Many studies have demonstrated a connection between the fibrillin matrix and TGFβ signalling, but at present the mechanistic basis for this link is unclear. An interaction between the C-terminus of Latent TGFβ Binding Protein 1 (LTBP1) and the N-terminus of fibrillin1 has previously been identified, and may have the potential to directly link the fibrillin matrix to TGFβ signalling. To investigate the structural basis for this interaction, several multi-domain fragments of fibrillin1 and LTBP1 were expressed prokaryotically and refolded in vitro. After initial characterisation to confirm folding, the structure, dynamics, and interdomain interactions of these fragments were investigated in more detail using NMR techniques. Domains in both LTBP1 and fibrillin1 appear to demonstrate folds consistent with homologous structures, and while the LTBP1 C-terminal cbEGF14-TB3-EGF3-cbEGF15 region contains many flexible linkers and few interdomain interactions, the fibrillin1 EGF2-EGF3-hyb1-cbEGF1 region appears rigid, with interfaces forming between all domains present. SPR studies were used to demonstrate binding between distinct LTBP1 and fibrillin fragments, suggesting interactions between multiple domains are involved in the LTBP1-fibrillin1 interaction. The binding sites involved were then mapped to specific residues using HSQC titration studies, and structural models for the LTBP1-fibrillin1 interaction were generated based on these data. Predictions from these models were used to target residues for site-directed mutagenesis, based on their potential involvement in salt bridges, and when certain residues were replaced with those of opposite charge, reductions in binding could be seen in the SPR assay. These key residues were consistent with a particular model of the LTBP1-fibrillin1 interaction, as derived from the HSQC titration data. The conservation of potential binding site residues through deuterostome evolution also supports an important biological role for the LTBP-fibrillin interaction.

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第22134号 / 生博第421号 / 新制||生||55(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 上村 匡, 教授 荒木 崇, 教授 井垣 達吏 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

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Disruptions in cardiac development cause congenital heart disease, the most prevalent and deadly congenital malformation. Genetic and environmental factors are thought to contribute to these defects, however molecular mechanisms remain largely undefined. Recent work highlighted potential roles of chromatin- modifying enzymes in congenital heart disease pathogenesis. Histone deacetylases, a class of chromatin-modifying enzymes, have developmental importance and recognized roles in the mature heart. This thesis aimed to characterize functions of Hdac3 in cardiac development. We found loss of Hdac3 in the primary heart field causes precocious progenitor cell differentiation, resulting in hypoplastic ventricular walls, ventricular septal defect, and mid- gestational lethality. In primary heart field progenitors, Hdac3 interacts with, deacetylates, and functionally suppresses transcription factor Tbx5. Furthermore, a disease-associated Tbx5 mutation disrupts this interaction, rendering Tbx5 hyperacetylated and hyperactive. By contrast, deletion of Hdac3 in second heart field progenitors bypasses these defects, instead causing malformations in the outflow tract and semilunar valves, with lethality prior to birth. Affected semilunar valves and outflow tract vessels exhibit extracellular matrix and EndMT defects and activation of the Tgfβ1 signaling pathway. In normal second heart field development, Hdac3 represses Tgfβ1 transcription, independent of its deacetylase activity, by recruiting the PRC2 methyltransferase complex to methylate the Tgfβ1 promoter. Importantly, knockouts of Hdac3 in differentiated cardiac cells do not fully recapitulate the progenitor-specific knockout phenotypes. These results illustrate spatiotemporal roles of Hdac3, both deacetylase-dependent and deacetylase-independent, in cardiac development, suggesting that dysregulation of Hdac3 in cardiac progenitor cells could be a contributing factor in congenital heart disease pathogenesis.

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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)

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Introduction : La prééclampsie (PE) est un trouble hypertensif caractérisé par une tension artérielle au-dessus de 140/90 mmHg et touche 2 à 8% des grossesses globalement. À ce jour, la plupart des médicaments ne peuvent traiter ou prévenir la PE. La thrombospondine-1 (THBS1) est un facteur anti-angiogénique et un activateur principal du TGF-β, ce qui pourrait également être impliqué dans l'altération de l'angiogenèse dans les placentas prééclamptiques. Objectif : Notre objectif est de décrire l'expression de la THBS1 et de tester l’effet d’un inhibiteur, le peptide LSKL, sur la vascularisation placentaire dans les placentas d'un modèle murin de PE (in vivo) et sur la capacité angiogénique des cellules endothéliales placentaires humaines (in vitro). Méthodes : L'expression de THBS1 a été mesurée dans les placentas de souris par western blot et dans les placentas humains par immunohistochimie (IHC). Des cellules endothéliales placentaires (pECs) extraites de placentas humains et des souris enceintes hétérozygotes femelles surexprimant la rénine et l’angiotensinogène humaines (hR+A+) sont traitées avec LSKL, ou son peptide témoin SLLK (cellules : 30 μmol/L pendant24h ; animal 129 μmol/mL s.c. au jour de gestation 14,5). Des pEC ont été soumises à des conditions contrôle (8% d’oxygène) ou hypoxie (0,5% d’oxygène) ou thrombine (10 unités/mL) pendant 24 h. L’angiogenèse cellulaire a été évaluée sur matrigel, les protéines évaluées par western blot et la localisation de la THBS1 ainsi que la vascularisation placentaire par IHC. Résultats : Le traitement avec LSKL augmente le nombre de tubes formées dans les pECs exposées à la thrombine ou à l’hypoxie. L’inhibiteur de la voie canonique du TGF-β, ALK5/Smad2/3 (SB-505124, SB), n’a pas modifié la capacité de formation de tubes, contrairement à l’inhibiteur de la voie non canonique, TAK1/p38 ((5Z) -7-oxozeaenol, (OXO) qui a réduit la capacité angiogénique (p<0,05). Chez le modèle de souris de PE, LSKL a significativement amélioré la vascularisation placentaire, évaluée par le contenu des cellules endothéliales CD31+ marquées par IHC (p<0,05), qui s’est accompagnée d’une phosphorylation réduite de TAK1 et ERK dans ce tissu. Conclusion : Nos résultats indiquent une augmentation de l’expression de la THBS1 dans les vaisseaux et cellules endothéliales placentaires des grossesses atteintes de PE, ainsi qu’un effet pro-angiogénique placentaire du LSKL par inhibition de la vie non-canonique du TGF-β. Ces résultats contribueront à identifier de nouvelles cibles thérapeutiques capables d'améliorer la vascularisation placentaire dans PE. / Introduction: Pre-eclampsia (PE) is a hypertensive disorder characterized by blood pressure above 140/90 mmHg and affects 2% to 8% of pregnancies globally. To date, most drugs cannot treat or prevent PE. Thrombospondin-1 (THBS1) is an anti-angiogenic factor and a major activator of TGF-β, which may also be involved in impaired angiogenesis in pre-eclamptic placentas. Objective: Our aim is to describe the expression of THBS1 and to test the effect of its inhibitor, LSKL peptide, on placental vascularization in placentas from a mouse model of PE (in vivo) and on the angiogenic capacity of human placental endothelial cells (in vitro). Methods: THBS1 expression was measured in placentas of mouse by western blot and in human placentas by immunohistochemistry (IHC). Placental endothelial cells (pECs) extracted from hu-man placentas and pregnant female heterozygous mice overexpressing human renin and angio-tensinogen (hR+A+) were treated with LSKL or its control peptide SLLK (cells: 30 μmol/L for 24h; animals: 129 μmol/mL subcutaneously on gestation day 14.5). pECs were subjected to control conditions (8% oxygen) or hypoxia (0.5% oxygen) or thrombin (10 units/mL) for 24 hours. Cellular angiogenesis was assessed on Matrigel, proteins were evaluated by western blot, and the locali-zation of THBS1 as well as placental vascularization were examined by immunohistochemistry (IHC). Results: Treatment with LSKL increased the number of tubes formed in pECs exposed to thrombin or hypoxia. The inhibitor of the canonical TGF-β pathway, ALK5/Smad2/3 (SB-505124, SB), did not alter tube formation capacity, unlike the inhibitor of the non-canonical pathway, TAK1/p38 ((5Z)-7-oxozeaenol, (OXO)), which reduced angiogenic capacity. In the mouse model of PE, LSKL significantly improved placental vascularization, assessed by CD31+ endothelial cell content marked by IHC, which was accompanied by reduced phosphorylation of TAK1 and ERK in this tissue. Conclusion: Our results indicate an increase in THBS1 expression in the vessels and placental endothelial cells of pregnancies affected by PE, as well as a pro-angiogenic effect of LSKL through the inhibition of the non-canonical TGF-β pathway. These findings contribute to identifying new therapeutic targets capable of improving placental vascularization in PE.