K.T. Lee

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.

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.

Advanced Coronary Atherosclerosis in Swine Produced by Combination of Balloon-Catheter Injury and Cholesterol Feeding

Abstract There has been a constant demand for an animal model of advanced coronary atherosclerosis produced in a short period. It was known that mild atherosclerosis could be produced in various vascular beds by high cholesterol diets. However, it requires considerable time to produce advanced atherosclerotic lesions by diets alone. The current study was designed to investigate effects of combination of a balloon-denudation procedure and high cholesterol diet in swine. We could produce advanced atherosclerotic lesions in coronary arteries in cholesterol-fed swine within a short period of time by inserting a balloon-catheter via carotid artery into the coronary artery, inflating it so as to distend the lumen, and pulling it back quickly, which resulted in extensive denudation of the endothelium of coronary arteries. An atherosclerosis-like lesion develops at the site with eventual narrowing of the lumen leading to myocardial ischemia, myocardial infarction, and occasionally sudden death, thus resembling in many aspects human coronary artery disease. Among 22 swine studied, 10 had severe atherosclerosis with virtual occlusion of proximal portion of either or both coronary arteries and developed myocardial infarction within 2–3 months. Seven of these died suddenly, probably due to arrhythmia. This model should be appropriate for studies where advanced coronary atherosclerosis with its complications is needed.

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.

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.

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: XIII. Mitogenic and cytotoxic effects of a hyperlipidemic (HL) diet on cells in advanced lesions in the abdominal aortas of swine fed an HL diet for 270–345 days

The abdominal aortas of five groups of young male Yorkshire swine were studied: (1) 0-day baseline group; (2) hyperlipidemic (HL) group with ballooning; (3) mash group with ballooning; (4) mash group without ballooning; and (5) HL group without ballooning. The last four groups were injected with [3H]thymidine at 270 days and sacrificed subsequently in subsets at intervals up to 75 days in order to study births and deaths (or loss by migration) among cells over the period 270-345 days. However, only in the HL-ballooned group were there enough swine for the isotopic data to be useful for most purposes. In the 0-day baseline group there were 6 +/- 2 X 10(6) cells in intimal cell masses (ICM); in the 270- to 345-day mash group without ballooning the number was 10 +/- 2 X 10(6), which is not a statistically significant increase over 0-day. This supports the hypothesis that in the normal state births and deaths (or loss by migration) among cells in ICM are nearly balanced at least up to 1 year of age. In the 270- to 345-day mash group with ballooning there were 61 +/- 12 X 10(6) cells in the ICM. Thus a single episode of deendothelialization results in tremendous hyperplasia of ICM. However, even the largest ICM (atherosclerotic lesion) in this group showed essentially no necrosis. In the 270- to 345-day HL group with ballooning there were 108 +/- 17 X 10(6) cells in the ICM turned atherosclerotic lesions. In addition an average of one-third of the lesion volume was occupied by lipid-rich, calcific necrotic debris. Thus the HL diet appears to have associated with it both mitogenic and cytotoxic influences on ICM cells. In the 270- to 345-day HL group not ballooned there were 130 +/- 30 X 10(6) lesion cells and lesions were somewhat more extensive and necrotic than in the HL-ballooned group, probably because the former group included by chance more hyperresponders (as regards serum cholesterol values) to the HL diet than the latter. Regardless of this, the data suggest that in this particular model of advanced atherosclerosis the balloon-injury stimulus to proliferation and the HL-diet stimulus are neither additive nor synergistic.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

Dietary lipids and thrombosis: Factors in blood that account for increased “binding power” in rats fed thrombogenic diets☆

Abstract In previous studies with rats we observed that certain thrombogenic diets resulted in a stronger clot (greater binding power as measured in the thrombelastograph) and prolonged clot-lysis times as compared to simple stock diets. Furthermore, there was a close correlation between the plasma fibrinogen concentration and the binding power. In an attempt to investigate further the previously observed relationship between plasma fibrinogen and binding power, varying amounts of purified fibrinogen were added to plasma with normal fibrinogen levels, and the binding powers were measured in the thrombelastograph. It was found that the binding power was directly proportional to the fibrinogen concentration in the plasma. The experiment was carried out further to determine how early changes in various coagulation factors begin to take place after initiation of high-fat diets. Fibrinogen and prothrombin levels began to rise as early as within 1 week after the administration of high-fat diets. The changes were accompanied by a significant increase of serum cholesterol levels. By the 18th day prothrombin and fibrinogen concentrations doubled their control values, and the increase in serum cholesterol was more than tenfold.