京都大学 / マギル大学 / 新制・課程博士 / 博士(ゲノム医学) / 甲第24203号 / 医博JD第1号 / 新制||医||JD1(附属図書館) / 京都大学大学院医学研究科京都大学マギル大学ゲノム医学国際連携専攻 / (主査)教授 稲垣 暢也, 教授 YOUSSEFIAN Shohab, 准教授 Majewski Jacek (マギル大学), 准教授 Gravel Simon (マギル大学), 教授 Gagneur Julien (ミュンヘン工科大学) / 学位規則第4条第1項該当 / Doctor of Philosophy in Human Genetics / Kyoto University / McGill University / DFAM

Idiopathic Pulmonary Fibrosis (IPF) is an interstitial lung disease with no known cause or cure. IPF has an incidence of 75/1,000,000 of the population, predominately in men over the age of 60. This relatively rare disease develops in a chronic and progressive way, ultimately leading to death within two to five years of diagnosis. Our use of translatable methodologies in the bleomycin mouse model of IPF led to the novel identification of the similarities between the average percent loss of lung function in previous human clinical trials to that of our mouse model data. There is no treatment for IPF outside of lung transplantation, therefore our goal is to develop a protein therapy to halt the progression of IPF. B6_BP_dslf is a small, 93.36 kDa minibinder protein with a nanomolar affinity to αvβ6, an integrin of therapeutic potential for IPF when inhibited by halting αvβ6/TGF-β signaling. Our hypothesis is that B6_BP_dslf will halt the progression of pulmonary fibrosis induced in a mouse model of IPF. To test this hypothesis, a de novo design method was used resulting in the B6_BP_dslf minibinder having high β unit selectivity and nanomolar affinity for αvβ6, and maintenance of its secondary structure after aerosolization. These attributes led to testing in the bleomycin mouse model for IPF as an inhaled therapy. We found that B6_BP_dslf inhalation by mice with induced pulmonary fibrosis had reduced pathogenesis through the quantification of biomarkers for αvβ6/TGF-β mediated fibrosis, lowered histopathological scores, and improved lung function. These positive results from standard biochemical analysis and clinically translatable methods show that BP_B6_dslf has clinical potential as an inhaled therapy for IPF. Additionally, we tested the use of lung function tests in an animal model of chronic obstructive pulmonary disease (COPD), using secondhand smoke exposure to induce the disease and to identify inflammatory pathways. We found that smoke exposure increased inflammatory signaling through receptors for advanced glycation end-products, and inhibition of these receptors using a novel therapy of semi-synthetic glycosaminoglycan ethers (SAGEs) reduced inflammation and improved lung function. Together, the data from two different lung disease models supports the use of lung function as a preclinical efficacy variable for experimental drugs. The combination of biochemical and functional assessments of B6_BP_dslf and SAGEs gives weight to their therapeutic potential.

La Fibrose Pulmonaire Idiopathique (FPI) est une maladie dévastatrice, d’étiologie inconnue, qui reste pour le moment incurable. Cette maladie est caractérisée par l’accumulation de fibroblastes et de protéines de la matrice extracellulaire dans les espaces aériens distaux aboutissant à une destruction alvéolaire et à une altération des propriétés mécaniques du poumon. La physiopathologie de la FPI est mal connue mais de nombreuses études suggèrent que la réactivation des voies impliquées dans le développement contribue à l’accumulation de la matrice extra-cellulaire et au comportement anormal des cellules épithéliales et des fibroblastes.La voie Hedgehog (HH) joue un rôle crucial dans le développement embryonnaire. Dans le développement pulmonaire fœtal, la voie HH est impliquée dans les interactions épithélium-fibroblaste et contrôle la prolifération et la différenciation du mésenchyme. La voie HH a été impliquée dans la fibrogénèse, notamment dans le foie et le rein.L’objectif de cette thèse a été de caractériser la voie HH dans la fibrose pulmonaire chez l’homme et dans un modèle de fibrose induite par la bléomycine chez la souris.Nous avons démontré que la voie HH est réactivée dans les tissus pulmonaires de patients atteints de FPI et dans le modèle de fibrose pulmonaire chez la souris. Nous avons montré que le TGF-β1 activait la voie HH dans les fibroblastes pulmonaires humains et que l’inhibition pharmacologique de la voie HH au niveau des facteurs GLI inhibait l’effet du TGF-β1 in vitro. Par contre, ces inhibiteurs ne protégent pas les cellules épithéliales alvéolaires de la transition épithélio-mésenchymateuse induite par le TGF-β1. In vivo, chez la souris, nous avons montré que le traitement par des inhibiteurs de Smoothened ne protégeait pas du développement de la fibrose tandis que le GANT61, un inhibiteur de l’interaction des GLI avec l’ADN, inhibait la fibrose.En conclusion, nos résultats démontrent l’implication de la voie HH dans la fibrose pulmonaire et ouvrent des perspectives thérapeutiques nouvelles. / Idiopathic Pulmonary Fibrosis (IPF ) is a devastating disease of unknown etiology, which no efficient treatment. This disease is characterized by the accumulation of fibroblasts and extracellular matrix proteins in the distal airways resulting to the destruction of alveoli and alteration of mechanical properties of the lung. The pathogenesis of IPF is not well known but many studies suggest that reactivation of pathways involved in the development, contributes to the accumulation of extracellular matrix and the abnormal behavior of epithelial cells and fibroblasts.The Hedgehog pathway (HH) plays a crucial role in embryonic development. In the fetal lung development, the HH pathway is involved in the epithelial-fibroblast interactions and controls the proliferation and differentiation of the mesenchyme. The HH pathway has been implicated in the fibrogenesis, particularly in the liver and kidney.The aim of this thesis was to characterize the HH pathway in pulmonary fibrosis in humans and in a model of bleomycin-induced fibrosis in mice.We demonstrated that the HH pathway is reactivated in lung tissue of IPF patients and in the model of pulmonary fibrosis in mice. We have shown that TGF-β1 activated the HH pathway in human lung fibroblasts and that the pharmacological inhibition of the HH pathway at the level of GLI transcription factors, inhibited the effect of TGF-β1 in vitro. By contrast, these inhibitors did not protect alveolar epithelial cells from TGF-β1-induced epithelial-mesenchymal transition. In vivo, we have shown that treatment with Smoothened inhibitors did not protect mice from the development of fibrosis while GANT61, an inhibitor of the GLI interaction with DNA, inhibited fibrosis .In conclusion, our results demonstrate the involvement of the HH pathway in pulmonary fibrosis and open new therapeutic perspectives.

No description available.

Fibroproliferative disorders are the leading cause of morbidity and mortality worldwide, with one specific group of fibroproliferative disorders being interstitial lung diseases (ILD). Idiopathic pulmonary fibrosis is the most common ILD; however its pathogenesis is not entirely understood. What is known is that there is repetitive cellular injury preceding the fibrotic remodeling in the lungs that contributes to the irreversible deposition of extracellular matrix (ECM) proteins. Myofibroblasts that accumulate at the site of injury are thought to be the key drivers of ECM deposition and are often associated in the disease. Although it is poorly understood how these immune cells differentiate in the lung, one hypothesis suggests the role of alternatively activated profibrotic macrophages in this process. The data presented in this thesis suggest that there are a presence of UPR and macrophage proteins in the lungs of IPF patients and the UPR may be necessary in the polarization of alternatively activated macrophages. / Thesis / Master of Science (MSc)

Rationnel : L’implication établie des progéniteurs endothéliaux circulants dans les phénomènes de néovascularisation chez l’adulte a stimulé la recherche de thérapeutiques angiogènes basées sur la greffe de ces cellules. Deux types cellulaires au phénotype endothélial sont actuellement définis entre autres par leur cinétique d’apparition en culture : les progéniteurs précoces (CFU-EC ou CAC) et tardifs (ECFC). Notre équipe a montré que l’injection thérapeutique de cellules mononucléées de moelle osseuse (BM-MNC) permettait la néovascularisation du site ischémié chez des patients atteints d’artériopathie des membres inférieurs, et que les néovaisseaux formés avaient le phénotype d’ECFC. Nous avons dans un premier temps mesuré les concentrations de différentes protéines modulant l’angiogenèse, chez des patients atteints de pathologies ischémiques et cardiovasculaires, ou impliquant des anomalies vasculaires associées à la fibrose. Ainsi, le transforming growth factor - β1 (TGF-β1) dans la fibrose pulmonaire idiopathique (FPI), la thrombospondine-1 (TSP-1) dans l’artériopathie des membres inférieurs (AMI), et le placental growth factor (PlGF) chez les patients atteints de pathologies cardiovasculaires [syndrome coronarien aigu (SCA), ou patients devant subir une chirurgie de la valve ou un pontage coronarien], se sont distingués comme potentiel biomarqueur plasmatique dans ces pathologies, et ont été étudiés dans la biologie des ECFC humaines.Résultats : Le taux plasmatique de TGF-β1 est augmenté chez les patients atteints de FPI par rapport à la population contrôle ; il a un effet pro-angiogène in vivo (vascularisation des implants de Matrigel®) et in vitro (prolifération et migration des ECFC) via les récepteurs ALK-1, ALK-5 et TGF-βRII. Le taux plasmatique de TSP-1 est augmenté chez les patients artéritiques par rapport à la population contrôle. Par ailleurs les néovaisseaux formés de patients artéritiques ayant été traités par injection locale de BM-MNC expriment la TSP-1. Dans les modèles murins de Matrigel®-plugs et d’ischémie du membre inférieur (IMI), la TSP-1 induit une diminution de la vascularisation des implants ainsi qu’une diminution de la revascularisation du membre ischémié. In vitro, la TSP-1 augmente l’adhésion via un mécanisme N-Terminal dépendant, et diminue le potentiel angiogène (prolifération et migration) des ECFC via sa liaison au récepteur CD47, ce qui active la voie de signalisation SDF-1/CXCR4. Le taux plasmatique de PlGF est augmenté chez les patients atteints de SCA par rapport à 2 populations contrôles ; il est également augmenté chez les patients ayant subit une chirurgie cardiaque. Les PlGF-1 et -2 potentialisent la tubulogenèse des ECFC in vitro via la phosphorylation du récepteur VEGFR1. Cet effet est aboli lorsque le VEGFR1 est inhibé par ARN interférence ou par le composé chimique « 4321 ». De plus ce composé « 4321 » inhibe la vascularisation des implants de Matrigel®, ainsi que la revascularisation du membre ischémié dans le modèle d’IMI.Conclusions : Le TGF-β1 joue un rôle dans le remodelage vasculaire de la FPI via les ECFC ; la TSP-1 est un potentiel biomarqueur de l’angiogenèse induite par les ECFC dans l’AMI ; l’inhibition de la voie PlGF/VEGFR1 module la tubulogenèse induite par les ECFC, cellules impliquées dans la formation de nouveaux vaisseaux. Nous avons ainsi mis en évidence 3 protéines modulant l’angiogenèse dans 3 contextes pathologiques différents, caractérisés par un remodelage vasculaire et où les ECFC sont impliquées dans leurs mécanismes physiopathologiques. Ces 3 protéines se présentent donc comme de potentiels biomarqueurs plasmatiques, modulant les propriétés angiogènes des ECFC et pouvant influencer leur efficacité en tant que produit de thérapie cellulaire. Ces protéines jouent un rôle probable dans l’équilibre homéostatique au décours des pathologies concernées. / Rationale: The pro-angiogenic capacities of endothelial progenitor cells are now well established, and their involvement in neovascularization events in adults has stimulated the research in the field of angiogenic therapy based on transplant of these cells. Current data converge towards the notion of two cell types with endothelial phenotype, defined at least by their kinetics of appearance in culture: early endothelial progenitor cells (CFU-EC or CAC) and late (ECFC). Our team has shown that the therapeutic injection of bone marrow mononuclear cells (BM-MNC) led to neovascularization of the ischemic site in patients with critical limb ischemia, and that the new vessels formed bore the phenotype of ECFC. We initially measured the concentrations of different proteins modulating angiogenesis in patients with ischemic and cardiovascular diseases, or involving vascular abnormalities associated with fibrosis. Thus, the transforming growth factor - ß1 (TGF-ß1) in idiopathic pulmonary fibrosis, the thrombospondin-1 (TSP-1) in peripheral artery disease, and the placental growth factor (PlGF) in patients with cardiovascular diseases [acute coronary syndrome (ACS), patients undergoing valve surgery or coronary artery bypass surgery], emerged as potential plasmatic biomarkers in these pathological settings, and have been studied in the biology of human ECFC.Results: TGF-ß1 plasma level is increased in patients with idiopathic pulmonary fibrosis (IPF) compared to the control population; it exerts a pro-angiogenic effect in vivo (vascularization of Matrigel ®-plugs) and in vitro (proliferation and migration of ECFC) via ALK-1, ALK-5 and TGF-ßRII receptors. TSP-1 plasma level is increased in patients with peripheral artery disease (PAD) compared to the control population. In addition, the new vessels formed in PAD patients treated by local injection of BM-MNC express TSP-1. In murine models of Matrigel ®-plugs and hindlimb ischemia, TSP-1 induces a decrease in plugs vascularization and impaired revascularization of ischemic limb. In vitro, TSP-1 increases ECFC adhesion via an N-terminal dependent mechanism and reduces their angiogenic potential (proliferation and migration) via its binding to CD47 receptor, which activates the SDF-1/CXCR4 signaling pathway. PlGF plasma level is increased in ACS patients compared with the control population and stable angina patients and is also increased in patients undergoing cardiac surgery. PlGF-1 and -2 potentiate ECFC tubulogenesis in vitro via phosphorylation of the VEGFR1 receptor. This effect was abolished when the ECFC VEGFR1 is inhibited by RNA interference or by the chemical compound "4321". In addition this compound "4321" inhibits the vascularization of Matrigel ®-plugs, and revascularization of the ischemic limb in the hindlimb ischemia model.Conclusions: TGF-ß1 is involved in the IPF vascular remodeling through ECFC; TSP-1 is a potential biomarker of angiogenesis induced by ECFC in PAD; the inhibition of the PlGF/VEGFR1 pathway modulates ECFC tubulogenesis, cells involved in the formation of new vessels. We thus identified three proteins that modulate angiogenesis in three different pathological settings characterized by a vascular remodeling and where ECFC are involved in their pathophysiology. These three proteins therefore state as potential plasmatic biomarkers, modulating ECFC angiogenic properties and are able to influence their efficacy as a cell therapy product. These plasmatic biomarkers likely play a role in the homeostasis of those pathologies progress.

La fibrose pulmonaire idiopathique (FPI) est de pronostic sombre et sans traitement efficace. Elle est caractérisée par un début sous pleural et la présence de myofibroblastes responsables de la synthèse excessive de la matrice extracellulaire. La voie de signalisation du Transforming Growth Factor (TGF)-β1, facteur clé de la genèse de la fibrose et sa progression, passe par les Smads, notamment Smad4. Le TGF-β1 induit la différenciation des fibroblastes pulmonaires et des cellules épithéliales et mésothéliales en myofibroblastes. AB-crystallin est une protéine de choc thermique surexprimée dans la fibrose du foie, du rein et la fibrose vasculaire. Elle peut être induite par le TGF-β1. Dans ce travail, nous avons étudié le rôle d’αB-crystallin dans la fibrose pleurale et pulmonaire. Nous montrons qu’αB-crystallin est surexprimée dans les poumons et la plèvre de patients atteints de FPI. In vivo, dans trois modèles de fibrose pulmonaire (bléomycine, surexpression de TGF-β1 ou d’IL-1β) les souris KO pour αB-crystallin sont protégées de la fibrose avec une inhibition de la voie du TGF-β. In vitro, dans les cellules épithéliales, mésothéliales ou les fibroblastes, αB-crystallin augmente la localisation nucléaire de Smad4. En interagissant avec TIF1γ, responsable de l’export nucléaire de Smad4, elle favorise la séquestration nucléaire de Smad4 et son activité pro-fibrosante. Au contraire, son inhibition permet la formation du complexe Smad4/TIF1γ et l’export nucléaire de Smad4 inhibant son activité. Ce travail montre l’importance d’αB-crystallin dans la fibrose pleuro-pulmonaire et son rôle sur la voie du TGF-. AB-crystallin pourrait être une cible thérapeutique de la FPI. / Idiopathic pulmonary fibrosis (IPF) has no effective current treatment. It is characterized by a sub-pleural onset and the presence of myofibroblasts, responsible for the excessive extracellular matrix synthesis. Transforming Growth Factor (TGF)-β1 is considered as the major profibrotic cytokine. Its signaling pathway occurs through the Smads proteins, including Smad4. TGF-β1 allows the differentiation of lung fibroblasts and epithelial and mesothelial cells into myofibroblasts. AB-crystallin is a small heat shock protein overexpressed in liver, renal and vascular fibrosis and can be induced by TGF-β1. In this study, we assessed the role of αB-crystallin in pleural and pulmonary fibrosis. We show that αB-crystallin is overexpressed in the lung and the pleura of IPF patients. In vivo, in three pulmonary fibrosis models (bleomycin, TGF-β1 or IL-1β overexpression) αB-crystallin KO mice are protected from fibrosis with an inhibition of the TGF-β pathway. In vitro, in epithelial and mesothelial cells or fibroblasts, αB-crystallin increases Smad4 nuclear localization. Interacting with TIF1γ, responsible for the nuclear export of Smad4, it promotes the nuclear sequestration of Smad4 and thus its profibrotic activity. Instead, αB-crystallin inhibition allows the formation of the Smad4/TIF1γ complex and promotes Smad4 nuclear export an profibrotic activity. This work shows the importance of αB-crystallin in pleuro-pulmonary fibrosis and its role on the TGF-β1 pathway. AB-crystallin appears as a putative therapeutic target for IPF.

Idiopathic Pulmonary Fibrosis (IPF) is a debilitating respiratory disorder that is characterized by a progressive decline in lung function. Originating through unknown etiology, it is essentially an unchecked wound healing response that causes the build-up of excessive scar tissue in the lung interstitial tissue with a heavy toll on the patient’s respiratory capacity. Pro-fibrotic alternatively activated macrophages (M2) have been linked as an important contributor to the fibrotic remodeling of the lung. Previous Ask research indicates that targeting M2 macrophages is possible through the use of the Dectin-1 receptor, a transmembrane cell surface receptor found in high abundance on M2 macrophages. Activating the Dectin-1 receptor through the use of beta-glucan, a ligand the receptor has a high affinity for, initiates a pro-inflammatory response within the naturally immunosuppressive macrophage and can alter its activity to be less fibrogenic. Our data suggest that M2 polarization of naïve macrophages can be inhibited in vitro by beta-glucan microparticles. Additionally, we have found that polarized M2 macrophages adopt M1-like characteristics when treated with beta-glucan microparticles, in a process that is largely Dectin-1 dependent. M2 cell surface marker CD206, increased levels of which are associated with rapidly progressing IPF, shows significantly decreased frequency of expression in M2 macrophages treated with beta-glucan microparticles. Our assessment for cell-specific uptake of beta-glucan microparticles suggests an important role of the Dectin-1 receptor for significantly increased uptake in murine wild-type M2 macrophages relative to their Dectin-1 knockout counterpart. The use of beta-glucan microparticles as a potential anti-fibrotic therapeutic was assessed in the bleomycin model of fibrotic lung disease. Mice given bleomycin and treated with beta-glucan displayed decreased soluble collagen content and TGFB expression within lung homogenate relative to fibrotic bleomycin control mice. Overall, these results provide insight into the use of beta-glucan as a potential activity modulator of macrophage function in IPF and the possibility of its use as a therapeutic. / Thesis / Master of Science (MSc)

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with worsening respiratory symptoms and physiological impairment. Pulmonary fibrosis is a chronic lung disease characterized by forming scar tissue (fibrosis) in the lungs. Alternatively activated macrophages (M2) known as pro-fibrotic are known to contribute to the fibrotic remodeling of the lung. In addition to the polarization of slices from naïve to pro-fibrotic, the addition of anti-fibrotic therapeutics reprogram slices back to a naïve condition. To polarize the slices, naïve slices are incubated with a previously investigated method in the lab known as the polarization cocktail. The polarization cocktail can be achieved by adding of IL-4, IL-6 and IL-13 to naïve(M0) slices in the Precision Cut lung slice (PCLS) model. For the therapeutic model, slices are incubated with the polarization cocktail and subsequently with the therapeutic. Our results have shown that the precision cut lung slice model can mimic previously investigated in-vivo experiments with the polarization cocktail. Secondly, the addition of therapeutics result in the slices exhibiting lower amounts of M2 markers and arginase activity concluding the model is suitable for the polarization and reprogramming of macrophages. / Thesis / Master of Science in Medical Sciences (MSMS)