PPAR-γ agonist pioglitazone regulates dendritic cells immunogenicity mediated by DC-SIGN via the MAPK and NF-κB pathways.

Dendritic cell-specific ICAM-3 grabbing non-integrin (DC-SIGN) is a dendritic cell-specific lectin which participates in dendritic cell (DC) trafficking, antigen uptake and DC-T cell interactions at the initiation of immune responses. This study investigated whether peroxisome proliferator-activated receptor-gamma (PPAR-γ) activation in human DCs regulates the immunogenicity of DCs mediated by DC-SIGN and exploited the possible molecular mechanisms, especially focused on the signaling pathways of mitogen-activated protein kinases (MAPK) and nuclear factor-κB (NF-κB). Here, we show that the PPAR-γ agonist pioglitazone decreased DC adhesion and transmigration, and DC stimulation of T cell proliferation mediated by DC-SIGN dependent on activation of PPAR-γ, although it increased DC endocytosis independent of PPAR-γ activation. Furthermore, PPAR-γ activation by pioglitazone in DCs down-regulated the expression of DC-SIGN, which was mediated by modulating the balance of the signaling pathways of extracellular signal-regulated kinase, c-Jun N-terminal kinase and NF-κB, but not p38 MAPK. Therefore, we conclude that PPAR-γ activation in human DCs regulates the immunogenicity of DCs mediated by DC-SIGN via the pathways of MAPK and NF-κB. These findings may support the important role of these mediators in the regulation of DC-mediated inflammatory and immunologic processes.

Xiaoxianggou attenuates atherosclerotic plaque formation in endogenous high Ang II ApoE(-/-) mice via the inhibition of miR-203 on the expression of Ets-2 in endothelial cells.

BACKGROUND: Atherosclerosis is a chronic immune-inflammatory disorder and one of the leading causes responsible for cardiovascular morbidity and mortality. Traditional Chinese medicine treatment with multi-targets has shown prospects for the therapeutic effect on atherosclerosis. Thus, this study aims to investigate whether xiaoxianggou has benefit for reducing the atherosclerotic plaque area in endogenous high Ang II ApoE(-/-) mice and investigated the underlying mechanisms. METHODS: Endogenous high Ang II ApoE(-/-) mice model was generated by using two kidney one clip (2K1C). All mice were treated by intragastric administration with xiaoxianggou two times a week for 16 weeks. En face plaque area was analyzed by oil-red O staining. Serum anti-OxLDL antibodies were measured by ELISA assay. Expression of miR-203 and Ets-2 were evaluated using qRT-RCR and western blotting analysis, respectively. RESULTS: This study revealed that xiaoxianggou treatment dose-dependently reduced the atherosclerotic plaque area and serum autoantibodies against oxLDL, elevated miR-203 expression and reduced Ets-2 expression in endogenous high Ang II ApoE(-/-) mice. In primary arterial ECs, Xiaoxianggou reverses the reduced miR-203 expression and the elevated Ets-2 expression induced by AngII, which was further recovered by miR-203 inhibitor. Additionally, miR-203 regulated the expression of Ets-2 by targeting Ets-2-3' UTR. Moreover, miR-203 inhibitor reversed the reduction of atherosclerotic lesion area induced by Xiaoxianggou. CONCLUSIONS: These findings present that xiaoxianggou plays an anti-atherosclerotic role in endogenous high Ang II ApoE(-/-) mice model, which is partly due to its antioxidant actions against atherosclerosis and the inhibition of miR-203 on the expression of Ets-2 in endothelial cells.

Distinct MAPK pathways are involved in IL-23 production in dendritic cells cocultured with NK cells in the absence or presence of angiotensin II.

Accumulated evidence suggests that the crosstalk between dendritic cells (DCs) and natural killer (NK) cells enhances each other's capacity, and results in the production of a variety of soluble factors. However, little is known about the effect of DC-NK crosstalk in interleukin-23 (IL-23) production. In the present study we show that DC-NK coculture caused a high expression of IL-23, angiotensin II (Ang II) alone moderately increased IL-23 production in DCs, but decreased IL-23 secretion in the DC-NK coculture system. We found that Ang II does not influence DC maturation in DC-NK crosstalk. We next investigated the mitogen-activated protein kinase (MAPK) pathway in DCs. We found that Ang II increased IL-23 production through the extracellular signal-related kinase (ERK) pathway. All three MAPK members c-Jun N-terminal kinase (JNK), ERK and p38 were involved in IL-23 production in the DC-NK coculture system. In the presence of Ang II, only the JNK pathway seems to play a role in IL-23 production in DCs cocultured with NK. These data suggest that distinct MAPK pathways are involved in IL-23 production in DCs in response to different stimuli. This work demonstrates for the first time that IL-23 is produced in the DC-NK coculture system, and that Ang II is involved in DC-NK crosstalk. This data will act as a resource that allows further exploitation of role of immune response in atherosclerosis.

Changes in proteomics profile during maturation of marrow-derived dendritic cells treated with oxidized low-density lipoprotein.

Increasing evidence suggests that dendritic cells (DCs) and oxidized low-density lipoprotein (ox-LDL) participate in atherosclerosis. However, few data on the molecular mechanisms of this process are available. To address this question, we used iTRAQ labeling followed by LC-MS/MS analysis to identify many proteins that changed markedly during the maturation of dendritic cells stimulated with ox-LDL. Among a total of 781 identified proteins, 93 were upregulated and 100 were downregulated. The major and significant changes in upregulated proteins were that ox-LDL not only affected the levels of intracellular cathepsins G, Z, D and S, but also significantly enhanced cathepsin S secretion by the treated cells. Our results may provide clues for a more comprehensive understanding the pathogenesis of atherosclerosis.

Ox-LDL can enhance the interaction of mice natural killer cells and dendritic cells via the CD48-2B4 pathway.

The importance of the interaction between natural killer (NK) cells and dendritic cells (DCs) in the expansion of antiviral and antitumor immune responses is well documented; however, limited information on NK/DC interaction during atherosclerosis is available. Inflammation plays an important role in the development of atherosclerosis, and oxidized low-density lipoprotein (ox-LDL) is believed to play a critical role in the development and progression of atherosclerosis. In this study, we developed a NK/DC coculture system to examine the role of ox-LDL in modulating the interaction of mice NK cells and DCs. Fresh NK cells were cocultured with DCs in the absence or presence of ox-LDL. We examined the cytokines released during the interaction. This report provides the first evidence of an enhancement effect by ox-LDL on the NK/DC crosstalk. Notably, we found that ox-LDL significantly promoted the interaction of NK cells and DCs via CD48-2B4 contact-dependent mechanisms. These findings highlight the importance of NK/DCs crosstalk in atherosclerosis and provide new information about the possible mechanisms of atherosclerosis.

Angiotensin-(1-7) enhances angiotensin II induced phosphorylation of ERK1/2 in mouse bone marrow-derived dendritic cells.

It is well known that angiotensin-(1-7) (Ang-(1-7)) counterbalances vasoconstrictive and proliferative functions of angiotensin II (Ang II), some of those actions are via inhibition of Ang II induced activation of mitogen-activated protein kinases(MAPK). This study investigated the effects of Ang-(1-7) on Ang II-mediated cell signaling pathways in mouse bone marrow-derived dendritic cells (DC). The expression of receptor Mas and angiotensin-converting enzyme-related carboxypeptidase (ACE2) mRNA was examined by reverse transcription-polymerase chain reaction (RT-PCR); activation of MAPK was detected by immunoblotting after incubation of dendritic cells with Ang II in the presence or absence of Ang-(1-7), valsartan, PD123319, and D-Ala(7)-Ang-(1-7). Ang II rapidly (5 min, 10(-7) mol/L) stimulated phosphorylation of extracellular signal-related kinase (ERK1/2); this effect was partially inhibited by Ang II type 1 (AT1) receptor antagonist valsartan and significantly attenuated by Ang II type 2 (AT2) receptor antagonist PD123319. Ang-(1-7) alone also induced phosphorylation of ERK1/2; co-treatment of Ang-(1-7) and Ang II markedly enhanced ERK1/2 phosphorylation, the enhancement was eliminated by the Ang-(1-7) receptor antagonist D-Ala(7)-Ang-(1-7). Both Ang-(1-7) and Ang II had no effect on p38 and c-Jun N-terminal kinase (JNK) phosphorylation. In conclusion, Ang II stimulates ERK1/2 phosphorylation via AT2 receptor in mouse DC, Ang-(1-7) enhances this effect. Generation of Ang-(1-7) by DC could thereby counteract on the pro-inflammatory function of locally generated Ang II.

[Pioglitazone inhibits dendritic cell adhesion and transmigration through down-regulation of dendritic cell-specific intercellular adhesion molecule-3 grabbing nonintegrin].

OBJECTIVE: To investigate the effect of pioglitazone (Pio) on dendritic cell-(DC) specific intercellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN) expression in DCs and explore the possible mechanism of Pio inhibiting DC adhesion and transmigration. METHODS: DCs derived from human peripheral blood mononuclear cells were cultured and divided into 6 groups: blank control group, Pio 0.1 micromol/L group, Pio 1.0 micromol/L group, Pio 10 micromol/L group, GW9662, a peroxisome proliferator activated receptor (PPAR)-gamma antagonist, 10 micromol/L group, and GW9662 10 micromol/L + Pio 10 micromol/L group. Western blotting was used to detect the protein expression of DC-SIGN 24 h later. Human umbilical vein endothelial cells (HUVECs) were obtained and co-cultured with the DCs undergoing different treatments. Immunofluorescence test was used to detect the protein expression of DC-SIGN. DCs labeled with 5-chloromethylfluorescein diacetate (CMFDA) were added into the monocellular layer of fused ECs. Blank DCs and DCs pretreated with anti-DC-SIGN antibody were used as blank and experimental groups. Laser confocal microscopy was used to observe the adhesion ability of the DCs. HUVECs were inoculated into the upper chamber of Transwell plate and CMFDA-labeled DCs of above mentioned groups were added to the mono-cellular layer of these ECs. Serum-free culture medium with monocyte chemoattractant protein-1 was added into the lower chamber. Eight hours later the transmigration ability was observed. RESULTS: Western blotting showed that the DC-SIGN protein expression levels of the DCs of the Pio 1.0 micromol/L and Pio 10 micromol/L group were 0.96 +/- 0.09 and 0.80 +/- 0.08 respectively, both significantly lower than that of the blank control group (1.25 +/- 0.23, P < 0.05 and P < 0.01); and the DC-SIGN protein expression level of the GW9662 10 micromol/L + Pio 10 micromol/L group was 1.10 +/- 0.12, significantly higher than that of the Pio 10 micromol/L group (P < 0.05). Immunofluorescence test showed that the DC-SIGN protein expression levels of the DCs of the Pio 1.0 micromol/L and Pio 10 micromol/L group were 22.3 +/- 5.4 and 14.4 +/- 2.3 respectively, both significantly lower than that of the control group (29.5 +/- 5.1, P < 0.05 and P < 0.01), and the DC-SIGN protein expression level of the GW9662 10 micromol/L + Pio 10 micromol/L group was 24.9 +/- 4.3, significantly higher than that of the Pio 10 micromol/L group (P < 0.01), and not significantly different from that of the blank control group (P > 0.05). The adhesion rates of the Pio 1.0 micromol/L and Pio 10 micromol/L groups were 10.8% +/- 2.0% and 7.6% +/- 1.5% respectively, both significantly lower than that of the control group (13.4% +/- 2.1%, P < 0.05 and P < 0.01); and the adhesion rate of the GW9662 + Pio 10 micromol/L group was 12.1% +/- 1.9%, significantly higher than that of the Pio 10 micromol/L group (P < 0.01), and not significantly different from that of the blank control group (P > 0.05). The transmigration inhibition rate of DCs of the Pio 0.1, 1.0, and 10 micromol/L groups were 4.1%, 12.9%, and 17.2% compared with the transmigration rate of the blank control group (P < 0.05, P < 0.05, and P < 0.01). The transmigration rate of the GW9662 + Pio 10 micromol/L group was significantly higher than that of the Pio 10 micromol/L group (P < 0.05), and not significantly different from that of the blank control group (P > 0.05). The transmigration rate of the anti-DC-SIGN intervention group was lower by 17.8% than that of the control group (P < 0.01). CONCLUSION: Pio down-regulates the DC-SIGN protein expression and inhibits DC adhesion and transmigration through the pathway of PPAR-gamma.

Homocysteine at pathophysiological concentrations enhances binding of dendritic cells to endothelial cells mediated by DC-SIGN.

Elevated plasma homocysteine (Hcy) is an independent risk factor for atherosclerosis, which is recognized as inflammatory and immune responses. The purpose of this study was to investigate the effect of Hcy on the interaction between dendritic cells (DCs) and endothelial cells (ECs) by upregulating the expression of DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN) in cultured DCs. The immunophenotype of Hcy-treated DCs was monitored by flow cytometry. Then, they were coincubated with cultured human umbilical vein endothelial cells, and adhesion of DCs to ECs, and migration of DCs through an endothelial monolayer growing on the insert of a transwell plate, were assessed using a confocal microscope and a multi-detection microplate reader. The expression of DC-SIGN on Hcy-stimulated DCs was assessed by Western blot and immunofluorescence staining. The presence of Hcy did not change the phenotype of immature and mature DCs. Hcy promoted adhesion of DCs to ECs and migration in a concentration-dependent fashion. This effect was inhibited by an anti-DC-SIGN monoclonal antibody. The expression of DC-SIGN on DCs was significantly upregulated by Hcy in a concentration-dependent manner. Taken together, our results show for the first time that Hcy can potentiate the adhesion of DCs to ECs and migration by upregulating the expression of DC-SIGN on DCs, suggesting a novel role of Hcy in the pathogenesis of human vascular disease.

[Delayed electrical uncoupling is involved in kappa-opioid receptor activation -induced cardioprotective effect in the isolated rat heart].

AIM: To determine whether activation of kappa-opioid receptor with U50,488H, a selective kappa-opioid receptor agonist, produces any changes in electrical uncoupling during prolonged ischemia and whether these changes in electrical uncoupling is associated with the cardioprotection induced by kappa-opioid receptor activation, and to explore the possible mechanism. METHODS: (1) To observe the effect of U50,488H (10(-7), 10(-6), 3 x10(-6) and 10(-5) mol/L), a selective kappa-opioid receptor agonist, or with a selective kappa-opioid receptor antagonist nor-BNI (5 x 10(-6) mol/L), or with a mitochondrial K(ATP) channel inhibitor 5-HD on myocardium during ischemia/reperfusion in isolated perfused rat heart. Parameters of measurements include hemodynamic data, formazan content, heart rate, coronary flow, and lactate dehydrogenase (LDH). (2) To examine the effect of U50,488H of different concentration on electrical coupling parameters (including onset of uncoupling, plateau time, slope, and fold increase in r1) during 70 min myocardial ischemia in isolated perfused rat heart. RESULTS: (1) Pretreatment with U50,488H concentration dependently increased formazan content and reduced LDH release induced by 30 min of ischemia and 120 min of reperfusion. (2) The onset of electrical uncoupling and plateau time during prolonged ischemia was delayed by kappa-opioid receptor activation with U50,488H. (3) Linear regression analysis shown that the increase in formazan content and decrease in LDH release produced by kappa-opioid receptor activation was associated with delayed electrical uncoupling during prolonged ischemia. (4) The effects of U50,488H on formazan content, LDH release and on electrical coupling were abolished by nor-BNI, or 5-HD. CONCLUSION: This results demonstrate that the onset of electrical uncoupling during prolonged ischemia is delayed by kappa-opioid receptor activation with a selective kappa-opioid receptor agonist U50,488H, and that delayed electrical uncoupling is associated with the cardioprotection induced by kappa-opioid receptor activation with U50,488H. These effects of kappa-opioid receptor activation with U50,488H are mediated by mitochondrial K(ATP) channels.

Delayed uncoupling is related to cardioprotection induced by kappa-agonist U-50,488H in rat heart.

OBJECTIVES: To determine whether the kappa-opioid receptor agonist U50,488H affects electrical uncoupling during prolonged ischemia and, if so, whether the changes are associated with its cardioprotective action. DESIGN: The isolated rat heart was perfused in a Langendorff apparatus. Formazan content, lactate dehydrogenase (LDH) and hemodynamic parameters were measured to confirm the cardioprotective effect of U50,488H. The effects of U50,488H on electrical coupling during prolonged ischemia were also measured. RESULTS: U50,488H concentration-dependently increased formazan content and reduced LDH release, and the ameliorating effect of 10(-5) mol/L U50,488H was abolished by 5 x 10(-6) mol/L nor-binaltorphimine (nor-BNI), a selective kappa-opioid receptor antagonist, or 10(-4) mol/L 5-hydroxydecanoate (5-HD), a selective mitochondrial ATP-sensitive K(+) (K(ATP)) channel blocker. The onset of electrical uncoupling during prolonged ischemia was delayed by U50,488H, and the delay was not only abolished, but also advanced by nor-BNI or 5-HD relative to the control group. CONCLUSIONS: These results demonstrate that delayed uncoupling during prolonged ischemia is associated with the cardioprotection of U50,488H, and these effects of U50,488H are mediated by mitochondrial K(ATP) channels.

Delayed uncoupling contributes to the protective effect of heptanol against ischaemia in the rat isolated heart.

1. It is known that infusion of the gap junction uncoupler heptanol, before ischaemia or during reperfusion, limits myocardial infarct size. However, whether this cardiac effect is linked to the effect of heptanol on communication across gap junctions has not been elucidated. The aims of the present study were to examine the effect of heptanol on infarct size, arrhythmias and myocardial tissue resistance and to assess whether changes in electrical coupling correlate with cardiac protection. 2. Rat isolated, perfused hearts were subjected to a 24 min infusion of heptanol (0.05, 0.1, 0.5 or 1.0 mmol/L) followed by 20 min regional ischaemia and 60 min reperfusion, or by 70 min global no-flow ischaemia. The effective refractory period, action potential and conduction velocity were measured in papillary muscles from the right ventricle. Heptanol markedly decreased arrhythmia scores during ischaemia and reperfusion, as well as reducing infarct size to a degree similar to that induced by ischaemic preconditioning. In the prolonged ischaemia model, heptanol delayed the onset of uncoupling, increased time to plateau and decreased the maximal rate of uncoupling during ischaemia. Ischaemic preconditioning had similar effects on these parameters. In papillary muscle, heptanol reduced the conduction velocity of the action potential in a dose-dependent manner, but had no significant effect on resting potential, action potential amplitude, action potential duration, maximal upstroke of depolarization or effective refractory period. 3. These results demonstrate that treatment with the gap junction uncoupler heptanol confers cardioprotection against ischaemia and this effect is related to delayed electrical uncoupling during prolonged ischaemia.

The Effects of κ-Opioid Receptor Stimulation on Electrical Coupling during Ischemia in the Perfused Isolated Rat Heart.

Two series of experiments were performed in the perfused isolated rat heart to determine whether stimulation of κ-opioid receptor with U50,488H, a selective κ-opioid receptor agonist, produces any changes in electrical coupling during prolonged ischemia and whether these changes in electrical coupling is associated with the cardioprotection induced by U50,488H. It was found that U50,488H concentration dependently increased formazan content and reduced lactate dehydrogenase (LDH) release induced by 30 min of ischemia and 120 min of reperfusion, and the ameliorating effect of 10-5mol/L U50,488H was abolished by 5x10-6mol/L nor-binaltorphimine (nor-BNI), a selective Κ-opioid receptor antagonist, or 10-4mol/L 5-hydroxydecanoate (5-HD), a selective mitochondrial ATP-sensitive K+(KATP) channels blocker. The onset of electrical uncoupling during prolonged ischemia was delayed by U50,488H, and delaying effect was not only abolished, but also advanced by nor-BNI or 5-HD compared with control group. These results demonstrate that delayed electrical uncoupling is associated with the cardioprotection induced by U50,488H. These effects of U50,488H are mediated by mitochondrial KATPchannels.

The Effects of Heptanol on Electrical Coupling during Ischemia in the Perfused Isolated Rat Heart.

The aims of the present study were to examine the effect of heptanol on electrical coupling during ischemia, and to assess whether changes in electrical coupling by heptanol is associated with its cardiac protection. Perfused isolated rat hearts were subjected to a 24 min infusion of heptanol (0.05, 0.1, 0.5 or 1.0 mmol/L) followed by 70 min of global no-flow ischemia or by 20 min of regional ischemia and 60 min of reperfusion. Heptanol markedly decreased arrhythmia scores during ischemia and reperfusion as well as reduced infarct size to a degree similar to that induced by ischemic preconditioning. In the prolonged ischemia model, heptanol delayed the onset of uncoupling, increased time to plateau, and decreased the maximal rate of uncoupling during ischemia. Ischemic preconditioning had similar effects on these parameters. These results demonstrate that treatment with the gap junction uncoupler heptanol confers cardioprotection against ischemia, and this effect is related to delayed electrical uncoupling during prolonged ischemia.