@phdthesis{Upcin2022,
  author    = {Upcin, Berin},
  title     = {Contribution of vascular adventitia-resident progenitor cells to new vessel formation in \(ex\) \(vivo\) 3D models},
  doi       = {10.25972/OPUS-25507},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-255070},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {Ongoing research to fight cancer, one of the dominant diseases of the 21st century has led to big progress especially when it comes to understanding the tumor growth and metastasis. This includes the discovery of the molecular mechanisms of tumor vascularization, which is critically required for establishment of tumor metastasis. Formation of new blood vessels is the first step in tumor vascularization. Therefore, understanding the molecular and cellular basis of tumor vascularization attracted a significant effort studying in biomedical research. The blood vessels for supplying tumor can be formed by sprouting from pre-existing vessels, a process called angiogenesis, or by vasculogenesis, that is de novo formation of blood vessels from not fully differentiated progenitor cell populations. Vasculogenic endothelial progenitor cells (EPCs) can either be activated from populations in the bone marrow reaching the pathological region via the circulation or they can be recruited from local reservoirs. Neovessel formation influences tumor progression, hence therapeutic response model systems of angiogenesis/vasculogenesis are necessary to study the underlying mechanisms. Although, initially the research in this area focused more on angiogenesis, it is now well understood that both angiogenesis and postnatal vasculogenesis contribute to neovessel formation in adult under both most pathological as well as physiological conditions. Studies in the last two decades demonstrate that in addition to the intimal layer of fully differentiated mature endothelial cells (ECs) and various smaller supplying vessels (vasa vasorum) that can serve as a source for new vessels by angiogenesis, especially the adventitia of large and medium size blood vessels harbors various vascular wall-resident stem and progenitor cells (VW-SPCs) populations that serve as a source for new vessels by postnatal vasculogenesis. However, little is known about the potential role of VW-SPCs in tumor vascularization. To this end, the present work started first to establish a modified aortic ring assay (ARA) using mouse aorta in order to study the contribution of vascular adventitia-resident VW-SPCs to neovascularization in general and in presence of tumor cells. ARA is already established an ex vivo model for neovascularization allows to study the morphogenetic events of complex new vessel formation that includes all layers of mature blood vessels, a significant advantage over the assays that employ monolayer endothelial cell cultures. Moreover, in contrast to assays employing endothelial cells monocultures, both angiogenic and vasculogenic events take place during new vessel formation in ARA although the exact contribution of these two processes to new vessel formation cannot be easily distinguished in conventional ARA. Thus, in this study, a modified protocol for the ARA (mdARA) was established by either removing or keeping the aortic adventitia in place. The mdARA allows to distinguish the role of VW-SPCs from those of other aortic layers. The present data show that angiogenic sprouting from mature aortic endothelium was markedly delayed when the adventitial layer was removed. Furthermore, the network between the capillary-like sprouts was significantly reduced in absence of aortic adventitia. Moreover, the stabilization of new sprouts by assembling the NG2+ pericyte-like cells that enwrapped the endothelial sprouts from the outside was improved when the adventitial layer remained in place. Next, mimicking the tumor-vessel adventitia-interaction, multicellular tumor spheroids (MCTS) and aortic rings (ARs) with or without adventitia of C57BL/6-Tg (UBC-GFP) mice were confronted within the collagen gel and cultured ex vivo. This 3D model enabled analysis of the mobilization, migration and capillary-like sprouts formation by VW-SPCs within tumor-vessel wall-interface in comparison to tumor-free side of the ARs. Interestingly, while MCTS preferred the uptake of single vascular adventitia-derived cells, neural spheroids were directly penetrated by capillary-like structures that were sprouted from the aortic adventitia. In summary, the model established in this work allows to study new vessel formation by both postnatal vasculogenesis and angiogenesis under same conditions. It can be applied in various mouse models including reporter mouse models, e.g. Cxcr1 CreER+/mTmG+/- mice, in which GFP-marked macrophages of the vessel wall were directly observed as they mobilized from their niche and migrated into collagen gel. Another benefit of the model is that it can be used for testing different factors such as small molecules, growth factors, cytokines, and drugs with both pro- and anti-angiogenic/vasculogenic effects.},
  language  = {en}
}
@phdthesis{Koeniger2021,
  author    = {K{\"o}niger, Tobias},
  title     = {The Vessel Wall and Beyond: Characterization of Myeloid Progenitors in the Adult Mouse Brain},
  doi       = {10.25972/OPUS-18646},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-186465},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {After almost two decades of extensive research, some controversy has remained regarding the self-renewal of resident macrophages of the central nervous system (CNS). Concurrently, the vessel wall has emerged as a potentially ubiquitous niche for stem and progenitor cells, including committed macrophage precursors. It is conceivable that their occurrence in the CNS might explain the brain-resident hematopoietic potential, which has repeatedly been observed but not yet characterized in detail. In this work, the presence of hematopoietic progenitors inside and outside the vessel wall was studied in the adult mouse brain, as well as their possible contribution to the resident macrophage pool. An immunohistological analysis did not corroborate CD45+ SCA-1+ macrophage progenitors, which have been characterized in peripheral arteries, in the circle of Willis. Accordingly, the ex vivo culture of CNS vessels did not provide evidence for de novo formation of macrophages, but for the extensive proliferative capacity of mature cells. However, when analyzing whole brain suspensions in colony-forming unit (CFU) assays, rare Iba1- Cx3cr1- (immature) clonogenic cells were detected, which were enriched at the cerebral surface/meninges and differentiated into macrophages in culture. Intravenous antibody injection and cell sorting confirmed their residence behind the blood-brain barrier. Intriguingly, brain-derived CFUs produced a unique pattern of colony types compared to cells from bone marrow (BM) or blood. Still they displayed the same immunophenotype as BM-resident myeloid progenitors (CD45lo, LIN-, SCA-1-, IL7Rα-, c-KIT+) and could be further stratified into a progenitor hierarchy giving rise to all erythro-myeloid cell types in vitro. This similarity was substantiated by labeling of their progeny in Flt3Cre x Rosa26mT/mG mice, which indicated a descendance from hematopoietic stem cells. While forced repopulation of brain macrophages using the CSF-1R inhibitor PLX5622 did not point to a role of progenitors in in vivo microglia/macrophage maintenance, recent advances in hematology imply that they might be involved in CNS immunosurveillance. In conclusion, though there was no evidence for adventitial macrophage precursors in the CNS, this study confirms the presence of myeloid progenitors in the adult brain and provides the anatomical and phenotypical details necessary to elucidate their relevance in neuroinflammation.},
  subject      = {Gehirn},
  language  = {en}
}