Induced Macrophage Foam Cell Formation Research Articles

Inflammatory corpuscle AIM2 facilitates macrophage foam cell formation by inhibiting cholesterol efflux protein ABCA1

The inflammatory corpuscle recombinant absents in melanoma 2 (AIM2) and cholesterol efflux protein ATP binding cassette transporter A1(ABCA1) have been reported to play opposing roles in atherosclerosis (AS) plaques. However, the relationship between AIM2 and ABCA1 remains unclear. In this study, we explored the potential connection between AIM2 and ABCA1 in the modulation of AS by bioinformatic analysis combined with in vitro experiments. The GEO database was used to obtain AS transcriptional profiling data; screen differentially expressed genes (DEGs) and construct a weighted gene co-expression network analysis (WGCNA) to obtain AS-related modules. Phorbol myristate acetate (PMA) was used to induce macrophage modelling in THP-1 cells, and ox-LDL was used to induce macrophage foam cell formation. The experiment was divided into Negative Control (NC) group, Model Control (MC) group, AIM2 overexpression + ox-LDL (OE AIM2 + ox-LDL) group, and AIM2 short hairpin RNA + ox-LDL (sh AIM2 + ox-LDL) group. The intracellular cholesterol efflux rate was detected by scintillation counting; high-performance liquid chromatography (HPLC) was used to detect intracellular cholesterol levels; apoptosis levels were detected by TUNEL kit; levels of inflammatory markers (IL-1β, IL-18, ROS, and GSH) were detected by ELISA kits; and levels of AIM2 and ABCA1 proteins were detected by Western blot. Bioinformatic analysis revealed that the turquoise module correlated most strongly with AS, and AIM2 and ABCA1 were co-expressed in the turquoise module with a trend towards negative correlation. In vitro experiments demonstrated that AIM2 inhibited macrophage cholesterol efflux, resulting in increased intracellular cholesterol levels and foam cell formation. Moreover, AIM2 had a synergistic effect with ox-LDL, exacerbating macrophage oxidative stress and inflammatory response. Silencing AIM2 ameliorated the above conditions. Furthermore, the protein expression levels of AIM2 and ABCA1 were consistent with the bioinformatic analysis, showing a negative correlation. AIM2 inhibits ABCA1 expression, causing abnormal cholesterol metabolism in macrophages and ultimately leading to foam cell formation. Inhibiting AIM2 may reverse this process. Overall, our study suggests that AIM2 is a reliable anti-inflammatory therapeutic target for AS. Inhibiting AIM2 expression may reduce foam cell formation and, consequently, inhibit the progression of AS plaques.

OxLDL-Induced Foam Cell Formation Inhibitory Activity of Pepsin Hydrolysate of Ark Shell (Scapharca subcrenata (Lischke, 1869)) in RAW264.7 Macrophages

Inhibitory effect of ark shell (Scapharca subcrenata (Lischke, 1869)) proteolytic hydrolysates (ASHs) on oxidized low-density lipoprotein (oxLDL)-induced macrophage foam cell formation was investigated. Two types of ASHs were prepared by Alcalase® and pepsin, ASAH (ark shell-Alcalase® hydrolysates), and ASPH (ark shell-pepsin hydrolysate). Oil Red O staining results showed that ASPH suppressed foam cell formation and lipid accumulation more than ASAH in oxLDL-induced foam cell formation of RAW264.7 macrophages. ASPH reduced the levels of total cholesterol, cholesterol ester, and free cholesterol in oxLDL-treated RAW264.7 macrophages. It was found that ASPH increased cholesterol efflux and decreased cholesterol influx rate. In this regard, protein expressions of CD36 and scavenger receptor class A1 (SR-A1) for cholesterol influx and ATP-binding cassette transporter A1 and G1 (ABCA1 and ABCG1) for cholesterol efflux were investigated. ASPH treatment resulted in an increase of ABCA1 and ABCG1 expression but downregulated CD36 and SR-A1 expression. Furthermore, ASPH suppressed production of proinflammatory cytokines, including tumor necrosis factor-α and interleukin-6 and -1β, through regulating nuclear factor-kappa B (NF-κB) in oxLDL-induced foam cell formation of RAW264.7 macrophages. Taken together, our data indicate that ASPH might be a useful ingredient in functional foods for ameliorating atherosclerosis by preventing foam cell formation.

Highly Oxygenated Labdane Diterpenoids from Stevia rebaudiana and Their Anti-Atherosclerosis Activities.

Five unknown labdane diterpenoids Stevelins A-E (1-5), three known labdane diterpenoids (6-8) and three labdane norditerpenoids (9-11) were isolated from the Stevia rebaudiana. The structures were determined primarily via NMR spectroscopic data and HR-ESI-MS experiments. X-ray crystallography using CuKα radiation was used to determine the absolute configurations of 1, and the absolute configurations of 2-5 were deduced by electronic circular dichroism (ECD) calculations. The potential anti-atherosclerosis activities of all compounds were evaluated by measuring their inhibitory effects on the macrophage foam cell formation. As a result, most isolated compounds could significantly inhibit oxidized low-density lipoprotein (ox-LDL)-induced macrophage foam cell formation, which suggests that these compounds may be promising candidates in the treatment for atherosclerosis.

Baicalein targets CD36 to prevent foam cell formation by suppressing the excessive uptake of oxLDL and accelerating ABCA1-mediated cholesterol efflux in oxLDL-induced THP-1 macrophages

• Baicalein can prevent oxLDL-induced foam cell formation in THP-1 macrophages. • Baicalein inhibits oxLDL uptake by preventing the binding of CD36 to oxLDL. • The binding of baicalein to CD36 triggers the Src-JNK-ABCA1 signalling pathway. • Baicalein enhances cholesterol efflux to avoid cholesterol accumulation. Oxidized low-density lipoprotein (oxLDL)-induced macrophage foam cell formation plays an important role in atherosclerosis progression. Baicalein is a constituent of Scutellaria baicalensis. Herein, we investigated the effect of baicalein on macrophage foam cell formation and the underlying mechanism. The THP-1 macrophage foam cell was established by oxLDL stimulation and the effects of baicalein on cholesterol uptake and efflux were analysed by molecular docking, ELISA, immunofluorescence, western blot, the inhibitor and siRNA experiment. The results showed that baicalein suppressed oxLDL-induced cholesterol accumulation. Mechanistically, baicalein reduced oxLDL uptake through competitive inhibiting the binding of CD36 to the epitope structure of oxLDL. Moreover, the binding of baicalein to CD36 enhanced the cholesterol efflux via the CD36-Src-JNK-ABCA1 signalling pathway. Meanwhile, Src and JNK inhibitors reversed the baicalein-induced reduction in cholesterol accumulation. In conclusion, these findings suggested that baicalein as a food supplement could inhibit macrophage foam cell formation and play an anti-atherosclerosis effect.

Phthalate promotes atherosclerosis through interacting with long-non coding RNA and induces macrophage foam cell formation and vascular smooth muscle damage

BackgroundPhthalates are commonly used in variety of plastic products. Previously it has been revealed that di (2-ethylhexyl) phthalate (DEHP), as the most common member of the class of phthalates, may disturb cholesterol homeostasis and deregulate the inflammatory response, and leading to accelerate the atherosclerosis process. In this regard, the aim of the current study is to explore the underlying mechanism of DEHP-induced atherosclerosis through the increasing of foam cell formation and Vascular Smooth Muscle Cells (VSMCs) damage via the interaction of long-non coding RNA (GAS5) and miR-145–5p. MethodsApoE−/− mice were used to evaluate the in vivo study. RAW264.7 and VSMCs were used to evaluate the effect of DEHP on formation of foam cell, cell proliferation, and cell damage in vitro. Animals were treated with DEHP (5% w/w of food) orally and cells were treated with medium containing of 100 μM DEHP; qRT-PCR, Western blotting, flowcytometry, IHC, oil red O, BODIPY, and autophagic vacuoles assay were used to evaluate the effect of DEHP on formation of atherosclerosis. ResultsDEHP significantly accelerated the formation of atherosclerosis in mice and alter the lipid profile in mice. In addition, after treating VSMCs with DEHP, GAS5 was significantly up-regulated and miR-145–5p was down-regulated. In VSMCs treated with DEHP, we observed that GAS5 could be used as the competing endogenous RNA (ceRNA) of miR-145–5p to regulate the proliferation and apoptosis of VSMCs; and the expression of GAS5 was correlated with the expression of miR-145–5p. DEHP increased the ox-LDL uptake by macrophage and increasing the formation of foam cells. Besides, GAS5 knocking down reversed the effect of DEHP on foam cell formation and ox-LDL uptake. ConclusionDEHP could accelerate the atherosclerosis process through increasing VSMCs damage and formation of macrophage foam cell by increasing lipid uptake though down regulating lncRNA GAS5 and altering in regulation of miR-145–5p.

Abstract 225: Lack Of Map Kinase Phosphatase-5 In Macrophages Protects Ldlr-null Mice Against Atherogenesis

Objective: Mitogen-activated protein kinases (MAPKs), including JNK, ERK, and p38 MAPK, are critical in regulating the expression of various pro-inflammatory and anti-inflammatory cytokines and chemokines. Previous work has suggested that the absence of MAP Kinase Phosphatase-5 (MKP-5) inhibits oxidized low-density lipoprotein (oxLDL)-induced macrophage foam cell formation without influencing the MAKP activation. The current study is to determine the role of macrophage MKP-5 in the pathogenesis of atherosclerosis and underlying mechanisms. Approach and Results: Nine-week-old male congenic MKP-5 deficient (MKP-5 -/- ) and C57Bl/6J control (WT) mice on an LDL receptor knock-out (LDLR -/- ) background were fed with a high-fat diet containing 1.25% cholesterol (HFD) for 14 weeks. Global deficiency of MKP-5 attenuated atherosclerotic plaque formation without altering the lipid profile in vivo. To further elucidate the macrophage-specific effect of MKP-5 in atherogenesis, Lethally irradiated LDLR -/- mice were transplanted with WT or MKP-5 -/- bone marrow and subjected to high-fat feeding. Mice transplanted with MKP-5 -/- bone marrow developed smaller atherosclerotic lesions accompanied by decreased lipid deposition and macrophage content compared to WT. Lack of MKP-5 in macrophages reduced plasma levels of interleukin-1α (IL-1α) and IL-7, elevated anti-inflammatory cytokines IL-1 receptor antagonist (IL-1rn), and IL-4. Mechanistically, lack of MKP-5 in macrophages inhibited ox-LDL-induced foam cell formation through enhanced cholesterol efflux mediated by increased expression of ATP-binding cassette transporters ABCA1 and ABCG1. Conclusions: These data suggest that the myeloid MKP-5 deficiency reduces atherosclerosis progression and foam cell formation by ameliorating cholesterol efflux and inhibiting inflammation.

Investigation of the effective components inhibited macrophage foam cell formation in Ophiopogonis Radix

Ethnopharmacological relevanceOphiopogonis Radix, the commonly used traditional Chinese medicine in clinic for treating cardiovascular diseases, is returned to the stomach, lung and heart meridian. It is reported to nourish yin, moisten lung and is used to treat heart yin deficiency syndromes and asthenia of heart and lung, which indicated that Ophiopogonis Radix may have a protective effect on heart disorders. Atherosclerosisis is an important process in the development of cardiovascular diseases and abnormal lipid deposition induced macrophage foam cells is its crucial foundation. Our previous study showed the extract of Ophiopogonis Radix (EOR) ameliorates atherosclerosis in vitro. However, it may protect against cardiovascular diseases through inhibiting macrophage foam cell formation and its potential effective components and mechanisms are still unclear. Aim of the studyOur study aimed to investigate the effect of Ophiopogonis Radix on macrophage foam cell formation and its potential active constituents and mechanisms. Materials and methodsOx-LDL induced macrophage cells were employed to evaluate the effect of Ophiopogonis Radix on macrophage foam cell formation. Then the potential active constituents inhibited formation of macrophage foam cells were screened by biospecific cell extraction and its underlying mechanisms were also explored by Western blot. ResultsThe extract of Ophiopogonis Radix was found to significantly inhibit macrophage foam cell formation, evidenced by the decrease of TG and TC and Oil Red O staining analysis in macrophage cells, which indicated that EOR reduced the formation of macrophage foam cells. At the same time, EOR was showed to increase antioxidant capacity in macrophage cells. After treatment with EOR, two potential active components interacted with macrophage foam cells specifically were identified to inhibit macrophage foam cell formation including methylophiopogonanone A and methylophiopogonanone B. Methylophiopogonanone A was then proved to decrease the expression of CD36, Lox-1 and SREBP2, increase the expression of ABCA1 obviously, while the expression of ABCG1 and SREBP1 had no changes. ConclusionsIn our study, Ophiopogonis Radix was found to protect against atherosclerosis through suppressing ox-LDL induced macrophage foam cell formation and two potential compounds were identified by biospecific cell extraction including methylophiopogonanone A and methylophiopogonanone B. Moreover, methylophiopogonanone A was proved to inhibit foam cells through reducing uptake, synthesis and increasing efflux, which may provide guidance and reference for application of Ophiopogonis Radix and investigation of the effective components of TCMs.

Cobalt Chloride Induces Macrophage Foam Cell Formation: A Chemical Hypoxia Model for Anti-Atherosclerotic Drug Screening.

Macrophages would engulf circulating oxidized (ox)- low-density lipoprotein and form lipid droplet-laden foam cells. Macrophage foam cells are considered an important therapeutic target of atherosclerosis. The aim of the study was to investigate a hypoxic foam cell model for anti-atherosclerotic drug screening using the chemical hypoxia-mimicking agent cobalt chloride (CoCl2). The oil red O stating results showed that treatment with CoCl2 could induce lipid accumulation and lead to cell transformation to spindle-shaped and lipid-rich foam cells in RAW 264.7 macrophages. Incubation with 150 μM CoCl2 for 24 h significantly increased the area of intracellular lipid droplets in macrophages, compared with the control group. Our findings indicate that CoCl2-triggered macrophage foam cells should be a potential in vitro hypoxia model for atherosclerosis drug discovery.

Identification and functional characterization of a biflavone as a novel inhibitor of TRPV4‐dependent proatherogenic processes in macrophages

Atherosclerosis, a chronic inflammatory disease, is the major cause of mortality and morbidity in the United States. Studies suggest that arterial stiffness is a marker and a risk factor for atherosclerosis. Recent reports from our laboratory showed that TRPV4 (transient receptor potential channel of the vanilloid subfamily 4), a mechanosensitive ion channel, plays a role in oxidized LDL (oxLDL)‐induced macrophage foam cell formation, a critical process in atherogenesis. We performed a high‐throughput screening for antagonists of TRPV4 from a library of 2000 natural compounds using a fluorometric imaging plate reader (FLIPR)‐based Ca2+ influx assay. We identified ginkgetin, a biflavone, as a novel small molecule chemical inhibitor of TRPV4. Primary and secondary screening was performed with mouse bone marrow derived macrophages (BMDMs) to assess the ability of ginkgetin and other candidates to inhibit TRPV4‐dependent Ca2+ influx. In our hand’s, ginkgetin demonstrated an IC50 of 0.5 μM for inhibition of TRPV4‐dependent Ca2+ influx. Furthermore, we found that antagonism of TRPV4 by ginkgetin blocks oxLDL‐induced macrophage foam cell formation. Moreover, we found that ginkgetin treatment 1) attenuated oxLDL uptake but not its cell surface binding in BMDMs, 2) inhibited lipopolysaccharide‐induced phosphorylation of JNK, and 3) inhibited oxLDL‐induced expression of proinflammatory cytokines. Taken together, our results show that ginkgetin is a novel inhibitor of TRPV4‐mediated proatherogenic processes in macrophages.Support or Funding InformationThis work was supported by NIH (1R01EB024556‐01) and NSF (CMMI‐1662776) grants to Shaik O. Rahaman.

Role of pyruvate kinase M2 in oxidized LDL-induced macrophage foam cell formation and inflammation

Pyruvate kinase M2 (PKM2) links metabolic and inflammatory dysfunction in atherosclerotic coronary artery disease; however, its role in oxidized LDL (Ox-LDL)-induced macrophage foam cell formation and inflammation is unknown and therefore was studied. In recombinant mouse granulocyte-macrophage colony-stimulating factor-differentiated murine bone marrow-derived macrophages, early (1-6 h) Ox-LDL treatment induced PKM2 tyrosine 105 phosphorylation and promotes its nuclear localization. PKM2 regulates aerobic glycolysis and inflammation because PKM2 shRNA or Shikonin abrogated Ox-LDL-induced hypoxia-inducible factor-1α target genes lactate dehydrogenase, glucose transporter member 1, interleukin 1β (IL-1β) mRNA expression, lactate, and secretory IL-1β production. PKM2 inhibition significantly increased Ox-LDL-induced ABCA1 and ABCG1 protein expression and NBD-cholesterol efflux to apoA1 and HDL. PKM2 shRNA significantly inhibited Ox-LDL-induced CD36, FASN protein expression, DiI-Ox-LDL binding and uptake, and cellular total cholesterol, free cholesterol, and cholesteryl ester content. Therefore, PKM2 regulates lipid uptake and efflux. DASA-58, a PKM2 activator, downregulated LXR-α, ABCA1, and ABCG1, and augmented FASN and CD36 protein expression. Peritoneal macrophages showed similar results. Ox-LDL induced PKM2- SREBP-1 interaction and FASN expression in a PKM2-dependent manner. Therefore, this study suggests a role for PKM2 in Ox-LDL-induced aerobic glycolysis, inflammation, and macrophage foam cell formation.

Geniposide protects against ox-LDL-induced foam cell formation through inhibition of MAPKs and NF-kB signaling pathways.

Atherosclerosis (AS) is characterized by the significant accumulation of low-density lipoprotein (LDL)-cholesterol in macrophages that reside in the vessel wall and the resultant inflammatory response. Therefore, inhibition of LDL-induced inflammation is a promising interference for AS. Many traditional Chinese medicine prescriptions have been developed for AS treatment. Geniposide (GEN) is an iridoid glycoside mainly found in Gardenia jasminoides fruit. Although GEN has previously been shown to possess anti-atherosclerotic activities, its effects on the formation of macrophage-derived foam cells remain poorly characterized. In our current study, we demonstrated that GEN could significantly inhibit oxidized light-density lipoprotein (ox-LDL) induced macrophage foam cell formation and the expression of pro-inflammatory cytokines in a dose-dependent manner. In addition, treatment of GEN in bone-marrow derived macrophages repressed iNOS expression and NO expression. GEN could also alleviate ox-LDL-dependent up-regulation of CD36 expression by blocking the phosphorylation of p38 MAPK, ERK, JNK and NF-kB p65. The results of our current study demonstrate that GEN exhibits significant therapeutic effects against ox-LDA-induced foam cell formation and inflammation. Therefore, GEN is promising agent for treating AS.

Chemerin-9, a potent agonist of chemerin receptor (ChemR23), prevents atherogenesis.

Plasma levels of chemerin, an adipocytokine produced from the adipose tissues and liver, are associated with metabolic syndrome and coronary artery disease (CAD). Chemerin and its analog, chemerin-9, are known to bind to their receptor, ChemR23. However, whether chemerin and chemerin-9 affect atherogenesis remains to be elucidated. We investigated the expression of chemerin and ChemR23 in human coronary arteries and cultured human vascular cells. The effects of chemerin and chemerin-9 on atheroprone phenomena were assessed in human THP1 monocytes, human umbilical vein endothelial cells (HUVECs), and human aortic smooth muscle cells (HASMCs) and aortic lesions in Apoe-/- mice. In patients with CAD, a small amount of ChemR23, but not chemerin, was expressed within atheromatous plaques in coronary arteries. Chemerin and ChemR23 were expressed at high levels in THP1 monocytes, THP1-derived macrophages, and HUVECs; however, their expression in HASMCs was weak. Chemerin and chemerin-9 significantly suppressed the tumor necrosis factor-α (TNF-α)-induced mRNA expression of adhesion and pro-inflammatory molecules in HUVECs. Chemerin and chemerin-9 significantly attenuated the TNF-α-induced adhesion of THP1 monocytes to HUVECs and macrophage inflammatory phenotype. Chemerin and chemerin-9 suppressed oxidized low-density lipoprotein (oxLDL)-induced macrophage foam cell formation associated with down-regulation of CD36 and up-regulation of ATP-binding cassette transporter A1 (ABCA1). In HASMCs, chemerin and chemerin-9 significantly suppressed migration and proliferation without inducing apoptosis. In the Apoe-/- mice, a 4-week infusion of chemerin-9 significantly decreased the areas of aortic atherosclerotic lesions by reducing intraplaque macrophage and SMC contents. Our results indicate that chemerin-9 prevents atherosclerosis. Therefore, the development of chemerin analogs/ChemR23 agonists may serve as a novel therapeutic target for atherosclerotic diseases.

Exosomal CagA derived from Helicobacter pylori-infected gastric epithelial cells induces macrophage foam cell formation and promotes atherosclerosis

BackgroundSeroepidemiological studies have highlighted a positive relation between CagA-positive Helicobacter pylori (H. pylori), atherosclerosis and related clinic events. However, this link has not been well validated. The present study was designed to explore the role of H. pylori PMSS1 (a CagA-positive strain that can translocate CagA into host cells) and exosomal CagA in the progression of atherosclerosis. MethodsTo evaluate whether H. pylori accelerates or even induces atherosclerosis, H. pylori-infected C57/BL6 mice and ApoE−/− mice were maintained under different dietary conditions. To identify the role of H. pylori-infected gastric epithelial cells-derived exosomes (Hp-GES-EVs) and exosomal CagA in atherosclerosis, ApoE−/− mice were given intravenous or intraperitoneal injections of saline, GES-EVs, Hp-GES-EVs, and recombinant CagA protein (rCagA). FindingsCagA-positive H. pylori PMSS1 infection does not induce but promotes macrophage-derived foam cell formation and augments atherosclerotic plaque growth and instability in two animal models. Meanwhile, circulating Hp-GES-EVs are taken up in aortic plaque, and CagA is secreted in Hp-GES-EVs. Furthermore, the CagA-containing EVs and rCagA exacerbates macrophage-derived foam cell formation and lesion development in vitro and in vivo, recapitulating the pro-atherogenic effects of CagA-positive H. pylori. Mechanistically, CagA suppresses the transcription of cholesterol efflux transporters by downregulating the expression of transcriptional factors PPARγ and LXRα and thus enhances foam cell formation. InterpretationThese results may provide new insights into the role of exosomal CagA in the pathogenesis of CagA-positive H. pylori infection-related atherosclerosis. It is suggested that preventing and eradicating CagA-positive H. pylori infection could reduce the incidence of atherosclerosis and related events.

Functional analysis of a biflavone, as a novel inhibitor of TRPV4‐dependent macrophages foam cell formation

Cardiovascular disease is the number one cause of death in United States, and atherosclerosis, a chronic inflammatory disease, is the most dominant underlying pathology. Macrophages, an essential cell type in atherogenesis, are known to directly respond to changes to their surrounding mechanical environment. As TRPV4 (transient receptor potential channel of the vanilloid subfamily) channels can be sensitized by changes in matrix stiffness, we hypothesize that, TRPV4 may upregulate inflammatory/atherogenic macrophage functions. We performed a high‐throughput screening for antagonists of TRPV4 from a library of 2000 compounds using a fluorometric imaging plate reader (FLIPR)‐based Ca2+ influx assay; we identified ginkgetin, a biflavone, as a novel antagonist to TRPV4. Primary and secondary screening was performed with normal human dermal fibroblasts and mouse bone marrow derived macrophages (BMDMs) to assess the ability of ginkgetin and other candidates to inhibit TRPV4‐elicited Ca2+ influx after TRPV4 activation with its selective agonist GSK1016790. In our hand's, ginkgetin demonstrated an IC50 of 0.5 μM for inhibition of TRPV4‐elicited Ca2+ influx. We show that antagonism of TRPV4 channel by ginkgetin blocks oxidized LDL (oxLDL)‐induced macrophage foam cell formation in a concentration dependent manner. Mechanistically, we show that ginkgetin attenuated oxLDL uptake but not its cell surface binding in BMDMs. Altogether, our results show that ginkgetin is a novel inhibitor of TRPV4‐mediated macrophage foam cell formation by modulating uptake of oxLDL.Support or Funding InformationAHA (13SDG17310007), Startup grant from University of Maryland, NIH (1R01EB024556‐01), and NSF (CMMI‐1662776) grants to Shaik O RahamanThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Sterol regulatory element binding protein (SREBP) -1 mediates oxidized low-density lipoprotein (oxLDL) induced macrophage foam cell formation through NLRP3 inflammasome activation

Macrophage foam cell formation (FCF) has long been known to play a critical role during atherosclerotic plaque development. In the presence of atherogenic molecules such as oxidized low-density lipoprotein (oxLDL) macrophages accumulate massive amounts of lipid through uptake. However, in the presence of oxLDL mechanism of dysregulated lipid homeostasis in the macrophages remains largely unknown. Herein we have investigated the role of Sterol regulatory element binding protein (SREBP)-1 in oxLDL-induced inflammation and altered lipid homeostasis in macrophages. The U937 monocytes and monocyte-derived macrophages (MDMs) were stimulated with different doses of oxLDL. MTT assay to study the effect of oxLDL on cell viability, Oil-Red-O (ORO) staining to observe cytosolic lipid accumulation, semi-quantitative PCR and Western blotting to analyze mRNA and protein expressions, respectively, and spectrophotometric assay to measure the lipid synthesizing enzyme's activity were performed. Our results indicate that oxLDL increased proliferation in monocytes and decreased the viability in MDMs in a time- and dose-dependent manner. The oxLDL (100 μg/ml) enhanced lipid accumulation via increased expressions of SREBP-1 and its downstream proteins such as fatty acid synthase (FAS) and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) at both RNA and protein levels in monocytes as well as in MDMs. Inhibiting SREBP-1 by a synthetic inhibitor prevented excessive lipid accumulation by downregulating the expression of its downstream proteins. Further, oxLDL increased reactive oxygen species (ROS) levels, NLRP3 inflammasome activation and active interleukin 1β (IL-1β) release in both the cell types. The oxLDL-induced NLRP3 could be responsible for SREBP-1 and downstream proteins overexpression as siRNA silencing of NLRP3 decreased SERBP-1 levels. In summary, we have demonstrated that SREBP-1 could be a key player in oxLDL-induced excessive lipid accumulation leading to macrophage FCF via ROS-mediated NLRP3/IL-1β/SREBP-1 pathway.

Dan-Lou Prescription Inhibits Foam Cell Formation Induced by ox-LDL via the TLR4/NF-κB and PPARγ Signaling Pathways.

Atherosclerosis is the major worldwide cause of mortality for patients with coronary heart disease. Many traditional Chinese medicine compound prescriptions for atherosclerosis treatment have been tried in patients. Dan-Lou prescription, which is improved from Gualou-Xiebai-Banxia decoction, has been used to treat chest discomfort (coronary atherosclerosis) for approximately 2,000 years in China. Although the anti-inflammatory activities of Dan-Lou prescription have been proposed previously, the mechanism remains to be explored. Based on the interaction between inflammation and atherosclerosis, we further investigated the effect of Dan-Lou prescription on macrophage-derived foam cell formation and disclosed the underlying mechanisms. In the oxidative low-density lipoprotein (ox-LDL) induced foam cells model using murine macrophage RAW 264.7 cells, the ethanol extract from Dan-Lou prescription (EEDL) reduced ox-LDL uptake and lipid deposition by inhibiting the protein and mRNA expression of Toll-like receptor (TLR)4 and scavenger receptor (SR)B1. After stimulation with ox-LDL, the metabolic profile of macrophages was also changed, while the intervention of the EEDL mainly regulated the metabolism of isovalerylcarnitine, arachidonic acid, cholesterol, aspartic acid, arginine, lysine, L-glutamine and phosphatidylethanolamine (36:3), which participated in the regulation of the inflammatory response, lipid accumulation and cell apoptosis. In total, 27 inflammation-related gene targets were screened, and the biological mechanisms, pathways and biological functions of the EEDL on macrophage-derived foam cells were systemically analyzed by Ingenuity Pathway Analysis system (IPA). After verification, we found that EEDL alleviated ox-LDL induced macrophage foam cell formation by antagonizing the mRNA and protein over-expression of PPARγ, blocking the phosphorylation of IKKα/β, IκBα and NF-κB p65 and maintaining the expression balance between Bax and Bcl-2. In conclusion, we provided evidences that Dan-Lou prescription effectively attenuated macrophage foam cell formation via the TLR4/NF-κB and PPARγ signaling pathways.

Polyphenols from Ilex latifolia Thunb. (a Chinese bitter tea) exert anti-atherosclerotic activity through suppressing NF-κB activation and phosphorylation of ERK1/2 in macrophages.

Ilex latifolia Thunb is a kind of herbal tea and widely consumed as a functional tea beverage in Asian countries. In this study, polyphenols were extracted from I. latifolia and the major compounds were identified by liquid chromatography-mass spectrometry (LC-MS), then the effect on oxidized low-density lipoprotein (ox-LDL)-induced macrophage foam cell formation was investigated. Results showed that the polyphenols could significantly inhibit ox-LDL-induced macrophage foam cell formation and suppress lipid droplet accumulation and cholesterol uptake in RAW 264.7 cells. Additionally, the secretion of pro-inflammatory cytokines, such as tumor necrosis factor (TNF-α), interleukin (IL)-1β, IL-6 and inducible nitric oxide synthase (iNOS), was significantly inhibited. Moreover, the polyphenols could suppress the expression of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) and clusters of differentiation 36 (CD 36), which were receptors for ox-LDL. Mechanistically, I. latifolia polyphenols could inhibit macrophage foam cell formation by suppressing NF-κB activation and phosphorylation of ERK1/2.

Tanshindiol C inhibits oxidized low-density lipoprotein induced macrophage foam cell formation via a peroxiredoxin 1 dependent pathway

NF-E2-related factor 2 (Nrf2) has been shown to be protective in atherosclerosis. The loss of Nrf2 in macrophages enhances foam cell formation and promotes early atherogenesis. Tanshindiol C (Tan C) is isolated from the root of Salvia miltiorrhiza Bge., a traditional Chinese medicine that has been used for the treatment of several cardiovascular diseases for many years. This study was aimed to test the potential role of Tan C against macrophage foam cell formation and to explore the underlying mechanism. Firstly, we observed that Tan C markedly suppressed oxidized low-density lipoprotein (oxLDL) induced macrophage foam cell formation. Then, we found that Tan C was an activator of both Nrf2 and Sirtuin 1 (Sirt1) in macrophages. Nrf2 and Sirt1 synergistically activated the transcription of anti-oxidant peroxiredoxin 1 (Prdx1) after Tan C treatment. More important, we demonstrated that silencing of Prdx1 promoted oxLDL-induced macrophage foam cell formation. Prdx1 upregulated adenosine triphosphate-binding cassette (ABC) transporter A1 (ABCA1) expression and decreased intracellular lipid accumulation. Furthermore, Tan C ameliorated oxLDL induced macrophage foam cell formation in a Prdx1-dependent manner. These observations suggest that Tan C protects macrophages from oxLDL induced foam cell formation via activation of Prdx1/ABCA1 signaling and that Prdx1 may be a novel target for therapeutic intervention of atherosclerosis.

Recombinant Human Thioredoxin-1 Protects Macrophages from Oxidized Low-Density Lipoprotein-Induced Foam Cell Formation and Cell Apoptosis.

Oxidized low-density lipoprotein (ox-LDL)-induced macrophage foam cell formation and apoptosis play critical roles in the pathogenesis of atherosclerosis. Thioredoxin-1 (Trx) is an antioxidant that potently protects various cells from oxidative stress-induced cell death. However, the protective effect of Trx on ox-LDL-induced macrophage foam cell formation and apoptosis has not been studied. This study aims to investigate the effect of recombinant human Trx (rhTrx) on ox-LDL-stimulated RAW264.7 macrophages and elucidate the possible mechanisms. RhTrx significantly inhibited ox-LDL-induced cholesterol accumulation and apoptosis in RAW264.7 macrophages. RhTrx also suppressed the ox-LDL-induced overproduction of lectin-like oxidized LDL receptor (LOX-1), Bax and activated caspase-3, but it increased the expression of Bcl-2. In addition, rhTrx markedly inhibited the ox-LDL-induced production of intracellular reactive oxygen species (ROS) and phosphorylation of p38 mitogen-activated protein kinases (MAPK). Furthermore, anisomycin (a p38 MAPK activator) abolished the protective effect of rhTrx on ox-LDL-stimulated RAW264.7 cells, and SB203580 (a p38 MAPK inhibitor) exerted a similar effect as rhTrx. Collectively, these findings indicate that rhTrx suppresses ox-LDL-stimulated foam cell formation and macrophage apoptosis by inhibiting ROS generation, p38 MAPK activation and LOX-1 expression. Therefore, we propose that rhTrx has therapeutic potential in the prevention and treatment of atherosclerosis.

Intermittent hypoxia induces murine macrophage foam cell formation by IKK-β-dependent NF-κB pathway activation

Obstructive sleep apnea (OSA) is a common sleep disorder characterized by intermittent hypoxia (IH). Clinical studies have previously shown that OSA is an independent risk factor for atherosclerosis. Atherogenicity in OSA patients has been assumed to be associated with the NF-κB pathways. Although foam cells are considered to be a hallmark of atherosclerosis, how IH as in OSA affects their development has not been fully understood. Therefore, we hypothesized that IH induces macrophage foam cell formation through NF-κB pathway activation. To test this hypothesis, peritoneal macrophages collected from myeloid-restricted IKK-β-deleted mice were incubated with native LDL and exposed to either IH or normoxia. After exposure, NF-κB pathway activity and intracellular cholesterol were measured. In control macrophages, IH significantly increased NF-κB pathway activity by 93% compared with normoxia (P < 0.05). However, such response to IH was diminished by IKK-β deletion (increased by +31% compared with normoxia; P = 0.64), suggesting that IKK-β is critical for IH-induced NF-κB pathway activation. Likewise, in control macrophages, total cholesterol was increased in IH compared with normoxia (65.7 ± 3.8 μg/mg cellular protein and 53.2 ± 1.2, respectively; P < 0.05). However, this IH-induced foam cell formation was disappeared when IKK-β was deleted (52.2 ± 1.2 μg/mg cellular protein for IH and 46.3 ± 1.7 for normoxia; P = 0.55). This IH-mediated effect still existed in macrophages without LDL receptor. Taken together, our findings show that IH activates the IKK-β-dependent NF-κB pathway and that this, in turn, induces foam cell formation in murine macrophages.