Significant Increase In mRNA Research Articles

Role of IL-13Rα2 in modulating IL-13-induced MUC5AC and ciliary changes in healthy and CRSwNP mucosa.

The IL-13 receptor α2 (IL-13Rα2) is a receptor for IL-13 which has conflicting roles in mediating IL-13 responses in the lower airway, with little known about its impact on upper airway diseases. We sought to investigate the expression of IL-13 receptors, IL-13Rα1 and IL-13Rα2, in chronically inflamed nasal epithelium, and explore IL-13-induced signaling pathways in an invitro model of human nasal epithelial cells (hNECs). The protein and mRNA expression levels of IL-13 and its receptors in nasal biopsies of patients with nasal polyps (NP) and healthy controls were evaluated. We investigated goblet cell stimulation with mucus hypersecretion induced by IL-13 (10ng/mL, 72hours) treatment in hNECs using a pseudostratified epithelium in air-liquid interface (ALI) culture. There were significant increases in IL-13, IL-13Rα1, and IL-13Rα2 mRNA and protein levels in NP epithelium with healthy controls as baseline. MUC5AC mRNA positively correlated with IL-13Rα2 (r=.5886, P=.002) but not with IL-13Rα1 in primary hNECs. IL-13 treatment resulted in a significant increase in mRNA and protein levels of IL-13Rα2 only in hNECs. IL-13 treatment induced an activation of extracellular signal-regulated kinases (ERK)1/2 and an upregulation of C-JUN, where the IL-13-induced effects on hNECs could be attenuated by ERK1/2 inhibitor (50μmol/L) or dexamethasone (10-4 -10-7 mol/L) treatment. IL-13Rα2 has a potential role in IL-13-induced MUC5AC and ciliary changes through ERK1/2 signal pathway in the nasal epithelium. IL-13Rα2 may contribute to airway inflammation and aberrant remodeling which are the main pathological features of CRSwNP.

Xanthohumol exerts protective effects in liver alterations associated with aging.

Aging is associated with a deregulation of biological systems that lead to an increase in oxidative stress, inflammation, and apoptosis, among other effects. Xanthohumol is the main preylated chalcone present in hops (Humulus lupulus L.) whose antioxidant, anti-inflammatory and chemopreventive properties have been shown in recent years. In the present study, the possible protective effects of xanthohumol on liver alterations associated with aging were evaluated. Male young and old senescence-accelerated prone mice (SAMP8), aged 2 and 10 months, respectively, were divided into four groups: non-treated young, non-treated old, old treated with 1mg/kg/day xanthohumol, and old treated with 5mg/kg/day xanthohumol. Male senescence-accelerated resistant mice (SAMR1) were used as controls. After 30 days of treatment, animals were sacrificed and livers were collected. mRNA (AIF, BAD, BAX, Bcl-2, eNOS, HO-1, IL-1β, NF-κB2, PCNA, sirtuin 1 and TNF-α) and protein expressions (BAD, BAX, AIF, caspase-3, Blc-2, eNOS, iNOS, TNF-α, IL1β, NF-κB2, and IL10) were measured by RT-PCR and Western blotting, respectively. Mean values were analyzed using ANOVA. A significant increase in mRNA and protein levels of oxidative stress, pro-inflammatory and proliferative markers, as well as pro-apoptotic parameters was shown in old non-treated SAMP8 mice compared to the young SAMP8group and SAMR1 mice. In general, age-related oxidative stress, inflammation and apoptosis were significantly decreased (p < 0.05) after XN treatment. In most cases, this effect was dose-dependent. XN was shown to modulate inflammation, apoptosis, and oxidative stress in aged livers, exerting a protective effect in hepatic alterations.

Implication of the PI3K/Akt/mTOR Pathway in the Process of Incompetent Valves in Patients with Chronic Venous Insufficiency and the Relationship with Aging.

Chronic venous insufficiency (CVI) is a multifactorial disease, commonly caused by valvular incompetence (clinically diagnosed by venous reflux) and venous hypertension. The incidence of these factors clearly increases with patient age, and aging is one of the risk factors involved. The activity of the PI3K/Akt/mTOR pathway is considered fundamental in vascular pathologies, and understanding its involvement would help in the development of possible therapeutic targets. This is an observational, analytical, and prospective cohort study that reviewed 110 patients with CVI scheduled to undergo stratified saphenectomy. They were distributed according to the presence (R = 81) or absence (NR = 29) of valvular incompetence (venous reflux) diagnosed clinically. Each of the groups was further divided according to age, with a cutoff point of 50 years (NR < 50 = 13, NR ≥ 50 = 16, R < 50 = 32, and R ≥ 50 = 49). The involvement of the PI3K/Akt/mTOR pathway, as well as that of HIF-1α and HIF-2α and of CD4+, CD8+, and CD19+ cells and mastocytes, was assessed. Saphenous vein tissue samples obtained during surgery were processed for RT-qPCR and immunohistochemistry. Patients with venous reflux showed a significant increase in mRNA and protein expression levels for PI3K/mTOR and HIF-1α/HIF-2α. The number of mast cells was significantly elevated in the R group. In distribution by age, PI3K/Akt/mTOR and HIF-1α were significantly higher in R < 50 patients. Furthermore, these patients had a significant increase in the number of CD4+, CD8+, and CD19+ cells and mastocytes in the saphenous vein wall. These findings provide a basis for the possible existence of changes in PI3K/Akt/mTOR pathway expression in young patients, with potential accelerated asynchronous aging that is enhanced by CVI.

Polyamine concentration is increased in thoracic ascending aorta of patients with bicuspid aortic valve.

Polyamines are cationic molecules synthesized via a highly regulated pathway, obtained from the diet or produced by the gut microbiota. They are involved in general molecular and cellular phenomena that play a role also in vascular disease. Bicuspid aortic valve (BAV) is a congenital malformation associated to a greater risk of thoracic ascending aorta (TAA) aneurysm, whose pathogenesis is not yet well understood. We focused on differential analysis of key members of polyamine pathway and on polyamine concentration in non-dilated TAA samples from patients with either stenotic tricuspid aortic valve (TAV) or BAV (diameter≤45mm), vs. normal aortas from organ donors, with the aim of revealing a potential involvement of polyamines in early aortopathy. Changes of gene expression in TAA samples were evaluated by RT-PCR. Changes of ornithine decarboxylase 1 (ODC1), a key enzyme in polyamine formation, and cationic amino acid transporter 1 (SLC7A1/CAT-1) expression were analyzed also by Western blot. ODC1 subcellular localization was assessed by immunohistochemistry. Polyamine concentration in TAA samples was evaluated by HPLC. BAV TAA samples showed an increased concentration of putrescine and spermidine vs. TAV and donor samples, together with a decreased mRNA level of polyamine anabolic enzymes and of the putative polyamine transporter SLC7A1/CAT-1. The catabolic enzyme spermidine/spermine N1-acetyltransferase 1 showed a significant mRNA increase in TAV samples only, together with a decreased concentration of spermine. The decreased expression of SLC7A1/CAT-1 and ODC1 mRNAs in BAV corresponded to increased or unchanged expression of the respective proteins. ODC was located mainly in smooth muscle cell (SMC) nucleus in TAV and donor samples, while it was present also in SMC cytoplasm in BAV samples, suggesting its activation. In conclusion, BAV, but not TAV non-dilated samples show increased polyamine concentration, accompanied by the activation of a regulatory negative feedback mechanism.

Ginsenoside Rg1 improves ischemic brain injury by balancing mitochondrial biogenesis and mitophagy

Purpose: To study the effects of ginsenoside Rg1 on mitochondrial dysfunction induced by ischemic stroke.Methods: Human neuroblastoma SK-N-SH cells, subjected to oxygen-glucose deprivation (OGD), were divided into six groups: control group, OGD group, 3 OGD + Rg1 groups (6.25, 12.5 and 25 μM), and Rg1 (25 μM) group. Apoptosis rate, intracellular production of reactive oxygen species (ROS), and mitochondrial transmembrane potential (MTP) in the OGD cells treated with different concentrations of Rg1 were determined. The mRNA and protein expression levels of mitochondrial biogenesis-related transcription factors and autophagy-related proteins were determined by reat time-polymerase chain reaction (RT-PCR) and Western blotting.Results: ROS production was significantly increased in OGD SK-N-SH cells (p &lt; 0.01), but this was reversed by Rg1 treatment (p &lt; 0.05). Rg1-treated cells had significantly higher MTP when compared with OGD cells (p &lt; 0.01). Rg1 treatment led to significant increases in mRNA and protein expression levels of PGC1-α, NRF-1, and TFAM-1 (p &lt; 0.01). Moreover, Rg1 treatment inhibited apoptosis in SKN- SH cells, and up-regulated autophagy-related proteins in t neuronal injury model. Treatment with autophagy inhibitors decreased the mitochondrial protective effects exerted by Rg1 in OGD SK-N-SH cells.Conclusion: Rg1 improves mitochondrial dysfunction by regulating autophagy in mitochondria. Thus, it may offer protection from brain injuries caused by cerebral ischemia. Keywords: Cerebral ischemia, Ginsenoside Rg1, Mitochondrial dysfunction, Mitophagy

Abstract 21011: Crispr/ndcas9 Adenoviral Vector System as a Tool for Promoting Mitochondrial Biogenesis in Cardiomyocytes

Mitochondrial dysfunction contributes to heart failure. Our recent work revealed that genes in mitochondrial biogenesis and quality control, such as the peroxisome proliferator activated receptor gamma coactivator-1α(PGC-1α) and mitochondrial transcription factor A(TFAM), are downregulated in a rodent model of heart failure. Here, we apply CRISPR/Cas9 technology as a transcriptional activator to target sequences upstream of the start site to promote transcription of PGC-1α or TFAM. Since cardiomyocytes are difficult to transduce, we developed an adenoviral gene delivery system for expression of the CRISPR/Cas9 activating complex and a targeting gRNA to modulate expression of any gene of interest in differentiated cardiomyocytes, with our initial focus on activating the metabolic gene program. We use nuclease-dead Cas9 (ndCas9) fused to an activation domain VP64 and guide RNAs for activation of target genes. Proof-of-principle evidence was obtained by activating a cyclic AMP response element (CRE)-driven luciferase reporter gene and activation of endogenous CRE-regulable genes (PCK1 and PGC-1α). Next, we constructed ndCas9-gRNA adenoviral vectors to target PGC-1α and TFAM in the rat genome to determine if transcription of these genes can be increased. Functional effects of viral gene transduction in H9C2 cells and neonatal rat ventricular myocytes are assessed by determining mitochondrial DNA copy number (mtDNA)/genomic DNA ratio, and mtDNA quantity by picogreen labeling. Results: We have successfully upregulated the luciferase gene and endogenous CRE-regulated gene PCK1 in the same reporter cell line with ndCas9-VP64, as evidenced by significant increases in mRNA and protein expression levels. We have constructed and delivered adenoviral vectors with ndCas9 that are efficiently expressed by the host cells (NRVMs and H9c2 cells) and showed increased expression of PGC1a and TFAM in H9c2 cells. Importantly, an increase in mtDNA copy number was evoked by TFAM activation. For the first time, we show that the CRISPR/ndCas9 system can be used as a powerful tool for modulating transcription of native genes involved in mitochondrial biogenesis in cardiac cells, opening the door to developing novel therapeutic approaches for heart failure

Study of the Role of CREB, BDNF, and VGF Neuropeptide in Long Term Antidepressant Activity of Crocin in the Rat Cerebellum.

Antidepressant activity of crocin, saffron main component, has been established before. Based on previous study, it is suggested that elevation in the levels of BDNF (brain-derived neurotrophic factor), CREB (cAMP response element binding) and VGF neuropeptide could be considered as one probable molecular mechanisms involved in antidepressant activity of long term crocin administration in the rat hippocampus. In this study we further investigated whether the antidepressant activity of crocin in long term administration was associated with alteration in these factors in the rat cerebellum. Crocin (12.5, 25 and 50 mg/kg/day) and imipramine (10 mg/kg/day) were administered interaperitoneally for 21 days to rats. At the end of experiment, animals were sacrificed and cerebellums were dissected. BDNF, VGF, CREB, and phospho-CREB (P-CREB) protein and mRNA levels in the rat cerebellum were evaluated using Western blot and quantitative reverse transcription-polymerase chain reaction (qRT-PCR). In the current study significant increases in mRNA and protein levels of VGF, CREB and (BDNF) in long term crocin treatment were not observed in the rat cerebellum. Although a slight increase was observed in protein level of P-CREB compared to normal saline, but it was not significant. It is concluded that antidepressant activity of crocin might be partially mediated to CREB. Moreover, other factors rather than BDNF and VGF neuropeptides may alter following long term crocin treatment in the cerebellum. To understand the precise mechanism of crocin antidepressant effects in the cerebellum, longer duration of crocin treatment in further studies is recommended.

Intracranial Injection of Dengue Virus Induces Interferon Stimulated Genes and CD8+ T Cell Infiltration by Sphingosine Kinase 1 Independent Pathways.

We have previously reported that the absence of sphingosine kinase 1 (SK1) affects both dengue virus (DENV) infection and innate immune responses in vitro. Here we aimed to define SK1-dependancy of DENV-induced disease and the associated innate responses in vivo. The lack of a reliable mouse model with a fully competent interferon response for DENV infection is a challenge, and here we use an experimental model of DENV infection in the brain of immunocompetent mice. Intracranial injection of DENV-2 into C57BL/6 mice induced body weight loss and neurological symptoms which was associated with a high level of DENV RNA in the brain. Body weight loss and DENV RNA level tended to be greater in SK1-/- compared with wildtype (WT) mice. Brain infection with DENV-2 is associated with the induction of interferon-β (IFN-β) and IFN-stimulated gene (ISG) expression including viperin, Ifi27l2a, IRF7, and CXCL10 without any significant differences between WT and SK1-/- mice. The SK2 and sphingosine-1-phosphate (S1P) levels in the brain were unchanged by DENV infection or the lack of SK1. Histological analysis demonstrated the presence of a cellular infiltrate in DENV-infected brain with a significant increase in mRNA for CD8 but not CD4 suggesting this infiltrate is likely CD8+ but not CD4+ T-lymphocytes. This increase in T-cell infiltration was not affected by the lack of SK1. Overall, DENV-infection in the brain induces IFN and T-cell responses but does not influence the SK/S1P axis. In contrast to our observations in vitro, SK1 has no major influence on these responses following DENV-infection in the mouse brain.

Cytochrome P450 26A1 modulates natural killer cells in mouse early pregnancy.

Cytochrome P450 26A1 (CYP26A1) has a spatiotemporal expression pattern in the uterus, with a significant increase in mRNA and protein levels during peri‐implantation. Inhibiting the function or expression of CYP26A1 can cause pregnancy failure, suggesting an important regulatory role of CYP26A1 in the maintenance of pregnancy. However, little is known about the exact mechanism involved. In this study, using a pCR3.1‐cyp26a1 plasmid immunization mouse model and a Cyp26a1‐MO (Cyp26a1‐specific antisense oligos) knockdown mouse model, we report that the number of Dolichos biflorus agglutinin (DBA) lectin‐positive uterine natural killer (uNK) cells was reduced in pCR3.1‐cyp26a1 plasmid immunized and Cyp26a1‐MO‐treated mice. In contrast, the percentage of CD3− CD49b+ NK cells in the uteri from the treatment group was significantly higher than that of the control group in both models. Similarly, significantly up‐regulated expression of CD49b (a pan‐NK cell marker), interferon gamma, CCL2, CCR2 (CCL2 receptor) and CCL3 were detected in the uteri of pCR3.1‐cyp26a1‐ and Cyp26a1‐MO‐treated mice. Transcriptome analysis suggested that CYP26A1 might regulate NK cells through chemokines. In conclusion, the present data suggest that silencing CYP26A1 expression/function can decrease the number of uNK cells and significantly increase the percentage of CD3− CD49b+ NK cells in the uteri of pregnant mice. These findings provide a new line of evidence correlating the deleterious effects of blocking CYP26A1 in pregnancy with the aberrant regulation of NK cells in the uterus.

Hypoxia-Inducible Factor-1α Mediates Increased Sympathoexcitation via Glutamatergic N-Methyl-d-Aspartate Receptors in the Paraventricular Nucleus of Rats With Chronic Heart Failure.

Increased sympathetic outflow is a major contributor to the progression of chronic heart failure (CHF). Potentiation of glutamatergic tone has been causally related to the sympathoexcitation in CHF. Specifically, an increase in the N-methyl-d-aspartate-type 1 receptor (NMDA-NR1) expression within the paraventricular nucleus (PVN) is critically linked to the increased sympathoexcitation during CHF. However, the molecular mechanism(s) for the upregulation of NMDA-NR1 remains unexplored. We hypothesized that hypoxia via hypoxia-inducible factor 1α (HIF-1α) might contribute to the augmentation of the NMDA-NR1-mediated sympathoexcitatory responses from the PVN in CHF. Immunohistochemistry staining, mRNA, and protein for hypoxia-inducible factor 1α were upregulated within the PVN of left coronary artery-ligated CHF rats. In neuronal cell line (NG108-15) in vitro, hypoxia caused a significant increase in mRNA and protein for HIF-1α (2-fold) with the concomitant increase in NMDA-NR1 mRNA, protein levels, and glutamate-induced Ca+ influx. Chromatin immunoprecipitation assay identified HIF-1α binding to NMDA-NR1 promoter during hypoxia. Silencing of HIF-1α in NG108 cells leads to a significant decrease in expression of NMDA-NR1, suggesting that expression of HIF-1α is necessary for the upregulation of NMDA-NR1. Consistent with these observations, HIF-1α silencing within the PVN abrogated the increased basal sympathetic tone and sympathoexcitatory responses to microinjection of NMDA in the PVN of rats with CHF. These results uncover a critical role for HIF-1 in the upregulation of NMDA-NR1 to mediate sympathoexcitation in CHF. We conclude that subtle hypoxia within the PVN may act as a metabolic cue to modulate sympathoexcitation during CHF.

Abstract P188: (Pro)renin Receptor (PRR) Mediates High Fat Diet-induced Hypertension via Upregulation of Epithelial Sodium Channel &amp; Aquaporin-2

We hypothesized that the renal prorenin receptor (PRR) mediates high fat diet (HFD)-induced hypertension via enhancing expression of epithelial sodium channel (α-ENaC) and aquaporin-2 (AQP-2). C57BL/6 mice underwent right nephrectomy (n=12) and allocated to receive regular diet (RD, 12 kcal% fat, n=6) or a high-fat diet (HFD, 45 kcal% fat, n=6) for 10 weeks. HFD group received left renal subcapsular interstitial administration of PRR shRNA (n=3) or vehicle (n=3). BW, food and water intake, BP, UV and UNaV, renal interstitial fluid (RIF) angiotensin II (Ang II), and renal expressions of PRR, α-ENaC, AQP-2 were monitored. At baseline, there were no significant differences in BW, food and water intake, BP, UV or UNaV between different animal groups. At the end of the study, HFD mice had significant increase in food intake, systolic blood pressure (HFD 154.6 ± 2.181 mmHg, vs. RD 112.21 ± 4.684 mmHg, P&lt;0.0.05), BW (HFD 43.2 ± 1.125 gm, vs. RD 31.9 ± 0.89654 gm, P&lt;0.0.05), and significant reduction in UV (HFD 0.16 ± 0.042 ml, vs. RD 0.41 ± 0.061 ml, P&lt;0.0.05) and UNaV (HFD 26.02 ± 3.652 μmol/day vs. RD 46.32 ± 5.236 μmol/day, P&lt;0.0.05). Compared to RD, there were significant increases in mRNA and protein expressions of PRR (64% and 40%, P&lt;0.01), α-ENaC (85% and 75%, P&lt;0.05), AQP-2 (105% and 80%, P&lt;0.05) in HFD alone mice respectively. Compared to HFD alone, HFD+ PRR shRNA treatment caused significant reductions in BP (108 ± 13.88 mmHg vs HFD 154.6 ± 2.181 mmHg), mRNA and protein expressions of PRR (33% and 50%, P&lt;0.01), α-ENaC (49% and 56%, P&lt;0.05), AQP-2 (30% and 29%, P&lt;0.05) respectively. There were no changes in RIF Ang II between different animal groups. We conclude that PRR mediates HFD-induced hypertension via enhancing renal α-ENaC and AQP-2 expression independent of Angiotensin II.

015 Norepinephrine (NE) biases Langerhans cell (LC) antigen presentation to T cells toward a Th17 response via actions on endothelial cells

We now report that exposure of primary dermal microvascular endothelial cells (pDMECs) to NE induces them to bias LC antigen presentation to CD4+ T cells toward generation of Th17 cells. BALB/c pDMECs (1x104/well) were plated in 96-well round bottom plates in medium containing 10-8 M to 10-5 M NE. After 3 h, plates were washed 4 times and 1x104 BALB/c LCs and 2x105 CD4+ T cells from DO11.10 mice (BALB/c background) were added per well followed by addition of a fragment of chicken ovalbumin (cOVA323-339) to 10 μM in 200 μl of medium. DO11.10 mice express transgenes in their T cell receptors that recognize cOVA323-339. After 48 h, supernatants were assayed for cytokine content by ELISA. Addition of NE-exposed pDMECs significantly enhanced IL-6 and IL-17A production while inhibiting interferon-γ (IFNγ) and IL-22 release compared to wells containing non-treated pDMECs. When T cells were harvested from these cultures and analyzed by FACS for intracellular cytokines, increased numbers of cells containing IL-17A were observed along with a decrease in cells containing IFNγ. RNA was extracted from T cells from these cultures. By quantitative RT-PCR the presence of NE-treated pDMECs led to a significant increase in mRNA for IL-17A and RORγT while IFNγ, IL-22 and T-bet mRNA were decreased. Similar experiments in which Transwell inserts were used to separate pDMECs from LCs and responding T cells showed that the effect on cytokine release did not depend on cell-cell contact. Thus, NE can influence the outcome of antigen presentation toward a Th17-type immune response through actions on ECs that induce release of a soluble mediator(s) mediating the effects. As dermal blood vessels and lymph nodes are associated with sympathetic nerves, we hypothesize that, when the sympathetic nervous system is activated by stress, NE release biases antigen presentation towards the Th17 pole via actions on ECs. This may exacerbate psoriasis and, perhaps, other inflammatory skin disorders involving Th17 cells. This pathway may prove to be a therapeutic target.

Isolation and characterization of extracellular vesicles secreted by pre-pubertal Sertoli cells

Recent studies have shown that extracellular vesicles (Ev) are an important mechanism of intercellular communication. In fact, Ev may contain proteins, DNA and mRNA. In particular, the latter play an important role in various biological processes including regulation and cell differentiation [1]. Sertoli cells (SC), previously considered as a mere “sustentacular cell”, were re-evalued in their functions and elevated to the rank of a “sentinel” in spermatogenesis due to production of trophic, differentiation and immune-modulators factors. Porcine pre-pubertal SC, isolated by our method [2], upon 48 hours culture, SC were stimulated with recombinant a-follitropin (rFSH) or FSH + testosterone (T) to evaluate both the presence in the medium of SC-derived Ev (SC-Ev) and SC-Ev content, in terms of mRNA for Anti-Mullerian hormone (AMH), inhibin B, Androgen Binding Protein (ABP) and FSH-receptor (FSH-r), by RT-PCR. SEM analysis highlighted the presence of SC-Nv in culture medium with mean diameters < 149 nm. We have also demonstrated, within the SC-Ev, significant increase in mRNA for AMH, ABP and FSH-r after both FSH and FSH+T stimulation, as compared to unstimulated SC-Ev. Differently from unstimulated SC-Ev, mRNA inhibin B levels were unchanged in FSH-stimulated SC-Ev, and increased after FSH+T stimulation. Interestingly, an opposite trend was shown in mRNA secretion, in control SC monolayer where, we demonstrated a decrease of AMH and FSH-r mRNA (after both stimulations with FSH or FSH + T) and an increase of inhibin B mRNA. On the contrary, mRNA ABP levels, in SC monolayer, decreased after stimulation with FSH but were unchanged in the presence of FSH+T. For the first time in the Literature, our work has shown the presence of SC-Nv containing AMH, inhibin B, ABP and FSH-r mRNA regulated by FSH with or without T. This result may suggest that other testicular cells could produce factors that, until now, were considered an exclusive SC secretory product.This work was supported by Mr.Gary Harlem (Altucell Inc. 3 Astor Court, Dix Hills, New York, NY) and Merck-Serono (London, UK).

Protective activity of crocin against indomethacin-induced gastric lesions in rats.

The present study was designed to elucidate the mechanism(s) of the gastro-protective effect of crocin against indomethacin-induced gastric lesions. Crocin or pantoprazole was administered to rats 30min before indomethacin. Five hours later, the animals were killed and their stomachs were removed and examined macroscopically. Samples of gastric mucosa were collected for microscopic evaluation, mRNA expression of caspase-3, inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 was quantified by RT-PCR, and protein levels of COX-1, COX-2, iNOS and caspase-3 were assessed by Western blotting. The pH, volume of gastric effluent and antioxidant activity were measured in 5 separate groups of rats following pylorus ligation. Indomethacin induced significant increases in mRNA and protein expression of iNOS and caspase-3 and increased MDA levels, and reduced the pH of the gastric effluent and protein and mRNA expression of COX-2 and protein expression of COX-1 and mucus content associated with gastric ulceration. Crocin and pantoprazole significantly inhibited mRNA and protein expression of iNOS, caspase-3 and MDA, and reduced mucus content induced by indomethacin. However, unlike pantoprazole, crocin failed to increase COX-1 and pH, but had variable increasing effects on mRNA and protein expression of COX-2. Macroscopic and microscopic observations showed that mucosal erosions induced by indomethacin were significantly inhibited by pantoprazole and crocin. These findings suggest that crocin exerts its gastro-protective effects mainly by inhibition of MDA, reduction in iNOS and caspase-3, and inhibition of the reduction in mucus content induced by indomethacin. Crocin is a novel agent that has potential in the prevention of ulceration induced by NSAIDs.

MiRNA-regulated gene expression differs in celiac disease patients according to the age of presentation.

Celiac disease is an intestinal disease which shows different symptoms and clinical manifestations among pediatric and adult patients. These variations could be imputable to age-related changes in gut architecture and intestinal immune system, which could be characterized by gene expression differences possibly regulated by miRNAs. We analyzed a panel of miRNAs and their target genes in duodenal biopsies of Marsh 3AB and 3C pediatric celiac patients, compared to controls. Moreover, to assess variation of expression in plasma samples, we evaluated circulating miRNA levels in controls and patients at diagnosis or on gluten-free diet. We detected a decreased miR-192-5p expression in celiac patients, but no variations in NOD2 and CXCL2, targets previously identified in adults. Conversely, we detected a significant increase in mRNA and protein levels of another target, MAD2L1, protein related to cell cycle control. miR-31-5p and miR-338-3p were down-regulated and their respective targets, FOXP3 and RUNX1, involved in Treg function, resulted up-regulated in celiac patients. Finally, we detected, in celiac patients, an increased expression of miR-21-5p, possibly caused by a regulatory loop with its putative target STAT3, which showed an increased activation in Marsh 3C patients. The analysis of plasma revealed a trend similar to that observed in biopsies, but in presence of gluten-free diet we could not detect circulating miRNAs values comparable to controls. miRNAs and their gene targets showed an altered expression in duodenal mucosa and plasma of celiac disease pediatric patients, and these alterations could be different from adult ones.

Simvastatin Increases Fibulin-2 Expression in Human Coronary Artery Smooth Muscle Cells via RhoA/Rho-Kinase Signaling Pathway Inhibition.

The composition and structure of the extracellular matrix (ECM) in the vascular wall and in the atherosclerotic plaque are important factors that determine plaque stability. Statins can stabilize atherosclerotic plaques by modulating ECM protein expression. Fibulins are important components of the ECM. We evaluated the in vitro effect of simvastatin on the expression of fibulin-1, -2, -4 and -5 in human coronary artery smooth muscle cells (SMCs) and the mechanisms involved. Cells were incubated with simvastatin (0.05–1 μM), mevalonate (100 and 200 μM), geranylgeranyl pyrophosphate (GGPP) (15 μM), farnesyl pyrophosphate (FPP) (15 μM), the Rho kinase (ROCK) inhibitor Y-27632 (15 and 20 μM), the Rac-1 inhibitor (another member of Rho family) NSC23766 (100 μM), arachidonic acid (a RhoA/ROCK activator, 25–100 μM) and other fatty acids that are not activators of RhoA/ROCK (25–100 μM). Gene expression was analyzed by quantitative real-time PCR, and fibulin protein levels were analyzed by western blotting and ELISA. Simvastatin induced a significant increase in mRNA and protein levels of fibulin-2 at 24 hours of incubation (p<0.05), but it did not affect fibulin-1, -4, and -5 expression. Mevalonate and GGPP were able to reverse simvastatin’s effect, while FPP did not. In addition, Y-27632, but not NSC23766, significantly increased fibulin-2 expression. Furthermore, activation of the RhoA/ROCK pathway with arachidonic acid decreased fibulin-2 mRNA. Simvastatin increased mRNA levels and protein expression of the ECM protein fibulin-2 through a RhoA and Rho-Kinase-mediated pathway. This increase could affect the composition and structure of the ECM.

72. Enhancement of Immune Responses to Measles Virotherapy with aPD-1 Therapy for Improved Efficacy in Glioblastoma Therapy

Glioblastoma (GBM) is one of the most lethal malignancies with a median survival of 12-16 months. There is urgent need for improved therapies. Use of the measles virus (MV) Edmonston vaccine strain lineage for GBM virotherapy is a promising direction due to the excellent safety record and demonstrated pre-clinical efficacy in multiple patient derived orthotopic models. GBM have been shown to be immune suppressive, subverting both the innate (da Fonseca et.al. 2013) and adaptive immune systems (Wei et.al. 2010). GBM have been shown to express PD-L1, MHC-1, and down regulate MHC-II compared to non-neoplastic tissue, thereby aiding in immune evasion and suppression. Furthermore GBM can induce PD-L1 expression on tumor associated macrophages and increase PD-1 expression on peripheral blood T cells, further bolstering an immune suppressive environment. Anti PD-1 therapy with mAB (aPD-1) blocks the binding of PD-1 to its ligands, thereby preventing T cell inactivation, and was previously reported to have synergistic effects in combination with radiation therapy in GBM (Zeng et.al. 2013). We therefore sought to combine aPD-1 therapy and MV therapy of GBM to enhance immune responses and improve therapeutic outcome. Herein we present our preliminary findings. We first sought to characterize PD-L1 expression on our primary patient derived GBM and murine GBM cells and found a significant up regulation of PD-L1 and HLA-ABC upon IFN-γ stimulation, indicating functionality of these immune evading mechanisms. Next we determined if T cells trafficked to GBM following MV therapy and whether aPD-1 therapy could affect T cell influx as assessed by MRI. Briefly, C57/Blk6 mice bearing GL261 syngeneic orthotopic GBM were treated with MV-EGFR, UV-MV-EGFR, or MV-EGFR+aPD-1 on days 5 and 9 post tumor implantation. Day 11 post tumor implantation mice received tail-vein injections of monocrystalline iron oxide particles conjugated to CD4 and CD8 mAB. MRI 24 hrs later demonstrated a significant influx of T cells into the brains of mice treated with MV-EGFR+aPD-1. To examine the effect of aPD-1 on the immune system in vitro, we established co-cultures of patient derived primary GBM39 or murine GL261 GBM cells, overlaid with BV2 microglia. GBM39 cells were infected with MV-GFP and murine GL261 cells were infected with MV-EGFR (MOI=3). Cultures were treated with either 1 or 20mg/ml of aPD-1 and overlaid with BV2. Twenty four hours later the co-cultures were harvested and rtQPCR performed: results showed a significant increase in mRNA for IFN-α, IFN-β in both GBM39 and GL261 co-culture models when aPD-1 was present at 1μg/mL. aPD-1 also increased BV2 migration towards MV-GFP infected GBM39 and MV-EGFR infected GL261 cells (MOI=3) in a transwell system. Lastly, survival analysis of mice bearing orthotopic syngeneic GL261 GBM treated with MV-EGFR (2×105 TCID50 × 4 doses), aPD-1 (10mg/kg × 3 doses), MV-EGFR+ aPD-1 or untreated showed a significant prolongation of survival for mice treated with MV-EGFR+aPD-1 compared to the other treatment groups (P=0.009, 0.038, and 0.003 respectively). Additional in vitro and in vivo experiments are ongoing. Funding by: Brain SPORE (P50 CA108961), R01 CA154348, T32 NS 07494.

The Thiazide-Sensitive Co-Transporter Promotes the Development of Sodium Retention in Mice with Diet-Induced Obesity

Background/Aims: Intravascular volume expansion due to sodium retention is involved in the pathogenesis of obesity-related hypertension. Institution of high fat diet (HFD) feeding leads to an initial state of positive sodium balance due to enhanced tubular reabsorption of sodium, but which tubular sodium transporters are responsible for this remains undefined. Methods: C57/Bl6 mice were fed control or HFD for 3 weeks. Blood pressures were recorded by tail cuff method. Sodium transporter expression and phosphorylation were determined by Western blotting. In vivo activity of NCC was determined using natriuretic responses to hydrochlorothiazide. Expression of NCC mRNA was determined using qPCR. Results: At 3 weeks HFD mice had significant weight gains compared to control mice, but blood pressures were not yet elevated. There were no changes in expression or phosphorylation of the bumetanide-sensitive cotransporter, NKCC2, or in expression of subunits of the amiloride-sensitive ion channel, ENaC. However, there were significant increases in mRNA and protein expression of the thiazide-sensitive co-transporter, NCC, in kidneys from HFD mice. Consistent with this, HFD mice had increased in vivo activity of NCC. Conclusions: Increased expression of NCC promotes the sodium loading response to institution of HFD feeding before onset of hypertension.

Redox regulation of mitophagy in the lung during murine Staphylococcus aureus sepsis

Oxidative mitochondrial damage is closely linked to inflammation and cell death, but low levels of reactive oxygen and nitrogen species serve as signals that involve mitochondrial repair and resolution of inflammation. More specifically, cytoprotection relies on the elimination of damaged mitochondria by selective autophagy (mitophagy) during mitochondrial quality control. This aim of this study was to identify and localize mitophagy in the mouse lung as a potentially upregulatable redox response to Staphylococcus aureus sepsis. Fibrin clots loaded with S. aureus (1×107 CFU) were implanted abdominally into anesthetized C57BL/6 and B6.129X1-Nfe2l2tm1Ywk/J (Nrf2−/−) mice. At the time of implantation, mice were given vancomycin (6mg/kg) and fluid resuscitation. Mouse lungs were harvested at 0, 6, 24, and 48h for bronchoalveolar lavage (BAL), Western blot analysis, and qRT-PCR. To localize mitochondria with autophagy protein LC3, we used lung immunofluorescence staining in LC3–GFP transgenic mice. In C57BL/6 mice, sepsis-induced pulmonary inflammation was detected by significant increases in mRNA for the inflammatory markers IL-1β and TNF-α at 6 and 24h, respectively. BAL cell count and protein also increased. Sepsis suppressed lung Beclin-1 protein, but not mRNA, suggesting activation of canonical autophagy. Notably sepsis also increased the LC3-II autophagosome marker, as well as the lung׳s noncanonical autophagy pathway as evidenced by loss of p62, a redox-regulated scaffolding protein of the autophagosome. In LC3–GFP mouse lungs, immunofluorescence staining showed colocalization of LC3-II to mitochondria, mainly in type 2 epithelium and alveolar macrophages. In contrast, marked accumulation of p62, as well as attenuation of LC3-II in Nrf2-knockout mice supported an overall decrease in autophagic turnover. The downregulation of canonical autophagy during sepsis may contribute to lung inflammation, whereas the switch to noncanonical autophagy selectively removes damaged mitochondria and accompanies tissue repair and cell survival. Furthermore, mitophagy in the alveolar region appears to depend on activation of Nrf2. Thus, efforts to promote mitophagy may be a useful therapeutic adjunct for acute lung injury in sepsis.

Blockage of potassium channel inhibits proliferation of glioma cells via increasing reactive oxygen species.

The potassium (K+) channel plays an important role in the cell cycle and proliferation of tumor cells, while its role in brain glioma cells and the signaling pathways remains unclear. We used tetraethylammonium (TEA), a nonselective antagonist of big conductance K+ channels, to block K+ channels in glioma cells, and antioxidant N-acetyl-l-cysteine (NAC) to inhibit production of intracellular reactive oxygen species (ROS). TEA showed an antiproliferation effect on C6 and U87 glioma cells in a time-dependent manner, which was accompanied by an increased intracellular ROS level. Antioxidant NAC pretreatment reversed TEA-mediated antiproliferation and restored ROS level. TEA treatment also caused significant increases in mRNA and protein levels of tumor-suppressor proteins p53 and p21, and the upregulation was attenuated by pretreatment of NAC. Our results suggest that K+ channel activity significantly contributes to brain glioma cell proliferation via increasing ROS, and it might be an upstream factor triggering the activation of the p53/p21Cip1-dependent signaling pathway, consequently leading to glioma cell cycle arrest.