Cholesteryl ester hydrolysis in J774 macrophages occurs in the cytoplasm and lysosomes.
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The relationship of cholesteryl ester hydrolysis to the physical state of the cholesteryl ester in J774 murine macrophages was explored in cells induced to store cholesteryl esters either in anisotropic (ordered) inclusions or isotropic (liquid) inclusions. In contrast to other cell systems, the rate of cholesteryl ester hydrolysis was faster in cells containing anisotropic inclusions than in cells containing isotropic inclusions. Two contributing factors were identified. Kinetic analyses of the rates of hydrolysis are consistent with a substrate competition by co-deposited triglyceride in cells with isotropic inclusions. In addition, hydrolysis of cholesteryl esters in cells with anisotropic droplets is mediated by both cytoplasmic and lysosomal lipolytic enzymes, as shown by using the lysosomotropic agent, chloroquine, and an inhibitor of neutral cholesteryl ester hydrolase, umbelliferyl diethylphosphate. In cells containing anisotropic inclusions, hydrolysis was partially inhibited by incubation in media containing either chloroquine or umbelliferyl diethylphosphate. Together, chloroquine and umbelliferyl diethylphosphate completely inhibited hydrolysis. However, when cells containing isotropic inclusions were incubated with umbelliferyl diethylphosphate, cholesteryl ester hydrolysis was completely inhibited, but chloroquine had no effect. Transmission electron microscopy demonstrated a primarily lysosomal location for lipid droplets in cells with anisotropic droplets and both non-lysosomal and lysosomal populations of lipid droplets in cells with isotropic droplets. These results support the conclusion that there is a lysosomal component to the hydrolysis of stored cholesteryl esters in foam cells. (+info)
Specific interaction of oxidized low-density lipoprotein with macrophage-derived foam cells isolated from rabbit atherosclerotic lesions.
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Interaction of oxidized LDL (OxLDL) with macrophage-derived foam cells is one of the key events in the development and progression of atherosclerosis. To study this interaction, macrophage-derived foam cells were isolated from rabbit atherosclerotic lesions and the expression of scavenger receptors for OxLDL was examined. Atherosclerosis was induced in rabbits by denudation of the large arteries, followed by a hypercholesteremic diet. Macrophage-derived foam cells, characterized by immunostaining with an RAM-11 antibody (a macrophage marker), contained a high content of intracellular lipid. Maximal binding of radiolabeled OxLDL to isolated macrophage-derived foam cells (1652+/-235 ng 125I-OxLDL/mg of cell protein) was 20-fold higher compared with Bmax values of monocytes. Levels of association of OxLDL to macrophage-derived foam cells isolated from atherosclerotic lesions 12 weeks after denudation were >3-fold higher compared with the levels expressed by macrophage-derived foam cells isolated after 6 weeks. Association of 125I-OxLDL could be completely blocked by OxLDL, and partially by acetylated LDL and polyinosinic acid, indicating the presence of a specific binding site for OxLDL on macrophage-derived foam cells. The induction of scavenger receptors for OxLDL on macrophage-derived foam cells during the development of atherosclerosis, as described in this study, may facilitate the lipid accumulation in macrophage-derived foam cells, as observed in advanced atherosclerotic lesions. (+info)
The role of interleukin 12 in the development of atherosclerosis in ApoE-deficient mice.
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The cytokine profile of atherosclerotic aortas from apoE-deficient mice was assessed by reverse transcriptase-polymerase chain reaction. The results clearly showed that the expression of mRNA for IL-12p40 was evident in aortas from 3-month-old apoE-deficient mice. The mRNA for IL-10 was detected in aorta from these mice at the age of 6 months, indicating that expression of IL-12 is earlier than that of IL-10 in these animals. Concurrent with IL-12p40, the mRNA for the T-cell cytokine IFN-gamma, but not IL-4, was detected in aortas of mice at young and old ages. Both in situ hybridization and immunostaining further demonstrated the localization of IL-12 in macrophages of atherosclerotic lesions. Immunohistochemistry also demonstrated the expression of costimulatory molecules B7-1 and B7-2 in macrophages, suggesting that activation of T lymphocytes by macrophages may occur via surface antigens in lesions. When the immunoglobulin isotype of the antioxidized LDL antibodies in sera of apoE-deficient mice was determined, it revealed that both IgM and IgG were present. Furthermore, IgG2a is predominant and comprises approximately 50% of the antioxidized LDL IgG in sera from young mice (3 months), but decreased to lower levels (35%) in older mice (6 months). Daily administration of IL-12 led to an increase in serum levels of antioxidized LDL antibodies and accelerated atherosclerosis in young apoE-deficient mice compared with control mice injected with PBS alone. Taken together, these data suggest that IL-12 plays an active role in regulating the immune response during the early phase of atherosclerosis in apoE-deficient mice. (+info)
MCP-1 deficiency reduces susceptibility to atherosclerosis in mice that overexpress human apolipoprotein B.
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The earliest recognizable atherosclerotic lesions are fatty streaks composed of lipid-laden macrophages (foam cells). Circulating monocytes are the precursors of these foam cells, but the molecular mechanisms that govern macrophage trafficking through the vessel wall are poorly understood. Monocyte chemoattractant protein-1 (MCP-1), a member of the chemokine (chemotactic cytokine) family, is a potent monocyte agonist that is upregulated by oxidized lipids. Recent studies in hypercholesterolemic mice lacking apo E or the low-density lipoprotein receptor have suggested a role for MCP-1 in monocyte recruitment to early atherosclerotic lesions. To determine if MCP-1 is critically involved in atherogenesis in the setting of elevated physiological plasma cholesterol levels, we deleted the MCP-1 gene in transgenic mice expressing human apo B. Here we report that the absence of MCP-1 provides dramatic protection from macrophage recruitment and atherosclerotic lesion formation in apo B transgenic mice, without altering lipoprotein metabolism. Taken together with the results of earlier studies, these data provide compelling evidence that MCP-1 plays a critical role in the initiation of atherosclerosis. (+info)
Depletion of pre beta 1LpA1 and LpA4 particles by mast cell chymase reduces cholesterol efflux from macrophage foam cells induced by plasma.
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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. (+info)
Effects of NTE-122, a novel acyl-CoA:cholesterol acyltransferase inhibitor, on cholesterol esterification and high-density lipoprotein-induced cholesterol efflux in macrophages.
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We investigated the effects of a novel acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor, NTE-122 (trans-1,4-bis[[1-cyclohexyl-3-(4-dimethylamino phenyl)ureido]methyl]cyclohexane), on ACAT activities in macrophages originating from several species and high-density lipoprotein (HDL)-induced cholesterol efflux in phorbol 12-myristate 13-acetate (PMA)-treated THP-1 cells. NTE-122 inhibited cell-free ACAT activities in human PMA-treated THP-1 cells and mouse J774.1 cells with IC50 values of 0.88 and 360 nM, respectively. NTE-122 competively inhibited the ACAT activity in PMA-treated THP-1 cells. NTE-122 also inhibited cellular ACAT activities in PMA-treated THP-1 cells, rat peritoneal macrophages and J774.1 cells with IC50 values of 3.5, 84 and 6800 nM, respectively. Furthermore, NTE-122 prevented cholesterol accumulation in PMA-treated THP-1 cells incubated with acetylated low density lipoprotein, simultaneously with HDL, while it caused accumulation of a significant amount of free cholesterol in the absence and even in the presence of HDL. NTE-122 also enhanced HDL-induced cholesterol efflux from established foam cells converted from PMA-treated THP-1 cells. These results suggest that NTE-122, capable of inhibiting macrophage ACAT activity in humans more strongly than those in the other species, exhibits anti-atherogenic effects by preventing the foam cell formation and enhancing the foam cell regression in humans. (+info)
Modified LDLs induce and bind to membrane ruffles on macrophages.
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Macrophage foam cell formation in vitro requires uptake of modified low density lipoproteins (LDL) such as acetylated LDL (AcLDL) and moderately oxidized LDL (OxLDL), or beta-migrating very low density lipoprotein (betaVLDL), a naturally occurring lipoprotein. Incubation ofmacrophages with AcLDL and OxLDL resulted in stimulation of membrane ruffle formation, while betaVLDL primarily resulted in increased numbers of microvilli. Time-lapse Allen video enhanced contrast differential interference contrast (AVEC-DIC) light microscopy and correlative whole mount intermediate-voltage transmission electron microscopy (IVEM) was used to examine the dynamics ofAcLDL stimulated membrane ruffling and membrane ruffle ultrastructure. Stereo 3D surface replicas confirmed that AcLDL bound to these AcLDL-induced membrane ruffles. Quantification of the plasma membrane surface area after incubation with AcLDL, betaVLDL or LDL confirmed that AcLDL stimulated membrane ruffling, while betaVLDL and LDL stimulated microvilli formation. These studies suggest that modified LDLs induce circular membrane ruffles and modified LDLs bind to these ligand-induced membrane ruffles. (+info)
Modified LDLs are internalized by macrophages in part via macropinocytosis.
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Macrophage foam cell formation in vitro requires uptake of modified low density lipoproteins (LDL) such as acetylated LDL (AcLDL) and moderately oxidized LDL (OxLDL). Macrophages incubated with AcLDL and OxLDL, but not LDL, showed increased membrane ruffling as seen with time-lapse phase contrast video light microscopy. Modified LDLs stimulated circular membrane ruffles between 2 and 10 min after incubation. These membrane ruffles were readsorbed into the plasma membrane between 5 and 15 min later. Phase-bright macropinosomes formed at the base of the stimulated membrane ruffles. The fluid-phase marker lucifer yellow labeled the modified LDL stimulated macropinosomes. Modified LDLs stimulate fluid-phase uptake by 1.5-fold to threefold as measured with 14C-sucrose uptake. Transmission electron microscopy showed that gold conjugated AcLDL and OxLDL bound preferentially to membrane ruffles and were present in macropinosomes (diameter >0.2 pm) underneath these membrane ruffles. AcLDL and OxLDL were also present in clathrin-coated pits and endosomes. These studies suggest that modified lipoproteins stimulate macropinocytosis. AcLDL and OxLDL are partially internalized by macropinocytosis and partially internalized via clathrin-coated pit endocytosis. (+info)