J. Schmee

Modification of lipoprotein patterns and retardation of atherogenesis by a fish oil supplement to a hyperlipidemic diet for swine

We have studied the effect of addition of 30 ml cod liver oil (FO) daily to a highly atherogenic butter (BT) diet for swine on lesion development in the coronary arteries and aorta, plasma lipoprotein (LP) patterns, plasma levels of thiobarbituric acid-reactive substances (TBARS) and on tritiated thymidine-labeling indices ([3H]TdR LI) of smooth muscle cells (SMC) and monocyte/macrophages (M/M phi) in the atherosclerotic lesions. Seventeen male Yorkshire swine (11.1 +/- 0.4 kg) were divided into 3 groups: BT (n = 6), BT + FO (n = 6) and mash (n = 5). They were fed the respective diets for 4 months. Terminally, fasting plasma was obtained and cholesterol contents were determined in various fractions of lipoproteins separated by density gradient ultracentrifugation, Pevikon block electrophoresis and immunoelectrophoresis. Apoprotein (B, A-I, E and C) contents of the plasma and lipoprotein fractions were determined by polyacrylamide gel electrophoresis and densitometry of gels stained with Coomassie blue. Swine were injected intramuscularly with 0.5 mCi/kg of [3H]TdR 2 h before death. The aorta and coronary arteries were perfusion fixed in situ under anesthesia. Samples were obtained for microscopic morphometry, autoradiography and immunohistochemistry from distal abdominal aorta, thoracic aorta, and proximal coronary arteries; left main (LM), left anterior descending (LAD), left circumflex (LCX), right main (RM), and right coronary artery (RCA). On the BT diet without FO there was extensive atherosclerotic (AS) lesion development, which was drastically reduced by the addition of FO to the BT diet in all sites by from 71 to 94%. The overall plasma cholesterol (CH) levels were reduced only modestly by the FO (816 +/- 64 to 629 +/- 14 mg/dl) but the distribution of CH in the various lipoprotein classes was remarkably altered. The CH in the large lipoprotein molecules containing both B and E apoproteins was reduced from 488 +/- 84 to 204 +/- 17 mg/dl by the FO with an almost corresponding increase in the conventional LDL molecules containing apo B only (158 +/- 29 to 344 +/- 15 mg/dl). We offer the hypothesis that the large apo B,E containing molecules are much more atherogenic than the smaller apo B containing molecules. This hypothesis is supported by a highly significant correlation between extent of lesion development in all arterial sites and plasma levels of CH in apo B,E containing lipoproteins. Plasma TBARS were elevated by the BT + FO diet but seemed to have no significant effect on the lesions.(ABSTRACT TRUNCATED AT 400 WORDS)

Dietary fish oil added to a hyperlipidemic diet for swine results in reduction in the excessive number of monocytes attached to arterial endothelium

Modest numbers of blood monocytes become attached at least temporarily to the endothelium of large arteries in normal swine fed low fat, low cholesterol diets. These numbers are increased several fold when the swine are fed a high saturated fat, high cholesterol atherogenic diet (BT). The main objective of this portion of a broader study was to see if the addition of fish oil (30 ml) to a BT diet (BT + FO) could prevent the increase in attached monocytes induced over arterial endothelium in BT fed swine. Six BT, 6 BT + FO and 5 control mash (MA) swine fed the respective diets for 4 months before killing were available for the current study. Other aspects of this experiment have been presented previously which in brief are that BT + FO resulted in retardation of atherosclerotic lesion development and a shift in lipoprotein components from predominantly apolipoprotein B,E containing with the BT diet to predominantly apo B only with BT + FO. There was a significant positive correlation between lesion development and apo B,E lipoproteins. In the current study we determined by scanning electron microscopy on the first portion of the left anterior descending coronary artery after perfusion fixation under pressure the number of monocytes per mm2 attached over or not over visible lesions. We also determined monocyte percentages in the circulating blood and analyzed the correlation of the numbers of attached monocytes and blood monocyte percentages with various lipoprotein components reported previously.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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 masses (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: lipid accumulation was present by 14 days and increased thereafter; the lipid was mostly in SMC but percentage wise the monocyte-macrophages were involved as much or more, cell division activity was increased 3-4-fold by 49 days, 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), 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.

Comparison of effects of fish oil and corn oil supplements on hyperlipidemic diet induced atherogenesis in swine

The addition of a fish oil supplement rich in n - 3 unsaturated fatty acids to a high cholesterol, high saturated fat (BT) diet for swine has been shown previously to result in modest lowering of plasma cholesterol levels and in marked retardation of atherogenesis. It has been suggested that the effect was due to the change in polyunsaturated (PUFA) to saturated fatty acid ratios (P/S) and that a supplement of PUFA of the n - 6 series might have the same effect as the fish oil. We have tested this hypothesis in swine fed an atherogenic diet by comparing the effect of a fish oil supplement producing a P/S ratio of 0.28 to that of corn oil in the same amount producing a ratio of 0.46. The P/S ratio of the atherogenic diet without supplements was 0.16. Thirteen young male Yorkshire swine were fed either BT alone (n = 4), BT + cod liver oil (n = 4) or BT + corn oil (n = 5) for 6 months and then killed for quantitative studies of atherosclerosis in the aortas and coronary arteries including lesion areas, number of lesion cells, and number of monocytes attached to endothelium. Plasma cholesterol levels were determined periodically and lipoproteins were separated terminally by density gradient ultracentrifugation, Pevikon block electrophoresis and immunoelectrophoresis. The fish oil supplement resulted in a 30% reduction in time-weighted average plasma cholesterol levels, and a marked shift in terminal lipoprotein patterns from predominantly apo B and E containing ones to predominantly apo B only ones. Atherogenesis was reduced by the fish oil supplement as judged by several morphometric criteria including size of lesions, number of lesion cells, and number of monocytes attached to lesion endothelium. The corn oil supplement produced no significant reductions in any of these variables from those in swine fed the atherogenic BT diet without the supplement. We conclude that the n - 3 fatty acid rich fish oil supplemented diet retarded atherogenesis, but that this effect was not shared by the corn oil supplemented diet which had an even higher P/S ratio.

Intimal cell mass-derived atherosclerotic lesions in the abdominal aorta of hyperlipidemic swine. Part 2. Investigation of endothelial cell changes and leukocyte adherence associated with early smooth muscle cell proliferative activity.

We have investigated several aspects of endothelial cell (EC) behavior during the initiation and early development of intimal cell mass (ICM)-derived atherosclerotic lesions in the distal abdominal aortas of young swine fed hyperlipidemic (HL) diets for 0, 14, 49, or 90 days. By scanning electron microscopy no breaks in endothelial integrity or other abnormalities were observed even at 90 days on diet after lesions were well established. Also, counts of leukocytes adherent to the endothelium by both scanning and light microscopy revealed no greater numbers in HL than in mash control swine. Estimates of individual EC losses over ICM-lesions in the HL swine (based on calculations from tritiated thymidine labeling indices and EC growth rates determined by counts) suggested a loss of approximately one per 100 EC/day. This loss over ICM lesion was not significantly greater than that over ICM in mash controls, but was significantly greater than that of EC not over ICM lesions in the same HL animal. In any event, the estimated loss seems to be too trivial for the endothelial barrier to be compromised even transiently in a biologically significant fashion. A significant correlation was observed between tritiated thymidine labeling indices of cells within the ICM lesions and of those of the overlying EC that was not observed with the ICM in the controls. Possibly the positive correlation may be a result of the abluminal surface of the overlying EC being exposed to the abnormal milieu of the ICM lesion. It is emphasized that results reported here apply only to ICM-derived lesions at an early stage of development and that they do not contradict results obtained by others with other lesion types such as those derived from monocytes. Furthermore, in later stages of development of ICM-derived lesions in the same model we know that extensive endothelial cell damage can be demonstrated. Also, functional changes in endothelial permeability may have been present in early stages that would not have been detected with the methods used in this study.

Atherosclerotic lesions in coronary arteries of hyperlipidemic swine. Part 2. Endothelial cell kinetics and leukocyte adherence associated with early lesions.

The role of endothelial cells (EC) in the development and progression of early swine intimal cell mass (ICM)-derived coronary artery lesions in 55 swine fed either a mash or hyperlipidemic diet for 14, 49 or 90 days was investigated. Characteristics studied were endothelial cell turnover (using tritiated thymidine autoradiography), adhesion of leukocytes (presumably chiefly monocytes) to endothelium, and the presence or absence of endothelial cell denudation. The major findings were: An increased adherence of leucocytes to endothelial cells over ICM-lesions in the HL-90 day group compared to the corresponding mash value at 90 days as well as to that of each of the other HL and mash groups. Significant positive correlation between the labeling indices (LI) of endothelial cells lying over coronary artery lesion cells and the labeling indices of the underlying ICM-lesion cells; also, a significant positive correlation between the labeling indices of endothelial cells over lesions of the abdominal aorta and those of the coronary arteries. At 90 days the endothelial cell LI over lesions in the HL group was significantly higher than the corresponding values in the mash group. Since the EC increase rates by growth in the two groups are also significantly different, the differences in LIs reflect at least in part EC growth differences and no strong conclusion can be made regarding possible cell turnover differences. No frank endothelial denudation was found. The findings suggest that in swine coronary arteries participation by monocytes from circulating blood is a factor in the early progression of the lesion as well as smooth muscle cell proliferation.

Intimal cell masses in the abdominal aortas of swine fed a low-fat, low-cholesterol diet for up to twelve years of age

The normal subendothelial intima of large arteries in man, swine and most other species is a variegated structure from birth onwards. In some regions it contains only a few scattered cells; in others there may be a continuous single layer of cells; and in still others the cells pile up to form what we have called intimal cell masses (ICM). The cells in the normal ICM are mostly smooth muscle cells although there is also a small resident population of monocyte-like cells. We have been studying the ICM in swine with emphasis on the abdominal aorta. We have found that atherosclerotic lesions in the abdominal aorta of swine induced by high-fat high-cholesterol diets begin by a hyperplastic reaction of the smooth muscle cells in the ICM and progress to form large lesions characterized by extensive regions of lipid-rich calcific necrotic debris similar to advanced lesions in man. Because of the putative key role of the ICM in atherogenesis we think that it is important to learn as much as possible about their natural history under conditions as normal as possible. In this report we present data on ICM in the abdominal aortas of 34 male and female Hormel miniature swine maintained on a low-fat low-cholesterol diet for up to 12 years of age. The ICM grow slowly with aging and in the distal portion of the aorta account for an average of 9% in the male and 15% in the female of the total cells in the aortic wall (intima + media).(ABSTRACT TRUNCATED AT 250 WORDS)

Association of plasma intermediate density lipoproteins with atherogenic intimal proliferative activity in abdominal aortas of hyperlipidemic swine

In an earlier study of swine fed hyperlipidemic (HL) diets containing either butter or corn oil we found wide differences in atherogenic intimal proliferative responses among the swine, especially in the corn oil group, that could not be entirely accounted for by differences in serum cholesterol levels. We hypothesized that there might be differences in some lipoprotein constituents other than total cholesterol that might better account for the differences in intimal proliferative responses. In the current experiment, 11 swine were fed HL diets containing butter (n = 5) or corn oil (n = 6). At 90 days on HL diet, plasma was obtained for lipoprotein fractionation. At 120 days the swine were killed and the lesion areas and number of nuclear profiles in the intima or lesions in multiple cross-sections of the distal abdominal aorta were determined under light microscopy. Tritiated thymidine labeling indices of the intima or lesion cells were also determined. On comparing the butter and corn oil groups, only IDL-cholesterol and tritiated thymidine labeling indices of the cells in the lesions were significantly different. Based on the number of nuclear profiles per cross-section (Np/Cx) 7 were classified as high intimal proliferative responders (5 butter, 2 corn oil) and 4 as low responders (4 corn oil). The high-proliferative responders had plasma IDL levels 6-fold greater than those of the low responders with no overlaps. LDL, VLDL and HDL were not significantly different between the high- and low-proliferative responders. Among correlation coefficients between Np/Cx and plasma lipoprotein variables for all swine (n = 11), IDL cholesterol level was best correlated with Np/Cx. However, in the high responder group LDL values showed a highly significant correlation with the amount of intimal proliferation.

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.