Ceramides reduce CD36 cell surface expression and oxidised LDL uptake by monocytes and macrophages

Oxidised LDL accumulates in macrophages following scavenger receptor (SR) uptake. The expression of the SR, CD36, is increased by oxidised LDL. The signalling molecule, ceramide, can modulate intracellular peroxides and increase lipid peroxidation. Ceramide also accumulates in atherosclerotic plaques. Thus, we have examined whether ceramide can modulate CD36 expression and function in human monocyte/macrophages. Addition of synthetic short chain ceramides or the action of sphingomyelinase to generate physiological long chain ceramides in situ caused significant reductions in CD36 expression by monocytes/macrophages which was not due to inhibition of mRNA expression. Inhibition of proteasomal degradation using lactacystin had no effect on CD36 expression, however, flow cytometric analysis of permeabilised cells suggested an intracellular trafficking blockade. Ceramide treated monocytes/macrophages showed dose dependent reduction in oxidised LDL uptake. Taken together, it is suggested that ceramide blocks the transport of CD36 to the membrane of monocytes/macrophages, thereby preventing uptake of oxidised LDL. © 2006 Elsevier Inc. All rights reserved.

Synthetic ceramides inhibit CD36 expression in U937 cells through a reduction in cytosolic peroxide

Ceramide (a sphingolipid) and reactive oxygen species (ROS) are each partly responsible for the intracellular signal transduction of a variety of physiological, pharmacological or environmental agents. It has been reported that synthesis of ceramide and ROS are intimately linked, and show reciprocal regulation. The levels of ceramide are reported to be elevated in atherosclerotic plaques providing circumstantial evidence for a pro-atherogenic role for ceramide. Indeed, LDL may be important sources of ceramide from sphingomyelin, where it promotes LDL aggregation. Using synthetic, short chain ceramides to mimic the cellular responses to fluctuations in natural endogenous ceramides, we have investigated ceramide effects on both intracellular redox state (as glutathione and ROS) and redox-sensitive gene expression, specifically the scavenger receptor CD36 (using RT-PCR and flow cytometry), in U937 monocytes and macrophages. We describe that the principal redox altering properties of ceramide are to lower cytosolic peroxide and to increase mitochondrial ROS formation, where growth arrest of U937 monocytes is also observed. In addition, cellular glutathione was depleted, which was independent of an increase in glutathione peroxidase activity. Examination of the effects of ceramide on stress induced CD36 expression in macrophages, revealed a dose dependent reduction in CD36 mRNA and protein levels, which was mimicked by N-acetyl cysteine. Taken together, these data suggest that ceramides differentially affect ROS within different cellular compartments, and that loss of cytosolic peroxide inhibits expression of the redox sensitive gene, CD36. This may attenuate both the uptake of oxidised LDL and the interaction of HDL with macrophages. The resulting sequelae in vivo remain to be determined.

Synthetic ceramides induce growth arrest or apoptosis by altering cellular redox status

Reactive oxygen species (ROS) and ceramide are each partly responsible for the signal transduction of a variety of extracellular agents. Furthermore, the application of synthetic, short-chain ceramides mimics the cellular responses to these extracellular agents. However, the significance of ROS involvement in ceramide signaling pathways is poorly understood. Here we describe that the (cellular responses to C2-/C6-ceramide of growth arrest in U937 monocytes and apoptosis in Jurkat T-cells are preceded by a rise in mitochondrial peroxide production. In Jurkat T-cells, this is associated with a large time- and dose-dependent loss of cellular glutathione. However, in U937 monocytes, glutathione loss is transient. Differences in the magnitude and kinetics of this alteration in cellular redox state associate with discrete outcomes, namely growth arrest or apoptosis. © 2002 Elsevier Science (USA). All rights reserved.

Ceramide and reactive oxygen species (ROS) as signal transduction molecules in inflammation

Reactive oxygen species (ROS) and the sphingolipid ceramide are each partly responsible for the intracellular signal transduction of a variety of physiological, pharmacological or environmental agents. Furthermore, the enhanced production of many of these agents, that utilise ROS and ceramide as signalling intermediates, is associated with the aetiologies of several vascular diseases (e.g. atherosclerosis) or disorders of inflammatory origin (e.g. rheumatoid arthritis; RA). Excessive monocyte recruitment and uncontrolled T cell activation are both strongly implicated in the chronic inflammatory responses that are associated with these pathologies. Therefore the aims of this thesis are (1) to further elucidate the cellular responses to modulations in intracellular ceramide/ROS levels in monocytes and T cells, in order to help resolve the mechanisms of progression of these diseases and (2) to examine both existing agents (methotrexate) and novel targets for possible therapeutic manipulation. Utilising synthetic, short chain ceramide to mimic the cellular responses to fluctuations in natural endogenous ceramide or, stimulation of CD95 to induce ceramide formation, it is described here that ceramide targets and manipulates two discrete sites responsible for ROS generation, preceding the cellular responses of growth arrest in U937 monocytes and apoptosis in Jurkat T-cells. In both cell types, transient elevations in mitochondrial ROS generation were observed. However, the prominent redox altering effects appear to be the ceramide-mediated reduction in cytosolic peroxide, the magnitude of which dictates in part the cellular response in U937 monocytes, Jurkat T-cells and primary human peripheral blood resting or PHA-activated T-cells in vitro. The application of synthetic ceramides to U937 monocytes for short (2 hours) or long (16 hours) treatment periods reduced the membrane expression of proteins associated with cell-cell interaction. Furthermore, ceramide treated U937 monocytes demonstrated reduced adhesion to 5 or 24 hour LPS activated human umbilical vein endothelial cells (HUVEC) but not resting HUVEC. Consequently it is hypothesised that the targeted treatment of monocytes from patients with cardiovascular diseases with short chain synthetic ceramide may reduce disease progression. Herein, the anti-inflammatory and immunosuppressant drug, methotrexate, is described to require ROS production for the induction of cytostasis or cytotoxicity in U937 monocytes and Jurkat T-cells respectively. Further, ROS are critical for methotrexate to abrogate monocyte interaction with activated HUVEC in vitro. The histological feature of RA of enhanced infiltration, survivability and hyporesponsiveness of T-cells within the diseased synovium has been suggested to arise from aberrant signalling. No difference in the concentrations of endogenous T-cell ceramide, the related lipid diacylglycerol (DAG) and cytosolic peroxide ex vivo was observed. TCR activation following PHA exposure in vitro for 72 hours did not induced maintained perturbations in DAG or ceramide in T-cells from RA patients or healthy individuals. However, T-cells from RA patients failed to upregulate cytosolic peroxide in response to PHA, unlike those from normals, despite expressing identical levels of the activation marker CD25. This inability to upregulate cytosolic peroxide may contribute to the T-cell pathology associated with RA by affecting the signalling capacity of redox sensitive biomolecules. These data highlight the importance of two distinctive cellular pools of ROS in mediating complex biological events associated with inflammatory disease and suggest that modulation of cellular ceramides represents a novel therapeutic strategy to minimise monocyte recruitment.

The functional effects of ceramide on the monocyte CD36 scavenger receptor and NADPH oxidase

Atherosclerosis is the principal cause of death in the United States, Europe and much of Asia. During the last decade, inflammation has been suggested to play a key role in the development of atherosclerosis. Reactive oxygen species (ROS) released during inflammation additionally oxidize LDL, which is subsequently taken up in an unregulated way through scavenger receptors on macrophages to form foam cells, the hallmark of atherosclerotic lesions. Previous work has shown that the lipid ceramide, which is found in aggregated LDL and in atherosclerotic plaques, decreases intracellular peroxide most likely through reducing NADPH oxidase activity. Ceramide is an important component of membrane microdomains called lipid rafts which are important for membrane protein function. Endogenous ceramide enhances lipid raft f'ormation and alters theirs composition. NADPH oxidase membrane subunits cytochrome b558 (which includes gp91) strongly associates with lipid rafts Therefore present study investigated whether short chain ceramides reduce NADPH oxidase in U937 monocytes by disrurting the membrane component of NADPH oxidase. Results showed that C2 ceramide alters the distribution of raft marker, flottillin and the raft environment. NADPH oxidase membrane component gp9J phox and cytosolic component p47 phox were identified in rafts. C2 ceramide reduces both gp91 and p47 phox in rafts, which leads to the decrease of peroxide production by NADPH oxidase. Ceramide is also an important second messenger involved in many different signaling pathways associated with atherogenesis from the activation of sphingomyelinase (SMase). It has been reported that SMase enhances LDL receptor mediated LDL endocytosis. However, no study has been done to investigate the effect of ceramide on scavenger receptors such as CD36 and oxidized LDL (OxLDL) uptake. CD36 is the major recertor far OxLDL. Reduced CD36 expression results in less foam cell formation and less atherosclerotic lesion without disrupting the clearance of OxLDL from plasma. This thesis shows that ceramides significantly reduce CD36 surface expression on U937 monocytes, macrophages and human primary monocytes. This effect is seen using both synthetic short chain ceramide and SMase catalysed long chain ceramide treatment. To investigate whether the effect of ceramide on CD36 is functional, OxLOL uptake was measured in ceramide treated cells. Ceramide reduces the uptake of OxLOL by both U937 monocytes and PMA-differentiated macrophages. The mechanism of ceramide reduction of CD36 expression was studied by measuring the surface antigen using flow cytometry and fluorescence microscopy, whole cellular CD36 expression and shedding of C036 by Western blotting of cell lysates and cell culture supernatants and mRNA level of CD36 using RT-PCR. Ceramide reduces shedding of CD36, activates mRNA expression of CD36 and induces intracellular CD36 accumulation probably through retaining the receptor inside cells. In summary, ceramides modulate several of the processes involved in LOL oxidation and uptake by CD36 receptors on monocytes/macrophages in a way which may protect against atherosclerosis.

A comparison of five lipid extraction solvent systems for lipidomic studies of human LDL

Lipidome profile of fluids and tissues is a growing field as the role of lipids as signaling molecules is increasingly understood, relying on an effective and representative extraction of the lipids present. A number of solvent systems suitable for lipid extraction are commonly in use, though no comprehensive investigation of their effectiveness across multiple lipid classes has been carried out. To address this, human LDL from normolipidemic volunteers was used to evaluate five different solvent extraction protocols [Folch, Bligh and Dyer, acidified Bligh and Dyer, methanol (MeOH)-tert-butyl methyl ether (TBME), and hexane-isopropanol] and the extracted lipids were analyzed by LC-MS in a high-resolution instrument equipped with polarity switching. Overall, more than 350 different lipid species from 19 lipid subclasses were identified. Solvent composition had a small effect on the extraction of predominant lipid classes (triacylglycerides, cholesterol esters, and phosphatidylcholines). In contrast, extraction of less abundant lipids (phosphatidylinositols, lyso-lipids, ceramides, and cholesterol sulfates) was greatly influenced by the solvent system used. Overall, the Folch method was most effective for the extraction of a broad range of lipid classes in LDL, although the hexane-isopropanol method was best for apolar lipids and the MeOH-TBME method was suitable for lactosyl ceramides. Copyright © 2013 by the American Society for Biochemistry and Molecular Biology, Inc.

Fatty acids, monocytes and ageing

Elevated free fatty acids (FFA) are a feature of ageing and a risk factor for metabolic disorders such as cardiovascular disease (CVD) and type-2 diabetes (T2D). Elevated FFA contribute to insulin resistance, production of inflammatory cytokines and expression of adhesion molecules on immune cells and endothelial cells, risk factors for CVD and T2D. Molecular mechanisms of FFA effects on monocyte function and how FFA phenotype is affected by healthy ageing remain poorly understood. This thesis evaluated the effects of the two major FFA in plasma, oleate and palmitate on monocyte viability, cell surface antigen expression, and inflammatory activation in THP-1 monocytes. Palmitate but not oleate increased cell surface expression of CD11b and CD36 after 24h, independent of mitochondrial superoxide, but dependent on de novo synthesis of ceramides. LPS-mediated cytokine production in THP-1 monocytes was enhanced and decreased following incubation with palmitate and oleate respectively. In a model of monocyte-macrophage differentiation, palmitate induced a pro-inflammatory macrophage phenotype which required de novo ceramide synthesis, whilst oleate reduced cytokine secretion, producing a macrophage with enhanced clearance apoptotic cells. Plasma fatty acid analysis in young and mid-life populations revealed age-related increases in both the SFA and MUFA classes, especially the medium and very long chain C14 and C24 fatty acids, which were accompanied by increases in the estimated activities of desaturase enzymes. Changes were independently correlated with increased PBMC CD11b, plasma TNF-a and insulin resistance. In conclusion, the pro-atherogenic phenotype, enhanced LPS responses in monocytes, and pro-inflammatory macrophage in the presence of palmitate but not oleate is reliant upon de novo ceramide synthesis. Age-related increases in inflammation, cell surface integrin expression are related to increases in both the MUFA and SFA fatty acids, which in part may be explained by altered de novo fatty acid synthesis.

Top-down lipidomics of low density lipoprotein reveal altered lipid profiles in advanced chronic kidney disease

This study compared the molecular lipidomic profi le of LDL in patients with nondiabetic advanced renal disease and no evidence of CVD to that of age-matched controls, with the hypothesis that it would reveal proatherogenic lipid alterations. LDL was isolated from 10 normocholesterolemic patients with stage 4/5 renal disease and 10 controls, and lipids were analyzed by accurate mass LC/MS. Top-down lipidomics analysis and manual examination of the data identifi ed 352 lipid species, and automated comparative analysis demonstrated alterations in lipid profi le in disease. The total lipid and cholesterol content was unchanged, but levels of triacylglycerides and N -acyltaurines were signifi cantly increased, while phosphatidylcholines, plasmenyl ethanolamines, sulfatides, ceramides, and cholesterol sulfate were signifi cantly decreased in chronic kidney disease (CKD) patients. Chemometric analysis of individual lipid species showed very good discrimination of control and disease sample despite the small cohorts and identifi ed individual unsaturated phospholipids and triglycerides mainly responsible for the discrimination. These fi ndings illustrate the point that although the clinical biochemistry parameters may not appear abnormal, there may be important underlying lipidomic changes that contribute to disease pathology. The lipidomic profi le of CKD LDL offers potential for new biomarkers and novel insights into lipid metabolism and cardiovascular risk in this disease. -Reis, A., A. Rudnitskaya, P. Chariyavilaskul, N. Dhaun, V. Melville, J. Goddard, D. J. Webb, A. R. Pitt, and C. M. Spickett. Topdown lipidomics of low density lipoprotein reveal altered lipid profi les in advanced chronic kidney disease. J. Lipid Res. 2015.