oxLDL In Macrophages Research Articles

The atheroprotective role of fucoidan involves the reduction of foam cell formation by altering cholesterol flux-associated factors in macrophages

Atherosclerosis is characterized by the accumulation of lipid-laden cells in the arterial walls, resulting from dysregulation of cholesterol homeostasis in the macrophage, triggered by oxidized low-density lipoprotein (oxLDL). Previous studies have shown that fucoidan, a sulfated polysaccharide from brown seaweeds, has several atheroprotective activities, however, the mechanism of fucoidan protection is not fully understood. Thus, we investigated the effect of fucoidan on atherogenesis in apolipoprotein E-deficient (ApoE−/−) mice, on oxLDL uptake by macrophages, and on the expression of the flux-associated scavenger receptors by macrophages. Also, we examined the absorption and biodistribution of orally administered fucoidan. ApoE−/− mice fed on a cholesterol-rich diet supplemented with 1% fucoidan showed reduced dyslipidemia and atherosclerosis. Fucoidan was detected in blood and peripheral tissue after gavage, suggesting that it can exert direct systemic effects. In vitro, fucoidan reduced macrophage oxLDL uptake, which resulted in lower foam cell formation. This effect was associated with downregulation of the cholesterol influx-associated scavenger receptor (SR)-A expression, and upregulation of the cholesterol efflux-associated SR-B1 expression. In conclusion, fucoidan prevented oxLDL-mediated foam cell formation in macrophages by downregulating SR-A1/2 and by up-regulating SR-B1.

Oxidized low-density lipoprotein stimulates CD206 positive macrophages upregulating CD44 and CD133 expression in colorectal cancer with high-fat diet.

BACKGROUNDOxidized low-density lipoprotein (ox-LDL), which is abnormally increased in the serum of colorectal cancer (CRC) patients consuming a high-fat diet (HFD), may be one of the risk factors for the development of CRC. Ox-LDL exerts a regulatory effect on macrophages and may influence CRC through the tumor microenvironment. The role of ox-LDL in CRC remains unclear.AIMTo investigate the role of ox-LDL through macrophages in HFD associated CRC.METHODSThe expression of ox-LDL and CD206 was detected in colorectal tissues of CRC patients with hyperlipidemia and HFD-fed mice by immunofluorescence. We stimulated the macrophages with 20 μg/mL ox-LDL and assessed the expression levels of CD206 and the cytokines by cell fluorescence and quantitative polymerase chain reaction. We further knocked down LOX-1, the surface receptor of ox-LDL, to confirm the function of ox-LDL in macrophages. Then, LoVo cells were co-cultured with the stimulated macrophages to analyze the CD44 and CD133 expression by western blot.RESULTSThe expression of ox-LDL and the CD206 was significantly increased in the stroma of colorectal tissues of CRC patients with hyperlipidemia, and also upregulated in the HFD-fed mice. Moreover, an increased level of CD206 and decreased level of inducible nitric oxide synthase were observed in macrophages after ox-LDL continuous stimulation. Such effects were inhibited when the surface receptor LOX-1 was knocked down in macrophages. Ox-LDL could induce CD206+ macrophages, which resulted in high expression of CD44 and CD133 in co-cultured LoVo cells.CONCLUSIONOx-LDL stimulates CD206+ macrophages to upregulate CD44 and CD133 expression in HFD related CRC.

Identification and functional analysis of a biflavone as a novel inhibitor of transient receptor potential vanilloid 4-dependent atherogenic processes

Atherosclerosis, a chronic inflammatory disease of large arteries, is the major contributor to the growing burden of cardiovascular disease-related mortality and morbidity. During early atherogenesis, as a result of inflammation and endothelial dysfunction, monocytes transmigrate into the aortic intimal areas, and differentiate into lipid-laden foam cells, a critical process in atherosclerosis. Numerous natural compounds such as flavonoids and polyphenols are known to have anti-inflammatory and anti-atherogenic properties. Herein, using a fluorometric imaging plate reader-supported Ca2+ influx assay, we report semi high-throughput screening-based identification of ginkgetin, a biflavone, as a novel inhibitor of transient receptor potential vanilloid 4 (TRPV4)-dependent proatherogenic and inflammatory processes in macrophages. We found that ginkgetin (1) blocks TRPV4-elicited Ca2+ influx into macrophages, (2) inhibits oxidized low-density lipoprotein (oxLDL)-induced foam cell formation by suppressing the uptake but not the binding of oxLDL in macrophages, and (3) attenuates oxLDL-induced phosphorylation of JNK2, expression of TRPV4 proteins, and induction of inflammatory mRNAs. Considered all together, the results of this study show that ginkgetin inhibits proatherogenic/inflammatory macrophage function in a TRPV4-dependent manner, thus strengthening the rationale for the use of natural compounds for developing therapeutic and/or chemopreventive molecules.

Identification of IgG1 isotype phosphorylcholine antibodies for the treatment of inflammatory cardiovascular diseases.

BackgroundPhosphorylcholine (PC) is an important pro‐inflammatory damage‐associated molecular pattern. Previous data have shown that natural IgM anti‐PC protects against cardiovascular disease. We aimed to develop a monoclonal PC IgG antibody with anti‐inflammatory and anti‐atherosclerotic properties.MethodsUsing various techniques PC antibodies were validated and optimized. In vivo testing was performed in a femoral artery cuff model in ApoE3*Leiden mice. Safety studies are performed in rats and cynomolgus monkeys.ResultsA chimeric anti‐PC (PC‐mAb(T15), consisting of a human IgG1 Fc and a mouse T15/E06 Fab) was produced, and this was shown to bind specifically to epitopes in human atherosclerotic tissues. The cuff model results in rapid induction of inflammatory genes and altered expression of genes associated with ER stress and choline metabolism in the lesions. Treatment with PC‐mAb(T15) reduced accelerated atherosclerosis via reduced expression of endoplasmic reticulum stress markers and CCL2 production. Recombinant anti‐PC Fab fragments were identified by phage display and cloned into fully human IgG1 backbones creating a human monoclonal IgG1 anti‐PC (PC‐mAbs) that specifically bind PC, apoptotic cells and oxLDL. Based on preventing macrophage oxLDL uptake and CCL2 production, four monoclonal PC‐mAbs were selected, which to various extent reduced vascular inflammation and lesion development. Additional optimization and validation of two PC‐mAb antibodies resulted in selection of PC‐mAb X19‐A05, which inhibited accelerated atherosclerosis. Clinical grade production of this antibody (ATH3G10) significantly attenuated vascular inflammation and accelerated atherosclerosis and was tolerated in safety studies in rats and cynomolgus monkeys.ConclusionsChimeric anti‐PCs can prevent accelerated atherosclerosis by inhibiting vascular inflammation directly and through reduced macrophage oxLDL uptake resulting in decreased lesions. PC‐mAb represents a novel strategy for cardiovascular disease prevention.

Hexarelin attenuates atherosclerosis via inhibiting LOX-1-NF-κB signaling pathway-mediated macrophage ox-LDL uptake in ApoE-/- mice

Growth hormone secretagogues (GHS) have been proved to exert protective effects on the cardiovascular system, while their potential beneficial effects on macrophages in atherosclerosis (AS) are rarely been clarified. This study aimed to demonstrate whether hexarelin, a synthetic peptidyl GHS, can suppress AS progression via regulating the function of macrophages. AS was induced by chronic (3 months) feeding with high lipid diet in ApoE-/- mice. Mice were treated either with hexarelin (100 μg/kg s.c., q.d. for 3 months) (AS + Hex group) or saline (AS group). Age-matched C57BL/6 J mice were used as normal controls. AS and related signaling molecules in aortic tissues and RAW264.7 macrophages were identified with variant methods including histological staining, ELISA, western blotting, confocal microscopy and flow cytometry.AS significantly developed in ApoE−/− mice fed with high lipids diet. Hexarelin decreased serum TC, TG and LDL-c, increased serum HDL-c and attenuated the formation of atherosclerotic plaques and neointima compared with the AS group. Hexarelin decreased the aortic expressions of CD68 and LOX-1 which were elevated in the AS group. Hexarelin increased GHSR expression, suppressed ox-LDL uptake and LOX-1 expression and inhibited nuclear factor-kappa B (NF-κB) activation both in the aorta of ApoE−/− mice and in RAW264.7 macrophages. We conclude that hexarelin effectively attenuates AS progression in ApoE−/− mice by modulating circulatory lipids profile and inhibiting macrophage ox-LDL uptake via suppressing the LOX-1-NF-κB signaling pathway. The study supports the perspective of hexarelin as an anti-AS drug.

The Role Of Endothelial And Macrophage Scavenger Receptor Stabilin 1 And 2 In Early Atherosclerosis Development In Zebrafish

Atherosclerosis, the main underlying cause of cardiovascular disease and related morbidities, is a multifactorial disease in which liver endothelial cells and macrophages have shown to play an important role. Recently, it has been demonstrated that oxidized phospholipids in oxidized LDL (oxLDL) are pro-inflammatory and pro-atherogenic in hypercholesterolaemic mice. Multiple scavenger receptors are known for oxLDL. They form a superfamily of membrane bound receptors that bind and internalize different types of ligands including other modified lipoproteins, endogenous proteins and pathogens. Several studies, including a microarray study of our research group, have already identified scavenger receptors stabilin-1 and -2 (STAB1 and STAB2) as a risk factor for atherosclerosis, and therefore as potential targets for treatment. An upcoming and powerful model organism to study early atherogenesis and that allows non-invasive cell tracking studies is the zebrafish. Recent studies have demonstrated the possibility to induce (early) atherosclerosis in zebrafish. We have used Crispr/Cas9 mutagenesis to generate zebrafish stab1 and stab2 mutants. Our results show that oxLDL clearance by zebrafish scavenger endothelial cells (homologous to liver sinusoidal endothelial cells) is diminished in stab2 zebrafish mutants and is absent in stab1/2 double mutants. In stabilin mutants, macrophage oxLDL uptake is increased, suggesting a protective role for stabilins in foam cell formation. To study this further, we are currently performing an atherosclerosis study to examine foam cell formation and vascular lipid accumulation in stabilin mutant zebrafish.

Inhibitory effect of BF523 from Ilex hainanensis on ox LDL-induced foam cells formation

Cardio-cerebral vascular disease induced by atherosclerosis is a serious cause of human health. The pathogenesis of AS is very complex,and the oxidized low-density lipoprotein( ox LDL) induced foam cells formation is considered to be the most important cytological change in AS. Based on the definition of " TCM chemical biology",we clarified the chemical composition of Ilex hainanensis,the effective substances of I. hainanensis on the activity of anti-AS were screened. Then we found that saponin BF523 had the good inhibitory effect on foam cell formation. In this research,we studied the BF523 as the research object to clarify the molecular target of the active compound of I. hainanensis by foam cell formation model. The results showed that BF523 significantly inhibited the oxidation of ox LDL-induced macrophage foaming and decreased the lipid content in macrophages. BF523 had inhibited the phagocytosis of ox LDL in macrophages by reducing the mRNA and protein levels of scavenger receptor CD36,thereby inhibiting the occurrence and development of AS. These findings not only clarified the mechanism of the inhibition of foam cell formation by saponin BF523,but also provided a useful exploration for the enrichment of the theory of " TCM chemical biology".

Fibroblast growth factor 21 regulates foam cells formation and inflammatory response in Ox-LDL-induced THP-1 macrophages.

Macrophages are paramount to the initiation and procession of atherosclerosis, thus targeting macrophages in the progress of atherosclerosis is indispensable. Therefore, we perform in vitro experiments to investigate the effects of FGF-21 on macrophages in the progress of atherosclerosis. First, we use phorbol-12-myristate-13-acetate (PMA), a phorbol ester, to induce THP-1 cells into macrophages as macrophages model. After that we use Ox-LDL to induce macrophages into foam cells and simultaneously administrate with FGF-21 or not to determine whether FGF-21 has effects on foam cells formation and related inflammatory response. Wound healing results show that FGF-21 can inhibit macrophage migration. Oil Red-O stain, immunofluorescence and flow cytometer results show that FGF-21 can repress cholesterol accumulation in macrophages thereby inhibit foam cells formation and these effects can be abolished by FGFR inhibitor. Moreover, real-time PCR results showed that FGF-21 significantly reduces expression of inflammatory factors including IL-1α, IL-6 and TNF-α and this effect can be abolished by FGFR inhibitor. Furthermore, to determine the mechanism of FGF-21 regulates inflammatory response in Ox-LDL-induced THP-1 macrophages, western blotting results show that after treatment of Ox-LDL in macrophages, NF-κB signaling pathway is activated but FGF-21 can significantly inhibit this pathway. In addition, FGF-21 also regulates some regulators of lipid metabolism after treatment of Ox-LDL in macrophages. Above all, our findings demonstrate that FGF-21 can regulate foam cells formation, macrophage migration, inflammatory response and lipid metabolism in Ox-LDL-induced THP-1 macrophages.

IRhom2 promotes atherosclerosis through macrophage inflammation and induction of oxidative stress

Atherosclerosis is a complex chronic inflammatory disease that is characterized by the formation of lipid-rich plaques on the inner walls of the arteries. Inactive rhomboid protein 2 (iRhom2) was recently determined as a necessary regulator for the shedding of tumor necrosis factor-alpha (TNF-α) in immune cells. In the present study, we aimed to explore the effects of iRhom2 on the inflammatory response and oxidative stress induced by low-density lipoprotein (ox-LDL) in RAW264.7 and THP-1-derived macrophages. The expression levels of iRhom2 were also investigated in apolipoprotein E knockout (ApoE−/-) mice fed a high-fat diet (HFD). iRhom2 was significantly induced by ox-LDL in macrophages, as confirmed by Western blotting and RT-qPCR analysis. Furthermore, iRhom2 knockdown showed significant suppressive effects on the activation of ox-LDL-induced RAW264.7 and THP-1-derived macrophages through reducing TACE and TNFR2 expressions, and the inactivation of the IκBα/NF-κB signaling pathway. A reduction in reactive oxygen species (ROS) generation, malondialdehyde (MDA) levels, and nitric oxide (NOX) activity and an increase in glutathione peroxidase (GSH-Px) activity were determined in the absence of iRhom2 expression. In addition, the NF-E2 related factor-2 (Nrf-2)/heme oxygenase-1 (HO-1) pathway was also upregulated in ox-LDL-treated macrophages subjected to iRhom2 inhibition. Moreover, suppression of iRhom2 expression inactivated PI3K/AKT pathway activation, contributing to ROS reduction in ox-LDL-stimulated macrophages. iRhom2 was also significantly expressed in ApoE−/- mice fed HFD. Finally, we observed increased serum levels of TNF-α, TNFR1, and TNFR2 in patients with coronary artery atherosclerosis as compared to healthy volunteers. In conclusion, our findings suggested that iRhom2 played a key role in the pathogenesis of atherosclerosis, and that iRhom2 might be a potential therapeutic target against atherosclerosis.

Inhibition of the CD36 receptor reduces visceral fat accumulation and improves insulin resistance in obese mice carrying the BDNF-Val66Met variant

Obesity-induced metabolic dysfunctions increase the risk for vascular diseases, including type II diabetes and stroke. Managing obesity is of interest to address the worldwide health problem; however, the role of genetic variability in human obesity development and specific targets for obesity-related metabolic disease have not been thoroughly studied. A SNP in the brain-derived neurotropic factor (BDNF) gene that results in the substitution of a valine with a methionine at codon 66 (Val66Met) occurs with a high frequency in humans. This study addressed the effect of genetic variability in developing obesity and the efficacy of the inhibition of cluster of differentiation 36 (CD36), a multifunctional receptor implicated in obesity and insulin resistance, in WT mice and mice with the BDNF Val66Met variant. CD36 inhibition by salvionolic acid B (SAB) in diet-induced obese WT mice reduced visceral fat accumulation and improved insulin resistance. The benefit of SAB was abrogated in CD36 knockout mice, showing the specificity of SAB. In addition, mice with the Val66Met variant in both alleles (BDNFM/M) fed a high-fat diet exhibited extreme obesity with increased CD36 gene and protein levels in macrophages. Chronic SAB treatment in BDNFM/M mice significantly decreased visceral fat accumulation and improved insulin resistance. Notably, the effect of SAB was greater in the extremely obese BDNFM/M mice compared with the WT mice. The study demonstrated a link between BDNF Val66Met and elevated CD36 expression and suggested that CD36 inhibition may be a potential strategy to improve metabolic dysfunctions and to normalize risk factors for vascular diseases in the obese population.

Construction of CD36 gene silencing cell lines by lentivirus-mediated RNA interference and the effect on protein expression of caveolin-1

CD36, the major scavenger receptor, is intimately involved in the uptake of oxLDL in macrophages. To further study the function of CD36 in macrophages, we constructed CD36 gene silence cell lines (J774A.1) by lentivirus-mediated RNA interference technique, and analyzed the effect of CD36 in caveolin-1 protein expression. At first, 5 shRNA fragments were designed and synthesized according to the coding sequence (CDS) region of CD36 gene. Next, the CD36-shRNA was inserted into lentiviral vector to yield pLKO.1-CD36-shRNA plasmid. After DNA sequencing, the pLKO.1-CD36-shRNA plasmid and psiCHECK-II-CD36 were co-transfected into the 293T cells to screen the efficient CD36-shRNA. The efficient CD36-shRNA plasmid and the helper plasmid were co-transfected into the 293T cells to package the lentivirus, and then infected the J774A.1 cells. After screening by puromycin, CD36 gene silence cell lines (J774A.1) was established. Western blotting and confocal fluorescence microscopy results showed that the CD36 silencing efficiency in the gene silence cell line was 90%. Accompanied by a decrease in CD36 protein on cell surface, oxLDL binding to CD36 was significantly inhibited, indicating that the CD36 gene silence cell line is successfully established. Finally, the oxLDL stimulation and inhibitor experiments results showed that the CD36 knockdown significantly suppresses the phosphorylation of JNK and ERK, thereby inhibiting the oxLDL-induced caveolin-1 protein expression, demonstrating that CD36 modulates the caveolin-1 protein expression through the JNK/ERK-mediated signaling transduction.

JNK1 Mediates Lipopolysaccharide-Induced CD14 and SR-AI Expression and Macrophage Foam Cell Formation.

Foam cell formation is the key process in the development of atherosclerosis. The uptake of oxidized low-density lipoprotein (oxLDL) converts macrophages into foam cells. We recently reported that lipopolysaccharide (LPS)-induced foam cell formation is regulated by CD14 and scavenger receptor AI (SR-AI). In this study, we employed pharmaceutical and gene knockdown approaches to determine the upstream molecular mediators, which control LPS-induced foam cell formation. Our results demonstrated that the specific c-Jun N-terminal kinase (JNK) pathway inhibitor, SP600125, but neither the specific inhibitor of extracellular signaling-regulated kinase (ERK) kinase MEK1/2, U0126, nor the specific inhibitor of p38 MAPK, SB203580, significantly blocks LPS-induced oxLDL uptake, suggesting that the JNK pathway is the upstream mediator of LPS-induced oxLDL uptake/foam cell formation. To address whether JNK pathway mediates LPS-induced oxLDL uptake is due to JNK pathway-regulated CD14 and SR-AI expression, we assessed whether the pharmaceutical inhibitor of JNK influences LPS-induced expression of CD14 and SR-AI. Our results indicate that JNK pathway mediates LPS-induced CD14 and SR-AI expression. To conclusively address the isoform role of JNK family, we depleted JNK isoforms using the JNK isoform-specific siRNA. Our data showed that the depletion of JNK1, but not JNK2 blocked LPS-induced CD14/SR-AI expression and foam cell formation. Taken together, our results reveal for the first time that JNK1 is the key mediator of LPS-induced CD14 and SR-AI expression in macrophages, leading to LPS-induced oxLDL uptake/foam cell formation. We conclude that the novel JNK1/CD14/SR-AI pathway controls macrophage oxLDL uptake/foam cell formation.

Ox-LDL increases microRNA-29a transcription through upregulating YY1 and STAT1 in macrophages.

Macrophages and oxidized low-density lipoprotein (ox-LDL) have been verified playing vital roles in the pathogenesis of atherosclerosis (AS). Previous studies demonstrated that microRNA-29a (miR-29a) was upregulated in many atherogenic process and cells, thus acting as an important participant in AS. But the detailed regulation mechanism of miR-29a in AS has not been fully understood. In our study, we demonstrated a positive feedback loop of ox-LDL-SRA-miR-29a. Furthermore, we found that YY1 and STAT1 were upregulated in ox-LDL-stimulating macrophages followed by translocation in the nucleus and binding to the transcriptional promoter region of miR-29a, thus leading to the increase of miR-29a expression. In addition, we demonstrated that JAK1/2 signaling was involved in miR-29a upregulation. Finally, we found that miR-29a played important roles in the secretion of proinflammation factors and lipid uptake in macrophages. We uncovered the molecular mechanism and provide novel insights into the function and regulatory network of miR-29a expression regulated by ox-LDL in macrophages.

Inhibitory effects of Dioscin on atherosclerosis and foam cell formation in hyperlipidemia rats.

Macrophage-derived foam cells are well known for their key role in development of atherosclerosis (AS). The present study aimed to examine whether dioscin exerts anti-atherosclerotic activity and inhibits foam cell formation. A high-fat induced AS model and ox-LDL treated macrophages were established and received treatment of dioscin. Anti-atherosclerotic activity in vivo was assessed by atherosclerotic lesions size and aortic lipid contents. Macrophage formed foam cells were positively identified by oil red o staining. Moreover, the expression of LOX-1 and NF-κB in aorta tissue and macrophages was examined by western blotting assay. Our results showed that dioscin not only reduced the levels of plasma lipid, TNF-a, IL-1β and IL-6, but also inhibited atherosclerotic development in AS rats, as evidenced by decreased atherosclerotic lesions size and aortic lipid level. In vitro study revealed dioscin directly reduced foam cell formation, decreased intracellular cholesterol accumulation and lowered TNF-a, IL-1β and IL-6 secretion in ox-LDL treated macrophages. Interestingly, further work found dioscin significantly reduced expression of LOX-1 and NF-κB in the aortic tissue and ox-LDL treated macrophages. In summary, our study was the first to confirm anti-atherosclerotic activity of dioscin in vivo and vitro. Moreover, the other important finding is dioscin mediated ox-LDL/LOX-1/NF-κB regulated contributions to the attenuate macrophage ox-LDL uptake and AS.

DNA methylation in cystathionine-γ-lyase (CSE) gene promoter induced by ox-LDL in macrophages and in apoE knockout mice

Recent studies suggest that epigenetic alterations such as DNA methylation control many aspects of monocytes/macrophages and participate in the pathogenesis of atherosclerosis, a lipid-driven inflammatory disorder. Our and other groups demonstrated that dysregulation of cystathionine γ-lyase (CSE) -hydrogen sulfide (H2S) pathway was involved in monocyte/macrophages-mediated inflammation and atherosclerosis. However, it remains unknown whether altered cse methylation in macrophages may play a role in linking CSE-H2S dysregulation and atherosclerosis. In the present study, we showed that plasma H2S and H2S production in the peritoneal macrophages of apolipoprotein knockout (apoE−/−) mice gradually decreased with ages, and were also lower than that in control mice at 12 weeks older. Moreover, CSE mRNA expressions decreased while DNA methyltransferase (DNMT) expressions increased in the peritoneal macrophages isolated from apoE−/− mice, compared to age-matched wildtype mice. Similar observations were obtained in an in vitro study. In oxidized low-density lipoprotein (ox-LDL)-treated raw264.7 macrophages, cse transcription was down-regulated while the expression and activity of DNMT was up-regulated, associated with enhanced DNA methylation in cse promoter. Suppression of DNMT with its inhibitor or siRNA reversed the decrease of CSE mRNA. Therefore, our data suggest that DNA hypermethylation of CpG rich region in cse promoter might contribute to the decrease of cse transcription and H2S production in macrophages, and thus contribute to atherosclerosis development.

AMP-activated protein kinase attenuates oxLDL uptake in macrophages through PP2A/NF-κB/LOX-1 pathway

The differentiation of macrophages into lipid-laden foam cells is a hallmark in early-stage atherosclerosis. The developmental role of adenosine monophosphate-activated protein kinase (AMPK) in a transformation of foam cells, especially in macrophage cholesterol uptake that remains undetermined. Here we demonstrate that AMPK activation in response to IMM-H007 or AICAR resulted in a decrease in macrophage cholesterol uptake and thus inhibited foam cell formation in macrophages mediated by oxidized low-density lipoprotein (oxLDL). This functional change was caused by a downregulation of mRNA and protein expression of LOX-1 but not other scavenger receptors, including scavenger receptor-A (SR-A), CD36 and scavenger receptor-BI (SR-BI). The expression of LOX-1 was regulated by AMPK activation induced decreased phosphorylation of nuclear transcription factor NF-κB, since siRNA interference or dominant negative AMPK overexpression significantly promotes Ser536 dephosphorylation of NF-κB p65 and thus increases LOX-1 expression. Moreover, pharmacological AMPK activation was shown to promote protein phosphatase 2A (PP2A) activity and the specific PP2A inhibitor, okadaic acid, could prevent the effects of IMM-H007 or AICAR on NF-κB and LOX-1. In vivo, pharmacological AMPK activation reduced the lesion size of atherosclerosis and the expression of LOX-1 in aortas in apolipoprotein E-deficient mice. Our current findings suggest a novel mechanism of LOX-1 regulation by AMPK to attenuate macrophage oxLDL uptake and atherosclerosis.

Celery Seed Extract Blocks Peroxide Injury in Macrophages via Notch1/NF-κB Pathway.

Oxidized low-density lipoprotein (ox-LDL)-induced macrophage foam cell formation and injury is one of the major atherogenic factors. This study is aimed to investigate the protective effect of celery seed extract (CSE) on ox-LDL-induced injury of macrophages and the underlying signaling pathway. RAW264.7 macrophages were pre-incubated with CSE for 24 h, followed by stimulation with ox-LDL. Oil red O staining and enzymatic colorimetry indicated CSE significantly lessened lipid droplets and total cholesterol (TC) content in ox-LDL-injured macrophages. ELISA revealed that CSE decreased the secretion of inflammatory cytokine TNF-α and IL-6 by 12-27% and 5-15% respectively. MTT assay showed CSE promoted cell viability by 16-40%. Cell apoptosis was also analyzed by flow cytometry and laser scanning confocal microscope and the data indicated CSE inhibited ox-LDL-induced apoptosis of macrophages. Meanwhile, western blot analysis showed CSE suppressed NF-κBp65 and notch1 protein expressions stimulated by ox-LDL in macrophages. These results suggest that CSE inhibits ox-LDL-induced macrophages injury via notch1/NF-κB pathway.

Berberine derivatives reduce atherosclerotic plaque size and vulnerability in apoE(-/-) mice.

Background and aimsOur previous in vitro and clinical work has demonstrated anti-inflammatory effects of berberine (BBR), but the clinical application of BBR is limited by its poor bioavailability. Derivatives of BBR have been suggested to have enhanced bioavailability compared to BBR. In this study, we tested whether BBR derivatives, compared with BBR, had superior beneficial effects on atherosclerotic plaques in apoE−/− mice, and defined possible molecular mechanisms underlying such effects.MethodsMacrophages were pretreated with BBR and its derivatives, dihydroberberine (dhBBR) and 8,8-dimethyldihydroberberine (Di-MeBBR), before incubation with oxLDL. Cell surface EMMPRIN expression was measured by flow cytometry and Western blotting, and phospho-(p)-p38, p-JNK, nuclear NFκB p65, and phospho-p65 were measured by Western blotting. ApoE−/− mice fed with the Western diet for 16 weeks were treated with BBR, dhBBR and Di-MeBBR 16 weeks. Aortic atherosclerotic lesion size, plaque matrix proteins, and EMMPRIN and other inflammatory factors were measured using Oil Red O Staining, Masson’s trichromestaining and immunohistochemical staining and real-time PCR.ResultsCompared with BBR, dhBBR and Di-MeBBR significantly reduced EMMPRIN expression, which was associated with a greater inhibition of p-p38, p-JNK, nuclear NFκB p65 and phospho-p65 induced by oxLDL in macrophages. dhBBR and Di-MeBBR, but not BBR, reduced atherosclerotic plaque size and improved plaque stability indicated by increased α-smooth muscle actin and collagen content, and thicker fibrous caps. dhBBR and Di-MeBBR reduced expression of EMMPRIN, CD68, and NFκB p65, and Di-MeBBR also reduced expression of matrix metalloproteinase-9, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 in aortic plaques.ConclusionsThese results have demonstrated that BBR derivatives, dhBBR and Di-MeBBR, are superior to BBR in inhibiting inflammation and reducing plaque size and vulnerability.

Curcumin inhibits monocyte chemoattractant protein-1 expression and enhances cholesterol efflux by suppressing the c-Jun N-terminal kinase pathway in macrophage.

To investigate the effect of curcumin on monocyte chemoattractant protein 1 (MCP-1) production and reverse cholesterol transport (RCT) in macrophage induced by oxidation low-density lipoprotein (ox-LDL), and to identify the signal pathways involved. The macrophages were treated with ox-LDL and various concentrations of curcumin simultaneously. The MCP-1 expression was measured by enzyme-linked immunosorbent assay. The apoAI-mediated cholesterol efflux was measured by (3)H-cholesterol-labeled counting radioactivity. The activation of intracellular signaling pathways was studied by Western blotting. Curcumin decreased the production of MCP-1 induced by ox-LDL in macrophages. MCP-1 expression was restrained by the inhibition of c-Jun N-terminal kinase (JNK) pathway (SP600125) and NF-κB pathway (BAY11-7082). Curcumin suppressed the phosphorylation of JNK and activation of NF-κB. Curcumin also enhanced RCT via up-regulating the expression of liver X receptor alpha (LXRα), ATP-binding cassette transporter A1 (ABCA1) and scavenger receptor class B type I (SR-BI). Additionally, the inhibition of JNK (SP600125) increased cholesterol efflux and increased the expression of ABCA1 and SR-BI, but had no effect on LXRα. Curcumin suppresses MCP-1 production induced by ox-LDL via the JNK pathway and NK-κB pathway, while enhances cholesterol efflux in macrophage via suppressing the JNK pathway and activating the LXR-ABCA1/SR-BI pathway, which indicate that the vascular protective effect of curcumin is related to anti-inflammation and anti-atherosclerosis.

Endothelial overexpression of LOX-1 increases plaque formation and promotes atherosclerosis in vivo

Lectin-like oxLDL receptor-1 (LOX-1) mediates the uptake of oxidized low-density lipoprotein (oxLDL) in endothelial cells and macrophages. However, the different atherogenic potential of LOX-1-mediated endothelial and macrophage oxLDL uptake remains unclear. The present study was designed to investigate the in vivo role of endothelial LOX-1 in atherogenesis. Endothelial-specific LOX-1 transgenic mice were generated using the Tie2 promoter (LOX-1TG). Oxidized low-density lipoprotein uptake was enhanced in cultured endothelial cells, but not in macrophages of LOX-1TG mice. Six-week-old male LOX-1TG and wild-type (WT) mice were fed a high-cholesterol diet (HCD) for 30 weeks. Increased reactive oxygen species production, impaired endothelial nitric oxide synthase activity and endothelial dysfunction were observed in LOX-1TG mice as compared with WT littermates. LOX-1 overexpression led to p38 phosphorylation, increased nuclear factor κB activity and subsequent up-regulation of vascular cell adhesion molecule-1, thereby favouring macrophage accumulation and aortic fatty streaks. Consistently, HCD-fed double-mutant LOX-1TG/ApoE(-/-) displayed oxidative stress and vascular inflammation with higher aortic plaques than ApoE(-/-) controls. Finally, bone marrow transplantation experiments showed that endothelial LOX-1 was sufficient for atherosclerosis development in vivo. Endothelial-specific LOX-1 overexpression enhanced aortic oxLDL levels, thereby favouring endothelial dysfunction, vascular inflammation and plaque formation. Thus, LOX-1 may serve as a novel therapeutic target for atherosclerosis.