The endothelial angiopoietin-Tie signaling pathway in development and disease

The vascular system consists of hierarchical networks of blood vessels and lymphatic vessels. The main function of the blood vasculature is to transport oxygen and nutrients to tissues, whereas the unidirectional lymphatic network is required mainly for maintenance of tissue fluid homeostasis and lipid absorption. Growth of new blood and lymphatic vessels, called angiogenesis and lymphangiogenesis, is required during embryonic development and in physiological processes in adults. Malfunction of the vascular systems is associated with various diseases such as cancer, inflammation and neovascular eye diseases. The functions of the vascular system are controlled by endothelial cells (ECs) lining the blood and lymphatic vessels. ECs are regulated by many growth factors including the endothelial angiopoietin (Ang) growth factor -Tie receptor signaling system. The two transmembrane tyrosine kinase receptors Tie1 and Tie2 form a receptor complex for angiopoietin growth factors, which regulate angiogenesis and lymphangiogenesis. Ang1 and Ang2 act as agonistic and context-dependent agonistic/antagonistic ligands of Tie2, respectively. However, Tie1 does not bind to angiopoietins. Instead, Tie1 participates in Ang-Tie2 signaling by forming a heterocomplex with Tie2. Ang-Tie signaling is important in vascular development and it controls pathological angiogenesis as well as vascular remodeling and integrity in inflammatory conditions. For example, increased Ang2 concentrations predict poor patient survival in severe diseases. Overall, the Ang-Tie pathway can be considered to be a potential therapeutic target. However, little is known about the function of Tie1 and the regulation of the context-dependency of Ang2. In this thesis, we investigated the function of Tie1 in embryonic lymphatic vascular development, postnatal sprouting angiogenesis and pathological angiogenesis. We also aimed to understand how Tie1 regulates angiopoietin signaling and the function of angiopoietins in inflammation. Furthermore, we studied the function of Ang2 in neuroinflammation. Constitutive Tie1-deletion results in embryonic lethality due to hemorrhage, edema and disruption of the microvasculature. Edema observed in Tie1-deleted embryos led us to hypothesize that Tie1 contributes to lymphatic vessel development. Indeed, we found that Tie1 was expressed in earliest lymphatic structures in developing embryos, called lymph sacs. The lymph sacs were fragmented in Tie1-deleted embryos. Analysis of different timepoints during development of Tie1-deleted embryos revealed that the edema and malformed lymph sacs precede the appearance of hemorrhages in these embryos. These results indicate that Tie1 is essential for normal lymphatic vessel development and that the lymphatic vessels are more sensitive to the loss of Tie1 than the blood vessels. We next investigated the function of Tie1 in tumor angiogenesis. EC-specific deletion of Tie1 reduced tumor angiogenesis and growth due to increased EC apoptosis. Tie1 deletion also reduced postnatal sprouting angiogenesis in the retina through activation of the Notch pathway. Importantly, Tie1 deletion did not affect healthy vasculature in adult mice. Tie1 deletion inhibited tumor growth as effectively as blockers targeting the vascular endothelial growth factor receptor (VEGFR) system, which are currently used in the clinic. However, no additive effects were observed when these two treatment strategies were combined, whereas additive inhibition of tumor growth was observed when Tie1 deletion was combined with angiopoietin inhibition. Combinatorial targeting of Tie1 and Ang2 also reduced angiogenesis in the retina, suggesting that targeting Tie1 in combination with Ang2 can improve anti-angiogenic therapy. We next analyzed the role of Tie1 in angiopoietin signaling and function in inflammation. We found that angiopoietin stimulation promoted Tie1-Tie2 interaction and Tie1 was required for normal Tie2 trafficking. Tie1 deletion in mice reduced Ang1- and Ang2-induced vascular remodeling. Furthermore, Tie1-deficiency reduced Ang1-induced activation of Tie2 and downstream signaling and inhibited the Tie2 agonistic activity of Ang2. In lipopolysaccharide (LPS)-induced inflammation, the extracellular domain of Tie1 was rapidly cleaved and this was associated with reduced Tie2 activation and loss of Ang2 agonistic activity. Our results thus indicated that Tie1 is an important regulator of Ang-Tie2 signaling and agonistic function of Ang2 is decreased in inflammation. Finally, we investigated the function of Ang2 in neuroinflammation. We found that Ang2 was induced in experimental autoimmune encephalomyelitis (EAE), a rodent model of multiple sclerosis (MS). Ang2 overexpression exacerbated the severity of EAE whereas its blockade ameliorated EAE. Similarly, treatment of the mice with ABTAA (Ang2-binding and Tie2-activating antibody), increased Tie2 phosphorylation and this reduced EAE severity, indicating that Tie2 activation is beneficial in this model. Our study showed that in neuroinflammation, Ang2 regulates the expression of EC adhesion molecules, blood brain barrier (BBB) integrity and recruitment of leukocytes into the central nervous system (CNS), as well as the pro-inflammatory polarization of CNS myeloid cells. These data implicate a role for Ang2 in autoimmune neuroinflammation. Overall, this thesis provides insight into the function of Tie1 and Ang2. Our studies reveal previously unknown roles of Tie1 in lymphatic vessel development, postnatal sprouting angiogenesis as well as in pathological tumor angiogenesis. We found that Tie1 is a critical component of the angiopoietin-Tie2 signaling and in inflammation its cleavage is associated with reduced agonistic function of Ang2. Furthermore, our work provides novel possibilities for therapeutic targeting of Ang2 in neuroinflammation. A deeper understanding of the functions of Tie1 and Ang2 in disease pathogenesis should aid in the development of therapies targeting the Ang-Tie signaling system.