CDX1 is an important molecular mediator of Barrett's metaplasia

The molecular pathogenesis of Barrett's metaplasia (BM) of the esophagus is poorly understood. The change to an intestinal phenotype occurs on a background of esophagitis due to refluxing acid and bile. CDX1, an important regulator of normal intestinal development, was studied as a potential key molecule in the pathogenesis of BM. CDX1 mRNA and protein were universally expressed in all samples of BM tested but not in normal esophageal squamous or gastric body epithelia. This tissue-specific expression was attributable to the methylation status of the CDX1 promoter. Conjugated bile salts and the inflammatory cytokines TNF-alpha and IL-1beta were all found to increase CDX1 mRNA expression in vitro. These effects were primarily mediated by NF-kappaB signaling but only occurred when the CDX1 promoter was unmethylated or partially methylated. The data suggest that CDX1 is a key molecule linking etiological agents of BM to the development of an intestinal phenotype. Although the initial trigger for CDX1 promoter demethylation is not yet identified, it seems likely that demethylation of its promoter may be the key to the induction and maintenance of CDX1 expression and so of the BM phenotype.     

N-乙酰半胱氨酸通过AMPK/SIRT1途径抑制缺氧诱导的大鼠脑血管内皮细胞损伤

目的研究N-乙酰半胱氨酸(NAC)对缺氧诱导脑血管内皮细胞损伤的调节作用及分子机制。方法选择SD大鼠分离培养脑血管内皮细胞,分为常氧组、缺氧组、0. 5 NAC组(缺氧+0. 5 mol/L NAC)、1. 0 NAC组(缺氧+1. 0 mol/L NAC)、NAC+8-bAMP组(缺氧+1. 0 mol/L NAC+1. 0 mol/L 8-bAMP)。采用MTS法检测细胞增殖活力,采用TUNEL染色检测凋亡率,采用试剂盒检测氧化应激指标,采用Western blot检测凋亡基因、AMPK/SIRT1通路分子的表达量。结果缺氧组细胞OD490值、T-AOC含量及B淋巴细胞瘤2(Bcl-2)、p-AMP活化蛋白激酶(pAMPK)、去乙酰化酶Sirtuin1(SIRT1)表达量均明显低于常氧组;缺氧组细胞凋亡率、细胞中活性氧(ROS)、丙二醛(MDA)、8-羟基脱氧鸟苷(8-OHDG)含量及bcl-2相关X蛋白(bax)、细胞色素C(Cyt-C)、含半胱氨酸的天冬氨酸蛋白水解酶3(Caspase-3)的表达量均明显高于常氧组。0. 5 NAC组、1. 0 NAC组细胞OD490值、T-AOC量及Bcl-2、pAMPK、SIRT1表达量均明显高于缺氧组; 0. 5 NAC组、1. 0 NAC组细胞凋亡率、ROS、MDA、8-OHDG含量及Caspase-3、Cyt-C、Bax的表达量均明显低于缺氧组。NAC+8-bAMP组细胞OD490值、T-AOC及Bcl-2、p-AMPK、SIRT1表达量均明显低于1. 0 NAC组; NAC+8-bAMP组凋亡率、ROS、MDA、8-OHDG含量及Caspase-3、Cyt-C、Bax的表达量均明显高于1. 0 NAC组。结论 NAC能够通过激活AMPK/SIRT1通路来减轻氧化应激及线粒体凋亡介导的脑血管内皮细胞损伤。     

IL-1 is an important mediator for microcirculatory changes in endotoxin-induced intestinal mucosal damage

Although small intestine is frequently injured in endotoxin shock, the exact pathological sequence has not been fully understood. The major objective of this study is to elucidate the role of interleukin (IL)-1 in endotoxin-induced microcirculatory disturbance of rat small intestine. Mucosal and submucosal microvessels of the rat ileum were observed by intravital microscope with a high speed video camera system and the attenuating effect of E5090, an inhibitor of IL-1 generation, on endotoxin-induced intestinal microcirculatory disturbances was investigated. Endotoxin infusion produced significant mucosal damage, but before these morphological changes became significant, microvascular stasis in villi, decreased red blood cell velocity, and increased leukocyte adherence to venular walls were observed in intestinal microcirculatory beds 30 min after endotoxin administration. Intestinal IL-1 levels were also significantly increased at that time. Endotoxin treatment enhanced chemiluminescence activity from neurophils and rapidly mobilized CD18 on leukocytes. E5090, which suppressed the IL-1 production in intestinal mucosa, attenuated the microcirculatory disturbances induced by endotoxin, and significantly reduced the subsequent mucosal damage. E5090 also attenuated the increased chemiluminescence activity and CD18 expression on leukocytes. In conclusion, the production of IL-1 is enhanced in the intestinal mucosa during endotoxin infusion. IL-1 may be an important mediator of microcirculatory changes, including decreased red blood cell velocity and increased leukocyte sticking and its activation, leading to the mucosal damage.     

CD157 is an important mediator of neutrophil adhesion and migration

CD157, a glycosylphosphatidylinositol (GPI)-anchored protein encoded by a member of the CD38 NADase/ADP-ribosyl cyclase gene family, is expressed on the surface of most human circulating neutrophils. This work demonstrates that CD157 is a receptor that induces reorganization of the cytoskeleton and significant changes in cell shape, and that signals mediated by CD157 act through modulation of cytosolic Ca(2+) concentration. These signals are independent of the products of CD157's enzymatic activities (ie, cyclic adenosine diphosphate [ADP]-ribose and ADP-ribose). Indeed, the enzymatic activities of CD157 in circulating neutrophils as well as in dimethyl sulfoxide (DMSO)-differentiated (CD157(+)/CD38(-)) HL-60 cells, are hardly detectable. This work also shows that the receptorial activity relies on cross-talk between CD157 and beta(2) integrin. CD157 localizes in GM1-enriched lipid rafts and, upon activation, it migrates to the uropod, a structure specialized in motility and adhesive functions. Indeed, CD157 is involved in adhesion to extracellular matrix proteins and in chemotaxis induced in vitro by formyl-methionyl-leucyl-phenylalanine (fMLP). These findings were consistent with the results obtained in neutrophils from patients with paroxysmal nocturnal hemoglobinuria (PNH), in which CD157 is deficient. These neutrophils showed constant defects in adhesion and migration. Our data attribute specific and crucial roles to CD157 in the regulation of innate immunity during inflammation.     

Osteopontin is an important mediator of alcoholic liver disease via hepatic stellate cell activation

AIM: To investigate over-expression of Osteopontin (OPN) pathway expression and mechanisms of action in human alcoholic liver disease (ALD), in vivo and in vitro acute alcohol models.METHODS: OPN pathway was evaluated in livers from patients with progressive stages of human ALD and serum from drinkers with and without liver cirrhosis. In vitro stellate LX2 cells exposed to acute alcohol and in vivo in acute alcoholic steatosis mouse models were also investigated for OPN pathway expression and function. WT and OPN^-/- mice were administered an acute dose of alcohol and extent of liver injury was examined by histopathology and liver biochemistry after 16-24 h. The causative role of OPN was studied in OPN knockout animals and in vitro in stellate LX2 cells, utilizing siRNA, aptamer and neutralizing antibodies to block OPN and OPN pathway. OPN pathway expression and downstream functional consequences were measured for signaling by Western blotting, plasmin activation by spectrophotometric assays and cell migration by confocal imaging and quantitation.RESULTS: OPN expression positively correlated with disease severity in patients with progressive stages of ALD. In vivo, associated with alcoholic steatosis, a single dose of acute alcohol significantly increased hepatic OPN mRNA and protein, and a cleaved OPN form in a dose dependent manner. OPN mRNA and secreted OPN also increased in parallel with activation of LX2 stellate cells within 4 h of a single dose of alcohol. Expression of OPN receptors, αvβ3-integrin and CD44, increased in human ALD, and in vivo and in vitro with alcohol administration. This was accompanied by downstream phosphorylation of Akt and Erk, increased mRNA expression of several fibrogenesis, fibrinolysis and extracellular matrix pathway genes, plasmin activation and hepatic stellate cell (HSC) migration. Inhibition of OPN and OPN-receptor mediated signaling partially inhibited alcohol-induced HSC activation, plasmin activity and cell migration.CONCLUSION: OPN is a key mediator of the alcohol-induced effects on hepatic stellate cell functions and liver fibrogenesis.     

Macrophage migration inhibitory factor is an important mediator in the pathogenesis of gastric inflammation in rats

BACKGROUND & AIMS: Macrophage migration inhibitory factor (MIF) has been shown to play a pivotal role in inflammatory and immune-mediated diseases. This study investigates the role of MIF in gastric inflammation. METHODS: Expression of MIF was examined in a rat gastric ulcer model induced by acetic acid, and the functional role of MIF in acute gastric ulcer was investigated by administration of a neutralizing anti-MIF antibody. RESULTS: MIF messenger RNA and protein were markedly up-regulated in acute gastric ulcer, which correlated with the accumulation of macrophages (P < 0.001) and neutrophils (P < 0.05) at the site of inflammation. Macrophages, like neutrophils, were the major inflammatory cells infiltrating the ulcer base and they strongly expressed inducible nitric oxide synthase. However, macrophages, not neutrophils, were a rich source of MIF production in acute gastric ulcer. In vivo and in vitro blockade of MIF with the neutralizing anti-MIF antibody significantly inhibited the marked up-regulation of MIF, tumor necrosis factor alpha, inducible nitric oxide synthase, and intercellular adhesion molecule-1. This was associated with the marked inhibition of macrophage (70% reduced) and neutrophil (60% reduced) accumulation and activation, thus reducing ulcer sizes and attenuating ulceration. CONCLUSIONS: This study has shown that MIF was markedly up-regulated during acute gastric ulcer. Inhibition of acute gastric ulcer by blockade of MIF indicates that MIF is a key inflammatory mediator and plays a pathogenic role in gastric inflammation.     

Tumour necrosis factor alpha is an important mediator of portal and systemic haemodynamic derangements in alcoholic hepatitis

BACKGROUND: The role of proinflammatory cytokines in the pathogenesis of portal hypertension is unclear. AIMS AND METHODS: This study tests the hypothesis that tumour necrosis factor alpha (TNF-alpha) is an important mediator of the circulatory disturbances in alcoholic hepatitis (AH) and evaluates the acute and short term effect of a single infusion of the monoclonal chimeric anti-TNF-alpha antibody (Infliximab) on portal and systemic haemodynamics in 10 patients with severe biopsy proven AH. Cardiovascular haemodynamics, hepatic venous pressure gradient (HVPG), and hepatic and renal blood flow were measured before, 24 hours after Infliximab, and prior to hospital discharge. RESULTS: Serum bilirubin (p<0.05), C reactive protein (p<0.001), and white cell count (p<0.01) were reduced significantly, as were plasma levels of interleukin (IL)-6 and IL-8 after treatment. Of the 10 patients, nine were alive at 28 days. Mean HVPG decreased significantly at 24 hours (23.4 (2.8) to 14.3 (1.9) mm Hg; p<0.001) with a sustained reduction prior to discharge (12.8 (1.9) mm Hg; p<0.001). Mean arterial pressure and systemic vascular resistance increased significantly (p<0.001and p<0.01, respectively), mirrored by a reduction in cardiac index (5.9 (0.5) to 4.7 (0.5) l/min/m(2); p<0.05) prior to discharge. Hepatic and renal blood flow also increased significantly (506.2 (42.9) to 646.3 (49.2) ml/min (p=0.001) and 424.3 (65.12) to 506.3 (85.7) ml/min (p=0.001), respectively) prior to discharge. CONCLUSION: The results of this study illustrate that anti-TNF-alpha treatment in AH patients produces a highly significant, early, and sustained reduction in HVPG, possibly through a combination of a reduction in cardiac output and intrahepatic resistance. In addition, there was a reduction in hepatic inflammation and improved organ blood flow, suggesting an important role for TNF-alpha in mediating the circulatory disturbances in AH.     

DJ-1 is critical for mitochondrial function and rescues PINK1 loss of function

Mutations or deletions in PARKIN/PARK2, PINK1/PARK6, and DJ-1/PARK7 lead to autosomal recessive parkinsonism. In Drosophila, deletions in parkin and pink1 result in swollen and dysfunctional mitochondria in energy-demanding tissues. The relationship between DJ-1 and mitochondria, however, remains unclear. We now report that Drosophila and mouse mutants in DJ-1 show compromised mitochondrial function with age. Flies deleted for DJ-1 manifest similar defects as pink1 and parkin mutants: male sterility, shortened lifespan, and reduced climbing ability. We further found poorly coupled mitochondria in vitro and reduced ATP levels in fly and mouse DJ-1 mutants. Surprisingly, up-regulation of DJ-1 can ameliorate pink1, but not parkin, mutants in Drosophila; cysteine C104 (analogous to C106 in human) is critical for this rescue, implicating the oxidative functions of DJ-1 in this property. These results suggest that DJ-1 is important for proper mitochondrial function and acts downstream of, or in parallel to, pink1. These findings link DJ-1, pink1, and parkin to mitochondrial integrity and provide the foundation for therapeutics that link bioenergetics and parkinsonism.     

IL-1beta is an essential mediator of the antineurogenic and anhedonic effects of stress

Stress decreases neurogenesis in the adult hippocampus, and blockade of this effect is required for the actions of antidepressants in behavioral models of depression. However, the mechanisms underlying these effects of stress have not been identified. Here, we demonstrate an essential role for the proinflammatory cytokine IL-1beta. Administration of IL-1beta or acute stress suppressed hippocampal cell proliferation. Blockade of the IL-1beta receptor, IL-1RI, by using either an inhibitor or IL-1RI null mice blocks the antineurogenic effect of stress and blocks the anhedonic behavior caused by chronic stress exposure. In vivo and in vitro studies demonstrate that hippocampal neural progenitor cells express IL-1RI and that activation of this receptor decreases cell proliferation via the nuclear factor-kappaB signaling pathway. These findings demonstrate that IL-1beta is a critical mediator of the antineurogenic and depressive-like behavior caused by acute and chronic stress.     

The post-endocytotic fate of the gonadotropin receptors is an important determinant of the desensitization of gonadotropin responses

Internalization of the ligand/receptor complexes is a consequence of the activation of the gonadotropin receptors. Since the recycling or degradation of the internalized receptors results in the maintenance or loss of cell surface receptors respectively and this contributes to the loss of responsiveness, we hypothesized that the fate of the internalized receptors could be an important component of desensitization. We examined this hypothesis using the wild-type and mutants of the human LH (hLHR) receptors and follitropin receptors expressed in MA-10 and KK-1 cells respectively. The receptor mutants were chosen because they are routed mostly to a lysosomal degradation pathway whereas the wild-type receptors are recycled back to the surface. We have shown that agonist stimulation of cells expressing the mutant receptors results in a more pronounced loss of cell surface receptors and agonist responses than stimulation of cells expressing the wild-type receptors. We concluded that receptor recycling promotes the maintenance of cell surface receptors and preserves hormonal responsiveness. This property of the hLHR is likely to be physiologically important because there at least two hLHR-expressing tissues in pregnant women, the maternal corpus luteum and the fetal Leydig cells, where a loss of hormonal responsiveness induced by the elevated levels of human chorionic gonadotropin that occur during pregnancy is not desirable.     

RNA binding activity of the recessive parkinsonism protein DJ-1 supports involvement in multiple cellular pathways

Parkinson's disease (PD) is a major neurodegenerative condition with several rare Mendelian forms. Oxidative stress and mitochondrial function have been implicated in the pathogenesis of PD but the molecular mechanisms involved in the degeneration of neurons remain unclear. DJ-1 mutations are one cause of recessive parkinsonism, but this gene is also reported to be involved in cancer by promoting Ras signaling and suppressing PTEN-induced apoptosis. The specific function of DJ-1 is unknown, although it is responsive to oxidative stress and may play a role in the maintenance of mitochondria. Here, we show, using four independent methods, that DJ-1 associates with RNA targets in cells and the brain, including mitochondrial genes, genes involved in glutathione metabolism, and members of the PTEN/PI3K cascade. Pathogenic recessive mutants are deficient in this activity. We show that DJ-1 is sufficient for RNA binding at nanomolar concentrations. Further, we show that DJ-1 binds RNA but dissociates after oxidative stress. These data implicate a single mechanism for the pleiotropic effects of DJ-1 in different model systems, namely that the protein binds multiple RNA targets in an oxidation-dependent manner.     

Prokineticin 2 is an endangering mediator of cerebral ischemic injury

Stroke causes brain dysfunction and neuron death, and the lack of effective therapies heightens the need for new therapeutic targets. Here we identify prokineticin 2 (PK2) as a mediator for cerebral ischemic injury. PK2 is a bioactive peptide initially discovered as a regulator of gastrointestinal motility. Multiple biological roles for PK2 have been discovered, including circadian rhythms, angiogenesis, and neurogenesis. However, the role of PK2 in neuropathology is unknown. Using primary cortical cultures, we found that PK2 mRNA is up-regulated by several pathological stressors, including hypoxia, reactive oxygen species, and excitotoxic glutamate. Glutamate-induced PK2 expression is dependent on NMDA receptor activation and extracellular calcium. Enriched neuronal culture studies revealed that neurons are the principal source of glutamate-induced PK2. Using in vivo models of stroke, we found that PK2 mRNA is induced in the ischemic cortex and striatum. Central delivery of PK2 worsens infarct volume, whereas PK2 receptor antagonist decreases infarct volume and central inflammation while improving functional outcome. Direct central inhibition of PK2 using RNAi also reduces infarct volume. These findings indicate that PK2 can be activated by pathological stimuli such as hypoxia-ischemia and excitotoxic glutamate and identify PK2 as a deleterious mediator for cerebral ischemia.     

The Parkinson's disease gene DJ-1 is also a key regulator of stroke-induced damage

Recent evidence has indicated that common mechanisms play roles among multiple neurological diseases. However, the specifics of these pathways are not completely understood. Stroke is caused by the interruption of blood flow to the brain, and cumulative evidence supports the critical role of oxidative stress in the ensuing neuronal death process. DJ-1 (PARK7) has been identified as the gene linked to early-onset familial Parkinson's disease. Currently, our work also shows that DJ-1 is central to death in both in Vitro and in Vivo models of stroke. Loss of DJ-1 increases the sensitivity to excitotoxicity and ischemia, whereas expression of DJ-1 can reverse this sensitivity and indeed provide further protection. Importantly, DJ-1 expression decreases markers of oxidative stress after stroke insult in Vivo, suggesting that DJ-1 protects through alleviation of oxidative stress. Consistent with this finding, we demonstrate the essential role of the oxidation-sensitive cysteine-106 residue in the neuroprotective activity of DJ-1 after stroke. Our work provides an important example of how a gene seemingly specific for one disease, in this case Parkinson's disease, also appears to be central in other neuropathological conditions such as stroke. It also highlights the important commonalities among differing neuropathologies.     

The effect of hypoxia-inducible factor 1-alpha on hypoxia-induced apoptosis in primary neonatal rat ventricular myocytes

Aim

To study the role of hypoxia-inducible factor 1-alpha (HIF-1α) on hypoxia-induced apoptosis in primary neonatal rat ventricular myocytes.

Methods

Primary neonatal rat ventricular myocytes were exposed to hypoxia for 24 hours. HIF-1α activity was suppressed by treating the cells with 3-(5′-hydroxymethyl-2′-furyl)-1-benzyl indazole (YC-1). The degree of cell apoptosis was assessed by Hoechst 33258 DNA staining. The levels of HIF-1α and the pro-apoptotic proteins Bnip3, Bax and Bad were measured with western blotting.

Results

On exposure to hypoxia, there was an increase in the expression levels of HIF-1α, and the pro-apoptotic protein Bnip3 was upregulated. Suppression of HIF-1α activity by YC-1 treatment was followed by blockade of hypoxia-induced apoptosis and Bnip3 expression; however, the changes in the levels of Bax and Bad expression were unclear.

Conclusion

Acute hypoxia enhanced primary neonatal rat ventricular myocyte apoptosis through the activation of HIF-1α and a mechanism that perhaps involved Bnip3. Targeting HIF-1α may be a new strategy for reducing the degree of hypoxia-induced apoptosis in ventricular myocytes.