Dragoslava Vesselinovitch

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.

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.

Regression of atherosclerosis in rabbits

Abstract The regression of rabbit atherosclerosis by means of a low-fat diet and hyperoxia either alone or in combination with cholestyramine or estrogen was investigated. During the induction period of 14 weeks, moderate to severe atherosclerosis was produced in male New Zealand rabbits by feeding them a high-cholesterol diet. The rabbits were then divided into various treatment groups. During an additional 10 week regression phase, all animals except those in one group were fed a low-fat diet. Serum lipids, which increased considerably during the induction period, were reduced by all treatments but most effectively by a low-fat diet combined with cholestyramine or with hyperoxia plus cholestyramine. There was little evidence for regression of lesions resulting from a low-fat diet alone. Evidence for regression of lesions was most apparent in rabbits treated with a combination of a low-fat diet and hyperoxia with either cholestyramine and/or estrogen. These results suggest a substantial additive beneficial effect of the combined therapy.

The effect of estradiol on the proliferation of rabbit aortic medial tissue culture cells induced by hyperlipemic serum

The outgrowths of medial explants of thoracic aorta from New Zealand rabbits were used to study the influence of estrogen on cell proliferation. After 5-6 weeks of rapid growth in Basal Eagle Medium (BME) supplemented with 10% normal rabbit serum, such cultures reached a stationary phase during which they showed little mitotic activity and little further increase in surface area. Replacement of 5% of the normal serum with hyperlipemic rabbit serum resulted in a stimulation of these stationary cultures into a phase of renewed proliferation, which was measured directly as increase in cell culture size and by [3H]thymidine incorporation visualized by autoradiography. The addition of estrogen (estradiol, Progynon, Schering Corp.) in a concentration of 0.02 microgram/ml to the culture medium inhibited the proliferative effect induced by the hyperlipemic serum. On the other hand it had no effect on the growth rate of such explant cultures during their rapid growth phase if added at the time of explantation for 6 weeks. This would indicate that the inhibition of the hyperlipemic serum-induced proliferation by estrogen is not due to a toxic effect on mitosis in general. Cells exposed to estrogen tended to have larger amounts of intracellular lipid as visualized by oil red O staining. Moreover, prolonged exposure to estrogen resulted in a significant decrease in stainable collagen and elastin in these cultures.

A dynamic pathology of atherosclerosis

Abstract Currently available evidence indicates that atherosclerosis of large and medium arteries is associated with the influx and trapping of low-density plasma lipoprotein within the myointimal and medial cells of the arterial wall. Many factors, including hyperlipemia, hemodynamic arterial injury and hypoxia, hasten and facilitate this process, but too little is known of the factors and mechanisms responsible. The role of local platelet aggregation in initiating localized vascular injury and increased vascular permeability is yet to be fully clarified. Blood vessels can no longer be viewed as mere blood conduits. They are composed mainly of actively metabolizing smooth muscle cells, which have a wide range of metabolic potentialities, summarized diagrammatically in Figure 6. An understanding of the metabolism of these cells is central for an appreciation of the pathogenesis of atherosclerosis for it is within these cells that low-density lipoprotein is first entrapped and accumulates. These cells, especially in the deeper layers of the inner media, exist on the brink of hypoxia and are therefore particularly susceptible to a variety of noxious stimuli. The exact identity of the factors responsible for the initiation of a particular smooth muscle cell response to injury remains unknown. However, from the experimental evidence of the characteristic smooth muscle cell response to various food fats it seems likely that certain components of the low-density lipoproteins in appropriate experimental animals elicit the typical vascular response associated with the feeding of a given food fat. Identification of the lipoprotein components important for the development of a particular cellular reaction pattern is a major problem in atherosclerosis research. A thorough study of the enzymatic potentialities and responses of smooth muscle cells is necessary for at least three reasons: 1.(1) Detailed knowledge of the metabolic characteristics of these cells will be provided. 2.(2) Such studies may also uncover a suitable quantitative measure of the incipient atherosclerotic process—a measure which would be most useful for experimental elucidation of the early pathogenesis of this disease. 3.(3) It is through such studies that we can hope to gain a better appreciation of the potentialities for dealing with this serious health hazard.

Can atherosclerotic plaques regress? Anatomic and biochemical evidence from nonhuman animal models.

For at least 60 years, spotty and poorly documented evidence has suggested that atherosclerotic disease in humans might be reversible. Little direct evidence was available until researchers demonstrated that rather advanced atherosclerotic lesions in experimental animals could show marked improvement after blood-lipid-reducing regimens that were often combined with other measures, such as increased ambient oxygen and estradiol therapy. In fact, this combination was used in this laboratory to produce one of the first effective regression studies in the rabbit model. In more recent studies in this laboratory, abundant evidence has been obtained that the advanced, eccentric, largely intimal lesions produced in the rhesus monkey are substantially reversible, and the much more inflammatory, concentric, and often transmural atheroarteritis induced by the same atherogenic ration in the cynomolgus monkeys is much more resistant to effective and beneficial regression. This unusual reaction appears to be due to the circulating immune complexes that participate in the pathogenesis of atherosclerosis in these cynomolgus monkeys, as well as possibly in a number of humans. The evidence for this phenomenon, as well as the varying effects of the lesions induced with contrasting food fats, is summarized in this presentation. Some of the time-related effects of varying interventions when the lesions are studied at 4-month intervals in rhesus and cynomolgus monkeys are also discussed. Other factors that may influence regression are also considered.