Pleural Mesothelial Cells Research Articles

Lethal (2) giant larvae regulates pleural mesothelial cell polarity in pleural fibrosis

Pleural fibrosis is barely reversible and the underlying mechanisms are poorly understood. Pleural mesothelial cells (PMCs) which have apical-basal polarity play a key role in pleural fibrosis. Loss of cell polarity is involved in the development of fibrotic diseases. Partition defective protein (PAR) complex is a key regulator of cell polarity. However, changes of PMC polarity and PAR complex in pleural fibrosis are still unknown. In this study, we observed that PMC polarity was lost in fibrotic pleura. Next we found increased Lethal (2) giant larvae (Lgl) bound with aPKC and PAR-6B competing against PAR-3A in PAR complex, which led to cell polarity loss. Then we demonstrated that Lgl1 siRNA prevented cell polarity loss in PMCs, and Lgl1 conditional knockout (ER-Cre+/−Lgl1flox/flox) attenuated pleural fibrosis in a mouse model. Our data indicated that Lgl1 regulates cell polarity of PMCs, inhibition of Lgl1 and maintenance of cell polarity in PMCs could be a potential therapeutic treatment approach for pleural fibrosis.

TLR4 and AT1 receptor contribute to tuberculous pleural fibrosis through NOX4‐ERK‐ROS signal pathway

Tuberculous pleurisy sometimes results in pleural fibrosis that could cause respiratory failure. Pleural mesothelial cells (PMC) play an important role in pleural fibrosis. Several studies suggest that reactive oxygen species (ROS) produced by NOXs have been shown to initiate lung fibrosis, but the detailed mechanisms about TB pleurisy-associated fibrosis remain to be not well understood. We aimed to investigate whether activation of TLR4 and renin angiotensin system (RAS) contributes to NOX4-ROS signaling in tuberculous pleural fibrosis. In vitro human PMC experiments were performed to assess the role of NOX4 -ROS associated signaling in heat-killed mycobacterium tuberculosis (HKMT) induced pleural fibrosis. We found that HKMT induces collagen I synthesis and NOX4-ROS expression in pleural mesothelial cells and NOX4 mediates HKMT-induced collagen -1 synthesis and epithelial-mesenchymal transition (EMT) via activation ERK signaling We also observed that the inhibition of TLR4 and AT1 receptor reduce HKMT-induced NOX4 – ROS production and collagen synthesis. In pleural mesothelial cells, HKMT upregulated Angiotensin Converting Enzyme (ACE) levels and TLR4 inhibitor pretreatment reduced ACE levels. These results suggest crosstalk between TLR4 and AT1 in tuberculous pleural fibrosis. In conclusion, these results suggest that NOX4-ERK-ROS mediates HKMT induced pleural fibrosis and EMT via TLR4 receptor and AT1 receptor. TLR4-NOX4-ROS signal pathway may a novel therapeutic target for intervention in tuberculous pleural fibrosis. Support or Funding Information This work was supported by National research Foundatin of Korea (NRF) grant funded by the Korea government (NRF-2017R1C1B501789, NRF-2017M3A9E8033225). This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Silver Nanoparticles Alter Cell Adhesion and Proliferation of Sheep Primary Mesothelial Cells.

Human exposure to engineered nanoparticles has been linked to pleural effusion, inflammation and fibrosis. Silver nanoparticles (AgNPs) are widely used in medical and domestic products, increasing the risk of occupational and domestic exposure. We assessed the influence of AgNPs on adhesion and proliferation of sheep primary pleural mesothelial cells. Cells were used for cell adhesion (90 min) and proliferation experiments (3 days) while exposed to 20 nm and 60 nm AgNPs (0.2 μg/ml and 2 μg/ml) using colorimetric assays. Exposure to 0.2 μg/ml of 20 nm and 60 nm AgNPs significantly increased cell adhesion, while at 2 μg/ml this effect was not elicited. Cell proliferation was significantly increased by both 20 nm and 60 nm AgNPs at 0.2 μg/ml, while at 2 μg/ml this effect was only elicited by the 60 nm AgNPs. AgNPs alter the adhesive and proliferative properties of primary pleural mesothelial cells.

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Inhibition of angiotensin II and calpain attenuates pleural fibrosis

Pleural fibrosis is associated with various inflammatory processes such as tuberculous pleurisy and bacterial empyema. There is currently no ideal therapeutic to attenuate pleural fibrosis. Some pro-fibrogenic mediators induce fibrosis through inflammatory processes, suggesting that blockage of these mediators might prevent pleural fibrosis. The MeT-5A human pleural mesothelial cell line (PMC) was used in this study as an in vitro model of fibrosis; and intra-pleural injection of bleomycin with carbon particles was used as an in vivo mouse model of pleural fibrosis. Calpain knockout mice, calpain inhibitor (calpeptin), and angiotensin (Ang) II type 1 receptor (AT1R) antagonist (losartan) were evaluated in prevention of experimental pleural fibrosis. We found that bleomycin and carbon particles induced calpain activation in cultured PMCs. This in vitro response was associated with increased collagen-I synthesis, and was blocked by calpain inhibitor or AT1R antagonist. Calpain genetic or treatment with calpeptin or losartan prevented pleural fibrosis in a mouse model induced by bleomycin and carbon particles. Our findings indicate that Ang II signaling and calpain activation induce collagen-I synthesis and contribute to fibrotic alterations in pleural fibrosis. Inhibition of Ang II and calpain might therefore be a novel strategy in treatment of pleural fibrosis.

Inhibition of Glycogen Synthase Kinase 3β Blocks Mesomesenchymal Transition and Attenuates Streptococcus pneumonia–Mediated Pleural Injury in Mice

Pleural loculation affects about 30,000 patients annually in the United States and in severe cases can resolve with restrictive lung disease and pleural fibrosis. Pleural mesothelial cells contribute to pleural rind formation by undergoing mesothelial mesenchymal transition (MesoMT), whereby they acquire a profibrotic phenotype characterized by increased expression of α-smooth muscle actin and collagen 1. Components of the fibrinolytic pathway (urokinase plasminogen activator and plasmin) are elaborated in pleural injury and strongly induce MesoMT invitro. These same stimuli enhance glycogen synthase kinase (GSK)-3β activity through increased phosphorylation of Tyr-216 in pleural mesothelial cells and GSK-3β mobilization from the cytoplasm to the nucleus. GSK-3β down-regulation blocked induction of MesoMT. Likewise, GSK-3β inhibitor 9ING41 blocked induction of MesoMT and reversed established MesoMT. Similar results were demonstrated in a mouse model of Streptococcus pneumoniae-induced empyema. Intraperitoneal administration of 9ING41, after the induction of pleural injury, attenuated injury progression and improved lung function (lung volume and compliance; P<0.05 compared with untreated and vehicle controls). MesoMT marker α-smooth muscle actin was reduced in 9ING41-treated mice. Pleural thickening was also notably reduced in 9ING41-treated mice (P<0.05). Collectively, these studies identify GSK-3β as a newly identified target for amelioration of empyema-related pleural fibrosis and provide a strong rationale for further investigation of GSK-3β signaling in the control of MesoMT and pleural injury.

Oxidized regenerated cellulose induces pleural thickening in patients with pneumothorax: possible involvement of the mesothelial-mesenchymal transition.

The pleural covering technique, i.e., wrapping a part of or the entire surface of the lung with oxidized regenerative cellulose (ORC), reinforces visceral pleura through pleural thickening for patients with pneumothorax and cystic lung diseases. However, it remains undetermined how ORC induces pleural thickening. A histopathological examination was performed for lung specimens from patients who had recurrent pneumothoraces after pleural covering and re-operation (n=5). To evaluate the influence of ORC on the pleura in vitro, we used MeT-5A cells (a human pleural mesothelial cell line). Pleural thickening was confirmed in all lung specimens examined. Three months after covering, the thickened pleura showed inflammatory cell infiltration, proliferation of myofibroblasts, and expression of fibronectin and TGF-β. However, after 1year, those findings virtually disappeared, and the thickened pleura was composed mainly of abundant collagen. When MeT-5A cells were cultured in ORC-immersed medium, their morphology changed from a cobblestone to spindle-shaped appearance. The expression of E-cadherin decreased, whereas that of N-cadherin, α-smooth muscle actin, and fibronectin increased, suggesting mesothelial-mesenchymal transition (Meso-MT). Our results suggest that Meso-MT may be involved as a mechanism of pleural thickening induced by pleural covering with ORC.

Lipoteichoic acid upregulates plasminogen activator inhibitor-1 expression in parapneumonic effusions.

Parapneumonic effusion (PPE) is commonly caused by Gram-positive bacteria (GPB) and often presents with pleural loculation, which is characterized by overproduction of plasminogen activator inhibitor (PAI)-1. Lipoteichoic acid (LTA), a surface adhesion molecule of GPB, binds to the pleural mesothelium and triggers inflammation. However, the effects of LTA on PAI-1 expression in PPE and underlying mechanisms remain unclear. Thirty consecutive patients with PPE were enrolled, including uncomplicated culture negative (CN, n = 11), Gram-negative bacteria (GNB, n = 7) and GPB (n = 12) groups stratified by pleural fluid characteristics and bacteriology, and the effusion PAI-1 levels were measured. In addition, human pleural mesothelial cells (PMC) were treated with LTA and the expression of PAI-1 and activation of signalling pathways were assayed. The median levels of PAI-1 were significantly higher in GPB (160.5 ng/mL) and GNB (117.0 ng/mL) groups than in the uncomplicated CN (58.0 ng/mL) group. In human PMC, LTA markedly upregulated PAI-1 mRNA and protein expression and enhanced elaboration of Toll-like receptor 2 (TLR2). Furthermore, LTA increased c-Jun N-terminal kinase (JNK) phosphorylation, induced activating transcription factor 2 (ATF2)/c-Jun nuclear translocation and activated PAI-1 promoter activity. Pretreatment with TLR2 siRNA significantly inhibited LTA-induced JNK phosphorylation and PAI-1 protein expression. Culture-positive PPE, especially that caused by GPB, has a significantly higher level of PAI-1 than uncomplicated CN PPE. LTA upregulates PAI-1 expression through activation of TLR2/JNK/activator protein 1 (AP-1) pathway in human PMC. Better understanding of the modulation of PAI-1 synthesis by LTA in PPE may provide potential therapies for infected pleural effusions.

Exposure to nano-size titanium dioxide causes oxidative damages in human mesothelial cells: The crystal form rather than size of particle contributes to cytotoxicity

Exposure to nanoparticles such as carbon nanotubes has been shown to cause pleural mesothelioma similar to that caused by asbestos, and has become an environmental health issue. Not only is the percutaneous absorption of nano-size titanium dioxide particles frequently considered problematic, but the possibility of absorption into the body through the pulmonary route is also a concern. Nevertheless, there are few reports of nano-size titanium dioxide particles on respiratory organ exposure and dynamics or on the mechanism of toxicity.In this study, we focused on the morphology as well as the size of titanium dioxide particles. In comparing the effects between nano-size anatase and rutile titanium dioxide on human-derived pleural mesothelial cells, the anatase form was shown to be actively absorbed into cells, producing reactive oxygen species and causing oxidative damage to DNA. In contrast, we showed for the first time that the rutile form is not easily absorbed by cells and, therefore, does not cause oxidative DNA damage and is significantly less damaging to cells. These results suggest that with respect to the toxicity of titanium dioxide particles on human-derived mesothelial cells, the crystal form rather than the particle size has a greater effect on cellular absorption. Also, it was indicated that the difference in absorption is the primary cause of the difference in the toxicity against mesothelial cells.

KIF5A transports collagen vesicles of myofibroblasts during pleural fibrosis

Fibrosis involves the production of extracellular matrix proteins in tissues and is often preceded by injury or trauma. In pleural fibrosis excess collagen deposition results in pleural thickening, increased stiffness and impaired lung function. Myofibroblasts are responsible for increased collagen deposition, however the molecular mechanism of transportation of procollagen containing vesicles for secretion is unknown. Here, we studied the role of kinesin on collagen-1 (Col-1) containing vesicle transportation in human pleural mesothelial cells (HPMCs). Among a number of cargo transporting kinesins, KIF5A was notably upregulated during TGF-β induced mesothelial-mesenchymal transition (MesoMT). Using superresolution structured illumination microscopy and the DUO-Link technique, we found that KIF5A colocalized with Col-1 containing vesicles. KIF5A knock-down significantly reduced Col-1 secretion and attenuated TGF-β induced increment in Col-1 localization at cell peripheries. Live cell imaging revealed that GFP-KIF5A and mCherry-Col-1 containing vesicles moved together. Kymography showed that these molecules continuously move with a mean velocity of 0.56 μm/sec, suggesting that the movement is directional but not diffusion limited process. Moreover, KIF5A was notably upregulated along with Col-1 and α-smooth muscle actin in pleural thickening in the carbon-black bleomycin mouse model. These results support our hypothesis that KIF5A is responsible for collagen transportation and secretion from HPMCs.

TLR2 contributes to trigger immune response of pleural mesothelial cells against Mycobacterium bovis BCG and M. tuberculosis infection

Mycobacterium tuberculosis is a causative agent leading to pleural effusion, characterized by the accumulation of fluid and immune cells in the pleural cavity. Although this phenomenon has been described before, detailed processes or mechanisms associated with the pleural effusion are still not well understood. Pleural mesothelial cells (PMCs) are specialized epithelial cells that cover the body wall and internal organs in pleural cavity playing a central role in pleural inflammation. Toll-like receptors are expressed in various cell types including mesothelial cells and initiate the recognition and defense against mycobacterial infection. In the present study, we investigated direct immune responses of PMCs against two mycobacterial strains, M. bovis vaccine strain Bacille Calmette-Guérin (BCG) and M. tuberculosis virulent strain H37Rv, and the role of TLR2 in such responses. Infection with BCG and H37Rv increased the production of IL-6, CXCL1, and CCL2 in WT PMCs, which was partially impaired in TLR2-deficient cells. In addition, the activation of NF-κB and MAPKs induced by BCG and H37Rv was suppressed in TLR2-deficient PMCs, as compared with the WT cells. TLR2 deficiency led to the decrease of nitric oxide (NO) production through the delayed gene expression of iNOS in PMCs. TLR2 was also shown to be essential for optimal expression of cellular adhesion molecules such as ICAM-1 and VCAM-1 in PMCs in response to BCG and H37Rv. These findings strongly suggest that TLR2 participates in mycobacteria-induced innate immune responses in PMCs and may play a role in pathogenesis of tuberculosis pleural effusion.

Evidence for pleural epithelial-mesenchymal transition in murine compensatory lung growth.

In many mammals, including rodents and humans, removal of one lung results in the compensatory growth of the remaining lung; however, the mechanism of compensatory lung growth is unknown. Here, we investigated the changes in morphology and phenotype of pleural cells after pneumonectomy. Between days 1 and 3 after pneumonectomy, cells expressing α-smooth muscle actin (SMA), a cytoplasmic marker of myofibroblasts, were significantly increased in the pleura compared to surgical controls (p < .01). Scanning electron microscopy of the pleural surface 3 days post-pneumonectomy demonstrated regions of the pleura with morphologic features consistent with epithelial-mesenchymal transition (EMT); namely, cells with disrupted intercellular junctions and an acquired mesenchymal (rounded and fusiform) morphotype. To detect the migration of the transitional pleural cells into the lung, a biotin tracer was used to label the pleural mesothelial cells at the time of surgery. By post-operative day 3, image cytometry of post-pneumonectomy subpleural alveoli demonstrated a 40-fold increase in biotin+ cells relative to pneumonectomy-plus-plombage controls (p < .01). Suggesting a similar origin in space and time, the distribution of cells expressing biotin, SMA, or vimentin demonstrated a strong spatial autocorrelation in the subpleural lung (p < .001). We conclude that post-pneumonectomy compensatory lung growth involves EMT with the migration of transitional mesothelial cells into subpleural alveoli.

Silver nanoparticles alter the permeability of sheep pleura and of sheep and human pleural mesothelial cell monolayers

Nanoparticles have been implicated in the development of pleural effusions in exposed factory workers while in experimental animal studies it has been shown that they induce inflammation, fibrosis and carcinogenesis in the pleura. The scope of this study was to investigate the direct effects of silver nanoparticles exposure on the membrane permeability of sheep parietal pleura, of primary sheep pleural cell monolayers and on a human mesothelial cell line. Our findings suggest that acute (30min) exposure increases the pleural permeability ex vivo, while longer (24h) exposure in vivo leads to late decrease of the pleural cell monolayers permeability.

KIF5A is Responsible for Collagen Transport of Myofibroblasts during Pleural Fibrosis

Fibrosis is the production of extracellular matrix proteins in tissues and often proceeded by injuries. In pleural fibrosis excess collagen deposition occurs, which results in increased stiffness and thickening of pleura during tissue rearrangements. Myofibroblasts are responsible for oversecretion of collagen, however the molecular mechanism of transportation of procollagen containing vesicles for secretion is unknown. Here, we studied the role of kinesin on collagen-1 containing vesicle transportation in human pleural mesothelial cells (HPMCs). Among a number of cargo transporting kinesins, KIF5A was notably upregulated during TGF-β induced mesothelial-mesenchymal transition (MesoMT). Using superresolution structured illumination microscopy (SIM) and DUO-Link technique, we found KIF5A notably colocalizes with collagen-1 containing vesicles. KIF5A knock-down (KD) by specific siRNA significantly reduced collagen-1 secretion but not plasminogen activator inhibitor 1 (PAI-1). KIF5A KD notably attenuated TGF-β induced increase in collagen-1 localization at cell peripheries. Live cell imaging with GFP-KIF5A and mCherry-collagen-1 showed that KIF5A and collagen-1 containing vesicles moved together. Kymograph showed that these molecules continuously move with mean velocity of 0.56 μm/sec, suggesting that the movement is directional but not diffusion process. Moreover, KIF5A was notably upregulated along with collagen-1 and α-smooth muscle actin (α-SMA) (MesoMT marker) in thickening pleura of carbon-black bleomycin mouse model. All the results support that KIF5A is responsible for collagen transportation and secretion from HPMCs.

MiR-18a-5p Inhibits Sub-pleural Pulmonary Fibrosis by Targeting TGF-β Receptor II

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive lung disease that typically leads to respiratory failure and death within 3-5 years of diagnosis. Sub-pleural pulmonary fibrosis is a pathological hallmark of IPF. Bleomycin treatment of mice is a an established pulmonary fibrosis model. We recently showed that bleomycin-induced epithelial-mesenchymal transition (EMT) contributes to pleural mesothelial cell (PMC) migration and sub-pleural pulmonary fibrosis. MicroRNA (miRNA) expression has recently been implicated in the pathogenesis of IPF. However, changes in miRNA expression in PMCs and sub-pleural fibrosis have not been reported. Using cultured PMCs and a pulmonary fibrosis animal model, we found that miR-18a-5p was reduced in PMCs treated with bleomycin and that downregulation of miR-18a-5p contributed to EMT of PMCs. Furthermore, we determined that miR-18a-5p binds to the 3' UTR region of transforming growth factor β receptor II (TGF-βRII) mRNA, and this is associated with reduced TGF-βRII expression and suppression of TGF-β-Smad2/3 signaling. Overexpression of miR-18a-5p prevented bleomycin-induced EMT of PMC and inhibited bleomycin-induced sub-pleural fibrosis in mice. Taken together, our data indicate that downregulated miR-18a-5p mediates sub-pleural pulmonary fibrosis through upregulation of its target, TGF-βRII, and that overexpression of miR-18a-5p might therefore provide a novel approach to the treatment of IPF.

Heme Oxygenase-1 Deficiency Diminishes Methicillin-Resistant Staphylococcus aureus Clearance Due to Reduced TLR9 Expression in Pleural Mesothelial Cells

Methicillin Resistant Staphylococcus aureus (MRSA) cause pneumonia and empyema thoraces. TLR9 activation provides protection against bacterial infections and Heme oxygenase-1 (HO-1) is known to enhance host innate immunity against bacterial infections. However, it is still unclear whether HO-1 regulates TLR-9 expression in the pleura and modulates the host innate defenses during MRSA empyema. In order to determine if HO-1 regulates host innate immune functions via modulating TLR expression, in MRSA empyema, HO-1+/+ and HO-1-/- mouse pleural mesothelial cells (PMCs) were infected with MRSA (1:10, MOI) in the presence or absence of Cobalt Protoporphyrin (CoPP) and Zinc Protoporphyrin (ZnPP) or CORM-2 (a Carbon monoxide donor) and the expression of mTLR9 and mBD14 was assessed by RT-PCR. In vivo, HO-1+/+ and HO-1-/- mice were inoculated with MRSA (5x106 CFU) intra-pleurally and host bacterial load was measured by CFU, and TLR9 expression in the pleura was determined by histochemical-immunostaining. We noticed MRSA inducing differential expression of TLR9 in HO-1+/+ and HO-1 -/- PMCs. In MRSA infected HO-1+/+ PMCs, TLR1, TLR4, and TLR9 expression was several fold higher than MRSA infected HO-1-/- PMCs. Particularly TLR9 expression was very low in MRSA infected HO-1-/- PMCs both in vivo and in vitro. Bacterial clearance was significantly higher in HO-1+/+ PMCs than compared to HO-1-/- PMCs in vitro, and blocking TLR9 activation diminished MRSA clearance significantly. In addition, HO-1-/- mice were unable to clear the MRSA bacterial load in vivo. MRSA induced TLR9 and mBD14 expression was significantly high in HO-1+/+ PMCs and it was dependent on HO-1 activity. Our findings suggest that HO-1 by modulating TLR9 expression in PMCs promotes pleural innate immunity in MRSA empyema.

Pleural inhibition of the caspase-1/IL-1β pathway diminishes profibrotic lung toxicity of bleomycin.

BackgroundIdiopathic and toxic pulmonary fibrosis are severe diseases starting classically in the subpleural area of the lung. It has recently been suggested that pleural mesothelial cells acquire a myofibroblast phenotype under fibrotic conditions induced by TGF-β1 or bleomycin. The importance and role of inflammation in fibrogenesis are still controversial. In this work, we explored the role of IL-1β/caspase-1 signaling in bleomycin lung toxicity and in pleural mesothelial cell transformation.MethodsC57BL/6 mice were intravenously injected with either bleomycin or nigericin or NaCl as control. In vitro, the Met5A cell line was used as a model of human pleural mesothelial cells.ResultsIntravenous injections of bleomycin induced lung fibrosis with histologically-proven peripheral distribution, collagen accumulation in the pleural and subpleural area, and overexpression of markers of myofibroblast transformation of pleural cells which migrated into the lung. These events were associated with an inflammatory process with an increase in neutrophil recruitment in pleural lavage fluid and increased caspase-1 activity. TGF-β1 was also overexpressed in pleural lavage fluid and was produced by pleural cells following intravenous bleomycin. In this model, local pleural inhibition of IL-1β with the IL-1β inhibitor anakinra diminished TGF-β1 and collagen accumulation. In vitro, caspase-1 inhibition interfered with Met5A cell transformation into the myofibroblast-like phenotype induced by bleomycin or TGF-β1. Moreover, nigericin, a caspase-1 activator, triggered transformation of Met5A cells and its intra-pleural delivery induced fibrogenesis in mice.ConclusionsWe demonstrated, after intravenous bleomycin injection in mice, the role of the pleura and highlighted the key role of IL-1β/caspase-1 axis in this fibrogenesis process.Electronic supplementary materialThe online version of this article (doi:10.1186/s12931-016-0475-8) contains supplementary material, which is available to authorized users.

Quercetin and Cisplatin combined treatment altered cell cycle and mitogen activated protein kinase expressions in malignant mesotelioma cells.

BackgroundMalignant mesothelioma is a locally aggressive and highly lethal neoplasm of pleural, peritoneal and pericardial mesothelial cells without successful therapy. Previously, we reported that Quercetin in combination with Cisplatin inhibits cell proliferation and activates caspase-9 and -3 enzymes in different malignant mesothelioma cell lines. Moreover, Quercetin + Cisplatin lead to accumulation of both SPC111 and SPC212 cell lines in S phase.MethodsIn present work, 84 genes involved in cell growth and proliferation have analysed by using RT2-PCR array system and protein profile of mitogen activated protein kinase (MAPK) family proteins investigated by western blots.ResultsOur results showed that Quercetin and Quercetin + Cisplatin modulated gene expression of cyclins, cyclin dependent kinases and cyclin dependent kinases inhibitors. In addition genes involved in JNK, p38 and MAPK/ERK pathways were up regulated. Moreover, while p38 and JNK phosphorylations were increased, ERK phosphorylations were decreased after using Quercetin + Cisplatin.ConclusionThis research has clarified our previous results and detailed mechanism of anti-carcinogenic potential of Quercetin alone and incombination with Cisplatin on malignant mesothelioma cells.

Abstract 3892: Unique pleural vs peritoneal mesothelioma exosomal signature: does mesothelial cell susceptibility to asbestos matter

Abstract Malignant mesothelioma (MM) is a cancer of mesothelial cells of the pleura or peritoneum caused exclusively by asbestos exposure. It is a deadly cancer which is only diagnosed at terminal stages, possibly due to the long latency period of development and lack of biomarkers for early diagnosis. Both pleural and peritoneal mesothelial cells can develop MM in response to asbestos, however, pleural MM (85%) is more common than peritoneal MM (15%). To get an insight of this difference we first isolated exosomes from pleural (Hmeso, H2373) and peritoneal (ORT) MM cells and performed proteomic analysis. We found that 45 proteins are common and specific to MM cells derived from pleural cavity (Hmeso/H2373) while 86 proteins are specific to ORT cells lines derived from peritoneal cavity. On the other hand, the non-transformed mesothelial cell line, LP9, showed a completely different profile with 269 proteins secreted in exosomes not shared with MM models. This led us to hypothesize that mesothelial cells of two different locations may have different susceptibility to asbestos, accounting for differences observed in pleural and peritoneal MM. To test this hypothesis we exposed human primary pleural and peritoneal mesothelial cells to asbestos for 8 h, and performed massively parallel sequencing on the RNA. Gene expression analysis performed on the RNA-Seq data showed a higher magnitude of responses from pleural mesothelial cells as compared to peritoneal mesothelial cells for the same genes. In addition, at a p&amp;lt;0.05 and 2-fold threshold, unique genes expressed in pleural mesothelium were pro-inflammatory genes known to be involved in MM tumorigenesis. Taken together, results from our study suggest that exosomal cargo from pleural and peritoneal MM cells bear unique signatures which may be due to the different responses of pleural and peritoneal mesothelial cells to asbestos. Future directions involve assessing miRNA cargo from exosomes of pleural and peritoneal MM cells and identifying potential biomarker candidates. This work is supported by grants from NIH (RO1 ES021110), DoD (W81XWH-14-1-0199) and the University of Vermont Cancer Center/Lake Champlain Cancer Research Organization. Citation Format: Arti Shukla, Phillip Munson, Joyce K. Thompson, Julie Dragon, Maximilian B. MacPherson, Hector Peinado. Unique pleural vs peritoneal mesothelioma exosomal signature: does mesothelial cell susceptibility to asbestos matter. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3892.

Activation of calpain by renin-angiotensin system in pleural mesothelial cells mediates tuberculous pleural fibrosis.

Pleural fibrosis is defined as an excessive deposition of extracellular matrix (ECM) components that results in destruction of the normal pleural tissue architecture. It can result from diverse inflammatory conditions, especially tuberculous pleurisy. Pleural mesothelial cells (PMCs) play a pivotal role in pleural fibrosis. Calpain is a family of calcium-dependent endopeptidases, which plays an important role in ECM remodeling. However, the role of calpain in pleural fibrosis remains unknown. In the present study, we found that tuberculous pleural effusion (TPE) induced calpain activation in PMCs and that inhibition of calpain prevented TPE-induced collagen-I synthesis and cell proliferation of PMCs. Moreover, our data revealed that the levels of angiotensin (ANG)-converting enzyme (ACE) were significantly higher in pleural fluid of patients with TPE than those with malignant pleural effusion, and ACE-ANG II in TPE resulted in activation of calpain and subsequent triggering of the phosphatidylinositol 3-kinase (PI3K)/Akt/NF-κB signaling pathway in PMCs. Finally, calpain activation in PMCs and collagen depositions were confirmed in pleural biopsy specimens from patients with tuberculous pleurisy. Together, these studies demonstrated that calpain is activated by renin-angiotensin system in pleural fibrosis and mediates TPE-induced collagen-I synthesis and proliferation of PMCs via the PI3K/Akt/NF-κB signaling pathway. Calpain in PMCs might be a novel target for intervention in tuberculous pleural fibrosis.