protein and concomitantly fed a lipid-rich cholesterol-poor diet, developed coronary arterial lesions that were different both in quality and distribution from those in rabbits fed the same lipid-rich diet but not injected with foreign protein (group I ) and similar in distribution and proliferative character to those in rabbits injected with foreign protein and fed a lipid-poor diet (group 11). Thus, as in the previous experiments, there was strong evidence that the lesions of group 111 represented immunologically induced lesions that had acquired lipid and evolved as atherosclerosis. As in the previous experiments, aortas of rabbits fed a lipid-rich cholesterol-poor diet and concomitantly injected with foreign serum protein (group 111) exhibited significantly greater atherosclerotic change than did those of animals fed the same diet (group I) but without injections of foreign serum protein.

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The results of these experiments differed from those described earlier, in that the fatty-proliferative lesions of group 111 in the later experiments bore closer, often marked, resemblance to chronic human atherosclerosis (FIGURES5-7). Among the many characteristics shared with human atherosclerosis were lipid-filled foam cells clustered deep in the intima, fatty hyaline change with little or no elastification, necrosis with pooled lipid, and cholesterol clefts deep in the intima and media with overlying fibromuscular caps or jackets, vascularization, occasional focal calcification of the intima, and rarely an overlying recent thrombus. Other changes in the rabbit arteries that were similar to human atherosclerosis included segmental medial degenerative change, scarring, and thinning and cellular proliferation and infiltrative change and fibrosis in the adventitia. In group 111, changes like those in the coronary

FIGURE 6. Coronary atherosclerosis induced in a rabbit by repeated injections of foreign serum protein and hyercholesterolemia that resulted from feeding a semisynthetic lipid-rich cholesterol-poor diet for 9 months; Weigert-hematoxylin and eosin. x96. (From Minick & Murphy." By permission of The American Journalof Pathology.)

arteries occurred in arteries at many other sites. In contrast, the arterial lesions of group I, which occurred primarily in small intramyocardial arteries and only rarely in large coronary arteries, bore little resemblance to human coronary atherosclerosis. The results of the experiments discussed earlier in this paper demonstrated that lipid accumulated at sites of immunologic injury to arteries and led to atherosclerosis that, in some instances, closely resembled that in man. In the same experiments, we found that immunologic injury without hypercholesterolemia led to fibromuscular intimal thickening without The latter change closely resembled the fibromuscular intimal thickening without lipid that occurs in man's arteries and is frequently referred to as diffuse intimal thickening (FIGURE4). It could not be determined in those experiments whether lipid had accumulated only acutely at sites of recent im-

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munologic injury to arteries or whether fibromuscular intimal thickening, which resulted from immunologic injury induced during previous weeks or months, had preferentially accumulated lipid and led to atherosclerosis. Although observations in man and other animals strongly suggest that earlier acquired fibromuscular intimal thickening without evident lipid may predispose to later developing a t h e r o ~ c l e r o s i s , ~there ~ - ~ ~is little direct experimental evidence to support this hypothesis. In fact, some investigators have shown that, under certain experimental conditions, fibromuscular intimal thickening may be resistant to later developing a t h e r o s c l e r ~ s i s . ~ ' - ~ ~ Experiments to be described were designed to determine whether immunologically induced fibromuscular intimal thickening in rabbits, which closely resembles

FIGURE7. Coronary atherosclerosis with areas of necrosis that contain atheromatous gruel and cholesterol clefts in a rabbit that received repeated injections of foreign serum protein and was fed the semisynthetic lipid-rich diet; Weigert-hematoxylin and eosin. x 210. (From Minick & Murphy." By permission of The American Journalof Parho/ogy.)

diffuse intimal thickening in man, would preferentially accumulate lipid and lead to a t h e r o ~ c l e r o s i s .In ~ ~these experiments, four groups of rabbits were fed a lipid-poor diet, and concomitant immunologic arterial injury was induced by multiple intravenous injections of horse serum. The resultant arterial lesions were allowed to heal for 40-80 days after the last serum injection. To determine whether lipid would preferentially accumulate a t such sites of intimal thickening, rabbits of groups IIIA and IIIB were fed cholesterol-supplemented diets that commenced either 40 or 80 days after the last serum injection. To assess arterial changes induced at the sites of immune injury, rabbits of groups IIA and IIB were continuously fed a diet low in lipid and were sacrificed at intervals after the last serum injection comparable to those used in groups IIIA and IIIB. To assess the effect of cholesterol feeding alone, two other groups, IA and IB, were fed the cholesterol-supplemented diet for 80 days.

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As in the previously described experiments, cholesterol feeding alone (groups IA and IB) induced fatty lesions in small intramyocardial coronary arteries. The lesions did not resemble human coronary atherosclerosis. Rabbits of groups IIA and IIB, which received four injections of horse serum and then were sacrificed a t intervals that ranged from 8 to 21 weeks after the last serum injection, had coronary arterial lesions equally distributed among large, medium, and small arteries. The lesions were characterized by fibromuscular intimal thickening with no lipid (FIGURE8). These proliferative lesions resembled the fibromuscular intimal thickening, o r the socalled diffuse intimal thickening, commonly found in man’s arteries. Rabbits of groups IIIA and IIIB, which received injections of horse serum and a cholesterolsupplemented diet after an interval of 40 o r 80 days, had coronary arterial lesions dis-

F I G U R E8. Musculoelastic intimal thickening in mesenteric artery of a rabbit repeatedly injected with foreign serum

protein over several months and fed a cholesterol-supplementeddiet that began 40 days after the last serum injection. Arterial change is characteristic of those seen in rabbits repeatedly injected with foreign serum protein and fed diets low in lipid but is also seen in some arteries of rabbits fed lipid-supplemented diets and injected w i t h foreign serum protein; Weigert-hematoxylin and eosin. x 120. (From Hardin et By permission of The American Journal of Pathology.)

tributed like those in group 11, that is, in approximately equal numbers in large, medium, and small arteries. Lesions in this group were of two types. Some were characterized by fibromuscular intimal thickening like that seen in rabbits injected with foreign serum protein and fed diets low in lipid. The majority, however, were fatty-proliferative. These latter lesions were similar to the serum-induced proliferative arterial lesions of group I1 in distribution and also with respect to histologic 8 & 9). These lesions character, except for the presence of lipid deposits (FIGURES were virtually identical to those in other large muscular arteries, and many closely resembled atherosclerosis in man. Thus, in the experiments reported here, immunologically induced arterial intimal thickening without evident lipid later accumulated lipid preferentially in the presence of hypercholesterolemia and evolved as

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atherosclerosis. The results of these experiments support the hypothesis that, in man, sites of fibromuscular intimal thickening that result from one or several instances of immunologic injury may continue to have increased avidity for lipid for weeks, months, or perhaps even years after the initial injury. Thus, we have produced by means of repeated immunologic injury and lipid-rich diets an animal model of atherosclerosis that in several important respects resembles athero-arteriosclerosis in man. First, immunologic reactions commonly occur in human populations. Second, the hypercholesterolemia necessary to induce these lesions is of the same order of magnitude as that of adult humans in the United States. Third, arterial lesions induced by repeated injections of foreign serum protein in rabbits concomitantly fed a lipid-rich diet are histologically very similar to those of

FIGURE 9. Atherosclerotic change in a different segment of the artery shown in FIGURE 8; Weigert-hematoxylin and eosin. x78. (From Hardin et uL31 By permission of The American Journal of Pathology.)

human arteriosclerosis. Fourth, marked involvement of the major coronary arteries is often a prominent feature of atherosclerosis both in man and in rabbits that received the lipid-rich diet and repeated injections of foreign serum. Finally, as in atherosclerosis in man, arterial lesions characterized by fibromuscular intimal thickening without manifest lipid are a part of the spectrum of arteriosclerosis seen in these rabbits. As in man, there is evidence to indicate that such areas of intimal thickening may later preferentially accumulate lipid and evolve as atherosclerosis. Because the arterial lesions induced in rabbits of these experiments bear such close resemblance to human atherosclerosis, it becomes especially important to study the early lesions in rabbits to learn more about the pathogenesis of atherosclerosis in man. Recently, we have examined sites of immunologic arterial injury that

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FIGURE 10. Scanning electron photomicrograph of endothelial surface of coronary artery of rabbit that received two intravenous injections of horse serum and cholesterol-supplemented diet. Platelets adhere to the endothelial surface in area of junction between endothelial cells. Note the opening between endothelial cells near adherent platelet (arrow). x 2250. (From Minick.”’ By permission of Advances in Experimental Medicine and Biology.)

result from immune complex disease by the relatively new technique of scanning electron In these investigations, we have examined the endothelial surface of the proximal portion of coronary arteries in rabbits that received two injections of foreign serum protein at 16-day intervals and were sacrificed 7-14 days after the last injection. Some rabbits were fed cholesterol-supplemented diets, and other rabbits were fed diets low in lipid. Endothelial changes induced in rabbits by immunologic arterial injury alone appear similar to those induced in other rabbits by the

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synergy of immunologic arterial injury and lipid-rich diets. In both groups, there are distinct changes on the surface of coronary arteries in areas of experimentally induced immunologic injury (FIGURE10). Platelet aggregates and individual platelets loosely adhere to the endothelium, particularly within junctions between endothelial cells. In adjacent areas, platelets are present in openings between endothelial cells. Similarly, leukocytes adhere to the endothelium and are present within small openings between endothelial cells. In other areas, large numbers of leukocytes fill gaps in the endothelium. Occasionally, we have observed areas in which the endothelium is denuded. Such areas are frequently, but not always, covered with leukocytes and platelets. Other partially

FIGURE11. Half-micron thick section of Epon@-embedded blocks of main coronary artery of transplanted rabbit heart that survived for 18 days. Partially

desquamated endothelium and platelet thrombus overlie disrupted internal elastic lamina. The intima is not thickened, and the subjacent media is intact. Lymphocytes and plasma cells are present in the adventitia;iron hematoxylin. x 137.

denuded areas are covered by markedly attenuated cells that may represent either regenerating endothelial cells or perhaps smooth muscle cells. We have utilized transmission electron microscopy to examine sites of immunologic arterial injury due to graft rejection.34 In early arterial lesions, that is, lesions without intimal thickening or appreciable medial change, we have observed platelet plugs in widened intracellular junctions between endothelial cells, adherence of partially degranulated platelets and apparently normal platelets and leukocytes to endothelium, and sloughing of endothelium with platelets adhering to underlying 11 & 12). In some of these areas, platelets are found basement membrane (FIGURES well beneath the internal elastic lamina within the subjacent media. In more ad-

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vanced lesions, proliferating intimal and medial cells without lipid, in addition to foam cells, often have characteristics of smooth muscle cells. Thus, observations of ostensibly early or evolving lesions that result from immunologically induced injury due either to immune complex disease or to graft rejection indicate that in both instances, endothelial injury may precede both the intimal thickening and the lipid accumulation. The results of experiments described herein demonstrate that the synergy of immunologic injury induced by serum sickness or graft rejection can lead to atherosclerosis in rabbits that, both with regard to its distribution in the arterial tree and in its morphology, resembles human atherosclerosis. The synergy of modest dietary hypercholesterolemia like that in Western man, average serum cholesterol concentra-

F I G U R E12. Electron photomicrograph of portion of large coronary artery illustrated in FIGURE10. Endothelial cell has partially lifted away from subjacent arterial wall, and platelets have reacted with exposed collagen and basement membrane. x 20,000.

tions between 200 and 250 mg%, and recurrent immunologic injury protracted over a period of many months led to atherosclerosis in rabbits that bore striking resemblance to some stages of human atherosclerosis. Repeated injections of foreign serum protein or graft rejection in rabbits fed diets low in lipid led to fibromuscular intimal thickening without manifest lipid. In other experiments, it was shown that in the presence of elevated serum cholesterol, sites of immunologically induced fibromuscular intimal thickening accumulate lipid preferentially as compared to the normal arterial wall. Preliminary results of more recent experiments indicate that endothelial damage and interaction of platelets and leukocytes with the surface of the artery are important early changes in the pathogenesis of the atherosclerosis induced in these rabbits. Because this rabbit atherosclerosis bears such close resem-

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blance to atherosclerosis in man, it is reasonable to suggest that similar changes in the endothelium may be important early changes in human atherosclerosis. I t is becoming increasingly evident that injury to the arterial wall is probably a primary causative factor in atherosclerosis. Rossle hypothesized3 and Haust recently supportedg the view that atherosclerosis is in essence an inflammatory process in which arterial injury, local reaction, and repair occur in sequence with local degenerative phenomena. We, similarly, look upon atherosclerosis as a condition in which inflammation and degenerative processes occur in reaction to various injuries and are modified in varying degree by insudated blood-borne elements, including lipid in particular. Changes in endothelial permeability are an essential feature in the inflammatory process, and increased permeability to and/or changes in the transport of lipoproteins may be necessary to the development of atheromas. There is a rapidly growing body of evidence to support the hypothesis that vasc'ular injury, and, in particular, endothelial injury, may be an early event in the pathogenesis of atherosclerosis. This hypothesis is based on the premise that normal endothelium acts as a barrier to a substance(s) in the blood that, upon exposure to vascular smooth muscle, promotes smooth muscle proliferation in the media and migration of proliferating smooth muscle cells to the intima, where they contribute to fibromuscular intimal thickening.45 Considerable experimental evidence obtained in vivo and in vitro supports the concept that endothelial injury may be an important early factor in arteriosclerosis. Fibomuscular intimal thickening occurs at arterial branch sites that are subjected to hemodynamic stress, a form of mechanical injury. Moreover, there is evidence for increased endothelial turnover in these locations, presumably a result of endothelial injury and repair.46 Arterial injury induced by stripping endothelium with a balloon catheter, a technique said to leave the subjacent wall intact, will lead to accumulation of platelets and leukocytes on the lining of arteries and subsequently to rapidly developing fibromuscular intimal thickening both in rabbits and in nonhuman prim a t e ~ . ' ~Moreover, .~~ evidence obtained from in vitro studies of arterial smooth muscle suggests that low-density lipoproteins, and, in particular, low-density lipoproteins from hyperlipoproteinemic animals, will lead to smooth muscle proliferation in tissue ~ u l t u r e . Recently, ~ ~ . ~ ~ it has also been shown that protein liberated from platelets may cause proliferation of smooth muscle cells in tissue Thus, endothelial injury associated with increased endothelial permeability and platelet adherence may expose the underlying arterial smooth muscle to low-density lipoproteins and proteins liberated from platelets, resulting in smooth muscle proliferation and fibromuscular intimal thickening. Changes in endothelial structure and permeability are also an essential feature in the pathogenesis of arterial injury and arterial inflammation that result from immunologic arterial injury. In immune complex disease, one of the animal models of immunologic arterial injury used in these experiments, there is strong indirect evidence to indicate that the deposition of immune complexes and the ensuing necrosis of the arterial wall and accompanying inflammation result from changes in endothelial permeability triggered by the release of vasoactive amines from platelets and leuko~ y t e ~ . Colloidal ~ ~ J ~ carbon . ~ ~ accumulates in arteries of animals with immune complex disease, which indicates increased permeability of endothelium to this macromolecule.4QMoreover, anthihistamines and platelet depletion will result in a striking reduction in the incidence of arterial lesions that result from immune complex disease. Endothelial injury is also believed to be an essential feature in the pathogenesis of at least some instances of arterial injury that result from graft rejection, particularly that induced by hyperacute graft rejection. Results of preliminary experiments reported here furnish direct evidence that endothelial injury is an im-

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portant early event in lesions of immunologic vascular injury due to immune complex disease and chronic graft rejection. These sites of endothelial injury appear to precede changes of fibromuscular intimal thickening that, in the presence of increased serum lipid, may evolve as atherosclero~is.~’ In searching for causes of arterial injury that may lead to atherosclerosis in man, it has been proposed that allergy to antigens in infecting microorganisms and vaccines, foreign serum, antibiotics and other drugs, tobacco, foodstuffs, and the individual’s own tissues may be causative of much arterial disease that can evolve as atherosclerosis. Persistent hepatitis B antigenemia is associated with an increased incidence of v a ~ c u l i t i s and ~ ~ ~intimal ~’ hyperplasia, some of which may evolve as atherosclerosis. Viral antigens are also thought to be important in the genesis of vascular disease in other animals, and some of this vascular disease may also evolve as atherosclerosis. Patterson et al. observed that preexisting spontaneously occurring vascular disease predisposed to the deposition of lipid and genesis of atherosclerosis in chickens fed cholesterol-supplemented diets. They suggested that the initial arterial lesions in these chickens may result from the viral agent of Marek’s disease, now known to be a herpes Sponor from an allergic reaction to the taneously developing arteriosclerosis is known to occur in squirrel monkeys.5s Vasculitis and a high incidence of glomerulonephritis occurs in these nonhuman primatess6 and may be a result of immunologic injury. It is reasonable to suggest that arteriosclerosis in these monkeys evolves from lipid deposits at sites of vasculitis. In man, other antigens may also be important. Serum antibodies to heat-dried cows’ milk and boiled egg white are increased in the sera of patients with coronary heart disease and myocardial infar~tion.~’ Harkavy and Perlman have shown that patients with coronary disease who smoked have an increased incidence of positive skin tests to tobacco leaf protein, as compared to smokers without coronary artery disease.ss Antibodies directed against tissue antigens may be of importance in the rapidly developing arteriosclerosis seen in cardiac homotransplants and in the coronary arteritis and premature coronary atherosclerosis seen in some cases of lupus erythematosus. It has been suggested that the increased incidence and severity of vascular disease in diabetes are due to antibodies to insulin or to immune complex disease that results from the increased incidence of organ-specific antibodies to thyroid and gastric antigen^.^^*^^ In many of these instances, the association between immunologic reaction to various foreign and self-antigens and arteriosclerosis is admittedly speculative and may be fortuitous. It is possible, however, that immunologic reaction to these and other antigens may lead to repeated or protracted immunologic arterial injury and local reactive changes that may favor repeated or protracted deposition of bloodbone elements, in particular lipid, in the arterial walls and thus lead to atherosclerosis. This possibility is supported by the results of experiments reported here.

REFERENCES N. N. 1933. Experimental arteriosclerosis in animals. I n Arteriosclerosis: A Survey of the Problem. C. V. Cowdry, Ed.: 271-322. Macmillan & Co. New York, N.Y. 2. DUFF, G. L. 1935. Experimental cholesterol arteriosclerosis and its relationship to human arteriosclerosis. Arch. Pathol. 20: 80-123, 259-304. 3. ROSSLE, R . 1944. Uber die Serosen Entzundungen der Organe. Virchows Arch. Pathol. Anat. 311:252-284. 4. HASS,G. L. 1955. Observations on vascular structure in relation to human and experimental arteriosclerosis. I n Symposium on Atherosclerosis.: 2 4 3 2 . National Academy of Sciences. Washington, D.C. I.

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