Serum amyloid A is not incorporated into HDL during HDL biogenesis.

Liver-derived serum amyloid A (SAA) is present in plasma where it is mainly associated with HDL and from which it is cleared more rapidly than are the other major HDL-associated apolipoproteins. Although evidence suggests that lipid-free and HDL-associated forms of SAA have different activities, the pathways by which SAA associates and disassociates with HDL are poorly understood. In this study, we investigated SAA lipidation by hepatocytes and how this lipidation relates to the formation of nascent HDL particles. We also examined hepatocyte-mediated clearance of lipid-free and HDL-associated SAA. We prepared hepatocytes from mice injected with lipopolysaccharide or an SAA-expressing adenoviral vector. Alternatively, we incubated primary hepatocytes from SAA-deficient mice with purified SAA. We analyzed conditioned media to determine the lipidation status of endogenously produced and exogenously added SAA. Examining the migration of lipidated species, we found that SAA is lipidated and forms nascent particles that are distinct from apoA-I-containing particles and that apoA-I lipidation is unaltered when SAA is overexpressed or added to the cells, indicating that SAA is not incorporated into apoA-I-containing HDL during HDL biogenesis. Like apoA-I formation, generation of SAA-containing particles was dependent on ABCA1, but not on scavenger receptor class B type I. Hepatocytes degraded significantly more SAA than apoA-I. Taken together, our results indicate that SAA's lipidation and metabolism by the liver is independent of apoA-I and that SAA is not incorporated into HDL during HDL biogenesis.

Hepatocytes direct the formation of a pro-metastatic niche in the liver.

The liver is the most common site of metastatic disease1. Although this metastatic tropism may reflect the mechanical trapping of circulating tumour cells, liver metastasis is also dependent, at least in part, on the formation of a 'pro-metastatic' niche that supports the spread of tumour cells to the liver2,3. The mechanisms that direct the formation of this niche are poorly understood. Here we show that hepatocytes coordinate myeloid cell accumulation and fibrosis within the liver and, in doing so, increase the susceptibility of the liver to metastatic seeding and outgrowth. During early pancreatic tumorigenesis in mice, hepatocytes show activation of signal transducer and activator of transcription 3 (STAT3) signalling and increased production of serum amyloid A1 and A2 (referred to collectively as SAA). Overexpression of SAA by hepatocytes also occurs in patients with pancreatic and colorectal cancers that have metastasized to the liver, and many patients with locally advanced and metastatic disease show increases in circulating SAA. Activation of STAT3 in hepatocytes and the subsequent production of SAA depend on the release of interleukin 6 (IL-6) into the circulation by non-malignant cells. Genetic ablation or blockade of components of IL-6-STAT3-SAA signalling prevents the establishment of a pro-metastatic niche and inhibits liver metastasis. Our data identify an intercellular network underpinned by hepatocytes that forms the basis of a pro-metastatic niche in the liver, and identify new therapeutic targets.

Serum amyloid A binds to fibrin(ogen), promoting fibrin amyloid formation.

Complex associations exist between inflammation and thrombosis, with the inflammatory state tending to promote coagulation. Fibrinogen, an acute phase protein, has been shown to interact with the amyloidogenic ß-amyloid protein of Alzheimer's disease. However, little is known about the association between fibrinogen and serum amyloid A (SAA), a highly fibrillogenic protein that is one of the most dramatically changing acute phase reactants in the circulation. To study the role of SAA in coagulation and thrombosis, in vitro experiments were performed where purified human SAA, in concentrations resembling a modest acute phase response, was added to platelet-poor plasma (PPP) and whole blood (WB), as well as purified and fluorescently labelled fibrinogen. Results from thromboelastography (TEG) suggest that SAA causes atypical coagulation with a fibrin(ogen)-mediated increase in coagulation, but a decreased platelet/fibrin(ogen) interaction. In WB scanning electron microscopy analysis, SAA mediated red blood cell (RBC) agglutination, platelet activation and clumping, but not platelet spreading. Following clot formation in PPP, the presence of SAA increased amyloid formation of fibrin(ogen) as determined both with auto-fluorescence and with fluorogenic amyloid markers, under confocal microcopy. SAA also binds to fibrinogen, as determined with a fluorescent-labelled SAA antibody and correlative light electron microscopy (CLEM). The data presented here indicate that SAA can affect coagulation by inducing amyloid formation in fibrin(ogen), as well as by propelling platelets to a more prothrombotic state. The discovery of these multiple and complex effects of SAA on coagulation invite further mechanistic analyses.

High-density lipoprotein inhibits serum amyloid A-mediated reactive oxygen species generation and NLRP3 inflammasome activation.

Serum amyloid A (SAA) is a high-density apolipoprotein whose plasma levels can increase more than 1000-fold during a severe acute-phase inflammatory response and are more modestly elevated in chronic inflammation. SAA is thought to play important roles in innate immunity, but its biological activities have not been completely delineated. We previously reported that SAA deficiency protects mice from developing abdominal aortic aneurysms (AAAs) induced by chronic angiotensin II (AngII) infusion. Here, we report that SAA is required for AngII-induced increases in interleukin-1ß (IL-1ß), a potent proinflammatory cytokine that is tightly controlled by the Nod-like receptor protein 3 (NLRP3) inflammasome and caspase-1 and has been implicated in both human and mouse AAAs. We determined that purified SAA stimulates IL-1ß secretion in murine J774 and bone marrow-derived macrophages through a mechanism that depends on NLRP3 expression and caspase-1 activity, but is independent of P2X7 nucleotide receptor (P2X7R) activation. Inhibiting reactive oxygen species (ROS) by N-acetyl-l-cysteine or mito-TEMPO and inhibiting activation of cathepsin B by CA-074 blocked SAA-mediated inflammasome activation and IL-1ß secretion. Moreover, inhibiting cellular potassium efflux with glyburide or increasing extracellular potassium also significantly reduced SAA-mediated IL-1ß secretion. Of note, incorporating SAA into high-density lipoprotein (HDL) prior to its use in cell treatments completely abolished its ability to stimulate ROS generation and inflammasome activation. These results provide detailed insights into SAA-mediated IL-1ß production and highlight HDL's role in regulating SAA's proinflammatory effects.

Serum Amyloid A Is an Exchangeable Apolipoprotein.

Objective- SAA (serum amyloid A) is a family of acute-phase reactants that have proinflammatory and proatherogenic activities. SAA is more lipophilic than apoA-I (apolipoprotein A-I), and during an acute-phase response, <10% of plasma SAA is found lipid-free. In most reports, SAA is found exclusively associated with high-density lipoprotein; however, we and others have reported SAA on apoB (apolipoprotein B)-containing lipoproteins in both mice and humans. The goal of this study was to determine whether SAA is an exchangeable apolipoprotein. Approach and Results- Delipidated human SAA was incubated with SAA-free human lipoproteins; then, samples were reisolated by fast protein liquid chromatography, and SAA analyzed by ELISA and immunoblot. Both in vitro and in vivo, we show that SAA associates with any lipoprotein and does not remain in a lipid-free form. Although SAA is preferentially found on high-density lipoprotein, it can exchange between lipoproteins. In the presence of CETP (cholesterol ester transfer protein), there is greater exchange of SAA between lipoproteins. Subjects with diabetes mellitus, but not those with metabolic syndrome, showed altered SAA lipoprotein distribution postprandially. Proteoglycan-mediated lipoprotein retention is thought to be an underlying mechanism for atherosclerosis development. SAA has a proteoglycan-binding domain. Lipoproteins containing SAA had increased proteoglycan binding compared with SAA-free lipoproteins. Conclusions- Thus, SAA is an exchangeable apolipoprotein and increases apoB-containing lipoproteins' proteoglycan binding. We and others have previously reported the presence of SAA on low-density lipoprotein in individuals with obesity, diabetes mellitus, and metabolic syndrome. We propose that the presence of SAA on apoB-containing lipoproteins may contribute to cardiovascular disease development in these populations.

Serum amyloid A3 is pro-atherogenic.

BACKGROUND AND AIMS: Serum amyloid A (SAA) predicts cardiovascular events. Overexpression of SAA increases atherosclerosis development; however, deficiency of two of the murine acute phase isoforms, SAA1.1 and SAA2.1, has no effect on atherosclerosis. SAA3 is a pseudogene in humans, but is an expressed acute phase isoform in mice. The goal of this study was to determine if SAA3 affects atherosclerosis in mice. METHODS: ApoE-/- mice were used as the model for all studies. SAA3 was overexpressed by an adeno-associated virus or suppressed using an anti-sense oligonucleotide approach. RESULTS: Over-expression of SAA3 led to a 4-fold increase in atherosclerosis lesion area compared to control mice (p = 0.01). Suppression of SAA3 decreased atherosclerosis in mice genetically deficient in SAA1.1 and SAA2.1 (p < 0.0001). CONCLUSIONS: SAA3 augments atherosclerosis in mice. Our results resolve a previous paradox in the literature and support extensive epidemiological data that SAA is pro-atherogenic.

Serum amyloid A3 is a high density lipoprotein-associated acute-phase protein.

Serum amyloid A (SAA) is a family of acute-phase reactants. Plasma levels of human SAA1/SAA2 (mouse SAA1.1/2.1) can increase ≥1,000-fold during an acute-phase response. Mice, but not humans, express a third relatively understudied SAA isoform, SAA3. We investigated whether mouse SAA3 is an HDL-associated acute-phase SAA. Quantitative RT-PCR with isoform-specific primers indicated that SAA3 and SAA1.1/2.1 are induced similarly in livers (∼2,500-fold vs. ∼6,000-fold, respectively) and fat (∼400-fold vs. ∼100-fold, respectively) of lipopolysaccharide (LPS)-injected mice. In situ hybridization demonstrated that all three SAAs are produced by hepatocytes. All three SAA isoforms were detected in plasma of LPS-injected mice, although SAA3 levels were ∼20% of SAA1.1/2.1 levels. Fast protein LC analyses indicated that virtually all of SAA1.1/2.1 eluted with HDL, whereas ∼15% of SAA3 was lipid poor/free. After density gradient ultracentrifugation, isoelectric focusing demonstrated that ∼100% of plasma SAA1.1 was recovered in HDL compared with only ∼50% of SAA2.1 and ∼10% of SAA3. Thus, SAA3 appears to be more loosely associated with HDL, resulting in lipid-poor/free SAA3. We conclude that SAA3 is a major hepatic acute-phase SAA in mice that may produce systemic effects during inflammation.

Impact of individual acute phase serum amyloid A isoforms on HDL metabolism in mice.

The acute phase (AP) reactant serum amyloid A (SAA), an HDL apolipoprotein, exhibits pro-inflammatory activities, but its physiological function(s) are poorly understood. Functional differences between SAA1.1 and SAA2.1, the two major SAA isoforms, are unclear. Mice deficient in either isoform were used to investigate plasma isoform effects on HDL structure, composition, and apolipoprotein catabolism. Lack of either isoform did not affect the size of HDL, normally enlarged in the AP, and did not significantly change HDL composition. Plasma clearance rates of HDL apolipoproteins were determined using native HDL particles. The fractional clearance rates (FCRs) of apoA-I, apoA-II, and SAA were distinct, indicating that HDL is not cleared as intact particles. The FCRs of SAA1.1 and SAA2.1 in AP mice were similar, suggesting that the selective deposition of SAA1.1 in amyloid plaques is not associated with a difference in the rates of plasma clearance of the isoforms. Although the clearance rate of SAA was reduced in the absence of the HDL receptor, scavenger receptor class B type I (SR-BI), it remained significantly faster compared with that of apoA-I and apoA-II, indicating a relatively minor role of SR-BI in SAA's rapid clearance. These studies enhance our understanding of SAA metabolism and SAA's effects on AP-HDL composition and catabolism.

Serum amyloid A impairs the antiinflammatory properties of HDL.

HDL from healthy humans and lean mice inhibits palmitate-induced adipocyte inflammation; however, the effect of the inflammatory state on the functional properties of HDL on adipocytes is unknown. Here, we found that HDL from mice injected with AgNO3 fails to inhibit palmitate-induced inflammation and reduces cholesterol efflux from 3T3-L1 adipocytes. Moreover, HDL isolated from obese mice with moderate inflammation and humans with systemic lupus erythematosus had similar effects. Since serum amyloid A (SAA) concentrations in HDL increase with inflammation, we investigated whether elevated SAA is a causal factor in HDL dysfunction. HDL from AgNO3-injected mice lacking Saa1.1 and Saa2.1 exhibited a partial restoration of antiinflammatory and cholesterol efflux properties in adipocytes. Conversely, incorporation of SAA into HDL preparations reduced antiinflammatory properties but not to the same extent as HDL from AgNO3-injected mice. SAA-enriched HDL colocalized with cell surface-associated extracellular matrix (ECM) of adipocytes, suggesting impaired access to the plasma membrane. Enzymatic digestion of proteoglycans in the ECM restored the ability of SAA-containing HDL to inhibit palmitate-induced inflammation and cholesterol efflux. Collectively, these findings indicate that inflammation results in a loss of the antiinflammatory properties of HDL on adipocytes, which appears to partially result from the SAA component of HDL binding to cell-surface proteoglycans, thereby preventing access of HDL to the plasma membrane.

Serum Amyloid A Facilitates Early Lesion Development in Ldlr-/- Mice.

BACKGROUND: Atherosclerosis is a chronic inflammatory disorder, and several studies have demonstrated a positive association between plasma serum amyloid A (SAA) levels and cardiovascular disease risk. The aim of the study was to examine whether SAA has a role in atherogenesis, the underlying basis of most cardiovascular disease. METHODS AND RESULTS: Mice globally deficient in acute-phase isoforms Saa1 and Saa2 (Saa(-/-)) were crossed to Ldlr(-/-) mice (Saa(-/-)Ldlr(-/-)). Saa(-/-)Ldlr(-/-) mice demonstrated a 31% reduction in lesional area in the ascending aorta but not in the aortic root or innominate artery after consuming a high-fat, high-cholesterol Western-type diet for 6 weeks. The lesions were predominantly macrophage foam cells. The phenotype was lost in more mature lesions in mice fed a Western-type diet for 12 weeks, suggesting that SAA is involved in early lesion development. The decreased atherosclerosis in the Saa(-/-)Ldlr(-/-) mice occurred despite increased levels of blood monocytes and was independent of plasma lipid levels. SAA is produced predominantly by hepatocytes and macrophages. To determine which source of SAA may have a dominant role in lesion development, bone marrow transplantation was performed. Ldlr(-/-) mice that received bone marrow from Saa(-/-)Ldlr(-/-) mice had slightly reduced ascending aorta atherosclerosis compared with Saa(-/-)Ldlr(-/-) mice receiving bone marrow from Ldlr(-/-) mice, indicating that the expression of SAA by macrophages may have an important influence on atherogenesis. CONCLUSIONS: The results indicate that SAA produced by macrophages promotes early lesion formation in the ascending aorta.

Deficiency of Endogenous Acute-Phase Serum Amyloid A Protects apoE-/- Mice From Angiotensin II-Induced Abdominal Aortic Aneurysm Formation.

OBJECTIVE: Rupture of abdominal aortic aneurysm (AAA), a major cause of death in the aged population, is characterized by vascular inflammation and matrix degradation. Serum amyloid A (SAA), an acute-phase reactant linked to inflammation and matrix metalloproteinase induction, correlates with aortic dimensions before aneurysm formation in humans. We investigated whether SAA deficiency in mice affects AAA formation during angiotensin II (Ang II) infusion. APPROACH AND RESULTS: Plasma SAA increased ≈60-fold in apoE(-/-) mice 24 hours after intraperitoneal Ang II injection (100 µg/kg; n=4) and ≈15-fold after chronic 28-day Ang II infusion (1000 ng/kg per minute; n=9). AAA incidence and severity after 28-day Ang II infusion was significantly reduced in apoE(-/-) mice lacking both acute-phase SAA isoforms (SAAKO; n=20) compared with apoE(-/-) mice (SAAWT; n=20) as assessed by in vivo ultrasound and ex vivo morphometric analyses, despite a significant increase in systolic blood pressure in SAAKO mice compared with SAAWT mice after Ang II infusion. Atherosclerotic lesion area of the aortic arch was similar in SAAKO and SAAWT mice after 28-day Ang II infusion. Immunostaining detected SAA in AAA tissues of Ang II-infused SAAWT mice that colocalized with macrophages, elastin breaks, and enhanced matrix metalloproteinase activity. Matrix metalloproteinase-2 activity was significantly lower in aortas of SAAKO mice compared with SAAWT mice after 10-day Ang II infusion. CONCLUSIONS: Lack of endogenous acute-phase SAA protects against experimental AAA through a mechanism that may involve reduced matrix metalloproteinase-2 activity.

Deficiency of endogenous acute phase serum amyloid A does not affect atherosclerotic lesions in apolipoprotein E-deficient mice.

OBJECTIVE: Although elevated plasma concentrations of serum amyloid A (SAA) are associated strongly with increased risk for atherosclerotic cardiovascular disease in humans, the role of SAA in the pathogenesis of lesion formation remains obscure. Our goal was to determine the impact of SAA deficiency on atherosclerosis in hypercholesterolemic mice. APPROACH AND RESULTS: Apolipoprotein E-deficient (apoE(-/-)) mice, either wild type or deficient in both major acute phase SAA isoforms, SAA1.1 and SAA2.1, were fed a normal rodent diet for 50 weeks. Female mice, but not male apoE-/- mice deficient in SAA1.1 and SAA2.1, had a modest increase (22%; P≤0.05) in plasma cholesterol concentrations and a 53% increase in adipose mass compared with apoE-/- mice expressing SAA1.1 and SAA2.1 that did not affect the plasma cytokine levels or the expression of adipose tissue inflammatory markers. SAA deficiency did not affect lipoprotein cholesterol distributions or plasma triglyceride concentrations in either male or female mice. Atherosclerotic lesion areas measured on the intimal surfaces of the arch, thoracic, and abdominal regions were not significantly different between apoE-/- mice deficient in SAA1.1 and SAA2.1 and apoE-/- mice expressing SAA1.1 and SAA2.1 in either sex. To accelerate lesion formation, mice were fed a Western diet for 12 weeks. SAA deficiency had effect neither on diet-induced alterations in plasma cholesterol, triglyceride, or cytokine concentrations nor on aortic atherosclerotic lesion areas in either male or female mice. In addition, SAA deficiency in male mice had no effect on lesion areas or macrophage accumulation in the aortic roots. CONCLUSIONS: The absence of endogenous SAA1.1 and 2.1 does not affect atherosclerotic lipid deposition in apolipoprotein E-deficient mice fed either normal or Western diets.

The Impairment of Macrophage-to-Feces Reverse Cholesterol Transport during Inflammation Does Not Depend on Serum Amyloid A.

Studies suggest that inflammation impairs reverse cholesterol transport (RCT). We investigated whether serum amyloid A (SAA) contributes to this impairment using an established macrophage-to-feces RCT model. Wild-type (WT) mice and mice deficient in SAA1.1 and SAA2.1 (SAAKO) were injected intraperitoneally with (3)H-cholesterol-labeled J774 macrophages 4 hr after administration of LPS or buffered saline. (3)H-cholesterol in plasma 4 hr after macrophage injection was significantly reduced in both WT and SAAKO mice injected with LPS, but this was not associated with a reduced capacity of serum from LPS-injected mice to promote macrophage cholesterol efflux in vitro. Hepatic accumulation of (3)H-cholesterol was unaltered in either WT or SAAKO mice by LPS treatment. Radioactivity present in bile and feces of LPS-injected WT mice 24 hr after macrophage injection was reduced by 36% (P < 0.05) and 80% (P < 0.001), respectively. In contrast, in SAAKO mice, LPS did not significantly reduce macrophage-derived (3)H-cholesterol in bile, and fecal excretion was reduced by only 45% (P < 0.05). Injection of cholesterol-loaded allogeneic J774 cells, but not syngeneic bone-marrow-derived macrophages, transiently induced SAA in C57BL/6 mice. Our study confirms reports that acute inflammation impairs steps in the RCT pathway and establishes that SAA plays only a minor role in this impairment.

SAA does not induce cytokine production in physiological conditions.

SAA has been shown to have potential proinflammatory properties in inflammatory diseases such as atherosclerosis. These include induction of tumor necrosis factor α, interleukin-6, and monocyte chemoattractant protein 1 in vitro. However, concern has been raised that these effects might be due to use of recombinant SAA with low level of endotoxin contaminants or its non-native forms. Therefore, physiological relevance has not been fully elucidated. In this study, we investigated the role of SAA in the production of inflammatory cytokines. Stimulation of mouse monocyte J774 cells with lipid-poor recombinant human SAA and purified SAA derived from cardiac surgery patients, but not ApoA-I and ApoA-II, elicited pro-inflammatory cytokines like granulocyte colony stimulating factor (G-CSF). However, HDL-associated SAA failed to stimulate production of these cytokines. Using neutralizing antibodies against toll like receptor (TLR) 2 and 4, we could evaluate that TLR 2 is responsible for G-CSF production by lipid-poor SAA. To confirm these data in vivo, we expressed mouse SAA in SAA deficient C57BL/6 mice using an adenoviral vector. G-CSF was identically expressed in SAA-Adenoviral infected mice as well as in control null-Adenoviral mice at the early time points (4-8h) and could not be detected in plasma 24h after infection when plasma SAA levels were maximally elevated, indicating that adenoviral vector rather than SAA affected G-CSF levels. Taken together, our findings suggest that lipid-poor SAA, but not HDL-associated SAA, stimulates G-CSF production and this stimulation is mediated through TLR 2 in J774 cells. However, its physiological role in vivo remains ambiguous.

Nascent HDL formation in hepatocytes and role of ABCA1, ABCG1, and SR-BI.

To study the mechanisms of hepatic HDL formation, we investigated the roles of ABCA1, ABCG1, and SR-BI in nascent HDL formation in primary hepatocytes isolated from mice deficient in ABCA1, ABCG1, or SR-BI and from wild-type (WT) mice. Under basal conditions, in WT hepatocytes, cholesterol efflux to exogenous apoA-I was accompanied by conversion of apoA-I to HDL-sized particles. LXR activation by T0901317 markedly enhanced the formation of larger HDL-sized particles as well as cellular cholesterol efflux to apoA-I. Glyburide treatment completely abolished the formation of 7.4 nm diameter and greater particles but led to the formation of novel 7.2 nm-sized particles. However, cells lacking ABCA1 failed to form such particles. ABCG1-deficient cells showed similar capacity to efflux cholesterol to apoA-I and to form nascent HDL particles compared with WT cells. Cholesterol efflux to apoA-I and nascent HDL formation were slightly but significantly enhanced in SR-BI-deficient cells compared with WT cells under basal but not LXR activated conditions. As in WT but not in ABCA1-deficient hepatocytes, 7.2 nm-sized particles generated by glyburide treatment were also detected in ABCG1-deficient and SR-BI-deficient hepatocytes. Our data indicate that hepatic nascent HDL formation is highly dependent on ABCA1 but not on ABCG1 or SR-BI.

Serum amyloid A facilitates the binding of high-density lipoprotein from mice injected with lipopolysaccharide to vascular proteoglycans.

OBJECTIVE: Levels of serum amyloid A (SAA), an acute-phase protein carried on high-density lipoprotein (HDL), increase in inflammatory states and are associated with increased risk of cardiovascular disease. HDL colocalizes with vascular proteoglycans in atherosclerotic lesions. However, its major apolipoprotein, apolipoprotein A-I, has no proteoglycan-binding domains. Therefore, we investigated whether SAA, which has proteoglycan-binding domains, plays a role in HDL retention by proteoglycans. METHODS AND RESULTS: HDL from control mice and mice deficient in both SAA1.1 and SAA2.1 (SAA knockout mice) injected with bacterial lipopolysaccharide (LPS) was studied. SAA mRNA expression in the liver and plasma levels of SAA increased dramatically in C57BL/6 mice after LPS administration, although HDL cholesterol did not change. Fast protein liquid chromatography analysis showed most of the SAA to be in HDL. Mass spectrometric analysis indicated that HDL from LPS-injected control mice had high levels of SAA1.1/2.1 and reduced levels of apolipoprotein A-I. HDL from LPS-injected control mice demonstrated high-affinity binding to biglycan relative to normal mouse HDL. In contrast, HDL from LPS-injected SAA knockout mice showed very little binding to biglycan, consistent with SAA facilitating the binding of HDL to vascular proteoglycans. CONCLUSION: SAA enrichment of HDL under inflammatory conditions plays an important role in the binding of HDL to vascular proteoglycans.

Scavenger receptor SR-BI in macrophage lipid metabolism.

OBJECTIVE: To investigate the mechanisms by which macrophage scavenger receptor BI (SR-BI) regulates macrophage cholesterol homeostasis and protects against atherosclerosis. METHODS AND RESULTS: The expression and function of SR-BI was investigated in cultured mouse bone marrow-derived macrophages (BMM). SR-BI, the other scavenger receptors SRA and CD36 and the ATP-binding cassette transporters ABCA1 and ABCG1 were each distinctly regulated during BMM differentiation. SR-BI levels increased transiently to significant levels during culture. SR-BI expression in BMM was reversibly down-regulated by lipid loading with modified LDL; SR-BI was shown to be present both on the cell surface as well as intracellularly. BMM exhibited selective HDL CE uptake, however, this was not dependent on SR-BI or another potential candidate glycosylphosphatidylinositol anchored high density lipoprotein binding protein 1 (GPIHBP1). SR-BI played a significant role in facilitating bidirectional cholesterol flux in non lipid-loaded cells. SR-BI expression enhanced both cell cholesterol efflux and cholesterol influx from HDL, but did not lead to altered cellular cholesterol mass. SR-BI-dependent efflux occurred to larger HDL particles but not to smaller HDL(3). Following cholesterol loading, ABCA1 and ABCG1 were up-regulated and served as the major contributors to cholesterol efflux, while SR-BI expression was down-regulated. CONCLUSION: Our results suggest that SR-BI plays a significant role in macrophage cholesterol flux that may partly account for its effects on atherogenesis.

ATP binding cassette G1-dependent cholesterol efflux during inflammation.

ATP binding cassette transporter G1 (ABCG1) mediates the transport of cellular cholesterol to HDL, and it plays a key role in maintaining macrophage cholesterol homeostasis. During inflammation, HDL undergoes substantial remodeling, acquiring lipid changes and serum amyloid A (SAA) as a major apolipoprotein. In the current study, we investigated whether remodeling of HDL that occurs during acute inflammation impacts ABCG1-dependent efflux. Our data indicate that lipid free SAA acts similarly to apolipoprotein A-I (apoA-I) in mediating sequential efflux from ABCA1 and ABCG1. Compared with normal mouse HDL, acute phase (AP) mouse HDL containing SAA exhibited a modest but significant 17% increase in ABCG1-dependent efflux. Interestingly, AP HDL isolated from mice lacking SAA (SAAKO mice) was even more effective in promoting ABCG1 efflux. Hydrolysis with Group IIA secretory phospholipase A(2) (sPLA(2)-IIA) significantly reduced the ability of AP HDL from SAAKO mice to serve as a substrate for ABCG1-mediated cholesterol transfer, indicating that phospholipid (PL) enrichment, and not the presence of SAA, is responsible for alterations in efflux. AP human HDL, which is not PL-enriched, was somewhat less effective in mediating ABCG1-dependent efflux compared with normal human HDL. Our data indicate that inflammatory remodeling of HDL impacts ABCG1-dependent efflux independent of SAA.

High-density lipoprotein suppresses the type I interferon response, a family of potent antiviral immunoregulators, in macrophages challenged with lipopolysaccharide.

BACKGROUND: High-density lipoprotein (HDL) protects the artery wall by removing cholesterol from lipid-laden macrophages. However, recent evidence suggests that HDL might also inhibit atherogenesis by combating inflammation. METHODS AND RESULTS: To identify potential antiinflammatory mechanisms, we challenged macrophages with lipopolysaccharide, an inflammatory microbial ligand for Toll-like receptor 4. HDL inhibited the expression of 30 (277 of 911) of the genes normally induced by lipopolysaccharide, microarray analysis revealed. One of its major targets was the type I interferon response pathway, a family of potent viral immunoregulators controlled by Toll-like receptor 4 and the TRAM/TRIF signaling pathway. Unexpectedly, the ability of HDL to inhibit gene expression was independent of macrophage cholesterol stores. Immunofluorescent studies suggested that HDL promoted TRAM translocation to intracellular compartments, which impaired subsequent signaling by Toll-like receptor 4 and TRIF. To examine the potential in vivo relevance of the pathway, we used mice deficient in apolipoprotein A-I, the major protein of HDL. After infection with Salmonella typhimurium, a Gram-negative bacterium that expresses lipopolysaccharide, apolipoprotein A-I-deficient mice had 6-fold higher plasma levels of interferon-ß, a key regulator of the type I interferon response, than did wild-type mice. CONCLUSIONS: HDL inhibits a subset of lipopolysaccharide-stimulated macrophage genes that regulate the type I interferon response, and its action is independent of sterol metabolism. These findings raise the possibility that regulation of macrophage genes by HDL might link innate immunity and cardioprotection.