Feng-Ming Ho

Signal transduction for inhibition of inducible nitric oxide synthase and cyclooxygenase-2 induction by capsaicin and related analogs in macrophages

Although capsaicin analogs might be a potential strategy to manipulate inflammation, the mechanism is still unclear. In this study, the effects and action mechanisms of vanilloid analogs on iNOS and COX‐2 expression were investigated in RAW264.7 macrophages. Capsaicin and resiniferatoxin (RTX) can inhibit LPS‐ and IFN‐γ‐mediated NO production, and iNOS protein and mRNA expression with similar IC50 values of around 10 μM. Capsaicin also transcriptionally inhibited LPS‐ and PMA‐induced COX‐2 expression and PGE2 production. However, this effect exhibited a higher potency (IC50: 0.2 μM), and RTX failed to elicit such responses at 10 μM. Interestingly, we found that capsazepine, a competitive TRPV1 antagonist, did not prevent the inhibition elicited by capsaicin or RTX. Nevertheless, it mimicked vanilloids in inhibiting iNOS/NO and COX‐2/PGE2 induction with an IC50 value of 3 μM. RT–PCR and immunoblotting analysis excluded the expression of TRPV1 in RAW264.7 macrophages. The DNA binding assay demonstrated the abilities of vanilloids to inhibit LPS‐elicited NF‐κB and AP‐1 activation and IFN‐γ‐elicited STAT1 activation. The reporter assay of AP‐1 activity also supported this action. The kinase assay indicated that ERK, JNK, and IKK activation by LPS were inhibited by vanilloids. In conclusion, vanilloids can modulate the expression of inflammatory iNOS and COX‐2 genes in macrophages through interference with upstream signalling events of LPS and IFN‐γ. These findings provide new insights into the potential benefits of the active ingredient in hot chilli peppers in inflammatory conditions.

Soluble decoy receptor 3 induces angiogenesis by neutralization of TL1A, a cytokine belonging to tumor necrosis factor superfamily and exhibiting angiostatic action

TL1A is a member of the tumor necrosis factor superfamily and plays an important role in regulating endothelial cell apoptosis. A previous study shows TL1A is able to interact with death receptor 3 and decoy receptor 3 (DcR3). Here, we demonstrate that DcR3 is able to induce angiogenesis in human umbilical vein endothelial cells (HUVECs). DcR3 promotes HUVEC proliferation and migration and up-regulates matrix metalloproteinase-2 mRNA expression and enzyme activity. Furthermore, DcR3 enhances EC differentiation into cord vascular-like structures in vitro, as well as neovascularization in vivo. The effects of DcR3 on HUVECs are also mimicked by anti-TL1A and antideath receptor 3 antibodies. In contrast, human aortic endothelial cells, which do not express TL1A, are not responsive to DcR3 treatment, including cell proliferation, migration, and angiogenic differentiation. These data demonstrate DcR3 might not only help tumor cells to escape immune surveillance but also induce angiogenesis by blocking TL1A action in endothelial cells. The pathological role of DcR3 in promoting cancer progress raises the possibility to target DcR3 for antiangiogenic therapy in the future.

The anti-inflammatory carbazole, LCY-2-CHO, inhibits lipopolysaccharide-induced inflammatory mediator expression through inhibition of the p38 mitogen-activated protein kinase signaling pathway in macrophages

The present study was undertaken to investigate the anti‐inflammatory effects of a synthetic compound, LCY‐2‐CHO, on the expression of inducible nitric oxide synthase (iNOS), COX‐2, and TNF‐α in murine RAW264.7 macrophages. Within 1–30 μM, LCY‐2‐CHO concentration‐dependently inhibited lipopolysaccharide (LPS)‐induced nitric oxide (NO), prostaglandin E2 (PGE2), and tumor necrosis factor‐α (TNF‐α) formation, with IC50 values of 2.3, 1, and 0.8 μM, respectively. Accompanying inhibition of LPS‐induced iNOS, cyclooxygenase‐2 (COX‐2), and pro‐TNF‐α proteins was observed. Reverse transcription‐polymerase chain reaction (RT–PCR) and promoter analyses indicated that iNOS expression was inhibited at the transcriptional level (IC50=2.3 μM), that inhibition of COX‐2 expression only partially depended on gene transcription (IC50=7.6 μM), and that TNF‐α transcription was unaffected. Transcriptional assays revealed that activation of AP‐1, but not NF‐κB, was concomitantly blocked by LCY‐2‐CHO. Our results showed that LCY‐2‐CHO was capable of interfering with post‐transcriptional regulation, altering the stability of COX‐2 and TNF‐α mRNAs. Since the 3′‐untranslated region (3′ UTR) of both COX‐2 and TNF‐α mRNA contains a p38 mitogen‐activated protein kinase (MAPK)‐regulated element involved in mRNA stability, we assessed the effect of LCY‐2‐CHO on p38 MAPK. Our data clearly indicated an inhibition (IC50=1.7 μM) of LPS‐mediated p38 MAPK activity, but not of extracellular signal‐regulated kinase (ERK) or c‐Jun N‐terminal kinase (JNK) activity. However, kinase assays ruled out a direct inhibition of p38 MAPK action. The selective p38 MAPK inhibitor, SB203580, inhibited the promoter activities of iNOS and COX‐2 rather than that of TNF‐α. In conclusion, LCY‐2‐CHO downregulates inflammatory iNOS, COX‐2, and TNF‐α gene expression in macrophages through interfering with p38 MAPK and AP‐1 activation.

Inhibition of lipopolysaccharide‐induced inducible nitric oxide synthase and cyclooxygenase‐2 gene expression by 5‐aminoimidazole‐4‐carboxamide riboside is independent of AMP‐activated protein kinase

Recent studies suggest AMP‐activated protein kinase (AMPK), an enzyme involved in energy homeostasis, might be a novel signaling pathway in regulating inflammatory response, but the precise intracellular mechanisms are not fully understood. In this study, we have demonstrated that 5‐aminoimidazole‐4‐carboxamide riboside (AICAR), an activator of AMPK, inhibited lipopolysaccharide (LPS)‐induced protein expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase‐2 (COX‐2) in macrophages and microglial cells at the gene transcription level. Data obtained from electrophoretic mobility shift assay (EMSA) and promoter activity assay have further confirmed the ability of AICAR to block LPS‐mediated NF‐κB, AP‐1, CREB, and C/EBPβ activation. However, AICAR did not affect LPS‐mediated IKK, ERK, and p38 activation. Regardless of the ability of AICAR to activate AMPK, the inhibitory effects of AICAR on iNOS and COX‐2 expression were not associated with AMPK. An adenosine kinase inhibitor 5′‐iodotubercidin, which effectively abolished AMPK activation caused by AICAR, did not reverse the anti‐inflammatory effect of AICAR. Moreover, another AMPK activator metformin was not able to mimic the effects of AICAR. Direct addition of AICAR in EMSA assay interrupted binding of NF‐κB, CREB, and C/EBPβ to specific DNA elements. Taken together, this study demonstrates that the anti‐inflammatory effects of AICAR against LPS‐induced iNOS and COX‐2 gene transcription are not associated with AMPK activation, but might be resulting from the direct interference with DNA binding to transcription factors. J. Cell. Biochem. 103: 931–940, 2008.

Decoy Receptor 3 Increases Monocyte Adhesion to Endothelial Cells via NF-κB-Dependent Up-Regulation of Intercellular Adhesion Molecule-1, VCAM-1, and IL-8 Expression

Decoy receptor 3 (DcR3), a soluble receptor for FasL, LIGHT and TL1A, is highly expressed in cancer cells. We show that pretreatment of HUVECs with DcR3 enhances the adhesion of THP-1 and U937 cells and primary monocytes. A similar stimulatory effect of DcR3 on THP-1 adhesion was also observed in human microvascular endothelial cells (HMVECs). Flow cytometry and ELISA showed that DcR3-treated HUVECs exhibited significant increases in ICAM-1 and VCAM-1 expression. We also demonstrate the ability of DcR3 to stimulate the secretion of IL-8 by HUVECs. RT-PCR and reporter assays revealed that the expression of adhesion molecules and IL-8 are regulated at the level of gene transcription. Experiments with pyrrolidine dithiocarbamate indicated the involvement of an NF-κB signaling pathway. DcR3 was found to induce IκB kinase activation, IκB degradation, p65 nuclear translocation, and NF-κB DNA-binding activity. The enhancement by DcR3 of cell adhesion to HUVECs was not mimicked by the TL1A-Ab, which has been shown in our previous work to be a neutralizing Ab against TL1A, thereby inducing HUVECs angiogenesis. Moreover, DcR3-induced cell adhesion could be detected in human aortic endothelial cells (ECs) in which TL1A expression is lacking. Together, our data demonstrate that DcR3 increases monocyte adhesion to ECs via NF-κB activation, leading to the transcriptional up-regulation of adhesion molecules and IL-8 in ECs. This novel action appears not to be due to TL1A neutralization, but occurs through an as yet undefined target(s). This study implicates DcR3 in the relationship between inflammation and cancer development.

The antioxidant effects of quercetin metabolites on the prevention of high glucose-induced apoptosis of human umbilical vein endothelial cells.

Diabetes mellitus is an important risk factor for CVD. A previous study showed that high glucose induced the apoptosis of human umbilical vein endothelial cells (HUVEC) via the sequential activation of reactive oxygen species, Jun N-terminal kinase (JNK) and caspase-3. The apoptosis cascade could be blocked by ascorbic acid at the micromolar concentration (100 microm). In addition to ascorbic acid, quercetin, the most abundant dietary flavonol, has been recently actively studied in vascular protection effects due to its antioxidant effect at low micromolar concentrations (10-50 microm). Quercetin sulfate/glucuronide, the metabolite of quercetin in blood, however, has been rarely evaluated. In the present study, we investigated the effect of quercetin sulfate/glucuronide on the prevention of high glucose-induced apoptosis of HUVEC. HUVEC were treated with media containing high glucose (33 mm) in the presence or absence of ascorbic acid (100 microm) or quercetin sulfate/glucuronide (100 nm, 300 nm and 1 microm). For the detection of apoptosis, a cell death detection ELISA assay was used. The level of intracellular H2O2 was measured by flow cytometry. JNK and caspase-3 were evaluated by a kinase activity assay and Western blot analysis. The results showed that high glucose-induced apoptosis was inhibited by quercetin sulfate/glucuronide in a dose-dependent manner. The effect of quercetin sulfate/glucuronide on H2O2 quenching, inhibition of JNK and caspase-3 activity at the nanomolar concentration (300 nm) was similar to that of ascorbic acid at the micromolar concentration (100 microm). The findings of the present study may shed light on the pharmacological application of quercetin in CVD.

Proteasome inhibitors up-regulate haem oxygenase-1 gene expression: requirement of p38 MAPK (mitogen-activated protein kinase) activation but not of NF-kappaB (nuclear factor kappaB) inhibition

Regulation of intracellular protein stability by the ubiquitin-dependent proteasome system plays a crucial role in cell function. HO-1 (haem oxygenase) is a stress response protein, which confers cytoprotection against oxidative injury and provides a vital function in maintaining tissue homoeostasis. In the present study, we found a novel action of proteasome inhibitors MG132 and MG262 on HO-1 induction, and characterized the underlying mechanisms. MG132 (> or =0.1 microM) treatment resulted in a marked time- and concentration-dependent induction of the steady-state level of HO-1 mRNA in RAW264.7 macrophages, followed by a corresponding increase in HO-1 protein. Actinomycin D and cycloheximide inhibited MG132-responsive HO-1 protein expression, indicating a requirement for transcription and de novo protein synthesis. The involvement of signal pathways in MG132-induced HO-1 gene expression was examined using chemical inhibitors. Antioxidant N -acetylcysteine and SB203580, an antioxidant and inhibitor of p38 MAPK (mitogen-activated protein kinase), abolished MG132-inducible HO-1 expression. Furthermore, MG132 activated the p38 MAPK pathway. The half-life of HO-1 protein was prolonged by MG132, indicating that the upregulation of HO-1 by proteasome inhibitor is partially attributable to the inhibition of protein degradation. MG132 can ablate IkappaBalpha degradation and NF-kappaB (nuclear factor kappaB) activation induced by lipopolysaccharide, similar to the effect of another NF-kappaB inhibitor pyrrolidine dithiocarbamate. We found HO-1 upregulation by MG132 and pyrrolidine dithiocarbamate is unrelated to their inhibition of NF-kappaB, since leptomycin B, another NF-kappaB inhibitor, did not elicit similar induction of HO-1. Taken together, we found a novel effect of proteasome inhibitor on induction of HO-1 expression. This action is ascribed to the activation of the p38 MAPK pathway, but is not dependent on NF-kappaB inhibition.

Hemocompatible Control of Sulfobetaine-Grafted Polypropylene Fibrous Membranes in Human Whole Blood via Plasma-Induced Surface Zwitterionization

In this work, the hemocompatibility of zwitterionic polypropylene (PP) fibrous membranes with varying grafting coverage of poly(sulfobetaine methacrylate) (PSBMA) via plasma-induced surface polymerization was studied. Charge neutrality of PSBMA-grafted layers on PP membrane surfaces was controlled by the low-pressure and atmospheric plasma treatment in this study. The effects of grafting composition, surface hydrophilicity, and hydration capability on blood compatibility of the membranes were determined. Protein adsorption onto the different PSBMA-grafted PP membranes from human fibrinogen solutions was measured by enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies. Blood platelet adhesion and plasma clotting time measurements from a recalcified platelet-rich plasma solution were used to determine if platelet activation depends on the charge bias of the grafted PSBMA layer. The charge bias of PSBMA layer deviated from the electrical balance of positively and negatively charged moieties can be well-controlled via atmospheric plasma-induced interfacial zwitterionization and was further tested with human whole blood. The optimized PSBMA surface graft layer in overall charge neutrality has a high hydration capability and keeps its original blood-inert property of antifouling, anticoagulant, and antithrmbogenic activities when it comes into contact with human blood. This work suggests that the hemocompatible nature of grafted PSBMA polymers by controlling grafting quality via atmospheric plasma treatment gives a great potential in the surface zwitterionization of hydrophobic membranes for use in human whole blood.

Oregonin inhibits lipopolysaccharide-induced iNOS gene transcription and upregulates HO-1 expression in macrophages and microglia

Oregonin isolated from Alnus formosana is a diarylheptanoid derivative, which appears to have antioxidative and anti‐inflammatory activities. In this study, our data demonstrated inhibitory actions of oregonin on the LPS‐induced iNOS protein in RAW264.7 macrophages and BV‐2 microglial cells. We also suggested that HO‐1 induction by oregonin might contribute to this action. Oregonin is able to dose‐dependently reduce NO production, iNOS protein and iNOS promoter activity stimulated by LPS in RAW264.7 and BV‐2 cells. Oregonin also showed inhibition of LPS‐mediated NF‐κB promoter activity and DNA‐binding ability, as well as p65 nuclear translocation and phosphorylation. However, oregonin had no effect on IKK activity. AP‐1 promoter activity and p38 MAPK activation but not PKC, ERK and JNK activation induced by LPS were attenuated by oregonin. Accompanying with iNOS protein reduction, moreover, we found that oregonin was able to induce HO‐1 protein level. Results using a CO donor, [Ru(CO)3Cl2]2 further showed the ability of CO in reduction of iNOS protein level induced by LPS through the blockade of NF‐κB and AP‐1. Taken together, these results provide new evidences into the anti‐inflammatory actions of oregonin, which include the inhibition of iNOS gene transcription via suppressing transcriptional activity of NF‐κB and AP‐1, as well as the upregulation of anti‐inflammatory molecule HO‐1. The HO‐1‐derived CO may also be involved in the suppressive effect on iNOS gene regulation.

Signaling pathways of LIGHT induced macrophage migration and vascular smooth muscle cell proliferation.

The biological actions of LIGHT, a member of the tumor necrosis factor superfamily, are mediated by the interaction with lymphotoxin‐β receptor (LTβR) and/or herpes virus entry mediator (HVEM). Previous study demonstrated high‐level expressions of LIGHT and HVEM receptors in atherosclerotic plaques. To investigate the role of LIGHT in the functioning of macrophages and vascular smooth muscle cells (VSMC) in relation to atherogenesis, we determined the effects of LIGHT on macrophage migration and VSMC proliferation. We found LIGHT through HVEM activation can induce both events. LIGHT‐induced macrophage migration was associated with activation of signaling kinases, including MAPKs, PI3K/Akt, NF‐κB, Src members, and FAK. Proliferation of VSMC was also shown relating to the activation of MAPKs, PI3K/Akt, and NF‐κB, which consequently led to alter the expression of cell cycle regulatory molecules. Down‐regulation of p21, p27, and p53, and inversely up‐regulation of cyclin D and RB hyper‐phosphorylation were demonstrated. In conclusion, LIGHT acts as a novel mediator for macrophage migration and VSMC proliferation, suggesting its involvement in the atherogenesis. J. Cell. Physiol. 209: 735–743, 2006.