DNA Array Analysis Research Articles

Highly Defined Induced Pluripotent Stem Cell Lines Mimic Donor Red Blood Cell Antigen Profiles for Therapeutic and Diagnostic Use.

Our group generated two induced pluripotent stem cell (iPSC) lines for in vitro red blood cell (RBC) production from blood donors with extensively known erythrocyte antigen profiles. One line was intended to give rise to RBCs for transfusions in patients with sickle cell disease (SCD), while the other was developed to create RBC panel reagents. Two blood donors were selected based on their RBC phenotypes, further complemented by high-throughput DNA array analysis to obtain a more comprehensive erythrocyte antigen profile. Enriched erythroblast populations from the donors' peripheral blood mononuclear cells were reprogrammed into iPSCs using nonintegrative plasmid vectors. The iPSC lines were characterized and subsequently subjected to hematopoietic differentiation. iPSC PB02 and iPSC PB12 demonstrated in vitro and invivo iPSC features and retained the genotype of each blood donor's RBC antigen profile. Colony-forming cell assays confirmed that iPSC PB02 and iPSC PB12 generated hematopoietic progenitors. These two iPSC lines were generated with defined erythrocyte antigen profiles, self-renewal capacity, and hematopoietic differentiation potential. With improvements in hematopoietic differentiation, these cells could potentially be more efficiently differentiated into RBCs in the future. They could serve as a complementary approach for obtaining donor-independent RBCs and addressing specific demands for blood transfusions.

Increased chemokine ligand 26 expression and its involvement in epithelial-mesenchymal transition in the endometrium with adenomyosis

ObjectiveAdenomyosis is a gynecologic disorder characterized by symptoms of dysmenorrhea, abnormal uterine bleeding, and infertility. This study aimed to analyze the expression profiles of key inflammatory cytokines in the endometrium with adenomyosis and their involvement in epithelial-mesenchymal transition (EMT). Study DesignEndometrial tissues collected from premenopausal women with (n = 3) or without (n = 3) adenomyosis during the secretory phase were subjected to DNA array analysis to examine inflammatory cytokines. The gene and protein expression levels were re-evaluated by reverse transcription-polymerase chain reaction (n = 19) and immunohistochemistry (n = 56). Immunohistochemical analysis using the Histo-scores of chemokine ligand 26 (CCL26) and EMT-related factors was performed with uterine tissues resected for adenomyosis (n = 37), including those from patients treated with gonadotropin-releasing hormone agonist (GnRHa). An invasion assay was also performed using endometrial epithelial cells. ResultsDNA array results showed that CCL26, IL-1B, and CCL3 were upregulated. CCL26 mRNA expression was markedly higher in the endometrium with adenomyosis than in that without adenomyosis. Immunohistochemical analysis revealed that CCL26 expression was elevated in the epithelial cells of the basal layer of the endometrium with adenomyosis than in that without adenomyosis regardless of GnRHa treatment. In the basal layer of the endometrium with adenomyosis, CCL26 expression was positively correlated with neural-cadherin and ZEB1 expression; additionally, the cases with intrinsic-type adenomyosis had high expression levels of CCL26 and ZEB1. Exogenous CCL26 promoted the invasive activity of endometrial epithelial cells. ConclusionsCCL26, an inflammatory mediator, may be involved in the pathogenesis of adenomyosis by inducing EMT in the basal layer of the endometrium.

EXTH-34. ION CHANNELS AS A THERAPEUTIC TARGET IN PAEDIATRIC HIGH-GRADE GLIOMAS

Abstract Pediatric Glioblastoma Multiforme (GBM) is a leading cause of CNS cancer-related death in children. The scarcity of treatments for GBM has opened the field to new pathways in adults, such as tumour treating fields(TTF). Studies have identified the clinical benefit of electrotherapy in combination with chemotherapeutics, however the mechanistic action is unclear. Increasing evidence suggests that ion channels not only regulate electrical signalling of excitable cells, but are also crucial in the development/progression of brain tumours. Ion channels are essential in cell-cycle control, invasion and migration of cancer cells, therefore presenting as valuable therapeutic targets. Candidate ion channel genes(ICG) associated with high-grade glioma (HGG) were identified via analysis of inhouse and publicly available data sets. Expression patterns of selected ICGs were assessed along with clinician correlations.. Protein and RNA expression of target ICGs was assessed in pGBM cell lines/ TMAs. Finally, the Human ClariomTMS array was used for whole transcriptome gene expression analysis of paediatric GBM cell lines treated with tumour treating fields or low frequency electrical fields via deep brain stimulating electrodes. Paediatric brain tumour cells were exposed to genetic and pharmacological manipulation of CLIC1 and CLIC4 ion channels individually or in combination. We have shown that HGG exhibit increased expression of CLIC4 and CLIC1 at protein and RNA levels in both publically available data sets and inhouse cell lines / TMAs. Clinical correlation determined that high CLIC4 and CLIC1 expression was associated with poor overall survival. Further to this, DNA array analysis revealed a downregulation of CLIC1 and CLIC4 ion channels genes in KNS42 cells when treated with electrotherapy compared to untreated cells. Inhibition of CLIC1 and CLIC4 reduces Cl- flux across cell membranes and reduces cell proliferation. These data provide rationale that manipulation of ICGs will reduce the capacity of childhood brain tumours to proliferate and invade.

Trop-2 inactivates E-cadherin and drives colon cancer metastasis.

e15576 Background: Metastatic diffusion is the biggest hurdle for colon cancer (CRC) cure and the identification of decisive drivers of CRC metastasis is an urgent need in CRC care. Methods: The expression of target molecules in primary tumors and metastases was systematically assessed by DNA array and IHC analysis. Cell-cell adhesion capacity was quantified in 2D cell cultures and in HCT116 CRC cell spheroids. Pro-metastatic impact of wtTrop-2 and activated, tail-less Trop-2 (Δcyto) was assessed in vivo in orthotopic diffusion models of KM12SM CRC cells. Primary CRC and metastasis transcriptomes were analyzed for differential induction of EMT determinants. Kaplan–Meier plots were used to illustrate survival and metastatic relapse in independent case series of CRC patients. Results: Trop-2 was identified as uniquely upregulated in CRC models by transcriptome analysis. wtTrop-2 and ΔcytoTrop-2 were shown to induce cell migration, wound-healing and resistance to apoptosis induction . wtTrop-2 increased the metastatic capacity of KM12SM cells, raising metastasis diffusion from 45% for control cells to 90% for wtTrop-2 transfectants. The constitutively-active ΔcytoTrop-2 further boosted metastatic spreading, with metastatic livers reaching up to four times their normal size. Constitutive high expression of E-cadherin was revealed in cancer metastases. No evidence was obtained for transcriptional down-regulation of epithelial differentiation biomarkers. No induction of EMT transcription factors was observed in Trop-2-activated cells. Trop-2 tightly bound E-cadherin and caused its release from the cytoskeleton, for loss of cell-cell adhesion and activation of β-catenin. The Trop-2/E-cadherin/β-catenin-driven pro-metastatic program was recapitulated in CRC patients and was shown to impact on CRC metastatic relapse and overall patient survival. Conclusions: We identify Trop-2-driven functional inactivation of E-cadherin as a key driver of metastatic diffusion in CRC. These findings may pave the way for novel multi-marker personalized diagnostics and anti-cancer therapies. [Table: see text]

Gene expression and DNA methylation regulation of arsenic in mouse bladder tissues and in human urothelial cells.

According to a report of the International Agency for Research on Cancer, arsenic and inorganic arsenic compounds are classified into Group 1 carcinogens with regard to human health. Epidemiological studies indicate that arsenic is one of the main risk factors for the development of bladder cancer. In the present study, arsenic-altered gene expression in mouse bladder tissues and in human urothelial cells was compared. In the mouse model, sodium arsenite-induced mouse urothelial hyperplasia and intracellular inclusions were present. Following DNA array analysis, four genes with differential expression were selected for quantitative real-time PCR assay. The genes were the following: Cystathionine β-synthase (CBS), adenosine A1 receptor (ADORA1), metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and Wnt inhibitory factor 1 (Wif1). The results indicated a significant increase in the levels of Cbs and Adora1. The analysis of the DNA CpG methylation levels of the mouse Cbs and Adora1 genes revealed no significant change. In contrast to these observations, the four genes were further analyzed in the human normal urothelial cell line SV-HUC1. The data indicated that WIF1 gene expression was decreased by sodium arsenite, whereas this was not noted for CBS, MALAT1 and ADORA1. Sodium arsenite decreased mRNA and protein expression levels of the WIF1 gene. In addition, the methylation levels of the WIF1 gene were increased. Sodium arsenite inhibited cell proliferation and promoted cell migration as demonstrated in cell functional assays. The gene status was compared in 8 human urothelial cell lines, and WIF1 mRNA expression levels were determined to be higher, whereas DNA CpG methylation levels were lower in SV-HUC1 cells compared with those noted in the other 7 bladder cancer cell lines. In summary, the data indicated that sodium arsenite decreased WIF1 gene expression and promoted cell migration. The increased methylation levels of WIF1 DNA CpG could be a potential biomarker for bladder cancer.

Biobank Establishment and Sample Management in the Tohoku Medical Megabank Project.

The Tohoku Medical Megabank biobank (TMM biobank) is the first major population-based biobank established in Japan. The TMM biobank was established based on two population cohorts and is a reconstruction program from the Great East Japan Earthquake and Tsunami of 2011. The biobank stores more than 3.4 million tubes of biospecimens and associated health and analytic data obtained from approximately 150,000 TMM cohort participants between May 2013 and December 2018, and the TMM biobank currently shares high-quality specimens and data. Various biospecimens, including peripheral and cord blood mononuclear cells, buffy coat, plasma, serum, urine, breast milk and saliva have been collected in the TMM biobank. To minimize human error and maintain the quality of data and specimens, we have been utilizing laboratory information management system into various biobank procedures from registration to storage with various automation systems, such as liquid dispensing, DNA extraction and their storage. The biobank procedures for the quality management system (ISO 9001:2015) and information security management system (ISO 27001:2013) are certified by the International Organization for Standardization. The quality of our biobank samples fulfills the pre-analytical requirements for researchers conducting next-generation whole genome sequencing, DNA array analyses, proteomics, metabolomics, etc. We established analytical centers to conduct standard genomic and multiomic analyses in-house and share the generated data. Additionally, we generate thousands of Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines and proliferating T cells for functional studies. The TMM biobank serves as an indispensable infrastructure for academic, clinical and industrial research to actualize next-generation medicine in Japan.

Neonatal rotenone lesions cause onset of hyperactivity during juvenile and adulthood in the rat

Attention deficit hyperactivity disorder (ADHD) is characterized by behavioral and cognitive symptoms. Longitudinal studies demonstrated that the symptoms remains clinically significant for the majority of ADHD children into adulthood. Furthermore, a population-based birth cohort provided the initial evidence of adult ADHD that lacks a history of childhood ADHD. We previously demonstrated that neonatal exposure to bisphenol A, an environmental chemical caused hyperactivity in the juvenile. Here, we extend to examine other chemical such as rotenone, a dopaminergic toxins. Oral administration of rotenone (3mg/kg) into 5-day-old male Wistar rats significantly caused hyperactivity at adulthood (8∼11 weeks old; p<0.05). It was about 1.3∼1.4-fold more active in the nocturnal phase after administration of rotenone than control rats. Higher dose (16mg/kg) or repeated lower dose of rotenone (1mg/kg/day for 4days) caused hyperactivity in the juvenile. Furthermore, DNA array analyses showed that neonatal exposure to rotenone altered the levels of gene expression of several molecules related to apoptosis/cell cycle, ATPase, skeletal molecule, and glioma. Bivariate normal distribution analysis indicates no correlation in gene expression between a hyperactivity disorder model and a Parkinson’s disease model by rotenone.Thus, we demonstrate a rotenone models of ADHD whose onset varies during juvenile and adulthood.

An oxygen-permeable spheroid culture chip (Oxy chip) promotes osteoblastic differentiation of mesenchymal stem cells

Mesenchymal stem cells (MSCs) are being clinically used for bone and cartilage regeneration. However, the preparation of MSCs for implantation is still costly and time consuming, and controlling the differentiation of stem cells remains a challenge. Although much attention has been paid to three-dimensional cultures in the fields of tissue engineering and regenerative medicine, adequate oxygen supply remains a challenge for growing thicker and larger cellular constructs. To solve this problem, we have developed an oxygen-permeable spheroid culture device (Oxy chip) that enables direct oxygen supply to the cells. The aim of this study was to examine the effect of a three-dimensional culture and oxygenation to the cells on the differentiation of mouse MSC strain D1 cells. Our data demonstrated that MSCs grown in the Oxy chip differentiated into osteoblasts more quickly and efficiently than those grown in the conventional non-oxygen permeable chip and monolayer culture. DNA array and energy metabolism analyses revealed that the Oxy chip facilitated osteoblastic differentiation and aerobic glycolysis, rather than chondrogenic differentiation and anaerobic glycolysis. Together, we revealed for the first time that the oxygenation by the Oxy chip was effective on the osteoblastic differentiation and survival of three-dimensional cultured MSCs. This chip is useful for preparing differentiated cells and controlling the direction of differentiation of MSCs. Moreover, this approach may be useful for transitioning spheroid cultures as a therapy in regenerative medicine.

Metabolic role of dipeptidyl peptidase 4 (DPP4) in primary human (pre)adipocytes.

Dipeptidyl peptidase 4 (DPP4) is the target of the gliptins, a recent class of oral antidiabetics. DPP4 (also called CD26) was previously characterized in immune cells but also has important metabolic functions which are not yet fully understood. Thus, we investigated the function of DPP4 in human white preadipocytes and adipocytes. We found that both cell types express DPP4 in high amounts; DPP4 release markedly increased during differentiation. In preadipocytes, lentiviral DPP4 knockdown caused significant changes in gene expression as determined by whole-genome DNA-array analysis. Metabolic genes were increased, e.g. PDK4 18-fold and PPARγC1α (=PGC1α) 6-fold, and proliferation-related genes were decreased (e.g. FGF7 5-fold). These effects, contributing to differentiation, were not inhibited by the PPARγ antagonist T0070907. Vice versa, the PPARγ agonist pioglitazone induced a different set of genes (mainly FABP4). DPP4 knockdown also affected growth factor signaling and, accordingly, retarded preadipocyte proliferation. In particular, basal and insulin-induced ERK activation (but not Akt activation) was markedly diminished (by around 60%). This indicates that DPP4 knockdown contributes to adipocyte maturation by mimicking growth factor withdrawal, an early step in fat cell differentiation. In mature adipocytes, DPP4 becomes liberated so that adipose tissue may constitute a relevant source of circulating DPP4.

Abstract A108: TAS-115, a VEGFR/MET-targeted kinase inhibitor, potently suppresses HGF-induced aggressive bone and systemic metastasis in HGF knock-in mice bearing UM-UC-3 bladder tumors

Abstract Background: Invasive bladder cancer frequently shows bone and systemic metastasis. Thus, development of a drug that is effective not only against the primary lesion but also the metastatic lesions is needed for therapy of bladder cancer. The expression level of hepatocyte growth factor (HGF) is high in invasive bladder cancer patients and associated with poor outcomes. Moreover, several recent clinical reports show that HGF receptor (MET) expression level was related to cancer grade, stage, tumor size, and poor prognosis in invasive bladder cancer patients. Because HGF-MET signaling has shown a species difference between humans and mice, the usual human cancer xenotransplantation models using nude mice are inadequate to evaluate the effect of agents against tumor growth and metastasis driven by hHGF-MET paracrine signaling. Thus, hHGF knock-in (hHGF KI) mice were generated, and the antitumor efficacy of TAS-115, a MET/VEGFR-targeted kinase inhibitor, was evaluated in a systemic metastasis model of bladder cancer. Material and Methods: A luciferase-transfected human bladder cancer cell line (UM-UC-3-luc) was established. Cell proliferation, invasion, and gene expression analyses were conducted in UM-UC-3-luc after HGF stimulation using Cell titer Glo, a migration assay kit, and DNA array analyses. In the in vivo metastasis model, UM-UC-3-luc cells were injected into the left ventricular cavity of hHGF KI mice. Luciferase activity was measured under deep anesthesia once a week as an index of tumor growth. TAS-115 was administered orally once daily for 4 weeks, when photons emitted from tumor-transplanted regions reached the order of 107. Results: In vitro, HGF significantly enhanced proliferation and invasion of UM-UC-3-luc cells through MET activation. Based on DNA array analyses, several tumor metastasis and invasion-related genes (MMPs, chemokines, transcription factors, and phosphatases) were changed in UM-UC-3-luc cells after HGF stimulation. TAS-115 completely inhibited HGF-driven proliferation and invasion activity in UM-UC-3-luc cells and blocked HGF-induced expression changes of tumor metastasis and invasion-related genes in in vivo xenograft models. Tumorigenicity and metastasis of UM-UC-3-luc cells showed approximately 60% increase in hHGF KI mice compared with WT mice. The major metastatic sites were lower jaw bone, adrenal gland, lymph node, and ovary. Tumor metastasis-related genes in UM-UC-3-luc cell-bearing hHGF KI mice were also changed, as in the in vitro experiments. TAS-115 suppressed &amp;gt;80% of systemic metastasis of UM-UC-3-luc cells in both hHGF KI and WT mice without severe body weight loss. Notably, the more aggressive metastasis of UM-UC-3-luc cells in hHGF KI mice was clearly inhibited by TAS-115 treatment. TAS-115 inhibited phosphorylation of MET and regulated tumor metastasis-related gene expression in metastasized tumor tissue. Conclusions:HGF provided a more aggressive phenotype related to tumor progression to UM-UC-3-luc cells in this in vivo models. TAS-115, a VEGFR/MET-targeted kinase inhibitor, significantly suppressed tumor progression and systemic metastasis in UM-UC-3-luc cell-bearing hHGF KI mice. TAS-115 appears to be a novel therapeutic strategy for patients with advanced bladder cancer. Citation Format: Yukari Yamada, Hidenori Fujita, Yayoi Fujioka, Naomoto Harada, Akihiro Hashimoto, Tomonori Haruma, Shingo Tsuzi, Ryouto Fujita, Kenichi Matsuo, Teruhiro Utsugi, Kazuhiko Yonekura. TAS-115, a VEGFR/MET-targeted kinase inhibitor, potently suppresses HGF-induced aggressive bone and systemic metastasis in HGF knock-in mice bearing UM-UC-3 bladder tumors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A108.

Fermentation and alternative respiration compensate for NADH dehydrogenase deficiency in a prokaryotic model of DJ-1-associated Parkinsonism.

YajL is the closest prokaryotic homologue of Parkinson's disease-associated DJ-1, a protein of undefined function involved in the oxidative stress response. We reported recently that YajL and DJ-1 protect cells against oxidative stress-induced protein aggregation by acting as covalent chaperones for the thiol proteome, including the NuoG subunit of NADH dehydrogenase 1, and that NADH dehydrogenase 1 activity is negligible in the yajL mutant. We report here that this mutant compensates for low NADH dehydrogenase activity by utilizing NADH-independent alternative dehydrogenases, including pyruvate oxidase PoxB and d-amino acid dehydrogenase DadA, and mixed acid aerobic fermentations characterized by acetate, lactate, succinate and ethanol excretion. The yajL mutant has a low adenylate energy charge favouring glycolytic flux, and a high NADH/NAD ratio favouring fermentations over pyruvate dehydrogenase and the Krebs cycle. DNA array analysis showed upregulation of genes involved in glycolytic and pentose phosphate pathways and alternative respiratory pathways. Moreover, the yajL mutant preferentially catabolized pyruvate-forming amino acids over Krebs cycle-related amino acids, and thus the yajL mutant utilizes pyruvate-centred respiro-fermentative metabolism to compensate for the NADH dehydrogenase 1 defect and constitutes an interesting model for studying eukaryotic respiratory complex I deficiencies, especially those associated with Alzheimer's and Parkinson's diseases.

Derivation of iPSCs after Culture of Human Dental Pulp Cells under Defined Conditions

Human dental pulp cells (hDPCs) are a promising resource for regenerative medicine and tissue engineering and can be used for derivation of induced pluripotent stem cells (iPSCs). However, current protocols use reagents of animal origin (mainly fetal bovine serum, FBS) that carry the potential risk of infectious diseases and unwanted immunogenicity. Here, we report a chemically defined protocol to isolate and maintain the growth and differentiation potential of hDPCs. hDPCs cultured under these conditions showed significantly less primary colony formation than those with FBS. Cell culture under stringently defined conditions revealed a donor-dependent growth capacity; however, once established, the differentiation capabilities of the hDPCs were comparable to those observed with FBS. DNA array analyses indicated that the culture conditions robustly altered hDPC gene expression patterns but, more importantly, had little effect on neither pluripotent gene expression nor the efficiency of iPSC induction. The chemically defined culture conditions described herein are not perfect serum replacements, but can be used for the safe establishment of iPSCs and will find utility in applications for cell-based regenerative medicine.

New type of Sendai virus vector provides transgene-free iPS cells derived from chimpanzee blood.

Induced pluripotent stem cells (iPSCs) are potentially valuable cell sources for disease models and future therapeutic applications; however, inefficient generation and the presence of integrated transgenes remain as problems limiting their current use. Here, we developed a new Sendai virus vector, TS12KOS, which has improved efficiency, does not integrate into the cellular DNA, and can be easily eliminated. TS12KOS carries KLF4, OCT3/4, and SOX2 in a single vector and can easily generate iPSCs from human blood cells. Using TS12KOS, we established iPSC lines from chimpanzee blood, and used DNA array analysis to show that the global gene-expression pattern of chimpanzee iPSCs is similar to those of human embryonic stem cell and iPSC lines. These results demonstrated that our new vector is useful for generating iPSCs from the blood cells of both human and chimpanzee. In addition, the chimpanzee iPSCs are expected to facilitate unique studies into human physiology and disease.

Reversal of vasohibin-driven negative feedback loop of vascular endothelial growth factor/angiogenesis axis promises a novel antifibrotic therapeutic strategy for liver diseases

Reversal of vasohibin-driven negative feedback loop of vascular endothelial growth factor/angiogenesis axis promises a novel antifibrotic therapeutic strategy for liver diseases

Nicotinamide N-methyltransferase knockdown protects against diet-induced obesity

In obesity and type 2 diabetes, Glut4 glucose transporter expression is decreased selectively in adipocytes. Adipose-specific knockout or overexpression of Glut4 alters systemic insulin sensitivity. Here we show, using DNA array analyses, that nicotinamide N-methyltransferase (Nnmt) is the most strongly reciprocally regulated gene when comparing gene expression in white adipose tissue (WAT) from adipose-specific Glut4-knockout or adipose-specific Glut4-overexpressing mice with their respective controls. NNMT methylates nicotinamide (vitamin B3) using S-adenosylmethionine (SAM) as a methyl donor. Nicotinamide is a precursor of NAD(+), an important cofactor linking cellular redox states with energy metabolism. SAM provides propylamine for polyamine biosynthesis and donates a methyl group for histone methylation. Polyamine flux including synthesis, catabolism and excretion, is controlled by the rate-limiting enzymes ornithine decarboxylase (ODC) and spermidine-spermine N(1)-acetyltransferase (SSAT; encoded by Sat1) and by polyamine oxidase (PAO), and has a major role in energy metabolism. We report that NNMT expression is increased in WAT and liver of obese and diabetic mice. Nnmt knockdown in WAT and liver protects against diet-induced obesity by augmenting cellular energy expenditure. NNMT inhibition increases adipose SAM and NAD(+) levels and upregulates ODC and SSAT activity as well as expression, owing to the effects of NNMT on histone H3 lysine 4 methylation in adipose tissue. Direct evidence for increased polyamine flux resulting from NNMT inhibition includes elevated urinary excretion and adipocyte secretion of diacetylspermine, a product of polyamine metabolism. NNMT inhibition in adipocytes increases oxygen consumption in an ODC-, SSAT- and PAO-dependent manner. Thus, NNMT is a novel regulator of histone methylation, polyamine flux and NAD(+)-dependent SIRT1 signalling, and is a unique and attractive target for treating obesity and type 2 diabetes.

Abstract 089: Response Gene To Complement 32 Is Involved In Glucose Metabolism Disorder In Endothelial Cells And Mediated By P53

Response gene to complement 32 (RGC-32) was considered as an apoptosis-promoting factor. To explore the novel functions of RGC-32, a DNA array analysis was performed in Human Microvascular Endothelial Cells (HMEC) treated with SiRNA, which showed that glucose metabolism was one of most dramatically changed biological process. qPCR analysis also proved that many glucose metabolism related genes were changed dramatically in RGC32 o/e HMEC, one of which was Insulin-like growth factor binding protein 4(IGFBP4). We propose that RGC-32 may be a potential factor involved in the cycle of endothelial glucose metabolism besides glucose induced apoptosis. Then we found that expression of RGC-32 was dramatically increased in mice hearts and adipose tissue after high fat diet treatment, as well as in HMEC after high glucose (30mM) treatment. In high fat diet treated endothelial specific RGC-32 transgenic mice (VE-Cad/RGC-32), decreases in area under the curve (AUC) of glucose tolerance test were observed when compared to their wild type littermates. Upregulated P53 and downregulated IGFBP4 mRNA expression level were also observed in the heart of VE-Cad/RGC-32 mice.Furthermore, we found that P53 was upregulated in RGC-32 o/e HMEC and blocking of p53 was able to decrease the RGC-32-related induction of apoptotic cells. We also observed that RGC-32 can not only decrease IGFBP4 but also reverse the decrease of IGFBP4 in high glucose or insulin condition. Interestingly, after treated the cells with pifithrin-a, a specific P53 inhibitor, decreased IGFBP4 by RGC-32 was rescued. These results demonstrate that RGC-32 may have some kind of connections with glucose metabolism besides its well-known apoptosis-promoting characters and P53 may be a mediator of RGC-32 related glucose metabolism disorder as well as apoptosis.These findings will provide some clues for novel insights into the functions of RGC-32.

FRI0208 Prediction of treatment response of tocilizumab for rheumatoid arthritis with comprehensive gene expression analysis in peripheral blood mononuclear cells

BackgroundTocilizumab (TCZ) is a biological agent which is highly efficacious for rheumatoid arthritis (RA). Although predicting treatment response to TCZ can be especially beneficial since TCZ takes longer time to...

Hepatic Artery Reconstruction Prevents Ischemic Graft Injury, Inhibits Graft Rejection, and Mediates Long-term Graft Acceptance in Rat Liver Transplantation

BackgroundHepatic artery (HA) reconstruction is performed in the clinical liver transplantation. MethodsWe assessed the importance of HA reconstruction in the success of liver transplantation. Orthotopic liver transplantation was performed without immunosspression from Lewis (RT1l) to Lewis rats (syngeneic transplantation) as well as Lewis to BN (RT1n) rats (allogeneic transplantation) with or without HA reconstruction. We examined graft function, pathology, and mRNA levels using DNA arrays in both arterialized and nonarterialized liver grafts. ResultsIn Lewis-to-Lewis syngeneic grafts, both the arterialized and nonarterialized grafts survived >120 days with normal graft function. lnfiltration of CD3+ T cells and CD68+ macrophages, marked bile duct proliferation with apoptotic epithelial cells, and expansion and increasing fibrosis of portal areas were evident in the nonarterialized grafts at day 120, although preservation of architecture was noted in the arterialized grafts. DNA array analysis of nonarterialized syngeneic grafts demonstrated the upregulation of mRNA of cell death-related proteins, cell cycle–related proteins, and inflammation-related proteins than those in arterialized grafts. Moreover, the arterialized Lewis-to-BN allogeneic grafts could survive for a long time with less severe graft dysfunction than those in non-arterialized allogeneic grafts. ConclusionsHA reconstruction in liver transplantation inhibited hypoxic injury and subsequent inflammation and bile duct proliferation, prevented the augmentation of T-cell–and antibody-mediated rejection, and mediated long-term graft acceptance. HA reconstruction is essential factor in the success of liver transplantation.

Transcriptional and post-translational regulation of mouse cation transport regulator homolog 1

Recently, cation transport regulator homolog 1 (Chac1) has been identified as a novel pro-apoptotic factor in cells under endoplasmic reticulum (ER) stress. Of the three major ER stress sensors, it is suggested that ATF4 participates in the transcriptional regulation of Chac1 gene expression. The precise characterization of the Chac1 promoter, however, has not yet been elucidated. In this study, we detected the induction of Chac1 mRNA expression using DNA array analysis and RT-PCR of thapsigargin (Tg)-inducible genes in Neuro2a cells. Chac1 mRNA expression was also induced immediately following treatment with tunicamycin (Tm) and brefeldin A. Characterization of the mouse Chac1 promoter activity using a luciferase reporter assay revealed that the CREB/ATF element and amino acid response element in the mouse Chac1 promoter are functional and respond to Tm stimulation and ATF4 overexpression. Mutations in either element in the Chac1 promoter did not inhibit the responsiveness of this promoter to Tm and ATF4; however, mutations in both of these elements dramatically decreased the basal activity and response to ER stress stimuli. In addition to the transcriptional regulation, we found that Chac1 protein expression was only detected in the presence of MG132, a proteasome inhibitor, even though mouse Chac1 gene was transiently overexpressed in Neuro2a cells. Taken together, we are the first to demonstrate the transcriptional and post-translational regulation of Chac1 expression in a neuronal cell line.

Vascular smooth muscle cells isolated from adipose triglyceride lipase-deficient mice exhibit distinct phenotype and phenotypic plasticity

The alteration of triglyceride (TG) metabolism in vascular smooth muscle cells (SMC) is likely to be correlated with certain phenotype, though this has not been elucidated. Adipose triglyceride lipase (ATGL) exerts major TG catalytic activity in both adipotic and non-adipotic cells. In the present study, we isolated SMC from ATGL-deficient mice (ATGL−/−mSMC). ATGL−/−mSMC showed spontaneous TG accumulation with lower mitogenic response and smooth muscle actin (SMA) expression compared to ATGL +/+mSMC. Percentage of senescence-associated β-galactosidase positive cells was also increased in ATGL−/−mSMC. Real-time PCR followed by screening with focused DNA array analysis revealed up-regulated expression of glucokinase (1.7-fold), lipoprotein lipase (3.8-fold) and interleukin-6 (3.7-fold) and down-regulated expression of vascular endothelial growth factor-A (0.2-fold), type I collagen (0.5-fold), and transforming growth factor-β (0.4-fold) in ATGL−/−mSMC compared to ATGL+/+mSMC. Next, ectopic gene transfer of human ATGL was attempted using doxycycline (Dox)-regulatable myc-DDK-tagged adenovirus vector (AdvATGL). AdvATGL infection resulted in a reduction of TG accumulation with elevated mitogenic response and SMA expression, and decreased in senescent cell numbers in ATGL−/−mSMC. Moreover, deviated gene expression pattern in ATGL−/−mSMC was potentially corrected. Our data suggest that ATGL−/−mSMC have a distinct phenotype that may be related to vascular pathogenesis. Plasticity of SMC phenotypes correlated to lipid metabolism could be a therapeutic target.