H2O2 activates G protein, ? 12 to disrupt the junctional complex and enhance ischemia reperfusion injury.
ABSTRACT: The epithelial cell tight junction separates apical and basolateral domains and is essential for barrier function. Disruption of the tight junction is a hallmark of epithelial cell damage and can lead to end organ damage including renal failure. Herein, we identify G?12 activation by H(2)O(2) leading to tight junction disruption and demonstrate a critical role for G?12 activation during bilateral renal ischemia/reperfusion injury. Madin-Darby canine kidney (MDCK) cells with inducible G?12 (G?12-MDCK) and silenced G?12 (shG?12-MDCK) were subjected to ATP depletion/repletion and H(2)O(2)/catalase as models of tight junction disruption and recovery by monitoring transepithelial resistance. In ATP depleted cells, barrier disruption and recovery was not affected by G?12, but reassembly was accelerated by G?12 depletion. In contrast, silencing of G?12 completely protected cells from H(2)O(2)-stimulated barrier disruption, a response that rapidly occurred in control cells. H(2)O(2) activated Src and Rho, and Src inhibition (by PP2), but not Rho (by Y27632), protected cells from H(2)O(2)-mediated barrier disruption. Immunofluorescent and biochemical analysis showed that H(2)O(2) led to increased tyrosine phosphorylation of numerous proteins and altered membrane localization of tight junction proteins through G?12/Src signaling pathway. G?12 and Src were activated in vivo during ischemia/reperfusion injury, and transgenic mice with renal tubular QL?12 (activated mutant) expression were delayed in recovery and showed more extensive injury. Conversely, G?12 knockout mice were nearly completely protected from ischemia/reperfusion injury. Taken together, these studies reveal that ROS stimulates G?12 to activate injury pathways and identifies a therapeutic target for ameliorating ROS mediated injury.
Project description:The restoration of blood flow following thrombolytic therapy causes ischemia and reperfusion (I/R) injury leading to blood-brain barrier (BBB) disruption and subsequent brain edema in patients of ischemic stroke. Levo-tetrahydropalmatine (l-THP) occurs in Corydalis genus and some other plants. However, whether l-THP exerts protective role on BBB disruption following cerebral I/R remains unclear. Male C57BL/6N mice (23 to 28 g) were subjected to 90 min middle cerebral artery occlusion, followed by reperfusion for 24 h. l-THP (10, 20, 40 mg/kg) was administrated by gavage 60 min before ischemia. We found I/R evoked Evans blue extravasation, albumin leakage, brain water content increase, cerebral blood flow decrease, cerebral infarction and neurological deficits, all of which were attenuated by l-THP treatment. Meanwhile, l-THP inhibited tight junction (TJ) proteins down-expression, Src kinase phosphorylation, matrix metalloproteinases-2/9 (MMP-2/9) and caveolin-1 activation. In addition, surface plasmon resonance revealed binding of l-THP to Src kinase with high affinity. Then we found Src kinase inhibitor PP2 could attenuate Evans blue dye extravasation and inhibit the caveolin-1, MMP-9 activation, occludin down-expression after I/R, respectively. In conclusion, l-THP attenuated BBB injury and brain edema, which were correlated with inhibiting the Src kinase phosphorylation.
Project description:Ethanol is metabolized into acetaldehyde in most tissues. In this study, we investigated the synergistic effect of ethanol and acetaldehyde on the tight junction integrity in Caco-2 cell monolayers. Expression of alcohol dehydrogenase sensitized Caco-2 cells to ethanol-induced tight junction disruption and barrier dysfunction, whereas aldehyde dehydrogenase attenuated acetaldehyde-induced tight junction disruption. Ethanol up to 150?mM did not affect tight junction integrity or barrier function, but it dose-dependently increased acetaldehyde-mediated tight junction disruption and barrier dysfunction. Src kinase and MLCK inhibitors blocked this synergistic effect of ethanol and acetaldehyde on tight junction. Ethanol and acetaldehyde caused a rapid and synergistic elevation of intracellular calcium. Calcium depletion by BAPTA or Ca2+-free medium blocked ethanol and acetaldehyde-induced barrier dysfunction and tight junction disruption. Diltiazem and selective knockdown of TRPV6 or CaV1.3 channels, by shRNA blocked ethanol and acetaldehyde-induced tight junction disruption and barrier dysfunction. Ethanol and acetaldehyde induced a rapid and synergistic increase in reactive oxygen species by a calcium-dependent mechanism. N-acetyl-L-cysteine and cyclosporine A, blocked ethanol and acetaldehyde-induced barrier dysfunction and tight junction disruption. These results demonstrate that ethanol and acetaldehyde synergistically disrupt tight junctions by a mechanism involving calcium, oxidative stress, Src kinase and MLCK.
Project description:Disruption of intestinal epithelial tight junctions is an important event in the pathogenesis of ulcerative colitis. Dextran sodium sulfate (DSS) induces colitis in mice with symptoms similar to ulcerative colitis. However, the mechanism of DSS-induced colitis is unknown. We investigated the mechanism of DSS-induced disruption of intestinal epithelial tight junctions and barrier dysfunction in Caco-2 cell monolayers in vitro and mouse colon in vivo. DSS treatment resulted in disruption of tight junctions, adherens junctions and actin cytoskeleton leading to barrier dysfunction in Caco-2 cell monolayers. DSS induced a rapid activation of c-Jun N-terminal kinase (JNK), and the inhibition or knockdown of JNK2 attenuated DSS-induced tight junction disruption and barrier dysfunction. In mice, DSS administration for 4 days caused redistribution of tight junction and adherens junction proteins from the epithelial junctions, which was blocked by JNK inhibitor. In Caco-2 cell monolayers, DSS increased intracellular Ca(2+) concentration, and depletion of intracellular Ca(2+) by 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid tetrakis(acetoxymethyl ester) (BAPTA/AM) or thapsigargin attenuated DSS-induced JNK activation, tight junction disruption and barrier dysfunction. Knockdown of apoptosis signal-regulated kinase 1 (Ask1) or MKK7 blocked DSS-induced tight junction disruption and barrier dysfunction. DSS activated c-Src by a Ca2+ and JNK-dependent mechanism. Inhibition of Src kinase activity or knockdown of c-Src blocked DSS-induced tight junction disruption and barrier dysfunction. DSS increased tyrosine phosphorylation of occludin, zonula occludens-1 (ZO-1), E-cadherin and ?-catenin. SP600125 abrogated DSS-induced tyrosine phosphorylation of junctional proteins. Recombinant JNK2 induced threonine phosphorylation and auto-phosphorylation of c-Src. The present study demonstrates that Ca(2+)/Ask1/MKK7/JNK2/cSrc signalling cascade mediates DSS-induced tight junction disruption and barrier dysfunction.
Project description:Cerebral ischemia, or stroke, is widespread leading cause of death and disability. Surgical and pharmacological interventions that recover blood flow are the most effective treatment strategies for stroke patients. However, restoring the blood supply is accompanied by severe reperfusion injury, with edema and astrocyte end-feet disruption. Here, we report that the oral administration of CU06-1004 (previously Sac-1004), immediately after onset of ischemia/reperfusion (I/R), ameliorated cerebral damage. CU06-1004 stabilized blood?brain barrier by inhibiting the disruption of the tight junction-related protein zona occludens-1 and the cortical actin ring in endothelial cells (ECs) after I/R. Interestingly, CU06-1004 significantly suppressed astrocyte end-feet swelling following I/R, by reducing aquaporin 4 and connexin 43 levels, which mediates swelling. Furthermore, the degradation of ?1-integrin and ?-dystroglycan, which anchors to the cortical actin ring in ECs, was inhibited by CU06-1004 administration after I/R. Consistently, CU06-1004 administration following I/R also suppressed the loss of laminin and collagen type IV, which bind to the cortical actin ring anchoring proteins. Unlike the protective effects of CU06-1004 in ECs, astrocyte viability and proliferation were not directly affected. Taken together, our observations suggest that CU06-1004 inhibits I/R-induced cerebral edema and astrocyte end-feet swelling by maintaining EC junction stability. KEY MESSAGES: • CU06-1004 ameliorates I/R-induced cerebral injury. • EC junction integrity was stabilized by CU06-1004 treatment after I/R. • CU06-1004 reduces astrocyte end-feet swelling following I/R. • EC junction stability affects astrocyte end-feet structure maintenance after I/R.
Project description:Injury due to brain ischemia followed by reperfusion (I/R) may be an important therapeutic target in the era of thrombectomy. FTY720, a widely known sphingosine-1-phosphate receptor agonist, exerts various neuroprotective effects. The aim of this study was to examine the protective effect of FTY720 with respect to I/R injury, especially focusing on blood-brain barrier (BBB) protection and anti-inflammatory effects. Male rats were subjected to transient ischemia and administered vehicle or 0.5 or 1.5 mg/kg of FTY720 immediately before reperfusion. Positron emission tomography (PET) with [18F]DPA-714 was performed 2 and 9 days after the insult to serially monitor neuroinflammation. Bovine and rat brain microvascular endothelial cells (MVECs) were also subjected to oxygen-glucose deprivation (OGD) and reperfusion, and administered FTY720, phosphorylated-FTY720 (FTY720-P), or their inhibitor. FTY720 dose-dependently reduced cell death, the infarct size, cell death including apoptosis, and inflammation. It also ameliorated BBB disruption and neurological deficits compared to in the vehicle group. PET indicated that FTY720 significantly inhibited the worsening of inflammation in later stages. FTY720-P significantly prevented the intracellular redistribution of tight junction proteins but did not increase their mRNA expression. These results suggest that FTY720 can ameliorate I/R injury by protecting the BBB and regulating neuroinflammation.
Project description:Neuroinflammation mediated by activation of microglia and interruption of the blood-brain barrier (BBB) is an important factor that contributes to neuron death and infarct area diffusion in ischemia reperfusion injury. Finding novel molecules to regulate neuroinflammation is of significant clinical value. We have previously shown that adjudin, a small molecule compound known to possess antispermatogenic function, attenuates microglia activation by suppression of the NF-?B pathway. In this study we continued to explore whether adjudin could be neuroprotective by using the transient middle cerebral artery occlusion (tMCAO) model. Adjudin treatment after reperfusion significantly decreased the infarction volume and neuroscore compared to the vehicle group. Staining of CD11b showed that adjudin markedly inhibited microglial activation in both the cortex and the striatum, accompanied by a reduction in the expression and release of cytokines TNF-?, IL-1? and IL-6. Concomitantly, adjudin noticeably prevented BBB disruption after ischemia and reperfusion, as indicated by the reduction of IgG detection in the brain cortex and striatum versus the vehicle group. This finding was also corroborated by immunofluorescence staining and immunoblotting of tight junction-related proteins ZO-1, JAM-A and Occludin, where the reduction of these proteins could be attenuated by adjudin treatment. Moreover, adjudin obviously inhibited the elevated MMP-9 activity after stroke. Together these data demonstrate that adjudin protects against cerebral ischemia reperfusion injury, and we present an effective neuroinflammation modulator with clinical potential.
Project description:We have previously shown that increases in blood-brain barrier permeability represent an important component of ischemia-reperfusion related brain injury in the fetus. Pro-inflammatory cytokines could contribute to these abnormalities in blood-brain barrier function. We have generated pharmacological quantities of mouse anti-ovine interleukin-1? monoclonal antibody and shown that this antibody has very high sensitivity and specificity for interleukin-1? protein. This antibody also neutralizes the effects of interleukin-1? protein in vitro. In the current study, we hypothesized that the neutralizing anti-interleukin-1? monoclonal antibody attenuates ischemia-reperfusion related fetal blood-brain barrier dysfunction. Instrumented ovine fetuses at 127 days of gestation were studied after 30 min of carotid occlusion and 24h of reperfusion. Groups were sham operated placebo-control- (n=5), ischemia-placebo- (n=6), ischemia-anti-IL-1? antibody- (n=7), and sham-control antibody- (n=2) treated animals. Systemic infusions of placebo (0.154M NaCl) or anti-interleukin-1? monoclonal antibody (5.1±0.6 mg/kg) were given intravenously to the same sham or ischemic group of fetuses at 15 min and 4h after ischemia. Concentrations of interleukin-1? protein and anti-interleukin-1? monoclonal antibody were measured by ELISA in fetal plasma, cerebrospinal fluid, and parietal cerebral cortex. Blood-brain barrier permeability was quantified using the blood-to-brain transfer constant (Ki) with ?-aminoisobutyric acid in multiple brain regions. Interleukin-1? protein was also measured in parietal cerebral cortices and tight junction proteins in multiple brain regions by Western immunoblot. Cerebral cortical interleukin-1? protein increased (P<0.001) after ischemia-reperfusion. After anti-interleukin-1? monoclonal antibody infusions, plasma anti-interleukin-1? monoclonal antibody was elevated (P<0.001), brain anti-interleukin-1? monoclonal antibody levels were higher (P<0.03), and interleukin-1? protein concentrations (P<0.03) and protein expressions (P<0.001) were lower in the monoclonal antibody-treated group than in placebo-treated-ischemia-reperfusion group. Monoclonal antibody infusions attenuated ischemia-reperfusion-related increases in Ki across the brain regions (P<0.04), and Ki showed an inverse linear correlation (r= -0.65, P<0.02) with anti-interleukin-1? monoclonal antibody concentrations in the parietal cortex, but had little effect on tight junction protein expression. We conclude that systemic anti-interleukin-1? monoclonal antibody infusions after ischemia result in brain anti-interleukin-1? antibody uptake, and attenuate ischemia-reperfusion-related interleukin-1? protein up-regulation and increases in blood-brain barrier permeability across brain regions in the fetus. The pro-inflammatory cytokine, interleukin-1?, contributes to impaired blood-brain barrier function after ischemia in the fetus.
Project description:Disruption of the mucosal barrier following intestinal ischemia reperfusion (I/R) is life threatening in clinical practice. Mitochondrial dysfunction and oxidative stress significantly contribute to the early phase of I/R injury and amplify the inflammatory response. MitoQ is a mitochondrially targeted antioxidant that exerts protective effects following I/R injury. In the present study, we aimed to determine whether and how MitoQ protects intestinal epithelial cells (IECs) from I/R injury. In both in vivo and in vitro studies, we found that MitoQ pretreatment downregulated I/R-induced oxidative stress and stabilized the intestinal barrier, as evidenced by MitoQ-treated I/R mice exhibiting attenuated intestinal hyperpermeability, inflammatory response, epithelial apoptosis, and tight junction damage compared to controls. Mechanistically, I/R elevated mitochondrial 8-hydroxyguanine content, reduced mitochondrial DNA (mtDNA) copy number and mRNA transcription levels, and induced mitochondrial disruption in IECs. However, MitoQ pretreatment dramatically inhibited these deleterious effects. mtDNA depletion alone was sufficient to induce apoptosis and mitochondrial dysfunction of IECs. Mitochondrial transcription factor A (TFAM), a key activator of mitochondrial transcription, was significantly reduced during I/R injury, a phenomenon that was prevented by MitoQ treatment. Furthermore, we observed that thee protective properties of MitoQ were affected by upregulation of cellular antioxidant genes, including HO-1, NQO-1, and γ-GCLC. Transfection with Nrf2 siRNA in IECs exposed to hypoxia/reperfusion conditions partially blocked the effects of MitoQ on mtDNA damage and mitochondrial oxidative stress. In conclusion, our data suggest that MitoQ exerts protective effect on I/R-induced intestinal barrier dysfunction.
Project description:At the blood-brain barrier (BBB), laminin-?5 is predominantly synthesized by endothelial cells and mural cells. Endothelial laminin-?5 is dispensable for BBB maintenance under homeostatic conditions but inhibits inflammatory cell extravasation in pathological conditions. Whether mural cell-derived laminin-?5 is involved in vascular integrity regulation, however, remains unknown. To answer this question, we generated transgenic mice with laminin-?5 deficiency in mural cells (?5-PKO). Under homeostatic conditions, no defects in BBB integrity and cerebral blood flow (CBF) were observed in ?5-PKO mice, suggesting that mural cell-derived laminin-?5 is dispensable for BBB maintenance and CBF regulation under homeostatic conditions. After ischemia-reperfusion (MCAO) injury, however, ?5-PKO mice displayed less severe neuronal injury, including reduced infarct volume, decreased neuronal death, and improved neurological function. In addition, ?5-PKO mice also showed attenuated vascular damage (milder BBB disruption, reduced inflammatory cell infiltration, decreased brain edema, and diminished hemorrhagic transformation). Mechanistic studies revealed less severe tight junction protein (TJP) loss and pericyte coverage reduction in ?5-PKO mice after ischemia-reperfusion injury, indicating that the attenuated ischemic injury in ?5-PKO mice is possibly due to less severe vascular damage. These findings suggest that mural cell-derived laminin-?5 plays a detrimental role in ischemic stroke and that inhibiting its signaling may have a neuroprotective effect.
Project description:We examined whether p38 MAPK plays role in calcium oxalate monohydrate (COM) crystal-induced tight junction disruption. Polarized MDCK cells were pretreated with or without 20 ?M SB239063 (p38 MAPK inhibitor) for 2-h, and then incubated with 100 ?g/ml COM crystals for up to 48-h. Western blotting showed increased level of phospho-p38, not total p38, in COM-treated cells, whereas SB239063 pretreatment successfully maintained phospho-p38 at its basal level. COM crystals also caused decreased levels of two tight junction proteins, zonula occludens-1 (ZO-1) and occludin. Immunofluorescence study revealed disruption of tight junction, redistribution, and dissociation of ZO-1 and occludin. Moreover, transepithelial resistance (TER) showed defective barrier function, whereas Western blotting for Na(+)/K(+)-ATPase-?1 revealed defective fence function of tight junction in COM-treated cells. All these expression and functional defects were successfully prevented by SB239063 pretreatment. These findings indicate that COM crystals cause tight junction disruption in distal renal tubular epithelial cells through p38 MAPK activation.