Prostacyclin and its stable analogues are used to treat pulmonary arterial hypertension (PAH), a disease associated with abnormal smooth muscle cell (SMC) proliferation. These analogues are thought to mediate their anti proliferative effects through prostacyclin (IP) receptors linked to cyclic AMP (cAMP) generation, though other targets may be involved including peroxisome proliferator activated receptors (PPARs), transcription factors known to regulate cell growth. Thus the aim was to assess the role of the IP receptor and PPARy in mediating the anti-proliferative effects of prostacyclin analogues in HEK-293 cells stably expressing the IP receptor (HEK-293-IP) and in SMCs derived from normal and hypertensive lungs. In proliferation assays, the growth rate of HEK-293-IP cells was significantly decreased compared to cells expressing the empty vector. Furthermore, treprostinil and carbacyclin, and non receptor-dependent cAMP-elevating agents only inhibited proliferation in HEK-293-IP cells, suggesting the physical presence of the IP receptor is crucial in mediating the effects of both analogues and agents working downstream of the receptor. Protein kinase A and to a lesser extent, PPARy appear to be involved since antagonists of these two pathways partially reversed the anti-proliferative effects of treprostinil. Using a dual luciferase reporter gene assay, I demonstrated that analogues could activate PPARy via a novel IP receptor- dependent, cAMP-independent mechanism, likely to involve phosphorylation. In separate studies, pulmonary SMC derived from young idiopathic PAH patients replicated at a faster rate compared to control cells. RT-PCR and immnuostaining showed that PAH cells expressed fewer IP receptors, although treprostinil still inhibited SMC proliferation, albeit through a mechanism largely involving PPARy but not the IP receptor or cAMP. This contrasted to normal pulmonary SMC, where treprostinil inhibited proliferation via the IP receptor, cAMP and PPARy. In conclusion, the IP receptor appears to play an important role in regulating cell growth and mediating the effects of prostacyclin analogues in normal but not in pulmonary hypertensive SMC.

There is a high prevalence of co-morbid anxiety disorders in patients with chronic obstructive pulmonary disease (COPD). Although co-morbid anxiety impacts negatively upon health-related quality of life, physical functioning and healthcare utilisation, anxiety disorders remain significantly under recognised and undermanaged. One reason for this may be the lack of a validated disease-specific patient-reported screening tool and outcome measure. Existing scales may be limited by their inclusion of somatic items, which may overlap with symptoms of COPD or the side-effects of medications. This thesis aimed to develop a novel non-somatic self-report anxiety scale that can be used to screen for anxiety disorders and assess the severity of anxiety in patients with COPD. The Anxiety Inventory for Respiratory disease (AIR) was developed using a mixed methods approach to item development that incorporated both emic (interviews with 14 COPD patients) and etic (review of extant anxiety scales) perspectives to generate 16 novel items scored using a Likert-type response set. Patients and clinicians were involved in the development of the AIR to ensure that the scale is user-friendly and clinically relevant. Qualitative findings from the interviews also provide a unique insight into the experience of anxiety from the patients' perspective and support the non-somatic format of the AIR. The draft 16-item AIR was completed by 88 patients with COPD and refined through rigorous item and factor analysis. Six items were removed to create the final10-item AIR (score range 0-30). The reliability and validity of the AIR were examined in a sample of 56 COPD outpatients. The AIR proved to have excellent internal consistency in all phases (Cronbach's a = 0.92-0.95) and test-retest reliability (Intraclass correlation coefficient = 0.81). The AIR also demonstrated high convergent validity with the Hospital Anxiety and Depression Scale (Spearman's rho correlation = 0.91) and was able to discriminate between patients with and without anxiety disorders (p<O.OOl). Confirmatory factor analysis found that a two-factor model containing two intercorrelated factors (general anxiety and panic) provided the best fit. The AIR was able to accurately screen for anxiety disorders. The area under the curve (AUC) for the AIR based on the Patient Health Questionnaire anxiety screener was 0.96. A cut-off score of 15 produced a sensitivity of 0.93 and a specificity of 0.98. Although further research is required to validate the AIR in larger clinical populations, the findings presented in this thesis support the use of the scale as a reliable and valid marker of anxiety in patients with COPD. The AIR is also a promising screening tool for anxiety disorders, particularly panic disorder and generalised anxiety disorder in patients with COPD.

Although the pathogenesis of pulmonary fibrosis is incompletely understood, an accepted underlying molecular mechanism is the dysregulation of soluble mediators which regulate fibroblast function in the lung. One such mediator is prostaglandin E2 (PGE2), which is a potent inhibitor of fibroblast proliferation, collagen production, chemotaxis and myofibroblast differentiation. Levels of PGE2 have been shown to be decreased in bronchoalveolar lavage fluid (BALF) and lung fibroblasts from patients with pulmonary fibrosis. In lung fibroblasts this has been shown to be due to limited expression of the rate limiting enzyme in its biosynthesis, cyclooxygenase-2 (COX-2). However, there is currently no direct proof that limited expression of COX-2 and PGE2 contribute to the pathogenesis of pulmonary fibrosis. This thesis has used pharmacological and genetic inhibition of COX-2 to address the hypothesis that limited expression of COX-2 and PGE2 potentiates bleomycin-induced pulmonary fibrosis. Through the use of the highly selective COX-2 inhibitor NS398, this thesis demonstrates that bleomycin-induced PGE2 production in the lung is COX-2 mediated for at least 14 days following injury. However, administration of NS398 had no effect on the development of bleomycin-induced lung fibrosis in wild type (WT) mice. Unexpectedly, COX-2-/- mice showed a compensatory upregulation in PGE2 biosynthesis following bleomycin injury compared with both WT and COX-2-/- animals, which is evident in macrophage/monocytes but not fibroblasts derived from these mice. Lung homogenates showed increased expression of COX-1 in COX-2-/- mice compared with WT controls suggesting the compensatory synthesis is via increased expression of COX-1. COX-2-/- mice show an enhanced and persistent inflammatory response to bleomycin, however the fibrotic response to injury was unaltered compared with WT animals. In contrast, COX-2+/- mice showed reduced expression of COX-2 and subsequently limited induction of PGE2 following bleomycin injury. This resulted in an enhanced fibrotic response at day 28 with increased total lung collagen content compared with both WT and COX-2-/- mice (WT, 2.6 0.22; COX-2+/-, 3.77 0.12; COX-2-/-, 2.51 0.26 mg collagen/lung, p 0.001).

Pulmonary fibrosis is characterised by excessive deposition of extracellular matrix proteins within the lung parenchyma. Activation of the coagulation cascade occurs in this condition and thrombin levels are increased in bronchoalveolar lavage fluid (BALF) from patients with this disorder. In addition to its role in blood coagulation, thrombin exerts cellular effects, including promoting fibroblast proliferation, procollagen production and expression of connective tissue growth factor (CTGF) via activation of protease activated receptor-1 (PAR-1). This thesis examined the hypothesis that thrombin plays a role in promoting lung collagen accumulation in pulmonary fibrosis, via activation of PAR-1. To address this hypothesis, the effect of a direct thrombin inhibitor, UK-156406 on bleomycin-induced pulmonary fibrosis was examined in rats. In addition, the effect of bleomycin-induced pulmonary fibrosis in PAR-1 knockout (PAR-1-/-) and wild-type (WT) mice was evaluated. In rats, immunohistochemical expression of thrombin and PAR-1 were dramatically increased in the lung following bleomycin instillation, compared with saline-treated animals. Following bleomycin instillation, lung collagen doubled and was preceded by significant elevations in 1(I) procollagen and CTGF mRNA levels. In bleomycin-treated animals receiving an anticoagulant dose of UK-156046, lung collagen accumulation, 1(I) procollagen and CTGF mRNA levels were all significantly reduced. In WT mice given bleomycin, total lung collagen was increased but in bleomycin-instilled PAR-1-/- mice, lung collagen accumulation was significantly reduced biochemically and histologically. Furthermore, BALF total inflammatory cell number, total protein and CTGF mRNA levels were also significantly reduced in bleomycin-instilled PAR-1-/- mice compared with WT mice receiving bleomycin. In summary, this thesis shows that direct thrombin inhibition attenuates lung collagen accumulation in bleomycin-induced pulmonary fibrosis and also that PAR-1-/- mice are protected from bleomycin-induced lung injury. These data support the hypothesis that thrombin and PAR-1 play a critical role in experimental pulmonary fibrosis and that the pro-fibrotic effects of thrombin in this model, may be mediated, at least in part, via a CTGF-dependent mechanism.

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Pulmonary fibrosis is the end stage of a heterogeneous group of disorders, characterised by the excessive deposition of extracellular matrix proteins within the pulmonary interstitium. Current evidence suggests that the differentiation of fibroblasts into highly synthetic and contractile myofibroblasts plays a central role in the pathogenesis of pulmonary fibrosis. Uncontrolled coagulation activity with extravascular expression of tissue factor (TF) leading to the activation of coagulation zymogens, including FVII, FX and prothrombin, has been documented in the lungs of patients with pulmonary fibrosis. In addition to their role in blood clotting, coagulation proteinases induce multiple pro inflammatory and pro-fibrotic cellular effects via the activation of their major signalling receptors the PARs, including PAR!. Until recently, coagulation zymogens were thought to be exclusively derived from the circulation and then locally activated in response to tissue injury. This thesis shows for the first time that FX is locally upregulated in human and murine fibrotic lung tissue and that the alveolar epithelium represents a prominent cellular source of this protein. This thesis further reports that FXa is a potent inducer of the myofibroblast differentiation programme via PAR! and the transcriptional upregulation of thrombospondin-1 leading to the activation of TGF-P!. The work presented here further demonstrates that PAR1f TSP-1 and a-SMA co-localize to fibrotic foci in idiopathic pulmonary fibrosis and a direct causal link between FXa and the development of fibrosis was demonstrated by showing that a direct FXa inhibitor attenuated bleomycin-induced lung fibrosis. This thesis therefore identifies a novel pathogenic mechanism by which FXa, a central proteinase of the coagulation cascade, is locally upregulated and drives the fibrotic response to lung injury. These findings herald a paradigm shift in the current understanding of the tissue origin of excessive procoagulant activity and further place PAR! central to the crosstalk between pro-coagulant signalling and tissue remodelling.

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