京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21018号 / 医博第4364号 / 新制||医||1028(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 小川 誠司, 教授 柳田 素子, 教授 江藤 浩之 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Cardiovascular diseases have always been the leading cause of global morbidity and mortality. Aortic stenosis, which is a kind of cardiovascular disease has a high prevalence in elderlies that are 75 years and older. Currently, the only available treatment would be valve replacement surgery. Recently, a few studies have risen regarding the potential of extracellular vesicles to reduce the effects of aortic stenosis, hence allowing patients to opt for a non-life-threatening treatment in comparison to a surgical one. The goal within this study is to determine the pro-coagulability of extracellular vesicles (EVs) that were endogenously derived from human blood (patients and healthy individuals) and their effect on the coagulation cascade. This study was performed on cardiac spheroids that were formed through seeding human aortic endothelial cells in an ultra-low attachment 96-well plate for 96 hours. Spheroids were challenged with tumour necrosis factor-alpha (TNFα) for 24 hours prior to EVs incubation for 48 and 72 hours. The effects of EVs on these spheroids were observed in terms of their ability to induce tissue factor activity. There was no significant difference in the tissue factor activity between spheroids incubated with patient derived EVs or healthy individual derive EVs irrespective of TNFα challenge. To conclude, the results of this study were not significant to stipulate that extracellular vesicles are procoagulant. Hence, further research regarding their ability to reduce or rescue the effects of cardiovascular diseases needs to be performed.

Exosomer är extracellulära vesiklar i nanostorlek som frigörs från cellerna till den extracellulära matrisen. Exosomer är laddade med nukleinsyror, proteiner, och lipider, och fungerar som kommunikatorer mellan celler. Det har gjort dem mycket attraktiva for forskning inom terapi, diagnostik och transport av läkemedel. För att använda exosomer i kliniska tillämpningar behöver standardiserade metoder för isolering, rening och analys av exosomer att utvecklas.  Detta projekt syftar till att sätta upp en snabb metod som använder "multiangle light scattering" (MALS) i kombination med kromatografi for att bestämma storleken på exosomer. Olika storlekskromatografi (SEC)/jonbyte (IEX)-kolonner kommer att undersökas och användas for analys av storlekar på exosomer. Partikelstorleken som erhålls från MALS kommer sedan att verifieras med nanoparticle tracking analysis (NTA).  För SEC-MALS-analyserna eluerades exosomerna i dödvolymen. IEX-MALS-metoden separerade exosomerna enligt resultatet. Exosomer i storlek mellan 60-110 nm eluerades ut med 800 mM NaCl. Större exosomer och eventuella aggregat i storlek 120-200 nm eluerades ut med 1200 mN NaCl.  Resultatet visar att signalen från MALS indikerar var exosomer i kromatogrammen elueras ut och kan ge värdefull information om storleksfördelningen i en topp. SEC-MALS-resultatet är dock inte reproducerbart, eftersom det ibland sker en förändring i storleksfördelningen över toppen. Resultatet har också visat att arean for toppen i dödvolymen varierar mellan körningarna, vilket förmodligen orsakats av att exosomer interagerar med den stationära fasen eller filtret i kolonnen. Förskjutningen i storleksfördelning observerades också i IEX­MALS-metoden. Medelvärdet av partikelstorlekarna som beräknades från SEC-MALS överensstämmer med storlekarna beräknade med NTA. SEC-MALS-metoden behöver förbättras för att fa reproducerbara resultat. Den beräknade storleken från IEX-MALS stämde inte överens med storleken från NT A. Jonbytesanalysen skulle kunna upprepas och fraktioner skulle kunna analyseras med andra tekniker för att verifiera resultatet i framtida arbete. Effekten av den höga saltkoncentrationen på exosomerna behöver också undersökas ytterligare. / Exosomes are nanosized extracellular vesicles released from the cells into the extracellular space. Exosomes are loaded with nucleic acids, proteins, and lipids, and work as communicators among cells. This has made them very attractive for research in therapeutics, diagnostics and drug delivery applications. Standardised exosome isolation, purification, and analysis methods need to be developed to use exosomes in clinical applications.  This project aimed to set up a quick method using multiangle light scattering (MALS) combined with chromatography techniques to determine exosome sizes. Different size exclusion (SEC)/ion exchange (IEX) columns will be investigated and used to analyse exosome sizes. The particle size obtained from MALS will then be verified with nanoparticle tracking analysis (NTA).  For the SEC-MALS analysis, the exosomes were eluted in the void volume. The IEX-MALS method separated the exosome sample. The exosome eluted with 800 mM NaCl ranged between 60-110 nm in diameter. The exosomes eluted with 1200 mM NaCl ranged between 120-200 nm in diameter. The result shows that the light scattering intensity from MALS indicates where the exosomes elute in the chromatograms and gives valuable information about the size distribution in a peak. However, the SEC-MALS result is not reproducible, as sometimes, a shift in the size distribution over the peak occurs.  The result has also shown that the void peak area varies between the runs, caused mainly by the exosomes interacting with the resin or the column's filter. The shift in size distribution was also observed in the IEX-MALS method.  The average sizes calculated from SEC-MALS agree with the sizes calculated with NTA. The SEC-MALS method needs to be improved to obtain reproducible results. The calculated size from IEX-MALS did not agree with the size from NTA. The ion exchange analysis could be repeated and further analysed with other techniques to verify the result in future work. The effect of the high salt concentration on the exosomes also needs to be further investigated.

Despite progress in our understanding of the molecular drivers that propagate the overall process of metastasis, the adaptation of specific organs upon these molecular interactions for metastatic entry remains poorly understood. This is particularly true for liver metastases, the liver being a common site for metastatic disease, and metastatic hepatic tumors are more prominent than primary hepatocellular or biliary tumors. Liver metastases most commonly arise from colorectal cancer than any other cancer and constitute one of the most detrimental outcomes of cancer, characterized by poor prognosis, high mortality, and no effective therapies available other than surgical interventions. Since interactions between tumour cells and the tumour microenvironment play an important part in the engraftment, survival, and progression of the metastases, the discovery of new drivers of liver metastasis with the potential to become therapeutic and preventive targets is required to advance the care of liver metastasis patients as well as cancer patients at risk of metastatic spread to the liver. The alteration of the physical structure of the tissue is extremely important in the progression of malignant diseases, such as cancer metastasis, as it directly affects the extravasation and colonization of tumour cells. The major hurdles in liver metastasis research, stem not only from our insufficient understanding of the molecular mechanisms directing and mediating metastasis particularly to the liver but also from the limited number of pre-clinical models available that mimic human disease and enable the study of the complex interactions between tumor cells and the liver microenvironment. The liver metastatic process underlies the acquisition of key adaptations by tumor-derived factors and is determined by both tumour-intrinsic properties and the crosstalk between tumour cells and stromal cells in the liver. A normal functioning and structurally intact extracellular matrix (ECM) constitute a hostile “soil” for seeding tumor cells to colonize. Eventually, it is the ability of tumor cells to remodel the liver microenvironment and create a supportive niche for metastatic tumor cell survival and outgrowth that determines successful metastatic colonization. Among tumour-secreted factors, which are recognized as major contributors to the formation of pre-metastatic and metastatic niches, tumor-derived extracellular vesicles (EVs) have recently arisen as crucial players in cell-to-cell communication and in the remodeling of distant microenvironments that favor organ-specific metastasis. Therefore, we sought to determine the role of tumor-derived EVs in the modulation of the liver microenvironment and their specific contribution to supporting metastatic colonization of the liver. The preliminary step to this process was to establish a model system to identify EV-associated targets and their effect on the ECM remodelling. Immunohistochemical analyses of primary colon tumour (CRC) and secondary liver metastases (CRC liver MET) tissue samples from patients with CRC revealed higher stromal TIMP1 levels in CRC liver MET than in CRC. The elevated stromal TIMP1 signature in the invasive front was associated with poor progression-free survival in patients with CRC liver MET. Our characterisation of the CRC tumour-derived EVs showed TIMP1 enrichment in the EVs (TIMP1EV) compared to its parental cell. Using cultures of primary liver fibroblasts, we could demonstrate that TIMP1 enrichment in the CRC-EVs was associated with regulation of TIMP1 levels in the EV-conditioned liver fibroblasts. Using our optimized ex vivo 3D ECM remodelling assay, we observed that pre-conditioning the liver fibroblasts with EVs from CRC cells promotes ECM remodelling. In accordance with our cell line model, we showed that serum-derived TIMP1EV from CRC patients promotes ECM remodelling. Moreover, high serum TIMP1EV expression in CRC liver MET patients was significantly associated with poor overall survival. In addition, our data also indicated that the determination of EV-associated TIMP1 is superior for non-invasive diagnosis than the analysis of soluble TIMP1 from total serum. Finally, we showed that HSP90AA is constitutively bound to TIMP1EV and that targeting HSP90AA leads to TIMP1 downregulation and inhibits ECM-mediated remodelling. This study defining the contribution of extravesicular TIMP1 to liver metastasis brings a novel insight into the molecular mechanisms through which tumor-secreted factors packaged via EVs promote remodelling of the liver microenvironment. The clinical significance of overexpression of extravesicular TIMP1 in patients with colorectal liver metastases highlights its potential as a prognostic biomarker and therapeutic target. With further clinical studies, Heparin and HSP90 inhibitors targeting the EV mediated TIMP1 regulation could be a putative treatment strategy to treat colorectal liver metastases.:Table of Contents Abbreviations v 1. Introduction 1 1.1 Colorectal cancer 1 1.1.1. Incidence and mortality 1 1.1.1. Tumor staging 2 1.1.1. Pattern of distant metastases in colorectal cancer 5 1.2 Colorectal liver metastases 6 1.2.1 Current evaluation and treatment strategies for colorectal liver metastases 7 1.2.2 The liver metastasis cascade - a multi-step process 10 1.3 Tumor microenvironment 12 1.3.1 Tumour-stroma interactions 15 1.3.2 ECM remodelling and its role in CRC tumor progression 17 1.4 Extracellular vesicles 21 1.4.1 EV types 21 1.4.2 Biogenesis and secretion of EVs 22 1.4.3 Molecular composition of EVs 24 1.4.4 Biological functions of EVs 26 1.4.5 EVs in Tumor microenvironment 28 1.4.6 EVs in Tumor-fibroblast communication 29 1.4.7 Role of EVs in colorectal cancer 31 1.5 Tissue inhibitor of metalloproteinases (TIMP1) 35 1.5.1 TIMP1 in cancer 37 2. Background and Research Aims 39 3. Material and Methods 40 3.1 Material 40 3.1.1 Devices 40 3.1.2 Additional material and equipment 42 3.1.3 Fine chemicals 43 3.1.4 Biochemicals 45 3.1.5 Primary antibodies 46 3.1.6 Secondary antibodies 47 3.1.7 Nucleic acids 47 3.1.8 Consumables 50 3.1.9 Softwares 51 3.2 Methods 52 3.2.1 Patients 52 3.2.2 Immunohistochemistry 52 3.2.3 Hematoxylin eosin staining 54 3.2.4 Cell lines 54 3.2.5 Primary liver fibroblast cell lines 54 3.2.6 Passaging and freezing of cells 55 3.2.7 Revival of frozen cells 55 3.2.8 Cell counting 56 3.2.9 EV Isolation from CRC cell lines 56 3.2.10 Isolation of serum-derived EVs from liquid biopsies 56 3.2.11 Characterisation of EVs 57 3.2.12 Treatment of Fibroblasts with EVs 58 3.2.13 Stimulation of PFs with recombinant TIMP1 59 3.2.14 RNA isolation 59 3.2.15 cDNA synthesis 59 3.2.16 Quantitative Real-Time PCR (qRT-PCR) 60 3.2.17 Protein quantification 61 3.2.18 Immunoblotting and co-immunoprecipitation 61 3.2.19 ELISA 62 3.2.20 TIMP1 Knock-Out (KO) and Over-Expression (OE) 62 3.2.21 17 AAG and HSP90AA antibody treatment 63 3.2.22 3D ECM-remodelling assay 63 3.2.23 PKH staining 65 3.2.24 In vivo experiments 65 3.2.25 DAPI staining 66 3.2.26 Tissue explant model 66 3.2.27 Statistical analysis and reproducibility 67 4. Results 68 4.1 Identification of TIMP1 as target molecule 68 4.1.1 Identification of TIMP1 as a target through data mining 68 4.1.2 Localization pattern of TIMP1 in CRC and CRC liver MET 70 4.1.3 Invasion front-specific overexpression of TIMP1 in the stroma of patients with CRC liver MET is associated with poor progression-free survival (PFS) 72 4.2 Model system to study CRC-EV mediated ECM remodelling 73 4.2.1 Investigating the role of CRC- derived EVs in the evolution of colorectal liver metastases 73 4.2.2 Characterizsation of isolated EVs from the CRC cell lines 74 4.2.3 TIMP1 enrichment in EVs derived from CRC cell lines 75 4.2.4 CRC-derived TIMP1EV regulates TIMP1 levels in recipient fibroblasts 76 4.2.5 TIMP1EV mediated TIMP1 upregulation in the recipient fibroblast is an EV-mediated effect 79 4.2.6 Recombinant TIMP-1 induces TIMP1 levels in recipient pFs in a time- and concentration-dependent manner 81 4.2.7 Alteration of TIMP1 levels in HCT 116 cells translates into EVs but does not affect EV packaging. 83 4.2.8 TIMP1EV levels in CRC EVs determine TIMP1 levels in recipient fibroblasts 85 4.2.9 EV-mediated TIMP1 upregulation in pFs induces ECM remodelling 86 4.2.10 TIMP1 levels in the PFs influence the extent of ECM remodelling 88 4.3 Clinical significance of TIMP1EV 89 4.3.1 TIMP1 enriched in serum-derived EVs of CRC patients compared to healthy controls 89 4.3.2 Serum derived TIMP1EV from CRC patients regulate TIMP1 levels in primary liver fibroblasts 91 4.3.3 Serum derived TIMP1EV from CRC patients promote ECM remodelling 93 4.3.4 TIMP1EV exhibits superior stratification power compared to soluble TIMP1 in liquid biopsies 93 4.3.5 TIMP1EV is a non-invasive independent prognostic marker in colorectal liver metastases 94 4.4 Targeting TIMP1EV mediated ECM remodelling 97 4.4.1 TIMP1EV binds to HSP90AA 97 4.4.2 HSP90 inhibition interferes with TIMP1 protein stabilisation 99 4.4.3 17AAG attenuates TIMP1EV-mediated ECM remodelling 101 4.5 EVs derived from murine CRC cell lines regulate TIMP1 levels in recipient fibroblasts 104 4.6 Increased homing of CRC EVs to the liver compared to other organs 106 4.7 TIMPEV regulates TIMP1 levels in liver tissues 108 5. Discussion 112 5.1 TIMP1 Localization and its significance in liver metastases 112 5.2 Model system to study the role of CRC-EVs in liver metastasis 113 5.3 In-vitro model to study the pro-metastatic effects of TIMP1EV 114 5.4 Serum-derived extravesicular TIMP1 and its pro-metastatic functions underlying remodeling of the extracellular matrix 116 5.5 Clinical significance of TIMP1EV in colorectal liver metastases 117 5.6 Scope of HSP90 inhibitors in the prevention and treatment of CRC liver metastases...……………………………………………………………………………………..118 6. Future perspectives and concluding remarks 120 7. Graphical summary of the findings 122 Zusammenfassung 123 Summary 125 List of figures 127 List of Tables 129 References 130 Acknowledgements 163 Appendix 165

Yes / In recent years, tremendous progress has been made in the treatment of acute myocardial infarction (AMI), but pathological ventricular remodeling often causes survivors to suffer from fatal heart failure. Currently, there is no effective therapy to attenuate ventricular remodeling. Recently, nanoparticles-based drug delivery system is widely applied in biomedicine especially in cancer and liver fibrosis, owing to its excellent physical, chemical, and biological properties. Therefore, using nanoparticles as delivery vehicles of small molecules, polypeptides, etc to improve post-infarct ventricular remodeling are expected. In this review, we summarized the updated researches in this fast-growing area and suggested further works needed.

La leishmaniose est une zoonose à transmission vectorielle due au parasite protozoaire Leishmania ; des co-infections avec plusieurs espèces de Leishmania ont également été rapportées. Il a été démontré que les vésicules extracellulaires (VE) de ce parasite jouent un rôle dans l'infection précoce, ainsi que la propagation de la résistance in vitro aux médicaments. Peu de médicaments anti-Leishmania sont disponibles, et la résistance continue de croître chez ce parasite; il est donc impératif de comprendre la propagation de la résistance aux antileishmaniens. Nous avons exploré la capacité des VE xénogéniques de Leishmania à moduler la physiopathologie de l'infection et la sensibilité du parasite aux médicaments après contact in vivo. La co-inoculation de parasites et de VE provenant de souches/espèces de Leishmania présentant divers profils de résistance aux médicaments a été réalisée chez la souris. La physiopathologie et la charge parasitaire ont été suivies, et des tests de sensibilité aux médicaments effectués. Les résultats ont démontré que les VE de Leishmania infantum influencent la physiopathologie de Leishmania major dans le cadre in vivo. Nous avons également constaté que ces VE modulent la sensibilité aux médicaments de L. major après un contact in vivo dans un modèle d'infection précoce, entraînant une diminution significative de la sensibilité à l’antileishmanien antimoine. Nous démontrons ici pour la première fois que les VE des parasites xénogéniques peuvent participer à la propagation de la résistance aux médicaments entre les populations de parasites après un contact in vivo, ce qui pourrait expliquer en partie l'augmentation des taux d'échec des traitements contre Leishmania. / Leishmaniasis is a zoonotic disease caused by the protozoan parasite Leishmania, endemic to 98 countries and territories. There are several manifestations of leishmaniasis, some fatal if left untreated. Furthermore, co-infections with multiple species of Leishmania have also been reported. Extracellular vesicles (EVs) from Leishmania have been demonstrated to play a role in early infection, as well as spread of drug resistance in vitro. Few antileishmanial drugs are available, and drug resistance to those in use continues to grow; as such, there is an urgent need to better understand the spread of Leishmania drug resistance. In this study, the ability of xenogeneic Leishmania EVs to modulate infection pathophysiology and parasite drug sensitivity after in vivo contact was explored. Co-inoculation of parasites and purified EVs from strains/species of Leishmania with contrasting drug resistance profiles was performed in BALB/c mice. Pathophysiology and parasite burden were monitored, and drug-susceptibility testing performed on recovered parasites. Results demonstrated that EVs from Leishmania infantum influence pathophysiology of Leishmania major in in vivo experiments. These EVs were also found to modulate drug sensitivity of L. major after in vivo contact in a 6-hour infection model, leading to a highly significant decrease in susceptibility to antileishmanial antimony. Here it is demonstrated for the first time that EVs from xenogeneic parasites can participate directly in propagating drug resistance between parasite populations after in vivo contact. These findings may help explain current observations of rising rates of Leishmania treatment failure.

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Les vésicules extracellulaires telles que les microvésicules et les exosomes sont libérées lors de l’apoptose ou de l’activation cellulaire. Ce sont des médiateurs importants dans la communication intercellulaire, suggérant que ces vésicules pourraient jouer un rôle physiopathologique, en particulier dans les maladies cardiovasculaires. L'athérosclérose est une maladie inflammatoire chronique de la paroi artérielle qui résulte de l’interaction entre les lipoprotéines, les cellules inflammatoires, et les cellules vasculaires. L'infarctus du myocarde est une complication aiguë et grave de l'athérosclérose. La réaction inflammatoire post-infarctus joue un rôle central dans la formation de néovaisseaux sanguins et la cicatrisation. Cependant, les mécanismes de l’inflammation sont encore mal connus dans ces pathologies. Mon travail de thèse a porté sur les effets des vésicules extracellulaires isolées de tissus pathologiques sur les cellules inflammatoires. Nous avons montré dans un premier travail que les microvésicules s’accumulant dans les lésions d’athérosclérose humaines contribuent à la surcharge en cholestérol et en triglycérides des macrophages et facilitent la formation de cellules spumeuses. L’accumulation des lipides intracellulaires induite par ces microvésicules est contrebalancée par une augmentation de l’efflux du cholestérol associée à une activation d’ABCA1. Dans un deuxième travail, nous avons examiné les effets des vésicules produites dans le cœur post-infarctus sur la réponse inflammatoire. Nos résultats montrent : 1- une augmentation de la libération in situ des microvésicules majoritairement d’origine cardiomyocytaire et des exosomes 15 heures après infarctus ; 2- la stimulation de la production de VEGF monocytaire par les vésicules extracellulaires ; 3- l’incapacité en ce qui concerne les vésicules isolées de cœur diabétique infarci à reproduire cet effet sur les monocytes des souris contrôles. Afin de clarifier les déterminants de l’angiogenèse post-ischémique, nous avons également étudié les profils de miARNs des vésicules contrôles et diabétiques. Après infarctus du myocarde, l’expression de miR-126-3p et de miR-92a-3p est significativement diminuée dans les vésicules diabétiques en comparaison avec les vésicules contrôles. Par ailleurs, nous avons observé une augmentation de miR-126-3p et de miR-92a-3p respectivement dans les microvésicules et les exosomes chez les souris contrôles post-infarctus. En conclusion, ce travail apporte des éléments nouveaux sur les fonctions des vésicules extracellulaires générées localement dans les tissus inflammatoires, en particulier leur capacité à promouvoir la transformation des macrophages en cellules spumeuses dans la plaque. Par ailleurs, les vésicules isolées du cœur ischémique pourraient favoriser l’angiogenèse post-infarctus en stimulant la production de VEGF monocytaire. La disparition de cet effet bénéfique dans le diabète pourrait être associée à des modifications d’adressage des miARNs dans les vésicules extracellulaires au cours de cette pathologie. / Extracellular vesicles, such as microvesicles and exosomes, are released during cell apoptosis or activation. They are important mediators of intercellular communication, suggesting that these vesicles could play a pathophysiological role, especially in cardiovascular diseases. Atherosclerosis is a chronic inflammatory disease of the arterial wall which results from the interaction between lipoproteins, inflammatory cells, and vascular cells. Myocardial infarction is an acute and severe complication of atherosclerosis. The postinfarction inflammatory response plays a central role in the formation of new blood vessels and scarring. However, the mechanisms of inflammation are still poorly known in these pathologies. My thesis concerned the effects of extracellular vesicles isolated from pathological tissues on inflammatory cells. We showed in the first work that microvesicles accumulating in human atherosclerotic lesions contribute to cholesterol and triglyceride overload in macrophages and facilitate foam cell formation. The accumulation of the intracellular lipids induced by those microvesicles is offset by an increase in cholesterol efflux associated with activation of ABCA1. In the second study, we examined the effect of vesicles produced in the infarcted heart on the inflammatory response. Our results showed : 1- an increased release in situ of microvesicles mostly of cardiomyocyte origin and exosomes 15 hours after infarction ; 2- the stimulation of monocyte VEGF production by extracellular vesicles ; 3- the incapacity of diabetic vesicles isolated from infarcted heart to reproduce that effect on control mice monocytes. In order to clarify the determinants of postischemic angiogenesis, we also studied miRNA profiles of control and diabetic vesicles. After myocardial infarction, the expression level of miR-126-3p and miR-92a-3p was significantly decreased in diabetic vesicles compared to control vesicles. Furthermore, we observed an increased expression of miR-126-3p and miR-92a-3p respectively in the microvesicles and the exosomes isolated from control mice heart after myocardial infarction. In conclusion, this work provides new information on the functions of extracellular vesicles locally generated in inflamed tissues, particularly in promoting macrophage transformation into foam cells in the atherosclerotic plaque. Furthermore, vesicles isolated from ischemic heart could enhance postinfarction angiogenesis by stimulating monocyte VEGF production. The loss of this beneficial effect in diabetes may be associated with changes of miRNA cargo in extracellular vesicles in this pathology.