京都大学 / 新制・課程博士 / 博士(医学) / 甲第23380号 / 医博第4749号 / 京都大学大学院医学研究科医学専攻 / (主査)教授 山下 潤, 教授 木村 剛, 教授 YOUSSEFIAN Shohab / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Transforming growth factor-β1 (TGF-β1), an abundant growth factor in bone matrix, has been shown to be involved in bone formation and fracture healing. The mechanism of action of the osteogenic effect of TGF-β1 is not clearly understood. In this study, we found that the addition of TGF-β1 to murine osteoblastic MC3T3-E1 cells induced vascular endothelial growth factor (VEGF) mRNA production. VEGF mRNA levels reached a plateau within 2 h after the addition of TGF-β1. The induction was superinduced by cycloheximide and blocked by actinomycin D. Ro 31-8220, a protein kinase C inhibitor, abrogated the induction. In addition, curcumin, an inhibitor for transcription factor AP-1, also blocked the induction. Electrophoretic mobility shift assay revealed an enhanced binding of transcription factors AP-1 and NF-κB. Transient transfection experiment showed that VEGF promoter activity increased 3.6-fold upon TGF-β1 stimulation. Immunoblot analysis showed that the amount of secreted VEGF was elevated in the medium 4 h after TGF-β1 stimulation. Our results therefore suggest that at least part of the osteogenic activity of TGF-β1 may be attributed to the production of VEGF.

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.

Mesenchymal stem cells (MSCs) are bone marrow derived multipotent cells with the ability to self-renew and differentiate into multiple connective cell lineages. In vivo, MSCs travel from the bone-marrow to the inflammatory sites and actively participate in remodeling and regeneration process under the influence of soluble growth factors. Due to these inherent properties, MSCs have emerged as an ideal candidate for diverse regenerative therapeutic applications. The development of MSC-based therapies requires in vitro expansion of MSCs; however, MSC expansion results in phenotypical changes that have limited its efficacy upon reintroduction in vivo. In order to increase the efficacy of MSC-based therapeutics, it is critical for us to improve the current understanding of MSC interactions with its niche specific factors and explore new methods to enhance MSC function in vivo. We used tumor conditioned media, which contains soluble factors secreted by tumor cells in culture (TCM), and inflammatory niche-specific soluble factors, such as platelet derived growth factor (PDGF) and transforming growth factor-β1 (TGF-β1), to characterize the mechanical response of MSCs. The intracellular mechanical properties of MSCs were dramatically altered in response to soluble factors and MSCs displayed cytosolic stiffening in response to TCM and TGF-β1. Although PDGF treated cells did not elicit any mechanical response, blocking PDGF signaling with a small molecule inhibitor reversed the stiffening response in TGF-β1 treated cells, indicating crosstalk between these two pathways is essential in TGF-β1 mediated cell stiffening. Furthermore, a genome-wide microarray analysis revealed TGF-β1 dependent regulation of cytoskeletal actin-binding protein (ABP) genes. Actin crosslinking and bundling protein genes, which regulate cytosolic rheology through changes in semiflexible actin polymer meshworks, were upregulated with TGF-β1 treatment. Since TGF-β1 treatment profoundly altered the MSC phenotype after relatively short exposure times, we sought to understand if pretreated cells could sustain these enhanced characteristics leading to higher efficacy in vivo. We found that MSCs pretreated with TGF-β1 displayed enhanced adhesive properties while maintaining the expression profile of surface adhesion molecules even after removal of stimulus. Additionally, pretreated MSCs exposed to lineage specific induction media, demonstrated superior differentiation potential along multiple lineages. Based on the large number of sustained changes, TGF-β1 pretreated cells were used to treat full thickness skin wounds for in vivo wound healing model to determine their therapeutic efficacy. TGF-β1 pretreated MSCs increased wound closure rate and displayed enhanced migration of MSCs towards the center of the wound compared to the control cells. In conclusion, soluble factor pretreated MSCs with altered mechanical properties displayed significantly improved cell functions leading to highly efficient tissue regeneration in vivo. Mechanical priming of MSCs with niche specific factors prior to transplantation can become a viable strategy to maximize their therapeutic potential.

In the exploration into new therapeutic agents for human disease, medicinal herbs offer an enormous resource due to their wide range of biologically active components. However, because of these biologically active components, medicinal herbs can also have toxic side effects. The focus of this thesis is the effect of herbal therapies, both good and bad, on chronic kidney disease (CKD) and tubulointerstitial fibrosis. Tubulointerstitial fibrosis is considered one of the defining characteristics of CKD. In Chapter 1, the literature regarding the pathogenesis of tubulointerstitial fibrosis is reviewed, beginning with the mechanisms of its development, the main structural and functional features, and the molecular mediators. The structural features include activation of resident fibroblasts and transition of tubular epithelial cells into myofibroblasts, deposition of extracellular matrix proteins, increased apoptosis of normal cells of the renal nephron and development of tubular atrophy, increased renal oxidative stress, and hypoxia of renal tissues. Molecular mediators that are explored include angiotensin II, transforming growth factor-ß1 and numerous other cytokines and growth factors. Pharmacological manipulation of these features and their molecular mediators for regression of tubulointerstitial fibrosis is then discussed. Currently, the gold standard of therapy for people with CKD is blockade of the renin-angiotensin-aldosterone system with angiotensin converting enzyme inhibitors (ACEIs) and/or angiotensin receptor blockers (ARBs). Because of the complexity of the pathogenesis of renal fibrosis and the multiple mechanisms by which ACEIs and ARBs work, this portion of the thesis focuses on the qualities that additional agents should have to complement their actions. These additional agents could work by decreasing oxidative damage, by decreasing fibroblast numbers through apoptosis, through the interruption of inflammatory, fibrotic mediators, by increasing anti-inflammatory mediators or through other mechanisms. The literature review therefore continues with a discussion of the historical use of medicinal herbs in genitourinary pathologies and the known contributions that medicinal herbs can make to the treatment or development of tubulointerstitial fibrosis and CKD. From this review, a large number of herbs were identified as having traditional use in renal disorders or as being of interest, currently, to researchers of renal pathologies. However, much information is lacking regarding the mechanisms by which the hypothesised benefits occur, making it impossible to assess which herb(s) may offer valuable additive support or alternative treatments to the therapies currently given to people with CKD. Furthermore, there is a lack of information regarding toxicity of these herbs; many herbs have never been assessed in cell culture or in animal toxicity studies. It was apparent that preliminary in vitro work was necessary before in vivo pharmacological work could be undertaken. This thesis, therefore, aimed to test the following hypotheses: (1) That medicinal herbs used currently for treatment of renal dysfunction have high anti-oxidant properties that can be further enhanced by specific extraction processes; (2) That the in vitro testing of selected extracts from medicinal herbs, identified in (1), will reveal some anti-oxidant benefits or indications of toxicity that need careful analysis in animal studies; (3) That careful in vivo testing of specific toxic medicinal herbs identified in these leadup studies will define specific pathological processes that predict an outcome of CKD; and (4) That careful in vivo testing of selected medicinal herbs, used in conjunction with more conventional medicines for CKD, will show an additive benefit when used to ameliorate development of CKD induced using an established animal model. The subsequent laboratory work was designed to test the validity of these hypotheses and the results are then presented in Chapters that each comprise a publication. The aim of Chapter 2 was to present a systematic analysis of the oxidant properties of 55 medicinal herbs that have been used traditionally to treat kidney and urinary disorders or have been of recent interest to researchers of renal disorders. Since different extraction processes yield different constituents, each of the herbs was sequentially extracted with three solvents of decreasing polarity. An assay was performed on each of the fractions to determine the oxygen radical absorbance capacity. The aim of Chapter 3 was to test the benefit or otherwise of each of the three extracts of the chosen herbs using an in vitro cell study. Each extract was tested for potential toxic, apoptotic, mutagenic and antioxidant activity on normal mammalian renal tubular epithelial cells (NRK-52E). The effect of the extracts on renal fibroblasts (NRK-49F) was also analysed. Several specific hypotheses arose from the combination of the systematic analyses and the literature review regarding benefits and toxicities of a number of the extracts. The subsequent in vivo work was designed to test the validity of two of these hypotheses. The aim of Chapter 4 was to test the hypotheses developed from the results of the previous Chapter. The herb Dioscorea villosa had demonstrated extreme cytotoxicity to mammalian renal epithelial cells and had caused transdifferentiation of epithelial cells into fibroblasts. An in vivo rodent model was used to test chronic dosage with this herb and its toxicity and predisposition for induction of CKD verified. The aim of Chapter 5 was to determine whether a herbal preparation (Angelica sinensis and Astragalus membranaceus) that had some support from the literature and the results from Chapters 2 and 3, could complement the actions of ACEIs in a rodent model of renal fibrosis (unilateral ureteral obstruction). The combination of herbal medicines and the ACEI was significantly more effective than the ACEI alone in ameliorating several characteristics of CKD development. To conclude the thesis, Chapter 6 provides an overview discussion of the results and a critical analysis of the methods used. Further, Chapter 6 looks towards future experiments that are planned to further resolve issues of concern about effects on renal health from use of medicinal herbs. .

La fibrose pulmonaire (FP) idiopathique est une maladie sans traitement efficace caractérisée par une prolifération de myofibroblastes et par un départ sous-pleural suggérant un rôle de la plèvre. Le transforming growth factor (TGF)-ß1 induit un processus de transformation des cellules mésothéliales pleurales (CMP) en cellules de type myofibroblaste. Les protéines de choc thermique régulent la voie du TGF-ß1. L’importance de l’axe caspase-1/IL-1ß, a été décrite dans les modèles animaux de FP.La protéine de stress AlphaB-crystallin a été étudiée dans la FP au niveau des CMP et l’importance de l’axe caspase-1/IL-1ß a été recherchée au niveau des cellules structurales pulmonaires dans la toxicité de la bléomycine (BLM).aB-crystallin est surexprimée dans la FP idiopathique au niveau des CMP. Son inhibition empêche la transformation et la migration des CMP dans la fibrose pleuro-pulmonaire. Dans un modèle de FP induite par la BLM chez la souris, la voie caspase-1 est activée dans les régions pleurales. In vitro, la caspase-1 a un rôle crucial dans la transformation des CMP. Son activation induit une réaction fibrosante chez la souris. Dans une seconde partie, nous montrons qu’une forme déglycosylée de la BLM, incapable d’activer la caspase-1, n’induit pas de FP mais a une activité anti-cancéreuse. La déglyco-BLM n’entraine pas la pyroptose, mort caspase-1 dépendante, chez les cellules épithéliales alvéolaires. Nos résultats suggèrent qu’AlphaB-crystallin et la voie caspase-1/IL-1ß pourraient être des cibles thérapeutiques dans la FP idiopathique ou induite par la BLM. Nous apportons aussi une preuve de concept de l’utilisation de la déglyco-BLM comme alternative non toxique à la bléomycine. / Idiopathic Pulmonary Fibrosis (PF) is a rare and devastating disease without efficient treatment at this time. Idiopathic FP is characterized by accumulation of myofibroblasts and has a typical sub-pleural onset suggesting a role of the pleura in the disease. Transforming Growth Factor (TGF)-ß1 induces transformation of pleural mesothelial cells (PMC) into active cells exhibiting myofibroblast phenotype. Heat shock proteins can act as regulator of the TGF-ß1 signaling. A role for caspase-1/IL-1ß axis has already been described in animal models of PF.The heat shock protein AlphaB-crystallin has been studied in PF at the PMC level and the importance of caspase-1/IL-1ß axis has been investigated specifically in lung structural cells in the context of bleomycin (BLM) toxicity.AlphaB-crystallin is overexpressed by PMC during idiopathic PF. Its inhibition in mice interferes with PMC transformation and subsequent migration in pleuro-pulmonary fibrosis. In BLM-induced PF in mice, caspase-1 is activated in sub-pleural areas. In vitro, caspase-1 has a crucial role in the transformation process of PMC. Activation of caspase-1 triggers fibrotic response in mice. In a second part, we show that a deglycosylated form of BLM, which failed to promote caspase-1 activation, is unable to trigger PF but stills have an anti-tumor activity. Deglyco-BLM does not induce pyroptosis, a caspase-1 dependent cell death, in alveolar epithelial cells.Our data suggest that AlphaB-crystallin and caspase-1/IL-1ß could represent interesting therapeutic targets in idiopathic as well as BLM-induced PF. We also bring a proof of concept for the use of deglyco-BLM as a less toxic alternative to BLM in cancer therapy.

Σκοπός της παρούσας διατριβής ήταν η μελέτη των σηματοδοτικών μονοπατιών που εμπλέκονται στην επαγόμενη από IL-1β παραγωγή της MMP-1, μιας από τις κυριότερες μεταλλοπρωτεϊνάσες του εξωκυτταρικού χώρου, από ινοβλάστες ανθρώπου και η διερεύνηση του μηχανισμού της κατασταλτικής επίδρασης του TGF-β1 στην επαγόμενη από IL-1β παραγωγή της μεταλλοπρωτεϊνάσης αυτής. Η μελέτη πραγματοποιήθηκε σε ινοβλάστες αρθρικού υμένα ασθενών με οστεοαρθρίτιδα ή ρευματοειδή αρθρίτιδα, ρινικού πολύποδα και πνεύμονα. Βρέθηκε ότι η επαγωγική δράση της IL-1 πραγματοποιείται μέσω του μονοπατιού της PKC, ενώ σημαντικό ρόλο φαίνεται να παίζουν η ενεργοποίηση του NF-kB, αλλά και των μεταγραφικών παραγόντων της οικογένειας AP-1, μέσω των MAP κινασών. Το μονοπάτι, επίσης των κινασών τυροσίνης φαίνεται να συμμετέχει. Τα μονοπάτια αυτά φαίνεται ότι είναι κοινά στις ινοβλάστες, ανεξάρτητα από την προέλευση. Η δράση αυτή της IL-1β βρέθηκε ότι καταστέλλεται από το μονοπάτι cAMP/PKA στις ινοβλάστες αρθρικού υμένα και ρινικού πολύποδα όχι όμως και πνεύμονα. Ο TGF-β1 βρέθηκε ότι στις ινοβλάστες αρθρικού υμένα και ρινιού πολύποδα είχε κατασταλτική επίδραση στην επαγόμενη από IL-1β παραγωγή της MMP-1 ενώ σε αυτές από πνεύμονα είχε συνεργειακή. Η κατασταλτική δράση του TGF-β1 βρέθηκε ότι πραγματοποιείται μέσω ενεργοποίησης της PKA, κατά τρόπο ανεξάρτητο της παραγωγής cAMP. / The aim of present work was the study of signal tranduction pathways, which are implicated in IL-1β-induced production of MMP-1, one from the predominant metalloproteinases of extacellular matrix, from human fibroblasts, and the investigation of mechanism of suppressive effect of TGF-β1 on IL-1β-induced production of this metalloproteinases. The study was performed in fibroblasts with osteoarthrits or rheumatoid arthritis, nasal polyps and lung. It was found that the IL-1β-induced production of MMP-1 from fibroblasts, independently of their origin, is carried out via PKC pathway, while the activation of transcription factors NF-kB and AP-1, via MAP kinases, seems to play significant role. The tyrosine kinases pathway may also contributes. The IL-1β effect is suppressed from the cAMP/PKA pathway and nasal polyps fibroblasts but not in lung fibroblasts. TGF-β1 is able to antagonize the IL-1β-induced production of MMP-1 from synovial and nasal polyps fibroblasts, while in lung fibroblasts it exhibits synergistic effect. This suppressive effect of TGF-β1 is carried out via PKA activation, independently of cAMP production.

La fibrose pulmonaire peut être induite par différentes agressions comme certaines chimiothérapies et notamment la bléomycine. Elle est souvent idiopathique (FPI) sans étiologie retrouvée. Cette maladie, qui n’a pas de traitement efficace et un pronostic sombre, débute dans les régions sous-pleurales. Elle est caractérisée par la présence de myofibroblastes responsables de la synthèse de la matrice extracellulaire qui va s’accumuler de façon disproportionnée. Ce dépôt excessif de collagène conduira à une perte des fonctions mécaniques du poumon et une limitation des échanges gazeux. L’origine des myofibroblastes est encore discutée mais une hypothèse récente propose que les cellules épithéliales puissent, sous l’influence de Transforming Growth Factor (TGF)-β1, une cytokine majeure du processus fibrosant, se transformer en myofibroblastes par un procédé nommé transition épithélio-mésenchymateuse (EMT). Dans ce travail, nous nous sommes intéressés dans un premier temps au rôle de la plèvre dans l’initiation de la FPI en développant un nouveau modèle de fibrose pleurale, puis nous avons étudié l’implication de la petite protéine de choc thermique HSP27 dans l’EMT. Enfin, nous nous sommes intéressés à la toxicité d’un dérivé de la bléomycine, la déglyco-bléomycine. Nous avons montré dans le premier projet que la bléomycine, quelque soit son mode d’administration, pouvait, si elle était associée à la présence de nanoparticules de carbone dans l’espace pleural (particules présentes dans la fumée de cigarette et dans la pollution), induire une fibrose pleurale progressive. Cette fibrose pleurale ne se limitait pas à une accumulation de collagène au niveau de la plèvre mais se propageait dans les régions sous-pleurales. Cette invasion de la fibrose s’expliquait par la capacité des cellules mésothéliales à subir une EMT. Dans une deuxième étude nous avons montré qu’HSP27 était fortement surexprimée au niveau de la plèvre et dans le parenchyme pulmonaire lors de la fibrose pulmonaire idiopathique et également dans nos différents modèles animaux de fibrose pulmonaire et pleurale. Nous avons montré in vitro qu’HSP27 était aussi fortement surexprimée au cours de l’EMT et que sa surexpression seule suffisait à induire une EMT. Au contraire, l’inhibition de son expression in vitro bloquait l’induction d’une l’EMT par le TGF-β1 et in vivo empêchait le développement de la fibrose pleurale et la migration des cellules mésothéliales dans le parenchyme pulmonaire. Enfin dans la troisième partie de notre travail, nous avons démontré chez le rongeur que la déglyco-bléomycine, dérivé de la bléomycine, avait le même effet anti-tumoral que cet anticancéreux mais sans sa toxicité pulmonaire fibrosante. Notre travail met en lumière le rôle probable de la plèvre dans l’initiation de la fibrose pulmonaire idiopathique. Nous espérons que nos travaux sur HSP27 et sur la déglyco-bléomycine vont pouvoir rapidement déboucher sur des applications thérapeutiques chez l’homme. / Pulmonary fibrosis could be induced by a number of injuries like chemotherapy (i.e. bleomycin) or could be idiopathic (IPF). This disease has currently no treatment. IPF classically starts in sub-pleural areas and is characterized by the presence of myofibroblasts producing the extracellular matrix that will accumulated into the parenchyma resulting in impaired lung functions and respiratory failure. The origin of the myofibroblasts is still debated but one recent hypothesis suggests that epithelial cells could become myofibroblasts through the epithelial-to-mesenchymal transition (EMT). This process is initiated by Transforming Growth Factor (TGF)-β1 one of the most, if not the most important cytokine involved in fibrotic process. In this work we focused on the role of the pleura in the onset of IPF and developed a new pleural fibrosis model in mice. We studied the implication of the heat shock protein 27 (HSP27) in pulmonary fibrotic processes. We then focused on the pulmonary toxicity of the deglyco-bleomycin a product derived from the anticancerous bleomycin. We demonstrated in the first part of our work that bleomycin was able, in combination with the presence of carbon nanoparticules in the pleural space (found in cigarette’s smoke and pollution), to induce a progressive pleural fibrosis. This pleural fibrosis was associated with a progression of the disease within the subpleural area as observed in IPF patients. This invasion within the parenchyma was explained by the fact that mesothelial cells undergo an EMT. In the second part of our work, we showed that HSP27 was overexpressed in the parenchyma and the pleura of IPF patients and also in all our pleural and pulmonary fibrosis models in rodents. Furthermore we established in vitro that HSP27 was also overexpressed during EMT and that its overexpression was sufficient to induce EMT. Additionally HSP27 inhibition blocks TGF-β1 induced EMT in vitro and blocks pleural fibrosis development and mesothelial cells migration within the parenchyma in vivo. In the last project we demonstrated that deglyco-bleomycin had the same anti-tumoral effect than bleomycin in vivo and was devoided from its pulmonary toxicity. This work highlights the potential role of the pleura in initiating IPF and may open fields for the development of new therapeutics by preventing pulmonary fibrosis initiation but also progression.