Phagocytosis of apoptotic and necrotic thymocytes is inhibited by PAF-receptor antagonists and affects LPS-induced COX-2 expression in murine macrophages

There is evidence that apoptotic cells and oxidized low density lipoprotein (oxLDL) particles have common ligands on their surface consisting of oxidized phospholipids which bind to scavenger receptors in macrophages leading to phagocytosis. Some effects of oxLDL binding to its receptor(s) were shown to be inhibited by Platelet Activating Factor (PAF)-receptor antagonists. Thus, we investigated the effect of PAF-receptor antagonists on the phagocytosis of apoptotic, necrotic and viable thymocytes by murine peritoneal macrophages. It was found that phagocytosis of altered cells is significantly increased compared to viable cells, a phenomenon reversed by pre-treatment of macrophages with PAF-receptor antagonists (WEB2170 and CV3988), PAF or oxLDL. Phagocytosis of altered cells induced negligible expression of cyclooxygenase-2 (COX-2) but strongly potentiated the LPS-induced expression of this enzyme. This phenomenon was restricted to altered cells and was reversed by pre-treatment of macrophages with PAF-receptor antagonists. These findings indicate that apoptotic and necrotic cells share common ligands with PAF and oxLDL and suggest the involvement of PAF-like receptors in the enhanced clearance of these cells.     

Inflammatory platelet-activating factor-like phospholipids in oxidized low density lipoproteins are fragmented alkyl phosphatidylcholines.

Oxidation of human low density lipoprotein (LDL) generates proinflammatory mediators and underlies early events in atherogenesis. We identified mediators in oxidized LDL that induced an inflammatory reaction in vivo, and activated polymorphonuclear leukocytes and cells ectopically expressing human platelet-activating factor (PAF) receptors. Oxidation of a synthetic phosphatidylcholine showed that an sn-1 ether bond confers an 800-fold increase in potency. This suggests that rare ether-linked phospholipids in LDL are the likely source of PAF-like activity in oxidized LDL. Accordingly, treatment of oxidized LDL with phospholipase A(1) greatly reduced phospholipid mass, but did not decrease its PAF-like activity. Tandem mass spectrometry identified traces of PAF, and more abundant levels of 1-O-hexadecyl-2-(butanoyl or butenoyl)-sn-glycero-3-phosphocholines (C(4)-PAF analogs) in oxidized LDL that comigrated with PAF-like activity. Synthesis showed that either C(4)-PAF was just 10-fold less potent than PAF as a PAF receptor ligand and agonist. Quantitation by gas chromatography-mass spectrometry of pentafluorobenzoyl derivatives shows the C(4)-PAF analogs were 100-fold more abundant in oxidized LDL than PAF. Oxidation of synthetic alkyl arachidonoyl phosphatidylcholine generated these C(4)-PAFs in abundance. These results show that quite minor constituents of the LDL phosphatidylcholine pool are the exclusive precursors for PAF-like bioactivity in oxidized LDL.     

Dualism of oxidized lipoproteins in provoking and attenuating the oxidative burst in macrophages: role of peroxisome proliferator-activated receptor-gamma

Activation and deactivation of macrophages are of considerable importance during the development of various disease states, atherosclerosis among others. Macrophage activation is achieved by oxidized lipoproteins (oxLDL) and is determined by oxygen radical (ROS) formation. The oxidative burst was measured by flow cytometry and quantitated by oxidation of the redox-sensitive dye dichlorodihydrofluorescein diacetate. Short-time stimulation dose-dependently elicited ROS formation. Diphenylene iodonium prevented ROS formation, thus pointing to the involvement of a NAD(P)H oxidase in producing reduced oxygen species. In contrast, preincubation of macrophages with oxLDL for 16 h showed an attenuated oxidative burst upon a second contact with oxLDL. Taking into account that oxLDL is an established peroxisome proliferator-activated receptor-gamma (PPARgamma) agonist and considering the anti-inflammatory properties of PPARgamma, we went on and showed that a PPARgamma agonist such as ciglitazone attenuated ROS formation. Along that line, major lipid peroxidation products of oxLDL, such as 9- and 13-hydroxyoctadecadienoic acid, shared that performance. Supporting evidence that PPARgamma activation accounted for reduced ROS generation came from studies in which proliferator-activated receptor response element decoy oligonucleotides, but not a mutated oligonucleotide, supplied in front of oxLDL delivery regained a complete oxidative burst upon cell activation. We conclude that oxLDL not only elicits an oxidative burst upon first contact, but also promotes desensitization of macrophages via activation of PPARgamma. Desensitization of macrophages may have important consequences for the behavior of macrophages/foam cells in atherosclerotic lesions.     

Regulation of platelet-activating factor (PAF) activity in human diseases by phospholipase A2 inhibitors, PAF acetylhydrolases, PAF receptor antagonists and free radical scavengers.

The aim of this review is to present recent findings indicating the likely involvement of platelet-activating factor (PAF) in human diseases, and possible ways of alleviating its harmful effects. PAF is a potent proinflammatory mediator and promotes adhesive interactions between leukocytes and endothelial cells, leading to transendothelial migration of leukocytes, by a process of juxtacrine intercellular signalling. This process leads to activation of leukocytes and the release of reactive oxygen radicals, lipid mediators, cytokines and enzymes. These reaction products subsequently contribute to the pathological features of various inflammatory diseases. The reactive oxygen radicals cause low density lipoprotein (LDL) oxidation which mediates the development of atherosclerosis. Oxidized LDL may damage cellular and subcellular membranes, leading to tissue injury and cell death. Among the therapeutic approaches considered are agents that inhibit/degrade proinflammatory mediators and thereby have anti-inflammatory and/or anti-atherogenic potential. These include inhibitors of phospholipase A2 activity, PAF-acetylhydrolases, PAF antagonists and free radical scavengers/antioxidants, the latter protecting against oxidized LDL-induced cytotoxicity.     

Lipopolysaccharide-induced leukocyte lipid body formation in vivo: innate immunity elicited intracellular Loci involved in eicosanoid metabolism

Lipid bodies are rapidly inducible, specialized cytoplasmic domains for eicosanoid-forming enzyme localization, which we hypothesize to have specific roles in enhanced inflammatory mediator production during pathological conditions, including sepsis. However, little is known about the origins, composition, or functions of lipid bodies in vivo. We show that lipid body numbers were increased in leukocytes from septic patients in comparison with healthy subjects. Analogously, the intrathoracic administration of LPS into mice induced a dose- and time-dependent increase in lipid body numbers. Pretreatment with anti-CD14 or anti-CD11b/CD18 mAb drastically inhibited LPS-induced lipid body formation. Moreover, LPS failed to form lipid bodies in C3H/HeJ (TLR4 mutated) mice, demonstrating a requisite role for LPS receptors in lipid body formation. LPS-induced lipid body formation was also inhibited by the platelet-activating factor-receptor antagonists, suggesting a role for endogenous platelet-activating factor. The eicosanoid-forming enzymes, 5-lipoxygenase and cyclooxygenase-2, were immunolocalized within experimentally induced (LPS in mice) or naturally occurring (septic patients) lipid bodies. The proinflammatory cytokine involved in the pathogenesis of sepsis, TNF-alpha, was also shown to colocalize within lipid bodies. Prior stimulation of leukocytes to form lipid bodies enhanced the capacity of leukocytes to produce leukotriene B(4) and PGE(2). In conclusion, our studies indicate that lipid bodies formed after LPS stimulation and sepsis are sites for eicosanoid-forming enzymes and cytokine localization and may develop and function as structurally distinct, intracellular sites for paracrine eicosanoid synthesis during inflammatory conditions.     

Sensing environmental lipids by dendritic cell modulates its function

Because of its oxidative modification during the acute-phase response to an aggression, low density lipoprotein (LDL) can be regarded as a source of lipid mediators that can act both to promote and inhibit inflammation. This can be exemplified by the production of anti-inflammatory oxidized fatty acids and proinflammatory lysophosphatidylcholine (LPC) during LDL oxidation. We have shown previously that oxidized LDL (oxLDL) plays an active role at the interface between innate and adaptive immunity by delivering instructive molecules such as LPC, which promotes mature dendritic cell (DC) generation from differentiating monocytes. It is shown in this study that LPC affects the signaling pathway of peroxisome proliferator-activated receptors (PPARs). LPC-induced DC maturation is associated with complete inhibition of PPARgamma activity and up-regulation of the activity of an uncharacterized nuclear receptor that bind peroxisome proliferator response element. Oxidized fatty acids generated during LDL oxidation are natural ligands for PPARgamma and inhibit oxLDL- and LPC-induced maturation. Inhibition experiments with synthetic PPARgamma ligands suggested a PPARgamma-dependent and independent effect of LPC on DC maturation. Therefore, the relative amount of oxidized fatty acids and LPC influences the immunological functions of oxLDL on DC, in part by regulating the PPAR pathway. By sensing the biochemical composition of lipoprotein particles, the innate immune system may thus identify various endogenous signals that influence the immune response during the acute-phase reaction. The therapeutic emulsion intralipid also blocks LPC action on PPAR activity and DC maturation. Intralipid may thus be an alternative therapeutic strategy for some chronic inflammatory diseases.     

Oxidized low density lipoprotein inhibits interleukin-12 production in lipopolysaccharide-activated mouse macrophages via direct interactions between peroxisome proliferator-activated receptor-gamma and nuclear factor-kappa B

Lipopolysaccharide (LPS) increases the production of interleukin-12 (IL-12) from mouse macrophages via a kappaB site within the IL-12 p40 promoter. In this study, we found that oxidized low density lipoprotein (oxLDL) inhibited this LPS-stimulated production of IL-12 in a dose-dependent manner while native LDL did not. OxLDL inhibited p40 promoter activation in monocytic RAW264.7 cells transiently transfected with p40 promoter/reporter constructs, and the repressive effect mapped to a region in the p40 promoter containing a binding site for nuclear factor-kappaB (NF-kappaB) (p40-kappaB). Activation of macrophages by LPS in the presence of oxLDL resulted in markedly reduced binding to the kappaB site, as demonstrated by the electrophoretic mobility shift assays. In contrast, native LDL did not inhibit the IL-12 p40 promoter activation and NF-kappaB binding to the kappaB sites, suggesting that oxidative modification of LDL was crucial for the inhibition of NF-kappaB-mediated IL-12 production. 9-Hydroxyoctadecadienoic acid, a major oxidized lipid component of oxLDL, significantly inhibited IL-12 production in LPS-stimulated mouse macrophages and also suppressed NF-kappaB-mediated activation in IL-12 p40 promoter. The NF-kappaB components p50 and p65 directly bound peroxisome proliferator-activated receptor-gamma (PPAR-gamma) in vitro. In cotransfections of CV-1 and HeLa cells, PPAR-gamma inhibited the NF-kappaB transactivation in an oxLDL-dependent manner. From these results, we propose that oxLDL-mediated suppression of the IL-12 production from LPS-activated mouse macrophages may, at least in part, involve both inhibition of the NF-kappaB-DNA interactions and physical interactions between NF-kappaB and PPAR-gamma.     

Implication of lipoprotein associated phospholipase A2 activity in oxLDL uptake by macrophages

Recognition and uptake of oxidized LDL (oxLDL) by scavenger receptors of macrophages and foam cell formation are mediated by the oxidatively modified apolipoprotein B (ApoB) and lipid moiety of oxLDL. A great amount of oxidized phosphatidylcholine (oxPC) of oxLDL is hydrolyzed at the sn-2 position by lipoprotein associated phospholipase A₂ (Lp-PLA₂) to lysophosphatidylcholine and small oxidation products. This study examines the involvement of Lp-PLA₂ in the uptake of oxLDL by mouse peritoneal macrophages. LDL with intact Lp-PLA₂ activity [LDL (+)] and LDL with completely inhibited Lp-PLA₂ activity [LDL (-)] were subjected to oxidation with 5 μM CuSO₄ for 6 h [moderately oxLDL (MoxLDL)], or 24 h [heavily oxLDL (HoxLDL)] and peritoneal macrophages were incubated with these preparations. The uptake of MoxLDL(-) was about 30% increased compared with that of MoxLDL(+), and HoxLDL(-) uptake was about 20% increased compared with that of HoxLDL(+). Inhibition of Lp-PLA₂ activity had no effect on the uptake of ApoB-liposomes conjugates with ApoB isolated from MoxLDL(-), MoxLDL(+), HoxLDL(-), and HoxLDL(+). Liposomes prepared from the lipid extract of MoxLDL(-), MoxLDL(+), HoxLDL(-), and HoxLDL(+) exhibited a similar pattern to that observed in the uptake of the corresponding intact lipoproteins. This study suggests that the progressive inactivation of Lp-PLA₂ during LDL oxidation leads to an increased uptake of oxLDL by macrophages, which could be primarily attributed to the increased uptake of the oxidized phospholipids enriched lipid moiety of oxLDL.     

Adenovirus-mediated gene transfer of Lp-PLA2 reduces LDL degradation and foam cell formation in vitro

Oxidation of LDL generates biologically active platelet-activating factor (PAF)-like phospholipid derivatives, which have potent proinflammatory activity. These products are inactivated by lipoprotein-associated phospholipase A2 (Lp-PLA2), an enzyme capable of hydrolyzing PAF-like phospholipids. In this study, we generated an adenovirus (Ad) encoding human Lp-PLA2 and injected 10(8), 10(9), and 10(10) plaque-forming unit doses of Adlp-PLA2 and control AdlacZ intra-arterially into rabbits to achieve overexpression of Lp-PLA2 in liver and in vivo production of Lp-PLA2-enriched LDL. As a result, LDL particles with 3-fold increased Lp-PLA2 activity were produced with the highest virus dose. Increased Lp-PLA2 activity in LDL particles decreased the degradation rate in RAW 264 macrophages after standard in vitro oxidation to 60-80% compared with LDL isolated from LacZ-transduced control rabbits. The decrease was proportional to the virus dose and Lp-PLA2 activity. Lipid accumulation and foam cell formation in RAW 264 macrophages were also decreased when incubated with oxidized LDL containing the highest Lp-PLA2 activity. Inhibition of the Lp-PLA2 activity in the LDL particles led to an increase in lipid accumulation and foam cell formation. It is concluded that increased Lp-PLA2 activity in LDL attenuates foam cell formation and decreases LDL oxidation and subsequent degradation in macrophages.     

Differential effects of low-density lipoprotein and chylomicron remnants on lipid accumulation in human macrophages

The effects of low-density lipoprotein (LDL) and chylomicron remnants on lipid accumulation in human monocyte-derived macrophages (HMDMs) and in macrophages derived from the human monocyte cell line THP-1 were compared. The HMDMs or THP-1 macrophages were incubated with LDL, oxidized LDL (oxLDL), chylomicron remnant-like particles (CMR-LPs), or oxidized CMR-LPs (oxCMR-LPs), and the amount and type of lipid accumulated were determined. As expected, the lipid content of both cell types was increased markedly by oxLDL but not LDL, and this was due to a rise in cholesterol, cholesteryl ester (CE), and triacylglycerol (TG) levels. In contrast, both CMR-LPs and oxCMR-LPs caused a considerable increase in cellular lipid in HMDMs and THP-1 macrophages, but in this case there was a greater rise in the TG than in the cholesterol or CE content. Lipid accumulation in response to oxLDL, CMR-LPs, and oxCMR-LPs was prevented by the ACAT inhibitor CI976 in HMDMs but not in THP-1 macrophages, where TG levels remained markedly elevated. The rate of incorporation of [(3)H]oleate into CE and TG in THP-1 macrophages was increased by oxLDL, CMR-LPs, and oxCMR-LPs, but incorporation into TG was increased to a greater extent with CMR-LPs and oxCMR-LPs compared with oxLDL. These results demonstrate that both CMR-LPs and oxCMR-LPs cause lipid accumulation in human macrophages comparable to that seen with oxLDL and that oxidation of the remnant particles does not enhance this effect. They also demonstrate that a greater proportion of the lipid accumulated in response to CMR-LPs compared with oxLDL is TG rather than cholesterol or CE and that this is associated with a higher rate of TG synthesis. This study, therefore, provides further evidence to suggest that chylomicron remnants have a role in foam cell formation that is distinct from that of oxLDL.     

Oxidized low density lipoproteins downregulate LPS-induced platelet-activating factor receptor expression in human monocyte-derived macrophages: implications for LPS-induced nuclear factor-kappaB binding activity.

Monocytes/macrophages play a key role in atherogenesis due to their inflammatory properties including formation of lipid mediators such as platelet-activating-factor (PAF). We investigated the effect of oxidized low-density lipoprotein (oxLDL) on lipopolysaccharide (LPS)-induced PAF receptor (PAF-R) expression in human macrophages and the implication of the nuclear factor (NF)-kappaB in this regulation. LPS-treatment (1 microg.mL(-1)) of macrophages increased PAF binding and PAF-R mRNA expression by 56% (P < 0.05) and twofold (P < 0.01), respectively. In contrast, highly oxidized low-density lipoprotein [ox24hLDL; 100 microg.mL(-1); thiobarbituric acid reacting substances: 31 +/- 3 nmol equiv. malondialdehyde (MDA).mg protein LDL-1] diminished PAF-R expression (-69%; P < 0.05) and mRNA level (- 45%; P < 0.01). LPS pretreatment induced the activated form of p65 in the nuclear compartment of macrophages (detected by Western blotting) and NF-kappaB binding activity (by electrophoretic mobility shift assay). Treatment of macrophages with ox24hLDL suppressed the LPS-induced binding of NF-kappaB to DNA. In addition, treatment of macrophages with lysophosphatidylcholine (2 and 10 microM), a major component of oxLDL, inhibited the LPS-induced NF-kappaB binding to DNA and reduced PAF binding by 30 and 70%, respectively. In conclusion, oxLDL may downregulate PAF-R expression in human macrophages by inhibiting LPS-induced NF-kappaB binding to DNA.     

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.     

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.     

OxLDL up-regulates Niemann-Pick type C1 expression through ERK1/2/COX-2/PPARα-signaling pathway in macrophages

The Niemann-Pick type C1 (NPC1) is located mainly in the membranes of the late endosome/lysosome and controls the intracellular cholesterol trafficking from the late endosome/lysosome to the plasma membrane. It has been reported that oxidized low-density lipoprotein (oxLDL) can up-regulate NPC1 expression. However, the detailed mechanisms are not fully understood. In this study, we investigated the effect of oxLDL stimulation on NPC1 expression in THP-1 macrophages. Our results showed that oxLDL up-regulated NPC1 expression at both mRNA and protein levels in a dose-dependent and time-dependent manner. In addition, oxLDL also induced the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2). Treatment with oxLDL significantly increased cyclooxygenase-2 (COX-2) mRNA and protein expression in the macrophages, and these increases were suppressed by the ERK1/2 inhibitor PD98059 or ERK1/2 small interfering RNA (siRNA) treatment. OxLDL up-regulated the expression of peroxisome proliferator-activated receptor α (PPARα) at the mRNA and protein levels, which could be abolished by COX-2 siRNA or COX-2 inhibitor NS398 treatment in these macrophages. OxLDL dramatically elevated cellular cholesterol efflux, which was abrogated by inhibiting ERK1/2 and/or COX-2. In addition, oxLDL-induced NPC1 expression and cellular cholesterol efflux were reversed by PPARα siRNA or GW6471, an antagonist of PPARα. Taken together, these results provide the evidence that oxLDL can up-regulate the expression of the NPC1 through ERK1/2/COX-2/PPARα-signaling pathway in macrophages.     

Phospholipids in oxidized LDL not adducted to apoB are recognized by the CD36 scavenger receptor

Previous studies have shown that oxidation of low-density lipoprotein (oxLDL) results in its recognition by scavenger receptors on macrophages. Whereas blockage of lysyl residues on apoB-100 of oxLDL by lipid peroxidation products appears to be critical for recognition by the scavenger receptor class A (SR-A), modification of the lipid moiety has been suggested to be responsible for recognition by the scavenger class B receptor, CD36. We studied the recognition by scavenger receptors of oxidized LDL in which lysyl residues are blocked prior to oxidation through methylation [ox(m)LDL]. This permits us to minimize any contribution of modified apoB-100 to the recognition of oxLDL, but does not disrupt the native configuration of lipids in the particle. We found that ox(m)LDL was recognized by receptors on mouse peritoneal macrophages (MPM) almost as well as oxLDL. Ox(m)LDL was recognized by CD36-transfected cells but not by SR-A-transfected cells. Oxidized phospholipids (oxPC) transferred from oxLDL or directly from oxPC to LDL, conveyed recognition by CD36-transfected cells, confirming that CD36 recognized unbound oxidized phospholipids in ox(m)LDL. Collectively, these results suggest that oxPC not adducted to apoB within the intact oxLDL particle are recognized by the macrophage scavenger receptor CD36, that these lipids are not recognized by SR-A, and that they can transfer from oxidized to unoxidized LDL and induce CD36 recognition.     

Vav protein guanine nucleotide exchange factor regulates CD36 protein-mediated macrophage foam cell formation via calcium and dynamin-dependent processes

Atherosclerosis, a chronic inflammatory disease, results in part from the accumulation of modified lipoproteins in the arterial wall and formation of lipid-laden macrophages, known as "foam cells." Recently, we reported that CD36, a scavenger receptor, contributes to activation of Vav-family guanine nucleotide exchange factors by oxidatively modified LDL in macrophages. We also discovered that CD36-dependent uptake of oxidized LDL (oxLDL) in vitro and foam cell formation in vitro and in vivo was significantly reduced in macrophages deficient of Vav proteins. The goal of the present study was to identify the mechanisms by which Vav proteins regulate CD36-dependent foam cell formation. We now show that a Vav-dynamin signaling axis plays a critical role in generating calcium signals in mouse macrophages exposed to CD36-specific oxidized phospholipid ligands. Chelation of intracellular Ca(2+) or inhibition of phospholipase C-γ (PLC-γ) inhibited Vav activation (85 and 70%, respectively, compared with vehicle control) and reduced foam cell formation (approximately 75%). Knockdown of expression by siRNA or inhibition of GTPase activity of dynamin 2, a Vav-interacting protein involved in endocytic vesicle fission, significantly blocked oxLDL uptake and inhibited foam cell formation. Immunofluorescence microscopy studies showed that Vav1 and dynamin 2 colocalized with internalized oxLDL in macrophages and that activation and mobilization of dynamin 2 by oxLDL was impaired in vav null cells. These studies identified previously unknown components of the CD36 signaling pathway, demonstrating that Vav proteins regulate oxLDL uptake and foam cell formation via calcium- and dynamin 2-dependent processes and thus represent novel therapeutic targets for atherosclerosis.     

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.     

Oridonin attenuates atherosclerosis by inhibiting foam macrophage formation and inflammation through FABP4/PPARγ signalling

Both lipid accumulation and inflammatory response in lesion macrophages fuel the progression of atherosclerosis, leading to high mortality of cardiovascular disease. A therapeutic strategy concurrently targeting these two risk factors is promising, but still scarce. Oridonin, the bioactive medicinal compound, is known to protect against inflammatory response and lipid dysfunction. However, its effect on atherosclerosis and the underlying molecular mechanism remain elusive. Here, we showed that oridonin attenuated atherosclerosis in hyperlipidemic ApoE knockout mice. Meanwhile, we confirmed the protective effect of oridonin on the oxidized low-density lipoprotein (oxLDL)-induced foam macrophage formation, resulting from increased cholesterol efflux, as well as reduced inflammatory response. Mechanistically, the network pharmacology prediction and further experiments revealed that oridonin dramatically facilitated the expression of peroxisome proliferator-activated receptor gamma (PPARγ), thereby regulating liver X receptor-alpha (LXRα)-induced ATP-binding cassette transporter A1 (ABCA1) expression and nuclear factor NF-kappa-B (NF-κB) translocation. Antagonist of PPARγ reversed the cholesterol accumulation and inflammatory response mediated by oridonin. Besides, RNA sequencing analysis revealed that fatty acid binding protein 4 (FABP4) was altered responding to lipid modulation effect of oridonin. Overexpression of FABP4 inhibited PPARγ activation and blunted the benefit effect of oridonin on foam macrophages. Taken together, oridonin might have potential to protect against atherosclerosis by modulating the formation and inflammatory response in foam macrophages through FABP4/PPARγ signalling.