We have investigated the effect of various forms of phosphodiester cytidine-phosphate-guanosine oligodeoxynucleotides (CpG ODNs) on the production of pro-inflammatory cytokines and related genes in RAW 264.7 macrophages. Treatment with the CpG ODNs increased the expression of tumor necrosis factor alpha (TNF-alpha), IL-6, and inducible nitric oxide synthase but not interleukin-1beta (IL-1beta). We also investigated the effect of CpG ODNs on the expression of ATP-binding cassette transporter A1 (ABCA1) and G1 (ABCG1) genes which are known to facilitate cholesterol efflux from macrophages for anti-atherosclerosis. CpG 2006 significantly reduced the levels of ABCG1 mRNA as determined by real-time polymerase chain reaction, whereas ABCA1 mRNA level was not changed. Western blot analysis further confirmed the reduction of ABCG1 protein expression by CpG 2006. In addition, we also determined the protein level of peroxisome proliferator activated receptor gamma (PPARgamma), which is recognized as a transcriptional activator of ABC transporters, was also reduced by CpG 2006. Thus, these results suggest that ABCG1 is specifically down-regulated by CpG 2006 in a PPARgamma-dependent manner in macrophages.
ATP-Binding Cassette Transporters/drug effects/genetics/*metabolism ; Animals ; Atherosclerosis/metabolism ; Cholesterol/metabolism ; Cytokines/drug effects/metabolism ; Gene Expression Regulation ; Inflammation/*metabolism ; Interleukin-1beta/drug effects/metabolism ; Interleukin-6/metabolism ; Lipoproteins/drug effects/genetics/*metabolism ; Macrophages/*cytology/*metabolism ; Mice ; Nitric Oxide Synthase/drug effects/metabolism ; Oligodeoxyribonucleotides/*pharmacology ; PPAR gamma/genetics/*metabolism ; Tumor Necrosis Factor-alpha/drug effects/metabolism

The effects of androgen on lipid and the cardiovascular system are very important. The relationship between androgen and lipoprotein is rather complicated and influenced by many factors. The effects of endogenous androgen on the metabolism of lipoprotein vary with age, environment, nutrition and gender, while the effects of exogenous androgen on lipoprotein vary with different androgen preparations, administration methods and diseases to be treated. Androgen can impact the metabolism of lipoprotein, vascular endothelium, macrophage, vascular smooth muscle, angiotasis, blood coagulation, platelet and so on. The effects of polymorphism of the androgen receptor gene CAG on the cardiovascular system are important and yet somehow controversial and have to be further investigated.
Androgens ; pharmacology ; physiology ; Cardiovascular System ; drug effects ; Female ; Humans ; Lipoproteins ; metabolism ; Male ; Polymorphism, Genetic ; Receptors, Androgen ; genetics ; physiology

We have observed earlier that testosterone at physiological concentrations can stimulate tissue factor pathway inhibitor (TFPI) gene expression through the androgen receptor in endothelial cells. This study further investigated the impact of testosterone on TFPI levels in response to inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha). Cultured human umbilical vein endothelial cells were incubated in the presence or absence of testosterone or TNF-alpha. TFPI protein and mRNA levels were assessed by enzyme-linked immunosorbent assay and quantitative real-time reverse transcription polymerase chain reaction. To study the cellular mechanism of testosterone's action, nuclear factor-kappa B (NF-kappaB) translocation was confirmed by electrophoretic mobility shift assays. We found that after NF-kappaB was activated by TNF-alpha, TFPI protein levels declined significantly by 37.3% compared with controls (P < 0.001), and the mRNA levels of TFPI also decreased greatly (P < 0.001). A concentration of 30 nmol L(-1) testosterone increased the secretion of TFPI compared with the TNF-alpha-treated group. NF-kappaB DNA-binding activity was significantly suppressed by testosterone (P < 0.05). This suggests that physiological testosterone concentrations may exert their antithrombotic effects on TFPI expression during inflammation by downregulating NF-kappaB activity.
Androgens ; pharmacology ; Cells, Cultured ; Down-Regulation ; drug effects ; Drug Combinations ; Endothelium, Vascular ; drug effects ; metabolism ; Humans ; Infant, Newborn ; Lipoproteins ; genetics ; metabolism ; NF-kappa B p50 Subunit ; antagonists & inhibitors ; genetics ; RNA, Messenger ; metabolism ; Testosterone ; pharmacology ; Tumor Necrosis Factor-alpha ; pharmacology

To study the effects of alkaloids from Coptidis Rhizoma on low-density lipoprotein receptor (LDLR) mRNA expression and antihyperlipedemic levels. The LDLR mRNA expression were detected by real time fluorescence quantitative PCR, and the levels of total cholesterol (TC), triglyceride (TG), low density lipoprotein (LDL-c) and high-density lipoprotein cholesterol (HDL-c) in serum were measured at the first and last examination. The results show that, after the drug treatment, compared with the model group, each drug group showed a lipid-lowering effect. Especially, coptisine, palmatine, jatrorrhinze were significantly reduced TC, TG, LDL-c (P < 0.05, P < 0.01), and increased HDL-c (P < 0.01). In addition, they also increased mRNA expression of the LDLR in liver and HepG2 cells. The results showed that alkaloids from Coptidis Rhizoma can regulate lipid metabolism disorder, and coptisine have the best lipid-lowering effect.
Alkaloids ; administration & dosage ; Animals ; Cholesterol ; metabolism ; Cricetinae ; Drugs, Chinese Herbal ; administration & dosage ; Humans ; Hyperlipidemias ; drug therapy ; genetics ; metabolism ; Hypoglycemic Agents ; administration & dosage ; Lipid Metabolism ; drug effects ; Lipids ; blood ; Lipoproteins, LDL ; metabolism ; Mesocricetus ; Receptors, Lipoprotein ; genetics ; metabolism ; Triglycerides ; metabolism

Aortic valve calcification is a common disease in the elderly, but its cellular and molecular mechanisms are not clear. In order to verify the hypothesis that Wnt/β-catenin signaling pathway is involved in the process of calcification of aortic valve, porcine aortic valve interstitial cells (VICs) were isolated, cultured and stimulated with oxidized low density lipoprotein (ox-LDL) for 48 h to induce the differentiation of VICs into osteoblast-like cells. The key proteins and genes of Wnt/β-catenin signaling pathway, such as glycogen synthase kinase 3β (GSK-3β) and β-catenin, were detected by using Western blotting and real-time polymerase chain reaction (PCR). The results showed that the VICs managed to differentiate into osteoblast-like cells after the stimulation with ox-LDL and the levels of proteins and genes of GSK-3β and β-catenin were increased significantly in VICs after stimulation for 48 h (P<0.05). It is suggested that Wnt/β-catenin signaling pathway may play a key role in the differentiation of VICs into osteoblast-like cells and make great contribution to aortic valve calcification.
Alkaline Phosphatase ; genetics ; metabolism ; Animals ; Aortic Valve ; metabolism ; pathology ; Aortic Valve Stenosis ; Blotting, Western ; Bone Morphogenetic Protein 2 ; genetics ; metabolism ; Calcinosis ; Cell Differentiation ; drug effects ; genetics ; Cells, Cultured ; Gene Expression ; drug effects ; Glycogen Synthase Kinase 3 ; genetics ; metabolism ; Lipoproteins, LDL ; pharmacology ; Osteoblasts ; drug effects ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Swine ; Wnt Signaling Pathway ; genetics ; physiology ; beta Catenin ; genetics ; metabolism

BACKGROUNDLipoprotein-associated phospholipase A2 (Lp-PLA2) is mainly secreted by macrophages, serving as a specific marker of atherosclerotic plaque and exerting pro-atherogenic effects. It is known that high-density lipoprotein (HDL) plays an important role against atherosclerosis by inhibiting pro-inflammatory factors, however, the relationship between HDL and Lp-PLA2 remains elusive.

METHODSIn this study, reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, and a platelet-activating factor (PAF) acetylhydrolase assay were performed to determine the Lp-PLA2 mRNA level, protein expression and activity in human monocyte-derived macrophages upon HDL treatment of different concentrations and durations. To investigate the underlying mechanism of HDL-induced Lp-PLA2 action, pioglitazone, a peroxisome proliferator-activated receptor-γ (PPARγ) ligand, was introduced to human monocyte-derived macrophages and mRNA and protein levels of Lp-PLA2, as well as its activity, were determined.

RESULTSLp-PLA2 mRNA levels, protein expression and activity were significantly inhibited in response to HDL treatment in a dose and time dependent manner in human monocyte-derived macrophages. Pioglitazone treatment (1 - 10 ng/ml) upregulated the Lp-PLA2 mRNA level, protein expression and activity in human monocyte-derived macrophages, while the effects were markedly reversed by HDL. In addition, pioglitazone resulted in a significant increase in PPARγ phosphorylation in human monocyte-derived macrophages, which could be inhibited by HDL.

CONCLUSIONThese findings indicate that HDL suppresses the expression and activity of Lp-PLA2 in human monocyte-derived macrophages, and the underlying mechanisms may be mediated through the PPARγ pathway.

1-Alkyl-2-acetylglycerophosphocholine Esterase ; genetics ; metabolism ; Cells, Cultured ; Humans ; Lipoproteins, HDL ; pharmacology ; Macrophages ; drug effects ; enzymology ; metabolism ; PPAR gamma ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; drug effects ; genetics

BACKGROUNDRemnant-like lipoproteins (RLPs) have been demonstrated to accelerate the onset of endothelial progenitor cells (EPCs) senescence. Recent study has determined that microRNAs (miRNAs) were closely associated with cellular proliferation and senescence. This study aimed to examine whether RLPs lead to an alteration of miRNAs in senescent EPCs.

METHODSRLPs were prepared from plasma samples with immunoaffinity method. After 8 days of culture, EPCs were identified by flow cytometry analysis. Cells were incubated with RLPs for 72 hours. The senescent markers p16INK4a and senescence-associated beta-galactosidase (SA-β-gal) were detected by Western blotting analysis and β-gal staining assay, respectively. A human miRNA microarray containing 723 miRNAs was used to detect the expression profile of miRNAs in control and senescent EPCs. The result from the above microarray was qualified by RT-PCR assay.

RESULTSRLPs dose-dependently up-regulated the protein level of p16(INK4a) in EPCs, and RLPs at a concentration of 100 µg/ml induced a significant increase in the percentage of SA-β-gal-positive EPCs. Of 723 miRNAs, four miRNAs expressed differentially and significantly in RLPs-treated EPCs (P < 0.05), then their changes in expression were validated by real-time RT-PCR. Among them miR-148b and miR-155 were upregulated while miR-574-3p was down-regulated significantly when compared with control (P < 0.01).

CONCLUSIONSRLPs result in the onset of EPCs senescence. Senescent EPCs induced by RLPs exhibit a different profile of miRNAs. These three miR-148b and miR-155 and miR-574-3p reach a significant difference when compared with control, indicating that microRNA might take part in RLPs-induced EPCs senescence.

Blotting, Western ; Cells, Cultured ; Cellular Senescence ; drug effects ; genetics ; Cyclin-Dependent Kinase Inhibitor p16 ; metabolism ; Endothelial Cells ; cytology ; drug effects ; Flow Cytometry ; Humans ; Lipoproteins ; pharmacology ; MicroRNAs ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Stem Cells ; cytology ; drug effects

OBJECTIVETo observe effect of Huoxue injection on the expression of intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), the adherence of monocytes to endothelial cells, and the regulation role of nuclear factor kappa B (NF-kappaB) in cultured human umbilical vein endothelial cells (HUVEC) injury induced by the oxidized low-density lipoprotein (ox-LDL).

METHODThe ox-LDL (100 mg x L(-1)) was added to the cultured HUVEC to prepare the injury model of HUVEC. The adhesive percentage between HUVEC treated with ox-LDL and monocytes was determined by protein quantification. Expression of mRNA and protein of ICAM-1 and VCAM-1 were determined by RT-PCR and flow cytometry respectively. The percentage of positive cells and the ratio of nuclei and cytoplasm of NF-kappaB p65 staining in HUVEC the were examined by cell immunochemistry.

RESULTTreatment of HUVEC with ox-LDL for 12, 24 hours significantly increased adhesion of monocytes to HUVEC and enhanced the expressions of mRNA and protein of ICAM-1 and VCAM-1. The percentage of positive cells and the ratio of nuclei and cytoplasm of NF-kappaB p65 staining in HUVEC were significantly increased after treatment with ox-LDL for 24 hours. Huo Xue Injection could significantly inhibit the adhesion between monocyte and HUVEC, the expression of mRNA and protein of ICAM-1 and VCAM-1, and declined the percentage of positive cells and the ratio of nuclei and cytoplasm of NF-kappaB p65 staining in HUVEC. The effects were strengthened with increasing the deal of Huoxue injection.

CONCLUSIONHuoxue injection has an inhibitory effect on the adherence of monocytes to HUVEC, probably by way of down-regulating the expression of mRNA and protein of ICAM-1 and VCAM-1 in HUVEC. The mechanism is probably associated with inhibiting the activation of NF-kappaB p65 of HUVEC. The effects of Huoxue injection can bring about the protective effect to endothelial cells injury induced by ox-LDL.

Animals ; Cell Adhesion ; drug effects ; Cells, Cultured ; Dose-Response Relationship, Drug ; Drugs, Chinese Herbal ; administration & dosage ; pharmacology ; Endothelial Cells ; cytology ; drug effects ; metabolism ; Gene Expression Regulation ; drug effects ; Humans ; Injections ; Intercellular Adhesion Molecule-1 ; genetics ; Lipoproteins, LDL ; metabolism ; Monocytes ; cytology ; drug effects ; NF-kappa B ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Umbilical Veins ; cytology ; Vascular Cell Adhesion Molecule-1 ; genetics

OBJECTIVETo study the protective effect of panax notoginseng saponins (PNS) on human umbilical vascular endothelial cells (HUVECs) injury induced by oxidized low-density lipoprotein (ox-LDL) for investigating the mechanism of PNS in treating arteriosclerosis obliterans (ASO).

METHODSTaking the cultured HUVECs as target cells, ox-LDL was used to establish a model of injured HUVEC and it was then intervened by PNS. The morphologic changes of HUVEC were observed under light microscope; activity of cells was determined by MTT method; the adhesive percentage between ox-LDL treated HUVEC and monocyte detennined by protein quantification and the protein expression of intercellular adhesion molecule-1 (ICAM-1) determined by flow cytometry.

RESULTSAt the time points of HUVEC being treated with ox-LDL (100 mg/L) for 12 h and 24 h, significant injury of HUVEC was shown, its activity reduced, the adhesion rate with monocytes elevated, and the protein expression of ICAM-l in HUVEC increased significantly (P < 0.05, P < 0.01). PNS showed significant effect in reversing all the above changes, as compared with the control group (without PNS treaded), respective significant difference was shown in all the four indexes (P < 0.05, P < 0.01).

CONCLUSIONPNS has a protective effect on endothelial cells injury induced by ox-LDL,which may be one of its mechanisms in treating ASO. The protective effect of PNS is probably by way of down-regulating the expression of ICAM-1 in endothelial cells and inhibiting the adherence of monocytes to endothelial cells.

Arteriosclerosis ; drug therapy ; metabolism ; physiopathology ; Cell Adhesion ; drug effects ; Cells, Cultured ; Drugs, Chinese Herbal ; pharmacology ; Endothelium, Vascular ; cytology ; drug effects ; metabolism ; Gene Expression ; drug effects ; Humans ; Intercellular Adhesion Molecule-1 ; genetics ; metabolism ; Lipoproteins, LDL ; metabolism ; Panax notoginseng ; chemistry ; Umbilical Veins ; cytology ; drug effects ; metabolism

It has been hypothesized that blood infusion of reconstituted HDL (rHDL) is a possible therapeutic strategy for the treatment of coronary artery disese. To compare short-term anti-inflammatory activity of wildtype (WT) apoA-I and point mutants, each rHDL containing WT, V156K, or R173C was infused into apo-E deficient atherosclerotic mice. Each rHDL was injected via the tail vein at a dosage of 120 mg/kg of body weight in 0.4 ml of tris-buffered saline (TBS), and blood was then collected at 24 and 48 h post-injection. Although regression activity was observed in each of the rHDL infused groups, a 30% reduction in the lipid-stained area of the aortic sinus was observed in the V156K and R173C-rHDL groups when compared to that of the WT-rHDL group, and this reduction was well correlated with an approximately 60% reduction in the accumulation of macrophages in the lesion area. Additionally, the groups that received the V156K and R173C-rHDL treatments showed smaller increases in the GOT, GPT, interleukin-6, myeloperoxidase (MPO) and lipid hydroperoxide (LPO) serum levels than those that received the WT-rHDL treatment. In addition, the strongest serum paraoxonase and ferric reducing ability was observed in the V156K and R173C-rHDL groups. In vitro nitration and chlorination of apoA-I by MPO treatment revealed that V156K-rHDL and R173C-rHDL were less susceptible to chlorination. Furthermore, rHDL treatment inhibited cellular uptake of oxidized LDL by macrophage cells and the production of proatherogenic species in culture media. In conclusion, blood infusions of the rHDLs exerted in vivo regression activity with anti-inflammatory and antioxidant activity in apo-E deficient mice and THP-1 cells, especially in those that were treated with V156K and R173C apoA-I.
Animals ; Anti-Inflammatory Agents/immunology/*therapeutic use ; Apolipoprotein A-I/blood/genetics/immunology/*therapeutic use ; Apolipoproteins E/genetics ; Aryldialkylphosphatase/blood/metabolism ; Atherosclerosis/*drug therapy ; Cell Line ; Cell Membrane Permeability ; Cholesterol/blood/metabolism ; Humans ; Lipoproteins, HDL/genetics/immunology/*therapeutic use ; Lipoproteins, LDL/metabolism ; Macrophages/cytology ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Oxidation-Reduction/*drug effects ; Peroxidase/blood/metabolism ; Point Mutation