This thesis deals with the determination of biological activity of adiponectin, a recombinant protein using cell culture. First it was important to acquire the working skills for the cell culture of cell line 3T3-L1. An optimal concentration of inactivated fetal bovine serum in cell culture media was determined. A stimulation of the cell proliferation by HB-EGF, PDGF-BB and bFGF growth factors was observed at various concentration levels. Afterwards the biological activity of adiponectin was determined as an inhibition of growth stimulation with 5 ng/ml PDGF-BB. This biological activity assay for adiponectin was also conducted with lyophilized adiponectin and a growth factor bFGF (0.1 ng/ml). The lyophilization did not affect the biological activity of adiponectin.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18886号 / 医博第3997号 / 新制||医||1009(附属図書館) / 31837 / 京都大学大学院医学研究科医学専攻 / (主査)教授 三森 経世, 教授 戸口田 淳也, 教授 開 祐司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

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Sisteminė sklerozė (SSc) – viena sunkiausių ir fatališkiausių autoimuninių sisteminių reumatinių ligų, o bazinių vaistų stygius, šiai ligai gydyti, itin didelis. Į onkologinę klinikinę praktiką įdiegtas tirozinkinazių inhibitorius – imatinibo mezilatas(IM). IM blokuoja TGF-β ir PDGF intraląstelinio signalo perdavimą ir taip sąlygoja fibrozės prevenciją SSc pelių modelyje. Mums buvo svarbu išsiaiškinti, ar imatinibas gali turėti įtakos ne tik prevencijai, bet ir susiformavusiai fibrozei. Be to TGF-β ir PDGF blokavimas angiogenezėje, galėtų riboti daug žadančio fibrozės inhibitoriaus IM naudojimą gydant SSc. Darbo tikslas: įvertinti imatinibo mezilato poveikį fibrozės procesui ir endoteliui sisteminės sklerozės eksperimentiniuose modeliuose ir ląstelių kultūrose. Darbo uždaviniai: įvertinti imatinibo efektyvumą neuždegiminiame SSc modelyje ir patikrinti imatinibo mezilato efektyvumą uždegiminiame suformuotos fibrozės modelyje; ištirti, ar terapinės imatinibo mezilato koncentracijos daro neigiamą poveikį gyvybinėms endotelio funkcijoms; įvertinti imatinibo mezilato poveikį angiogenezės etapams. Mūsų gauti duomenys rodo, kad: IM ne tik sustabdė bet ir paskatino jau egzistuojančios (bleomicino sukeltos) odos firbrozės regresiją; IM ryškiai sumažino poodžio ir odos storį, bei normalizavo miofibroblastų skaičių Tsk-1 pelėse; IM neturėjo poveikio endotelio ląstelių bazinėms funkcijoms; IM neturėjo neigiamo poveikio angiogenezės etapams. / Systemic sclerosis (SSc) – is one of the most complicated and fatal systemic diseases, and the lack of effective therapy is very evident. The tyrosine kinase inhibitor imatinib mesylate (IM) was shown to inhibit TGF-β and PDGF signaling pathways and prevent the development of dermal fibrosis upon challenge with bleomycin in murine model of SSc. The aim of therapy is not only to stop disease progression, but even induce regression of preexisting fibrosis. On other hand, blocking TGF-β and PDGF signaling in angiogenesis might worsen the vascular manifestations of SSc. We found important to evaluate effectiveness of IM for the treatment of pre-established tissue fibrosis and to exclude that the anti-fibrotic effects of IM are complicated by inhibitory effects on endothelial cell functions. Aim of the study: assess the effect of IM on the process of fibrosis and endothelium in experimental models of systemic sclerosis and cell cultures. Objectives of the study: assess the effectiveness of IM on murine models of established fibrosis; evaluate if IM has an effect on basal functions of endothelial cells; assess effect of IM on the process of angiogenesis. We have shown that IM exerts potent antifibrotic effects in two different models of SSc. Imatinib was effective for prevention of fibrosis and for treatment of established dermal fibrosis. We’ve demonstrated that IM does not inhibit major functions of endothelial cells. Thus, IM might not augment further the preexisting vascular... [to full text]

Systemic sclerosis (SSc) – is one of the most complicated and fatal systemic diseases, and the lack of effective therapy is very evident. The tyrosine kinase inhibitor imatinib mesylate (IM) was shown to inhibit TGF-β and PDGF signaling pathways and prevent the development of dermal fibrosis upon challenge with bleomycin in murine model of SSc. The aim of therapy is not only to stop disease progression, but even induce regression of preexisting fibrosis. On other hand, blocking TGF-β and PDGF signaling in angiogenesis might worsen the vascular manifestations of SSc. We found important to evaluate effectiveness of IM for the treatment of pre-established tissue fibrosis and to exclude that the anti-fibrotic effects of IM are complicated by inhibitory effects on endothelial cell functions. Aim of the study: assess the effect of IM on the process of fibrosis and endothelium in experimental models of systemic sclerosis and cell cultures. Objectives of the study: assess the effectiveness of IM on murine models of established fibrosis; evaluate if IM has an effect on basal functions of endothelial cells; assess effect of IM on the process of angiogenesis. We have shown that IM exerts potent antifibrotic effects in two different models of SSc. Imatinib was effective for prevention of fibrosis and for treatment of established dermal fibrosis. We’ve demonstrated that IM does not inhibit major functions of endothelial cells. Thus, IM might not augment further the preexisting vascular... [to full text] / Sisteminė sklerozė (SSc) – viena sunkiausių ir fatališkiausių autoimuninių sisteminių reumatinių ligų, o bazinių vaistų stygius, šiai ligai gydyti, itin didelis. Į onkologinę klinikinę praktiką įdiegtas tirozinkinazių inhibitorius – imatinibo mezilatas(IM). IM blokuoja TGF-β ir PDGF intraląstelinio signalo perdavimą ir taip sąlygoja fibrozės prevenciją SSc pelių modelyje. Mums buvo svarbu išsiaiškinti, ar imatinibas gali turėti įtakos ne tik prevencijai, bet ir susiformavusiai fibrozei. Be to TGF-β ir PDGF blokavimas angiogenezėje, galėtų riboti daug žadančio fibrozės inhibitoriaus IM naudojimą gydant SSc. Darbo tikslas: įvertinti imatinibo mezilato poveikį fibrozės procesui ir endoteliui sisteminės sklerozės eksperimentiniuose modeliuose ir ląstelių kultūrose. Darbo uždaviniai: įvertinti imatinibo efektyvumą neuždegiminiame SSc modelyje ir patikrinti imatinibo mezilato efektyvumą uždegiminiame suformuotos fibrozės modelyje; ištirti, ar terapinės imatinibo mezilato koncentracijos daro neigiamą poveikį gyvybinėms endotelio funkcijoms; įvertinti imatinibo mezilato poveikį angiogenezės etapams. Mūsų gauti duomenys rodo, kad: IM ne tik sustabdė bet ir paskatino jau egzistuojančios (bleomicino sukeltos) odos firbrozės regresiją; IM ryškiai sumažino poodžio ir odos storį, bei normalizavo miofibroblastų skaičių Tsk-1 pelėse; IM neturėjo poveikio endotelio ląstelių bazinėms funkcijoms; IM neturėjo neigiamo poveikio angiogenezės etapams.

L’hypertension artérielle pulmonaire (HTAP) est une maladie rare définie par une augmentation de la pression artérielle pulmonaire moyenne due à un remodelage progressif des artérioles pulmonaires, menant à une défaillance du ventricule cardiaque droit. Le remodelage vasculaire est la conséquence d’une dysfonction endothéliale conduisant à une hyperprolifération et à un défaut d’apoptose des cellules vasculaires pulmonaires. Le récepteur NMDA (NMDAR), un récepteur au glutamate connu pour son rôle dans la plasticité neuronale et la transmission synaptique, a été récemment identifié comme acteur de ce remodelage vasculaire. Cependant, le sous-type de NMDAR impliqué n’est pas connu. Le développement de traitements potentiels ciblant le NMDAR nécessite de mieux comprendre quelles sous-unités du récepteur sont mobilisées dans la maladie. Dans la mesure où la sous-unité GluN2A est impliquée dans la survie des neurones et la sous-unité GluN2B dans leur mort, nous avons fait l’hypothèse que la composition des NMDARs vasculaires pulmonaires devait être dérégulée dans l’HTAP. Par conséquent, cette thèse a pour objectifs i) d’étudier la composition du NMDAR dans l’HTAP, ii) d’en identifier les conséquences fonctionnelles, et iii) d’explorer son intégration au sein de la physiopathologie de l’HTAP.Nous avons montré que l’expression de la sous-unité GluN2B est réduite dans les artères pulmonaires des patients HTAP comparés à des sujets non-HTAP, malgré l’augmentation de l’expression de la sous-unité obligatoire GluN1, suggérant une commutation de l’expression des NMDARs de type GluN2B vers d’autres sous-types. Nous avons également montré que les NMDARs de type GluN2B sont rapidement et transitoirement recrutés à la membrane des cellules musculaires lisses (CMLs) en réponse à un facteur de croissance, le PDGF, par l’intermédiaire des Src family kinases (SFKs). En utilisant un inhibiteur spécifique des NMDARs de type GluN2B, nous avons observé qu’ils réduisaient la prolifération et la migration dépendantes du PDGF, indiquant une boucle de rétrocontrôle négatif. Ces résultats suggèrent une signalisation croisée entre le PDGFR-β, les SFKs et les NMDARs de type GluN2B. Ainsi le déficit en NMDARs de type GluN2B chez les patients HTAP pourrait potentialiser la réponse proliférative et migratoire au PDGF, une voie suractivée dans l’HTAP. De plus, nous avons montré que les NMDARs de type GluN2A sont recrutés de façon prolongée à la membrane des CMLs lors d’une stimulation par le PDGF. Néanmoins, le rôle précis des récepteurs de type GluN2A dans l’HTAP reste à découvrir. Pour approfondir le rôle du NMDAR dans la physiopathologie de l’HTAP, nous avons mené une étude bio-informatique complémentaire afin de modéliser les voies de signalisation impliquant le NMDAR dans l’HTAP. Nous avons construit et connecté en réseau les bases de connaissance sur les acteurs de l’HTAP d’une part, et les voies de signalisation impliquant le NMDAR dans le système nerveux central d’autre part. Nous avons montré que ces réseaux positionnent le NMDAR au cœur de nombreuses voies de signalisation caractéristiques de l’HTAP, dont celle du PDGFR-β.Ainsi, nous avons montré que l’expression membranaire des récepteurs de type GluN2A et GluN2B est dérégulée dans l’HTAP, orientant probablement la réponse au glutamate dépendante du PDGF vers les récepteurs de type GluN2A. Les conséquences d’un tel déséquilibre sont l’augmentation de la prolifération et de la migration des CMLs vasculaires pulmonaires. De plus le manque de récepteurs de type GluN2B est une caractéristique physiopathologique nouvelle dans l’HTAP et dans la compréhension du mode d’action des NMDARs périphériques en général. Enfin, le NMDAR semble être un acteur central dans la physiopathologie de l’HTAP, interagissant avec de nombreuses voies de signalisation impliquées dans la maladie, suggérant de nouvelles pistes pour avancer dans la compréhension des mécanismes physiopathologiques de l’HTAP. / Pulmonary arterial hypertension (PAH) is a rare disease defined by an increase in mean pulmonary arterial pressure due to progressive obstruction of the small pulmonary arteries, leading to right heart failure and death. The vascular remodeling is a consequence of complex and multiple patho-mechanisms, including endothelial cells dysfunction and hyperproliferation of smooth muscle cells in the pulmonary vascular wall. The N-methyl-D-aspartate receptor (NMDAR), a glutamate receptor, has been recently identified as playing an active role in this vascular remodeling. It has been shown that in pulmonary arteries of PAH patients, NMDAR is overexpressed and overactivated and is involved in the proliferation and resistance to apoptosis of pulmonary vascular cells. However, the NMDAR subtype involved in this process remains unknown. The development of potential treatments targeting the NMDAR requires a better understanding of its subunit involvement in the disease. Since the GluN2A subunit is involved in the survival of neurons and the GluN2B subunit in their death, we hypothesized that the pulmonary vascular NMDAR subunit composition could be dysregulated in PAH. Therefore, in this thesis study we aimed to: i) study the composition of NMDAR in PAH, ii) explore its functional consequences in the pathophysiology of PAH, and iii) uncover its integration in the pathophysiology of PAH.We showed that the expression of the GluN2B subunit is reduced in the pulmonary arteries of PAH patients compared to non-PAH subjects. This occurs despite the overall increased expression of the obligatory GluN1 subunit, suggesting a switch from GluN2B-type receptors to, at least, another GluN2-type receptor. We also showed that in the presence of PDGF-BB, there is an immediate increase in the levels of phosphorylated Src family kinases (SFKs), associated to an increase in phosphorylated GluN2B (the active form) that were relocated to the cell membrane, suggesting the cross-talk between PDGF, SFKs and NMDAR. To validate the pathway, we inhibited the activation of PDGFR-β or SFKs, and in both cases the phosphorylation of GluN2B after PDGF stimulation was aborted. To assess the functional importance of this pathway, proliferation and “wound healing” tests were performed. The results clearly showed that selective inhibition of GluN2B, in the presence of PDGF, significantly increased both migration and proliferation of PASMCs. These results suggest that the lack of GluN2B-type receptors in PAH may potentiate SMC proliferative and migratory response to PDGF, a well-known overactivated pathway in PAH. In addition, we showed that GluN2A-type NMDARs arerecruited to the SMC membrane following PDGF stimulation, but the precise role of GluN2A-type NMDARs in PAH remains elusive. To further explore the crosstalk between the NMDAR and the PDGF receptor (PDGFR) pathways, we conducted a complementary bioinformatics study. To provide a model of the NMDAR signaling pathways in PAH we constructed and connected comprehensive knowledge bases of the actors involved in PAH on one hand and the signaling pathways involving NMDAR within the central nervous system on the other hand. Within these networks the NMDAR was revealed as a central downstream effector of the hallmark signaling pathways of PAH, including that of PDGFR.These results indicate that the membrane expression of GluN2A-type and GluN2B-type receptors is dysregulated in PAH, presumably switching the PDGF-dependent glutamate response towards GluN2A-type receptors. The consequences of such imbalance are the increased proliferation and migration of pulmonary vascular SMCs. Moreover, the lack of GluN2B-type NMDARs is a new feature in the pathophysiology of PAH and in the understanding of peripheral NMDA receptors in general. Besides, the NMDAR seems to be a central effector in PAH, interacting with multiple hallmark pathways of the disease, suggesting new tracks to further understanding the pathophysiology of PAH.

Recruitment of mural cells, i.e. pericytes and smooth muscle cells (SMC), is essential to improve the maturation of newly formed vessels. One of the major factors involved in this process is the endothelial cell-secreted Platelet-Derived Growth Factor BB (PDGF BB). Sonic hedgehog (Shh) has also been suggested to promote the formation of larger and more muscularized vessels, but the underlying mechanisms involved have not yet been elucidated. We first identified Shh as a target of PDGF BB and found that SMC respond to Shh not only by upregulating the Gli1-dependent canonical pathway, but also by activating ERK1/2 and PI3K-dependent non-canonical pathways. Moreover, we found that PDGF BB-induced SMC migration, involves Shh-dependent PI3K, ERK1/2 and Gli1 activation. In the mouse model of corneal angiogenesis, PDGF BB and Shh were expressed by endothelial cells and mural cells of VEGF-induced newly formed blood vessels, respectively. PDGF BB inhibition reduced Shh expression, confirming that Shh is a target of PDGF BB, as demonstrated by in vitro experiments. Finally, we found that inhibition of either PDGF BB or Shh signaling reduced NG2+ mural cell recruitment into neovessels and subsequently reduced the neo-vessel lifespan. In this work, we demonstrate, for the first time, that Shh is a key mediator of PDGF BB-induced mural cell migration and recruitment into neo-vessels and elucidates the molecular signaling pathway involved in this process. / Recruitment of mural cells, i.e. pericytes and smooth muscle cells (SMC), is essential to improve the maturation of newly formed vessels. One of the major factors involved in this process is the endothelial cell-secreted Platelet-Derived Growth Factor BB (PDGF BB). Sonic hedgehog (Shh) has also been suggested to promote the formation of larger and more muscularized vessels, but the underlying mechanisms involved have not yet been elucidated. We first identified Shh as a target of PDGF BB and found that SMC respond to Shh not only by upregulating the Gli1-dependent canonical pathway, but also by activating ERK1/2 and PI3K-dependent non-canonical pathways. Moreover, we found that PDGF BB-induced SMC migration, involves Shh-dependent PI3K, ERK1/2 and Gli1 activation. In the mouse model of corneal angiogenesis, PDGF BB and Shh were expressed by endothelial cells and mural cells of VEGF-induced newly formed blood vessels, respectively. PDGF BB inhibition reduced Shh expression, confirming that Shh is a target of PDGF BB, as demonstrated by in vitro experiments. Finally, we found that inhibition of either PDGF BB or Shh signaling reduced NG2+ mural cell recruitment into neovessels and subsequently reduced the neo-vessel lifespan. In this work, we demonstrate, for the first time, that Shh is a key mediator of PDGF BB-induced mural cell migration and recruitment into neo-vessels and elucidates the molecular signaling pathway involved in this process.

Die Studien der vorliegenden Habilitationsschrift hatten die Untersuchung der Abhängigkeit diffuser und tumorförmiger Hypervaskularisationen der Plazenta von angiogenen Wachstumsfaktoren zum Ziel. Kernaussagen sind, daß in Chorangiomen eine höhere Expression der Angiopoietine - 1 und - 2 vorliegt, bei morphologisch gleicher Rezeptorausstattung. Keine Differenzen zeigte die Expression von PDGF sowie seinem Rezeptor PDGF-ß. Bei Differenzierung solitärer und multipler Chorangiome (Chorangiomatosen) hinsichtlich klinischer Parameter sowie der Expression der angiogenen Wachstumsfaktoren Angiopoietin-1 und bFGF sowie hinsichtlich der Proliferationsrate konnten Unterschiede zwischen den plazentaren Läsionen deutlich werden. Die Expression der Wachstumsfaktoren war jeweils in Chorangiomatosen kräftiger als in solitären Veränderungen. Das Auftreten von Chorangiomatosen steht offensichtlich in Zusammenhang mit einer verstärkten Expression dieser Wachstumsfaktoren, die damit noch die ohnehin in Chorangiomen - gegenüber ortholog gereiftem Plazentaparenchym - gesteigerte Expression übertrifft. So deutet diese Studie nunmehr verstärkt auf eine Korrelation zwischen der Höhe der Wachstumsfaktorexpression und dem quantitativen Auftreten lokalisierter Angiopathien hin. Zudem ließen sich klinische Korrelationen aufzeigen. So lag das mütterliche Alter beim Auftreten lokalisierter hypervakularisierter Veränderungen deutlich über der bei unauffälligen Plazenten, ein Ergebnis, das vorherige Untersuchungen bestätigte. Geringe Differenzen das maternale Alter betreffend bestanden zwischen Chorangiomen und Chorangiomatosen. Hinsichtlich der Schwangerschaftskomplikationen fanden sich keine Unterschiede zwischen Chorangiomen und Chorangiomatosen. Begleitende Reifungsstörungen fanden sich auch in diesem Studienkollektiv, dahingehend, daß Chorangiome und häufiger noch Chorangiomatosen kombiniert mit diesen Auftreten. Untersuchungen zur Apoptose- und Proliferationsrate in Chorangiomen mit differierendem histologischen Subtyp erbrachten keinen Hinweis auf den Einfluß der Apoptose auf das Wachstum dieser tumorartigen Läsionen. In Chorangiomen zeigte sich im Vergleich zu regelhaft entwickeltem reifem Plazentagewebe eine leicht gesteigerte Proliferationsrate. Auffällig war einzig, daß Chorangiome endotheliomatösen Subtyps die höchste Proliferationsrate aufwiesen. Studien intrauteriner Todesfällen der frühen und mittleren Fetalperiode konnten zeigen, das diffuse Hypervaskularisationen offensichtlich reaktiver Natur sind. Untersuchungen zur Expression angiogener Wachstumsfaktoren zeigten, daß diese exprimiert werden und damit bereits an der Gefäßentwicklung der Plazenta in der Fetalperiode beteiligt sind. Unterschiede hinsichtlich der Expression der Wachstumsfaktoren in Plazenten mit Hypervaskularisationen und Kontrollproben fanden sich jedoch morphologisch nicht. Die gesteigerte Gefäßbildung in der frühen Fetalperiode ist offenbar eine reaktive Anpassung der Zotten auf die Hypoxie. Falldarstellungen zweier weiterer Plazentatumoren, eines inzidentellen Chorangiokarzinoms sowie eines Angiomyxoms der Nabelschnur erbrachten hinsichtlich der Gefäßkomponente in diesen Tumoren keine Abhängigkeit von den untersuchten Wachstumsfaktoren. Eine Studie von 136 Chorangiomen aus 22439 Plazenten ließen aussagefähigen Analysen der Häufigkeit, morphologischer Charakteristika und Korrelationen zwischen Chorangiomen und klinischen Befunden zu. So zeigten sich signifikante Abhängigkeiten zwischen dem Auftreten eines Chorangioms und dem mütterlichen Alter, der gestationsbedingten Hochdrucksymptomatik, der Parität, dem kindlichen Geschlecht und begleitender Reifungsstörungen der Plazenta. Insbesondere die Reifungsstörungen in Kombination zum Auftreten eines Chorangioms waren zuvor nie Gegenstand einer größeren wissenschaftlichen Studie. Einzelne Parameter fanden zwar sporadisch in einzelnen Publikationen mit wenigen Chorangiomen wissenschaftlich Beachtung, doch die dort publizierten Ergebnisse widersprachen sich vielfach. So ist die jetzt vorliegende Untersuchung in ihrer Größe und damit Aussagekraft sehr relevant. / The studies had the investigation of the dependence of vague and tumoric hypervascularisation of the placenta on angiogenic growth factors as a goal. Core statements are that in chorangiomas a higher expression of Angiopoietin-1 and - 2 is present, during morphologically same receptor equipment. The expression of PDGF as well as ist receptor did not show differences PDGF - beta. With differentiation of solitär and multiple chorangiomas (chorangiomatoses) regarding clinical parameters as well as the expression of the angiogenic growth factors Angiopoietin-1 and bFGF as well as regarding the proliferation rate could become clear differences between the placental lesions. The expression of the growth factors was in each case in chorangiomatoses stronger than in single changes. The occurrence of chorangiomatoses stands obviously in connection with a strengthened expression of these growth factors, which exceeds thereby still anyway the expression increased in chorangiomas - opposite ortholog matured placental tissue. Thus this study points now strengthened on a correlation between the height of the expression on growth factors and the quantitative occurrence of located angiopathia. Besides clinical correlations could be pointed out. Thus the maternal age was clearly with the occurrence of located such changes over with inconspicuous placentas, a result, which confirmed previous investigations. Small differences concerning the maternal age existed between chorangiomas and chorangiomatoses. Regarding the pregnancy complications were no differences between chorangiomas and chorangiomatoses. Accompanying maturing disturbances were in such a way also in this study collective, that chorangiomas combines and more frequently still chorangiomatoses with this occurrence. Investigations to the apoptoses and proliferation rate in chorangiomas with differing histological subtyp did not furnish a reference to the influence of the apoptoses on the growth of these tumor-like lesions. To chorangiomas an easily increased proliferation rate pointed itself compared with regularly developed ripe placental tissue. Remarkably it was only that chorangiomas with endotheliomatos subtyp the highest proliferation rate exhibited. Studies of intrauterin deaths of the early and middle fetal periode could show, which are vague hypervascularisation of obviously reactive nature. Investigations to the expression of angiogenic growth factors showed the fact that these are exprime and so that in the container development of the placenta in the fetal periode is involved already. Differences regarding the expression of the growth factors in plazentas with hypervascularisationen and inspection samples were not however morphologically. The increased container formation in the early fetal periode is obviously a reactive adjustment of the villous trees on the hypoxia. Drop representations of two further placenta tumors, a incidentially chorangiocarcinoma as well as a angiomyxoma of the umbilical cord did not furnish dependence on the examined growth factors regarding the container component in these tumors. Expressive analyses of the frequency, morphologic characteristics and correlations between chorangiomas and clinical findings permitted a study of 136 chorangiomas from 22439 plazentas. Thus significant dependence between the occurrence of a chorangioma and the maternal age, the gestational high pressure symptomatology, the parity, the childlike sex showed up and accompanying maturing disturbances of the placenta.

No description available.