Blood Brain Barrier (BBB) breakdown is a secondary form of brain injury which has yet to be fully elucidated mechanistically. Existing research suggests that breakdown of tight junction proteins ...between endothelial cells is a primary driver of increased BBB permeability following injury, and intercellular signaling between primary cells of the neurovascular unit: endothelial cells, astrocytes, and pericytes; contribute to tight junction restoration. To expound upon this body of research, we analyzed the effects of severely injured patient plasma on each of the cell types in monoculture and together in a triculture model for the transcriptional and translational expression of the tight junction proteins Claudins 3 and 5, (CLDN3, CLDN5) and Zona Occludens 1 (ZO-1). Conditioned media transfer studies were performed to illuminate the cell type responsible for differential tight junction expression. Our data show that incubation with 5% human ex vivo severely injured patient plasma is sufficient to produce a differential response in endothelial cell tight junction mRNA and protein expression. Endothelial cells in monoculture produced a significant increase of CLDN3 and CLDN5 mRNA expression, (3.98 and 3.51 fold increase vs. control respectively, p<0.01) and CLDN5 protein expression, (2.58 fold change vs. control, p<0.01), whereas in triculture, this increase was attenuated. Our triculture model and conditioned media experiments suggest that conditioned media from astrocytes and pericytes and a triculture of astrocytes, pericytes and endothelial cells are sufficient in attenuating the transcriptional increases of tight junction proteins CLDN3 and CLDN5 observed in endothelial monocultures following incubation with severely injured trauma plasma. This data suggests that inhibitory molecular signals from astrocytes and pericytes contributes to prolonged BBB breakdown following injury via tight junction transcriptional and translational downregulation of CLDN5.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Trauma/hemorrhagic shock is a complex physiological phenomenon that leads to dysregulation of many molecular pathways. For over a decade, hypertonic saline (HTS) has been used as an alternative ...resuscitation fluid in the setting of trauma/hemorrhagic shock. In addition to restoring circulating volume within the vascular space, studies have shown a positive immunomodulatory effect of HTS. Targeted studies have shown that HTS affects the transcription of several pro-inflammatory cytokines by inhibiting the NF-κB-IκB pathway in model cell lines and rats. However, few studies have been undertaken to assess the unbiased effects of HTS on the whole transcriptome. This study was designed to interrogate the global transcriptional responses induced by HTS and provides insight into the underlying molecular mechanisms and pathways affected by HTS. In this study, RNA sequencing was employed to explore early changes in transcriptional response, identify key mediators, signaling pathways, and transcriptional modules that are affected by HTS in the presence of a strong inflammatory stimulus. Our results suggest that primary human small airway lung epithelial cells (SAECS) exposed to HTS in the presence and absence of a strong pro-inflammatory stimulus exhibit very distinct effects on cellular response, where HTS is highly effective in attenuating cytokine-induced pro-inflammatory responses via mechanisms that involve transcriptional regulation of inflammation which is cell type and stimulus specific. HTS is a highly effective anti-inflammatory agent that inhibits the chemotaxis of leucocytes towards a pro-inflammatory gradient and may attenuate the progression of both the innate and adaptive immune response.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Hypertonic saline (HTS) has been used intravenously to reduce organ dysfunction following injury and as an inhaled therapy for cystic fibrosis lung disease. The role and mechanism of HTS inhibition ...was explored in the TNFα and IL-1β stimulation of pulmonary epithelial cells. Hyperosmolar (HOsm) media (400 mOsm) inhibited the production of select cytokines stimulated by TNFα and IL-1β at the level of mRNA translation, synthesis and release. In TNFα stimulated A549 cells, HOsm media inhibited I-κBα phosphorylation, NF-κB translocation into the nucleus and NF-κB nuclear binding. In IL-1β stimulated cells HOsm inhibited I-κBα phosphorylation without affecting NF-κB translocation or nuclear binding. Incubation in HOsm conditions inhibited both TNFα and IL-1β stimulated nuclear localization of interferon response factor 1 (IRF-1). Additional transcription factors such as AP-1, Erk-1/2, JNK and STAT-1 were unaffected by HOsm. HTS and sorbitol supplemented media produced comparable outcomes in all experiments, indicating that the effects of HTS were mediated by osmolarity, not by sodium. While not affecting MAPK modules discernibly in A549 cells, both HOsm conditions inhibit IRF-1 against TNFα or IL-1β, but inhibit p65 NF-kB translocation only against TNFα but not IL-1β. Thus, anti-inflammatory mechanisms of HTS/HOsm appear to disrupt cytokine signals at distinct intracellular steps.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
1 Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado Health Sciences Center, Denver, Colorado 2 Central Institute, Shino-Test Corporation, Sagamihara, Kanagawa, Japan 3 ...Department of Laboratory and Molecular Medicine, Faculty of Medicine, Kagoshima University, Kagoshima, Japan 4 Department of Medicine, Keio University, School of Medicine, Tokyo, Japan 5 Department of Internal Medicine, Chung Ang University College of Medicine, Seoul, Korea 6 Service d'Anesthesie-Réanimation et Unité Propre de Recherche de l'Enseignment Superieur-Equipe d'Accueil, Hospital de Bicêtre, Le Kremlin Bicetre, France 7 Department of Anesthesiology, Chonnam University Medical School, Gwangju, Korea
Submitted 21 September 2004
; accepted in final form 3 January 2005
High mobility group box 1 (HMGB1) is a novel late mediator of inflammatory responses that contributes to endotoxin-induced acute lung injury and sepsis-associated lethality. Although acute lung injury is a frequent complication of severe blood loss, the contribution of HMGB1 to organ system dysfunction in this setting has not been investigated. In this study, HMGB1 was detected in pulmonary endothelial cells and macrophages under baseline conditions. After hemorrhage, in addition to positively staining endothelial cells and macrophages, neutrophils expressing HMGB1 were present in the lungs. HMGB1 expression in the lung was found to be increased within 4 h of hemorrhage and then remained elevated for more than 72 h after blood loss. Neutrophils appeared to contribute to the increase in posthemorrhage pulmonary HMGB1 expression since no change in lung HMGB1 levels was found after hemorrhage in mice made neutropenic with cyclophosphamide. Plasma concentrations of HMGB1 also increased after hemorrhage. Blockade of HMGB1 by administration of anti-HMGB1 antibodies prevented hemorrhage-induced increases in nuclear translocation of NF- B in the lungs and pulmonary levels of proinflammatory cytokines, including keratinocyte-derived chemokine, IL-6, and IL-1 . Similarly, both the accumulation of neutrophils in the lung as well as enhanced lung permeability were reduced when anti-HMGB1 antibodies were injected after hemorrhage. These results demonstrate that hemorrhage results in increased HMGB1 expression in the lungs, primarily through neutrophil sources, and that HMGB1 participates in hemorrhage-induced acute lung injury.
high mobility group box 1; nuclear factor- B; neutrophils
Address for reprint requests and other correspondence: E. Abraham, Univ. of Colorado Health Sciences Center, 4200 E. Ninth Ave., Box C272, Denver, CO 80262 (E-mail: Edward.Abraham{at}uchsc.edu )
Poorly differentiated esophageal adenocarcinoma (PDEAC) has a dismal prognosis. Glypican-1(GPC-1) is known to be upregulated in several cancer types in contrast to healthy tissues, rendering it as a ...biomarker. Nevertheless, the potential therapeutic targeting of GPC-1 has not been explored in PDEAC. There is accumulating evidence that GPC-1, via upregulation of PI3K/Akt/ERK signaling, plays a crucial role in the progression and chemoresistance in cancer. Pictilisib, a class I pan PI3K inhibitor, has shown promising antitumor results in clinical trials, however, has not gained widespread success due to acquired drug resistance. This study investigated the role of GPC-1 in chemo-resistant PDEAC and appraises the impact of targeted silencing of GPC-1 on the antitumor effects of Pictilisib in PDEAC cell lines. Immunohistochemistry assays in PDEAC tissue specimens demonstrated a pronounced intensity of staining with GPC-1. Upregulation of GPC-1 was found to be correlated with advanced stage and poor prognosis. In-vitro studies examined the influence of GPC-1 knockdown and Pictilisib, both as individual agents and in combination, on cytotoxicity, cell cycle distribution, apoptosis, and gene expression profiles. Silencing GPC-1 alone showed significantly reduced cell viability, migration, colony formation, epithelial-mesenchymal transition, and stemness in PDEAC cells. Significantly, knockdown of GPC-1 combined with low-dose Pictilisib led to enhancement of cytotoxicity, cell cycle arrest, and apoptosis in ESO-26 and OE-33 cells. In the xenograft mouse model, the combination of Pictilisib and GPC-1 knockdown exhibited synergy. These findings suggest that GPC-1 represents a promising target to augment chemosensitivity in esophageal adenocarcinoma.
Lysophosphatidylcholines (lysoPCs) are effective polymorphonuclear neutrophil (PMN) priming agents implicated in transfusion‐related acute lung injury (TRALI). LysoPCs cause ligation of the G2A ...receptor, cytosolic Ca2+ flux, and activation of Hck. We hypothesize that lysoPCs induce Hck‐dependent activation of protein kinase C (PKC), resulting in phosphorylation and membrane translocation of 47 kDa phagocyte oxidase protein (p47phox). PMNs, human or murine, were primed with lysoPCs and were smeared onto slides and examined by digital microscopy or separated into subcellular fractions or whole‐cell lysates. Proteins were immunoprecipitated or separated by polyacrylamide gel electrophoresis and immunoblotted for proteins of interest. Wild‐type (WT) and PKCγ knockout (KO) mice were used in a 2‐event model of TRALI. LysoPCs induced Hck coprecipitation with PKCδ and PKCγ and the PKCδ:PKCγ complex also had a fluorescence resonance energy transfer (FRET)+ interaction with lipid rafts and Wiskott‐Aldrich syndrome protein family verprolin‐homologous protein 2 (WAVE2). PKCγ then coprecipitated with p47phox. Immunoblotting, immunoprecipitation (IP), specific inhibitors, intracellular depletion of PKC isoforms, and PMNs from PKCγ KO mice demonstrated that Hck elicited activation/Tyr phosphorylation (Tyr311 and Tyr525) of PKCδ, which became Thr phosphorylated (Thr507). Activated PKCδ then caused activation of PKCγ, both by Tyr phosphorylation (Τyr514) and Ser phosphorylation, which induced phosphorylation and membrane translocation of p47phox. In PKCγ KO PMNs, lysoPCs induced Hck translocation but did not evidence a FRET+ interaction between PKCδ and PKCγ nor prime PMNs. In WT mice, lysoPCs served as the second event in a 2‐event in vivo model of TRALI but did not induce TRALI in PKCγ KO mice. We conclude that lysoPCs prime PMNs through Hck‐dependent activation of PKCδ, which stimulates PKCγ, resulting in translocation of phosphorylated p47phox.
LysoPCs elicit Hck translocation, causing nPKC (PKCδ) to activate cPKC (PKCγ), which results in priming of neutrophil NADPH oxidase.
Neutrophils are critical initiators and effectors of the innate immune system and express Toll-like receptor 2 (TLR2) and TLR4. Although signaling through pathways involving phosphoinositide 3-kinase ...(PI3-K) and the downstream kinase Akt (protein kinase B) plays a central role in modulating neutrophil chemotaxis and superoxide generation in response to engagement of G protein-coupled receptors, the importance of these kinases in affecting inflammatory responses of neutrophils stimulated through TLR2 has not been examined. In these experiments, we found activation of Akt in neutrophils stimulated with the TLR2-specific ligands peptidoglycan and the lipopeptide tri-palmitoyl-S-glyceryl-Cys-Ser-(Lys)(4) that occurred earlier and was of greater magnitude than that present after exposure to the TLR4 agonist LPS. The release of the proinflammatory mediators TNF-alpha and macrophage inflammatory protein-2 was inhibited in a dose-dependent manner by PI3-K blockade. The IC(50) for inhibition of peptidoglycan-stimulated Akt activation and macrophage inflammatory protein-2 release correlated closely, indicating linkage of these two events. PI3-K blockade did not inhibit nuclear translocation of NF-kappa B, but did prevent Ser(536) phosphorylation of the p65 subunit of NF-kappa B, an event required for maximal transcriptional activity of NF-kappa B. Inhibition of PI3-K also prevented activation of p38 mitogen-activated protein kinase and extracellular receptor-activated kinase 1/2 in TLR2-stimulated neutrophils. These results demonstrate that the PI3-K-Akt axis occupies a central role in TLR2-induced activation of neutrophils.
Objective Invasive lung tumors are associated with intercellular adhesion molecule-1 (ICAM-1) expression. Secretory phospholipase A2 (sPLA2 ) enzymes produce inflammatory mediators that stimulate ...ICAM-1 expression, and upregulation of PLA2 activity can enhance metastasis. We hypothesize a link between sPLA2 activity, ICAM-1 expression, and tumor cell invasion. We propose that inhibition of sPLA2 modulates ICAM-1 expression in cancer cells and attenuates their invasiveness. Methods Human lung adenocarcinoma cells (A549) were treated with an ICAM-1 blocking antibody and assayed for invasion. Lung cancer cells (A549 and H358) were then treated with an sPLA2 inhibitor and evaluated by immunoblotting for ICAM-1 expression. Next cells (A549) treated with sPLA2 inhibitor were assayed for invasion. Finally, sPLA2 messenger RNA and protein expression were evaluated by quantitative reverse-transcriptase polymerase chain reaction and immunofluorescence microscopy, respectively. Statistical analysis was performed by the Student t test or analysis of variance, as appropriate. Results Antibody blockade of ICAM-1 decreased lung cancer cell invasion. sPLA2 inhibition significantly reduced ICAM-1 expression and invasion. sPLA2 inhibition also significantly decreased sPLA2 mRNA expression and immunofluorescent staining of sPLA2. Conclusions sPLA2 plays a significant role in mediating the inflammatory signals that induce ICAM-1 expression in lung cancer cells. Inhibition of the enzyme can significantly decrease ICAM-1 expression and subsequent cancer cell invasion. This lays the groundwork for further investigation into the cellular mechanisms of sPLA2 and its role in lung cancer.
Reactive oxygen species (ROS) can participate in cellular signaling and have been shown to modulate activation of the transcriptional regulatory factor NF-κB. However, the effects of ROS can differ ...in various cell populations. To examine the role of superoxide in neutrophil activation, we exposed resting neutrophils and neutrophils stimulated with LPS to paraquat, an agent that specifically increases intracellular superoxide concentrations. Culture of resting neutrophils with paraquat resulted in increased production of the proinflammatory cytokines TNF-α and MIP-2, enhanced degradation of IκB-α, and increased nuclear accumulation of NF-κB. Such effects of paraquat were due to intracellular superoxide (O
2
−) since they were blocked by the non-specific antioxidant N-acetyl cysteine and the cell permeable superoxide scavenger Tiron, but not by catalase, which facilitates the conversion of H
2O
2 to H
2O and O
2. Similar potentiating effects of paraquat were found in LPS-stimulated neutrophils. Exposure of neutrophils to paraquat also enhanced phosphorylation of Ser536 in the p65 subunit of NF-κB an event associated with increased transcriptional activity. Examination of kinases critical for LPS-stimulated gene expression showed that addition of paraquat to resting or LPS exposed neutrophils enhanced activation of p38 MAPK, but not that of Akt or ERK1/2. The potentiation of NF-κB translocation and proinflammatory cytokine production, but not of Ser536 p65 phosphorylation, by paraquat was dependent on activation of p38 MAPK. These results demonstrate that increased intracellular superoxide concentrations are proinflammatory in neutrophils, acting through a p38 MAPK dependent mechanism that results in enhanced nuclear accumulation of NF-κB and increased expression of NF-κB dependent proinflammatory cytokines.
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