Phosphatidylethanolamines modified by γ-ketoaldehyde (γKA) induce endoplasmic reticulum stress and endothelial activation.

Peroxidation of plasma lipoproteins has been implicated in the endothelial cell activation and monocyte adhesion that initiate atherosclerosis, but the exact mechanisms underlying this activation remain unclear. Lipid peroxidation generates lipid aldehydes, including the γ-ketoaldehydes (γKA), also termed isoketals or isolevuglandins, that readily modify the amine headgroup of phosphatidylethanolamine (PE). We hypothesized that aldehyde modification of PE could mediate some of the proinflammatory effects of lipid peroxidation. We found that PE modified by γKA (γKA-PE) induced THP-1 monocyte adhesion to human umbilical cord endothelial cells. γKA-PE also induced expression of adhesion molecules and increased MCP-1 and IL-8 mRNA in human umbilical cord endothelial cells. To determine the structural requirements for γKA-PE activity, we tested several related compounds. PE modified by 4-oxo-pentanal induced THP-1 adhesion, but N-glutaroyl-PE and C(18:0)N-acyl-PE did not, suggesting that an N-pyrrole moiety was essential for cellular activity. As the N-pyrrole headgroup might distort the membrane, we tested the effect of the pyrrole-PEs on membrane parameters. γKA-PE and 4-oxo-pentanal significantly reduced the temperature for the liquid crystalline to hexagonal phase transition in artificial bilayers, suggesting that these pyrrole-PE markedly altered membrane curvature. Additionally, fluorescently labeled γKA-PE rapidly internalized to the endoplasmic reticulum (ER); γKA-PE induced C/EBP homologous protein CHOP and BiP expression and p38 MAPK activity, and inhibitors of ER stress reduced γKA-PE-induced C/EBP homologous protein CHOP and BiP expression as well as EC activation, consistent with γKA-PE inducing ER stress responses that have been previously linked to inflammatory chemokine expression. Thus, γKA-PE is a potential mediator of the inflammation induced by lipid peroxidation.

Nonenzymatic free radical-catalyzed generation of 15-deoxy-Δ(12,14)-prostaglandin J2-like compounds (deoxy-J2-isoprostanes) in vivo.

15-Deoxy-Δ(12,14)-prostaglandin J2 (15-d-PGJ2) is a reactive cyclopentenone eicosanoid generated from the dehydration of cyclooxygenase-derived prostaglandin D2 (PGD2). This compound possesses an α,ß-unsaturated carbonyl moiety that can readily adduct thiol-containing biomolecules such as glutathione and cysteine residues of proteins via the Michael addition. Due to its reactivity, 15-d-PGJ2 is thought to modulate inflammatory and apoptotic processes and is believed to be an endogenous ligand for peroxisome proliferator-activated receptor-γ. However, the extent to which 15-d-PGJ2 is formed in vivo and the mechanisms that regulate its formation are unknown. Previously, we have reported the formation of PGD2 and PGJ2-like compounds, termed D2/J2-isoprostanes (D2/J2-IsoPs), produced in vivo by the free radical-catalyzed peroxidation of arachidonic acid (AA). Based on these findings, we investigated whether 15-d-PGJ2-like compounds are also formed via this nonenzymatic pathway. Here we report the generation of novel 15-d-PGJ2-like compounds, termed deoxy-J2-isoprostanes (deoxy-J2-IsoPs), in vivo, via the nonenzymatic peroxidation of AA. Levels of deoxy-J2-IsoPs increased 12-fold (6.4 ± 1.1 ng/g liver) in rats after oxidant insult by CCl4 treatment, compared with basal levels (0.55 ± 0.21 ng/g liver). These compounds may have important bioactivities in vivo under conditions associated with oxidant stress.

Triglyceride alters lysosomal cholesterol ester metabolism in cholesteryl ester-laden macrophage foam cells.

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.

Identification of intact oxidation products of glycerophospholipids in vitro and in vivo using negative ion electrospray iontrap mass spectrometry.

Free radical-induced oxidation products of polyunsaturated fatty acids esterified to phospholipids have been implicated in a number of human diseases including atherosclerosis and neurodegenerative diseases. Some of these phospholipid oxidation products have potent biological activities and likely contribute to human pathophysiological conditions. Oxidation products have also been used as markers of oxidative stress in vivo. Identification and quantification of phospholipid oxidation products are often performed by analyzing the oxidized free fatty acid moieties after hydrolysis from the phospholipids head groups by gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-mass spectrometry (LC-MS). We now describe the definitive identification of intact oxidized products of glycerophospholipids including glycerophosphatidylcholine (GPC), glycerophosphatidylethanolamine (GPE), and glycerophosphatidylserine (GPS) in vitro and in vivo using iontrap MS. For these analyses, the negative ions of the oxidation products of phospholipids are fragmented to MS(n) and unequivocal structural characterization is obtained based on collision-induced dissociation (CID) of the sn-2 carboxylate ion. This technique overcomes the need to hydrolyze fatty acids from phospholipids in the analysis. The method has been used to identify a number of oxidation products of glycerophospholipids including hydroxyeicosatetraenoates (HETEs) and isoprostanes (IsoPs) esterified to different classes of glycerophospholipids in vitro and in vivo. These studies thus provide a new approach to identify the intact oxidation products of glycerolphospholipids.

Lysosomal cholesterol accumulation inhibits subsequent hydrolysis of lipoprotein cholesteryl ester.

Human macrophages incubated for prolonged periods with mildly oxidized LDL (oxLDL) or cholesteryl ester-rich lipid dispersions (DISP) accumulate free and esterified cholesterol within large, swollen lysosomes similar to those in foam cells of atherosclerosis. The cholesteryl ester (CE) accumulation is, in part, the result of inhibition of lysosomal hydrolysis due to increased lysosomal pH mediated by excessive lysosomal free cholesterol (FC). To determine if the inhibition of hydrolysis was long lived and further define the extent of the lysosomal defect, we incubated THP-1 macrophages with oxLDL or DISP to produce lysosome sterol engorgement and then chased with acetylated LDL (acLDL). Unlike oxLDL or DISP, CE from acLDL normally is hydrolyzed rapidly. Three days of incubation with oxLDL or DISP produced an excess of CE in lipid-engorged lysosomes, indicative of inhibition. After prolonged oxLDL or DISP pretreatment, subsequent hydrolysis of acLDL CE was inhibited. Coincident with the inhibition, the lipid-engorged lysosomes failed to maintain an acidic pH during both the initial pretreatment and subsequent acLDL incubation. This indicates that the alterations in lysosomes were general, long lived, and affected subsequent lipoprotein metabolism. This same phenomenon, occurring within atherosclerotic foam cells, could significantly affect lesion progression.

Effects of cellular cholesterol loading on macrophage foam cell lysosome acidification.

Macrophages incubated with mildly oxidized low density lipoprotein (OxLDL), aggregated low density lipoprotein (AggLDL), or cholesteryl ester-rich lipid dispersions (DISPs) accumulate cholesterol in lysosomes followed by an inhibition of lysosomal cholesteryl ester (CE) hydrolysis. The variety of cholesterol-containing particles producing inhibition of hydrolysis suggests that inhibition may relate to general changes in lysosomes. Lysosome pH is a key mediator of activity and thus is a potential mechanism for lipid-induced inhibition. We investigated the effects of cholesterol accumulation on THP-1 macrophage lysosome pH. Treatment with OxLDL, AggLDL, and DISPs resulted in inhibition of the lysosome's ability to maintain an active pH and concomitant decreases in CE hydrolysis. Consistent with an overall disruption of lysosome function, exposure to OxLDL or AggLDL reduced lysosomal apolipoprotein B degradation. The lysosomal cholesterol sequestration and inactivation are not observed in cholesterol-equivalent cells loaded using acetylated low density lipoprotein (AcLDL). However, AcLDL-derived cholesterol in the presence of progesterone (to block cholesterol egression from lysosomes) inhibited lysosome acidification. Lysosome inhibition was not attributable to a decrease in the overall levels of vacuolar ATPase. However, augmentation of membrane cholesterol in isolated lysosomes inhibited vacuolar ATPase-dependent pumping of H+ ions into lysosomes. These data indicate that lysosomal cholesterol accumulation alters lysosomes in ways that could exacerbate foam cell formation and influence atherosclerotic lesion development.

Severely altered cholesterol homeostasis in macrophages lacking apoE and SR-BI.

Mice deficient in scavenger receptor class B type I (SR-BI) and apolipoprotein E (apoE) [double knockout (DKO) mice] develop dyslipidemia, accelerated atherosclerosis, and myocardial infarction, and die prematurely. We examined effects of apoE and SR-BI deficiency on macrophage cholesterol homeostasis. DKO macrophages had increased total cholesterol (TC) stores (220-380 microg/mg protein) compared with apoE-/- cells (40 microg/mg), showed significant lysosomal lipid engorgement, and increased their TC by 34% after exposure to HDL. DKO macrophages from apoE-/- mice reconstituted with DKO bone marrow showed less cholesterol accumulation (89 microg/mg), suggesting that the dyslipidemia of DKO mice explains part of the cellular cholesterol defect. However, analyses of DKO and apoE-/- macrophages from transplanted apoE-/- mice revealed a role for macrophage SR-BI, inasmuch as the TC in DKO macrophages increased by 10% in the presence of HDL, whereas apoE-/- macrophage TC decreased by 33%. After incubation with HDL, the free cholesterol (FC) increased by 29% in DKO macrophages, and decreased by 8% in apoE-/- cells, and only DKO cells had FC in large peri-nuclear pools. Similar trends were observed with apoA-I as an acceptor. Thus, the abnormal cholesterol homeostasis of DKO macrophages is due to the plasma lipid environment of DKO mice and to altered trafficking of macrophage cholesterol. Both factors are likely to contribute to the accelerated atherosclerosis in DKO mice.

Aggregated LDL and lipid dispersions induce lysosomal cholesteryl ester accumulation in 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. Co-treatment 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.