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Hypoxia and inflammation are closely intertwined phenomena. Critically ill patients often suffer from systemic inflammatory conditions and concurrently experience short-lived hypoxia. We evaluated the effects of short-term hypoxia on systemic inflammation, and show that it potently attenuates pro-inflammatory cytokine responses during murine endotoxemia. These effects are independent of hypoxia-inducible factors (HIFs), but involve augmented adenosine levels, in turn resulting in an adenosine 2B receptor-mediated post-transcriptional increase of interleukin (IL)-10 production. We translated our findings to humans using the experimental endotoxemia model, where short-term hypoxia resulted in enhanced plasma concentrations of adenosine, augmentation of endotoxin-induced circulating IL-10 levels, and concurrent attenuation of the pro-inflammatory cytokine response. Again, HIFs were shown not to be involved. Taken together, we demonstrate that short-term hypoxia dampens the systemic pro-inflammatory cytokine response through enhanced purinergic signaling in mice and men. These effects may contribute to outcome and provide leads for immunomodulatory treatment strategies for critically ill patients., Highlights • Short-term hypoxia attenuates the systemic pro-inflammatory cytokine response in vivo in both mice and men • The underlying mechanism involves adenosine 2B receptor-mediated enhanced production of anti-inflammatory interleukin-10 • These effects may contribute to outcome and provide leads for novel treatment strategies for critically ill patients Inflammation and short bouts of low oxygen levels are frequently encountered phenomena in severely ill patients admitted to the Intensive Care unit. This study investigated the effect of short-term low oxygen levels on the inflammatory response in both mice and humans. The results show that a short period of low levels of oxygen potently dampens inflammation, and identify the underlying mechanisms. These effects of low oxygen levels may contribute to the outcome of severely ill patients. Furthermore, the increased understanding of the underlying mechanisms provided by this study can be used to develop therapies to dampen inflammation in patients.

Cancer cell proliferation and insufficient blood supply can lead to the development of hypoxic areas in the tumor tissue. The adaptation to the hypoxic environment is mediated by a transcriptional complex called hypoxia-inducible factor (HIF). HIF protein levels are tightly controlled by oxygen-dependent prolyl hydroxylase domain proteins (PHDs). However, the precise roles of these enzymes in tumor progression and their downstream signaling pathways are not fully characterized. Here, we study PHD3 function in murine experimental osteosarcoma. Unexpectedly, PHD3 silencing in LM8 cells affects neither HIF-1&alpha, protein levels, nor the expression of various HIF-1 target genes. Subcutaneous injection of PHD3-silenced tumor cells accelerated tumor progression and was accompanied by dramatic phenotypic changes in the tumor vasculature. Blood vessels in advanced PHD3-silenced tumors were enlarged whereas their density was greatly reduced. Examination of the molecular pathways underlying these alterations revealed that platelet-derived growth factor (PDGF)-C signaling is activated in the vasculature of PHD3-deficient tumors. Silencing of PDGF-C depleted tumor growth, increased vessel density and reduced vessel size. Our data show that PHD3 controls tumor growth and vessel architecture in LM8 osteosarcoma by regulating the PDGF-C pathway, and support the hypothesis that different members of the PHD family exert unique functions in tumors.

Prolyl hydroxylase domain-containing proteins (PHDs) regulate the adaptation of cells to hypoxia. Pan-hydroxylase inhibition is protective in experimental colitis, in which PHD1 plays a prominent role. However, it is currently unknown how PHD1 targeting regulates this protection and which cell type(s) are involved. Here, we demonstrated that Phd1 deletion in endothelial and haematopoietic cells (Phd1(f/f)Tie2:cre) protected mice from dextran sulphate sodium (DSS)-induced colitis, with reduced epithelial erosions, immune cell infiltration, and colonic microvascular dysfunction, whereas the response of Phd2(f/+)Tie2:cre and Phd3(f/f)Tie2:cre mice to DSS was similar to that of their littermate controls. Using bone marrow chimeras and cell-specific cre mice, we demonstrated that ablation of Phd1 in haematopoietic cells but not in endothelial cells was both necessary and sufficient to inhibit experimental colitis. This effect relied, at least in part, on skewing of Phd1-deficient bone marrow-derived macrophages towards an anti-inflammatory M2 phenotype. These cells showed an attenuated nuclear factor-B-dependent response to lipopolysaccharide (LPS), which in turn diminished endothelial chemokine expression. In addition, Phd1 deficiency in dendritic cells significantly reduced interleukin-1 beta production in response to LPS. Taken together, our results further support the development of selective PHD1 inhibitors for ulcerative colitis, and identify haematopoietic cells as their primary target. Copyright (C) 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Ana M Meneses, Ben Wielockx Heisenberg Research Group, Department of Clinical Pathobiochemistry, Institute for Clinical Chemistry and Laboratory Medicine, Technische Universität Dresden, Dresden, Germany Abstract: Oxygen represents one of the major molecules required for the development and maintenance of life. An adequate response to hypoxia is therefore required for the functioning of the majority of living organisms and relies on the activation of the hypoxia-inducible factor (HIF) pathway. HIF prolyl hydroxylase domain-2 (PHD2) has long been recognized as the major regulator of this response, controlling a myriad of outcomes that range from cell death to proliferation. However, this enzyme has been associated with more pathways, making the role of this protein remarkably complex under distinct pathologies. While a protective role seems to exist in physiological conditions such as erythropoiesis; the picture is more complex during pathologies such as cancer. Since the regulation of this enzyme and its closest family members is currently considered as a possible therapy for various diseases, understanding the different particular roles of this protein is essential. Keywords: Hypoxia, PHD, HIF, inflammation, cancer

The main oxygen sensor hypoxia inducible factor (HIF) prolyl hydroxylase 2 (PHD2) is a critical regulator of tissue homeostasis during erythropoiesis, hematopoietic stem cell maintenance, and wound healing. Recent studies point toward a role for the PHD2‐erythropoietin (EPO) axis in the modulation of bone remodeling, even though the studies produced conflicting results. Here, we used a number of mouse strains deficient of PHD2 in different cell types to address the role of PHD2 and its downstream targets HIF‐1α and HIF‐2α in bone remodeling. Mice deficient for PHD2 in several cell lineages, including EPO‐producing cells, osteoblasts, and hematopoietic cells (CD68:cre‐PHD2f/f) displayed a severe reduction of bone density at the distal femur as well as the vertebral body due to impaired bone formation but not bone resorption. Importantly, using osteoblast‐specific (Osx:cre‐PHD2f/f) and osteoclast‐specific PHD2 knock‐out mice (Vav:cre‐ PHD2f/f), we show that this effect is independent of the loss of PHD2 in osteoblast and osteoclasts. Using different in vivo and in vitro approaches, we show here that this bone phenotype, including the suppression of bone formation, is directly linked to the stabilization of the α‐subunit of HIF‐2, and possibly to the subsequent moderate induction of serum EPO, which directly influenced the differentiation and mineralization of osteoblast progenitors resulting in lower bone density. Taken together, our data identify the PHD2:HIF‐2α:EPO axis as a so far unknown regulator of osteohematology by controlling bone homeostasis. Further, these data suggest that patients treated with PHD inhibitors or EPO should be monitored with respect to their bone status.

The tumor microenvironment plays a pivotal role during cancer development and progression. The balance between suppressive and cytotoxic responses of the tumor immune microenvironment has been shown to have a direct effect on the final outcome in various human and experimental tumors. Recently, we demonstrated that the oxygen sensor HIF-prolyl hydroxylase-2 (PHD2) plays a detrimental role in tumor cells, stimulating systemic growth and metastasis in mice. In our current study, we show that the conditional ablation of PHD2 in the hematopoietic system also leads to reduced tumor volume, intriguingly generated by an imbalance between enhanced cell death and improved proliferation of tumor cells. This effect seems to rely on the overall downregulation of protumoral as well as antitumoral cytokines. Using different genetic approaches, we were able to confine this complex phenotype to the crosstalk of PHD2-deficient myeloid cells and T-lymphocytes. Taken together, our findings reveal a multifaceted role for PHD2 in several hematopoietic lineages during tumor development and might have important implications for the development of tumor therapies in the future. What's new? Here the authors describe the complex role of the oxygen sensor PHD2 in tumor-associated immune cells in a conditional PHD2-deficient mouse line. They demonstrate that the observed reduced tumor volume is a consequence of opposing changes including the drastic down-regulation of numerous pro- as well as anti-tumoral genes and an imbalance between enhanced cell death and greater proliferation of tumor cells. They confined this complex phenotype to the crosstalk of PHD2-deficient myeloid cells and T-lymphocytes. The findings reveal a multifaceted role for PHD2 in hematopoietic lineages during tumor development and might have important implications for the development of tumor therapies.

Trained innate immunity fosters a sustained favorable response of myeloid cells to a secondary challenge, despite their short lifespan in circulation. We thus hypothesized that trained immunity acts via modulation of hematopoietic stem and progenitor cells (HSPCs). Administration of β-glucan (prototypical trained-immunity-inducing agonist) to mice induced expansion of progenitors of the myeloid lineage, which was associated with elevated signaling by innate immune mediators, such as IL-1β and granulocyte-macrophage colony-stimulating factor (GM-CSF), and with adaptations in glucose metabolism and cholesterol biosynthesis. The trained-immunity-related increase in myelopoiesis resulted in a beneficial response to secondary LPS challenge and protection from chemotherapy-induced myelosuppression in mice. Therefore, modulation of myeloid progenitors in the bone marrow is an integral component of trained immunity, which to date, was considered to involve functional changes of mature myeloid cells in the periphery., Cell, 172 (1-2), ISSN:0092-8674, ISSN:1097-4172

Background: Highly infiltrative growth and resistance to radiation as well as chemotherapy contribute to a poor prognosis of glioblastoma. To improve the survival of patients with glioblastoma, further research to uncover the complex signaling network is essential. Due to the central role of the signaling adaptor lamellipodin in nervous system development and cell migration, a function of lamellipodin in glioblastoma is conceivable. However, the specific function of lamellipodin in the invasion and radioresistance of glioblastoma is so far entirely unknown. Therefore, the present work investigates the invasion and radioresistance of glioblastoma cells and the underlying signaling mechanism under the influence of lamellipodin. Material and Methods: Expression of lamellipodin was evaluated in human astrocytes and nine glioblastoma cell lines by Western blot analysis. Localization of lamellipodin in glioblastoma cells was analyzed by applying immunofluorescence staining. The effects of lamellipodin silencing by siRNA on the glioblastoma characteristics invasion, survival, and residual DNA double strand breaks (DSB) upon X-ray irradiation, proliferation, apoptosis, and senescence were investigated. Alterations in molecular signaling were analyzed by phosphoproteome analysis upon control and lamellipodin depletion combined with/-out X-ray irradiation in A172 cells. Colony formation assays were performed after single and double knockdown of lamellipodin and the affected proteins to connect latter findings to clonogenic survival. Direct lamellipodin binding partners were explored using mass spectrometry analysis. Validation of protein-protein interaction was determined by immunoprecipitation and proximity ligation assay. Clonogenic survival assay was performed after triple knockdown of lamellipodin, previous proteins identified by phosphoproteome, and the direct interaction partner of lamellipodin. Hierarchical analysis was performed by analyzing the expression and phosphorylation of the determined proteins by immunoblot. Results: Lamellipodin was shown to be expressed and phosphorylated in varying amounts in the glioblastoma cell culture panel, with a preferential localization in membrane protrusions and the cytoplasm. The siRNA-specific depletion of lamellipodin tremendously decreased glioblastoma invasion and proliferation while exerting no impact on apoptosis or senescence. Moreover, seven of the nine studied glioblastoma cell lines were radiosensitized by lamellipodin silencing without affecting the number of residual DNA double strand breaks, while its overexpression improved radiation survival. Mechanistically, the loss of lamellipodin impaired the phosphorylation of nine proteins (EIF2A, EGFR, FOS, MKK6, NFKBIA, PRKAA2, RSK2, SRC, and TAK1), which are mostly implicated in the EGFR-MAPK signaling. The combinational silencing of lamellipodin and the relevant proteins achieved overall radiosensitization in A172 and U343MG cells. Furthermore, mass spectrometric analysis of lamellipodin immunoprecipitates demonstrated that the lamellipodin interactome alters in response to X ray irradiation conditions. RICTOR was confirmed as a direct linker of lamellipodin to the EGFR-MAPK signaling by immunoprecipitation and proximity ligation assay. In addition, triple depletion of lamellipodin, RICTOR, and EGFR resulted in similar degrees of radiosensitization as reported for lamellipodin knockdown, highlighting their superimposable role in glioblastoma radiation response. In line, lamellipodin silencing increased EGFR expression and phosphorylation, while lamellipodin phosphorylation was decreased upon EGFR deficiency. Conclusion: The results uncover the crucial function of lamellipodin for invasion, proliferation, and radiosensitivity of glioblastoma cells. Based on molecular analyses, lamellipodin was discovered as a determinant of EGFR signaling by interacting with RICTOR. Based on these data, the complexity of the signaling networks conducting radiation survival is broadened by adding the adaptor protein lamellipodin. Hintergrund: Ein stark infiltrierendes Wachstum und Resistenzen gegenüber Bestrahlung und Chemotherapie tragen zu einer schlechten Prognose des Glioblastoms bei. Um die Therapie des Glioblastoms zu verbessern, ist die weitere Erforschung des komplexen Signalnetzwerkes notwendig. Aufgrund der essentiellen Rolle des Signaladaptors Lamellipodin für die Entwicklung des Nervensystems und der Migration von Zellen ist eine Funktion von Lamellipodin im Glioblastom vorstellbar. Die spezifische Funktion von Lamellipodin in der Invasion und Strahlenresistenz des Glioblastoms ist allerdings bisher völlig unbekannt. Die vorliegende Arbeit untersucht daher die Invasion und Strahlenresistenz von Glioblastomzellen sowie den zugrundliegenden molekularen Mechanismus im Hinblick auf Lamellipodin. Material und Methoden: Die Expression von Lamellipodin wurde in humanen Astrozyten und neun Glioblastom-Zelllinien mittels Western Blot Analyse untersucht. Die Lokalisierung von Lamellipodin in Glioblastomzellen wurde mit Immunfluoreszenzfärbung analysiert. Die Auswirkungen eines Lamellipodin-Knockdown mittels siRNA auf die Glioblastom-Eigenschaften Invasion, Überleben und residuale DNA-Doppelstrangbrüche (DSB) bei Röntgenbestrahlung, Proliferation, Apoptose und Seneszenz wurden untersucht. Veränderungen in molekularen Signalwegen wurden durch Phosphoproteomanalyse nach Kontroll- und Lamellipodin-Knockdown in Kombination mit und ohne Röntgenbestrahlung in A172 Zellen analysiert. Koloniebildungsassays wurden nach einfachem und doppeltem Knockdown von Lamellipodin und den betroffenen Proteinen durchgeführt, um letztere Ergebnisse mit dem klonogenen Überleben in Verbindung zu bringen. Direkte Lamellipodin-Bindungspartner wurden mittels massenspektrometrischer Analyse untersucht. Die Validierung der Protein-Protein-Interaktion wurde durch Immunpräzipitation und Proximity Ligation Assay bestimmt. Ein klonogenes Überlebensassay wurde nach dreifachem Knockdown von Lamellipodin, durch das Phosphoproteom identifizierten Proteinen und dem direkten Interaktionspartner von Lamellipodin durchgeführt. Die hierarchische Analyse erfolgte durch Analyse der Expression und Phosphorylierung der ermittelten Proteine mittels Immunblot. Ergebnisse: Die Expression und Phosphorylierung von Lamellipodin war unterschiedlich in den Glioblastomzelllinien. Die siRNA-vermittelte Deletion von Lamellipodin verringerte die Invasion und Proliferation von Glioblastomzellen enorm, während die Reduktion von Lamellipodin keine Auswirkungen auf Apoptose oder Seneszenz hatte. Darüber hinaus wurden sieben der neun untersuchten Glioblastomzelllinien durch das Ausschalten von Lamellipodin radiosensibilisiert, ohne dass sich dies auf die Anzahl der residualen DNA-Doppelstrangbrüche auswirkte, während die Überexpression von Lamellipodin das Überleben nach Bestrahlung verbesserte. Mechanistisch beeinträchtigte der Verlust von Lamellipodin die Phosphorylierung von neun Proteinen (EIF2A, EGFR, FOS, MKK6, NFKBIA, PRKAA2, RSK2, SRC und TAK1), die hauptsächlich an der EGFR-MAPK-Signalübertragung beteiligt sind. Die kombinierte Ausschaltung von Lamellipodin und den relevanten Proteinen führte zu einer allgemeinen Radiosensibilisierung in den Zelllinien A172 und U343MG. Darüber hinaus zeigte die massenspektrometrische Analyse von Lamellipodin-Immunpräzipitaten, dass sich das Lamellipodin-Interaktom als Reaktion auf Röntgenbestrahlung verändert. RICTOR wurde durch Immunpräzipitation und Proximity Ligation Assay als direktes Bindeglied von Lamellipodin zum EGFR-MAPK-Signalweg identifiziert. Darüber hinaus führte die dreifache Deletion von Lamellipodin, RICTOR und EGFR zu einem ähnlichen Grad an Radiosensibilisierung wie der Knockdown von Lamellipodin, was ihre gemeinsame Rolle bei der Strahlenreaktion von Glioblastomen unterstreicht. Darüber hinaus erhöhte die Ausschaltung von Lamellipodin die EGFR-Expression und -Phosphorylierung, während die Lamellipodin-Phosphorylierung bei EGFR-Mangel verringert wurde. Schlussfolgerung: Die Ergebnisse decken die entscheidende Funktion von Lamellipodin für die Invasion, Proliferation und Radiosensitivität von Glioblastomzellen auf. Basierend auf molekularen Analysen wurde Lamellipodin als eine Determinante der EGFR-Signalübertragung durch Interaktion mit RICTOR identifiziert. Auf der Grundlage dieser Daten wird die Komplexität des Signalnetzwerks, welches das Überleben durch Strahlung reguliert, durch das Adaptorprotein Lamellipodin erweitert.

Regulated cell death (RCD) is indispensable for homeostasis and plays a crucial role in the pathophysiology of numerous diseases. Adrenocortical carcinomas (ACCs) represent a rare and highly malignant type of cancer. Currently, the most common therapeutic options include the complete surgical removal of the adrenal gland and/or the administration of mitotane, a derivative of the pesticide DDT. Yet patient survival remains poor and the mechanism of action of mitotane remains elusive. In this thesis it is demonstrated that the human ACC cell line NCI-H295R is sensitive to mitotane-induced cell death. In the first part, the involvement of three different RCD pathways, namely apoptosis, necroptosis and ferroptosis, in mitotane induced necrosis was investigated. To this end, different inhibitors were used, which were not able to block mitotane-induced cell death. When the medium was supplemented with insulin, transferrin, sodium selenite and linoleic acid (ITS+1) no cell death of the ACC cells was observed. This phenomenon was attributed to the presence of linoleic acid, since ITS supplementation lacking this component was not able to reverse mitotane-induced necrosis. Identification of new drug targets for alternative options of ACC treatment led to the investigation of key molecules involved in the pathways of necroptosis and ferroptosis. The receptor-interacting protein kinase 1 and 3 (RIPK1 and 3) and the mixed lineage kinase domain-like protein (MLKL) were considered as interesting targets given their crucial role in the execution of necroptosis. A western blot analysis of those molecules revealed the presence only of RIPK1, suggesting that the necroptosis machinery is not present in the NCI-H295R cells. Of interest, evaluation of the expression levels of glutathione peroxidase four (GPX4), one of the main inhibitory molecules of ferroptosis, showed a much higher expression in the ACC cells compared to the standard cell line used for studying ferroptosis, the human fibrosarcoma HT1080 cells. A hypothesis that the NCI-H295R cells are susceptible to ferroptosis induction was formed based on this finding. Compounds representative of all the four classes of ferroptosis inducers (FINs) were tested. Direct inhibition of GPX4 using the small compound RSL3, a type II FIN, led to high necrotic populations. Co-treatment with the ferroptosis inhibitor ferrostatin-1 (Fer-1) completely reversed RSL3-induced ferroptosis. Type IV FIN FINO2, that causes indirect loss of the enzymatic activity of GPX4, lead also to high necrotic populations, while Fer-1 prevented FINO2-induced ferroptosis. Data from public databases concerning gene methylation or mutation status of ACC tissues and normal human adrenal tissues was used to investigate potential key players of ferroptosis that might be either mutated or silenced in ACCs. Of note, glutathione peroxidases 3 and 5 (GPX3 and 5) were highly methylated, while the enzyme cystathionine gamma-lyase (CSE) involved in the transsulfuration pathway via the break down of cystathionine into cysteine and α-ketobutyrate and ammonia was found to be highly mutated. Collectively, these data point towards a high sensitivity of ACCs to ferroptosis induction. This could provide a new chapter for the therapeutic approaches of ACCs. Additionally, these findings provide a better understanding of the biology of this type of cancer that highly mutates or silences ferroptosis-related genes. The second part of this thesis focuses on the involvement of RCD in spontaneous cell death in isolated murine tubules. Existing literature points towards an involvement of necroptosis and ferroptosis pathways in the kidney in models of acute kidney injury (AKI). Acute tubular necrosis (ATN) represents a hallmark of AKI. While the work in the Linkermann lab has shown that isolated tubules perfused with type I FIN erastin undergo cell death in a “wave-of-death” manner, no deeper insights into the propagation of tubular necrotic injury exist. A protocol for isolation of murine kidney tubules was established, providing an ex-vivo model for investigation of tubular death. The absence of potentially confounding blood cells as well as immune cells was ensured by extensive washing steps as well as the use of collagenase. Visual observation and staining of isolated tubules with the nucleic acid stain SYTOX green revealed a spontaneous cell death in a “wave-of-death” manner. This wave was running in parallel with a calcium concentration change, indicating its involvement in the spontaneous necrosis. To investigate the potential involvement of mitochondria in this process, electron microscopy images were obtained from parts of the tubules with different levels of damage which revealed highly damaged and ballooned mitochondria. These data provided with a phenotypic characterisation of the spontaneous tubular necrosis. Aiming to approach this type of death genetically, necroptosis and pyroptosis deficient mice (MLKL/GSDMDDKO) were used. Comparison of the LDH release, used as a measure of necrosis, from isolated kidney tubules of the MLKL/GSDMDDKO mice and wild type (WT) mice showed no difference. This indicated that neither necroptosis nor pyroptosis are involved in the tubular necrosis. Therefore, the next step was to investigate the effects of Fer-1 at the levels of LDH of isolated tubules from WT mice. A significantly lower LDH release was observed in tubules treated with Fer-1 compared to the ones treated with vehicle. However, this reduction in the LDH release was not complete, suggesting that ferroptosis is only partially responsible for the spontaneous death of isolated tubules. The difference of male and female mice towards AKI sensitivity has been noted in the literature in that female mice are less susceptible compared to the male mice. Therefore, the next step was to investigate whether this protection of females can be observed at the level of isolated tubules. Indeed, the LDH release from tubules isolated from female mice was significantly less compared to the LDH release of tubules isolated from male mice. Based on the data obtained from isolated tubules from WT male mice treated with Fer-1, a similar experiment was performed with tubules isolated from WT female mice. No difference in the LDH release was observed between the Fer-1-treated tubules and the vehicle-treated ones, indicating that another cell death pathway might be involved. The most obvious difference between male and female organisms is the sex hormones. Whether testosterone or β-estradiol are responsible for the higher susceptibility or protection against cell death has been a debate over the last years. To test this hypothesis, three different cell lines were utilised. A pre-treatment of 16 h with either testosterone or β-estradiol was performed. Treatment with either type I FIN erastin or type II FIN RSL3 followed, and cells were analysed via flow cytometry. Data revealed protective effects of β-estradiol against ferroptosis induction. Next, the effects of β-estradiol in a simultaneous treatment with RSL3 were investigated. Interestingly the protective effects of the hormone were still observed. Among the metabolites of β-estradiol, 2-hydroxyestradiol (2-OHE2) has been reported to exert antioxidant effects. Therefore, 2-OHE2 was used in a simultaneous treatment with RSL3, and the obtained data showed that it was a much more potent inhibitor of necrotic cell death than β-estradiol even at lower concentrations. Collectively these data indicate that the lower susceptibility of female organisms towards cell death might be explained by the presence of β-estradiol and its more potent antioxidant metabolites. Such findings could change the way the two sexes are approached scientifically, while providing new insights on different therapeutic strategies between male and female organisms.

Background: A highly desmoplasmic microenvironment, the mutational landscape, and intra-tumoral heterogeneity contribute to the therapy resistance in pancreatic ductal adenocarcino-ma (PDAC). Due to higher precision, proton beam therapy is regarded beneficial compared to standard photon radiotherapy, although the role of radiotherapy is still ambiguous in PDAC. Cellular adhesion to the extracellular matrix (ECM) via integrins is well-known to mediate radi-ochemoresistance in various tumor entities. In PDAC, β1 integrins are associated with tumor progression. However, their role in radiochemoresistance is yet to be unraveled. Consequently, a comparative evaluation of cell survival and therapy sensitization after inhibition of β1 integ-rins or other survival-promoting protein kinases combined with either photon or proton irradia-tion and the underlying molecular mechanisms was carried out in this work. Materials and Methods: The expression of β1 integrins in PDAC and the correlation with pa-tient survival were assessed using publicly available patient data. The effect of β1 integrin inhi-bition by inhibitory antibodies or depletion on PDAC cell survival upon photon and proton irra-diation was explored using the tumoroid formation assay in three therapy-naïve and one radio-resistant PDAC cell lines grown in more physiological 3D laminin-rich ECM. Protein expression and cellular localization of β1 integrins were analyzed applying Western blot and immunofluo-rescence in the cell line panel. Basal molecular differences and those upon β1 integrin inhibi-tion were determined by a broad-spectrum kinome profiling in therapy-naïve and radioresistant 3D PDAC cultures. A potential interrelation of specific kinases with β1 integrin signaling in re-sponse to radiation was investigated in a single and double knockdown screen. The effects of photon and proton irradiation on PDAC cell survival of five 3D cultured PDAC cell lines were comparatively assessed using the 3D tumoroid formation assay. Molecular alterations upon both radiation types were identified by a broad-spectrum phosphoproteome analysis and Western blot. The exploitation of uniquely altered molecules and other signal transduction and DNA repair molecules for specific sensitization to photon and proton radiation was evaluated using specific biologicals in 3D cultured PDAC cell lines. Results: β1 integrins are overexpressed in PDAC compared to the normal pancreas, and the expression correlates with poorer patient survival. β1 integrin expression and localization var-ied cell line-dependently in the PDAC cell line panel. β1 integrin inhibition elicited an increase in radiosensitivity in all analyzed therapy-naïve and radioresistant 3D PDAC cultures, although less prominent in the latter. In line, the extent of kinase deregulation induced by AIIB2 was lower in the radioresistant 3D PDAC cultures. Double targeting of specific kinases with β1 in-tegrins further increased the radiosensitization in the therapy-naïve 3D PDAC cell cultures, whereas in the radioresistant counterpart, the effect was weak. Further, Erk2, PKD1, and CK1δ were revealed as potential interactors in the β1 integrin-mediated response to radiation. Proton irradiation showed a higher efficacy in the reduction of PDAC cell survival than photon irradia-tion. On the molecular level, irradiation with protons induced more phosphoproteomic altera-tions than photon radiation. Targeting of molecules uniquely altered upon irradiation failed to sensitize 3D PDAC cultures radiation type-specifically. However, a similar degree of radiosen-sitization for proton and photon irradiation in 3D PDAC cultures was observed upon targeting signal transduction and DNA repair proteins. Targeting non-homologous end joining (NHEJ)-specific proteins increased cellular radiosensitivity exceedingly for both radiation types in all 3D PDAC cell cultures. Conclusion: This work revealed a fundamental role of β1 integrins in intrinsic and acquired radioresistance of PDAC. However, to substantially overcome acquired radioresistance, further investigation is needed. Furthermore, a similar efficacy of proton and photon irradiation when combined with targeted therapies was demonstrated. These results suggest that multitargeting approaches based on targeting of β1 integrins or NHEJ-specific molecules combined with pho-ton or proton irradiation may turn out particularly promising. The strong radiosensitizing poten-tial of targeting these molecules may enable a more frequent use of radiotherapy for PDAC patient treatment. Hintergrund: Ein stark desmoplasmatisches Mikromilieu, das Mutationsprofil und die intra-tumorale Heterogenität tragen zur Therapieresistenz des Pankreaskarzinoms bei. Aufgrund der höheren Präzision wird die Protonentherapie als vorteilhaft gegenüber der Standard Pho-tonentherapie angesehen, obwohl ihre Wirksamkeit im Pankreaskarzinom noch ungewiss ist. Es ist bekannt, dass die zelluläre Adhäsion an die extrazelluläre Matrix (EZM) über Integrine die Radiochemoresistenz in verschiedenen Tumorentitäten vermittelt. Hierbei stehen β1 In-tegrine in Zusammenhang mit dem Fortschreiten der Erkrankung. Welche Rolle die β1 Integri-ne in der Radiochemoresistenz im Pankreaskarzinom spielen ist jedoch noch nicht bekannt. In dieser Arbeit wurde daher eine vergleichende Analyse des Zellüberlebens und der Therapie-empfindlichkeit nach Hemmung von β1 Integrinen oder anderen überlebensfördernden Pro-teinkinasen in Kombination mit Photonen- oder Protonenbestrahlung sowie der zugrundelie-genden molekularen Mechanismen durchgeführt. Material und Methoden: Die Expression von β1 Integrinen im Pankreaskarzinom und die Kor-relation mit dem Patientenüberleben wurden mittels öffentlich verfügbarer Patientendaten be-stimmt. Die Wirkung der β1-Integrin-Hemmung durch inhibitorische Antikörper oder mittels knockdown auf das Überleben von Pankreaskarzinomzellen bei Bestrahlung mit Photonen und Protonen wurde durch den Tumoroidbildungsassay in drei therapienaiven und einer strahlen-resistenten Pankreaskarzinomzelllinien untersucht. Diese wurden in physiologischer, 3D Lami-nin-reicher EZM kultiviert. Die Proteinexpression und die zelluläre Lokalisation von β1 Integri-nen wurden in allen Zelllinien mittels Western Blot und Immunfluoreszenz analysiert. Basale molekulare Unterschiede und solche nach Hemmung von β1 Integrinen wurden durch eine Breitspektrum-Kinomanalyse in therapienaiven und strahlenresistenten 3D-Pankreaskarzinomkulturen bestimmt. Eine mögliche Wechselbeziehung spezifischer Kinasen mit der β1 Integrin-vermittelten Strahlenantwort wurde in einem Einzel- und Doppel-knockdown Screen untersucht. Die Effekte der Bestrahlung mit Photonen und Protonen auf das Überleben von Pankreaskarzinomzellen wurden vergleichend anhand des Tumoroidbildungsassays in fünf 3D-kultivierten Pankreaskarzinomzelllinien bewertet. Durch beide Strahlungstypen indu-zierte molekulare Veränderungen wurden durch eine Breitspektrum-Phosphoproteomanalyse und mittels Western Blot identifiziert. Die Nutzung einzigartig veränderter Moleküle sowie an-derer Signaltransduktions- und DNA-Reparaturmoleküle zur spezifischen Sensibilisierung für Photonen- und Protonenstrahlung wurde untersucht. Dies erfolgte durch eine gezielte Hem-mung dieser Moleküle durch spezifische Biologika in 3D-kultivierten Pankreaskarzinomzellli-nien. Ergebnisse: β1 Integrine sind im Pankreaskarzinom im Vergleich zum gesunden Pankreas überexprimiert und ihre Expression korreliert negativ mit dem Patientenüberleben. Die Expres-sion und Lokalisierung der β1 Integrine variierte zelllinienabhängig in den Pankreaskarzinom-zelllinien. Die Hemmung der β1 Integrine führte zu einer Erhöhung der Strahlenempfindlichkeit in allen analysierten therapienaiven und strahlenresistenten 3D kultivierten Pankreaskarzinom-zelllinien, auch wenn diese in den letzteren weniger stark ausfiel. Auch der Grad der durch die Hemmung induzierten Deregulierung von Kinasen in den strahlenresistenten 3D Pankreaskar-zinomzellkulturen war geringer. Der gleichzeitige knockdown spezifischer Kinasen mit β1 In-tegrinen potenzierte die Strahlenempfindlichkeit in den therapienaiven 3D Pankreaskarzinom-zellkulturen, während diese im strahlenresistenten Pendant nur wenig beeinflusst wurde. Fer-ner wurden Erk2, PKD1 und CK1δ als potenziell beteiligte Kinasen in der durch β1 Integrine vermittelten Strahlenantwort entdeckt. Die Bestrahlung mit Protonen zeigte eine höhere Wirk-samkeit bei der Verringerung des Pankreaskarzinomzellüberlebens als die Photonenbestrah-lung. Auf molekularer Ebene induzierte die Protonenbestrahlung mehr Veränderungen im Phosphoproteom als die Photonenbestrahlung. Die gezielte Hemmung von einzigartig verän-derten Molekülen sensibilisierte die 3D Pankreaskarzinomzellkulturen nicht Strahlungsart-spezifisch. Jedoch konnte eine ähnliche Effizienz der beiden Strahlungstypen nach Hemmung gewisser Signaltransduktions- und DNA-Reparaturproteine in 3D Pankreaskarzinomzellkultu-ren gezeigt werden. Die zelluläre Strahlenempfindlichkeit gegenüber beiden Strahlungsarten wurde hierbei besonders durch die Hemmung spezifischer Proteine des non-homologous end joining (NHEJ) in allen 3D Pankreaskarzinomzellkulturen erhöht. Schlussfolgerung: Diese Arbeit konnte eine wesentliche Rolle von β1 Integrinen bei der intrinsischen und erworbenen Strahlenresistenz im Pankreaskarzinom ermitteln. Um die Strah-lenresistenz jedoch gänzlich zu überwinden, sind weitere Untersuchungen erforderlich. Des Weiteren wurde eine ähnliche Wirksamkeit von Protonen- und Photonenbestrahlung in Kombi-nation mit gezielten Therapien gezeigt. Diese Ergebnisse legen nahe, dass Multi-Targeting-Ansätze, die auf der Hemmung von β1 Integrinen oder NHEJ-spezifischen Molekülen in Kom-bination mit Photonen- oder Protonenbestrahlung basieren, ausgesprochen vielversprechend sein können. Angesichts des enormen Potentials zur Steigerung der Strahlenempfindlichkeit durch die Hemmung dieser Moleküle könnte eine häufigere Anwendung der Strahlentherapie bei Pankreaskarzinompatienten ermöglicht werden.

Summary The hematopoietic stem cell (HSC) compartment consists of a small pool of cells capable of replenishing all blood cells. Although it is established that the hematopoietic system is assembled as a hierarchical organization under steady-state conditions, emerging evidence suggests that distinct differentiation pathways may exist in response to acute stress. However, it remains unclear how different hematopoietic stem and progenitor cell subpopulations behave under sustained chronic stress. Here, by using adult transgenic mice overexpressing erythropoietin (EPO; Tg6) and a combination of in vivo, in vitro, and deep-sequencing approaches, we found that HSCs respond differentially to chronic erythroid stress compared with their closely related multipotent progenitors (MPPs). Specifically, HSCs exhibit a vastly committed erythroid progenitor profile with enhanced cell division, while MPPs display erythroid and myeloid cell signatures and an accumulation of uncommitted cells. Thus, our results identify HSCs as master regulators of chronic stress erythropoiesis, potentially circumventing the hierarchical differentiation-detour., Highlights • Chronic high EPO drives only HSCs to exhibit an exclusive erythroid progenitor profile • MPPs accumulate as uncommitted cells and display differential signatures, In this article, Wielockx and colleagues reveal that chronic erythropoietic stress induces distinct differences in the behavior of HSCs and their closely related MPPs. In particular, HSCs show greater commitment to an erythroid progenitor profile with heightened cell division, whereas MPPs are characterized by erythroid and immune signatures and an accumulation of uncommitted cells.

Hintergrund: Endothelzellen spielen sowohl in Homöostase als auch in Stresssituationen eine wesentliche Rolle bei der Regulation und der Erhaltung der hämatopoetischen Stammzellen (HSC). Viele Zytokine und Wachstumsfaktoren werden für die natürliche HSC-Aktivität benötigt und von den Endothelzellen exprimiert. Neben der Unterstützung der hämatopoetischen Stammzellaktivität stellen Endothelzellen ein Gefäßversorgungsnetzwerk zur Verfügung, um eine ausreichende Sauerstoffversorgung zu gewährleisten. Fragestellung: In der hier vorgestellten Arbeit habe ich die Hypothese verfolgt, dass eine Veränderung der Sauerstoffsensorik in Endothelzellen eine Modifizierung der Aktivität hämatopoetischer Stammzellen ermöglicht. Material und Methoden: Trotz des Wissens um die Bedeutung von Sauerstoff für die Endothelzellen fehlt uns das Verständnis dafür, wie die Sauerstoffsensorik der Endothelzellen die lokalen Knochenmark-Nischenzellen und die HSCs reguliert. Um einen Einblick in diesen Mechanismus zu erhalten, haben wir ein Mausmodell mit einem endothelzellenspezifischen Knockout des zentralen Sauerstoffsensors PHD2 entwickelt. Mit diesem in vivo-Ansatz habe ich versucht, den Einfluss von Veränderungen der Hypoxie-Signalwegproteine in Endothelzellen auf HSCs und ihre Nische zu untersuchen. Ergebnisse: Ich konnte in der vorliegenden Arbeit zeigen, dass die Morphologie der sinusförmigen Endothelzellen des Knochenmarks nach Verlust von PHD2 verändert ist. Außerdem konnte ich eine ausgeprägte Gefäßvasodilatation, begleitet von reduzierten hypoxischen Bereichen im angrenzenden Knochenmark beobachten. Zudem stellte ich fest, dass die Inaktivierung von PHD2 in Endothelzellen zu einem Rückgang des Knochenvolumens und zu einer Verminderung der an das Endothel angrenzenden Perizyten führt. Auffallend ist, dass sich gravierende Unterschiede in den hämatopoetischen Zellen der Peripherie zeigten, insbesondere war ein deutlicher Anstieg der zirkulierenden Leukozyten bei den KO-Mäusen zu erkennen. Dieser Phänotyp ist mit einer Vermehrung der hämatopoetischen Stamm- und Vorläuferzellen in Knochenmark und Milz verbunden. Darüber hinaus konnte ich zeigen dass die B-Zelldifferenzierung in der Milz vollständig zum Erliegen kommt, was zu einem signifikanten Rückgang der B-Zellen in der Marginalzone führt. Um die Funktionalität von Endothelzellen mit deletiertem PHD2 zu beurteilen, habe ich die Mäuse mit einer nicht-tödlichen Dosis ionisierender Strahlung behandelt. Die Analyse der endothelialen Zellregeneration im KO-Knochenmark zeigte eine Verminderung der Gefäßneubildung ohne Einfluss auf das gesamte Gefäßlumen im Vergleich zu unbehandelten Wurfgeschwistern. Außerdem stellte ich fest, dass sich in den ersten 3 Wochen nach der Bestrahlung bei Mäusen mit fehlendem PHD2 auf der Endothelzellenoberfläche das RBC-Kompartiments schneller regeneriert. Ich konnte ausschließen, dass dieser Effekt auf eine erhöhte Produktion von RBCs zurückzuführen ist, was zu der Hypothese führte, dass eine Verminderung des endothelialen PHD2 entweder zu einem längerfristigen Überleben der RBCs oder zu einer beeinträchtigten RBC-Clearance führt. Zusätzlich habe ich einen Anstieg in der Anzahl der quieszenten hämatopoetischen Vorläuferzellen bei Mäusen mit endothelialer PHD2-Deletion beobachtet, was darauf hindeutet, dass das endotheliale PHD2 Downstream-Signaling den Zellzyklus von hämatopoetischen Vorläuferzellen bei myeloablativen Stress beeinflusst. Abschließend konnte ich zeigen, dass ein Großteil der beobachteten Phänotypen bei KO-Mäusen durch den nachgeschalteten Transkriptionsfaktor HIF-2α vermittelt wird. Die Verwendung meiner selbstgenerierten Doppel-Knockout-Mauslinie, bei der erstmalig gleichzeitig PHD2 und HIF-2 in den Endothelzellen deletiert sind, führte zu eine vollständige Umkehrung des hämatopoetischen Phänotyps der bei den PHD2-Knockout-Mäusen im Steady-State beobachtet wurde. Zusätzlich wird der zuvor beobachtete signifikante Anstieg der Lymphozyten und der Rückgang der Erythrozyten- und Thrombozytenzahl in den Doppel-Knockout-Mäusen genetisch wiederhergestellt. Gleichzeitig reduziert sich die Entwicklung der marginalen B-Zellen im stationären Zustand wieder auf das Wildtyp-Niveau. Des Weiteren habe ich nach der Bestrahlung von Mäusen mit ionisierender Strahlung keine signifikanten Unterschiede zwischen WT und KO in ihrer hämatopoetischen Zellregeneration mehr feststellen können. Schlussfolgerungen: Zusammengenommen konnte ich in meiner Dissertation neue Eigenschaften von Hypoxie-Pathway-Proteinen in Endothelzellen mit Einfluss auf hämatopoetische Stamm- und Vorläuferzellen sowie auf verschiedene Kompartimente ihrer Nische demonstrieren; sowohl im stationären Zustand als auch nach Belastung durch Bestrahlung. Abschließend unterstreicht meine Arbeit die entscheidende Bedeutung der Sauerstoffsensorik im Knochenmark und gibt neue Einblicke in das Zusammenspiel zwischen dem Knochenmark-Endothel und dem hämatopoetischen System.:1 Introduction 1.1 Oxygen is necessary for survival of multicellular organisms 1.2 Oxygen sensing is necessary for induction of rapid cellular response 1.3 Endothelial and hematopoietic tissues together deliver oxygen 1.4 Hematopoietic stem cells give rise to blood cells 1.5 Regulation of HSCs is dependent on cells of the hematopoietic niche 1.6 Types of HSC regulation 1.7 Cancer therapy affects all highly proliferating cells 1.8 Irradiation stress disrupts hematopoietic stem cell niche signaling 1.9 Hypoxia induced signaling during recovery after irradiation 2 Aims of the thesis Aim 1: Determination of the role of endothelial PHD2 on the signaling towards the hematopoietic stem cells and its niche. 3 Materials and methods 3.1 Mice. 3.2 Histology, immunohistochemistry and immunofluorescence staining 3.2.1 Tissue processing prior to staining: 3.2.2 Staining: 3.3 Endothelial cell sorting 3.4 Mature hematopoietic cell isolation 3.5 Expression analysis 3.6 Hypoxyprobe 3.7 Quantitative image analysis 3.8 Bone structure analysis 3.9 Blood analysis 3.10 FACS analysis 3.11 Cell cycle analysis 3.12 RBC transfusion 3.13 Statistics. 4 Results 4.1 Determination of the role of endothelial PHD2 on the signaling towards the hematopoietic stem cells and its niche 4.1.1 Validation of the Flk1:cre line endothelial cell targeting 4.1.2 Characterization of the endothelial cell morphology and vessel function upon PHD2 inactivation 4.1.3 Loss of endothelial PHD2 leads to a decrease in bone marrow niche 4.1.4 Loss of PHD2 in endothelial cells leads to alterations of hematopoietic cells in the periphery 4.1.5 Significant increase of white blood cells in the periphery upon loss of endothelial PHD2 4.1.6 Loss of endothelial PHD2 does not impact the hematopoietic stem cells in the bone marrow. 4.1.7 Loss of PHD2 leads to increase in hematopoietic stem cells in the spleen 4.1.8 Loss of endothelial PHD2 impacts lineage-committed progenitors in bone marrow and the spleen 4.1.9 Loss of PHD2 in endothelial cells leads to an increase in frequency and activity of myeloid progenitors in bone marrow and the spleen 4.1.10 Loss of endothelial PHD2 impacts lymphocyte progenitors in secondary lymphatic organs but not in the bone marrow 4.2 Defining the impact of radiation exposure on the recovery of PHD2-deficient endothelial cells and its hematopoietic compartment. 4.2.1 Characterization of the non-lethal ionizing radiation damage to bone marrow endothelial and hematopoietic recovery 4.2.2 Loss of PHD2 from endothelium impacts endothelial recovery after myeloablative assault 4.2.3 Loss of PHD2 in endothelial cells and its subsequent effect on hematopoietic recovery following ionizing radiation exposure 4.2.4 Mechanism of increased RBC numbers is not due to an increase in hematopoietic stem cell frequency or activity in bone marrow or the spleen, 2 weeks after irradiation 4.3 Characterization of the influence of the transcription factor HIF-2α in mice lacking EC PHD2 4.3.1 The molecular signal transduction upon PHD2 inactivation is mediated by the HIF-2 transcription factor. 4.3.2 Loss of PHD2 and HIF-2α from endothelial cells partially rescues alterations in bone marrow endothelial cell morphology 4.3.3 Simultaneous loss of PHD2 and HIF-2α in endothelial cells completely reverses the hematopoietic phenotype observed in KO mice. 4.3.4 HIF-2α transcription factor induce signaling on endothelial cells that leads to marginal zone B cell impairment 4.3.5 Loss of PHD2 and subsequent stabilization of the HIF-2αtranscription factor is responsible for the increased recovery of RBCs following ionizing radiation 5 Discussion 6 References 7 List of abbreviations 8 Abstract 9 Zussamenfassung 10 Acknowledgements 11 Deklarations Background: Endothelial cells have an essential role in hematopoietic stem cell (HSC) regulation and maintenance during homeostasis and stress. Many cytokines and growth factors are required for normal HSC activity and are expressed by the endothelial cells. Along the support of hematopoietic stem cell activity, endothelial cells provide vessel delivery network to ensure proper oxygen delivery. Despite the importance of oxygen on endothelial cells, we lack the understanding of how oxygen sensing in endothelial cells regulates the local bone marrow niche cells and HSCs. Hypothesis: I have hypothesized that by modulating oxygen sensor in endothelial cells I will be able to modify the activity of hematopoietic stem cells Material and methods: To gain insight into this system, we developed a mouse model with an endothelial cell-specific knockout of the central oxygen sensor PHD2. Using this in vivo approach I sought to determine the impact of changes in hypoxia pathway proteins in endothelial cells on HSCs and their niche. Results: First, I revealed that the morphology of bone marrow sinusoidal endothelial cells is altered upon loss of PHD2. I observed prominent vessel vasodilation accompanied by reduced hypoxic areas in their adjacent marrow. Moreover, I determined that inactivation of PHD2 in endothelial cells led to a decrease in bone volume and pericytes adjacent to endothelium. Remarkably, I observed profound differences in the hematopoietic cells of the periphery. Specifically, I observed a profound increase in circulating leukocytes of KO mice. This phenotype was related to an increase in hematopoietic stem and progenitor cells in bone marrow and spleen. Moreover, I found a complete impairment of B cell differentiation in the spleen, which consequently led to a profound decrease in marginal zone B cells. To assess the functionality of endothelial cells lacking PHD2, I subjected the mice to a non-lethal dose of ionizing radiation. Analysis of endothelial cell recovery in KO bone marrow revealed a decrease in the formation of new vessels without an impact on the overall vascular lumen compared to WT littermates. Similarly, I found an enhanced recovery of the RBC compartment during the first 3 weeks after irradiation in mice lacking PHD2 on endothelial cells. I excluded the possibility that this effect was due to an increased RBC production, which led to hypothesis that inhibition of endothelial PHD2 results in prolonged RBC survival or impaired RBC clearance. Additionally, I observed an increase in quiescent hematopoietic progenitors cells in mice lacking PHD2 in endothelial cells that implies that endothelial PHD2 downstream signaling impact cycling of hematopoietic progenitors upon myeloablative stress. Finally, I demonstrated that a majority of the observed phenotypes in KO mice are mediated by the downstream HIF-2α transcription factor. Using my unique self-made double knockout mouse line simultaneously lacking PHD2 and HIF-2 in their endothelial cells, I was able to reveal a reversal of the hematopoietic phenotypes observed in the single PHD2 knockout mice during steady state. Additionally, the previously observed significant increase in lymphocyte and decrease in erythrocyte and thrombocyte numbers was genetically rescued in double knockout mice. Similarly, marginal zone B cell development returned to wild-type levels during steady state. Moreover, after subjecting mice to ionizing radiation I did not observe any significant differences between WT and KO in their hematopoietic cell recovery. Conclusions: Taken together, during my thesis I was able to demonstrate novel properties of hypoxia pathway proteins in endothelial cells having an impact on hematopoietic stem and progenitor cells as well as different compartments of their niche; both during steady state and radiation stress. In conclusion, my work underscores the critical importance of oxygen sensor signaling in the bone/bone marrow and provides new insight into the interplay between the bone marrow endothelium and the hematopoietic system.:1 Introduction 1.1 Oxygen is necessary for survival of multicellular organisms 1.2 Oxygen sensing is necessary for induction of rapid cellular response 1.3 Endothelial and hematopoietic tissues together deliver oxygen 1.4 Hematopoietic stem cells give rise to blood cells 1.5 Regulation of HSCs is dependent on cells of the hematopoietic niche 1.6 Types of HSC regulation 1.7 Cancer therapy affects all highly proliferating cells 1.8 Irradiation stress disrupts hematopoietic stem cell niche signaling 1.9 Hypoxia induced signaling during recovery after irradiation 2 Aims of the thesis Aim 1: Determination of the role of endothelial PHD2 on the signaling towards the hematopoietic stem cells and its niche. 3 Materials and methods 3.1 Mice. 3.2 Histology, immunohistochemistry and immunofluorescence staining 3.2.1 Tissue processing prior to staining: 3.2.2 Staining: 3.3 Endothelial cell sorting 3.4 Mature hematopoietic cell isolation 3.5 Expression analysis 3.6 Hypoxyprobe 3.7 Quantitative image analysis 3.8 Bone structure analysis 3.9 Blood analysis 3.10 FACS analysis 3.11 Cell cycle analysis 3.12 RBC transfusion 3.13 Statistics. 4 Results 4.1 Determination of the role of endothelial PHD2 on the signaling towards the hematopoietic stem cells and its niche 4.1.1 Validation of the Flk1:cre line endothelial cell targeting 4.1.2 Characterization of the endothelial cell morphology and vessel function upon PHD2 inactivation 4.1.3 Loss of endothelial PHD2 leads to a decrease in bone marrow niche 4.1.4 Loss of PHD2 in endothelial cells leads to alterations of hematopoietic cells in the periphery 4.1.5 Significant increase of white blood cells in the periphery upon loss of endothelial PHD2 4.1.6 Loss of endothelial PHD2 does not impact the hematopoietic stem cells in the bone marrow. 4.1.7 Loss of PHD2 leads to increase in hematopoietic stem cells in the spleen 4.1.8 Loss of endothelial PHD2 impacts lineage-committed progenitors in bone marrow and the spleen 4.1.9 Loss of PHD2 in endothelial cells leads to an increase in frequency and activity of myeloid progenitors in bone marrow and the spleen 4.1.10 Loss of endothelial PHD2 impacts lymphocyte progenitors in secondary lymphatic organs but not in the bone marrow 4.2 Defining the impact of radiation exposure on the recovery of PHD2-deficient endothelial cells and its hematopoietic compartment. 4.2.1 Characterization of the non-lethal ionizing radiation damage to bone marrow endothelial and hematopoietic recovery 4.2.2 Loss of PHD2 from endothelium impacts endothelial recovery after myeloablative assault 4.2.3 Loss of PHD2 in endothelial cells and its subsequent effect on hematopoietic recovery following ionizing radiation exposure 4.2.4 Mechanism of increased RBC numbers is not due to an increase in hematopoietic stem cell frequency or activity in bone marrow or the spleen, 2 weeks after irradiation 4.3 Characterization of the influence of the transcription factor HIF-2α in mice lacking EC PHD2 4.3.1 The molecular signal transduction upon PHD2 inactivation is mediated by the HIF-2 transcription factor. 4.3.2 Loss of PHD2 and HIF-2α from endothelial cells partially rescues alterations in bone marrow endothelial cell morphology 4.3.3 Simultaneous loss of PHD2 and HIF-2α in endothelial cells completely reverses the hematopoietic phenotype observed in KO mice. 4.3.4 HIF-2α transcription factor induce signaling on endothelial cells that leads to marginal zone B cell impairment 4.3.5 Loss of PHD2 and subsequent stabilization of the HIF-2αtranscription factor is responsible for the increased recovery of RBCs following ionizing radiation 5 Discussion 6 References 7 List of abbreviations 8 Abstract 9 Zussamenfassung 10 Acknowledgements 11 Deklarations

As graft source for lymphoma or leukemia treatment, hematopoietic stem and progenitor cells (HSPCs) have been the focus of translational medicine for decades. HSPCs are defined by their self-renewing capacity and their ability to give rise to all mature blood cells. They are found anchored to a specialized microenvironment in the bone marrow (BM) called the hematopoietic niche. HSPCs can be enriched by sorting them based on the presence of the surface antigen CD34 before clinical or tissue engineering use. As these cells represent a minority in most graft sources and the amount of applicable cells is limited, ex vivo expansion-cultures were established using cytokine cocktails or small molecules. However, in vitro culture of HSPCs as suspension-cultures result in heterogeneous cell populations with undefined cellular identities. In the BM niche, HSPCs are not exclusively maintained by cytokines but also by cell-matrix adhesions mediated by integrins (ITGs). Thus, β1 and β2 ITGs were found to promote initial contact of HSPCs with mesenchymal stromal cells (MSCs) and ITGβ3 expression was shown to be a marker for long-term repopulating HSPCs in vivo. Consequently, ex vivo remodeling of the BM niche using co-cultures of HSPCs and niche cells like MSCs came into spotlight and was proven to be a promising tool for stem cell expansion. However, in clinical and research applications, direct contact of two cell populations necessitates HSPC post-culture purification. To address these problems, we established a novel culture method for remodeling the BM extra cellular stroma in vitro wherein we used decellularized extracellular matrix (ECM) scaffolds derived from immortalized mesenchymal stromal cells (SCP-1). Such scaffolds were found to be highly reproducible and served as in vitro niche for HSPCs by being more effective for the expansion of CD34+ cells, compared to classical suspension cultures. ECMs were shown to consist of multiple proteins including fibronectins, collagens, and a major niche chemokine responsible for BM homing and retention of HSPCs in vivo, namely, stromal derived factor 1 (SDF-1). SDF-1 is known to be secreted by MSCs and is anchored to matrix proteins. This reveals that ECM scaffolds produced by SCP-1 cells are a naïve reconstructed microenvironment. When CD34+ cells were seeded, only around 20% of the cells adhered to the provided ECM scaffold. These cells recognized SDF-1 via C-X-C chemokine receptor type 4 (CXCR-4), as shown by laser scanning confocal microscopy. Thus, adhesive sides as they are present in the BM niche are provided. However, CD34+ cells isolated from G-CSF mobilized peripheral blood of healthy donors were found to be heterogenous with respect to adhesion capacity. Nonetheless, it was similar to HSPC co-cultures with SCP-1 cells as feeder layer. Therefore, we separated and analyzed two cell fractions, the adherent (AT-cells) and the non- adherent supernatant (SN-cells) cells. Other signals provided by the BM extracellular stroma to HSPCs are physical cues that control HSPC fate. HSPCs sense these physical features through focal contacts and accordingly remodel their morphological and biomechanical properties. Using real-time deformability cytometry (RT-DC) to uncover biomechanical phenotypes of freshly isolated HSPCs, SN-cells, AT-cells, and classical suspension cultured HSPCs in plastic culture dishes (PCD) were analyzed. We found freshly isolated cells to be less deformable and small. AT-cells displayed actin polymerization to stress fibers, and exhibited a stiffer mechanical phenotype compared to PCD-cultured or SN-cells. This might constitute the first hint of functional adaptation. Integrins are known to establish mechanosensing focal contacts. Thus, we analyzed ITG surface expression and identified ITGαIIb, ITGαV, and ITGβ3 to be enriched on AT-cells compared to freshly isolated cells or SN-cells. Active integrins need to form heterodimers consisting of one α- and one β subunit. Interestingly, the identified ITGs exclusively interact with each other to form RGD peptide receptors. RGD is a tripeptide consisting of the amino acids arginine, glycine, and aspartic acid and was identified as an adhesion sequence within fibronectin and other extracellular proteins. Consequently, we could confirm an important role for ITGαVβ3 in HSPC- ECM interaction with respect to adhesion and migration. However, we also identified ITGβ3 expression on a subset of CD34+ cells either freshly isolated or ECM cultured cells, as a marker for erythrocyte differentiation. These findings demonstrate that, in vitro, the ECM compartment acts as a regulator of HSPC fate and portray mechanical recognition as a potent driver of differentiation. In this context, targeted modulation of ECM scaffolds could enhance cell-ECM interactions and accelerate stem cell expansion or differentiation. These modulations could also provide further insights into HSPC-niche regulation. We demonstrate that ECMs derived from osteogenic differentiated SCP-1 cells increase HSPC expansion but do not lead to increased cell adhesion. As ECM adhesion preliminary alters HSPC function, we aimed at developing ECM scaffolds with increased adhesion capacity. Using lentiviral transduction, we generated a stable knock down of fibulin-1 in SCP-1 cells. Fibulin-1 is an ECM protein known to form anti-adhesion sites with fibronectin. However, we failed to increase adherent cell numbers or enhance HSPC expansion in the fibulin-1 knock down ECMs. Taken together, SCP-1 cell-derived ECM protein scaffolds provide an in vitro niche for HSPCs capable of stem cell expansion. Integrin mediated signaling altered the biomechanical and functional properties of HSPCs and hints at suspension cultures as being inappropriate to study the physiological aspects of HSPCs. Targeted modulation of ECM scaffolds could theoretically generate suitable ex vivo environments with the capacity to gain detailed insight into HSPC regulation within their niche. This will enhance the functionality of new biomaterials and will lead to improved regenerative therapies like BM transplantation.:List of contents I List of figures IV List of tables VI Abbreviations VII 1 Introduction 1 1.1 The stem cell microenvironment 3 1.1.1 The cellular endosteal bone marrow microenvironment 6 1.1.1.1 Mesenchymal stem/stromal cells 7 1.1.1.2 Hematopoietic stem and progenitor cells 8 1.1.2 Extracellular bone marrow microenvironment 10 1.1.2.1 Extracellular matrix 11 Chemokines and Cytokines 12 Cell adhesion to ECM 13 1.2 Native ex vivo ECM scaffolds 16 2 Aim of the study 19 3 Materials and methods 21 3.1 Materials 21 3.1.1 Chemicals and reagents 21 3.1.2 Kits 23 3.1.3 Media 24 3.1.4 Antibodies 24 3.1.5 Primers, sh-RNA sequences, and vectors 25 3.1.6 Equipment 26 3.1.7 Software 27 3.2 Methods 27 3.2.1 Cell preparation and culture 27 3.2.1.1 Mesenchymal stromal cells 27 3.2.1.2 Hematopoietic stem cells 28 3.2.1.3 Single cell picked clone 1 (SCP-1) cells 28 3.2.2 Generation of surface immobilized ECM preparations 29 3.2.2.1 Surface functionalization 29 3.2.2.2 ECM preparation 29 3.2.3 Flow cytometry and fluorescent activated cell sorting 30 3.2.4 Cell cycle analyses 30 3.2.5 Proliferation analyses 31 3.2.6 Colony forming unit cell assay (CFU-GEMM) 31 3.2.7 Migration assays 31 3.2.7.1 Transwell migration 31 3.2.7.2 Live cell migration 32 3.2.8 Confocal laser scanning microscopy 32 3.2.9 Real-time deformability cytometry (RT-DC) 32 3.2.10 Molecular biological methods 33 3.2.10.1 RNA isolation, reverse transcription, and PCR 33 3.2.10.2 Lentiviral shRNA transduction 34 3.2.10.3 Western blot 35 3.2.10.4 ELISA 36 3.2.11 Statistical analysis 37 4 Results 38 4.1 Extracellular matrix scaffolds for HSPCs 38 4.1.1 ECM properties 39 4.1.2 HSPC survival in ECM and PCD cultures 40 4.1.3 HSPC expansion in ECM and PCD cultures 41 4.2 HSPC morphological and mechanical adaptation to ECM 44 4.2.1 Actin polymerization and polarization 45 4.2.2 Biomechanical phenotype 46 4.3 Bioactive SDF-1 is incorporated in ECM scaffolds 49 4.3.1 CXCR4 polarization towards ECM 50 4.4 HSPC integrin expression and migration 52 4.4.1 Integrin surface expression on HSPC subsets 52 4.4.2 Focal contact formation 53 4.4.3 Integrin activation via ECM adhesion 55 4.4.4 Clonogenicity of ECM cultured HSPCs 57 4.4.5 HSPC migration when attached to ECM scaffolds 60 4.4.5.1 Reduced migratory behavior via ITGαVβ3 inhibition 61 4.4.5.2 SDF-1 induces migration but not adhesion 64 4.5 Targeted modulation of ECM scaffolds 65 4.5.1 Fibulin-1 knock down in SCP-1 cells 66 4.5.2 HSPC support of fibulin-1 reduced ECM scaffolds 70 5 Discussion 73 5.1 SCP-1 cells as a source for ECM scaffold production 74 5.2 Cell adhesion and focal contact formation 75 5.3 HSPC multilineage potential 78 5.4 ECM scaffold modulation 79 6 Summary 83 7 Zusammenfassung 86 Bibliography 89 Danksagung 108 Anlagen 110 Erklärung zur Eröffnung des Promotionsverfahrens [Formblatt 1.2.1] 110 Erklärung zur Einhaltung rechtlicher Vorschriften [Formblatt 1.1] 110

Mitochondria are essential for the viability of mammalian cells and provide a compartment for specific chemical reactions. Cellular respiration -- the main mitochondrial function -- is tightly connected with ROS production: the mitochondrial electron transport chain complexes I and III are the main ROS sources in mammalian cells. It has been reported that complex I and complex III activities are essential for cell cycle, apoptosis and stem cell differentiation (Spitkovsky et al., 2004; Varum et al., 2009; Lee et al., 2011; Ma et al., 2011; Tormos et al., 2012). In our work, we aimed to investigate the role of mitochondrial electron transport chain activity in the regulation of the differentiation potential and to unravel signaling pathways that could participate in this regulation. As a model, we used the P19 pluripotent stem cell line that can be easily differentiated into trophoblasts, expressing intermediate filaments cytokeratin 8/18, and neurons, which express cytoskeleton protein beta-III-tubulin. We first showed that both trophoblast and neural differentiation of P19 cells were accompanied by activation of cellular respiration. The analysis of respiratory chain complexes and supercomplexes, however, showed that undifferentiated P19 cells, as well as their differentiated derivatives did not differ in their respiratory machinery, including functional respirasomes. While undifferentiated cells did not use respiration as the main energy source, cellular respiration was activated during differentiation, indicating that oxidative metabolism was important for efficient differentiation. To investigate the potential role of mitochondrial electron transport chain activity we monitored the influence of a disrupted electron flow on the differentiation of P19 cells. We found that the activity of complex I and complex III influenced the differentiation potential of the pluripotent P19 cell line: the presence of complex I and complex III inhibitors rotenone, antimycin A, or myxothiazol increased the amount of cytokeratin 8/18+ cells during trophoblast differentiation, but almost completely prevented the formation of neuron-like beta-III-tubulin+ cells during neuron differentiation. Moreover, a low oxygen level (1 % O2 vs 21 % O2 in atmosphere) - the final electron acceptor - had the same effect on differentiation. These data suggest that mitochondrial electron transport chain activity contributes to the regulation of differentiation. The presence of complex I and complex III inhibitors, as well as oxygen scarcity, increase ROS production. We suggested that increased ROS level could explain the observed effects. By visualizing mitochondrial superoxide production with a specific dye – MitoSox - we confirmed that rotenone, antimycin A, myxothiazol, as well as low oxygen conditions, increased the superoxide level. These results suggest that the observed changes of the differentiation potential of P19 cells are associated with ROS production. To prove this idea, we differentiated P19 cells in presence of paraquat – a known ROS inducer. In line with our hypothesis paraquat promoted trophoblast differentiation. The received results suggest that the mitochondrial electron transport chain activity regulates differentiation through the ROS level. ROS are secondary messengers that participate in numerous processes including cell proliferation and differentiation. We aimed to predict the signal pathway that connects ROS level in stem cells and their differentiation potential. For this purpose, we performed a microarray analysis and compared the gene expression profiles of cells grown under hypoxia or in the presence of the complex III inhibitor myxothiazol with untreated control cells. The expression analysis revealed p53 as a transcriptional factor that impacts the differentiation potential in treated cells. p53 is a known redox-sensing molecule (Bigarella et al., 2014) that influences the differentiation potential through cell cycle control (Maimets et al., 2008). This observation is in line with our results and suggests that p53 may regulate the differentiation potential of P19 cells. We are planning to investigate the role of p53 signaling in the regulation of cell cycle and differentiation potential of P19 cell line.

Erythropoiesis must be tightly balanced to guarantee adequate oxygen delivery to all tissues in the body. This process relies predominantly on the hormone erythropoietin (EPO) and its transcription factor hypoxia inducible factor (HIF). Accumulating evidence suggests that oxygen-sensitive prolyl hydroxylases (PHDs) are important regulators of this entire system. Here, we describe a novel mouse line with conditional PHD2 inactivation (cKO P2) in renal EPO producing cells, neurons, and astrocytes that displayed excessive erythrocytosis because of severe overproduction of EPO, exclusively driven by HIF-2α. In contrast, HIF-1α served as a protective factor, ensuring survival of cKO P2 mice with HCT values up to 86%. Using different genetic approaches, we show that simultaneous inactivation of PHD2 and HIF-1α resulted in a drastic PHD3 reduction with consequent overexpression of HIF-2α-related genes, neurodegeneration, and lethality. Taken together, our results demonstrate for the first time that conditional loss of PHD2 in mice leads to HIF-2α–dependent erythrocytosis, whereas HIF-1α protects these mice, providing a platform for developing new treatments of EPO-related disorders, such as anemia.