Myocardin regulates fibronectin expression and secretion from human pleural mesothelial cells.

During the progression of pleural fibrosis, pleural mesothelial cells (PMCs) undergo a phenotype switching process known as mesothelial-mesenchymal transition (MesoMT). During MesoMT, transformed PMCs become myofibroblasts that produce increased extracellular matrix (ECM) proteins, including collagen and fibronectin (FN1) that is critical to develop fibrosis. Here, we studied the mechanism that regulates FN1 expression in myofibroblasts derived from human pleural mesothelial cells (HPMCs). We found that myocardin (Myocd), a transcriptional coactivator of serum response factor (SRF) and a master regulator of smooth muscle and cardiac muscle differentiation, strongly controls FN1 gene expression. Myocd gene silencing markedly inhibited FN1 expression. FN1 promoter analysis revealed that deletion of the Smad3-binding element diminished FN1 promoter activity, whereas deletion of the putative SRF-binding element increased FN1 promoter activity. Smad3 gene silencing decreased FN1 expression, whereas SRF gene silencing increased FN1 expression. Moreover, SRF competes with Smad3 for binding to Myocd. These results indicate that Myocd activates FN1 expression through Smad3, whereas SRF inhibits FN1 expression in HPMCs. In HPMCs, TGF-ß induced Smad3 nuclear localization, and the proximity ligation signal between Myocd and Smad3 was markedly increased after TGF-ß stimulation at nucleus, suggesting that TGF-ß facilitates nuclear translocation of Smad3 and interaction between Smad3 and Myocd. Moreover, Myocd and Smad3 were coimmunoprecipitated and isolated Myocd and Smad3 proteins directly bound each other. Chromatin immunoprecipitation assays revealed that Myocd interacts with the FN1 promoter at the Smad3-binding consensus sequence. The results indicate that Myocd regulates FN1 gene activation through interaction and activation of the Smad3 transcription factor.NEW & NOTEWORTHY During phenotype switching from mesothelial to mesenchymal, pleural mesothelial cells (PMCs) produce extracellular matrix (ECM) proteins, including collagen and fibronectin (FN1), critical components in the development of fibrosis. Here, we found that myocardin, a transcriptional coactivator of serum response factor (SRF), strongly activates FN1 expression through Smad3, whereas SRF inhibits FN1 expression. This study provides insights about the regulation of FN1 that could lead to the development of novel interventional approaches to prevent pleural fibrosis.

Role of ZIP kinase in development of myofibroblast differentiation from HPMCs.

During the development of pleural fibrosis, pleural mesothelial cells (PMCs) undergo phenotypic switching from differentiated mesothelial cells to mesenchymal cells (MesoMT). Here, we investigated how external stimuli such as TGF-ß induce HPMC-derived myofibroblast differentiation to facilitate the development of pleural fibrosis. TGF-ß significantly increased di-phosphorylation but not mono-phosphorylation of myosin II regulatory light chain (RLC) in HPMCs. An increase in RLC di-phosphorylation was also found at the pleural layer of our carbon black bleomycin (CBB) pleural fibrosis mouse model, where it showed filamentous localization that coincided with alpha smooth muscle actin (αSMA) in the cells in the pleura. Among the protein kinases that can phosphorylate myosin II RLC, ZIPK (zipper-interacting kinase) protein expression was significantly augmented after TGF-ß stimulation. Furthermore, ZIPK gene silencing attenuated RLC di-phosphorylation, suggesting that ZIPK is responsible for di-phosphorylation of myosin II in HPMCs. Although TGF-ß significantly increased the expression of ZIP kinase protein, the change in ZIP kinase mRNA was marginal, suggesting a posttranscriptional mechanism for the regulation of ZIP kinase expression by TGF-ß. ZIPK gene knockdown (KD) also significantly reduced TGF-ß-induced upregulation of αSMA expression. This finding suggests that siZIPK attenuates myofibroblast differentiation of HPMCs. siZIPK diminished TGF-ß-induced contractility of HPMCs consistent with siZIPK-induced decrease in the di-phosphorylation of myosin II RLC. The present results implicate ZIPK in the regulation of the contractility of HPMC-derived myofibroblasts, phenotype switching, and myofibroblast differentiation of HPMCs.NEW & NOTEWORTHY Here, we highlight that ZIP kinase is responsible for di-phosphorylation of myosin light chain, which facilitates stress fiber formation and actomyosin-based cell contraction during mesothelial to mesenchymal transition in human pleural mesothelial cells. This transition has a significant impact on tissue remodeling and subsequent stiffness of the pleura. This study provides insight into a new therapeutic strategy for the treatment of pleural fibrosis.

The mTORC2/SGK1/NDRG1 Signaling Axis Is Critical for the Mesomesenchymal Transition of Pleural Mesothelial Cells and the Progression of Pleural Fibrosis.

Progressive lung scarring because of persistent pleural organization often results in pleural fibrosis (PF). This process affects patients with complicated parapneumonic pleural effusions, empyema, and other pleural diseases prone to loculation. In PF, pleural mesothelial cells undergo mesomesenchymal transition (MesoMT) to become profibrotic, characterized by increased expression of α-smooth muscle actin and matrix proteins, including collagen-1. In our previous study, we showed that blocking PI3K/Akt signaling inhibits MesoMT induction in human pleural mesothelial cells (HPMCs) (1). However, the downstream signaling pathways leading to MesoMT induction remain obscure. Here, we investigated the role of mTOR complexes (mTORC1/2) in MesoMT induction. Our studies show that activation of the downstream mediator mTORC1/2 complex is, likewise, a critical component of MesoMT. Specific targeting of mTORC1/2 complex using pharmacological inhibitors such as INK128 and AZD8055 significantly inhibited transforming growth factor ß (TGF-ß)-induced MesoMT markers in HPMCs. We further identified the mTORC2/Rictor complex as the principal contributor to MesoMT progression induced by TGF-ß. Knockdown of Rictor, but not Raptor, attenuated TGF-ß-induced MesoMT in these cells. In these studies, we further show that concomitant activation of the SGK1/NDRG1 signaling cascade is essential for inducing MesoMT. Targeting SGK1 and NDRG1 with siRNA and small molecular inhibitors attenuated TGF-ß-induced MesoMT in HPMCs. Additionally, preclinical studies in our Streptococcus pneumoniae-mediated mouse model of PF showed that inhibition of mTORC1/2 with INK128 significantly attenuated the progression of PF in subacute and chronic injury. In conclusion, our studies demonstrate that mTORC2/Rictor-mediated activation of SGK1/NDRG1 is critical for MesoMT induction and that targeting this pathway could inhibit or even reverse the progression of MesoMT and PF.

Myo5b Transports Fibronectin-Containing Vesicles and Facilitates FN1 Secretion from Human Pleural Mesothelial Cells.

Pleural mesothelial cells (PMCs) play a central role in the progression of pleural fibrosis. As pleural injury progresses to fibrosis, PMCs transition to mesenchymal myofibroblast via mesothelial mesenchymal transition (MesoMT), and produce extracellular matrix (ECM) proteins including collagen and fibronectin (FN1). FN1 plays an important role in ECM maturation and facilitates ECM-myofibroblast interaction, thus facilitating fibrosis. However, the mechanism of FN1 secretion is poorly understood. We report here that myosin 5b (Myo5b) plays a critical role in the transportation and secretion of FN1 from human pleural mesothelial cells (HPMCs). TGF-ß significantly increased the expression and secretion of FN1 from HPMCs and facilitates the close association of Myo5B with FN1 and Rab11b. Moreover, Myo5b directly binds to GTP bound Rab11b (Rab11b-GTP) but not GDP bound Rab11b. Myo5b or Rab11b knockdown via siRNA significantly attenuated the secretion of FN1 without changing FN1 expression. TGF-ß also induced Rab11b-GTP formation, and Rab11b-GTP but not Rab11b-GDP significantly activated the actin-activated ATPase activity of Myo5B. Live cell imaging revealed that Myo5b- and FN1-containing vesicles continuously moved together in a single direction. These results support that Myo5b and Rab11b play an important role in FN1 transportation and secretion from HPMCs, and consequently may contribute to the development of pleural fibrosis.

Calponin 1 contributes to myofibroblast differentiation of human pleural mesothelial cells.

Pleural mesothelial cells (PMCs) can become myofibroblasts via mesothelial-mesenchymal transition (MesoMT) and contribute to pleural organization, fibrosis, and rind formation. However, how these transformed mesothelial cells contribute to lung fibrosis remains unclear. Here, we investigated the mechanism of contractile myofibroblast differentiation of PMCs. Transforming growth factor-ß (TGF-ß) induced marked upregulation of calponin 1 expression, which was correlated with notable cytoskeletal rearrangement in human PMCs (HPMCs) to produce stress fibers. Downregulation of calponin 1 expression reduced stress fiber formation. Interestingly, induced stress fibers predominantly contain α-smooth muscle actin (αSMA) associated with calponin 1 but not ß-actin. Calponin 1-associated stress fibers also contained myosin II and α-actinin. Furthermore, focal adhesions were aligned with the produced stress fibers. These results suggest that calponin 1 facilitates formation of stress fibers that resemble contractile myofibrils. Supporting this notion, TGF-ß significantly increased the contractile activity of HPMCs, an effect that was abolished by downregulation of calponin 1 expression. We infer that differentiation of HPMCs to contractile myofibroblasts facilitates stiffness of scar tissue in pleura to promote pleural fibrosis (PF) and that upregulation of calponin 1 plays a central role in this process.

DOCK2 Promotes Pleural Fibrosis by Modulating Mesothelial to Mesenchymal Transition.

Mesothelial to mesenchymal transition (MesoMT) is one of the crucial mechanisms underlying pleural fibrosis, which results in restrictive lung disease. DOCK2 (dedicator of cytokinesis 2) plays important roles in immune functions; however, its role in pleural fibrosis, particularly MesoMT, remains unknown. We found that amounts of DOCK2 and the MesoMT marker α-SMA (α-smooth muscle actin) were significantly elevated and colocalized in the thickened pleura of patients with nonspecific pleuritis, suggesting the involvement of DOCK2 in the pathogenesis of MesoMT and pleural fibrosis. Likewise, data from three different pleural fibrosis models (TGF-ß [transforming growth factor-ß], carbon black/bleomycin, and streptococcal empyema) consistently demonstrated DOCK2 upregulation and its colocalization with α-SMA in the pleura. In addition, induced DOCK2 colocalized with the mesothelial marker calretinin, implicating DOCK2 in the regulation of MesoMT. Our in vivo data also showed that DOCK2-knockout mice were protected from Streptococcus pneumoniae-induced pleural fibrosis, impaired lung compliance, and collagen deposition. To determine the involvement of DOCK2 in MesoMT, we treated primary human pleural mesothelial cells with the potent MesoMT inducer TGF-ß. TGF-ß significantly induced DOCK2 expression in a time-dependent manner, together with α-SMA, collagen 1, and fibronectin. Furthermore, DOCK2 knockdown significantly attenuated TGF-ß-induced α-SMA, collagen 1, and fibronectin expression, suggesting the importance of DOCK2 in TGF-ß-induced MesoMT. DOCK2 knockdown also inhibited TGF-ß-induced Snail upregulation, which may account for its role in regulating MesoMT. Taken together, the current study provides evidence that DOCK2 contributes to the pathogenesis of pleural fibrosis by mediating MesoMT and deposition of neomatrix and may represent a novel target for its prevention or treatment.

TGF-ß regulation of the uPA/uPAR axis modulates mesothelial-mesenchymal transition (MesoMT).

Pleural fibrosis (PF) is a chronic and progressive lung disease which affects approximately 30,000 people per year in the United States. Injury and sustained inflammation of the pleural space can result in PF, restricting lung expansion and impairing oxygen exchange. During the progression of pleural injury, normal pleural mesothelial cells (PMCs) undergo a transition, termed mesothelial mesenchymal transition (MesoMT). While multiple components of the fibrinolytic pathway have been investigated in pleural remodeling and PF, the role of the urokinase type plasminogen activator receptor (uPAR) is unknown. We found that uPAR is robustly expressed by pleural mesothelial cells in PF. Downregulation of uPAR by siRNA blocked TGF-ß mediated MesoMT. TGF-ß was also found to significantly induce uPA expression in PMCs undergoing MesoMT. Like uPAR, uPA downregulation blocked TGF-ß mediated MesoMT. Further, uPAR is critical for uPA mediated MesoMT. LRP1 downregulation likewise blunted TGF-ß mediated MesoMT. These findings are consistent with in vivo analyses, which showed that uPAR knockout mice were protected from S. pneumoniae-mediated decrements in lung function and restriction. Histological assessments of pleural fibrosis including pleural thickening and α-SMA expression were likewise reduced in uPAR knockout mice compared to WT mice. These studies strongly support the concept that uPAR targeting strategies could be beneficial for the treatment of PF.

NOX1 Promotes Mesothelial-Mesenchymal Transition through Modulation of Reactive Oxygen Species-mediated Signaling.

Pleural organization may occur after empyema or complicated parapneumonic effusion and can result in restrictive lung disease with pleural fibrosis (PF). Pleural mesothelial cells (PMCs) may contribute to PF through acquisition of a profibrotic phenotype, mesothelial-mesenchymal transition (MesoMT), which is characterized by increased expression of α-SMA (α-smooth muscle actin) and other myofibroblast markers. Although MesoMT has been implicated in the pathogenesis of PF, the role of the reactive oxygen species and the NOX (nicotinamide adenine dinucleotide phosphate oxidase) family in pleural remodeling remains unclear. Here, we show that NOX1 expression is enhanced in nonspecific human pleuritis and is induced in PMCs by THB (thrombin). 4-Hydroxy-2-nonenal, an indicator of reactive oxygen species damage, was likewise increased in our mouse model of pleural injury. NOX1 downregulation blocked THB- and Xa (factor Xa)-mediated MesoMT, as did pharmacologic inhibition of NOX1 with ML-171. NOX1 inhibition also reduced phosphorylation of Akt, p65, and tyrosine 216-GSK-3ß, signaling molecules previously shown to be implicated in MesoMT. Conversely, ML-171 did not reverse established MesoMT. NOX4 downregulation attenuated TGF-ß- and THB-mediated MesoMT. However, NOX1 downregulation did not affect NOX4 expression. NOX1- and NOX4-deficient mice were also protected in our mouse model of Streptococcus pneumoniae-mediated PF. These data show that NOX1 and NOX4 are critical determinants of MesoMT.

Glycogen Synthase Kinase-3ß Inhibition with 9-ING-41 Attenuates the Progression of Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease with a median survival of 3 years after diagnosis. Although the etiology of IPF is unknown, it is characterized by extensive alveolar epithelial cell apoptosis and proliferation of myofibroblasts in the lungs. While the origins of these myofibroblast appear to be diverse, fibroblast differentiation contributes to expansion of myofibroblasts and to disease progression. We found that agents that contribute to neomatrix formation and remodeling in pulmonary fibrosis (PF); TGF-ß, Factor Xa, thrombin, plasmin and uPA all induced fibroblast/myofibroblast differentiation. These same mediators enhanced GSK-3ß activation via phosphorylation of tyrosine-216 (p-Y216). Inhibition of GSK-3ß signaling with the novel inhibitor 9-ING-41 blocked the induction of myofibroblast markers; α-SMA and Col-1 and reduced morphological changes of myofibroblast differentiation. In in vivo studies, the progression of TGF-ß and bleomycin mediated PF was significantly attenuated by 9-ING-41 administered at 7 and 14 days respectively after the establishment of injury. Specifically, 9-ING-41 treatment significantly improved lung function (compliance and lung volumes; p < 0.05) of TGF-ß adenovirus treated mice compared to controls. Similar results were found in mice with bleomycin-induced PF. These studies clearly show that activation of the GSK-3ß signaling pathway is critical for the induction of myofibroblast differentiation in lung fibroblasts ex vivo and pulmonary fibrosis in vivo. The results offer a strong premise supporting the continued investigation of the GSK-3ß signaling pathway in the control of fibroblast-myofibroblast differentiation and fibrosing lung injury. These data provide a strong rationale for extension of clinical trials of 9-ING-41 to patients with IPF.

Increased expression of plasminogen activator inhibitor-1 (PAI-1) is associated with depression and depressive phenotype in C57Bl/6J mice.

Plasminogen activator inhibitor 1 (PAI-1), which is elevated in numerous disease states, has been implicated as a stress-related protein involved in the pathogenesis of depression. We measured PAI-1 in the plasma of healthy and depressed individuals and assessed plasminogen activator (PA) expression and regulation by PAI-1 in cultured normal human astrocytes (NHA). Elevated plasma PAI-1 levels were found in depressed patients. Brain tissues from depressed individuals also showed stronger expression of hippocampal PAI-1 by confocal imaging in comparison to healthy individuals. Using a lipopolysaccharide-induced inflammatory model of depression in mice, we measured PAI-1 in murine plasma and brain, by ELISA and immunohistochemistry, respectively. Similar elevations were seen in plasma but not in brain homogenates of mice exposed to LPS. We further correlated the findings with depressive behavior. Ex vivo experiments with NHA treated with proinflammatory cytokines implicated in the pathogenesis of depression showed increased PAI-1 expression. Furthermore, these studies suggest that urokinase-type plasminogen activator may serve as an astrocyte PA reservoir, able to promote cleavage of brain-derived neurotrophic factor (BDNF) during stress or inflammation. In summary, our findings confirm that derangements of PAI-1 variably occur in the brain in association with the depressive phenotype. These derangements may impede the availability of active, mature (m)BDNF and thereby promote a depressive phenotype.

Myocardin Is Involved in Mesothelial-Mesenchymal Transition of Human Pleural Mesothelial Cells.

Pleural fibrosis is characterized by severe inflammation of the pleural space and pleural reorganization. Subsequent thickening of the visceral pleura contributes to lung stiffness and impaired lung function. Pleural mesothelial cells (PMCs) can become myofibroblasts via mesothelial-mesenchymal transition (MesoMT) and contribute to pleural organization, fibrosis, and rind formation. However, the mechanisms that underlie MesoMT remain unclear. Here, we investigated the role of myocardin in the induction of MesoMT. Transforming growth factor ß (TGF-ß) and thrombin induced MesoMT and markedly upregulated the expression of myocardin, but not myocardin-related transcription factor A (MRTF-A) or MRTF-B, in human PMCs (HPMCs). TGF-ß stimulation notably induced the nuclear translocation of myocardin in HPMCs, whereas nuclear translocation of MRTF-A and MRTF-B was not observed. Several genes under the control of myocardin were upregulated in cells undergoing MesoMT, an effect that was accompanied by a dramatic cytoskeletal reorganization of HPMCs consistent with a migratory phenotype. Myocardin gene silencing blocked TGF-ß- and thrombin-induced MesoMT. Although myocardin upregulation was blocked, MRTF-A and MRTF-B were unchanged. Myocardin, α-SMA, calponin, and smooth muscle myosin were notably upregulated in the thickened pleura of carbon black/bleomycin and empyema mouse models of fibrosing pleural injury. Similar results were observed in human nonspecific pleuritis. In a TGF-ß mouse model of pleural fibrosis, PMC-specific knockout of myocardin protected against decrements in lung function. Further, TGF-ß-induced pleural thickening was abolished by PMC-specific myocardin knockout, which was accompanied by a marked reduction of myocardin, calponin, and α-SMA expression compared with floxed-myocardin controls. These novel results show that myocardin participates in the development of MesoMT in HPMCs and contributes to the pathogenesis of pleural organization and fibrosis.

Inhibition of Glycogen Synthase Kinase 3ß 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 in vitro. These same stimuli enhance glycogen synthase kinase (GSK)-3ß activity through increased phosphorylation of Tyr-216 in pleural mesothelial cells and GSK-3ß mobilization from the cytoplasm to the nucleus. GSK-3ß down-regulation blocked induction of MesoMT. Likewise, GSK-3ß 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-3ß as a newly identified target for amelioration of empyema-related pleural fibrosis and provide a strong rationale for further investigation of GSK-3ß signaling in the control of MesoMT and pleural injury.

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-ß 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-ß 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.

Organizing empyema induced in mice by Streptococcus pneumoniae: effects of plasminogen activator inhibitor-1 deficiency.

BACKGROUND: Pleural infection affects about 65,000 patients annually in the US and UK. In this and other forms of pleural injury, mesothelial cells (PMCs) undergo a process called mesothelial (Meso) mesenchymal transition (MT), by which PMCs acquire a profibrogenic phenotype with increased expression of α-smooth muscle actin (α-SMA) and matrix proteins. MesoMT thereby contributes to pleural organization with fibrosis and lung restriction. Current murine empyema models are characterized by early mortality, limiting analysis of the pathogenesis of pleural organization and mechanisms that promote MesoMT after infection. METHODS: A new murine empyema model was generated in C57BL/6 J mice by intrapleural delivery of Streptococcus pneumoniae (D39, 3 × 10(7)-5 × 10(9) cfu) to enable use of genetically manipulated animals. CT-scanning and pulmonary function tests were used to characterize the physiologic consequences of organizing empyema. Histology, immunohistochemistry, and immunofluorescence were used to assess pleural injury. ELISA, cytokine array and western analyses were used to assess pleural fluid mediators and markers of MesoMT in primary PMCs. RESULTS: Induction of empyema was done through intranasal or intrapleural delivery of S. pneumoniae. Intranasal delivery impaired lung compliance (p < 0.05) and reduced lung volume (p < 0.05) by 7 days, but failed to reliably induce empyema and was characterized by unacceptable mortality. Intrapleural delivery of S. pneumoniae induced empyema by 24 h with lung restriction and development of pleural fibrosis which persisted for up to 14 days. Markers of MesoMT were increased in the visceral pleura of S. pneumoniae infected mice. KC, IL-17A, MIP-1ß, MCP-1, PGE2 and plasmin activity were increased in pleural lavage of infected mice at 7 days. PAI-1(-/-) mice died within 4 days, had increased pleural inflammation and higher PGE2 levels than WT mice. PGE2 was induced in primary PMCs by uPA and plasmin and induced markers of MesoMT. CONCLUSION: To our knowledge, this is the first murine model of subacute, organizing empyema. The model can be used to identify factors that, like PAI-1 deficiency, alter outcomes and dissect their contribution to pleural organization, rind formation and lung restriction.

Tissue factor expression by myeloid cells contributes to protective immune response against Mycobacterium tuberculosis infection.

Tissue factor (TF) is a transmembrane glycoprotein that plays an essential role in hemostasis by activating coagulation. TF is also expressed by monocytes/macrophages as part of the innate immune response to infections. In the current study, we determined the role of TF expressed by myeloid cells during Mycobacterium tuberculosis (M. tb) infection by using mice lacking the TF gene in myeloid cells (TF(Δ) ) and human monocyte derived macrophages (MDMs). We found that during M. tb infection, a deficiency of TF in myeloid cells was associated with reduced inducible nitric oxide synthase (iNOS) expression, enhanced arginase 1 (Arg1) expression, enhanced IL-10 production and reduced apoptosis in infected macrophages, which augmented M. tb growth. Our results demonstrate that a deficiency of TF in myeloid cells promotes M2-like phenotype in M .tb infected macrophages. A deficiency in TF expression by myeloid cells was also associated with reduced fibrin deposition and increased matrix metalloproteases (MMP)-2 and MMP-9 mediated inflammation in M. tb infected lungs. Our studies demonstrate that TF expressed by myeloid cells has newly recognized abilities to polarize macrophages and to regulate M. tb growth.

Mesomesenchymal transition of pleural mesothelial cells is PI3K and NF-κB dependent.

Pleural organization follows acute injury and is characterized by pleural fibrosis, which may involve the visceral and parietal pleural surfaces. This process affects patients with complicated parapneumonic pleural effusions, empyema, and other pleural diseases prone to pleural fibrosis and loculation. Pleural mesothelial cells (PMCs) undergo a process called mesothelial mesenchymal transition (MesoMT), by which PMCs acquire a profibrotic phenotype characterized by cellular enlargement and elongation, increased expression of α-smooth muscle actin (α-SMA), and matrix proteins including collagen-1. Although MesoMT contributes to pleural fibrosis and lung restriction in mice with carbon black/bleomycin-induced pleural injury and procoagulants and fibrinolytic proteases strongly induce MesoMT in vitro, the mechanism by which this transition occurs remains unclear. We found that thrombin and plasmin potently induce MesoMT in vitro as does TGF-ß. Furthermore, these mediators of MesoMT activate phosphatidylinositol-3-kinase (PI3K)/Akt and NF-κB signaling pathways. Inhibition of PI3K/Akt signaling prevented TGF-ß-, thrombin-, and plasmin-mediated induction of the MesoMT phenotype exhibited by primary human PMCs. Similar effects were demonstrated through blockade of the NF-κB signaling cascade using two distinctly different NF-κB inhibitors, SN50 and Bay-11 7085. Conversely, expression of constitutively active Akt-induced mesenchymal transition in human PMCs whereas the process was blocked by PX866 and AKT8. Furthermore, thrombin-mediated MesoMT is dependent on PAR-1 expression, which is linked to PI3K/Akt signaling downstream. These are the first studies to demonstrate that PI3K/Akt and/or NF-κB signaling is critical for induction of MesoMT.

Thrombin down-regulates tissue factor pathway inhibitor expression in a PI3K/nuclear factor-κB-dependent manner in human pleural mesothelial cells.

Tissue factor pathway inhibitor (TFPI) is the primary inhibitor of the extrinsic coagulation cascade, and its expression is reported to be relatively stable. Various pathophysiologic agents have been shown to influence TFPI activity by regulating its expression or by modifying the protein. It is not clear how TFPI activity is regulated in normal physiology or in injury. Because thrombin and TFPI are locally elaborated in pleural injury, we sought to determine if thrombin could regulate TFPI in human pleural mesothelial cells (HPMCs). Thrombin significantly decreased TFPI mRNA and protein levels by > 70%. Thrombin-mediated down-regulation of TFPI promoted factor X activation by HPMCs. The ability of thrombin to significantly decrease TFPI mRNA and protein levels was maintained at nanomolar concentrations. Protease-activated receptor (PAR)-1, a mediator of thrombin signaling, is detectable in the mesothelium in human and murine pleural injury. PAR-1 silencing blocked thrombin-mediated decrements of TFPI in HPMCs. Thrombin activates PI3K/Akt and nuclear factor κB (NF-κB) signaling in HPMCs. Inhibition of PI3K (by PX-866) and NF-κB (by SN50) prevented thrombin-mediated TFPI mRNA and protein down-regulation. These are the first studies to demonstrate that thrombin decreases TFPI expression in HPMCs. Our findings demonstrate a novel mechanism by which thrombin regulates TFPI expression in HPMCs and promotes an unrestricted procoagulant response, and suggest that interactions between PI3K and NF-κB signaling pathways are linked in HPMCs and control TFPI expression. These findings raise the possibility that targeting this pathway could limit the ability of the mesothelium to support extravascular fibrin deposition and organization associated with pleural injury.

Plasminogen activator inhibitor-1 deficiency augments visceral mesothelial organization, intrapleural coagulation, and lung restriction in mice with carbon black/bleomycin-induced pleural injury.

Local derangements of fibrin turnover and plasminogen activator inhibitor (PAI)-1 have been implicated in the pathogenesis of pleural injury. However, their role in the control of pleural organization has been unclear. We found that a C57Bl/6j mouse model of carbon black/bleomycin (CBB) injury demonstrates pleural organization resulting in pleural rind formation (14 d). In transgenic mice overexpressing human PAI-1, intrapleural fibrin deposition was increased, but visceral pleural thickness, lung volumes, and compliance were comparable to wild type. CBB injury in PAI-1(-/-) mice significantly increased visceral pleural thickness (P < 0.001), elastance (P < 0.05), and total lung resistance (P < 0.05), while decreasing lung compliance (P < 0.01) and lung volumes (P < 0.05). Collagen, α-smooth muscle actin, and tissue factor were increased in the thickened visceral pleura of PAI-1(-/-) mice. Colocalization of α-smooth muscle actin and calretinin within pleural mesothelial cells was increased in CBB-injured PAI-1(-/-) mice. Thrombin, factor Xa, plasmin, and urokinase induced mesothelial-mesenchymal transition, tissue factor expression, and activity in primary human pleural mesothelial cells. In PAI-1(-/-) mice, D-dimer and thrombin-antithrombin complex concentrations were increased in pleural lavage fluids. The results demonstrate that PAI-1 regulates CBB-induced pleural injury severity via unrestricted fibrinolysis and cross-talk with coagulation proteases. Whereas overexpression of PAI-1 augments intrapleural fibrin deposition, PAI-1 deficiency promotes profibrogenic alterations of the mesothelium that exacerbate pleural organization and lung restriction.