Oswald Quehenberger

Lipidomics reveals a remarkable diversity of lipids in human plasma

The focus of the present study was to define the human plasma lipidome and to establish novel analytical methodologies to quantify the large spectrum of plasma lipids. Partial lipid analysis is now a regular part of every patients blood test and physicians readily and regularly prescribe drugs that alter the levels of major plasma lipids such as cholesterol and triglycerides. Plasma contains many thousands of distinct lipid molecular species that fall into six main categories including fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterols, and prenols. The physiological contributions of these diverse lipids and how their levels change in response to therapy remain largely unknown. As a first step toward answering these questions, we provide herein an in-depth lipidomics analysis of a pooled human plasma obtained from healthy individuals after overnight fasting and with a gender balance and an ethnic distribution that is representative of the US population. In total, we quantitatively assessed the levels of over 500 distinct molecular species distributed among the main lipid categories. As more information is obtained regarding the roles of individual lipids in health and disease, it seems likely that future blood tests will include an ever increasing number of these lipid molecules.

Characterization of CLA-1, a Human Homologue of Rodent Scavenger Receptor BI, as a Receptor for High Density Lipoprotein and Apoptotic Thymocytes

Recently, a murine scavenger receptor type B class I (SR-BI) was identified that binds high density lipoprotein (HDL) and mediates the selective uptake of cholesterol esters. The human CD36 and LIMPII analogous-1 (CLA-1) receptor shows high sequence homology with SR-BI, but their functional relationship has not been determined. Transfected cells expressing CLA-1 bound HDL with a K d of about 35 μg/ml, similar to the K d for HDL binding to rodent SR-BI. This binding resulted in an intracellular accumulation of HDL-derived [3H]cholesterol esters without internalization or degradation of 125I-apolipoprotein. CLA-1 was strongly expressed in the adrenal gland and was also abundant in liver and testis, suggesting that CLA-1, like SR-BI, could play a role in the metabolism of HDL. However, CLA-1 was also expressed in monocytes and, like SR-BI, recognized modified forms of low density lipoproteins as well as native LDL and anionic phospholipids. These findings suggest that CLA-1 might have additional physiological functions. We found that CLA-1 recognizes apoptotic thymocytes. Our results demonstrate that CLA-1, a close structural homologue of SR-BI, is also functionally related to SR-BI and may play an important role as a “docking receptor” for HDL in connection with selective uptake of cholesterol esters. An additional role in recognition of damaged cells is suggested by these studies.

Regulated Accumulation of Desmosterol Integrates Macrophage Lipid Metabolism and Inflammatory Responses

Inflammation and macrophage foam cells are characteristic features of atherosclerotic lesions, but the mechanisms linking cholesterol accumulation to inflammation and LXR-dependent response pathways are poorly understood. To investigate this relationship, we utilized lipidomic and transcriptomic methods to evaluate the effect of diet and LDL receptor genotype on macrophage foam cell formation within the peritoneal cavities of mice. Foam cell formation was associated with significant changes in hundreds of lipid species and unexpected suppression, rather than activation, of inflammatory gene expression. We provide evidence that regulated accumulation of desmosterol underlies many of the homeostatic responses, including activation of LXR target genes, inhibition of SREBP target genes, selective reprogramming of fatty acid metabolism, and suppression of inflammatory-response genes, observed in macrophage foam cells. These observations suggest that macrophage activation in atherosclerotic lesions results from extrinsic, proinflammatory signals generated within the artery wall that suppress homeostatic and anti-inflammatory functions of desmosterol.

Scavenger receptors, oxidized LDL, and atherosclerosis.

Abstract: Oxidized LDL (OxLDL) competes with oxidatively damaged and apoptotic cells for binding to mouse peritoneal macrophages, implying the presence of one or more common domains. However, the nature of the ligands involved has not been determined. Studies in this laboratory over the last several years provide evidence that oxidized phospholipids, present in OxLDL and also in the membrane of apoptotic cells, represent one such ligand. These oxidized phospholipids, either in the lipid phase of OxLDL or becoming attached covalently to apoprotein B during LDL oxidation, have been shown to play a major role in the binding of OxLDL to CD36 and to SR‐B1 expressed in transfected cells. The lipid and protein moieties compete with each other to some extent, indicating that they are binding to at least one common site. A monoclonal antibody selected because of its reactivity with OxLDL proved to be an antibody against oxidized phospholipids (but not native phospholipids). This antibody (EO6) blocked the uptake of OxLDL by CD36 and by SR‐B1 in transfected cells by as much as 80%; it also inhibited macrophage phagocytosis of apoptotic cells by about 40%. Thus, the persistence of receptors for OxLDL during evolution is probably accounted for by their role in recognition of ligands on the surfaces of oxidatively damaged or apoptotic cells. This has important implications in biology generally and specifically in atherogenesis, because apoptosis is a prominent feature of late lesions.

Mechanisms of hypochlorite injury of target cells.

HOCl, which is produced by the action of myeloperoxidase during the respiratory burst of stimulated neutrophils, was used as a cytotoxic reagent in P388D1 cells. Low concentrations of HOCl (10-20 microM) caused oxidation of plasma membrane sulfhydryls determined as decreased binding of iodoacetylated phycoerythrin. These same low concentrations of HOCl caused disturbance of various plasma membrane functions: they inactivated glucose and aminoisobutyric acid uptake, caused loss of cellular K+, and an increase in cell volume. It is likely that these changes were the consequence of plasma membrane SH-oxidation, since similar effects were observed with para-chloromercuriphenylsulfonate (pCMBS), a sulfhydryl reagent acting at the cell surface. Given in combination pCMBS and HOCl showed an additive effect. Higher doses of HOCl (greater than 50 microM) led to general oxidation of -SH, methionine and tryptophan residues, and formation of protein carbonyls. HOCl-induced loss of ATP and undegraded NAD was closely followed by cell lysis. In contrast, NAD degradation and ATP depletion caused by H2O2 preceded cell death by several hours. Formation of DNA strand breaks, a major factor of H2O2-induced injury, was not observed with HOCl. Thus targets of HOCl were distinct from those of H2O2 with the exception of glyceraldehyde-3-phosphate dehydrogenase, which was inactivated by both oxidants.

Mildly oxidized low density lipoprotein activates multiple apoptotic signaling pathways in human coronary cells.

Apoptosis of arterial cells induced by oxidized low density lipoproteins (OxLDL) is thought to contribute to the progression of atherosclerosis. However, most data on apoptotic effects and mechanisms of OxLDL were obtained with extensively oxidized LDL unlikely to occur in early stages of atherosclerotic lesions. We now demonstrate that mildly oxidized LDL generated by incubation with oxygen radical‐producing xanthine/xanthine oxidase (X/XO) induces apoptosis in primary cultures of human coronary endothelial and SMC, as determined by TUNEL technique, DNA laddering, and FACS analysis. Apoptosis was markedly reduced when X/XO‐LDL was generated in the presence of different oxygen radical scavengers. Apoptotic signals were mediated by intramembrane domains of both Fas and tumor necrosis factor (TNF) receptors I and II. Blocking of Fas ligand (FasL) reduced apoptosis by 50% and simultaneous blocking of FasL and TNF receptors by 70%. Activation of apoptotic receptors was accompanied by an increase of proapoptotic and a decrease in antiapoptotic proteins of the Bcl‐2 family and resulted in marked activation of class I and II caspases. Mildly oxidized LDL also activated MAP and Jun kinases and increased p53 and other transcription factors (ATF‐2, ELK‐1, CREB, AP‐1). Inhibitors of Map and Jun kinase significantly reduced apoptosis. Our results provide the first evidence that OxLDL‐induced apoptosis involves TNF receptors and Jun activation. More important, they demonstrate that even mildly oxidized LDL formed in atherosclerotic lesions may activate a broad cascade of oxygen radical‐sensitive signaling pathways affecting apoptosis and other processes influencing the evolution of plaques. Thus, we suggest that extensive oxidative modifications of LDL are not necessary to influence signal transduction and transcription in vivo.—Napoli, C., Quehenberger, O., de Nigris, F., Abete, P., Glass, C. K., Palinski, W. Mildly oxidized low density lipoprotein activates multiple apoptotic signaling pathways in human coronary cells. FASEB J. 14, 1996–2007 (2000)

The Human Plasma Lipidome

Biochemical analyses reveal that plasma contains over 600 structural forms of lipid molecules. This review summarizes known correlations between levels of lipid moieties and disease and describes potential therapeutic targets relevant to CVD, cancer, and neurologic disease.

Oxidized LDL reduces monocyte CCR2 expression through pathways involving peroxisome proliferator–activated receptor γ

The CCR2-mediated recruitment of monocytes into the vessel wall plays an important role in all stages of atherosclerosis. In recent studies, we have shown that lipoproteins can modulate CCR2 expression and have identified native LDL as a positive regulator. In contrast, oxidized LDL (OxLDL), which is mainly formed in the aortic intima, reduces CCR2 expression, promotes monocyte retention, and may cause pathological accumulation of monocytes in the vessel wall. We now provide evidence that OxLDL reduces monocyte CCR2 expression by activating intracellular signaling pathways that may involve peroxisome proliferator-activated receptor gamma (PPARgamma). Receptor-mediated uptake of the lipoprotein particle was required and allows for delivery of the exogenous ligand to the nuclear receptor. The suppression of CCR2 expression by OxLDL was mediated by lipid components of OxLDL, such as the oxidized linoleic acid metabolites 9-HODE and 13-HODE, known activators of PPARgamma. Modified apoB had no such effect. Consistent with a participation of the PPARgamma signaling pathway, BRL49653 reduced CCR2 expression in freshly isolated human monocytes ex vivo and in circulating mouse monocytes in vivo. These results implicate PPARgamma in the inhibition of CCR2 gene expression by oxidized lipids, which may help retain monocytes at sites of inflammation, such as the atherosclerotic lesion.

A Mouse Macrophage Lipidome

We report the lipidomic response of the murine macrophage RAW cell line to Kdo2-lipid A, the active component of an inflammatory lipopolysaccharide functioning as a selective TLR4 agonist and compactin, a statin inhibitor of cholesterol biosynthesis. Analyses of lipid molecular species by dynamic quantitative mass spectrometry and concomitant transcriptomic measurements define the lipidome and demonstrate immediate responses in fatty acid metabolism represented by increases in eicosanoid synthesis and delayed responses characterized by sphingolipid and sterol biosynthesis. Lipid remodeling of glycerolipids, glycerophospholipids, and prenols also take place, indicating that activation of the innate immune system by inflammatory mediators leads to alterations in a majority of mammalian lipid categories, including unanticipated effects of a statin drug. Our studies provide a systems-level view of lipid metabolism and reveal significant connections between lipid and cell signaling and biochemical pathways that contribute to innate immune responses and to pharmacological perturbations.

Chemokine Receptor CCR2 Expression and Monocyte Chemoattractant Protein-1–Mediated Chemotaxis in Human Monocytes A Regulatory Role for Plasma LDL

The subendothelial accumulation of macrophage-derived foam cells is one of the hallmarks of atherosclerosis. The recruitment of monocytes to the intima requires the interaction of locally produced chemokines with specific cell surface receptors, including the receptor (CCR2) for monocyte chemoattractant protein-1 (MCP-1). We have previously reported that monocyte CCR2 gene expression and function are effectively downregulated by proinflammatory cytokines. In this study we identified low density lipoprotein (LDL) as a positive regulator of CCR2 expression. Monocyte CCR2 expression was dramatically increased in hypercholesterolemic patients compared with normocholesterolemic controls. Similarly, incubation of human THP-1 monocytes with LDL induced a rapid increase in CCR2 mRNA and protein. By 24 hours the number of cell surface receptors was doubled, causing a 3-fold increase in the chemotactic response to MCP-1. The increase in CCR2 expression and chemotaxis was promoted by native LDL but not by oxidized LDL. Oxidized LDL rapidly downregulated CCR2 expression, whereas reductively methylated LDL, which does not bind to the LDL receptor, had only modest effects on CCR2 expression. A neutralizing anti-LDL receptor antibody prevented the effect of LDL, suggesting that binding and internalization of LDL were essential for CCR2 upregulation. The induction of CCR2 expression appeared to be mediated by LDL-derived cholesterol, because cells treated with free cholesterol also showed increased CCR2 expression. These data suggest that elevated plasma LDL levels in conditions such as hypercholesterolemia enhance monocyte CCR2 expression and chemotactic response and potentially contribute to increased monocyte recruitment to the vessel wall in chronic inflammation and atherogenesis.