TY - THES A1 - Mott, Kristina T1 - Regulation of platelet biogenesis in the native and myeloablated bone marrow niche T1 - Die Regulation der Thrombozytenbiogenese im nativen und myeloablatierten Knochenmark N2 - Megakaryocytes (MKs) are the largest cells of the hematopoietic system and the precursor cells of platelets. During proplatelet formation (PPF) bone marrow (BM) MKs extent large cytoplasmic protrusions into the lumen of sinusoidal blood vessels. Under homeostatic conditions PPF occurs exclusively in the direction of the sinusoid, while platelet generation into the marrow cavity is prevented. So far, the mechanisms regulating this process in vivo are still not completely understood, especially when PPF is deregulated during disease. This thesis investigated the mechanisms of PPF in native BM and after myeloablation by total body irradiation (TBI). First, we have identified a specialized type of BM stromal cells, so called CXCL12-abundant reticular (CAR) cells, as novel possible regulators of PPF. By using complementary high-resolution microscopy techniques, we have studied the morphogenetic events at the MK/vessel wall interface in new detail, demonstrating that PPF formation preferentially occurs at CAR cell-free sites at the endothelium. In the second part of this thesis, we analyzed the processes leading to BM remodeling in response to myeloablation by TBI. We used confocal laser scanning microscopy (CLSM) to study the kinetic of radiation-triggered vasodilation and mapped extracellular matrix (ECM) proteins after TBI. We could demonstrate that collagen type IV and laminin α5 are specifically degraded at BM sinusoids. At the radiation-injured vessel wall we observed ectopic release of platelet-like particles into the marrow cavity concomitantly to aberrant CAR cell morphology, suggesting that the balance of factors regulating PPF is disturbed after TBI. ECM proteolysis is predominantly mediated by the matrix metalloproteinase MMP9, as revealed by gelatin-zymography and by a newly established BM in situ zymography technique. In transgenic mice lacking MMP9 vascular recovery was delayed, hinting towards a role of MMP9 in vessel reconstitution after myeloablation. In a third series of experiments, we studied the irradiated BM in the context of hematopoietic stem cell transplantation (HSCT). By using mice as BM donors that ubiquitously express the fluorescent reporter protein dsRed we tracked engraftment of donor cells and especially MKs in the recipient BM. We found a distinct engraftment pattern and cluster formation for MKs, which is different from other blood cell lineages. Finally, we assessed platelet function after TBI and HSCT and were the first to demonstrate that platelets become massively hyporeactive, particularly upon stimulation of the collagen receptor GPVI. In summary, our findings shed light on the processes of PPF during health and disease which will help to develop treatments for aberrant thrombopoiesis. N2 - Megakaryozyten (MKs) sind die größten Zellen des hämatopoetischen Systems und die Vorläuferzellen der Blutplättchen. Während der Ausbildung von Proplättchen schnüren MKs im Knochenmark (KM) große zytoplasmatische Ausläufer in das Lumen der sinusoidalen Blutgefäße ab. Unter homöostatischen Bedingungen erfolgt die Proplättchenbildung ausschließlich in Richtung der Sinusoide, während die Thrombozytenbildung in die Knochenmarkkavität verhindert wird. Bislang sind die Mechanismen, die diesen Prozess in vivo steuern, noch nicht vollständig aufgeklärt insbesondere, wenn die Thrombozytenbiogenese unter Krankheitsbedingungen dereguliert ist. In dieser Arbeit wurden die Mechanismen der Thrombopoese im nativen Knochenmark und nach Myeloablation durch Ganzkörperbestrahlung (total body irradiation, TBI) untersucht. Zunächst haben wir einen spezialisierten Typ von KM-Stromazellen, die sog. CXCL12-abundant reticular (CAR) Zellen, als neue mögliche Regulatoren der Proplättchenbildung identifiziert. Durch den Einsatz komplementärer hochauflösender Mikroskopietechniken haben wir die morphogenetischen Vorgänge an der Schnittstelle zwischen MKs und Gefäßwand genauer untersucht und gezeigt, dass die Generierung von Proplättchen bevorzugt an CAR-Zell-freien Stellen am Endothel stattfindet. Im zweiten Teil dieser Arbeit analysierten wir die Prozesse, die zum Knochenmarkumbau nach Myeloablation durch TBI führen. Mit Hilfe von konfokaler Laser-Scanning-Mikroskopie untersuchten wir die Kinetik der strahleninduzierten Vasodilatation und kartierten die extrazelluläre Matrix (EZM) Proteine nach TBI. So konnten wir zeigen, dass Kollagen Typ IV und Laminin α5 spezifisch an den Knochenmarksinusoiden abgebaut werden. An der strahlengeschädigten Gefäßwand beobachteten wir die ektope Freisetzung plättchenartiger Partikel in die Knochenmarkkavität, die mit einer abnormalen CAR-Zellmorphologie einherging. Dies weist darauf hin, dass das Gleichgewicht der Faktoren, die die gerichtete Proplättchenbildung regulieren, nach TBI gestört ist. Die EZM-Proteolyse wird vor allem durch die Matrix-Metalloproteinase MMP9 vermittelt, was durch Gelatine-Zymographie und durch eine neu etablierte in situ Zymographie-Technik für das Knochenmark nachgewiesen wurde. Bei transgenen Mäusen, die defizient für MMP9 sind, war die Regeneration der Vaskulatur verzögert, was auf eine Rolle von MMP9 bei der Gefäßrekonstitution nach Myeloablation hindeutet. In einer dritten Versuchsreihe untersuchten wir das bestrahlte KM im Rahmen einer hämatopoetischen Stammzelltransplantation (HSZT). Mit Hilfe von Mäusen als KM-Spender, die ubiquitär das fluoreszierende Reporterprotein dsRed exprimieren, verfolgten wir das Anwachsen von Spenderzellen und insbesondere von MKs im Empfängerknochenmark. Wir beobachteten ein eindeutiges Muster bzw. Clusterbildung für anwachsende MKs, die sich von anderen Blutzelllinien unterschied. Schließlich untersuchten wir die Funktion von Thrombozyten nach TBI und HSZT und konnten als erste zeigen, dass Thrombozyten eine massive Hyporeaktivität ausbilden, insbesondere nach Stimulation des Kollagenrezeptors GPVI. Zusammenfassend geben unsere Ergebnisse Aufschluss über die Prozesse der Thrombopoese im nativen und pathologischen Knochenmark, was zur Entwicklung von Therapien zu Behandlung von defekter Thrombozytenbiogenese beitragen wird. KW - Knochenmark KW - Knochenmarktransplantation KW - Megakaryozyt KW - Thrombozyt KW - Ganzkörperbestrahlung KW - bone marrow KW - myeloablation KW - thrombopoiesis Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-289630 ER - TY - JOUR A1 - Elgheznawy, Amro A1 - Öftering, Patricia A1 - Englert, Maximilian A1 - Mott, Kristina A1 - Kaiser, Friederike A1 - Kusch, Charly A1 - Gbureck, Uwe A1 - Bösl, Michael R. A1 - Schulze, Harald A1 - Nieswandt, Bernhard A1 - Vögtle, Timo A1 - Hermanns, Heike M. T1 - Loss of zinc transporters ZIP1 and ZIP3 augments platelet reactivity in response to thrombin and accelerates thrombus formation in vivo JF - Frontiers in Immunology N2 - Zinc (Zn2+) is considered as important mediator of immune cell function, thrombosis and haemostasis. However, our understanding of the transport mechanisms that regulate Zn2+ homeostasis in platelets is limited. Zn2+ transporters, ZIPs and ZnTs, are widely expressed in eukaryotic cells. Using mice globally lacking ZIP1 and ZIP3 (ZIP1/3 DKO), our aim was to explore the potential role of these Zn2+ transporters in maintaining platelet Zn2+ homeostasis and in the regulation of platelet function. While ICP-MS measurements indicated unaltered overall Zn2+ concentrations in platelets of ZIP1/3 DKO mice, we observed a significantly increased content of FluoZin3-stainable free Zn2+, which, however, appears to be released less efficiently upon thrombin-stimulated platelet activation. On the functional level, ZIP1/3 DKO platelets exhibited a hyperactive response towards threshold concentrations of G protein-coupled receptor (GPCR) agonists, while immunoreceptor tyrosine-based activation motif (ITAM)-coupled receptor agonist signalling was unaffected. This resulted in enhanced platelet aggregation towards thrombin, bigger thrombus volume under flow ex vivo and faster in vivo thrombus formation in ZIP1/3 DKO mice. Molecularly, augmented GPCR responses were accompanied by enhanced Ca2+ and PKC, CamKII and ERK1/2 signalling. The current study thereby identifies ZIP1 and ZIP3 as important regulators for the maintenance of platelet Zn2+ homeostasis and function. KW - platelets KW - zinc KW - ZIP KW - thrombin KW - signaling KW - thrombosis Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-320154 VL - 14 ER - TY - JOUR A1 - Wagner, Nicole A1 - Mott, Kristina A1 - Upcin, Berin A1 - Stegner, David A1 - Schulze, Harald A1 - Ergün, Süleyman T1 - CXCL12-abundant reticular (CAR) cells direct megakaryocyte protrusions across the bone marrow sinusoid wall JF - Cells N2 - Megakaryocytes (MKs) release platelets into the lumen of bone marrow (BM) sinusoids while remaining to reside within the BM. The morphogenetic events of this complex process are still not fully understood. We combined confocal laser scanning microscopy with transmission and serial block-face scanning electron microscopy followed by 3D-reconstruction on mouse BM tissue sections. These analyses revealed that MKs in close vicinity to BM sinusoid (BMS) wall first induce the lateral retraction of CXCL12-abundant reticular (CAR) cells (CAR), followed by basal lamina (BL) degradation enabling direct MK-sinusoidal endothelial cells (SECs) interaction. Subsequently, an endothelial engulfment starts that contains a large MK protrusion. Then, MK protrusions penetrate the SEC, transmigrate into the BMS lumen and form proplatelets that are in direct contact to the SEC surface. Furthermore, such processes are induced on several sites, as observed by 3D reconstructions. Our data demonstrate that MKs in interaction with CAR-cells actively induce BMS wall alterations, including CAR-cell retraction, BL degradation, and SEC engulfment containing a large MK protrusion. This results in SEC penetration enabling the migration of MK protrusion into the BMS lumen where proplatelets that are adherent to the luminal SEC surface are formed and contribute to platelet release into the blood circulation. KW - megakaryocytes KW - microvasculature KW - CXCL12-abundant reticular (CAR)-cells Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-234180 SN - 2073-4409 VL - 10 IS - 4 ER -