Oxidative modification of lipoproteins is implicated in the occurrence and development of atherosclerotic lesions. Earlier studies have elucidated on the mechanisms of foam cell formation and lipid accumulation in these lesions, which is mediated by scavenger receptor-mediated endocytosis of oxidized low-density lipoprotein (oxLDL). Mounting clinical evidence has supported the involvement of oxLDL in cardiovascular diseases. High-density lipoprotein (HDL) is known as anti-atherogenic; however, recent studies have shown circulating oxidized HDL (oxHDL) is related to cardiovascular diseases. A modified structure of oxLDL, which was increased in the plasma of patients with acute myocardial infarction, was characterized. It had two unique features: (1) a fraction of oxLDL accompanied oxHDL, and (2) apoA1 was heavily modified, while modification of apoB, and the accumulation of oxidized phosphatidylcholine (oxPC) and lysophosphatidylcholine (lysoPC) was less pronounced. When LDL and HDL were present at the same time, oxidized lipoproteins actively interacted with each other, and oxPC and lysoPC were transferred to another lipoprotein particle and enzymatically metabolized rapidly. This brief review provides a novel view on the dynamics of oxLDL and oxHDL in circulation.

Oxidized low-density lipoprotein (oxLDL) and oxidized high-density lipoprotein (oxHDL), known as risk factors for cardiovascular disease, have been observed in plasma and atheromatous plaques. In a previous study, the content of oxidized phosphatidylcholine (oxPC) and lysophosphatidylcholine (lysoPC) species stayed constant in isolated in vivo oxLDL but increased in copper-induced oxLDL in vitro. In this study, we prepared synthetic deuterium-labeled 1-palmitoyl lysoPC and palmitoyl-glutaroyl PC (PGPC), a short chain-oxPC to elucidate the metabolic fate of oxPC and lysoPC in oxLDL in the presence of HDL. When LDL preloaded with d₁₃-lysoPC was mixed with HDL, d₁₃-lysoPC was recovered in both the LDL and HDL fractions equally. d₁₃-LysoPC decreased by 50% after 4 h of incubation, while d₁₃-PC increased in both fractions. Diacyl-PC production was abolished by an inhibitor of lecithin-cholesterol acyltransferase (LCAT). When d₁₃-PGPC-preloaded LDL was incubated with HDL, d₁₃-PGPC was transferred to HDL in a dose-dependent manner when both LCAT and lipoprotein-associated phospholipase A₂ (Lp-PLA₂) were inhibited. Lp-PLA₂ in both HDL and LDL was responsible for the hydrolysis of d₁₃-PGPC. These results suggest that short chain-oxPC and lysoPC can transfer between lipoproteins quickly and can be enzymatically converted from oxPC to lysoPC and from lysoPC to diacyl-PC in the presence of HDL.

Neutrophil extracellular traps (NETs) are found in patients with various diseases, including cardiovascular diseases. We previously reported that copper-oxidized low-density lipoprotein (oxLDL) promotes NET formation of neutrophils, and that the resulting NETs increase the inflammatory responses of endothelial cells. In this study, we investigated the effects of high-density lipoproteins (HDL) on NET formation. HL-60-derived neutrophils were treated with phorbol 12-myristate 13-acetate (PMA) and further incubated with oxLDL and various concentrations of HDL for 2 h. NET formation was evaluated by quantifying extracellular DNA and myeloperoxidase. We found that the addition of native HDL partially decreased NET formation of neutrophils induced by oxLDL. This effect of HDL was lost when HDL was oxidized. We showed that oxidized phosphatidylcholines and lysophosphatidylcholine, which are generated in oxLDL, promoted NET formation of PMA-primed neutrophils, and NET formation by these products was completely blocked by native HDL. Furthermore, we found that an electronegative subfraction of LDL, LDL(–), which is separated from human plasma and is thought to be an in vivo oxLDL, was capable of promoting NET formation. These results suggest that plasma lipoproteins and their oxidative modifications play multiple roles in promoting NET formation, and that HDL acts as a suppressor of this response.

Lipid droplets (LDs) are intracellular organelles that are ubiquitous in many types of cells. The LD core consists of triacylglycerols (TGs) surrounded by a phospholipid monolayer and surface proteins such as perilipin 2 (PLIN2). Although TGs accumulate in the phospholipid bilayer of the endoplasmic reticulum (ER) and subsequently nascent LDs buds from ER, the mechanism by which LD proteins are transported to LD particles is not fully understood. Sar1 is a GTPase known as a regulator of coat protein complex Ⅱ (COPⅡ) vesicle budding, and its role in LD formation was investigated in this study. HuH7 human hepatoma cells were infected with adenoviral particles containing genes coding GFP fused with wild-type Sar1 (Sar1 WT) or a GTPase mutant form (Sar1 H79G). When HuH7 cells were treated with oleic acid, Sar1 WT formed a ring-like structure around the LDs. The transient expression of Sar1 did not significantly alter the levels of TG and PLIN2 in the cells. However, the localization of PLIN2 to the LDs decreased in the cells expressing Sar1 H79G. Furthermore, the effects of Sar1 on PLIN2 localization to the LDs were verified by the suppression of endogenous Sar1 using the short hairpin RNA technique. In conclusion, it was found that Sar1 has some roles in the intracellular distribution of PLIN2 to LDs in liver cells.

Intracellular lipid droplets (LDs) are ubiquitous organelles found in many cell types. During mitosis, membranous organelles, including mitochondria, are divided into small pieces and transferred to daughter cells; however, the process of LD transfer to daughter cells is not fully elucidated. Herein, we investigated the behavior of LDs during mitosis in HuH7 human hepatoma cells. While fragments of the Golgi apparatus were scattered in the cytosol during mitosis, intracellular LDs retained their size and spherical morphology as they translocated to the two daughter cells. LDs were initially distributed throughout the cell during prophase but positioned outside the spindle in metaphase, aligning at the far sides of the centrioles. A similar distribution of LDs during mitosis was observed in another hepatocarcinoma HepG2 cells. When the spindle was disrupted by nocodazole treatment or never in mitosis gene A-related kinase 2A knockdown, LDs were localized in the area outside the chromosomes, suggesting that spindle formation is not necessary for LD localization at metaphase. The amount of major LD protein perilipin 2 reduced while LDs were enriched in perilipin 3 during mitosis, indicating the potential alteration of LD protein composition. Conclusively, the behavior of LDs during mitosis is distinct from that of other organelles in hepatocytes.

Background: Neutrophils eject their DNA strings and cellular proteins into the extracellular space upon treatment with various stimulants. In the present study, we examined the effects of four major oxidized cholesterol metabolites on DNA release from granulocytes. Methods and Results: When oxysterols were added to HL-60-derived granulocytes stimulated with phorbol 12-myristate 13-acetate (PMA), they suppressed the release of DNA and myeloperoxidase from the cells. Among the four oxysterols tested, 7-ketocholesterol was the most effective. Addition of the same concentration of 7-ketocholesterol did not induce any cytotoxic effects, as evaluated based on the release of lactate dehydrogenase and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazoliumbromide (MTT) assays. DNA release from human peripheral blood neutrophils after PMA stimulation was also suppressed by 7-ketocholesterol. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis was used to quantify sterol content in the cells. The addition of oxysterols increased the cellular content of the corresponding compounds by more than 10-fold compared to those at baseline. Treatment of HL-60-derived granulocytes with methyl-β-cyclodextrin that removes sterol compounds from the membranes increased DNA release from the cells in a dose-dependent manner. Conclusions: These results suggest that oxysterols have suppressive effects on DNA release from granulocytes stimulated with PMA.