Atherosclerosis, 61 (1987) 127-142 Elsevier Scientific Publishers Ireland, Ltd.

127

ATH 03993

Development of intracellular lipid deposits in the lipid-laden cells of atherosclerotic lesions

A cytochemical and ultrastructural study

Florea Lupu, Ion Danaricu and Nicolae Simionescu Instiiute of Cellular Biology and Pathology, Bucharest- 79691 (Romania)

(Received 30 October, 1986) (Revised, received 19 March, 1987)

(Accepted 23 March, 1987)

Summary

In atherosclerotic lesions of rabbits fed a cholesterol-rich diet, the lipid deposits of foam cells derived from monocytes, smooth muscle and endothelial cells were studied by physical, cytochemical and ultrastructural methods. Beginning with the third week of diet, the lipid material that could be visualized at the light microscope level by Oil red 0 and Nile red staining was progressively accumulated in the intimal cells of the atherosclerotic lesions. In the early stages of foam cell formation, the deposits occurred especially as intracytoplasmic non-membrane bound lipid inclusions (lipid droplets). In polarizing mi- croscopy these appeared as a mixture of iso-, and anisotropic material. The latter were birefringent and showed an axial symmetry with a black cross image, suggesting that the lipids were in a liquid crystalline state. In chemically-fixed specimens, the content of lipid inclusions was preserved in various degrees. In freeze-fractured preparations they displayed a layered onion-like arrangement with smooth cleavage faces surrounding an amorphous core. Upon incubation with filipin, that specifically binds to 3/3_hydroxysterols, the peripheral layers of the inclusions were labeled, revealing the existence of unesterified cholesterol. In the advanced stages of foam cell formation, lipids were additionally accumulated in the lysosomal compartment as polymorphic multilamellar structures concentrically arranged, with cleavage faces devoid of intralamellar particles. The presence of acid phosphatase showed that these features were modified lysosomes and were tentatively named lysosomal lipid bodies. In the latest stages examined cholesterol crystals developed within lysosomal lipid bodies usually enclosed in multilamellar structures. This lipid coat may represent the place of crystal formation and presumably acts as barrier for the turnover of the crystalline cholesterol. thus impeding plaque regression.

Key words: Intracellular lipid deposits; Foam cells; Lipid inclusions; Lysosomal lipid bodies; Multilamel- lar lipid structures: Cholesterol crystals; Cytochemistry; Atherosclerosis

This work was supported by the Ministry of Education, Correspondence to: Florea Lupu, Ph. D., Institute of Cellu- Romania and by the National Institutes of Health (USA) lar Biology and Pathology, 8, B.P. Hasdeu Street, Bucharest- Grant HL-26343 79691, Romania

0021-9150/87/$03.50 0 1987 Elsevier Scientific Publishers Ireland, Ltd.

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Introduction

In the prelesional stages of hypercholestero- lemic atherogenesis in the rabbit, lipoprotein-de- rived lipids accumulate in the extracellular space of the arterial intima as uni-, bi-, or multilamellar liposome-like structures rich in unesterified cholesterol [l]. At later stages, the lipid deposits appear predominantly as intracellular lipid inclu- sions (droplets) occupying most of the cell body of monocyte-derived macrophages and smooth muscle cells [2-61. In advanced fatty streaks, such lipid deposition may also involve some endothelial cells [7].

The intracellular lipid deposits can be found in various forms. The most common is the lipid droplet, actually a non-membrane bound inclu- sion, characteristically rich in cholesteryl esters [6,8]. The other types described are represented by a variety of lamellar lipid structures [9], some of which express lysosomal enzyme activities [5], and/or contain crystalline cholesterol [5]. Though some studies were reported [lo], the dynamics of lipid deposit formation and their possible correla- tive development are still unclear.

In the present study we have applied physical, histochemical, ultrastructural and cytochemical methods to characterize the intracellular lipids in the arterial wall of Chinchilla rabbits fed a hyper- lipidemic diet. By morphometric analysis we have analyzed the sequence of events in lipid deposit formation within foam cells (derived from smooth muscle cell, endothelial cells, and especially from macrophages), in order to understand the process of intracellular lipid accumulation and its role in the progression or regression of the atherosclerotic plaque. Our findings bring some novel data on (a) the appearance of various types of intracellular lipid deposits in freeze-fracture preparations, (b) localization at electron microscopic level of free cholesterol with specific probes (filipin and toma- tine), (c) possible formation and morphogenetic relations between various types of lipid deposits.

Material and methods

Animals Twenty six young adult male Chinchilla rab-

bits, 2-3 kg body weight, were fed for l-20 weeks

a diet containing 0.5% cholesterol and 5% butter. Animals were killed weekly under anesthesia with 0.3 g/kg body weight chloral hydrate in- traperitoneally injected.

Reagents Dulbecco’s phosphate-buffered saline (PBS)

supplemented with minimal essential medium (MEM) and amino acids was purchased from Gibco Laboratories (Grand Island Biological Co., Grand Island, NY). Filipin was a gift from Dr. J.E. Grady (Upjohn Co., Kalamazoo, MO). Di- methylsulfoxide (DMSO), tomatine, /3-glycero- phosphate grade I and cytidine 5’-monophosphate were obtained from Sigma Chemicals Co., St. Louis, MO, and tannic acid (AR code no. 1764) from Mallinckrodt Inc., St. Louis, MO. Oil red 0 was purchased from Serva, Heidelberg, West Germany and Nile red was prepared from Nile blue chloride (Loba Chemie, Vienna, Austria) according to [ll].

Tissue collection Under anesthesia, the chest was opened and the

heart and thoracic aorta were rapidly removed. Segments of the aortic arch, thoracic aorta and atrioventricular valves were briefly washed in PBS and fixed for light and electron microscopy.

Light Microscopy

Samples were fixed by immersion in 10% for- maldehyde in PBS for 1 h at 22 o C, then frozen in isopentane cooled with liquid nitrogen and sec- tioned in a cryostat microtome.

Nile red staining Sections were incubated with 1 yg/ml Nile red

in PBS for 5 min, rinsed in PBS and examined by fluorescence microscopy using rhodamine filters.

Oil red 0 staining Sections were washed in propyleneglycol for 2

min, immersed for 30 min in 0.5% Oil red 0 in propyleneglycol, washed 3 times in 70% pro- pyleneglycol, 15 min in running water and stained with Harris’ hematoxylin for 5 min.

Polarizing microscopy Sections mounted in 87% glycerol were

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examined with a polarizing microscope (Carl Zeiss, Jena, East Germany) with the polarizer and analyzer Nicol’s prisms crossed at 80 ‘.

Electron Microscopy

For cytochemical localization of unesterified cholesterol we used filipin and tomatine as specific probes [12-151; the lysosomal compartment was identified by the acid phosphatase reaction [16,17]. For studying lipids, in contrast to chemically-fixed specimens, the freeze-fracture preparation pre- sents important advantages because it prevents the lipid extraction during sample processing and pro- vides a 3-dimensional aspect of the structures [18]. Moreover, making use of the property of layered lipidic structures to cleave at the hydrophobic surface level, freeze-fracture represents an excel- lent method for visualizing the lamellar lipid organization.

All samples were first prefixed by rapid immer- sion in 2% glutaraldehyde in 0.1 M HCl/sodium cacodylate buffer, pH 7.4, for 10 min at 22OC.

Sterol binding probes Specimens prefixed with aldehyde were in-

cubated with 0.02% filipin in 2.5% glutaraldehyde in 0.1 M HCl/sodium cacodylate buffer for 30-60 min at 22’C or with 0.05% tomatine in the same buffer and conditions. Then tissue blocks were processed for either thin sectioning or freeze-frac- ture.

Thin sections Samples incubated or not with the sterol bind-

ing probes were further fixed for 90 min in 2.5% glutaraldehyde, postfixed in 1% osmium tetroxide for 90 min at 4” C and mordanted with 1% tannic acid; all solutions were in 0.1 M HCl/sodium cacodylate buffer pH 7.2 [19]. Some specimens were fixed with the triple fixative freshly prepared on ice by mixing 3 volumes of 5% paraformalde- hyde, 2 volumes of 4% 0~0, and 1 volume of saturated solution of lead citrate, all in 0.1 M HCl/sodium cacodylate buffer, pH 7.2 [20]. Other samples were fixed only with 6% 0~0, in 0.1 M veronal-acetate buffer, pH 7.4, for 90 n-tin at 4” C. All specimens were dehydrated in graded ethanol and embedded in Epon 812. Sections cut with a

diamond knife on Reichert Om 3 or American Optical Ultracut microtomes were stained with uranyl acetate and lead citrate. Some thick sec- tions were stained with Richardson’s dye and ex- amined by light microscopy.

Freeze-fracture Specimen cryoprotection was done by treat-

ment for 2 h with 25% glycerol in 0.1 M HCl/sodium cacodylate buffer, pH 7.2. The sam- ples were then frozen by rapid immersion in Freon-22 cooled with liquid nitrogen. Fracturing and replicating were performed in a Balzers BAF 301 apparatus. The replicas cleaned in chlo- roform/methanol (1: 1) mixture and in Chlorox or chromic acid were rinsed in bidistill&i water and mounted on Formvar-coated 150 mesh grids.

Detection of acid phosphatase The glutaraldehyde-fixed specimens were sec-

tioned at 40 pm thickness with a Smith-Farquhar TC-2 tissue chopper (Sorvall Instruments, Du Pont, Newtown, CT, U.S.A.). Sections were in- cubated at 30-35O C for 2-3 h in 1.25% & glycerophosphate grade I in 0.2 M Tris-maleate buffer containing 0.1% lead nitrate [16] or in 1% 5’-cytidyl monophosphate in 0.025 M acetate buffer containing 0.3% lead nitrate 1171. The sam- ples were further prepared for thin section elec- tron microscopy. Thin sections and freeze-fracture replicas were examined in a Philips 400-HM elec- tron microscope.

R6Sllt.9

At the light microscopic level, the atheromatous aortic iiitima usually contained lipid-laden cells derived from macrophages, smooth muscle cells and in advanced stages also from some endothelial cells (Fig. la-c). Brief incubation of fixed sections with an aqueous solution of Nile red revealed a large number of spherical cytoplasmic structures (Fig. la) which corresponded to lipid droplets as seen in phase contrast and in Gil red 0 stained sections (Fig. lb). By polarizing microscopy, the tissue sections exhibited a large number of aniso- tropic inclusions appearing as bright spherical fea- tures divided by a black cross image (Fig. lc). The orientation of the latter did not change when the

Fig. 1. Microscopic views of an atherosclerotic plaque in thoracic aorta after 4 months of diet: a and b: specimens stained with Nile red (combined fluorescence and phase contrast microscopy), and oil red 0, respectively; c: in polarizing microscopy many lipid inclusions appear as anisotropic structures, presenting black cross images (arrow); CI: X 224; b: X 350; c: X 800

microscopic stage was rotated through 360 ‘. At the ultrastructural level, the deposits ap-

peared as lipid inclusions (droplets), lysosomal lipid bodies (Fig. 2) and/or cholesterol crystals.

The lipid inclusions represented the most fre- quent type of lipid deposits and were the first to appear during the formation of a foam cell. Such non-membranous inclusions became visible after 3-4 weeks of diet in macrophages, 4-5 weeks in smooth muscle cells and 12 weeks in some endo- thelial cells (Table 1). In thin sections, although many vacuoles looked empty (assumed to be ex- tracted during preparation for electron mi- croscopy), some maintained in part their lipidic content with a heterogeneous organization (Fig. 3a-c). Some inclusions, displayed a continuous

homogeneous osmiophilic rim surrounding an ex- tracted core. Others had an osmiophilic core but the peripheral layer was largely extracted, whereas others showed an almost completely preserved content. In freeze-fracture replicas the lipid inclu- sions displayed an onion-like arrangement made up of a variable number of concentric lamellae, with a smooth fracture face when cleaved through a plane close to the surface. In cross fracture, the core of the inclusion seemed to be amorphous (Fig. 4a). Upon filipin incubation, the peripheral layers of the inclusion appeared crenated resem- bling the characteristic aspect of a filipin-affected membrane (Fig. 3a-c). The existence of typical f&pin-sterol complexes (fsc) at the lipid inclusion periphery could be also observed in freeze-fracture

Fig. 2. Electron micrograph of lipid laden macrophages in an atheromatous aortic plaque of hyperlipidemic rabbits at 3 months of diet. Lipid accumulation appears in the cytoplasm as lipid inclusions (li), and multilamellar structures identified as lysosomal lipid bodies (lb). x 13 500

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TABLE 1

DISTRIBUTION OF INTRACELLULAR LIPID DEPOSITS IN FOAM CELLS OF DIFFERENT ORIGIN AT VARIOUS STAGES OF DIET-INDUCED ATHEROGENESIS (PERCENTAGE VALUES)

For each foam cell type and period of diet lipid deposits found in 20 low magnification ( X 9000) electron micrographs were counted. li, lipid inclusions; lb, lysosomal lipid bodies; cc, cholesterol crystals.

Cells of origin

Monocytes

Smooth muscle cells

Endothelial cells

Forms of lipid deposits

li lb cc Total

Ii lb cc Total

li lb cc Total

Month of diet

1 2

100 81.32 12.68

loo 100

100 100

100 100

-

3 4 5 6

68.24 52.14 44.09 29.24 28.85 36.46 30.18 33.13

2.91 11.40 25.73 37.03 100 100 100 loo

76.62 63.53 55.42 46.32 22.34 26.05 18.98 13.85

1.04 10.42 25.68 39.83 100 100 loo 100

98.14 94.79 89.53 4.11

1.86 5.21 6.36 100 100 100

Fig. 3. Heterogeneity of lipid inclusions (li) as revealed by thin section electron microscopy (aortic lesions at 1 month of diet). (a): some present a continuous osmiophilic rim (arrowheads), (b) others exhibit an osmiophilic core (oc), the peripheric layers being largely extracted, (c) whereas in others the lipidic content is partially and randomly preserved. In all pictures the deformations induced by filipin-sterol complexes (fsc) can be observed around the lipid inclusion periphery. (I: X 29000; b: X 29ooO; c: X 28 350

Fig. 4. II and b: Lipid inclusions in freeze fracture replicas (aortic lesions at 1 month of diet). 0: When cleaved, lipid inclusions (Ii) show an onion-like layered structure with a less organized core (c). For comparison, a true bilayered membrane (arrows) displaying intramembranous particles can be distinguished within the cytoplasm. b: After filipin incubation, filipin-sterol complexes (fsc) can be observed around the inclusion periphery. (I: x70400; b: x70400.

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replicas (Fig. 4b) indicating the existence of a pool of unesterified cholesterol associated with the droplet periphery.

The lysosomal lipid bodies were generally very well preserved in specimens treated with tannic acid as mordant (Fig. 5). Under various shape and size, these bodies showed a lamellar organization with variable degrees of packing. The layered arrangement was particularly conspicuous in freeze-fracture replicas (Fig. 6). The smooth clea-

vage faces were devoid of intralamellar particles. The lysosomal lipid bodies were commonly posi- tive for acid phosphatase; the amount of this reaction product decreased in parallel with the increasing number of lipidic lamellae (Fig. 7a-c). These structures were visible after 8-12 weeks of diet and constituted almost half of the total lipid deposits occurring in macrophages and lipid-laden smooth muscle cells. Such types of deposits were very rarely found in endothelial cells.

Fig. 5. Aortic lesion at 4 months of diet. Thin section from a filipin-incubated specimen showing multilamellar lipid structures. While lb lack characteristic filipin-sterol complexes (fsc), lipid inclusion (li) periphery is heavily marked by these deformations. Inset: Occasionally one can observe multilayered structures which develop filipin-sterol complexes (fsc) but only in their peripheral layers (arrowheads). X 66CHM; inset: X 78 000.

Fig. 6. Freeze fracture replica of a multilamellar lipid structure cytochemically identified to be lysosomal lipid bodies (lb) (aortic lesions at 3 months of diet). Note the large, very smooth surfaces of the lipidic lamellae. x 56400.

The intracellular cholesterol crystals appeared after lo-16 weeks of diet and became frequent in the advanced stages of fatty streak formation (16-20 weeks of diet) (Figs. 8, 10). As revealed by both thin sections and freeze-fracture preparations (Figs. 8, 9) the forming cholesterol crystals were commonly enclosed in the multilamellar structures designated as lysosomal lipid bodies. The crystal content was usually extracted during preparation for thin sectioning but was rather well preserved in freeze-fracture specimens. The filipin treatment induced specific deformations (fsc) of the crystal coat, revealing the presence of unesterified cholesterol at this level (Fig. 8~).

Discussion

Some morphological aspects of the intracellular lipid droplets as revealed by light microscopy [21-241, transmission [9,10,25,26] and scanning electron microscopy [24,27-301 were previously published. Using ultrastructural and cytochemical techniques we obtained additional data about the formation and organization of the various forms of intracellular lipid deposition during atherogene- sis. Our observations showed the existence of three types of intracellular lipid deposits which may appear gradually in the atherosclerotic foam cells in our model: lipid inclusions, lysosomal lipid

136

Fig. 7. Aortic lesion at 3 months of diet. Accumulation of lipids into lysosomes. II: Lysosomes rich in acid phosphatase reaction product (ap) contain a small amount of lipidic material probably phospholipids (pl) and free cholesterol. b and c: The gradual decrease on acid phosphatase (ap) parallels the augmented lipid deposition (~1). The lipidic material (presumably phospholipids and free cholesterol) organized in tightly packed lamellae entrapped the acid phosphatase reaction product (arrows). (I: X107800; b: x60300; c: x63400.

Fig. 8. (I-C: Low magnification (u) and high magnification (b) and (c) of intracellular cholesterol crystals at 4 months of diet. ‘I crystal content was extracted during specimen preparation but around the crystal one can notice a multilamellar lipidic coat (mlc) After filipin incubation, the lamellae are characteristically crenated by filipin-sterol complexes (fsc) (which do not show up specimens not treated with filipin). cc, cholesterol crystal; li, lipid inclusion. a: ~70800; b: X98ooO; c: X 66000.

bodies, and cholesterol crystals - each type dis- appearing after 3-4 weeks of cholesterol diet in playing characteristic structural and cytochemical the monocyte-derived macrophages, then in features. smooth muscle cells and after 16-20 weeks also in

The lipid inclusion represents chronologically some endothelial cells. These inclusions cons list the first form of intracellular lipid deposition, mainly of cholesterol esters [31] resulting from 1 :he

le . c: in

Fig. 9. Freeze fracture view of a cholesterol crystal (cc) enclosed in a multilamellar coat (arrows) (aortic lesions at 5 months of diet). Ii, lipid inclusion; lb a multilamellar lipid structure representing a lysosomal lipid body. x 70400.

cytoplasmic esterification of cholesterol by acyl ters) that is responsible for the anisotropy observed CoA: cholesterol acyltransferase (ACAT) [32,33]. in the polarized microscopy. This spatial organiza- The shape and the layered arrangement of the tion may be controlled by physicochemical mecha- inclusion may be the result of a successive packing nisms, such as the hydrophobic effect of lipid of constituent molecules (mainly cholesterol es- molecules [34]. The amorphous core may represent

Fig. 10. Intracellular lipid deposits in advanced atherosclerotic lesions (5-6 months of diet). Note the existence of a large number of cholesterol crystals (cc), and lipid inclusions (li). Upon tomatine treatment, characteristic tomatine-sterol spicules (arrowheads) can be distinguished next to some lipid droplets and cholesterol crystals. X 29600.

the liquid phase of cholesterol esters which was this type of deposit. The filipin binding studies predicted to exist in the inclusion center [30]. The revealed that the peripheral layers of the inclusion various degrees of lipid extraction during prepara- contain a significant amount of unesterified tion for electron microscopy suggest the existence cholesterol uniformly distributed around its con- of some differences in the chemical composition tour at the interface with the cytoplasm. Some or/and physicochemical organization of lipids in thin section cytochemical studies using f&pin were

140

previously carried out by MC Gookey and Ander- son [6] in an in vitro study on peritoneal macro- phases transformed into foam cells: their findings showed the existence of a free cholesterol pool, focally localized to one inclusion pole. Using dig- itonin to identify the free cholesterol in isolated aortic foam cells, Shio et al. [5] reported that while the cytoplasmic droplets were not digitonin-reac- tive, a considerable number of digitonin-sterol complexes occurred in lipid-loaded lysosomes.

Among the sterol binding agents, filipin has the advantage of inducing small, discrete deforma- tions of the lipid layered structures, but has a rather poor penetration through tightly packed layers [13,14]. As all saponins, tomatine induces the formation of large spicules which disturb the membrane organization [13]. However by its prop- erty to interact with unesterified cholesterol even outside membranous structures, tomatine is a val- uable tool for the localization of 3@-hydroxysterols also in locations not related to membranes or membrane-bound organelles. The smooth surfaces of lysosomal lipid bodies suggest that the phos- pholipids disposed in liquid crystalline bilayers represent their major component. The lack of in- tralamellar particles might be indicative of the absence of translamellar proteins enclosed in the lipid layers. Treatment with filipin results in poor labeling, the filipin-induced deformations appear- ing only in the outer layers of the multilamellar structures; however, the tomatine treatment in- duced the formation of a large number of spicules, revealing a rich content of unesterified cholesterol trapped within the lipid layers. Similar results were reported by Shio et al. [5] using digitonin as sterol probe. The small number of fsc is probably due to the reduced accessibility of the label to the cholesterol molecules. The tight lipid packing and the rigidity of these layers probably constitute a barrier to the insertion of filipin molecules into the deeper layers.

As demonstrated by the presence of acid phos- phatase activity, these polymorphic multilamellar lipid structures actually represent modified lipid- loaded lysosomes. The gradual decrease of lyso- somal enzyme which parallels the lipid accumula- tion suggests a progressive depletion of lysosomal hydrolases as a result of an overwhelming lipid invasion. In lysosomes, the non-degraded lipid

material is organized in concentrically packed lame&r structures which contain phospholipids, unesterified cholesterol and probably cholesteryl esters.

The cholesterol crystals appear in the later stages of lipid deposition. It is unanimously recog- nized that the formation of cholesterol crystals represents the last stage in the evolution of a lipid deposition in the atherosclerotic lesion. Both in thin section and freeze-fracture preparations we observed that the cholesterol crystals were en- closed in multilamellar lipidic structures which probably contain mainly phospholipids in a liquid crystalline state. The sterol probes used showed the presence also of unesterified cholesterol within these lipidic layers. The occurrence of a multi- layered lipid coat at the crystal periphery suggests that the lysosome-derived multilamellar structures may be the place of crystal formation. This may result from a saturation of lipid-overloaded lyso- somes with free cholesterol, leading to the prccipi- tation of the excess cholesterol as cholesterol monohydrate crystals [30,34,35].

It can be speculated that in the early stages of foam cell formation, after the excess endocytosed &VLDL is degraded in lysosomes, the free cholesterol passing through lysosomal membrane into the cytosol is reesterified and progressively stored as inclusions of cholesteryl esters, mostly cholesteryl oleate. These are separated from the cytoplasm by a coat of unesterified cholesterol and probably a certain amount of phospholipid.

When lysosomal hydrolytic capability is over- whelmed by the excess of incoming lipoproteins, phospholipids and unesterified cholesterol are progressively accumulated within the fading lyso- some as multilamellar structures. In more ad- vanced stages, these become the sites where cholesterol in a fluid state is converted into mono- hydrate cholesterol crystals. As a working hy- pothesis, a schematic representation of the possi- ble pathways for the uptake and intracellular lipid deposition with subsequent formation of the three types of such deposits is shown in Fig. 11.

However, we cannot completely rule out the possibility that the formation of the lipidic layers may alternatively represent a part of the defence reaction of the cell against the toxic effects of cholesterol [1,36]. Katz et al. [37] showed that the

+ contains cholesteryl esters, free cholesterol, triglycerides, and phospholtptds

**contans phospholipids, free cholesterol, apo 6 [ref. 361

Fig. 11. Possible pathways for the uptake and intracellular deposition of lipids (working hypothesis). (1) Chemically-modi- fied lipoproteins (lp) can enter the cell mainly by receptor-mediated endocytosis (scavenger receptor) via endo- cytic vesicles (ev)-endosomes (e)-lysosomes (I): within the latter lp are degraded to amino acids and cholesteryl esters which are hydrolysed by an acid hpase to free cholesterol. This enters the cytoplasm where it is reesterified by acyl CoA: cholesterol acyltransferase (ACAT) as cholesteryl oleate that is progressively stored in non-membrane bound lipid inclusions (li). A thin layer of free cholesterol (thick bands) is deposited at the interface between the concentric layers of cholesteryl ester and the cytosol. (2) Although conceivable, it is still uncertain whether and how much free cholesterol (c) can reach the cytoplasmic pool by direct transfer from lp to plasma membrane. (3) Lipoprotein-derived material organized mostly as extracellular liposomes (el) may be phagocytosed by large vacuoles (v), internalized as phagosomes (p) which upon fusion with lysosomes become acid phosphatase (ap)-positive and take polymorphic patterns of phospholipid (ph) multilamellar lysosomal lipid bodies (Ilb). Some of these may originate directly from lipid-laden lysosomes. Increasing accumulation of free cholesterol within these structures leads to saturation and precipitation as monohydrate cholesterol crystals (cc) ini- tially surrounded by several layers of phospholipids (ph) which diminish markedly around large crystal. (Not to scale).

turnover of crystalline cholesterol is much slower, almost undetectable, as compared with other physical states of the free cholesterol (liquid and liquid crystalline), being relatively inert and al-

141

most impossible to be mobilized in an in vivo situation. This emphasizes that cholesterol crystals constitute a most important obstacle to plaque regression. The multilamellar structures surround- ing the crystal may represent one of the physical barriers which inhibits the equilibration of cholesterol concentration between crystals and the surrounding fluid, as predicted by Katz et al. [37].

Acknowledgements

The authors are grateful for the excellent tech- nical assistance provided by M. State (experi- ments), M. Misici, A. Bitir, V. Barbulescu (mi- crotomy), E. Stefan (photography), C. Neacsu (graphic work) and D. Neacsu (editing and typ- ing).

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