Human Pleural Mesothelial Cell Line Research Articles

VEGF/Src signaling mediated pleural barrier damage and increased permeability contributes to subpleural pulmonary fibrosis

The distribution of fibrosis in idiopathic pulmonary fibrosis (IPF) is subpleural with basal predominance. Alveolar epithelial cell was considered as the key cell in the initial phase of IPF. However, the idea of activation and damage of alveolar epithelial cells is very difficult to explain why fibrosis distributes in the subpleural area. In this study, human pleural mesothelial cell (PMC) line and primary rat PMC was used as in vitro model. Intraperitoneal injection of bleomycin was used for making a pulmonary fibrosis model. The integrity of cultured monolayer PMCs was determined by transepithelial electric resistance (TEER). Pleural permeability was estimated by measuring paracellular transport of fluorescein isothiocyanate (FITC)-conjugated dextran. Changes in lung tissue of patients with IPF were analyzed by Masson’s and immunofluorescence staining. We found bleomycin induced PMCs damage and increased PMCs permeability; increased PMCs permeability aggravated bleomycin-induced subpleural inflammation and pulmonary fibrosis. Moreover, bleomycin was found to activate VEGF/Src signaling which increased PMCs permeability. In vivo, inhibition of VEGF/Src signaling prevented bleomycin-induced subpleural pulmonary fibrosis. At last, activation of VEGF/Src signaling was confirmed in subpleural area in patients with IPF. Taken together, our findings indicate that VEGF/Src signaling mediated pleural barrier damage and increased permeability which contributes to subpleural pulmonary fibrosis.

Patient-derived and artificial ascites have minor effects on MeT-5A mesothelial cells and do not facilitate ovarian cancer cell adhesion.

The presence of ascites in the peritoneal cavity leads to morphological and functional changes of the peritoneal mesothelial cell layer. Cells loose cell-cell interactions, rearrange their cytoskeleton, activate the production of fibronectin, and change their cell surface morphology in a proinflammatory environment. Moreover, ovarian cancer cell adhesion has been shown to be facilitated by these changes due to increased integrin- and CD44-mediated binding sites. In this study, the biological responsiveness of the human pleural mesothelial cell line MeT-5A to patient-derived and artificial ascites was studied in vitro and adhesion of ovarian cancer cells, i.e. SKOV-3 cells, investigated. Changes were mainly observed in cells exposed to artificial ascites containing higher cytokine concentrations than patient-derived ascites. Interestingly, reduced cell-cell interactions were already observed in untreated MeT-5A cells and effects on tight junction protein expression and permeability upon exposure to ascites were minor. Ascites induced upregulation of CDC42 effector protein 2 expression, which affects stress fiber formation, however significant F-actin reorganization was not observed. Moreover, fibronectin production remained unchanged. Analysis of mesothelial cell surface characteristics showed upregulated expression of intercellular adhesion molecule 1, slightly increased hyaluronic acid secretion and decreased microvillus expression upon exposure to ascites. Nevertheless, the observed changes were not sufficient to facilitate adhesion of SKOV-3 cells on MeT-5A cell layer. This study revealed that MeT-5A cells show a reduced biological responsiveness to the presence of ascites, in contrast to published studies on primary human peritoneal mesothelial cells.

Transmembrane protein 88 inhibits transforming growth factor-β1-induced-extracellular matrix accumulation and epithelial-mesenchymal transition program in human pleural mesothelial cells through modulating TGF-β1/Smad pathway

Pleural fibrosis is an irreversible pathological process occurred in the development of several lung diseases. TMEM88 is a member of transmembrane (TMEM) family and has been found to be involved in the regulation of fibrogenesis. However, the role of TMEM88 in pleural fibrosis remains unknown. In this study, we aimed to explore the role of TMEM88 in pleural fibrosis in vitro using transforming growth factor-β1 (TGF-β1)-induced human pleural mesothelial cell line MeT-5A cells. Our results showed that the expression levels of TMEM88 were downregulated in pleural fibrosis tissues and TGF-β1-treated Met-5A cells. Overexpression of TMEM88 inhibited the proliferation of Met-5A cells under TGF-β1 stimulation. In addition, TMEM88 overexpression prevented TGF-β1-induced extracellular matrix (ECM) accumulation and epithelial-mesenchymal transition (EMT) in Met-5A cells with decreased expression levels of Col I and fibronectin, increased levels of cytokeratin-8 and E-cadherin, as well as decreased levels of vimentin and α-SMA. Furthermore, overexpression of TMEM88 inhibited the expression of TGF-β receptor I (TβRI) and TβRII and suppressed the phosphorylation of Smad2 and Smad3 in Met-5A cells. In conclusion, these results indicated that TMEM88 exhibited an anti-fibrotic activity in pleural fibrosis via inhibiting the activation of TGF-β1/Smad signaling pathway.

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.

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.

Co-Transcriptomes of Initial Interactions In Vitro between Streptococcus Pneumoniae and Human Pleural Mesothelial Cells.

Streptococcus pneumoniae (Spn) is a major causative organism of empyema, an inflammatory condition occurring in the pleural sac. In this study, we used human and Spn cDNA microarrays to characterize the transcriptional responses occurring during initial contact between Spn and a human pleural mesothelial cell line (PMC) in vitro. Using stringent filtering criteria, 42 and 23 Spn genes were up-and down-regulated respectively. In particular, genes encoding factors potentially involved in metabolic processes and Spn adherence to eukaryotic cells were up-regulated e.g. glnQ, glnA, aliA, psaB, lytB and nox. After Spn initial contact, 870 human genes were differentially regulated and the largest numbers of significant gene expression changes were found in canonical pathways for eukaryotic initiation factor 2 signaling (60 genes out of 171), oxidative phosphorylation (32/103), mitochondrial dysfunction (37/164), eIF4 and p70S6K signaling (28/142), mTOR signaling (27/182), NRF2-mediated oxidative stress response (20/177), epithelial adherens junction remodeling (11/66) and ubiquitination (22/254). The cellular response appeared to be directed towards host cell survival and defense. Spn did not activate NF-kB or phosphorylate p38 MAPK or induce cytokine production from PMC. Moreover, Spn infection of TNF-α pre-stimulated PMC inhibited production of IL-6 and IL-8 secretion by >50% (p<0.01). In summary, this descriptive study provides datasets and a platform for examining further the molecular mechanisms underlying the pathogenesis of empyema.

106P PROTEOMIC ANALYSIS SHOWED HEAT SHOCK 70 KDA PROTEIN AND 78 KDA GLUCOSE-REGULATED PROTEIN WERE UP-REGULATED IN HUMAN MALIGNANT PLEURAL MESOTHELIOMA CELLS

Malignant pleural mesothelioma (MPM) is a malignant neoplasm arising from mesothelial cells existing in pleura. It is difficult to identify a crucial symptom in patients with MPM, and there is no standard curative therapy for this malignancy. The incidence of MPM is increasing worldwide, and many patients can not survive more than one year after diagnosis. MPM is an asbestos-related disease, and it is believed that the incidence is closely related to the amount and time of exposure to asbestos. Since the latency period after exposure to asbestos is very long, the incidence may increase over the next two decades. Therefore, it is important to understand the biology of MPM, and to find a molecular target for its therapy. Three of human malignant pleural mesothelioma (MPM) cell lines (NCIH28, NCI-H2052, NCI-H2452) and a human pleural mesothelial cell line (MeT-5A) were analyzed by proteomics with the use of twodimensional gel electrophoresis (2-DE) and liquid chromatographytandem mass spectrometry (LC-MS/MS). From the results of 2-DE between MeT-5A and three MPM cell lines, 9 protein spots showed increased expression levels in MPM cells compared to MeT-5A. These spots were analyzed by LC-MS/MS analysis, and identified by a peptide sequence tag. Identified increased spots included 78 kDa glucose regulated protein (GRP78), heat shock 70 kDa protein (HSP70), protein disulfide isomerase A3, annexin A3, peroxiredoxin-2 and 14 kDa phosphohistidine phosphatase. Western blotting using specific antibodies against GRP78 and HSP70 confirmed the elevated expression level of GRP78 and HSP70 in MPM cell lines compared to MeT-5A cells. These results suggest that GRP78 and HSP70 expression is likely to be associated with the tumorigenesis of MPM, and they may be expectable as biomarkers or therapeutic targets for MPM. Disclosure: All authors have declared no conflicts of interest.

107P IMPACT OF PREINTERVENTIONAL PLASMA FIBRINOGEN AS A PROGNOSTIC MARKER IN MALIGNANT PLEURAL MESOTHELIOMA

Malignant pleural mesothelioma (MPM) is a malignant neoplasm arising from mesothelial cells existing in pleura. It is difficult to identify a crucial symptom in patients with MPM, and there is no standard curative therapy for this malignancy. The incidence of MPM is increasing worldwide, and many patients can not survive more than one year after diagnosis. MPM is an asbestos-related disease, and it is believed that the incidence is closely related to the amount and time of exposure to asbestos. Since the latency period after exposure to asbestos is very long, the incidence may increase over the next two decades. Therefore, it is important to understand the biology of MPM, and to find a molecular target for its therapy. Three of human malignant pleural mesothelioma (MPM) cell lines (NCIH28, NCI-H2052, NCI-H2452) and a human pleural mesothelial cell line (MeT-5A) were analyzed by proteomics with the use of twodimensional gel electrophoresis (2-DE) and liquid chromatographytandem mass spectrometry (LC-MS/MS). From the results of 2-DE between MeT-5A and three MPM cell lines, 9 protein spots showed increased expression levels in MPM cells compared to MeT-5A. These spots were analyzed by LC-MS/MS analysis, and identified by a peptide sequence tag. Identified increased spots included 78 kDa glucose regulated protein (GRP78), heat shock 70 kDa protein (HSP70), protein disulfide isomerase A3, annexin A3, peroxiredoxin-2 and 14 kDa phosphohistidine phosphatase. Western blotting using specific antibodies against GRP78 and HSP70 confirmed the elevated expression level of GRP78 and HSP70 in MPM cell lines compared to MeT-5A cells. These results suggest that GRP78 and HSP70 expression is likely to be associated with the tumorigenesis of MPM, and they may be expectable as biomarkers or therapeutic targets for MPM. Disclosure: All authors have declared no conflicts of interest.

Histone Deacetylase Inhibitor m-Carboxycinnamic Acid bis-Hydroxamide Attenuates Plasminogen Activator Inhibitor–1 Expression in Human Pleural Mesothelial Cells

Plasminogen activator inhibitor-1 (PAI-1), primarily up-regulated by transforming growth factor (TGF)-β, is essential in the development of fibrosis. Histone deacetylase (HDAC) was shown to modulate gene expression and fibrogenesis in various tissues. However, the implications of HDAC in terms of PAI-1 expression and pleural fibrosis remain unclear. In this study, we examined the effects of m-carboxycinnamic acid bis-hydroxamide (CBHA), a hybrid-polar HDAC inhibitor, on the TGF-β1-induced expression of PAI-1 in a human pleural mesothelial cell line (MeT-5A). MeT-5A cells were treated with TGF-β1 in the presence or absence of CBHA. We assayed the expression and stability of PAI-1 mRNA and protein, PAI-1 promoter activity, the activation of Smad signaling, the protein-protein interactions of Smads with transcriptional cofactors Sp1 and coactivator p300, and the expression of the mRNA-stabilizing protein nucleolin. The results indicate that CBHA significantly inhibited TGF-β1-induced PAI-1 mRNA and protein expression, and attenuated PAI-1 promoter activity in MeT-5A cells. CBHA abrogated TGF-β1-induced Smad4 nuclear translocation, but not Smad2/3 activation. Furthermore, the association of Smad4 with p300, but not with Sp1, was disrupted by CBHA. Alternatively, CBHA suppressed TGF-β1-induced nucleolin expression, and thereby destabilized PAI-1 mRNA and decreased PAI-1 protein concentrations. These findings suggest that the inhibition of HDAC activity by CBHA may attenuate PAI-1 expression through the modulation of cellular signaling at multiple levels. Given the down-regulating effect of CBHA on PAI-1 expression, HDAC inhibitors should be tested further in animal models as potential therapeutic agents for pleural fibrosis.

The Effect of Tissue Plasminogen Activator on TGF-β1 Pre-Treated Human Mesothelial Cell Line

Background: In an effort to find alternative therapeutic agents to prevent excessive fibrosis as a sequela to complicated parapneumonic effusion or empyema, we examined the effect of tissue plasminogen activator (tPA) as a fibrinolytic agent combined with talc or transforming growth factor (TGF)-β1 in a human pleural mesothelial cell line, MeT-5A. Methods: MeT-5A cells were stimulated with various doses of talc, doxycycline or TGF-β1 for 24 h and then were treated with tPA for an additional 24 h. Cell viability was measured by MTT assay. The production of interleukin (IL)-8 and vascular endothelial growth factor (VEGF) in the culture supernatants was measured by ELISA. Real-time PCR was carried out for measurement of type I collagen mRNA. Results: MeT-5A cells treated with talc showed a dose-dependent increase in production of IL-8. Talc also increased production of type I collagen mRNA at low doses, but talc did not influence the induction of VEGF. Addition of tPA to talc-stimulated cells showed further increases in the production of IL-8, but tPA did not influence the production of VEGF or type I collagen mRNA. TGF-β1 increased the production of both VEGF and collagen type I mRNA, both of which were effectively inhibited by additional tPA treatment in MeT-5A cells. Conclusion: TGF-β1 is a potent inducer of collagen synthesis without induction of IL-8 in MeT-5A cells. Addition of tPA after TGF-β1 stimulation inhibited further fibrosis by direct inhibition of collagen mRNA synthesis as well as by inhibition of VEGF production.

P66 Histone deacetylase inhibitor CBHA attenuates the expression of plasminogen activator inhibitor-1 in human pleural mesothelial cells

BackgroundPlasminogen activator inhibitor-1 (PAI-1), primarily up-regulated by transforming growth factor (TGF)-β, is essential for development of fibrosis. Histone deacetylases (HDACs) have been shown to modulate gene expression and fibrogenesis in...

Proteomic differential display analysis identified upregulated astrocytic phosphoprotein PEA‐15 in human malignant pleural mesothelioma cell lines

We performed proteomic differential display analysis of human malignant pleural mesothelioma (MPM) cell lines and a human pleural mesothelial cell line by using 2-DE and LC-MS/MS. The human MPM cell lines were NCI-H28, NCI-H2052 and NCI-H2452, and the human pleural mesothelial cell line was MeT-5A. Between MeT-5A and NCI-H2052, we found 38 protein spots whose expression levels were different, from the results of 2-DE; 28 protein spots appeared higher, and 10 other protein spots lower in NCI-H2052 than in MeT-5A. These spots were analyzed by LC-MS/MS analysis and identified by a peptide sequence tag. However, from the results of 2-DE of the other cell lines, there was only one consistently upregulated protein, astrocytic phosphoprotein PEA-15, in all three MPM cell lines. Western blotting using specific antibodies against PEA-15 confirmed the elevated expression level of PEA-15 in all three MPM cell lines compared with MeT-5A cells and normal pleura tissues from patients. PEA-15 was knocked down in NCI-H2052 cells, and the proliferation of PEA-15-silenced NCI-H2052 cells was suppressed 7-15% compared with negative control cells. These results suggest that PEA-15 expression is likely to be associated with the tumorigenesis of MPM.

Dynasore, a Dynamin Inhibitor, Induces PAI-1 Expression in MeT-5A Human Pleural Mesothelial Cells

Plasminogen activator inhibitor-1 (PAI-1) is a primary regulator of plasminogen activation that plays an essential role in regulating the physiological thrombotic/fibrinogenic balance. The elevation of PAI-1 expression by human pleural mesothelial cells has been reported to contribute to pleural fibrosis and pleurodesis. In this study, we examined the effects on PAI-1 expression of dynasore, a cell-permeable inhibitor of dynamin, and its mechanisms in a human pleural mesothelial cell line (MeT-5A). The results indicated that dynasore enhanced transforming growth factor (TGF)-beta(1)- and TNF-alpha-induced PAI-1 protein expression in a concentration-dependent manner. Furthermore, dynasore significantly up-regulated PAI-1 protein and its messenger RNA expressions. Interestingly, Smad2/3 activation was induced by TGF-beta(1) but not by dynasore. Among signaling inhibitors, a c-Jun NH(2)-terminal kinase (JNK) inhibitor (SP600125) markedly attenuated dynasore-stimulated PAI-1 protein production. Consistently, dynasore strongly increased JNK phosphorylation. On the other hand, there was no enhancement effect by dynasore on TGF-beta(1)-induced matrix metalloproteinase-2 activation. These findings suggest that dynasore may stimulate PAI-1 protein expression and enhance TGF-beta(1) activity through activation of JNK-mediated signaling in human pleural mesothelial cells. Given the profibrotic effect of dynasore, further in vivo studies may be conducted to evaluate its potential as a pleurodesing agent.

The pathogenetic role of immunoglobulin G from patients with systemic lupus erythematosus in the development of lupus pleuritis.

To investigate the pathophysiological effect of immunoglobulin G (IgG) from systemic lupus erythematosus (SLE) patients on pleural mesothelial cells and related mechanisms. Serum IgG from 28 lupus patients and 13 healthy controls was purified by protein-G affinity chromatography. The concentrations of anti-dsDNA-, anti-histone- and/or anti-nucleohistone-containing IgGs were determined by enzyme-linked immunosorbent assay (ELISA). Lupus patients were divided into an active (n = 12) and an inactive group (n = 16) on the basis of the SLE Disease Activity Index (SLEDAI). The binding of IgG to a human pleural mesothelial cell line (MeT-5A) under different conditions, including pretreatment with DNase and preincubation with exogenous histone, DNA or nucleohistone, was examined using flow cytometry and cellular ELISA. The effect of IgG on MeT-5A cell proliferation was studied using an MTT assay. Gene expression and protein synthesis for interleukin 1beta (IL-1beta), monocyte chemoattractant protein 1 (MCP-1) and transforming growth factor beta1 (TGF-beta1) in MeT-5A cells were determined using reverse transcription-polymerase chain reaction and ELISA. The binding of IgG to MeT-5A cells was higher in the active lupus group than the inactive lupus group (P = 0.047) and controls (P = 0.003). The binding decreased in both lupus groups following pretreatment of MeT-5A cells with DNase. The binding of IgG to MeT-5A cells was greater by 112% in the active lupus group after preincubation with histone (P < 0.001), but not with DNA or nucleohistone. Exposure of MeT-5A cells to IgG from either lupus group induced cell proliferation when compared with IgG from healthy controls (P = 0.04). Gene expression and protein synthesis of MCP-1, TGF-beta1 and IL-1beta in MeT-5A cells were significantly increased after incubation with IgG from patients with active lupus when compared with IgG from the inactive lupus and control groups (P < 0.01). The concentration of anti-dsDNA antibodies correlated with the binding of IgG to MeT-5A cells and the synthesis of cytokines by MeT-5A cells. The serum level of anti-histone antibodies in the active lupus group was higher than that in the inactive group (P = 0.015) and the serum concentration correlated with cell binding and MCP-1 production. IgG from lupus patients can bind to MeT-5A cells and the binding is modulated by DNA or histone. Binding of anti-dsDNA-containing IgG to MeT-5A cells induces the synthesis of proinflammatory cytokines. Our findings suggest that the binding of anti-dsDNA antibodies, particularly the IgG isotype, to pleural mesothelium plays a direct pathogenetic role in inducing inflammatory injury in the serositis of SLE.