Uptake of oxLDL and IL-10 production by macrophages requires PAFR and CD36 recruitment into the same lipid rafts

Macrophage interaction with oxidized low-density lipoprotein (oxLDL) leads to its differentiation into foam cells and cytokine production, contributing to atherosclerosis development. In a previous study, we showed that CD36 and the receptor for platelet-activating factor (PAFR) are required for oxLDL to activate gene transcription for cytokines and CD36. Here, we investigated the localization and physical interaction of CD36 and PAFR in macrophages stimulated with oxLDL. We found that blocking CD36 or PAFR decreases oxLDL uptake and IL-10 production. OxLDL induces IL-10 mRNA expression only in HEK293T expressing both receptors (PAFR and CD36). OxLDL does not induce IL-12 production. The lipid rafts disruption by treatment with βCD reduces the oxLDL uptake and IL-10 production. OxLDL induces co-immunoprecipitation of PAFR and CD36 with the constitutive raft protein flotillin-1, and colocalization with the lipid raft-marker GM1-ganglioside. Finally, we found colocalization of PAFR and CD36 in macrophages from human atherosclerotic plaques. Our results show that oxLDL induces the recruitment of PAFR and CD36 into the same lipid rafts, which is important for oxLDL uptake and IL-10 production. This study provided new insights into how oxLDL interact with macrophages and contributing to atherosclerosis development

CXCL16 and oxLDL are induced in the onset of diabetic nephropathy

Diabetic nephropathy (DN) is a major cause of end-stage renal failure worldwide. Oxidative stress has been reported to be a major culprit of the disease and increased oxidized low density lipoprotein (oxLDL) immune complexes were found in patients with DN. In this study we present evidence, that CXCL16 is the main receptor in human podocytes mediating the uptake of oxLDL. In contrast, in primary tubular cells CD36 was mainly involved in the uptake of oxLDL. We further demonstrate that oxLDL down-regulated α3-integrin expression and increased the production of fibronectin in human podocytes. In addition, oxLDL uptake induced the production of reactive oxygen species (ROS) in human podocytes. Inhibition of oxLDL uptake by CXCL16 blocking antibodies abrogated the fibronectin and ROS production and restored α3 integrin expression in human podocytes. Furthermore we present evidence that hyperglycaemic conditions increased CXCL16 and reduced ADAM10 expression in podocytes. Importantly, in streptozotocin-induced diabetic mice an early induction of CXCL16 was accompanied by higher levels of oxLDL. Finally immunofluorescence analysis in biopsies of patients with DN revealed increased glomerular CXCL16 expression, which was paralleled by high levels of oxLDL. In summary, regulation of CXCL16, ADAM10 and oxLDL expression may be an early event in the onset of DN and therefore all three proteins may represent potential new targets for diagnosis and therapeutic intervention in DN

Oxidized LDL induces alternative macrophage phenotype through activation of CD36 and PAFR

OxLDL is recognized by macrophage scavenger receptors, including CD36; we have recently found that Platelet-Activating Factor Receptor (PAFR) is also involved. Since PAFR in macrophages is associated with suppressor function, we examined the effect of oxLDL on macrophage phenotype. It was found that the presence of oxLDL during macrophage differentiation induced high mRNA levels to IL-10, mannose receptor, PPARγ and arginase-1 and low levels of IL-12 and iNOS. When human THP-1 macrophages were pre-treated with oxLDL then stimulated with LPS, the production of IL-10 and TGF-β significantly increased, whereas that of IL-6 and IL-8 decreased. In murine TG-elicited macrophages, this protocol significantly reduced NO, iNOS and COX2 expression. Thus, oxLDL induced macrophage differentiation and activation towards the alternatively activated M2-phenotype. In murine macrophages, oxLDL induced TGF-β, arginase-1 and IL-10 mRNA expression, which were significantly reduced by pre-treatment with PAFR antagonists (WEB and CV) or with antibodies to CD36. The mRNA expression of IL-12, RANTES and CXCL2 were not affected. We showed that this profile of macrophage activation is dependent on the engagement of both CD36 and PAFR. We conclude that oxLDL induces alternative macrophage activation by mechanisms involving CD36 and PAFR

Oxidized low-density lipoprotein inhibits hepatitis C virus cell entry in human hepatoma cells.

Cell entry of hepatitis C virus, pseudoparticles (HCVpp) and cell culture grown virus (HCVcc), requires the interaction of viral glycoproteins with CD81 and other as yet unknown cellular factors. One of these is likely to be the scavenger receptor class B type I (SR-BI). To further understand the role of SR-BI, we examined the effect of SR-BI ligands on HCVpp and HCVcc infectivity. Oxidized low-density lipoprotein (oxLDL), but not native LDL, potently inhibited HCVpp and HCVcc cell entry. Pseudoparticles bearing unrelated viral glycoproteins or bovine viral diarrhea virus were not affected. A dose-dependent inhibition was observed for HCVpp bearing diverse viral glycoproteins with an approximate IC50 of 1.5 microg/mL apolipoprotein content, which is within the range of oxLDL reported to be present in human plasma. The ability of lipoprotein components to bind to target cells associated with their antiviral activity, suggesting a mechanism of action which targets a cell surface receptor critical for HCV infection of the host cell. However, binding of soluble E2 to SR-BI or CD81 was not affected by oxLDL, suggesting that oxLDL does not act as a simple receptor blocker. At the same time, oxLDL incubation altered the biophysical properties of HCVpp, suggesting a ternary interaction of oxLDL with both virus and target cells. In conclusion, the SR-BI ligand oxLDL is a potent cell entry inhibitor for a broad range of HCV strains in vitro. These findings suggest that SR-BI is an essential component of the cellular HCV receptor complex

Porphyromonas gingivalis lipopolysaccharide alters atherosclerotic-related gene expression in oxidized low-density-lipoprotein-induced macrophages and foam cells

The molecular mechanism between atherosclerosis formation and periodontal pathogens is not clear although positive correlation between periodontal infections and cardiovascular diseases has been reported. Objective: To determine if atherosclerosis related genes were affected in foam cells during and after its formation by P. gingivalis lipopolysaccharide (LPS) stimulation. Methods: Macrophages from human THP-1 monocytes were treated with oxidized low density lipoprotein (oxLDL) to induce the formation of foam cells. P. gingivalis LPS was added to cultures of either oxLDL-induced macrophages or foam cells. The expression of atherosclerosis related genes was assayed by quantitative real time PCR and the protein production of granulocyte-macrophage colony-stimulating factor（GM-CSF）, monocyte chemotactic protein-1 (MCP-1), IL-1β, IL-10 and IL-12 was determined by ELISA. Nuclear translocation of NF-κB P65 was detected by immunocytochemistry and western blot was used to evaluate IKB-α degradation to confirm the NF-κB pathway activation. Results: P. gingivalis LPS stimulated atherosclerosis related gene expression in foam cells and increased oxLDL induced expression of chemokines, adhesion molecules, growth factors, apoptotic genes, and nuclear receptors in macrophages. Transcription of the pro-inflammatory cytokines IL-1β and IL-12 was elevated in response to LPS in both macrophages and foam cells, whereas the anti-inflammatory cytokine IL-10 was not affected. Increased NF-κB pathway activation was also observed in LPS and oxLDL co-stimulated macrophages. Conclusion: P. gingivalis LPS appears to be an important factor in the development of atherosclerosis by stimulation of atherosclerosis related gene expression in both macrophages and foam cells via activation of the NF-κB pathway

Deletion of TSPO resulted in change of metabolomic profile in retinal pigment epithelial cells

Age-related macular degeneration is the main cause of vision loss in the aged population worldwide. Drusen, extracellular lesions formed underneath the retinal pigment epithelial (RPE) cells, are a clinical feature of AMD and associated with AMD progression. RPE cells support photoreceptor function by providing nutrition, phagocytosing outer segments and removing metabolic waste. Dysfunction and death of RPE cells are early features of AMD. The translocator protein, TSPO, plays an important role in RPE cholesterol efflux and loss of TSPO results in increased intracellular lipid accumulation and reactive oxygen species (ROS) production. This study aimed to investigate the impact of TSPO knockout on RPE cellular metabolism by identifying the metabolic differences between wildtype and knockout RPE cells, with or without treatment with oxidized low density lipoprotein (oxLDL). Using liquid chromatography mass spectrometry (LC/MS), we differentiated several metabolic pathways among wildtype and knockout cells. Lipids amongst other intracellular metabolites were the most influenced by loss of TSPO and/or oxLDL treatment. Glucose, amino acid and nucleotide metabolism was also affected. TSPO deletion led to up-regulation of fatty acids and glycerophospholipids, which in turn possibly affected the cell membrane fluidity and stability. Higher levels of glutathione disulphide (GSSG) were found in TSPO knockout RPE cells, suggesting TSPO regulates mitochondrial-mediated oxidative stress. These data provide biochemical insights into TSPO-associated function in RPE cells and may shed light on disease mechanisms in AMD

Effects of oxidized low density lipoprotein, lipid mediators and statins on vascular cell interactions

The integrin heterodimer CD11b/CD18 (alpha M beta 2, Mac-1, CR3) expressed on monocytes or polymorphonuclear leukocytes (PMN) is a receptor for iC3b, fibrinogen, heparin, and for intercellular adhesion molecule (ICAM)-1 on endothelium, crucially contributing to vascular cell interactions in inflammation and atherosclerosis. In this report, we summarize our findings on the effects of lipid mediators and lipid-lowering drugs. Exposure of endothelial cells to oxidized low density lipoprotein (oxLDL) induces upregulation of ICAM-1 and increases adhesion of monocytic cells expressing Mac-1. Inhibition experiments show that monocytes use distinct ligands, i.e. ICAM-1 and heparan sulfate proteoglycans for adhesion to oxLDL-treated endothelium. An albumin-transferable oxLDL activity is inhibited by the antioxidant pyrrolidine dithiocarbamate (PDTC), while 8-epi-prostaglandin F2 alpha (8-epi-PGF2 alpha) or lysophosphatidylcholine had no effect, implicating yet unidentified radicals. Sequential adhesive! and signaling events lead to the firm adhesion of rolling PMN on activated and adherent platelets, which may occupy areas of endothelial denudation. Shear resistant arrest of PMN on thrombin-stimulated platelets in flow conditions requires distinct regions of Mac-1, involving its interactions with fibrinogen bound to platelet alpha llb beta 3, and with other platelet ligands. Both arrest and adhesion strengthening under flow are stimulated by platelet-activating factor and leukotriene B4, but not by the chemokine receptor CXCR2. We tested whether Mac-1-dependent monocyte adhesiveness is affected by inhibitors of hydroxy-methylglutaryl-Coenzyme A reductase (statins) which improve morbidity and survival of patients with coronary heart disease. As compared to controls, adhesion of isolated monocytes to endothelium ex vivo was increased in patients with hypercholesterolemia. Treatment with statins decreased total and low density lipoprotein (LDL) cholesterol plasma levels, surface expression of Mac-1, and resulted in a dramatic reduction of Mac,mediated monocyte adhesion to endothelium. The inhibition of monocyte adhesion was reversed by mevalonate but not LDL in vitro,indicating that isoprenoid precursors are crucial for adhesiveness of Mac-1. Such effects may crucially contribute to the clinical benefit of statins, independent of cholesterol-lowering, and may represent a paradigm for novel, anti-inflammatory mechanisms of action by this class of drugs

The relationship between oxidised LDL, endothelial progenitor cells and coronary endothelial function in patients with CHD

Objective The balance between coronary endothelial dysfunction and repair is influenced by many protective and deleterious factors circulating in the blood. We studied the relationship between oxidised low-density lipoprotein (oxLDL), circulating endothelial progenitor cells (EPCs) and coronary endothelial function in patients with stable coronary heart disease (CHD). Methods 33 patients with stable CHD were studied. Plasma oxLDL was measured using ELISA, coronary endothelial function was assessed using intracoronary acetylcholine infusion and EPCs were quantified using flow cytometry for CD34+/KDR+ cells. Results Plasma oxLDL correlated positively with the number of EPCs in the blood (r=0.46, p=0.02). There was a positive correlation between the number of circulating EPCs and coronary endothelial function (r=0.42, p=0.04). There was no significant correlation between oxLDL and coronary endothelial function. Conclusions Plasma levels of oxLDL are associated with increased circulating EPCs in the blood of patients with CHD, which may reflect a host-repair response to endothelial injury. Patients with stable CHD had a high prevalence of coronary endothelial dysfunction, which was associated with lower numbers of circulating EPCs, suggesting a mechanistic link between endothelial dysfunction and the pathogenesis of atherosclerosis