Procoagulant Signalling In Lung Injury And Fibrosis

Coagulation proteinases, such as thrombin, exert a plethora of cellular effects via activation of proteinase-activated receptors (PARs). Abnormal activation of the coagulation cascade has been widely implicated in the pathology of fibroproliferative lung disease, including the most common form idiopathic pulmonary fibrosis (IPF). The high-affinity thrombin receptor PAR-1 is over-expressed in the lungs of IPF patients, particularly by the hyperplastic epithelium and fibroblasts within fibrotic foci. Moreover, viral infections are implicated in aberrant coagulation activation and accumulating evidence points to viruses as contributory factors to acute exacerbations that lead to deterioration of lung function in IPF patients. This thesis focuses on investigating and extending our current understanding of the role of PAR-1 in driving lung injury and fibrosis. The data show that primary human lung fibroblasts and microvascular endothelial cells express abundant PAR-1 and elicit robust calcium signalling responses following thrombin stimulation. In contrast, normal human alveolar and bronchial epithelial cells express low levels of PAR-1 but high levels of PAR-2. Exposure of the alveolar epithelial cell line, A549, to TGFβ increases PAR-1 expression and functional responses but this was not seen in freshly isolated primary human alveolar and bronchial epithelial cells. The role of TGFβ and pro-coagulant signalling responses was also investigated in an in vivo model of viral infection on a background of pulmonary fibrosis. Herpesvirus infection in the fibrotic lung promoted robust inflammatory responses characterized by extensive pulmonary consolidation but did not lead to an increase in lung collagen deposition. Importantly, viral infection was associated with the local upregulation of key components of the extrinsic coagulation pathway. Although PAR-1 is known to contribute to lung injury and fibrosis in a single hit experimental model of pulmonary fibrosis by promoting the activation of latent TGFβ, systemic targeting of TGFβ and the coagulation cascade does not impact on inflammation induced by viral infection in the fibrotic lung

Identification of an active metabolite of PAR-1 antagonist RWJ-58259 and synthesis of analogues to enhance its metabolic stability

The discontinuation of PAR-1 antagonist RWJ-58259 beyond use as a biological probe is most likely due to it's short half-life in vivo. However, retention of significant in vivo activity beyond the point where most of the RWJ-58259 had been consumed implies the generation of an active metabolite. Herein we describe the biological activity of a predicted metabolite of RWJ-58259 and the synthesis of analogues designed to enhance the metabolic stability of RWJ-58259

Synthesis of novel and potent vorapaxar analogues

Vorapaxar is a first-in-class PAR-1 antagonistic drug based on the ent-himbacine scaffold. Detailed in this article are enantioselective and racemic routes to various novel vorapaxar analogues. Biological testing revealed these compounds to have moderate to excellent potencies against PAR-1 with the most potent analogue demonstrating an IC50 of 27 nM

The anti-fibrotic effect of inhibition of TGFβ-ALK5 signalling in experimental pulmonary fibrosis in mice is attenuated in the presence of concurrent γ-herpesvirus infection.

Journal ArticleTGFβ-ALK5 pro-fibrotic signalling and herpesvirus infections have been implicated in the pathogenesis and exacerbation of pulmonary fibrosis. In this study we addressed the role of TGFβ-ALK5 signalling during the progression of fibrosis in a two-hit mouse model of murine γ-herpesvirus 68 (MHV-68) infection on the background of pre-existing bleomycin-induced pulmonary fibrosis. Assessment of total lung collagen levels in combination with ex vivo micro-computed tomography (µCT) analysis of whole lungs demonstrated that MHV-68 infection did not enhance lung collagen deposition in this two-hit model but led to a persistent and exacerbated inflammatory response. Moreover, µCT reconstruction and analysis of the two-hit model revealed distinguishing features of diffuse ground-glass opacities and consolidation superimposed on pre-existing fibrosis that were reminiscent of those observed in acute exacerbation of idiopathic pulmonary fibrosis (AE-IPF). Virally-infected murine fibrotic lungs further displayed evidence of extensive inflammatory cell infiltration and increased levels of CCL2, TNFα, IL-1β and IL-10. Blockade of TGFβ-ALK5 signalling attenuated lung collagen accumulation in bleomycin-alone injured mice, but this anti-fibrotic effect was reduced in the presence of concomitant viral infection. In contrast, inhibition of TGFβ-ALK5 signalling in virally-infected fibrotic lungs was associated with reduced inflammatory cell aggregates and increased levels of the antiviral cytokine IFNγ. These data reveal newly identified intricacies for the TGFβ-ALK5 signalling axis in experimental lung fibrosis, with different outcomes in response to ALK5 inhibition depending on the presence of viral infection. These findings raise important considerations for the targeting of TGFβ signalling responses in the context of pulmonary fibrosis.MRCNovartis CASE studentshi

TGFβ upregulates PAR-1 expression and signalling responses in A549 lung adenocarcinoma cells.

The major high-affinity thrombin receptor, proteinase activated receptor-1 (PAR-1) is expressed at low levels by the normal epithelium but is upregulated in many types of cancer, including lung cancer. The thrombin-PAR-1 signalling axis contributes to the activation of latent TGFβ in response to tissue injury via an αvβ6 integrin-mediated mechanism. TGFβ is a pleiotropic cytokine that acts as a tumour suppressor in normal and dysplastic cells but switches into a tumour promoter in advanced tumours. In this study we demonstrate that TGFβ is a positive regulator of PAR-1 expression in A549 lung adenocarcinoma cells, which in turn increases the sensitivity of these cells to thrombin signalling. We further demonstrate that this effect is Smad3-, ERK1/2- and Sp1-dependent. We also show that TGFβ-mediated PAR-1 upregulation is accompanied by increased expression of integrin αv and β6 subunits. Finally, TGFβ pre-stimulation promotes increased migratory potential of A549 to thrombin. These data have important implications for our understanding of the interplay between coagulation and TGFβ signalling responses in lung cancer.Medical Research Council UK (MRC) CASE studentship with Novartis awarded to RCC, MRC Centenary Award awarded to NS and RCC, and MRC Career Development Award G0800340 to CJS

Growth, entropy and commutativity of algebras satisfying prescribed relations

In 1964, Golod and Shafarevich found that, provided that the number of relations of each degree satisfy some bounds, there exist infinitely dimensional algebras satisfying the relations. These algebras are called Golod-Shafarevich algebras. This paper provides bounds for the growth function on images of Golod-Shafarevich algebras based upon the number of defining relations. This extends results from [32], [33]. Lower bounds of growth for constructed algebras are also obtained, permitting the construction of algebras with various growth functions of various entropies. In particular, the paper answers a question by Drensky [7] by constructing algebras with subexponential growth satisfying given relations, under mild assumption on the number of generating relations of each degree. Examples of nil algebras with neither polynomial nor exponential growth over uncountable fields are also constructed, answering a question by Zelmanov [40]. Recently, several open questions concerning the commutativity of algebras satisfying a prescribed number of defining relations have arisen from the study of noncommutative singularities. Additionally, this paper solves one such question, posed by Donovan and Wemyss in [8].Comment: arXiv admin note: text overlap with arXiv:1207.650

Author Correction: Abnormal contractility in human heart myofibrils from patients with dilated cardiomyopathy due to mutations in TTN and contractile protein genes

Correction to: Scientific Reportshttps://doi.org/10.1038/s41598-017-13675-8, published online 01 November 201

Examining the Theoretical Framework of Behavioral Activation for Major Depressive Disorder: Smartphone-Based Ecological Momentary Assessment Study

Background: Behavioral activation (BA), either as a stand-alone treatment or as part of cognitive behavioral therapy, has been shown to be effective for treating depression. The theoretical underpinnings of BA derive from Lewinsohn et al's theory of depression. The central premise of BA is that having patients engage in more pleasant activities leads to them experiencing more pleasure and elevates their mood, which, in turn, leads to further (behavioral) activation. However, there is a dearth of empirical evidence about the theoretical framework of BA.Objective: This study aims to examine the assumed (temporal) associations of the 3 constructs in the theoretical framework of BA.Methods: Data were collected as part of the "European Comparative Effectiveness Research on Internet-based Depression Treatment versus treatment-as-usual" trial among patients who were randomly assigned to receive blended cognitive behavioral therapy (bCBT). As part of bCBT, patients completed weekly assessments of their level of engagement in pleasant activities, the pleasure they experienced as a result of these activities, and their mood over the course of the treatment using a smartphone-based ecological momentary assessment (EMA) application. Longitudinal cross-lagged and cross-sectional associations of 240 patients were examined using random intercept cross-lagged panel models.Results: The analyses did not reveal any statistically significant cross-lagged coefficients (all P>.05). Statistically significant cross-sectional positive associations between activities, pleasure, and mood levels were identified. Moreover, the levels of engagement in activities, pleasure, and mood slightly increased over the duration of the treatment. In addition, mood seemed to carry over, over time, while both levels of engagement in activities and pleasurable experiences did not.Conclusions: The results were partially in accordance with the theoretical framework of BA, insofar as the analyses revealed cross-sectional relationships between levels of engagement in activities, pleasurable experiences deriving from these activities, and enhanced mood. However, given that no statistically significant temporal relationships were revealed, no conclusions could be drawn about potential causality. A shorter measurement interval (eg, daily rather than weekly EMA reports) might be more attuned to detecting potential underlying temporal pathways. Future research should use an EMA methodology to further investigate temporal associations, based on theory and how treatments are presented to patients.</p