Cell Barrier Disruption Research Articles

LACTOBACILLUS MURINUS ACTIVATES THE ARYL HYDROCARBON RECEPTOR TO ATTENUATE TNF-ALPHA-INDUCED PRO-INFLAMMATORY RESPONSES IN HUMAN INTESTINAL EPITHELIAL CELLS

Abstract BACKGROUND Anti-TNF-alpha therapy is an established treatment modality for inflammatory bowel disease (IBD). Yet up to 30% of patients do not respond to anti-TNF-alpha therapy (primary non-responders), and almost 50% of responders lose clinical efficacy over time (secondary non-responders). Furthermore, numerous safety concerns are associated with the long-term use of anti-TNF-alpha agents. Anti-TNF-alpha therapy safety concerns and the increased incidence of non-responders have driven the need to develop innovative strategies to treat IBD. OBJECTIVE A potential alternative to anti-TNF-alpha therapy is the use of novel probiotics capable of attenuating the damaging effects of TNF-alpha on the intestinal epithelium. We hypothesized that Lactobacillus murinus attenuates TNF-alpha induced pro-inflammatory responses and decreases barrier disruption in human intestinal epithelial cells (Caco-2) by activating the aryl hydrocarbon receptor (AhR). METHODS We used Caco-2 cells grown on Transwell inserts to model the human intestinal epithelial barrier. Transepithelial Electrical Resistance (TEER) was measured to evaluate the formation of tight junctions. Once TEER reached 500 ohms per cm2, the cells were considered to have formed tight junctions. To induce pro-inflammatory responses, Caco-2 cell monolayers were treated with increasing doses of TNF-alpha (0.1, 1 and 10 ng/ml). Caco-2 cell monolayers were also treated with L. murinus to test its ability to activate AhR. Two defined ligands for AhR were used as controls i.e., 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 6-formylindolo[3,2-b] carbazole (FICZ). Next, we pre-treated the Caco-2 cells with either L. murinus or defined AhR ligands for 12 hours. After pre-treatment, monolayers were stimulated with TNF-alpha for 24 hours. IL-8 and TEER were quantified as endpoints to determine treatment efficacy. RESULTS TNF-alpha stimulation decreased the TEER and induced the secretion of IL-8 in a dose-dependent manner thus, providing a model of intestinal inflammation. AhR was significantly activated in monolayers treated with L. murinus as well as with defined AhR ligands. Furthermore, pre-treating the cells with L. murinus or with endogenous AhR ligand attenuated the TNF-alpha induced pro-inflammatory response and decreased epithelial barrier disruption in Caco-2 cells. CONCLUSIONS These results indicate that TNF-alpha induced damage to the gut wall can be attenuated using novel probiotics such L. murinus or endogenous AhR ligand. These data suggest that AhR could be a novel drug target for treating IBD. These results also indicate that L. murinus may be a potential therapeutic to mitigate or treat IBD without the use of anti-TNF-alpha agents.

Hydroxyxanthone ameliorates IL1β-induced epithelial barrier disruption in colonic-like cells by down-regulation of p-MLC expression

Hydroxyxanthones, the synthetic form of xanthones that predominantly found in mangosteen (Garcinia mangostana), have been indicated as a potent anti-inflammatory action. This study aimed to examine the action of various HDX forms in preventing a tight junction (TJ) barrier disruption induced by TNF-α or IL-1β through the inhibition of p-MLC expression in Caco-2 cell model. TJ barrier function was determined by transepithelial electrical resistance (TER) and flux measurement of fluorescein isothiocyanate-dextran (FD-4). 1-monoHDX or 1,3-diHDX (10 μM) promoted TER but not FD-4 permeability in the absence of cytokines. Pretreatment with 1-monoHDX at 10 μM for 24 h prevented TNF-α or IL-1β effect on TJ permeability. p-MLC protein expression by Western blot analysis, which was induced by IL-1β was inhibited with all HDXs. Tricyclic scaffold as the basic skeleton of all HDXs may be a potential candidate therapeutic for treatment of diseases-related leaky gut.

Prenatal Maternal Stress Exacerbates Experimental Colitis of Offspring in Adulthood.

The prevalence of inflammatory bowel disease (IBD) is increasing worldwide and correlates with dysregulated immune response because of gut microbiota dysbiosis. Some adverse early life events influence the establishment of the gut microbiota and act as risk factors for IBD. Prenatal maternal stress (PNMS) induces gut dysbiosis and perturbs the neuroimmune network of offspring. In this study, we aimed to investigate whether PNMS increases the susceptibility of offspring to colitis in adulthood. The related index was assessed during the weaning period and adulthood. We found that PNMS impaired the intestinal epithelial cell proliferation, goblet cell and Paneth cell differentiation, and mucosal barrier function in 3-week-old offspring. PNMS induced low-grade intestinal inflammation, but no signs of microscopic inflammatory changes were observed. Although there was no pronounced difference between the PNMS and control offspring in terms of their overall measures of alpha diversity for the gut microbiota, distinct microbial community changes characterized by increases in Desulfovibrio, Streptococcus, and Enterococcus and decreases in Bifidobacterium and Blautia were induced in the 3-week-old PNMS offspring. Notably, the overgrowth of Desulfovibrio persisted from the weaning period to adulthood, consistent with the results observed using fluorescence in situ hybridization in the colon mucosa. Mechanistically, the fecal microbiota transplantation experiment showed that the gut microbiota from the PNMS group impaired the intestinal barrier function and induced low-grade inflammation. The fecal bacterial solution from the PNMS group was more potent than that from the control group in inducing inflammation and gut barrier disruption in CaCo-2 cells. After treatment with a TNF-α inhibitor (adalimumab), no statistical difference in the indicators of inflammation and intestinal barrier function was observed between the two groups. Finally, exposure to PNMS remarkably increased the values of the histopathological parameters and the inflammatory cytokine production in a mouse model of experimental colitis in adulthood. These findings suggest that PNMS can inhibit intestinal development, impair the barrier function, and cause gut dysbiosis characterized by the persistent overgrowth of Desulfovibrio in the offspring, resulting in exacerbated experimental colitis in adulthood.

2′-Fucosyllactose impacts the expression of mucus-related genes in goblet cells and maintains barrier function of gut epithelial cells

Cost-effective microbial biosynthesis of 2′-fucosyllactose (2′-FL) allows its application in infant formula. The specific effects of 2′-FL on the gastrointestinal immune barrier are still largely unknown. Here, we quantified and compared the effects of HMOs isolated from human milk, 2′-FL/lactose (Lac), and 2′-FL on the expression of the mucus associated genes MUC2, TFF3, RETLB and the Golgi sulfotransferases, CHST5, and GAL3ST5, in human goblet cells. We also determined whether these compounds have protective effects on A23187-induced barrier disruption of human T84 gut epithelial cells in vitro. The impact of isolated HMOs and 2′-FL/Lac on the mRNA expression of the mucus-related genes was minor while pure 2′-FL significantly induced GAL3ST2 and CHST5. Isolated HMOs, 2′-FL/Lac and 2′-FL all prevented A23187-induced barrier disruption in human T84 cells. Our findings indicate that 2′-FL modulates the secretory function of goblet cells and protects gut epithelial cells.

Protective effects of FK 506 against haemorrhagic shock-induced microvascular hyperpermeability.

Microvascular hyperpermeability, the excessive leakage of fluid and proteins from the intravascular space to the interstitium, is a devastating clinical concern in haemorrhagic shock (HS), sepsis, burn and so forth. Previous studies have shown that HS-induced microvascular hyperpermeability is associated with activation of the mitochondria-mediated 'intrinsic' apoptotic signalling cascade and caspase-3 mediated disruption of the endothelial cell barrier. In this study, our objective was to test if FK506, an immunomodulator that is also known to protect mitochondria, would protect barrier functions and decrease vascular hyperpermeability following HS by acting on this pathway. FK506 (25µM) was given 10 minutes before the shock period in a rat model of HS. The HS model was a non-traumatic/fixed pressure model of hypovolemic shock developed by withdrawing blood to reduce the mean arterial pressure to 40mm Hg for 60minutes. The mesenteric post-capillary venules were monitored for changes in permeability using intravital microscopic imaging. The changes in mitochondrial transmembrane potential (MTP) were determined using the cationic dye 5,5',6,6' tetrachoro-1,1',3,3' tetraethyl benzimidazolyl carbocyanine iodide (JC-1), that was superfused on the mesenteric vasculature followed by intravital imaging. The mesenteric caspase-3 activity was measured fluorometrically. Haemorrhagic shock induced a significant increase in hyperpermeability compared to the sham-control group and FK506 treatment decreased HS-induced hyperpermeability significantly (P<.05). FK506 dampened HS-induced loss of MTP and elevation of caspase-3 activity significantly (P<.05). FK506 has protective effects against HS-induced microvascular hyperpermeability. The maintenance of the MTP and protection against caspase-3 mediated endothelial cell barrier disruption are possible mechanisms by which FK506 attenuates HS-induced hyperpermeability. FK506, currently used in clinical settings as an immunomodulator, needs to be explored further for its therapeutic usefulness against HS-induced vascular hyperpermeability and associated complications.

IL-36α/IL-36RA/IL-38 signaling mediates inflammation and barrier disruption in human corneal epithelial cells under hyperosmotic stress

To explore the distinct expression and diverse roles of IL-36 cytokines in dry eye disease using an in vitro hyperosmolarity model of human corneal epithelial cells (HCECs). Primary HCECs were cultured from fresh donor limbal explants. Hyperosmolarity model was established by switching HCECs from isosmotic (312mOsM) to hyperosmotic medium (350-500 mOsM) alone or with addition of rhIL-36RA or rhIL-38 for 2-48h. Some cultures were treated with IL-36α (1-10ng/ml) with or without rhIL-36RA or rhIL-38. Gene expression was detected by RT-qPCR; and protein production and barrier disruption were evaluated by ELISA and/or immunofluorescent staining. IL-36 cytokines were differential expressed in primary HCECs. Among 3 pro-inflammatory agonists, IL-36α, but not IL-36β and IL-36γ, was distinctly induced at osmolarity-dependent manner while two antagonist IL-36RA and IL-38 were significantly suppressed in HCECs exposed to hyperosmotic stress. IL-36α increased to 4.4-fold in mRNA and 6.9-fold at protein levels (116.0±36.33pg/ml vs 16.79±6.51pg/ml in controls) by 450 mOsM, but dramatically inhibited by addition of rhIL-36RA or rhIL-38. Exogenous rhIL-36α stimulated expression of TNF-α and IL-1β at mRNA and protein levels and disrupted tight junction proteins ZO-1 and occludin. However, rhIL-36RA or rhIL-38 suppressed TNF-α and IL-1β production and protected HCECs from barrier disruption in response to IL-36α or hyperosmolarity. Our findings demonstrate that the stimulated pro-inflammatory IL-36α with the suppressed antagonists IL-36RA and IL-38 is a novel mechanism by which hyperosmolarity induces inflammation in dry eye. IL-36RA and IL-38 may have a therapeutic potential in dry eye.

Urban Particulate Matters May Affect Endoplasmic Reticulum Stress and Tight Junction Disruption in Nasal Epithelial Cells.

Exposure to airborne urban particulate matter (UPM) has been closely related to the development and aggravation of respiratory disease, including sinonasal disorders. The aims of this study were to investigate the effect of UPM on nasal epithelial tight junctions (TJs) and mucosal barrier function and delineate the underlying mechanism by using both in vitro and in vivo models. In this study, human nasal epithelial cells (hNECs) and BALB/c mice were exposed to UPMs. UPM 1648a and 1649 b were employed. TJ and endoplasmic reticulum (ER) stress marker expression was measured using western blot analysis and immunofluorescence. TJ integrity and nasal epithelial barrier function were evaluated by transepithelial electric resistance (TER) and paracellular flux. In addition, the effects of N-acetyl-L-cysteine (NAC) on UPM-induced nasal epithelial cells were investigated. UPM significantly impaired the nasal epithelial barrier, as demonstrated by decreased protein expression of TJ and ER stress markers in human nasal epithelial cells. This finding was in parallel to reduced transepithelial electrical resistance and increased fluorescein isothiocyanate-dextran permeability. Pretreatment with NAC decreased the degree of UPM-mediated ER stress and restored nasal epithelial barrier disruption in human nasal epithelial cells (hNEC) and the nasal mucosa of experimental animals. These data suggest that UPMs may induce nasal epithelial barrier dysfunction by targeting TJs and ER stress could be related in this process. Based on these results, we suggest that suppression of this process with an inhibitor targeting ER stress responses could represent a novel promising therapeutic target in UPM-induced sinonasal disease.

SOCS3–microtubule interaction via CLIP-170 and CLASP2 is critical for modulation of endothelial inflammation and lung injury

Proinflammatory cytokines such as IL-6 induce endothelial cell (EC) barrier disruption and trigger an inflammatory response in part by activating the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway. The protein suppressor of cytokine signaling-3 (SOCS3) is a negative regulator of JAK-STAT, but its role in modulation of lung EC barrier dysfunction caused by bacterial pathogens has not been investigated. Using human lung ECs and EC-specific SOCS3 knockout mice, we tested the hypothesis that SOCS3 confers microtubule (MT)-mediated protection against endothelial dysfunction. SOCS3 knockdown in cultured ECs or EC-specific SOCS3 knockout in mice resulted in exacerbated lung injury characterized by increased permeability and inflammation in response to IL-6 or heat-killed Staphylococcus aureus (HKSA). Ectopic expression of SOCS3 attenuated HKSA-induced EC dysfunction, and this effect required assembled MTs. SOCS3 was enriched in the MT fractions, and treatment with HKSA disrupted SOCS3–MT association. We discovered that—in addition to its known partners gp130 and JAK2—SOCS3 interacts with MT plus-end binding proteins CLIP-170 and CLASP2 via its N-terminal domain. The resulting SOCS3–CLIP-170/CLASP2 complex was essential for maximal SOCS3 anti-inflammatory effects. Both IL-6 and HKSA promoted MT disassembly and disrupted SOCS3 interaction with CLIP-170 and CLASP2. Moreover, knockdown of CLIP-170 or CLASP2 impaired SOCS3–JAK2 interaction and abolished the anti-inflammatory effects of SOCS3. Together, these findings demonstrate for the first time an interaction between SOCS3 and CLIP-170/CLASP2 and reveal that this interaction is essential to the protective effects of SOCS3 in lung endothelium.

Activation of the mechanosensitive Ca2+ channel TRPV4 induces endothelial barrier permeability via the disruption of mitochondrial bioenergetics

Mechanical ventilation is a life-saving intervention in critically ill patients with respiratory failure due to acute respiratory distress syndrome (ARDS), a refractory lung disease with an unacceptable high mortality rate. Paradoxically, mechanical ventilation also creates excessive mechanical stress that directly augments lung injury, a syndrome known as ventilator-induced lung injury (VILI). The specific mechanisms involved in VILI-induced pulmonary capillary leakage, a key pathologic feature of VILI are still far from resolved. The mechanoreceptor, transient receptor potential cation channel subfamily V member 4, TRPV4 plays a key role in the development of VILI through unresolved mechanism. Endothelial nitric oxide synthase (eNOS) uncoupling plays an important role in sepsis-mediated ARDS so in this study we investigated whether there is a role for eNOS uncoupling in the barrier disruption associated with TRPV4 activation during VILI. Our data indicate that the TRPV4 agonist, 4α-Phorbol 12,13-didecanoate (4αPDD) induces pulmonary arterial endothelial cell (EC) barrier disruption through the disruption of mitochondrial bioenergetics. Mechanistically, this occurs via the mitochondrial redistribution of uncoupled eNOS secondary to a PKC-dependent phosphorylation of eNOS at Threonine 495 (T495). A specific decoy peptide to prevent T495 phosphorylation reduced eNOS uncoupling and mitochondrial redistribution and preserved PAEC barrier function under 4αPDD challenge. Further, our eNOS decoy peptide was able to preserve lung vascular integrity in a mouse model of VILI. Thus, we have revealed a functional link between TRPV4 activation, PKC-dependent eNOS phosphorylation at T495, and EC barrier permeability. Reducing pT495-eNOS could be a new therapeutic approach for the prevention of VILI.

A Novel Serine Protease Inhibitor PE-BBI Ameliorates Cockroach Extract-Mediated Airway Epithelial Barrier Dysfunction.

Epithelial barrier dysfunction, characteristic of allergic airway disease may be, at least in part, due to the action of allergen-associated protease activities. Cockroach allergy is a major global health issue, with cockroaches containing considerable serine trypsin-like protease (TLP) activity. The present study sought to evaluate two novel protease inhibitors (PE-BBI and pLR-HL), recently isolated from amphibian skin secretions, for their potential to neutralise cockroach TLP activity and to determine any protective effect on cockroach-induced airway epithelial barrier disruption. Inhibitor potencies against the cockroach-associated activities were determined using a fluorogenic peptide substrate-based activity assay. 16HBE14o- cells (16HBE; a bronchial epithelial cell line) were treated with cockroach extract (CRE) in the presence or absence of the compounds in order to assess cell viability (RealTime Glo luminescent assay) and epithelial barrier disruption (transepithelial resistance and paracellular dextran flux). PE-BBI potently and selectively inhibited CRE TLP activity (pIC50 -8), but not host (16HBE) cell surface activity, which conferred protection of 16HBE cells from CRE-induced cell damage and barrier disruption. Novel protease inhibitor strategies such as PE-BBI may be useful for the treatment of allergic airway disease caused by cockroach proteases.

S100A6 is a positive regulator of PPP5C-FKBP51-dependent regulation of endothelial calcium signaling.

ISOC is a cation current permeating the ISOC channel. In pulmonary endothelial cells, ISOC activation leads to formation of inter-endothelial cell gaps and barrier disruption. The immunophilin FK506-binding protein 51 (FKBP51), in conjunction with the serine/threonine protein phosphatase 5C (PPP5C), inhibits ISOC . Free PPP5C assumes an autoinhibitory state, which has low "basal" catalytic activity. Several S100 protein family members bind PPP5C increasing PPP5C catalytic activity in vitro. One of these family members, S100A6, exhibits a calcium-dependent translocation to the plasma membrane. The goal of this study was to determine whether S100A6 activates PPP5C in pulmonary endothelial cells and contributes to ISOC inhibition by the PPP5C-FKBP51 axis. We observed that S100A6 activates PPP5C to dephosphorylate tau T231. Following ISOC activation, cytosolic S100A6 translocates to the plasma membrane and interacts with the TRPC4 subunit of the ISOC channel. Global calcium entry and ISOC are decreased by S100A6 in a PPP5C-dependent manner and by FKBP51 in a S100A6-dependent manner. Further, calcium entry-induced endothelial barrier disruption is decreased by S100A6 dependent upon PPP5C, and by FKBP51 dependent upon S100A6. Overall, these data reveal that S100A6 plays a key role in the PPP5C-FKBP51 axis to inhibit ISOC and protect the endothelial barrier against calcium entry-induced disruption.

Sphingosine-1-phosphate receptor-independent lung endothelial cell barrier disruption induced by FTY720 regioisomers.

RationaleVascular permeability is a hallmark of acute respiratory distress syndrome (ARDS) and ventilator-induced lung injury pathobiology; however, the mechanisms underlying this vascular dysregulation remain unclear, thereby impairing the development of desperately needed effective therapeutics. We have shown that sphingosine-1-phosphate (S1P) and 2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol (FTY720) analogues are useful tools for exploring vascular barrier regulation mechanisms.ObjectiveTo experimentally define the effects of FTY720 regioisomers on lung endothelial cell barrier regulation.MethodsSpecific barrier-regulatory receptor and kinase inhibitors were utilized to probe signaling mechanisms involved in FTY720 regioisomer-mediated human lung endothelial cell barrier responses (trans-endothelial electrical resistance, TER). Docking simulations with the S1P1 receptor were performed to further evaluate FTY720 regioisomer signaling.ResultsFTY720 regioisomers produced potent endothelial cell barrier disruption reflected by declines in TER alterations. Pharmacologic inhibition of Gi-coupled S1P receptors (S1P1, S1P2, S1P3) failed to alter FTY720 regioisomer-mediated barrier disruption; findings that were corroborated by docking simulations demonstrating FTY720 regiosomers were repelled from S1P1 docking, in contrast to strong S1P1 binding elicited by S1P. Inhibition of either the barrier-disrupting PAR-1 receptor, the VEGF receptor, Rho-kinase, MAPK, NFkB, or PI3K failed to alter FTY720 regioisomer-induced endothelial cell barrier disruption. While FTY720 regioisomers significantly increased protein phosphatase 2 (PP2A) activity, PP2A inhibitors failed to alter FTY720 regioisomer-induced endothelial cell barrier disruption.ConclusionsTogether, these results imply a vexing model of pulmonary vascular barrier dysregulation in response to FTY720-related compounds and highlight the need for further insights into mechanisms of vascular integrity required to promote the development of novel therapeutic tools to prevent or reverse the pulmonary vascular leak central to ARDS outcomes.

Inhibitory effects of cudratricusxanthone O on particulate matter-induced pulmonary injury

ABSTRACT Particulate matter 2.5 (PM2.5), aerodynamic diameter ≤ 2.5 μm, is the primary air pollutant that plays the key role for lung injury resulted from the loss of vascular barrier integrity. Cudratricusxanthone O (CTXO) is a novel xanthone compound isolated from the root of Cudrania tricuspidata Bureau. Here, we investigated the beneficial effects of CTXO against PM-induced lung endothelial cell (EC) barrier disruption and pulmonary inflammation. Permeability, leukocyte migration, activation of proinflammatory proteins, generation of reactive oxygen species (ROS), and histology were examined in PM2.5-treated ECs and mice. CTXO significantly scavenged PM2.5-induced ROS and inhibited the ROS-induced activation of p38 mitogen-activated protein kinase (MAPK). Concurrently, CTXO activated Akt, which helped maintain endothelial integrity. Furthermore, CTXO reduced vascular protein leakage, leukocyte infiltration, and proinflammatory cytokine release in the bronchoalveolar lavage fluid in PM-induced lung tissues. These results indicated that CTXO may exhibit protective effects against PM-induced inflammatory lung injury and vascular hyperpermeability.

Pulmonary Protective Functions of Rare Ginsenoside Rg4 on Particulate Matter-induced Inflammatory Responses

Inhalation of fine particulate matter (PM2.5) is associated with an increase in lung injury caused by the loss of integrity of the vascular barrier. The rare ginsenoside Rg4 is a main protopanaxatriol type ginsenoside of black ginseng (BG). The aim of this study was to investigate the beneficial effects of Rg4 on PM-induced lung endothelial cell (EC) barrier disruption and pulmonary inflammation. Permeability, leukocyte migration, activation of proinflammatory proteins, generation of reactive oxygen species (ROS), and histology were examined in PM2.5-treated EC and mice. Rg4 significantly scavenged PM2.5-induced ROS, inhibited ROS-induced activation of p38 mitogen-activated protein kinase (MAPK), activated Akt in purified pulmonary endothelial cells, which helped maintain endothelial integrity. Further, Rg4 reduced vascular protein leakage, leukocyte infiltration, and proinflammatory cytokine release in bronchoalveolar lavage fluids in PM-induced mouse lung tissues. Data suggested that Rg4 might exhibit protective effects in PM-induced inflammatory lung injury and vascular hyperpermeability.

Pharmacological stimulation of G–protein coupled receptor 40 alleviates cytokine-induced epithelial barrier disruption in airway epithelial Calu-3 cells

Impairment of airway tight junctions induced by elevated levels of proinflammatory cytokines is implicated in the pathogenesis of inflammatory airway diseases. Pharmacological stimulation of G–protein coupled receptor (GPR) 40, a receptor of polyunsaturated fatty acids, have recently been shown to promote tight junction assembly in airway epithelial cells under non-inflammatory conditions. However, roles of GPR40 in regulating airway epithelial integrity in response to inflammatory insults are unknown. This study was aimed to investigate the effect of GPR40 stimulation on proinflammatory cytokine (TNFα and IL–1β)-induced tight junction disruption in human airway epithelial Calu–3 cells using GW9508, a GPR40 agonist. We found that stimulation of GPR40 by GW9508 attenuated the cytokine–induced airway epithelial barrier leakage as analyzed by measurements of transepithelial electrical resistance and transepithelial flux of fluorescently labeled dextran (molecular weight of 4 kDa). Furthermore, GW9508 prevented the cytokine-induced dislocalization of zonula occludens (ZO)–1, occludin and claudin–1. The barrier-protective effect of GW9508 was abolished by a GPR40 antagonist, but not a GPR120 antagonist. Immunofluorescence staining of NF–ĸB indicated that GW9508 had no effect on cytokine-induced NF–ĸB activation. Intriguingly, GW9508 inhibited cytokine-induced airway epithelial barrier disruption through suppression of extracellular signal–regulated kinase (ERK) phosphorylation in a phospholipase C (PLC) and calcium/calmodulin–dependent protein kinase kinase beta (CaMKKβ)-dependent manner. Collectively, this study uncovered the novel role of GPR40 in preventing cytokine–induced tight junction disruption in airway epithelial cells through mechanisms involving PLC–CaMKKβ-mediated suppression of ERK signaling. Pharmacological stimulation of GPR40 may be beneficial in the treatment of airway diseases.

Critical role of autophagy regulator Beclin1 in endothelial cell inflammation and barrier disruption

Recent studies have implicated autophagy in several inflammatory diseases involving aberrant endothelial cell (EC) responses, such as acute lung injury (ALI). However, the mechanistic basis for a role of autophagy in EC inflammation and permeability remain poorly understood. In this study, we impaired autophagy by silencing the essential Beclin1 autophagy gene in human pulmonary artery EC. This resulted in reduced expression of proinflammatory genes in response to thrombin, a procoagulant and proinflammatory mediator whose concentration is elevated in many diseases including sepsis and ALI. These (Beclin1-depleted) cells also displayed a marked decrease in NF-κB activity secondary to impaired DNA binding of RelA/p65 in the nucleus, but exhibited normal IκBα degradation in the cytosol. Further analysis showed that Beclin1 knockdown was associated with impaired RelA/p65 translocation to the nucleus. Additionally, Beclin1 knockdown attenuated thrombin-induced phosphorylation of RelA/p65 at Ser536, a critical event necessary for the transcriptional activity of RelA/p65. Beclin1 silencing also protected against thrombin-induced EC barrier disruption by preventing the loss of VE-cadherin at adherens junctions. Moreover, Beclin1 knockdown reduced thrombin-induced phosphorylation/inactivation of actin depolymerizing protein Cofilin1 and thereby actin stress fiber formation required for EC permeability as well as RelA/p65 nuclear translocation. Together, these data identify Beclin1 as a novel mechanistic link between autophagy and EC dysfunction (inflammation and permeability).

Inhibitory effects of collismycin C and pyrisulfoxin A on particulate matter-induced pulmonary injury

BackgroundInhalation of fine particulate matter (PM2.5) is associated with elevated pulmonary injury caused by the loss of vascular barrier integrity. Marine microbial natural products isolated from microbial culture broths were screened for pulmonary protective effects against PM2.5. Two 2,2′-bipyridine compounds isolated from a red alga-associated Streptomyces sp. MC025—collismycin C (2) and pyrisulfoxin A (5)—were found to inhibit PM2.5-mediated vascular barrier disruption. PurposeTo confirm the inhibitory effects of collismycin C and pyrisulfoxin A on PM2.5-induced pulmonary injury Study designIn this study, we investigated the beneficial effects of collismycin C and pyrisulfoxin A on PM-induced lung endothelial cell (EC) barrier disruption and pulmonary inflammation. MethodsPermeability, leukocyte migration, proinflammatory protein activation, reactive oxygen species (ROS) generation, and histology were evaluated in PM2.5-treated ECs and mice. ResultsCollismycin C and pyrisulfoxin A significantly scavenged PM2.5-induced ROS and inhibited the ROS-induced activation of p38 mitogen-activated protein kinase as well as activated Akt, which helped in maintaining endothelial integrity, in purified pulmonary endothelial cells. Furthermore, collismycin C and pyrisulfoxin A reduced vascular protein leakage, leukocyte infiltration, and proinflammatory cytokine release in the bronchoalveolar lavage fluid of PM-treated mice. ConclusionThese data suggested that collismycin C and pyrisulfoxin A might exert protective effects on PM-induced inflammatory lung injury and vascular hyperpermeability.

Sphingosine 1-phosphate receptor 1 regulates cell-surface localization of membrane proteins in endothelial cells

The endothelial cell (EC) barrier disruption has been implicated in vascular leakage and pulmonary edema. Many reports have shown that the EC barrier dysfunction is regulated by the sphingosine-1-phophate (S1P)/S1P receptor-1 (S1PR1) axis. Identifying downstream effectors for the S1P/S1PR1 axis in pulmonary vasculature has been limited by mixed populations in vitro cultures that do not retain physiological EC phenotype and complex of tedious proteomics. In this study, we used a combination of in vivo biotinylation and liquid chromatograph tandem mass spectrometry on three mouse models of S1pr1 expression, namely normal, knockout (KO) and high, to identify EC membrane proteins whose cell-surface expression is S1pr1-dependent. EC-specific KO of S1pr1 caused severe pulmonary vascular disruption and reduction of many membrane proteins on ECs. Using the MaxQuant software we were able to identify novel membrane targets of S1pr1, for instance, Cd105 and Plvap, by comparison with their membrane expressions among the three EC model systems. Moreover, regulation of Cd105 and Plvap by S1pr1 were validated with Western blot and immunostaining in vivo and in vitro. Our data suggest that S1pr1 dictates cell-surface localization of several apical membrane proteins in ECs. Our results are insightful for development of novel therapeutics to specifically target EC barrier function.

Inhibitory effects of protopanaxatriol type ginsenoside fraction (Rgx365) on particulate matter-induced pulmonary injury

ABSTRACTInhalation of fine particulate matter (PM2.5) is associated with elevated pulmonary injury attributed to the loss of vascular barrier integrity. Black ginseng (BG), steamed 9 times and dried ginseng, and its major protopanaxatriol type ginsenosides (ginsenoside Rg4, Rg6, Rh4, Rh1, and Rg2) exhibited various biological activities including anti-septic, anti-diabetic, wound healing, immune-stimulatory, and anti-antioxidant activity. The aim of this study was to investigate the beneficial effects of Rgx365 (a protopanaxatriol type rare ginsenosides fraction) on PM-induced lung endothelial cell (EC) barrier disruption and pulmonary inflammation. Permeability, leukocyte migration, activation of proinflammatory proteins, generation of reactive oxygen species (ROS), and histology were examined in PM2.5-treated EC and mice. Rgx365 significantly scavenged PM2.5-induced ROS, inhibited ROS-induced activation of p38 mitogen-activated protein kinase (MAPK), activated Akt in purified pulmonary EC, which helped maintain endothelial integrity. Further, Rgx365 reduced vascular protein leakage, leukocyte infiltration, and proinflammatory cytokine release in bronchoalveolar lavage fluids in PM-induced mouse lung tissues. Data suggested that Rgx365 might exhibit protective effects in PM-induced inflammatory lung injury and vascular hyperpermeability.

Elafibranor interrupts adipose dysfunction-mediated gut and liver injury in mice with alcoholic steatohepatitis.

Background: Reversal of alcohol-induced peroxisome proliferator-activated receptor (PPAR) α (PPARα) and PPARδ dysfunction has been reported to decrease the severity of alcoholic steatohepatitis (ASH). Autophagy is essential for cell survival and tissue energy homeostasis. Emerging evidence indicates that alcohol-induced adipose tissue (AT) autophagy dysfunction contributes to injury in the intestine, liver, and AT of ASH. Methods: The effects and mechanisms of dual PPARα/δ agonist elafibranor on autophagy stimulation were investigated using mice with ASH. Results: C57BL/6 mice on ethanol diet showed AT dysfunction, disrupted intestinal barrier, and ASH, which was accompanied by alcohol-mediated decrease in PPARα, PPARδ, and autophagy levels in intestine, liver, and AT. Chronic treatment with elafibranor attenuated AT apoptosis and inflammation by restoration of tissue PPARα, PPARδ, and autophagy levels. In ASH mice, alcohol-induced AT dysfunction along with increased fatty acid (FA) uptake and decreased free FA (FFA) release from AT was inhibited by elafibranor. The improvement of AT autophagy dysfunction by elafibranor alleviated inflammation and apoptosis-mediated intestinal epithelial disruption in ASH mice. Acute elafibranor incubation inhibited ethanol-induced ASH-mice-sera-enhanced autophagy dysfunction, apoptosis, barrier disruption, and intracellular steatosis in Caco-2 cells and primary hepatocytes (PHs). Conclusion: Altogether, these findings demonstrated that the PPARα/δ agonist, elafibranor, decreased the severity of liver injury by restoration of alcohol-suppressed AT autophagy function and by decreasing the release of apoptotic markers, inflammatory cytokines, and FFA, thereby reducing intestinal epithelium disruption and liver inflammation/apoptosis/steatosis in ASH mice. These data suggest that dual PPAR agonists can serve as potential therapeutic agents for the management of ASH.