M. Kroms

Experimental atherosclerosis in rhesus monkeys: I. Gross and light microscopy features and lipid values in serum and aorta☆

Abstract Forty-five rhesus monkeys were divided into five groups fed four different high-fat diets and a stock diet. The duration of feeding varied from 33 to 70 weeks. All monkeys fed the high-fat diets developed aortic proliferative lesions composed predominantly of spindle-shaped cells, most of them laden with lipid. The thickest of these proliferative lesions measured 0.85 mm, and most were thinner, suggesting that in the monkey receiving these high-fat diets some proliferative lesions can reach this range of thickness before undergoing necrosis. Atheromatous aortic lesions characterized by necrosis and the accumulation of lipid debris were found only in monkeys receiving high-fat diets for 70 weeks. In monkeys receiving a diet in which the lipid component was 30% peanut oil and 5% cholesterol, the fatty acid composition of the aorta was similar tothat in humans developing coronary artery atherosclerosis, in that the fatty acids showing the greatest accumulation were oleic and linoleic. The largest number of atheromatous lesions, however, was associated with a diet otherwise identical to the peanut oil-cholesterol diet but containing as its lipid component 30% butter and 5% cholesterol. This latter diet also resulted in higher serum lipid levels and higher aortic lipid levels per milligram of DNA than did peanut oil diet. High-fat diets fed to rhesus monkeys appear to produce both proliferative and atheromatous aortic lesions similar to those seen in the human as regards both light microscopy features and lipid composition. Work is now in progress assessing some of the metabolic features of these lesions.

Glucose degradation in normal and atherosclerotic aortic intima-media

Abstract Pathways of glucose degradation and 0 2 uptake were measured in intimamedia of normal swine, rabbits and monkeys, and in atherosclerotic intima-media of rabbits and monkeys. In the normal aortas most of the glucose was degraded to lactic acid, with very little entering the Krebs cycle or pentose shunt systems. Only small amounts ended up as lipid, RNA-DNA-protein, or glycogen. In the atherosclerotic aortas, glucose utilization more than doubled per mg DNA, but again virtually all of it was converted to lactic acid. The 0 2 uptake per mg DNA sharply increased in the atherosclerotic tissue, but no more glucose entered the Krebs cycle than in the normal artery tissue. It thus appears that neither in normal nor in atherosclerotic artery wall is glucose an important substrate for oxidative phosphorylation.

Uptake, oxidation, and esterification of free fatty acids in normal and atherosclerotic rabbit aorta.

Abstract Intimamedia tissue from atherosclerotic rabbits showed a 15-fold increase in the amount of [U- 14 C]palmitic acid oxidized to CO 2 compared to normal intimamedia. The findings suggest that oxidation of free fatty acids may at least in part contribute to the increased oxygen uptake seen in atherosclerotic tissue. The concentration of endogenous free fatty acids in atherosclerotic tissue was much higher than in normal tissue. The higher level may have been partly due to the increased levels of serum free fatty acid in the atherosclerotic rabbits, and to the increased permeability of atherosclerotic intimamedia to free fatty acids demonstrated in vitro . The tissue from atherosclerotic rabbit aortas exhibited a different pattern of fatty acid esterification than did that obtained from control aortas. In atherosclerotic tissue more cholesterol ester than triglyceride was formed from palmitate. In determining the specific activity of the free fatty acid tissue pool(s), equilibration of medium and tissue free fatty acid was found to be slow. In addition, the amount of free fatty acid in aorta was substantial and somewhat variable from animal to animal. Therefore, it was necessary to base calculations of fatty acid metabolic pathways on the specific activity of the palmitate in the tissue and not on that of the medium.

Aortic respiration and glycolysis in the pre-proliferative phase of diet-induced atherosclerosis in swine

Summary Cholesterol or stock diets were fed to fourteen pairs of miniature swine for periods varying from 3 to 112 days. Measurements of respiration, aerobic and anaerobic net lactic acid production and lipid content were done in grossly normal intima-media from the upper thoracic aorta in both dietary groups. Intima-media respiration was significantly higher per unit of DNA in the cholesterol-fed swine than in stock-fed swine. This was so even before any grossly visible atherosclerotic lesions were apparent and before there was any light microscopy or biochemical evidence of cell proliferation, but when pre-proliferative changes were presumably present. The aerobic and anaerobic net lactic acid production however were the same in the aortic tissue of both the cholesterol and stock-fed groups. Both total cholesterol and phospholipid were increased in concentration in the grossly normal intima-media after 44 days of feeding the cholesterol diet. The increased tissue respiration before the appearance of atherosclerotic lesions characterized by smooth muscle cell proliferation may mean that the intima media of the swine is synthesizing more ATP or high energy intermediates. That there might be an increased demand for energy in the artery associated with cholesterol diets in swine is suggested by electron microscopy and autoradiographic studies of the pre-proliferative phase of cholesterol-induced atherosclerosis in the same animals as were used in this experiment.

Mosaicism in female hybrid hares heterozygous for glucose-6-phosphate dehydrogenase (G-6-PD): I. General properties of a hybrid hare model with special reference to atherogenesis

Abstract Hybrid hares ( Lepus europaeus male x Lepus timidus female) were fed intermittently with a cholesterol-containing diet for 3 to 13 months. Their aortas were evaluated for extent of lesions, and for glucose-6-phosphate dehydrogenase (G-6-PD) isozyme patterns in lesions and in normal-appearing aortic media. The red cells of all animals were assayed above 50% for T (the slower-moving form of the enzyme, corresponding to type B in black women, and contributed in the cross by the L. timidus dam). The lesions and normal aortic tissues showed a similar bias toward type T, although values fell slightly below 50% in a few cases. The average percentage of T in lesions and in normal tissues from the same aorta differed significantly in some of the animals, suggesting that cells with one type of active X chromosomes might have some selective advantage over the other in the abnormal environment of the atherosclerotic lesion. The putative selective advantage would have to be related to genes on the X chromosome other than that for G-6-PD, since deviations toward T-type G-6-PD were found in some hares and toward E in others. One hare was fed for 6 months with 25-hydroxycholesterol as well as cholesterol. In this animal (and only in this one) monotypic samples were obtained from lesions, even more frequently from grossly normal regions of the aorta, and from uterus and liver. The monotypes were 100% E in liver, but 100% T in normal aorta, aortic lesions, and uterus. Skin, heart, kidney, spleen, and lung samples were all ditypic. In addition to developing aortic lesions readily on a cholesterol diet, these hybrids show marked stenotic lesions in the extramyocardial coronaries, and are therefore tentatively considered a promising model for the study of atherogenesis.

Effects of methyl prednisolone and colchicine on the development of aortic atherosclerosis in swine

The effect of methyl prednisolone and colchicine on the development of both the early proliferative and advanced atherosclerotic lesion in swine aorta was studied. In order to accelerate the development of atherosclerosis, the abdominal aortic endothelium was partially denuded by a balloon before the animals were placed on either a moderate or severe hypercholesterolemic diet. Neither drug in either dietary group inhibited the development of atherosclerosis. Swine receiving methyl prednisolone and severe hypercholesterolemic diet actually had a significantly greater number of the advanced necrotic lesions and more arterial calcification than the group receiving the atherogenic diet alone. In addition, the thoracic aorta of swine receiving the moderate hypercholesterolemic diet and methyl prednisolone showed larger amounts of lipid than did the non-drug fed control group. In swine receiving the moderate hypercholesterolemic diet, methyl prednisolone significantly raised serum cholesterol levels. Colchicine only slightly worsened the atherosclerosis in swine aorta and had no effect on serum cholesterol levels.

A method for isolating aortic mitochondria exhibiting high respiratory control.

Abstract A method for isolating tightly coupled mitochondria from an intima-media strip of swine aorta has been devised. The most important differences from previous methods were the use of a special device for mincing the aorta into a fine gruel before the homogenization, the incubation of the homogenate with elastase before centrifugation, and the use of an electrolyte-based medium for homogenization and centrifugation of the tissue rather than sucrose. The method resulted in the recovery of enough mitochondria from a single aorta so that a number of determinations could be performed. The respiratory control ratios using succinate, glutamate, glutamatemalate, and pyruvate-malate were 4.3, 8.2, 8.8, and 9.2, respectively. β-OH butyrate functioned very poorly as an electron donor under the conditions used in this experiment. Judging from their functional characteristics after rather rigorous treatment during isolation, swine aortic mitochondria, like cardiac mitochondria, are relatively resistant to damage.

Mosaicism in female hybrid hares heterozygous for glucose-6-phosphate dehydrogenase: VI. Production of monotypism in the aortas of 4 of 10 mosaic hares Fed cholesterol oxidation products☆

The normal aortic tissue of black women heterozygous for glucose-6-phosphate dehydrogenase (G-6-PD) usually consists of two cell phenotypes (mosaicism). By electrophoresis two G-6-PD types are demonstrated (ditypism). Advanced atherosclerotic lesions from such women not infrequently yield samples displaying only one G-6-PD type (monotypism). Possible causes for the monotypism include (1) monoclonal origin of the lesion and (2) selective growth and/or survival advantage of one phenotype over the other. We have been attempting to produce monotypism in the aortas of a hybrid hare model that displays G-6-PD mosaicism in the normal state. In the current study 14 G-6-PD mosaic hares were fed a high-cholesterol diet for 6 to 17 months. All developed extensive moderately severe (as compared to humans) atherosclerotic lesions. No monotypic samples were found. Ten hares were fed (in addition to the standard high-cholesterol diet) small doses of one of two cholesterol oxidation products (25-hydroxycholesterol or triol) for 6 to 21 months. Four of the ten developed monotypic foci in either atherosclerotic lesions or normal aortic tissue or both. The reason for giving the oxidation products was because they are cytotoxic for cells in vitro and one (25-hydroxycholesterol) had been shown to be more toxic for one phenotype than the other with cultured fibroblasts from the mosaic hares (not infrequently resulting in the culture becoming monotypic). We suggest that the monotypism observed in vivo was probably produced by a mechanism similar to that operating in vitro.

Effect of thrombogenic and atherogenic diets on aspects of hepatic energy metabolism in rats

Abstract The purposes of this study were to learn if any abnormalities of hepatic energy metabolism existed in rats receiving two different atherogenic and thrombogenic diets, a butter-cholesterol and peanut oil-cholesterol diet. It has been shown previously that after 14 days of feeding such rats have decreased hepatic protein synthesis and display abnormal circulating coagulation factors synthesized in the liver. After only 21 days of feeding, hepatic mitochondria in these rats display a 3-fold increase in membrane-associated cholesterol, with an altered mitochondrial fatty acid pattern. Electron microscopy of mitochondria within the liver of rats receiving these diets for 21 days revealed no obvious abnormalities; after isolation, however, mitochondria from rats receiving the butter-cholesterol diet (which induced the greatest accumulation of cholesterol) revealed structural changes, including separation of the outer from the inner membrane. At the same time, isolated hepatic mitochondria had a significantly lower oxygen uptake than hepatic mitochondria from stock-fed rats, but oxidative phosphorylation and respiratory control ratios were similar in both groups. After 90 days of feeding one of the diets (butter-cholesterol diet that is primarily thrombogenic in rats) the respiratory control ratio was decreased when β-OH butyrate was used as the substrate. To further investigate energy metabolism in these livers adenine nucleotide levels were measured after 14 and 28 days of dietary feeding. The levels of ADP suggested that the livers of rats fed these high-cholesterol diets have a decreased rate of mitochondrial synthesis of ATP, confirming the results of the first portion of the study. In spite of the evidence of decreased ATP synthesis in these livers, levels of ATP either per g of wet weight or per total liver were no lower in the experimental diet-fed group than in stock-fed rats. The finding of normal ATP levels in the face of decreased ATP synthesis suggested decreased ATP utilization, a suggestion that was confirmed by examination of ATPADP ratios. This series of experiments has shown that by 14–28 days, when there are abnormalities of hepatic protein synthesis and of circulating coagulation factors synthesized in the livers of rats fed thrombogenic or atherogenic diets, there are also abnormalities of energy metabolism present. These later abnormalities, however, in our opinion do not appear to be of such a nature or extent that they can be called the primary causes of the hepatic protein abnormalities.

Aortic mitochondrial function in experimentally induced atherosclerosis in swine and rabbits

Abstract An improved method for isolating aortic mitochondria with good respiratory control has been developed. Using this method, mitochondria were isolated from atherosclerotic and normal aortic intima-media tissue in both swine and rabbits. No differences could be found in State 3 O 2 uptake, acceptor control ratios or ADP O ratios in mitochondria from the atherosclerotic vs normal tissue in either species. The study suggests that mitochondrial function in the proliferative (non-necrotic) phase of atherosclerosis is not impaired.