This thesis reviews the current literature on idiopathic pulmonary fibrosis (IPF), a progressive, scarring lung condition largely affecting older adults that is experiencing an increasing incidence in the U.S. and abroad. Two troubling clinical aspects of IPF are the difficulty of timely diagnosis and uncertain progression once diagnosed. The need for early detection is driven by the condition’s median survival rate post-diagnosis of about 3 years. Environmental and familial risk factors are important predictors of IPF, but cannot alone determine who is at risk for the condition. High-resolution computed tomography is currently the best non-invasive diagnostic tool, but many efforts are now underway to identify biological markers, which may aid not only in diagnosis, but illuminate both susceptibility and progression of the disease. Although the pathogenesis of IPF remains unclear, a compelling correlation has surfaced between the mechanics of IPF and herpes virus infection, which also may lead to a biological marker for the condition. Likewise, some genetic factors have shown promise in revealing pathogenesis and possible diagnosis. The only treatment currently available to ameliorate IPF is lung transplantation, but it is a last resort effort. In terms of pharmaceutical treatment, the most significant development has been the recent approval and use of two anti-fibrotic drugs, pirfenidone and nintedanib, that appear to slow the progression of the disease, but do not eliminate the fibrotic condition that impairs patients’ breathing. As efforts progress in addressing affirmative treatments for IPF, there is consensus that not enough is being done to address palliative and psychological needs of IPF. In sum, a review of the current literature suggests tremendous accomplishments have made in treating what remains a fatal condition, but much work remains to truly understand how and why IPF occurs, and whether, short of lung transplantation, there are treatments that can improve, not just maintain, patients’ health.

IPF is a progressive disease, characterized by dysregulated fibrosis of the extracellular matrix (ECM). The pathobiology of the disease is still unknown, and the median survival post-diagnosis is about 3-5 years. The two current US FDA approved drugs for IPF (nintedanib and pirfenidone) slow, but fail to reverse, disease progression. There is cumulating research that suggests the ECM is an active player in fibrosis. In this thesis, we summarized the current knowledge of ECM-cell interactions in the context of pulmonary fibrosis. To gain more mechanistic insight into the ECM characteristics that dictate cell behavior, we established a 3D ECM ex vivo system to assess the nonfibrotic and fibrotic ECM’s effect on fibroblasts. The ECM appears to promote both pathological and physiological cellular changes, depending on its structural and compositional properties. We also used this 3D ex vivo system as a preclinical tool to test the effect of directly inhibiting mechanotransduction in the fibrotic ECM – fibroblast profibrotic relationship. Lastly, since the fibrotic ECM seems to play a key role in progressive fibrosis, we evaluate if researchers are appropriately using the bleomycin model by starting interventions after ECM fibrosis is established. Over the past decade in the field, there has been an overall improvement in the appropriate therapeutic timing. In the preventative studies, however, there is still an inadequate characterization of inflammation. There is also poor transparency of preclinical-bleomycin data for clinically tested interventions for IPF. Addressing these shortcomings may improve the utility of the model at predicting an intervention’s success in clinical trials. These findings illustrate the ECM’s role in driving pulmonary fibrosis. Therefore, the ECM should be further investigated to understand disease progression, and reproduced in preclinical models to test interventions. This will improve the transition of pathobiological findings into efficient drug development for this devastating disease. / Thesis / Candidate in Philosophy / Idiopathic pulmonary fibrosis (IPF) (idiopathic - unknown cause; pulmonary - lungs; fibrosis - scarring) is characterized by progressive scarring of the lung extracellular matrix (ECM). The ECM is an organ’s backbone that provides structural and biochemical support to surrounding cells. Continued ECM scarring can lead to difficulty breathing, cough, and ultimately death. The cause of IPF is unknown, however, studies suggest that the scarred ECM can promote further scarring, and cause disease progression. In this thesis, we summarized the current knowledge of how the ECM interacts with cells. Using a 3D model we see that depending on the ECM’s structure and composition, it can promote both disease and healthy cellular changes. Lastly, we evaluate if researchers are appropriately using the bleomycin model (most common preclinical model for pulmonary fibrosis) by testing interventions after ECM fibrosis is established. We propose changes to improve its usefulness as a preclinical tool for IPF.

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease of unknown etiology, in which excessive accumulation of scar tissue in the interstitial spaces of the lung obstruct normal pulmonary function. Currently, the only curative treatment is lung transplantation. While pharmaceutical therapeutics have been recently approved for use in IPF in 2014, they are still unable to provide a truly curative treatment. While genetic risk factors have been identified, the most commonly occurring mutation is only detected in approximately 38% of IPF patients, leaving an uncertainty in the very existence of a common genetic factor in IPF. Cigarette smoke and other environmental particulates have been significantly linked to the diagnosis of IPF, implicating an initial immunological response to trigger the pathogenesis of IPF. Nintedanib, a potent tyrosine kinase receptor inhibitor was first developed in 1998 as a candidate for cancer treatment. Investigation of its effects in fibrosis in the past few decades has led to a significant discovery of its application in IPF. Nintedanib significantly inhibits the fibrotic activity of fibrotic myofibroblasts in the lungs by inhibiting signaling cascades necessary for cell proliferation and progression of the disease. However, nintedanib falls short in that it cannot fully inhibit the advancement of the disease and mortality rates of IPF still remain high. Pirfenidone, the other currently available pharmaceutical therapeutic, was discovered in 1976 as a potent inhibitor of inflammation. Subsequent experiments further reviled its potency as an anti-fibrotic drug. After decades of research, pirfenidone’s mechanism of antifibrotic characteristics were revealed as a potent inhibitor of fibrocyte recruitment and chemotaxis, and as an inhibitor of transcription growth factor beta (a growth factor heavily implicated in the activity of myofibroblasts) mediated pathways. However, like nintedanib, pirfenidone fails as a curative treatment, only delaying the progression of the disease. In the search for new molecular targets for pharmaceutical therapy, forkhead box M1 (FOXM1), programmed cell death protein-1 (PD-1), and prostaglandin E2, have been identified to play a mediatory role in many of the pathways involved in myofibroblast activity. Many of these targets have also been identified in other disease models such as cancer and immunological inflammatory disease. Avasimibe has been recently identified as a potent inhibitor of aldo-ketoreductase through a FOXM1 mediated pathway. Its molecular mechanism in osteosarcoma cancer disease model may prove to be a novel pharmaceutical therapeutic for IPF. BI 853250, a novel focal adhesion kinase (FAK) inhibitor also demonstrates potential to be a new pharmaceutical therapeutic for IPF patients. Exploring signaling pathways that involve these newly found targets and collaborative research with cancer and immunological diseases shows promise in providing steps to cure IPF in the future.

ROGDI, a novel gene, locates on human¡¦s chromosome 16p13.3. According to Gene Ontology Annotation database, ROGDI is related to hemopoiesis and positive regulation of cell proliferation. In order to investigate the function of this novel gene in pulmonary fibrosis, fibrotic models in vivo and in vitro were created. Mice which received single intra-tracheal bleomycin injection were sacrificed on various intervals. Rogdi and other pro-fibrotic mediators, including CCL2 and TGF-£]1, were up-regulated in the early phase(< 10 days). On contrary, the anti-fibrotic mediators IL-10, IFN-£^ and heme oxygenase(HO)-1 were up-regulated in the late phase(> 10 days). The precursor microRNA 21 (miR-21) was up-regulated as the fibrotic severity increased. The human embryonic fibroblasts(WI-38 cells) showed fibrogenic phenotype and up-regulation of precursor miR-21 and ROGDI after bleomycin treatment. Human embryonic fibroblasts transfected by coding sequence of ROGDI showed up-regulated precursor miR-21 and £\-SMA compared to those transfected by empty vectors after bleomycin treatment. Two signaling molecules related to positive regulation of cell proliferation, Akt and Erk, showed over-expressed after ROGDI transfection and bleomycin treatment compared to those with empty vector transfection. Our results imply that ROGDI is up-regulated in pulmonary fibrosis and turns fibroblasts into fibrogenic phenotype through positive regulation of miR-21. The increase of precursor, but not primary miR-21, after ROGDI transfection and bleomycin treatment indicates that ROGDI may regulate the TGF-£] signaling pathway in human embryonic fibroblasts. Our results support that ROGDI is a novel gene for pulmonary fibrosis and warrants for further investigation. £[

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19573号 / 医博第4080号 / 新制||医||1013(附属図書館) / 32609 / 京都大学大学院医学研究科医学専攻 / (主査)教授 伊達 洋至, 教授 小池 薫, 教授 瀬原 淳子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(社会健康医学) / 甲第24808号 / 社医博第132号 / 新制||社医||12(附属図書館) / 京都大学大学院医学研究科社会健康医学系専攻 / (主査)教授 川上 浩司, 教授 西浦 博, 教授 平井 豊博 / 学位規則第4条第1項該当 / Doctor of Public Health / Kyoto University / DFAM

Introdução: A fibrose pulmonar idiopática (FPI) é uma grave doença pulmonar crônica com sintomas de dispneia progressiva, resultando na diminuição da capacidade de exercício, impactando negativamente na qualidade de vida relacionada à saúde (QVRS). A reabilitação pulmonar (RP) melhora a capacidade funcional (CF) com redução dos sintomas, porém na FPI avançada seus efeitos e magnitudes são pouco conhecidos. Objetivo: Avaliar o impacto da RP sobre a QVRS e a CF em pacientes portadores de FPI. Métodos: Estudo de coorte retrospectiva em que foram revisados dados de 56 prontuários de pacientes em lista de transplante de pulmão com diagnóstico de FPI de acordo com o consenso da American Toracic Society 2011, submetidos a 12 semanas (36 sessões) de RP ambulatorial entre o período de janeiro de 2008 a outubro de 2012. Foram avaliadas a CF e a QVRS através do teste de caminhada de seis minutos (TC6) e do questionário 36-item short-form survey, SF36, respectivamente, antes e imediatamente após a RP. Resultados: Vinte e sete pacientes foram incluídos no estudo, 16 (61%) gênero masculino com idade média de 53 ±13 anos. Dezoito pacientes (68%) tinham diagnóstico histológico por biópsia pulmonar com padrão de pneumonia intersticial usual (PIU), com tempo médio de diagnóstico de 3 ±1,7 anos. Quanto à classificação da dispneia pela escala modified Medical Research Council (mMRC) basal, 59% dos pacientes foram classificados entre 3-4. Houve aumento significativo na distância percorrida de 393 ±88 metros para 453 ±90 metros (p<0,001). As medianas de dispneia sofreram diminuição significativa (p=0,01) na escala do mMRC de 2 (IC95%: 1-4) para 1 (IC95%: 1-4) e de 5 (Mín/Máx:1-10) para 3 (Mín/Máx:0-10) no BORG de dispneia no final do TC6. Apesar de caminharem maiores distâncias, a fadiga em membros inferiores foi menor com uma mediana de 2 (Mín/Máx:0-10) para 1 (Mín/Máx:0-9) (p=0,02). Houve aumento em 5 dos 8 domínios, porém somente a capacidade funcional foi significativa de 26 (IC95%: 19-33) para 37 (IC95%: 27-48) (p<0,05); os demais domínios não tiveram significância estatística. Conclusão: Observaram-se nestes pacientes aumentos da CF, com diminuição dos sintomas dispneia e fadiga; o que não se refletiu em melhora clínica na QVRS em portadores de FPI em lista de transplante de pulmão após um programa de RP. / Introduction: Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease with severe symptoms of progressive dyspnea, resulting in decreased exercise capacity, negatively impacting the health-related quality of life (HRQL). Pulmonary rehabilitation (PR) improves functional capacity (FC) with reduction in symptoms, but in advanced IPF, its effects and magnitudes are unknown. Objective: To evaluate the impact of PR and in HRQL and in FC of patients with IPF. Methods: Coorte study with a retrospective review of data from 56 medical records of patients on lung transplant list diagnosed with IPF according to the American Toracic Society 2011 consensus, submitted to 12 weeks (36 sessions) of outpatient RP between January 2008 and October 2012. The FC and the HRQL were assessed through a six-minute walk test (6MWT) and the 36-item short-form survey (SF36) respectively before and immediately after PR. Results: Twenty-seven patients were included in the study, 16 (61%) male with a mean age of 53 ± 13 years. Eighteen patients (68%) had histologic diagnosis by lung biopsy compatible with usual interstitial pneumonia (UIP), with median time from diagnosis of 3 ± 1.7 years. Regarding the classification of the dyspnea in the modified Medical Research Council (mMRC) scale, 59% of patients were classified between 3-4. There was a significant increase in the distance covered from 393 ± 88 meters to 453 ± 90 meters (p <0.001). The baseline medians of dyspnea had a significant decrease (p = 0.01) in the mMRC scale from 2 (CI 95%: 1-4) to 1 (CI 95%: 1-4) and the median decreased from 5 (Min/Max: 1-10) to 3 (Min/Max :0-10) in the Borg dyspnea index at the end of the 6MWT. Although the patients walked greater distances, they had less fatigue in the legs, with a median decrease from 2 (Min/Max: 0-10) to 1 (Min/Max: 0-9) (p = 0.02). There was an increase in 5 of the 8 domains, but only the functional capacity was significant: from 26 (CI95%: 19-33) to 37 (CI95%: 27-48) (p <0.05), while the remaining areas were not statistically significant. Conclusion: We observed increases of FC in these patients, with decreased symptoms of dyspnea and fatigue; which were not reflected in clinical improvement in HRQL of patients with IPF on lung transplant list after a PR program.

Background Idiopathic pulmonary fibrosis (IPF) is one of a number of interstitial lung diseases (ILDs) that result in extensive and chronic pulmonary fibrosis. In IPF pathology, immunological dysfunction has been identified as a contributing factor to the ongoing fibrotic process, implicating cells and mechanisms of both the innate and humoral immune response. Due to the complex and diverse range of cells and mediators involved in IPF, the pathology is still poorly understood. Evidence of complement activation through the classical pathway in IPF lungs implies a role for IgG in the pathology. The active IgG in IPF may be autoreactive in nature, as IgG that target antigens of alveolar epithelial cells have been. Two autoantibodies in IPF, anti-periplakin IgG and anti-Hsp72 IgG, have been associated with poorer prognoses in IPF patients. The association of anti-Hsp72 IgG with IPF patient outcomes has not been validated and little work has been done to study the underlying mechanisms of autoantibodies in IPF pathogenesis. Hypothesis Anti-Hsp72 IgG is associated with poorer outcomes in IPF, and may induce alveolar macrophages to exhibit a pro-fibrotic phenotype. Aims The aims were to:  Optimise an ELISA for anti-Hsp72 IgG detection and determine any association of anti-Hsp72 IgG with IPF patient outcomes  Determine the location of anti-Hsp72 IgG producing cells and detect if Hsp72-IgG complexes are present in IPF patients’ lungs  Explore a potential underlying pro-fibrotic mechanism through which anti-Hps72 IgG modulates macrophage function. Results The presence of anti-Hsp72 IgG was determined in ILD patient and healthy control bronchoalveolar lavage fluid (BALf) and serum. A novel anti-Hsp72 IgG ELISA was developed and optimised and then compared against a commercial anti-Hsp72 IgGAM ELISA which became available during the PhD. Progression in IPF was defined by a decrease of ≥10% vital capacity (VC) over twelve months. Serum anti-Hsp72 IgG(AM) did not associate with changes in VC over 12 months. In contrast, BALf anti-Hsp72 IgG(AM) concentrations were elevated in IPF non-progressors. Patients with high BALf anti-Hsp72 IgGAM, had improved survival compared patient with low anti-Hsp72 IgGAM (adjusted HR 0.39, 95% CI 0.16-0.92; p=0.032) In contrast there was no association between anti-Hsp72 IgG and survival. Detection of anti-Hsp72 IgG subtypes in the serum and BALf of IPF patients revealed no significant difference in anti-Hsp72 IgG subtype detection levels between progressors and non-progressors. BALf anti-Hsp72 IgG1 levels were associated with a significantly lower rate of decline in VC over twelve months than patients with no detectable anti-Hsp72 IgG1. The presence of Hsp72-IgG complexes was confirmed by detection in purified IgG from IPF patient BALf. Immuno-histological detection of C4d deposition in the lungs of IPF patients coincided in areas of Hsp72 expression in alveolar epithelium. Summary These findings do not validate serum and-Hsp72 IgG as a biomarker for IPF. They support a role for anti-Hsp72 IgG in IPF, but associate with decreased rates of lung function decline and increased patient survival. Data also suggests that the decreased rate of decline may be related to specific anti-Hsp72 IgG subtype expression. The immune-histological data further suggests that anti-Hsp72 IgG may be targeting Hsp72 expressed by lung epithelium. Therefore these findings support a role for immunological dysfunction in IPF, but further work is required to determine the underlying mechanism.

Idiopathic pulmonary fibrosis (IPF) is an irreversible disease of unknown etiology that involves progressive scarring of the lung tissue, leading to respiratory failure and death.1 IPF is thought to develop from repetitive lung injury and aberrant wound healing that leads to the generation of fibrous tissue rather than restoration of normal tissue.2 It has been suggested in mice that vascular leak after lung injury contributes to the development of lung fibrosis.2,3 Gadofosveset is an intravascular enhancing, gadolinium-based contrast agent used with magnetic resonance imaging (MRI) to assess a variety of biological processes in vivo because it can reversibly bind to albumin.13-14 Gadofosveset has been used to assess endothelial permeability and function, as it diffuses through the vessel walls via leaky neovessels and damaged endothelium.15 Our research group has developed a new method to assess disease activity in IPF patients using gadofosveset-enhanced lung MRI. In unpublished work, we have demonstrated that this technique can be used to generate an albumin extravasation index (AEI), and we have found that this is significantly and diffusely increased in the lung of patients with idiopathic pulmonary fibrosis compared to healthy controls.16 The AEI is a measure of the change in signal intensity post-contrast minus pre-contrast in predefined regions of interest (ROIs) in the lung parenchyma divided by post- minus pre-contrast signal intensity in the ROI in the aorta. In this study, we compared the AEI in patients with IPF to healthy control (HC) subjects and evaluated the correlation between the AEI and high-resolution computed tomography (HRCT) and pulmonary function testing (PFT). We found that IPF subjects had increased AEI values compared with HC subjects. While not statistically significant, AEI was more strongly correlated with fibrosis (interstitial abnormalities) than ground-glass (alveolar abnormalities) on HRCT. Furthermore, there was a possible correlation between AEI and change in percent predicted forced expiratory volume in the first second (FEV1), forced vital capacity (FVC), and diffusion capacity of carbon monoxide adjusted for hemoglobin (DLCO) [Hb]. Our results demonstrate that AEI calculations from gadofosveset-enhanced lung MRI are a surrogate measure of vascular leak and can potentially serve as an alternative method for predicting the clinical course and severity of IPF through its correlation with fibrosis on HRCT and pulmonary function.

Les microARN sont des petits ARN non codants d’une vingtaine de nucléotides qui ont pour fonction de réguler l’expression des gènes en se fixant sur l’extrémité 3’UTR d’ARNm cibles, permettant ainsi leur dégradation ou l’arrêt de leur traduction en protéines. A ce jour, de nombreuses études ont montré l’implication des microARN dans divers processus physiologiques ou pathologiques ; leur rôle dans la réponse de l’organisme aux substances toxiques environnementales commence à être évoqué.La FPI appartient au groupe des pneumopthies interstitielles diffuses idiopathique dont elle est la forme la plus fréquente. Elle se caractérise par la présence de foyers de prolifération de fibroblastes/myofibroblastes responsables d’une production excessive de matrice extracellulaire, une destruction progressive et irréversible de l’architecture pulmonaire entrainant la perte de la fonction respiratoire. L’agression répétée de l’épithélium respiratoire par des composés chimiques environnementaux (ou xénobiotiques) est fortement suspectée dans le déclenchement de la FPI.Le premier objectif de mes travaux de recherche a été d’identifier des microARN susceptibles d’intervenir dans la pathogenèse de la fibrose pulmonaire idiopathique (FPI) et de préciser la (les) fonction(s) de ces microARN d’intérêt. Pour atteindre cet objectif, nous avons étudié deux microARN, le miR-199a-5p et le miR-214-3p qui présentaient la particularité d’être significativement surexprimés dans les poumons de souris souffrant de fibrose pulmonaire. L’analyse systématique des profils d’expression des gènes de fibroblastes surexprimant le miR-199a-5p et le miR-214-3p nous a permis d’identifier un grand nombre de gènes qui étaient significativement modulés par ces deux microARN. Nous avons pu établir l’implication respective du miR-199a-5p et du miR-214-3p dans la régulation de la voie profibrotique TGFβ et dans l’apoptose des fibroblastes pulmonaires médiée par le Fas-ligand. L’ensemble de nos résultats nous permet de suggérer l’utilisation de molécules ciblées contre ces 2 microARN dans le traitement de la FPI.Le second objectif de mes travaux de recherche a été d’identifier le modèle cellulaire in vitro le plus proche du tissu pulmonaire afin d’étudier l’impact des composés toxiques environnementaux sur la pathogenèse des maladies respiratoires et, en particulier, de la FPI. Pour cela, nous avons comparé les profils d’expression génique de l’ensemble des protéines impliquées dans le métabolisme et l’éliminations des xénobiotiques, de 10 lignées cellulaires et de 4 cultures primaires de cellules épithéliales bronchiques humaines, à ceux précédemment observés par notre équipe dans les tissus broncho-pulmonaires humains. L’exposition du modèle cellulaire le plus pertinent à des polluants atmosphériques permettra d’identifier les microARN associés à la toxicité pulmonaire de ces composés chimiques et de vérifier si ces microARN régulent des voies de signalisations communes à celles impliquées dans la pathogenèse de la FPI. / MicroRNAs are small non-coding RNAs with about 20 nucleotides that regulate gene expression by binding to the 3' UTR end of target mRNAs, thus allowing their degradation or stopping their translation into proteins. To date, many studies have shown the involvement of microRNAs in various physiological or pathological processes; their role in the body's response to environmental toxic substances is beginning to be mentioned. It is characterized by the presence of fibroblast/myofibroblast proliferation foci responsible for excessive extracellular matrix production, progressive and irreversible destruction of lung architecture leading to loss of respiratory function. The repeated aggression of the respiratory epithelium by environmental (or xenobiotic) chemicals is strongly suspected in the initiation of IVF. The first objective of my research was to identify microRNAs that may be involved in the pathogenesis of idiopathic pulmonary fibrosis (IVF) and to specify the function(s) of these microRNAs of interest. To achieve this objective, we studied two microRNAs, miR-199a-5p and miR-214-3p, which had the particularity of being significantly overexpressed in the lungs of mice with pulmonary fibrosis. Systematic analysis of the expression profiles of fibroblast genes overexpressing miR-199a-5p and miR-214-3p allowed us to identify a large number of genes that were significantly modulated by these two microRNAs. We were able to establish the respective involvement of miR-199a-5p and miR-214-3p in the regulation of the profibrotic pathway TGFβ and in Fas-ligand mediated apoptosis of pulmonary fibroblasts. The second objective of my research was to identify the in vitro cellular model closest to lung tissue in order to study the impact of environmental toxic compounds on the pathogenesis of respiratory diseases and, in particular, of IVF. To do this, we compared the gene expression profiles of all proteins involved in the metabolism and elimination of xenobiotics, 10 cell lines and 4 primary cultures of human bronchial epithelial cells, with those previously observed by our team in human bronchopulmonary tissues. Exposure of the most relevant cellular model to air pollutants will identify the microRNAs associated with the pulmonary toxicity of these chemical compounds and verify whether these microRNAs regulate signaling pathways common to those involved in the pathogenesis of IVF.