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The physical state of cholesteryl esters (CE) in the arterial-smooth-muscle-derived foam cells may contribute to the documented reduction in CE hydrolysis. The physical state of CE may also provide a potential enhancing mechanism for increased CE accumulation. To explore these concepts, we therefore examined the influence of alterations in CE and triacylglycerol (TG) content and their fatty acid composition on the thermotropic behaviour of these lipids by differential scanning calorimetry (d.s.c.). After exposure to cationized LDL (cLDL) or after infection with herpes simplex virus type I (HSV), smooth-muscle cells accumulated significant amounts of CE. The CE/TG ratio was significantly higher in cells treated with cLDL compared with HSV infection. TG content was unaffected by either treatment. However, the fatty acid profile of both CE and TG was significantly different between treatment groups, with the polyunsaturated fatty acid/saturated fatty acid (PUFA/SFA) ratio being significantly higher in cLDL-treated cells than in HSV-infected cells. The d.s.c.-generated thermograms of intact cells revealed that neutral lipids of both treatment groups were in the isotropic-liquid state, similar to the state of lipids derived from 'fatty streak' types of atherosclerotic lesions. Differences in the thermograms between HSV-infected and cLDL-treated cells can be ascribed to differences in the CE content and the fatty acid composition of CE and TG (PUFA/SFA ratio). Polarizing optical microscopy revealed the presence of isotropic lipids in both groups. Biochemical and physicochemical data confirm the lysosomal localization of engorged CE, and indicate that the cellular isotropic CE in these foam cells are in a physical state which favours enzymic hydrolysis.

Beta-migrating very low density lipoproteins (beta--VLDL) and chemically modified low density lipoproteins (LDL), labeled with the fluorescent probe 3,3'-dioctadecylindocarbocyanine (Dil), were used to determine whether foam cells from atherosclerotic lesions have lipoprotein receptors similar to those of macrophages. The advantage of the Dil probe is that it produces a brilliant fluorescence due to its retention by the cells. When cells grown from intimal-medial explants of atherosclerotic rabbit aortas were incubated with Dil-labeled beta-VLDL, both smooth muscle cells and foam cells became fluorescent. However, when large amounts of unlabeled LDL were added to compete with the beta-VLDL for binding to the LDL receptors, Dil-labeled beta-VLDL binding to smooth muscle cells was blocked, and only the foam cells became highly fluorescent, due to the presence of specific receptors for beta-VLDL on those cells. The foam cells in the explants also became fluorescent when incubated with Dil-labeled acetoacetylated (AcAc) LDL. The same fluorescently labeled foam cells avidly bound IgG-coated erythrocytes. It was possible to inhibit the binding of the Dil-labeled AcAc LDL to aortic foam cells by the addition of fucoidin, a known inhibitor of modified LDL binding to macrophages. Foam cells that became fluorescent were similar to cells grown from the explants that contained an abundance of Oil red O positive lipid droplets and demonstrated high acid lipase activity. These results suggest that macrophages were the precursors of the foam cells derived from explants of atherosclerotic rabbit aortas. One difference we noted between these foam cells and macrophages, however, was that foam cells from the explants retained their cholesteryl esters under the same culture conditions that resulted in the loss of lipid from macrophages loaded with lipoproteins in vitro. The existence of receptors for beta-VLDL on aortic foam cells supports the contention that dietary cholesterol-induced beta-VLDL may be atherogenic lipoproteins in vivo.

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.

We investigated the modifications that diet-induced hypercholesterolemia, in rabbit, can produce in the cell surface charge and chemistry of arterial endothelium (E) and blood monocytes (M). Weekly, up to 8 weeks, after blood samples were taken for lipid analysis and blood cell preparation, the vasculature was washed free of blood and the endothelial luminal surface (ES) exposed to cytochemical probes for detecting charged groups, sialoconjugates and oligosaccharides. After fixation in situ, specimens collected from lesion-prone regions (aortic arch and coronary artery) and vena cava, were processed for electron microscopy. Morphometric analysis of tracer distribution on endothelium of nonlesional and lesional areas occurring in various stages of structural alterations, showed a remarkable resistance of the cell coat to very high level of serum cholesterol. In nonlesional zones the E surface charge and glycoconjugates were not significantly changed. In lesional areas, including those with forming fatty streaks, while cationic sites, galactosyl-, and N-acetyl-galactosaminyl residues were not altered whereas mannosyl moieties increased in density. A reduction in anionic groups and sialoconjugates appeared only after advanced extracellular and intracellular accumulation of lipoprotein-derived material and stromal proliferation developed in the intima. Moreover, these ES changes were usually restricted to the relatively rare E cells heavily loaded with lipid inclusions. The modulations were generally paralleled by comparable variations in the M surface. Regardless the extent of surface charge reduction, monocytes continued to migrate and foam cells to egress from the vessel wall. The results suggest that the onset and progression of early intimal lesions are not preceded but followed by significant restricted alterations in cell surface charge and glycoconjugates of arterial endothelium and monocytes.

We have examined foam cell accumulation in abdominal and thoracic aortic segments of swine with experimentally induced atherosclerosis. Young (less than 1-year-old) male swine were divided into four groups that were fed control, lard (20%), lard (20%) plus cholesterol (1%), and regression (3 months cholesterol-lard followed by 3 months control) diets for 6 months. Aortas were removed from animals and enzymatically dissociated. Foam cells were detected by specifically staining their cholesteryl ester inclusions with the fluorescent dye filipin. Flow cytometry was used to quantify the number of foam cells in each aortic cell suspension. Cholesterol-lard feeding increased serum cholesterol levels 6-fold and induced a substantial increase in both abdominal and thoracic foam cell densities. Lesion development in cholesterol-lard-fed animals, as assessed by macroscopic evaluation of aortas, correlated with foam cell accumulation as determined by flow cytometry. Accumulation of foam cells was extremely variable from animal to animal and did correlate significantly with serum cholesterol but not with serum triglyceride levels. Interestingly, although serum cholesterol levels increased 1.5-fold in swine fed the lard diet, foam cells did not increase significantly as compared to control animals. Animals placed on a control diet following 3 months of the cholesterol-lard diet showed a trend toward lower foam cell densities as compared to animals fed the cholesterol-lard diet for the entire 6 months. Flow cytometric analysis of filipin-stained aortic foam cells provides a new means to evaluate atherosclerotic lesion development.

The search for foam cells in the urinary sediment of patients with possible renal xanthogranulomatosis can constitute a harmless and useful diagnostic method for this frequently occurring renal disease, which previously has been diagnosed with certainty only by histological examination or biopsy. Of 5 cases studied the search was positive in 80 per cent.

The ultrastructural pathology of the stored materials in lipid storage cells, particularly of macrophagic nature, in various disorders of sphingolipidosis was investigated. Cell morphology of the lipid storage cells was largely divided into two groups; one had peculiar cell morphology, such as Gaucher cells or globoid cells, and the other showed the appearance of foam cells. These cytological characteristics of the lipid storage cells were closely related to the ultrastructural configuration of lipid storage inclusions. By transmission electron microscopy, the fundamental structures of the stored materials were classified into two types; tubular and lamellar. The tubular structures were formed by accumulation of ceramide or monohexosyl ceramide, whereas the lamellar structures were formed by accumulation of larger sphingolipids than monohexosyl ceramide. These tubular structures were proven to consist of multilayers of lamellae, which are considered fundamentally similar to the lamellar structures. Almost all the lipid storage inclusions are considered to be of lysosomal origin, because of their encirclement by a single unit membrane and localization of acid phosphatase activity, and participation of heterophagic or autophagic mechanisms as for the development of the inclusions may be noted. Besides, the occurrence of secondary lipid storage was pointed out in some disorders of sphingolipidosis.

We have quantified using flow cytometry foam cells of aortas from normolipidemic swine varying in age from 6 months to 12 years. These swine were maintained throughout their lives on a low-fat, cholesterol-free diet. Intimal-medial tissues removed from the swine aortas were enzymatically dissociated to prepare Formalin-fixed cell suspensions. Foam cells were labeled by specific staining of their intracellular cholesteryl ester using the fluorescent dye filipin. This was carried out by first removing cellular unesterified cholesterol with ethanol, then enzymatically hydrolyzing cellular cholesteryl ester, and finally staining with filipin the unesterified cholesterol derived from hydrolysis of cholesteryl ester. Results of flow cytometric analysis indicated that thoracic foam cell densities of female swine became more variable and tended to increase with age. It appeared that there were two subgroups of female swine with either high or low levels of thoracic foam cells. A similar finding was not observed for abdominal foam cells in female swine nor for thoracic or abdominal foam cells in the smaller number of older male swine. Abdominal foam cell densities in younger males, however, also appeared to be comprised of low and high foam cell groups. Foam cell densities did not correlate with serum cholesterol or triglyceride levels. However, a genetic basis for some of the variability in foam cell densities among these animals was suggested by the observation that females with high thoracic foam cell densities had greater commonality among ancestors than did females with low thoracic foam cell densities.

A series of biochemical and morphological studies has focused on the properties and origins of lipid laden foam cells in experimentally induced atherosclerosis in rabbits. Lipids inclusions present in these cells occupy half or more of the cytoplasmic volume and are of two kinds: cytoplasmic lipid droplets composed predominantly of cholesteryl esters and lysosomes in which substantial quantities of free cholesterol have accumulated. The foam cells exhibit some properties of macrophages but not others. They possess high levels of acid hydrolases and catalase and Fc membrane receptors can be detected on their surface. Only about one third of the foam cells, however, exhibit C3 receptors and few if any of the cells appear to contain or secrete lysozyme.

A total of 13 forty-day-old male Japanese quails had free access to a atherogenic diet containing 15% corn oil and 2% cholesterol or commercial basal diet for 3 months. Birds fed basal diet showed no significant intimal lesions. These birds had two types of cells, i.e. smooth muscle cell and fibroblast-like cell, in the tunica media of the ascending aorta. While fat-fed birds showed marked lipid-rich intimal lesions in the ascending aorta but not in the abdominal aorta. Some macrophage-derived foam cells, which were stained for lysozyme and OKM1, were demonstrated in the superficial portion of the thickened intima. The majority of the cells in the lipid-rich thickened intima showed ultrastructural character of fibroblast-like cells with or without lipid droplets. Alpha-1-antichymotrypsin was positive for fibroblast-like cells in the thickened intima but not for those in the tunica media of the ascending aorta. These results suggest that metaplasia of the medial fibroblast-like cells is responsible for the development of atherosclerosis in the quail.