**8.1 Overview of the Tie2/Angiopoietin axis**

Tie2 is a receptor tyrosine kinase first identified on vascular endothelial cells [61]. There are multiple components to the Tie2 signaling pathway where the angiopoietin ligands serve as binding partners [59]. In addition to its expression on endothelial cells, Tie2 is expressed in TEMs, hematopoietic stem cells, neutrophils, eosinophils, and some muscle satellite cells [59, 62]. Angiopoietin 1 (Angpt1) is primarily expressed in platelets and perivascular cells, while Angiopoietin 2 (Angpt2) is expressed in endothelial cells [63]. Expression of both Angiopoietins has also been demonstrated in hematopoietic stem cells and some immune cell types including monocyte/macrophages [64, 65]. Angpt1 serves as a Tie2 agonist, activating this receptor and increasing endothelial vessel stability [59]. However, the function of Angiopoietin 2 (Angpt2) is more variable. Some studies have shown that Angpt2 can act as either an agonist or antagonist of Tie2 depending on cellular context, and increased expression of Angpt2 has been demonstrated in multiple disease states [59]. Angpt2 has been found to act as a Tie2 agonist in the context of decreased Angpt1 signaling, absence of Tie1/Tie2 heterocomplexes, or inhibition of vascular endothelial protein tyrosine phosphatase (VE-PTP) in the endothelium [59, 66, 67]. However, the dominant role of Angpt2 and/or these cocomplexes in TBI has not been established. This ligand has repeatedly been shown to act as an antagonist in the setting of inflammation [68]. Although less studied than its counterpart, Tie1 has also been found to interact with Tie2 to promote Tie2/Angiopoietin interactions in vascular remodeling [59, 69]. The interactions between Tie2, Tie1, Angpt1, and Angpt2 have a profound influence on cell survival and vascular permeability [59, 61].

The downstream cellular effects of Tie2 binding with an Angiopoietin ligand can vary widely with cellular context. This is partially due to the differing effects of Angpt1 vs. Angpt2—Angpt1 binding has been shown to oppose the effects of inflammatory cytokines and decrease vascular permeability, while Angpt2 has been found to increase vascular permeability in a number of inflammatory models [59]. Binding patterns of these two ligands with Tie2 are distinct from each other, which may contribute to their differing effects. The fibrinogen-like domain of Angpt1 binds an immunoglobulin domain of Tie2, which may help Angpt1 increase cluster formation and cross-phosphorylation of Tie2 upon binding [68]. In contrast, Angpt2 has a slightly different amino acid sequence in the fibrinogen-like domain

and is also more likely to form dimers than oligomers. These structural differences may contribute to the different effects of the two ligands [68]. The central importance of clustering in Tie2 activation is confirmed by the results of one study that used an anti-Angpt2 antibody to cluster Angpt2. The clustering of Angpt2 caused it to act as an agonist to Tie2 rather than an antagonist, resulting in decreased vascular permeability and increased organ protection in the setting of sepsis [70]. Once Tie2 is activated, multiple downstream signaling pathways could be involved as effectors. Specifically, the Akt/PI3K (phosphatidylinositol 3 kinase) pathway has been implicated as a downstream effector of Tie2. This pathway is critical for cell survival and M2 macrophage polarization [61, 71, 72]. In the context of inflammation, Tie2 activation is decreased by a variety of mechanisms, (1) Angpt2 can be released from endothelial cells and competitively inhibits Angpt1/Tie2 binding, (2) overall expression of Tie2 and Angpt1 may be decreased, or (3) the extracellular domain of Tie2 can be cleaved [68]. The decrease in Tie2-Akt/PI3K signaling up-regulates Angpt2. This creates a feedback loop that further decreases Tie2 signaling [68]. The overall effect of the increasing endothelial-derived Angpt2 signaling is an increase in vascular permeability and amplification of inflammatory processes; however, these effects in the brain have not been established following TBI-induced neuroinflammation [63].

While expression of Tie2 has been most studied in endothelial cells, Tie2 has also been shown to be expressed in hematopoietic cell types. The role of Tie2 has been studied in hematopoietic stem cells and a subset of monocytes in addition to vascular and lymphatic endothelial cells [59]. Interestingly, Tie2 expression has also been demonstrated on neutrophils—Angiopoietin 1 has been shown to interact with Tie2 on neutrophils to promote neutrophil migration [73]. The role of Tie2 in macrophages has been increasingly recognized for its importance in tumorigenesis and inflammation. This critical function of Tie2 signaling will be discussed in the following sections of this review.
