Hideshige Imai

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.

Cerebral atherosclerosis in swine: role of necrosis in progression of diet-induced lesions from proliferative to atheromatous stage.

Summary In recent studies of the pathogenesis of atherosclerosis, we have sequentially categorized the atheroslcerotic lesions into (1) preproliferative, (2) proliferative, and (3) atheromatous phases. This report based on electron microscopy observations is mainly concerned with the cellular and subcellular events occurring in transition from the proliferative to the atheromatous phase with emphasis on the possible role of necrosis. We also paid special attention to the internal elastica, endothelial cells and smooth muscle cells of the inner media at all stages. Materials for study consisted of middle cerebral arteries of young swine fed atherogenic or stock diet for 160 days. Atheroslcerotic lesions in these arteries appeared to be in a transitional state from the proliferative to atheromatous phase. They consisted of collections of bizarre smooth muscle cells, fibrillary and nonfibrillary materials including dissolved neutral lipids, dense and laminated phospholipids, cholesterol clefts, and fine reticulated material consistent with mucopolysaccharides. There were also cellular degenerative changes that can be classified into (1) pyknosis and karyorrhexis and (2) cytolysis, and, in addition, focal accumulation of lipid-rich debris which is the hallmark of atheroma. Viable smooth muscle cells responded by either phagocytizing or sequestering the products of cell death. Based on the transitional forms from recognizable smooth muscle cells to disintegrated products of cell death, and also on the lack of identifying features suggestive of other origin, most and perhaps all of the dead cells in the early atheromata appeared to have been smooth muscle cells and the major portion of atheromatous debris probably originated from necrosis of smooth muscle cells. Deposition of cholesterol-rich material from the extracellular fluid in the necrotic foci probably occurs as the lesion progresses, adding to the mass. The endothelial cells had changes suggestive of disturbed metabolism in both lesion and nonlesion areas perhaps leading to increased permeability, and resulting in accelerated transport of blood consituents from the lumen to deeper layers. Although the internal elastica was well developed in the middle cerebral arteries, it often had erosion effects in the diet-induced lesions apparently leading to wide and numerous gaps with loss of sharp demarcation between the intima and media. Such erosion effects and fragmentation of internal elastica were associated with adjacent smooth muscle cells with exaggerated basement membrane. Study of the control specimens revealed in a few instances miniature foci of degeneration and necrosis in the inner media, but without visible collections of lipid-rich debris. Although an extensive search was required to find these degenerative foci in the control inner media, the fact that they were present at all is suggestive of a precarious situation for these cells, which would make them relatively vulnerable to the effect of noxious agents.

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.

Ultrastructural studies of absorption of methoxychlor in the jejunal mucosa of the rat

Abstract Thirty-four male adult Wistar rats were given by gastric tubing either a single dose of 400 mg/kg or three consecutive daily doses of 1,600 mg/kg of methoxychlor dissolved in corn oil or suspended in aqueous polyvinylpyrrolidone. Controls were given equal amounts of vehicle alone. Specimens of the jejunal mucosa for light and electron microscopy were taken at 1 hour after the single dose or 24 hours after the last of three daily administrations. Material, probably derived from methoxychlor was identified as intra- and intercellular densities that were different from those derived from the vehicles. The route of absorption of all administered materials through the mucosa was largely similar except that the mixtures containing methoxychlor seemed to traverse to the intercellular spaces immediately after the passage through the luminal border in addition to the usual transport through the endoplasmic reticulum towards the Golgi apparatus. Furthermore, the absorption of methoxychlor was characterized by salient distention of vesicles and intercellular spaces that appeared nearly empty and larger than necessary to accommodate methoxychlor products. Overall cellular and organellar changes associated with absorption of methoxychlor at 1 hour and on the fourth day comprised a similar accentuation of asynchronous deterioration of organelles. Special reference was made to the occasional coexistence of intact and swollen mitochondria in the same columnar cells of the fasted controls and fed animals.

Differential counts by electron microscopy of cell types in normal intimal cell masses in swine abdominal aortas

Hyperlipidemic diet-induced atherosclerotic lesions show a predilection for locating at the site of normal collections of intimal cells in the swine abdominal aorta. This implies that preexisting intimal cell masses (ICM) evolve into early diet-induced lesions. This study characterizes in detail the distal abdominal aortic ICM of young normal swine as a basis for a better understanding of lesion development. Seventeen male Yorkshire swine were used, from neonates to 20 weeks of age. Only three of six neonates showed multilayered ICM, the remaining three showed scattered individual cells or a single layer of cells. All older swine had multilayered ICM at a predictable ventral site corresponding to Evans blue in vivo staining. The majority of cells counted by electron microscopy were smooth muscle (89 to 94%), the remaining were macrophages (1 to 8%) and poorly differentiated cells (3 to 5%). Weanling and older swine showed high proportions of contractile smooth muscle cells. Ergastoplasm-rich smooth muscle cells comprised nearly half of the neonatal population. Stainable lipids were demonstrable in 20-week-olds, but not in younger swine. No foam cells were seen, but cells containing a few droplets totaled 2%; macrophages comprised a high proportion of these. Pyknotic dead cells were rare, being found in the neonate and 20-week-old swine. Mitoses were observed in two ergastoplasm-rich smooth muscle cells in the neonate and one each in contractile smooth muscle cells of a 14- and a 20-week-old swine. Saddlebag-shaped, atypical-appearing cells were present in all age groups, involving all three cell types, and ranging from 1 to 3%. These represented either bridged nuclei and cytoplasm or more likely artefactual distortion of relatively normal cells.

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.

Mosaicism in female hybrid hares heterozygous for glucose-6-phosphate dehydrogenase: IV. Aortic atherosclerosis in hybrid hares fed alternating cholesterol-supplemented and nonsupplemented diets

Hybrid hares provide a model for induction of aortic atherosclerosis. The basic dietary regimen consisted of cholesterol-supplemented and nonsupplemented commercial pellets alternating at 1-week intervals for up to 18 months. Controls were fed nonsupplemented pellets. Control aorta features included branch cushions, intimal thickenings unrelated to branches, and several longitudinal layers of smooth muscle in the outer media. Branch cushions extended longitudinally in the main vessel, and had multidirectional, streamlike bundles of smooth muscle in the intima and media. Intimal thickenings unrelated to branches occurred as longitudinal patches, most frequently in the ventral quadrant. The adjacent media had a regular array of cells. Raised lesions concentrated at branch orifices and in the ventral quadrant. Lesions involved up to 80% of the thoracic and 40% of the abdominal aorta by 5 months. Predominant lesions consisted of superficial fibromuscular tissue and deeper foam cells and stainable lipids. Some of these lesions were atheromata with fibrous cap. Fibromuscular plaques, 120 to 150 micron thick, had a few superficial foam cells and hydropic degeneration. Foam-cell lesions were also observed. Data obtained suggest the importance of branch cushions and intimal thickenings unrelated to branches, and of the periods on nonsupplemented pellets.

Coronary arterial heart disease in hybrid hares fed alternating cholesterol-enriched and normal diets

At one-week intervals hybrid hares (Lepus europaeus X Lepus timidus) were fed supplemented or nonsupplemented commercial pellets. Supplements were 1% cholesterol (USP-grade) and 6% peanut oil. In addition, the normal nonsupplemented diet was fed for a month when they appeared weak or when serum cholesterol values exceeded 1,000 mg/dl at monthly examinations. At the end of 1 year on such an alternating regimen, the hares appeared well nourished. Atherosclerotic lesions were severe and involved the epicardial and intramyocardial segments of the coronary arteries as well as the thoracic and abdominal aorta. Coronary arterial lesions were characterized by fibromuscular thickening with stainable lipids, necrosis, and calcification. Intramyocardial arterioles were diseased with fibromuscular plaques rich in foam cells. Transmural infarcts of the left ventricle occurred distal to the stenotic arterial lesions. Exact mechanisms are unknown for the development of such functionally significant coronary atherosclerosis. Among the possible pathogenetic factors, however, the alternating schedule seems most important. Such a regimen resulted in fluctuating, mild to moderate hypercholesterolemia and relatively good nutritional state of hybrid hares.

Pulmonary acariasis in rhesus monkeys: electron microscopy study

Abstract Lungs of six untreated rhesus monkeys were examined by light and electron microscopy in order to characterize the responses due to the infestation by Pneumonyssus simicola . The lesions were characterized by slight fibrosis with bronchiolitis, peribronchiolitis, and pneumonitis adjacent to the affected bronchioles. Pigmented and nonpigmented materials peculiar to the infestation by the mite were classified into three types, namely, (1) silver to gold colored, needle-like bodies with medium electron density; (2) tan to yellow brown, granular, birefringent granules with the tinctorial characteristics of lipochromes, which could be subdivided by electron microscopy into (a) extremely dense particles that disintegrated under high pH and electron beams, and (b) sickle-like to rhomboid, fine filamentous bodies; and (3) nonpigmented, sudanophilic, and PAS-positive vesicles that were, by electron microscopy, a mixture of homogeneously gray and flocculent materials. Most of these bodies were found in macrophages. Poorly differentiated, mononuclear interstitial cells were the second prominent ultrastructural feature. These cells showed hyperplasia and apparent transition to macrophages. Other cell types noted were plasma cells, lymphocytes, eosinophiles, multinucleated giant cells, and mast cells.