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Because of its critical role in HDL formation, significant efforts have been devoted to studying apolipoprotein A-I (APOA1) structural transitions in response to lipid binding. To assess the requirements for the conformational freedom of its termini during HDL particle formation, we generated three dimeric APOA1 molecules with their termini covalently joined in different combinations. The dimeric (d)-APOA1C-N mutant coupled the C-terminus of one APOA1 molecule to the N-terminus of a second with a short alanine linker, whereas the d-APOA1C-C and d-APOA1N-N mutants coupled the C-termini and the N-termini of two APOA1 molecules, respectively, using introduced cysteine residues to form disulfide linkages. We then tested the ability of these constructs to generate reconstituted HDL by detergent-assisted and spontaneous phospholipid microsolubilization methods. Using cholate dialysis, we demonstrate WT and all APOA1 mutants generated reconstituted HDL particles of similar sizes, morphologies, compositions, and abilities to activate lecithin:cholesterol acyltransferase. Unlike WT, however, the mutants were incapable of spontaneously solubilizing short chain phospholipids into discoidal particles. We found lipid-free d-APOA1C-N and d-APOA1N-N retained most of WT APOA1’s ability to promote cholesterol efflux via the ATP binding cassette transporter A1, whereas d-APOA1C-C exhibited impaired cholesterol efflux. Our data support the double belt model for a lipid-bound APOA1 structure in nascent HDL particles and refute other postulated arrangements like the “double super helix.” Furthermore, we conclude the conformational freedom of both the N- and C-termini of APOA1 is important in spontaneous microsolubilization of bulk phospholipid but is not critical for ABCA1-mediated cholesterol efflux.

7-Ketocholesterol (7KC) is a toxic oxysterol that is associated with many diseases and disabilities of aging, as well as several orphan diseases. 7KC is the most common product of a reaction between cholesterol and oxygen radicals and is the most concentrated oxysterol found in the blood and arterial plaques of coronary artery disease patients as well as various other disease tissues and cell types. Unlike cholesterol, 7KC consistently shows cytotoxicity to cells and its physiological function in humans or other complex organisms is unknown. Oxysterols, particularly 7KC, have also been shown to diffuse through membranes where they affect receptor and enzymatic function. Here, we will explore the known and proposed mechanisms of pathologies that are associated with 7KC, as well speculate about the future of 7KC as a diagnostic and therapeutic target in medicine.

This report details the lipid composition of nascent HDL (nHDL) particles formed by the action of the ATP binding cassette transporter A1 (ABCA1) on apolipoprotein A-I (apoA-I). nHDL particles of different size (average diameters of ∼12, 10, 7.5, and

In late-stage atherosclerosis, much of the cholesterol in macrophage foam cells resides within enlarged lysosomes. Similarly, human macrophages incubated in vitro with modified LDLs contain significant amounts of lysosomal free cholesterol and cholesteryl ester (CE), which disrupts lysosomal function similar to macrophages in atherosclerotic lesions. The lysosomal cholesterol cannot be removed, even in the presence of strong efflux promoters. Thus, efflux of sterol is prevented. In the artery wall, foam cells interact with triglyceride-rich particles (TRPs) in addition to modified LDLs. Little is known about how TRP metabolism affects macrophage cholesterol. Therefore, we explored the effect of TRP on intracellular CE metabolism. Triglyceride (TG), delivered to lysosomes in TRP, reduced CE accumulation by 50%. Increased TG levels within the cell, particularly within lysosomes, correlated with reductions in CE content. The volume of cholesterol-engorged lysosomes decreased after TRP treatment, indicating cholesterol was cleared. Lysosomal TG also reduced the cholesterol-induced inhibition of lysosomal acidification allowing lysosomes to remain active. Enhanced degradation and clearance of CE may be explained by movement of cholesterol out of the lysosome to sites where it is effluxed. Thus, our results show that introduction of TG into CE-laden foam cells influences CE metabolism and, potentially, atherogenesis.—Ullery-Ricewick, J. C., B. E. Cox, E. E. Griffin, and W. G. Jerome. Triglyceride alters lysosomal cholesterol ester metabolism in cholesteryl ester-laden macrophage foam cells.

Macrophage foam cells in atherosclerotic lesions accumulate substantial cholesterol stores within large, swollen lysosomes. Previous studies with mildly oxidized low density lipoprotein (OxLDL)-treated THP-1 macrophages suggest an initial buildup of free cholesterol (FC), followed by an inhibition of lysosomal cholesteryl ester (CE) hydrolysis and a subsequent lysosomal accumulation of unhydrolyzed lipoprotein CE. We examined whether other potential sources of cholesterol found within atherosclerotic lesions could also induce similar lysosomal accumulation. Biochemical analysis combined with microscopic analysis showed that treatment of THP-1 macrophages with aggregated low density lipoprotein (AggLDL) or CE-rich lipid dispersions (DISP) produced a similar lysosomal accumulation of both FC and CE. Cotreatment with an ACAT inhibitor, CP113,818, confirmed that the CE accumulation was primarily the result of the inhibition of lysosomal CE hydrolysis. The rate of unhydrolyzed CE buildup was more rapid with DISP than with AggLDL. However, with both treatments, FC appeared to accumulate in lysosomes before the inhibition in hydrolysis and CE accumulation, a sequence shared with mildly OxLDL.Thus, lysosomal accumulation of FC and CE can be attributable to more general mechanisms than just the inhibition of hydrolysis by oxidized lipids.

Microsomal triglyceride transfer protein (MTP) is essential for the assembly of apolipoprotein B-containing lipoproteins. Within the endoplasmic reticulum, it transfers lipid from the membrane to the forming lipoprotein. Recent evidence suggests that it may also function within the Golgi apparatus. To address this hypothesis, we developed a polyclonal antibody to MTP and used it in a series of studies on mouse liver and McArdle-RH7777 (McA) cells. Western blot analysis demonstrated the presence of MTP within mouse hepatic-Golgi apparatus-rich fractions. In addition, in vitro lipid transfer assays demonstrated the presence of triglyceride transfer activity within the Golgi fractions. Immunohistochemical studies with mouse liver demonstrated the presence of MTP within all hepatocytes, but not in nonparenchymal cells. The subcellular location of MTP in McA cells was investigated using confocal microscopy. MTP colocalized with the trans-Golgi network (TGN) 38 and Golgi SNARE (soluble N-ethylmalemide-sensitive factor attachment protein receptor) of 28 kDa (GS28), markers for the trans- and cis-Golgi apparatus, respectively. Morphometric analyses indicated that ∼17% of the MTP signal colocalized with the TGN38, while 33% of the trans-Golgi marker colocalized with the MTP. Approximately 17% of the MTP signal colocalized with the GS28, whereas 53% of the cis-Golgi marker colocalized with the MTP.The results provide unequivocal evidence for the location of MTP within the Golgi apparatus, and further highlight the importance of this organelle in the assembly of lipoproteins.

In atherosclerotic lesions, macrophages store lipid in cytoplasmic inclusions and lysosomes. Regression studies show that lysosomal lipid is not as easily cleared as cytoplasmic inclusion lipid. Macrophages enriched with mildly oxidized low density lipoprotein (oxLDL) accumulate cholesteryl ester (CE) and free cholesterol (FC) in lysosomes. We examined whether lysosomal stores of cholesterol from oxLDL are cleared from THP-1 and mouse macrophages. As in previous studies, oxLDL-enriched THP-1 macrophages accumulated substantial lysosomal cholesterol. Surprisingly, less than 12% of oxLDL-derived lysosomal CE was cleared to efficient FC acceptors (e.g., cyclodextrins, apolipoprotein/phosphatidylcholine vesicles, and fetal bovine serum). Filipin staining showed that lysosomes of oxLDL-treated THP-1 cells contained FC, and despite removal of most of the cell FC (70–80%) by incubation with cyclodextrins, filipin staining of FC in lysosomes did not diminish. Also, when THP-1 macrophages were incubated with [3H]CE oxLDL, 73–76% of the [3H]CE was retained in a lysosomal hydrolysis resistant pool. In contrast, greater than 90% of acetylated low density lipoprotein (acLDL) [3H]CE was hydrolyzed. Furthermore, [3H]FC liberated from oxLDL [3H]CE was released at a slower rate to cyclodextrins than was [3H]FC from acLDL [3H]CE. In contrast, only 27% of oxLDL [3H]CE was resistant to hydrolysis in mouse macrophages, and the [3H]FC generated from oxLDL and acLDL [3H]CE was released to cyclodextrins at similar rates. We conclude that lack of hydrolysis and efflux of oxLDL cholesterol is not exclusively inherent in oxLDL, but also requires specific cell factors present in one cell type but not the other. —Yancey, P. G., and W. G. Jerome. Lysosomal cholesterol derived from mildly oxidized low density lipoprotein is resistant to efflux.

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.—Avart, S. J., D. W. Bernard, W. G. Jerome, and J. M. Glick. Cholesteryl ester hydrolysis in J774 macrophages occurs in the cytoplasm and lysosomes. J. Lipid Res. 1999. 40: 405–414.

Lipid accumulation within macrophages is a major sequelae of atherosclerosis. Much of this lipid accumulation occurs within large, swollen lysosomes. We analyzed lipid accumulation in cultured macrophages using oxidized or acetylated low density lipoprotein (LDL) as the loading agent. Pigeon macrophages incubated for 48 h with mildly oxidized pigeon LDL (TBARS = 5–10 nmol/mg protein) showed significant increases in cellular cholesterol compared with untreated controls. Forty-eight percent of the increased cholesterol occurred as unesterified cholesterol. Treated cells had lipid-swollen lysosomes similar to those of atherosclerotic foam cells. The increase in lysosomal lipid was accompanied (correlation coefficient of 0.96) by increases in acid phosphatase staining cisternae of the Golgi and trans-Golgi network (TGN). THP-1 macrophages incubated with oxidized LDL showed similar lysosomal loading and Golgi/TGN hypertrophy. In contrast, macrophages incubated with acetylated LDL accumulated significant amounts of cholesterol but the increase occurred as cholesteryl ester (81% in pigeons) within cytoplasmic droplets and there was no associated increase in acid phosphatase-containing cisternae of Golgi or TGN. The correlation in both pigeon and THP-1 macrophages of oxidized LDL-induced lysosomal lipid accumulation and Golgi hypertrophy suggests a linkage of these two phenomena. This implicates intracellular membrane trafficking as a possible defect in foam cells of the atherosclerotic lesion.—Jerome, W. G., C. Cash, R. Webber, R. Horton, and P. G. Yancey. Lysosomal lipid accumulation from oxidizied low density lipoprotein is correlated with hypertrophy of the Golgi apparatus and trans-Golgi network. J. Lipid Res. 1998. 39: 1362–1371.

Macrophage foam cells of atherosclerotic lesions store lipid in lysosomes and cytoplasmic inclusions. Oxidized low density lipoprotein (oxLDL) has been proposed to be the atherogenic particle responsible for the free and esterified cholesterol stores in macrophages. Currently, however, there is a paucity of data showing that oxLDL can induce much cholesterol accumulation in cells. The present studies compare the ability of mildly oxLDL (TBARS = 5 to 10 nmols/mg LDL protein) with acetylated LDL to induce free cholesterol (FC) and esterified cholesterol (EC) accumulation in pigeon, THP-1, and mouse macrophages. Mildly oxLDL stimulated high levels of loading comparable to acLDL where the cellular cholesterol concentrations ranged from 160 to 420 μg/mg cell protein with EC accounting for 52–80% of the cholesterol. Pigeon and THP-1 macrophages stored most (60–90%) of oxLDL cholesterol (both FC and EC) in lysosomes, and the bulk (64–88%) of acLDL cholesterol in cytoplasmic inclusions. Consistent with lysosomal accumulation, cholesterol esterification was 75% less in THP-1 macrophages enriched with oxLDL cholesterol compared with acLDL. Furthermore, addition of an acyl-CoA:cholesterol acyltransferase inhibitor did not significantly affect either cholesterol loading or the percent distribution of FC and EC in THP-1 and pigeon cells incubated with oxLDL. Surprisingly, mouse macrophages stored most of oxLDL (71%) and acLDL (83%) cholesterol within cytoplasmic inclusions. Also, in mouse macrophages, esterification paralleled cholesterol loading, and was 3-fold more in oxLDL treated cells compared with acLDL treated cells. Inhibition of ACAT led to a 62% and 90% reduction in the %EC in oxLDL and acLDL treated mouse macrophages, respectively. The results demonstrate that mildly oxidized low density lipoprotein (oxLDL) stimulates macrophage foam cell formation and lipid engorgement of lysosomes. However, the fate of oxLDL cholesterol markedly differs in macrophages of different species.—Yancey, P. G., and W. G. Jerome. Lysosomal sequestration of free and esterified cholesterol from oxidized low density lipoprotein in macrophages of different species. J. Lipid Res. 1998. 39: 1349–1361.

Dr. Mary Blair Bowers is recognized as a pioneer in biological electron microscopy, contributing significantly to the study of cell cytoskeleton and motility. She played a crucial role in advancing specimen preparation techniques for studying biological samples under an electron beam. Dr. Bowers' career spanned from her early work at Harvard University to her groundbreaking research at the National Heart Institute of NIH, focusing on cell structure and function relationships. Her dedication to microscopy education and service to various scientific organizations, including the Microscopy Society of America, highlights her lasting impact on the field. [Extracted from the article]