Response Of Mesothelial Cells Research Articles

The Response of Mesothelial Cells to Fibrotic Stress Is Influenced by Carnitine Acetyltransferase (CrAT)

The Response of Mesothelial Cells to Fibrotic Stress Is Influenced by Carnitine Acetyltransferase (CrAT)

Matrix metalloproteinase-10 deficiency has protective effects against peritoneal inflammation and fibrosis via transcription factor NFκΒ pathway inhibition

One of the most common causes of discontinued peritoneal dialysis is impaired peritoneal function. However, its molecular mechanisms remain unclear. Previously, by microarray analysis of mouse peritoneum, we showed that MMP (matrix metalloproteinase)-10 expression is significantly increased in mice with peritoneal fibrosis, but its function remains unknown. Chlorhexidine gluconate (CG) was intraperitoneally injected to wild-type and MMP-10 knockout mice to induce fibrosis to elucidate the role of MMP-10 on peritoneal injury. We also examined function of peritoneal macrophages and mesothelial cells obtained from wild-type and MMP-10 knockout mice, MMP-10-overexpressing macrophage-like RAW 264.7 cells and MeT-5A mesothelial cells, investigated MMP-10 expression on peritoneal biopsy specimens, and the association between serum proMMP-10 and peritoneal solute transfer rates determined by peritoneal equilibration test on patients. MMP-10 was expressed in cells positive for WT1, a mesothelial marker, and for MAC-2, a macrophage marker, in the thickened peritoneum of both mice and patients. Serum proMMP-10 levels were well correlated with peritoneal solute transfer rates. Peritoneal fibrosis, inflammation, and high peritoneal solute transfer rates induced by CG were all ameliorated by MMP-10 deletion, with reduction of CD31-positive vessels and VEGF-A-positive cells. Expression of inflammatory mediators and phosphorylation of NFκΒ subunit p65 at S536 were suppressed in both MMP-10 knockout macrophages and mesothelial cells in response to lipopolysaccharide stimulation. Overexpression of MMP-10 in RAW 264.7 and MeT-5A cells upregulated pro-inflammatory cytokines with phosphorylation of NFκΒ subunit p65. Thus, our results suggest that inflammatory responses induced by MMP-10 are mediated through the NFκΒ pathway, and that systemic deletion of MMP-10 ameliorates peritoneal inflammation and fibrosis caused by NFκΒ activation of peritoneal macrophages and mesothelial cells.

Restoration of CPT1A-mediated fatty acid oxidation in mesothelial cells protects against peritoneal fibrosis.

Background: Peritoneal dialysis (PD) is limited by gradual fibrotic remodeling in the peritoneum, a process involving profibrotic response of mesothelial cells. However, the role of fatty acid oxidation (FAO) and carnitine palmitoyltransferase 1A (CPT1A) in this process remains unexplored. Methods: FAO and CPT1A expression were characterized in mesothelial cells from patients on long-term PD and from a mouse model of PD using multiple experimental methods, including single-cell sequencing, seahorse assay, real-time quantitative PCR, Western blot, and immunofluorescence staining. Overexpression of CPT1A was achieved in a human mesothelial cell line and in primary mouse mesothelial cells. Finally, genetic and pharmacological manipulations of CPT1A were performed in a mouse model of PD. Results: Herein, FAO and CPT1A expression were reduced in mesothelial cells from patients on long-term PD, which negatively correlated with expression of fibrogenic markers in these cells. This was corroborated in PD mice, as well as in mouse and human mesothelial cells incubated with transforming growth factor (TGF) β1. CPT1A overexpression in mesothelial cells, which prevented TGFβ1-induced suppression of mitochondrial respiration, restored cellular ATP levels and downregulated the expression of fibrogenic markers. Furthermore, restoration of FAO by overexpressing CPT1A in PD mice reversed profibrotic phenotype in mesothelial cells and reduced fibrotic lesions in the peritoneum. Treatment with the CPT1A activator C75 induced similar therapeutic benefit in PD mice. In contrast, inhibition of FAO with a CPT1 inhibitor caused more severe fibrosis in PD mice. Conclusions: A defective FAO is responsible for the profibrotic response of mesothelial cells and thus the peritoneal fibrogenesis. This aberrant metabolic state could be improved by modulating CPT1A in mesothelial cells, suggesting FAO enhancement in mesothelial cells is a potential treatment of peritoneal fibrosis.

Polycaprolactone/Chitosan Composite Nanofiber Membrane as a Preferred Scaffold for the Culture of Mesothelial Cells and the Repair of Damaged Mesothelium.

Mesothelial cells are specific epithelial cells lining the serosal cavity and internal organs. Nonetheless, few studies have explored the possibility to culture mesothelial cells in a nanostructure scaffold for tissue engineering applications. Therefore, this study aims to fabricate nanofibers from a polycaprolactone (PCL) and PCL/chitosan (CS) blend by electrospinning, and to elucidate the effect of CS on the cellular response of mesothelial cells. The results demonstrate that a PCL and PCL/CS nanofiber membrane scaffold could be prepared with a comparable fiber diameter (~300 nm) and porosity for cell culture. Blending CS with PCL influenced the mechanical properties of the scaffold due to interference of PCL crystallinity in the nanofibers. However, CS substantially improves scaffold hydrophilicity and results in a ~6-times-higher cell attachment rate in PCL/CS. The mesothelial cells maintain high viability in both nanofiber membranes, but PCL/CS provides better maintenance of cobblestone-like mesothelial morphology. From gene expression analysis and immunofluorescence staining, the incorporation of CS also results in the upregulated expression of mesothelial marker genes and the enhanced production of key mesothelial maker proteins, endorsing PCL/CS to better maintain the mesothelial phenotype. The PCL/CS scaffold was therefore chosen for the in vivo studies, which involved transplanting a cell/scaffold construct containing allograft mesothelial cells for mesothelium reconstruction in rats. In the absence of mesothelial cells, the mesothelium wound covered with PCL/CS showed an inflammatory response. In contrast, a mesothelium layer similar to native mesothelium tissue could be obtained by implanting the cell/scaffold construct, based on hematoxylin and eosin (H&E) and immunohistochemical staining.

Impact of Factors Secreted by Tumor Cells on Response of Pleural Mesothelial Cells to Different Sclerosing Agents in an In Vitro Model.

BackgroundChemical pleurodesis is one of the major therapeutic options for patients with recurrent malignant pleural effusion. Mesothelial cells are considered to play a pivotal role in the response to different chemical compounds (sclerosants) used for pleurodesis. Malignant cells might have an impact on the mesothelial response to applied sclerosing agents and, in consequence, on the efficacy of pleurodesis. We aimed to evaluate the impact of cancer cell paracrine on mesothelial cell response to different sclerosing agents.Material/MethodsThe study used mesothelial cell (MeT-5A) cultures stimulated with sclerosing agents (talc, doxycycline, iodopovidone, and TGF-β for 24 h) in the presence or absence of supernatants from adenocarcinoma cultures (HCC827). The mesothelial mRNA expression and protein levels of IL-6, IL-8, and TGF-β was assessed. Further, lung fibroblasts were cultured with and without cell supernatants from previously established cell cultures for 24 h. Then, concentration of soluble collagen was evaluated in culture supernatants.ResultsThe exposure of mesothelial cells to sclerosants decreased the concentration of IL-6 and IL-8 protein. The addition of mediators secreted by adenocarcinoma altered the inflammatory response of the mesothelial cells to sclerosing agents. IL-8 concentration in cultures stimulated with talc and adenocarcinoma supernatant was higher compared to cultures stimulated with talc only. The exposure of lung fibroblasts to supernatant from mesothelial cell (with or without adenocarcinoma) did not affect collagen secretion.ConclusionsAn addition of soluble factors produced by adenocarcinoma altered the inflammatory response of the pleural mesothelial cells after stimulation with sclerosing agents. Our observations suggest that the tumor paracrine effect affects biological pathways of pleurodesis.

MicroRNA 148a Suppresses Tuberculous Fibrosis by Targeting NOX4 and POLDIP2

Extracellular matrix production by pleural mesothelial cells in response to Mycobacterium tuberculosis contributes to tuberculous fibrosis. NOX4 is involved in the pathogenesis of tuberculous fibrosis. In this study, we evaluated whether NOX4 gene-targeting microRNAs showed protective effects in tuberculosis fibrosis. TargetScan prediction software was used to identify candidate microRNAs that bind the 3′ UTRs of NOX4, and microRNA-148a (miR-148a) was selected as the best miRNA candidate. A repressed and forced expression assay in Met5A cells was performed to investigate the causal relationship between miR-148a and NOX4. The role of miR-148a in tuberculous pleural fibrosis was studied using a murine model of Mycobacterium bovis bacillus Calmette–Guérin (BCG) pleural infection. Heat-killed M. tuberculosis (HKMT) induces NOX4 and POLDIP2 expression. We demonstrated the inhibitory effect of miR-148a on NOX4 and POLDIP2 expression. The increased expression of miR-148a suppressed HKMT-induced collagen-1A synthesis in PMC cells. In the BCG pleurisy model, miR-148a significantly reduced fibrogenesis and epithelial mesenchymal transition. High levels of miR-148a in tuberculous pleural effusion can be interpreted as a self-limiting homeostatic response. Our data indicate that miR-148a may protect against tuberculous pleural fibrosis by regulating NOX4 and POLDIP2.

Primary human mesothelial cell culture in the evaluation of the inflammatory response to different sclerosing agents used for pleurodesis.

The mechanisms of chemical pleurodesis are still not fully explained. We aimed to evaluate the feasibility of using primary biopsy‐derived human mesothelial cells to establish an in vitro culture and to assess the response of pleural mesothelial cells to different sclerosing agents. Talc, povidone‐iodine, doxycycline, and TGF‐β were used at different doses to stimulate pleural mesothelial cells. After 6 and 24 h, mRNA expression of interleukin (IL)‐1β, IL‐6, IL‐8, TGF‐β, MCP‐1, IL‐17A, and MMP9 was measured in cultured cells, and the protein level of IL‐1β, IL‐6, and IL‐8 was measured in the culture supernatant. The most pronounced response was observed after talc exposure. It was expressed as an increase in IL‐1β concentration in culture supernatant after 24 h of higher talc dose stimulation compared to 6 h of stimulation (17.14 pg/ml [11.96–33.32 pg/ml] vs. 1.84 pg/ml [1.81–1.90 pg/ml], p = 0.02). We showed that culture pleural mesothelial cells isolated from pleura biopsy specimens is feasible. Inflammatory responses of mesothelial cells to different sclerosants were highly variable with no consistent pattern of mesothelium reaction neither in terms of different sclerosing agents nor in the time of the most significant reaction. We demonstrated that pro‐inflammatory mesothelial response includes an increase in IL‐1β mRNA expression and protein production. This may suggest the role of IL‐1β in the formation and maintenance of the inflammatory response during pleurodesis.

Caveolin1 and YAP drive mechanically induced mesothelial to mesenchymal transition and fibrosis

Despite their emerging relevance to fully understand disease pathogenesis, we have as yet a poor understanding as to how biomechanical signals are integrated with specific biochemical pathways to determine cell behaviour. Mesothelial-to-mesenchymal transition (MMT) markers colocalized with TGF-β1-dependent signaling and yes-associated protein (YAP) activation across biopsies from different pathologies exhibiting peritoneal fibrosis, supporting mechanotransduction as a central driving component of these class of fibrotic lesions and its crosstalk with specific signaling pathways. Transcriptome and proteome profiling of the response of mesothelial cells (MCs) to linear cyclic stretch revealed molecular changes compatible with bona fide MMT, which (i) overlapped with established YAP target gene subsets, and were largely dependent on endogenous TGF-β1 signaling. Importantly, TGF-β1 blockade blunts the transcriptional upregulation of these gene signatures, but not the mechanical activation and nuclear translocation of YAP per se. We studied the role therein of caveolin-1 (CAV1), a plasma membrane mechanotransducer. Exposure of CAV1-deficient MCs to cyclic stretch led to a robust upregulation of MMT-related gene programs, which was blunted upon TGF-β1 inhibition. Conversely, CAV1 depletion enhanced both TGF-β1 and TGFBRI expression, whereas its re-expression blunted mechanical stretching-induced MMT. CAV1 genetic deficiency exacerbated MMT and adhesion formation in an experimental murine model of peritoneal ischaemic buttons. Taken together, these results support that CAV1-YAP/TAZ fine-tune the fibrotic response through the modulation of MMT, onto which TGF-β1-dependent signaling coordinately converges. Our findings reveal a cooperation between biomechanical and biochemical signals in the triggering of MMT, representing a novel potential opportunity to intervene mechanically induced disorders coursing with peritoneal fibrosis, such as post-surgical adhesions.

Transcriptome analysis of signaling pathways of human peritoneal mesothelial cells in response to different osmotic agents in a peritoneal dialysis solution

BackgroundGlucose is a primary osmotic agent in peritoneal dialysis (PD) solutions, but its long-term use causes structural alteration of the peritoneal membrane (PM). Hyperbranched polyglycerol (HPG) is a promising alternative to glucose. This study was designed to compare the cellular responses of human peritoneal mesothelial cells (HPMCs) to these two different osmotic agents in a hypertonic solution using transcriptome analysis.MethodsCultured HPMCs were repeatedly exposed to HPG-based or Physioneal 40 (PYS, glucose 2.27%) hypertonic solutions. Transcriptome datasets were produced using Agilent SurePrint G3 Human GE 8 × 60 microarray. Cellular signaling pathways were examined by Ingenuity Pathway Analysis (IPA). Protein expression was examined by flow cytometry analysis and Western blotting.ResultsThe HPG-containing solution was better tolerated compared with PYS, with less cell death and disruption of cell transcriptome. The levels of cell death in HPG- or PYS- exposed cells were positively correlated with the number of affected transcripts (HPG: 128 at day 3, 0 at day 7; PYS: 1799 at day 3, 212 at day 7). In addition to more affected “biosynthesis” and “cellular stress and death” pathways by PYS, both HPG and PYS commonly affected “sulfate biosynthesis”, “unfolded protein response”, “apoptosis signaling” and “NRF2-mediated oxidative stress response” pathways at day 3. PYS significantly up-regulated HLA-DMB and MMP12 in a time-dependent manner, and stimulated T cell adhesion to HPMCs.ConclusionThe lower cytotoxicity of hypertonic HPG solution is in agreement with its transient and minimal impact on the pathways for the “biosynthesis of cell constituents” and the “cellular stress and death”. The significant up-regulation of HLA-DMB and MMP12 by PYS may be part of its initiation of immune response in the PM.

Toll/IL-1 domain-containing adaptor inducing IFN-β (TRIF) mediates innate immune responses in murine peritoneal mesothelial cells through TLR3 and TLR4 stimulation

Mesothelial cells are composed of monolayer of the entire surface of serosal cavities including pleural, pericardial, and peritoneal cavity. Although mesothelial cells are known to express multiple Toll-like receptors (TLRs) which contribute to trigger innate immune responses against infections, the precise molecular mechanism remains still unclear. In the present study, we investigated the role of Toll/IL-1 domain-containing adaptor inducing IFN-β (TRIF), one of the two major TLRs–adaptor molecules, on innate immune response induced by TLR3 and TLR4 stimulation in murine peritoneal mesothelial cells (PMCs). TRIF was strongly expressed in PMCs and its deficiency led to impaired production of cytokines and chemokines by poly I:C and LPS in the cells. Activation of NF-κB or MAPKs through poly I:C and LPS stimulation was reduced in TRIF-deficient PMCs as compared to the WT cells. TRIF was also necessary for optimal nitric oxide synthesis and gene expression of inducible nitric oxide synthase (iNOS) and IFN-β in PMCs in response to poly I:C and LPS. Furthermore, both Escherichia coli and Pseudomonas aeruginosa induced high level of IL-6, CXCL1, and CCL2 production in PMCs, which was significantly impaired by TRIF deficiency. These results demonstrated that TRIF is required for optimal activation of innate immune responses in mesothelial cells against microbial infections.

Causes and Pathophysiology of Malignant Pleural Mesothelioma

Malignant pleural mesothelioma (MPM) results from the neoplastic transformation of pleural mesothelial cells. Asbestos exposure is a major risk factor for MPM, but epidemiologic studies demonstrated the occurrence of MPM in populations exposed to other fibers, and an excess of MPM in populations occupationally exposed to man-made vitreous fibers and previously to asbestos. The development of nanotechnologies also raises some concern about the potential health effects of new particles of high aspect ration, such as carbon nanotubes. Toxicological studies investigated the mechanism of asbestos-induced transformation of mesothelial cells, and molecular analyses defined the genomic and physiopathological changes in MPM. These findings allowed identifying some key events accounting for the neoplastic process. This article summarizes the known and suspected causes of MPM, the cellular events and responses of mesothelial cells to asbestos fibers and the alterations of key genes and regulatory pathways involved in the pathological mechanism.

Injury-induced inflammation and inadequate HSP expression in mesothelial cells upon repeat exposure to dual-chamber bag peritoneal dialysis fluids.

Peritoneal dialysis fluids (PDFs) may induce inadequate heat-shock protein (HSP) expression and injury-related inflammation in exposed mesothelial cells. The aim of this study was to relate cellular injury to these cellular responses in mesothelial cells following repeated exposure to 3 commercial PDFs with different biocompatibility profiles. Primary cultures of human peritoneal mesothelial cells (HPMC) were exposed to a 1:2 mixture of cell culture medium and CAPD2 (single-chamber bag PDF; Fresenius, Bad Homburg, Germany), Physioneal (dual-chamber bag PDF; Baxter, Deerfield, IL, USA) or Balance (dual-chamber bag PDF, Fresenius) for up to 10 days exposure time (4 dwells). Supernatant was analyzed for LDH, IL-6, and IL-8, cells for HSP-72 expression, and protein content. PDF exposure resulted in a biphasic pattern of cell damage switching from an earlier phase with increased injury by single-chamber PDF to a delayed phase with increased susceptibility to dual-chamber PDF. Sterile inflammation was related to LDH release over time and could be reproduced by exposure to necrotic cellular material. PDF exposure resulted in low HSP-72 expression in all tested PDFs. Exposure to single-chamber as well as to dual-chamber bag PDFs induce increased vulnerability of mesothelial cells to repeated exposure of the same solution. These effects were delayed with dual-chamber PDFs. Injury-induced inflammation and impaired HSP expression upon PDF exposure might initiate a vicious cycle with progredient mesothelial cell damage upon repeated PDF exposure. Certainly, interventional studies and translation of these results into the in vivo system is needed.

Cross-Omics Comparison of Stress Responses in Mesothelial Cells Exposed to Heat- versus Filter-Sterilized Peritoneal Dialysis Fluids

Recent research suggests that cytoprotective responses, such as expression of heat-shock proteins, might be inadequately induced in mesothelial cells by heat-sterilized peritoneal dialysis (PD) fluids. This study compares transcriptome data and multiple protein expression profiles for providing new insight into regulatory mechanisms. Two-dimensional difference gel electrophoresis (2D-DIGE) based proteomics and topic defined gene expression microarray-based transcriptomics techniques were used to evaluate stress responses in human omental peritoneal mesothelial cells in response to heat- or filter-sterilized PD fluids. Data from selected heat-shock proteins were validated by 2D western-blot analysis. Comparison of proteomics and transcriptomics data discriminated differentially regulated protein abundance into groups depending on correlating or noncorrelating transcripts. Inadequate abundance of several heat-shock proteins following exposure to heat-sterilized PD fluids is not reflected on the mRNA level indicating interference beyond transcriptional regulation. For the first time, this study describes evidence for posttranscriptional inadequacy of heat-shock protein expression by heat-sterilized PD fluids as a novel cytotoxic property. Cross-omics technologies introduce a novel way of understanding PDF bioincompatibility and searching for new interventions to reestablish adequate cytoprotective responses.

Lipopolysaccharide (LPS)-induced autophagy is involved in the restriction of Escherichia coliin peritoneal mesothelial cells

BackgroundHost cell autophagy is implicated in the control of intracellular pathogen. Escherichia coli (E.coli) is the most common organism caused single-germ enterobacterial peritonitis during peritoneal dialysis. In this study, we investigated autophagy of peritoneal mesothelial cells and its role in defense against E.coli.ResultsAutophagy in human peritoneal mesothelial cell line (HMrSV5) was induced by lipopolysaccharide (LPS) in a dose-dependent and time-dependent way, which was demonstrated by increased expression of Beclin-1 and light chain 3 (LC3)-II, the accumulation of punctate green fluorescent protein-LC3, and a higher number of monodansylcadaverine-labeled autophagic vacuoles. After incubation of HMrSV5 cells with E.coli following LPS stimulation, both the intracellular bactericidal activity and the co-localization of E.coli (K12-strain) with autophagosomes were enhanced. Conversely, blockade of autophagy with 3-methyladenine, wortmannin or Beclin-1 small-interfering RNA (siRNA) led to a significant reduction in autophagy-associated protein expression, attenuation of intracellular bactericidal activity, and reduced co-localization of E.coli with monodansylcadaverine-labeled autophagosomes. In addition, treatment of HMrSV5 cells with LPS caused a dose-dependent and time-dependent increase in Toll-like receptor 4 (TLR4) expression. Both knockdown of TLR4 with siRNA and pharmacological inhibition of TLR4 with Polymyxin B significantly decreased LPS-induced autophagy. Furthermore, TLR4 siRNA attenuated remarkably LPS-induced intracellular bactericidal activity.ConclusionsOur findings demonstrated for the first time that LPS-induced autophagy in peritoneal mesothelial cells could enhance the intracellular bactericidal activity and the co-localization of E.coli with autophagosomes. The activation of TLR4 signaling was involved in this process. These results indicate that LPS-induced autophagy may be a cell-autonomous defense mechanism triggered in peritoneal mesothelial cells in response to E.coli infection.

Critical role of Toll‐like receptor 2 in Bacteroides fragilis‐mediated immune responses in murine peritoneal mesothelial cells

In this study, the role of Toll-like receptor 2 (TLR2) in immune responses of murine peritoneal mesothelial cells against Bacteroides fragilis was investigated. Enzyme linked immunosorbent assay was used to measure cytokines and chemokines. Activation of nuclear factor κB (NF-κB-α) and mitogen-activated protein kinases (MAP kinases) was investigated by western blot analysis. B. fragilis induced production of interleukin-6, chemokine (C-X-C motif) ligand 1 (CXCL1) and chemokine (C-C motif) ligand 2 (CCL2) in wild type peritoneal mesothelial cells; this was impaired in TLR2-deficient cells. In addition, in response to B. fragilis, phosphorylation of inhibitory NF-κB-α and c-Jun N-terminal kinase mitogen-activated protein kinase (MAPK) was induced in wild type mesothelial cells, but not in TLR2-deficient cells,. Inhibitor assay revealed that NF-κB and MAPKs are essential for B. fragilis-induced production of CXCL1 and CCL2 in mesothelial cells. These findings suggest that TLR2 mediates immune responses in peritoneal mesothelial cells in response to B. fragilis.

New mechanism of elevated CA125 in heart failure: The mechanical stress and inflammatory stimuli initiate CA125 synthesis

Carbohydrate antigen 125 has been known as a membrane-associated mucin. It has found application as a tumor marker that may be elevated in the blood of some patients with ovarian cancer, or other benign conditions. Recently, increased serum carbohydrate antigen 125 levels have been documented in patients with heart failure. However, little is known about the pathophysiologic mechanism. It has been found that carbohydrate antigen 125 will be shed from surfaces of mesothelial cells in response to mechanical stress such as fluid overload, and inflammatory stimuli. High carbohydrate antigen 125 levels are closely related to the presence of serosal fluid and positively correlated with serum TNF-α, IL-6 and IL-10 levels in heart failure patients. This leads to proposed hypothesis that the mechanical stress and inflammatory stimuli both initiate carbohydrate antigen 125 synthesis. This finding suggests that carbohydrate antigen 125 might be a promising biomarker to evaluate the risk stratification of heart failure and monitor the process of therapy.

The mechanism of pleural inflammation by long carbon nanotubes: interaction of long fibres with macrophages stimulates them to amplify pro-inflammatory responses in mesothelial cells

Carbon nanotubes (CNT) are high aspect ratio nanoparticles with diameters in the nanometre range but lengths extending up to hundreds of microns. The structural similarities between CNT and asbestos have raised concern that they may pose a similar inhalation hazard. Recently CNT have been shown to elicit a length-dependent, asbestos-like inflammatory response in the pleural cavity of mice, where long fibres caused inflammation but short fibres did not. However the cellular mechanisms governing this response have yet to be elucidated. This study examined the in vitro effects of a range of CNT for their ability to stimulate the release of the acute phase cytokines; IL-1β, TNFα, IL-6 and the chemokine, IL-8 from both Met5a mesothelial cells and THP-1 macrophages. Results showed that direct exposure to CNT resulted in significant cytokine release from the macrophages but not mesothelial cells. This pro-inflammatory response was length dependent but modest and was shown to be a result of frustrated phagocytosis. Furthermore the indirect actions of the CNT were examined by treating the mesothelial cells with conditioned media from CNT-treated macrophages. This resulted in a dramatic amplification of the cytokine release from the mesothelial cells, a response which could be attenuated by inhibition of phagocytosis during the initial macrophage CNT treatments. We therefore hypothesise that long fibres elicit an inflammatory response in the pleural cavity via frustrated phagocytosis in pleural macrophages. The activated macrophages then stimulate an amplified pro-inflammatory cytokine response from the adjacent pleural mesothelial cells. This mechanism for producing a pro-inflammatory environment in the pleural space exposed to long CNT has implications for the general understanding of fibre-related pleural disease and design of safe nanofibres.

NFAT5 Contributes to Osmolality-Induced MCP-1 Expression in Mesothelial Cells

Increased expression of the C-C chemokine monocyte chemoattractant protein-1 (MCP-1) in mesothelial cells in response to high glucose concentrations and/or high osmolality plays a crucial role in the development of peritoneal fibrosis during continuous ambulatory peritoneal dialysis (CAPD). Recent studies suggest that in kidney cells osmolality-induced MCP-1 upregulation is mediated by the osmosensitive transcription factor, nuclear factor of activated T cells 5 (NFAT5). The present study addressed the question of whether activation of NFAT5 by hyperosmolality, as present in PD fluids, contributes to MCP-1 expression in the mesothelial cell line Met5A. Hyperosmolality, induced by addition of glucose, NaCl, or mannitol to the growth medium, increased NFAT5 activity and stimulated MCP-1 expression in Met5A cells. siRNA-mediated knockdown of NFAT5 attenuated osmolality-induced MCP-1 upregulation substantially. Hyperosmolality also induced activation of nuclear factor-κB (NF-κB). Accordingly, pharmacological inhibition of NF-κB significantly decreased osmolality-induced MCP-1 expression. Taken together, these results indicate that high osmolalities activate the transcription factor NFAT5 in mesothelial cells. NFAT5 in turn upregulates MCP-1, likely in combination with NF-κB, and thus may participate in the development of peritoneal fibrosis during CAPD.

Peritoneal Dialysis Fluid Induces P38-Dependent Inflammation in Human Mesothelial Cells

Noninfectious upregulation of proinflammatory pathways in mesothelial cells may represent an integral part of their stress response upon exposure to peritoneal dialysis fluids (PDF). The aim of this study was to evaluate the role of the stress-inducible mitogen-activated protein kinase (MAPK) p38 in regulation of inflammatory and stress responses in mesothelial cells following in vitro exposure to PDF. Human peritoneal mesothelial cells were exposed to Dianeal PD4 or Physioneal (Baxter AG, Vienna, Austria) containing 1.36% glucose and then allowed to recover. Phosphorylation of p38, induction of heat shock protein-70 (HSP70), release of lactate dehydrogenase (LDH), secretion of interleukin (IL)-8, gene transcription, and mRNA stability were assessed with and without the MAPK p38 inhibitor SB203580. Exposure to Dianeal resulted in phosphorylation of p38 within 30 minutes (309% of control, p < 0.05) and increased IL-8 release (370% of control, p < 0.05), HSP70 expression (151% of control, p < 0.05), and LDH release (180% of control, p < 0.05). Exposure to Physioneal resulted in attenuated changes in IL-8, HSP70, and LDH. Addition of the p38 inhibitor SB203580 to Dianeal resulted in dampened IL-8 release (-55%; p < 0.05) and basal HSP70 expression, and unchanged LDH release. Effects of p38 on IL-8 were at transcriptional, posttranscriptional, and translational levels. These data confirm concordant p38-dependent upregulation of IL-8 and HSP70 following exposure to bioincompatible PDF. The MAPK p38 pathway therefore links proinflammatory processes and the cellular stress response in human peritoneal mesothelial cells.

Sulforaphane and Rebamipide Protect Small Intestinal Mucosa From Indomethacin-Induced Injury Through Different Mechanisms in Mice In Vivo

Background and Aims: Postoperative peritoneal adhesion formation frequently occurs and remains a major problem after abdominal surgery. Daikenchuto (TU-100), a traditional Japanese oral medicine, prevents peritoneal adhesion after abdominal surgery. However, the precise mechanism of action is still unclear. We recently reported that 6-shogaol (6SG) and hydroxy α-sanshool (HAS), the main ingredients in TU-100, are absorbed in humans and can enhance the endogenous anti-fibrotic and anti-inflammatory peptide, adrenomedullin (ADM). The peritoneal fibrinolytic system is crucial for peritoneal adhesion formation. The present study assessed the mechanism of ADM production and the fibrinolytic responses of mesothelial cells to inflammatory factors to determine the anti-adhesion mechanism of TU-100. Methods: Test samples were added to cultures of the rat mesothelial cell line 4/ 4RM-4. After 6 to 24 h, the levels of ADM, tissue plasminogen activator (tPA), and plasminogen activator inhibitor-1 (PAI-1) in the culture supernatants were measured by EIA and RT-PCR. The active ingredients of TU-100 in the blood of rats that were orally administered TU-100 were measured by LC-Ms/Ms. Results: ADM production by 4/4RM-4 cells was significantly increased upon the addition of 6SG and HAS, which were efficiently absorbed into the circulation within 30 minutes after oral administration. Because these compounds activate the transient receptor potential (TRP) V1 and A1 channels, the TRPA1 agonist allyl isothiocyanate (AITC) and TRPV1 agonist capsaicin (CAP) were evaluated in the ADM production test. AITC, but not CAP, was active and increased ADM production. This activity of 6SG, HAS and AITC was significantly abolished by also adding the TRPA1 antagonist HC-030031. RT-PCR analysis demonstrated that 4/4RM-4 cells expressed messenger RNA for TRPA1 but not TRPV1. Inflammatory mediators IL-6, TNFα, and lipopolysaccharide (LPS) upregulated PAI-1, but not tPA, production by 4/4RM-4 cells. Conversely, ADM downregulated PAI-1 without affecting tPA, and moreover inhibited IL-6 and TNFα production by immune cells. Conclusions: ADM can restore the imbalance in the fibrinolytic