Bone Marrow Angiopoietin/Tie signaling in the control of hematopoietic stem cells

Abstract

Hematopoietic stem cells (HSC) primarily reside in the bone marrow (BM) and possess the ability of self-renewal and differentiation to any progenitor or mature blood cell through hematopoiesis. Adult HSCs are found in specialized bone marrow niches that are essential for the regulation of quiescence, mobilization and differentiation of HSCs. Multiple studies have attempted to shed light on the complex signaling pathways between stromal and hematopoietic cells of the niche at steady state, inflammation and disease. Endothelial cells (EC) and perivascular stromal niches are known to illicit paracrine signals for the control of HSC maintenance and function. Curative transplantation approaches dwell on the effective activation and mobilization of hematopoietic stem and progenitor cells (HSPC) to the blood circulation. Current pharmaceutical approaches involve the use of mobilizing agent granulocyte-colony stimulating factor (G-CSF). The Angiopoietin/Tie (Ang/Tie) signaling pathway is essential for embryonic blood and lymphatic vessel development and maturation as well as vessel homeostasis in the adult. Few studies attempted to investigate Ang/Tie signaling in the BM stem cell niche. Angiopoietin-1 (Ang1) has been studied in the context of HSC maintenance and quiescence in the BM. It has also been shown that Ang1/Tie2 signaling is important for vascular recovery following BM irradiation. Besides the well-established role of Ang/Tie signaling in EC, Tie2 receptor is also known for its expression in HSPC. These findings led to the hypothesis that Ang/Tie signaling might impact HSPC in the bone marrow niche. The present study investigated Angiopoietin-2 (Ang2) in vivo in the context of HSPC activation, egress and mobilization to the periphery. For this purpose, both genetic approaches as well as pharmaceutical inhibition of Ang2 were employed. Although Ang2 did effect HSPC egress at steady state, Ang2KO mice demonstrated a delayed and reduced HSPC mobilization to the periphery upon G-CSF stimulation. Further dissection of the phenotype revealed that the absence of Ang2 hindered the prompt activation of HSPC rather than the process of mobilization. The bone marrow vasculature and its function seemed unaffected by Ang2 at steady state and upon G-CSF mobilization. Further assessment of Ang2 function on HSPC was carried out in the context of hematopoietic reconstitution upon lethal irradiation. The reduced capability of immune cell reconstitution in Ang2KO mice confirmed the ligand’s importance in replenishing the BM. The next focus of the thesis was elucidating the roles of Tie1 receptor on ECs in the BM in vivo. The investigation of HSPC egress and G-CSF-induced mobilization revealed fewer HSPCs in the periphery. Functional assays on blood vessels revealed that subtle changes in the vasculature are responsible for the reduced ability of HSPC mobilization in Tie1iECKO mice. Finally, since Tie1 receptor is not only expressed in ECs but also in HSPCs, this study involved the investigation of the receptor’s role in progenitor colony formation in vitro and BM reconstitution in vivo. Colony forming unit (CFU) assays revealed that Tie1 deletion on HSPC (Tie1KO) reduced the cells' ability to form differentiated colonies. Serial and competitive transplantations in mice confirmed the reduced ability of Tie1-deleted HSPC to repopulate the myeloid BM compartment of lethally irradiated mice. The present thesis sheds lights on the interactions of blood vessels and HSPCs from an Ang/Tie-centric perspective. The experiments have unraveled the contribution of the context-dependent partial agonist Ang2, and the orphan receptor Tie1 in HSPC egress, mobilization and bone marrow reconstitution. These new discoveries are important in elucidating Ang/Tie signaling in the BM and potentially contribute to HSPC mobilization research for the treatment of hematological malignancies.