Increased NF-κB Phosphorylation Research Articles

Involvement of CKS1B in the anti-inflammatory effects of cannabidiol in experimental stroke models

We have previously demonstrated that treatment with cannabidiol (CBD) ameliorates mitochondrial dysfunction and attenuates neuronal injury in rats following cerebral ischemia. However, the role of CBD in the progression of ischemic stroke-induced inflammation and the molecules involved remain unclear. Here, we found that CBD suppressed the production of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), reduced the activation of microglia, ameliorated mitochondrial deficits, and decreased the phosphorylation of nuclear factor κ-B (NF-κB) in BV-2 cells subjected to oxygen-glucose deprivation/reoxygenation (OGD/R). Cyclin-dependent kinase regulatory subunit 1B (CKS1B) expression was decreased in BV-2 cells following OGD/R and this reduction was blocked by treatment with CBD. Knockdown of CKS1B increased the activation of microglia and enhanced the production of IL-1β and TNF-α in BV-2 cells treated with CBD. Moreover, CKS1B knockdown exacerbated mitochondrial deficits and increased NF-κB phosphorylation. CBD treatment also ameliorated brain injury, reduced neuroinflammation, and enhanced the protein levels of mitochondrial transcription factor A and CKS1B in rats following middle cerebral artery occlusion/reperfusion. These data identify CKS1B as a novel regulator of neuroinflammation; and reveals its involvement in the anti-inflammatory effects of CBD. Interventions targeting CKS1B expression are potentially promising for treating in ischemic stroke.

Toll-like receptor 4 signaling pathway in sensory neurons mediates remifentanil-induced postoperative hyperalgesia via transient receptor potential ankyrin 1.

Background: Remifentanil-induced postoperative hyperalgesia (RIH) refers to a state of hyperalgesia or aggravated pre-existing pain after remifentanil exposure. There has been considerable interest in understanding and preventing RIH. However, the mechanisms responsible for RIH are still not completely understood. Toll-like receptor 4 (TLR4), a classic innate immune receptor, has been detected in sensory neurons and participates in various nociceptive conditions, whereas its role in RIH remains unclear. Transient receptor potential ankyrin 1 (TRPA1) always serves as a nociceptive channel, whereas its role in RIH has not yet been investigated. This study aimed to determine whether the TLR4 signaling pathway in sensory neurons engaged in the development of RIH and the possible involvement of TRPA1 during this process. Methods: A rat model of remifentanil-induced postoperative hyperalgesia (RIH) was established, which presented decreased paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL). The mRNA and protein expression levels of TLR4, phosphorylated NF-κB, and TRPA1 in the dorsal root ganglion (DRG) from RIH model were analyzed by real-time PCR, western blot, and immunofluorescence. The TLR4 antagonist TAK-242 and the TRPA1 antagonist HC-030031 were applied to determine the role of sensory neuron TLR4 signaling and TRPA1 in RIH. Results: Compared with control, PWMT and PWTL were significantly decreased in RIH model. Moreover, the mRNA and protein expression of TLR4 and TRPA1 in DRG were upregulated after remifentanil exposure together with increased NF-κB phosphorylation. TLR4 antagonist TAK-242 mitigated mechanical pain in RIH together with downregulated expression of TLR4, phosphorylated NF-κB, and TRPA1 in DRG neurons. In addition, TRPA1 antagonist HC-030031 also alleviated mechanical pain and decreased TRPA1 expression in RIH without affecting TLR4 signaling in DRG. Conclusions: Taken together, these results suggested that activation of TLR4 signaling pathway engaged in the development of RIH by regulating TRPA1 in DRG neurons. Blocking TLR4 and TRPA1 might serve as a promising therapeutic strategy for RIH.

Agomelatine improves streptozotocin-induced diabetic nephropathy through melatonin receptors/SIRT1 signaling pathway.

Diabetic nephropathy (DN) is a major cause of end-stage renal disease (ESRD). Agomelatine, a melatonin receptor agonist, has a potent anti-inflammatory activity. The current study aimed to determine the ameliorative anti-inflammatory effect of agomelatine against DN. We used 10% fructose with streptozotocin (STZ) to induce DN in male Wistar rats. Diabetic rats were treated with agomelatine in presence or absence of melatonin receptor antagonist (luzindole) or Sirtuin1 (SIRT1) inhibitor (EX527). SIRT1 expression was measured by qRT-PCR and immunohistochemical analysis. The expression of nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB), 5'adenosine monophosphate-activated protein kinase (AMPK), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion protein-1 (VCAM-1), and monocyte chemoattractant protein-1 (MCP-1) were measured using ELISA. Histological assessment was performed using hematoxylin and eosin-stained renal sections. Fructose and STZ treatment induced diabetes, insulin resistance, and renal damage accompanied by reduced SIRT1 expression, increased NFκB activation, and decreased AMPK phosphorylation in the kidney. Agomelatine treatment improved kidney histology and function and upregulated SIRT1 expression (2-fold). Inhibition of melatonin receptors and SIRT1 activity increased NFκB phosphorylation (2.13 and 1.98-folds, respectively), reduced AMPK activation (0.51 and 0.53-folds, respectively), increased inflammatory markers ICAM-1 (2.16 and 2.23-folds, respectively), VCAM-1 (2.19 and 2.26-folds, respectively), and MCP-1(2.84 and 3.12-folds, respectively), and inhibited the ameliorative effect of agomelatine on kidney structure and function. Our findings reveal the ameliorative anti-inflammatory activity of agomelatine against STZ-induced DN and this effect is SIRT1- and melatonin receptor-dependent. Therefore, agomelatine may be beneficial to prevent the development of ESRD from diabetes mellitus.

Human Amniotic Membrane Mesenchymal Stem Cell-Synthesized PGE2 Exerts an Immunomodulatory Effect on Neutrophil Extracellular Trap in a PAD-4-Dependent Pathway through EP2 and EP4.

Human amniotic membrane mesenchymal stem cells (hAM-MSC) secrete a myriad of components with immunosuppressive activities. In the present research, we aimed to describe the effect of prostaglandin E2 (PGE2) secreted by hAM-MSCs on neutrophil extracellular trap (NET) release and to characterize the role of its receptors (EP2/EP4) in PAD-4 and NFκB activity in neutrophils. Human peripheral blood neutrophils were ionomycin-stimulated in the presence of hAM-MSC conditioned medium (CM) treated or not with the selective PGE2 inhibitor MF-63, PGE2, EP2/EP4 agonists, and the selective PAD-4 inhibitor GSK-484. NET release, PAD-4, and NFκB activation were analyzed. Ionomycin induced NET release, which was inhibited in the presence of hAM-MSC-CM, while CM from hAM-MSCs treated with MF-63 prevented NET release inhibition. PGE2 and EP2/EP4 agonists, and GSK-484 inhibited NET release. EP2/EP4 agonists and GSK-484 inhibited H3-citrullination but did not affect PAD-4 protein expression. Finally, PGE2 and EP2/EP4 agonists and GSK-484 increased NFκB phosphorylation. Taken together, these results suggest that hAM-MSC exert their immunomodulatory activities through PGE2, inhibiting NET release in a PAD-4-dependent pathway. This research proposes a new mechanism by which hAM-MSC exert their activities when modulating the innate immune response and inhibiting NET release.

Hepatocyte-specific deletion of Nlrp6 in mice exacerbates the development of non-alcoholic steatohepatitis

ObjectivePrevious studies have established that deficiency in Nucleotide-binding and oligomerization domain (NOD)-like receptor family pyrin domain containing 6 (Nlrp6) changes the configuration of the gut microbiota, which leads to hepatic steatosis. Here, we aimed to determine the hepatic function of Nlrp6 in lipid metabolism and inflammation and its role in the development of non-alcoholic steatohepatitis (NASH). MethodsNlrp6Loxp/Loxp and hepatocyte-specific Nlrp6-knockout mice were fed a high-fat diet (HFD) or methionine-choline deficient (MCD) diet to induce fatty liver or steatohepatitis, respectively. Primary hepatocytes were isolated to further explore the underlying mechanisms in vitro. In addition, we used adenovirus to overexpress Nlrp6 in ob/ob mice to demonstrate its role in NASH. ResultsHepatic Nlrp6 expression was downregulated in NASH patients and in obese mice. Hepatocyte-specific Nlrp6 deficiency promoted HFD- or MCD diet-induced lipid accumulation and inflammation, whereas Nlrp6 overexpression in ob/ob mice had beneficial effects. In vitro studies demonstrated that knockdown of Nlrp6 aggravated hepatic steatosis and inflammation in hepatocytes, but its overexpression markedly attenuated these abnormalities. Moreover, both in vitro and in vivo study demonstrated that Nlrp6 inhibited Cd36‐mediated lipid uptake. Nlrp6 deficiency-enhanced fatty acid uptake was blocked by a Cd36 inhibitor in hepatocytes. Nlrp6 ablation increased the expression of proinflammatory cytokines, likely as a result of increased NF-κB phosphorylation and activation. Mechanistically, Nlrp6 promoted the degradation of transforming growth factor-β-activated kinase 1 (TAK1)-binding protein 2/3 (TAB2/3) via a lysosomal-dependent pathway, which suppressed NF-κB activation. ConclusionsNlrp6 may play a key role in the pathological process of NASH by inhibiting Cd36 and NF-κB pathways. It may be a potential therapeutic target for NASH.

Ketamine inhibits LPS-mediated BV2 microglial inflammation via NMDA receptor blockage.

Microglial inflammation leads to the upregulation of proinflammatory cytokine and proinflammatory enzyme expression, resulting in inflammation-induced neuronal cell apoptosis. Ketamine, an anesthetic mostly used in critical patients, has been reported to possess neuroprotective effects. However, the potential mechanism is still not well understood. In the present study, we investigated how ketamine attenuates lipopolysaccharide (LPS)-mediated BV2 cell inflammation. LPS upregulated proinflammatory cytokine and proinflammatory enzyme expression, increased NF-κB phosphorylation and nuclear translocation, and augmented calcium (Ca2+ )/calmodulin-dependent protein kinase II (CaMK II) phosphorylation and Ca2+ levels in BV2 cells. Ketamine could reverse these LPS-induced effects. Furthermore, AP5, an inhibitor of NMDA receptors, inhibited LPS-induced inflammatory effects in BV2 cells, which was similar to the effects of ketamine. Moreover, these effects of ketamine against LPS-mediated inflammation in BV2 cells could be reversed by D-serine, an activator of NMDA receptors. The present study suggests that ketamine, by inhibiting NMDA receptors, attenuating Ca2+ levels, and inhibiting CaMK II phosphorylation, NF-κB phosphorylation and nuclear translocation, may ameliorate LPS-mediated inflammation in BV2 cells.

MDM2 inhibitor ameliorates cisplatin-induced nephropathy via NFκΒ signal inhibition.

Cisplatin is a platinum‐containing chemotherapeutic drug, which is widely used and highly effective. While effective against tumors, its use is limited by severe side effects such as nephrotoxicity and bone marrow suppression. Murine double minute 2 (MDM2) is the E3 ubiquitin ligase of the tumor suppressor gene, p53, and inhibition of MDM2 can suppress tumor cell growth. However, independent of p53, MDM2 acts as a co‐transcription factor for nuclear factor‐κB (NFκB), whose signaling can be involved in cisplatin‐induced tubular injury. We therefore examined the effects of MDM2 inhibitor on cisplatin cytotoxicity. In order to induce acute kidney injury and to investigate MDM2 inhibitory effects, we injected cisplatin into rats with or without the MDM2 inhibitor, DS‐5272, and analyzed kidney physiology/histology and NFκB signaling. Serum creatinine was significantly lower in the DS‐5272 group than in the vehicle group on day 3 (0.55 ± 0.069 vs 0.70 ± 0.072 mg/dL, P < 0.05). DS‐5272 also significantly decreased kidney injury molecule‐1 (KIM‐1) expression, improved tubular injury, and decreased apoptotic cells. Western blotting showed that cisplatin increased NFκB phosphorylation in kidneys, which was significantly suppressed by DS‐5272. In vitro, we treated HEK 293 cells with cisplatin, in the absence or presence of DS‐5272, and examined cytotoxicity and NFκB transcriptional activity. DS‐5272 co‐treatment reduced both cisplatin‐induced cell death and NFκB transcriptional activity. Collectively, these findings suggest that DS‐5272 can ameliorate cisplatin nephrotoxicity via NFκB signal inhibition.

Attenuation of ventilation-induced diaphragm dysfunction through toll-like receptor 4 and nuclear factor-κB in a murine endotoxemia model

Mechanical ventilation (MV) is often used to maintain life in patients with sepsis and sepsis-related acute lung injury. However, controlled MV may cause diaphragm weakness due to muscle injury and atrophy, an effect termed ventilator-induced diaphragm dysfunction (VIDD). Toll-like receptor 4 (TLR4) and nuclear factor-κB (NF-κB) signaling pathways may elicit sepsis-related acute inflammatory responses and muscle protein degradation and mediate the pathogenic mechanisms of VIDD. However, the mechanisms regulating the interactions between VIDD and endotoxemia are unclear. We hypothesized that mechanical stretch with or without endotoxin treatment would augment diaphragmatic structural damage, the production of free radicals, muscle proteolysis, mitochondrial dysfunction, and autophagy of the diaphragm via the TLR4/NF-κB pathway. Male C57BL/6 mice, either wild-type or TLR4-deficient, aged between 6 and 8 weeks were exposed to MV (6 mL/kg or 10 mL/kg) with or without endotoxemia for 8 h. Nonventilated mice were used as controls. MV with endotoxemia aggravated VIDD, as demonstrated by the increases in the expression levels of TLR4, caspase-3, atrogin-1, muscle ring finger-1, and microtubule-associated protein light chain 3-II. In addition, increased NF-κB phosphorylation and oxidative loads, disorganized myofibrils, disrupted mitochondria, autophagy, and myonuclear apoptosis were also observed. Furthermore, MV with endotoxemia reduced P62 levels and diaphragm muscle fiber size (P < 0.05). Endotoxin-exacerbated VIDD was attenuated by pharmacologic inhibition with a NF-κB inhibitor or in TLR4-deficient mice (P < 0.05). Our data indicate that endotoxin-augmented MV-induced diaphragmatic injury occurs through the activation of the TLR4/NF-κB signaling pathway.Mechanical ventilation may cause diaphragm weakness due to muscle injury and atrophy. This study shows that endotoxin-augmented mechanical ventilation-induced diaphragmatic injury occurs through the activation of toll-like receptor 4 and nuclear factor-κB signaling pathways. These findings may aid in the development of treatments for patients with sepsis.

Glucagon-like peptide-1 exerts anti-inflammatory effects on mouse colon smooth muscle cells through the cyclic adenosine monophosphate/nuclear factor-&amp;kappa;B pathway in vitro

BackgroundIntestinal smooth muscle cells (SMCs) undergo substantial morphological, phenotypic, and contractile changes during inflammatory bowel disease (IBD). SMCs act as a source and target for different inflammatory mediators, however their role in IBD pathogenesis is usually overlooked. Glucagon-like peptide-1 (GLP-1) is an incretin hormone reported to exert multiple anti-inflammatory effects in different tissues including the gastrointestinal tract through various mechanisms.AimThe aim of this research is to explore the effect of GLP-1 analog exendin-4 on the expression and secretion of inflammatory markers from mouse colon smooth muscle cells (CSMCs) after stimulation with lipopolysaccharide (LPS).Materials and methodsFreshly isolated CSMCs from male BALB/c mice were cultured in DMEM and treated with vehicle, LPS (1 μg/mL), LPS+exendin-4 (50 nM), or LPS+exendin-4 (100 nM) for 24 h. Expression of inflammatory cytokines was then evaluated by antibody array membrane.ResultsCSMCs showed basal expression of several cytokines which was enhanced with the induction of inflammation by LPS. However, exendin-4 (50 and 100 nM) significantly (p<0.05) reduced the expression of multiple cytokines including tumor necrosis factor-α (TNF-α), interleukin-1α (IL-1α), T cell activation gene-3 (TCA-3), stromal cell-derived factor-1 (SDF-1), and macrophage colony stimulating factor (M-CSF). To confirm these results, expression of these cytokines was further assessed by enzyme-linked immunosorbent assay and real-time polymerase chain reaction and similar results were also observed. Moreover, secretion of TNF-α and IL1-α into the conditioned media was significantly downregulated by exendin-4 when compared to LPS-treated cells. Furthermore, LPS increased NF-κB phosphorylation, while exendin-4 significantly reduced levels of NF-κB phosphorylation.ConclusionThese data indicate that GLP-1 analogs can exert significant anti-inflammatory effects on CSMCs and can potentially be used as an adjunct treatment for inflammatory bowel conditions.

The transcriptional repressor Gfi1 prevents lupus autoimmunity by restraining TLR7 signaling.

The transcriptional repressor growth factor independence 1 (Gfi1) is important in myeloid and lymphoid differentiation. In the current study we evaluated the involvement of Gfi1 in systemic lupus erythematosus (SLE). We found that Genista mice, which carry a hypomorphic mutation in the gfi1 gene or Gfi1-deficient (Gfi1-/- ) mice develop signs of spontaneous lupus autoimmunity, including increased serum levels of IgM and IgG2a, autoantibodies against RNA and DNA, glomerular immunodeposits and increased frequencies of plasmablasts, germinal center (GC) B cells and age-associated B cells (ABCs). On the contrary, Genista mice deprived of TLR7 did not show any of these phenotypes, suggesting that the observed lupus autoimmunity in Genista mice is TLR7-dependent. Moreover, Genista mice showed an increased activation of dendritic cells (DCs), B and T cells that was dependent on TLR7 for DCs and B cells, but not for T cells. Upon TLR7 or TLR4 stimulation Genista DCs produced increased amounts of TNF, IL-6 and IFN-β and showed increased NF-κB phosphorylation and IRF7 nuclear translocation, suggesting that Gfi1 controls the NF-κB and type I IFN signaling pathway downstream of TLRs. Our data reveal that Gfi1 plays a critical role in the prevention of spontaneous lupus autoimmunity by negatively regulating TLR7 signaling.

Extracellular Mitochondria and Mitochondrial Components Act as Damage-Associated Molecular Pattern Molecules in the Mouse Brain

Mitochondria and mitochondrial debris are found in the brain's extracellular space, and extracellular mitochondrial components can act as damage associated molecular pattern (DAMP) molecules. To characterize the effects of potential mitochondrial DAMP molecules on neuroinflammation, we injected either isolated mitochondria or mitochondrial DNA (mtDNA) into hippocampi of C57BL/6 mice and seven days later measured markers of inflammation. Brains injected with whole mitochondria showed increased Tnfα and decreased Trem2 mRNA, increased GFAP protein, and increased NFκB phosphorylation. Some of these effects were also observed in brains injected with mtDNA (decreased Trem2 mRNA, increased GFAP protein, and increased NFκB phosphorylation), and mtDNA injection also caused several unique changes including increased CSF1R protein and AKT phosphorylation. To further establish the potential relevance of this response to Alzheimer's disease (AD), a brain disorder characterized by neurodegeneration, mitochondrial dysfunction, and neuroinflammation we also measured App mRNA, APP protein, and Aβ1-42 levels. We found mitochondria (but not mtDNA) injections increased these parameters. Our data show that in the mouse brain extracellular mitochondria and its components can induce neuroinflammation, extracellular mtDNA or mtDNA-associated proteins can contribute to this effect, and mitochondria derived-DAMP molecules can influence AD-associated biomarkers.

Probiotic Saccharomyces boulardii CNCM I-745 prevents outbreak-associated Clostridium difficile-associated cecal inflammation in hamsters.

C. difficile infection (CDI) is a common debilitating nosocomial infection associated with high mortality. Several CDI outbreaks have been attributed to ribotypes 027, 017, and 078. Clinical and experimental evidence indicates that the nonpathogenic yeast Saccharomyces boulardii CNCM I-745 (S.b) is effective for the prevention of CDI. However, there is no current evidence suggesting this probiotic can protect from CDI caused by outbreak-associated strains. We used established hamster models infected with outbreak-associated C. difficile strains to determine whether oral administration of live or heat-inactivated S.b can prevent cecal tissue damage and inflammation. Hamsters infected with C. difficile strain VPI10463 (ribotype 087) and outbreak-associated strains ribotype 017, 027, and 078 developed severe cecal inflammation with mucosal damage, neutrophil infiltration, edema, increased NF-κB phosphorylation, and increased proinflammatory cytokine TNFα protein expression. Oral gavage of live, but not heated, S.b starting 5 days before C. difficile infection significantly reduced cecal tissue damage, NF-κB phosphorylation, and TNFα protein expression caused by infection with all strains. Moreover, S.b-conditioned medium reduced cell rounding caused by filtered supernatants from all C. difficile strains. S.b-conditioned medium also inhibited toxin A- and B-mediated actin cytoskeleton disruption. S.b is effective in preventing C. difficile infection by outbreak-associated via inhibition of the cytotoxic effects of C. difficile toxins.

Abstract 3279: Expression of chemerin and chemerin receptors in neuroblastoma: implications in tumorigenesis

Abstract Chemerin is an adipokine and immunomodulating factor that promotes chemotaxis of immature dendritic cells, natural killer cells, macrophages and endothelial cells. Secreted as prochemerin with low activity, it can be C-terminally processed by different proteases expressed by a broad range of cell types and tissues. The resulting isoforms vary in receptor affinity and biological activity and are natural ligands for the G protein-coupled receptors (GPCRs) CMKLR1, GPR1 and CCLR2. To date, the activation of CMKLR1 (chemokine-like receptor 1) by chemerin and its role in metabolism and metabolic disorders as well as inflammation is best understood. Neuroblastoma (NB) is a malignancy of the sympathetic nervous system and the most common extracranial solid pediatric tumor. Several chemoattractant GPCRs have been suggested to promote tumor progression, angiogenesis and metastasis in NB. Although for some cancers a potential function has been suggested, the role of chemerin and its receptors in the NB tumor microenvironment remains unknown. In our study, the screening of microarray databases and analysis of neuroblastoma expression data showed a correlation between high CMKLR1, GPR1 and CCLR2 expression and a reduction in the overall survival probability. Expression of CMKLR1, GPR1, and chemerin was shown in nine neuroblastoma cell lines using RT-PCR, Western blot and immunocytochemistry. Furthermore, chemerin and CMKLR1 were also detected in neuroblastoma tumor tissue by immunohistochemistry. Stimulation of NB cell lines with active chemerin induced calcium mobilization and increased phosphorylation of MEK1/2 and ERK1/2 indicating an activation of the MAPK signaling pathway. Furthermore, chemerin stimulation led to increased NF-κB phosphorylation and translocation to the nucleus. The induction of NF-κB mediated signaling was observed by luciferase reporter assay. TNFα, IL-1β or serum stimulation increased chemerin protein expression and secretion in neuroblastoma cells. To assess the functional significance of chemerin and its receptors in neuroblastoma tumorigenesis, cell clones overexpressing or silenced for CMKLR1/ Chemerin/ GPR1 are used in NB animal models. Citation Format: Conny Tuemmler, Igor Snapkov, Ugo L. Moens, Per Kogner, John Inge Johnsen, Baldur Sveinbjørnsson. Expression of chemerin and chemerin receptors in neuroblastoma: implications in tumorigenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3279. doi:10.1158/1538-7445.AM2015-3279

Calcineurin Alpha Inhibition Induces Nox2 Up‐regulation via NFκB

Although essential for preventing transplant rejection, prolonged calcineurin inhibitor (CNI) use induces nephrotoxicity. We showed that mice lacking the alpha catalytic isoform of calcineurin (CnAα) have a phenotype that mirrors CNI nephrotoxicity. Other studies implicate NADPH oxidase‐2 (Nox2) in the pathogenesis. This study seeks to determine if CnAα and Nox2 function in a common pathway. To determine the mechanism of CNI inhibition‐mediated increases in renal Nox2, WT mice were treated with cyclosporine A (CsA) for 6 weeks. In addition, Nox2 expression and activity were examined in CnAα‐/‐ and CnAβ‐/‐ mice and renal fibroblasts. In kidneys, co‐IP reveals that CsA treatment reduces CnAα association with its binding partner, calmodulin. Consistent with previous data, CnAβ and calmodulin association is not observed. Additionally, reduced CnAα activation is accompanied by increased Nox2 expression and ROS levels, as assessed by Western blot and Amplex Red. Consistently, renal Nox2 expression and ROS levels are increased in CnAα‐/‐ mice but not CnAβ‐/‐. Surprisingly, NFAT transcriptional activity is unchanged in CnAα‐/‐ mice compared to WT and CnAβ‐/‐, as assessed by luciferase activity. However, increased NFκB phosphorylation is observed. In CnAα‐/‐ cells, NFκB inhibition with CAPE attenuates enhanced Nox2 expression and ROS levels. In conclusion, these data indicate that CsA‐induced CnAα inhibition and subsequent NFκB activation stimulates Nox2 up‐regulation. Therapeutic approaches using CNI that do not target CnAα could ameliorate oxidative damage associated with CNI nephrotoxicity.NIH‐T32 (CRW), NIH‐R01 (RSH), VA‐MERIT (JLG)

Abstract 174: Roles of Pericyte NADPH Oxidase 4 in Acute Brain Ischemia

Pericytes exist abundantly in the brain and compose the neurovascular unit. It has been elucidated that pericytes play a key role in the formation and maintenance of the blood-brain barrier (BBB), thus being considered to play significant roles in brain ischemia. The NADPH oxidase (Nox) family proteins are a major source of reactive oxygen species (ROS). We have reported previously that Nox4 is abundantly expressed in pericytes among the Nox family. Our goal was to elucidate the roles of Nox4 in brain pericytes during acute brain ischemia. We confirmed by quantitative PCR that Nox4 was abundantly expressed in human cultured brain microvascular pericytes (HBMPC) and was significantly upregulated by hypoxia. We produced a mouse middle cerebral artery occlusion (MCAO) stroke model and examined the expression of Nox4 in the brain. Immunofluorescent double labeling demonstrated that Nox4 expression was upregulated in microvessels particularly in peri-infarct areas and was co-stained with PDGFRβ, a pericyte marker. In order to elucidate the role of Nox4 in brain pericyte during brain ischemia, we generated mice with human Nox4 overexpression using a promoter of SM22α, a pericyte/smooth muscle cell marker (Tg-Nox4). We confirmed that SM22α was expressed in mouse brain pericytes by co-immunostaining with PDGFRβ. We isolated microvessels from Tg-Nox4 brain and confirmed that human Nox4 mRNA was highly expressed. In MCAO model, the infarct volume was significantly larger in Tg-Nox4 than in littermate controls. Confocal microscopy demonstrated that IgG leakage in peri-infarct areas was significantly increased in Tg-Nox4, suggesting that Nox4 overexpression in pericytes enhanced BBB breakdown during acute brain ischemia. To elucidate the mechanisms, we induced adenovirus-mediated overexpression of Nox4 in HBMPC. We demonstrated that Nox4 overexpression increased NFκB phosphorylation and MMP9 expression in the cells.In conclusion, Nox4 may be a major source of ROS in brain pericytes and is upregulated directly by hypoxia in peri-infarct areas during acute brain ischemia. Pericyte Nox4 may enhance BBB breakdown through the activation of NFκB-MMP9 signaling during acute brain ischemia.

MAPK mediates inflammatory response and cell death in rat pulmonary microvascular endothelial cells in an ischemia–reperfusion model of lung transplantation

Hypoxia-reoxygenation of cultured macrovascular endothelial cells is used to study ischemia-reperfusion (IR)-related cellular and molecular changes; however, these models do not accurately depict events in pulmonary microvascular endothelial cells (PMVECs) during conventional lung retrieval and transplantation. We used rat PMVECs in a new non-hypoxic cell-based lung transplantation model to assess these events. To simulate cold storage, rat PMVECs were preserved in 95% O2-5% CO2 at 4°C for 6 hours in low-potassium dextran solution. Dishes were warmed for 1 hour to room temperature for simulating implantation. Medium was added at 37°C in 50% O2-5% CO2-45% N2 to simulate reperfusion. Additional PMVECs were transfected with siRNA targeting mitogen-activated protein kinases (MAPKs) and then subjected to simulated IR. MAPKs and NF-κB were activated during simulated reperfusion, and AP-1 was activated during ischemia and reperfusion. Increased malondialdehyde levels were found during cold ischemia, and apoptosis and production of IL-1β, IL-6, and TNF-α were observed during reperfusion. Silencing of MAPKs attenuated oxidative stress, inflammation and apoptosis. Silencing of JNK and p38 decreased NF-κB phosphorylation and increased inhibitor of NF-κB (IκB)α levels. Knockdown of ERK1/2 increased NF-κB phosphorylation but had no effect on IκBα expression. Silencing of JNK and ERK1/2, but not p38, decreased AP-1 phosphorylation. Exposing rat PMVECs to simulated non-hypoxic IR caused lipid peroxidation, inflammation and apoptosis, which required MAPK-mediated NF-κB and AP-1 activation and distinct regulation of MAPKs by these 2 transcription factors. This model could be used to uncouple mechanisms of IR and evaluate potential therapeutics in alleviating IR injury.

Modulation of mercury-induced mitochondria-dependent apoptosis by glycine in hepatocytes

Mercury (Hg) is one of the universal environmental pollutants and is responsible for various organ pathophysiology including oxidative stress-induced hepatic disorders. In the present study, we aimed to explore the protective role of glycine in Hg-induced cytotoxicity and cell death in murine hepatocytes. Exposure of mercury (20 μM), in the form HgCl2 for 1 h, significantly enhanced the ALT and ALP leakage, increased reactive oxygen species production, reduced cell viability and distorted the antioxidant status of hepatocytes. Flow cytometric analyses shows that Hg-induced apoptotic death in hepatocytes. Mechanism of this pathophysiology involves reduced mitochondrial membrane potential, variations in Bcl-2/Bad proteins, activation of caspases and cleavage of PARP protein. In addition, Hg distinctly increased NF-κB phosphorylation in association with IKKα phosphorylation and IκBα degradation. Concurrent treatment with glycine (45 mM), however, reduced Hg-induced oxidative stress, attenuated the changes in NF-κB phosphorylation and protects hepatocytes from Hg-induced apoptotic death. Hg also distinctly increased the phosphorylation of p38, JNK and ERK mitogen-activated protein kinase (MAPKs). Glycine treatment suppressed these apoptotic events, signifying its protective role in Hg-induced hepatocyte apoptosis as referred by reduction of p38, JNK and ERK MAPK signaling pathways. Results suggest that glycine can modulate Hg-induced oxidative stress and apoptosis in hepatocytes probably because of its antioxidant activity and functioning via mitochondria-dependent pathways and could be a beneficial agent in oxidative stress-mediated liver diseases.