Vav family Rho guanine nucleotide exchange factors regulate CD36-mediated macrophage foam cell formation

Lipid-laden macrophages or "foam cells" are the primary components of the fatty streak, the earliest atherosclerotic lesion. Although Vav family guanine nucleotide exchange factors impact processes highly relevant to atherogenesis and are involved in pathways common to scavenger receptor CD36 signaling, their role in CD36-dependent macrophage foam cell formation remains unknown. The goal of the present study was to determine the contribution of Vav proteins to CD36-dependent foam cell formation and to identify the mechanisms by which Vavs participate in the process. We found that CD36 contributes to activation of Vav-1, -2, and -3 in aortae from hyperlipidemic mice and that oxidatively modified LDL (oxLDL) induces activation of macrophage Vav in vitro in a CD36 and Src family kinase-dependent manner. CD36-dependent uptake of oxLDL in vitro and foam cell formation in vitro and in vivo was significantly reduced in Vav null macrophages. These studies for the first time link CD36 and Vavs in a signaling pathway required for macrophage foam cell formation.     

A CD36-dependent signaling cascade is necessary for macrophage foam cell formation

Accumulation of macrophage foam cells in atherosclerotic blood vessel intima is a critical component of atherogenesis mediated by scavenger receptor-dependent internalization of oxidized LDL. We demonstrated by coimmunoprecipitation and pull-down assays that the macrophage scavenger receptor CD36 associates with a signaling complex containing Lyn and MEKK2. The MAP kinases JNK1 and JNK2 were specifically phosphorylated in macrophages exposed to oxLDL. Using cells isolated from SRA, TLR2, or CD36 null mice, and phospholipid ligands specific for either SRA or CD36, we showed that JNK activation was mediated by CD36. Both foam cell formation and activation of JNK2 in hyperlipidemic mice were diminished in the absence of CD36. Furthermore, inhibition of Src or JNK blocked oxLDL uptake and inhibited foam cell formation in vitro and in vivo. These findings show that a specific CD36-dependent signaling pathway initiated by oxLDL is necessary for foam cell formation and identify potential targets for antiatherosclerosis therapy.     

CD9 tetraspanin interacts with CD36 on the surface of macrophages: a possible regulatory influence on uptake of oxidized low density lipoprotein

CD36 is a type 2 scavenger receptor with multiple functions. CD36 binding to oxidized LDL triggers signaling cascades that are required for macrophage foam cell formation, but the mechanisms by which CD36 signals remain incompletely understood. Mass spectrometry analysis of anti-CD36 immuno-precipitates from macrophages identified the tetraspanin CD9 as a CD36 interacting protein. Western blot showed that CD9 was precipitated from mouse macrophages by anti-CD36 monoclonal antibody and CD36 was likewise precipitated by anti-CD9, confirming the mass spectrometry results. Macrophages from cd36 null mice were used to demonstrate specificity. Membrane associations of the two proteins on intact cells was analyzed by confocal immunofluorescence microscopy and by a novel cross linking assay that detects proteins in close proximity (<40 nm). Functional significance was determined by assessing lipid accumulation, foam cell formation and JNK activation in wt, cd9 null and cd36 null macrophages exposed to oxLDL. OxLDL uptake, lipid accumulation, foam cell formation, and JNK phosphorylation were partially impaired in cd9 null macrophages. The present study demonstrates that CD9 associates with CD36 on the macrophage surface and may participate in macrophage signaling in response to oxidized LDL.     

Inhibition of Glutathione Production Induces Macrophage CD36 Expression and Enhances Cellular-oxidized Low Density Lipoprotein (oxLDL) Uptake

The glutathione (GSH)-dependent antioxidant system has been demonstrated to inhibit atherosclerosis. Macrophage CD36 uptakes oxidized low density lipoprotein (oxLDL) thereby facilitating foam cell formation and development of atherosclerosis. It remains unknown if GSH can influence macrophage CD36 expression and cellular oxLDL uptake directly. Herein we report that treatment of macrophages with l-buthionine-S,R-sulfoximine (BSO) decreased cellular GSH production and ratios of GSH to glutathione disulfide (GSH/GSSG) while increasing production of reactive oxygen species. Associated with decreased GSH levels, macrophage CD36 expression was increased, which resulted in enhanced cellular oxLDL uptake. In contrast, N-acetyl cysteine and antioxidant enzyme (catalase or superoxide dismutase) blocked BSO-induced CD36 expression as well as oxLDL uptake. In vivo, administration of mice with BSO increased CD36 expression in peritoneal macrophages and kidneys. BSO had no effect on CD36 mRNA expression and promoter activity but still induced CD36 protein expression in macrophages lacking peroxisome proliferator-activated receptor γ expression, suggesting it induced CD36 expression at the translational level. Indeed, we determined that BSO enhanced CD36 translational efficiency. Taken together, our study demonstrates that cellular GSH levels and GSH/GSSG status can regulate macrophage CD36 expression and cellular oxLDL uptake and demonstrate an important anti-atherogenic function of the GSH-dependent antioxidant system by providing a novel molecular mechanism.     

Minimally oxidized LDL inhibits macrophage selective cholesteryl ester uptake and native LDL-induced foam cell formation

Scavenger receptor-mediated uptake of oxidized LDL (oxLDL) is thought to be the major mechanism of foam cell generation in atherosclerotic lesions. Recent data has indicated that native LDL is also capable of contributing to foam cell formation via low-affinity receptor-independent LDL particle pinocytosis and selective cholesteryl ester (CE) uptake. In the current investigation, Cu²⁺-induced LDL oxidation was found to inhibit macrophage selective CE uptake. Impairment of selective CE uptake was significant with LDL oxidized for as little as 30 min and correlated with oxidative fragmentation of apoB. In contrast, LDL aggregation, LDL CE oxidation, and the enhancement of scavenger receptor-mediated LDL particle uptake required at least 3 h of oxidation. Selective CE uptake did not require expression of the LDL receptor (LDL-R) and was inhibited similarly by LDL oxidation in LDL-R^−/− versus WT macrophages. Inhibition of selective uptake was also observed when cells were pretreated or cotreated with minimally oxidized LDL, indicating a direct inhibitory effect of this oxLDL on macrophages. Consistent with the effect on LDL CE uptake, minimal LDL oxidation almost completely prevented LDL-induced foam cell formation. These data demonstrate a novel inhibitory effect of mildly oxidized LDL that may reduce foam cell formation in atherosclerosis.     

Oxidation of low-density lipoproteins induces amyloid-like structures that are recognized by macrophages

The macrophage scavenger receptor CD36 plays a key role in the initiation of atherosclerosis through its ability to bind to and internalize oxidized low-density lipoproteins (oxLDL). Prompted by recent findings that the CD36 receptor also recognizes amyloid fibrils formed by beta-amyloid and apolipoprotein C-II, we investigated whether the oxidation of low-density lipoproteins (LDL) generates characteristic amyloid-like structures and whether these structures serve as CD36 ligands. Our studies demonstrate that LDL oxidized by copper ions, 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH), or ozone react with the diagnostic amyloid dyes thioflavin T and Congo Red and bind to serum amyloid P component (SAP), a universal constituent of physiological amyloid deposits. X-ray powder diffraction patterns for native LDL show a diffuse powder diffraction ring with maximum intensity corresponding to an atomic spacing of approximately 4.7 A, consistent with the spacing between beta-strands in a beta-sheet. Ozone treatment of LDL generates an additional diffuse powder diffraction ring with maximum intensity indicating a spacing of approximately 9.8 A. This distance is consistent with the presence of cross-beta-structure, a defining characteristic of amyloid. Evidence that these cross-beta-amyloid structures in oxLDL are recognized by macrophages is provided by the observation that SAP strongly inhibits the association and internalization of (125)I-labeled copper-oxidized LDL by peritoneal macrophages. The ability of SAP to bind to amyloid-like structures in oxLDL and prevent lipid uptake by macrophages highlights the potential importance of these structures and suggests an important preventative role for SAP in foam cell formation and early-stage atherosclerosis.     

Uptake of Oxidized Low Density Lipoprotein by CD36 Occurs by an Actin-dependent Pathway Distinct from Macropinocytosis

The class B scavenger receptor CD36 has numerous ligands that include modified forms of low density lipoprotein, fibrillar amyloid, apoptotic cells, and Plasmodium falciparum-infected red blood cells, linking this molecule to atherosclerosis, Alzheimer disease, malaria, and other diseases. We studied the signaling events that follow receptor engagement and lead to CD36 and ligand internalization. We show that oxidized low density lipoprotein or antibody-induced clustering of CD36 triggers macropinocytosis and internalization of the receptor-ligand complex. Remarkably, however, CD36 internalization is independent of macropinocytosis and occurs by a novel endocytic mechanism that depends on actin, but not dynamin. This actin-driven endocytosis requires the activation Src family kinases, JNK, and Rho family GTPases, but, unlike macropinocytosis, it is not affected by inhibitors of phosphatidylinositol 3-kinase or Na/H exchange. Manipulation of this unique mode of internalization may prove helpful in the prevention and management of the wide range of diseases in which CD36 is implicated.Uptake and storage of cholesterol by macrophages are key contributors to the formation of atherosclerotic plaque. Endothelial cells, seemingly activated by the deposition of modified forms of low-density lipoprotein (LDL),³ release chemokines that recruit macrophages into the vascular intima. Infiltrated macrophages can then readily oxidize and take up the modified LDL. Accumulation of lipids derived from oxidized LDL (oxLDL) transforms macrophages into foam cells, which release excess cytokines, triggering an inflammatory cascade. In addition, foam cells express proteases and other factors that contribute to plaque rupture and subsequent thrombosis.OxLDL particles are recognized by a variety of receptors, including the class A scavenger receptor SR-A and the class B scavenger receptor CD36. CD36 is thought to be responsible for ∼50% of oxLDL uptake by murine and human macrophages (1–3). In addition, this protein mediates cholesterol uptake from high density lipoprotein (4) and is also a receptor for internalization of oxidized high density lipoprotein (5).CD36 encodes a protein with two transmembrane domains located near the N and C termini, leaving only short cytoplasmic tails at each end. Despite having small intracellular domains, engagement of CD36 by its cognate ligands triggers signaling reactions that lead to the internalization of the resulting complex. However, the precise pathways that are activated and the specific mode of internalization remain unclear.Jones and Willingham (6) demonstrated that, in macrophages, modified LDL stimulates ruffling activity and the formation of phase-bright macropinosomes. By transmission electron microscopy they found that gold-conjugated modified LDL associated with ruffles and was present within macropinosomes. These observations underlie the widely accepted view that uptake of modified LDL occurs by macropinocytosis. However, Zeng et al. (7) showed that internalized DiI-oxLDL and CD36 were found in moderately sized cytoplasmic structures that co-localized with a glycosylphosphatidylinositol-anchored protein, suggesting uptake via lipid raft endocytosis. Moreover, Sun et al. (8) reported that uptake of oxLDL by CD36 was independent of actin but dependent on dynamin. The results of these two studies are not easily reconciled with mediation by macropinocytosis, an actin-dependent process that generates large vacuoles, and they suggest instead that CD36 is internalized by a more conventional endocytic pathway.It is not clear whether the apparent discrepancy stems from the engagement of different receptors in the different biological systems used in these studies. Jones and Willingham used macrophages, whereas Zeng et al. and Sun et al. studied, respectively, Chinese hamster ovary and COS cells heterologously transfected with CD36. The types and abundance of receptors capable of binding modified LDL in all likelihood differ greatly in these systems, and heterologous (over)expression in immortalized cells is liable to produce results of questionable biological relevance.In view of this uncertainty and considering the important and versatile roles of CD36, we set out to reexamine the mode of internalization of this receptor. We used both primary and cultured macrophages and selectively targeted CD36 using specific antibodies. The responses triggered by selective CD36 cross-linking were also compared with those elicited by oxLDL. We show that clustering CD36 initiates a signaling cascade that results in the activation of both macropinocytosis and internalization of CD36. Remarkably, however, CD36 internalization is largely independent of macropinocytosis and occurs by a novel dynamin-independent, actin-driven process that requires activation of Src family and c-Jun N-terminal kinases.     

Lipopolysaccharide-induced gene expression in murine macrophages is enhanced by prior exposure to oxLDL

Uptake of modified lipoproteins by macrophages results in the formation of foam cells. We investigated how foam cell formation affects the inflammatory response of macrophages. Murine bone marrow-derived macrophages were treated with oxidized LDL (oxLDL) to induce foam cell formation. Subsequently, the foam cells were activated with lipopolysaccharide (LPS), and the expression of lipid metabolism and inflammatory genes was analyzed. Furthermore, gene expression profiles of foam cells were analyzed using a microarray. We found that prior exposure to oxLDL resulted in enhanced LPS-induced tumor necrosis factor (TNF) and interleukin-6 (IL-6) gene expression, whereas the expression of the anti-inflammatory cytokine IL-10 and interferon-beta was decreased in foam cells. Also, LPS-induced cytokine secretion of TNF, IL-6, and IL-12 was enhanced, whereas secretion of IL-10 was strongly reduced after oxLDL preincubation. Microarray experiments showed that the overall inflammatory response induced by LPS was enhanced by oxLDL loading of the macrophages. Moreover, oxLDL loading was shown to result in increased nuclear factor-kappaB activation. In conclusion, our experiments show that the inflammatory response to LPS is enhanced by loading of macrophages with oxLDL. These data demonstrate that foam cell formation may augment the inflammatory response of macrophages during atherogenesis, possibly in an IL-10-dependent manner.     

A novel family of atherogenic oxidized phospholipids promotes macrophage foam cell formation via the scavenger receptor CD36 and is enriched in atherosclerotic lesions

The macrophage scavenger receptor CD36 plays an important role in binding and uptake of oxidized forms of low-density lipoprotein (LDL), foam cell formation, and lesion development during atherosclerosis. The structural basis of CD36-lipoprotein ligand recognition is an area of intense interest. In a companion article we reported the characterization of a structurally conserved family of oxidized choline glycerophospholipids (oxPC(CD36)) that serve as novel high affinity ligands for cells stably transfected with CD36, mediating recognition of multiple oxidized forms of LDL (Podrez, E. A., Poliakov, E., Shen, Z., Zhang, R., Deng, Y., Sun, M., Finton, P., Shan, L., Gugiu, B., Fox, P. L., Hoff, H. F., Salomon, R. G., and Hazen, S. L. (July 8, 2002) J. Biol. Chem. 277, 10.1074/jbc.M203318200). Here we use macrophages from wild-type and CD36 null mice to demonstrate that CD36 is the major receptor on macrophages mediating recognition of oxPC(CD36) species when presented (+/- plasma) in pure form, within PC bilayers in small unilamellar vesicles, and within liposomes generated from lipid extracts of native LDL. We also show that oxPC(CD36) promote CD36-dependent recognition when present at only a few molecules per particle, resulting in macrophage binding, uptake, metabolism, cholesterol accumulation, and foam cell formation. Finally, using high performance liquid chromatography with on-line electrospray ionization tandem mass spectrometry (LC/ESI/MS/MS), we demonstrate that oxPC(CD36) are generated in vivo and are enriched in atherosclerotic lesions. Collectively, our data suggest that formation of this novel family of oxidized phospholipids participates in CD36-mediated recognition of oxidized lipoproteins and foam cell formation in vivo.     

Sulfo-N-succinimidyl Oleate (SSO) Inhibits Fatty Acid Uptake and Signaling for Intracellular Calcium via Binding CD36 Lysine 164: SSO ALSO INHIBITS OXIDIZED LOW DENSITY LIPOPROTEIN UPTAKE BY MACROPHAGES*

FAT/CD36 is a multifunctional glycoprotein that facilitates long-chain fatty acid (FA) uptake by cardiomyocytes and adipocytes and uptake of oxidized low density lipoproteins (oxLDL) by macrophages. CD36 also mediates FA-induced signaling to increase intracellular calcium in various cell types. The membrane-impermeable sulfo-N-hydroxysuccinimidyl (NHS) ester of oleate (SSO) irreversibly binds CD36 and has been widely used to inhibit CD36-dependent FA uptake and signaling to calcium. The inhibition mechanism and whether SSO modification of CD36 involves the FA-binding site remain unexplored. CHO cells expressing human CD36 were SSO-treated, and the protein was pulled down, deglycosylated, and resolved by electrophoresis. The CD36 band was extracted from the gel and digested for analysis by mass spectrometry. NHS derivatives react with primary or secondary amines on proteins to yield stable amide or imide bonds. Two oleoylated peptides, found only in SSO-treated samples, were identified with high contribution and confidence scores as carrying oleate modification of Lys-164. Lysine 164 lies within a predicted CD36 binding domain for FA and oxLDL. CHO cells expressing CD36 with mutated Lys-164 had impaired CD36 function in FA uptake and FA-induced calcium release from the endoplasmic reticulum, supporting the importance of Lys-164 for both FA effects. Furthermore, consistent with the importance of Lys-164 for oxLDL binding, SSO inhibited oxLDL uptake by macrophages. In conclusion, SSO accesses Lys-164 in the FA-binding site on CD36, and initial modeling of this site is presented. The data suggest competition between FA and oxLDL for access to the CD36 binding pocket.     

oxHDL decreases the expression of CD36 on human macrophages through PPARγ and p38 MAP kinase dependent mechanisms

CD36, belongs to class B scavenger receptor family, is a macrophage receptor for oxidized low-density lipoprotein (oxLDL) and has been proven to play a critical role in atherosclerotic foam cell formation. In addition, CD36 expression is regulated by many factors including oxLDL and HDL. A recent study suggests that CD36 can also bind with oxidized high-density lipoprotein (oxHDL). However, the direct role of oxHDL in atherosclerosis is still not clear and it is not known whether oxHDL has any influence on the expression of CD36 in macrophages. Here, we performed experiments to investigate the effect of oxHDL on the expression of CD36 on human peripheral blood monocytes–macrophages and the possible mechanisms. Our results suggest that the uptake of oxHDL by CD36 on macrophages accelerates foam cell formation. In addition, oxHDL can down-regulate both the mRNA and surface protein expression of CD36 on human peripheral macrophages in vitro. oxHDL increased the mRNA expression and protein phosphorylation of peroxisome proliferators-activated receptor-γ (PPARγ). Using different mitogen-activated protein kinase (MAPK) inhibitors, we demonstrated that oxHDL regulated CD36 and PPARγ expression in a p38-MAP kinase dependent mechanism.     

OxLDL-mediated survival of macrophages does not require LDL internalization or signalling by major pattern recognition receptors

Macrophages play a key role in the pathogenesis of atherosclerosis, in part by destabilizing plaques. We and others have shown that low concentrations of oxidized LDL (oxLDL) inhibit macrophage apoptosis. As oxLDL is present in lesions, this may be a mechanism by which macrophage populations in the intima are expanded. We have previously shown that oxLDL activates prosurvival signalling pathways such as the phosphoinositide 3-kinase (PI3K) pathway in bone marrow derived macrophages (BMDMs). However, little is known about more upstream signalling events especially at the receptor level. The endocytic pattern recognition receptors (PRRs), scavenger receptor A (SR-A) and CD36, are the main receptors on macrophages for uptake of oxLDL and are therefore important in foam cell formation. The signalling PRRs such as toll-like receptor (TLR) 2 and 4 also bind some types of oxLDL. This study was done to determine if any of the known PRRs are required for the anti-apoptotic effects of oxLDL in BMDMs. To do this, we tested the effect of oxLDL on viability of BMDMs lacking both SR-A and CD36 or lacking TLR2, TLR4, CD14, FcγRIIb, or RAGE. Our results indicate that none of these receptors are essential for activating the oxLDL prosurvival pathway. Furthermore, we show that the anti-apoptotic effect is not dependent on the uptake of oxLDL.     

Endoplasmic reticulum stress controls M2 macrophage differentiation and foam cell formation

Macrophages are essential in atherosclerosis progression, but regulation of the M1 versus M2 phenotype and their role in cholesterol deposition are unclear. We demonstrate that endoplasmic reticulum (ER) stress is a key regulator of macrophage differentiation and cholesterol deposition. Macrophages from diabetic patients were classically or alternatively stimulated and then exposed to oxidized LDL. Alternative stimulation into M2 macrophages lead to increased foam cell formation by inducing scavenger receptor CD36 and SR-A1 expression. ER stress induced by alternative stimulation was necessary to generate the M2 phenotype through JNK activation and increased PPARγ expression. The absence of CD36 or SR-A1 signaling independently of modified cholesterol uptake decreased ER stress and prevented the M2 differentiation typically induced by alternative stimulation. Moreover, suppression of ER stress shifted differentiated M2 macrophages toward an M1 phenotype and subsequently suppressed foam cell formation by increasing HDL- and apoA-1-induced cholesterol efflux indicating suppression of macrophage ER stress as a potential therapy for atherosclerosis.     

Oxidized LDL/CD36 interaction induces loss of cell polarity and inhibits macrophage locomotion

Cell polarization is essential for migration and the exploratory function of leukocytes. However, the mechanism by which cells maintain polarity or how cells revert to the immobilized state by gaining cellular symmetry is not clear. Previously we showed that interaction between oxidized low-density lipoprotein (oxLDL) and CD36 inhibits macrophage migration; in the current study we tested the hypothesis that oxLDL/CD36-induced inhibition of migration is the result of intracellular signals that regulate cell polarity. Live cell imaging of macrophages showed that oxLDL actuated retraction of macrophage front end lamellipodia and induced loss of cell polarity. Cd36 null and macrophages null for Vav, a guanine nucleotide exchange factor (GEF), did not show this effect. These findings were caused by Rac-mediated inhibition of nonmuscle myosin II, a cell polarity determinant. OxLDL induced dephosphorylation of myosin regulatory light chain (MRLC) by increasing the activity of Rac. Six-thioguanine triphosphate (6-thio-GTP), which inhibits Vav-mediated activation of Rac, abrogated the effect of oxLDL. Activation of the Vav-Rac-myosin II pathway by oxidant stress may induce trapping of macrophages at sites of chronic inflammation such as atherosclerotic plaque.     

Macrophage receptors responsible for distinct recognition of low density lipoprotein containing pyrrole or pyridinium adducts: models of oxidized low density lipoprotein

Oxidation of low density lipoproteins (LDL) induced by incubation with Cu(2+) ions results in the formation of a heterogeneous group of aldehydic adducts on lysyl residues (Lys) of apolipoprotein B (apoB) that are thought to be responsible for the uptake of oxidized LDL (oxLDL) by macrophages. To define the structural and chemical criteria governing such cell recognition, we induced two modifications of lysines in LDL that mimic prototypic adducts present in oxLDL; namely, epsilon-amino charge-neutralizing pyrrolation by treatment with 2,5-hexanedione (hdLDL), and epsilon-amino charge-retaining pyridinium formation via treatment with 2,4,6-trimethylpyrylium (tmpLDL). Both modifications led to recognition by receptors on mouse peritoneal macrophages (MPM). To assess whether the murine scavenger receptor class A-I (mSR-A) was responsible for recognition of hdLDL or tmpLDL in MPM, we measured binding at 4 degrees C and degradation at 37 degrees C of these modified forms of (125)I-labeled LDL by mSR-A-transfected CHO cells. Although uptake and degradation of hdLDL by mSR-A-transfected CHO cells was quantitatively similar to that of the positive control, acLDL, tmpLDL was not recognized by these cells. However, both tmpLDL and hdLDL were recognized by 293 cells that had been transfected with CD36. In the human monocytic cell line THP-1 that had been activated with PMA, uptake of tmpLDL was significantly inhibited by blocking monoclonal antibodies to CD36, further suggesting recognition of tmpLDL by this receptor. Macrophage uptake and degradation of LDL oxidized by brief exposure to Cu(2+) was inhibited more effectively by excess tmpLDL and hdLDL than was more extensively oxidized LDL, consistent with the recognition of the former by CD36 and the latter primarily by SR-A.Collectively, these studies suggest that formation of specific pyrrole adducts on LDL leads to recognition by both the mSR-A and mouse homolog of CD36 expressed on MPM, while formation of specific pyridinium adducts on LDL leads to recognition by the mouse homolog of CD 36 but not by mSR-A. As such, these two modifications of LDL may represent useful models for dissecting the relative contributions of specific modifications on LDL produced during oxidation, to the cellular uptake of this heterogeneous ligand.     

Co-stimulation of PAFR and CD36 is required for oxLDL-induced human macrophages activation

The oxidative process of LDL particles generates molecules which are structurally similar to platelet-activating factor (PAF), and some effects of oxidized LDL (oxLDL) have been shown to be dependent on PAF receptor (PAFR) activation. In a previous study, we showed that PAFR is required for upregulation of CD36 and oxLDL uptake. In the present study we analyzed the molecular mechanisms activated by oxLDL in human macrophages and the contribution of PAFR to this response. Human adherent monocytes/macrophages were stimulated with oxLDL. Uptake of oxLDL and CD36 expression were determined by flow cytometry; MAP kinases and Akt phosphorylation by Western blot; IL-8 and MCP-1 concentration by ELISA and mRNA expression by real-time PCR. To investigate the participation of the PI3K/Akt pathway, Gαi-coupled protein or PAFR, macrophages were treated with LY294002, pertussis toxin or with the PAFR antagonists WEB2170 and CV3988, respectively before addition of oxLDL. It was found that the addition of oxLDL to human monocytes/macrophages activates the PI3K/Akt pathway which in turn activates the MAPK (p38 and JNK). Phosphorylation of Akt requires the engagement of PAFR and a Gαi-coupled protein. The upregulation of CD36 protein and the uptake of oxLDL as well as the IL-8 production are dependent on PI3K/Akt pathway activation. The increased CD36 protein expression is dependent on PAFR and Gαi-coupled protein. Transfection studies using HEK 293t cells showed that oxLDL uptake occurs with either PAFR or CD36, but IL-8 production requires the co-transfection of both PAFR and CD36. These findings show that PAFR has a pivotal role in macrophages response to oxLDL and suggest that pharmacological intervention at the level of PAFR activation might be beneficial in atherosclerosis.     

Oxidized low-density lipoprotein-induced foam cell formation is mediated by formyl peptide receptor 2

The increased level of LDL and its modification into oxLDL has been regarded as an important risk factor for the development of cardiovascular diseases such as atherosclerosis. Although some scavenger receptors including CD36 and RAGE have been considered as target receptors for oxLDL, involvement of other receptors should be investigated for oxLDL-induced pathological responses. In this study, we found that oxLDL-induced foam cell formation was inhibited by formyl peptide receptor 2 (FPR2) antagonist WRW⁴. oxLDL also stimulated calcium signaling and chemotactic migration in FPR2-expressing RBL-2H3 cells but not in vector-expressing RBL-2H3 cells. Moreover, oxLDL stimulated TNF-α production, which was also almost completely inhibited by FPR2 antagonist. Our findings therefore suggest that oxLDL stimulates macrophages, resulting in chemotactic migration, TNF-α production, and foam cell formation via FPR2 signaling, and thus likely contributes to atherogenesis.     

Binding and uptake of differently oxidized low density lipoprotein in mouse peritoneal macrophages and THP-1 macrophages: involvement of negative charges as well as oxidation-specific epitopes

Oxidatively modified low-density lipoprotein (LDL) has been found in vivo, and oxidized LDL (oxLDL) could bind to scavenger receptors, leading to foam cell formation. Macrophages bear a number of different scavenger receptors for oxLDL, and macrophages of different origins may have a different scavenger receptor repertoire. In addition, LDL oxidized to different degrees may differ in the ability to bind macrophage scavenger receptors. In this study, we characterized the patterns of the binding and uptake of differently oxidized LDL in mouse peritoneal macrophages (MPM) and human THP-1 macrophages, and the influence of negative charge and oxidation-specific epitopes in oxLDL on these processes. Thresholds of increased binding and uptake in MPM were found when LDL was oxidized to the degrees with a relative electrophoretic mobility (REM) of 2.6 (minor threshold) and 3.0 (major threshold), corresponding to 49 and 57%, respectively, of the loss of free amino groups in these oxLDL. There was no threshold for the binding of oxLDL to THP-1 macrophages, while for uptake, a major threshold with REM of 3.0 (57% free amino groups lost) was found. The presence of the F(ab')(2) fragments of the monoclonal antibody OB/04, which was raised against copper-oxidized LDL, led to the reduction of the binding and uptake, respectively, of Eu(3+)-oxLDL (REM:3.6) in MPM by 31 and 29%, and by 19 and 22% in THP-1 macrophages. It is concluded that LDL oxidized to different degrees binds differently to macrophages, and the patterns of binding and uptake are different for MPM and human THP-1 macrophages. Both, the negative charge and the oxidation-specific epitopes of oxLDL are involved in these processes.     

Oxidized LDL immune complexes induce release of sphingosine kinase in human U937 monocytic cells

The transformation of macrophages into foam cells is a critical event in the development of atherosclerosis. The most studied aspect of this process is the uptake of modified LDL through the scavenger receptors. Another salient aspect is the effect of modified LDL immune complexes on macrophages activation and foam cell formation. Macrophages internalize oxidized LDL immune complexes (oxLDL-IC) via the Fc-gamma receptor and transform into activated foam cells. In this study we examined the effect of oxLDL-IC on sphingosine kinase 1 (SK1), an enzyme implicated in mediating pro-survival and inflammatory responses through the generation of the signaling molecule sphingosine-1-phosphate (S1P). Intriguingly, oxLDL-IC, but not oxLDL alone, induced an immediate translocation and release of SK1 into the conditioned medium as evidenced by fluorescence confocal microscopy. Immunoblot analysis of cell lysates and conditioned medium revealed a decrease in intracellular SK1 protein levels accompanied by a concomitant increase in extracellular SK1 levels. Furthermore, measurement of S1P formation showed that the activity of cell-associated SK decreased in response to oxLDL-IC compared to oxLDL alone, whereas the activity of SK increased extracellularly. Blocking oxLDL-IC binding to Fc-gamma receptors resulted in decreased levels of extracellular S1P. The data also show that cell survival of human U937 cells exposed to oxLDL-IC increased compared to oxLDL alone. Exogenously added S1P further increased cell survival induced by oxLDL-IC. Taken together, these findings indicate that S1P may be generated extracellularly in response to modified LDL immune complexes and may therefore promote cell survival and prolong cytokine release by activated macrophages.     

Oxidized LDL induces phosphorylation of non-muscle myosin IIA heavy chain in macrophages

Oxidized LDL (oxLDL) performs critical roles in atherosclerosis by inducing macrophage foam cell formation and promoting inflammation. There have been reports showing that oxLDL modulates macrophage cytoskeletal functions for oxLDL uptake and trapping, however, the precise mechanism has not been clearly elucidated. Our study examined the effect of oxLDL on non-muscle myosin heavy chain IIA (MHC-IIA) in macrophages. We demonstrated that oxLDL induces phosphorylation of MHC-IIA (Ser1917) in peritoneal macrophages from wild-type mice and THP-1, a human monocytic cell line, but not in macrophages deficient for CD36, a scavenger receptor for oxLDL. Protein kinase C (PKC) inhibitor-treated macrophages did not undergo the oxLDL-induced MHC-IIA phosphorylation. Our immunoprecipitation revealed that oxLDL increased physical association between PKC and MHC-IIA, supporting the role of PKC in this process. We conclude that oxLDL via CD36 induces PKC-mediated MHC-IIA (Ser1917) phosphorylation and this may affect oxLDL-induced functions of macrophages involved in atherosclerosis. [BMB Reports 2015; 48(1): 48-53]