Presence Of High-density Lipoprotein Research Articles

Abstract 1040: Investigating The Role Of Lipoproteins In Macrophage Er Stress

Atherosclerosis, a major cause of cardiovascular disease (CVD), is an inflammatory disease defined by the progressive buildup of lipid-laden macrophages and cholesterol-filled plaques. Low-density lipoprotein (LDL) and high-density lipoprotein (HDL) play critical roles in the pathogenesis of atherosclerosis due to their roles in transporting cholesterol between tissues and cell types, including macrophages. Epidemiological studies have shown that high levels of HDL-cholesterol (HDL-C) are correlated with decreased CVD. However, HDL-C raising therapeutics failed to show clinical benefits, suggesting that HDL function may be a better indicator of CVD risk. Recent studies from our lab demonstrated that HDL modified by reactive aldehydes has impaired cardioprotective functions in macrophages. Other studies have also shown that oxidized LDL triggers ER stress in vascular cells and macrophages, a process that can be blunted by the presence of HDL. Therefore, we tested the hypothesis that HDL modified by reactive aldehydes triggers ER stress in macrophages, with the goal of identifying mechanisms by which dysfunctional HDL contributes to atherosclerosis progression. Primary murine macrophages were incubated with native or modified forms of lipoproteins and ER stress markers (XBP1, BiP, p-eIF2a, ATF6) were assessed by immunoblot analysis or semi-quantitative PCR. Through a comprehensive and extensive analysis, our findings led us to discover three novel findings in primary murine macrophages: (i) oxLDL alone was unable to induce ER stress; (ii) co-incubation with oxLDL or HDL in the presence of thapsigargin (an inducer of ER stress) resulted in increased and prolonged expression of ER stress markers as compared to cells treated with thapsigargin alone; and (iii) neither HDL nor HDL modified by reactive aldehydes was able blunt the ER stress induced by thapsigargin in the presence or absence of oxLDL. In summary, our highly systematic approach to assess the role of lipoproteins in mediating ER stress in primary macrophages generated convincing data that refutes previously-published findings, thus resulting in an inability to make definitive conclusions on the role of HDL modification in ER stress.

Decreased Efficiency of Very-Low-Density Lipoprotein Lipolysis Is Linked to Both Hypertriglyceridemia and Hypercholesterolemia, but It Can Be Counteracted by High-Density Lipoprotein.

Impaired triglyceride-rich lipoprotein plasma catabolism is considered the most important factor for hypertriglyceridemia development. The aim of this study was to evaluate the impact of hypercholesterolemia and hypertriglyceridemia on the efficiency of lipoprotein lipase (LPL)-mediated very-low-density lipoprotein (VLDL)-triglyceride lipolysis and the role of high-density lipoprotein (HDL) in this process. Subjects with no history of cardiovascular disease (CVD) and untreated with lipid-lowering agents were recruited into the study and divided into normolipidemic, hypercholesterolemic, and hyperlipidemic groups. VLDL was isolated from serum and incubated with LPL in the absence or presence of HDL. For the hypercholesterolemic and hyperlipidemic groups, a significantly lower percentage of hydrolyzed VLDL-triglyceride was achieved compared to the normolipidemic group (p < 0.01). HDL enhanced the lipolysis efficiency in the hypercholesterolemic and hyperlipidemic groups on average by ~7% (p < 0.001). The lowest electrophoretic mobility of the VLDL remnants indicating the most effective lipolysis was obtained in the normolipidemic group (p < 0.05). HDL presence significantly reduced the electrophoretic mobility of the VLDL remnants for the hypercholesterolemic and hyperlipidemic groups (p < 0.05). The results of our study indicate that VLDL obtained from hypercholesterolemic and hyperlipidemic subjects are more resistant to lipolysis and are additional evidence of the need for early implementation of hypocholesterolemic treatment, already in asymptomatic CVD subjects.

Effect of High-Density Lipoprotein from Healthy Subjects and Chronic Kidney Disease Patients on the CD14 Expression on Polymorphonuclear Leukocytes.

In uremic patients, high-density lipoprotein (HDL) loses its anti-inflammatory features and can even become pro-inflammatory due to an altered protein composition. In chronic kidney disease (CKD), impaired functions of polymorphonuclear leukocytes (PMNLs) contribute to inflammation and an increased risk of cardiovascular disease. This study investigated the effect of HDL from CKD and hemodialysis (HD) patients on the CD14 expression on PMNLs. HDL was isolated using a one-step density gradient centrifugation. Isolation of PMNLs was carried out by discontinuous Ficoll-Hypaque density gradient centrifugation. CD14 surface expression was quantified by flow cytometry. The activity of the small GTPase Rac1 was determined by means of an activation pull-down assay. HDL increased the CD14 surface expression on PMNLs. This effect was more pronounced for HDL isolated from uremic patients. The acute phase protein serum amyloid A (SAA) caused higher CD14 expression, while SAA as part of an HDL particle did not. Lipid raft disruption with methyl-β-cyclodextrin led to a reduced CD14 expression in the absence and presence of HDL. HDL from healthy subjects but not from HD patients decreased the activity of Rac1. Considering the known anti-inflammatory effects of HDL, the finding that even HDL from healthy subjects increased the CD14 expression was unexpected. The pathophysiological relevance of this result needs further investigation.

Low-Density Lipoproteins, High-Density Lipoproteins (HDL), and HDL-Associated Proteins Differentially Modulate Chronic Myelogenous Leukemia Cell Viability.

Cellular lipid metabolism, lipoprotein interactions, and liver X receptor (LXR) activation have been implicated in the pathophysiology and treatment of cancer, although findings vary across cancer models and by lipoprotein profiles. In this study, we investigated the effects of human-derived low-density lipoproteins (LDL), high-density lipoproteins (HDL), and HDL-associated proteins apolipoprotein A1 (apoA1) and serum amyloid A (SAA) on markers of viability, cholesterol flux, and differentiation in K562 cells-a bone marrow-derived, stem-like erythroleukemia cell model of chronic myelogenous leukemia (CML). We further evaluated whether lipoprotein-mediated effects were altered by concomitant LXR activation. We observed that LDL promoted higher K562 cell viability in a dose- and time-dependent manner and increased cellular cholesterol concentrations, while LXR activation by the agonist TO901317 ablated these effects. LXR activation in the presence of HDL, apoA1 and SAA-rich HDL suppressed K562 cell viability, while robustly inducing mRNA expression of ATP-binding cassette transporter A1 (ABCA1). HDL and its associated proteins additionally suppressed mRNA expression of anti-apoptotic B-cell lymphoma-extra large (BCL-xL), and the erythroid lineage marker 5'-aminolevulinate synthase 2 (ALAS2), while SAA-rich HDL induced mRNA expression of the megakaryocytic lineage marker integrin subunit alpha 2b (ITGA2B). Together, these findings suggest that lipoproteins and LXR may impact the viability and characteristics of CML cells.

The structure of apoA‐II on HDL reveals novel insights into its regulation of lipoprotein composition and function

High‐density lipoproteins (HDL) are compositionally and functionally diverse. HDL can host at least 95 different proteins and studies suggest many of these interactions are governed by specific conformations of HDL's primary scaffold protein, apolipoprotein (apo)A‐I. Previously, we showed that the presence of HDL's second most abundant scaffold protein, apoA‐II, impacts particle composition and function; presumably through interactions with apoA‐I. The goal of this study was to determine the structural conformation of apoA‐II on HDL to understand how its presence impacts HDL metabolism.We used chemical cross‐linking and mass spectrometry to derive spatial information on homogenous populations of recombinant HDL (rHDL) generated in vitro. The rHDL was created using a mixture of wild‐type and isotopically‐labeled apoA‐I to unambiguously identify the molecular span of the cross‐linked peptides; i.e. within a molecule of apoA‐I (intramolecular) or between two molecules of apoA‐I (intermolecular). Two rHDL populations were studied: (i) rHDL that contained only apoA‐I (LpA‐I) and (ii) rHDL containing both apoA‐I and apoA‐II (LpA‐I/A‐II).Cross‐linking and SDS‐PAGE showed both populations contained two molecules of apoA‐I while the LpA‐I/A‐II rHDL contained a single dimer of apoA‐II. Analysis by negative stain electron microscopy revealed both populations were discoidal in shape with no differences in size. The intermolecular cross‐linking pattern between apoA‐I molecules were consistent with the “double‐belt” model of apoA‐I on HDL and, surprisingly, the presence of apoA‐II didn't appear to perturb the pattern. However, we observed strong cross‐links between apoA‐II and the C‐terminus of apoA‐I.In a parallel approach, native LpA‐I and LpA‐I/A‐II HDL was isolated from human plasma using immunoaffinity and size exclusion chromatography. Shotgun proteomics and cross‐linking revealed accessory proteins were mostly associated with LpA‐I HDL and interactions were primarily localized to the N‐ and C‐termini of apoA‐I. As with the rHDL, we found apoA‐II specifically interacts with the C‐terminus of apoA‐I in the LpA‐I/A‐II particles. Taken together, this suggests that apoA‐II's compositional and functional effects on HDL are exerted via its interaction with the C‐terminus of apoA‐I possibly limiting its availability to interact with the accessory proteins. We have gone on to generate preliminary all‐atoms models of apoA‐II on HDL to illustrate this structural mechanism.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 602: High-Density Lipoprotein Protects Macrophages Against Apoptosis Through BH3-only Bcl-2 Family Member Bim

Macrophage apoptosis contributes to the formation of necrotic cores within atherosclerotic lesions, increasing the susceptibility of atherosclerotic plaques to rupture and the risk of cardiovascular events. In advanced atherosclerotic plaques, lipid-laden foam cells are exposed to a diverse number of apoptosis inducers, including prolonged endoplasmic reticulum (ER) stress. The pro-apoptotic BH3-only protein Bim has been reported to mediate apoptosis in response to ER stress induction in a variety of cells including macrophages. Treatment of macrophages with high-density lipoprotein (HDL) protects them against apoptosis induced by a variety of stressors. We hypothesize that HDL mediated protection against macrophage apoptosis occurs via pathways that regulate Bim activity. To test this, thioglycollate-elicted peritoneal macrophages were treated in lipoprotein deficient culture with different apoptosis inducers in the absence or presence of HDL. Cell apoptosis was assessed by cleaved caspase 3 staining and terminal deoxynucleotidal transferase dUTP nick end labeling. Treatment of mouse peritoneal macrophages in culture with 50μg/ml of human HDL protected against apoptosis induced by tunicamycin (P&lt;0.05) without affecting ER stress markers, Grp78, Grp94 and CHOP. Peritoneal macrophages from mice deficient in Bim were less susceptible to apoptosis induced by treatment with either tunicamycin or 7-ketocholesterol. Furthermore, treatment with HDL provided no additional protection to apoptosis in Bim KO macrophages. Our results suggest that protection of macrophage apoptosis by HDL is regulated through pathways involving the activity of BH3-only Bcl-2 family member Bim.

High-density lipoprotein inhibits human M1 macrophage polarization through redistribution of caveolin-1.

Monocyte-derived macrophages are critical in the development of atherosclerosis and can adopt a wide range of functional phenotypes depending on their surrounding milieu. High-density lipoproteins (HDLs) have many cardio-protective properties including potent anti-inflammatory effects. We investigated the effects of HDL on human macrophage phenotype and the mechanisms by which these occur. Human blood monocytes were differentiated into macrophages in the presence or absence of HDL and were then induced to either an inflammatory macrophage (M1) or anti-inflammatory macrophage (M2) phenotype using LPS and IFN-γ or IL-4, respectively. HDL inhibited the induction of macrophages to an M1-phenotype, as evidenced by a decrease in the expression of M1-specific cell surface markers CD192 and CD64, as well as M1-associated inflammatory genes TNF-α, IL-6 and MCP-1 (CCL2). HDL also inhibited M1 function by reducing the production of ROS. In contrast, HDL had no effect on macrophage induction to the M2-phenotype. Similarly, methyl-β-cyclodextrin, a non-specific cholesterol acceptor also suppressed the induction of M1 suggesting that cholesterol efflux is important in this process. Furthermore, HDL decreased membrane caveolin-1 in M1 macrophages. We confirmed that caveolin-1 is required for HDL to inhibit M1 induction as bone marrow-derived macrophages from caveolin-1 knockout mice continued to polarize into M1-phenotype despite the presence of HDL. Moreover, HDL decreased ERK1/2 and STAT3 phosphorylation in M1 macrophages. We concluded that HDL reduces the induction of macrophages to the inflammatory M1-phenotype via redistribution of caveolin-1, preventing the activation of ERK1/2 and STAT3.

Lipoprotein-induced phenoloxidase-activity in tarantula hemocyanin

Phenoloxidases play vital roles in invertebrate innate immune reactions, wound closure and sclerotization processes in arthropods. In chelicerates, where phenoloxidases are lacking, phenoloxidase-activity can be induced in the oxygen carrier hemocyanin in vitro by proteolytic cleavage, incubation with the artificial inducer SDS, or lipids. The role of protein–protein interaction has up to now received little attention. This is remarkable, as lipoproteins — complexes of proteins and lipids — are present at high concentrations in arthropod hemolymph. We characterized the three lipoproteins present in tarantula hemolymph, two high-density lipoproteins and one very high-density lipoprotein, and show that the two high-density lipoproteins have distinct structures: the more abundant high-density lipoprotein is an ellipsoid particle with axes of ~22.5nm and ~16.8nm, respectively. The second high-density lipoprotein, present only in trace amount, is a large discoidal lipoprotein with a diameter of ~38.4nm and an on-edge thickness of ~7.1nm. We further demonstrate that the interaction between lipoproteins and hemocyanin induces phenoloxidase activity in hemocyanin, and propose that this activation is due to protein–protein interaction rather than protein–lipid interaction, as neither lipid micelles nor lipid monomers were found to be activating. Activation was strongest in the presence of high-density lipoproteins; very high-density lipoproteins were found to be non-activating. This is the first time that the ability of lipoproteins to induce phenoloxidase activity of hemocyanin has been demonstrated, thus adding novel aspects to the function of lipoproteins apart from their known role in nutrient supply.

High-density lipoprotein and atherosclerosis: Roles of lipid transporters.

Various previous studies have found a negative correlation between the risk of cardiovascular events and serum high-density lipoprotein (HDL) cholesterol levels. The reverse cholesterol transport, a pathway of cholesterol from peripheral tissue to liver which has several potent antiatherogenic properties. For instance, the particles of HDL mediate to transport cholesterol from cells in arterial tissues, particularly from atherosclerotic plaques, to the liver. Both ATP-binding cassette transporters (ABC) A1 and ABCG1 are membrane cholesterol transporters and have been implicated in mediating cholesterol effluxes from cells in the presence of HDL and apolipoprotein A-I, a major protein constituent of HDL. Previous studies demonstrated that ABCA1 and ABCG1 or the interaction between ABCA1 and ABCG1 exerted antiatherosclerotic effects. As a therapeutic approach for increasing HDL cholesterol levels, much focus has been placed on increasing HDL cholesterol levels as well as enhancing HDL biochemical functions. HDL therapies that use injections of reconstituted HDL, apoA-I mimetics, or full-length apoA-I have shown dramatic effectiveness. In particular, a novel apoA-I mimetic peptide, Fukuoka University ApoA-I Mimetic Peptide, effectively removes cholesterol via specific ABCA1 and other transporters, such as ABCG1, and has an antiatherosclerotic effect by enhancing the biological functions of HDL without changing circulating HDL cholesterol levels. Thus, HDL-targeting therapy has significant atheroprotective potential, as it uses lipid transporter-targeting agents, and may prove to be a therapeutic tool for atherosclerotic cardiovascular diseases.

35: Sterile inflammation: Surface LIPIDS on STIMULATED T cells that induce cytokine production in human monocytes/macrophages

Imbalance in cytokine homeostasis plays an important part in the pathogenesis of chronic/sterile inflammatory diseases such as multiple sclerosis and rheumatoid arthritis. Stimulated T cells exert a pathological effect through direct cellular contact with monocytes/macrophages, inducing a massive up-regulation of IL-1β and TNF in the latter cells. This mechanism that is likely to be relevant to chronic/sterile inflammation is specifically inhibited by high-density lipoproteins (HDL). Here we determine that cytokine induction is due to lipids present in membranes of stimulated T cells. Total lipids from plasma membrane were extracted from HUT-78 cells and peripheral blood T lymphocytes stimulated and unstimulated by PHA/PMA. Lipids extracted from stimulated HUT-78 and stimulated blood T lymphocytes induced IL-1β, sIL-1Ra and TNF production in monocytes, whereas lipids from unstimulated cells did not. Pro-inflammatory cytokine production was inhibited in the presence of HDL. These results indicated that lipids were the active factors that induced cytokine production upon direct cellular contact activation of monocytes. The activity was abolished when lipids were treated with methylamine suggesting that sphingolipids were not involved in monocyte activation. Preliminary identification of surface lipids implicated in the induction of cytokine production in monocytes was performed. Surface lipids of stimulated and unstimulated HUT-78 cells were separated in a Synergy 4 μm MAX-RP 18A column and mass spectrometry analysis of active fractions was carried out. Results suggest the involvement of T cell lysophospholipids and phosphatidic acids in the induction of cytokine production in human monocytes. Our results demonstrate that stimulated T cells display bioactive lipids at their surface which induce cytokine production in human monocytes. The identification of these lipids might open the way to new therapeutic approaches in chronic/sterile inflammatory disorders such as rheumatoid arthritis and multiple sclerosis.

24(S)‐hydroxycholesterol is actively eliminated from neuronal cells by ABCA1

High cholesterol turnover catalyzed by cholesterol 24-hydroxylase is essential for neural functions, especially learning. Because 24(S)-hydroxycholesterol (24-OHC), produced by 24-hydroxylase, induces apoptosis of neuronal cells, it is vital to eliminate it rapidly from cells. Here, using differentiated SH-SY5Y neuron-like cells as a model, we examined whether 24-OHC is actively eliminated via transporters induced by its accumulation. The expression of ABCA1 and ABCG1 was induced by 24-OHC, as well as TO901317 and retinoic acid, which are ligands of the nuclear receptors liver X receptor/retinoid X receptor (LXR/RXR). When the expression of ABCA1 and ABCG1 was induced, 24-OHC efflux was stimulated in the presence of high-density lipoprotein (HDL), whereas apolipoprotein A-I was not an efficient acceptor. The efflux was suppressed by the addition of siRNA against ABCA1, but not by ABCG1 siRNA. To confirm the role of each transporter, we analyzed human embryonic kidney 293 cells stably expressing human ABCA1 or ABCG1; we clearly observed 24-OHC efflux in the presence of HDL, whereas efflux in the presence of apolipoprotein A-I was marginal. Furthermore, the treatment of primary cerebral neurons with LXR/RXR ligands suppressed the toxicity of 24-OHC. These results suggest that ABCA1 actively eliminates 24-OHC in the presence of HDL as a lipid acceptor and protects neuronal cells.

Abstract 133: Hemodynamic Environment-dependent Effects of Human HDL and oxHDL on Vessel Wall Inflammation

High density lipoprotein (HDL) concentration is inversely correlated with cardiovascular disease and exerts atheroprotective effects on endothelial (EC) and smooth muscle cells (SMC). Oxidation of LDL (oxLDL) is a well-appreciated driver of atherosclerosis. What is less well known is that HDL can become oxidized (oxHDL) by myeloperoxidase, which may negate its beneficial effects. Further, previous mechanistic studies of HDL were performed on static culture ECs or SMCs that do not take into consideration in vivo hemodynamics; a critical regulator of vascular phenotype. Regional hemodynamics prime EC/SMCs towards either an atheroprotective phenotype or, in regions such as bifurcations, towards an atheroprone, pro-inflammatory phenotype. We assessed the role of the hemodynamic environment in modulating the differential response of vascular cells to human HDL or oxHDL from normal donors. To test this, HDL was added to EC/SMC cocultures primed by atheroprotective or atheroprone shear stresses derived from the human carotid bifurcation or an “Advanced Inflammatory” condition that combines atheroprone shear stress with additional atherogenic factors such as oxLDL and TNFα. In the presence of HDL, ECs under Advanced Inflammatory conditions reduced pro-inflammatory (NFkB Activity, IL6, COX2), and oxidative stress (SOD2), but did not have an effect in baseline atheroprotective and atheroprone conditions. Many of the anti-inflammatory responses of endothelial cells were retained when HDL was oxidized. Conversely, while HDL had minimal effect on SMC gene expression, oxHDL was found to reduced the atheroprotective phenotype by potently inducing oxidative and inflammatory cytokine genes/secreted proteins (IL1B, IL6). Overall, this study illustrates the importance of reproducing relevant in vivo environments in order to understand HDL-mediated mechanisms in vascular biology in vitro. This study supports that oxidation dysfunctionally transforms HDL from an atheroprotective to an inflammatory and intrinsically oxidative particle and might damage otherwise healthy regions of the vasculature. Thus, measurement of circulating HDL without consideration of HDL composition and oxidation may limit its predictive value for cardiovascular risk.

Endothelial Lipase Modulates Pressure Overload–Induced Heart Failure Through Alternative Pathway for Fatty Acid Uptake

Lipoprotein lipase has been considered as the only enzyme capable of generating lipid-derived fatty acids for cardiac energy. Endothelial lipase is another member of the triglyceride lipase family and hydrolyzes high-density lipoproteins. Although endothelial lipase is expressed in the heart, its function remains unclear. We assessed the role of endothelial lipase in the genesis of heart failure. Pressure overload-induced cardiac hypertrophy was generated in endothelial lipase(-/-) and wild-type mice by ascending aortic banding. Endothelial lipase expression in cardiac tissues was markedly elevated in the early phase of cardiac hypertrophy in wild-type mice, whereas lipoprotein lipase expression was significantly reduced. Endothelial lipase(-/-) mice showed more severe systolic dysfunction with left-ventricular dilatation compared with wild-type mice in response to pressure overload. The expression of mitochondrial fatty acid oxidation-related genes, such as carnitine palmitoyltransferase-1 and medium-chain acyl coenzyme A dehydrogenase, was significantly lower in the heart of endothelial lipase(-/-) mice than in wild-type mice. Also, endothelial lipase(-/-) mice had lower myocardial adenosine triphosphate levels than wild-type mice after aortic banding. In cultured cardiomyocytes, endothelial lipase was upregulated by inflammatory stimuli, whereas lipoprotein lipase was downregulated. Endothelial lipase-overexpression in cardiomyocytes resulted in an upregulation of fatty acid oxidation-related enzymes and intracellular adenosine triphosphate accumulation in the presence of high-density lipoprotein. Endothelial lipase may act as an alternative candidate to provide fatty acids to the heart and regulate cardiac function. This effect seemed relevant particularly in the diseased heart, where lipoprotein lipase action is downregulated.

Influence of LRP-1 and Apolipoprotein E on Amyloid-β Uptake and Toxicity to Cerebrovascular Smooth Muscle Cells

Vascular deposition of amyloid-β (Aβ) leads to the death of cerebrovascular smooth muscle cells (CVSMCs) in cerebral amyloid angiopathy (CAA). Apolipoprotein E (APOE) genotype influences the severity of CAA and development of vasculopathic complications. We have studied the relationship between uptake of Aβ into human CVSMCs and cell death, and the influence of ApoE isoforms on this process. We found that both Aβ42 and Aβ40 were taken up by human CVSMCs, and that this uptake-particularly that of Aβ42-caused disruption to smooth muscle actin and cell death. Uptake of Aβ42 was partially blocked by the addition of receptor-associated protein (RAP), implicating low-density lipoprotein receptor-related protein-1 (LRP-1) as the cell surface receptor. RAP significantly reduced the death of CVSMCs exposed to Aβ42. In further experiments, CVSMCs were exposed to Aβ42 in the presence of the different isoforms of exogenous ApoE and high-density lipoprotein (HDL). All three isoforms of ApoE in the presence of HDL (HDL-ApoE) reduced the uptake of fluorescein-tagged Aβ42 but only HDL-ApoE3 significantly decreased cell death. We conclude that HDL-ApoE3, acting as an Aβ chaperone molecule, significantly reduces the death of CVSMCs that result from LRP-1-mediated uptake of Aβ. This may contribute to the differential effects of APOE genotype on severity of CAA and the risk of rupture of Aβ-laden blood vessels.

P084 Bioactive lipids at the surface of stimulated T cells induce cytokine production in human monocytes

Introduction Imbalance in cytokine homeostasis plays an important part in the pathogenesis of chronic inflammatory diseases such as multiple sclerosis and rheumatoid arthritis. Stimulated T cells exert a pathological effect through direct cellular contact with monocytes/macrophages, inducing a massive up-regulation of IL-1 β and TNF in the latter cells. This mechanism that is likely to be relevant to chronic/sterile inflammation is specifically inhibited by high-density lipoproteins (HDL). Here we determine that cytokine induction is due to lipids present in membranes of stimulated T cells. Methods Chloroform:methanol or methyl-ter-butylether extraction of lipids from membranes isolated from PMA/PHA-stimulated and unstimulated HUT-78 cells and peripheral blood T lymphocytes. Methylamine treatment. Mass spectrometry analysis of total lipids. Induction of cytokine production/expression in isolated human blood monocytes. Results Total lipids isolated from membranes of stimulated HUT-78 cells and T lymphocytes induced IL-1 β , sIL-1Ra and TNF production in monocytes, whereas aqueous fraction was inactive. Pro-inflammatory cytokine production was inhibited in the presence of HDL. These results indicated that lipids were the active factors that induced cytokine production upon direct cellular contact activation of monocytes by stimulated T cells. The activity was abolished when lipids were treated with methylamine suggesting that sphingolipids were not involved in monocyte activation. Comparison of mass spectrometry profiles of total lipids from membranes of stimulated and unstimulated HUT-78 cells suggested the involvement of lysophospholipids in cytokine induction. Conclusion Our results demonstrate that stimulated T cells display bioactive lipids at their surface which induce cytokine production in human monocytes. The identification of these lipids might open the way to new therapeutic approaches in chronic/sterile inflammatory disorders such as rheumatoid arthritis and multiple sclerosis.

Identification of Fibrin Clot-Bound Plasma Proteins

Several proteins are known to bind to a fibrin network and to change clot properties or function. In this study we aimed to get an overview of fibrin clot-bound plasma proteins. A plasma clot was formed by adding thrombin, CaCl2 and aprotinin to citrated platelet-poor plasma and unbound proteins were washed away with Tris-buffered saline. Non-covalently bound proteins were extracted, separated with 2D gel electrophoresis and visualized with Sypro Ruby. Excised protein spots were analyzed with mass spectrometry. The identity of the proteins was verified by checking the mass of the protein, and, if necessary, by Western blot analysis. Next to established fibrin-binding proteins we identified several novel fibrin clot-bound plasma proteins, including α2-macroglobulin, carboxypeptidase N, α1-antitrypsin, haptoglobin, serum amyloid P, and the apolipoproteins A-I, E, J, and A-IV. The latter six proteins are associated with high-density lipoprotein particles. In addition we showed that high-density lipoprotein associated proteins were also present in fibrinogen preparations purified from plasma. Most plasma proteins in a fibrin clot can be classified into three groups according to either blood coagulation, protease inhibition or high-density lipoprotein metabolism. The presence of high-density lipoprotein in clots might point to a role in hemostasis.

Pre‐synaptic defects of NPC1‐deficient hippocampal neurons are not directly related to plasma membrane cholesterol

Imbalances in brain cholesterol homeostasis have been observed in several neurodegenerative diseases. In Niemann-Pick Type C (NPC) disease, mutations in NPC1 or NPC2 lead to endosomal cholesterol accumulation, neuronal dysfunction and death. Cholesterol in synaptic plasma membranes influences membrane fluidity, curvature, and protein function, and its depletion may adversely affect synaptic vesicle cycling. We have investigated pre-synaptic function in primary hippocampal neurons with altered cholesterol distribution because of NPC1 deficiency or cyclodextrin treatment. In NPC1-deficient neurons grown in serum-free medium, plasma membrane cholesterol was reduced and total synaptic vesicle release during prolonged stimulation was attenuated. In NPC1-deficient neurons cultured in the presence of high-density lipoproteins, plasma membrane cholesterol markedly increased, but the defects in synaptic vesicle release in NPC1-deficient neurons were exacerbated. Treatment with 1 mM methyl-beta-cyclodextrin acutely depleted plasma membrane cholesterol in wild-type neurons to levels below those in NPC1 deficiency, but did not alter synaptic vesicle exo- or endocytosis. Defects only became apparent when higher methyl-beta-cyclodextrin concentrations were used. Our data indicate that synaptic vesicle release can tolerate some degree of plasma membrane cholesterol depletion and suggest that the pre-synaptic defects in NPC1-deficient neurons are not solely caused by a reduction of plasma membrane cholesterol.

Expression of reverse cholesterol transport proteins ATP-binding cassette A1 (ABCA1) and scavenger receptor BI (SR-BI) in the retina and retinal pigment epithelium

Aims:Excessive lipid accumulation in Bruch’s membrane (BrM) is a hallmark of ageing, the major risk factor for age-related macular degeneration (AMD). Retinal pigment epithelial (RPE) cells may utilise reverse cholesterol...

Genotypic Effect of ABCG1 Gene Promoter -257T&gt;G Polymorphism on Coronary Artery Disease Severity in Japanese Men

ATP-binding cassette transporter G1 (ABCG1) is a cholesterol transporter that plays a role in cholesterol efflux in the presence of high-density lipoprotein (HDL). Moreover, HDL-mediated cholesterol efflux is increased in cultured ABCG1 overexpressed cells; however, the physiological roles of ABCG1 and its contribution to atherosclerosis in humans remain unclear. The effect of ABCG1 -257T>G mutation on transcription activity was determined by a reporter assay. One hundred nine Japanese men with coronary artery disease (CAD) were analyzed. ABCG1 -257T>G polymorphism was assessed by mutation-selective PCR methods to identify T/T, T/G, and G/G genotypes. The reporter assay showed that ABCG1 transcription activity was significantly lower (p<0.01) in the G allele of -257T>G polymorphism compared with that in the T allele. Clinically, there were no significant differences in serum triglyceride and total, LDL, and HDL cholesterol levels, or other risk factors. Logistic regression analysis revealed significant additive and dominant effects on the frequency of patients with multi-vessel disease compared with single-vessel disease (T/T vs. T/G vs. G/G, odds ratio:2.1, p=0.027; T/T vs. T/G and. G/G, odds ratio:3.5, p=0.005). This is the first report to show that a novel ABCG1 -257T>G promoter polymorphism influences CAD severity in Japanese men.

Stimulated T cells generate microparticles, which mimic cellular contact activation of human monocytes: differential regulation of pro- and anti-inflammatory cytokine production by high-density lipoproteins

Imbalance in cytokine homeostasis plays an important part in the pathogenesis of chronic inflammatory diseases such as multiple sclerosis and rheumatoid arthritis. We demonstrated that T cells might exert a pathological effect through direct cellular contact with human monocytes/macrophages, inducing a massive up-regulation of the prototypical proinflammatory cytokines IL-1beta and TNF. This mechanism that might be implicated in chronic inflammation is specifically inhibited by high-density lipoproteins (HDL). Like many other stimuli, besides proinflammatory cytokines, the contact-mediated activation of monocytes induces the production of cytokine inhibitors such as the secreted form of the IL-1 receptor antagonist (sIL-1Ra). The present study demonstrates that stimulated T cells generate microparticles (MP) that induce the production of TNF, IL-1beta, and sIL-1Ra in human monocytes; the production of TNF and IL-1beta but not that of sIL-1Ra is inhibited in the presence of HDL. The results were similar when monocytes were stimulated by whole membranes of T cells or soluble extracts of the latter. This suggests that MP carry similar monocyte-activating factors to cells from which they originate. Thus, by releasing MP, T cells might convey surface molecules similar to those involved in the activation of monocytes by cellular contact. By extension, MP might affect the activity of cells, which are usually not in direct contact with T cells at the inflammatory site. Furthermore, this study demonstrates that HDL exert an anti-inflammatory effect in nonseptic activation of human monocytes, not only by inhibiting the production of IL-1beta and TNF but also, by leaving sIL-1Ra production unchanged.