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 119 / Die 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 119
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:87971 |
| Date | 13 November 2023 |
| Creators | Gaete Alvarez, Diana Estefania |
| Contributors | Wielockx, Ben, Erdmann, Kati, Technische Universität Dresden |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, doc-type:doctoralThesis, info:eu-repo/semantics/doctoralThesis, doc-type:Text |
| Rights | info:eu-repo/semantics/openAccess |
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