Nana Yang

Endoplasmic reticulum stress promotes macrophage-derived foam cell formation by up-regulating cluster of differentiation 36 (CD36) expression.

Background: Endoplasmic reticulum (ER) stress is involved in the pathogenesis of atherosclerosis. Results: Pharmacological manipulation and siRNA treatment to reduce ER stress mitigate ox-LDL-induced CD36 up-regulation, which is promoted synergistically by ER stress inducer. Conclusion: ER stress promotes macrophage-derived foam cell formation by up-regulating CD36. Significance: ER stress-mediated macrophage-derived foam cell formation may be a novel target in the prevention of atherosclerosis. Oxidized low-density lipoprotein (ox-LDL) up-regulates CD36, a scavenger receptor responsible for macrophage uptake of ox-LDL without limitation. However, the precise underlying mechanism is not completely understood. Our previous study has demonstrated that ox-LDL induces endoplasmic reticulum (ER) stress in macrophages. The goal of this study was to explore the exact relationship between ER stress and macrophage-derived foam cell formation and whether ER stress would be involved in ox-LDL-induced CD36 up-regulation. Our results showed that ox-LDL-induced lipid accumulation in macrophages was promoted synergistically by ER stress inducer tunicamycin (TM), while attenuated by ER stress inhibitor 4-phenylbutyric acid (PBA). Ox-LDL caused CD36 up-regulation with concomitant activation of ER stress as assessed by phosphorylation of inositol-requiring kinase/endonuclease-1 (IRE-1) and protein kinase-like ER kinase (PERK), up-regulation of X-box-binding protein 1 (XBP1) and glucose-regulated protein 78 (GRP 78), and nuclear translocation of activating transcription factor 6 (ATF6). TM not only up-regulated CD36 alone but also synergized with ox-LDL to increase CD36 expression. Alleviation of ER stress with PBA and siRNA against ATF6, IRE1, and GRP78 mitigated ox-LDL-induced CD36 protein up-regulation. Moreover, administration of apoE−/− mice with PBA suppressed the up-regulation of CD36, phospho-IRE1, and GRP78 in macrophage-dense atherosclerotic lesions and in peritoneal macrophages. Additionally, CD36 silencing attenuated ox-LDL-induced nuclear translocation of ATF6, phosphorylation of IRE1 and up-regulation of XBP1 and GRP78. These data indicate that CD36-mediated ox-LDL uptake in macrophages triggers ER stress response, which, in turn, plays a critical role in CD36 up-regulation, enhancing the foam cell formation by uptaking more ox-LDL.

Quercetin protects macrophages from oxidized low-density lipoprotein-induced apoptosis by inhibiting the endoplasmic reticulum stress-C/EBP homologous protein pathway.

Quercetin (QUE), a member of the bioflavonoid family, has been proposed to have antioxidative, anti-inflammatory and antihypertensive properties. This study was designed to investigate the protective effect of QUE on oxidized low-density lipoprotein (ox-LDL)-induced cytotoxicity in RAW264.7 macrophages and specifically the endoplasmic reticulum (ER) stress-C/EBP homologous protein (CHOP) pathway-mediated apoptosis. Our results showed that treatment with QUE (20, 40 and 80 μmol/L) significantly attenuated ox-LDL-induced cholesterol accumulation in macrophages and foam cell formation in a dose-dependent manner. Similar to tunicamycin (TM), a classical ER stress inducer, ox-LDL reduced cell viability and induced apoptosis in RAW264.7 macrophages. The cytotoxic effects of ox-LDL and TM were significantly inhibited by QUE treatment. Interestingly, we found that QUE also significantly suppressed the ox-LDL- and TM-induced activation of ER stress signaling events, including the phosphorylation of inositol-requiring enzyme 1 (IRE1), translocation of activating transcription factor 6 (ATF6) from the cytoplasm to the nucleus and upregulation of X-box-binding protein 1. In addition, exposure of RAW264.7 macrophages to ox-LDL or TM resulted in a significant increase in the expression of CHOP, a transcription factor regulated by IRE1 and ATF6 under conditions of ER stress, as well as a decrease in Bcl-2 transcript and protein concentrations. QUE blocked these effects in a dose-dependent manner. These data indicate that QUE can protect RAW264.7 cells from ox-LDL-induced apoptosis and that the mechanism at least partially involves its ability to inhibit the ER stress-CHOP signaling pathway.

Niacin inhibits vascular inflammation via downregulating nuclear transcription factor-κB signaling pathway.

The study aimed to investigate the effect of niacin on vascular inflammatory lesions in vivo and in vitro as well as its lipid-regulating mechanism. In vivo study revealed that niacin downregulated the levels of inflammatory factors (IL-6 and TNF-α) in plasma, suppressed protein expression of CD68 and NF-κB p65 in arterial wall, and attenuated oxidative stress in guinea pigs that have been fed high fat diet. In vitro study further confirmed that niacin decreased the secretion of IL-6 and TNF-α and inhibited NF-κB p65 and notch1 protein expression in oxLDL-stimulated HUVECs and THP-1 macrophages. Moreover, niacin attenuated oxLDL-induced apoptosis of HUVECs as well. In addition, niacin significantly lessened lipid deposition in arterial wall, increased HDL-C and apoA levels and decreased TG and non-HDL-C levels in plasma, and upregulated the mRNA amount of cholesterol 7α-hydroxylase A1 in liver of guinea pigs. These data suggest for the first time that niacin inhibits vascular inflammation in vivo and in vitro via downregulating NF-κB signaling pathway. Furthermore, niacin also modulates plasma lipid by upregulating the expression of factors involved in the process of reverse cholesterol transport.

Walk-run training improves the anti-inflammation properties of high-density lipoprotein in patients with metabolic syndrome.

CONTEXT Metabolic syndrome (MetS) is a constellation of cardiovascular risk factors, including central obesity, dysglycemia, hypertension, and dyslipidemia. The anti-inflammatory properties of high density lipoprotein (HDL) can be compromised in MetS. Exercise is recognized as an important factor in the prevention and treatment of MetS. OBJECTIVE This study was designed to investigate whether walk/run training without any specific diet could enhance anti-inflammation capacity of HDL from MetS patients. DESIGN This was a case control study. SETTING The study was conducted in a Zhoudian community, Taian. PATIENTS Thirty nine patients with MetS were recruited and divided into a control group (n = 12) remaining in an untrained state and exercise group (n = 27) performing a 10-week walk/run training program. MAIN OUTCOME MEASURES The anti-inflammation capacities of HDL3 (HDL subfractions) from MetS patients with or without exercise were investigated by co-incubating with TNF- α-injured endothelial cells in vitro. RESULTS The training did not influence serum lipoprotein level in MetS patients and cholesterol efflux capacity of circulating HDL. However, walk/run training increased paraoxonase-1 (PON1) activity and decreased the levels of malondialdehyde in either serum or isolated HDL from MetS patients prominently. More importantly, HDL3 isolated from MetS patients with 10 weeks training protected endothelial cells against tumor necrosis factor-a (TNF-a) -induced injury, decreased monocyte chemotactic protein-1 levels in media and vascular cell adhesion molecule-1 expression markedly. Furthermore, HDL3 isolated from MetS patients with walk/run training inhibited the TNF-á-induced monocyte adhesion to endothelial cells and obviously increased nitric oxide production by activating endothelial nitric oxide synthase. CONCLUSION Walk/run training leads to a significant improvement in HDL anti-inflammation capacity in subjects with MetS without restricted diet, the mechanism underlying which at least partially is due to increased PON1 activity in HDL, NO production, and eNOS expression in endothelial cells.

Minimally modified low-density lipoprotein induces macrophage endoplasmic reticulum stress via toll-like receptor 4

Minimally modified low-density lipoprotein (mm-LDL) induces intimal foam cell formation, which is promoted by endoplasmic reticulum stress (ERS), a cross-point to link cellular processes with multiple risk factors that exist in all stages of atherosclerosis. However, it remains unclear whether mm-LDL-induced lipid accumulation in macrophages involves ERS and its underlying mechanisms. We showed that mm-LDL induced the accumulation of lipid droplets in RAW264.7 macrophages with increased free cholesterol in the endoplasmic reticulum, which was markedly attenuated by pretreatment with an antibody against toll-like receptor 4 (TLR4). Additionally, mm-LDL stimulated the transport of Cy3-labeled activating transcription factor 6 (ATF6), a key sensor to the unfolded protein response (UPR), from cytoplasm into nucleus. The expression of phosphorylated inositol-requiring enzyme 1 (p-IRE1), another sensor to the UPR, and its two downstream molecules, X box binding protein 1 and glucose-regulated protein 78 (GRP78), were significantly upregulated by mm-LDL. The alterations induced by mm-LDL were all significantly inhibited by antibodies against TLR4 or CD36. In addition, the upregulation of p-IRE1 and GRP78 and the nuclear translocation of ATF6 induced by mm-LDL were significantly attenuated by TLR4 siRNA. These results suggest that mm-LDL may induce free cholesterol accumulation in the endoplasmic reticulum and subsequently stimulate ERS and activate the UPR signaling pathway mediated by ATF6 and IRE1 in macrophages, a process that is potentially mediated by TLR4.

D4F alleviates macrophage-derived foam cell apoptosis by inhibiting CD36 expression and ER stress-CHOP pathway.

This study was designed to explore the protective effect of D4F, an apoA-I mimetic peptide, on oxidized LDL (ox-LDL)-induced endoplasmic reticulum (ER) stress-CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) pathway-mediated apoptosis in macrophages. Our results showed that treating apoE knockout mice with D4F decreased the serum ox-LDL level and apoptosis in atherosclerotic lesions with concomitant downregulation of cluster of differentiation 36 (CD36) and inhibition of ER stress. In vitro, D4F inhibited macrophage-derived foam cell formation. Furthermore, like ER stress inhibitor 4-phenylbutyric acid (PBA), D4F inhibited ox-LDL- or tunicamycin (TM, an ER stress inducer)-induced reduction in cell viability and increase in lactate dehydrogenase leakage, caspase-3 activation, and apoptosis. Additionally, like PBA, D4F inhibited ox-LDL- or TM-induced activation of ER stress response as assessed by the reduced nuclear translocation of activating transcription factor 6 and the decreased phosphorylation of protein kinase-like ER kinase and eukaryotic translation initiation factor 2α, as well as the downregulation of glucose-regulated protein 78 and CHOP. Moreover, D4F mitigated ox-LDL uptake by macrophages and CD36 upregulation induced by ox-LDL or TM. These data indicate that D4F can alleviate the formation and apoptosis of macrophage-derived foam cells by suppressing CD36-mediated ox-LDL uptake and subsequent activation of the ER stress-CHOP pathway.

Pigment epithelium-derived factor alleviates endothelial injury by inhibiting Wnt/β-catenin pathway

BackgroundOxidized low-density lipoprotein (ox-LDL) can induce endothelial injury and plays a vital role in the procession and development of atherosclerosis. Little is known regarding whether Wnt/β-catenin pathway is involved in ox-LDL-induced endothelial injury or whether it further promotes atherosclerosis via increased oxidative stress. This study aimed to investigate the role of Wnt/β-catenin pathway in ox-LDL-induced vascular endothelial injury and determine whether pigment epithelium-derived factor (PEDF) could alleviate ox-LDL-induced endothelial injury by inhibiting Wnt/β-catenin pathway.MethodsInjury of human umbilical vein endothelial cells (HUVECs) was evaluated with an MTT assay, by monitoring lactate dehydrogenase (LDH) release and determining the apoptotic ratio. The expression of β-catenin (non-phosphorylated-β-catenin), disheveled-1 (Dvl-1) and Cyclin D1 was analyzed with western blotting and quantitative real-time PCR. Oxidative stress status was assessed by measuring the levels of reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD) and nitric oxide (NO).ResultsExposure of HUVECs to ox-LDL led to a decrease in cell viability and an increase in LDH release and apoptosis with concomitant enhancement of oxidative stress, as assessed by increased ROS and MDA generation, as well as decreased SOD activity and NO levels. Similar to lithium chloride (LiCl, a Wnt/β-catenin pathway activator), ox-LDL up-regulated the expression of β-catenin, Dvl-1 and Cyclin D1, markers of Wnt/β-catenin pathway activation. However, ox-LDL-induced activation of Wnt/β-catenin pathway, as well as ox-LDL-induced cell injury and oxidative stress, were synergistically promoted by LiCl and mitigated by Dickkopf 1 (DKK-1), an inhibitor of Wnt/β-catenin pathway. Additionally, ox-LDL-induced HUVEC injury and apoptosis, oxidative stress and activation of Wnt/β-catenin pathway were suppressed by PEDF, while they were further strengthened by a small interfering RNA of PEDF.ConclusionWnt/β-catenin pathway may mediate ox-LDL-induced endothelial injury via oxidative stress, and PEDF ameliorates endothelial injury by suppressing Wnt/β-catenin pathway and subsequently reducing oxidative stress.

Exogenous supplement of N-acetylneuraminic acid ameliorates atherosclerosis in apolipoprotein E-deficient mice

BACKGROUND AND AIMS Previous studies investigating the correlation between plasma sialic acid and the severity of atherosclerosis present conflicting results. In atherosclerosis patients, plasma levels of N-acetylneuraminic acid (NANA) are increased; however, the underlying mechanisms have not yet been clarified. We assume the increased NANA level may be a compensatory mechanism due to oxidative stress and/or inflammation. The aim of this study is to investigate whether supplementation of NANA could attenuate the progression of atherosclerosis. METHODS Exogenous NANA was used to determine its effect on apolipoprotein E-deficient (apoE(-/-)) mice taking natural quercetin as a positive control. The effect of NANA on lipid lowering, antioxidant activity and anti-inflammation was investigated by methods of molecular biology. RESULTS 1) NANA administration decreased 18.9% of the atherosclerotic plaque formation in the aorta and 26.7% of the lipid deposition in the liver of high-fat diet apoE(-/-) mice; 2) notably, NANA treatment reduced 62.6% of the triglyceride by improving lipoprotein lipase activity; 3) NANA lowered 17.5% of the plasma total cholesterol by up-regulating reverse cholesterol transport (RCT)-related protein expression such as ATP-binding cassette transporter (ABC) G1 and ABCG5 in liver or small intestine; 4) NANA administration notably decreased oxidative stress by increasing antioxidant enzymes activity and protein expression of paraoxonase 1 and 2; 5) NANA markedly reduced tumour necrosis factor-α and intercellular adhesion molecule-1 expression in aorta and liver. CONCLUSIONS NANA exhibited triglyceride lowering, anti-oxidation, and RCT promoting activities, and therefore NANA supplementation may be a new strategy for prevention and treatment of atherosclerosis.

Synthesis and cardiovascular protective effects of quercetin 7-O-sialic acid.

Oxidative stress and inflammation play important roles in the pathogenesis of cardiovascular disease (CVD). Oxidative stress‐induced desialylation is considered to be a primary step in atherogenic modification, and therefore, the attenuation of oxidative stress and/or inflammatory reactions may ameliorate CVD. In this study, quercetin 7‐O‐sialic acid (QA) was synthesized aiming to put together the cardiovascular protective effect of quercetin and the recently reported anti‐oxidant and anti‐atherosclerosis functions of N‐acetylneuraminic acid. The biological efficacy of QA was evaluated in vitro in various cellular models. The results demonstrated that 50 μM QA could effectively protect human umbilical vein endothelial cells (HUVEC, EA.hy926) against hydrogen peroxide‐ or oxidized low‐density lipoprotein‐induced oxidative damage by reducing the production of reactive oxygen species. QA attenuated hydrogen peroxide‐induced desialylation of HUVEC and lipoproteins. QA decreased lipopolysaccharide‐induced secretion of tumour necrosis factor‐α (TNF‐α) and monocyte chemoattractant protein‐1 (MCP‐1), and it significantly reduced the expression of intercellular adhesion molecule‐1, vascular cell adhesion molecule‐1, TNF‐α and MCP‐1. Furthermore, QA effectively promoted cholesterol efflux from Raw 264.7 macrophages to apolipoprotein A‐1 and high‐density lipoprotein by up‐regulating ATP‐binding cassette transporter A1 and G1, respectively. Results indicated that the novel compound QA exhibited a better capacity than quercetin for anti‐oxidation, anti‐inflammation, cholesterol efflux promotion and biomolecule protection against desialylation and therefore could be a candidate compound for the prevention or treatment of CVD.

D4F alleviates macrophage-derived foam cell apoptosis by inhibiting the NF-κB-dependent Fas/FasL pathway

This study was designed to explore the protective effect of D4F, an apolipoprotein A-I mimetic peptide, on nuclear factor-κB (NF-κB)-dependent Fas/Fas ligand (FasL) pathway-mediated apoptosis in macrophages induced by oxidized low-density lipoprotein (ox-LDL). Our results showed that ox-LDL induced apoptosis, NF-κB P65 nuclear translocation and the upregulation of Fas/FasL pathway-related proteins, including Fas, FasL, Fas-associated death domain proteins (FADD), caspase-8 and caspase-3 in RAW264.7 macrophages, whereas silencing of Fas blocked ox-LDL-induced macrophage apoptosis. Furthermore, silencing of P65 attenuated macrophage apoptosis and the upregulation of Fas caused by ox-LDL, whereas P65 expression was not significantly affected by treatment with Fas siRNA. D4F attenuated the reduction of cell viability and the increase in lactate dehydrogenase leakage and apoptosis. Additionally, D4F inhibited ox-LDL-induced P65 nuclear translocation and upregulation of Fas/FasL pathway-related proteins in RAW264.7 cells and in atherosclerotic lesions of apoE−/− mice. However, Jo2, a Fas-activating monoclonal antibody, reversed the inhibitory effect of D4F on ox-LDL-induced cell apoptosis and upregulation of Fas, FasL and FADD. These data indicate that NF-κB mediates Fas/FasL pathway activation and apoptosis in macrophages induced by ox-LDL and that D4F protects macrophages from ox-LDL-induced apoptosis by suppressing the activation of NF-κB and the Fas/FasL pathway.