Chinese olive (Canarium album L.) fruit regulates glucose utilization by activating AMP-activated protein kinase.

A growing body of evidence demonstrates obesity-induced insulin resistance is associated with the development of metabolic diseases. This study was designed to investigate ethyl acetate fraction of Chinese olive fruit extract (CO-EtOAc)-mediated attenuation of obesity and hyperglycemia in a mouse model. About 60% HFD-fed mice were treated intragastrically with CO-EtOAc for last 6 weeks, and body weight, blood biochemical parameters as well as hepatic inflammation response were investigated. Our results showed that CO-EtOAc treatment significantly reduced the formation of hepatic lipid droplets, body weight gain, blood glucose, and improved serum biochemical parameters in HFD-induced obese and insulin resistant mice. We further explored the molecular mechanism underlying the blood glucose modulating effect of CO-EtOAc using L6 myotubes model. We conclude that CO-EtOAc effectively increases the glycogen content and glucose uptake by stimulating the membrane translocation of glucose transporter 4. In addition, CO-EtOAc depolarizes the mitochondrial membrane and decreases the mitochondrial oxygen consumption, which may result in AMPK activation and the consequent mitochondrial fission. This study shows that CO-EtOAc prevents the development of obesity in mice fed with HFD and is also capable of stimulating glucose uptake. The possible mechanism might be due to the effects of CO-EtOAc on activation of AMPK and promotion of mitochondrial fission.

Structural and functional characterization of ß2 -glycoprotein I domain 1 in anti-melanoma cell migration.

We previously found that circulating ß2 -glycoprotein I inhibits human endothelial cell migration, proliferation, and angiogenesis by diverse mechanisms. In the present study, we investigated the antitumor activities of ß2 -glycoprotein I using structure-function analysis and mapped the critical region within the ß2 -glycoprotein I peptide sequence that mediates anticancer effects. We constructed recombinant cDNA and purified different ß2 -glycoprotein I polypeptide domains using a baculovirus expression system. We found that purified ß2 -glycoprotein I, as well as recombinant ß2 -glycoprotein I full-length (D12345), polypeptide domains I-IV (D1234), and polypeptide domain I (D1) significantly inhibited melanoma cell migration, proliferation and invasion. Western blot analyses were used to determine the dysregulated expression of proteins essential for intracellular signaling pathways in B16-F10 treated with ß2 -glycoprotein I and variant recombinant polypeptides. Using a melanoma mouse model, we found that D1 polypeptide showed stronger potency in suppressing tumor growth. Structural analysis showed that fragments A and B within domain I would be the critical regions responsible for antitumor activity. Annexin A2 was identified as the counterpart molecule for ß2 -glycoprotein I by immunofluorescence and coimmunoprecipitation assays. Interaction between specific amino acids of ß2 -glycoprotein I D1 and annexin A2 was later evaluated by the molecular docking approach. Moreover, five amino acid residues were selected from fragments A and B for functional evaluation using site-directed mutagenesis, and P11A, M42A, and I55P mutations were shown to disrupt the anti-melanoma cell migration ability of ß2 -glycoprotein I. This is the first study to show the therapeutic potential of ß2 -glycoprotein I D1 in the treatment of melanoma progression.

Chinese olive extract ameliorates hepatic lipid accumulation in vitro and in vivo by regulating lipid metabolism.

Chinese olive contains plenty of polyphenols, which possess a wide range of biological actions. In this study, we aimed to investigate the role of the ethyl acetate fraction of Chinese olive fruit extract (CO-EtOAc) in the modulation of lipid accumulation in vitro and in vivo. In cellular studies, CO-EtOAc attenuated oleic acid-induced lipid accumulation; we then elucidated the molecular mechanisms of CO-EtOAc in FL83B mouse hepatocytes. CO-EtOAc suppressed the mRNA levels of fatty acid transporter genes (CD36 and FABP) and lipogenesis genes (SREBP-1c, FAS, and ACC1), but upregulated genes that govern lipolysis (HSL) and lipid oxidation (PPARα, CPT-1, and ACOX). Moreover, CO-EtOAc increased the protein expression of phosphorylated AMPK, ACC1, CPT-1, and PPARα, but downregulated the expression of mature SREBP-1c and FAS. AMPK plays an essential role in CO-EtOAc-mediated amelioration of lipid accumulation. Furthermore, we confirmed that CO-EtOAc significantly inhibited body weight gain, epididymal adipose tissue weight, and hepatic lipid accumulation via regulation of the expression of fatty acid transporter, lipogenesis, and fatty acid oxidation genes and proteins in C57BL/6 mice fed a 60% high-fat diet. Therefore, Chinese olive fruits may have the potential to improve the metabolic abnormalities associated with fatty liver under high fat challenge.

Chinese Olive (Canarium album L.) Fruit Extract Attenuates Metabolic Dysfunction in Diabetic Rats.

Hyperglycemia and dysregulation of lipid metabolism play a crucial role in metabolic dysfunction. The aims of present study were to evaluate the ameliorative effect of the ethyl acetate fraction of Chinese olive fruit extract (CO-EtOAc) on high-fat diet (HFD) and streptozotocin (STZ)-induced diabetic rats. CO-EtOAc, rich in gallic acid and ellagic acid, could markedly decreased the body weight and epididymal adipose mass. In addition, CO-EtOAc increased serum HDL-C levels, hepatic GSH levels, and antioxidant enzyme activities; lowered blood glucose, serum levels of total cholesterol (TC), triglycerides (TG), bile acid, and tumor necrosis factor alpha (TNFα); and reduced TC and TG in liver. We further demonstrated that CO-EtOAc mildly suppressed hepatic levels of phosphorylated IRS-1, TNF-α, and IL-6, but enhanced Akt phosphorylation. The possible mechanisms of cholesterol metabolism were assessed by determining the expression of genes involved in cholesterol transportation, biosynthesis, and degradation. It was found that CO-EtOAc not only inhibited mRNA levels of SREBP-2, HMG-CoAR, SR-B1, and CYP7A1 but also increased the expression of genes, such as ABCA1 and LDLR that governed cholesterol efflux and cholesterol uptake. Moreover, the protein expressions of ABCA1 and LDLR were also significantly increased in the liver of rats supplemented with CO-EtOAc. We suggest that Chinese olive fruit may ameliorate metabolic dysfunction in diabetic rats under HFD challenge.

The methanol-ethyl acetate partitioned fraction from Chinese olive fruits inhibits cancer cell proliferation and tumor growth by promoting apoptosis through the suppression of the NF-κB signaling pathway.

Chinese olives (Canarium album L.) have historically been used for medicinal purposes rather than commercially for oil. In this report, we reveal that the methanol-ethyl acetate partitioned fraction from Chinese olive fruits (MEO), of which ellagic acid accounted for 12%, exhibited profound anti-proliferative activities in the human colon cancer cell line, HCT116. Additionally, oral administration of MEO remarkably inhibited the tumor growth of subcutaneously implanted CT26 cells, a mouse colon carcinoma cell line, in BALB/c mice. Treatment with MEO induced a significant increase in the percentage of apoptotic cells and resulted in poly(ADP-ribose) polymerase (PARP) cleavage, suggesting that MEO inhibits cancer cell proliferation by promoting apoptosis. Our study also showed that MEO exerted the most potent effect on the inhibition of NF-κB-mediated signaling among the partitioned fractions from Chinese olives. This process employed the use of reporter-based bio-platforms that are capable of detecting the activation of NF-κB. In addition, phosphorylation of NF-κB signaling-associated proteins, IKKα/ß, IκBα, and p65, was reduced in MEO-incubated cancer cells, indicating that MEO suppresses NF-κB activation. Moreover, MEO treatment significantly suppressed lipopolysaccharide (LPS)-induced cancer cell proliferation, demonstrating that MEO promotes cancer cell apoptosis through the inhibition of the NF-κB signaling pathway. In summary, our findings demonstrate that the methanol-ethyl acetate partitioned fraction from Chinese olive fruits inhibits cancer cell proliferation and tumor growth by promoting apoptosis through the suppression of NF-κB signaling. Therefore, the Chinese olive fruit has promising potential in cancer treatment.

ß2-Glycoprotein I Inhibits Vascular Endothelial Growth Factor-Induced Angiogenesis by Suppressing the Phosphorylation of Extracellular Signal-Regulated Kinase 1/2, Akt, and Endothelial Nitric Oxide Synthase.

Angiogenesis is the process of new blood vessel formation, and it plays a key role in various physiological and pathological conditions. The ß2-glycoprotein I (ß2-GPI) is a plasma glycoprotein with multiple biological functions, some of which remain to be elucidated. This study aimed to identify the contribution of 2-GPI on the angiogenesis induced by vascular endothelial growth factor (VEGF), a pro-angiogenic factor that may regulate endothelial remodeling, and its underlying mechanism. Our results revealed that ß2-GPI dose-dependently decreased the VEGF-induced increase in endothelial cell proliferation, using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and the bromodeoxyuridine (BrdU) incorporation assays. Furthermore, incubation with both ß2-GPI and deglycosylated ß2-GPI inhibited the VEGF-induced tube formation. Our results suggest that the carbohydrate residues of ß2-GPI do not participate in the function of anti-angiogenesis. Using in vivo Matrigel plug and angioreactor assays, we show that ß2-GPI remarkably inhibited the VEGF-induced angiogenesis at a physiological concentration. Moreover, ß2-GPI inhibited the VEGF-induced phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), Akt, and endothelial nitric oxide synthase (eNOS). In summary, our in vitro and in vivo data reveal for the first time that ß2-GPI inhibits the VEGF-induced angiogenesis and highlights the potential for ß2-GPI in anti-angiogenic therapy.

Modulation of microRNA Expression in Subjects with Metabolic Syndrome and Decrease of Cholesterol Efflux from Macrophages via microRNA-33-Mediated Attenuation of ATP-Binding Cassette Transporter A1 Expression by Statins.

Metabolic syndrome (MetS) is a complicated health problem that encompasses a variety of metabolic disorders. In this study, we analyzed the relationship between the major biochemical parameters associated with MetS and circulating levels of microRNA (miR)-33, miR-103, and miR-155. We found that miRNA-33 levels were positively correlated with levels of fasting blood glucose, glycosylated hemoglobin A1c, total cholesterol, LDL-cholesterol, and triacylglycerol, but negatively correlated with HDL-cholesterol levels. In the cellular study, miR-33 levels were increased in macrophages treated with high glucose and cholesterol-lowering drugs atorvastatin and pitavastatin. miR-33 has been reported to play an essential role in cholesterol homeostasis through ATP-binding cassette transporter A1 (ABCA1) regulation and reverse cholesterol transport. However, the molecular mechanism underlying the linkage between miR-33 and statin treatment remains unclear. In the present study, we investigated whether atorvastatin and pitavastatin exert their functions through the modulation of miR-33 and ABCA1-mediated cholesterol efflux from macrophages. The results showed that treatment of the statins up-regulated miR-33 expression, but down-regulated ABCA1 mRNA levels in RAW264.7 cells and bone marrow-derived macrophages. Statin-mediated ABCA1 regulation occurs at the post-transcriptional level through targeting of the 3'-UTR of the ABCA1 transcript by miR-33. Additionally, we found significant down-regulation of ABCA1 protein expression in macrophages treated with statins. Finally, we showed that high glucose and statin treatment significantly suppressed cholesterol efflux from macrophages. These findings have highlighted the complexity of statins, which may exert detrimental effects on metabolic abnormalities through regulation of miR-33 target genes.

Differential regulation of protein expression in response to polyunsaturated fatty acids in the liver of apoE-knockout mice and in HepG2 cells.

BACKGROUND: Polyunsaturated fatty acids (PUFAs) are nutrients necessary for life. The liver is the essential metabolic center, which aids in maintaining health via diverse biological actions. In the present work, a proteomics study was conducted with an aim to provide new insights into PUFA-regulated hepatic protein expression in apoE-knockout mice. Additionally, we investigated how n-3 PUFAs influence cytokine-challenge by using HepG2 cells as a model. RESULTS: Through the proteomic analysis using 2-dimensional electrophoresis and mass spectrometry, we found that 28, 23, 14, and 28 hepatic proteins were up-regulated at least a two-fold difference in intensity compared with the control group in mice treated with the docosahexaenoic acid, eicosapentaenoic acid, arachidonic acid, and linoleic acid, respectively. In contrast, 12 hepatic proteins were down-regulated with a ratio value of less than 0.5 compared to their control counterparts by these four fatty acids. All of the altered proteins were then sorted according to their biochemical properties related to metabolism, redox stress/inflammation, enzymatic reactions, and miscellaneous functions. The results provide evidence that PUFAs may act as either pro-inflammatory or anti-inflammatory agents. Cytokine-challenged HepG2 cells were used to reveal the anti-inflammatory function of n-3 PUFAs. The results showed that interleukin (IL)-1ß combined with IL-6 induced C-reactive protein (CRP) mRNA expression and its protein secretion by HepG2 cells. The CRP promoter activity was significantly increased in the IL-6-treated cells, whereas IL-1ß alone had no effect. However, IL-1ß and IL-6 acted synergistically to further enhance CRP promoter activities. Furthermore, n-3 PUFAs inhibited nuclear factor-κB (NF-κB) activation and the phosphorylation of the nuclear signal transducer and activator of transcription 3 (STAT3) during cytokine-induced CRP production. CONCLUSION: This study indicates that PUFAs induced changes in the hepatic protein profile in vivo. Furthermore, n-3 PUFAs exert their anti-inflammatory properties through differential molecular mechanisms in hepatic cells. These results provide novel information regarding the roles of PUFAs in the liver at the tissue and cellular levels.

Transient receptor potential vanilloid type 1 is vital for (-)-epigallocatechin-3-gallate mediated activation of endothelial nitric oxide synthase.

SCOPE: Epigallocatechin-3-gallate (EGCG), the most abundant catechin of green tea, has beneficial effects on physiological functions of endothelial cells (ECs), yet the detailed mechanisms are not fully understood. In this study, we investigated the role of transient receptor potential vanilloid type 1 (TRPV1), a ligand-gated nonselective calcium channel, in EGCG-mediated endothelial nitric oxide (NO) synthase (eNOS) activation and angiogenesis. METHODS AND RESULTS: In ECs, treatment with EGCG time-dependently increased the intracellular level of Ca(2+) . Removal of extracellular calcium (Ca(2+) ) by EGTA or EDTA or inhibition of TRPV1 by capsazepine or SB366791 abrogated EGCG-increased intracellular Ca(2+) level in ECs or TRPV1-transfected HEK293 cells. Additionally, EGCG increased the phsophorylation of eNOS at Ser635 and Ser1179, Akt at Ser473, calmodulin-dependent protein kinase II (CaMKII) at Thr286 and AMP-activated protein kinase (AMPK) at Thr172, all abolished by the TRPV1 antagonist capsazepine. EGCG-induced NO production was diminished by pretreatment with LY294002 (an Akt inhibitor), KN62 (a CaMKII inhibitor), and compound C (an AMPK inhibitor). Moreover, blocking TRPV1 activation prevented EGCG-induced EC proliferation, migration, and tube formation, as well as angiogenesis in Matrigel plugs in mice. CONCLUSION: EGCG may trigger activation of TRPV1-Ca(2+) signaling, which leads to phosphorylation of Akt, AMPK, and CaMKII; eNOS activation; NO production; and, ultimately, angiogenesis in ECs.

Docosahexaenoic acid and eicosapentaenoic acid suppress adhesion molecule expression in human aortic endothelial cells via differential mechanisms.

SCOPE: Dietary PUFAs modulate the progression of cardiovascular disease, but the underlying mechanisms within vascular cells remain unclear. The aim of this study was to investigate the biological function and regulatory mechanisms of PUFAs in LPS-activated human aortic endothelial cells (HAECs). METHODS AND RESULTS: To simulate the in vivo conditions of atherosclerosis, we have established an in vitro model in which THP-1 monocytes adhere to HAECs. Our results showed that n-3 PUFAs docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) remarkably attenuated the adhesion of THP-1 cells to HAECs, probably through inhibiting the expression of VCAM-1 and ICAM-1. Using lipid raft isolation and confocal microscopy, we found that DHA and EPA suppressed the translocation of TLR4 into lipid rafts. Furthermore, DHA and EPA inhibited the ubiquitination and translocation of TRAF6, and the phosphorylation of TAK1, p38, and IκBα. We demonstrated that DHA reduced the phosphorylation of PKR, but EPA increased the expression of A20. Additionally, silencing of A20 reversed the inhibitory effect of EPA on the expression of adhesion molecules. CONCLUSION: Our study revealed differential signaling pathways modulated by n-3 PUFAs in LPS-stimulated HAECs. These signaling pathways are potential targets for the prevention of atherosclerotic progression.

EGb-761 prevents ultraviolet B-induced photoaging via inactivation of mitogen-activated protein kinases and proinflammatory cytokine expression.

BACKGROUND: EGb-761 is an antioxidant and anticarcinogen; however, its role as a photoprotector remains unknown. OBJECTIVE: To determine whether EGb-761 photoprotects human dermal fibroblasts and BALB/c mice skin against ultraviolet B (UVB) light irradiation. METHODS: To simulate chronic photodamage, shaved BALB/c mice were exposed to UVB irradiation (90mJ/cm(2)) thrice weekly for 3 months. EGb-761 (2mg/cm(2)) was topically applied 1h before irradiation to evaluate its effect. The mechanisms by which EGb-761 protects the skin from photodamage were evaluated by immunohistochemical analysis, enzyme-linked immunosorbent assay (ELISA), and Western blotting. RESULTS: In BALB/c mice, the signs of photoaging or photodamage, such as coarse wrinkle formation, epidermal hyperplasia, and elastic fiber degeneration, markedly reduced with the topical application of EGb-761. Western blot and ELISA revealed that the activation of MMP-1 in cultured fibroblasts markedly diminished after pretreatment with EGb-761. In addition, EGb-761 inhibited UVB-induced overexpression by the fibroblasts of the proinflammatory cytokines, such as interleukin (IL)-1α, IL-1ß, IL-6, and tumor necrosis factor-α. The phosphorylation of the mitogen-activated protein kinase (MAPK) signal transduction pathway components, including extracellular signal-regulated kinase, C-Jun N-terminal kinase, and p38, which are induced by UV irradiation, was significantly inhibited in vivo and in vitro. EGb-761 also diminished the generation of UVB-induced reactive oxygen species (ROS). CONCLUSIONS: EGb-761 photoprotects mice and cultured fibroblasts, inhibits the UVB-induced phosphorylation of MAPK pathway components, and reduces the expression of the proinflammatory cytokines by suppressing ROS generation. Thus, topically applied EGb-761 may be a promising photoprotective agent.

Establishment of reporter platforms capable of detecting NF-κB mediated immuno-modulatory activity.

Cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) are transcriptional targets of nuclear factor kappa B (NF-κB) that are involved in inflammatory responses. The aim of this study is to develop a method for efficiently detecting inflammation modulatory activities. Here we established RAW264.7 macrophage cells stably expressing a luciferase reporter gene directed by iNOS or COX-2 promoter. Lipopolysaccharide (LPS) treatment stimulated the luciferase activity which paralleled with increased iNOS and COX-2 mRNA levels determined by RT-q-PCR. The LPS-stimulated luciferase activity was blocked by NF-κB inhibitor CAPE and by nobiletin, an anti-inflammatory natural product from citrus peels. We have applied the platforms to screen various mushroom species; analysis by scatter plot revealed a strong correlation to the results obtained by ELISA-based detection of TNF-α. Together we have established luciferase reporter systems sensitive to NF-κB-dependent iNOS and COX-2 activation, which provides an alternative screening method for identifying food components with immune-modulatory activities.

The therapeutic potential and mechanisms of action of quercetin in relation to lipopolysaccharide-induced sepsis in vitro and in vivo.

Sepsis caused by Gram-negative bacterial infection is characterized by extensive inflammatory cytokine production, which leads to multiple organ failure and a high lethality rate. Therefore, compounds that are able to alleviate profound inflammatory responses may have therapeutic potential in relation to sepsis. Quercetin, one of the flavonoids found widely in the human diet, has been reported to have many health benefits, but the mechanisms underlying its biological effects remain obscure. In the present study, our aim was to investigate the molecular mechanisms by which quercetin inhibits lipopolysaccharide (LPS)-induced pro-inflammatory cytokine production and to evaluate the capacity of quercetin to attenuate the mortality rate in a mice model of lethal sepsis. Our results show that quercetin significantly attenuates LPS-induced production of tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) in RAW264.7 macrophages. The LPS-stimulated phosphorylations of the inhibitors of κB kinase (IKKs), Akt, and c-Jun N-terminal kinase (JNK) are also inhibited by quercetin. Quercetin causes a significant reduction in the phosphorylation and degradation of inhibitor of κBα (IκBα) and in the nuclear level of nuclear factor-κB (NF-κB), the latter being associated with decreased NF-κB binding activity. Most importantly, acute administration of quercetin reduces the lethality rate and circulating levels of TNF-α and IL-1ß in C57BL/6J mice with endotoxemia induced by LPS, whereas chronic dietary supplementation with quercetin shows no inhibitory effect on serum TNF-α and IL-1ß levels. These findings provide clues that quercetin may be a promising agent for the prevention of systemic inflammatory diseases such as sepsis.

Docosahexaenoic acid and eicosapentaenoic acid reduce C-reactive protein expression and STAT3 activation in IL-6-treated HepG2 cells.

C-reactive protein (CRP), an acute phase protein in humans, is predominantly produced by hepatocytes in response to interleukin-6 (IL-6). Several epidemiological studies have reported that dietary intake of n-3 polyunsaturated fatty acids (n-3 PUFAs) is inversely associated with serum CRP concentration. However, the molecular mechanism by which n-3 PUFAs reduce the serum CRP level in HepG2 cells remains unclear. The aims of this study were to examine the effect of the n-3 PUFAs, docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA), on the modulation of IL-6-induced CRP expression and to explore its possible mechanisms. We demonstrated that DHA and EPA inhibited IL-6-induced CRP protein and mRNA expression, as well as reduced CRP promoter activity in HepG2 cells. Knockdown of Signal Transducer and Activator of Transcription 3 (STAT3) and CCAAT box/Enhancer-Binding Protein ß (C/EBPß) by small interfering RNAs (siRNAs) significantly decreased IL-6-induced CRP promoter activity. Gel electrophoresis mobility shift assays (EMSA) showed that pretreatment with DHA and EPA decreased IL-6-induced STAT3 DNA binding activity but not C/EBPß. By western blot analysis, DHA and EPA inhibited IL-6-induced STAT3 phosphorylation but not ERK1/2 or C/EBPß. The suppression of the phosphorylation of STAT3 by DHA and EPA was further verified by immunofluorescence staining. Taken together, our results demonstrate that DHA and EPA are able to reduce IL-6-induced CRP expression in HepG2 cells via an inhibition of STAT3 activation. This mechanism, which explains the inhibitory effect of n-3 PUFAs on the CRP expression, provides new insights into the beneficial anti-inflammatory effect of n-3 PUFAs.

ß2-glycoprotein I inhibits VEGF-induced endothelial cell growth and migration via suppressing phosphorylation of VEGFR2, ERK1/2, and Akt.

ß(2)-glycoprotein I (ß(2)-GPI) is a plasma glycoprotein with diverse functions, but the impact and molecular effects of ß(2)-GPI on vascular biology are as yet unclear. Based on the limited information available on the contribution of ß(2)-GPI to endothelial cells, we investigated the effect of ß(2)-GPI on cell growth and migration in human aortic endothelial cells (HAECs). The regulation of ß(2)-GPI as part of intracellular signaling in HAECs was also examined. Vascular endothelial growth factor (VEGF) is a pro-angiogenic factor that may regulate endothelial functions. We found that ß(2)-GPI dose-dependently inhibited VEGF-induced endothelial cell growth using the 3-(4,5-dimethylthiazol-2-yl)-2,5-dipenyl tetrazolium bromide assay and cell counts. Using wound healing and Boyden chamber assays, ß(2)-GPI remarkably reduced VEGF-increased cell migration at the physiological concentration. Furthermore, ß(2)-GPI suppressed VEGF-induced phosphorylation of VEGF receptor 2 (VEGFR2), extracellular signal-regulated kinase 1/2 (ERK1/2), and Akt. These results suggest that ß(2)-GPI plays an essential role in the down-regulation of VEGF-induced endothelial responses and may be a useful component for anti-angiogenic therapy.

Hyperglycemia accelerates ATP-binding cassette transporter A1 degradation via an ERK-dependent pathway in macrophages.

An elevation in blood glucose concentration leads to increased risk of developing diabetes-associated atherosclerotic cardiovascular disease due to an excessive accumulation of cholesterol in arterial macrophages. ATP-binding cassette transporter A1 (ABCA1) is an atheroprotective protein that mediates the export of cholesterol from macrophages. The present study aims to investigate the effect of hyperglycemia on the regulation of ABCA1 expression and to explore its underlying mechanisms of regulation in macrophages. Our results show that high glucose activates the extracellular signal-regulated kinases (ERK) signaling pathway via reactive oxygen species (ROS) production, which in turn down-regulates ABCA1 mRNA and protein expression. This down-regulation is mediated by accelerating ABCA1 mRNA and protein degradation in macrophages exposed to high concentrations of glucose. Our results provide evidence for the first time that hyperglycemia inhibits ABCA1 expression by ERK-modulated ABCA1 mRNA and protein stability. Overall, these results provide a mechanism for hyperglycemia-induced reduction in ABCA1 expression, which suggests a promising strategy for the treatment of diabetes-associated atherosclerosis.

Molecular mechanism of curcumin on the suppression of cholesterol accumulation in macrophage foam cells and atherosclerosis.

SCOPE: Curcumin, a potent antioxidant extracted from Curcuma longa, confers protection against atherosclerosis, yet the detailed mechanisms are not fully understood. In this study, we examined the effect of curcumin on lipid accumulation and the underlying molecular mechanisms in macrophages and apolipoprotein E-deficient (apoE⁻/⁻) mice. METHODS AND RESULTS: Treatment with curcumin markedly ameliorated oxidized low-density lipoprotein (oxLDL)-induced cholesterol accumulation in macrophages, which was due to decreased oxLDL uptake and increased cholesterol efflux. In addition, curcumin decreased the protein expression of scavenger receptor class A (SR-A) but increased that of ATP-binding cassette transporter (ABC) A1 and had no effect on the protein expression of CD36, class B receptor type I (SR-BI), or ATP-binding cassette transporter G1 (ABCG1). The downregulation of SR-A by curcumin was via ubiquitin-proteasome-calpain-mediated proteolysis. Furthermore, the curcumin-induced upregulation of ABCA1 was mainly through calmodulin-liver X receptor α (LXRα)-dependent transcriptional regulation. Curcumin administration modulated the expression of SR-A, ABCA1, ABCG1, and SR-BI in aortas and retarded atherosclerosis in apoE⁻/⁻ mice. CONCLUSION: Our findings suggest that inhibition of SR-A-mediated oxLDL uptake and promotion of ABCA1-dependent cholesterol efflux are two crucial events in suppression of cholesterol accumulation by curcumin in the transformation of macrophage foam cells.

Quercetin enhances ABCA1 expression and cholesterol efflux through a p38-dependent pathway in macrophages.

ATP-binding cassette transporter A1 (ABCA1) plays a crucial role in exporting cholesterol from macrophages, a function relevant to its involvement in the prevention of atherosclerosis. Quercetin, one of flavonoids, has been described to reduce atherosclerotic lesion formation. This study is aimed to investigate the effect of quercetin on regulation of ABCA1 expression and to explore its underlying mechanisms in macrophages. The results show that quercetin markedly enhanced cholesterol efflux from macrophages in a concentration-dependent manner, which was associated with an increase in ABCA1 mRNA and protein expression. Remarkably, quercetin is able to stimulate the phosphorylation of p38 by up to 234-fold at 6 h via an activation of the transforming growth factor ß-activated kinase 1 (TAK1) and mitogen-activated kinase kinase 3/6 (MKK3/6). Inhibition of p38 with a pharmacological inhibitor or small hairpin RNA (shRNA) suppressed the stimulatory effects of quercetin on ABCA1 expression and cholesterol efflux. Moreover, knockdown of p38 reduced quercetin-enhanced ABCA1 promoter activity and the binding of specificity protein 1 (Sp1) and liver X receptor α (LXRα) to the ABCA1 promoter using chromatin immunoprecipitation assays. These findings provide evidence that p38 signaling is essential for the regulation of quercetin-induced ABCA1 expression and cholesterol efflux in macrophages.

ß2-Glycoprotein I inhibits endothelial cell migration through the nuclear factor κB signalling pathway and endothelial nitric oxide synthase activation.

ß2-GPI (ß2-glycoprotein I) is a plasma glycoprotein ascribed with an anti-angiogenic function; however, the biological role and molecular basis of its action in cell migration remain unknown. The aim of the present study was to assess the contribution of ß2-GPI to HAEC (human aortic endothelial cell) migration and the details of its underlying mechanism. Using wound healing and Boyden chamber assays, we found that ß2-GPI inhibited endothelial cell migration, which was restored by its neutralizing antibody. NF-κB (nuclear factor κB) inhibitors and lentiviral siRNA (small interfering RNA) silencing of NF-κB significantly attenuated the inhibitory effect of ß2-GPI on cell migration. Moreover, ß2-GPI was found to induce IκBα (inhibitor of NF-κB) phosphorylation and translocation of p65 and p50. We further demonstrated that mRNA and protein levels of eNOS [endothelial NO (nitric oxide) synthase] and NO production were all increased by ß2-GPI and these effects were remarkably inhibited by NF-κB inhibitors and siRNAs of p65 and p50. Furthermore, ß2-GPI-mediated inhibition of cell migration was reversed by eNOS inhibitors and eNOS siRNAs. The findings of the present study provide novel insight into the ability of ß2-GPI to inhibit endothelial cell migration predominantly through the NF-κB/eNOS/NO signalling pathway, which indicates a potential direction for clinical therapy in vascular diseases.

ß Common receptor integrates the erythropoietin signaling in activation of endothelial nitric oxide synthase.

Erythropoietin (EPO), the key hormone for erythropoiesis, also increases nitric oxide (NO) bioavailability in endothelial cells (ECs), yet the definitive mechanisms are not fully understood. Increasing evidence has demonstrated that ß common receptor (ßCR) plays a crucial role in EPO-mediated non-hematopoietic effects. We investigated the role of ßCR in EPO-induced endothelial NO synthase (eNOS) activation in bovine aortic ECs (BAECs) and the molecular mechanisms involved. Results of confocal microscopy and immunoprecipitation analyses revealed that ßCR was colocalized and interacted with EPO receptor (EPOR) in ECs. Inhibition of ßCR or EPOR by neutralizing antibodies or small interfering RNA abolished the EPO-induced NO production. Additionally, blockage of ßCR abrogated the EPO-induced increase in the phosphorylation of eNOS, Akt, Src, or Janus kinase 2 (JAK2). Immunoprecipitation analysis revealed that treatment with EPO increased the interaction between ßCR and eNOS, which was suppressed by inhibition of Src, JAK2, or Akt signaling with specific pharmacological inhibitors. Furthermore, EPO-induced EC proliferation, migration, and tube formation were blocked by pretreatment with ßCR antibody and Src, JAK2, or PI3K/Akt inhibitors. Moreover, in vivo experiments showed that EPO increased the level of phosphorylated eNOS, Src, JAK2, and Akt, as well as ßCR-eNOS association in aortas and promoted the angiogenesis in Matrigel plug, which was diminished by ßCR or EPOR neutralizing antibodies. Our findings suggest that ßCR may play an integrative role in the EPO signaling-mediated activation of eNOS in ECs.