R.F. Scott

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.

Experimental atherosclerosis in rhesus monkeys: I. Gross and light microscopy features and lipid values in serum and aorta☆

Abstract Forty-five rhesus monkeys were divided into five groups fed four different high-fat diets and a stock diet. The duration of feeding varied from 33 to 70 weeks. All monkeys fed the high-fat diets developed aortic proliferative lesions composed predominantly of spindle-shaped cells, most of them laden with lipid. The thickest of these proliferative lesions measured 0.85 mm, and most were thinner, suggesting that in the monkey receiving these high-fat diets some proliferative lesions can reach this range of thickness before undergoing necrosis. Atheromatous aortic lesions characterized by necrosis and the accumulation of lipid debris were found only in monkeys receiving high-fat diets for 70 weeks. In monkeys receiving a diet in which the lipid component was 30% peanut oil and 5% cholesterol, the fatty acid composition of the aorta was similar tothat in humans developing coronary artery atherosclerosis, in that the fatty acids showing the greatest accumulation were oleic and linoleic. The largest number of atheromatous lesions, however, was associated with a diet otherwise identical to the peanut oil-cholesterol diet but containing as its lipid component 30% butter and 5% cholesterol. This latter diet also resulted in higher serum lipid levels and higher aortic lipid levels per milligram of DNA than did peanut oil diet. High-fat diets fed to rhesus monkeys appear to produce both proliferative and atheromatous aortic lesions similar to those seen in the human as regards both light microscopy features and lipid composition. Work is now in progress assessing some of the metabolic features of these lesions.

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.

Coronary arteries of children and young adults: A comparison of lipids and anatomic features in New Yorkers and East Africans☆

Abstract Lipid classes (including fatty acids), crude protein, and calcium were measured in the coronary arteries of a total of 246 New Yorkers and East Africans from stillborn to 39 years of age. In subjects stillborn to 19 years of age a histological comparison of the arteries in the two groups was made. 1. (1) Starting at the end of the second decade New Yorker coronary arteries accumulate increasing amounts of cholesterol, in contrast to the findings in age-matched East Africans. 2. (2) The kinds of lipids (including fatty acids) in the coronary arteries of New Yorkers and East Africans were in general similar. This suggests that (as far as lipids are concerned), the type of arteriosclerosis is the same in both groups; the same conclusion is suggested by light microscopy observations. 3. (3) The selective accumulation of certain cholesterol esters and not of other lipids in the coronary artery suggests that the process is a dynamic one, and does not depend on simple filtration from the blood.

Glucose degradation in normal and atherosclerotic aortic intima-media

Abstract Pathways of glucose degradation and 0 2 uptake were measured in intimamedia of normal swine, rabbits and monkeys, and in atherosclerotic intima-media of rabbits and monkeys. In the normal aortas most of the glucose was degraded to lactic acid, with very little entering the Krebs cycle or pentose shunt systems. Only small amounts ended up as lipid, RNA-DNA-protein, or glycogen. In the atherosclerotic aortas, glucose utilization more than doubled per mg DNA, but again virtually all of it was converted to lactic acid. The 0 2 uptake per mg DNA sharply increased in the atherosclerotic tissue, but no more glucose entered the Krebs cycle than in the normal artery tissue. It thus appears that neither in normal nor in atherosclerotic artery wall is glucose an important substrate for oxidative phosphorylation.

Population dynamics of arterial cells during atherogenesis: VIII. Separation of the roles of injury and growth stimulation in early aortic atherogenesis in swine originating in pre-existing intimal smooth muscle cell masses

Abstract This is a study of focal masses of smooth muscle cells that are found normally in the intima of large and medium sized arteries from intrauterine life to old age in man and most experimental animals. These intimal cellular masses are of special interest because they appear to be sites of predilection for atherogenesis. The experimental animals used in this study were young swine; intimal cellular masses were those in the abdominal aorta; arterial cell population changes were studied from approximately the 8th to the 16th weeks of life and comparisons were made between normolipidemic and hyperlipidemic swine; methods included overall cell counts and isotopic techniques designed to trace cells through multiple divisions. Fifteen swine were given [ 3 H]thymidine on approximately the 60th day of life. Five were sacrificed on the 75th day as a baseline group; five were fed a hyperlipidemic diet from the 75th to the 135th day of life and the remaining five were continued on a conventional low-cholesterol mash diet until the 135th day, when both groups were sacrificed. Total smooth muscle cells in intimal cellular masses of the abdominal aorta averaged 1.59 million in the 75-day-old baseline group; 2.06 million in the 135-day-old mash-fed group; and 4.73 million in the 135-day-old hyperlipidemic group. This tripling in the number of cells in intimal cellular masses of hyperlipidemic swine (as compared to no significant increase in the corresponding value in mash-fed swine) during the initial 60 days on the diet occurred prior to development of overt gross lesions and probably represents the earliest phase of atherogenesis. In the isotopic portion of the study, we attempted to trace the behavior of the original cell population of the intimal cellular masses (re smooth muscle cell births and deaths) for the 60 days in the two dietary groups and to identify differences that accounted for the 2- to 3-fold greater increase of cells in the hyperlipidemic diet group. In regard to cell loss (probably by death) in the mash-fed group, 39% of the cells present at the onset were lost (without surviving progeny) over the 60 days as compared with 40% in the hyperlipidemic group—hence no significant difference. In regard to percentage of original cells surviving but not dividing in 60 days in the mash-fed group, the value was 30% and in the hyperlipidemic no non-dividers were detected; thus more cells were recruited from non-dividers into the active dividing population in the hyperlipidemic group. As regards the 60-day dividing population in the mash dict group, 31% of the original cells divided one or more times with an average of 3.66 live progeny per dividing cell; in the hyperlipidemic group 60% divided one or more times with an average of 5.27 live progeny per divided cell. Thus, the hyperlipidemic diet produced a mitogenic effect on the smooth muscle cells of the intimal cellular masses manifested by significantly larger numbers of cells in the 60-day dividing population and by significantly more divisions per dividing cell (and hence more progeny) than in the mash-fed group. Smooth muscle cell losses were negligible in the media of both groups; numbers of cells were increased considerably in both by growth as expected and no differences were observed in this respect between the two groups. The cell losses in the intimal cellular masses, though apparently not related to the diet at this stage of atherogenesis, probably accounted for localization of the diet-related changes to the intimal cellular masses in the hyperlipidemic swine.

Population dynamics of arterial cells during atherogenesis: VII. Comparison of loss of endothelial cells over abdominal aortic intimal cellular masses (ICM) with that over non-ICM areas in swine fed a hyperlipidemic diet for 60 days☆

Abstract One of the earlies events in the development of an intimal atherosclerotic lesion may be an alteration in the endothelial cell barrier, allowing the entry of injurious or mitogenic substances, resulting in proliferation of arterial smooth muscle cells. Intimal smooth muscle cell proliferation in atherosclerosis appears to begin in intimal cellular masses (ICM), which are normally occurring intimal structures made up of 2 to 10 layers of smooth muscle cells found predominantly in relation to branching points of arteries. If endothelial cell loss is a precursor of overt atherosclerotic lesions, the loss should be greater over ICM than over areas showing no ICM. This study presents evidence suggesting that in swine the endothelial cell barrier is compromised over intimal cell masses more than in non-ICM regions prior to development of overt atherosclerotic lesions. The method used was to label swine aortic endothelium and ICM using tritiated thymidine. All swine were then placed on a low-fat, low-cholesterol diet for 15 days, at which time five baseline animals were sacrificed; the remaining five were placed on a hyperlipidemic (HL) diet and sacrificed 60 days later. The rate of endothelial cell growth, division patterns, and loss of labeled endothelial cells in the HL swine was determined by comparing the findings in this group with the baseline group. Smooth muscle cells of ICM and non-ICM medial regions were similar in regard to labeling indices in the baseline swine and in regard to division patterns in 60-day HL diet swine, except one which was an active lesion. None of the remaining ICM were active lesions and thus were suitable for study of the covering endothelial cells in what can be assumed to be the prodromal stage of lesion development. The approximate area of the abdominal aorta occupied by intimal cell mass was considerably more than we had anticipated, averaging more than 20%. The labeling indices and endothelial cell division patterns over ICM and non-ICM in the baseline swine were similar. In contrast, the labeling index was lower, and grain count changes indicated more endothelial cell divisions over ICM areas than over non-ICM areas in the hypercholesterolemic swine. Calculation showed that the percentage of endothelial cell loss over ICM in the HL swine was more than twice as great as endothelial cell loss over non-ICM areas. The results of this study suggest the possibility that in the assumed prodromal phase of experimental aortic atherosclerosis in young swine, the endothelial cell barrier may be compromised selectively over areas of predilection for the future development of atherosclerotic lesions. This occurs before there is any evidence of focal increase in intimal smooth muscle cell proliferation, which we regard as the earliest detectable stage of lesion development.

Uptake, oxidation, and esterification of free fatty acids in normal and atherosclerotic rabbit aorta.

Abstract Intimamedia tissue from atherosclerotic rabbits showed a 15-fold increase in the amount of [U- 14 C]palmitic acid oxidized to CO 2 compared to normal intimamedia. The findings suggest that oxidation of free fatty acids may at least in part contribute to the increased oxygen uptake seen in atherosclerotic tissue. The concentration of endogenous free fatty acids in atherosclerotic tissue was much higher than in normal tissue. The higher level may have been partly due to the increased levels of serum free fatty acid in the atherosclerotic rabbits, and to the increased permeability of atherosclerotic intimamedia to free fatty acids demonstrated in vitro . The tissue from atherosclerotic rabbit aortas exhibited a different pattern of fatty acid esterification than did that obtained from control aortas. In atherosclerotic tissue more cholesterol ester than triglyceride was formed from palmitate. In determining the specific activity of the free fatty acid tissue pool(s), equilibration of medium and tissue free fatty acid was found to be slow. In addition, the amount of free fatty acid in aorta was substantial and somewhat variable from animal to animal. Therefore, it was necessary to base calculations of fatty acid metabolic pathways on the specific activity of the palmitate in the tissue and not on that of the medium.

Aortic respiration and glycolysis in the pre-proliferative phase of diet-induced atherosclerosis in swine

Summary Cholesterol or stock diets were fed to fourteen pairs of miniature swine for periods varying from 3 to 112 days. Measurements of respiration, aerobic and anaerobic net lactic acid production and lipid content were done in grossly normal intima-media from the upper thoracic aorta in both dietary groups. Intima-media respiration was significantly higher per unit of DNA in the cholesterol-fed swine than in stock-fed swine. This was so even before any grossly visible atherosclerotic lesions were apparent and before there was any light microscopy or biochemical evidence of cell proliferation, but when pre-proliferative changes were presumably present. The aerobic and anaerobic net lactic acid production however were the same in the aortic tissue of both the cholesterol and stock-fed groups. Both total cholesterol and phospholipid were increased in concentration in the grossly normal intima-media after 44 days of feeding the cholesterol diet. The increased tissue respiration before the appearance of atherosclerotic lesions characterized by smooth muscle cell proliferation may mean that the intima media of the swine is synthesizing more ATP or high energy intermediates. That there might be an increased demand for energy in the artery associated with cholesterol diets in swine is suggested by electron microscopy and autoradiographic studies of the pre-proliferative phase of cholesterol-induced atherosclerosis in the same animals as were used in this experiment.