R.A. Florentin

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.

“Preatheroma” phase of coronary atherosclerosis in man

Abstract “Preatheroma” is a term which we apply to the proliferative changes in the arterial wall which develop prior to the occurrence of atheroma. The changes are characterized chiefly by smooth muscle cell proliferation with extracellular fibrillar and nonfibrillar material. The preatheromatous lesion is similar to the atheroma in that it is a focal intimal lesion in the sense that some areas of the intima are involved to a greater degree than others. Preatheroma is more extensive in populations known to develop extensive atheromatous changes, such as North Americans, in contrast to certain Africans in whom atherosclerosis is seldom severe even in old age. Finally, atheroma is always preceded by an appreciable degree of preatheromatous changes. In our opinion, preatheroma is an integral part of the atherosclerotic process and a precursor of atheroma.

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.

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.

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.

Alterations in population dynamics of arterial smooth muscle cells during atherogenesis: I. Activation of interphase cells in cholesterol-fed swine prior to gross atherosclerosis demonstrated by “postpulse salvage labeling”

Abstract The effect of a high-cholesterol diet on the cell dynamics of swine aortic smooth muscle was investigated by pulse labeling in vivo with 8 H-thymidine autoradiography, and the determination of nuclear grain-count distributions. The shift in grain-count distributions with time after labeling was analyzed with the aid of a mathematical model. The conclusions inferred were that: (1) the initially labeled cells virtually all completed their partial cell cycle from S phase through mitosis within 2 days, and gave no evidence of further division up to 30 days; (2) control and cholesterol-fed animals did not differ in this respect; and (3) the known effect of cholesterol diet on labeling index is accounted for by newly recruited G 0 cells or by acceleration of slowly moving G 1 cells. This was demonstrated by postpulse labeling, presumably resulting from reutilization of label salvaged from dead cells in the intestine and elsewhere which continued for at least 7 days. These conclusions were supported by estimation of the G 2 population withthe aid of microspectrophotometry, by a detailed analysis of grain counts and mitoses during the first 2 days with the help of successive 5-hour colchicine collections, and by a comparison of labeling in carotid artery cells in which one artery was excluded from the systemic circulation during the initial pulsing period and subsequently readmitted to the circulation. Since no observations were made beyond 30 days, it was not possible to decide whether the diet ultimately did affect the generation time of the pulse-labeled cells, nor whether the cells newly recruited by the diet resulted from activation of G 0 cells or from acceleration of G 1 cells. However, it was possible with the help of preliminary data on the S period and some further mathematical analysis to estimate a range of values for the generation time and for the fraction of cells in G 0 .

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.

Population dynamics of arterial smooth muscle cells: V. Cell proliferation and cell death during initial 3 months in atherosclerotic lesions induced in swine by hypercholesterolemic diet and intimal trauma

Abstract The sine qua non of the lesion of atherosclerosis in man and experimental animals is excessive focal accumulation of modified smooth muscle cells in the intima of arteries. In advanced stages extensive necrosis with accumulation of lipid-rich debris is a prominent feature. In swine fed hypercholesterolemic (HC) diets focal atherosclerotic lesions are produced, but they progress slowly and do not develop frank necrosis until they have been on HC diet for many months or even years. The atherosclerotic process can be greatly accelerated by traumatizing the arterial intima with a ballon-catheter in addition to feeding the HC diet. Within a few months thick atherosclerotic lesions with extensive necrosis and calcification can be produced. In regard to arterial cell population dynamics, we showed in the first part of the current study that under the specified experimental conditions, lesions produced in young swine by balloon-catheter trauma and HC diet have at least four features in common with lesions produced in young swine by HC diet alone. (1) Multiple cells are involved in the initiation of the lesions (i.e., they are not monoclonal in origin). (2) The cells do not divide in a completely random fashion. (3) The division pattern is consistent with polyclonal origin with considerable heterogeneity as regards growth rate. (4) Most and perhaps all lesion cells were in the dividing population during the periods that were studied. The differences observed between development of lesions produced by HC diet alone and those produced by HC diet plus ballon-catheter intimal trauma were quantitative and not qualitative, i.e., the lesions produced by the latter procedure had a greater rate of cell multiplication and proceeded more rapidly to the necrotic stage than did those produced by HC diet alone. In the second part of the current study, we have devised a method for calculating cell deaths in the atherosclerotic lesions produced by HC diet plus intimal trauma. In the period 60–97 days on HC diet which was studied we found that circa 40% of the lesion cells died during the perireplicative and/or perimitotic period of the cell cycle. The high cell death rate was offset in part by a high cell birth rate. As indicated by tritiated thymidine ( 3 HTdR) labeling indices, approximately four times as many cells were synthesizing DNA at the time of observation as in the adjacent normal appearing media. Obviously the rate of growth of the atherosclerotic lesions that were studied was determined by the balance between excessive cell birth and death rates.

Comparison of the amount of coronary arteriosclerosis in autopsied East Africans and New Yorkers

Abstract A comparison of the degree of coronary arteriosclerosis and number of myocardial infarcts in 117 autopsied East Africans and 137 New Yorkers was made. In forty-three subjects from each series (thirty-four men and nine women) matched for age and sex, the amount of coronary arteriosclerosis was generally much higher in the New York autopsy series, although the East Africans were not free from arteriosclerosis, and some overlapping occurred between the two groups. In this same matched group there were seventeen myocardial infarcts among the Americans, while none were found in the East African autopsy series. Comparative studies of blood and tissue lipid patterns, as well as clot lysis in the two groups, are now being carried out in an attempt to obtain other information pertaining to these findings.