Kupffer cell-derived prostaglandin E(2) is involved in alcohol-induced fat accumulation in rat liver

Destruction of Kupffer cells with gadolinium chloride (GdCl(3)) and intestinal sterilization with antibiotics diminished ethanol-induced steatosis in the enteral ethanol feeding model. However, mechanisms of ethanol-induced fatty liver remain unclear. Accordingly, the role of Kupffer cells in ethanol-induced fat accumulation was studied. Rats were given ethanol (5 g/kg body wt) intragastrically, and tissue triglycerides were measured enzymatically. Kupffer cells were isolated 0-24 h after ethanol, and PGE(2) production was measured by ELISA, whereas inducible cyclooxygenase (COX-2) mRNA was detected by RT-PCR. As expected, ethanol increased liver triglycerides about threefold. This increase was blunted by antibiotics, GdCl(3), the dihydropyridine-type Ca(2+) channel blocker nimodipine, and the COX inhibitor indomethacin. Ethanol also increased PGE(2) production by Kupffer cells about threefold. This increase was also blunted significantly by antibiotics, nimodipine, and indomethacin. Furthermore, tissue triglycerides were increased about threefold by PGE(2) treatment in vivo as well as by a PGE(2) EP(2)/EP(4) receptor agonist, whereas an EP(1)/EP(3) agonist had no effect. Moreover, permeable cAMP analogs also increased triglyceride content in the liver significantly. We conclude that PGE(2) derived from Kupffer cells, which are activated by ethanol, interacts with prostanoid receptors on hepatocytes to increase cAMP, which causes triglyceride accumulation in the liver. This mechanism is one of many involved in fatty liver caused by ethanol.     

Ethanol-dependent induction of bilirubin UDP-glucuronosyl-transferase in rat liver is mediated by Kupffer cells

Recently we have reported that bilirubin UDP-glucuronosyltransferase (UGT1A1) is induced in rat liver by chronic ethanol treatment. Several studies have shown that Kupffer cells play a central role in the mediation of various hepatic effects of chronic alcohol consumption. In the present work, the participation of Kupffer cells in the ethanol dependent induction of UGT1A1 was investigated. A group of rats was pretreated with gadolinium chloride, a known Kupffer-cell-depleting agent. We compared the effect of chronic ethanol ingestion on UGT1A1 expression in the liver of normal and gadolinium chloride treated rats. The effect of ethanol on bilirubin glucuronidation was completely prevented in Kupffer cell deficient rats. The western and northern blot analyses showed that the increase of both the protein and mRNA of UGT1A1 was prevented in these animals. These results suggest that Kupffer cells play a major role in the mediation of ethanol-stimulated induction of UGT1A1 in liver parenchymal cells.     

Ischaemic preconditioning modulates the activity of Kupffer cells during in vivo reperfusion injury of rat liver

This work was performed to elucidate further the main cellular events underlying the protective effect of ischaemic preconditioning in an in vivo rat liver model of 90 min ischaemia followed by 30 min reperfusion. A significant attenuation of the various aspects of post-ischaemic injury, namely necrosis and the levels of hydrogen peroxide and 5- and 15-hydroperoxyeicosatetraenoic acids, was afforded by the prior application of a short cycle of ischaemia/reperfusion (10 + 10 min) or when rats were previously treated with gadolinium chloride. However, when preconditioning was applied on Kupffer cell-depleted livers, no additional level of ischaemic tolerance was obtained. In terms of cellular pathology, this result could be suggestive of Kupffer cells as the target of the preconditioning phenomenon during the warm ischaemia/reperfusion injury. Accordingly, modulation of Kupffer cell activity was associated with a well-preserved hepatocyte integrity, together with low levels of pro-oxidant generation during reperfusion. As activated Kupffer cells can generate and release potentially toxic substances, their modulation by ischaemic preconditioning could help to provide new surgical and/or pharmacological strategies to protect the liver against reperfusion damage.     

Nonsynaptic glycine release is involved in the early KCC2 expression

The cation‐chloride co‐transporters are important regulators of the cellular Cl^‐ homeostasis. Among them the Na⁺‐K⁺?2Cl^? co‐transporter (NKCC1) is responsible for intracellular chloride accumulation in most immature brain structures, whereas the K⁺‐Cl^? co‐transporter (KCC2) extrudes chloride from mature neurons, ensuring chloride‐mediated inhibitory effects of GABA/glycine. We have shown that both KCC2 and NKCC1 are expressed at early embryonic stages (E11.5) in the ventral spinal cord (SC). The mechanisms by which KCC2 is prematurely expressed are unknown. In this study, we found that chronically blocking glycine receptors (GlyR) by strychnine led to a loss of KCC2 expression, without affecting NKCC1 level. This effect was not dependent on the firing of Na⁺ action potentials but was mimicked by a Ca²⁺‐dependent PKC blocker. Blocking the vesicular release of neurotransmitters did not impinge on strychnine effect whereas blocking volume‐sensitive outwardly rectifying (VSOR) chloride channels reproduced the GlyR blockade, suggesting that KCC2 is controlled by a glycine release from progenitor radial cells in immature ventral spinal networks. Finally, we showed that the strychnine treatment prevented the maturation of rhythmic spontaneous activity. Thereby, the GlyR‐activation is a necessary developmental process for the expression of functional spinal motor networks. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 764–779, 2016     

Role of endogenous regucalcin in nuclear regulation of regenerating rat liver: suppression of the enhanced ribonucleic acid synthesis activity

The role of endogenous regucalcin in the regulation of ribonucleic acid (RNA) synthesis activity in the nucleus of normal and regenerating rat livers was investigated. Nuclear RNA synthesis was measured by the incorporation of [(3)H]-uridine 5'-triphosphate into the nuclear RNA in vitro. The presence of regucalcin (0.25 or 0.5 microM) in the reaction mixture caused a significant decrease in nuclear RNA synthesis of normal rat liver. alpha-Amanitin (10(-8)-10(-6) M), an inhibitor of RNA polymerase II and III, decreased significantly nuclear RNA synthesis activity. The effect of regucalcin (0.25 microM) in decreasing nuclear RNA synthesis activity was not seen in the presence of alpha-amanitin (10(-6) M). The calcium chloride (10 microM)-increased nuclear RNA synthesis activity was significantly suppressed by the addition of regucalcin (0.25 microM). RNA synthesis activity was significantly enhanced in the nuclei of regenating rat liver obtained at 24, 48, or 72 h after partial hepatectomy. This enhancement was significantly inhibited in the presence of PD98059 (10(-5) M), staurosporine (10(-6) M), or vanadate (10(-3) M). Western analysis of the nuclei of regenerating liver obtained at 24, 48, or 72 h after partial hepatectomy showed a significant increase in regucalcin protein as compared with that of sham-operated rats. The presence of anti-regucalcin monoclonal antibody (25 or 50 ng/ml) in the reaction mixture caused a significant increase in nuclear RNA synthesis activity of normal rat liver. This increase was completely blocked by the addition of regucalcin (1.0 microM). The effect of anti-regucalcin monoclonal antibody (50 ng/ml) in increasing nuclear RNA synthesis activity was significantly enhanced in the nuclei of regenerating liver obtained at 24, 48, or 72 h after partial hepatectomy. This enhancement was significantly suppressed by the addition of alpha-amanitin (10(-6) M), PD98059 (10(-5) M), staurosporine (10(-6) M), or vanadate (10(-3) M) in the reaction mixture. The present study demonstrates that endogenous regucalcin has a suppressive effect on the enhancement of RNA synthesis activity in the nucleus of regenerating rat liver with proliferative cells.     

Cross-talk between human mesenchymal stem/progenitor cells (MSCs) and rat hippocampal slices in LPS-stimulated cocultures: the MSCs are activated to secrete prostaglandin E2

Mesenchymal stem/progenitor cells (MSCs) improve functional outcome in a number of disease models through suppression of inflammation. However, their effects on neuroinflammation are unknown. In this study, we show that MSCs suppress endotoxin-induced glial activation in organotypic hippocampal slice cultures (OHSCs). Lipopolysaccharide-stimulated OHSCs activated MSCs to increase the expression of cyclo-oxygenase-2 and produce prostaglandin E2. MSC-derived prostaglandin E2, then suppressed pro-inflammatory cytokine production by the OHSCs. Together, the results suggest the potential anti-inflammatory mechanism of MSCs in models of disease and support earlier observations that MSCs may offer a therapy for neuroinflammation produced by trauma or disease.     

Embryonic liver fodrin involved in hepatic stellate cell activation and formation of regenerative nodule in liver cirrhosis

Transforming growth factor (TGF) β(1) plays a critical role in liver fibrosis. Previous studies demonstrated embryonic liver fodrin (ELF), a β-spectrin was involved in TGF-β/Smad signalling pathway as Smad3/4 adaptor. Here we investigate the role of ELF in pathogenesis of liver cirrhosis. In carbon tetrachloride (CCl(4))-induced mice model of liver cirrhosis, ELF is up-regulated in activated hepatic stellate cells (HSCs), and down-regulated in regenerative hepatocytes of cirrhotic nodules. In activated HSCs in vitro, reduction of ELF expression mediated by siRNA leads to the inhibition of HSC activation and procollagen I expression. BrdU assay demonstrates that down-regulation of ELF expression does not inhibit proliferation of activated HSCs in vitro. Immunostaining of cytokeratin 19 and Ki67 indicates that regenerative hepatocytes in cirrhotic liver are derived from hepatic progenitor cells (HPC). Further study reveals that HPC expansion occurs as an initial phase, before the reduction of ELF expression in regenerative hepatocytes. Regenerative hepatocytes in cirrhotic liver show the change in proliferative activity and expression pattern of proteins involved in G1/S transition, which suggests the deregulation of cell cycle in regenerative hepatocytes. Finally, we find that ELF participates in TGF-β/Smad signal in activated HSCs and hepatocytes through regulating the localization of Smad3/4. These data reveal that ELF is involved in HSC activation and the formation of regenerative nodules derived from HPC in cirrhotic liver.     

Induction of rat alpha 2-macroglobulin in vivo and in hepatocyte primary cultures: synergistic action of glucocorticoids and a Kupffer cell-derived factor

Turpentine injection into rats elicits enhanced secretion of acute phase proteins including alpha 2-macroglobulin (alpha 2M). Hypophysectomized rats, however, do not respond in this way unless dexamethasone is given together with turpentine. On the other hand, dexamethasone injection alone did not result in an induction of alpha 2M synthesis. When a medium of Kupffer cell cultures was added to hepatocytes, a dose-dependent stimulation of alpha 2M synthesis of up to 4-fold after 10-12 h was observed. However, the presence of low concentrations (10(-9)M) of dexamethasone was essential for the stimulatory effect. We conclude that the acute phase induction of alpha 2M in hepatocytes requires the synergistic action of glucocorticoids and a non-dialysable factor secreted by Kupffer cells.     

Delayed production of arachidonic acid contributes to the delay of proteinase-activated receptor-1 (PAR1)-triggered prostaglandin E2 release in rat gastric epithelial RGM1 cells

Proteinase-activated receptor-1 (PAR1), upon activation, exerts prostanoid-dependent gastroprotection, and increases prostaglandin E(2) (PGE(2)) release through cyclooxygenase-2 (COX-2) upregulation in rat gastric mucosal epithelial RGM1 cells. However, there is a big time lag between the PAR1-triggered PGE(2) release and COX-2 upregulation in RGM1 cells; that is, the former event takes 18 h to occur, while the latter rapidly develops and reaches a plateau in 6 h. The present study thus aimed at clarifying mechanisms for the delay of PGE(2) release after PAR1 activation in RGM1 cells. Although a PAR1-activating peptide, TFLLR-NH(2), alone caused PGE(2) release at 18 h, but not 6 h, TFLLR-NH(2) in combination with arachidonic acid dramatically enhanced PGE(2) release even for 1-6 h. TFLLR-NH(2) plus linoleic acid caused a similar rapid response. CP-24879, a Δ(5)/Δ(6)-desaturase inhibitor, abolished the PGE(2) release induced by TFLLR-NH(2) plus linoleic acid, but not by TFLLR-NH(2) alone. The TFLLR-NH(2)-induced PGE(2) release was not affected by inhibitors of cytosolic phospholipase A(2) (cPLA(2)), Ca(2+)-independent PLA(2) (cPLA(2)) or secretory PLA(2) (sPLA(2)), but was abolished by their mixture or a pan-PLA(2) inhibitor. Among PLA(2) isozymes, mRNA of group IIA sPLA(2) (sPLA(2)-IIA) was upregulated following PAR1 stimulation for 6-18 h, whereas protein levels of PGE synthases were unchanged. These data suggest that the delay of PGE(2) release after COX-2 upregulation triggered by PAR1 is due to the poor supply of free arachidonic acid at the early stage in RGM1 cells, and that plural isozymes of PLA(2) including sPLA(2)-IIA may complementarily contribute to the liberation of free arachidonic acid.     

Syncytiotrophoblast‐derived extracellular vesicles carry apolipoprotein‐E and affect lipid synthesis of liver cells in vitro

In normal pregnancy, hepatic metabolism adaptation occurs with an increase in lipid biosynthesis. Placental shedding of syncytiotrophoblast‐derived extracellular vesicles (STBEVs) into the maternal circulation constitutes a major signalling mechanism between foetus and mother. We investigated whether STBEVs from normal pregnant women might target liver cells in vitro and induce changes in lipid synthesis. This study was performed at the Nuffield Department of Women''s & Reproductive Health, Oxford, UK. STBEVs were obtained by dual‐lobe placental perfusion from 11 normal pregnancies at term. Medium/large and small STBEVs were collected by ultracentrifugation at 10,000g and 150,000g, respectively. STBEVs were analysed by Western blot analysis and flow cytometry for co‐expression of apolipoprotein‐E (apoE) and placental alkaline phosphatase (PLAP). The uptake of STBEVs by liver cells and the effect on lipid metabolism was evaluated using a hepatocarcinoma cell line (HepG2 cells). Data were analysed by one‐way ANOVA and Student''s t test. We demonstrated that: (a) STBEVs carry apoE; (b) HepG2 cells take up STBEVs through an apoE‐LDL receptor interaction; (c) STBEV incorporation into HepG2 cells resulted in (i) increased cholesterol release (ELISA); (ii) increased expression of the genes SQLE and FDPS (microarray) involved in cholesterol biosynthesis; (iii) downregulation of the CLOCK gene (microarray and PCR), involved in the circadian negative control of lipid synthesis in liver cells. In conclusion, the placenta may orchestrate the metabolic adaptation of the maternal liver through release of apoE‐positive STBEVs, by increasing lipid synthesis in a circadian‐independent fashion, meeting the nutritional needs of the growing foetus.     

Immediate prostaglandin E2 synthesis in rat 3Y1 fibroblasts following vasopressin V1a receptor stimulation

Arginine vasopressin (AVP) induces immediate prostaglandin E(2) (PGE(2)) production in rat 3Y1 fibroblasts. Judging from effects of several inhibitors, cytosolic phospholipase A(2)alpha (cPLA(2)alpha) and cyclooxygenase-1 (COX-1) were mainly involved in this reaction. The antagonist of vasopressin receptor V1a, and not that of V2, inhibited the AVP-induced PGE(2) synthesis, indicating that AVP activates cPLA(2)alpha through V1a receptor. Treatment of 3Y1 cells with AVP resulted in transient activation of p44/42 mitogen-activated protein kinase (MAPK) and cPLA(2)alpha, and phosphatidylinositol 3-kinase (PI3K) inhibitor blocked not only AVP-induced PGE(2) synthesis but also MAPK activation, suggesting that PI3K is involved in the AVP-induced MAPK and cPLA(2)alpha activation, which initiates the production of PGE(2). These results suggest that PGE(2) generated by the stimulation of AVP probably modulates the physiological effects of AVP.     

The SCP2‐thiolase‐like protein (SLP) of Trypanosoma brucei is an enzyme involved in lipid metabolism

Bioinformatics studies have shown that the genomes of trypanosomatid species each encode one SCP2‐thiolase‐like protein (SLP), which is characterized by having the YDCF thiolase sequence fingerprint of the Cβ2‐Cα2 loop. SLPs are only encoded by the genomes of these parasitic protists and not by those of mammals, including human. Deletion of the Trypanosoma brucei SLP gene (TbSLP) increases the doubling time of procyclic T. brucei and causes a 5‐fold reduction of de novo sterol biosynthesis from glucose‐ and acetate‐derived acetyl‐CoA. Fluorescence analyses of EGFP‐tagged TbSLP expressed in the parasite located the TbSLP in the mitochondrion. The crystal structure of TbSLP (refined at 1.75 Å resolution) confirms that TbSLP has the canonical dimeric thiolase fold. In addition, the structures of the TbSLP‐acetoacetyl‐CoA (1.90 Å) and TbSLP‐malonyl‐CoA (2.30 Å) complexes reveal that the two oxyanion holes of the thiolase active site are preserved. TbSLP binds malonyl‐CoA tightly (K_d 90 µM), acetoacetyl‐CoA moderately (K_d 0.9 mM) and acetyl‐CoA and CoA very weakly. TbSLP possesses low malonyl‐CoA decarboxylase activity. Altogether, the data show that TbSLP is a mitochondrial enzyme involved in lipid metabolism. Proteins 2016; 84:1075–1096. © 2016 Wiley Periodicals, Inc.     

Suppressive role of endogenous regucalcin in the enhancement of deoxyribonucleic acid synthesis activity in the nucleus of regenerating rat liver

The role of endogenous regucalcin in the regulation of deoxyribonucleic acid (DNA) synthesis activity in the nuclei of regenerating rat liver after partial hepatectomy was investigated. The addition of regucalcin (0.25 and 0.5 microM) in the reaction mixture caused a significant decrease in the nuclear DNA synthesis activity of normal rat liver. This decrease was also seen in the presence of Ca2+-chelator EGTA (0.4 mM), indicating that the effect of regucalcin is not related to nuclear Ca2+. Nuclear DNA activity was significantly increased in the presence of anti-regucalcin monoclonal antibody (10-50 ng/ml) in the reaction mixture. The effect was completely abolished by the addition of regucalcin (0.5 microM). Nuclear DNA synthesis activity was significantly increased at 24, 48, and 72 h after partial heptectomy. The effect of anti-regucalcin monoclonal antibody (25 ng/ml) in increasing nuclear DNA synthesis activity was significantly enhanced at 24 and 48 h after partial hepatectomy. The presence of staurospone (10(-6) M), trifluoperazine (2 x 10(-5) M), or PD98059 (10(-5) M) in the reaction mixture caused a significant decrease in DNA synthesis activity in the nuclei obtained at 24 after partial hepateactomy. The effect of these inhibitors in the presence of anti-regucalcin monoclonal antibody (25 ng/ml) was greater than that in the absence of the antibody. The present study suggests that endogenous regucalcin plays a suppressive role in the enhancement of nuclear DNA synthesis activity in regenerating liver with cell proliferation after partial hepatectomy in rats.     

Plasma leukocyte cell-derived chemotaxin 2 is associated with the severity of systemic inflammation in patients with sepsis

The aim of the present study was to determine the correlations between leukocyte cell-derived chemotaxin 2 (LECT2) and inflammation-related variables in human inflammatory disease. Plasma samples from 23 septic patients who had been admitted to the intensive care unit (ICU) of our institution and 31 volunteers were used. Plasma LECT2 concentrations were examined retrospectively and compared with those of various inflammatory cytokines and routine laboratory data. The LECT2 concentrations of the septic patients at the time of ICU entry (5.3 ± 4.1 ng/mL) were significantly lower than those of the volunteers (19.7 ± 3.4 ng/mL) and these concentrations had significantly increased by the time of ICU discharge. Individual analyses showed that the LECT2 concentrations of all 19 patients had increased by the time of ICU discharge. A combination of LECT2 and C-reactive protein (CRP) concentrations was capable of discriminating the acute and recovery phases of sepsis to a degree similar to those of the combinations of CRP concentration and percentage of neutrophils, CRP concentration and percentage of immature white blood cells, or CRP and interleukin-6 concentrations. Thus, the LECT2 concentration correlates with the severity of systemic inflammation in patients with sepsis. LECT2 may be a reliable diagnostic indicator of human inflammatory diseases.     

Notch is the key factor in the process of fetal liver stem/progenitor cells differentiation into hepatocytes

Cell transplantation is efficient method to therapy end-stage liver disease (ESLD). How to punctually induce stem cell differentiation into hepatocyte is still a challenge. Notch plays important roles in embryonic development and cell differentiation. However, during the differentiation process from fetal liver stem/progenitor cells (FLSPCs) to mature hepatocytes, the contribution of Notch, especially which Notch receptor is primarily responsible, is unknown. First, specific Notch receptor responsible for FLSPCs differentiation was identified. On both tissue level and cell level, we found that Notch3 was the only receptor greater expressed in liver tissue at embryonic day (ED) 14 and FLSPCs, compared with the adult liver and BRL cells, respectively. Second, morphological phenotypic and functional aspects were analyzed to evaluate whether Notch inhibition by GSIs (γ-secretase inhibitors, inhibitor of Notch) promotes the differentiation of FLSPCs into hepatocytes. Results showed that N-[N-(3, 5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) as GSIs was able to induce FLSPCs differentiation into hepatocytes. The differentiated FLSPCs showed similar morphology to mature hepatocytes, expressed hepatic markers indicative of a mature developmental stage, and displayed similar functionality to mature hepatocytes. The differentiation efficiency by GSIs was similar to that by hepatocyte growth factor (HGF) induction. More specifically, as the differentiation of FLSPCs progressed towards hepatocytes, the expression of Notch3 was gradually down-regulated, consistent with the down-regulation of other stem cell markers. These findings imply that Notch3 may not only be a regulator of FLSPCs differentiation into hepatocytes, but also be a potential marker of FLSPCs.     

Platelet-derived growth factor-induced arachidonic acid release for enhancement of prostaglandin E(2) synthesis in human gingival fibroblasts pretreated with interleukin-1beta

Platelet-derived growth factor (PDGF) is a biological mediator for connective tissue cells and plays a critical role in a wide variety of physiological and pathological processes. We here investigated the effect of PDGF on arachidonic acid release and prostaglandin E(2) (PGE(2)) synthesis in human gingival fibroblasts (HGF). PDGF induced arachidonic acid release in a time- and dose-dependent manner, and simultaneously induced a transient increase in intracellular Ca(2+) concentration ([Ca(2+)](i)), but less provoked PGE(2) release and cyclooxygenase-2 (COX-2) mRNA expression. When [Ca(2+)](i) was increased by Ca(2+)-mobilizing reagents, arachidonic acid release was increased. The PDGF-induced arachidonic acid release and increase in [Ca(2+)](i) were prevented by a tyrosine kinase inhibitor. On the other hand, in the HGF pre-stimulated with interleukin-1beta (IL-1beta), PDGF clearly increased PGE(2) release. The PDGF-induced PGE(2) release was inhibited by a tyrosine kinase inhibitor. In the HGF pretreated with IL-1beta, arachidonic acid strongly enhanced PGE(2) release and COX-2 mRNA expression. These results suggest that PDGF stimulates arachidonic acid release by the increase in [Ca(2+)](i) via tyrosine kinase activation, and which contributes to PGE(2) production via COX-2 expression in HGF primed with IL-1beta.     

Concurrent blockade of free radical and microsomal prostaglandin E synthase-1-mediated PGE(2) production improves safety and efficacy in a mouse model of amyotrophic lateral sclerosis

J. Neurochem. (2012) 122, 952-961. ABSTRACT: While free radicals and inflammation constitute major routes of neuronal injury occurring in amyotrophic lateral sclerosis (ALS), neither antioxidants nor non-steroidal anti-inflammatory drugs have shown significant efficacy in human clinical trials. We examined the possibility that concurrent blockade of free radicals and prostaglandin E(2) (PGE(2) )-mediated inflammation might constitute a safe and effective therapeutic approach to ALS. We have developed 2-hydroxy-5-[2-(4-trifluoromethylphenyl)-ethylaminobenzoic acid] (AAD-2004) as a derivative of aspirin. AAD-2004 completely removed free radicals at 50?nM as a potent spin-trapping molecule and inhibited microsomal PGE(2) synthase-1 (mPGES-1) activity in response to both lipopolysaccharide-treated BV2 cell with IC(50) of 230?nM and recombinant human mPGES-1 protein with IC(50) of 249?nM in vitro. In superoxide dismutase 1(G93A) transgenic mouse model of ALS, AAD-2004 blocked free radical production, PGE(2) formation, and microglial activation in the spinal cords. As a consequence, AAD-2004 reduced autophagosome formation, axonopathy, and motor neuron degeneration, improving motor function and increasing life span. In these assays, AAD-2004 was superior to riluzole or ibuprofen. Gastric bleeding was not induced by AAD-2004 even at a dose 400-fold higher than that required to obtain maximal therapeutic efficacy in superoxide dismutase 1(G93A) . Targeting both mPGES-1-mediated PGE(2) and free radicals may be a promising approach to reduce neurodegeneration in ALS and possibly other neurodegenerative diseases.     

Cyclo-oxygenase-1 and -2 differently contribute to prostaglandin E2 synthesis and lipid peroxidation after in vivo activation of N-methyl-D-aspartate receptors in rat hippocampus

Using intracerebral microdialysis, we reported previously that acute in vivo activation of NMDA glutamate receptors triggers rapid and transient releases of prostaglandin E2 (PGE2) and F2-isoprostane 15-F(2t)-IsoP in the hippocampus of freely moving rats. The formation of the two metabolites--produced through cyclo-oxygenase (COX) enzymatic activity and free radical-mediated peroxidation of arachidonic acid (AA), respectively,--was prevented by the specific NMDA antagonist MK-801, and was largely dependent on COX-2 activity. Here, we demonstrate that besides COX-2, which is the prominent COX isoform in the brain and particularly in the hippocampus, the constitutive isoform, COX-1 also contributes to prostaglandin (PG) synthesis and oxidative damage following in vivo acute activation of hippocampal NMDA glutamate receptors. The relative contribution of the two isoforms is dynamically regulated, as the COX-2 selective inhibitor NS398 immediately prevented PGE2 and 15-F(2t)-IsoP formation during the application of NMDA, whereas the COX-1 selective inhibitor SC560 was effective only 1 h after agonist infusion. Our data suggest that, although COX-2 is the prominent isoform, COX-1 activity may significantly contribute to excitotoxicity, particularly when considering the amount of lipid peroxidation associated with its catalytic cycle. We suggest that both isoforms should be considered as possible therapeutic targets to prevent brain damage caused by excitotoxicity.     

Phosphorylation of Dab2 is involved in inhibited VEGF‐VEGFR‐2 signaling induced by downregulation of syndecan‐1 in glomerular endothelial cell

Disabled‐2 (Dab2) and PAR‐3 (partitioning defective 3) are reported to play critical roles in maintaining retinal microvascular endothelial cells biology by regulating VEGF‐VEGFR‐2 signaling. The role of Dab2 and PAR‐3 in glomerular endothelial cell (GEnC) is unclear. In this study, we found that, no matter whether with vascular endothelial growth factor (VEGF) treatment or not, decreased expression of Dab2 could lead to cell apoptosis by preventing activation of VEGF‐VEGFR‐2 signaling in GEnC, accompanied by reduced membrane VEGFR‐2 expression. And silencing of PAR‐3 gene expression caused increased apoptosis of GEnC by inhibiting activation of VEGF‐VEGFR‐2 signaling and membrane VEGFR‐2 expression. In our previous research, we found that the silencing of syndecan‐1 gene expression inhibited VEGF‐VEGFR‐2 signaling by modulating internalization of VEGFR‐2. And our further research demonstrated that downregulation of syndecan‐1 lead to no significant change in the expression of Dab2 and PAR‐3 both at messenger RNA and protein levels in GEnC, while phosphorylation of Dab2 was significantly increased in GEnC transfected with Dab2 small interfering RNA (siRNA) compared with control siRNA. Atypical protein kinase C (aPKC) could induce phosphorylation of Dab2, thus negatively regulating VEGF‐VEGFR‐2 signaling. And we found that decreased expression of syndecan‐1 lead to activation of aPKC, and aPKC inhibitor treatment could block phosphorylation of Dab2 in GEnC. Besides, aPKC inhibitor treatment could activate VEGF‐VGEFR‐2 signaling in GEnC transfected with syndecan‐1 siRNA in a dose‐dependent manner. In conclusion, we speculated that phosphorylation of Dab2 is involved in preventing activation of VEGF‐VEGFR‐2 signaling in GEnC transfected with syndecan‐1 siRNA. This provides a new target for the therapy of GEnC injury and kidney disease.