TLR4/NF-κB signaling contributes to chronic unpredictable mild stress-induced atherosclerosis in ApoE-/- mice.

OBJECTIVE: Chronic stress is an important risk factor for atherosclerotic diseases. Our previous studies have shown that chronic unpredictable mild stress (CUMS) accelerates atherosclerosis and up-regulates TLR4/NF-κB expression in apoE-/- mice. However, TLR4/NF-κB signaling whether directly contributes to the development of atherosclerosis in CUMS mice is unclear. We hypothesized that the interference of TLR4/NF-κB can ameliorate CUMS-induced inflammation and atherosclerosis in apoE-/- mice. METHODS: ApoE-/- mice were exposed to 12 weeks CUMS. Ad-siRNA TLR4 was given by tail vein injection (10 µl/mouse, every 5 days), and PDTC (an inhibitor of NF-κB) was given by intraperitoneal injection (60 mg/kg, once a day). Plasma corticosterone concentrations were determined by solid-phase 125I radioimmunoassay. Atherosclerosis lesions in aortic sinuses were evaluated and quantified by IMAGEPRO PLUS. Western blotting was used to detect the expression of TLR4, NF-κB, and IL-1ß in aortas of the mice. Plasma lipid profiles, IL-1ß, TNF-α, and MCP-1 were measured by ELISA. RESULTS: Our results indicated that CUMS apoE-/- mice treatment with siRNA TLR4 significantly decreased atherosclerosis and down-regulated TLR4, NF-κB, and inflammatory cytokines. PDTC also remarkably reduced atherosclerosis and the levels of IL-1ß, TNF-α and MCP-1 in plasma. However, Treatment with siRNA TLR4 or PDTC had no effect on plasma corticosterone levels, and lipid profiles. CONCLUSIONS: TLR4/NF-κB pathway may participate in CUMS-induced atherosclerosis through activation of proinflammatory cytokines in apoE-/- mice. Our data may provide a new potential therapeutic target for prevention of CUMS -induced atherosclerosis.

Psychological stress, immune response, and atherosclerosis.

It is well known that psychological stress is associated with increased atherosclerosis. This response is mainly mediated by altered immune reactions due to either activation or depression of the hypothalamic-pituitary-adrenal (HPA) regulatory feed back mechanisms that influence both the vascular endothelium function and the recruitment of circulating monocytes and their conversion to foam cells. Although the detailed mechanisms behind these processes are not well understood, it has been assumed that expression of pro- and anti-inflammatory cytokines by stress hormones, such as catecholamines and corticosteroids, maybe involved. In this review, we focus on evidences that various immunological factors are transformed under prolonged psychological stress by causing vascular low-grade inflammation. A better understanding of the bidirectional communication between the neuroendocrine and immune systems may contribute to new treatment strategies.

Oxidized low-density lipoprotein activates adipophilin through ERK1/2 signal pathway in RAW264.7 cells.

It has been reported that oxidized low-density lipoprotein (Ox-LDL) can increase the expression of adipophilin. However, the detailed mechanisms are not fully understood. The aim of this study was to investigate the mechanism of Ox-LDL on adipophilin expression and the intracellular lipid droplet accumulation. A mouse macrophage-like cell line, RAW264.7, was used throughout, and it was found that Ox-LDL induced adipophilin expression in a dose-dependent manner. Moreover, Ox-LDL induced peroxisome proliferator-activated receptor-gamma (PPARgamma) expression and PPARgamma-specific inhibitor T0070907 abrogated Ox-LDL-induced adipophilin expression, but specific agonist GW1929 not. Furthermore, Ox-LDL induced phosphorylation of ERK1/2, and ERK1/2-specific inhibition by PD98059 suppressed the Ox-LDL-induced PPARgamma and adipophilin expression. The results showed that ERK1/2 or PPARgamma-specific inhibition decreased the amounts of intracellular lipid droplets. Meanwhile, the PPARgamma-specific agonist increased intracellular lipid droplets. These results suggested that Ox-LDL-induced increase in adipophilin level via ERK1/2 activation is one of the mechanisms of inducing greater amounts of intracellular lipid droplets in RAW264.7 cells, which indicated that adipophilin is involved in atherosclerotic progression.

Effects of chronic mild stress on the development of atherosclerosis and expression of toll-like receptor 4 signaling pathway in adolescent apolipoprotein E knockout mice.

Here, we investigated the effect of chronic mild stress (CMS) on the development of atherosclerosis as well as the expression of Toll-like receptors (TLRs) signaling pathway in adolescent apolipoprotein E knockout (apoE-/-) mice. Mice were subjected to daily CMS for 0, 4, and 12 weeks, respectively. To identify the expression of Toll-like receptor 4 signaling pathway in adolescent apolipoprotein E knockout mice subjected to CMS, we compared gene expression in aortas of stressed and unstressed mice using TLRs signaling pathway real-time PCR microarrays consisting of 87 genes. We found that atherosclerosis lesions both in aortic tress and sinuses of CMS mice were significantly increased linearly in response to duration of CMS exposure. Among 87 genes analyzed, 15 genes were upregulated in stressed mice, especially TLR4, myeloid differentiation factor 88 (MyD88), and IL-1beta, and 28 genes were downregulated compared with nonstressed mice. CMS mice demonstrated markedly increased aortic atherosclerosis that were associated with significant increases in levels of expression of TLR4, MyD88, nuclear factor kappaB (NF-kappaB), MCP-1, IL-1beta, TNF-alpha, and sICAM-1. Taken together, our results suggest an important role for TLR4 signaling pathway in atherosclerosis in a CMS mouse model.

Mast cell degranulator compound 48-80 promotes atherosclerotic plaque in apolipoprotein E knockout mice with perivascular common carotid collar placement.

BACKGROUND: Study of the relationship between mast cells and atherosclerosis is mostly dependent on pathological observation and cytology experiments. To investigate the effects of mast cells degranulation on plaque and their possible mechanisms we used apolipoprotein E knockout mice which had been placed perivascular common carotid collar with mast cells degranulator compound 48-80. METHODS: Forty apolipoprotein E knockout mice were fed a western-type diet and operated on with placement of perivascular right common carotid collar. Four weeks after surgery, the mice were intraperitoneally injected with compound 48-80 (0.5 mg/kg) or D-Hanks every other day for 4 times. The serum lipids and activity of tryptase were measured. Tissue sections were stained with hematoxylin and eosin. Corresponding sections were stained with toluidine blue and immunohistochemically with antibodies against macrophage-specific antigen, alpha-smooth muscle actin, interleukin-1beta and von Willebrand factor. Simultaneously, basic fibroblast growth factor was detected by in situ hybridization and immunofluorescence. RESULTS: No pathological change was observed in common carotid non-collar placement but atherogenesis in common carotid collar placement of both groups. There was a significant increase in plaque area ((5.85+/-0.75) x 10(4) vs (0.86+/-0.28) x 10(4) microm(2), P<0.05), the degree of lumen stenosis ((81+/-15)% vs (41+/-12)%, P<0.05), the activity of tryptase in serum ((0.57+/-0.13) U/L vs (0.36+/-0.10) U/L, P<0.05), and the percentage of degranulated mast cells ((80.6+/-17.8)% vs (13.5+/-4.1)%, P<0.05). The expressions of macrophage-specific antigen, alpha-smooth muscle actin, interleukin-1beta, basic fibroblast growth factor and the density of neovessel in plaque were more in the compound 48-80 group than in the control group. CONCLUSIONS: Perivascular common carotid collar placement can promote atherosclerotic plaque formation in apolipoprotein E knockout mice. Compound 48-80 increases plaque area and the degree of lumen stenosis by the mechanism that compound 48-80 promotes proliferation of smooth muscle cells and aggregation of macrophages. Compound 48-80 promotes angiogenesis in plaque. The mechanism is potentially that compound 48-80 increases the expressions of basic fibroblast growth factor mRNA and protein in plaque. Compound 48-80 enhances the expression of interleukin-1beta in plaque.

Phosphorylation of beta-actin by protein kinase C-delta in camptothecin analog-induced leukemic cell apoptosis.

AIM: This study was conducted to reveal new proteins involved in acute myeloid leukemia (AML) cell apoptosis. METHODS: Using camptothecin analog NSC606985- induced leukemic U937 cell apoptosis as a model, this study performed a differential proteomic analysis during apoptosis induction. The significantly modulated protein was underwent further investigation in the apoptotic process. RESULTS: We found that beta-actin protein presented two different spots on the two-dimensional electrophoresis (2-DE) map, which shared similar molecular weight and different pI. Those two spots demonstrated contrary changes (disappeared on the basic-end and increased on the acid-end spot) during apoptosis induction, although the total level of beta-actin kept constant. This observation was further confirmed by immunoblot analysis on 2-DE gel. When NSC606985-treated cell lysate was incubated with alkaline phosphotase, beta-actin on the basic-end spot was restored, indicating increased phosphorylation of beta-actin during NSC606985- induced apoptosis. Moreover, the polymerization of actin also decreased after NSC606985 treatment. The increased beta-actin phosphorylation and decreased actin polymerization was antagonized by pre-treatment of rottlerin, a specific protein kinase C-delta (PKC delta) inhibitor. CONCLUSION: All these results indicate that beta-actin was phosphorylated during apoptosis induction, which was mediated by activated PKC delta.

Oxidized high-density lipoprotein promotes maturation and migration of bone marrow derived dendritic cells from C57BL/6J mice.

OBJECTIVE: To explore the influence of oxidized high-density lipoprotein (oxHDL) on the maturation and migration of bone marrow-derived dendritic cells (BMDCs) from C57BL/6J mice. METHODS: The C57BL/6J mice bone marrow cell suspension was prepared and purified. Recombinant granulocyte-macrophage colony-stimulating factor (rmGM-CSF) and recombinant interleukin-4 (rmIL-4) were used to promote monocytes to differentiate and suppress lymphocytes. Then 50 microg/mL oxHDL was added to stimulate BMDCs, using 50 microg/mL high-density lipoprotein (HDL) as homologous protein control, PBS as negative control, and 1 microg/mL lipopolysaccharide (LPS) as positive control. The CD86 and MHCII expression rates were detected with fluorescence-activated cell sorting (FACS). Liquid scintillation counting (LSC) was used in mixed lymphocyte reactions (MLRs) to reflect the ability of BMDCs in stimulating the proliferation of homologous T cells. Levels of cytokines IL-12 and IL-10 were detected by ELISA. The cell migration was evaluated with the transwell system. RESULTS: Compared with PBS group, the expressions of CD86 and MHCII, counts per minute of MLRs, secretion of IL-12 and IL-10, and number of migrated cells in oxHDL group and LPS group significantly increased (all P<0.05), while the increment was less in oxHDL group than LPS group. The number of migrated cells in oxHDL group was about twice of that in HDL group. CONCLUSION: OxHDL may promote the maturation and migration of BMDCs in vitro.

Opposite effects of WEB2086 on angiogenesis in atheromas and ischemic hindlimb of apoE gene deficient mice.

BACKGROUND: Our previous research has suggested that platelet activating factor receptor was related to atherosclerosis. The present study investigated the effect of a platelet activating factor receptor antagonist-WEB2086 on angiogenesis in aortal plaque and ischemic hindlimb of apolipoprotein E-deficient mice. METHODS: Eight-week-old apolipoprotein E-deficient mice were fed with a 0.15% cholesterol diet to develop advanced lesions. At age 32 weeks unilateral hindlimb ischemia was surgically induced and the mice were divided into two groups: with or without WEB2086 mixed with their drinking water (4.3 mg in 100 ml). At age 40 weeks blood was collected from the orbit for measurement of serum lipids and an enzyme linked immunosorbent assay was used to determine platelet activating factor and oxidized low density lipoprotein in the gastrocnemius and aorta. Whole-Mount CD31 stain and plaque-associated sprouting have been used to estimate angiogenesis in plaque from the aorta and laser Doppler perfusion imaging and immunohistochemical expression of von Willebrand factor have been used to estimate angiogenesis in ischemic hindlimb. RESULTS: The lipid composition of serum was not different between the groups. However, the amount of platelet activating factor and oxidized low density lipoprotein detected in the aorta was significantly higher than that in the gastrocnemius of ischemic hindlimb. The ratio of lesion to aorta levels was significantly reduced by administration of WEB2086, (31.52 +/- 6.18)% vs (55.58 +/- 8.34)%, P < 0.01. The mean density of intimal capillaries in atherosclerotic plaque, (31.13 +/- 9.20)% vs (57.74 +/- 11.28)%, P < 0.01, and the mean number of sprouts per aorta were significantly reduced, 183.92 +/- 34.17 vs 392.54 +/- 76.79, P < 0.01, in the WEB2086 group. Blood flow (0.85 +/- 0.12 vs 0.45 +/- 0.06, P < 0.01) and capillary density of ischemic hindlimb (1.18 +/- 0.17 vs 0.53 +/- 0.09, P < 0.01) were markedly increased in apolipoprotein E-deficient mice treated with WEB2086 versus controls. CONCLUSION: The study provides evidence that WEB2086 can inhibit angiogenesis in atherosclerotic plaque but promote it in ischemic hindlimb.

Construction and functional analysis of a lentiviral expression vector containing a scavenger receptor (SR-PSOX) that binds uniquely phosphatidylserine and oxidized lipoprotein.

The aim of this study is to construct a lentiviral expression vector containing a scavenger receptor (SR-PSOX) that binds with uniquely phosphatidylserine and oxidized lipoprotein with six histidine tags and to investigate the function of SR-PSOX in atherosclerosis. We utilize the ViraPower lentiviral expression system which was efficient to deliver in vitro or in vivo the target gene into dividing and non-dividing mammalian cells using an enhanced biosafety replication-incompetent lentivirus. The blunt-end sequence was amplified using the reverse transcription-polymerase chain reaction and directional TOPO cloning reaction. Through a pair of the cytomegalovirus forward primer and the reverse primer of SR-PSOX, the correct clones were identified by polymerase chain reaction and sequencing. The ViraPower packaging mix and SR-PSOX-pLenti6/V5 TOPO expression plasmid were co-transfected into the 293FT cell line using Lipofectamine 2000. The expression of endogenous and exogenous SR-PSOX as well as tumor necrosis factor (TNF)-alpha protein in various foam cell models at different time points were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blot and indirect immunofluorescence assay. Western blot and immunofluorescence analysis confirmed that the expressions of SR-PSOX and TNF-alpha protein were upregulated in foam cell models. Our data suggested that the overexpression of recombinant human SR-PSOX protein can promote foam cell formation and upregulate the expression of the inflammatory factor TNF-alpha.

Effect of apolipoprotein A-I on ATP binding cassette transporter A1 degradation and cholesterol efflux in THP-1 macrophage-derived foam cells.

Cholesterol-loaded macrophage foam cells are a central component of atherosclerotic lesions. ATP binding cassette transporter A1 (ABCA1), the defective molecule in Tangier disease, mediates the efflux of phospholipid and cholesterol from cells to apolipoprotein A-I (apoA-I), reversing foam cell formation. This study investigated the effect of apoA-I on ABCA1 degradation and cholesterol efflux in THP-1 macrophage-derived foam cells. After exposure of the cultured THP-1 macrophage-derived foam cells to apoA-I for different time, cholesterol efflux, ABCA1 mRNA and protein levels were determined by FJ-2107P type liquid scintillator, RT-PCR and Western blot, respectively. The mean ABCA1 fluorescence intensity on THP-1 macrophage-derived foam cells was detected by flow cytometry. Results showed that apoA-I markedly increased ABCA1-mediated cholesterol efflux from THP-1 macrophage-derived foam cells. This was accompanied by an increase in the content of ABCA1. ApoA-I did not alter ABCA1 mRNA abundance. Significantly, thiol protease inhibitors increased the level of ABCA1 protein and slowed its decay in THP-1 macrophage-derived foam cells, whereas none of the proteosome-specific inhibitor lactacystin, other protease inhibitors, or the lysosomal inhibitor NH4Cl showed such effects. The apoA-I-mediated cellular cholesterol efflux was enhanced by thiol protease inhibitors. Our results suggested that thiol protease inhibitors might provide an alternative way to upregulate ABCA1 protein. This strategy is especially appealing since it may mimic the stabilizing effect of the natural ligands apoA-I.

Oxidized LDL upregulated ATP binding cassette transporter-1 in THP-1 macrophages.

AIM: To study the effect of oxidized low density lipoprotein (ox-LDL) on ATP binding cassette transporter A1 (ABCA1) in THP-1 macrophages. METHODS: After exposing the cultured THP-1 macrophages to ox-LDL for different periods, cholesterol efflux was determined by FJ-2107P type liquid scintillator. ABCA1 mRNA and protein level were determined by reverse trancriptase-polymerase chain reaction (RT-PCR) and Western blot, respectively. The cholesterol level in THP-1 macrophage foam cells was detected by high performance liquid chromatography. RESULTS: ox-LDL elevated ABCA1 in both protein and mRNA levels and increased apolipoprotein (apo) A-I-mediated cholesterol efflux in a time- and dose-dependent manner. 22(R)-hydroxycholesterol and 9-cis-retinoic acid did significantly increase cholesterol efflux in THP-1 macrophage foam cells (P<0.05), respectively. Both of them further promoted cholesterol efflux (P<0.01). As expected, liver X receptor (LXR) agonist decreased content of esterified cholesterol in the macrophage foam cells compared with control, whereas only a slight decrease of free cholesterol was observed. LXR activity was slightly increased by oxidized LDL by 12 % at 12 h compared with 6 h. However, LXR activity was increased about 1.8 times at 24 h, and oxidized LDL further increased LXR activity by about 2.6 times at 48 h. CONCLUSION: ABCA1 gene expression was markedly increased in cholesterol-loaded cells as a result of activation of LXR/RXR. ABCA1 plays an important role in the homeostasis of cholesterol in the macrophages.

[Effects of oleate on ATP binding cassette transporter A1 expression and cholesterol efflux in THP-1 macrophage-derived foam cells].

To study the effect of oleate on ATP binding cassette transporter A1 (ABCA1) expression and cholesterol efflux in THP-1 macrophage-derived foam cells, after exposure of the cultured THP-1 macrophage-derived foam cells to oleate for different time, cholesterol efflux was determined by FJ-2107P type liquid scintillator. ABCA1 mRNA and its protein level were determined by RT-PCR and Western blot, respectively. The mean ABCA1 fluorescence intensity of THP-1 macrophage-derived foam cells was detected by flow cytometry. The results showed that oleate markedly inhibited ABCA1-mediated cholesterol efflux from THP-1 macrophage-derived foam cells. This was accompanied by a reduction in the membrane content of ABCA1. Oleate did not alter ABCA1 mRNA abundance, indicating that decreased ABCA1 transcription, enhanced mRNA decay, or impaired translation efficiency did not account for these inhibitory effects. Oleate, however, increased ABCA1 turnover when protein synthesis was blocked by cycloheximide. Oleate reduces cholesterol efflux and the level of ABCA1 protein in THP-1 macrophage-derived foam cells.

A lipoprotein lipase-promoting agent, NO-1886, improves glucose and lipid metabolism in high fat, high sucrose-fed New Zealand white rabbits.

The synthetic compound NO-1886 is a lipoprotein lipase activator that lowers plasma triglycerides and elevates high-density lipoprotein cholesterol (HDL-C). Recently, the authors found that NO-1886 also had an action of reducing plasma glucose in high-fat/high-sucrose diet-induced diabetic rabbits. In the current study, we investigated the effects of NO-1886 on insulin resistance and beta-cell function in rabbits. Our results showed that high-fat/high-sucrose feeding increased plasma triglyceride, free fatty acid (FFA), and glucose levels and decreased HDL-C level. This diet also induced insulin resistance and impairment of acute insulin response to glucose loading. Supplementing 1% NO-1886 into the high-fat/high-sucrose diet resulted in decreased plasma triglyceride, FFA, and glucose levels and increased HDL-C level. The authors also found a clear increased glucose clearance and a protected acute insulin response to intravenous glucose loading by NO-1886 supplementation. These data suggest that NO-1886 suppresses the elevation of blood glucose in rabbits induced by feeding a high-fat/high-sucrose diet, probably through controlling lipid metabolism and improving insulin resistance.

[Establishment and identification of the gene cell line transfected aldose reductase like gene and its drug resistance to drugs containing carbonyl group].

OBJECTIVE: To investigate the relationship between aldose reductase like protein (ARL-1) gene overexpressed in HCC cells and drug-resistance of the cell to drugs containing carbonyl group. METHODS: To establish ARL-1 stable expression positive cell line, eukaryotic expression vectors containing ARL-1 gene cDNA were transfected into Hep cell mediated by lipofect AMINE. The positive monoclones were determined by PCR and RT-PCR, respectively. Then MTT assay was used to study the drug resistance ability of the cells to drugs containing carbonyl after incubating three days with those drugs. RESULTS: After ARL-1 gene transfection mediated by lipofect AMINE, one positive monoclonal cell overexpressing ARL-1 gene was selected. Compared with the control cell group, drug resistance ability of the positive cells to ADM and MMC which contain carbonyl group increased 2.3 and 3.17 fold, respectively (t=6.39, P=0.016 in ADM group and t=30.06, P=0.001 in MMC group). In the same time, drug resistance ability to 5-FU which has no carbonyl group had no statistical difference between positive monoclonal cell group and control cell group (t=0.684, P=0.531). CONCLUSIONS: The Hep ARL-1 positive cell line with stable expression of ARL-1 gene has been established successfully and the up-regulation of ARL-1 gene may plays an important role in drug resistance of the cells to anticancer drugs containing carbonyl group.

A diet high in saturated fat and sucrose alters glucoregulation and induces aortic fatty streaks in New Zealand White rabbits.

A new and convenient animal model for studying peripheral vascular and coronary artery disease in diabetes was established in this study. Male New Zealand White rabbits weighing approximately 2 kg were divided into 2 groups: a normal control group fed standard laboratory chow and a diabetogenic diet-fed group received a high-fat/high-sucrose diet. The high-fat/high-sucrose diet (contained 10% lard and 37% sucrose) feeding was maintained for 6 months. Plasma total cholesterol, high-density lipoprotein (HDL) cholesterol, triglyceride, superoxide dismutase, nitric oxide, nitric oxide synthase, insulin, and glucose were quantitated at monthly or bimonthly intervals. The aortic fatty streak lesions were quantified following lipid staining with Sudan IV. The aortic samples were observed by electron microscopy. High plasma triglyceride and glucose concentrations were induced. At the end of 6 months, the aortic fatty streak lesions were present in the animals' vascular specimens. As far as we know, this is the first report that demonstrates that New Zealand White rabbits can develop obvious aortic fatty streaks by feeding a high-fat/high-sucrose diet. Our results suggest that New Zealand White rabbits fed a high-fat/high-sucrose diet would provide a convenient model for studying peripheral vascular-and coronary artery disease in diabetes.