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Foam cell formation is an important event in atherosclerosis. Fisetin, a bioflavonoid, has been identified to possess anti-inflammatory, antilipidemic, and anticancerous properties; however, its role as a lipid homeostasis regulator in macrophages, specifically in the presence of metabolic stressors such as oxidized low-density lipoprotein (oxLDL) is not well understood. In this study, we have investigated the role of fisetin in preventing oxLDL-induced macrophage foam cell formation. U937-derived macrophages were stimulated with oxLDL with or without fisetin for varied time points, and various parameters were assessed including cell viability by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay; reactive oxygen species (ROS) by dichlorofluorescin diacetate assay; lipid accumulation by Oil Red O staining; and expression of NLR family pyrin domain containing 3 (NLRP3), sterol regulatory element-binding protein (SREBP)-1, and associated downstream proteins 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR) and fatty acid synthase (FAS) by quantitative reverse transcription polymerase chain reaction (RT-qPCR) and immunoblotting. Functionality of FAS enzyme was determined using enzyme activity assay. Docking studies were performed to determine the in silico interaction between NLRP3 and fisetin. The results showed that fisetin up to the dose of 10 µM did not alter cell viability but at the same dose could decrease the accumulation of lipids in macrophages and prevented foam cell formation. Fisetin could also ameliorate and reduce oxLDL-induced upregulation of SREBP-1 and thereby the expression of its downstream lipid synthesis genes HMGCR and FAS and inhibited ROS-induced NLRP3 inflammasome activation. In conclusion, fisetin could inhibit foam cell formation by blocking oxLDL-induced ROS formation and subsequent NLRP3 activation, thereby inhibiting SREBP-1 and its downstream genes including FAS and HMGCR.

Aims Our previous study demonstrated that Ca2+ influx through the Orai1 store-operated Ca2+ channel in macrophages contributes to foam cell formation and atherosclerosis via the calcineurin–ASK1 pathway, not the classical calcineurin–nuclear factor of activated T-cell (NFAT) pathway. Moreover, up-regulation of NFATc3 in macrophages inhibits foam cell formation, suggesting that macrophage NFATc3 is a negative regulator of atherogenesis. Hence, this study investigated the precise role of macrophage NFATc3 in atherogenesis. Methods and results Macrophage-specific NFATc3 knockout mice were generated to determine the effect of NFATc3 on atherosclerosis in a mouse model of adeno-associated virus-mutant PCSK9-induced atherosclerosis. NFATc3 expression was decreased in macrophages within human and mouse atherosclerotic lesions. Moreover, NFATc3 levels in peripheral blood mononuclear cells from atherosclerotic patients were negatively associated with plaque instability. Furthermore, macrophage-specific ablation of NFATc3 in mice led to the atherosclerotic plaque formation, whereas macrophage-specific NFATc3 transgenic mice exhibited the opposite phenotype. NFATc3 deficiency in macrophages promoted foam cell formation by potentiating SR-A- and CD36-meditated lipid uptake. NFATc3 directly targeted and transcriptionally up-regulated miR-204 levels. Mature miR-204-5p suppressed SR-A expression via canonical regulation. Unexpectedly, miR-204-3p localized in the nucleus and inhibited CD36 transcription. Restoration of miR-204 abolished the proatherogenic phenotype observed in the macrophage-specific NFATc3 knockout mice, and blockade of miR-204 function reversed the beneficial effects of NFATc3 in macrophages. Conclusion Macrophage NFATc3 up-regulates miR-204 to reduce SR-A and CD36 levels, thereby preventing foam cell formation and atherosclerosis, indicating that the NFATc3/miR-204 axis may be a potential therapeutic target against atherosclerosis.

Elevated levels of lipoprotein(a) [Lp(a)] have been identified as an independent and causal risk factor for atherosclerotic cardiovascular disease (ASCVD) and, more recently, calcific aortic valve disease (CAVD). CAVD is a slow, progressive disorder presenting as severe trileaflet calcification known as aortic valve stenosis (AS) that impairs valve motion and restricts ventricular outflow. AS afflicts 2% of the aging population (≥ 65 years) and tends to be quite advanced by the time it presents clinical symptoms of exertional angina, syncope, or heart failure. Currently, the only effective clinical therapy for AS patients is surgical or transcatheter aortic valve replacement. Evidence is accumulating that Lp(a) can exacerbate pathophysiological processes in CAVD, specifically, endothelial dysfunction, formation of foam cells, and promotion of a pro-inflammatory state. In the valve milieu, the pro-inflammatory effects of Lp(a) are manifested in valve thickening and mineralization through pro-osteogenic signaling and changes in gene expression in valve interstitial cells that is primarily facilitated by the oxidized phospholipid content of Lp(a). In AS pathogenesis, an incomplete understanding of the role of Lp(a) at the molecular level and the absence of appropriate animal models are barriers for the development of specific and effective clinical interventions designed to mitigate the role of Lp(a) in AS. However, the advent of effective therapies that dramatically lower Lp(a) provides the possibility of the first medical treatment to halt AS progression.

Cholesterol efflux from macrophages is the first step of reverse cholesterol transport (RCT), whose increase inhibits cholesterol accumulation and foam cell formation to suppress atherogenesis. Hesperetin has been reported to exert several protective effects on cardiovascular diseases, while little is known about the role of hesperetin and its underlying mechanism in macrophage foam cell formation. In this study, we sought to investigate the potential effects of hesperetin on foam cell formation and cholesterol efflux by using human macrophages, focusing on liver X receptor alpha (LXRα) and AMPK. We found that hesperetin treatment reduced foam cell formation, intracellular cholesterol levels and the cholesterol esterification rate, and increased cholesterol efflux in THP-1 macrophages. Hesperetin increased the levels of LXRα protein and its targets, including ABCA1, ABCG1, SR-BI, and phosphorylated-AMPK. Meanwhile, the hesperetin-induced increase in LXRα expression was further increased by the AMPK agonist and inhibited by an AMPK inhibitor. Meanwhile, hesperetin increased the levels of LXRα mRNA and its target genes, all of which were decreased in cells transfected with the AMPKα1/α2 small interfering RNA (siRNA). Furthermore, the hesperetin-induced inhibition of foam cell formation and promotion of cholesterol efflux were decreased by transfection of AMPKα1/α2 siRNA. In conclusions, We are the first to report that hesperetin activate AMPK in THP-1-derived macrophages. This activation upregulats LXRα and its targets, including ABCA1, ABCG1 and SR-BI, which significantly inhibits foam cell formation and promotes cholesterol efflux. Our results highlight the therapeutic potential of hesperetin to possibly reduce foam cell formation. This new mechanism might contribute the anti-atherogenic effects of hesperetin.

Objective: Atherosclerosis is accumulation of lipids and extracellular matrix in the arterial wall. TIMPs (tissue inhibitor of metalloproteinases) can impact plaque deposition by regulating ECM (extracellular matrix) turnover. TIMP4 also influences lipid metabolism and smooth muscle cell (SMC) proliferation. We investigated the role of TIMP4 in atherosclerosis. Approach and Results: Mice lacking low-density lipoprotein receptor ( Ldlr −/− ) and Timp4 ( Timp4 −/− / Ldlr −/− ) were fed high-fat diet (HFD) or regular laboratory diet. After 3 or 6 months, HFD-fed male and female Timp4 −/− / Ldlr −/− mice exhibited higher plaque density in the abdominal aorta (but not in aortic valves, arch, thoracic aorta) compared with Ldlr −/− mice. Although plasma lipid and cholesterol levels were lower in Timp4 −/− / Ldlr −/− -HFD, cholesterol content in the abdominal aorta was higher along with elevated inflammatory cytokines, MMP (matrix metalloproteinase) activities, CD68 + /calponin + macrophage-like SMCs in Timp4 −/− / Ldlr −/− -HFD compared with Ldlr −/− -HFD mice. In vitro, oxidized LDL (low-density lipoprotein) markedly increased CD68 expression, reduced SMC markers, increased lipid uptake, and reduced cholesterol efflux protein ABCA1 (ATP-binding cassette transporter A1) in Timp4 −/− / Ldlr −/− compared with Ldlr −/− primary SMCs from abdominal, but not thoracic aorta. TIMP4 expression in the abdominal aorta (in vivo) and its corresponding SMCs (in vitro) was ≈2-fold higher than in the thoracic aorta and SMCs; TIMP4 levels decreased following HFD. Timp4 -deficiency in bone marrow–derived macrophages did not alter their foam cell formation capacity. Conclusions: TIMP4 protects against plaque deposition in the abdominal aorta independent of plasma cholesterol levels. TIMP4 prevents proteolytic degradation of ABCA1 in SMCs, hindering cholesterol accumulation and transdifferentiation to macrophage-like foam cells, representing a novel negative regulator of atherosclerosis.

Accumulation of cholesterol‐laden macrophage foam cells characteristic of early stage atherosclerotic lesions. Phenethyl isothiocyanate (PEITC) is a naturally occurring isothiocyanate found in cruciferous vegetables that has reported a variety of activities including antioxidant and anti‐inflammatory properties. However, the protective effect of PEITC on foam cell formation and its precise mechanism is not yet clear. Therefore, we investigated whether PEITC suppresses foam cell formation and regulates the expression of genes related to lipid accumulation, cholesterol efflux, and inflammation in THP‐1 derived‐macrophages. We exposed THP‐1 derived‐macrophages to oxidized low‐density lipoprotein (ox‐LDL) (20 μg/mL) and lipopolysaccharide (LPS) (500 ng/ml) to mimic foam cell formation. Here, PEITC downregulated the expression of lectin‐like oxidized low‐density lipoprotein receptor‐1 (LOX‐1), cluster of differentiation 36 (CD36), scavenger receptor A1 (SR‐A1), and nuclear factor‐κB (NF‐κB), while upregulated ATP binding cassette subfamily A member 1 (ABCA1)/liver‐X‐receptor α (LXR‐α)/peroxisome proliferator‐activated receptor gamma (PPARγ) and sirtuin 1 (SIRT1) expression compared to co‐treated with ox‐LDL and LPS. Taken together, PEITC, at least in part, inhibits foam cell formation and reduces lipid accumulation in foam cells. Therefore, we suggest that PEITC may be a potential candidate for the treatment and prevention of vascular inflammation and atherosclerosis., PEITC control foam cell formation by promoting cholesterol efflux, reducing lipid accumulation, and modulating the SIRT1‐NF‐κB signaling pathway.

Ethyl gallate (EG) is a well-known constituent of medicinal plants, but its effects on atherosclerosis development are not clear. In the present study, the anti-atherosclerosis effects of EG and the underlying mechanisms were explored using macrophage cultures, zebrafish and apolipoprotein (apo) E deficient mice. Treatment of macrophages with EG (20 μM) enhanced cellular cholesterol efflux to HDL, and reduced net lipid accumulation in response to oxidized LDL. Secretion of monocyte chemotactic protein-1 (MCP-1) and interleukin-6 (IL-6) from activated macrophages was also blunted by EG. Fluorescence imaging techniques revealed EG feeding of zebrafish reduced vascular lipid accumulation and inflammatory responses in vivo. Similar results were obtained in apoE-/- mice 6.5 months of age, where plaque lesions and monocyte infiltration into the artery wall were reduced by 70% and 42%, respectively, after just 6 weeks of injections with EG (20 mg/kg). HDL-cholesterol increased 2-fold, serum cholesterol efflux capacity increased by ∼30%, and the levels of MCP-1 and IL-6 were reduced with EG treatment of mice. These results suggest EG impedes early atherosclerosis development by reducing the lipid and macrophage-content of plaque. Underlying mechanisms appeared to involve HDL cholesterol efflux mechanisms and suppression of pro-inflammatory cytokine secretion.

Coronary heart disease (CHD) with atherosclerosis is the leading cause of death worldwide. ABCA1 and ABCG1 promote cholesterol efflux to suppress foam cell generation and reduce atherosclerosis development. Long noncoding RNAs (lncRNAs) are emerging as a unique group of RNA transcripts that longer than 200 nucleotides and have no protein-coding potential. Many studies have found that lncRNAs regulate cholesterol efflux to influence atherosclerosis development. ABCA1 is regulated by different lncRNAs, including MeXis, GAS5, TUG1, MEG3, MALAT1, Lnc-HC, RP5-833A20.1, LOXL1-AS1, CHROME, DAPK1-IT1, SIRT1 AS lncRNA, DYNLRB2-2, DANCR, LeXis, LOC286367, and LncOR13C9. ABCG1 is also regulated by different lncRNAs, including TUG1, GAS5, RP5-833A20.1, DYNLRB2-2, ENST00000602558.1, and AC096664.3. Thus, various lncRNAs are associated with the roles of ABCA1 and ABCG1 on cholesterol efflux in atherosclerosis regulation. However, some lncRNAs play dual roles in ABCA1 expression and atherosclerosis, and the functions of some lncRNAs in atherosclerosis have not been investigated in vivo. In this article, we review the roles of lncRNAs in atherosclerosis and focus on new insights into lncRNAs associated with the roles of ABCA1 and ABCG1 on cholesterol efflux and the potential of these lncRNAs as novel therapeutic targets in atherosclerosis.

Foam cells are one of the major cellular components of atherosclerotic plaques, within which the trace of periodontal pathogens has also been identified in recent studies. In line with these findings, the correlation between periodontitis and atherosclerotic cardiovascular incidences has been repetitively supported by evidence from a number of experimental studies. However, the direct role of periodontal pathogens in altered cellular signaling underlying such cardiovascular events has not been clearly defined. To determine the role of periodontal pathogens in the pathogenesis of atherosclerosis, especially in the evolution of macrophages into foam cells, we monitored the pattern of lipid accumulation within macrophages in the presence of periodontal pathogens, followed by characterization of these lipids and investigation of major molecules involved in lipid homeostasis. The cells were stained with the lipophilic fluorescent dye BODIPY 493/503 and Oil Red O to characterize the lipid profile. The amounts of Oil Red O–positive droplets, representing neutral lipids, as well as fluorescent lipid aggregates were prominently increased in periodontal pathogen–infected macrophages. Subsequent analysis allowed us to locate the accumulated lipids in the endoplasmic reticulum. In addition, the levels of cholesteryl ester in periodontal pathogen–infected macrophages were increased, implying disrupted lipid homeostasis. Further investigations to delineate the key messengers and regulatory factors involved in the altered lipid homeostasis have revealed alterations in cholesterol efflux–related enzymes, such as ABCG1 and CYP46A1, as contributors to foam cell formation, and increased Ca2+ signaling and reactive oxygen species (ROS) production as key events underlying disrupted lipid homeostasis. Consistently, a treatment of periodontal pathogen–infected macrophages with ROS inhibitors and nifedipine attenuated the accumulation of lipid droplets, further confirming periodontal pathogen–induced alterations in Ca2+ and ROS signaling and the subsequent dysregulation of lipid homeostasis as key regulatory events underlying the evolution of macrophages into foam cells.

Objective Plasminogen/plasmin is a serine protease system primarily responsible for degrading fibrin within blood clots. Plasminogen mediates its functions by interacting with plasminogen receptors on the cell surface. H2B, one such plasminogen receptor, is found on the surface of several cell types including macrophages. Both basic and clinical studies support the role of plasminogen in the process of foam cell formation (FCF), a hallmark of atherosclerosis. Growing evidence also implicates serine protease-activated receptors (PARs) in atherosclerosis. These receptors are also found on macrophages, and plasmin is capable of activating PAR1 and PAR4. The goal of this study was to determine the extent of H2B's contribution to plasminogen-mediated FCF by macrophages and if PARs are involved in this process. Approach and results Treating macrophages with plasminogen increases their oxidized low-density lipoprotein uptake and plasminogen-mediated foam cell formation (Plg-FCF) significantly. The magnitude of Plg-FCF correlates with cell-surface expression of the H2B level. H2B blockade or downregulation reduces Plg-FCF, whereas its overexpression or high endogenous levels increases Plg-FCF. Modulating PAR1 level in mouse macrophages affects Plg-FCF. Activation/overexpression of PAR1 increases and its blockade/knockdown reduces this response. Confocal imaging indicates that both H2B and PAR1 colocalize with clathrin coated pits on the surface of macrophages, and reducing expression of clathrin or interfering with the clathrin-coated pits integrity reduces Plg-FCF. Conclusion Our data indicate that the magnitude of Plg-FCF by macrophages is proportional to the H2B levels and demonstrate for the first time that PAR1 is involved in this process and that the integrity of clathrin-coated pits is required for the full effect of Plg-induced FCF.

MicroRNA-328 (miR-328) was reported to protect against atherosclerosis, but its role in foam cell formation remains unknown. The aim of this study was to investigate the effect of miR-328-5p on macrophage lipid accumulation and the underlying mechanisms. The results showed that miR-328-5p expression was robustly decreased in oxidized low-density lipoprotein (ox-LDL)-treated macrophages. Treatment of human acute monocytic leukemia cel (THP-1) macrophage-derived foam cells with a miR-328-5p mimic markedly increased [3 H]-cholesterol efflux, inhibited lipid droplet accumulation, and decreased intracellular total cholesterol (TC), free cholesterol (FC) and cholesteryl ester (CE) contents. Upregulation of miR-328-5p also reduced the expression of histone deacetylase 3 (HDAC3) but increased the levels of ATP-binding cassette transporter A1 (ABCA1) in THP-1 macrophage-derived foam cells. Mechanistically, miR-328-5p inhibited HDAC3 expression by directly targeting its 3'UTR, thereby promoting ABCA1 expression and the subsequent cholesterol efflux. Furthermore, miR-328-5p mimic treatment did not affect the uptake of Dil-ox-LDL or the expression of scavenger receptor-A (SR-A), thrombospondin receptor (CD36) and ABCG1. Taken together, these findings suggest that miR-328-5p alleviates macrophage lipid accumulation through the HDAC3/ABCA1 pathway.

Disruption of the intracellular lipid balance leading to cholesterol accumulation is one of the features of cells that participate in the development of atherosclerotic lesions. Evidence form our laboratory indicates that anti-inflammatory cyclopentenone prostaglandins (cyPGs) of A- and J-family deviate lipid metabolism from the synthesis of cholesterol and cholesteryl esters to the synthesis of phospholipids in foam-cell macrophages. cyPGs possessing an α,β-unsaturated cyclopentane ring are highly electrophilic substances able to promptly react with reactive cysteines of intracellular molecules through Michael addition. On the other hand, HMG-CoA reductase (HMGCR), the enzyme responsible for the rate-limiting step in cholesterol biosynthesis, presents critically reactive cysteines at the entry of catalytic domain, particularly Cys561, that could be target of cyPG inhibition. In the present study, we showed that cyPGs (but not other non-α,β-unsaturated PGs) physically interact with HMGCR, in a dithiothreitol- and β-mercaptoethanol-sensitive way, and block the activity of the catalytic subunit of the enzyme (IC50 for PGA2 = 0.17 μM). PGA2 inhibits HMGCR activity in cultured rat and human macrophages/macrophage-foam cells and leads to enhanced expression of HMGCR protein, as observed with statins. In cell culture models, PGA2 effectively inhibits the reductase at non-toxic doses (e.g., 1 μM) that block cell proliferation thus suggesting that part of the well-known antiproliferative effect of PGA2 may be due to its ability of blocking HMGCR activity, as cells cannot proliferate without a robust cholesterogenesis. Therefore, besides the powerfully anti-inflammatory and antiproliferative effects, the anticholesterogenic effects of PGA2 should be exploited in atherosclerosis therapeutics.

Macrophage autophagy is a highly anti-atherogenic process that promotes the catabolism of cytosolic lipid droplets (LDs) to maintain cellular lipid homeostasis. Selective autophagy relies on tags such as ubiquitin and a set of selectivity factors including selective autophagy receptors (SARs) to label specific cargo for degradation. Originally described in yeast cells, “lipophagy” refers to the degradation of LDs by autophagy. Yet, how LDs are targeted for autophagy is poorly defined. Here, we employed mass spectrometry to identify lipophagy factors within the macrophage foam cell LD proteome. In addition to structural proteins (e.g., PLIN2), metabolic enzymes (e.g., ACSL) and neutral lipases (e.g., PNPLA2), we found the association of proteins related to the ubiquitination machinery (e.g., AUP1) and autophagy (e.g., HMGB, YWHA/14-3-3 proteins). The functional role of candidate lipophagy factors (a total of 91) was tested using a custom siRNA array combined with high-content cholesterol efflux assays. We observed that knocking down several of these genes, including Hmgb1, Hmgb2, Hspa5, and Scarb2, significantly reduced cholesterol efflux, and SARs SQSTM1/p62, NBR1 and OPTN localized to LDs, suggesting a role for these in lipophagy. Using yeast lipophagy assays, we established a genetic requirement for several candidate lipophagy factors in lipophagy, including HSPA5, UBE2G2 and AUP1. Our study is the first to systematically identify several LD-associated proteins of the lipophagy machinery, a finding with important biological and therapeutic implications. Targeting these to selectively enhance lipophagy to promote cholesterol efflux in foam cells may represent a novel strategy to treat atherosclerosis. Abbreviations: ADGRL3: adhesion G protein-coupled receptor L3; agLDL: aggregated low density lipoprotein; AMPK: AMP-activated protein kinase; APOA1: apolipoprotein A1; ATG: autophagy related; AUP1: AUP1 lipid droplet regulating VLDL assembly factor; BMDM: bone-marrow derived macrophages; BNIP3L: BCL2/adenovirus E1B interacting protein 3-like; BSA: bovine serum albumin; CALCOCO2: calcium binding and coiled-coil domain 2; CIRBP: cold inducible RNA binding protein; COLGALT1: collagen beta(1-O)galactosyltransferase 1; CORO1A: coronin 1A; DMA: deletion mutant array; Faa4: long chain fatty acyl-CoA synthetase; FBS: fetal bovine serum; FUS: fused in sarcoma; HMGB1: high mobility group box 1; HMGB2: high mobility group box 2: HSP90AA1: heat shock protein 90: alpha (cytosolic): class A member 1; HSPA5: heat shock protein family A (Hsp70) member 5; HSPA8: heat shock protein 8; HSPB1: heat shock protein 1; HSPH1: heat shock 105kDa/110kDa protein 1; LDAH: lipid droplet associated hydrolase; LIPA: lysosomal acid lipase A; LIR: LC3-interacting region; MACROH2A1: macroH2A.1 histone; MAP1LC3: microtubule-associated protein 1 light chain 3; MCOLN1: mucolipin 1; NBR1: NBR1, autophagy cargo receptor; NPC2: NPC intracellular cholesterol transporter 2; OPTN: optineurin; P/S: penicillin-streptomycin; PLIN2: perilipin 2; PLIN3: perilipin 3; PNPLA2: patatin like phospholipase domain containing 2; RAB: RAB, member RAS oncogene family; RBBP7, retinoblastoma binding protein 7, chromatin remodeling factor; SAR: selective autophagy receptor; SCARB2: scavenger receptor class B, member 2; SGA: synthetic genetic array; SQSTM1: sequestosome 1; TAX1BP1: Tax1 (human T cell leukemia virus type I) binding protein 1; TFEB: transcription factor EB; TOLLIP: toll interacting protein; UBE2G2: ubiquitin conjugating enzyme E2 G2; UVRAG: UV radiation resistance associated gene; VDAC2: voltage dependent anion channel 2; VIM: vimentin

Lipid metabolism disorder and inflammatory response are considered to be the major causes of atherosclerogenesis. Astragalin, the most important functional component of flavonoid obtained from persimmon leaves, has the hypolipidemic effects. However, it is unknown, how astragalin protects against atherosclerosis. The aim of this study was to observe the effects of astragalin on cholesterol efflux and inflammatory response and to explore the underlying mechanisms. Our results showed that astragalin upregulated the expression of ATP-binding cassette transporters A1 and G1 (ABCA1 and ABCG1), promoted cholesterol efflux, and suppressed foam cell formation. Inhibition of the PPARγ/LXRα pathway abrogated the promotive effects of astragalin on both transporter expression and cholesterol efflux. In addition, treatment of astragalin markedly decreased the secretion of inflammatory factors, including interleukin 6, monocyte chemotactic protein 1, tumor necrosis factor α, and interleukin 1β. Mechanistically, astragalin upregulated ABCA1 and ABCG1 expression, which in turn reduced TLR4 surface levels and inhibited NF-κB nuclear translocation. Consistently, astragalin reduced atherosclerotic plaque area in apoE-/- mice. Taken together, these findings suggest that astragalin protects against atherosclerosis by promoting ABCA1- and ABCG1-mediated cholesterol efflux and inhibiting proinflammatory mediator release.

Objective The involvement of lysosomal integral membrane protein 2 (LIMP2) in cholesterol transport and formation of foam cells under the infection of Porphyromonas gingivalis (P. gingivalis) is yet to be elucidated. The current study verified the role and explored the mechanism of LIMP2 in promoting foam cell formation by P. gingivalis. Background An association between periodontitis and atherosclerosis (AS) has been established. P. gingivalis is a key pathogen of periodontitis that promotes foam cell formation by regulating activities of CD36 scavenger receptors expressed on the macrophages. LIMP2, a member of CD36 superfamily, is involved in cholesterol efflux. However, whether LIMP2 is involved in the formation of foam cells promoted by P. gingivalis remains unclear. Methods The formation of foam cells was examined by Oil Red O staining. The knockdown of limp2 was identified by qRT-PCR. The accumulation of cholesterol was monitored by Cholesterol Assay Kit. The location of P. gingivalis was visualized by confocal microscopy. Cathepsin L activity was monitored with Magic Red Cathepsin L Assay Kit. The key genes and pathways in P. gingivalis-infected macrophages were explored by RNA sequencing. The protein level was investigated by Western blotting. Results Porphyromonas gingivalis increases foam cells formation and upregulates the expression of LIMP2 in foam cells. The knockdown of limp2 decreases the number of foam cells and increases cholesterol export, which is related to lysosomal functions. In addition, the interaction between LIMP2 and caveolin-1(CAV1) might contribute to this process, and NF-κB and JNK activity is required for increased expression of P. gingivalis-induced LIMP2. Conclusions This study suggested that LIMP2 is involved in the foam cells formation facilitated by P. gingivalis, which favors a close connection between periodontitis and atherosclerosis (AS).

Objective Cardiovascular disease (CVD) is the leading cause of death in systemic lupus erythematosus (SLE). B cells play a key role in the pathogenesis of lupus, and anti-BAFF therapy has been approved for use in SLE. Since mature B cells also promote atherosclerosis, we undertook this study to evaluate, in a mouse model and in SLE patients, whether BAFF neutralization has an atheroprotective effect in SLE. Methods The effect of BAFF on atherosclerosis associated with lupus was investigated in the atherosclerosis/lupus-prone apolipoprotein E-knockout D227K mouse model and in a cohort of SLE patients. Mice were treated with a blocking anti-BAFF monoclonal antibody (mAb), while fed a standard chow diet. Carotid plaque and carotid intima-media thickness were assessed by ultrasound at baseline and during follow-up in SLE patients who were asymptomatic for CVD. Results Anti-BAFF mAb in ApoE-/- D227K mice induced B cell depletion, efficiently treated lupus, and improved atherosclerosis lesions (21% decrease; P = 0.007) in mice with low plasma cholesterol levels but worsened the lesions (17% increase; P = 0.06) in mice with high cholesterol levels. The atheroprotective effect of the BAFF-BAFF receptor signaling inhibition on B cells was counterbalanced by the proatherogenic effect of the BAFF-TACI signaling inhibition on macrophages. In SLE patients, blood BAFF levels were associated with subclinical atherosclerosis (r = 0.26, P = 0.03). Anti-BAFF mAb treatment had a differential effect on the intima-media thickness progression in SLE patients depending on body mass index. Conclusion Depending on the balance between lipid- and B cell-induced proatherogenic conditions, anti-BAFF could be detrimental or beneficial, respectively, to atherosclerosis development in SLE.

Foam cell formation and inflammation in macrophages contribute to the development of atherosclerosis (AS). Clematichinenoside AR (AR) is a major active ingredient extracted from the traditional Chinese herb Clematis chinensis and has potent pharmacological effects on various diseases, including AS. However, little is known about the exact role and mechanism of AR in AS. RAW264.7 macrophages were exposed to oxidized low-density lipoprotein (ox-LDL) to induce AS in vitro. Cell viability was assessed by the CCK-8 assay. Foam cell formation was detected by Oil Red staining. Cholesterol levels were determined by corresponding commercial kits. The expression of inflammatory cytokines was detected by ELISA. Western blot and immunofluorescence assays were employed to detect the expression of corresponding genes. The results indicated that AR treatment inhibited the formation of foam cells and cholesterol accumulation but promoted cholesterol efflux by upregulating ABCA1/ABCG1 in ox-LDL-induced RAW264.7 macrophages. In addition, AR decreased the production of inflammatory cytokines by blunting the activation of the NLRP3 inflammasome and inducing autophagy. However, these effects of AR were weakened by the autophagy inhibitor bafilomycin A1 but were similar to those produced by the autophagy activator rapamycin. Collectively, our study provides novel insights into the beneficial effects of AR on promoting cholesterol efflux as well as inhibiting foam cell formation and inflammation by regulating autophagy, thus identifying AR as a promising therapeutic agent for the treatment of AS.

The uptake of modified low-density lipoprotein (LDL) and the accumulation of lipid droplets induce the formation of vascular smooth muscle cells (VSMCs)-derived foam cells, thereby promoting the development and maturation of plaques and accelerating the progression of atherosclerosis. Celastrol is a quinine methide triterpenoid isolated from the root bark of traditional Chinese herb Tripterygium wilfordii. It possesses various biological properties, including anti-obesity, cardiovascular protection, anti-inflammation, etc. In the present study, we found that celastrol significantly reduced lipid accumulation induced by oxidized LDL (ox-LDL) in VSMCs. Mechanistically, celastrol up-regulated adenosine triphosphate-binding cassette transporter A1 (ABCA1) expression through activating liver X receptor α (LXRα) expression, which contributed to inhibit lipid accumulation in VSMCs. Meanwhile, celastrol decreased lipid accumulation by triggering autophagy in VSMCs. Therefore, these findings supported celastrol as a potentially effective agent for the prevention and therapy of atherosclerosis.

In this study, two homogeneous polysaccharides (PFC-1 and PFC-2) having anti-atherosclerotic activity were isolated from Fructus Corni. PFC-1 and PFC-2 were 1,6-α-glucans with the molecular weight of 4.4 kDa and 82.0 kDa, respectively. In the in vitro experiments, PFC-1 and PFC-2 showed significant inhibitory effects on the cholesterol accumulation in RAW264.7 macrophages induced by oxidized low-density lipoproteins (ox-LDL), and the inhibitory rate of PFC-2 was 81.62%. Apolipoprotein E-deficient (ApoE−/−) mice fed high-fat diet (HFD) were used to evaluate the anti-atherosclerotic effects of PFC-2 in vivo. The aortic root lipid area decreased by 55.01% in the PFC-2-administered group as compared to the model group. PFC-2 decreased the levels of serum low-density lipoprotein cholesterol, total cholesterol, triglycerides, and malondialdehyde, increased the superoxide dismutase activity, and reduced the contents of lipid and macrophages in the aortic sinus plaque in ApoE−/− mice fed with HFD. Furthermore, PFC-2 markedly inhibited the expression of type A1 scavenger receptor (SR-A1) and cluster of differentiation 36 (CD36) in ox-LDL-treated macrophages. Taken together, 1,6-α-glucans from Fructus Corni showed significant anti-atherogenic effect, and the mechanism is related to enhanced antioxidant activity of the ApoE−/− mice and down-regulated the expression of SR-A1 and CD36 proteins in macrophages.

Previous studies have shown that the occurrence of atherosclerosis is closely related to changes of α2, 6-sialic acid transferase I (ST6Gal-I). Bace1 has been identified as a protease responsible for the cleavage and secretion of Golgi-resident ST6Gal-I. There have been only a few attempts to clarify the direct connection between Bace1 and atherosclerosis. The purpose of this study was to investigate the relationship between Bace1 gene and atherosclerosis. Expressions of Bace1 protein and mRNA in ApoE−/− mice fed on high-fat diet were evaluated and the development of atherosclerosis was assessed in Bace1−/− mice fed on high-fat diet. In vitro, the expression of Bace1 gene was detected in foam cell model and the formation of foam cells was examined after knocking down Bace1 by siRNA. We observed a significant increase in Bace1 expression in the aortic root in the model of atherosclerosis in ApoE−/−mice. The expression of Bace1 protein and mRNA levels had a remarkable increase in high-fat group. After knocking out the Bace1 gene, serum lipid levels were significantly lower and intimal thickness was obvious thinner than those in wild-type mice with high-fat diet. Expression of Bace1 protein and mRNA levels were significantly elevated in foam cell. The formation of foam cells was blocked when Bace1 was knocked down by siRNA interferes. Our results suggested that elevated Bace1 gene had a positive role in the progression of atherosclerosis. Affecting the glycosyltransferase may be one of its mechanisms.

The pregnane X receptor (PXR) is a nuclear receptor that can be activated by numerous drugs and xenobiotic chemicals. PXR thereby functions as a xenobiotic sensor to coordinately regulate host responses to xenobiotics by transcriptionally regulating many genes involved in xenobiotic metabolism. We have previously reported that PXR has pro-atherogenic effects in animal models, but how PXR contributes to atherosclerosis development in different tissues or cell types remains elusive. In this study, we generated an LDL receptor-deficient mouse model with myeloid-specific PXR deficiency (PXR(ΔMye)LDLR(−/−)) to elucidate the role of macrophage PXR signaling in atherogenesis. The myeloid PXR deficiency did not affect metabolic phenotypes and plasma lipid profiles, but PXR(ΔMye)LDLR(−/−) mice had significantly decreased atherosclerosis at both aortic root and brachiocephalic arteries compared with control littermates. Interestingly, the PXR deletion did not affect macrophage adhesion and migration properties, but reduced lipid accumulation and foam cell formation in the macrophages. PXR deficiency also led to decreased expression of the scavenger receptor CD36 and impaired lipid uptake in macrophages of the PXR(ΔMye)LDLR(−/−) mice. Further, RNA-Seq analysis indicated that treatment with a prototypical PXR ligand affects the expression of many atherosclerosis-related genes in macrophages in vitro. These findings reveal a pivotal role of myeloid PXR signaling in atherosclerosis development and suggest that PXR may be a potential therapeutic target in atherosclerosis management.

Cholesterol efflux from macrophages is the initial step of reverse cholesterol transport, an important process for high-density lipoprotein-mediated atheroprotection. G protein-coupled receptor (GPR) 120, which functions as long-chain fatty acid receptor, is well known for its anti-inflammatory and insulin-sensitizing function in macrophages. However, the role of GPR120 on macrophage foam cell formation, the hallmark of atherosclerotic plaques, has not been verified. In this study, we found for the first time that stimulation of GPR120 by its agonist GW9508 elevated the expression of ATP-binding cassette transporters (ABC) A1 and ABCG1 in THP-1 macrophage-derived foam cells and Raw264.7 macrophages, and promoted ABCA1- and ABCG1-mediated cholesterol efflux and reduced cellular cholesteryl ester (CE) content as well. In addition, GPR120 activation was accompanied with the stimulation of AMPK pathway in macrophages; however, the effect of GPR120 on macrophage cholesterol efflux was largely abolished by AMPK inhibition. Moreover, the AMPK activity and the expression of ABCA1 and ABCG1 were markedly abrogated by knockdown of GPR120, or application of phospholipase C (PLC) inhibitor, calcium chelator, or CaMKK inhibitor. Because only free cholesterol can be effluxed from macrophages, we found that activation of AMPK could lead to increase both neutral CEs hydrolysis by upregulation of neutral cholesterol ester hydrolase expression and acid CEs hydrolysis by activation of ULK1. In conclusion, these results demonstrated that GPR120 facilitated ABCA1- and ABCG1-mediated cholesterol efflux through activation of PLC/Ca2+ /CaMKK/AMPK signaling pathway, which induced CE hydrolysis and elevated the expression of ABCA1 and ABCG1 in macrophages.

Vascular smooth muscle cells (VSMCs) are an important source of foam cells in atherosclerosis. The mechanism for VSMC-derived foam cell formation is, however, poorly understood. Here, we demonstrate that the P2RY12/P2Y12 receptor is important in regulating macroautophagy/autophagy and VSMC-derived foam cell formation in advanced atherosclerosis. Inhibition of the P2RY12 receptor ameliorated lipid accumulation and VSMC-derived foam cell formation in high-fat diet-fed apoe(-/-) mice (atherosclerosis model) independent of LDL-c levels. Activation of the P2RY12 receptor blocked cholesterol efflux via PI3K-AKT, while genetic knockdown or pharmacological inhibition of the P2RY12 receptor inhibited this effect in VSMCs. Phosphoproteomic analysis showed that the P2RY12 receptor regulated the autophagy pathway in VSMCs. Additionally, activation of the P2RY12 receptor inhibited MAP1LC3/LC3 maturation, SQSTM1 degradation, and autophagosome formation in VSMCs. Genetic knockdown of the essential autophagy gene Atg5 significantly attenuated P2RY12 receptor inhibitor-induced cholesterol efflux in VSMCs. Furthermore, activation of the P2RY12 receptor led to the activation of MTOR through PI3K-AKT in VSMCs, whereas blocking MTOR activity (rapamycin) or reducing MTOR expression reversed the inhibition of cholesterol efflux mediated by the P2RY12 receptor in VSMCs. In vivo, inhibition of the P2RY12 receptor promoted autophagy of VSMCs through PI3K-AKT-MTOR in advanced atherosclerosis in apoe(-/-) mice, which could be impeded by an autophagy inhibitor (chloroquine). Therefore, we conclude that activation of the P2RY12 receptor decreases cholesterol efflux and promotes VSMC-derived foam cell formation by blocking autophagy in advanced atherosclerosis. Our study thus suggests that the P2RY12 receptor is a therapeutic target for treating atherosclerosis. Abbreviations: 2-MeSAMP: 2-methylthioadenosine 5′-monophosphate; 8-CPT-cAMP: 8-(4-chlorophenylthio)-adenosine-3ʹ,5ʹ-cyclic-monophosphate; ABCA1: ATP binding cassette subfamily A member 1; ABCG1: ATP binding cassette subfamily G member 1; ACTB: actin beta; ADPβs: adenosine 5′-(alpha, beta-methylene) diphosphate; ALs: autolysosomes; AMPK: AMP-activated protein kinase; APOA1: apolipoprotein A1; APs: autophagosomes; ATG5: autophagy related 5; ATV: atorvastatin; AVs: autophagic vacuoles; CD: chow diet; CDL: clopidogrel; CQ: chloroquine; DAPI: 4ʹ,6-diamidino-2-phenylindole; dbcAMP: dibutyryl-cAMP; DIL-oxLDL: dioctadecyl-3,3,3,3-tetramethylin docarbocyanine-oxLDL; EIF4EBP1/4E-BP1: eukaryotic translation initiation factor 4E binding protein 1; EVG: elastic van gieson; HE: hematoxylin-eosin; HDL: high-density lipoprotein; HFD: high-fat diet; KEGG: Kyoto Encyclopedia of Genes and Genomes; LDL-c: low-density lipoprotein cholesterol; LDs: lipid droplets; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; Masson: masson trichrome; MCPT: maximal carotid plaque thickness; MK2206: MK-2206 2HCL; NBD-cholesterol: 22-(N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl] amino)-23,24-bisnor-5-cholen-3β-ol; OLR1/LOX-1: oxidized low density lipoprotein receptor 1; ORO: oil Red O; ox-LDL: oxidized low-density lipoprotein; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy; TIC: ticagrelor; ULK1: unc-51 like autophagy activating kinase 1; VSMCs: vascular smooth muscle cells

Background Atherosclerosis (AS) is the basis of cardiovascular diseases, characterized by chronic inflammatory and lipid metabolism disorders. Although the anti-inflammatory effect of Klotho in AS has been clearly shown, its lipid-lowering effect is unclear. In this study, we examined the effects of recombinant Klotho (Re-KL) protein on lipid accumulation in foam cells. Methods THP-1 cells were exposed to 100 nM phorbol myristate acetate for 24 h and then to oxidized low-density lipoprotein (ox-LDL; 80 mg/mL) to induce foam cell formation. Subsequently, the foam cells were incubated with Re-KL and/or DKK1, an inhibitor of the Wnt/β-catenin pathway. Results Oil red O staining and cholesterol intake assay revealed that the foam cell model was constructed successfully. Pre-treatment of the foam cells with Re-KL decreased total cholesterol level, up-regulated the expression of ATP binding cassette transporter A1 (ABCA1) and G1 (ABCG1), and down-regulated the expression of acyl coenzyme a-cholesterol acyltransferase 1 (ACAT1) and members of the scavenger family (SR-A1 and CD36). In addition, the expression of Wnt/β-catenin pathway-related proteins in foam cells was significantly decreased by the stimulus of Re-KL. Interestingly, the effect of Re-KL was similar to that of DKK1 on foam cells. Conclusions The Re-KL-induced up-regulation of reverse cholesterol transport capacity promotes cholesterol efflux and reduces lipid accumulation by suppressing the Wnt/β-catenin pathway in foam cells.

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.

Atherosclerosis is a chronic inflammation condition resulting from the interaction between lipoproteins, monocyte-derived macrophages, T lymphocytes, and other cellular elements in the arterial wall. Macrophage-derived foam cells play a key role in both early and advanced stage of atherosclerosis. Previous studies have shown that berberine could inhibit foam cell formation and prevent experimental atherosclerosis. However, its underlying molecular mechanisms have not been fully clarified. In this study, we explored the cholesterol-lowering effects of berberine in macrophage-derived foam cells and investigated its possible mechanisms in prevention and treatment of atherosclerosis. Here, we demonstrated that berberine could inhibit atherosclerosis in apolipoprotein E-deficient mice and induce cholesterol reduction as well as decrease the content of macrophages. Berberine can regulate oxLDL uptake and cholesterol efflux, thus suppresses foam cell formation. Mechanisms study showed that berberine can suppress scavenger receptor expression via inhibiting the activity of AP-1 and upregulate ATP-binding cassette transporter via activating Nrf2/HO-1 signaling in human macrophage. In summary, berberine significantly inhibits atherosclerotic disease development by regulating lipid homeostasis and suppressing macrophage foam cell formation.

Atherosclerosis is regarded as a chronic progressive inflammatory disease and is a basic pathophysiological process in coronary artery disease which is life threatening in clinic. The formation of foam cell plays a key role in the pathogenesis of atherosclerosis. OxLDL is a significant factor in progression of coronary artery disease. Our studies have demonstrated that USP14 promotes cancer development and mediates progression of cardiac hypertrophy and LPS‐induced inflammation. However, the underlying mechanism of USP14 is unknown. In this study, we found that the inhibition of USP14 significantly suppressed the oxLDL uptake, subsequently decreased the foam cell formation. Surprisingly, USP14 has an effect on the expression of CD36 but not SR‐A, ABCA1, Lox‐1, ABCG1 and SR‐Bl. Furthermore, USP14 stabilizes CD36 protein via cleaving the ubiquitin chain on CD36. Blocking CD36 activation using antibody‐dependent blocking assay remarkably attenuated the function of USP14 on the formation of foam cell. In summary, our results suggested that the inhibition of USP14 decreases foam cell formation by down‐regulating CD36‐mediated lipid uptake and provides a potential therapeutic target for atherosclerosis.

Background and Purpose: Atherosclerosis is vascular disease of chronic inflammation and lipid disorder, which is a major cause of coronary heart disease. Foam cell formation is key progress during the atherosclerosis development. Insulin-like growth factor (IGF)-1 is a growth hormone that plays a crucial role in growth, metabolism, and homeostasis. Previous studies have demonstrated that increase in circulating IGF-1 can reduce atherosclerotic burden. However, active IGF-1 is characterized with poor tissue retention and is at a very low level in circulation system. Therefore, supplementation of exogenous IGF-1 to restore the physiological level is a promising approach to inhibit atherosclerosis. In this study, we develop a self-assembling, anti-inflammatory drug-modified peptide derived from IGF-1 to mimic IGF-1 bioactivity and simultaneously with an anti-inflammatory property for the treatment of atherosclerosis. Methods: ApoE-/- mice were subcutaneously (s.c.) injected with the different hydrogels or natural IGF-1 protein solution per week and simultaneously fed a high-fat diet for 16 weeks. Atherosclerotic lesion formation and stability were assessed after treatment. Moreover, peritoneal macrophage and serum samples were collected to determine lipid profile and inflammatory cytokines. Concurrently, we determined the effect of bifunctional supramolecular nanofibers/hydrogel on cholesterol efflux, foam cell formation, phenotypic transformation of VSMC to macrophage-like cells, and macrophage polarization in vitro or in vivo. Results: Bifunctional supramolecular nanofibers/hydrogel for the treatment of atherosclerosis was formed by a short peptide consisting of a tetrapeptide SSSR from C-region of growth factor IGF-1, an anti-inflammatory drug naproxen (Npx), and a powerful self-assembling D-peptide DFDF. The resulting hydrogel of Npx-DFDFGSSSR (Hydrogel 1, H1) possessed both the anti-inflammatory and IGF-1 mimicking properties, and it efficiently promoted the expression of ABCA1 and ABCG1, thereby significantly reducing cholesterol accumulation in macrophages and preventing foam cell formation. Moreover, H1 markedly inhibited the transformation of vascular smooth muscle cells (VSMCs) into macrophage-like cells which also contributed to foam cell formation. In addition, H1 significantly reduced the inflammatory response in vitro and in vivo. Most importantly, the IGF-1 mimetic peptide showed comparable performance to IGF-1 in vivo and inhibited atherosclerosis by markedly reducing lesion area and enhancing plaque stability. Conclusions: Our study provides a novel supramolecular nanomaterial to inhibit pathological progress of atherosclerosis through regulating cholesterol efflux and inflammation, which may contribute to the development of a promising nanomedicine for the treatment of atherosclerosis in the clinic.

This review discusses formation of reactive halogen species (RHS) catalyzed by myeloperoxidase (MPO), an enzyme mostly present in leukocytes. An imbalance between the RHS production and body’s ability to remove or neutralize them leads to the development of halogenative stress. RHS reactions with proteins, lipids, carbohydrates, and antioxi-dants in the content of low-density lipoproteins (LDLs) of the human blood are described. MPO binds site-specifically to the LDL surface and modifies LDL properties and structural organization, which leads to the LDL conversion into proatherogenic forms captured by monocytes/macrophages, which causes accumulation of cholesterol and its esters in these cells and their transformation into foam cells, the basis of atherosclerotic plaques. The review describes the biomarkers of MPO enzymatic activity and halogenative stress, as well as the involvement of the latter in the development of atherosclerosis.

Cholesterol biosynthetic intermediates, such as lanosterol and desmosterol, are emergent immune regulators of macrophages in response to inflammatory stimuli or lipid overloading, respectively. However, the participation of these sterols in regulating macrophage functions in the physiological context of atherosclerosis, an inflammatory disease driven by the accumulation of cholesterol-laden macrophages in the artery wall, has remained elusive. Here, we report that desmosterol, the most abundant cholesterol biosynthetic intermediate in human coronary artery lesions, plays an essential role during atherogenesis, serving as a key molecule integrating cholesterol homeostasis and immune responses in macrophages. Depletion of desmosterol in myeloid cells by overexpression of 3β-hydroxysterol Δ(24)-reductase (DHCR24), the enzyme that catalyzes conversion of desmosterol to cholesterol, promotes the progression of atherosclerosis. Single-cell transcriptomics in isolated CD45(+)CD11b(+) cells from atherosclerotic plaques demonstrate that depletion of desmosterol increases interferon responses and attenuates the expression of antiinflammatory macrophage markers. Lipidomic and transcriptomic analysis of in vivo macrophage foam cells demonstrate that desmosterol is a major endogenous liver X receptor (LXR) ligand involved in LXR/retinoid X receptor (RXR) activation and thus macrophage foam cell formation. Decreased desmosterol accumulation in mitochondria promotes macrophage mitochondrial reactive oxygen species production and NLR family pyrin domain containing 3 (NLRP3)–dependent inflammasome activation. Deficiency of NLRP3 or apoptosis-associated speck-like protein containing a CARD (ASC) rescues the increased inflammasome activity and atherogenesis observed in desmosterol-depleted macrophages. Altogether, these findings underscore the critical function of desmosterol in the atherosclerotic plaque to dampen inflammation by integrating with macrophage cholesterol metabolism and inflammatory activation and protecting from disease progression.

Mycobacteriosis, mostly resulting from Mycobacterium tuberculosis (MTb), nontuberculous mycobacteria (NTM), and Mycobacterium leprae (M. leprae), is the long-standing granulomatous disease that ravages several organs including skin, lung, and peripheral nerves, and it has a spectrum of clinical-pathologic features based on the interaction of bacilli and host immune response. Histiocytes in infectious granulomas mainly consist of infected and uninfected macrophages (Mφs), multinucleated giant cells (MGCs), epithelioid cells (ECs), and foam cells (FCs), which are commonly discovered in lesions in patients with mycobacteriosis. Granuloma Mφ polarization or reprogramming is the crucial appearance of the host immune response to pathogen aggression, which gets a command of endocellular microbe persistence. Herein, we recapitulate the current gaps and challenges during Mφ polarization and the different subpopulations of mycobacteriosis.

In atherosclerotic lesions, vascular smooth muscle cells (VSMCs) represent half of the foam cell population, which is characterized by an aberrant accumulation of undigested lipids within lysosomes. Loss of lysosome function impacts VSMC homeostasis and disease progression. Understanding the molecular mechanisms underlying lysosome dysfunction in these cells is, therefore, crucial. We identify cholesteryl hemiazelate (ChA), a stable oxidation end-product of cholesteryl-polyunsaturated fatty acid esters, as an inducer of lysosome malfunction in VSMCs. ChA-treated VSMCs acquire a foam-cell-like phenotype, characterized by enlarged lysosomes full of ChA and neutral lipids. The lysosomes are perinuclear and exhibit degradative capacity and cargo exit defects. Lysosome luminal pH is also altered. Even though the transcriptional response machinery and autophagy are not activated by ChA, the addition of recombinant lysosomal acid lipase (LAL) is able to rescue lysosome dysfunction. ChA significantly affects VSMC proliferation and migration, impacting atherosclerosis. In summary, this work shows that ChA is sufficient to induce lysosomal dysfunction in VSMCs, that, in ChA-treated VSMCs, neither lysosome biogenesis nor autophagy are triggered, and, finally, that recombinant LAL can be a therapeutic approach for lysosomal dysfunction.

Mesenchymal stem cell (MSC) therapy has potential applications in treating atherosclerosis and coronary heart disease (CAD). Previous studies have demonstrated that MSCs are the most preferable sources of therapeutic exosomes, which carry long non‑coding RNAs and participate in the progression of atherosclerosis. The results of our previous bioinformatics study demonstrated that the levels of LOC100129516 were significantly upregulated in peripheral blood mononuclear cells obtained from patients with CAD. However, the biological role of LOC100129516 in the development of atherosclerosis remains to be elucidated. In the present study, THP‑1 cells were treated with oxidized low‑density lipoproteins to induce foam cell formation in vitro. Reverse transcription‑quantitative PCR (RT‑qPCR) was performed to detect the levels of LOC100129516 in THP‑1 macrophage‑derived foam cells. In addition, an in vivo model of atherosclerosis was established using Apolipoprotein E (ApoE) knockout (ApoE‑/‑) mice. The results of the RT‑qPCR assays demonstrated that the levels of LOC100129516 were upregulated in THP‑1 macrophage‑derived foam cells. MSC‑derived exosomes were able to deliver small interfering (si)‑LOC100129516 to THP‑1 cells to reduce the levels of LOC100129516. Moreover, transfection of si‑LOC100129516 via exosomal delivery significantly decreased the levels of total cholesterol (TC), free cholesterol and cholesterol ester in THP‑1 macrophage‑derived foam cells. Exosomal‑mediated delivery of si‑LOC100129516 decreased TC levels and low‑density lipoprotein levels in the ApoE‑/‑ atherosclerosis mouse model. Mechanistically, exosomal‑mediated delivery of si‑LOC100129516 promoted cholesterol efflux by activating the peroxisome proliferator‑activated receptor γ (PPARγ)/liver X receptor α (LXRα)/phospholipid‑transporting ATPase ABCA1 (ABCA1) signaling pathway in vitro and in vivo. Collectively, these results suggested that exosomal‑mediated delivery of si‑LOC100129516, in which the exosomes were derived from MSCs, promoted cholesterol efflux and suppressed intracellular lipid accumulation, ultimately alleviating the progression of atherosclerosis via stimulation of the PPARγ/LXRα/ABCA1 signaling pathway.

Oxidized LDL (oxLDL) plays a pivotal role on atherosclerosis development, mainly in the formation of lipid-laden macrophage "foam cells". As a consequence, substances that can modulate LDL oxidation have a pharmacological and therapeutic relevance. Based in previous findings showing the ability of Syzigium cumini leaf extract (ScExt) in preventing LDL oxidation in vitro, this study was aimed to assess the effects of ScExt on oxLDL-mediated toxicity in murine J774 macrophages-like cells. For biochemical analyses, LDL isolated from fresh human plasma and oxidized with CuSO4 was incubated with ScExt pre-treated macrophages. Our results demonstrated that ScExt was efficient in preventing the overproduction of reactive oxygen/nitrogen species (ROS/RNS), the loss of macrophage's viability and the foam cells formation induced by oxLDL. These protective effects of ScExt make it a promising antioxidant for future trials toward atherogenesis.

Polybrominated diphenyl ethers (PBDEs) are used worldwide in brominated flame retardants. Although due to the forbiddance of their application, PBDEs continuously exist in the environment due to their persistence. Therefore, it is important to expand the understanding of their potential toxicities and human risks. The underlying cardiovascular toxicological mechanisms of PBDEs are still largely unknown. Our previous studies indicated that PBDE quinone-type metabolite (PBDEQ) exposure causes reactive oxygen species (ROS)-driven cytotoxicity and various types of programmed cell death. Here, we first reported PBDEQ exposure induces atherosclerosis progression in bone marrow-derived macrophages (BMDMs) isolated from wild-type C57BL/6 or CD36-/- mice and J774A.1 macrophage models. First, we found that PBDEQ exposure induced lipid accumulation in oxidized low-density lipid (Ox-LDL)-treated J774A.1 macrophages. Consistently, in J774A.1 macrophages, PBDEQ exposure resulted in NLR family pyrin domain containing 3 (NLRP3) inflammasome activation and pyroptosis. CD36, a scavenger receptor responsible for the mediation of Ox-LDL uptake, was upregulated upon PBDEQ treatment. On the contrary, genetic knockout of CD36 or CD36 silencing by small interfering RNA efficiently attenuates PBDEQ-promoted lipid accumulation in BMDMs and J774A.1 macrophages. These findings highlight the effect of CD36 on the cardiovascular toxicity of PBDEs, which provides a better understanding of the pro-atherosclerosis effect of PBDEs.

The nuclear factor of activated T-cells 5 (NFAT5) is a transcriptional regulator of macrophage activation and T-cell development, which controls stabilizing responses of cells to hypertonic and biomechanical stress. In this study, we detected NFAT5 in the media layer of arteries adjacent to human arteriosclerotic plaques and analyzed its role in vascular smooth muscle cells (VSMCs) known to contribute to arteriosclerosis through the uptake of lipids and transformation into foam cells. Exposure of both human and mouse VSMCs to cholesterol stimulated the nuclear translocation of NFAT5 and increased the expression of the ATP-binding cassette transporter Abca1, required to regulate cholesterol efflux from cells. Loss of Nfat5 promoted cholesterol accumulation in these cells and inhibited the expression of genes involved in the management of oxidative stress or lipid handling, such as Sod1, Plin2, Fabp3, and Ppard. The functional relevance of these observations was subsequently investigated in mice fed a high-fat diet upon induction of a smooth muscle cell-specific genetic ablation of Nfat5 (Nfat5(SMC)-/- ). Under these conditions, Nfat5(SMC)-/- but not Nfat5fl/fl mice developed small, focal lipid-rich lesions in the aorta after 14 and 25 weeks, which were formed by intracellular lipid droplets deposited in the sub-intimal VSMCs layer. While known for being activated by external stimuli, NFAT5 was found to mediate the expression of VSMC genes associated with the handling of lipids in response to a cholesterol-rich environment. Failure of this protective function may promote the formation of lipid-laden arterial VSMCs and pro-atherogenic vascular responses.

The formation of fat-laden foam cells plays an important role in the initiation and progression of atherosclerosis (AS). Amentoflavone (AF) is found in various traditional Chinese medicines, such as ginkgo biloba, which are used to treat cardiovascular diseases (CVDs). We aimed to explore the potential effects and mechanisms of AF on lipid accumulation, and its possible application in atherosclerotic cardiovascular disease (ASCVD). Cellular models of lipid accumulation were established by treatment of HUASMCs and THP-1 cells with oxidized low-density lipoprotein (ox-LDL). Cell viability, lipid accumulation, and ox-LDL uptake were assessed. Small interfering RNAs (siRNAs) and overexpression plasmids were used to reveal the hierarchical correlations of regulatory pathways. AF reduced the lipid accumulation and ox-LDL uptake induced by ox-LDL, and reduced the expression levels of cluster of differentiation 36 (CD36) and peroxisome proliferator-activated receptor gamma (PPARγ) proteins, while the expression level of ATP binding cassette subfamily A member 1 (ABCA1) increased. Knockdown of PPARγ or CD36 with siRNAs prevented ox-LDL-induced lipid accumulation. Overexpression of CD36 or PPARγ promoted the lipid accumulation induced by ox-LDL and eliminated the effect of AF on ox-LDL-induced lipid accumulation. Overall, AF prevents ox-LDL-induced lipid accumulation by suppressing the PPARγ/CD36 signaling pathway.

Background Accumulation of foam cells in the neointima represents an early stage of atherosclerosis. 1-trifluoromethoxyphenyl-3-(1-propionylpiperidine-4-yl) urea (TPPU), a novel soluble epoxide hydrolase inhibitor (sEHi), effectively elevates epoxyeicosatrienoic acid (EET) levels. The effects of EETs on macrophages foam cells formation are poorly understood.Methods and Results:Incubation of foam cells with TPPU markedly ameliorate cholesterol deposition in oxidized low-density lipoprotein (oxLDL)-loaded macrophages by increasing the levels of EETs. Notably, TPPU treatment significantly inhibits oxLDL internalization and promotes cholesterol efflux. The elevation of EETs results in a decrease of class A scavenger receptor (SR-A) expression via downregulation of activator protein 1 (AP-1) expression. Additionally, TPPU selectively increases protein but not the mRNA level of ATP-binding cassette transporter A1 (ABCA1) through the reduction of calpain activity that stabilizes the protein. Moreover, TPPU treatment reduces the cholesterol content of macrophages and inhibits atherosclerotic plaque formation in apolipoprotein E-deficient mice. These changes induced by TPPU are dependent on heme oxygenase-1 (HO-1) activation. Conclusions The present study findings elucidate a precise mechanism of regulating cholesterol uptake and efflux in macrophages, which involves the prevention of atherogenesis by increasing the levels of EETs with TPPU.

Several studies have demonstrated that allergic asthma can induce atherosclerosis formation in mice. Moreover, allergic asthma and atherosclerosis have been shown to be strongly associated with dyslipidemia. In this study, we investigated the underlying mechanism of allergic asthma-aggravated atherosclerosis-induced cholesterol metabolism disorder in asthmatic apoE-/- mice. We found that allergic asthma increased the expression of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) in the liver and CD36 in the aorta during the acute and advanced stages of atherosclerosis, respectively. These results indicate that cholesterol biosynthesis is increased during acute atherosclerosis and cholesterol uptake and foam cell formation is increased during advanced atherosclerosis. Simvastatin administration significantly ameliorated the aortic root lesion size of asthmatic mice and significantly decreased HMGCR and CD36 expression. However, the expression of the low-density lipoprotein receptor and ATP-binding cassette transporter A1 was markedly increased, indicating that the beneficial effect of statins in allergic asthma and coronary artery disease was mediated, at least in part, by decreasing cholesterol biosynthesis and foam cell formation. In conclusion, allergic asthma aggravates atherosclerosis by regulating cholesterol metabolism in apoE-/- mice. Allergic asthma selectively promotes cholesterol biosynthesis in acute atherosclerosis and increases foam cell formation in advanced atherosclerosis.

Background and aims Aberrant fucosylation, such as α-1,6 fucosylation catalyzed by fucosyltransferase 8 (Fut8), is associated with reduced cell migration and is responsible for cholesterol-enriched foam cell accumulation in the intima in the early stage of atherosclerosis. The current study evaluated the impact of glycosyltransferases on foam cell migration induced by lysophosphatidic acid (LPA) and its potential mechanism. Methods The mobility of foam cells was evaluated via transwell and scratch assays. The expression of Fut8 and α-1,6 fucosylation of proteins were assessed by RT-PCR, Western blotting, etc. Overexpression of Fut8 was used to explore the direct relationship between Fut8 and foam cell migration. Dual luciferase reporter assay was performed to determine whether the regulation of Fut8 by LPA occurred at the transcriptional level. Binding of hepatocyte nuclear factor 1-alpha (HNF1α) to the Fut8 promoter was assessed by electrophoretic mobility shift assay and chromatin immunoprecipitation assay. Results We found that the migration capacity of foam cells induced by LPA was significantly decreased. Fut8 and α-1,6 fucosylation showed the most obvious decline after treatment with 200 μM LPA for 24 h. Overexpression of Fut8 was able to restore the foam cell migration capacity. Another important finding was that the LPA1 and LPA3 (LPA1,3) receptors were involved in the regulation of Fut8. It is interesting to note that LPA led to a decrease in Fut8 gene transcription activity, and HNF1α transcription factor played a positive role in downregulation of Fut8 promoter activity. Conclusions Our results strongly indicated that the LPA-LPA1, 3 receptor-HNF1α pathway is involved in the downregulation of Fut8, leading to diminished foam cell migration.

Background Increased macrophage and foam cell apoptosis during early atherogenesis retards plaque progression by impeding foam cell formation, suppressing inflammation and limiting lesion cellularity. Our previous in vitro study in THP1 macrophages demonstrated that Terminalia Arjuna (TA) attenuates dual-specificity phosphatase1 (DUSP1), a key negative regulator of JNK/P38MAPK signaling cascade, the branch also implicated in the UPR (unfolded protein response)-CHOP-mediated apoptotic pathway; however this pathway has not been explored so far in the presence of TA. Therefore, we aimed to elucidate the pro-apoptotic effect of aqueous bark extract of TA (aqTAE) on macrophage and foam cells and the underlying mechanism associated with it. Methods THP1 cells were initially differentiated into macrophages with phorbol-12-myristate-13-acetate (PMA) (100 ng/ml) for 24 h, followed by ox-LDL (100 μg/ml) treatment for another 24 h to induce foam cell formation. Thereafter, macrophages and ox-LDL- treated cells were incubated with aqTAE (100 μg/ml) for the next 24 h. Further, Oil Red O (ORO) staining, CD36 expression profiling, apoptotic assay and transcriptional and translational expression of ER-stress markers i.e., X-box binding protein 1 (XBP1) and C/EBP homologous protein (CHOP) were performed for elucidating the potential mechanism underlying TA-induced macrophage and foam cell apoptosis. Results We demonstrated that ox-LDL treatment significantly increased lipid accumulation and upregulated CD36 expression, indicating foam cell formation; while the addition of aqTAE resulted in a significant decline in ORO positive cells, and suppression of CD36 expression in ox-LDL-stimulated macrophages, suggestive of reduced formation of lipid-laden foam cells. Further, aqTAE treatment alone and in combination with oxidized low-density lipoprotein (ox-LDL) stimulus, significantly attenuated CD36 expression; increased apoptosis; and augmented the expression of UPR regulatory proteins including XBP1 and CHOP, and similar observations were noted when cells were treated with ox-LDL alone. These findings indicate that TA promotes macrophage and foam cell apoptosis via enhancing UPR-mediated activation of JNK/p38MAPK-CHOP pathway in a DUSP1-dependent manner, implying a possible interplay between ox-LDL-induced ER stress- and TA-mediated MAPK signaling. Conclusion Our data shows that aqTAE inhibits foam cell formation, as well as promotes macrophage and foam cell apoptosis by augmenting UPR- JNK/p38MAPK-CHOP signaling cascade via inhibiting DUSP1. These findings provide novel mechanistic insight into the anti-atherogenic potential of TA, which may prove beneficial against early-stage atherosclerotic lesions.

The contribution of soluble epoxide hydrolase (sEH) to atherosclerosis has been well defined. However, less is understood about the role of sEH and its underlying mechanism in the cholesterol metabolism of macrophages. The expression of sEH protein was increased in atherosclerotic aortas of apolipoprotein E‐deficient mice, primarily in macrophage foam cells. Oxidized low‐density lipoprotein (oxLDL) increased sEH expression in macrophages. Genetic deletion of sEH (sEH −/−) in macrophages markedly exacerbated oxLDL‐induced lipid accumulation and decreased the expression of ATP‐binding cassette transporters‐A1 (ABCA1) and apolipoprotein AI‐dependent cholesterol efflux following oxLDL treatment. The down‐regulation of ABCA1 in sEH −/− macrophages was due to an increase in the turnover rate of ABCA1 protein but not in mRNA transcription. Inhibition of phosphatase activity, but not hydrolase activity, of sEH decreased ABCA1 expression and cholesterol efflux following oxLDL challenge, which resulted in increased cholesterol accumulation. Additionally, oxLDL increased the phosphatase activity, promoted the sEH‐ABCA1 complex formation and decreased the phosphorylated level of ABCA1 at threonine residues. Overexpression of phosphatase domain of sEH abrogated the oxLDL‐induced ABCA1 phosphorylation and further increased ABCA1 expression and cholesterol efflux, leading to the attenuation of oxLDL‐induced cholesterol accumulation. Our findings suggest that the phosphatase domain of sEH plays a crucial role in the cholesterol metabolism of macrophages.

Nitrogen mustard (NM) is a vesicant known to target the lung, causing acute injury which progresses to fibrosis. Evidence suggests that activated macrophages contribute to the pathologic response to NM. In these studies, we analyzed the role of lung lipids generated following NM exposure on macrophage activation and phenotype. Treatment of rats with NM (0.125 mg/kg, i.t.) resulted in a time-related increase in enlarged vacuolated macrophages in the lung. At 28 days postexposure, macrophages stained positively for Oil Red O, a marker of neutral lipids. This was correlated with an accumulation of oxidized phospholipids in lung macrophages and epithelial cells and increases in bronchoalveolar lavage fluid (BAL) phospholipids and cholesterol. RNA-sequencing and immunohistochemical analysis revealed that lipid handling pathways under the control of the transcription factors liver-X receptor (LXR), farnesoid-X receptor (FXR), peroxisome proliferator-activated receptor (PPAR)-ɣ, and sterol regulatory element-binding protein (SREBP) were significantly altered following NM exposure. Whereas at 1–3 days post NM, FXR and the downstream oxidized low-density lipoprotein receptor, Cd36, were increased, Lxr and the lipid efflux transporters, Abca1 and Abcg1, were reduced. Treatment of naïve lung macrophages with phospholipid and cholesterol enriched large aggregate fractions of BAL prepared 3 days after NM exposure resulted in upregulation of Nos2 and Ptgs2, markers of proinflammatory activation, whereas large aggregate fractions prepared 28 days post NM upregulated expression of the anti-inflammatory markers, Il10, Cd163, and Cx3cr1, and induced the formation of lipid-laden foamy macrophages. These data suggest that NM-induced alterations in lipid handling and metabolism drive macrophage foam cell formation, potentially contributing to the development of pulmonary fibrosis.

Foam cells are specialized types of cells which predominate the necrotic core of atherosclerotic plaque. Recently, autophagy-mediated cholesterol efflux from foam cells has been proposed as a beneficial therapy for atherosclerosis. The purpose of this study was to delineate the underlying molecular mechanism of oligomeric proanthocyanidins (OPC) and epigallocatechin gallate (EGCG) induced autophagy of foam cells and associated cholesterol efflux. The oxidized low-density lipoprotein induced foam cells demonstrated impaired autophagy flux through the downregulated expressions of LC3BII/LC3BI, autophagy related gene-5, Class III phosphoinositide 3 kinase (Class III PI3K), Beclin1, ABCA1, and ABCG1 with concomitant increase in the expressions of protein 62, Class I phosphoinositide 3 kinase, Akt, and mammalian target of rapamycin. However, these effects were significantly abolished by treatment with OPC and EGCG through activation of autophagy flux via Class III PI3K/Beclin1 and with upregulated expression of transporter proteins ABCA1 and ABCG1. Furthermore, the cholesterol efflux process in the foam cells was activated by lysosomal acid lipase and cathepsin D facilitated lipolysis of lipid droplets. Taken together, our data demonstrate that OPC and EGCG treatment stimulated the coordinated activation of autophagy and cholesterol efflux through Class III PI3K/Beclin1 pathway in foam cells, suggesting a promising therapeutic strategy against atherosclerosis.

Lonicera caerulea berry polyphenols (LCBP) are known to reduce cholesterol accumulation. Currently, it is unknown whether LCBP can activate Sirtuin 1 (SIRT1) to regulate the formation of RAW264.7 macrophage foam cells. In this study, the effect of LCBP on lipid accumulation in macrophages was evaluated. Fluorescently labeled ox-LDL and 25-NBD cholesterol were used to detect the ox-LDL uptake and cholesterol outflow rate from macrophages. Gene silencing was performed using siRNA to detect changes in the expression of the ATP-binding cassette transporter A1 (ABCA1), sterol regulatory element-binding protein 2 (SREBP2), and SIRT1 proteins using Western blotting, and changes in the expression of miR-33 were detected by real-time polymerase chain reaction. The results showed that treatment with 80 μg/mL LCBP significantly inhibited the accumulation of lipids in RAW264.7 macrophages induced by ox-LDL and reduced intracellular cholesterol levels by activating SIRT1 to enhance the expression of ABCA1, a cholesterol efflux gene, but not independent effect. Of the three key LCBP components investigated, chlorogenic acid was found to activate SIRT1 and regulate the expression of the cholesterol-related factors ABCA1, SREBP2, and miR-33; cyanidin-3-glucoside and catechins were effective to a lesser extent. Our results suggest a novel hypolipidemic mechanism of LCBP.

Far beyond hemostasis and thrombosis, significant evidence has indicated the critical role of platelets in atherosclerosis. SDF-1 is among the pro-inflammatory chemokines that are increased in platelets of patients with coronary artery disease (CAD). The goal of the current work is to identify the in vitro effect of platelets from either CAD patients or healthy volunteers on the induction of macrophages and foam cells.The expression of SDF-1 on platelet surfaces in CAD patients and healthy volunteers was investigated using flow cytometry. We also evaluated the CXCR4/CXCR7 expression on monocytes from buffy coats of healthy volunteers. The effect of platelets from CAD patients and healthy volunteers on differentiation of monocytes and foam cell formation was evaluated using Oil Red O (ORO) staining. Flow cytometry and real-time PCR were also employed to evaluate surface markers and mRNA expression of genes involved in this process after co-culture of platelets with monocytes.Monocytes in co-culture with platelets acquired a spindleshape appearance and ORO-positive lipid droplets. In addition, platelets could induce CD163 expression, as an important marker of M2 macrophage, and upregulate the mRNA expression of theOur results indicate that platelets from CAD patients could provoke monocyte differentiation into macrophages with an M2 phenotype, which in turn may participate in an atheroprotective process.Hemostaz ve trombozun çok ötesinde, önemli kanıtlar trombositlerin aterosklerozdaki kritik rolüne işaret etmektedir. SDF-1 koroner arter hastalığı (KAH) olan hastaların trombositlerinde artan proenflamatuvar kemokinler arasında yer almaktadır. Bu çalışmanın amacı, KAH olan hastalar ve sağlıklı gönüllülerin trombositlerinin makrofajlar ve köpük hücrelerinin indüksiyonunda in vitro etkilerini belirlemektir.KAH olan hastalar ve sağlıklı gönüllülerde trombosit yüzeyindeki SDF-1 ekspresyonu akım sitometri ile incelendi. Biz ayrıca sağlıklı gönüllülerin beyaz kan hücrelerinin bir tabakasından elde edilen monositlerde CXCR4/CXCR7 ekspresyonunu değerlendirdik. KAH olan hastalar ve sağlıklı gönüllülerin trombositlerinin monositler ve köpük hücreleri oluşumu üzerine etkisi Oil Red O (ORO) boyası kullanılarak değerlendirildi. Yüzey belirteçlerini ve trombositlerin monositlerle ortak kültürü sonrası süreçteki genlerin mRNA ekspresyonunu değerlendirmek için ayrıca akım sitometri ve gerçek zamanlı polimeraz zincir reaksiyonu çalışıldı.Trombositler ile ortak kültüre edilen monositler iğ şeklinde görünüm ve ORO-pozitif yağ damlacıkları kazandılar. Ayrıca, trombositler M2 makrofajın önemli bir belirteçi olan CD163 ekspresyonunu indüklediler ve monositlerdekiBulgularımız, KAH olan hastaların trombositlerinin, monositlerin M2 fenotipinde makrofajlara farklılaşmasına neden olduklarını ve böylece ateroprotektif sürece iştirak ettiklerini göstermektedir.

Objective— Smooth muscle cells (SMCs) are the most abundant cells in human atherosclerotic lesions and are suggested to contribute at least 50% of atheroma foam cells. In mice, SMCs contribute fewer total lesional cells. The purpose of this study was to determine the contribution of SMCs to total foam cells in apolipoprotein E-deficient (ApoE −/− ) mice, and the utility of these mice to model human SMC foam cell biology and interventions. Approach and Results— Using flow cytometry, foam cells in the aortic arch of ApoE −/− mice were characterized based on the expression of leukocyte-specific markers. Nonleukocyte foam cells increased from 37% of total foam cells in 27-week-old to 75% in 57-week-old male ApoE −/− mice fed a chow diet and were ≈70% in male and female ApoE −/− mice following 6 weeks of Western diet feeding. A similar contribution to total foam cells by SMCs was found using SMC-lineage tracing ApoE −/− mice fed the Western diet for 6 or 12 weeks. Nonleukocyte foam cells contributed a similar percentage of total atheroma cholesterol and exhibited lower expression of the cholesterol exporter ABCA1 (ATP-binding cassette transporter A1) when compared with leukocyte-derived foam cells. Conclusions— Consistent with previous studies of human atheromas, we present evidence that SMCs contribute the majority of atheroma foam cells in ApoE −/− mice fed a Western diet and a chow diet for longer periods. Reduced expression of ABCA1, also seen in human intimal SMCs, suggests a common mechanism for formation of SMC foam cells across species, and represents a novel target to enhance atherosclerosis regression.

Sortilin is closely associated with hyperlipidemia and the risk of atherosclerosis (AS). The role of sortilin and the underlying mechanism in peripheral macrophage are not fully understood. In this study, we investigated the effect of macrophage sortilin on ATP-binding cassette transporter A1 (ABCA1) expression, ABCA1-mediated cholesterol efflux, and aortic AS. Macrophage sortilin expression was upregulated by oxidized low-density lipoproteins (ox-LDLs) in both concentration- and time-dependent manners. Its expression reached the peak level when cells were incubated with 50 μg/ml ox-LDL for 24 h. Overexpression of sortilin in macrophage reduced cholesterol efflux, leading to an increase in intracellular total cholesterol, free cholesterol, and cholesterol ester. Sortilin was found to bind with ABCA1 protein and suppress macrophage ABCA1 expression, resulting in a decrease in cholesterol efflux from macrophages. The inhibitory effect of sortilin in cholesterol efflux was partially reversed by treatment with chloroquine, a lysosomal inhibitor. On the contrary, the ABCA1 protein level and ABCA1-mediated cholesterol efflux is increased by sortilin short hairpin RNA transfection. The fecal and biliary cholesterol 3H-sterol from cholesterol-laden mouse peritoneal macrophage was reduced by sortilin overexpression through lentivirus vector (LV)-sortilin in low-density lipoprotein receptor knockout mice, which was prevented by co-treatment with chloroquine. Treatment with LV-sortilin reduced plasma high-density lipoprotein and increased plasma ox-LDL levels. Accordingly, aortic lipid deposition and plaque area were exacerbated, and ABCA1 expression was reduced in mice in response to infection with LV-sortilin alone. These effects of LV-sortilin were partially reversed by chloroquine. Sortilin enhances lysosomal degradation of ABCA1 protein and suppresses ABCA1-mediated cholesterol efflux from macrophages, leading to foam cell formation and AS development.