The Development Of A Cortical Bone Turnover Model: Parathyroid Hormone Effects In The Rabbit

Osteoporosis (OP) is a disease of progressive bone loss that is intimately linked with bone remodeling, a lifelong process of turnover essential for the maintenance of bone microarchitecture. Remodeling involves processes of coupled bone resorption and formation, carried out by complex cellular groupings known as basic multicellular units (BMUs). Imbalances in remodeling, where bone resorption exceeds formation, leads to elevations in cortical porosity and an increased risk of OP. Understanding the spatio-temporal coordination of BMUs is the key to elucidating the mechanisms underpinning cortical bone loss associated with OP and developing therapeutic approaches aimed at improving the balance between BMU phases to minimize bone loss. Much of the research on this subject has been largely theoretical or in silico thus far due to the lack of direct empirical evidence. Combining the use of high-resolution synchrotron based micro-computed tomography (micro-CT), a powerful imaging tool capable of characterizing cortical porosity in three-dimensional (3D) space, with the appropriate animal model has the potential to directly track individual BMUs and assess their behaviours in vivo. Rabbits are suitable model systems since they are the smallest laboratory animals to exhibit cortical bone remodeling like humans, however, remodeling rates in their cortices are generally low and approaches aimed at increasing remodeling have largely focused on trabecular bone. Therefore, there exists a need to develop an operationally effective cortical bone model given the growing recognition of the role cortical bone microarchitecture plays in bone loss and fragility in OP. The primary aim of this thesis was to establish a cortical porosity model in the rabbit using parathyroid hormone (PTH), a known inducer of cortical remodeling at intermittent levels. The experiment described in this thesis is presented as one aspect of a larger study characterizing numerous rabbit-based models of cortical bone loss (ovariectomy and glucocorticoid induced), all of which are compared to a control. After one month of intermittent PTH administration, cortical bone microarchitectural changes in the distal tibiae of rabbits were assessed ex vivo using micro-CT at a resolution of 10 μm and dynamic histomorphometry. Trabecular bone microarchitecture of the proximal tibial epiphysis was assessed secondarily. Results indicated that cortical porosity was substantially elevated in PTH rabbits relative to controls, and this was associated with increases in remodeling rate and cortical pore size. Despite bone losses intracortically, bone formation was evident on endosteal and periosteal surfaces of the tibiae and the rate of new bone deposition was increased in BMUs. Furthermore, a pattern of bone gain was observed in the proximal tibial epiphysis indicated by changes in micro-CT derived trabecular microarchitectural parameters. Notably, remodeling induced by PTH appeared to be distinct from that of the other rabbit-based models of bone loss in this larger study, with regards to cortical porosity and trabecular bone structural changes, warranting further investigations into these intriguing PTH-driven effects. Overall, the PTH rabbit model provides an effective and novel platform for developing future mechanistic studies aimed at testing a number of hypotheses directly related to bone remodeling regulation, which would ultimately enhance our understanding of the complex and dynamic nature of cortical bone in health and disease

A bone marrow-on-a-chip that maintains hematopoietic regenerative capacity in vitro

Thesis (Ph.D.)--Boston UniversityThe bone marrow niche is composed of a complex set of cellular, chemical, structural and physical cues that are required to maintain viability and function of the hematopoietic system. [1-5]. The source of all differentiated blood cells, the hematopoietic stem cell (HSC), is housed within the protective confines of the bone marrow where the complex microenvironment regulates its ability to undergo self-renewal or to differentiate into all of the mature functional blood cell types that constitute the hematopoietic system [4-7]. Engineering an artificial bone marrow that reconstitutes the critical inductive cues of naturally occurring bone marrow in vivo that maintains them in vitro could lead to new models of hematopoietic diseases, as well as enable expansion of bone marrow for therapeutic transplantation and manufacturing of differentiated blood cell replacements. It has proven difficult, however, to identify or combine the correct set of biomaterials and biological signals necessary to recreate the complex bone marrow microenvironment or to maintain functional, multi-potent, self-renewing HSCs in culture [8-13]. Here, we describe a microfluidic bone marrow-on-a-chip created in vivo by combining microsystems and tissue engineering strategies to produce bone that contains a complex bone marrow niche. The hematopoietic compartment of the engineered bone marrow (eBM) has a distribution of HSCs, hematopoietic progenitor cells, and differentiated blood cell types that is virtually identical to natural marrow. Moreover, these hematopoietic populations are retained in normal proportions and the HSCs maintain their full regenerative capacity when the eBM is explanted and cultured in the microfluidic bone marrow chip in vitro. After four days of culture on-chip, hematopoietic cells isolated from the eBM engrafted a lethally-irradiated mouse, reconstituted the compromised bone marrow, and fully restored all differentiated blood cell lineages. Preliminary work with human umbilical cord blood (hCB) suggests that the bone marrow-on-a-chip platform may be extended beyond the mouse to support human HSCs and hematopoietic progenitors in vitro. This ability to engineer a complex bone marrow niche that is capable of maintaining functional HSCs offers new tools for expansion of cells for transplantation, manufacturing. differentiated blood cells, evaluation of drug efficacy and toxicities, and study of hematopoietic diseases

The Methodological and Diagnostic Applications of Micro-CT to Palaeopathology: A Quantitative Study of Porotic Hyperostosis

The purpose of this dissertation was to assess the value of micro-CT to palaeopathology for the non-destructive analysis of orbital and cranial porotic hyperostosis, common lesions observed in many archaeological skeletal collections. The objectives of this study were to: 1) identify palaeoepidemiological trends in the prevalence of porotic hyperostosis that may support differential diagnoses, 2) evaluate the reproducibility and reliability of two-dimensional (2D) and three-dimensional (3D) methods of micro-CT data collection for the quantitative analysis of bone microarchitecture, and 3) quantitatively evaluate orbital and cranial porotic hyperostosis to determine the value of micro-CT methods for understanding disease pathogenesis and improving the differential diagnosis of these lesions. Sixty-six individuals obtained from four skeletal collections were assessed macroscopically using published methods of visual analysis as well as quantitatively using micro-CT methods. Structural indices used to quantify bone microarchitecture included bone volume density (BV/TV), specific bone surface (BS/BV), trabecular thickness (Tb.Th.), trabecular number (Tb.N.), and trabecular spacing (Tb.Sp.) (Hildebrand et al. 1999). The results of the visual analysis demonstrated an age-related trend in the prevalence of porotic hyperostosis, supporting previous hypotheses that this condition has an onset in childhood (e.g. Stuart-Macadam 1985). The micro-CT results illustrated that the most reliable and reproducible method for quantifying bone microarchitecture was a 3D volume of interest (VOI) method that maximized VOI size. Three-dimensional methods using VOIs of a uniform size were recommended with caution, and 2D VOI methods did not provide consistent observer agreement. The analysis of orbital porotic hyperostosis demonstrated significant changes (p \u3c 0.05) to bone microarchitecture in the advanced stages of disease pathogenesis, but not in the early or healing stages. The results for cranial porotic hyperostosis demonstrated significant changes only in the light stage. These results suggest that orbits are differentially and more significantly affected than the cranial vault likely due to structural differences between the bones of the skull. Changes to bone microarchitecture included an overall loss of trabecular bone and an increase in thinned, gracile trabeculae. Considering these findings within the clinical literature a differential diagnosis that includes anaemic conditions was supported. The identified palaeoepidemiological context also supported this differential diagnosis. Overall, the application of 3D micro-CT methods is of significant value for elucidating the process of disease pathogenesis and supporting current differential diagnoses of porotic hyperostosis in archaeological skeletal remains

Discovery of First-in-Class Small Molecule Agonists of the RXFP2 Receptor as Therapeutic Candidates for Osteoporosis

Osteoporosis is a chronic bone disease characterized by decreased bone mass and increased risk of developing fractures, predominantly observed in the elderly. The pathophysiological cause of the disease is a decrease in the activity of the bone-forming cells (osteoblasts) that alters bone remodeling in favor of bone resorption, leading to a decrease in bone mass. Recent studies identified the relaxin family peptide receptor 2 (RXFP2), the G protein-coupled receptor (GPCR) for insulin-like 3 peptide (INSL3), as an attractive target expressed in osteoblast cells to increase bone formation. The goal of this dissertation is to discover and characterize small molecule agonists of RXFP2 that are stable and can be delivered orally to promote bone growth. Several low molecular weight compounds were identified as agonists of the RXFP2 receptor using a cAMP high-throughput screen of the NCATS small molecule library. An extensive structure-activity relationship campaign resulted in highly potent and efficient full RXFP2 agonists. The selectivity and specificity of these compounds for human and mouse RXFP2 was shown in counter-screens against the related relaxin receptor RXFP1 and other GPCRs. Using a series of RXFP2/RXFP1 chimeric receptors, in silico modeling and RXFP2 point mutants, we established that the compounds are allosteric agonists of the RXFP2 receptor and identified the GPCR transmembrane domains as the specific region for compound interaction. We also showed that the candidate compounds promoted mineralization in primary human osteoblasts and had low cytotoxicity in various cell types. The compound with the highest activity in vitro was selected for pharmacokinetics profiling in mice, showing oral bioavailability and bone exposure. Moreover, an efficacy study in wild-type female mice treated orally with the lead compound showed a significant increase of the vertebral trabecular number and thickness compared to vehicle treated controls. Overall, our study has successfully identified and characterized the first-in-class small molecule series of RXFP2 agonists, which may lead to the development of a new class of orally bioavailable drugs for the treatment of diseases associated with bone loss

A Novel In Vivo Synchrotron Radiation Micro-CT Imaging Platform For The Direct Tracking Of Remodeling Events In Cortical Bone

Throughout life, bone tissue continuously alters its microarchitecture in response to microdamage and other stimuli through remodeling. Specialized cellular groupings, Basic Multicellular Units (BMUs), conduct remodeling through ‘coupling’ bone resorption to formation. Osteoclasts within a BMU’s cutting cone create a localized cylindrical space which osteoblasts concentrically refill, creating a secondary osteon (a.k.a. Haversian system). Continual production of secondary osteons by multitudes of BMUs creates a vast interconnected vascular network that permeates the cortex of bone, and therefore, BMUs are essential components in the overall maintenance of bone health. However, with increasing age or diseased states, such as osteoporosis (OP), remodeling can destabilize where resorbed bone is not entirely replaced (unbalanced) and/or where BMUs become ‘uncoupled’ preventing initiation of the bone formation following resorption. This increases porosity and thins cortices, leading to fragile, brittle bones much more susceptible to fracture. BMU behavior has never been replicated in vitro nor directly observed in vivo. The resorptive characteristics of BMUs, such as Longitudinal Erosion Rate (LER) – the rate of the advance of the cutting cone over time – are particularly poorly understood as our current understanding is inferred from indirect histological assessment of bone formation. Critically, BMUs have never been imaged in 4D (3D over time) due to limitations imposed by the radiation dose associated with conventional absorption-based imaging. This thesis explores in-line phase contrast synchrotron radiation micro- CT (SR micro-CT) as means of overcoming the limitations of conventional imaging. The goal was to develop a novel pre-clinical (animal) platform capable of directly tracking individual BMUs. The specific objectives of my thesis research were: 1) develop an in vivo imaging protocol to target individual BMU remodeling events within rabbit tibiae cortical bone to permit longitudinal imaging, using in-line phase contrast SR micro-CT; 2) Within rabbits, implement OP models of ovariectomy, glucocorticoids, a combination thereof and parathyroid hormone (PTH) to elevate cortical bone remodeling rates and, thus, the ability to observe BMU behavior on a large scale; and 3) directly measure BMU LER in 4D for the first time. A novel SR micro-CT protocol capable of detecting cortical porosity without any apparent radiation impacts was successfully developed on the BioMedical Imaging and Therapy Beamline of the Canadian Light Source. Compared to sham controls, elevated remodeling was found for all the OP models. PTH induced the highest rate of remodeling and it was selected as the model for direct assessment of LER. Through a novel co-registration technique, where in vivo SR micro-CT and follow-up ex vivo micro-CT scans acquired two weeks later were combined, LER (23.79 µm/day) was directly assessed for the first time. This novel platform establishes a means of investigating BMU spatio- temporal behavior and thus has great potential to advance our understanding of the role of remodeling in bone aging, adaptation, and disease

Analysis of Subchondral Bone and Microvessels Using a Novel Vascular Perfusion Contrast Agent and Optimized Dual-Energy Computed Tomography

Osteoarthritis (OA), is a chronic debilitating disease that affects millions of individuals and is characterized by the degeneration of joint subchondral bone and cartilage. These tissue degenerations manifest as joint pain, limited range of joint motion, and overall diminished quality of life. Currently, the exact mechanism(s) and cause(s) by which OA initiates and progresses remain unknown. The multi-factorial complex nature of OA (i.e. age, diabetes, obesity, and prior injuries have all been shown to play a role in OA) contributes to the current lack of a cure or effective long-term treatment for OA. One re-emerging and interesting hypothesis revolves around the delicate homeostatic microvascular environment around the cartilage – an avascular tissue. The absence of blood vessels within cartilage stresses the importance of nutrient and oxygen delivery from the neighbouring synovium and subchondral bone. Currently, the effects of changes in the subchondral bone microvessel density on cartilage health remain unknown due to the difficulties in simultaneously studying dense bone and the associated small microvessels. Computed tomography (CT) is widely used in the diagnosis of OA, as the use of x-rays provide detailed images of the bone degeneration associated with OA. However, the study of microvessels using CT has been exceptionally difficult due to their small (\u3c 10 µm) size, lack of contrast from neighbouring soft tissues, and proximity to dense bone. The purpose of this thesis was to develop a novel dual-energy micro-computed tomography (DECT) compatible vascular perfusion contrast agent and the associated instrumentation to optimize DECT on pre-clinical, cone-beam micro-CT scanners. The combination of these two techniques would facilitate the simultaneous visualization and quantification of subchondral bone and microvessels within the bone underlining the cartilage (i.e. distal femoral epiphysis and proximal tibial epiphysis) of rats that have undergone an OA-induced surgery. Results gained from this study will further provide information into the role that microvessels may play in OA

Development of Macromolecular Prodrug Conjugates for the Treatment of Inflammatory Diseases

Inflammation is the complex biological response to the stimuli triggered by various factors like pathogens, damaged cells, or irritants. Constant uncontrolled acute inflammation may become chronic conditions, leading to significant tissue damage, contributing to a series of chronic inflammatory diseases. Our lab has been working on the different types of inflammatory diseases and has developed multiple macromolecular prodrug conjugates for the better therapeutic efficacy and reduced toxicity. The general approach we have taken is to incorporate small molecules containing monomers into water-soluble and biocompatible polymers, such as N-(2-Hydroxypropyl) methacrylamide (HPMA) copolymers. The superior and the sustained efficacy of those macromolecular prodrug may due to their Extravasation through Leaky Vasculature and subsequent Inflammatory cell-mediated Sequestration (ELVIS mechanism) at the inflammatory site. To extend of our prodrug application, in my research, (1) we investigated whether the prodrug copolymer system we designed could ameliorate injury-induced inflammation. We established traumatic brain injury model in mice by the controlled cortical impact. We successfully ameliorated the neuroinflammation caused by the traumatic brain injury using HPMA Dexamethasone copolymer conjugates. We also reduced the complication caused by traumatic brain injury such as systemic osteopenia. (2) We utilized HPMA Tofacitinib copolymer conjugates (P-Tofa) to ameliorate the inflammation at the colon in a mouse ulcerative colitis model. We proved its improved therapeutic efficacy by targeting and retaining at the colon tissue. (3) To further reduce the toxicity of systemic administrated therapeutic agents while increasing the targeting property, we designed a local drug delivery system named Local Extracorporeal Vascular Circuit (LEVC). We minimized the systemic exposure of the drug and achieved superior local accumulation of the drug in ischemia-reperfusion injury mouse model. Collectively, the results from these systematic studies provide us the insight of the wider application of these prodrugs in the treatment of inflammatory diseases

Endothelial HIF-2alpha controls Cellular Migration in the Bone Marrow

Establishment and maintenance of the blood system relies on the cellular and spatial organization of bone marrow. In the BM niche, sinusoidal endothelial cells (SECs) are mainly located in the trabecular zone of the metaphysis area of long bones. SECs present a fenestrated structure characterized by high permeability, low shear rates, and oxygen pressure. The hypoxic environment surrounding SECs is needed for the movement and engraftment of hematopoietic cells, but it might also facilitate the homing of malignant cells. SECs’ adaptive response to hypoxia depends on the Hypoxia Inducible Factors (HIFs) promoting angiogenesis, hematopoiesis, and other processes. The upstream regulator of HIFs, the prolyl hydroxylase domain-2 (PHD2) is considered the key cellular oxygen sensor. Our group has shown the crucial regulatory effects that PHD2 has on different physiological and pathological settings. During steady state, PHD2 modulates proliferation and mobilization of hematopoietic progenitor cells (HSCPs), as well as bone metabolism. On the other hand, PHD2 is crucial for neutrophil motility affecting their extravasation during arthritis. We have also demonstrated that loss of PHD2 in different humans and mouse tumor cell lines, as well as in myeloid cells and T-lymphocytes where it impairs tumor development. Others have shown that heterozygous deletion of PHD2 in tumor ECs can reduce distant metastasis. Due to the intricate interplay between the different players of the PHD2/HIFs axis, as well as their effect on other signaling pathways, the positive or negative impact of the hypoxia pathways has been shown to be cell type and context dependent. In the present study, we analyzed the role of hypoxia pathway proteins, mainly PHD2, in ECs in the bone/BM niche, as well as its consequent influence on the environment during physiological and pathological scenarios. We have demonstrated that endothelial PHD2 has a profound intrinsic effect on vessel morphology and functionality, affecting the crucial communication between ECs and the bone/BM niche. Further, using different transgenic mouse lines, we have identified the BM endothelial cells (BM-ECs) PHD2/HIF-2α axis directly increasing leukocytosis via vascular cell adhesion protein 1 (VCAM-1) protein downregulation. Moreover, during steady state conditions we have discovered a novel regulatory effect that PHD2 exerts on VCAM-1 by increasing miR-126-3p transcription, a well-known inhibitor of VCAM-1 expression. Lastly, the PHD2/HIF-2/miR-126-3p/VCAM-1 axis not only influenced the myeloid cell intravasation towards circulation but also increased the extravasation and homing of metastatic breast carcinoma cells into the bone/BM tissue, increasing tumor burden throughout the bone. BM-ECs PHD2 offers a protective role against tumor homing cells in the bone/BM while serving as an important regulator in the communication between endothelium and BM niche. Concluding Remarks: Loss of PHD2 in ECs generates profound changes in vessel function and in the hematopoietic compartment leading to increase myelopoiesis resulting in leukocytosis. The above-mentioned effect occurs in a HIF-2α-dependent manner. PHD2/HIF-2α also transcribed in an increase of miR-126-3p expression leading to VCAM-1 downregulation. Process that resulted in increased leukocytes in circulation due to hematopoiesis dysregulation. The novel PHD2/HIF-2/miR-126-3p/VCAM-1 axis enhanced extravasation and homing of metastatic breast carcinoma cells into the bone/BM tissue, increasing tumor burden throughout the bone.:Introduction 8 The Endothelium 9 Endothelial barrier. 9 Leukocyte transendothelial migration. 11 Intracellular mechanism for activation of adhesion molecules. 12 Cellular migration in pathological conditions. 13 Tumor Dissemination: Metastasis. 14 Metastatic cascade. 15 The bone is a preferential site for malignant cells arrival. 16 Bone/BM physiology influences metastasis. 17 The endosteal niche. 18 The perivascular niche. 19 Hypoxia pathway proteins impact on metastasis. 20 Thesis Aims 23 Aim 1: Characterize the role of the hypoxia pathway proteins in bone vasculature and the impact on the niche. 24 Aim 2: Analyze the genetic changes that resulted from PHD2 deletion in BM-ECs and their impact on the cross-talk communication with the different BM-niches. 24 Aim 3: Investigate the impact of PHD2 and downstream regulators (HIF-1/2α) on vessel functionality. 25 Materials and Methods 26 Mice. 27 Histology: tissue processing and immunofluorescence staining 28 Bone tissue processing. 28 Staining of Bone cryosections. 28 Induced skin inflammatory model. 29 Staining of ears treated with PMA. 29 Vascular morphology quantification. 29 Microscopy 31 Antibodies used for immunofluorescence. 31 Bone analysis. 31 Bone µCT measurements and analysis 31 Tartrate-resistant acid phosphatase (TRAP) staining 32 Blood and BM analysis. 32 Sysmex. 32 Flow cytometry. 32 BM cell extraction from bones. 32 BM cells staining. 33 Meso Scale Discovery (MSD) 35 Evans Blue assay. 35 BM soup ELISA. 35 RNAseq of CD31+ EMCN+ BM-ECs 36 BM-EC cell sorting for RNAseq. 36 RNA extraction and qPCRs 36 Tumor model. 36 Tumor breast carcinoma cells. 36 Tumor homing model. 37 Statistical analyses. 37 Results 38 PHD2 Conditional Knockout from Endothelial Cell Compartment. 39 Further P2EC mice characterization. 41 Transgenic deletion of PHD2 showed slight developmental retardation. 41 Spleen size showed not to be affected by deletion endothelial PHD2. 41 P2EC mice displayed increased vessel leakiness. 42 Endothelial PHD2 deletion does not affect lung endothelial cells. 43 BM-ECs PHD2-HIF-2α axis modulates leukocytosis and vessel morphology. 44 HIF-2α modulates P2EC leukocytosis and thrombocytopenia. 44 BM-ECs PHD2 deficient mice hinder vessel morphology in a HIF-2α dependent manner. 44 Endothelial PHD2-deficient mice exhibit perturbed hematopoiesis. 45 P2EC mice early progenitor displayed reduced total cell number, but frequency remained unchanged. 46 P2EC mice favor differentiation of committed progenitors with a myeloid bias. 48 P2EC mice significantly reduced the numbers and frequency of megakaryocyte/erythroid progenitor’s linage. 48 PHD2-HIF-2α deletion restored normal hematopoiesis. 50 P2EC vascular functionality during pathological conditions. 53 Endothelial PHD2 modulates leukocyte migration during localized inflammation. 53 Endothelial PHD2 shapes bone/BM tumor homing. 55 Tumor homing in the bone: generation of an early metastatic model. 56 Early metastasis limitation. 58 Endothelial PHD2 modulates tumor colonization to the bone/BM. 59 Simultaneous deletion of PHD2 and HIF-1 in BM-ECs worsen tumor metastasis to bone. 61 Simultaneous deletion of PHD2 and HIF-2 in BM-ECs showed no differences in tumor homing. 62 Deep sequencing of PHD2 deficient BM-ECs. 63 BM EC from P2EC mice display enriched leukocyte migration gene signatures. 63 P2EC mice presented genetic dysfunction in the integrin-binding system. 64 P2EC steady-state VCAM-1 expression is HIF-2α dependent. 66 BM-ECs VCAM-1 + is regulated by PHD2 through HIF-2α. 66 PHD2-dependent downregulation of VCAM-1 does not affect VE-cadherin expression. 68 BM pro-inflammatory cytokines do not contribute to VCAM-1 lower expression. 69 During steady-state, loss of VCAM-1 increased frequency BM resident mature cells. 69 BM-ECs VCAM-1 deficient mice 71 VCAM1EC mice developed leukocytosis. 71 VCAM1EC does not exhibit significant changes in hematopoiesis. 73 P2EC vessel morphology is independent of downregulation of VCAM-1 74 VCAMEC mice showed increased tumor homing in the diaphysis. 75 PHD2-HIF-2 regulatory effect on VCAM-1 is modulated by mir-126-3p. 76 HIF-2α regulates mir-126 expression in PHD2 deficient BM-ECs. 77 BM-ECs PHD2 influence bone homeostasis. 78 Loss of BM-ECs PHD2 lead to increase Osteoclast numbers and activity. 79 Osteoclast differentiation and activity could be independent of OBs. 79 Loss of PHD2 in BM-ECs leads to osteoclastogenesis. 80 BM resident Tcell CD8+ could be Increasing Osteoclast Activation. 82 Discussion. 83 Mouse Model: Conditional Deletion of Endothelial PHD2. 85 Endothelial PHD2 Modulates Myelopoiesis. 86 BM-EC PHD2 regulates vessel morphology and functionality under steady-state independent of VCAM-1. 88 Endothelial VCAM-1 downregulation does not impaired neutrophil migration during inflammation. 88 BM-ECs PHD2 is a Gatekeeper of Tumor Homing in the Bone. 89 HIF-2α dependent Mir-126 activation leads to VCAM-1 downregulation 91 Endothelial PHD2 controls Osteoclastogenesis independent of BM RANKL. 94 References 96 List of Abbreviations 107 Summary 109 Zusammenfassung 111 Acknowledgements 113 Deklaration 114 Appendix 118 List of Figures. 118 List of tables 119Die Organisation und Aufrechterhaltung des Blutsystems hängt von der zellulären und räumlichen Organisation des Knochenmarks ab. In der BM-Nische befinden sich, hauptsächlich in der Trabekelzone des Metaphysenbereichs langer Knochen, die sinusoidale Endothelzellen (SECs). SECs weisen eine gefensterte Struktur auf, die von hoher Durchlässigkeit, geringer Scherrate und Sauerstoffdruck geprägt ist. Das hypoxische Milieu der SECs ist notwendig für Bewegung und Einwanderung der hämatopoetischen Zellen und könnte dies ebenso für bösartige Zellen begünstigen. Die Anpassung der SECs an Hypoxie hängt von den Hypoxia Inducible Factors (HIFs) ab. HIFs fördern die Angiogenese, die Hämatopoese und andere Prozesse. Die prolyl hydorxylase domain-2 (PHD2) ist der vorgeschaltete Regulator der HIFs und gilt als wichtigster zellulärer Sauerstoffsensor. Unsere Gruppe konnte zeigen welche zentralen regulatorischen Effekte die PHD2 auf verschiedene physiologische und pathophysiologische Mechanismen ausübt. Im Gleichgewichtszustand moduliert PHD2 Proliferation und Mobilisation der hämatopoetischen Vorläuferzellen (HSCPs) sowie den Knochenmetabolismus. Auf der einen Seite spielt PHD2 eine entscheidende Rolle bei der Motilität der Neutrophilen und beeinflusst daher die Extravasation bei Arthritis. Wir konnten außerdem zeigen, dass der Verlust von PHD2 in verschiedenen humanen und murinen Tumorzelllinien, sowie in myeloischen Zellen und T-Lymphozyten die Tumorentwicklung beeinträchtigt. In anderen Arbeiten wurde gezeigt, dass eine heterozygote PHD2-Deletion in Tumor-ECs eine Fernmetastasierung reduziert. In Anbetracht der komplizierten Wechselwirkung zwischen den verschiedenen Komponenten der PHD2/HIF-Signalkaskade sowie deren Effekte auf andere Signalkaskaden, übt sich in Abhängigkeit des Zelltyps und des Kontexts ein positiver oder negativer Einfluss auf die Hypoxie-Signalwege aus. In der vorliegenden Studie haben wir die Rolle von Proteinen des Hypoxiewegs, hauptsächlich PHD2, in den ECs der Knochen-/BM-Nische sowie deren daraus resultierenden Einfluss auf die Umwelt in physiologischen und pathologischen Szenarien analysiert. Wir konnten zeigen, dass das endotheliale PHD2 einen tiefgreifenden intrinsischen Effekt auf die Gefäßmorphologie und Funktionalität besitzt und damit entscheidend die Kommunikation zwischen ECs und der Knochen-/BM-Nische beeinflusst. Weiterhin konnte unter Nutzung verschiedener transgener Muslinien identifiziert werden, dass die Knochenmarksendothelzellen (BM-ECs) PHD2/HIF-2α-Achse direkt die Myelopoese, durch eine Herabsetzung der vascular cell adhesion protein 1 (VCAM-1) -Expression, steigert. Darüber hinaus haben wir einen neuartigen regulatorischen Effekt der PHD2 auf das VCAM-1 entdeckt. Hierbei wird die Expression des VCAM-1 Inhibitors miR-126-3p gesteigert. Des Weiteren beeinflusst die PHD2/HIF-2/miR-126-3p/VCAM-1 Achse nicht nur die Intravasation der Myloidzellen in Richtung des Kreislaufs, sondern auch eine Steigerung der Extravastion und Einwanderung von metastasierenden Brustkarzinomzellen in die Knochen/BM-Gewebe und steigert somit die Tumorlast in den Knochen. In Anbetracht klinischer Versuche der Krebsbehandlung mit PHD2-Inhibitoren, bietet BM-ECs PHD2 einen schützenden Effekt gegen Tumorzelleinwanderung in die Knochen/BM, während es gleichzeitig als ein wichtiger Regulator in der Kommunikation zwischen Endothelium und der BM-Nische dient. Abschließende Bemerkungen: Der Verlust von PHD2 in ECs führt zu tiefgreifenden Veränderungen in der Gefäßfunktion und im hämatopoetischen Kompartiment, was zu einer verstärkten Myelopoese und damit zu Leukozytose führt. Die oben erwähnte Wirkung tritt in einer HIF-2α-abhängigen Weise auf. PHD2/HIF-2α führte auch zu einem Anstieg der miR-126-3p-Expression, was zu einer Herunterregulierung von VCAM-1 führte. Dieser Prozess führte zu einer erhöhten Anzahl von Leukozyten im Blutkreislauf aufgrund einer Dysregulation der Hämatopoese. Die neuartige PHD2/HIF-2/miR-126-3p/VCAM-1-Achse förderte die Extravasation und Ansiedlung von metastatischen Brustkrebszellen im Knochen/BM-Gewebe, wodurch die Tumorlast im gesamten Knochen erhöht wurde.:Introduction 8 The Endothelium 9 Endothelial barrier. 9 Leukocyte transendothelial migration. 11 Intracellular mechanism for activation of adhesion molecules. 12 Cellular migration in pathological conditions. 13 Tumor Dissemination: Metastasis. 14 Metastatic cascade. 15 The bone is a preferential site for malignant cells arrival. 16 Bone/BM physiology influences metastasis. 17 The endosteal niche. 18 The perivascular niche. 19 Hypoxia pathway proteins impact on metastasis. 20 Thesis Aims 23 Aim 1: Characterize the role of the hypoxia pathway proteins in bone vasculature and the impact on the niche. 24 Aim 2: Analyze the genetic changes that resulted from PHD2 deletion in BM-ECs and their impact on the cross-talk communication with the different BM-niches. 24 Aim 3: Investigate the impact of PHD2 and downstream regulators (HIF-1/2α) on vessel functionality. 25 Materials and Methods 26 Mice. 27 Histology: tissue processing and immunofluorescence staining 28 Bone tissue processing. 28 Staining of Bone cryosections. 28 Induced skin inflammatory model. 29 Staining of ears treated with PMA. 29 Vascular morphology quantification. 29 Microscopy 31 Antibodies used for immunofluorescence. 31 Bone analysis. 31 Bone µCT measurements and analysis 31 Tartrate-resistant acid phosphatase (TRAP) staining 32 Blood and BM analysis. 32 Sysmex. 32 Flow cytometry. 32 BM cell extraction from bones. 32 BM cells staining. 33 Meso Scale Discovery (MSD) 35 Evans Blue assay. 35 BM soup ELISA. 35 RNAseq of CD31+ EMCN+ BM-ECs 36 BM-EC cell sorting for RNAseq. 36 RNA extraction and qPCRs 36 Tumor model. 36 Tumor breast carcinoma cells. 36 Tumor homing model. 37 Statistical analyses. 37 Results 38 PHD2 Conditional Knockout from Endothelial Cell Compartment. 39 Further P2EC mice characterization. 41 Transgenic deletion of PHD2 showed slight developmental retardation. 41 Spleen size showed not to be affected by deletion endothelial PHD2. 41 P2EC mice displayed increased vessel leakiness. 42 Endothelial PHD2 deletion does not affect lung endothelial cells. 43 BM-ECs PHD2-HIF-2α axis modulates leukocytosis and vessel morphology. 44 HIF-2α modulates P2EC leukocytosis and thrombocytopenia. 44 BM-ECs PHD2 deficient mice hinder vessel morphology in a HIF-2α dependent manner. 44 Endothelial PHD2-deficient mice exhibit perturbed hematopoiesis. 45 P2EC mice early progenitor displayed reduced total cell number, but frequency remained unchanged. 46 P2EC mice favor differentiation of committed progenitors with a myeloid bias. 48 P2EC mice significantly reduced the numbers and frequency of megakaryocyte/erythroid progenitor’s linage. 48 PHD2-HIF-2α deletion restored normal hematopoiesis. 50 P2EC vascular functionality during pathological conditions. 53 Endothelial PHD2 modulates leukocyte migration during localized inflammation. 53 Endothelial PHD2 shapes bone/BM tumor homing. 55 Tumor homing in the bone: generation of an early metastatic model. 56 Early metastasis limitation. 58 Endothelial PHD2 modulates tumor colonization to the bone/BM. 59 Simultaneous deletion of PHD2 and HIF-1 in BM-ECs worsen tumor metastasis to bone. 61 Simultaneous deletion of PHD2 and HIF-2 in BM-ECs showed no differences in tumor homing. 62 Deep sequencing of PHD2 deficient BM-ECs. 63 BM EC from P2EC mice display enriched leukocyte migration gene signatures. 63 P2EC mice presented genetic dysfunction in the integrin-binding system. 64 P2EC steady-state VCAM-1 expression is HIF-2α dependent. 66 BM-ECs VCAM-1 + is regulated by PHD2 through HIF-2α. 66 PHD2-dependent downregulation of VCAM-1 does not affect VE-cadherin expression. 68 BM pro-inflammatory cytokines do not contribute to VCAM-1 lower expression. 69 During steady-state, loss of VCAM-1 increased frequency BM resident mature cells. 69 BM-ECs VCAM-1 deficient mice 71 VCAM1EC mice developed leukocytosis. 71 VCAM1EC does not exhibit significant changes in hematopoiesis. 73 P2EC vessel morphology is independent of downregulation of VCAM-1 74 VCAMEC mice showed increased tumor homing in the diaphysis. 75 PHD2-HIF-2 regulatory effect on VCAM-1 is modulated by mir-126-3p. 76 HIF-2α regulates mir-126 expression in PHD2 deficient BM-ECs. 77 BM-ECs PHD2 influence bone homeostasis. 78 Loss of BM-ECs PHD2 lead to increase Osteoclast numbers and activity. 79 Osteoclast differentiation and activity could be independent of OBs. 79 Loss of PHD2 in BM-ECs leads to osteoclastogenesis. 80 BM resident Tcell CD8+ could be Increasing Osteoclast Activation. 82 Discussion. 83 Mouse Model: Conditional Deletion of Endothelial PHD2. 85 Endothelial PHD2 Modulates Myelopoiesis. 86 BM-EC PHD2 regulates vessel morphology and functionality under steady-state independent of VCAM-1. 88 Endothelial VCAM-1 downregulation does not impaired neutrophil migration during inflammation. 88 BM-ECs PHD2 is a Gatekeeper of Tumor Homing in the Bone. 89 HIF-2α dependent Mir-126 activation leads to VCAM-1 downregulation 91 Endothelial PHD2 controls Osteoclastogenesis independent of BM RANKL. 94 References 96 List of Abbreviations 107 Summary 109 Zusammenfassung 111 Acknowledgements 113 Deklaration 114 Appendix 118 List of Figures. 118 List of tables 11