Tie1 (tyrosine kinase with immunoglobulin and epidermal growth factor-like domains 1) was first reported in 1992 along with its related receptor Tie2 (Qu and Baldwin 2013). These Tie receptors are co-expressed in the endothelium, the innermost layer of cells lining blood and lymphatic vasculature, as well as all the chambers of the heart. Tie1 and Tie2 appear to be mutually expressed in all endothelia, as no study to date has identified a subpopulation of endothelial cells that exclusively expresses just one Tie receptor. However, their expression and activity are differentially regulated, as is evident in the lymphatic endothelium where Tie1 expression predominates and Tie2 expression is relatively diminished (Shen et al. 2014). Null mutations of Tie1 result in vascular defects and embryonic demise, and conditional mutagenesis is beginning to document important roles for Tie1 in postnatal angiogenesis. Tie1 remains an orphan receptor with no known ligand, while angiopoietin (Ang) molecules bind Tie2. As such, Tie1 is largely characterized through its context-dependent modulation of Tie2 signaling. Yet Tie1 has unique functions as a mediator of endothelial cell activity and vascular behavior, although its signaling mechanisms remain enigmatic.
Structure and Expression
Tie1 is expressed primarily in endothelial and hematopoietic cells (Qu and Baldwin 2013). Due to its expression in blood and lymphatic vascular systems, which extend throughout the body to provide nutrients to cells and remove cellular waste, respectively, Tie1 mRNA is detected in many developing organs in the human and mouse including the lungs, liver, heart, and brain (Korhonen et al. 1992). After birth, Tie1 is expressed in the pulmonary and retinal vasculature undergoing angiogenesis (formation of new blood vessels from existing vasculature) and remodeling (D’Amico et al. 2014; Savant et al. 2015; Woo and Baldwin 2011). Likewise, Tie1 expression persists in lymphatic endothelial cells (LECs) during postnatal restructuring of the lymphatic network (Qu et al. 2015). To maintain vascular quiescence in the adult, Tie1 is grossly downregulated compared to neonatal expression (Savant et al. 2015). However, Tie1 expression persists in angiogenic locations, such as maturing ovarian follicles and sites of wound healing (Korhonen et al. 1992) and in [neovascularizing tumors] neovascularizing tumors (D’Amico et al. 2014).
The first glimpses of Tie1’s unique roles came about as a result of global knockout studies in mice (see Qu and Baldwin 2013 for review). While Tie2 and its ligand Ang1 are necessary for the creation of the vascular network, Tie1 is requisite for vascular integrity and endothelial cell survival later in development. Without Tie1, the blood vasculature leaks, resulting in edema and localized hemorrhaging. Tie1-depleted embryos die between embryonic day 13.5 and birth, depending on the genetic background. Unlike Tie2 and Ang1, Tie1 is dispensable for early heart chamber development. Yet, loss of both Tie receptors causes a more severe cardiovascular phenotype with earlier demise (E8.5–E9.5) than that seen in embryos with deletion of either gene alone. These results suggest that although they have distinct roles in regulating endothelial cells during development, there is a critical Tie1-Tie2 genetic interaction. Consistent with this model, mice with high levels of both Tie1 and Tie2 mRNA are more resistant to infection by Ebola virus, presumably due to increased vascular integrity, repair, and/or maintenance (Rasmussen et al. 2014).
Conditional inactivation of Tie1 from the blood vasculature at later timepoints unveils its importance in normal and pathological angiogenesis. During angiogenesis, tip cells from existing blood vessels sprout and invade the surrounding area, followed by stalk cells which will form the lumen of new vessels. Savant et al. (2015) show Tie1 is upregulated in tip cells, whereas the adjacent stalk cells express both Tie1 and Tie2. Retinal angiogenesis, which occurs after birth, is disrupted with conditional deletion of endothelial Tie1 (D’Amico et al. 2014). It appears that Tie1 coordinates retinal vascular sprouting through the Tie2-Ang2 axis, as the defects in retinal angiogenesis in mice with conditional removal of Tie1 are exacerbated by blocking the Tie2 agonist, Ang2 (D’Amico et al. 2014) or by also deleting Tie2 from the endothelium (Savant et al. 2015). Consistent with its role in promoting retinal vascularization, ablation of Tie1 from adult mouse endothelia inhibits tumor angiogenesis and, subsequently, tumor growth (D’Amico et al. 2014). Together, these data highlight the importance of Tie1, not only in embryonic vascular development but also during active periods of angiogenesis in the postnatal period.
Tie1 is important for development of the lymphatic vascular system as well. Indeed, LECs are more sensitive to decreases in Tie1 expression than endothelial cells of the blood vasculature. Mice with an estimated 20% of normal Tie1 expression survive to late gestation, or even to adulthood in a few cases, with no defect in blood vessel development (Qu et al. 2010). However, the patterning and organization of the lymphatic system is perturbed. Normally, the primitive lymphatic system undergoes hierarchical remodeling with formation of intraluminal valves to ensure efficient, unidirectional collection and transport of lymphatic fluid to venous circulation during embryogenesis and through adulthood. Specific deletion of Tie1 from LECs confirms that it orchestrates lymphatic vessel remodeling and patterning as well as lymphatic valve (LV) specification and formation (Qu et al. 2015). Loss of Tie1 from the lymphatic network is incompatible with life, resulting in abnormal accumulation of lymphatic fluid and postnatal demise. Loss of the Tie1 endodomain causes a similar lymphatic phenotype, underlining the importance of Tie1 intracellular functions in LECs (Shen et al. 2014).
Flow-Dependent Regulation of Tie1 Expression
Consistent with its role in vascular remodeling and angiogenesis, Tie1 expression is regulated by flow. Laminar flow, like that in quiescent vessels, lowers Tie1 expression (Woo et al. 2011). However, when endothelial cells are exposed to non-laminar, disturbed flow like that found at vasculature branch points, Tie1 is upregulated (Woo et al. 2011). Notably, atherogenic plaques are prone to form on endothelia subjected to turbulent flow, such as the arterial vessel wall. Woo et al. (2011) found that loss of Tie1 resulted in a dose-dependent reduction of atheromas in a mouse model of atherosclerosis. Moreover, there was a loss of inflammatory adhesion molecules associated with atherosclerotic plaque development. In vitro studies also show that Tie1 is necessary for expression of pro-inflammatory genes (Woo and Baldwin 2011). In addition to being located at the site of plaque formation, Tie1 is expressed within the plaque, suggesting it may promote neovascularization and stabilization (Woo and Baldwin 2011). Together, these data suggest that Tie1 has a threefold part in atherosclerosis through its flow-regulated changes in expression, promotion of inflammatory markers, and the presence within vascularizing plaques.
Tie1 is also required for flow-dependent processes during lymphatic development, the hierarchical vessel remodeling, and development of LVs (Qu et al. 2015). Intraluminal valves typically form at vessel branch points of the remodeling lymphatic network, where disturbed flow stimulates the expression of master regulators of LV initiation (Kazenwadel et al. 2015; Sweet et al. 2015). Once fully formed, mature LV structure and function is also contingent on proper lymph flow (Kazenwadel et al. 2015). Tie1 is not only prominently expressed within developing LVs; embryonic deletion of Tie1 from the lymphatic endothelium disrupts LV specification and development (Qu et al. 2015). What is more, Tie1 expression persists in mature LVs and loss of lymphatic Tie1 results in LV agenesis (Qu et al. 2015). Taken together, it is tempting to speculate that Tie1 acts as mechanotransducer, as its expression is sensitive to changes in flow and loss of Tie1 disrupts flow-mediated processes in both lymphatic and blood vascular systems.
The underlying molecular mechanisms of Tie1 in endothelial cells remain elusive. As mentioned previously, much of Tie1 activity is characterized through its effects on the more defined Tie2/Ang signaling cascade (reviewed in Eklund et al. 2017). Tie1 is able to form heteromeric receptor complexes with Tie2 in the absence of angiopoietin ligands, yet Ang1 and Ang4 binding to Tie2 can also facilitate receptor interaction (Seegar et al. 2010; Woo and Baldwin 2011). Further complicating the signaling mechanism, angiopoietin stimulation of Tie1-Tie2 complex formation can also depend on a third transmembrane receptor, integrin β1 (Korhonen et al. 2016). Once the heteromeric Tie receptor complex is formed, Tie1 can be activated via phosphorylation of its intracellular domain by stimulation with Ang1, Ang4, or Comp-Ang1 (a synthetic, stable Ang1) (Eklund et al. 2017; Savant et al. 2015; Woo and Baldwin 2011).
Depending on the cellular context, Tie1 negatively or positively modulates Tie2 signaling. For instance, Tie1 phosphorylation is associated with reduced Tie2 signaling in endothelial progenitor cells (Woo and Baldwin 2011). Likewise, silencing Tie1, thereby preventing Tie1-Tie2 interactions, augments Ang1- and Ang3-mediated Tie2 phosphorylation (Eklund et al. 2017; Seegar et al. 2010). Conversely, (D’Amico et al. 2014; Korhonen et al. 2016) report that the agonistic actions of Ang1 and Ang2 on Tie2 rely on Tie1. Also, stimulation with a stable chimeric Tie1 protein containing its intracellular domain fused to colony stimulating factor 1 results in Tie1 autophosphorylation and activation of the Tie2 signaling cascade (Kontos et al. 2002). Savant et al. (2015 Cell Report) have helped clarify the roles of Tie1 in modulating Ang-Tie2 signaling during angiogenesis by parsing out Tie1 functions in phenotypically different endothelial cells. They found that expression of Tie1 in sprouting tip cells during angiogenesis is necessary for inhibition of Tie2 through cytosolic sequestration (Savant et al. 2015). Yet, in the trailing stalk cells which are proliferative and differentiating, Tie1 expression is required to maintain Tie2 activity. Thus, it seems promising that as Tie1 is explored in specific contexts within the endothelial milieu, its effects on Tie2/Ang signaling will be better understood.
Whether Tie1 signals independent of its association with Tie2 is controversial. As noted above, there is markedly reduced Tie2 in the lymphatic vasculature. However, Tie1 is abundantly expressed in lymphatics and is required for lymphatic development. Interestingly, the defects caused by LEC-specific deletion of Tie1 (Qu et al. 2015) are phenotypically recapitulated in Ang2-null mutants (Eklund et al. 2017). Additionally, the abnormalities in the Ang2 mutants can be rescued by placing the Ang1 gene into the Ang2 locus (Eklund et al. 2017), suggesting a possible unique agonistic role for Ang2/Tie1 in lymphangiogenesis.
Another avenue of Tie1 regulation is the proteolytic cleavage of the extracellular domain (ECD). Proteolytic Tie1 ECD shedding occurs via exposure of endothelial cells to chemicals, shear stress at physiological levels, and inflammatory cytokines like VEGF and TNFα (Woo and Baldwin 2011). Once Tie1 is cleaved, less phosphorylated Tie1 is present at the cell surface, but the truncated intracellular portion endures in the cytosol (Woo and Baldwin 2011). The cytosolic Tie1 endodomain remains for hours and can associate with other proteins, including Tie2 and the tyrosine phosphatase and adapter protein, Shp2 (Marron et al. 2000; Woo and Baldwin 2011). This proteolytic processing places additional layers of complexity onto Tie1 signaling, and the functions of its cleaved ECD in the bloodstream or the truncated cytosolic endomain are not yet mapped out.
Inflammation-induced shearing of the Tie1 ectodomain compromises vascular integrity through modulation of Ang/Tie2 activities. Once the Tie1 ectodomain is cleaved, Tie2 activity and expression are reduced, Ang1 expression is downregulated, and Ang2 levels increase (Korhonen et al. 2016). Ang2, although upregulated, has less agonist effects and may become an antagonist of Tie2 during inflammation (Eklund et al. 2017; Korhonen et al. 2016). Accordingly, it has been suggested that during baseline conditions, Tie1 is necessary to maintain Tie2 signaling and promote endothelial stability. However, under inflammatory conditions, Tie1 ectodomain shedding is a means to mitigate Ang activation of Tie2, causing loss of vascular integrity (Eklund et al. 2017; Korhonen et al. 2016).
The past 25 years of research have laid the groundwork to more deeply interrogate Tie1’s multifaceted roles in the development of the blood and lymphatic vascular systems, as well as its pathological functions in active endothelial populations. The fact that no definitive ligand has been identified for Tie1 remains a major roadblock in harnessing potential mechanisms that enhance or attenuate Tie1-mediated signaling for therapeutic benefit. The context-dependent nature of Tie1 signaling has further complicated the unique role this receptor plays in pathological processes. Clearly, further understanding of the situational roles Tie1 plays in normal and abnormal processes will aid in development of therapeutic strategies and determine the utility of Tie1 as a biomarker.
For example, Tie1 is involved in acute and chronic inflammation. Patients with acute Puumala virus (a type of hanta virus) infection have higher concentrations of cleaved Tie1 ectodomain in their serum (Korhonen et al. 2016). In chronic inflammation, Tie1 expression is increased in inflamed tissues of patients with rheumatoid arthritis and osteoarthritis and in mouse models of arthritis (Malik et al. 2010; Woo and Baldwin 2011). Strikingly, treating arthritic mice with a variant of the Tie1 extracellular domain alleviates arthritic symptoms through inhibition of Ang1 and VEGF (Malik et al. 2010). These studies imply that the Tie1 ECD can be a measurable marker of disease and may be manipulated for therapeutic purposes.
Several anticancer agents that target Ang-Tie are in clinical trials (www.clinicaltrials.gov). In humans with metastatic colorectal cancer, lower levels of Ang2 expression correlate with better responses to anti-VEGF antibodies with chemotherapy (Jeltsch et al. 2013). Furthermore, combinatorial therapy, i.e., blocking VEGFA and angiopoietins, improves vascular integrity, inhibits metastasis, and causes regression of tumor vessels in mice compared to monotherapy (Jeltsch et al. 2013). Tie1 is a potent regulator of angiogenesis, and high levels of Tie1 in gastric adenocarcinoma are inversely related to patient survival (Woo and Baldwin 2011). Based on the known interplay and dependence between Ang-Tie2-Tie1 during angiogenesis, anti-Tie1 compounds may be created and added to therapeutic cocktails in the future to inhibit tumor growth and metastasis.
Insights from mouse models place Tie1 as a key coordinator of molecular events necessary for proper lymphatic valve formation and maintenance. In humans, dysregulation of lymphatic drainage due to defects in lymphangiogenesis or lymphatic valves results in lymphedema (Alitalo 2011). Lymphedema can also occur after damage to existing structures caused by trauma, surgery, or infection (Alitalo 2011). Loss-of-function mutations in human Foxc2, a key regulator of lymphatic valve development, cause lymphedema-distichiasis, an autosomal dominant disorder characterized in part by limb lymphedema (OMIM #153400). In the mouse, Tie1 is necessary for lymphatic expression of Foxc2 (Qu et al. 2015). Further investigation is needed to determine if the role of Tie1 in regulating LV formation is conserved in humans and if it can be utilized to alleviate lymphedema.
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