Regulation of peroxisome proliferator-activated receptor-gamma-mediated gene expression. A new mechanism of action for high density lipoprotein

Cellular cholesterol content reflects a balance of lipid influx by lipoprotein receptors and endogenous synthesis and efflux to cholesterol acceptor particles. The beneficial effect of high density lipoprotein (HDL) in protecting against the development of cardiovascular disease is thought to be mediated predominately through its induction of cellular cholesterol efflux and "reverse cholesterol transport" from peripheral tissues to the liver. We tested the hypothesis that HDL could inhibit cellular lipid accumulation by modulating expression of peroxisome proliferator-activated receptor-gamma (PPARgamma)-responsive genes. To this end, we evaluated expression of two PPARgamma-responsive genes, CD36, a receptor for oxidized low density lipoprotein, and aP2, a fatty acid-binding protein. HDL decreased expression of macrophage CD36 and aP2 in a dose-dependent manner. HDL also decreased aP2 expression in fibroblasts, reduced accumulation of lipid, and slowed differentiation of fibroblasts into adipocytes. HDL stimulated mitogen-activated protein (MAP) kinase activity, and inhibition of CD36 expression was blocked by co-incubation with a MAP kinase inhibitor. HDL increased expression of PPARgamma mRNA and protein, induced translocation of PPARgamma from the cytoplasm to the nucleus, and increased PPARgamma phosphorylation. Our data demonstrate that despite induction and translocation of PPARgamma in response to HDL, MAP kinase-mediated phosphorylation of PPARgamma inhibited expression of PPARgamma-responsive genes and suggest mechanisms by which HDL may inhibit cellular lipid accumulation.     

The role of lipolysis in mediating the proinflammatory effects of very low density lipoproteins in mouse peritoneal macrophages

Hypertriglyceridemia is an important risk factor for atherosclerosis, especially in obesity. Macrophages are one of the primary cell types involved in atherogenesis and are thought to contribute to lesion formation through both lipid accumulation and proinflammatory gene expression. In this study, we sought to determine the direct impact of triglyceride (TG)-rich VLDL-induced lipid accumulation on macrophage proinflammatory processes. Incubation of mouse peritoneal macrophages with 100 microg/ml VLDL for 6 h led to 2.8- and 3.7-fold increases in intracellular TGs and FFAs, respectively (P < 0.05). The inflammatory proteins tumor necrosis factor-alpha, interleukin-1beta, monocyte chemoattractant protein-1, intercellular adhesion molecule-1, matrix metalloproteinase 3 (MMP3), and macrophage inflammatory protein-1alpha (MIP-1alpha) were all upregulated by at least 2-fold (P < 0.05) in a dose-dependent manner in VLDL-treated macrophages. The increase in inflammatory gene expression coincided with the phosphorylation of the mitogen-activated protein kinase (MAPK) pathway members extracellular signal-regulated kinase (ERK) 1/2, stress-activated protein kinase/c-Jun NH2-terminal kinase, and p38 MAPK and was ameliorated by U0126, an inhibitor of ERK1/2. Inhibition of extracellular TG hydrolysis with tetrahydrolipstatin (Orlistat) resulted in the absence of intracellular TG and FFA accumulation and was accompanied by the amelioration of ERK1/2 phosphorylation and MIP-1alpha gene expression. These data indicate that VLDL hydrolysis, and the subsequent accumulation of intracellular FFAs and TGs, plays a substantive role in mediating the proinflammatory effects of VLDL. These data have important implications for the direct proatherogenic effects of VLDL on macrophage-driven atherosclerosis.     

A pyrene based fluorescence approach to study conformation of apolipoprotein E3 in macrophage-generated nascent high density lipoprotein

Apolipoprotein E3 (apoE3) is an anti-atherogenic apolipoprotein with the ability to exist in lipid-free and lipoprotein-associated states. During atherosclerosis, its function in promoting cholesterol efflux from macrophages via the ATP-binding cassette transporter A1 (ABCA1) takes a prominent role, leading to generation of nascent high density lipoprotein (nHDL) particles. The objective of this study is to understand the conformation adopted by apoE3 in macrophage-generated nHDL using a fluorescence spectroscopic approach involving pyrene. Pyrene-labeled recombinant human apoE3 displayed a robust ability to stimulate ABCA1-mediated cholesterol efflux from cholesterol-loaded J774 macrophages (which do not express apoE), comparable to that elicited by unlabeled apoE3. The nHDL recovered from the conditioned medium revealed the presence of apoE3 by immunoblot analysis. A heterogeneous population of nHDL bearing exogenously added apoE3 was generated with particle size varying from ∼12 to ∼19 nm in diameter, corresponding to molecular mass of ∼450 to ∼700 kDa. The lipid: apoE3 ratio varied from ∼60:1 to 10:1. A significant extent of pyrene excimer emission was noted in nHDL, indicative of spatial proximity between Cys112 on neighboring apoE3 molecules similar to that noted in reconstituted HDL. Cross-linking analysis using Cys-specific cross-linkers revealed the predominant presence of dimers. Taken together the data indicate a double belt arrangement of apoE molecules on nHDL. A similar organization of the C-terminal tail of apoE on nHDL was noted when pyrene-apoEA277C(201–299) was used as the cholesterol acceptor. These studies open up the possibility of using exogenously labeled apoE3 to generate nHDL for structural and conformational analysis.     

Nascent high density lipoproteins formed by ABCA1 resemble lipid rafts and are structurally organized by three apoA-I monomers

This report details the lipid composition of nascent HDL (nHDL) particles formed by the action of the ATP binding cassette transporter A1 (ABCA1) on apolipoprotein A-I (apoA-I). nHDL particles of different size (average diameters of ～ 12, 10, 7.5, and <6 nm) and composition were purified by size-exclusion chromatography. Electron microscopy suggested that the nHDL were mostly spheroidal. The proportions of the principal nHDL lipids, free cholesterol, glycerophosphocholine, and sphingomyelin were similar to that of lipid rafts, suggesting that the lipid originated from a raft-like region of the cell. Smaller amounts of glucosylceramides, cholesteryl esters, and other glycerophospholipid classes were also present. The largest particles, ～ 12 nm and 10 nm diameter, contained ～ 43% free cholesterol, 2-3% cholesteryl ester, and three apoA-I molecules. Using chemical cross-linking chemistry combined with mass spectrometry, we found that three molecules of apoA-I in the ～ 9-14 nm nHDL adopted a belt-like conformation. The smaller (7.5 nm diameter) spheroidal nHDL particles carried 30% free cholesterol and two molecules of apoA-I in a twisted, antiparallel, double-belt conformation. Overall, these new data offer fresh insights into the biogenesis and structural constraints involved in forming nascent HDL from ABCA1.     

Adverse effects on macrophage lipid transport and survival by high density lipoprotein from patients with coronary heart disease

High density lipoprotein (HDL)–macrophage interactions have the potential to modulate macrophage function in a beneficial way to prevent the development of lipid‐loaded foam cell formation in atherosclerosis. Although HDL is atheroprotective, it can become dysfunctional in chronic inflammatory conditions and increase cardiovascular risk. Here, we examined the effect of dysfunctional‐HDL from patients with coronary artery disease, on macrophage function in comparison to functional‐HDL from controls. Exposure of macrophages to dysfunctional‐HDL for 24 h resulted significant increase in cellular oxidative stress, cholesterol, and cytotoxicity. It also stimulated mitochondrial membrane depolarization, DNA damage, apoptosis, and cleavage of poly (ADP‐ribose) polymerase, which are characteristics of proapoptotic pathways. In contrast, functional‐HDL treatment maintained cholesterol homeostasis, essential membrane potential, DNA integrity, and cell survival. These results demonstrate that HDL from coronary artery disease (CAD) patient promotes proatherogenic effects that in turn trigger macrophage apoptosis, an important feature in atherogenesis and thereby providing new insight in our understanding of the atherogenic mechanisms.     

Over-activation of NMDA receptors promotes ABCA1 degradation and foam cell formation

ATP-binding cassette transporter A1 (ABCA1) is an essential regulator of intracellular cholesterol efflux. Secreted cholesterol binds to lipid-free apolipoprotein A-I (apoA-I) in peripheral blood to constitute high-density lipoprotein cholesterol (HDL) complexes. ABCA1 protein on the surface of macrophages acts as a crucial controller in preventing cholesterol accumulation. Importantly, ABCA1 is unstable and easily degraded via a series of biochemical activities, including but not limited to calpain-mediated and ubiquitin-proteasome system-mediated processes. How accelerated ABCA1 degradation impacts disordered lipid metabolism in macrophages and foam cell formation is unclear. N-methyl d-aspartate receptors (NMDARs) are ionotropic glutamate receptors with high calcium permeability. Calcium influx via NMDARs activates downstream signaling pathways. Over-activation of NMDARs stimulated by NMDA contributes to dysfunctional lipid metabolism in macrophages and foam cell formation via promotion of calpain-mediated ABCA1 proteolysis. However, increased NMDAR activity does not affect liver X receptor expression or ABCA1 mRNA levels. Following NMDA receptor silencing or calpain inhibition, NMDA treatment did not reduce ABCA1 protein levels, nor caused lipid accumulation in macrophages. In addition, NMDAR over-activation activates NF-κB signaling to promote IL-1β and IL-6 macrophage marker expression. However, NMDAR silencing and calpain inhibition reduce inflammatory macrophage responses. In summary, our study suggests that NMDAR activation reduces surface ABCA1 protein, promotes lipid accumulation, and induces the production and secretion of many inflammatory mediators in macrophages, possibly through enhanced calpain-mediated ABCA1 protein degradation. Thus, the NMDAR receptor may be a novel pharmacologic target for atherosclerosis therapy.     

Apolipoprotein E derived HDL mimetic peptide ATI-5261 promotes nascent HDL formation and reverse cholesterol transport in vitro

Modulation of the reverse cholesterol transport (RCT) pathway may provide a therapeutic target for the prevention and treatment of atherosclerotic cardiovascular disease (CVD). In the present study, we evaluated a novel 26-amino acid apolipoprotein mimetic peptide (ATI-5261) designed from the carboxyl terminal of apoE, in its ability to mimic apoA-I functionality in RCT in vitro. Our data shows that nascent HDL-like (nHDL) particles generated by incubating cells over-expressing ABCA1 with ATI-5261 increase the rate of specific ABCA1 dependent lipid efflux, with high affinity interactions with ABCA1. We also show that these nHDL particles interact with membrane micro-domains in a manner similar to nHDL apoA-I. These nHDL particles then interact with the ABCG1 transporter and are remodeled by plasma HDL-modulating enzymes. Finally, we show that these mature HDL-like particles are taken up by SR-BI for cholesterol delivery to liver cells. This ATI-5621-mediated process mimics apoA-I and may provide a means to prevent cholesterol accumulation in the artery wall. In this study, we propose an integrative physiology approach of HDL biogenesis with the synthetic peptide ATI-5261. These experiments provide new insights for potential therapeutic use of apolipoprotein mimetic peptides.     

The ω-carboxyl group of 7-ketocholesteryl-9-carboxynonanoate mediates the binding of oxLDL to CD36 receptor and enhances caveolin-1 expression in macrophages

CD36 signal transduction modulates the uptake of oxidized low-density lipoprotein (oxLDL) and foam cell formation. We previously observed that 7-ketocholesteryl-9-carboxynonanoate (oxLig-1), the lipid moiety of oxLDL, activates the CD36-Src-JNK/ERK1/2 signalling pathway. In this study, we assessed the role of the ω-carboxyl group in the binding of oxLig-1 to CD36 and investigated whether the binding of the ω-carboxyl group to CD36 triggers CD36-mediated signalling, thereby resulting in the upregulation of caveolin-1 expression. Our results showed that oxLig-1 bound to CD36 and that the ω-carboxyl group was critical for this binding. Furthermore, immunoprecipitation and Western blot analyses showed that interaction between the ω-carboxyl group of oxLig-1 and CD36 triggered intracellular Src-JNK/ERK1/2 signal transduction. Moreover, the binding of the ω-carboxyl group to CD36 induced caveolin-1 expression and translocation to the membrane in macrophages. Additionally, inhibitors of Src, JNK and ERK and siRNA targeting CD36 and NF-κB significantly suppressed the enhanced caveolin-1 expression induced by oxLig-1. In conclusion, these observations suggest that oxLig-1 is a critical epitope of oxLDL that mediates the binding of oxLDL to CD36 and activates downstream Src-JNK/ERK1/2-NF-κB signal transduction, resulting in upregulation of caveolin-1 expression in macrophages.     

Disruption of CD36 impairs cytokine response to Plasmodium falciparum glycosylphosphatidylinositol and confers susceptibility to severe and fatal malaria in vivo

CD36 is a scavenger receptor that has been implicated in malaria pathogenesis as well as innate defense against blood-stage infection. Inflammatory responses to Plasmodium falciparum GPI (pfGPI) anchors are believed to play an important role in innate immune response to malaria. We investigated the role of CD36 in pfGPI-induced MAPK activation and proinflammatory cytokine secretion. Furthermore, we explored the role of this receptor in an experimental model of acute malaria in vivo. We demonstrate that ERK1/2, JNK, p38, and c-Jun became phosphorylated in pfGPI-stimulated macrophages. In contrast, pfGPI-induced phosphorylation of JNK, ERK1/2, and c-Jun was reduced in Cd36(-/-) macrophages and Cd36(-/-) macrophages secreted significantly less TNF-alpha in response to pfGPI than their wild-type counterparts. In addition, we demonstrate a role for CD36 in innate immune response to malaria in vivo. Compared with wild-type mice, Cd36(-/-) mice experienced more severe and fatal malaria when challenged with Plasmodium chabaudi chabaudi AS. Cd36(-/-) mice displayed a combined defect in cytokine induction and parasite clearance with a dysregulated cytokine response to infection, earlier peak parasitemias, higher parasite densities, and higher mortality rates than wild-type mice. These results provide direct evidence that pfGPI induces TNF-alpha secretion in a CD36-dependent manner and support a role for CD36 in modulating host cytokine response and innate control of acute blood-stage malaria infection in vivo.     

Oxidized low density lipoprotein decreases macrophage expression of scavenger receptor B-I

Scavenger receptor class B type I (SR-BI) has recently been identified as a high density lipoprotein (HDL) receptor that mediates bidirectional flux of cholesterol across the plasma membrane. We have previously demonstrated that oxidized low density lipoprotein (OxLDL) will increase expression of another class B scavenger receptor, CD36 (Han, J., Hajjar, D. P., Febbraio, M., and Nicholson, A. C. (1997) J. Biol. Chem. 272, 21654-21659). In studies reported herein, we evaluated the effects of OxLDL on expression of SR-BI in macrophages to determine how exposure to this modified lipoprotein could alter SR-BI expression and cellular lipid flux. OxLDL decreased SR-BI expression in a dose- and time-dependent manner. Incubation with OxLDL had no effect on the membrane distribution of SB-BI, and it decreased expression of both cytosolic and membrane protein. Consistent with its effect on SR-BI protein expression, OxLDL decreased SR-BI mRNA in a dose-dependent manner. The ability of OxLDL to decrease SR-BI expression was dependent on the degree of LDL oxidation. OxLDL decreased both [(14)C]cholesteryl oleate/HDL uptake and efflux of [(14)C]cholesterol to HDL in a time-dependent manner. Incubation of macrophages with 7-ketocholesterol, but not free cholesterol, also inhibited expression of SR-BI. Finally, we demonstrate that the effect of OxLDL on SR-BI is dependent on the differentiation state of the monocyte/macrophage. These results imply that in addition to its effect in inducing foam cell formation in macrophages through increased uptake of oxidized lipids, OxLDL may also enhance foam cell formation by altering SR-BI-mediated lipid flux across the cell membrane.     

Interleukin-10 Facilitates Both Cholesterol Uptake and Efflux in Macrophages

Foam cell formation is a hallmark event during atherosclerosis. The current paradigm is that lipid uptake by scavenger receptor in macrophages initiates the chronic proinflammatory cascade and necrosis core formation that characterize atherosclerosis. We report here that a cytokine considered to be anti-atherogenic, interleukin-10 (IL10), promotes cholesterol uptake from modified lipoproteins in macrophages and its transformation into foam cells by increasing the expression of scavenger receptor CD36 and scavenger receptor A. Although uptake of modified lipoproteins is considered proatherogenic, we found that IL10 also increases cholesterol efflux from macrophages to protect against toxicity of free cholesterol accumulation in the cell. This process was PPARγ-dependent and was mediated through up-regulation of ABCA1 (ATP-binding cassette transporter A1) protein expression. Importantly, expression of inflammatory molecules, such as tumor necrosis factor-α, intercellular adhesion molecule-1, and MMP9 as well as apoptosis were dramatically suppressed in lipid-laden foam cells treated with IL10. The notion that IL10 can mediate both the uptake of cholesterol from modified lipoproteins and the efflux of stored cholesterol suggests that the process of foam cell formation is not necessarily detrimental as long as mechanisms of cholesterol efflux and transfer to an exogenous acceptor are functioning robustly. Our results present a comprehensive antiatherogenic role of IL10 in macrophages, including enhanced disposal of harmful lipoproteins, inhibition of inflammatory molecules, and reduced apoptosis.     

Enhanced Hepatic apoA-I Secretion and Peripheral Efflux of Cholesterol and Phospholipid in CD36 Null Mice

CD36 facilitates oxidized low density lipoprotein uptake and is implicated in development of atherosclerotic lesions. CD36 also binds unmodified high and very low density lipoproteins (HDL, VLDL) but its role in the metabolism of these particles is unclear. Several polymorphisms in the CD36 gene were recently shown to associate with serum HDL cholesterol. To gain insight into potential mechanisms for these associations we examined HDL metabolism in CD36 null (CD36^−/−) mice. Feeding CD36^−/− mice a high cholesterol diet significantly increased serum HDL, cholesterol and phospholipids, as compared to wild type mice. HDL apolipoproteins apoA-I and apoA-IV were increased and shifted to higher density HDL fractions suggesting altered particle maturation. Clearance of dual-labeled HDL was unchanged in CD36^−/− mice and cholesterol uptake from HDL or LDL by isolated CD36^−/− hepatocytes was unaltered. However, CD36^−/− hepatocytes had higher cholesterol and phospholipid efflux rates. In addition, expression and secretion of apoA-I and apoA-IV were increased reflecting enhanced PXR. Similar to hepatocytes, cholesterol and phospholipid efflux were enhanced in CD36^−/− macrophages without changes in protein levels of ABCA1, ABCG1 or SR-B1. However, biotinylation assays showed increased surface ABCA1 localization in CD36^−/− cells. In conclusion, CD36 influences reverse cholesterol transport and hepatic ApoA-I production. Both pathways are enhanced in CD36 deficiency, increasing HDL concentrations, which suggests the potential benefit of CD36 inhibition.     

Acrolein Impairs the Cholesterol Transport Functions of High Density Lipoproteins

High density lipoproteins (HDL) are considered athero-protective, primarily due to their role in reverse cholesterol transport, where they transport cholesterol from peripheral tissues to the liver for excretion. The current study was designed to determine the impact of HDL modification by acrolein, a highly reactive aldehyde found in high abundance in cigarette smoke, on the cholesterol transport functions of HDL. HDL was chemically-modified with acrolein and immunoblot and mass spectrometry analyses confirmed apolipoprotein crosslinking, as well as acrolein adducts on apolipoproteins A-I and A-II. The ability of acrolein-modified HDL (acro-HDL) to serve as an acceptor of free cholesterol (FC) from COS-7 cells transiently expressing SR-BI was significantly decreased. Further, in contrast to native HDL, acro-HDL promotes higher neutral lipid accumulation in murine macrophages as judged by Oil Red O staining. The ability of acro-HDL to mediate efficient selective uptake of HDL-cholesteryl esters (CE) into SR-BI-expressing cells was reduced compared to native HDL. Together, the findings from our studies suggest that acrolein modification of HDL produces a dysfunctional particle that may ultimately promote atherogenesis by impairing functions that are critical in the reverse cholesterol transport pathway.     

Regulation of human polymorphonuclear leukocytes functions by the neuropeptide pituitary adenylate cyclase-activating polypeptide after activation of MAPKs

Anti-inflammatory activities of pituitary adenylate cyclase-activating protein (PACAP) are mediated in part through specific effects on lymphocytes and macrophages. This study shows that in human polymorphonuclear neutrophils (PMNs), PACAP acts as a proinflammatory molecule. In PMNs, vaso-intestinal peptide/PACAP receptor 1 (VPAC-1) was the only receptor found to be expressed by RT-PCR. Using VPAC-1 Ab, we found that VPAC-1 mRNA was translated into proteins. In PMNs, PACAP increases cAMP, inositol triphosphate metabolites, and calcium. It activates two of the three members of the MAPK superfamily, the ERK and the stress-activated MAPK p38. U73122, an inhibitor of phospholipase C (PLC), inhibits PACAP-induced ERK activation, whereas p38 MAPK phosphorylation was unaffected. Using specific pharmalogical inhibitors of ERK (PD098059) and p38 MAPK (SB203580), we found that PACAP-mediated calcium increase was ERK and PLC dependent and p38 independent. PACAP primes fMLP-associated calcium increase; it also primes fMLP activation of the respiratory burst as well as elastase release, these last two processes being ERK and PLC dependent and p38 MAPK independent. PACAP also increases membrane expression of CD11b and release of lactoferrin and metallo proteinase-9 (MMP-9). These effects were PLC dependent (CD 11b, lactoferrin, MMP-9), ERK dependent (CD 11b, lactoferrin, MMP-9), and p38 dependent (CD11b, lactoferrin). We conclude that PACAP is a direct PMN activator as well as an effective PMN priming agent that requires PLC, ERK, and p38 MAPK activities.     

Estrogen receptor-alpha mediates gender differences in atherosclerosis induced by HIV protease inhibitors

As part of highly active antiretroviral therapy, protease inhibitor treatment has significantly increased the lifespan of human immunodeficiency virus (HIV)-infected individuals. Many patients, however, develop negative side effects, including premature atherosclerosis. We have previously demonstrated that in male low density lipoprotein receptor (LDL-R) null mice, HIV protease inhibitors induce atherosclerotic lesions and cholesterol accumulation in macrophages in the absence of changes in plasma lipid levels. We determined that these increases were due to an up-regulation of the scavenger receptor, CD36. In the present study, we examined the effects of HIV protease inhibitors in female LDL-R null mice. Female mice given ritonavir and amprenavir (23 and 10 microg/mouse/day, respectively) developed fewer atherosclerotic lesions than males. Furthermore, peritoneal macrophages isolated from ritonavir-treated females had reduced levels of cholesterol accumulation as compared with males, and CD36 protein levels were increased to a significantly lesser degree in females than in males. To investigate the molecular mechanisms of this gender difference, we examined the effect of genetically removing estrogen receptor-alpha (ERalpha). In female mice lacking both LDL-R and ERalpha, the protective effect of gender was lost. Additionally, the reduced levels of cholesterol accumulation in macrophages observed in females was reversed. Furthermore, the absence of ERalpha resulted in increased expression of CD36 protein in a macrophage-specific manner in mice treated with ritonavir. These data demonstrate that ERalpha is directly involved in the regulation of cholesterol metabolism in macrophages and plays an important role in the gender differences observed in HIV protease inhibitor-induced atherosclerosis.     

Wogonin promotes cholesterol efflux by increasing protein phosphatase 2B-dependent dephosphorylation at ATP-binding cassette transporter-A1 in macrophages

Wogonin, one component in Scutellaria baicalensis Georgi extracts, has several beneficial properties for cancers and inflammatory diseases. However, the efficacy of wogonin in cholesterol metabolism of macrophages remains unknown. In macrophages, cholesterol uptake is controlled by scavenger receptors (SR-A and CD36) and cholesterol efflux by SR-BI, ATP-binding cassette transporter-A1 (ABCA1) and ABCG1. In the present study, we investigated the effect and underlying molecular mechanism of wogonin on the formation of macrophage foam cells by murine J774.A1 macrophages. Wogonin attenuated oxidized low-density lipoprotein (oxLDL)-induced cholesterol accumulation in macrophages. The binding of oxLDL to macrophages and protein expression of SR-A and CD36 were not affected by wogonin. Wogonin enhanced cholesterol efflux and increased the protein level of ABCA1 without affecting the protein expression of SR-BI or ABCG1. Inhibition of ABCA1 by pharmacological inhibitor 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid disodium salt or neutralizing antibody abolished this suppressive effect of wogonin on lipid accumulation. Moreover, the up-regulation of ABCA1 protein by wogonin resulted from a decrease in degradation rate of ABCA1 protein, with no effect on ABCA1 mRNA expression. This reduction in ABCA1 degradation was due to increased protein phosphatase 2B (PP2B)-mediated ABCA1 dephosphorylation, as evidenced by increased interaction between ABCA1 and PP2B; pharmacological inhibition of PP2B would prevent wogonin-induced ABCA1 protein expression, dephosphorylation and attenuation of lipid accumulation. Collectively, wogonin increases the protein stability of ABCA1 via PP2B-mediated dephosphorylation, thus leading to reduced cholesterol accumulation in macrophage foam cells.     

Lysosomal cholesterol accumulation in macrophages leading to coronary atherosclerosis in CD38−/− mice

The disruption in transportation of oxLDL‐derived cholesterol and the subsequent lipid accumulation in macrophages are the hallmark events in atherogenesis. Our recent studies demonstrated that lysosomal Ca²⁺ messenger of nicotinic acid adenine dinucleotide phosphate (NAADP), an enzymatic product of CD38 ADP‐ribosylcyclase (CD38), promoted lipid endocytic trafficking in human fibroblast cells. The current studies are designed to examine the functional role of CD38/NAADP pathway in the regulation of lysosomal cholesterol efflux in atherosclerosis. Oil red O staining showed that oxLDL concentration‐dependently increased lipid buildup in bone marrow‐derived macrophages from both wild type and CD38^?/?, but to a significant higher extent with CD38 gene deletion. Bodipy 493/503 fluorescence staining found that the deposited lipid in macrophages was mainly enclosed in lysosomal organelles and largely enhanced with the blockade of CD38/NAADP pathway. Filipin staining and direct measurement of lysosome fraction further revealed that the free cholesterol constituted a major portion of the total cholesterol segregated in lysosomes. Moreover, in situ assay disclosed that both lysosomal lumen acidity and the acid lipase activity were reduced upon cholesterol buildup in lysosomes. In CD38^?/? mice, treatment with Western diet (12 weeks) produced atherosclerotic damage in coronary artery with striking lysosomal cholesterol sequestration in macrophages. These data provide the first experimental evidence that the proper function of CD38/NAADP pathway plays an essential role in promoting free cholesterol efflux from lysosomes and that a defection of this signalling leads to lysosomal cholesterol accumulation in macrophages and results in coronary atherosclerosis in CD38^?/? mice.     

CD36 Is a Novel Serum Amyloid A (SAA) Receptor Mediating SAA Binding and SAA-induced Signaling in Human and Rodent Cells

Serum amyloid A (SAA) is a major acute phase protein involved in multiple physiological and pathological processes. This study provides experimental evidence that CD36, a phagocyte class B scavenger receptor, functions as a novel SAA receptor mediating SAA proinflammatory activity. The uptake of Alexa Fluor® 488 SAA as well as of other well established CD36 ligands was increased 5–10-fold in HeLa cells stably transfected with CD36 when compared with mock-transfected cells. Unlike other apolipoproteins that bind to CD36, only SAA induced a 10–50-fold increase of interleukin-8 secretion in CD36-overexpressing HEK293 cells when compared with control cells. SAA-mediated effects were thermolabile, inhibitable by anti-SAA antibody, and also neutralized by association with high density lipoprotein but not by association with bovine serum albumin. SAA-induced cell activation was inhibited by a CD36 peptide based on the CD36 hexarelin-binding site but not by a peptide based on the thrombospondin-1-binding site. A pronounced reduction (up to 60–75%) of SAA-induced pro-inflammatory cytokine secretion was observed in cd36^−/− rat macrophages and Kupffer cells when compared with wild type rat cells. The results of the MAPK phosphorylation assay as well as of the studies with NF-κB and MAPK inhibitors revealed that two MAPKs, JNK and to a lesser extent ERK1/2, primarily contribute to elevated cytokine production in CD36-overexpressing HEK293 cells. In macrophages, four signaling pathways involving NF-κB and three MAPKs all appeared to contribute to SAA-induced cytokine release. These observations indicate that CD36 is a receptor mediating SAA binding and SAA-induced pro-inflammatory cytokine secretion predominantly through JNK- and ERK1/2-mediated signaling.