DRUG DEVELOPMENT RESEARCH 60:29–35 (2003) DDR

Research Overview Proteinase-Activated Receptors and the Pathophysiology of Pulmonary Fibrosis Rachel C. Chambersn Centre for Respiratory Research, University College London, London, England

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ABSTRACT Interest in the role of proteinase-activated receptors (PARs) in the pathogenesis of fibrotic lung disease was fuelled by two central observations: first, the recognition that the cascade is activated in a number of these disorders; and second, the long-standing observation that , a principal regulator of coagulation, is a potent mitogen for fibroblasts, the key cell type responsible for the production of interstitial collagens in the fibrotic lung. This article will briefly review this evidence and focus on recent studies suggesting an important role for PAR1-mediated cellular responses in the pathophysiology of lung fibrosis. Drug Dev. Res. 60:29–35, 2003. c 2003 Wiley-Liss, Inc.

Key words: PARs; pulmonary fibrosis; bleomycin

ACTIVATION OF THE COAGULATION CASCADE IN recruited inflammatory cells into the injured lung FIBROTIC LUNG DISEASE injury, are thought to play a major role [reviewed in Pulmonary fibrosis is not a single disease but Chambers and Laurent, 1997]. In addition to classic represents the end stage of a heterogeneous group of fibrogenic mediators, it is increasingly recognized that disorders, of known and unknown cause, in which the coagulation cascade may also play an important role excessive deposition of collagen and other extracellular in fuelling the fibrotic response. Indeed, activation of matrix proteins within the pulmonary interstitium leads the coagulation cascade, which is manifest as either to progressive loss of lung function. There are currently increased thrombin levels, tissue factor–factor VIIa no adequate therapies, and it has been estimated that complexes or tissue factor expression and the resultant there are more than 3,000 deaths a year in the United extravascular deposition of fibrin, is a characteristic Kingdom from the most common form, cryptogenic feature of a number of respiratory diseases associated fibrosing alveolitis (CFA; also known as idiopathic with excessive deposition of connective tissue proteins. pulmonary fibrosis, IPF) alone [Hubbard et al., 1996]. These diseases include CFA/IPF [Imokawa et al., The pathogenesis of lung fibrosis remains incompletely 1997], pulmonary fibrosis associated with systemic understood, but current hypotheses propose that sclerosis [Ohba et al., 1994; Hernandez Rodriguez epithelial and/or endothelial injury leads to an ex- et al., 1995], chronic lung disease of prematurity [Dik aggerated fibroproliferative response during which fibroblasts transdifferentiate, proliferate, and increase Contract grant sponsors: the Wellcome Trust and the Medical Research Council, United Kingdom. extracellular matrix protein production. n In terms of the mediators involved in driving the Correspondence to: Rachel C. Chambers, Centre for Respiratory Research, University College London, 5 University fibrotic response, several potent fibrogenic cytokines Street, London WC1E 6JJ UK, England. and growth factors, including transforming growth E-mail: [email protected] factor-b (TGF-b) and platelet-derived growth factor Published online in Wiley InterScience (www.interscience. (PDGF), released from resident lung cells and wiley.com) DOI: 10.1002/ddr.10317

c 2003 Wiley-Liss, Inc. 30 CHAMBERS et al., 2003], and cryptogenic organising pneumonia role for this receptor in the pathogenesis of lung [Peyrol et al., 1990]. Extensive interstitial and intraal- fibrosis. veolar deposition of fibrin is also a characteristic feature in acute lung injury/adult respiratory distress ACTIVATION OF THE COAGULATION CASCADE IN syndrome (ARDS), in which rapid fibroproliferation EXPERIMENTALLY INDUCED LUNG FIBROSIS and matrix synthesis can lead to the development of The notion that inappropriate activation of the extensive fibrotic lesions. In the latter condition, coagulation cascade contributes to lung fibrosis is excessive procoagulant activity is mainly attributable supported by studies of animal models of lung injury to tissue factor–factor VII/VIIa complexes associated and fibrosis. The most widely used is that induced after with alveolar macrophages, in the face of reduced intratracheal instillation of the antineoplastic agent, levels of endogenous anticoagulants, including antith- bleomycin, to mice or rats. Instillation of this agent into rombin III, , and protein S [recently reviewed the lung mimics some of the features of human fibrotic in Idell, 2003]. In patients with IPF, reduced protein C lung disease in that it leads to acute lung injury, plasma activity is further associated with abnormal collagen exudation, recruitment of inflammatory cells (predo- turnover in the intraalveolar space [Yasui et al., 2000]. minantly , lymphocytes and macrophages), and eventually activation and proliferation of fibro- blasts and fibrosis. Intraalveolar and parenchymal fibrin ACTIVATORS OF PARs IN LUNG FIBROSIS formation is initiated by tissue factor [Olman et al., All four currently known PAR receptors are 1995] and active coagulation proteinases associated expressed in the lung, and collectively the proteinases with alveolar macrophages and epithelial cells, as well of the coagulation cascade can target all of them [see as fibroblasts within fibroproliferative foci [Howell et Riewald and Ruf, this Special Issue of Drug Develop- al., 2001]. Immunohistochemical studies performed in ment Research 59(4):400]. Thrombin is considered to our laboratory further revealed that PAR1 and active be one of the major physiologic activators of PAR1 [Vu thrombin colocalise to alveolar macrophages in inflam- et al., 1991]. Factor Xa, which is immediately upstream matory and fibroproliferative foci, as well as to of thrombin in the coagulation cascade, can activate interstitial spindle-shaped fibroblasts (Fig. 1). A direct PAR1 and PAR2, depending on the cell type, whereas causal role for the coagulation cascade in the tissue factor–factor VIIa complexes activate PAR2 pathogenesis of lung fibrosis in this model was [Bono et al., 2000; Riewald et al., 2001]. Transient provided by research performed in our laboratory ternary tissue factor–factor VIIa-Xa complexes activate and other laboratories that demonstrated that , PAR1 and PAR2 with greater efficiency than the activated protein C, or a direct inhibitor of thrombin individual proteinases, indicating that tissue factor– proteolytic activity attenuated the fibrotic response factor VIIa initiated coagulation in the vasculature is obtained [Piguet et al., 1996; Yasui et al., 2000; Howell inseparably linked to PAR1 and PAR2 activation and et al., 2001]. cell signalling [Riewald and Ruf, 2001]. More recently, CONTRIBUTION OF THE PROCOAGULANT VERSUS PAR1 has also been shown to be activated by protein C bound to its receptor EPCR on endothelial cells PAR-MEDIATED CELLULAR EFFECTS IN LUNG [Riewald et al., 2002] and by , a proteinase of FIBROSIS the fibrinolytic pathway [Pendurthi et al., 2000]. Other One of the most interesting challenges for major physiologic activators of PAR2 that may be researchers working in this area centers on establishing relevant to lung fibrosis include [Belham et al., the relative contribution of the procoagulant (e.g., 1996], [Akers et al., 2000], membrane-type fibrin generation) versus the PAR-mediated cellular serine proteinase-1 (MT-SP-1) [Takeuchi et al., 2000], effects of coagulation proteinases in these models and and proteinase 3 [Uehara et al., 2002]. The in human fibrotic lung disease. Early mechanistic physiologic functions of PAR3, which acts as a studies focussing on the contribution of fibrin to both for thrombin signalling [Nakanishi-Matsui et al., 2000], acute lung injury and progression to fibrosis provided and of PAR4, a more general serine proteinase evidence that fibrin and fibrinogen degradation pro- receptor, are also coming to light. Recent evidence ducts can influence tissue inflammation and repair by obtained in vitro suggests that PAR4, which is also acting as a provisional structural matrix and a source of activated by G [Sambrano et al., 2000], may fibrogenic mediators for infiltrating fibroblasts and play a role in lung inflammatory responses [Asoka- inflammatory cells [recently reviewed in Idell, 2003]. nanthan et al., 2002]. However, the primary focus of Studies performed in genetically modified mice this article will be on discussing the potential role of in which the fibrinolytic capacity of the lung was PAR1, because there is growing in vivo evidence for a either up- or downregulated by knocking-out or PARs AND PULMONARY FIBROSIS 31

Fig. 1. Thrombin and proteinase-activated receptor 1 (PAR1) are localised to the same cell types in bleomycin-induced lung fibrosis. a and b: Normal rat lung stained for active thrombin at low ( Â 400) and high power ( Â 1,000). There is only very weak staining associated with alveolar macrophages. c ( Â 400) and d ( Â 1,000): Corresponding section for thrombin from rats with bleomycin-induced lung fibrosis. Thrombin is localised to macrophages in inflammatory and fibroproliferative foci, as well as to spindle-shaped fibroblasts. e ( Â 400) and f ( Â 1,000): Localisation of PAR1 in the normal lung. The lung parenchyma is negative but the bronchial epithelium is positive. g and h: Corresponding sections for PAR1 from rats with bleomycin-induced fibrosis. As observed for thrombin, PAR1 is localised to macrophages in inflammatory and fibroproliferative foci, as well as to spindle-shaped fibroblasts. Reprinted with permission from Howell et al., 2001. The figure is presented in grey scale. For a color version of this figure, please go to: http://www.inflammation-calgary.com 32 CHAMBERS overexpressing inhibitor-1 pro- have been shown to promote both lung collagen vided further in vivo support to the notion that fibrin production and cell proliferation in vitro [Cairns and persistence promotes lung collagen accumulation Walls, 1997; Akers et al., 2000]. However, in our [Eitzman et al., 1996]. However, recent reports that experience with cultured lung fibroblasts, PAR2- fibrinogen knockout mice are not protected in this mediated fibroblast mitogenic responses are much model [Hattori et al., 2000] suggest that although reduced compared with the potent mitogenic effects extravascular fibrin contributes to lung inflammation obtained with PAR1 agonists [Blanc-Brude et al., and dysfunction in response to acute lung injury, it may unpublished communication]. Finally, although the promote but is not required for experimentally induced receptor systems remain to be established, thrombin lung fibrosis. is also a chemoattractant for lung fibroblasts [Dawes et In terms of the importance of PAR-mediated al., 1993] and may, therefore, also contribute to the cellular effects of coagulation proteinases, there are pathogenesis of lung fibrosis by recruiting fibroblasts to several potential pathways by which coagulation sites of lung injury from neighbouring environments. proteinases may contribute to the pathogenesis of lung fibrosis. These will now be considered in further detail. PROFIBROTIC EFFECTS OF PAR1 ACTIVATION ARE MEDIATED VIA SECONDARY GROWTH FACTORS ROLE OF PAR1 IN INFLUENCING LUNG FIBROBLAST Current evidence suggests that the profibrotic FUNCTION effects of thrombin and factor Xa are not mediated Extensive in vitro studies using selective PAR1 after PAR1 activation directly but rather via the activating peptides and cells derived from PAR1- induction of secondary mediators. For example, the deficient mice have provided compelling evidence that mitogenic effects of thrombin for lung fibroblasts are PAR1 is the major receptor by which thrombin exerts mediated via both the induction of PDGF-AA and its potent stimulatory effects on lung fibroblast upregulation of the PDGF a-receptor [Ohba et al., proliferation [Trejo et al., 1996] and extracellular 1994]. Thrombin is also a mitogen for endothelial cells, matrix production [Chambers et al., 1998]. The and a novel thrombin response element has been mitogenic effects of thrombin are likely to play an identified in the PDGF-B chain promoter important role in expanding the fibroblast population in [Scarpati and DiCorleto, 1996]. More, recently our the injured lung. In support of this notion, we have laboratory and others have shown that PAR1 activation shown that the mitogenic potential of bronchoalveolar by thrombin and factor Xa leads to rapid and dramatic lavage fluid, obtained from patients with pulmonary induction of connective tissue growth factor (CTGF) fibrosis associated with systemic sclerosis, for cultured [Chambers et al., 2000; Pendhurti et al., 2000]. CTGF lung fibroblasts was significantly reduced in the is a fibroblast mitogen, chemoattractant, and promoter presence of the thrombin inhibitor, hirudin [Hernan- of connective tissue deposition [reviewed in Groten- dez Rodriguez et al., 1995]. PAR1 is also the receptor dorst, 1997]. It is also induced in response to TGF-b, by which thrombin has more recently been shown to and there is evidence that it may mediate some of promote the transformation of fibroblasts into highly TGF-b’s potent profibrotic effects and act as a cofactor activated smooth muscle a-actin-positive myofibro- for TGF-b receptor binding and signalling [Abreu et blasts [Bogatkevich et al., 2001]Fthe predominant al., 2002]. Thrombin also induces the expression or fibroblast phenotype present in active fibrotic lesions. release of these fibrogenic mediators by other cell Recent unpublished data from our laboratory further types, including macrophages (PDGF-AA) [Tani et al., support a critical role for PAR1 in mediating the 1997] and epithelial cells (CTGF) [Riewald et al., stimulatory effects of factor Xa on fibroblast prolifera- 2001]. Both PDGF and CTGF levels are increased in tion and procollagen production [Blanc-Brude et al., the lungs of patients with fibrotic lung disease [Homma unpublished communication]. The importance of PAR1 et al., 1995; Allen et al., 1999], as well as in the in influencing fibroblast function and cell signalling in bleomycin model [Tani et al., 1997; Lasky et al., 1998], response to factor Xa is further supported by recent so that the induction of these growth factors after PAR1 studies from Shaun Coughlin’s laboratory showing that activation may contribute to the pathogenesis of this PAR1 accounts for virtually all factor Xa-induced condition via both autocrine and paracrine mechan- phosphoinositide hydrolysis in fibroblasts [Camerer et isms. Indirect in vivo support for CTGF as a potential al., 2002]. Although current evidence suggests that downstream mediator of thrombin’s profibrotic effects PAR1 is the major receptor by which coagulation was recently obtained in experiments performed in our proteinases influence fibroblast function, lung fibro- laboratory showing that the characteristic and coordi- blasts also express PAR2, and activation of PAR2 by nate increases in lung procollagen and CTGF mRNA mast cell tryptase, as well as selective PAR-2 agonists, levels in response to bleomycin injury were attenuated PARs AND PULMONARY FIBROSIS 33 in rats given a direct thrombin inhibitor [Howell et al., Confirmation that PAR1-mediated inflammatory 2002]. responses could be important in the setting of human disease was provided by the observation that PAR1- deficient mice are partially protected from experimen- ROLE OF PARs IN LUNG INFLAMMATION tally induced crescentic glomerulonephritis [Cunning- In addition to effects on fibroblast function, ham et al., 2000]. In contrast, in our bleomycin studies, activation of PAR1 by coagulation proteinases may although direct thrombin inhibition attenuated lung contribute to the development of lung fibrosis via other collagen accumulation in this model, inflammatory cell cell-mediated responses. As discussed above, increased recruitment was unaffected [Howell et al., 2001]. This pulmonary vascular permeability is a common feature does not exclude the possibility that once recruited, of both acute lung injury/ARDS and chronic fibrotic thrombin may induce inflammatory cells to release lung disease and is thought to be a major mechanism potent profibrotic mediators. As already mentioned, leading to leak of coagulation proteinases and fibrin thrombin and PAR1 are present on the surface of deposition in the extravascular space [reviewed in alveolar macrophages in the bleomycin model [Howell Idell, 2003]. Thrombin exerts potent stimulatory et al., 2001 and Figure 1] and thrombin has been effects on endothelial cell permeability and contraction reported to induce the release of PDGF by cultured in vitro, and direct intravenous infusion of thrombin alveolar macrophages isolated from bleomycin-treated has been shown to increase pulmonary vascular rats [Tani et al., 1997]. Whether this is PAR mediated permeability in experimental models [reviewed in remains to be established. Siflinger-Birnboim and Johnson, 2003]. A critical role for PAR1 in mediating these effects was provided by SUMMARY AND CONCLUSIONS studies showing that thrombin-induced increase in pulmonary microvascular permeability is abrogated in There is increasing evidence that activation of the coagulation cascade with the resultant extravasation of lung organ cultures from PAR1 knockout mice [Vogel et al., 2000]. active coagulation proteinases into alveolar tissue An additional mechanism by which coagulation may play an important role in the pathogenesis of proteinases may contribute to tissue fibrosis is via their lung fibrosis. Studies in the bleomycin model ability to influence tissue inflammatory responses have provided some insights into the mechanism by [please see article by Vergnolle, this Special Issue of which coagulation proteinases may contribute to the Drug Development Research 59(4):375]. Thrombin disease process. Current evidence suggests an impor- induces the release of potent proinflammatory media- tant role for the PAR1-mediated profibrotic effects of tors implicated in the pathogenesis of pulmonary thrombin in this model: (1) the lack of protection of fibrosis, including, amongst others, monocyte chemo- fibrinogen-null mice, (2) no evidence of a role for tactic protein, interleukin (IL)-1b, IL-6, and IL-8, by a thrombin in inflammatory cell recruitment in this number of resident lung cells, including fibroblasts, model, and (3) the coordinate attenuation of CTGF epithelial cells, as well as monocytes/macrophages and and procollagen mRNA levels in rats given the vascular endothelial cells [Colotta et al., 1994; Sower et thrombin inhibitor. Studies confirming the importance al., 1995; Ueno et al., 1996; Ludwicka-Bradley et al., of PAR1 using PAR1-knockout mice and selective PAR1 2000; Naldini et al., 2002]. Thrombin further influ- antagonists are currently ongoing and should provide ences inflammatory cell trafficking by inducing the further valuable information on the potential of PAR1 expression of endothelial cell adhesion molecules, antagonists as novel antifibrotic agents. Strategies including P-selectin and ICAM-1 [Sugama et al., aimed at interfering with PAR1-mediated profibrotic 1992]. There is a wealth of in vitro evidence suggesting responses of coagulation proteinases would circumvent adverse hemostatic complications associated with that PAR1 is a major receptor involved in mediating the proinflammatory effects of thrombin [Sower et al., traditional anticoagulant therapy and will be of 1995; Ludwicka-Bradley et al., 2000; Naldini et al., particular appeal for use in critically ill patients (e.g., 2002]. 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