Lysosomes of the Arterial Wall: IV. Cytochemical Localization of Acid Phosphatase and Catalase in Smooth Muscle Cells and Foam Cells from Rabbit Atheromatous Aorta

Cytochemical methods for acid phosphatase and catalase were applied to atheromatous aortas from cholesterol-fed rabbits. Whole tissue, partially digested aortic slices and isolated cells were used for the study. Present in the atheromatous lesions were smooth muscle cells in all stages of foamy transformation, from virtually normal appearing smooth muscle cells to severely altered cells with pronounced lipid accumulation. The results with the acid phosphatase method show that lysosomes increase both in size and in number as the smooth muscle cells become foam cells. In normal appearing smooth muscle cells, acid phosphatase reaction product was found in stacked cisternae of the Golgi apparatus and in small vesicles located in the Golgi region and distributed throughout the cytoplasm. In foam cells, reaction product was found in membrane-limited vacuoles of varying size which typically contained membranous debris or myelin-like figures together with massive lipid deposits. No reaction was seen in “free” cytoplasmic lipid droplets lacking a surrounding membrane. These results confirm and extend previous biochemical findings indicating that, in the cholesterol-fed rabbit, the change from normal smooth muscle cell to foam cell is accompanied by marked physical and chemical changes of the lysosomes, including their progressive overloading with cholesteryl ester. Small diaminobenzidine-positive particles were present in normal smooth muscle cells and in those at all stages of foamy transformation. These particles were more frequent in foam cells, in agreement with the marked increase in catalase activity detected biochemically in these cells.     

Early atherogenesis in the White Carneau pigeon. III. Lipid accumulation in nascent foam cells.

The role of lysosomes in aortic atherogenesis in White Carneau pigeons was examined by means of acid phosphatase cytochemistry. Foam cells were the major constituent of nascent atherosclerotic lesions in pigeons fed a 0.5% cholesterol diet for either 5 or 10 weeks. Seventy-four percent of foam cell lipid from animals at 5 weeks was in cytoplasmic droplets. The remaining lipid appeared in secondary lysosomes. After 10 weeks of cholesterol feeding, lysosomal lipid accounted for 73% of the lipid volume. The lipid accumulation correlated with increases in both size and number of lysosomes. An average of 2.4 lysosomes per 10(4) cu mu of cytoplasm was observed at 5 weeks. This value doubled by 10 weeks. The average lysosome diameter also increased between 5 and 10 weeks from 2.2 mu to 5.75 mu. Concomitantly, the complexity of lysosomes increased from simple, spherical organelles at 5 weeks to complex, multichambered organelles at 10 weeks. In contrast, lipid storage within cytoplasmic lipid droplets did not change either in size or in number. These observations suggest that by 5 weeks lipid storage within cytoplasmic droplets was maximized, and continued increases in lipid stores occurred predominantly through lysosomal loading.     

Subcellular fractionation of arterial smooth muscle cells laden with lipid following incubation with low density lipoproteins and chloroquine

Lipid accumulation was induced in pig aortic smooth muscle cells in culture by incubating them with LDL and the lysosomotropic agent chloroquine, as an in vitro model of lipid accumulation in atherosclerosis. The cells were homogenized and subjected to a conventional low-speed centrifugation to prepare a postnuclear supernatant. This supernatant was then subjected to analytical subcellular fractionation by sucrose density gradient centrifugation. This showed that in normal smooth muscle cells most of the cholesterol was in the plasma membrane. When postnuclear supernatants of cells that had been incubated with LDL, either with or without chloroquine, were applied to the density gradient, there was no change in the distribution of cholesterol. The distribution of cholesterol became denser, however, when postnuclear supernatants of cells that had been incubated with chloroquine alone were studied, whereas the equilibrium density of the plasma membrane was not increased to the same extent. The cholesterol in these postnuclear supernatants was probably contained in both the plasma membrane and in the lysosomes. The distribution of chloroquine in the density gradients was consistent with a lysosomal localization. Incubation with chloroquine alone preferentially reduced the activity of the lysosomal component of β-glucuronidase compared to its endoplasmic reticulum component. The ratio of the activity of the cytosolic to the mitochondrial forms of malate dehydrogenase in cells incubated with chloroquine, either with or without LDL, was also decreased. When the cells were incubated with LDL and chloroquine together, lipid droplets, at least some of which were membrane-bound, and large autophagic vacuoles developed in the cells. When homogenates of these cells were subjected to low-speed centrifugation to prepare a postnuclear supernatant, all of the large autophagic vacuoles were sedimented into the nuclear fraction, together with most of the cholesteryl ester and chloroquine and most of the acid hydrolase activities. Therefore whole homogenates of smooth muscle cells, rather than postnuclear supernatants, were applied to the sucrose density gradients. When the cells had been incubated with LDL and chloroquine together, most of the cholesteryl ester and chloroquine and part of the nonesterified cholesterol were then found to be associated with lysosomes. These lipid-laden lysosomes probably correspond to the autophagic vacuoles seen in the cells by electron microscopy. The density of the lipid-laden lysosomes was increased considerably compared to the lysosomes of normal cells. This may have been due to either the high chloroquine content of the lysosomes, to an enhanced permeability of the lysosomal membrane to sucrose, or to the presence of partially degraded cell organelles within the lysosomes due to the autophagy caused by chloroquine. The distributions of cholesterol and cholesteryl ester had a small shoulder of low density, which may possibly correspond to the lipid droplets seen within the cells by electron microscopy. Thus, when large-scale lipid accumulation is induced in smooth muscle cells by incubating them with LDL and chloroquine together, the large majority of the cholesterol and cholesteryl ester that accumulates in the cells is contained within the autophagic vacuoles rather than within the lipid droplets.     

Enhancement of cholesterol and cholesteryl ester accumulation in re-endothelialized aorta.

The purpose of the experiments reported here was to determine chemically the character and quantity of lipid in re-endothelialized and de-endothelialized areas of rabbit aortas. The aortas of 22 rabbits, Groups I and II, were de-endothelialized with a balloon catheter, and the rabbits were maintained on a lipid-poor diet for 4 weeks. Thirteen rabbits, Group II, were then fed an egg-supplemented diet for 6 weeks. Nine rabbits, Group I, were continued on the lipid-poor diet for an additional 6 weeks. Control rabbits with uninjured aortas were fed a lipid-poor diet for 10 weeks (Group III) or an egg-supplemented diet for 6 weeks (Group IV). Nonesterified cholesterol and fatty acids, cholesteryl esters, triacylglycerols, and squalene were quantitated in re-endothelialized and de-endothelialized aorta by thin-layer chromatography and fluorometric analysis. The results indicate 1) that there was approximately three times as much nonesterified cholesterol and cholesteryl ester in re-endothelialized aorta of Groups I and II as compared with adjacent de-endothelialized aorta and 2) that in re-endothelialized aorta of Group II the amount of total cholesterol correlated with serum cholesterol concentration in contrast to adjacent de-endothelialized aorta, with no correlation over a range of nearly 900 mg/100 ml. These studies indicate that the presence of endothelium favors accumulation of aortic cholesteryl esters. The results suggest that arterial lipid accumulation is not simply a result of passive filtration but may result from metabolic differences in the re-endothelialized neointima.     

Morphological characteristics of lipid accumulation in liver-constituting cells of acid lipase deficiency rats (Wolman's disease model rats)

Lysosomal acid lipase is a hydrolase essential for the intracellular degradation of cholesteryl esters and triglycerides. In the laboratory, rats with congenital deficiency of lysosomal acid lipase and marked accumulation of cholesteryl ester, cholesterol free and triglyceride in livers (Wolman's disease rat or Yoshida rat) that corresponded to human Wolman's disease were found and maintained. The morphological characteristics of accumulated lipids in the livers of affected rats were examined also. Many small lipid droplets and lipid crystals were found in the cytoplasms of hepatocytes and ED1-positive and ED2-positive foamy Kupffer's cells, respectively. Electron microscopically, many electron-lucent lipid droplets with limiting membrane were found in hepatocytes. Foamy Kupffer's cells had many multivesicular bodies with limiting membrane, which contained crivilinear bodies, lipid droplets and crystal clefts. At areas of aggregation of foamy Kupffer's cells forming islets, there were many desmin-positive Ito cells. Small lipid droplets with limiting membrane were also found in the cytoplasm of Ito cells and endothelial cells. These findings, which were obtained by morphological methods, indicated that triglyceride and both cholesteryl ester and free cholesterol accumulated in lipolysosomes mainly in hepatocytes and Kupffer's cells, respectively, and suggest that lysosomal acid lipase could participate in dissolution of the membrane.     

Arterial prostaglandins and lysosomal function during atherogenesis. II. Isolated cells of diet-induced atherosclerotic aortas of rabbit

This report validates and expands further the interpretation of our findings on prostaglandins and lysosomes in rabbit aortic homogenates (see paper I of this series) to enzymatically isolated and separated aortic cell populations during atherogenesis. Evidence is provided by which isolated arterial cells may be considered representative of in situ increases of diseased aortic tissue prostaglandin I2 and E2 levels, as well as lysosomal acid hydrolase activities and total cholesterol content based on DNA. Increasing latency of aortic lysosomal N-acetyl-beta-glucosaminidase activity was confirmed and correlated with increasing severity of atherosclerosis, in parallel to increasing levels of prostaglandin I2 but not increasing levels of prostaglandin E2. Ultrastructural observations also confirmed aortic intracellular lipid accumulation within lysosomes and as lipid droplets. Consistent with these relationships, separated low density, lipid-filled aortic cells were especially increased in total (197%) and latent (15%) lysosomal acid hydrolase activities, catalase activity (274%), total cholesterol (151%), and in both prostaglandin I2 (67%) and E2 (325%) levels based on DNA, as compared to control aortic cells or more normal-appearing high-density diseased aortic smooth muscle cells; high-density diseased aortic cells were increased in prostaglandin E2 but similar in latent acid hydrolase activity compared to control aortic cells. Since the total cholesterol content of rabbit atherosclerotic aortas was evidenced more intracellularly (75%) than extracellularly (25%) in this study, the association of increased prostaglandin I2 and E2 levels with low-density lipid-filled cells suggest the participation of these prostaglandins in the genesis of aortic foam cells during arterial lipid accumulation in rabbit atherosclerosis. The association of increasing prostaglandin I2 levels and increasing latent lysosomal N-acetyl-beta-glucosaminidase activities also implicates a possible relationship between this prostaglandin and lysosomal membranes of aortic cells, either primary or secondary to intralysosomal lipid accumulation.     

Lipid characterization in an animal model of atherosclerosis using NMR spectroscopy and imaging

High resolution 1H and 13C NMR spectroscopic measurements including 1H/13C 2D correlation and magnetic resonance imaging (MRI) studies, have been carried out on intact rabbit aortic tissues ex vivo using animals fed both normal and high cholesterol diets. The results show that 1H and 13C NMR spectroscopy can distinguish mobile lipids and can differentiate between normal triglyceride content and cholesterol-enriched lipids, in intact tissue, There were considerable differences in the level of deposition of cholesteryl esters in animals all fed on the same diet. Confirmation is presented of temperature-dependent differences in mobility and organization between the triglycerides found in control tissue and the cholesteryl esters found in aortas from high lipid diet animals. Water-suppressed MRI showed evidence of lipid accumulation in the aortas of high cholesterol diet rabbits. It is concluded that the hypercholesterolaemic rabbit model of atherosclerosis, coupled with such NMR methods, may offer a noninvasive method of monitoring disease development, allowing the evaluation of the effect of therapeutic agents on the progress of atherosclerosis.     

Lysosomal lipid accumulation in vascular smooth muscle cells

Using an inverted culture technique, the accumulation of lipid within vascular smooth muscle cells incubated with lipid droplets was studied. Initially, lipid was found exclusively within cytoplasmic inclusions but, as accumulation continued, lysosomes became the predominant site of lipid storage. After 3 hr of incubation, 84% of lipid was within lysosomes. This lysosomal lipid accumulation produced a tripling of the average size of lysosomes and resulted in lysosomes with complex, multilobed shapes. In contrast, although the number of cytoplasmic inclusions increased with lipid loading, individual inclusions maintained a spherical shape and a consistent diameter of 1-1.3 microns. Concomitant with changes in cellular lipid storage, incubation with lipid droplets induced development of an anastomosing network of acid phosphatase-containing tubules which were spatially related to sites of lysosomal lipid accumulation. Thus lipid accumulation produced ultrastructural alterations in a number of metabolic compartments. Similar alterations in the intracellular compartmentalization of acquired lipid have been demonstrated in foam cells during atherogenesis and have been hypothesized to have profound effects on lipid metabolism and disease progression.     

Virus-induced atherosclerosis. Herpesvirus infection alters aortic cholesterol metabolism and accumulation.

Infection of normocholesterolemic, specific-pathogen-free chickens with Marek's disease herpesvirus (MDV) has been shown histologically to lead to chronic atherosclerosis like that in humans. The development of herpesvirus-induced atherosclerosis in vivo and the presence of specific Marek's antigen within aortic cells suggested that MDV infection may modify lipid metabolism and lead to significant lipid accumulation. Experiments reported herein were designed to determine the types and quantity of lipid present in aortas from MDV-infected and uninfected chickens between 2 and 8 months of age following infection and assess one possible mechanism of lipid accumulation by evaluating the effect of MDV infection on aortic cholesterol and cholesteryl ester (CE) metabolism. Chromatographic-fluorometric analyses indicated that at 4 and 8 months of age after MDV inoculation, MDV-infected animals had a significant (P less than 0.05) two-fold to threefold increase in total aortic lipid accumulation characterized by significant increases in cholesterol, CE, triacylglycerol, and phospholipid as compared with aortas from uninfected animals. At 8 months of age, similar increases in aortic lipid accumulation were observed in MDV-infected animals as compared with those animals vaccinated with turkey herpesvirus and later challenged with MDV. CE synthetic activity was increased significantly by 50% at 4 months of age in the MDV-infected group as compared with the uninfected group, which could explain the initial increase in CE accumulation. By 8 months of age, the authors also observed a twofold increase in CE synthetic activity and a 30% and 80% reduction in lysosomal and cytoplasmic CE hydrolytic activities, respectively, in aortas of MDV-infected chickens as compared to controls. Moreover, infection with MDV blocked the activation of cytoplasmic CE hydrolytic activity by dibutyryl cyclic AMP or exogenous cyclic AMP-dependent protein kinase. Taken together, these results suggest that lipid accretion in aortas of MDV-infected chickens results, in part, from alterations in cholesterol/CE metabolism during early stages of the disease. These findings support the hypothesis that human atherosclerosis may result from specific herpesvirus infection which can alter lipid metabolism and lead to lipid accretion.     

The subcellular biochemistry of human arterial lesions. I. Biochemical constituents and marker enzymes in diseased and unaffected portions of human aortic specimens.

Atheromatous lesions (including media beneath) and uninvolved media from neighboring areas of the same vessel were dissected from 23 human aortas obtained at autopsy. After tissue slices were taken for histological assessment, homogenates were prepared and the levels of DNA, protein, hydroxyproline, free and esterified cholesterol, and eight enzymes known to be associated with various subcellular organelles [N-acetyl-β-glucosaminidase, β-galactosidase, cathepsin C, acid cholesteryl esterase (lysosomes); malate dehydrogenase (mitochondria); neutral α-glucosidase (endoplasmic reticulum); 5′-nucleotidase (plasma membrane); catalase (“microperoxisomes”)] were compared between lesions and uninvolved media preparations. No difference was found in the contents of the two samples in DNA, protein, malate dehydrogenase, neutral α-glucosidase, or 5′-nucleotidase. In contrast, the lesions were significantly enriched in catalase activity (67%) and in each of the acid hydrolases (55 to 126%) as well as in free (300%) and esterified (822%) cholesterol, based on DNA, compared to uninvolved media from the same vessels. Increases in both catalase and acid hydrolases correlated significantly with increases in total, free and esterified cholesterol contents of the preparations. Changes in the acid hydrolase specific activities were also correlated significantly with each other. Increased catalase and acid hydrolase activities in human atheromas are similar to previous findings in aortas of experimental animals fed a cholesterol-rich diet and suggest the participation of peroxisomes and lysosomes in the cellular response to lipid accumulation in atherosclerosis.Because the analyses were conducted on aortas taken at autopsy, modelexperiments on aortas of rabbit cadavers were also employed to determine the extent to which postmortem changes may have affected the levels of enzyme activities and chemical constituents surveyed. These studies showed only minor changes occur during the first 48 hr after death. Investigations of the latency of N-acetyl-β-glucosaminidase in postmortem human and rabbit aortas indicated that lysosomes of arterial cells remain intact for a considerable time after death.     

The pathology of Tangier disease. A light and electron microscopic study.

Tangier disease (deficiency of high density plasma lipoproteins) is characterized clinically by: low levels of plasma cholesterol; enlarged, orange-yellow to yellow-gray tonsils and, frequently, peripheral neuropathy. Histologic and ultrastructural studies were made of various tissues from 5 patients with Tangier disease, and comparisons were made of these findings with those in the 12 other patients thus far known to have this disease. Deposits of cholesteryl esters were found in: reticuloendothelial cells (foam cells) in tonsils, bone marrow, skin and jejunal submucosa; Schwann cells in peripheral nerves and myenteric plexus; and in nonvascular smooth muscle cells. These deposits appeared electron lucent and intensely birefringent, varied from spherical to crystalline in shape, often were extensively confluent throughout large areas of cytoplasm, and were not limited by membranes. Certain foam cells in bone marrow also contained membrane-limited clusters of lipid particles resembling chylomicrons. The foam cells in Tangier disease differ morphologically from those in numerous lysosomal enzyme deficiency states, particularly Wolman's disease and cholesteryl ester stroage disease, and in proliferative diseases of the reticuloendothelial system in which cholesteryl esters also accumulate in abnormal histiocytes. Morphologic and biochemical data suggest several hypotheses to explain the accumulation of cholesteryl esters in tissues of patients with Tangier disease. Among these hypotheses, the most likely are considered to be the presence in plasma of abnormal lipoprotein particles that are subject to phagocytic removal by reticuloendothelial cells, and the failure of a process that normally removes locally synthesized cholesterol from cells to plasma. (Am J Pathol 78:101-158, 1975)     

Lysosomal hydrolysis of lipids in a cell culture model of smooth muscle foam cells

Rabbit aortic smooth muscle cells take up lipid droplets when they are presented using an inverted culture technique. These droplets were localized in secondary lysosomes as demonstrated by staining for acid phosphatase. Initially, 69% of the cell volume was occupied by lipid, and 94% of the lipid was in lysosomes. After a 24-hr clearance period, the cell volume occupied by lipid decreased to 53%, although there was no change in the fraction of cell lipid that was in lysosomes. To confirm that hydrolysis of droplet lipid was occurring in lysosomes, cultures were exposed to medium containing Sandoz 58-035, an inhibitor of acyl CoA:cholesterol acyl transferase, for 24 hr in the presence and absence of chloroquine, ammonium chloride, or methylamine. Although the hydrolysis of cholesteryl oleate was sensitive to these lysosomotropic agents, the hydrolysis of triolein was not. Using reconstituted LDL containing cholesteryl oleate and triolein, we demonstrated that the hydrolyses of cholesteryl oleate and triolein were equally sensitive to the lysosomotropic agents when the cells were not loaded with droplet lipid. However, in cells loaded with lipid, hydrolysis of LDL cholesteryl ester was sensitive to the lysosomotropic agents but hydrolysis of triolein was not. We therefore conclude that both droplet lipids were hydrolyzed in lysosomes, and we attribute the failure of the lysosomotropic agents to inhibit fully the hydrolysis of droplet triolein to the presence of a large mass of free fatty acids in the lysosome that maintains a sufficiently low pH to sustain the triglyceridase activity, but not the cholesteryl esterase activity, of the lysosomal acid lipase.     

Endothelium modifies the altered metabolism of the injured aortic wall.

Results of previous experiments in this laboratory indicate that lipids, especially cholesterol and cholesteryl ester, preferentially accumulate in re-endothelialized, as compared with de-endothelialized, areas of aorta (Am J Pathol 1980, 99:81-104). In the experiments reported here, the hypothesis that this lipid accumulation results from alterations in arterial wall metabolism induced by injury and modified by endothelium was tested. Activities of the two cholesterol-ester-metabolizing enzymes acyl CoA: cholesterol acyltransferase and acid cholesteryl esterase were assayed in uninjured aortas and in de-endothelialized and re-endothelialized areas of balloon-catheter-injured aortas from normocholesterolemic and hypercholesterolemic rabbits. Activities of marker enzymes for major cell organelles were also assayed. Our results indicate that acyl CoA: cholesterol acyltransferase activity was similarly increased in re-endothelialized and de-endothelialized areas of injured aortas. Activity of acid cholesteryl esterase was also increased; however, it was significantly less in re-endothelialized as compared with de-endothelialized areas. Activities of several marker enzymes were changed in injured aortas, particularly in de-endothelialized as compared with re-endothelialized areas. These findings suggest that 1) injury predisposes to general metabolic changes in the aorta that are modified by endothelium and 2) increased cholesteryl ester accumulation in re-endothelialized aortas occurs at least in part from increased synthesis and decreased hydrolysis.     

Early extracellular and cellular lipid deposits in aorta of cholesterol-fed rabbits.

Subendothelial accumulation of extracellular liposomes rich in unesterified cholesterol has been described as an early feature of atherosclerosis induced by cholesterol feeding in rabbits. Beta-very-low-density lipoproteins, however, the presumed source of atherogenic lipid in this animal model, contain mostly esterified cholesterol. The purpose of this study was to test for the presence of extracellular neutral lipid deposits consistent with esterified cholesterol, by employing new electron microscopic techniques. Rabbits were fed 0.5% cholesterol, 5% butter for 0, 1, 2, and 4 weeks. The lipid-preserving ultrastructural techniques showed, in control and atherosclerotic rabbit arteries, neutral lipid droplets adherent to the endothelial luminal surface. After 1 to 2 weeks, subendothelial extracellular deposits of mostly membranous lipid appeared; these deposits contained variable amounts of neutral lipid. At the same time, cytoplasmic neutral lipid droplets appeared in smooth muscle cells and in a small number of subendothelial macrophagelike cells. After 4 weeks, monocytic infiltration and macrophage foam cell development were prominent, but abundant extracellular lipid deposits also were found. Therefore, in arteries of cholesterol-fed rabbits, deposition of membranous and neutral lipid in the extracellular space and neutral lipid accumulation in resident arterial cells are early and probably independent events, both occurring before monocytic infiltration of the arterial intima.     

Lipid Accumulation and Ultrastructural Change Within the Aortic Wall During Early Spontaneous Atherogenesis

To identify the initial period and type of lipid accumulation during spontaneous atherosclerosis, quantitative chromatographic profiles of major lipid classes in upper thoracic aortas (non-lesion areas) and celiac artery cushions (lesion areas) were obtained from atherosclerosis-susceptible White Carneau (WC) and atherosclerosis-resistant Show Racer (SR) pigeons from 1 day to 6 months of age. Thoracic aortas of WC and SR pigeons contained similar amounts of cholesterol, nonesterified fatty acids, triacylglycerols, cholesteryl esters, phospholipids, and hydrocarbon at each age studied. However, celiac sites in WCs contained more total lipid than corresponding SR sites at 6 weeks and 6 months of age. This initial increase at 6 weeks in WCs was characterized by invreased concentrations of nonesterified saturated fatty acids. By 6 months of age, WC celiac cushions had greater concentrations of each lipid class except hydrocarbon than did SR cushions. This initial lipid accumulation was accompanied by ultrastructural changes within the arterial wall, which included the presence of extracellular, vesiclelike structures and extensive accumulation of basal lamina-like material between cells. This material was not present in aortic regions that are not predisposed to lesion formation. This material increased by 6 months of age in the enlarging WC fibromuscular intimal cushions. These morphologic changes paralleled the quantitative lipid increases and represented the first morphologic changes detectable at this site. Age-related changes in arterial lipid content and ultrastructure in SRs are different from those related to early spontaneous atherogenesis in WCs.     

Effects of lipids on the stability of lysosomes in vitro. I. Effects of egg lecithin, lysolecithin, sphingomyelin, and cholesterol on the stability of isolated aortic and hepatic lysosomes of swine.

The effect of lipids on the stability of isolated aortic and hepatic lysosomes of swine was studied using ultrasonicated aqueous lipid dispersions. Egg lecithin labilized aortic lysosomes whereas addition of free cholesterol to the lecithin medium prevented this labilization. Sphingomyelin showed little effect on aortic lysosomes. Phospholipids, such as egg lecithin and lysophosphatidylcholine, also showed a labilizing effect on isolated hepatic lysosomes. Free cholesterol inhibited the labilizing effect of these phospholipids. On the other hand, cholesteryl esters were without effect, presumably due to low affinity for phospholipids.     

Chemical and physiochemical comparison of advanced atherosclerotic lesions of similar size and cholesterol content in cholesterol-fed New Zealand White and Watanabe Heritable Hyperlipidemic rabbits

Watanabe Heritable Hyperlipidemic (WHHL) and cholesterol-fed New Zealand White (CH-FED NZW) rabbits were sacrificed at 15 months of age or after 16 weeks of cholesterol feeding, respectively. During the experimental period, the arterial walls of both the CH-FED NZW and WHHL rabbits were exposed to similar amounts of cholesterol and the lesions which developed at the aortic arch had similar intimal thicknesses, total lipid and cholesterol content. However, the lesions of the WHHL rabbits morphologically resembled human plaques, and contained lipid in the form of smectic liquid crystalline droplets and cholesterol monohydrate crystals. The CH-FED NZW rabbits had lesions which were fatty streak-like, containing liquid crystalline cholesteryl ester droplets but few crystals. The aortic arch intimas of the CH-FED NZW rabbits contained significantly more cholesteryl ester, and less unesterified cholesterol and triglyceride, than those of the WHHL rabbits. The intimal compositions of the two rabbit models did not overlap. Analysis of the compositions predicted precipitation of cholesterol monohydrate crystals in the WHHL but not the CH-FED NZW. The physical state of the deposited cholesterol esters was similar in both with about half being in smectic liquid crystalline form at body temperature. Since the size and total lipid content of the lesions of the CH-FED NZW and WHHL rabbits were similar, we suggest that the greater time of exposure to hypercholesterolemia was important in the formation of cholesterol monohydrate crystal-containing plaques in the aortic arch of the WHHL rabbits.     

Fine structure of the human gallbladder with cholesterosis with special reference to the mechanism of lipid accumulation.

Eight excised gallbladders with cholesterosis were studied by light and electron microscopy. Lipid droplets were found not only in the submucosa, but also in the infranuclear cytoplasm of epithelial cells. These contained well developed mitochondria and agranular reticulum. Lipid droplets were seen to form in the agranular reticulum. Macrophages were often present between the epithelial cells and the submucosa and contained numerous processes and well developed cell organelles, abundant lysosomes and lipid droplets. With the advance of lipid deposition, macrophages were filled with lipid droplets and became foam cells. It is suggested that these may become too large and rigid to pass through the endothelium of lymph vessels or that the lumen of the lymph vessels may be obstructed by large foam cells resulting in the destruction of these vessels and the accumulation of foam cells in the submucosa.     

Lipoprotein degradation and cholesterol esterification in primary cell cultures of rabbit atherosclerotic lesions.

Lipoprotein metabolism and cholesterol accumulation in atherosclerotic lesions was studied using enzymatically isolated primary cell cultures from aortas of rabbits made atherosclerotic by cholesterol feeding. The cultures consisted of macrophages and smooth muscle cells, thus resembling, in composition, fatty streak lesions. The mean (+/- SD) cholesteryl ester content of the dispersed cells was 1059 +/- 445 micrograms/mg cell protein, but it declined steeply during 1 week in primary culture. The uptake of low-density lipoprotein (LDL), beta-migrating very low-density lipoprotein (beta-VLDL), and acetylated LDL (acetyl-LDL), labeled with 125I or with the fluorescent probe 1,1'-dioctadecyl-3,3,3',3'- tetramethylindocarbocyanine (DiI), was studied in 2-day-old primary cultures. DiI-acetyl-LDL was avidly taken up by the macrophages and, to a lesser extent, by some smooth muscle cells. The uptake of DiI-beta-VLDL by the macrophages was weaker and less homogeneous than that of DiI-acetyl-LDL. The degradation rates of 125I-labeled beta-VLDL, LDL and acetyl-LDL were 135 +/- 54, 195 +/- 20, and 697 +/- 14 ng/mg cell protein/8 hours, respectively. Incubation with unlabeled acetyl-LDL enhanced the incorporation of [3H]oleate into cholesteryl esters and increased the cellular cholesteryl ester content. These results suggest that arterial macrophages and, to some extent, smooth muscle cells from cholesterol-fed rabbits actively metabolize acetyl-LDL and are thus capable of accumulating cholesteryl esters by uptake of modified forms of LDL.     

Lysosome lipid storage disorder in NCTR-BALB/c mice. II. Morphologic and cytochemical studies.

Electron-microscopic and cytochemical studies were carried out on tissues of NCTR-BALB/c mice. These mice are affected with a neurovisceral genetic disorder involving excessive tissue accumulation of lipid. Distinctive polymorphic intracellular inclusions, bounded by a membrane and containing lamellated bodies, were found in many cells of liver, spleen, lung, kidney, intestine, lymph nodes, and brain. The inclusions transformed reticuloendothelial cells into massive foam cells. Acid phosphatase cytochemical studies performed on sections of liver demonstrated that the inclusions were lysosomes. Fixation of liver in the presence of digitonin produced "spicules" in the inclusions characteristic of digitonin-cholesterol complexes. Clefts of cholesterol crystals were seen in the inclusions in liver, spleen, and lung. We conclude that the NCTR-BALB/c mice are affected by a lysosome lipid storage disease and that cholesterol is a major storage product.