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PubMed2001

Phenotype determination of anti-GM3 positive cells in atherosclerotic lesions of the human aorta. Hypothetical role of ganglioside GM3 in foam cell formation.

Earlier we reported that atherosclerotic plaques contain cells which were specifically and very intensively stained with anti-GM3 antibodies although no GM3 positive cells were detected in the normal non-diseased arterial intima. Because of their lipid inclusions, GM3 positive cells in atherosclerotic lesions seemed to be foam cells but their origin needed clarification. Using an immunohistochemical technique in the present work, we showed that some of these foam cells contained CD68 antigen. However, the most intense accumulation of GM3 occurred in the areas composed of foam cells which did not stain with any cell type-specific antibodies, including antibodies to macrophages (anti-CD68) and smooth muscle cells (anti-smooth muscle alpha-actin), perhaps, because the cell type-specific antigens were lost during the transformation of intimal cells into foam cells. Ultrastructural analysis of the areas where foam cells overexpressed GM3 demonstrated that some foam cells lacked both a basal membrane and myofilaments but contained a large number of secondary lysosomes and phagolysosomes, morphological features which might indicate their macrophage origin. Other foam cells contained a few myofilaments and fragments of basal membrane around their plasmalemmal membrane, suggesting a smooth muscle cell origin. These observations indicate that accumulation of excessive amounts of GM3 occurs in different cell types transforming into foam cells. We suggest that up-regulation of GM3 synthesis in intimal cells might be an essential event in foam cell formation. Shedding of a large number of membrane-bound microvesicles from the cell surface of foam cells was observed in areas of atherosclerotic lesions corresponding to extracellular GM3 accumulation. We speculate that extracellularly localised GM3 might affect the differentiation and modification of intimal cells in atherosclerotic lesions.

Adult↗

PubMed1978

Glomerular foam cells in kidney allograft.

Foam cells are occasionally encountered in renal glomeruli in cases of nephrotic syndrome, however, they were not described so far in renal allografts. The authors found the glomerular foam cells in a cadaveric kidney transplant which survived 7 months after transplantation in a 35-year-old man. Electron microscopy revealed the mesangial cells to be loaded with lipids in this case. The formation of the glomerular foam cells is generally referred to hyperlipidemia, but its cause remains obscure in the presented case.

Adult↗

PubMed1999

Depletion of pre beta 1LpA1 and LpA4 particles by mast cell chymase reduces cholesterol efflux from macrophage foam cells induced by plasma.

Exposure of the LpA1-containing particles present in HDL3 and plasma to a minimal degree of proteolysis by the neutral protease chymase from exocytosed rat mast cell granules (granule remnants) leads to a reduction in the high-affinity component of cholesterol efflux from macrophage foam cells. In this study, we demonstrate for the first time, a role for mast cell chymase in the depletion of the lipid-poor minor components of HDL that are specifically involved in reverse cholesterol transport as initial acceptors of cellular cholesterol. Thus, addition of proteolytically active granule remnants or human skin chymase to cholesterol-loaded macrophages of mouse or human origin incubated with human apoA1, ie, a system in which prebeta1LpA1 is generated, resulted in a sharp reduction in the high-affinity cholesterol efflux promoted by apoA1. As determined by nondenaturing 2-dimensional polyacrylamide gradient gel electrophoresis, the granule remnants effectively depleted the prebeta1LpA1, but not the alphaLpA1, in HDL3 and in plasma during incubation at 37 degrees C for <1 hour. Incubation of plasma with granule remnants for 1 hour also led to near disappearance of the LpA4-1 and LpA4-2 particles, but did not affect the distribution of the apoA2-containing lipoproteins present in the plasma. We conclude that the reduced ability of granule remnant-treated HDL3 and granule remnant-treated plasma to induce cholesterol efflux from macrophage foam cells is caused by selective depletion by mast cell chymase of quantitatively minor A1- and A4-containing subpopulations of HDL. Because these particles, ie, prebeta1LpA1 and LpA4, are efficient acceptors of cholesterol from cell surfaces, their depletion by mast cells may block the initiation of reverse cholesterol transport in vivo and thereby favor foam cell formation in the arterial intima, the site of atherogenesis.

Animals↗

PubMed1999

Clinicopathological characteristics of interstitial foam cells in membranous nephropathy.

BACKGROUND: Interstitial foam cells are occasionally observed in various renal diseases, and they have been reported to belong to the monocyte/macrophage (M phi) lineage and to be associated with heavy proteinuria and hyperlipidemia. We investigated the characteristics of interstitial foam cells and their association with proteinuria and hyperlipidemia in idiopathic membranous nephropathy (MN). METHODS: Patients with MN (N = 320) were divided into two groups: group I consisted of 51 patients with interstitial foam cells, and group II consisted of the other 269 without foam cells. We compared clinical parameters and the findings of an immunohistochemical study using monoclonal antibodies to various types of leukocytes and adhesion molecules. RESULTS: The age at renal biopsy, the degree of proteinuria, serum levels of lipids, and other clinical parameters except for sex ratio were not different between the two groups. The ratio of nephrotic patients was compatible between groups I (56.9%) and II (52.8%). All interstitial foam cells were positive for CD68 and 25F9, which are markers for M phi and mature M phi, respectively, but were negative for CD3 or cytokeratin. Interstitial infiltrating cells were positive for CD68 and CD3 but were negative for 25F9. Furthermore, most of interstitial foam cells were positive for both leukocyte function associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1), but not for ICAM-3 (the third ligand for LFA-1). By contrast, most of infiltrating nonfoamy M phi s were positive for ICAM-3 and LFA-1, however, ICAM-1 was observed on only some of them. CONCLUSION: These results suggest that interstitial foam cells in MN may not depend on proteinuria nor hyperlipidemia directly. The accumulation of foam cells, which have characteristics of mature M phi, may be related to ICAM-1 as a ligand of LFA-1, whereas infiltration of nonfoamy M phi s has a close relationship with ICAM-3. Thus, the formation of interstitial foam cells may be related to the phenotypical transformation of M phi s.

Adult↗

PubMed2005

Monocyte recruitment and foam cell formation in atherosclerosis.

Atherosclerosis is a chronic immune-inflammatory disease in which the interactions of monocytes with activated endothelium are crucial events leading to atherosclerotic alteration of the arterial intima. In early atherosclerosis, monocytes migrate into the subendothelial layer of the intima where they differentiate into macrophages or dendritic cells. In the subendothelial space enriched with atherogenic lipoproteins, most macrophages transform into foam cells. Foam cells aggregate to form the atheromatous core and as this process progresses, the atheromatous centres of plaques become necrotic, consisting of lipids, cholesterol crystals and cell debris. This review highlights some aspects of monocyte recruitment and foam cell formation in atherosclerosis.

Aorta↗

PubMed1991

Foam cells of the rabbit atherosclerotic plaque arrested in metaphase by colchicine show a macrophage phenotype.

Proliferative activity of smooth muscle cells and foam cells characterizes experimental atherosclerotic plaques as they first appear. Immunohistochemical and ultrastructural methods were applied to cells arrested in metaphase by colchicine and the phenotype of cells in mitosis was detected. Most of the metaphase arrested FC found in aortic plaques of cholesterol fed New Zealand rabbits were positive to the anti-macrophage monoclonal antibody and negative to the anti-smooth muscle actin monoclonal antibody. Moreover, most of the metaphase blocked FC had the ultrastructural features of macrophages. These preliminary results further strengthen previous observations on rabbit plaques that the FC pool is mainly constituted by macrophages and show, for the first time, that the dimension of this pool depends not only on migration of circulating monocytes but also on the in situ proliferation of macrophages.

Animals↗

PubMed1998

Morphologic and immunophenotypic characterization of foam cells in endometrial lesions.

The detection of foam cells in cervicovaginal smears obtained from postmenopausal women suggests the possibility of an endometrial lesion. Ultrastructural studies have suggested that foam cells represent endometrial stromal cells but the histogenesis of these cells has not been firmly established. To investigate the origin and diagnostic significance of foam cells, we analyzed the morphology and immunophenotype of these cells in endometrial tissue specimens and correlated the findings with cervical smears obtained within the preceding 6 months. Selected biopsies containing foam cells were evaluated using four well-characterized macrophage markers: KP-1(CD68), HAM 56, MAC 387, and lysozyme. Foam cells were found in 11 (38%) of 29 simple hyperplasias, 7 (50%) of 14 complex hyperplasias, 6 (50%) of 12 complex atypical hyperplasias, 21 (70%) of 30 adenocarcinomas, 1 (4%) of 25 samples with stromal breakdown, and 0 of 30 specimens showing normal cycling endometrium. Foam cells were also found in smears preceding the histologic diagnosis of 2 (13%) simple hyperplasias, 2 (25%) complex hyperplasias, 3 (43%) complex atypical hyperplasias, 5 (28%) adenocarcinomas, 5 (28%) cases of stromal breakdown, and 0 of 8 normal tissue specimens examined. Foam cells were immunoreactive with at least 2 of the 3 macrophage-specific antibodies in all 21 biopsies studied. Our results suggest that foam cells phenotypically represent macrophages and not endometrial stromal cells. Foam cells are identified in a significant percentage of cervical smears and endometrial tissue specimens obtained from women with endometrial pathology. The morphology and immunophenotype of foam cells, however, does not appear to be useful in distinguishing benign endometrial stromal breakdown, endometrial hyperplasia, and endometrial adenocarcinoma.

Adenocarcinoma↗

PubMed1989

Triglyceride and cholesteryl ester hydrolysis in a cell culture model of smooth muscle foam cells.

Cultured rabbit aortic smooth muscle cells were converted to foam cells by exposure to sonicated lipid droplets of defined composition using an inverted culture technique. Uptake of the lipid droplets by the cells was shown to be dependent on the time of exposure to the droplets and on the mass of droplets presented to the cells. A comparison of the hydrolysis of triolein and cholesteryl oleate by cells that had been exposed to isotropic lipid droplets containing equimolar amounts of the two lipids revealed that the rate of hydrolysis of triglyceride was 3 to 4 times faster than that for cholesteryl ester. The hydrolysis of cholesteryl oleate from cells loaded with the isotropic droplets was approximately 1.5 times as fast as that from cells loaded with anisotropic droplets containing only cholesteryl oleate. A comparison of the hydrolysis of cholesteryl ester in the presence and absence of Sandoz compound 58-035, an inhibitor of acyl CoA:cholesterol acyl transferase, by cells loaded with isotropic droplets showed that about 30% of the free cholesterol liberated by hydrolysis was reesterified regardless of the mass of free cholesterol. We conclude that cultured smooth muscle cells have a greater capacity to hydrolyze triglyceride than cholesteryl ester, and that the rate of hydrolysis of cholesteryl ester appears to be related to the physical state of the droplet in which the cholesteryl ester is stored. In addition, it appears that the smooth muscle cells have a cholesteryl ester cycle that is inefficient in the reesterification of excess free cholesterol.

Animals↗

PubMed1999

Crystallization of free cholesterol in model macrophage foam cells.

-The present study examined free cholesterol (FC) crystallization in macrophage foam cells. Model foam cells (J774 or mouse peritoneal macrophages [MPMs]) were incubated with acetylated low density lipoprotein and FC/phospholipid dispersions for 48 hours, resulting in the deposition of large stores of cytoplasmic cholesteryl esters (CEs). The model foam cells were then incubated for up to 5 days with an acyl-coenzyme A:cholesterol acyltransferase (ACAT) inhibitor (CP-113,818) in the absence of an extracellular FC acceptor to allow intracellular accumulation of FC. FC crystals of various shapes and sizes formed in the MPMs but not in the J774 macrophages. Examination of the MPM monolayers by microscopy indicated that the crystals were externalized rapidly after formation and thereafter continued to increase in size. Incubating J774 macrophages with 8-(4-chlorophenylthio)adenosine 3':5'-cyclic monophosphate (CPT-cAMP) in addition to CP-113,818 caused FC crystal formation as a consequence of CPT-cAMP stimulation of CE hydrolysis and inhibition of cell growth. In addition, 2 separate cholesterol phases (liquid-crystalline and cholesterol monohydrate) in the plane of the membrane bilayer were detected after 31 hours of ACAT inhibition by the use of small-angle x-ray diffraction of J774 macrophage foam cells treated with CPT-cAMP. Other compounds reported to inhibit ACAT, namely progesterone (20 microgram/mL) and N-acetyl-D-sphingosine (c(2)-ceramide, 10 microgram/mL), induced cellular toxicity in J774 macrophage foam cells and FC crystallization when coincubated with CPT-cAMP. Addition of the extracellular FC acceptors apolipoproteins (apo) E and A-I (50 microgram/mL) reduced FC crystal formation. In MPMs, lower cell density and frequent changes of medium were conducive to crystal formation. This may be due to "dilution" of apoE secreted by the MPMs and is consistent with our observation that the addition of exogenous apoE or apoA-I inhibits FC crystal formation in J774 macrophage foam cells cotreated with CP-113,818 plus CPT-cAMP. These data demonstrate that FC crystals can form from the hydrolysis of cytoplasmic stores of CEs in model foam cells. FC crystal formation can be modulated by the addition of extracellular FC acceptors or by affecting the cellular rate of CE hydrolysis. This process may contribute to the formation of FC crystals in atherosclerotic plaques.

Animals↗

PubMed1995

Prevention of cholesteryl ester accumulation in P388D1 macrophage-like cells by increased cellular vitamin E depends on species of extracellular cholesterol. Conventional heterologous non-human cell cultures are poor models of human atherosclerotic foam cell formation.

Since the cellular role of the antioxidative vitamins in the formation of foam cells has not yet been studied in detail, we investigated the effect of alpha-tocopherol and ascorbic acid loading of P388D1 macrophage-like cells on their cholesterol and cholesteryl ester levels and their response to the exposure to different lipoproteins. alpha-Tocopherol loading, but not ascorbic acid loading, of P388D1 cells strongly reduced their cellular cholesteryl ester/cholesterol ratio (the crucial indicator of foam cell formation) when fetal calf serum was the only extracellular source of cholesterol. Balance studies suggest that this effect of alpha-tocopherol was mainly due to a reduced uptake of fetal-calf-serum-derived cholesterol. alpha-Tocopherol loading, however, did not reduce the cholesteryl ester/cholesterol ratio when human unmodified low-density lipoprotein (LDL) was added to culture medium containing fetal calf serum. Thus, the uptake of fetal-calf-serum-derived cholesterol was competitively reduced by human LDL, the uptake of which remained unaffected by alpha-tocopherol. Similarly, alpha-tocopherol loading did not prevent cholesteryl ester formation induced by human LDL either oxidized with Cu2+, ultraviolet light or HOCl, or modified by acetylation, aggregation or by malondialdehyde treatment. The present experimental conditions lacked any pro-oxidative burden, since (a) ascorbic acid, either alone or combined with alpha-tocopherol, did not affect cellular cholesteryl ester levels, (b) foam cell formation was not a linear function of the degree of oxidative LDL modification, and (c) alpha-tocopherol lacked specific effects on oxidatively modified LDL. Thus, the reduction of cellular cholesteryl esters by alpha-tocopherol in the absence of human unmodified LDL was hardly due to common antioxidative properties of vitamin E. In conclusion, the present observation that a desirable alpha-tocopherol effect on the cholesteryl ester balance in mouse-tumor-derived P388D1 cells strongly depended on the species of extracellular cholesterol carrier, cautions against premature generalizations of conventional non-human cell culture data.

Animals↗

PubMed2002

Characterization of foam cells in nipple aspirate fluid.

Foam cells with abundant vacuolated cytoplasm are prominent in most samples of spontaneous nipple discharge, nipple aspirate fluid, and ductal lavage. Although several investigators have attempted to characterize these cells, there is no consensus about whether these cells are derived entirely from macrophages or from both ductal epithelial cells and macrophages. Using immunocytochemical methods, we studied 20 paired specimens of nipple aspirate fluid containing abundant foam cells obtained from the involved breast of women with in situ or invasive carcinoma and from the contralateral normal breast. We used a cocktail of anticytokeratin antibodies including AE1, AE3, and CAM5.2 and the macrophage marker KP1 (CD68). In addition, we examined samples by electron microscopy. The foam cells were consistently negative for cytokeratin and positive for CD68. In every case electron microscopy of these cells revealed irregular outlines with short cytoplasmic processes. The cytoplasm was abundant and contained numerous lysosomes, a small Golgi complex, lipid droplets, mitochondria, and short profiles of rough endoplasmic reticulum. There was no evidence, however, of cell junctions or tonofilaments. The immunocytochemical and electron microscopic findings of our study together clearly support a macrophage derivation for foam cells in nipple aspirate fluid.

Antigens, CD↗

PubMed2006

A CD36-dependent signaling cascade is necessary for macrophage foam cell formation.

Accumulation of macrophage foam cells in atherosclerotic blood vessel intima is a critical component of atherogenesis mediated by scavenger receptor-dependent internalization of oxidized LDL. We demonstrated by coimmunoprecipitation and pull-down assays that the macrophage scavenger receptor CD36 associates with a signaling complex containing Lyn and MEKK2. The MAP kinases JNK1 and JNK2 were specifically phosphorylated in macrophages exposed to oxLDL. Using cells isolated from SRA, TLR2, or CD36 null mice, and phospholipid ligands specific for either SRA or CD36, we showed that JNK activation was mediated by CD36. Both foam cell formation and activation of JNK2 in hyperlipidemic mice were diminished in the absence of CD36. Furthermore, inhibition of Src or JNK blocked oxLDL uptake and inhibited foam cell formation in vitro and in vivo. These findings show that a specific CD36-dependent signaling pathway initiated by oxLDL is necessary for foam cell formation and identify potential targets for antiatherosclerosis therapy.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Protein expressions in macrophage-derived foam cells: comparative analysis by two-dimensional gel electrophoresis.

AIM: To study the overall protein expression changes induced by oxidized low-density lipoprotein (ox-LDL) in the U937 foam cells. METHODS: Foam cell model was established by incubating the human monoblastic leukemia (U937) cells with ox-LDL. Each protein samples in U937 control cells and U937 foam cells were separated by two-dimensional gel electrophoresis (2-DE). After the gels were stained by silver staining method, the images were analyzed by PDquest 2D-image-analysis software (Bio-Rad). Some of the spots were available via the Internet with links to the U937 proteomic map provided from the ExPASy Proteomics server. RESULTS: Using 2-DE, the overall protein map was obtained, in which 150 spots were matched with the control gel (match ratio: 75 %). Compared with U937 cells, 37 spots significantly changed in the foam cells (P<0.05), among which the expression levels in 28 spots increased and those in 9 spots decreased. Especially, 8 spots in U937 cells were absent in the foam cells, while 11 spots in the foam cells were absent in the control cells. CONCLUSION: The changed protein profiles induced by ox-LDL in U937 foam cells were established to support the functional studies on the macrophage-derived foam cells in atherosclerotic pathological states.

Cells, Cultured↗

PubMed1996

Phagocytic activation induces formation of platelet-activating factor in human monocyte-derived macrophages and in macrophage-derived foam cells. Relevance to the inflammatory reaction in atherogenesis.

Monocyte-derived macrophages and macrophage-derived foam cells in arterial tissue may undergo phagocytic activation and thereby contribute to an inflammatory reaction. We have investigated the effect of phagocytic activation on the formation of platelet-activating factor (1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine, PAF-acether, PAF), a proinflammatory phospholipid, in human monocyte-derived macrophages (macrophages) and in cholesterol-loaded macrophage foam cells (foam cells). Adherent human monocyte-derived macrophages were transformed into foam cells upon incubation with acetylated low-density lipoproteins (Ac-LDL). Such foam cells characteristically displayed a markedly increased content of cholesteryl esters compared with macrophages (4.3 +/- 1.3 microgram/microgram DNA and 0.2 +/- 0.3 microgram/microgram DNA, n = 5, respectively). After phagocytic stimulation with serum-opsonized zymosan (OPZ), both macrophages and foam cells synthesized PAF transiently with maximal production (0.5-1.1 pmol PAF/microgram DNA, n = 5, corresponding to 4.0-8.8 pmol PAF/10(6) cells, as assessed by bioassay) occurring approximately 15 min after stimulation. A major fraction of the synthesized PAF remained cell-associated; such PAF was composed mainly of the hexadecyl (16:0 PAF, approximately 75%) and the octadecenyl (18:1 PAF) species and of trace amounts of octadecyl (18:0 PAF), as assessed by reverse-phase liquid chromatography. Addition of exogenous 16:0 lyso-PAF alone triggered PAF formation (0.9-1.7 pmol PAF/microgram DNA, after 15 min of cellular stimulation); simultaneous cellular stimulation with OPZ and 16:0 lyso-PAF increased PAF formation in an additive manner. Acetyltransferase, the enzyme which acetylates the precursor lyso-PAF and transforms it into PAF, displayed elevated activity both in macrophages and in foam cells, attaining 83-240 pmol PAF formed per min per mg DNA (n = 4); such elevated activity was not increased by OPZ-stimulation. The activity of acetylhydrolase, the PAF-degrading enzyme, was similar in macrophages and in foam cells, and varied between 120 pmol and 320 pmol PAF degraded per min per mg DNA (n = 5). Cell-associated acetylhydrolase activity was increased significantly by 40+/-15 % (P < 0.003, n = 5) after 15 - 30 min of activation with OPZ compared with non-stimulated cells and may account for the rapid decrease in cellular PAF content observed approximately 30 min after stimulation. These studies have established that metabolism of PAF in foam cells closely resembles that in macrophages, and thus PAF metabolism is largely independent of cellular cholesterol content. Moreover our data are consistent with the hypothesis that both macrophages and macrophage-derived foam cells upon phagocytic-activation constitute a significant transient source of PAF at inflammatory sites in the arterial intima where this phospholipidic mediator may exert potent proatherogenic and prothrombotic effects.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗

PubMedDate not supplied

Foam cells in explants of atherosclerotic rabbit aortas have receptors for beta-very low density lipoproteins and modified low density lipoproteins.

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.

Animals↗

PubMed1995

Mast cell granule remnants carry LDL into smooth muscle cells of the synthetic phenotype and induce their conversion into foam cells.

We report the effect of mast cells on the uptake of LDL by smooth muscle cells (SMCs) and their conversion into foam cells in vitro. The mast cells were stimulated to exocytose their cytoplasmic secretory granules, and the granule remnants formed were recovered from the extracellular fluid and added to cultures of SMCs of either the synthetic or contractile phenotype in LDL-containing medium. In the presence but not in the absence of granule remnants, SMCs of the synthetic phenotype took up LDL with ensuing stimulation of intracellular cholesteryl ester synthesis and cytoplasmic accumulation of neutral lipid droplets. Using methylated LDL (mLDL), a modified species of LDL that binds to granule remnants but not to LDL receptors, we demonstrated that this uptake (leading to foam cell formation) occurred only when LDL was bound to granule remnants. After the addition of colloidal gold-LDL and granule remnants to the incubation system, electron microscopy revealed that within phagosomes of the SMCs there were granule remnants (diameter, 0.5 to 1 micron) coated with LDL, confirming that LDL had been carried into the cells with the remnants. SMCs of the contractile phenotype were less efficient than their synthetic counterparts at phagocytosing LDL-coated granule remnants and were not converted into foam cells. This difference in the rate of phagocytosis of granule remnants was present even in the absence of LDL, revealing that the more active phagocytosis by SMCs of the synthetic phenotype was not specifically related to uptake of lipids but rather reflected a general phenotype characteristic of these cells. These observations indicate a phagocytic mechanism by which SMCs of the synthetic phenotype are converted into cholesteryl ester-filled foam cells, and they also suggest that degranulation of mast cells plays a role in the development of fatty streak lesions.

Animals↗

PubMed1987

Method for detection and isolation of cholesteryl ester-containing "foam" cells using flow cytometry.

Accumulation of cholesteryl ester within vascular cells is a defining characteristic of atherosclerotic lesions. Therefore, it is of interest to be able to monitor this critical event in the development of atherosclerosis. With this objective in mind, we have developed a method for the detection of cholesteryl ester-containing cells (i.e., foam cells) in cell suspensions prepared from enzymatically dissociated aortas. Cholesteryl ester in aortic cells was selectively stained with the fluorescent dye filipin. Because filipin binds to unesterified cholesterol but not to esterified cholesterol, it was necessary first to remove unesterified cholesterol from cells by ethanol extraction so that its presence would not interfere with the specific detection of cholesteryl ester. Then unesterified cholesterol made available by enzymatic hydrolysis of cellular cholesteryl ester could be specifically stained with filipin. The filipin-stained cell suspensions were analyzed using flow cytometry. With a flow cytometer it was possible to detect and sort cholesteryl ester-containing cells onto glass slides for microscopic analysis. Cell suspensions prepared from either grossly normal or atherosclerotic swine aortas contained cells with cholesteryl ester inclusions. As expected, these cells were more numerous in the atherosclerotic aortas. Cells with higher levels of fluorescence contained more numerous cholesteryl ester inclusions. Flow cytometric detection of cholesteryl ester-containing cells should be generally useful in studies of cellular cholesterol metabolism as well as in specific studies of cellular cholesterol accumulation in atherosclerotic vessels.

Animals↗

PubMed2005

Intracellular-free calcium dynamics and F-actin alteration in the formation of macrophage foam cells.

The formation of macrophage foam cells, which is the key event in atherosclerosis, occurs by the uptake of oxidized low-density lipoprotein (Ox-LDL) via the scavenger receptor (CD36) pathway. Ca(2+) plays an important role in atherosclerosis. However, in the spatiotemporal view, the correlation between kinetic changes of intracellular-free calcium ([Ca(2+)](i)) and the cellular dysfunctions in the formation of macrophage foam cells has not yet been studied in detail. By the use of confocal laser scanning microscope and flow cytometer, we have detected Ca(2+) dynamics, the assembly of F-actin, and the expression of CD36 under the exposure of U937-derived macrophages to Ox-LDL. The uptake of Ox-LDL significantly increased [Ca(2+)](i) in U937-derived macrophages in both acute and chronic treatments (P<0.01). In particular, the increases of the induced [Ca(2+)](i) were different in the presence or absence of extracellular Ca(2+) under acute exposure. A time-dependent rise in F-actin assembly and CD36 expression at 12 and 24h was induced, respectively, by Ox-LDL. The spatiotemporal increases of [Ca(2+)](i) induced by Ox-LDL probably have the key effect on the early phrase in the formation of macrophage foam cells.

Actins↗