Mash-fed Swine Research Articles

Endothelial cell denudation, labelling indices and monocyte attachment in advanced swine coronary artery lesions

This study shows that frank endothelial denudation, as measured by scanning electron microscopy, is associated with coronary artery atherosclerotic lesions in swine fed hyperlipidemic diets for 9 or 18 months. The largest areas of endothelial cell denudation were found to be associated with the most advanced atherosclerotic lesions, and lesser areas of denudation were present in less severely atherosclerotic arteries. Overall, the study suggests that frank endothelial denudation in swine coronaries occurs secondarily to advanced lesion development, and is proportionate to the amount of lesion present. In contrast attachment of monocytes to the endothelial surface was not proportionate to the amount of lesion present. While the number of attached monocytes after 9 or 18 months of a hyperlipidemic diet was much greater than in the mash-fed swine, there was no difference between the two hyperlipidemic groups. In both hyperlipidemic groups, the attached monocytes were concentrated over lesion areas. By scanning electron microscopy, most attached monocytes in these perfused coronary arteries showed a slender foot process extending into a gap between endothelial cells. Endothelial cell turnover, as measured by tritiated thymidine labelling indices, increased with the amount of coronary artery atherosclerosis. However, it did not become significantly greater than in mash-fed swine until advanced lesions developed.

Atherosclerotic lesions in the coronary arteries of hyperlipidernic swine Part 1. Cell increases, divisions, losses and cells of origin in first 90 days on diet

The intima of the proximal portion of the coronary arteries of young swine is normally thickened by accumulations of cells about 90% of which are smooth muscle cells (SMC) and about 10% are of probable monocyte origin. Extracellular components such as collagen and elastic tissue are also present but we have chosen to emphasize their cellular nature by calling the regions of thickened intima, intimal cell masse (ICM). We have previously shown that atherosclerotic lesions produced in the coronary arteries of swine by 90 days of feeding a hyperlipidemic (HL) diet arise almost exclusively in the normally occurring ICM. We are reporting here a study of the pathogenesis of these lesions following killing at 0, 14, 49 and 90 HL diet days with comparisons between ICM in control mash-fed swine and ICM-lesions in the HL swine. We found that in the ICM: (1) lipid accumulation was present by 14 days and increased thereafter; the lipid was mostly in SMC but percentagewise the monocyte-macrophages were involved as much or more, (2) cell division activity was increased 3–4-fold by 49 days, (3) cell numbers in ICM were similar in HL and control swine at 49 days but were about 6-fold greater in the HL swine at 90 days, (now in ICM-lesions), (4) at 90 days, circa 90% of the cells appeared to be of SMC and circa 10% of monocyte origin both in the ICM-lesions of the HL swine and in the normal ICM of the controls. The data suggest but do not prove that early lipid accumulation precedes increased cell divisions especially among the SMC component and this in turn precedes increased numbers of cells in the ICM. Although SMC constitute the major cell component of the ICM-lesion at 90 days, the monocyte-macrophage-like cells also increase in number as a result of the HL diet and constitute a small but definite minor component. One possible explanation for the increased cell division activity is that one of the lipid constituents is acting as mitogen; another possibility is that the effect of a well known mitogen such as platelet-derived growth factor is enhanced by the lipid; another is that the monocytes are being stimulated to produce monocyte-derived growth factor. In any event in the very early stage of atherogenesis in the coronary arteries in these experiments excessive proliferation of resident SMC in the ICM appears to be the predominant feature.

Endothelial cell labeling indices in swine aortas in relation to intimal cell mass-derived atherosclerotic lesions

In the swine abdominal aorta, most atherosclerotic lesions arise from naturally occuring collections of intimal cells called intimal cell masses. The main objective of this study was to determine if the tritiated thymidine labeling index of endothelial cells lying over intimal cell masses was greater than that of endothelial cells not over intimal cell masses. A higher endothelial cell labeling index over intimal cell masses would indicate possibly a greater turnover in the area. Such a finding would suggest possible transient but repeated breaks in the endothelial cell barrier that might contribute to the initiation of the atherosclerotic lesion in intimal cell masses. In 2 groups of mash-fed swine, 2 and 11 months old, no differences were found in labeling indices of endothelial cells lying over or not over intimal cell mases. It appears that the initiation of atherosclerosis in swine abdominal aorta is not dependent upon pre-existing altered endothelial cell kinetics as measured by tritiated thymidine labeling indices. The labeling index of cells within the intimal cell mass was significantly higher than in the media; this also is obviously not dependent upon an increased labeling index of the covering endothelial cells. Another group of swine were killed at 11 months of age after receiving a hyperlipidemic diet for 270 days. The labeling index of endothelial cells over atherosclerotic lesions was significantly higher than the labeling index of endothelial cells not over lesions in these swine, and also significantly higher than that of endothelial cells over intimal cell masses in the 11-month old mash-fed group. It is concluded that the increased endothelial cell labeling index over atherosclerotic lesions may be secondary to the lesion itself, and not due to the direct effect of the hyperlipidemic blood.

Intimal cell mass-derived atherosclerotic lesions in the abdominal aorta of hyperlipidemic swine Part 1. Cell of origin, cell divisions and cell losses in first 90 days on diet

Atherosclerotic lesions may originate and develop in a variety of ways. In this study we are focusing our attention on atherosclerotic lesions arising in normally occurring intimal cell masses (ICM) in the abdominal aortas of hyperlipidemic (HL) swine. Times chosen for study were 0, 14, 49 and 90 days on HL diet; mash-fed swine were used as controls. Total numbers of cells in the ICM of HL and mash swine were similar at 14 and 49 days; by 90 days the number of cells had increased dramatically in the HL swine to 8-fold greater than control values. Changes present at 49 days and thus preceding increase in cell numbers included extensive intracellular lipid accumulation with by count nearly half of the ICM cells involved and elevated tritiated thymidine labeling indices (LI) 4-fold greater than control. Differential cell counts by transmission electron microscopy were made on the ICM lesions in the HL swine at 49 and 90 days. More than 95% of all cells were smooth muscle cells (SMC), with relatively few monocytes being present. Calculations from the LI and total cell counts showed that the entire increase in cell numbers could be accounted for by divisions among the resident SMC in the ICM. Further calculations suggested that cell losses (deaths) from the ICM were minimal. Scanning electron microscopy studies reported elsewhere revealed no loss of endothelial integrity. The results suggest: (1) that the lesions arise by stimulation of the resident SMC in the ICM to hyperplastic activity, (2) that the role of monocytes in the early development of these lesions is minimal if any, (3) that in view of the intact endothelium platelets are not likely to play an important role, (4) that ICM cell death is not a major factor, (5) that the most likely candidate for the cell growth stimulatory role (? mitogen) is some component(s) of the excess lipid that accumulates in the ICM.

Some effects of a grain-based mash diet on cholesterol metabolism in swine

The aim of the first part of this study was to find out in swine how much fat could be included in a cholesterol-free semi-purified diet without resulting in changes in serum cholesterol levels or hepatic microsomal HMG-CoA reductase activities from those in swine fed a grain-based mash diet. Graded levels of peanut oil from 5 to 40% by calories were included in a casein-carbohydrate-cellulose based diet and effects compared with those in swine fed mash (10% fat) for 2 weeks. Serum cholesterol levels were essentially unaffected by any of the diets. However, in the semipurified diet-fed swine at all levels of dietary fat hepatic microsomal HMG-CoA reductase activities were reduced to values circa 25% of those in the mash-fed swine. This is as great as the reduction from mash levels of activity produced by high fat, high-cholesterol hyperlipidemic diets. The aim of the second part of the study was to find out in terms of whole body cholesterol balance difference in the effects of a cholesterol-free, low-fat (9%) semi-purified diet and a mash diet that might account for the differences in hepatic microsomal HMG-CoA reductase activities. The principal difference proved to be in the fecal output of bile acids (908 ± 86 mg/day on mash vs 292 ± 11 mg/day on a low-fat semi-purified diet). Thus the higher hepatic microsomal HMG-CoA reductase activities in the mash fed swine are presumably in response to the greater loss of steroids. The mixed natural fibers in the grain-based mash diet, as compared to cellulose only in the other, probably accounts for the increased fecal output of bile acids.

Effect of clofibrate, cholestyramine, cholesterol and feeding pattern on the diurnal variation of cholesterol 7 alpha-hydroxylation in swine

A rhythmic change in hepatic microsomal cholesterol 7 alpha-hydroxylase activity in young male Yorkshire swine fed either low-fat, low-cholesterol mash or high-fat, high-cholesterol diets in a single daily meal at 9:00 am has been established. In mash-fed swine, dual peaks were observed; the first peak with 2-fold increase in the enzyme activity over that of 9:00 am at 2:00 pm, and another peak appeared at midnight. In swine fed a high-fat, high-cholesterol diet, the enzyme activity peaked only at 2:00 pm, then the enzyme activity gradually returned to the base line. The total daily activities were not significantly different between mash (one meal) and high-fat, high-cholesterol (one meal) groups. When swine were fed mash in three divided meals daily, the peak seemed virtually to disappear. But the daily total activity integrated for 24 hr was the same as that of swine fed single mash meals. Addition of cholestyramine in mash (one meal) increased amplitude of the diurnal peak, but the rhythmic pattern was not altered. Addition of clofibrate in mash (one meal) swine did not affect the amplitude nor pattern of enzyme activities. Both cholestyramine and clofibrate treatments reduced serum cholesterol concentrations after 2 weeks.

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.

Effect of cholestyramine in normo-lipidemic swine

The effects of cholestyramine on cholesterol balance parameters, bile acids and the enzyme activities of HMG-CoA reductase and cholesterol 7α-hydroxylase were studied in mash-fed young male Yorkshire swine. In addition, our previous results on cholestyramine effect in swine fed hyperlipidemic diet were compared with the results of the current study. Eight swine were divided into two groups: four were given 12 g cholestyramine daily for 2 weeks, and the other four served as the control. Serum cholesterol levels were monitored weekly. Feces were collected before and during the cholestyramine treatment for neutral and acidic steroid measurements and determination of other cholesterol balance parameters. Terminally, gallbladder bile was collected and bile acid composition and an in vitro cholestyramine adsorption study were carried out. Cholestyramine treatment in mash-fed swine resulted in the following significant changes: 1. (1) Whole body cholesterol synthesis increased to well above the mash level. 2. (2) Neutral steroid excretion was increased. 3. (3) The proportions of the more polar bile acid were increased in the bile and fecal bile acid excretion was increased with the increase accounted for by lithocholic acid. 4. (4) In vitro adsorption by cholestyramine of gallbladder bile acids from cholestyramine-treated swine was less than that of bile from untreated swine. This was considered to be the result of relative and/or absolute increases in the primary bile acids (more polar bile acids) in cholestyramine-treated swine. 5. (5) In vivo, cholestyramine removed very little bile acids as compared to that in vitro (31 vs 438 mg/g cholestyramine) suggesting the presence of competing factors in the intestine, e.g., fatty acids. 6. (6) Hepatic microsomal HMG-CoA reductase and cholesterol-7-α-hydroxylase activities reflected the fecal excretions of the steroids. However, ileal microsomal HMG-CoA reductase activities were not affected by cholestyramine treatment. On the basis of results from this study combined with those from previous studies, we conclude that: (1) the adsorption of bile acids on cholestyramine may be interfered with by the other dietary constituents such as fatty acids, and (2) cholestyramine treatment increases the relative ratio as well as absolute amount of primary bile acids and such bile acids may be adsorbed less effectively than other bile acids.

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

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 [ 3H]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.

Lack of effect of clofibrate on hepatic HMG-CoA reductase activity in young swine in the postabsorptive state

Effects of clofibrate treatment of 7 days duration were studied on hepatic HMG-CoA (β-hydroxy-β-methylglutaryl Coenzyme A) reductase (EC 1.1.1.34) activity and on acetate incorporation into cholesterol by tissue slices from liver and ileum in 29 young male mash-fed swine in the postabsorptive state. Swine treated with either 2 or 10 gm of clofibrate daily showed marked reductions in serum cholesterol levels. Unlike the large decrease observed in rats, hepatic HMG-CoA reductase activity was not significantly reduced in either treated group. Acetate incorporation into cholesterol by liver and ileal slices was similarly unaffected by clofibrate. These results and our previous cholesterol balance studies of clofibrate-treated swine suggest that the drug does not inhibit cholesterol synthesis in these animals. The mechanism of action of clofibrate in swine may be different from that in rats.

An evaluation of some of the potential immediate sources of cholesterol for bile acid synthesis in swine

An experiment with cholesterol-fed and mash-fed swine, untreated or treated with hypocholesterolemic drugs, was terminated with a single dietary pulse of radioactive cholesterol followed by a seven-day period of observation for a balance study.The appearance of radioactivity in fecal bile acids was consistent with a transit time of three days or less. Comparison of measured quantity and specific activity of fecal bile acids with quantity and specific activities calculated on the assumption that serum cholesterol is the more or less immediate precursor cast doubt on this very assumption. The inconsistency remains if calculations are based on free cholesterol only or ester cholesterol only.With the data available, the simplest assumption leading to reasonable agreement with observation was that the precursor is a mixture (unweighted average) of dietary and endogenously synthesized cholesterol.