@phdthesis{Mott2023,
  author    = {Mott, Kristina},
  title     = {Regulation of platelet biogenesis in the native and myeloablated bone marrow niche},
  doi       = {10.25972/OPUS-28963},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-289630},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {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.},
  subject      = {Knochenmark},
  language  = {en}
}
@phdthesis{Heib2022,
  author    = {Heib, Tobias},
  title     = {The role of Rho GTPases in megakaryopoiesis and thrombopoiesis},
  doi       = {10.25972/OPUS-21664},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-216645},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {Platelets, derived from megakaryocytes (MKs) in the bone marrow (BM), are small, anucleated cells that circulate in the bloodstream and are critical for thrombosis and hemostasis. Megakaryo- and subsequent thrombopoiesis are highly orchestrated processes involving the differentiation and maturation of MKs from hematopoietic stem cells (HSCs), after which MKs are able to release platelets into the bloodstream, a process termed proplatelet formation (PPF). Here, the MK penetrates the endothelial lining and releases cytoplasmic portions (proplatelets) into the blood stream, which finally mature into platelets within the ciruculation. PPF requires an extensive crosstalk as well as a tight regulation of the MK cytoskeleton, in which small GTPases of the Rho family, such as RhoA and Cdc42 are critically involved and play opposing roles. MK and platelet specific Cdc42 or RhoA-deficiency in mice results in macrothrombocytopenia. Moreover, RhoA deficient mice displayed a frequent mislocalization of entire MKs into BM sinusoids, a finding that was reverted upon concomitant lack of Cdc42 but accompanied by an aggravated macrothrombocytopenia. Whether receptors are involved in the process of transendothelial MK migration, however, remained unclear. In the first part of this thesis, a centrifugation-based method ('spin isolation') to harvest murine BM cells was established, which not only reduces experimental time, costs and animals but is also highly suitable for studies on primary and in vitro cultured BM-derived cells. The spin isolation was used particularly for MK studies during the course of the thesis. In the second part of this thesis, a MK- and platelet-specific RhoA/Cdc42 double-deficiency was shown to result in reduced expression of a variety of MK-specific glycoproteins and cytoskeletal regulators of importance during MK maturation and PPF, a phenotype culminating in virtually abolished platelet biogenesis. We thus uncovered that RhoA/Cdc42-regulated gene expression is a prerequisite for cytoplasmic MK maturation, but dispensable for endomitosis. In the third part of this thesis we analyzed mice double-deficient in RhoA and prominent MK receptors which are potentially involved in the regulation of PPF in the BM environment. We were able to show that integrins as well as the inhibitory receptor G6b-B are dispensable for transendothelial migration of RhoA-deficient MKs. Surprisingly however, the myelofibrosis and concomitant osteosclerosis observed in G6b-B single-deficient mice was attenuated in RhoA/G6b B double-deficient mice, thus implying an important role of RhoA during myelofibrotic disease progression. BM transplantation experiments furthermore revealed that not only the macrothrombocytopenia but also the transmigration of RhoA-deficient MKs is due to cell-intrinsic defects and not related to possible Pf4-Cre off-target effects in non-hematopoietic cells. In the last part of this study we demonstrated that the new approach for MK- and platelet-specific gene ablatation using Gp1ba-Cre deleter mice is associated with intrinsic MK defects and in addition results in insufficient depletion of RhoA compared to the Pf4-Cre model, positioning the latter still as the gold standard for studying MK biology.},
  subject      = {Megakaryozyt},
  language  = {en}
}