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.     

Early atherogenesis in White Carneau pigeons. II. Ultrastructural and cytochemical observations.

The addition of cholesterol (0.5%) to the diet of White Carneau pigeons induces site specific, temporally predictable, atherosclerotic lesions. The earliest lesions, which occurred after 3 weeks, were small (less than 2500 sq mu in surface area) and were composed primarily of macrophage foam cells (94% of lesion volume). With a prolonged time on the diet the lesions expanded due to increases in the number and size of foam cells, increases in the amount of extracellular space, and influx of smooth muscle cells. Macrophage foam cells in advanced lesions composed 61% of the lesion volume, smooth muscle cells 25%, and extracellular space 14%. Concurrent with the alteration in the constituency of the lesion, redistribution of lipid within foam cells was noted. Lipid in small lesions was primarily cytoplasmic (88%), with the remaining 12% in acid-phosphatase-positive secondary lysosomes. In more advanced lesions, 34% of the lipid was cytoplastic and 66% was lysosomal. The changes in large lesions appeared to be a function of lesion age, because at the growing edge of large lesions both composition and lipid distribution resembled those of small early lesions.     

Lysosomal alterations during coronary atherosclerosis in the pigeon: correlative cytochemical and three-dimensional HVEM/IVEM observations

Lysosomal changes have been implicated as one of the major factors contributing to the progression and complications of atherosclerosis, and recently foam cell formation has been correlated with increases in several acid hydrolases. To explore at the subcellular level relationships among lesion progression, cellular lipid accumulation, and lysosomal change, atherosclerotic lesions from hypercholesterolemic White Carneau pigeons have been studied through combined ultrastructural cytochemistry and stereo (three-dimensional) high-voltage electron microscopy. Lysosomal enzyme activity in the prelesion intima and in foam cells of early lesions was in discrete lysosomes of macrophage foam cells. Foam cell lipid at the early stages was primarily (72%) in cytoplasmic droplets, which formed a three-dimensional network with the small (0.25-0.8 microM in diameter), reaction-positive lysosomes suspended at the vertices of a cytoplasmic lattice that delineated individual lipid pools. Concomitant with lesion progression and increasing complexity, foam cell lysosome number, size, and complexity increased. The complexity was characterized by lysosome lipid accumulation (60% of cell lipid) and the fusion of lysosomes to form multilobulated organelles in which the acid phosphatase reaction product typically was circumferential to the lysosomal lipid core. The involvement of lysosomes climaxed in the more advanced region of lesions with foam cells in which the bulk of cytoplasmic volume was occupied by large (15-20 microM in diameter), multicompartmental, lipid-containing lysosomes. It is suggested that this progressive involvement of lysosomes is responsible for cell and tissue necroses characteristic of advanced lesions.     

Early atherogenesis in White Carneau pigeons: effect of a short-term regression diet

The intracellular distribution of lipid shifts from cytoplasmic to lysosomal localization during the progression of atherosclerosis. It has been suggested that this shift may relate to regressability of lesions. The effects of reducing plasma cholesterol on the regression of early cholesterol-induced atherosclerosis were evaluated. Most small, early lesions disappeared after 5 weeks on the regression regimen. Larger lesions, however, did not change in extent even following 10 weeks regression. Although large lesions were not reduced in size under the regression conditions, total cytoplasmic lipid decreased. Paradoxically, the size of residual intracellular lipid deposits increased. The structural features of these remaining deposits suggest that they were lipid-filled lysosomes. Leukocyte adherence to endothelium, which increases 10- to 20-fold during progression, returned to control levels over most areas of large lesions. Levels of adherent leukocytes remained elevated, however, over small lesions and at the edges of larger lesions. Our data indicate that regression is not a uniform process, but rather, even in early lesions, varies within separate intimal microdomains. In addition, our data suggest that part of the difference may reside in differential partitioning of lipid into lysosomes.     

TWO MALE SIBLINGs' CASES OF SPECIFIC LIPIDOSIS FOUND BY ELECTRON MICROSCOPY

Biopsy materials obtained from cervical lymph nodes and liver tissues of two male siblings, 5 years and 10 months old and 1 year and 11 months old, were studied electron microscopically. The results were summarized as follows: 1) Marked accumulation of lipid droplets, probably neutral fat, and cholesterol clefts in reticulum cells of lymph node, Kupffer cells and hepatic cells was the most prominent feature in these biopsies. 2) These lipids were found in both lysosomes and cytoplasmic matrices. Many of the lysosomes were considered to be autolysosomes or autophagosomes, because they contained fibrillar components, endoplasmic reticulum, glycogen particles and degraded organelles. It was suggested that these lipids might have been synthesized in lysosomes. 3) This disorder seems not to be included in the category of inborn lysosomal disease, and is considered as an unclassified one.     

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.     

Characterization of lipid-laden aortic cells from cholesterol-fed rabbits. III. Intracellular localization of cholesterol and cholesteryl ester.

The subcellular sites of accumulation of cholesterol and cholesteryl esters in rabbit atheromatous cells, were investigated by morphologic and biochemical techniques. Electron microscopy of lipid-filled cells in situ in atheromatous aortas of cholesterol-fed rabbits revealed lipid accumulation in the cytoplasm as lipid droplets and within lysosomes in the form of lipid globules, membranous whorls, and crystals. When such cells were isolated from the rabbit aortas by enzymic digestion, and then treated with Flickinger's aldehyde fixative containing 0.2 per cent digitonin, characteristic digitonide-lipid complexes ("spicules") were observed in discrete sites of the cytoplasm distinct from the cytoplasmic droplets. If these cells were first stained cytochemically for acid phosphatase and then treated with digitonin-aldehyde fixative, enzyme reaction product was found associated with the spicules indicating that the lysosomes of the atheromatous cells possess digitonin-reactive lipid. Subcellular fractionation of isolated rabbit aortic foam cells by sucrose density gradient centrifugation demonstrated the coequilibration of most of the intracellular unesterified cholesterol with low density lysosomes. Some cholesteryl ester was also associated with low density lysosomes, although most was found in a lipid droplet fraction of very low density. Together the results indicate that in rabbit atheromatous cells, lysosomes are the site of accumulation of intracellular cholesterol in excess of that structurally associated with membranes and that both cytoplasmic droplets and lysosomes are depot sites for cholesteryl esters.     

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.     

Pigeon monocyte/macrophage lysosomes during beta VLDL uptake. Induction of acid phosphatase activity. A model for complex arterial lysosomes.

Lysosomes have long been implicated as a factor contributing to the progression and complication of atherosclerosis. The authors' laboratory previously has shown that lysosomal ultrastructure in arterial macrophage foam cells is altered as primary lysosomes give rise to large pleiomorphic organelles on lipid accumulation during lesion progression. To further explore the subcellular alterations in lysosomes and associated organelles during foam cell formation, three-dimensional (3D) intermediate voltage electron microscopy was used to examine monocyte-derived macrophages (monocyte/macrophages) during early in vitro uptake of beta migrating very-low-density lipoproteins (beta VLDL). Lysosomes were identified using acid phosphatase cytochemistry, and in control cells these organelles constituted 3.5% of the total cytoplasmic volume. Both primary and secondary lysosomes were observed. Upon beta VLDL uptake, the total volume of acid-phosphatase-positive organelles increased threefold over 30 minutes, and the reaction product was found in three additional morphologically distinct structures: tubular lysosomes, membrane stacks, and endoplasmic reticulum with widened cisternae. The proportion of the cell occupied by each of the five acid-phosphatase-positive organelles was quantitated at 10 minutes, 30 minutes, 1 hour, and 4 hours of beta VLDL incubation, and their relative abundance was compared with controls that were processed either with no lipoprotein challenge or albumin incubation for 1 hour. Secondary lysosomes compartment volume peaked at 30 minutes; over the ensuing 3.5 hours, however, the reaction progressively shifted to three new membrane-limited locations. Our observations document the complex 3D organization and spacial relationships among the acid-phosphatase-positive structures induced by lipoprotein uptake. The 3D organization patterns for acid-phosphatase-positive lysosomes in lipoprotein-stimulated pigeon monocyte/macrophages were similar in several aspects to the complex lysosomes previously observed in the macrophages of pigeon arterial lesions.     

Ultrastructural lesions of ovine GM1 gangliosidosis.

Ovine GM1 gangliosidosis, an inherited disease of sheep with deficiencies of beta-galactosidase and alpha-neuraminidase, storage of GM1 ganglioside, asialo-GM1 and neutral long chain oligosaccharides in the brain, autosomal recessive inheritance, and histopathologic lesions typical of lysosomal storage diseases, has been described recently. Selected tissues from two sheep with the condition and an age-matched control were examined by transmission electron microscopy to characterize the ultrastructural lesions. In all central and peripheral neurons, the majority of the cytoplasmic space was occupied by membrane-limited enlarged bodies judged to be lysosomes, with a resultant displacement of normal organelles. The neuronal lysosomes usually contained stacks and concentric whorls of lamellae of stored material with a periodicity of 25 to 75 nM. Individual lamellae consisted of fine, multilayered (three to 10, and occasionally more) bands. Less commonly, enlarged neuronal lysosomes contained fibrillogranular or electron dense material. Central nervous system microglia and peripheral nervous system satellite cells had less extensive storage of similar material within enlarged lysosomes, whereas oligodendrocytes, astrocytes, and Schwann cells were relatively unaffected. Hepatocytes and renal epithelial cells also had storage of less quantity than neurons, but within even larger lysosomes. In contrast to neuronal storage material, visceral storage consisted of vesicles containing fibrillogranular or electron dense material within a mostly electron lucent matrix with only occasional lamellae. Kupffer cells and macrophages from bone marrow were affected similarly to but less severely than hepatocytes and renal epithelial cells, whereas hematopoietic cells and chondrocytes were unaffected. Both neuronal and visceral storage were evident, but the neuronal storage was much more extensive.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ultrastructural changes in the renal papillary cells of rats during maintenance and repair of profound potassium depletion.

In rats with long-term diet-induced potassium depletion, the cytoplasm of markedly enlarged papillary cells was mainly occupied by membrane-bound droplets, many of which acquired massive proportions. Both free and attached ribosomes were decreased, the Golgi apparatus was inconspicuous and there was a paucity of mitochondria. Despite the overwhelming accumulation of droplets with concomitant loss of normal metabolic organelles, cell death did not occur. With potassium repletion, the organelles readily proliferated regardless of the progression of droplet dissolution. The shrinkage of the droplets was accompanied either by disintegration of the limiting membrane or by disappearance of the constituents within an intact membrane. Microtubules were conspicuous in many of the cells undergoing gradual reduction in size. These cytoplasmic changes in renal medullary cells of rats during long-term potassium depletion and immediate post-repletion periods essentially represented the consequences of maintenance and repair of a storage process.     

Alterations in the fine structure of the prostate and seminal vesicle of rats treated with cyproterone acetate.

Young adult male rats were treated with daily injections of 10 mg of cyproterone acetate for periods up to 16 weeks. Samples of the ventral prostate and the seminal vesicle were studied with the light and electron microscopes. Alterations visible with the light microscope included decreases in cell size, cytoplasmic basophilia, the size of the nucleolus, and the amount of luminal secretory material. Ultrastructural changes in the epithelium of both glands involved mainly the organelles that participate in the formation of secretions. Large declines were observed in the abundance of rough endoplasmic reticulum, size of the Golgi apparatus, and number of secretory vacuoles. Lipid droplets accumulated in the seminal vesicle epithelium, and lysosomes were numerous in both glands. Changes were first observed microscopically in the seminal vesicle after one week and in the prostate after two or three weeks. Maximal development of the alteration occurred after treatment for approximately eight weeks.     

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.     

Aminoglutethimide-induced lysosomal changes in adrenal gland in mice

Aminoglutethimide is a steroidogenesis inhibitor and inhibits a cholesterol side-chain cleavage enzyme (CYP11A1) that converts cholesterol to pregnenolone in mitochondria. We investigated histopathological changes induced by 5-day administration of AG in mice. Cytoplasmic vacuoles of various sizes and single cell necrosis were found in zona fasciculata cells in AG-treated mice. Some vacuoles were positive for adipophilin, whereas others were positive for lysosome-associated membrane protein-2 on immunohistochemical staining, indicating they were enlarged lipid droplets and lysosomes, respectively. Electron microscopy revealed enlarged lysosomes containing damaged mitochondria and lamellar bodies in zona fasciculata cells, and they were considered to reflect the intracellular protein degradation processes, mitophagy and lipophagy. From these results, we showed that AG induces excessive lipid accumulation and mitochondrial damage in zona fasciculata cells, which leads to an accelerated lysosomal degradation in mice.     

IMMUNOELECTRON MICROSCOPIC LOCALIZATION OF THYROGLOBULIN (TG) IN THE THYROID GLAND IN BASEDOW'S DISEASE

An electron microscopic immunohistochemical localization of thyroglobulin (TG) using PAP methods from Epok-812 embedded tissues has been made in human thyroids obtained from 15 patients with treated Basedow's disease aged 13 to 43 years. Follicular cells vary considerably in shape and content of cytoplasmic organelles depending mainly upon the size of follicles. Reaction product for TG coincided well with previous investigations studied by electron microscopic autoradiography and is strongly positive in most follicular lumina having no relation to their size, reabsorbed colloid droplets or small subapical vesicles. The reabsorbed colloid droplets are rarely observed in cells surrounding distended follicles and more frequently appeared in small follicles. The reabsorbed colloid droplets after fusion with lysosomes show a variable amount of reaction products from intense deposition to a complete absence of product indicating hydrolysis of TG by lysosomal enzymes. There is no reaction product in rough endoplasmic reticulum or Golgi apparatus in spite of positive reaction product in the subapical vesicles. These subapical vesicles are composed of both endocytotic and exocytotic vesicles.     

Alterations in the fine structure of the prostate and seminal vesicle of rats treated with cyproterone acetate

Young adult male rats were treated with daily injections of 10 mg of cyproterone acetate for periods up to 16 weeks. Samples of the ventral prostate and the seminal vesicle were studied with the light and electron microscopes. Alterations visible with the light microscope included decreases in cell size, cytoplasmic basophila, the size of the nucleolus, and the amount of luminal secretory material. Ultrastructural changes in the epithelium of both glands involved mainly the organelles that participate in the formation of secretions. Large declines were observed in the abundance of rough endoplasmic reticulum, size of the Golgi apparatus, and number of secretory vacuoles. Lipid droplets accumulated in the seminal vesicle epithelium, and lysosomes were numerous in both glands. Changes were first observed microscopically in the seminal vesicle after one week and in the prostate after two or three weeks. Maximal development of the alterations occurred after treatment for approximately eight weeks.     

Lysosomal degradation of cell organelles. III. Uptake and disappearance in Kupffer cells of intravenously injected isotope-labeled mitochondria and microsomes in vivo and in vitro.

14C-leucine and 3H-glycerol-labeled microsomes and mitochondria were intravenously injected into a series of highly inbred rats. The uptake and disappearance of the organelles were followed in a crude liver lysosomal fraction and in serum. Approximately half of the injected dose was recovered in the liver, and only smaller amounts were found in lungs, kidneys, spleen, and heart. The clearance in serum was more rapid for microsomes (t1/2, 5 to 15 minutes) than for mitochondria (t1/2 30 to 60 minutes). Both organelles showed a biphasic type of disappearance curve consistent with the two-phase theory of phagocytosis: attachment and engulfment. The estimated half-life for mitochondria of the liver was in the range of 3 to 4 hours, whereas that of the microsomes was considerably longer, or 8 hours. There was an increase of trichloroacetic acid-soluble material in the crude lysosomal fraction up to 2 hours after injection of glycerol-labeled microsomes, whereas the peak was reached at 60 minutes after 14C-leucine labeling. In vitro hydrolysis rate of hydrolysis. Experiments with Kupffer cells previously labeled with Thorotrast and biochemical assay of hydrolysis indicated that there was a lag phase of approximately 10 to 20 minutes before the phagosomes gained acid hydrolases, presumably by fusion with lysosomes. It is concluded at somewhat different rates. The remnants from lipid degradation, in comparison with protein degradation, seem to remain for a longer period within the lysosomal apparatus. These results are compatible with the concept that lysosomes represent an important, and at the present the only well defined locus for organelle turnover.     

Lysosome lipid storage disorder in NCTR-BALB/c mice. III. Isolation and analysis of storage inclusions from liver.

Livers of NCTR-BALB/c mice, affected by excessive accumulation of cholesterol and phospholipid, were fractionated by sucrose density gradient centrifugation. Lysosomes of very low density (rho = 1.05 - 1.08) were found, which by electron microscopy appeared identical to the storage inclusions seen in fixed tissues. These lysosomes could be purified about 10-fold over the original homogenate, and represented 4% of the total protein and 30-40% of the liver acid hydrolase content. The preparations were nearly free of mitochondrial, endoplasmic reticulum, and plasma membrane contamination. The lysosomes were laden with cholesterol and phospholipid. Cholesterol (greater than 97% unesterified) accounted for half of the total lipid, and sphingomyelin accounted for another 20%. Phosphatidylcholine and phosphatidylethanolamine were also present in substantial quantities. All of the excess cholesterol and sphingomyelin of liver could be attributed to the low density lysosomes. Lysosomal acid sphingomyelinase activity, measured with a synthetic substrate, was found to be 10-60% of BALB/c mouse control levels in liver, spleen, and cerebellum, while two other lysosomal enzymes, N-acetyl-beta-glucosaminidase and beta-glucuronidase, were increased 2-8-fold in the same tissues. These data and the morphologic observations of the preceding paper establish that the disorder affecting NCTR-BALB/c mice is a lysosome storage disease. We propose several possible mechanisms to explain the cholesterol and phospholipid overloading of lysosomes. The specific gene defect remains to be established.     

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.     

The lipid-rich core region of human atherosclerotic fibrous plaques. Prevalence of small lipid droplets and vesicles by electron microscopy.

Abundant extracellular lipid deposits are associated with cell necrosis and tissue weakening in the core region of human atherosclerotic fibrous plaques. The ultrastructural morphology of the core region, previously undefined because of lipid extraction artifacts, was studied with the aid of new osmium-thiocarbohydrazide-osmium and osmium-tannic acid-paraphenylenediamine sequences for tissue processing. Small droplets of neutral lipid (30 to 400 nm profile diameter) and lipid vesicles with aqueous centers accounted for more than 90% of the area occupied by lipid-rich structures in the core region. No foam cells were present. Cholesterol crystals, lipid droplets of a size similar to those in foam cells (0.4 to 6 mu), and larger neutral lipid deposits (greater than 6 mu) together occupied less than 10% of the total area of lipid structures. Abundant lipid vesicles were associated with the nearby presence of cholesterol crystals, whereas small lipid droplets were predominant in areas without crystals. Many droplets had surface defects in the form of pits and vesicular blebs. These morphologic findings are explained most concisely by postulating direct accumulation of extracellular lipid from interstitial lipoproteins as a major process in core region formation. Moreover, a dynamic state of ongoing physical/metabolic transformation of extracellular lipid deposits is suggested.