W.A. Thomas

Atherosclerosis in rabbits: Architectural and subcellular alterations of smooth muscle cells of aortas in response to hyperlipemia

Abstract Rabbits have been extensively used in the past for experimental production of arterial lesions by simple short-term cholesterol feeding. However, most lesions thus produced have been composed principally of foam cells and have seldom developed necrosis and complications such as calcification, ulceration, and thrombosis, in sharp contrast to human atherosclerotic lesions. Some recent experiments have indicated that by introducing certain types of dietary regimen non-necrotic proliferative arterial lesions and also necrotic intimal lesions strikingly similar to human atheroma can be produced in rabbits. However, the necrotic lesions were presumably transformed from foam cell lesions and the sequence of events does not appear to have been precisely the same as in man. The current study was carried out in an attempt to (1) produce by dietary means atherosclerotic lesions in rabbits with a pathogenetic sequence of events more closely approximating those in man, and (2) study by electron microscopy changes that might occur in architectural arrangement and subcellular components of smooth muscle cells of the inner media and of the intimal masses in the early stages of atherosclerosis. By feeding rabbits high fat-cholesterol diets, non-necrotic, and necrotic atherosclerotic lesions closely resembling human atheroclerosis have been produced within 12 weeks. The most prominent feature of non-necrotic lesions produced was the smooth muscle cells in either the mature or immature form or in any stage of maturity between the two extremes. Smooth muscle cells containing a large amount of lipid and macrophages containing variable amounts of fat were far more common in rabbit lesions than in most human lesions. In man non-necrotic atherosclerotic lesions consisting largely of smooth muscle cells appear to progress to necrotic atherosclerotic lesions. In the current study this occurred in only one rabbit, probably because of the short duration of experiments. In the atherosclerotic lesions of these rabbits cells that appear to be extremely active exist side by side with cells appearing much less active, which may even contain degenerative elements. The direct effect of the diet may be to depress and damage smooth muscle cells of the vessel wall with the “active cells” appearing as a secondary phenomenon in response to products released by injured cells. Another possibility is that the direct effect of the diet is to stimulate the metabolism of smooth muscle cells of the intima with damage of some occurring as a result of excessive stimulation. Other explanations are also possible but all must take into account the metabolism of smooth muscle cells of inner media and intima. In both man and rabbits changes in the internal elastica and the inner media appear concomitantly with intimal changes. The changes in the internal elastica include widening of the fenestrae making the intima and inner media essentially one unit in many areas. Changes in the smooth muscle cell of the inner media parallel those of the smooth muscle cell in the intima and include distention with lipid and appearance of fibroblast-like smooth muscle cells. Many smooth muscle cells actually lie within the fenestrae of the internal elastica leading to a speculation that smooth muscle cells migrate between the inner media and intima, perhaps in both directions. The essence of the pathogenesis of atherosclerosis in both man and rabbits would appear to be the direct or indirect response of smooth muscle cells in the intima and inner media to excess lipids. Difference in response between rabbits and man seem to be largely a matter of degree. Qualitative differences in response of the arterial wall due to species specificity may be present but there is little or no evidence to support this idea. With the information available the rabbit would appear to be as suitable for the study of certain fundamental aspects of atherosclerosis as any other mammal in spite of the fact that he is primarily a herbivore.

Experimental atherosclerosis in rhesus monkeys: II. Cellular elements of proliferative lesions and possible role of cytoplasmic degeneration in pathogenesis as studied by electron microscopy

Abstract The proliferative (non-necrotic) lesions of experimental atherosclerosis in the rhesus monkeys reported here are similar to those of the human in that they are composed primarily of mature smooth muscle cells, most of them containing lipid. In addition, there were small numbers of primitive cells, fibroblast-like cells, and smooth muscle cells of apparent intermediate maturity. This variety of morphologic classes of cells appears to reflect maturation of smooth muscle cells within the intimal lesion. Accompanying these cells were cells tremendously distended with lipid that could not be identified as to their histogenesis. They were probably either distorted smooth muscle cells or macrophages. Many foci suggesting cellular degeneration were found scattered throughout the proliferative lesions, while only a few were found in stock-fed monkeys. The increased number of cells in the proliferative lesions showing degenerative changes were apparently the indirect or direct result of feeding high-fat diets. Whatever their cause, it is likely that these foci of intracellular degeneration enhanced the development of the intimal lesion by stimulating the cells to proliferate.

Effects of a soy protein product on serum and tissue cholesterol concentratins in swine fed high-fat, high-cholesterol diets

Abstract Hypocholesterolemic effect of a soy protein product was studied in swine fed a high-fat, high-cholesterol diet. In the first experiment, a group of swine were fed 42% butter (by calories) and 1055 mg cholesterol daily, with casein as the source for protein, for 6 weeks and this diet resulted in moderately high serum cholesterol concentrations (219 ± 33 mg/dl). Another group fed the same diet except with soy protein product as the protein source instead of casein showed virtual normocholesterolemia at the end (107 ± 3 mg/dl). Cholesterol balance was studied under non-steady state conditions using methods designed for this purpose. Reflecting the serum cholesterol concentration, the total body cholesterol concentration (excluding CNS) was also significantly lower in soy protein group. However, parameters of cholesterol balance, such as fecal neutral and acidic steroid excretions, dietary cholesterol absorption, and whole body cholesterol synthesis were studied and no differences were demonstrated between the casein- and soy protein-fed swine. The experiment was repeated and in Experiment II virtually the same results were obtained. When swine were given the same high-fat, high-cholesterol diets with 1 2 casein + 1 2 soy protein or casein + soy protein, hypocholesterolemic effects were also observed. Therefore, such action is probably caused principally by soy protein per se rather than simply by replacement of casein by soy protein. Addition of dl -methionine to soy protein containing diet did not alter the hypocholesterolemic effect of soy protein indicating that the effect was not the result of methionine deficiency. In conclusion, we can state that the hypocholesterolemic action of soy protein was clearly demonstrated in swine fed a high-fat, high-cholesterol diet, but that the mechanism of action is yet to be established.

Rapid production of advanced atherosclerosis in swine by a combination of endothelial injury and cholesterol feeding

Abstract The aim of the current study was to find out whether a combination of a balloon-endothelial-cell-denudation procedure and cholesterol feeding would result in more rapid growth of atherosclerotic lesions in the abdominal aorta of swine than if either were used alone. The results far exceeded our expectation. The two procedures appear to act synergistically. In the first 2 or 3 mo lesions are patchy and scattered but by 6 mo they become confluent so that practically the entire abdominal aorta is covered with thick lipid-rich atherosclerotic lesions. The lesions produced by 6 mo have many of the characteristics of advanced human lesions.

Modification of lipoprotein patterns and retardation of atherogenesis by a fish oil supplement to a hyperlipidemic diet for swine

We have studied the effect of addition of 30 ml cod liver oil (FO) daily to a highly atherogenic butter (BT) diet for swine on lesion development in the coronary arteries and aorta, plasma lipoprotein (LP) patterns, plasma levels of thiobarbituric acid-reactive substances (TBARS) and on tritiated thymidine-labeling indices ([3H]TdR LI) of smooth muscle cells (SMC) and monocyte/macrophages (M/M phi) in the atherosclerotic lesions. Seventeen male Yorkshire swine (11.1 +/- 0.4 kg) were divided into 3 groups: BT (n = 6), BT + FO (n = 6) and mash (n = 5). They were fed the respective diets for 4 months. Terminally, fasting plasma was obtained and cholesterol contents were determined in various fractions of lipoproteins separated by density gradient ultracentrifugation, Pevikon block electrophoresis and immunoelectrophoresis. Apoprotein (B, A-I, E and C) contents of the plasma and lipoprotein fractions were determined by polyacrylamide gel electrophoresis and densitometry of gels stained with Coomassie blue. Swine were injected intramuscularly with 0.5 mCi/kg of [3H]TdR 2 h before death. The aorta and coronary arteries were perfusion fixed in situ under anesthesia. Samples were obtained for microscopic morphometry, autoradiography and immunohistochemistry from distal abdominal aorta, thoracic aorta, and proximal coronary arteries; left main (LM), left anterior descending (LAD), left circumflex (LCX), right main (RM), and right coronary artery (RCA). On the BT diet without FO there was extensive atherosclerotic (AS) lesion development, which was drastically reduced by the addition of FO to the BT diet in all sites by from 71 to 94%. The overall plasma cholesterol (CH) levels were reduced only modestly by the FO (816 +/- 64 to 629 +/- 14 mg/dl) but the distribution of CH in the various lipoprotein classes was remarkably altered. The CH in the large lipoprotein molecules containing both B and E apoproteins was reduced from 488 +/- 84 to 204 +/- 17 mg/dl by the FO with an almost corresponding increase in the conventional LDL molecules containing apo B only (158 +/- 29 to 344 +/- 15 mg/dl). We offer the hypothesis that the large apo B,E containing molecules are much more atherogenic than the smaller apo B containing molecules. This hypothesis is supported by a highly significant correlation between extent of lesion development in all arterial sites and plasma levels of CH in apo B,E containing lipoproteins. Plasma TBARS were elevated by the BT + FO diet but seemed to have no significant effect on the lesions.(ABSTRACT TRUNCATED AT 400 WORDS)

Short-term feeding of unsaturated versus saturated fat in the production of atherosclerosis in the rat☆

Abstract Complex diets containing either 40% peanut oil, corn oil, or butter were fed to weanling rats for 3 months. The rats fed the peanut oil-containing diets developed intimal spindle cell lesions similar in many respects to early human atherosclerotic lesions. Rats receiving the butter-containing diets developed foam cell lesions which do not resemble human atherosclerosis. Under electron microscopy the spindle cells were of the smooth muscle type. They appeared more undifferentiated than the smooth muscle cells seen in aortic lesions in rats fed butter-containing diets for 12 months in a previous experiment. The primitive nature of the cells in the present experiment may have been due to their age, their unsaturated fatty acid environment, or other factors. The difference in serum fatty acids was possibly an important factor determining the type of lesion produced, since both the peanut oil- and butter-fed rats had much the same degree of hyperlipemia as shown by similar serum cholesterol and triglyceride levels. Both the peanut oil- and butter-fed rats showed a sharp drop in the relative percentage of serum arachidonic acid, even though the peanut oil-fed group had excessively high levels of serum linoleates. The absolute level of serum arachidonic acid, however, changed very little.

DISTRIBUTION OF INTIMAL SMOOTH MUSCLE CELL MASSES AND THEIR RELATIONSHIP TO EARLY ATHEROSCLEROSIS IN THE ABDOMINAL AORTAS OF YOUNG SWINE

In the abdominal aortas of young mash-fed swine, intimal cell masses (pads, cushions) are located predominantly away from blood vessel orifices. They are found scattered throughout the aorta but nevertheless have a definite pattern of distribution. In the distal one half of the abdominal aorta, they are more frequent in the ventral quandrant than in the dorsal or either lateral quadrant. In the proximal half, intimal cell masses are more frequent in the dorsal quadrant. When experimental atherosclerosis is induced in the abdominal aortas of young swine by either a hypercholesterolemic diet or by aortic ballooning followed by a hypercholesterolemic diet, the distribution of early lesions is similar. The lesions are found predominantly in quadrants where intimal cell masses were found to be most frequent in the control group of swine. The results suggest that most of the lesions, though not necessarily all, arose from pre-existing intimal cell masses beneath the aortic surface.

Increased steroid excretion in swine fed high-fat, high-cholesterol diet with soy protein

Abstract Hypocholesterolemic mechanism of soy protein when added to high-fat, high-cholesterol (HC) diet as compared to casein was studied in young male Yorkshire swine (10 kg) in two experiments. Three soy protein products were used: Soy Protein A, B, and C. Soy Protein A is Pro-Lean™ (Miles Laboratories) and contains 62.2% protein. Soy Protein B is more purified than Soy Protein A and contains 92% protein. Soy Protein C is the same as Soy Protein A, except that it contains less salt than Soy Protein A. The first experiment with 43 swine was designed to observe: (1) the effects of two soy protein products (Soy Protein A and B) vs casein on serum cholesterol concentrations and hepatic microsomal HMG-CoA reductase activities when added to a mash diet, and (2) the effect of more purified soy protein (Soy Protein B) when added to an HC diet. The second experiment with 10 swine was designed to compare serum cholesteol concentrations and fecal steroid excretions on an individual basis in two groups of swine fed either HC with Soy Protein C or HC with casein diet for 4 weeks and switching the diets for 2 weeks. 1. 1. Neither of the soy protein products, A or B, affected serum cholesterol levels when added to mash. Similarly, no changes were noted when casein was added to mash. 2. 2. Total hepatic microsomal HMG-CoA reductase activities were not altered by the addition of either Soy Protein A or casein to mash. The activities of the enzyme were reduced by 70% in the group in which soy protein was used in an HC diet as compared to the activities of the enzyme with the groups fed mash alone or mash plus Soy Protein A. 3. 3. All three soy protein products were hypocholesterolemic when added to HC diet. 4. 4. The effect of soy protein on lowering serum cholesterol levels as compared with casein in swine fed high-fat, high-cholesterol diet appears to be due to the increases in fecal steroid excretions not counter-balanced by a concomitant increase in cholesterol synthesis. However, the mechanism of such increases in steroid excretions is not known.

Cerebral atherosclerosis in swine: role of necrosis in progression of diet-induced lesions from proliferative to atheromatous stage.

Summary In recent studies of the pathogenesis of atherosclerosis, we have sequentially categorized the atheroslcerotic lesions into (1) preproliferative, (2) proliferative, and (3) atheromatous phases. This report based on electron microscopy observations is mainly concerned with the cellular and subcellular events occurring in transition from the proliferative to the atheromatous phase with emphasis on the possible role of necrosis. We also paid special attention to the internal elastica, endothelial cells and smooth muscle cells of the inner media at all stages. Materials for study consisted of middle cerebral arteries of young swine fed atherogenic or stock diet for 160 days. Atheroslcerotic lesions in these arteries appeared to be in a transitional state from the proliferative to atheromatous phase. They consisted of collections of bizarre smooth muscle cells, fibrillary and nonfibrillary materials including dissolved neutral lipids, dense and laminated phospholipids, cholesterol clefts, and fine reticulated material consistent with mucopolysaccharides. There were also cellular degenerative changes that can be classified into (1) pyknosis and karyorrhexis and (2) cytolysis, and, in addition, focal accumulation of lipid-rich debris which is the hallmark of atheroma. Viable smooth muscle cells responded by either phagocytizing or sequestering the products of cell death. Based on the transitional forms from recognizable smooth muscle cells to disintegrated products of cell death, and also on the lack of identifying features suggestive of other origin, most and perhaps all of the dead cells in the early atheromata appeared to have been smooth muscle cells and the major portion of atheromatous debris probably originated from necrosis of smooth muscle cells. Deposition of cholesterol-rich material from the extracellular fluid in the necrotic foci probably occurs as the lesion progresses, adding to the mass. The endothelial cells had changes suggestive of disturbed metabolism in both lesion and nonlesion areas perhaps leading to increased permeability, and resulting in accelerated transport of blood consituents from the lumen to deeper layers. Although the internal elastica was well developed in the middle cerebral arteries, it often had erosion effects in the diet-induced lesions apparently leading to wide and numerous gaps with loss of sharp demarcation between the intima and media. Such erosion effects and fragmentation of internal elastica were associated with adjacent smooth muscle cells with exaggerated basement membrane. Study of the control specimens revealed in a few instances miniature foci of degeneration and necrosis in the inner media, but without visible collections of lipid-rich debris. Although an extensive search was required to find these degenerative foci in the control inner media, the fact that they were present at all is suggestive of a precarious situation for these cells, which would make them relatively vulnerable to the effect of noxious agents.

Increased 3H-thymidine incorporation into endothelial cells of swine fed cholesterol for 3 days☆

Segments of abdominal aortas from swine fed either a chlesterol or a control diet for 1 or 3 days were incubated for 20 minutes in a medium containing 3H-thymidine. En face preparations of endothelium were then made and labeling indices determined. At 3 days the labeling indices were 2- to 3-fold greater in cholesterol-fed than in control swine.