Bis(monoacylglycero)phosphate accumulation in macrophages induces intracellular cholesterol redistribution, attenuates liver-X receptor/ATP-Binding cassette transporter A1/ATP-binding cassette transporter G1 pathway, and impairs cholesterol efflux.

OBJECTIVE: Endosomal signature phospholipid bis(monoacylglycero)phosphate (BMP) has been involved in the regulation of cellular cholesterol homeostasis. Accumulation of BMP is a hallmark of lipid storage disorders and was recently reported as a noticeable feature of oxidized low-density lipoprotein-laden macrophages. This study was designed to delineate the consequences of macrophage BMP accumulation on intracellular cholesterol distribution, metabolism, and efflux and to unravel the underlying molecular mechanisms. APPROACH AND RESULTS: We have developed an experimental design to specifically increase BMP content in RAW 264.7 macrophages. After BMP accumulation, cell cholesterol distribution was markedly altered, despite no change in low-density lipoprotein uptake and hydrolysis, cholesterol esterification, or total cell cholesterol content. The expression of cholesterol-regulated genes sterol regulatory element-binding protein 2 and hydroxymethylglutaryl-coenzyme A reductase was decreased by 40%, indicative of an increase of endoplasmic reticulum-associated cholesterol. Cholesterol delivery to plasma membrane was reduced as evidenced by the 20% decrease of efflux by cyclodextrin. Functionally, BMP accumulation reduced cholesterol efflux to both apolipoprotein A1 and high-density lipoprotein by 40% and correlated with a 40% decrease in mRNA contents of ATP-binding cassette transporter A1, ATP-binding cassette transporter G1, and liver-X receptor α and ß. Foam cell formation induced by oxidized low-density lipoprotein exposure was exacerbated in BMP-enriched cells. CONCLUSIONS: The present work shows for the first time a strong functional link between BMP and cholesterol-regulating genes involved in both intracellular metabolism and efflux. We propose that accumulation of cellular BMP might contribute to the deregulation of cholesterol homeostasis in atheromatous macrophages.

Bis(monoacylglycero)phosphate regulates oxysterol binding protein-related protein 11 dependent sterol trafficking.

Bis(Monoacylglycero) Phosphate (BMP) is a unique phospholipid localized in late endosomes, a critical cellular compartment in low density lipoprotein (LDL)-cholesterol metabolism. In previous work, we demonstrated the important role of BMP in the regulation of macrophage cholesterol homeostasis. BMP exerts a protective role against the pro-apoptotic effect of oxidized LDL (oxLDL) by reducing the production of deleterious oxysterols. As the intracellular sterol traffic in macrophages is in part regulated by oxysterol binding protein (OSBP) and OSBP-related proteins (ORPs), we investigated the role of ORP11, localized at the Golgi-late endosomes interface, in the BMP-mediated protection from oxLDL/oxysterol cytotoxicity. Stably silencing of ORP11 in mouse RAW264.7 macrophages via a shRNA lentiviruses system had no effect on BMP production. However, ORP11 knockdown abrogated the protective action of BMP against oxLDL induced apoptosis. In oxLDL treated control cells, BMP enrichment was associated with reduced generation of 7-oxysterols, while these oxysterol species were abundant in the ORP11 knock-down cells. Of note, BMP enrichment in ORP11 knock-down cells was associated with a drastic increase in free cholesterol and linked to a decrease of cholesterol efflux. The expression of ATP-binding cassette-transporter G1 (ABCG1) was also reduced in the ORP11 knock-down cells. These observations demonstrate a cooperative function of OPR11 and BMP, in intracellular cholesterol trafficking in cultured macrophages. We suggest that BMP favors the egress of cholesterol from late endosomes via an ORP11-dependent mechanism, resulting in a reduced production of cytotoxic 7-oxysterols.

In vitro oxidized HDL and HDL from type 2 diabetes patients have reduced ability to efflux oxysterols from THP-1 macrophages.

Oxidized LDL (OxLDL) that are enriched in products of lipid peroxidation including oxysterols have been shown to induce cellular oxidative stress and cytotoxicity therefore accelerating atheroma plaque formation. Upon oxLDL exposure of THP-1 macrophages, intracellular oxidation of LDL derived-cholesterol as well as endogenous cholesterol was increased. The oxysterols intracellularly produced were efficiently exported to HDL whereas apolipoprotein A1 was inefficient. These findings prompted us to investigate the consequences of modification of HDL by oxidation and glycation as observed in type 2 diabetes with respect to oxysterol and cholesterol efflux. We show that efflux of oxysterols was significantly impaired after in vitro oxidation and glycoxidation of HDL whereas glycation alone had no impact. Cholesterol efflux was only slightly decreased by oxHDL or glycoxidized HDL and not changed with glycated HDL. The defect of HDL towards oxysterol efflux was also observed with HDL isolated from diabetic subjects as compared to healthy controls. These findings support a deleterious cellular retention of oxysterols due to dysfunctional HDL in type 2 diabetes.

THP1 macrophages oxidized cholesterol, generating 7-derivative oxysterols specifically released by HDL.

Macrophages are well recognized as key pathophysiologic agents in many chronic inflammatory diseases, especially atherosclerosis. During atherogenesis process, low density lipoproteins (LDL) undergo oxidation (oxLDL) and become highly atherogenic as they induce a strong accumulation of cholesterol in subendothelial macrophages leading to the formation of foam cells, the major cellular component of fatty streaks. OxLDL are enriched in oxidation products of cholesterol called oxysterols involved in the regulation of cholesterol homeostasis, by their ability to induce cellular oxidative stress and cytotoxicity. Little is known about intracellular oxysterol production in macrophages. Using both radiochemical and mass analyzes, we showed that THP1 macrophages promote the intracellular oxidation of LDL derived-cholesterol as well as intracellular cholesterol, this later mechanism being enhanced by exposure with native or oxLDL. We demonstrated that in both THP1 and Raw 267.4 cells cholesterol oxidation occurs in the late endosomal compartment. Most oxysterols were produced by non-enzymatic routes (7-ketocholesterol and 7α/ß-hydroxycholesterol) but enzymatically formed 7α-, 27-hydroxycholesterol were also quantified. Incubation of THP1 macrophages with nLDL or oxLDL, induced a 2- and 100-fold increase in oxysterol production, respectively. Both oxysterols derived from LDL cholesterol and cellular cholesterol were readily exported to HDL whereas apoA1 was inefficient, showing that HDL plays a major role in the removal of excess oxysterols in THP1 macrophages.

Bis(monoacylglycero)phosphate reduces oxysterol formation and apoptosis in macrophages exposed to oxidized LDL.

Atherosclerosis is a major cardiovascular complication of diseases associated with increased oxidative stress that favors oxidation of circulating low density lipoproteins (LDLs). Oxidized LDL (oxLDL) is considered as highly atherogenic as it induces a strong accumulation of cholesterol in subendothelial macrophages leading to the formation of foam cells and emergence of atherosclerotic plaque. OxLDL is enriched in oxidation products of cholesterol called oxysterols, some of which have been involved in the ability of oxLDL to induce cellular oxidative stress and cytotoxicity, mainly by apoptosis. Little is known about the possible contribution of cell-generated oxysterols toward LDL-associated oxysterols in cellular accumulation of oxysterols and related apoptosis. Using both radiochemical and mass analyzes, we showed that oxLDL greatly enhanced oxysterol production by RAW macrophages in comparison with unloaded cells or cells loaded with native LDL. Most oxysterols were produced by non-enzymatic routes (7-ketocholesterol and 7α/ß-hydroyxycholesterol) but enzymatically formed 7α-, 25- and 27-hydroxycholesterol were also quantified. Bis(monoacylglycero)phosphate (BMP) is a unique phospholipid preferentially found in late endosomes. We and others have highlighted the role of BMP in the regulation of intracellular cholesterol metabolism/traffic in macrophages. We here report that cellular BMP accumulation was associated with a significantly lower production of oxysterols upon oxLDL exposure. Of note, potent pro-apoptotic 7-ketocholesterol was the most markedly decreased. OxLDL-induced cell cytotoxicity and apoptosis were consistently attenuated in BMP-enriched cells. Taken together, our data suggest that BMP exerts a protective action against the pro-apoptotic effect of oxLDL via a reduced production of intracellular pro-apoptotic oxysterols.