J.M. Reiner

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.

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.

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: 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 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.

Population dynamics of arterial cells during atherogenesis: X. Study of monotypism in atherosclerotic lesions of black women heterozygous for glucose-6-phosphate dehydrogenase (G-6-PD)☆

Abstract The problem of cellular population origin of atherosclerotic lesions—unicellular or multicellular, mutational or physiological—was investigated in mosaic aortas of black females heterozygous for the X-linked marker, glucose-6-phosphate dehydrogenase (G-6-PD), occurring in the electrophoretically distinguishable types A and B. After rejection of blood-contaminated, lesion-poor, or otherwise unsuitable postmortem material, 44 cases were studied in detail, a total of 617 lesions being screened. Subregions (samples) were taken from all lesions, and multiple samples were taken from many (on the average, more than two per lesion). All samples were checked histologically to exclude medial contamination. Numerous samples of normal-appearing media were obtained from each aorta as well. Lesion subregions were assayed for marker enzyme types, and classified as ditypic (types A and B both present as a mixture not resolved by the sampling procedure), monotypic A (no B enzyme detectable within experimental sensitivity), and monotypic B (no A enzyme detectable). In the majority of lesions, all subregions were ditypic. Almost half the aortas had lesions with no monotypic samples. Among the remainder, most lesions presented monotypic subregions in varying numbers, but with ditypic regions also present. A smaller number of lesions yielded only monotypic samples (from most of these, no more than two samples had been taken). In most aortas, the single type observed (A or B) was just one throughout, this being the majority type found in the corresponding normal media; in a smaller number of aortas, the single type was opposite to the medial bias. Finally, lesion thickness was measured in 287 lesions. In those cases, frequency of monotypism in creased with thickness. Various hypotheses about the mechanisms leading to these results are discussed. It is concluded inter alia that the data rule out an exclusive monoclonal (unicellular and mutational) origin of lesions, that X-linked allelic selection is probably an important factor in female heterozygotes, and that the as yet unidentified biochemical mechanism leading to growth potential heterogeneity in experimental animals is probably a major factor in atherogenesis in general.

Population dynamics of arterial smooth muscle cells: III. Inhibition by aortic tissue extracts of proliferative response to intimal injury in hypercholesterolemic swine☆

Abstract Previously we demonstrated in swine that a single ip injection of an aqueous extract of swine aortic tissue (containing less than a mg of protein) would inhibit entry of arterial smooth muscle cells into mitosis for at least 2 hr. In the current study we investigated the effect of repeated injections of aortic extract for periods up to 104 days on the development of intimal hyperplasia resulting from a combination of hypercholesterolemia and trauma producd by an intraarterial balloon. Quantitatively both the extent and the thickness of intimal lesions were found to be significantly less in aortic extract treated swine than in paired controls given only saline. Qualitative features did not appear to differ and no general or local toxic effects were observed.