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Background This clinical study investigates the role of 5-methoxytryptophan (5-MTP) in pediatric systemic lupus erythematosus (SLE), with a particular interest in lupus nephritis (LN). Patients and methods One hundred ten children with SLE were enrolled in the cohort study. Among the patients, seventy-seven (70%) had active LN and thirty-three (30%) were not present with LN during their first visit to the clinic. The diagnoses of LN were biopsy-proven. Serum samples were collected before and after administration of immunosuppressive medications to evaluate 5-MTP levels and regular laboratory data. Data were analyzed longitudinally. Results Before any treatment started, patients with active LN had significantly higher 5-MTP levels as compared to patients with no LN (1.021 ± 0.709 vs. 0.719 ± 0.606, P = 0.0456). Also, in patient with active LN, 5-MTP level was significant decreased after treatment, compared with the levels before treatment (1.021 ± 0.709 vs. 0.802 ± 0.597, P = 0.0484). Patients who reached complete remission also had significantly higher initial serum 5-MTP levels than that in patients with no remission (1.244 ± 0.784 vs. 0.846 ± 0.556, P = 0.0488). There was an overall reduction in 5-MTP levels after six months of immunosuppressive treatment, regardless of the disease outcome. Subgroup analysis further revealed a significantly higher 5-MTP level during the active stage of LN (1.127 ± 0.149 vs. 0.742 ± 0.092, P = 0.0384). Conclusion We demonstrated that serum 5-MTP level is positively correlated to the disease activity, prognosis, and remission status of pediatric LN in vivo.

Inflammation of the vascular microenvironment modulates distinct types of vascular cells, and plays important roles in promoting atherosclerosis, stenosis/restenosis, and vascular-related diseases. Nik-related kinase (Nrk), a member of the Ste20-type kinase family, has been reported to be selectively expressed in embryonic skeletal muscle. However, whether Nrk is expressed in adult vascular smooth muscle, and if it influences intimal hyperplasia is unclear. Here, we found that Nrk is abundantly expressed in cultured vascular smooth muscle cells (VSMC) and mouse arterial intima. Treatment of mouse VSMCs with lipopolysaccharide (LPS) or platelet-derived growth factor significantly reduced Nrk expression. In addition, expression of Nrk was significantly reduced in regions of neointimal formation caused by guide-wire carotid artery injuries in mice, as well as in human atherosclerotic tissues, when compared to normal vessels. We identified that expression of matrix metalloproteinases (MMP3, MMP8 and MMP12) and inflammatory cytokines/chemokines (CCL6, CCL8, CCL11, CXCL1, CXCL3, CXCL5 and CXCL9) are synergistically induced by Nrk siRNA in LPS-treated mouse VSMCs. Moreover, we found that resveratrol significantly impaired LPS- and Nrk siRNA-induced expression of MMP3, CCL8, CCL11, CXCL3 and CXCL5. These results suggested that Nrk may play important roles in regulating pathological progression of atherosclerosis or neointimal- hyperplasia-related vascular diseases.

Background Toll-like receptor-2 (TLR2) promotes vascular smooth muscle cell (VSMC) transdifferentiation to chondrocytes and calcification in a p38 MAPK-dependent manner. Vascular 5-methoxytryptophan (5-MTP) is a newly identified factor with anti-inflammatory actions. As 5-MTP targets p38 MAPK for its actions, we postulated that 5-MTP protects against vascular chondrogenesis and calcification. Methods High-fat diet-induced advanced atherosclerosis in mice were performed to investigate the effect of 5-MTP on atherosclerotic lesions and calcification. VSMCs were used to determine the role of 5-MTP in VSMC chondrogenic differentiation and calcification. Alizarin red S and Alcian blue staining were used to measure VSMC calcification and chondrogenic differentiation, respectively. Results 5-MTP was detected in aortic tissues of ApoE−/− mice fed control chow. It was reduced in ApoE−/− mice fed high-fat diet (HFD), but was restored in ApoE−/−Tlr2−/− mice, suggesting that HFD reduces vascular 5-MTP production via TLR2. Intraperitoneal injection of 5-MTP or its analog into ApoE−/− mice fed HFD reduced aortic atherosclerotic lesions and calcification which was accompanied by reduction of chondrogenesis and calcium deposition. Pam3CSK4 (Pam3), ligand of TLR2, induced SMC phenotypic switch to chondrocytes. Pretreatment with 5-MTP preserved SMC contractile proteins and blocked Pam3-induced chondrocyte differentiation and calcification. 5-MTP inhibited HFD-induced p38 MAPK activation in vivo and Pam3-induced p38 MAPK activation in SMCs. 5-MTP suppressed HFD-induced CREB activation in aortic tissues and Pam3-induced CREB and NF-κB activation in SMCs. Conclusions These findings suggest that 5-MTP is a vascular arsenal against atherosclerosis and calcification by inhibiting TLR2–mediated SMC phenotypic switch to chondrocytes and the consequent calcification. 5-MTP exerts these effects by blocking p38 MAPK activation and inhibiting CREB and NF-κB transactivation activity.

Simple Summary 5-methoxytryptophan (5-MTP) is a tryptophan (Trp) metabolite synthesized by hydroxyindole O-methyltransferase (HIOMT). Expression of HIOMT is decreased in various tumors. However, whether HIOMT expression and serum 5-MTP concentration associate with prognosis of hepatocellular carcinoma (HCC) remains unclear. The aim of this study was to analyze HCC tissue HIOMT mRNA and serum 5-MTP and determine their association with survival following therapeutic liver resection. We found a significant association of serum 5-MTP or tissue HIOMT and serum kynurenine (Kyn) with overall and relapse free (RF) survival of HCC. The combination of serum 5-MTP and Kyn is a potential prognostic biomarker of HCC. Abstract 5-methoxytryptophan (5-MTP) is a recently discovered tryptophan (Trp) metabolite with anti-inflammatory and tumor-suppressing actions. Its synthesis is catalyzed by hydroxyindole O-methyltransferase (HIOMT). HIOMT levels were reported to be decreased in some patients with colorectal, pancreatic and breast cancer. It is unclear whether tissue HIOMT levels is altered in hepatocellular carcinoma (HCC). It is also unclear whether serum 5-MTP concentration is influenced by HCC. In this study, 150 HCC and adjacent normal liver tissues and serum samples were obtained from the HCC biobank established by a prospective multicenter study. Serum samples from 47 healthy subjects were included as a reference. HIOMT mRNA was measured by real time PCR. Serum 5-MTP and selected Trp metabolites were analyzed by quantitative LC-MS. HCC tissue HIOMT mRNA levels adjusted for adjacent normal tissue HIOMT mRNA levels was associated with overall and relapse-free (RF) survival. Combined serum 5-MTP or tissue HIOMT mRNA and serum kynurenine (Kyn) analysis predicted prolonged overall and RF survival following liver resection. A high serum 5-MTP or tissue HIOMT mRNA and low serum Kyn is associated with long-term survival. In conclusion, tumor tissue HIOMT mRNA and serum 5-MTP are potential biomarkers of HCC, especially when analyzed in combination with serum Kyn.

Objective— Vascular smooth muscle cell (VSMC) transformation to an osteochondrogenic phenotype is an initial step toward arterial calcification, which is highly correlated with cardiovascular disease–related morbidity and mortality. TLR2 (Toll-like receptor 2) plays a pathogenic role in the development of vascular diseases, but its regulation in calcification of arteries and VSMCs remains unclear. We postulate that TLR2-mediated inflammation participates in mediating atherosclerotic arterial calcification and VSMC calcification. Approach and Results— We found that ApoE −/− Tlr2 −/− genotype in mice suppressed high-fat diet–induced atherosclerotic plaques formation during initiation but progressively lost its preventative capacity, compared with ApoE −/− mice. However, TLR2 deficiency prohibited high-fat diet–induced advanced atherosclerotic calcification, chondrogenic metaplasia, and OPG (osteoprotegerin) downregulation in the calcified lesions. Incubation of VSMCs in a calcifying medium revealed that TLR2 agonists significantly increased VSMC calcification and chondrogenic differentiation. Furthermore, TLR2 deficiency suppressed TLR2 agonist–mediated VSMC chondrogenic differentiation and consequent calcification, which were triggered via the concerted actions of IL (interleukin)-6–mediated RANKL (receptor activator of nuclear factor κB ligand) induction and OPG suppression. Inhibition experiments with pharmacological inhibitors demonstrated that IL-6–mediated RANKL induction is signaled by p38 and ERK1/2 (extracellular signal-regulated kinase 1/2) pathways, whereas the OPG is suppressed via NF-κB (nuclear factor κB) dependent signaling mediated by ERK1/2. Conclusions— We concluded that on ligand binding, TLR2 activates p38 and ERK1/2 signaling to selectively modulate the upregulation of IL-6–mediated RANKL and downregulation of OPG. These signaling pathways act in concert to induce chondrogenic transdifferentiation of VSMCs, which in turn leads to vascular calcification during the pathogenesis of atherosclerosis.

Rationale: One of the most common metabolic defects in cancers is the deficiency in arginine synthesis, which has been exploited therapeutically. Yet, challenges remain, and the mechanisms of arginine-starvation induced killing are largely unclear. Here, we sought to demonstrate the underlying mechanisms by which arginine starvation-induced cell death and to develop a dietary arginine-restriction xenograft model to study the in vivo effects. Methods: Multiple castration-resistant prostate cancer cell lines were treated with arginine starvation followed by comprehensive analysis of microarray, RNA-seq and ChIP-seq were to identify the molecular and epigenetic pathways affected by arginine starvation. Metabolomics and Seahorse Flux analyses were used to determine the metabolic profiles. A dietary arginine-restriction xenograft mouse model was developed to assess the effects of arginine starvation on tumor growth and inflammatory responses. Results: We showed that arginine starvation coordinately and epigenetically suppressed gene expressions, including those involved in oxidative phosphorylation and DNA repair, resulting in DNA damage, chromatin-leakage and cGAS-STING activation, accompanied by the upregulation of type I interferon response. We further demonstrated that arginine starvation-caused depletion of α-ketoglutarate and inactivation of histone demethylases are the underlying causes of epigenetic silencing. Significantly, our dietary arginine-restriction model showed that arginine starvation suppressed prostate cancer growth in vivo, with evidence of enhanced interferon responses and recruitment of immune cells. Conclusions: Arginine-starvation induces tumor cell killing by metabolite depletion and epigenetic silencing of metabolic genes, leading to DNA damage and chromatin leakage. The resulting cGAS-STING activation may further enhance these killing effects.

5-methoxytryptophan (5-MTP) is an endothelial factor with anti-inflammatory properties. It is synthesized from L-tryptophan via two enzymatic steps: tryptophan hydroxylase-1 (TPH-1) and hydroxyindole O-methyltransferase. Lipopolysaccharide (LPS) and pro-inflammatory cytokines suppress endothelial 5-MTP production by inhibiting TPH-1 expression. 5-MTP protects endothelial barrier function and promotes endothelial repair, while it blocks vascular smooth muscle cell migration and proliferation by inhibiting p38 MAPK activation. 5-MTP controls macrophage transmigration and activation by inhibiting p38 MAPK and NF-κB activation. 5-MTP administration attenuates arterial intimal hyperplasia, defends against systemic inflammation and prevents renal fibrosis in relevant murine models. Serum 5-MTP level is depressed in human sepsis as well as in mice with sepsis-like disorder. It is reduced in chronic kidney disease and acute myocardial infarction in humans. The reported data suggest that serum 5-MTP may be a theranostic biomarker. In summary, 5-MTP represents a new class of tryptophan metabolite which defends against inflammation and inflammation-mediated tissue damage and fibrosis. It may be a valuable lead compound for developing new drugs to treat complex human inflammatory disorders.

5-Methoxytryptophan (5-MTP) is a tryptophan metabolite with recently discovered anti-inflammatory and tumor-suppressing activities. Its synthesis is catalyzed by a hydroxyindole O-methyltransferase (HIOMT)-like enzyme. However, the exact identity of this HIOMT in human cells remains unclear. Human HIOMT exists in several alternatively spliced isoforms, and we hypothesized that 5-MTP–producing HIOMT is a distinct isoform. Here, we show that human fibroblasts and cancer cells express the HIOMT298 isoform as contrasted with the expression of the HIOMT345 isoform in pineal cells. Sequencing analysis of the cloned isoforms revealed that HIOMT298 is identical to the sequence of a previously reported truncated HIOMT isoform. Of note, HIOMT298 expression was reduced in cancer cells and tissues. Stable transfection of A549 cancer cells with HIOMT298 restored HIOMT expression to normal levels, accompanied by 5-MTP production. Furthermore, HIOMT298 transfection caused a tryptophan-metabolic switch from serotonin to 5-MTP production. To determine the in vivo relevance of this alteration, we compared growth and lung metastasis of HIOMT298-transfected A549 cells with those of vector- or untransfected A549 cells as controls in a murine xenograft model. Of note, the HIOMT298-transfected A549 cells exhibited slower growth and lower metastasis than the controls. Our findings provide insight into the crucial role of HIOMT298 in 5-MTP production in cells and in inhibiting cancer progression and highlight the potential therapeutic value of 5-MTP for managing cancer.

Advanced glycation end-products (AGEs) form during oxidative stress, which is increased in diabetes mellitus (DM). Uromodulin is a protein with a renal protective effect, and may be subject to glycation. The implications of uromodulin glycation and AGEs in the urine are not understood. Here, immunoprecipitation and liquid chromatography–mass spectrometry identified glycated uromodulin (glcUMOD) in the urine of 62.5% of patients with diabetic kidney disease (DKD), 20.0% of patients with non-diabetic chronic kidney disease (CKD), and no DM patients with normal renal function or healthy control participants; a finding replicated in a larger cohort of 84 patients with CKD in a case–control study (35 with DM, 49 without). Uromodulin forms high molecular weight polymers that associate with microvesicles and exosomes. Differential centrifugation identified uromodulin in the supernatant, microvesicles, and exosomes of the urine of healthy participants, but only in the supernatant of samples from patients with DKD, suggesting that glycation influences uromodulin function. Finally, the diagnostic and prognostic utility of measuring urinary glcUMOD concentration was examined. Urinary glcUMOD concentration was substantially higher in DKD patients than non-diabetic CKD patients. Urinary glcUMOD concentration predicted DKD status, particularly in patients with CKD stages 1–3a aged

Cardiovascular diseases remain the leading cause of morbidity and mortality worldwide, particularly among older adults. Despite the advent of medical technology, restenosis is still an issue after interventional procedures. Tryptophan metabolite 5-methoxytryptophan (5-MTP) has recently been shown to protect against systemic inflammatory responses. This study aimed to investigate the function and mechanisms of 5-MTP in interventional procedure-induced restenosis. We found that after mouse femoral artery denudation with a guide wire, 5-MTP accelerated recovery of endothelium in the denuded area and reduced vascular leakage and intimal thickening. 5-MTP increased endothelial cell proliferation in the denuded arteries and rescued TNF-α-reduced endothelial cell proliferation and migration, likely via maintaining vascular endothelial growth factor receptor 2 activation. In contrast, 5-MTP preserved differentiated phenotype of medial vascular smooth muscle cells (VSMCs) and decreased VSMC proliferation and migration. Furthermore, 5-MTP maintained expression levels of critical transcription factors for VSMC marker gene expressions via attenuated activation of p38 MAPK and NFκB-p65. Our findings uncover a novel protective mechanism of 5-MTP in restenosis. In response to denudation injury, 5-MTP attenuates intimal hyperplasia via concerted but opposing actions on endothelial cells and VSMCs. Taken together, our results suggest that 5-MTP is a valuable therapeutic target for arterial injury-induced restenosis.

Systemic inflammation has emerged as a key pathophysiological process that induces multiple organ injuries and causes serious human diseases. Despite substantial evidence supporting the role of diacylglycerols (DAG) in modulating chronic inflammation and chronic diseases, the potential mechanisms of its involvement in TLRs-mediated inflammation are still unclear. Here, we show that sn-1,2-diacylglycerols modulate LPS/TLR4-mediated inflammation in vitro and in vivo. ELISA and western blotting experiments indicated that sn-1,2-diacylglycerols suppress LPS-induced responses, including IL-6 and TNF-α production, and COX-2 expression in mouse RAW264.7 macrophages and human endothelial cells, in a dose-dependent manner. Using LPS-induced murine model of systemic inflammation, we show that sn-1,2-diacylglycerols block the cytokine storm, the expression of inflammatory mediators, and LPS-induced septic lung damage and mortality. sn-1,2-diacylglycerols reduce systemic inflammation by inhibiting LPS-induced p38 MAPK- and PI3K/AKT-mediated NF-κB activation in macrophages. These results suggest that exogenous DAG probably acts by blocking p38 MAPK or PI3K/AKT signal transduction, thereby down-regulating NF-κB activation and NF-κB-mediated transcription of genes encoding cytokines and pro-inflammatory oxidative enzymes. Our findings demonstrate that exogenous sn-1,2-diacylglycerol protects mice from LPS-induced lethal endotoxemia by suppressing TLR4-driven inflammatory responses, suggesting that 1,2-diacylglycerols may be used as dietary health supplements for the prevention or therapy of systemic inflammatory diseases.

Rationale: Systemic inflammation has emerged as a key pathophysiological process that induces multiorgan injury and causes serious human diseases. Endothelium is critical in maintaining cellular and inflammatory homeostasis, controlling systemic inflammation, and progression of inflammatory diseases. We postulated that endothelium produces and releases endogenous soluble factors to modulate inflammatory responses and protect against systemic inflammation. Objective: To identify endothelial cell–released soluble factors that protect against endothelial barrier dysfunction and systemic inflammation. Methods and Results: We found that conditioned medium of endothelial cells inhibited cyclooxgenase-2 and interleukin-6 expression in macrophages stimulated with lipopolysaccharide. Analysis of conditioned medium extracts by liquid chromatography–mass spectrometry showed the presence of 5-methoxytryptophan (5-MTP), but not other related tryptophan metabolites. Furthermore, endothelial cell–derived 5-MTP suppressed lipopolysaccharide-induced inflammatory responses and signaling in macrophages and endotoxemic lung tissues. Lipopolysaccharide suppressed 5-MTP level in endothelial cell-conditioned medium and reduced serum 5-MTP level in the murine sepsis model. Intraperitoneal injection of 5-MTP restored serum 5-MTP accompanied by the inhibition of lipopolysaccharide-induced endothelial leakage and suppression of lipopolysaccharide- or cecal ligation and puncture–mediated proinflammatory mediators overexpression. 5-MTP administration rescued lungs from lipopolysaccharide-induced damages and prevented sepsis-related mortality. Importantly, compared with healthy subjects, serum 5-MTP level in septic patients was decreased by 65%, indicating an important clinical relevance. Conclusions: We conclude that 5-MTP belongs to a novel class of endothelium-derived protective molecules that defend against endothelial barrier dysfunction and excessive systemic inflammatory responses.

We reported previously that human fibroblasts release 5-methoxytryptophan (5-MTP) which inhibits cancer cell COX-2 overexpression and suppresses cancer cell migration and metastasis. To determine whether fibroblasts block cancer cell epithelial mesenchymal transition (EMT) via 5-MTP, we evaluated the effect of Hs68 fibroblasts (HsFb) on A549 cancer cell EMT in a two-chamber system. Co-incubation of A549 with HsFb prevented TGF-β1-induced reduction of E-cadherin and increase in Snail and N-cadherin. Transfection of HsFb with tryptophan hydroxylase-1 siRNA, which inhibited tryptophan hydroxylase-1 protein expression and 5-MTP release in HsFb abrogated the effect of HsFb on A549 EMT. Direct addition of pure 5-MTP to cultured A549 cells followed by TGF-β1 prevented TGF-β1-induced reduction of E-cadherin, and elevation of Snail, vimentin and matrix metalloproteinase 9. Administration of 5-MTP to a murine xenograft tumor model reduced vimentin protein expression in the tumor tissues compared to vehicle control which was correlated with reduction of metastasis in the 5-MTP treated mice. Our experimental data suggest that 5-MTP exerted its anti-EMT actions through inhibition of p38 MAPK activation, p65/p50 NF-κB nuclear translocation and transactivation without the involvement of COX-2 or p300 histone acetyltransferase. Our findings indicate that fibroblasts release a tryptophan metabolite, 5-MTP, to reduce cancer cell EMT, migration, invasion and metastasis.

Studies suggest that gene mutation and carcinogen exposure contribute to lung tumorigenesis including a mutation of epidermal growth factor receptor (EGFR) and exposure to benzo[a]pyrene (BaP). However, the interaction between EGFR mutation and BaP exposure during lung tumorigenesis is unclear. Metabolomics has become an important tool in clinical research and has been utilized to help our understanding of mechanisms and to identify indicators of cancers. This study's aim was to identify the changes in metabolite profiles in mice associated with an EGFR exon 21 deletion and/or BaP treatment-induced lung tumorigenesis. While the EGFR mutation increased the incidence of lung adenoma in transgenic mice (EGFR mutant mice) at 32 weeks of age, exposure to BaP caused the onset of lung tumorigenesis in these mice as early as 16 weeks after exposure. Using a metabolomics strategy involving liquid chromatography-mass spectrometry in conjunction with principal component analysis and confirmation by liquid chromatography triple quadrupole tandem mass spectrometry, we demonstrated that the serum amino acid profiles of these mice were changed. A total of eight amino acid concentrations were lower in EGFR mutant mice than in wild-type mice at 32 weeks of age. Five amino acids were lower in tumor-bearing mice than in non-tumor-bearing EGFR mutant mice at 10th week post-treatment of BaP, namely phenylalanine, tyrosine, alanine, proline, and threonine. Our results suggest that gene mutation and carcinogen exposure-induced lung adenomas share some common mechanisms. Changes in serum amino acid profiles may be early indicators of lung tumorigenesis.

Vanadium is a transition metal widely distributed in the Earth&rsquo, s crust, and is a major contaminant in fossil fuels. Its pathological effect and regulation in atherosclerosis remain unclear. We found that intranasal administration of the vanadium derivative NaVO3 significantly increased plasma and urinary vanadium levels and induced arterial lipid accumulation and atherosclerotic lesions in apolipoprotein E-deficient knockout mice (ApoE&minus, /&minus, ) murine aorta compared to those in vehicle-exposed mice. This was accompanied by an increase in plasma reactive oxygen species (ROS) and interleukin 6 (IL-6) levels and a decrease in the vascular smooth muscle cell (VSMC) differentiation marker protein SM22&alpha, in the atherosclerotic lesions. Furthermore, exposure to NaVO3 or VOSO4 induced cytosolic ROS generation and IL-6 production in VSMCs and promoted VSMC synthetic differentiation, migration, and proliferation. The anti-oxidant N-acetylcysteine (NAC) not only suppresses IL-6 production and VSMC pathological responses including migration and proliferation but also prevents atherosclerosis in ApoE&minus, mice. Inhibition experiments with NAC and pharmacological inhibitors demonstrated that NaVO3-induced IL-6 production is signaled by ROS-triggered p38-mediated NF-&kappa, B-dependent pathways. Neutralizing anti-IL-6 antibodies impaired NaVO3-mediated VSMC migration and proliferation. We concluded that NaVO3 exposure activates the ROS-triggering p38 signaling to selectively induce NF-&kappa, B-mediated IL-6 production. These signaling pathways induce VSMC synthetic differentiation, migration, and proliferation, leading to lipid accumulation and atherosclerosis.

Background Concerns were raised about pneumonia development from benzodiazepines (BZDs) and Z-drugs, but direct evidence is limited, conflicting and without examining the highly susceptible patients with chronic kidney disease (CKD) nor specifying the risk for different drug utilizations. This study aimed to investigate whether use of BZDs and Z-drugs was each associated with an increased risk of pneumonia in a CKD population. Methods We performed a nested case-control study of 36,880 CKD patients analyzing the Taiwan National Health Insurance Database between 01/1/2000 and 12/31/2011. Among the study cohort, we identified 4,533 cases of pneumonia based on validated disease codes, chest x-ray examination, and prescriptions of respiratory antibiotics, and randomly selected 16,388 controls from risk sets, matched by sex, age, and number of CKD-related hospitalizations. All prescription filling records of BZDs and Z-drugs in the year before the event/index date were analyzed for cases and controls. Conditional logistic regressions were performed to estimate the odds ratios (ORs). Results Current use of BZDs was associated with a 1.31-fold (95% CI, 1.18–1.26) increased risk of pneumonia compared to nonuse, but not for recent and past use. The risk from current BZD use was confined to new initiation (adjusted OR, 2.47; 95% CI, 2.02–3.03) or use for ≤ 30 days, and elevated to 2.88-fold (95% CI, 1.87–4.42) with parenteral administration. New initiation and current short-term use of Z-drugs was associated with a 2.94-fold (95% CI, 1.65–5.26) and 1.75-fold (95% CI, 1.13–2.72) increased risk of pneumonia, respectively. The findings were robust to adoption of a case-crossover study that analyzed cases only. Conclusions Use of BZRAs is associated with an increased risk of pneumonia in CKD patients, especially for patients newly initiating BZDs or Z-drugs or those injected with BZDs. Physicians should exercise cautions for signs of pneumonia when prescribing BZDs or Z-drugs to CKD patients.

Focal adhesion kinase (FAK) plays an important role in vascular development, including the regulation of endothelial cell (EC) adhesion, migration, proliferation, and survival. 3&rsquo, deoxyadenosine (cordycepin) is known to suppress FAK expression, cell migration, and the epithelial&ndash, mesenchymal transition in hepatocellular carcinoma (HCC). However, whether cordycepin affects FAK expression and cellular functions in ECs and the specific molecular mechanism remain unclear. In this study, we found that cordycepin suppressed FAK expression and the phosphorylation of FAK (p-FAK) at Tyr397 in ECs. Cordycepin inhibited the proliferation, wound healing, transwell migration, and tube formation of ECs. Confocal microscopy revealed that cordycepin significantly reduced FAK expression and decreased focal adhesion number of ECs. The suppressed expression of FAK was accompanied by induced p53 and p21 expression in ECs. Finally, we demonstrated that cordycepin suppressed angiogenesis in an in vivo angiogenesis assay and reduced HCC tumor growth in a xenograft nude mice model. Our study indicated that cordycepin could attenuate cell proliferation and migration and may result in the impairment of the angiogenesis process and tumor growth via downregulation of FAK and induction of p53 and p21 in ECs. Therefore, cordycepin may be used as a potential adjuvant for cancer therapy.

The gut microbiota plays profound roles in host metabolism and the inflammatory response associated with the development of obesity. Dusp6-deficient mice have been shown to be resistant to diet-induced obesity, but the mechanism behind this remains unclear. 16S ribosomal RNA gene analysis demonstrated that dusp6-deficient mice harbour unique gut microbiota with resistance to diet-induced-obesity-mediated alteration of the gut microbiome. Using a germ-free mouse model, we found that faecal/gut microbiota derived from dusp6-deficient mice significantly increased energy expenditure and reduced weight gain in recipient wild-type mice fed on a high-fat diet. On analysis of the intestinal transcriptome of dusp6-deficient mice, we found that dusp6 deficiency mainly induced biological processes involved in metabolism and the extracellular matrix, particularly the peroxisome proliferator-activated receptor gamma (Pparγ) pathway and tight-junction genes. Furthermore, dusp6-deficient mice have a high-fat-diet-specific transcriptomic response to reverse the expression of genes associated with intestinal barrier functions and mucosal immunity involved in microbiome homeostasis. This study demonstrates that dusp6 deficiency is a strong genetic factor shaping gut microbiota, and that it confers obesity protection by ameliorating the gut microbiota response to diet-mediated stress.

This paper proposes a drowsiness-fatigue-detection (DFD) system for improving road safety, which consists of a wearable smart glasses, an in-vehicle infotainment system (a vehicular telematics device), an automotive diagnostic bridge, and a cloud-based platform, etc. The proposed DFD system can real-time detect driver's state for drowsiness or fatigue. When detecting drowsiness or fatigue situation occurrence, thus the rear lights of the vehicle will automatically be flickered and message will be sent to the cloud-based platform, concurrently. Hence, the proposed system can effectively achieve the purpose of road safety improvement.

Mutations of isocitrate dehydrogenase 1 (IDH1) and IDH2 in acute myeloid leukemia (AML) cells produce the oncometabolite R-2-hydroxyglutarate (R-2HG) to induce epigenetic alteration and block hematopoietic differentiation. However, the effect of R-2HG released by IDH-mutated AML cells on the bone marrow microenvironment is unclear. Here, we report that R-2HG induces IκB kinase-independent activation of NF-κB in bone marrow stromal cells. R-2HG acts via a reactive oxygen species/extracellular signal-regulated kinase (ERK)-dependent pathway to phosphorylate NF-κB on the Thr254 residue. This phosphorylation enhances the interaction of NF-κB and the peptidyl-prolyl cis-trans isomerase PIN1 and increases the protein stability and transcriptional activity of NF-κB. As a consequence, R-2HG enhances NF-κB-dependent expression of cytokines including IL-6, IL-8 and complement 5a to stimulate proliferation of AML cells. In addition, R-2HG also upregulates vascular endothelial adhesion molecule 1 and CXCR4 in stromal cells to enhance the contact between AML and stromal cells and attenuates chemotherapy-induced apoptosis. More importantly, we validated the R-2HG-activated gene signature in the primary bone marrow stromal cells isolated from IDH-mutated AML patients. Collectively, our results suggest that AML cell-derived R-2HG may be helpful for the establishment of a supportive bone marrow stromal niche to promote AML progression via paracrine stimulation.

Background Ventricular remodeling following myocardial infarction (MI) is closely associated with cyclooxygenase-2 (COX-2) expression. 5-methoxytryptophan (5-MTP) was reported to control COX-2 expression. Objectives To investigeate the association between 5-MTP and post-MI left ventricular remodeling. Methods This prospective study enrolled 26 non-diabetic patients with first-time ST segment elevation myocardial infarction (STEMI), and 58 controls. Levels of 5-MTP, N-terminal of pro-brain natriuretic peptide (NT-proBNP), aminoterminal propeptide of type III procollagen, matrix metalloproteinase-9, and tissue inhibitor of metalloproteinase-1 were measured at day 1, day 3, 3months, 6months, and 1year post-MI. Echocardiography was performed during the acute stage (within 72h) and 3months, 6months, and 1year post-MI. Results The STEMI patients had a significantly lower plasma 5-MTP level at day 1 which reached a nadir at 3months post-MI. The level of 5-MTP at day 3 post-MI was significantly correlated with the level of NT-proBNP 1year post-MI, suggesting that the level of plasma 5-MTP in the early phase after MI may predict subsequent cardiac stress and failure. Receiver operating characteristic curve analysis revealed that plasma 5-MTP had the best area under the curve value to predict plasma NT-proBNP 1year post-MI. Further analysis using net reclassification improvement and integrated discrimination improvement models confirmed that plasma 5-MTP at day 3 post-MI significantly improved the predictive power of each of the parameters. Conclusion In non-diabetic STEMI patients, plasma 5-MTP levels were associated with biomarkers of post-MI left ventricular remodeling and damage.

Toll-like receptor 4 (TLR4) is important in promoting inflammation and vascular smooth muscle cell (VSMC) migration, both of which contribute to atherosclerosis development and progression. But the mechanism underlying the regulation of TLR4 in VSMC migration remains unclear. Stimulation of VSMCs with LPS increased the cellular level of F-spondin which is associated with the regulation of proinflammatory cytokine production. The LPS-induced F-spondin expression depended on TLR4-mediated PI3K/Akt pathway. Suppression of F-spondin level by siRNA inhibited not only F-spondin expression but also LPS-induced phosphorylation of cAMP response element binding protein (CREB) and IL-6 expression, VSMC migration and proliferation as well as MMP9 expression. Moreover, suppression of CREB level by siRNA inhibited TLR4-induced IL-6 production and VSMC migration. Inhibition of F-spondin siRNA on LPS-induced migration was restored by addition of exogenous recombinant mouse IL-6. We conclude that upon ligand binding, TLR4 activates PI3K/Akt signaling to induce F-spondin expression, subsequently control CREB-mediated IL-6 production to promote VSMC migration. These findings provide vital insights into the essential role of F-spondin in VSMC function and will be valuable for developing new therapeutic strategies against atherosclerosis.

// Yen-Chun Ho 1,2 , Meng-Ling Wu 1 , Pei-Yu Gung 1 , Chung-Huang Chen 1 , Cheng-Chin Kuo 1,3 and Shaw-Fang Yet 1,2,3 1 Institute of Cellular and System Medicine, National Health Research Institutes, Zhunan, Taiwan 2 Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan 3 Graduate Institute of Basic Medical Science and Metabolomic Research Center, China Medical University, Taichung, Taiwan Correspondence to: Shaw-Fang Yet, email: // Keywords : aortic aneurysm, abdominal, heme oxygenase-1, oxidative stress, inflammation, Pathology Section Received : April 05, 2016 Accepted : September 02, 2016 Published : September 08, 2016 Abstract Abdominal aortic aneurysm (AAA) is a chronic but often fatal disease in elderly population. Heme oxygenase-1 (HO-1) is a stress response protein with antioxidative and anti-inflammatory properties. HO-1 has been shown to protect against atherogenesis and arterial intimal thickening. Emerging evidences suggest that AAA and arterial occlusive disease have distinct pathogenic mechanisms. Thus, in this study we investigated the role of HO-1 in angiotensin II-induced AAA formation in HO-1 +/+ apoE –/– and HO-1 –/– apoE –/– mice. We found that complete loss of HO-1 increased AAA incidence and rupture rate, and drastically increased aneurysmal area and severity, accompanied with severe elastin degradation and medial degeneration. Interestingly, we often observed not only AAA but also thoracic aortic aneurysm in HO-1 –/– apoE –/– mice. Furthermore, reactive oxygen species levels, vascular smooth muscle cell (VSMC) loss, macrophage infiltration, matrix metalloproteinase (MMP) activity were markedly enhanced in the aneurysmal aortic wall in HO-1 –/– apoE –/– mice. In addition, HO-1 –/– apoE –/– VSMCs were more susceptible to oxidant-induced cell death and macrophages from HO-1 –/– apoE –/– mice had aggravated responses to angiotensin II with substantial increases in inflammatory cytokine productions and MMP9 activity. Taken together, our results demonstrate the essential roles of HO-1 in suppressing the pathogenesis of AAA. Targeting HO-1 might be a promising therapeutic strategy for AAA.

Objective— Migration of vascular smooth muscle cells (VSMCs) from the media into intima contributes to the development of atherosclerosis. Gene deletion experiments implicate a role for toll-like receptor 2 (TLR2) in atherogenesis. However, the underlying mechanisms remain unclear. We postulate that TLR2 promotes VSMC migration by enhancing interleukin (IL)-6 production. Methods and Results— Migration assays revealed that TLR2 agonists promoted VSMC migration but not cell proliferation or viability. TLR2 deficiency or inhibition of TLR2 signaling with anti-TLR2 antibody suppressed TLR2 agonist–induced VSMC migration and IL-6 production, which was mediated via p38 mitogen-associated protein kinase and extracellular signal–regulated kinase 1/2 signaling pathways. Neutralizing anti−IL-6 antibodies impaired TLR2-mediated VSMC migration and formation of filamentous actin fiber and lamellipodia. Blockade of p38 mitogen-associated protein kinase or extracellular signal–regulated kinase 1/2 activation inhibited TLR2 agonist pam3CSK4-induced phosphorylation of cAMP response element−binding protein, which regulates IL-6 promoter activity through the cAMP response element site. Moreover, cAMP response element−binding protein small interfering RNA inhibited pam3CSK4-induced IL-6 production and VSMC migration. Additionally, Rac1 small interfering RNA inhibited pam3CSK4-induced VSMC migration but not IL-6 production. Conclusion— Our results suggest that on ligand binding, TLR2 activates p38 mitogen-associated protein kinase and extracellular signal–regulated kinase 1/2 signaling in VSMCs. These signaling pathways act in concert to activate cAMP response element−binding protein and subsequent IL-6 production, which in turn promotes VSMC migration via Rac1-mediated actin cytoskeletal reorganization.

5-Methoxytryptophan (5-MTP), a 5-methoxyindole metabolite of tryptophan metabolism, was recently shown to suppress inflammatory mediator-induced cancer cell proliferation and migration. However, the role of 5-MTP in vascular disease is unknown. In this study, we investigated whether 5-MTP protects against vascular remodeling following arterial injury. Measurements of serum 5-MTP levels in healthy subjects and patients with coronary artery disease (CAD) showed that serum 5-MTP concentrations were inversely correlated with CAD. To test the role of 5-MTP in occlusive vascular disease, we subjected mice to a carotid artery ligation model of neointima formation and treated mice with vehicle or 5-MTP. Compared with vehicle-treated mice, 5-MTP significantly reduced intimal thickening by 40% 4 weeks after ligation. BrdU incorporation assays revealed that 5-MTP significantly reduced VSMC proliferation both in vivo and in vitro. Furthermore, 5-MTP reduced endothelial loss and detachment, ICAM-1 and VCAM-1 expressions and inflammatory cell infiltration in the ligated arterial wall, suggesting attenuation of endothelial dysfunction. Signaling pathway analysis indicated that 5-MTP mediated its effects predominantly via suppressing p38 MAPK signaling in endothelial and VSMCs. Our data demonstrate a novel vascular protective function of 5-MTP against arterial injury-induced intimal hyperplasia. 5-MTP might be a therapeutic target for preventing and/or treating vascular remodeling.

Head and neck squamous cell carcinoma (HNSCC), the most common malignant neoplasm arising in the mucosa of the upper aerodigestive tract, remains a significant cause of morbidity worldwide. With respect to the cancer treatment, HNSCC has a moderately good survival rate, however, the disease often recurs, leading to a poor prognostic disease course and tends to fail in treatment. The metabolic properties of cancer cells diverge significantly from those of normal cells. Emerging evidence suggests these metabolic alterations are also linked to therapeutic resistance in cancer treatment. EGFR targeted agents currently approved or under investigation for HNSCC. We recently identified a novel EGFR tyrosine kinase inhibitor (EGFR-TKI), BPR3K007S0, and found that this compound significantly inhibited EGFR phosphorylation in EGFR-overexpressing HNSCC cells in vitro and in vivo than that of gefitinib. BPR3K007S0 significantly suppressed the expression of a wide range of metabolic genes, including metabolic-related transcription factors, glycolysis, TCA cycle, and pentose phosphate pathway genes. Collectively, we also found that BPR3K007S0 significantly decreased glycolytic and mitochondria respiratory capacity. Pharmacological and genetic manipulation demonstrated that c-Myc/hexokinase 2 axis was one of downstream effector in response to EGFR inhibition. Furthermore, we found that EGFR-TKIs were able to suppress succinate dehydrogenase A leading to reduce fumarate, an oncometabolite generates from Kreb cycle, and contributed to EGFR-TKIs mediated antitumor effect. Taken together, these results revealed that blockage of EGFR signaling repurposes tumor metabolism through suppression of hexokinase 2 and succinate dehydrogenase A, in glycolysis and Kreb cycle, respectively, and demonstrated added benefits to treatment of HNSCCs. Citation Format: Ching-Chuan Kuo, Jang-Yang Chang, Hsing-Pang Hsieh, Hsing-Jien Kung, Hsih-Huei Chang, Chih-Hsiang Huang, Cheng-Chin Kuo, Yi-Yu Ke. Blockage of EGFR signaling repurposes tumor metabolism through suppression of glycolysis and Kreb cycle in head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3944.

Pigeon liver malic enzyme was inactivated and cleaved at Asp141, Asp194, and Asp464 by the Cu2+-ascorbate system in acidic environment. Site-specific mutagenesis was performed at these putative metal-binding sites. Three point mutants, D141N, D194N, and D464N; three double mutants, D(141,194)N, D(194,464)N, and D(141,464)N; and a triple mutant, D(141,194,464)N; as well as the wild-type malic enzyme (WT) were successfully cloned and expressed in Escherichia coli cells. All recombinant enzymes, except the triple mutant, were purified to apparent homogeneity by successive Q-Sepharose and adenosine-2',5'-bisphosphate-agarose columns. The mutants showed similar apparent Km,NADP values to that of the WT. The Km,Mal value was increased in the D141N and D194N mutants. The Km,Mn value, on the other hand, was increased only in the D141N mutant by 14-fold, corresponding to approximately 1.6 kcal/mol for the Asp141-Mn2+ binding energy. Substrate inhibition by L-malate was only observed in WT, D464N, and D(141,464)N. Initial velocity experiments were performed to derive the various kinetic parameters. The possible interactions between Asp141, Asp194, and Asp464 were analyzed by the double-mutation cycles and triple-mutation box. There are synergistic weakening interactions between Asp141 and Asp194 in the metal binding that impel the D(141,194)N double mutant to an overall specificity constant [k(cat)/(Kd,Mn Km,Mal Km,NADP)] at least four orders of magnitude smaller than the WT value. This difference corresponds to an increase of 6.38 kcal/mol energy barrier for the catalytic efficiency. Mutation at Asp464, on the other hand, has partial additivity on the mutations at Asp141 and Asp194. The overall specificity constants for the double mutants D(194,464)N and D(141,464)N or the triple mutant D(141,194,464)N were decreased by only 10- to 100-fold compared to the WT. These results strongly suggest the involvement of Asp141 in the Mn2+-L-malate binding for the pigeon liver malic enzyme. The Asp194 and Asp464, which may be oxidized by nonspecific binding of Cu2+, are involved in the Mn2+-L-malate binding or catalysis indirectly by modulating the binding affinity of Asp141 with the Mn2+.

International audience; The CpG motif within bacterial DNA is a potent immuno-stimulatory moiety. Here, using a 2-D electrophoretic approach, we investigated the effect of synthetic oligodeoxynucleotide containing a B type CpG motif (CpG-B ODN) on the protein expression profile of swine peripheral blood mononuclear cells (PBMC). We found that several proteins including spondin 1, N-acetolactate alpha linked acidic dipeptidase; V kappa light chain, T cell receptor variable alpha chain, heat shock protein (Hsp) 60, Hsp70, KIAA0857 protein, and PNAS-146 were up-regulated in PBMC by CpG-B ODN stimulation. Further studies showed that CpG-B ODN-mediated Hsp60, Hsp70 and Hsp90 expressions were closely associated with the TLR9 signalling pathway. Pretreatment with inhibitors of Hsp70, Hsp90 and TLR9 all blocked the CpG-B ODN-mediated anti-apoptotic effect in swine PBMC. These results suggest that CpG-B ODN treatment of swine PBMC may enhance the expression of biologically active proteins, notably spondin 1, V kappa light chain, T cell receptor variable alpha chain and Hsps, which may play an important role in CpG-B ODN-mediated activation of immune responses and enhancement of swine PBMC survival.

The endothelial junction is tightly controlled to restrict the passage of blood cells and solutes. Disruption of endothelial barrier function by bacterial endotoxins, cytokines or growth factors results in inflammation and vascular damage leading to vascular diseases. We have identified 5-methoxytryptophan (5-MTP) as an anti-inflammatory factor by metabolomic analysis of conditioned medium of human fibroblasts. Here we postulated that endothelial cells release 5-MTP to protect the barrier function. Conditioned medium of human umbilical vein endothelial cells (HUVECs) prevented endothelial hyperpermeability and VE-cadherin downregulation induced by VEGF, LPS and cytokines. We analyzed the metabolomic profile of HUVEC conditioned medium and detected 5-MTP but not melatonin, serotonin or their catabolites, which was confirmed by enzyme-linked immunosorbent assay. Addition of synthetic pure 5-MTP preserved VE-cadherin and maintained barrier function despite challenge with pro-inflammatory mediators. Tryptophan hydroxylase-1, an enzyme required for 5-MTP biosynthesis, was downregulated in HUVECs by pro-inflammatory mediators and it was accompanied by reduction of 5-MTP. 5-MTP protected VE-cadherin and prevented endothelial hyperpermeability by blocking p38 MAPK activation. A chemical inhibitor of p38 MAPK, SB202190, exhibited a similar protective effect as 5-MTP. To determine whether 5-MTP prevents vascular hyperpermeability in vivo, we evaluated the effect of 5-MTP administration on LPS-induced murine microvascular permeability with Evans blue. 5-MTP significantly prevented Evans blue dye leakage. Our findings indicate that 5-MTP is a new class of endothelium-derived molecules which protects endothelial barrier function by blocking p38 MAPK.

Toll-like receptor 9 (TLR9) trafficking from the endoplasmic reticulum (ER) into endolysosomes is critical for eliciting cytidine-phosphate-guanosine (CpG) DNA-mediated immune responses. ADP-ribosylation factor 3 (ARF3) is a member of the Ras superfamily, which is crucial for a wide variety of cellular events including protein trafficking. In this study, we found that the inhibition of ARF3 by dominant mutants and siRNA impaired CpG oligodeoxynucleotide (ODN)-mediated responses whereas cells expressing the constitutively active ARF3 mutant enhanced CpG ODN-induced NF-κB activation and cytokine production. Further experiments with MyD88-overexpressing fibroblast cells transfected with a dominant-negative mutant and a constitutively active mutant of ARF3 demonstrated that ARF3 regulated CpG ODN-mediated signaling upstream of MyD88. Additional studies have shown that ARF3 inhibition impairs TLR9 trafficking from the ER into endolysosomes, thereby inhibiting the functional cleavage of TLR9, although it has no significant effect on CpG ODN uptake. Furthermore, activated ARF3 is associated with Unc93B1 and TLR9, suggesting that ARF3 conducts TLR9 trafficking by forming the TLR9-Unc93B1-ARF3 complex. Overall, our findings demonstrate that a novel ARF3 axis pathway mediates CpG ODN-induced responses by regulating TLR9 trafficking.

Vascular smooth muscle cells (VSMCs) in the blood vessel wall exhibit a differentiated phenotype; their main function is contraction and to regulate vascular tone. In response to injury, VSMCs undergo a phenotypic transition whereby they proliferate and migrate from the medial layer into the intima, contributing to lesion formation and atherosclerosis. 5-methoxytryptophan (5-MTP), a recently identified novel tryptophan metabolite, has been shown to inhibit cancer cell growth, migration, and cancer metastasis. We hypothesized that 5-MTP might play an analogous role in vascular disease as in tumorigenesis. To test our hypothesis, we subjected 12 weeks old C57BL/6 mice to a carotid artery cessation of blood flow model to induce neointima formation. Following surgery, mice were treated with vehicle (PBS) or 100 mg/kg of 5-MTP by intraperitoneal injection 3 times a week. Four weeks later, carotid arteries were harvested for histological analysis. H&E and elastin staining revealed robust neointima in ligated carotids of vehicle-treated mice. In contrast, 5-MTP significantly attenuated neointima formation. Given that proinflammatory cytokine interleukin 1 beta (IL-1β) is increased in the injured vessel wall, we examined whether IL-1β affected VSMC phenotypic modulation. Indeed, IL-1β downregulated VSMC marker SM α-actin expression and increased VSMC proliferation and migration. Importantly, in VSMCs, 5-MTP attenuated IL-1β-mediated SM α-actin downregulation, proliferation, and migration, suggesting a potential role of 5-MTP in controlling VSMC phenotypic modulation. Furthermore, 5-MTP inhibited IL-1β-mediated p38 MAPK activation. Inhibiting p38 MAPK activation by SB203580 dose-dependently decreased IL-1β-induced VSMC proliferation. Taken together, our results suggest an important role of 5-MTP in vascular disease.

Unmethylated CpG oligodeoxynucleotides (ODNs) activate immune responses in a TLR9-dependent manner. In this study, stimulation of mouse macrophages with CpG-B ODN increased cellular Hsp70 expression and prevented apoptosis induced by serum starvation or staurosporine treatment. CpG-B ODN-induced Hsp70 expression depended on TLR9, MyD88, and phosphatidylinositol 3-kinase. Inhibition of Hsp70 synthesis by an inhibitor (quercetin) or antisense hsp70 attenuated not only Hsp70 expression but also the anti-apoptotic capacity of CpG-B ODN. Ectopic expression of Hsp70 rescued the inhibitory effect of quercetin on CpG-B ODN-induced anti-apoptosis. Additional experiments demonstrated that quercetin and anti-sense hsp70 modulated CpG-B ODN-induced anti-apoptosis via a caspase-3-independent pathway by down-regulating the survival gene bcl-x(L) and by increasing translocation of apoptosis-inducing factor. These findings suggest that CpG-B ODN may up-regulate Hsp70 via a TLR9/MyD88/phosphatidylinositol 3-kinase pathway to increase Bcl-x(L) and to decrease apoptosis-inducing factor nuclear translocation, resulting in anti-apoptosis.