Robert W. Wissler

A Definition of Advanced Types of Atherosclerotic Lesions and a Histological Classification of Atherosclerosis A Report From the Committee on Vascular Lesions of the Council on Arteriosclerosis, American Heart Association

This report is the continuation of two earlier reports that defined human arterial intima and precursors of advanced atherosclerotic lesions in humans. This report describes the characteristic components and pathogenic mechanisms of the various advanced atherosclerotic lesions. These, with the earlier definitions of precursor lesions, led to the histological classification of human atherosclerotic lesions found in the second part of this report. The Committee on Vascular Lesions also attempted to correlate the appearance of lesions noted in clinical imaging studies with histological lesion types and corresponding clinical syndromes. In the histological classification, lesions are designated by Roman numerals, which indicate the usual sequence of lesion progression. The initial (type 1) lesion contains enough atherogenic lipoprotein to elicit an increase in macrophages and formation of scattered macrophage foam cells. As in subsequent lesion types, the changes are more marked in locations of arteries with adaptive intimal thickening. (Adaptive thickenings, which are present at constant locations in everyone from birth, do not obstruct the lumen and represent adaptations to local mechanical forces). Type II lesions consist primarily of layers of macrophage foam cells and lipid-laden smooth muscle cells and include lesions grossly designated as fatty streaks. Type III is the intermediate stage between type II and type IV (atheroma, a lesion that is potentially symptom-producing). In addition to the lipid-laden cells of type II, type III lesions contain scattered collections of extracellular lipid droplets and particles that disrupt the coherence of some intimal smooth muscle cells. This extracellular lipid is the immediate precursor of the larger, confluent, and more disruptive core of extracellular lipid that characterizes type IV lesions. Beginning around the fourth decade of life, lesions that usually have a lipid core may also contain thick layers of fibrous connective tissue (type V lesion) and/or fissure, hematoma, and thrombus (type VI lesion). Some type V lesions are largely calcified (type Vb), and some consist mainly of fibrous connective tissue and little or no accumulated lipid or calcium (type Vc).

A study of the immunohistochemical localization of serum lipoproteins and other plasma proteins in human atherosclerotic lesions

Abstract Multiple unfixed 4μ sections from 53 areas of 28 human aortas ranging from premature infants to a 94-year-old subjects were studied microscopically by using fluorescent-labeled rabbit antibodies to immunoelectrophoretically “pure” human plasma fractions, including LD lipoproteins and HD lipoproteins, albumin, gamma globulin, and fibrinogen. Some tissues were immersed in fixatives prior to staining. It was found that the fixation methods tested were not suitable for this type of investigation. The severity of the atherosclerotic lesions sampled varied from no gross evidence of disease, through fatty streak, fatty plaque with fibrous cap to complicated lesions. Adjacent cryostat sections from each aortic area were stained with Oil red O as well as Mallory picrofuchsin. Suitable control sections were also studied. Fluorescent anti-LD lipoproteins consistently stained diseased areas of aortic intima where lipid was revealed by Oil red O stain. The fluorescent anti-HD lipoprotein stained the diseased areas very faintly or not at all. The fluorescent anti-fibrinogen stained focal areas of intima which also contained lipoproteins. The fluorescent anti-albumin and anti-gamma globulin stained none of the lesions.

Ultrastructural and immunohistochemical studies of primary cultures of aortic medial cells

Abstract When small (2-mm) round explants of aortic media from Rhesus monkeys or rabbit are cultured in Eagles Basal Medium with 10% serum added cell growth starts in a monolayer within 1–2 weeks. Growth continues exponentially for 3–4 weeks. Elastin and acid mucopolysaccharide were demonstrated in electron micrographs. Mature collagen was not seen but vitamin C was not added to the culture medium. The proliferating cells were characterized as mature smooth muscle cells both ultrastructurally and by immunohistochemical methods. For the latter studies antibodies against smooth muscle cell actomyosin were labeled with horseradish peroxidase (HRP) using either difluoro-dinitrophenyl sulfone (FNPS) or glutaraldehyde as a coupling agent. After incubation with the labeled antibody, the cultures were stained for peroxidase with tetrachloridine to yield a brown reaction product against the yellow to light-tan background. With antiactomyosin antibody most cells were stained in a rather uniform fashion. Pretreatment of the cultures with unlabeled antibodies prevented positive staining and labeled normal gamma globulin did not stain the cells. Adventitia from the same aortas yielded rapidly growing control cultures which contained only a few cells which reacted positively with the anti-actomyosin. With the same immunohistochemical technique we have also obtained evidence for the uptake of low-density lipoproteins (LDL) by these cells using specific antibodies to LDL. As a control, antigenic proteins of high-density lipoproteins (HDL) have been used to immunize rabbits or chickens and their resulting antibodies have been similarly labeled. LDL apoprotein was present in sufficient quantities in these cells to be demonstrated by this method with either light or electron microscopy. The arterial medial cell has been implicated in atherogenesis and it is proposed that such cultures may serve as a model to measure the response of these cells to selected atherogenic stimuli.

Atherosclerosis in the rhesus monkey fed three food fats.

Abstract Three groups of 6 male Rhesus monkeys were fed one of three diets, each containing 2% cholesterol and 25% lipid, either corn oil, butterfat, or peanut oil, over a period of 50 weeks. These diets produced prompt elevation of serum lipids. The highest serum cholesterol concentrations were observed in animals fed butterfat and the levels in animals fed peanut oil and corn oil were similar and much lower. Gross examination of the arteries revealed a definite difference in the degree and the characteristics of aortic atherosclerosis among the three dietary groups. The butterfat diet produced severe aortic lesions, characterized by abundant lipid deposition and relatively little cell proliferation or collagen deposition. The most widespread and advanced atherosclerosis was observed in monkeys fed peanut oil. The aortic lesions in these animals were characterized by thick, fibrous plaques which were elevated to the point of apparent narrowing of the ostia of various vessels. In addition, the peanut oil ration produced not only the highest incidence of coronary artery involvement but also the most severe coronary narrowing. This was apparently due to prominent intimal cell proliferation associated with a particularly high content of collagen. In spite of similar blood lipid elevations, the corn oil fed monkeys showed fewer gross atheromatous lesions in all parts of the arterial system and there was relatively little intimal thickening and much less lipid in the lesions. This study adds to the growing body of experimental evidence in several animal species that the tissue components and the severity of atherosclerotic lesions can be greatly influenced by the food fat fed and that peanut oil is an unusually atherogenic fat, mainly because of the severe intimal proliferation and fibrosis that occur.

Cholesterol vehicle in experimental atherosclerosis Part II. Peanut oil

Abstract In 4 consecutive experiments, young adult, male, Dutch belted rabbits were fed diets, containing 6% of fats of varying fatty acid composition. These fats included coconut oil (CNO), corn oil (CO), peanut oil (PNO), and a special fat (PGF) simulated to resemble peanut oil minus arachidic and behenic acids. All diets contained 2% cholesterol and were fed to the rabbits for 8 weeks. Biochemical and histological findings were then compared. In the 4 experiments, the following characteristics were generally seen. The rabbits fed corn oil showed a slightly greater weight gain than did the other groups, with the group fed coconut oil exhibiting the least weight gain. The liver weight of the group fed coconut oil was consistently lowest, as was the liver cholesterol content of this group; the liver weight and liver cholesterol levels observed in the other 3 groups were comparable. Serum cholesterol levels were highest in the group fed the special fat and those fed corn oil. Visual grading of the prestained aortas showed the groups fed the special fat or corn oil to have lesions of comparable severity, but less severe than the lesions of the groups fed coconut or peanut oil, which were similar to each other. The gross visual and microscopic evaluations of the severity of aortic lesions indicated that the rabbits fed coconut or peanut oil had the most extensive, as well as the most frequent and severe, lesions. In contrast, the animals fed the special fat consistently showed much less aortic disease, no matter how the disease was evaluated; generally in these animals the aortic involvement was similar to that seen in the rabbits fed corn oil. The main characteristic of the lesions seen in rabbits fed peanut oil was a prominent intimal proliferation with a high proportion of collagen, whereas in animals fed coconut oil intimal proliferation was over the area of lipid deposition. The trend in severity and frequency of the coronary artery lesion was similar to that seen in the aorta. Intimal proliferation with lipid deposition was a characteristic finding, regardless of the lipid fed. These findings support the concept that arachidic and behenic acids, which are present in peanut oil, but not in the special fat, may be responsible, in part, for the unexpectedly greater atherogenic effect of peanut oil. However, our observations suggest that other factors, such as triglyceride structure, may also play an important role in atherogenesis.

Cholesterol vehicle in experimental atherosclerosis Part 13. Randomized peanut oil

Abstract Peanut oil (PNO) is more atherogenic than is corn oil (CO) when fed to rabbits in a diet containing 2 % cholesterol and 6 % fat. To test the hypothesis that the arachidic and behenic acid content of PNO is responsible for this observation, four groups of rabbits were fed 2 % cholesterol plus 6 % PNO, CO or two fats, prepared by interesterification (randomization) of CO and a special fat, PGF, with a fat containing arachidic and behenic acids. The new fats, CO/R and PGF/R, contained levels of arachidic and behenic acids similar to those found in PNO. Average atheromata (arch plus thoracic/2) for a series of 3 experiments were: PNO — 1.59; PGF/R — 1.41; CO/R — 1.34; CO — 1.20 and PGF — 1.17. The data suggested an effect beyond fatty acid composition. We then compared PNO, CO and autorandomized peanut oil, PNO/A. The fatty acid composition of PNO/A is identical with that of PNO, but its triglyceride structure is markedly different. Average atheromata for 3 experiments were: PNO — 1.88; PNO/A — 1.18 and CO — 1.17. The data clearly indicate that triglyceride structure of a fat, as well as its fatty acid composition, is a determining factor in its atherogenic potency.

Studies of regression of advanced atherosclerosis in experimental animals and man.

In general, the results to date in humans and experimental animals seem to indicate that substantial regression of advanced atherosclerosis is possible. The results also indicate that the advanced atherosclerotic lesions are much more likely to respond favorably if the serum cholesterol concentrations are reduced to the minimum that prevails in animals or people who consume a low-fat low-cholesterol diet. In human subjects and in rhesus monkeys, this value appears to be about 150 mg%. Under these circumstances, much of the lipid disappears from the plaques, and the remaining fibrous tissue and cells appear to condense and undergo remodeling, as they do in fracture or wound healing. Additional effort will be required to ascertain how rapidly and how much of the fiber proteins and calcium can be removed from the advanced plaques and to work out methods that will consistently produce regression of advanced atherosclerotic lesions in human subjects. This goal would appear to be worth working toward. Interruption of progression of atherosclerosis appears to be more easily achieved, and it also would appear to be a worthwhile goal. The diagram that is reproduced as FIGURE 2 presents the multiple methods of intervention in atherosclerosis that are now available to the physician and to the patient. To those of us who look on atherosclerosis as an almost completely preventable disease and one that is largely reversible, the following quotation from the perceptive essay by Lewis Thomas seems to be prophetic and most appropriate. An extremely complex and costly technology for the management of coronary heart disease has evolved, involving specialized ambulances and hospital units, all kinds of electronic gadgetry and whole platoons of new professional personnel to deal with the end results of coronary thrombosis. Almost everything offered today for the treatment of heat disease is at this level of technology, with the transplatned and artificial hearts as ultimate examples. When enough has been learned for us to know what really goes wrong in heart disease, we ought to be in a position to figure out ways to prevent or reverse the process; and when this happens, the current elaborate technology will be set to one side.

Stimulation of proliferation in stationary primary cultures of monkey and rabbit aortic smooth muscle cells: I. Effects of Lipoprotein Fractions of Hyperlipemic Serum and Lymph

Abstract Medial explants of thoracic aorta from rhesus monkey or rabbit show outgrowth within 5–10 days when cultured in 90% basal Eagles medium and 10% normal serum. After 5–6 weeks of rapid growth, cell colonies around the explant reach a stationary phase with low mitotic activity. Homologous hypercholesterolemic serum and lymph stimulate these stationary cultures into another proliferative phase, when 5% of the normal serum is replaced in the culture medium. Measurement of increase in colony size and evaluation of [ 3 H]thymidine incorporation by autoradiography serve to indicate increased proliferation. Low density lipoproteins (LDL) from hyperlipemic serum and lymph have the greatest stimulatory effect, while high density lipoproteins (HDL) and the lipid-free bottom fraction, as well as LDL from normal serum and lymph, have no effect. These stationary cultures are valuable for the study of agents suspected of having a stimulatory effect on cell proliferation of aortic lesions in vivo .

Risk Factors and Progression of Atherosclerosis in Youth

“The Pathobiological Determinants of Atherosclerosis in Youth” (PDAY), a unique research program initiated in 1985, has now developed into a detailed and comprehensive 15-center investigation of the arteries of more than 3,000 young persons who died between 15 and 34 years of age (Appendix A). The main objectives of this highly organized multicenter study are to compare the quantitatively evaluated risk factors for coronary heart disease including blood lipid values, evidence of smoking, indices of hypertension, tendency toward diabetes, etc., with the results of macroscopic and microscopic quantitation of severity and with microscopic components of developing atherosclerotic lesions in these young people. 1-5 This commentary will summarize some of the results which have been published in more than 75 full-length reports, with emphasis on the results obtained because of the unusual and frequently unique features of the study’s protocol, which was developed during a 10-year period before the study began. 6-17 Reported findings can be divided into two categories. Results in the first category are derived from the gross evaluation of the extent and severity of lesions. 18-28 Computer-assisted mapping was also applied to raised lesions traced out by the pathologists. 28,29 The second category of reported results comes from detailed micromorphometric, microchemical, and immunohistochemical quantitative data based on the major microscopic components and the classification of each lesion. 30-39 In fact, as the study progressed, it became possible to classify the four major types of intermediate lesions that are associated with different rates of progression of the atherosclerotic process in the aortas and coronary arteries of these young people as well as with certain risk factors. 40,41 Because lesions tend to increase in extent, numbers, and severity with age, it is assumed that this in-depth quantitative analysis of the atherosclerosis found in each of these cases from the 15–34 age group may give new insights into why some young people’s plaques seem to progress rapidly with age while others’ remain almost stationary, as well as a comparison of progression in various parts of the arterial tree. 20,22,30,42 Results of the study reflect the state of atherosclerosis development in young people living in the USA late in the 20th century. All case material came from forensic autopsies on young individuals who had no evidence of chronic debilitating disease and who succumbed suddenly to traumatic or other fatal episodes. The intervals between death and refrigeration of the body, as well as performance of the autopsy, were generally short. This has made it possible for the tissues and cells to be studied using a number of pathobiological methods appropriate for well-preserved human tissues. 31-41 In order to insure an adequate and representative autopsy population, nine of the centers selected, approved, and funded functioned as “collecting centers.” All nine are geographically and/or organizationally related to forensic laboratories where state and local regulations make it possible to collect and utilize small samples of tissue for research purposes. Most of the principal investigators leading the 15 centers have a long-standing interest and a record of productivity in the field of atherosclerosis research with an emphasis on the artery wall and the pathogenesis of atherosclerotic lesions. 5 Centers were selected during a period of approximately two years (1983–85) of intensive preliminary meetings of potential principal investigators. The overall plans took shape over a 10-year period (1975–85) during which a carefully prepared and detailed protocol as well as a detailed manual of procedures for the study were developed. 5,34,36 Furthermore, during this period the steering committee developed a standardized sampling strategy and procedures based on the results of an NIH-funded preliminary study by Dr. Frederick Cornhill, Director of the Biomedical Engineering Center at Ohio State University, and Dr. Herbert Stary, Professor of Pathology at Louisiana State University. This preliminary study established the patterns of lesion location most likely to develop in the aorta 5,29 and helped establish the best sites to study in the proximal coronary arteries. This commentary will conclude with a list of major opportunities for future studies afforded by the accumulated data and the currently unused PDAY samples of arteries and other tissues.

Reversal of advanced atherosclerosis in Rhesus monkeys: Part 1. Light-microscopic studies

Abstract The regression of atherosclerotic lesions in Rhesus monkeys was evaluated by means of a low-fat, low-cholesterol diet with or without N-γ-phenylpropyl-N-benzyloxy acetamide (W-1372). Moderate to severe aortic and coronary atherosclerosis was induced by feeding 4 groups of male monkeys a high-fat, high-cholesterol diet for 18 months, after which the first group was autopsied for assessment of the lesions. During a subsequent 18-month regression period, the second group of animals was fed a low-fat, low-cholesterol diet with W-1372, and the third group the low-fat, low-cholesterol diet without W-1372. A pair of monkeys (the fourth “group”) was fed an atherogenic diet throughout the experiment. Serum cholesterol, which increased about 5-fold during the induction period, returned to baseline values or below in the 2 treated groups. Evidence of regression of lesions was obtained in both these groups, but was most noticeable in the monkeys fed the low-fat, low-cholesterol diet without W-1372. The aortas of the animals treated with the low-fat, low-cholesterol diet with or without W-1372 showed about two-thirds as many lesions which were on the average about half as severe as those in the animals killed at 18 months. The coronary artery lesions showed a similar contrast, with the treated groups having about one-third to one-half as many lesions which were about one-half to two-thirds as severe. In both locations the differences in frequency and severity of arterial lesions were statistically significant when the reference group killed at 18 months was compared with the group treated with the low-fat, low-cholesterol diet without W-1372.