Suppression of Lipopolysaccharide-Induced IL-1ß Gene Expression by High-Molecular-Weight Adiponectin in RAW264.7 Macrophages.

Adiponectin is an abundant adipocytokine secreted by adipocytes. It exists in the plasma in its trimeric, hexameric, high-molecular-weight (HMW), and globular (a proteolytic product) isoforms. Adiponectin's anti-inflammatory effects on macrophages remain controversial. We have previously reported a simple and effective method for purifying native HMW adiponectin from human plasma. Here, we investigated whether native HMW adiponectin from human plasma has anti-inflammatory effects on macrophages. Pretreatment with human native HMW adiponectin inhibited lipopolysaccharide (LPS)-induced interleukin-1ß (IL-1ß) gene expression, but not tumor necrosis factor (TNF)-α expression. However, simultaneous treatment with HMW adiponectin and LPS did not inhibit IL-1ß expression. Further, HMW adiponectin pretreatment decreases glycogen synthase kinase-3ß (GSK-3ß) inactivation by abrogating LPS-induced Akt (Ser473) phosphorylation, which subsequently suppresses LPS-induced CCAAT/enhancer binding protein ß (C/EBPß) protein translation and nuclear translocation. However, HMW adiponectin pretreatment did not affect LPS-induced nuclear factor-kappaB (NF-κB) activation. These results suggest that HMW adiponectin mediates potent anti-inflammatory activities in macrophages by inhibiting its Akt-C/EBPß signaling pathway, thereby suppressing IL-1ß gene expression.

Expression of uterine lipocalin 2 and its receptor during early- to mid-pregnancy period in mares.

From previous cDNA subtraction studies analyzing gene expression in equine endometrium, high lipocalin 2 (LCN2) mRNA expression was found in the gravid endometrium. In the uterus, LCN2 may transport hydrophobic molecules and siderophores with iron, or may form a complex with another protein, however, the expression of uterine LCN2 beyond day 20 of equine pregnancy and its receptor has not been characterized. To study the expression and potential roles of uterine LCN2 from pre-implantation to mid-gestation period, stage-specific endometrial samples were obtained from day 13 (day 0 = ovulation) cyclic and days 13, 19, 25, and 60 to 131 pregnant mares. Expression of LCN2 mRNA increased in day 19 gravid endometrium and was abundant from day 60 onward. The expression of LCN2 mRNA was localized to the glandular epithelium. LCN2 protein was detected in day 25 gravid endometrium and luminal fluid, and the protein was localized to the glandular epithelium and luminal cavity, whereas LCN2 receptor expression was found in luminal and glandular epithelium and trophectoderm throughout the experimental period. The presence of matrix metalloproteinase-9 (MMP9) was also examined because MMP9 is known to form a complex with LCN2. Although MMP9 and LCN2 were both found in luminal fluid from day 25 pregnant uterus, the complex of these proteins was not detected. Localization of the receptor in the trophectoderm suggests that endometrial LCN2 could play a role in carrying small substances from the mother to fetus in the equine species.

Suppressive Effects of Glucose-Dependent Insulinotropic Polypeptide on Cardiac Hypertrophy and Fibrosis in Angiotensin II-Infused Mouse Models.

BACKGROUND: Activation of glucose-dependent insulinotropic polypeptide receptor (GIPR) has been shown to be protective against atherosclerosis. However, effects of GIP on the heart have remained unclear. To address this question, in vitro and in vivo experiments were conducted. METHODS AND RESULTS: In isolated mouse cardiomyocytes, GIPR mRNA was detected by reverse transcription-polymerase chain reaction, and GIP stimulation increased adenosine 3',5'-cyclic monophosphate production. In apolipoprotein E-knockout mice, infusion of angiotensin II (AngII; 2,000 ng·kg(-1)·min(-1)) significantly increased the heart weights, and co-administration of GIP (25 nmol·kg(-1)·day(-1)) reversed this increase (both P<0.01). In the left ventricular walls, GIP suppressed AngII-induced cardiomyocyte hypertrophy by 34%, apoptosis by 77%, and interstitial fibrosis by 79% (all P<0.01). Furthermore, GIP reduced AngII-induced expression of transforming growth factor-ß1 (TGF-ß1) and hypoxia inducible factor-1α. In wild-type mice, cardiac hypertrophy was induced by AngII to a lesser extent, and prevented by GIP. In contrast, GIP did not show any cardioprotective effect against AngII-induced cardiac hypertrophy in GIPR-knockout mice. In an in vitro experiment using mouse cardiomyocytes, GIP suppressed AngII-induced mRNA expression of B-type natriuretic peptide and TGF-ß1. CONCLUSIONS: It was demonstrated that cardiomyocytes represent a direct target of GIP action in vitro, and that GIP ameliorated AngII-induced cardiac hypertrophy via suppression of cardiomyocyte enlargement, apoptosis, and fibrosis in vivo. (Circ J 2016; 80: 1988-1997).

TRPM2 channels in alveolar epithelial cells mediate bleomycin-induced lung inflammation.

Lung inflammation is a major adverse effect of therapy with the antitumor drug bleomycin (BLM). Transient receptor potential melastatin 2 (TRPM2) is a Ca(2+)-permeable channel that is activated by oxidative stress through the production of ADP-ribose. We herein investigated whether TRPM2 channels contributed to BLM-induced lung inflammation. The intratracheal instillation of BLM into wild-type (WT) mice increased the number of polymorphonuclear leukocytes (PMNs) and inflammatory cytokine levels in the lung. Increases in inflammatory markers in WT mice were markedly reduced in trpm2 knockout (KO) mice, which demonstrated that the activation of TRPM2 channels was involved in BLM-induced lung inflammation. The expression of TRPM2 mRNA was observed in alveolar macrophages, alveolar epithelial cells, and lung fibroblasts. Actually, TRPM2 protein was expressed in lung tissues. Of these, TRPM2 channels in epithelial cells were activated by the addition of H2O2 following a BLM pretreatment, resulting in the secretion of macrophage inflammatory protein-2 (MIP-2). The H2O2-induced activation of TRPM2 by the BLM pretreatment was blocked by the poly(ADP-ribose) polymerase (PARP) inhibitors PJ34 and 3-aminobenzamide. The accumulation of poly(ADP-ribose) in the nucleus, a marker for ADP-ribose production, was strongly induced by H2O2 following the BLM pretreatment. Furthermore, administration of PRAP inhibitors into WT mice markedly reduced recruitment of inflammatory cells and MIP-2 secretion induced by BLM instillation. These results suggest that the induction of MIP-2 secretion through the activation of TRPM2 channels in alveolar epithelial cells is an important mechanism in BLM-induced lung inflammation, and the TRPM2 activation is likely to be mediated by ADP-ribose production via PARP pathway. TRPM2 channels may be new therapeutic target for BLM-induced lung inflammation.

Potentiated macrophage activation by acid sensing under low adiponectin levels.

Adiponectin can protect against inflammation; one of the mechanisms involves direct, inhibition of macrophages (MΦ). We postulated that adiponectin anti-sense transgenic (AsTg) mice raised in our laboratory are prone to inflammation because of systemic low adiponectin levels. The writhing response to acetic acid was utilized as an in vivo inflammatory model, and using Ca(2)(+), response to the acid was exploited in vitro to evaluate the function of resident peritoneal MΦ. The in vivo response to the acid was increased and the Ca(2)(+) response of MΦ was enhanced in AsTg mice, compared with those in wild type (WT) mice. In parallel with these enhanced responses, MΦ from AsTg mice augmented TNF-α and IL-6 mRNA expression. We further analyzed the enhancement in activity of MΦ from AsTg mice by acid sensing using specific inhibitors, amiloride for acid-sensing ion channels (ASICs) and KB-R7943 for Na(+)/Ca(2)(+) exchangers (NCXs). Our results indicated that in AsTg mice, the Ca(2)(+) response to the acid was facilitated in MΦ by a low threshold of ASIC1 and NCX1 molecules and the activity of these channel was possibly regulated by adiponectin.

A novel population of subepithelial platelet-derived growth factor receptor α-positive cells in the mouse and human colon.

Recently platelet-derived growth factor-α-positive cells (PDGFRα(+) cells), previously called "fibroblast-like" cells, have been described in the muscle layers of the gastrointestinal tract. These cells form networks and are involved in purinergic motor neurotransduction. Examination of colon from mice with enhanced green fluorescent protein (eGFP) driven from the endogenous Pdgfra (PDGFRα-eGFP mice) revealed a unique population of PDGFRα(+) cells in the mucosal layer of colon. We investigated the phenotype and potential role of these cells, which have not been characterized previously. Expression of PDGFRα and several additional proteins was surveyed in human and murine colonic mucosae by immunolabeling; PDGFRα(+) cells in colonic mucosa were isolated from PDGFRα-eGFP mice, and the gene expression profile was analyzed by quantitative polymerase chain reaction. We found for the first time that PDGFRα was expressed in subepithelial cells (subepithelial PDGFRα(+) cells) forming a pericryptal sheath from the base to the tip of crypts. These cells were in close proximity to the basolateral surface of epithelial cells and distinct from subepithelial myofibroblasts, which were identified by expression of α-smooth muscle actin and smooth muscle myosin. PDGFRα(+) cells also lay in close proximity to varicose processes of nerve fibers. Mouse subepithelial PDGFRα(+) cells expressed Toll-like receptor genes, purinergic receptor genes, 5-hydroxytryptamine (5-HT) 4 receptor gene, and hedgehog signaling genes. Subepithelial PDGFRα(+) cells occupy an important niche in the lamina propria and may function in transduction of sensory and immune signals and in the maintenance of mucosal homeostasis.

Expression of endometrial immune-related genes possibly functioning during early pregnancy in the mare.

Despite enormous efforts, biochemical and molecular mechanisms associated with equine reproduction, particularly processes of pregnancy establishment, have not been well characterized. Previously, PCR-selected suppression subtraction hybridization analysis was executed to identify unique molecules functioning in the equine endometrium during periods of pregnancy establishment, and granzyme B (GZMB) cDNA was found in the pregnant endometrial cDNA library. Because GZMB is produced from natural killer (NK) cells, endometrial expression of GZMB and immune-related transcripts were characterized in this study. The level of GZMB mRNA is higher in the pregnant endometrium than in non-pregnant ones. This expression was also confirmed through Western blot and immunohistochemical analyses. IL-2 mRNA declined as pregnancy progressed, while IL-15, IFNG and TGFB1 transcripts increased on day 19 and/or 25. Analyses of IL-4 and IL-12 mRNAs demonstrated the increase in these transcripts as pregnancy progressed. Increase in CCR5 and CCR4 mRNAs indicated that both Th1 and Th2 cells coexisted in the day 25 pregnant endometrium. Taken together, the endometrial expression of immune-related transcripts suggests that immunological responses are present even before the trophectoderm actually attaches to the uterine epithelial cells.

Effect of adiponectin on cardiac allograft vasculopathy.

BACKGROUND: The role of adiponectin (APN), an adipose tissue-specific secretory protein, on chronic rejection after cardiac transplantation in APN-sense transgenic mice (APN-SE) was evaluated. METHODS AND RESULTS: Heterotopic cardiac transplantation in major histocompatibility complex class II-mismatched mice was performed. B6.C-H-2(bm12)KhEg (Bm12) hearts were transplanted into APN-SE, and allografts were harvested at 8 weeks after transplantation. Quantitative polymerase chain reaction (PCR) and immunohistochemical staining showed that the expression of both AdipoR1 and AdipoR2 was induced in APN-SE recipients. Neointimal hyperplasia was significantly decreased in allografts transplanted into APN-SE (luminal occlusion, 8.9 ± 2.2%) compared to those transplanted into controls (49.4 ± 10.5%; P=0.011). APN-SE showed significantly reduced mRNA levels of interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-2, IL-6, and monocyte chemoattractant protein-1 (MCP-1) by quantitative PCR. Western blot analysis revealed that the protein levels of IFN-γ and MCP-1 were reduced in APN-SE recipients. Proliferation of smooth muscle cells stimulated with activated T cells was suppressed by APN addition, and this effect was canceled by treatment with an adenosine monophosphate-activated protein kinase (AMPK) inhibitor. CONCLUSIONS: APN plays a critical role in the attenuation of chronic rejection by suppressing inflammatory cytokine and chemokine expression and enhancing APN receptor expression. APN plays a beneficial role in reducing the progression of cardiac allograft vasculopathy through the AMPK pathway.

Adiponectin protects against doxorubicin-induced cardiomyopathy by anti-apoptotic effects through AMPK up-regulation.

AIMS: Adiponectin (APN) has been reported to protect against ischaemia-reperfusion injury and hypertrophy. However, few reports have investigated the cardioprotective effects of APN in doxorubicin (DOX)-induced cardiomyopathy; therefore, we studied the cardioprotective mechanisms of APN in this model. METHODS AND RESULTS: In an in vivo study, we quantified the cardiac pathohistology of C57BL/6 mice [wild-type (WT) mice], APN transgenic mice with high APN concentrations [APN transgenic sense (SE) mice], and those with reduced APN concentrations [APN transgenic antisense (AS) mice] after intraperitoneal injections of DOX (4 mg/kg) weekly for 6 weeks. The survival rate after 14 days was significantly increased in APN-SE mice (WT vs. APN-AS vs. APN-SE: 40 vs. 17 vs. 73%, P < 0.05). We assessed myocardial pathohistological changes and observed that fibrosis and apoptosis were significantly decreased in APN-SE mice compared with those of the other groups. We also assessed DOX-induced apoptotic mechanisms in vitro using cultured cardiomyocytes isolated from neonatal WT mice. The expression of adenosine monophosphate-activated protein kinase (AMPK) and anti-apoptotic factor Bcl-2 increased, but that of pro-apoptotic factor Bax decreased in cardiomyocytes treated with highly concentrated APN. The protective effects of APN were reversed by the addition of an AMPK inhibitor (dorsomorphin) to the culture medium. CONCLUSION: These data suggest that APN improved cardiac function through anti-apoptotic effects by up-regulation of AMPK in DOX-induced cardiomyopathy in mice.

Production of calcium maintenance factor Stanniocalcin-1 (STC1) by the equine endometrium during the early pregnant period.

A factor responsible for progression to pregnancy establishment in the mare has not been definitively characterized. To identify factors possibly involved in the establishment of equine pregnancy, the endometrium was collected from day 13 (day 0=day of ovulation) cyclic and day 13, 19 and 25 pregnant animals. From initial subtractive hybridization studies, a calcium regulating factor, Stanniocalcin-1 (STC1) mRNA, was found as a candidate molecule expressed uniquely in the pregnant endometrium. Endometrial expression of STC1 mRNA was noted on day 19 and was markedly increased in the day 25 gravid endometrium. STC1 protein was found in the extracts of day 25 gravid endometrium and immunochemically localized in the uterine glands. In addition, STC1 protein was detected in uterine flushing media collected from day 25 pregnant mares. High concentrations of estradiol-17 ß (E(2)) were detected in day 25 conceptuses. E(2) levels were much higher in the gravid endometrium than in other regions, whereas progesterone levels did not differ among the samples from different endometrial regions. Expression of STC1 mRNA, however, was not significantly upregulated in cultured endometrial explants treated with various concentrations of E(2) (0.01-100 ng/ml) with or without 10 ng/ml progesterone. These results indicate that an increase in STC1 expression appears to coincide with capsule disappearance in the conceptus, and suggest that STC1 from the uterine glands likely plays a role in conceptus development during the pregnancy establishment period in the mare.

Analytical evaluation of a high-molecular-weight (HMW) adiponectin chemiluminescent enzyme immunoassay.

BACKGROUND: The measurement of high-molecular-weight (HMW) adiponectin concentration provides valuable clinical information. However, the conventional ELISA method requires complicated and lengthy assay procedures to obtain assay results. METHODS: We prepared new assay reagents based on chemiluminescent enzyme immunoassay (CLEIA) on a fully-automated analyzer system using the same IH7 monoclonal antibody as for ELISA as solid phase and detection antibodies (CLEIA/cartridge-type and CLEIA/bottle-type). RESULTS: The assay range of both CLEIA reagents were from 0.20 to 15.00 µg/ml, and lower limit of detection and quantification were lower than 0.0928 and 0.1346 µg/ml in CLEIA/cartridge-type and in CLEIA/bottle-type reagents, respectively. A good correlation was observed between both reagents (y = 1.000x + 0.120). The imprecision test as % of coefficient variation in both reagents were less than 3.3% and recovery test showed the range from 100% to 109%. No or little interference of blood components was observed in both reagents. HMW adiponectin concentration measured by CLEIA reagents was approximately half that measured by the previous ELISA because of reevaluation using freshly and highly purified HMW adiponectin standard. CONCLUSION: The newly prepared CLEIA reagents are robust and adequate and can be used for the measurement of HMW adiponectin in the clinical laboratory.

Adiponectin does not bind to gelatin: a new and easy way to purify high-molecular-weight adiponectin from human plasma.

Human plasma contains three forms of adiponectin, a trimer, a hexamer, and a high-molecular-weight (HMW) multimer. We previously reported HMW adiponectin was a gelatin-binding protein of 28 kDa (GBP28), it having been purified due to its affinity to gelatin-Cellulofine (Nakano, Y., et al. Isolation and characterization of GBP28, a novel gelatin-binding protein purified from human plasma. J. Biochem. 1996. 120: 803-12). Although HMW adiponectin binds to gelatin-Cellulofine, it cannot bind to gelatin-Sepharose. Gelatin-Cellulofine was made of formyl-Cellulofine and gelatin, and we found that HMW adiponectin binds to reduced formyl-Cellulofine with similar affinity as to gelatin-Cellulofine. Through only two steps using reduced formyl-Cellulofine and DEAE-Sepharose, HMW adiponectin can be effectively purified from human plasma.

Association studies of the IL-23R gene in autoimmune thyroid disease in the Japanese population.

Autoimmune thyroid diseases (AITDs), including Graves' disease (GD) and Hashimoto's thyroiditis (HT), are caused by interplays of genetic factors and environmental triggers. Interleukin-23 and its receptor (IL-23R) guide T cells towards the Th17 phenotype. IL-23R single nucleotide polymorphisms (SNPs) have been shown to be associated with several autoimmune diseases, including Crohn's disease and rheumatoid arthritis, and Graves' ophthalmopathy (GO) in Caucasians. To determine whether variants in the IL-23R gene are associated with AITDs in Japanese, 464 Japanese AITD patients (290 with GD, 174 with HT) and 179 matched Japanese control subjects were genotyped for four SNPs spanning the IL-23R gene. SNPs rs11209026 and rs7530511 were genotyped using TaqMan allelic discrimination assays and SNPs rs2201841 and rs10889677 were genotyped using a fluorescent-based restriction fragment length polymorphism method. Case-control association studies were performed using the chi(2) and Fisher's exact tests with Yates correction. Of the four SNPs rs11209026 was non-polymorphic in our dataset. The other three SNPs were not associated with GD or GO or HT in our Japanese population. These results suggest that the IL-23R gene is associated with AITDs only in a specific ethnic group.

Prg1 is regulated by the basic helix-loop-helix transcription factor Math2.

Math2 (NEX-1/NeuroD6) is a member of the basic helix-loop-helix transcription factor family and is involved in neuronal differentiation and maturation. In this study, we identified the genes targeted by Math2 using DNA microarrays and cultured rat cortical cells transfected with Math2. Of the genes regulated by Math2, we focused on plasticity-related gene 1 (Prg1). Prg1 expression induced by Math2 was confirmed in cultured rat cortical cells and PC12 cells analyzed by real-time quantitative PCR. In the promoter region of rat Prg1, we identified four E-boxes [designated -E1 to -E4 (CANNTG)] recognized by the basic helix-loop-helix transcription factor. Using chromatin immunoprecipitation assays, we found that Math2 directly bound to at least one of these E-boxes. The Prg1 reporter assay showed that -E1 was critical for the regulation of Math2-mediated Prg1 expression. Investigation of the functional roles of Math2 and Prg1 in PC12 cells revealed that 72 h after transfection with either Math2 or Prg1, neurite length and number were significantly induced. Co-transfection with Prg1-siRNA completely inhibited Math2-mediated morphological changes. Our results suggest that Math2 directly regulates Prg1 expression and that the Math2-Prg1 cascade plays an important role in neurite outgrowth in PC12 cells.

Use of sample hematocrit value to correct blood tacrolimus concentration derived by microparticle enzyme immunoassay.

The quality of microparticle enzyme immunoassay (MEIA) for blood tacrolimus is guaranteed in samples with hematocrit (Ht) values of 25 to 45%. Because MEIA provides inaccurate blood tacrolimus concentrations in samples with Ht out of this range (i.e. <25% or >45%), correction of the calibration is required for therapeutic drug monitoring. The authors demonstrated previously that overestimated MEIA tacrolimus concentration could be corrected by modified, calibrated MEIA (cMEIA) using the original calibrator. Here, an equation was established to more easily derive a corrected tacrolimus concentration by calculation (MEIAcalc) using the Ht of each sample. The tacrolimus concentrations of 99 whole-blood samples with low Ht (<25%) were then tested by the 3 assay methods: MEIA, cMEIA, and MEIAcalc. MEIA gave a significantly higher blood concentration of tacrolimus (median 12.9 ng/ml, range 3.6-26.4 ng/ml) than did cMEIA (median 10.0 ng/ml, range 0.2-21.1 ng/ml, p<0.05). This overestimation was eliminated by using MEIAcalc. There was no difference in blood tacrolimus concentration between cMEIA and MEIAcalc (median 10.0 ng/ml, range 1.7-21.4 ng/ml). MEIAcalc can be used to correct the tacrolimus concentration in samples obtained from patients with unstable Ht values.

Adiponectin induces NF-kappaB activation that leads to suppression of cytokine-induced NF-kappaB activation in vascular endothelial cells: globular adiponectin vs. high molecular weight adiponectin.

Adiponectin circulates in plasma as various isoforms. However, the biological activity of each isoform has not been firmly established. High molecular weight (HMW) adiponectin may be the active form of adiponectin, while a proteolytic cleavage product of adiponectin, known as globular adiponectin (gAd), has recently been shown to activate vascular endothelial cells. We compared HMW adiponectin with gAd to investigate whether they could activate nuclear factor kappa B (NF-kappaB) and suppress cytokine-induced NF-kappaB activation in vascular endothelial cells. HMW adiponectin was found to activate NF-kB modestly compared to the activation observed with gAd. HMW adiponectin requires a shorter incubation period to demonstrate inhibition against tumour necrosis factor alpha (TNFalpha)-induced NF-kappaB activation, compared with gAd. gAd strongly activates NF-kappaB, thereby inducing the expression of various pro-inflammatory and adhesion molecule genes, and requires a longer incubation period to show inhibition against cytokine-induced NF-kappaB activation. Thus, HMW adiponectin might function to protect against inflammatory stimuli, while cleavage of adiponectin at inflammatory sites might enhance the inflammatory process.

High molecular weight adiponectin activates AMPK and suppresses cytokine-induced NF-kappaB activation in vascular endothelial cells.

Various isoforms of adiponectin circulate in the plasma. We purified high molecular weight (HMW) adiponectin from human plasma. HMW adiponectin was observed to activate AMP-activated protein kinase (AMPK), thereby increasing the phosphorylation of eNOS and NO production in endothelial cells. On the other hand, cells preincubated with HMW adiponectin had reduced TNFalpha-induced NF-kappaB activation. HMW adiponectin by itself was found to modestly activate NF-kappaB, which was significantly enhanced by inhibition of AMPK/eNOS activation. Thus, HMW adiponectin might have dual action, both pro and anti-inflammatory. An initial period of NF-kappaB activation by HMW adiponectin might be proinflammatory, but it could be counteracted by activation of AMPK/eNOS, which lead to a potential reduction in a second activation of NF-kappaB against inflammatory stimuli.

Inhibition by adiponectin of IL-8 production by human macrophages upon coculturing with late apoptotic cells.

Adiponectin, an adipocyte-derived hormone, reportedly suppresses the production of TNF-alpha and IL-6 by LPS-stimulated human or porcine macrophages, and the phagocytosis of microbeads by human macrophages. In this study, we used a high molecular weight form of adiponectin purified from human plasma to examine its effects on the phagocytosis of late apoptotic cells by human macrophages and the subsequent IL-8 production. Adiponectin suppressed both the phagocytosis of apoptotic cells and the IL-8 production. In contrast, adiponectin augmented both the phagocytosis of apoptotic cells and the IL-8 production in the presence of LPS. These results suggest that adiponectin is not an anti-inflammatory hormone but rather a dual modulator of innate responses.