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Extracellular Vesicles from Human Cardiac Cells As Future Allogenic Therapeutic Tool for Heart Diseases

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Extracellular Vesicles from Human Cardiac Cells As Future Allogenic Therapeutic Tool for Heart Diseases Dissertation EXTRACELLULAR VESICLES FROM HUMAN CARDIAC CELLS AS FUTURE ALLOGENIC THERAPEUTIC TOOL FOR HEART DISEASES. zur Erlangung des akademischen Grades Doctor rerum naturalium (Dr. rer. nat.) im Fach Biologie eingereicht an der Lebenswissenschaftlichen Fakultät der Humboldt-Universität zu Berlin von Dipl. Biochemikerin Christien M. Beez Präsidentin der Humboldt-Universität zu Berlin Prof. Dr.-Ing. Dr. Sabine Kunst Dekan der Lebenswissenschaftlichen Fakultät Prof. Dr. Bernhard Grimm GutachterInnen: 1. Prof. Dr. Martina Seifert 2. Prof. Dr. Hans-Dieter Volk 3. PD Dr. Irina Nazarenko Tag der mündlichen Verteidigung: 23.Februar.2021 The thesis was performed during July 2015 till December 2019 under the supervision of Prof. Dr. Martina Seifert at the Institute of Medical Immunology and the Berlin Institute of Health (BIH) Centre for Regenerative Therapies (BCRT), Charité-Universitätsmedizin Berlin, as graduate student of the Berlin-Brandenburg School for Regenerative Therapies (BSRT, DFG- Graduiertenschule 203). The work was funded by the Friede-Springer-Herz-Stiftung and the Einstein Foundation. „Ein Ei ist ein Ei“, sagte jener und nahm das größere. K. Tucholsky Abstract Extracellular vesicles (EVs) facilitate intercellular communication by transferring molecules from a donor to a recipient cell. It is proposed that EVs from regenerative cells as a therapeutic tool can help to overcome the leading role of cardiovascular diseases as cause of death. Accordingly, this thesis aimed to evaluate the suitability of EVs from regenerative human cardiac-derived adherent proliferating (CardAP) cells as an allogenic cell-free approach to treat heart diseases. For that purpose, we isolated EVs by differential centrifugation from the conditioned medium that was derived either in the presence or absence of a pro-inflammatory cytokine cocktail (IFNγ, TNFα, and IL-1β). Afterwards, isolated EVs were analysed in vitro for their phenotypical characteristics, therapeutic effects, and immunological compatibility. Isolated EVs from CardAP cells exhibited vesicular structures with diameters mostly of exosomes (d < 100 nm), known EV-associated proteins (e.g. tetraspanins) as well as miRNAs (e.g. miRNA 146-5p). Interestingly, cytokine stimulated EVs were observed to have significantly smaller diameters and a greater repertoire of transported miRNAs than their unstimulated counterpart. Nevertheless, both unstimulated as well as cytokine stimulated EVs were equally internalized by murine cardiac cells, namely HL-1 and MHEC5-T cells, and protected murine cardiomyocytes from reactive oxygen species or starvation induced apoptosis. Virus induced apoptosis was, however, only reduced by treatment with unstimu-lated EVs. Deviations between both EVs were also determined for their pro-angiogenic effect. Human umbilical vein endothelial cells (HUVECs) increased the release of different pro-angiogenic factors when treated with unstimulated EVs (e.g. VEGF) or cytokine stimulated EVs (e.g. IL-8). Nonetheless, both EVs were capable to amplify the tube formation capabilities of treated HUVECs. The exposure of both EVs to unstimulated peripheral blood mono-nuclear cells (PBMCs) did neither induce T cell activation, T cell proliferation nor the release of IFNγ. Additionally, isolated EVs demonstrated immune modulating features in induced immune responses of stimulated PBMCs as observed by diminished T cell proliferation, higher frequencies of regulatory T cells, and a weakened inflammatory milieu (e.g. decreased IFNγ concentrations). Moreover, it was shown that CD14+ cells are essential for this desired immune modulating effect of isolated EVs. In particular, isolated EVs interacted predominantly with CD14+ cells, which consequently acquired a regulatory immune phenotype (e.g. reduced expression of HLA-DR, increased expression of PD-L1). Moreover, EVs solely modulated induced immune responses of isolated T cells when EV-primed CD14+ cells were present. Overall, EVs derived from CardAP cells appear to convey beneficial characteristics that could contribute to an enhanced regeneration in damaged cardiac tissue by limiting unwanted inflammatory processes, enhancing angiogenesis, decreasing apoptosis and their immune-logical compatibility. Future in vivo studies are necessary to validate this indicated suitability of EVs as cell-free allogenic therapeutic approach. In addition, some molecules transported by EVs (e.g. galectin-1 or miRNA 302d-5p) are hypothesized to contribute to the observed beneficial features. In future, these molecules could be used to achieve a more efficient therapeutic approach by further selection of isolated EVs. Keywords: cardiac EVs, regenerative therapies, immunomodulation, angiogenesis Zusammenfassung Von einer Spenderzelle freigesetzten extrazellulären Vesikel (EVs) können wichtige Prozesse in einer Empfängerzelle beeinflussen. Es wird angenommen, dass durch die Behandlung mit EVs regenerativer Zellen die führende Stellung von Herz-Kreislauf-Erkrankungen als Todesursache reduziert werden könnte. In diesem Zusammenhang untersuchte die vorliegende Dissertation, ob EVs von regenerativen humanen kardialen adhärenten proliferierenden (CardAP) Zellen geeignet wären Herzerkrankungen in einem allogenen zellfreien Ansatz zu behandeln. Dazu wurden EVs aus dem konditionierten Medium von CardAP Zellen, die mit oder ohne pro- inflammatorischen Zytokin-Cocktail (IFNγ, TNFα und IL-1β) kultiviert wurden, durch Differentialzentrifugation gewonnen. Im Anschluss wurden die isolierten EVs in vitro bezüglich ihrer phänotypischen Eigenschaften, therapeutischen Wirkungen und immunologischen Verträglichkeit charakterisiert. Generell besaßen die isolierten EVs vesikuläre Strukturen, hauptsächlich mit Durchmessern von Exosomen (d <100 nm), bekannte EV-assoziierte Proteine (z.B. Tetraspanine) und miRNAs (z.B. miRNA 146-5p). Interessanterweise wiesen stimulierte EVs kleinere Durch- messer sowie ein größeres Repertoire an transportierten miRNAs gegenüber unstimulierten EVs auf. Dennoch wurden EVs beider Biogenesebedingungen gleichermaßen von kardialen Mauszellen internalisiert und sie reduzierten sowohl die durch reaktive Sauerstoffspezies als auch durch Nährstoffmangel induzierte Apoptose von murinen Kardiomyozyten. Im Gegen- satz dazu konnten ausschließlich unstimulierte EVs die Virus-induzierte Apoptose verringern. Ebenso zeigten humane Endothelzellen der Nabelschnurvene (HUVECs) Unterschiede in der Freisetzung proangiogener Faktoren nach Kontakt mit unstimulierten EVs (z.B. vaskuläre endotheliale Wachstumsfaktor) oder stimulierten EVs (z.B. IL-8). Allerdings konnten EVs beider Biogenesebedingungen die Netzwerkausbildung von HUVECs in vitro deutlich erhöhen. Immunzellen, die mononuklearen Zellen des peripheren Blutes (PBMCs), reagierten auf isolierte EVs weder mit einer Aktivierung noch einer Proliferation von T Zellen oder der Freisetzung von pro-inflammatorischem IFNγ. Allerdings, konnte die induzierte Immunantwort von PBMCs durch beide EVs moduliert werden, was sowohl die verminderte Proliferation von T Zellen, den erhöhten Anteil regulatorischer T Zellen und ein gemindertes entzündliches Milieu umfasste. Es wurde zudem gezeigt, dass die untersuchten EVs vor allem mit CD14+ Zellen in PBMC Kulturen interagierten und diese Zellen infolgedessen einen regulatorischen Immunphänotyp annahmen (z.B. verringerte Expression von HLA-DR, erhöhte Expression von PD-L1). Darüberhinaus konnte die induzierte Immunreaktion von isolierten T Zellen nur in Anwesenheit von EV-behandelten CD14+ Zellen moduliert werden. EVs von CardAP Zellen erscheinen durch ihre immunmodulativen, pro-angiogenen und anti- apoptotischen Effekte bei gleichzeitiger immunologischer Kompatibilität durchaus geeignet, geschädigtes Herzgewebe zu regenerieren, was in künftigen in vivo Studien zu validieren ist. Darüber hinaus wurden Moleküle (z.B. Galectin-1 oder miRNA 302d-5p) identifiziert, die vermutlich diese gewünschten Eigeneschaften vermitteln. Dies bietet die Möglichkeit EVs mit potentiell erhöhter Funktionalität spezifisch aufzureinigen. Schlagwörter: kardiale EVs, Regenerative Therapie, Immunmodulation, Angiogenese TABLE OF CONTENT LIST OF ABBREVIATIONS ............................................................................................................................. V LIST OF FIGURES ............................................................................................................................................ VI LIST OF TABLES ............................................................................................................................................ VIII 1. INTRODUCTION ............................................................................................................................ 1 1.1 CARDIOVASCULAR DISEASES ........................................................................................................... 1 1.2 WAYS TO MINIMIZE DAMAGES OF CVDS ...................................................................................... 1 1.2.1 Myocardial infarction and its untreated resolution ........................................................................ 2 1.2.2 Desired regenerative effects .............................................................................................................. 3 1.3 CELL THERAPY AS A REGENERATIVE APPROACH ........................................................................ 4 1.3.1 Autologous and allogenic cell therapy ............................................................................................
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