Dr Michael Schuliga

Transforming growth factor-beta (TGF-ß) is a major mediator of fibrotic diseases, including idiopathic pulmonary fibrosis (IPF). However, therapeutic global inhibition of TGF-ß is limited by unwanted immunosuppression and mitral valve defects. We performed an extensive literature search to uncover a little-known connection between TGF-ß signaling and casein kinase (CK) activity. We have examined the abundance of CK1 delta and epsilon (CK1d/e) in lung tissue from IPF patients and non-diseased controls, and investigated whether inhibition of CK1d/e with PF670462 inhibits pulmonary fibrosis. CK1d/e levels in lung tissue from IPF patients and non-diseased controls were assessed by immunohistochemistry. Anti-fibrotic effects of the CK1d/e inhibitor PF670462 were assessed in pre-clinical models, including acute and chronic bleomycin mouse models and in vitro experiments on spheroids made from primary human lung fibroblast cells from IPF and control donors, and human A549 alveolar-like adenocarcinoma-derived epithelial cells. Increased expression of CK1d and e in IPF lungs compared to non-diseased controls was accompanied by increased levels of the product, phospho-period 2. In vitro, PF670462 prevented TGF-ß-induced epithelial-mesenchymal transition. The stiffness of IPF-derived spheroids was reduced by PF670462 and TGF-ß-induced fibrogenic gene expression was inhibited. The CK1d/e inhibitor PF670462 administered systemically or locally by inhalation prevented both acute and chronic bleomycin-induced pulmonary fibrosis in mice. PF670462 administered in a 'therapeutic' regimen (day 7 onward) prevented bleomycin-induced lung collagen accumulation. Elevated expression and activity of CK1d and e in IPF and anti-fibrogenic effects of the dual CK1d/e inhibitor, PF670462, support CK1d/e as novel therapeutic targets for IPF.

Idiopathic pulmonary fibrosis (IPF) is a chronic fibrosing interstitial pneu monia of unknown cause with a median survival of only three years. Little is known about the mechanisms that precede the excessive collagen deposition seen in IPF, but cellular senescence has been strongly implicated in disease pathology. Senescence is a state of irreversible cell-cycle arrest accompanied by an abnormal secretory profile and is thought to play a critical role in both development and wound repair. Normally, once a senescent cell has contributed to wound repair, it is promptly removed from the environment via infiltrating immune cells. However, if immune clearance fails, the persistence of senescent cells is thought to drive disease pathology through their altered secretory profile. One of the major cell types involved in wound healing is fibroblasts, and senescent fibroblasts have been identified in the lungs of patients with IPF and in fibroblast cultures from IPF lungs. The question of what is driving abnormally high numbers of fibroblasts into senescence remains unanswered. The transcription factor signal transducer and activator of transcription 3 (STAT3) plays a role in a myriad of processes, including cell-cycle progression, gene transcription, as well as mitochondrial respiration, all of which are dysregulated during senescence. Activation of STAT3 has previously been shown to correlate with IPF progression and therefore is a potential molecular target to modify early-stage senescence and restore normal fibroblast function. This review summarizes what is presently known about fibroblast senescence in IPF and how STAT3 may contribute to this phenotype.

Fibrosis causes irreversible damage to lung structure and function in restrictive lung diseases such as idiopathic pulmonary fibrosis (IPF). Extravascular coagulation involving fibrin formation in the intra-alveolar compartment is postulated to have a pivotal role in the development of pulmonary fibrosis, serving as a provisional matrix for migrating fibroblasts. Furthermore, proteases of the coagulation and plasminogen activation (plasminergic) systems that form and breakdown fibrin respectively directly contribute to pulmonary fibrosis. The coagulants, thrombin and factor Xa (FXa) evoke fibrogenic effects via cleavage of the N-terminus of protease-activated receptors (PARs). Whilst the formation and activity of plasmin, the principle plasminergic mediator is suppressed in the airspaces of patients with IPF, localized increases are likely to occur in the lung interstitium. Plasmin-evoked proteolytic activation of factor XII (FXII), matrix metalloproteases (MMPs) and latent, matrix-bound growth factors such as epidermal growth factor (EGF) indirectly implicate plasmin in pulmonary fibrosis. Another plasminergic protease, urokinase plasminogen activator (uPA) is associated with regions of fibrosis in the remodelled lung of IPF patients and elicits fibrogenic activity via binding its receptor (uPAR). Plasminogen activator inhibitor-1 (PAI-1) formed in the injured alveolar epithelium also contributes to pulmonary fibrosis in a manner that involves vitronectin binding. This review describes the mechanisms by which components of the two systems primarily involved in fibrin homeostasis contribute to interstitial fibrosis, with a particular focus on IPF. Selectively targeting the receptor-mediated mechanisms of coagulant and plasminergic proteases may limit pulmonary fibrosis, without the bleeding complications associated with conventional anti-coagulant and thrombolytic therapies.

In lung injury and disease, including idiopathic pulmonary fibrosis (IPF), extravascular factor X is converted into factor Xa (FXa), a coagulant protease with fibrogenic actions. Extracellular annexin A2 binds to FXa, augmenting activation of the protease-activated receptor-1 (PAR-1). In this study, the contribution of annexin A2 in lung injury and fibrosis was investigated. Annexin A2 immunoreactivity was observed in regions of fibrosis, including those associated with fibroblasts in lung tissue of IPF patients. Furthermore, annexin A2 was detected in the conditioned media and an EGTA membrane wash of human lung fibroblast (LF) cultures. Incubation with human plasma (5% vol/vol) or purified FXa (15¿50 nM) evoked fibrogenic responses in LF cultures, with FXa increasing interleukin-6 (IL-6) production and cell number by 270 and 46%, respectively (P < 0.05, n = 5¿8). The fibrogenic actions of plasma or FXa were attenuated by the selective FXa inhibitor apixaban (10 µM, or antibodies raised against annexin A2 or PAR-1 (2 µg/ml). FXastimulated LFs from IPF patients (n = 6) produced twice as much IL-6 as controls (n = 10) (P < 0.05), corresponding with increased levels of extracellular annexin A2. Annexin A2 gene deletion in mice reduced bleomycin-induced increases in bronchoalveolar lavage fluid (BALF) IL-6 levels and cell number (*P < 0.05; n = 4¿12). Lung fibrogenic gene expression and dry weight were reduced by annexin A2 gene deletion, but lung levels of collagen were not. Our data suggest that annexin A2 contributes to lung injury and fibrotic disease by mediating the fibrogenic actions of FXa. Extracellular annexin A2 is a potential target for the treatment of IPF.

During asthma exacerbation, plasma circulating coagulant factor X (FX) enters the inflamed airways and is activated (FXa). FXa may have an important role in asthma, being involved in thrombin activation and an agonist of protease-activated receptor-1 (PAR-1). Extracellular annexin A2 and integrins are also implicated in PAR-1 signaling. In this study, the potential role of PAR-1 in mediating the effects of FXa on human airway smooth muscle (ASM) cell cytokine production and proliferation was investigated. FXa (5-50 nM), but not FX, stimulated increases in ASM IL-6 production and cell number after 24- and 48-hour incubation, respectively (P < 0.05; n = 5). FXa (15 nM) also stimulated increases in the levels of mRNA for cytokines (IL-6), cell cycle-related protein (cyclin D1), and proremodeling proteins (FGF-2, PDGF-B, CTGF, SM22, and P AI-1) after 3-hour incubation (P < 0.05; n = 4). The actions of FXa were insensitive to inhibition by hirudin (1 U/ml), a selective thrombin inhibitor, but were attenuated by SCH79797 (100 nM), a PAR-1 antagonist, or Cpd 22 (1 µM), an inhibitor of integrin-linked kinase. The selective targeting of PAR-1, annexin A2, or ß 1-integrin by small interfering RNA and/or by functional blocking antibodies also attenuated FXa-evoked responses. In contrast, the targeting of annexin A2 did not inhibit thrombin-stimulated ASM function. In airway biopsies of patients with asthma, FXa and annexin A2 were detected in the ASM bundle by immunohistochemistry. These findings establish FXa as a potentially important asthma mediator, stimulating ASM function through actions requiring PAR-1 and annexin A2 and involving integrin coactivation.

Pulmonary inflammation and tissue remodelling are common elements of chronic respiratory diseases such as asthma, chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), and pulmonary hypertension (PH). In disease, pulmonary mesenchymal cells not only contribute to tissue remodelling, but also have an important role in pulmonary inflammation. This review will describe the immunomodulatory functions of pulmonary mesenchymal cells, such as airway smooth muscle (ASM) cells and lung fibroblasts, in chronic respiratory disease. An important theme of the review is that pulmonary mesenchymal cells not only respond to inflammatory mediators, but also produce their own mediators, whether pro-inflammatory or pro-resolving, which influence the quantity and quality of the lung immune response. The notion that defective pro-inflammatory or pro-resolving signalling in these cells potentially contributes to disease progression is also discussed. Finally, the concept of specifically targeting pulmonary mesenchymal cell immunomodulatory function to improve therapeutic control of chronic respiratory disease is considered.

In asthma, basic fibroblast growth factor (FGF-2) plays an important (patho)physiological role. This study examines the effects of FGF-2 on the transforming growth factor-b (TGF-b)-stimulated differentiation of airway smooth muscle (ASM) cells in vitro. The differentiation of humanASMcells after incubation with TGF-b(100 pM)and/ or FGF-2 (300 pM) for 48 hours was assessed by increases in contractile protein expression, actin-cytoskeleton reorganization, enhancements in cell stiffness, and collagen remodeling. FGF-2 inhibited TGF-ß-stimulated increases in transgelin (SM22) and calponin gene expression (n = 15, P < 0.01) in an extracellular signal-regulated kinase 1/2 (ERK1/2) signal transduction-dependent manner. The abundance of ordered a-smooth muscle actin (a-SMA) filaments formed in the presence of TGF-b were also reduced by FGF-2, as was the ratio of F-actin to G-actin (n = 8, P < 0.01). Furthermore, FGF-2 attenuated TGF-ß-stimulated increases in ASM cell stiffness andtheASM-mediatedcontraction of lattices,composed of collagen fibrils (n = 5, P < 0.01). However, the TGF-ß-stimulated production of IL-6 was not influenced by FGF-2 (n=4, P.>.05), suggesting that FGF-2 antagonism is selective for the regulation of ASM cell contractile protein expression, organization, and function. Another mitogen, thrombin (0.3 U ml21), exerted no effect on TGF-ß-regulated contractile protein expression (n=8, P.>.05),a-SMA organization, or the ratio of F-actin to G-actin (n=4, P.>.05), suggesting that the inhibitory effect of FGF-2 is dissociated from its mitogenic actions. The addition of FGF-2, 24 hours after TGF-b treatment, still reduced contractile protein expression, even when the TGF-ß-receptor kinase inhibitor, SB431542 (10 mM), was added 1 hour before FGF-2. Weconclude that the ASM cell differentiation promoted by TGF-b is antagonized by FGF-2. A better understanding of the mechanism of action for FGF-2 is necessary to develop a strategy for therapeutic exploitation in the treatment of asthma. Copyright © 2013 by the American Thoracic Society.

Background and Purpose The conversion of plasminogen into plasmin by interstitial urokinase plasminogen activator (uPA) is potentially important in asthma pathophysiology. In this study, the effect of uPA-mediated plasminogen activation on airway smooth muscle (ASM) cell proliferation was investigated. Experimental Approach Human ASM cells were incubated with plasminogen (0.5-50 µg·mL-1) or plasmin (0.5-50 mU·mL-1) in the presence of pharmacological inhibitors, including UK122, an inhibitor of uPA. Proliferation was assessed by increases in cell number or MTT reduction after 48 h incubation with plasmin(ogen), and by earlier increases in [ 3H]-thymidine incorporation and cyclin D1 expression. Key Results Plasminogen (5 µg·mL-1)-stimulated increases in cell proliferation were attenuated by UK122 (10 µM) or by transfection with uPA gene-specific siRNA. Exogenous plasmin (5 mU·mL-1) also stimulated increases in cell proliferation. Inhibition of plasmin-stimulated ERK1/2 or PI3K/Akt signalling attenuated plasmin-stimulated increases in ASM proliferation. Furthermore, pharmacological inhibition of cell signalling mediated by the EGF receptor, a receptor trans-activated by plasmin, also reduced plasmin(ogen)-stimulated cell proliferation. Knock down of annexin A2, which has dual roles in both plasminogen activation and plasmin-signal transduction, also attenuated ASM cell proliferation following incubation with either plasminogen or plasmin. Conclusions and Implications Plasminogen stimulates ASM cell proliferation in a manner mediated by uPA and involving multiple signalling pathways downstream of plasmin. Targeting mediators of plasminogen-evoked ASM responses, such as uPA or annexin A2, may be useful in the treatment of asthma. © 2013 The British Pharmacological Society.

Plasminogen has a role in airway inflammation. Airway smooth muscle(ASM) cells cleave plasminogen into plasmin, a protease with proinflammatory activity. In this study, the effect of plasminogen on cytokine production by human ASM cells was investigated in vitro. Levels of IL-6 and IL-8 in the medium of ASM cells were increased byincubation with plasminogen (5-50 µg/ml) for 24 hours (P,0.05; n = 6-9), corresponding to changes in the levels of cytokine mRNA at 4 hours. The effects of plasminogen were attenuated by a2- antiplasmin (1 mg/ml), a plasmin inhibitor (P < 0.05; n = 6-12). Exogenous plasmin (5-15 mU/ml) also stimulated cytokine production (P < 0.05; n = 6-8) in a manner sensitive to serine-protease inhibition by aprotinin (10 KIU/ml). Plasminogen-stimulated cytokine production was increased in cells pretreated with basic fibroblast growth factor (300 pM) in a manner associated with increases in urokinase plasminogen activator expression and plasmin formation. The knockdown of annexin A2, a component of the putative plasminogen receptor comprised of annexin A2 and S100A10, attenuated plasminogen conversion into plasmin and plasmin-stimulated cytokine production byASMcells. Moreover, a role for annexin A2 in airway inflammation was demonstrated in annexin A2-/- mice in which antigen-induced increases in inflammatory cell number and IL-6 levels in the bronchoalveolar lavage fluid were reduced (P < 0.01; n = 10-14). In conclusion, plasminogen stimulates ASM cytokine production in a manner regulated by annexin A2. Our study shows for the first time that targeting annexin A2-mediated signaling may provide a novel therapeutic approach to the treatment of airway inflammation in diseases such as chronic asthma. Copyright © 2013 by the American Thoracic Society.

The plasminogen activation system (PAS) and the plasmin it forms have dual roles in chronic respiratory diseases including asthma, chronic obstructive pulmonary disease and interstitial lung disease. Whilst plasmin-mediated airspace fibrinolysis is beneficial, interstitial plasmin contributes to lung dysfunction because of its pro-inflammatory and tissue remodeling activities. Recent studies highlight the potential of fibrinolytic agents, including small molecule inhibitors of plasminogen activator inhibitor-1 (PAI-1), as treatments for chronic respiratory disease. Current data also suggest that interstitial urokinase plasminogen activator is an important mediator of lung inflammation and remodeling. However, further preclinical characterization of uPA as a drug target for lung disease is required. Here we review the concept of selectively targeting the contributions of PAS to treat chronic respiratory disease. © 2013 Elsevier Ltd. All rights reserved.