AIBP Limits Angiogenesis Through γ-Secretase-Mediated Upregulation of Notch Signaling.

RATIONALE: Angiogenesis improves perfusion to the ischemic tissue after acute vascular obstruction. Angiogenesis in pathophysiological settings reactivates signaling pathways involved in developmental angiogenesis. We showed previously that AIBP (apolipoprotein A-I [apoA-I]-binding protein)-regulated cholesterol efflux in endothelial cells controls zebra fish embryonic angiogenesis. OBJECTIVE: This study is to determine whether loss of AIBP affects angiogenesis in mice during development and under pathological conditions and to explore the underlying molecular mechanism. METHODS AND RESULTS: In this article, we report the generation of AIBP knockout (Apoa1bp-/-) mice, which are characterized of accelerated postnatal retinal angiogenesis. Mechanistically, AIBP triggered relocalization of γ-secretase from lipid rafts to nonlipid rafts where it cleaved Notch. Consistently, AIBP treatment enhanced DLL4 (delta-like ligand 4)-stimulated Notch activation in human retinal endothelial cells. Increasing high-density lipoprotein levels in Apoa1bp-/- mice by crossing them with apoA-I transgenic mice rescued Notch activation and corrected dysregulated retinal angiogenesis. Notably, the retinal vessels in Apoa1bp-/- mice manifested normal pericyte coverage and vascular integrity. Similarly, in the subcutaneous Matrigel plug assay, which mimics ischemic/inflammatory neovascularization, angiogenesis was dramatically upregulated in Apoa1bp-/- mice and associated with a profound inhibition of Notch activation and reduced expression of downstream targets. Furthermore, loss of AIBP increased vascular density and facilitated the recovery of blood vessel perfusion function in a murine hindlimb ischemia model. In addition, AIBP expression was significantly increased in human patients with ischemic cardiomyopathy. CONCLUSIONS: Our data reveal a novel mechanistic connection between AIBP-mediated cholesterol metabolism and Notch signaling, implicating AIBP as a possible druggable target to modulate angiogenesis under pathological conditions.

SYK regulates macrophage MHC-II expression via activation of autophagy in response to oxidized LDL.

Adaptive immunity, which plays an important role in the development of atherosclerosis, is mediated by major histocompatibility complex (MHC)-dependent antigen presentation. In atherosclerotic lesions, macrophages constitute an important class of antigen-presenting cells that activate adaptive immune responses to oxidized low-density lipoprotein (OxLDL). It has been reported that autophagy regulates adaptive immune responses by enhancing antigen presentation to MHC class II (MHC-II). In a previous study, we have demonstrated that SYK (spleen tyrosine kinase) regulates generation of reactive oxygen species (ROS) and activation of MAPK8/JNK1 in macrophages. Because ROS and MAPK8 are known to regulate autophagy, in this study we investigated the role of SYK in autophagy, MHC-II expression and adaptive immune response to OxLDL. We demonstrate that OxLDL induces autophagosome formation, MHC-II expression, and phosphorylation of SYK in macrophages. Gene knockout and pharmacological inhibitors of NOX2 and MAPK8 reduced OxLDL-induced autophagy. Using bone marrow-derived macrophages isolated from wild-type and myeloid-specific SYK knockout mice, we demonstrate that SYK regulates OxLDL-induced ROS generation, MAPK8 activation, BECN1-BCL2 dissociation, autophagosome formation and presentation of OxLDL-derived antigens to CD4(+) T cells. ldlr(-/-) syk(-/-) mice fed a high-fat diet produced lower levels of IgG to malondialdehyde (MDA)-LDL, malondialdehyde-acetaldehyde (MAA)-LDL, and OxLDL compared to ldlr(-/-) mice. These results provide new insights into the mechanisms by which SYK regulates MHC-II expression via autophagy in macrophages and may contribute to regulation of adaptive immune responses in atherosclerosis.

Disruption of mammalian target of rapamycin complex 1 in macrophages decreases chemokine gene expression and atherosclerosis.

RATIONALE: The mammalian target of rapamycin complex 1 inhibitor, rapamycin, has been shown to decrease atherosclerosis, even while increasing plasma low-density lipoprotein levels. This suggests an antiatherogenic effect possibly mediated by the modulation of inflammatory responses in atherosclerotic plaques. OBJECTIVE: Our aim was to assess the role of macrophage mammalian target of rapamycin complex 1 in atherogenesis. METHODS AND RESULTS: We transplanted bone marrow from mice in which a key mammalian target of rapamycin complex 1 adaptor, regulatory-associated protein of mTOR, was deleted in macrophages by Cre/loxP recombination (Mac-Rap(KO) mice) into Ldlr(-/-) mice and then fed them the Western-type diet. Atherosclerotic lesions from Mac-Rap(KO) mice showed decreased infiltration of macrophages, lesion size, and chemokine gene expression compared with control mice. Treatment of macrophages with minimally modified low-density lipoprotein resulted in increased levels of chemokine mRNAs and signal transducer and activator of transcription (STAT) 3 phosphorylation; these effects were reduced in Mac-Rap(KO) macrophages. Although wild-type and Mac-Rap(KO) macrophages showed similar STAT3 phosphorylation on Tyr705, Mac-Rap(KO) macrophages showed decreased STAT3Ser727 phosphorylation in response to minimally modified low-density lipoprotein treatment and decreased Ccl2 promoter binding of STAT3. CONCLUSIONS: The results demonstrate cross-talk between nutritionally induced mammalian target of rapamycin complex 1 signaling and minimally modified low-density lipoprotein-mediated inflammatory signaling via combinatorial phosphorylation of STAT3 in macrophages, leading to increased STAT3 activity on the chemokine (C-C motif) ligand 2 (monocyte chemoattractant protein 1) promoter with proatherogenic consequences.

Polyoxygenated cholesterol ester hydroperoxide activates TLR4 and SYK dependent signaling in macrophages.

Oxidation of low-density lipoprotein (LDL) is one of the major causative mechanisms in the development of atherosclerosis. In previous studies, we showed that minimally oxidized LDL (mmLDL) induced inflammatory responses in macrophages, macropinocytosis and intracellular lipid accumulation and that oxidized cholesterol esters (OxCEs) were biologically active components of mmLDL. Here we identified a specific OxCE molecule responsible for the biological activity of mmLDL and characterized signaling pathways in macrophages in response to this OxCE. Using liquid chromatography - tandem mass spectrometry and biological assays, we identified an oxidized cholesteryl arachidonate with bicyclic endoperoxide and hydroperoxide groups (BEP-CE) as a specific OxCE that activates macrophages in a TLR4/MD-2-dependent manner. BEP-CE induced TLR4/MD-2 binding and TLR4 dimerization, phosphorylation of SYK, ERK1/2, JNK and c-Jun, cell spreading and uptake of dextran and native LDL by macrophages. The enhanced macropinocytosis resulted in intracellular lipid accumulation and macrophage foam cell formation. Bone marrow-derived macrophages isolated from TLR4 and SYK knockout mice did not respond to BEP-CE. The presence of BEP-CE was demonstrated in human plasma and in the human plaque material captured in distal protection devices during percutaneous intervention. Our results suggest that BEP-CE is an endogenous ligand that activates the TLR4/SYK signaling pathway. Because BEP-CE is present in human plasma and human atherosclerotic lesions, BEP-CE-induced and TLR4/SYK-mediated macrophage responses may contribute to chronic inflammation in human atherosclerosis.

Development and application of a nonradioactive binding assay of oxidized low-density lipoprotein to macrophage scavenger receptors.

Macrophages play a key role in atherogenesis in part through excessive uptake of oxidized LDL (OxLDL) via scavenger receptors. Binding of OxLDL to macrophages has traditionally been assessed using radiolabeled OxLDL. To allow more efficient and convenient measurements, we developed a nonradioactive binding assay in which biotinylated OxLDL (Bt-OxLDL) is added to macrophages in 96-well microtiter culture plates under various conditions and the extent of binding is determined using solid phase chemiluminescent immunoassay techniques. As examples, we show that Bt-OxLDL displayed high and saturable binding to macrophages in contrast to Bt-LDL, which showed very low binding. In competition assays, unlabeled OxLDL and the anti-OxLDL monoclonal antibody E06 inhibited Bt-OxLDL binding to macrophages in a dose-dependent manner. Specific binding of Bt-OxLDL to ApoE/SR-A/CD36 triple knockout macrophages was reduced by 80% as compared with binding to macrophages from ApoE knockout mice. Binding of Bt-OxLDL to CD36 transfected COS-7 cells showed enhanced saturable binding compared with mock-transfected cells. This assay avoids the use of radioactivity and uses small amounts of materials. It can be used to study binding of OxLDL to macrophages and factors that influence this binding. The techniques described should be readily adaptable to study of other ligands, receptors, and cell types.

Control of angiogenesis by AIBP-mediated cholesterol efflux.

Cholesterol is a structural component of the cell and is indispensable for normal cellular function, although its excess often leads to abnormal proliferation, migration, inflammatory responses and/or cell death. To prevent cholesterol overload, ATP-binding cassette (ABC) transporters mediate cholesterol efflux from the cells to apolipoprotein A-I (apoA-I) and the apoA-I-containing high-density lipoprotein (HDL). Maintaining efficient cholesterol efflux is essential for normal cellular function. However, the role of cholesterol efflux in angiogenesis and the identity of its local regulators are poorly understood. Here we show that apoA-I binding protein (AIBP) accelerates cholesterol efflux from endothelial cells to HDL and thereby regulates angiogenesis. AIBP- and HDL-mediated cholesterol depletion reduces lipid rafts, interferes with VEGFR2 (also known as KDR) dimerization and signalling and inhibits vascular endothelial growth factor-induced angiogenesis in vitro and mouse aortic neovascularization ex vivo. Notably, Aibp, a zebrafish homologue of human AIBP, regulates the membrane lipid order in embryonic zebrafish vasculature and functions as a non-cell-autonomous regulator of angiogenesis. aibp knockdown results in dysregulated sprouting/branching angiogenesis, whereas forced Aibp expression inhibits angiogenesis. Dysregulated angiogenesis is phenocopied in Abca1 (also known as Abca1a) Abcg1-deficient embryos, and cholesterol levels are increased in Aibp-deficient and Abca1 Abcg1-deficient embryos. Our findings demonstrate that secreted AIBP positively regulates cholesterol efflux from endothelial cells and that effective cholesterol efflux is critical for proper angiogenesis.

MCP-1 binds to oxidized LDL and is carried by lipoprotein(a) in human plasma.

Lipoprotein oxidation plays an important role in pathogenesis of atherosclerosis. Oxidized low density lipoprotein (OxLDL) induces profound inflammatory responses in vascular cells, such as production of monocyte chemoattractant protein-1 (MCP-1) [chemokine (C-C motif) ligand 2], a key chemokine in the initiation and progression of vascular inflammation. Here we demonstrate that OxLDL also binds MCP-1 and that the OxLDL-bound MCP-1 retains its ability to recruit monocytes. A human MCP-1 mutant in which basic amino acids Arg-18 and Lys-19 were replaced with Ala did not bind to OxLDL. The MCP-1 binding to OxLDL was inhibited by the monoclonal antibody E06, which binds oxidized phospholipids (OxPLs) in OxLDL. Because OxPLs are carried by lipoprotein(a) [Lp(a)] in human plasma, we tested to determine whether Lp(a) binds MCP-1. Recombinant wild-type but not mutant MCP-1 added to human plasma bound to Lp(a), and its binding was inhibited by E06. Lp(a) captured from human plasma contained MCP-1 and the Lp(a)-associated endogenous MCP-1 induced monocyte migration. These results demonstrate that OxLDL and Lp(a) bind MCP-1 in vitro and in vivo and that OxPLs are major determinants of the MCP-1 binding. The association of MCP-1 with OxLDL and Lp(a) may play a role in modulating monocyte trafficking during atherogenesis.

Spleen tyrosine kinase regulates AP-1 dependent transcriptional response to minimally oxidized LDL.

Oxidative modification of low-density lipoprotein (LDL) turns it into an endogenous ligand recognized by pattern-recognition receptors. We have demonstrated that minimally oxidized LDL (mmLDL) binds to CD14 and mediates TLR4/MD-2-dependent responses in macrophages, many of which are MyD88-independent. We have also demonstrated that the mmLDL activation leads to recruitment of spleen tyrosine kinase (Syk) to TLR4 and TLR4 and Syk phosphorylation. In this study, we produced a macrophage-specific Syk knockout mouse and used primary Syk(-/-) macrophages in our studies. We demonstrated that Syk mediated phosphorylation of ERK1/2 and JNK, which in turn phosphorylated c-Fos and c-Jun, respectively, as assessed by an in vitro kinase assay. c-Jun phosphorylation was also mediated by IKKε. c-Jun and c-Fos bound to consensus DNA sites and thereby completed an AP-1 transcriptional complex and induced expression of CXCL2 and IL-6. These results suggest that Syk plays a key role in TLR4-mediated macrophage responses to host-generated ligands, like mmLDL, with subsequent activation of an AP-1 transcription program.

Oxidized cholesteryl esters and phospholipids in zebrafish larvae fed a high cholesterol diet: macrophage binding and activation.

A novel hypercholesterolemic zebrafish model has been developed to study early events of atherogenesis. This model utilizes optically transparent zebrafish larvae, fed a high cholesterol diet (HCD), to monitor processes of vascular inflammation in live animals. Because lipoprotein oxidation is an important factor in the development of atherosclerosis, in this study, we characterized the oxidized lipid milieu in HCD-fed zebrafish larvae. Using liquid chromatography-mass spectrometry, we show that feeding an HCD for only 2 weeks resulted in up to 70-fold increases in specific oxidized cholesteryl esters, identical to those present in human minimally oxidized LDL and in murine atherosclerotic lesions. The levels of oxidized phospholipids, such as 1-palmitoyl-2-oxovaleroyl-sn-glycero-3-phosphocholine, and of various lysophosphatidylcholines were also significantly elevated. Moreover, lipoproteins isolated from homogenates of HCD-fed larvae induced cell spreading as well as ERK1/2, Akt, and JNK phosphorylation in murine macrophages. Removal of apoB-containing lipoproteins from the zebrafish homogenates with an anti-human LDL antibody, as well as reducing lipid hydroperoxides with ebselen, resulted in inhibition of macrophage activation. The TLR4 deficiency in murine macrophages prevented their activation with zebrafish lipoproteins. Using biotinylated homogenates of HCD-fed larvae, we demonstrated that their components bound to murine macrophages, and this binding was effectively competed by minimally oxidized LDL but not by native LDL. These data provide evidence that molecular lipid determinants of proatherogenic macrophage phenotypes are present in large quantities in hypercholesterolemic zebrafish larvae and support the use of the HCD-fed zebrafish as a valuable model to study early events of atherogenesis.

Low doses of lipopolysaccharide and minimally oxidized low-density lipoprotein cooperatively activate macrophages via nuclear factor kappa B and activator protein-1: possible mechanism for acceleration of atherosclerosis by subclinical endotoxemia.

RATIONALE: Oxidized low-density lipoprotein (LDL) is an important determinant of inflammation in atherosclerotic lesions. It has also been documented that certain chronic infectious diseases, such as periodontitis and chlamydial infection, exacerbate clinical manifestations of atherosclerosis. In addition, low-level but persistent metabolic endotoxemia is often found in diabetic and obese subjects and is induced in mice fed a high-fat diet. OBJECTIVE: In this study, we examined cooperative macrophage activation by low levels of bacterial lipopolysaccharide (LPS) and by minimally oxidized LDL (mmLDL), as a model for subclinical endotoxemia-complicated atherosclerosis. METHODS AND RESULTS: We found that both in vitro and in vivo, mmLDL and LPS (Kdo2-LipidA) cooperatively activated macrophages to express proinflammatory cytokines Cxcl2 (MIP-2), Ccl3 (MIP-1alpha), and Ccl4 (MIP-1beta). Importantly, the mmLDL and LPS cooperative effects were evident at a threshold LPS concentration (1 ng/mL) at which LPS alone induced only a limited macrophage response. Analyzing microarray data with a de novo motif discovery algorithm, we found that genes transcribed by promoters containing an activator protein (AP)-1 binding site were significantly upregulated by costimulation with mmLDL and LPS. In a nuclear factor-DNA binding assay, the cooperative effect of mmLDL and LPS costimulation on c-Jun and c-Fos DNA binding, but not on p65 or p50, was dependent on mmLDL-induced activation of extracellular signal-regulated kinase (ERK) 1/2. In addition, mmLDL induced c-Jun N-terminal kinase (JNK)-dependent derepression of AP-1 by removing nuclear receptor corepressor (NCoR) from the chemokine promoters. CONCLUSIONS: The cooperative engagement of AP-1 and nuclear factor (NF)-kappaB by mmLDL and LPS may constitute a mechanism of increased transcription of inflammatory cytokines within atherosclerotic lesions.

12/15-lipoxygenase is required for the early onset of high fat diet-induced adipose tissue inflammation and insulin resistance in mice.

BACKGROUND: Recent understanding that insulin resistance is an inflammatory condition necessitates searching for genes that regulate inflammation in insulin sensitive tissues. 12/15-lipoxygenase (12/15LO) regulates the expression of proinflammatory cytokines and chemokines and is implicated in the early development of diet-induced atherosclerosis. Thus, we tested the hypothesis that 12/15LO is involved in the onset of high fat diet (HFD)-induced insulin resistance. METHODOLOGY/PRINCIPAL FINDINGS: Cells over-expressing 12/15LO secreted two potent chemokines, MCP-1 and osteopontin, implicated in the development of insulin resistance. We assessed adipose tissue inflammation and whole body insulin resistance in wild type (WT) and 12/15LO knockout (KO) mice after 2-4 weeks on HFD. In adipose tissue from WT mice, HFD resulted in recruitment of CD11b(+), F4/80(+) macrophages and elevated protein levels of the inflammatory markers IL-1beta, IL-6, IL-10, IL-12, IFNgamma, Cxcl1 and TNFalpha. Remarkably, adipose tissue from HFD-fed 12/15LO KO mice was not infiltrated by macrophages and did not display any increase in the inflammatory markers compared to adipose tissue from normal chow-fed mice. WT mice developed severe whole body (hepatic and skeletal muscle) insulin resistance after HFD, as measured by hyperinsulinemic euglycemic clamp. In contrast, 12/15LO KO mice exhibited no HFD-induced change in insulin-stimulated glucose disposal rate or hepatic glucose output during clamp studies. Insulin-stimulated Akt phosphorylation in muscle tissue from HFD-fed mice was significantly greater in 12/15LO KO mice than in WT mice. CONCLUSIONS: These results demonstrate that 12/15LO mediates early stages of adipose tissue inflammation and whole body insulin resistance induced by high fat feeding.

Lipoprotein accumulation in macrophages via toll-like receptor-4-dependent fluid phase uptake.

Toll-like receptor (TLR)4 recognizes microbial pathogens, such as lipopolysaccharide, and mediates lipopolysaccharide-induced proinflammatory cytokine secretion, as well as microbial uptake by macrophages. In addition to exogenous pathogens, TLR4 recognizes modified self, such as minimally oxidized low-density lipoprotein (mmLDL). Here we report that mmLDL and its active components, cholesteryl ester hydroperoxides, induce TLR4-dependent fluid phase uptake typical of macropinocytosis. We show that mmLDL induced recruitment of spleen tyrosine kinase (Syk) to a TLR4 signaling complex, TLR4 phosphorylation, activation of a Vav1-Ras-Raf-MEK-ERK1/2 signaling cascade, phosphorylation of paxillin, and activation of Rac, Cdc42, and Rho. These mmLDL-induced and TLR4- and Syk-dependent signaling events and cytoskeletal rearrangements lead to enhanced uptake of small molecules, dextran, and, most importantly, both native and oxidized LDL, resulting in intracellular lipid accumulation. An intravenous injection of fluorescently labeled mmLDL in wild-type mice resulted in its rapid accumulation in circulating monocytes, which was significantly attenuated in TLR4-deficient mice. These data describe a novel mechanism leading to enhanced lipoprotein uptake in macrophages that would contribute to foam cell formation and atherosclerosis. These data also suggest that cholesteryl ester hydroperoxides are an endogenous ligand for TLR4. Because TLR4 is highly expressed on the surface of circulating monocytes in patients with chronic inflammatory conditions, and cholesteryl ester hydroperoxides are present in plasma, lipid uptake by monocytes in circulation may contribute to the pathological roles of monocytes in chronic inflammatory diseases.

Vascular lipid accumulation, lipoprotein oxidation, and macrophage lipid uptake in hypercholesterolemic zebrafish.

Lipid accumulation in arteries induces vascular inflammation and atherosclerosis, the major cause of heart attack and stroke in humans. Extreme hyperlipidemia induced in mice and rabbits enables modeling many aspects of human atherosclerosis, but microscopic examination of plaques is possible only postmortem. Here we report that feeding adult zebrafish (Danio rerio) a high-cholesterol diet (HCD) resulted in hypercholesterolemia, remarkable lipoprotein oxidation, and fatty streak formation in the arteries. Feeding an HCD supplemented with a fluorescent cholesteryl ester to optically transparent fli1:EGFP zebrafish larvae in which endothelial cells express green fluorescent protein (GFP), and using confocal microscopy enabled monitoring vascular lipid accumulation and the endothelial cell layer disorganization and thickening in a live animal. The HCD feeding also increased leakage of a fluorescent dextran from the blood vessels. Administering ezetimibe significantly diminished the HCD-induced endothelial cell layer thickening and improved its barrier function. Feeding HCD to lyz:DsRed2 larvae in which macrophages and granulocytes express DsRed resulted in the accumulation of fluorescent myeloid cells in the vascular wall. Using a fluorogenic substrate for phospholipase A(2) (PLA(2)), we observed an increased vascular PLA(2) activity in live HCD-fed larvae compared to control larvae. Furthermore, by transplanting genetically modified murine cells into HCD-fed larvae, we demonstrated that toll-like receptor-4 was required for efficient in vivo lipid uptake by macrophages. These results suggest that the novel zebrafish model is suitable for studying temporal characteristics of certain inflammatory processes of early atherogenesis and the in vivo function of vascular cells.

Macrophages generate reactive oxygen species in response to minimally oxidized low-density lipoprotein: toll-like receptor 4- and spleen tyrosine kinase-dependent activation of NADPH oxidase 2.

Oxidative modification of low-density lipoprotein (LDL) plays a causative role in the development of atherosclerosis. In this study, we demonstrate that minimally oxidized LDL (mmLDL) stimulates intracellular reactive oxygen species (ROS) generation in macrophages through NADPH oxidase 2 (gp91phox/Nox2), which, in turn, induces production of RANTES and migration of smooth muscle cells. Peritoneal macrophages from gp91phox/Nox2(-/-) mice or J774 macrophages in which Nox2 was knocked down by small interfering RNA failed to generate ROS in response to mmLDL. Because mmLDL-induced cytoskeletal changes were dependent on Toll-like receptor (TLR)4, we analyzed ROS generation in peritoneal macrophages from wild-type, TLR4(-/-), or MyD88(-/-) mice and found that mmLDL-mediated ROS was generated in a TLR4-dependent, but MyD88-independent, manner. Furthermore, we found that ROS generation required the recruitment and activation of spleen tyrosine kinase (Syk) and that mmLDL also induced phospholipase PLCgamma1 phosphorylation and protein kinase C membrane translocation. Importantly, the phospholipase Cgamma1 phosphorylation was reduced in J774 cells expressing Syk-specific short hairpin RNA. Nox2 modulated mmLDL activation of macrophages by regulating the expression of proinflammatory cytokines interleukin-1beta, interleukin-6, and RANTES. We showed that purified RANTES was able to stimulate migration of mouse aortic smooth muscle cells and addition of neutralizing antibody against RANTES abolished the migration of mouse aortic smooth muscle cells stimulated by mmLDL-stimulated macrophages. These results suggest that mmLDL induces generation of ROS through sequential activation of TLR4, Syk, phospholipase Cgamma1, protein kinase C, and gp91phox/Nox2 and thereby stimulates expression of proinflammatory cytokines. These data help explain mechanisms by which endogenous ligands, such as mmLDL, can induce TLR4-dependent, proatherogenic activation of macrophages.

Cholesteryl ester hydroperoxides are biologically active components of minimally oxidized low density lipoprotein.

Oxidation of low density lipoprotein (LDL) occurs in vivo and significantly contributes to the development of atherosclerosis. An important mechanism of LDL oxidation in vivo is its modification with 12/15-lipoxygenase (LO). We have developed a model of minimally oxidized LDL (mmLDL) in which native LDL is modified by cells expressing 12/15LO. This mmLDL activates macrophages inducing membrane ruffling and cell spreading, activation of ERK1/2 and Akt signaling, and secretion of proinflammatory cytokines. In this study, we found that many of the biological activities of mmLDL were associated with cholesteryl ester (CE) hydroperoxides and were diminished by ebselen, a reducing agent. Liquid chromatography coupled with mass spectroscopy demonstrated the presence of many mono- and polyoxygenated CE species in mmLDL but not in native LDL. Nonpolar lipid extracts of mmLDL activated macrophages, although to a lesser degree than intact mmLDL. The macrophage responses were also induced by LDL directly modified with immobilized 12/15LO, and the nonpolar lipids extracted from 12/15LO-modified LDL contained a similar set of oxidized CE. Cholesteryl arachidonate modified with 12/15LO also activated macrophages and contained a similar collection of oxidized CE molecules. Remarkably, many of these oxidized CE were found in the extracts of atherosclerotic lesions isolated from hyperlipidemic apoE(-/-) mice. These results suggest that CE hydroperoxides constitute a class of biologically active components of mmLDL that may be relevant to proinflammatory activation of macrophages in atherosclerotic lesions.

The CC chemokine MCP-1 stimulates surface expression of CX3CR1 and enhances the adhesion of monocytes to fractalkine/CX3CL1 via p38 MAPK.

The membrane-anchored form of CX3CL1 has been proposed as a novel adhesion protein for leukocytes. This functional property of CX3CL1 is mediated through CX3CR1, a chemokine receptor expressed predominantly on circulating white blood cells. Thus far, it is still uncertain at what stage of the trafficking process CX3CR1 becomes importantly involved and how the CX3CR1-dependent adhesion of leukocytes is regulated during inflammation. The objective of this study was to examine the functional effects of chemokine stimulation on CX3CR1-mediated adhesion of human monocytes. Consistent with previous reports, our data indicate that the activity of CX3CR1 on resting monocytes is sufficient to mediate cell adhesion to CX3CL1. However, the basal, nonstimulated adhesion activity is low, and we hypothesized that like the integrins, CX3CR1 may require a preceding activation step to trigger firm leukocyte adhesion. Compatible with this hypothesis, stimulation of monocytes with MCP-1 significantly increased their adhesion to immobilized CX3CL1, under both static and physiological flow conditions. The increase of the adhesion activity was mediated through CCR2-dependent signaling and obligatory activation of the p38 MAPK pathway. Stimulation with MCP-1 also induced a rapid increase of CX3CR1 protein on the cell surface. Inhibition of the p38 MAPK pathway prevented this increase of CX3CR1 surface expression and blunted the effect of MCP-1 on cell adhesion, indicating a causal link between receptor surface density and adhesion activity. Together, our data suggest that a chemokine signal is required for firm CX3CR1-dependent adhesion and demonstrate that CCR2 is an important regulator of CX3CL1-dependent leukocyte adhesion.

The amino terminus and the third extracellular loop of CX3CR1 contain determinants critical for distinct receptor functions.

The G protein-coupled receptor CX3CR1 is a specific receptor for the CX3C chemokine fractalkine (CX3CL1 according to the new chemokine nomenclature). The aim of this study was to identify receptor elements that contribute independently to agonist binding and receptor activation. Targeted mutation of selected acidic amino acid residues demonstrated that the binding activity of CX3CR1 was critically dependent on the two negatively charged residues Asp25 and Glu254 located on the N-terminal domain and third extracellular loop, respectively. In addition, mutation of the uncharged polar residue Tyr14 in the amino terminus caused a reduction in the ligand binding affinity. In contrast, the three acidic residues Glu13, Asp16, and Asp266 did not contribute to ligand binding but were crucial for receptor activation. The mutant receptors E13A, D16A, and D266A bound fractalkine with high affinity but were unable to induce signaling events necessary to support chemotaxis. These acidic residues may engage in electrostatic interactions with basic residues on fractalkine that are necessary for receptor function but not for binding. Our data are consistent with a model of chemokine receptor activation consisting of a multi-step mechanism. Step one mediates the high-affinity fractalkine binding involving Tyr14, Asp25, and Glu254. The initial interaction then triggers the engagement of Glu13, Asp16, and Asp266, which are necessary for CX3CR1 activation.

Phosphocholine as a pattern recognition ligand for CD36.

We have previously shown that CD36 recognizes oxidation products of phospholipids on oxidized LDL (OxLDL) such as 1-palmitoyl-2-(5'-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC). The current study was designed to examine whether the phosphocholine (PC) headgroup in POVPC constitutes an obligatory binding target for CD36. To examine the contribution of PC in the binding of POVPC to CD36, we used well-defined synthetic oxidized phospholipids (OxPLs) cross-linked to BSA or to a hexapeptide. The OxPL adducts were then tested for their ability to bind to CD36-transfected cells and for their ability to inhibit OxLDL binding to CD36. Both POVPC-BSA and POVPC-peptide adducts were high-affinity ligands for CD36 and potent inhibitors of OxLDL binding. Enzymatic removal of the entire PC moiety of the POVPC-peptide, or of the choline headgroup alone, as well as substitution of the choline headgroup by ethanolamine abrogated the inhibitory activity of POVPC. Interestingly, PC by itself or cross-linked to BSA did not show any intrinsic competition activity. In conclusion, our data demonstrate that the PC headgroup of OxPL alone is sufficient for binding to CD36, but only if presented in the correct conformation as in OxPL of OxLDL or as in POVPC-peptide adducts.