The angiopoietin/Tie (ANG/Tie) receptor system controls developmental and tumor angiogenesis, inflammatory vascular remodeling, and vessel leakage. ANG1 is a Tie2 agonist that promotes vascular stabilization in inflammation and sepsis, whereas ANG2 is a context-dependent Tie2 agonist or antagonist. A limited understanding of ANG signaling mechanisms and the orphan receptor Tie1 has hindered development of ANG/Tie-targeted therapeutics. Here, we determined that both ANG1 and ANG2 binding to Tie2 increases Tie1-Tie2 interactions in a β 1 integrin-dependent manner and that Tie1 regulates ANG-induced Tie2 trafficking in endothelial cells. Endothelial Tie1 was essential for the agonist activity of ANG1 and autocrine ANG2. Deletion of endothelial Tie1 in mice reduced Tie2 phosphorylation and downstream Akt activation, increased FOXO1 nuclear localization and transcriptional activation, and prevented ANG1-and ANG2-induced capillary-to-venous remodeling. However, in acute endotoxemia, the Tie1 ectodomain that is responsible for interaction with Tie2 was rapidly cleaved, ANG1 agonist activity was decreased, and autocrine ANG2 agonist activity was lost, which led to suppression of Tie2 signaling. Tie1 cleavage also occurred in patients with hantavirus infection. These results support a model in which Tie1 directly interacts with Tie2 to promote ANG-induced vascular responses under noninflammatory conditions, whereas in inflammation, Tie1 cleavage contributes to loss of ANG2 agonist activity and vascular stability. The Journal of Clinical Investigation R E S E A R C H A R T I C L E3 4 9 6 jci.org Volume 126 Number 9 September 2016We found that angiopoietins promoted a direct interaction of Tie1 and Tie2 and that this interaction was regulated by integrin β 1 . ANG1-and ANG2-induced Tie2 activation and vascular remodeling were reduced or absent in mice where Tie1 was deleted in vascular endothelial cells. Tie1 deletion also attenuated ANG1-induced Akt activation and nuclear exclusion of FOXO1. We found that Tie1 was suppressed via ectodomain cleavage during acute inflammation and that this was associated with reduced agonistic activity of ANG2 and decreased Tie2 signaling. These results indicate that the agonist action of ANG2 is attenuated in inflammation and that Tie1 is an essential component of the angiopoietin signaling system that has potential for therapeutic targeting in disease. ResultsAngiopoietins induce direct interaction of Tie1 and Tie2. To investigate the dynamics of the Tie receptors in angiopoietin signaling, we transfected HUVECs with retroviral vectors expressing full-length (FL) Tie1 and Tie2, tagged on the C terminus with mCherry and GFP, respectively. Stimulation of FL-Tie2-GFP and FL-Tie1-mCherry expressing endothelial cells with COMP-Ang1 (CAng1) (49) or with recombinant human ANG2 induced colocalization of Tie1 and Tie2 in endothelial cell-cell junctions ( Figure 1A and Supplemental Video 1; supplemental material available online with this article; doi:10.1172/JCI84923DS1) (44,45,50). To investigat...

tributed to the design of experiments, data analysis, and interpretation of results. PB provided transgenic mice. DMM contributed to the design of experiments, data analysis, interpretation of results, and writing of the manuscript.

Primitive lymphatic vessels are remodeled into functionally specialized initial and collecting lymphatics during development. Lymphatic endothelial cell (LEC) junctions in initial lymphatics transform from a zipper-like to a button-like pattern during collecting vessel development, but what regulates this process is largely unknown. Angiopoietin 2 (Ang2) deficiency leads to abnormal lymphatic vessels. Here we found that an ANG2-blocking antibody inhibited embryonic lymphangiogenesis, whereas endothelium-specific ANG2 overexpression induced lymphatic hyperplasia. ANG2 inhibition blocked VE-cadherin phosphorylation at tyrosine residue 685 and the concomitant formation of button-like junctions in initial lymphatics. The defective junctions were associated with impaired lymph uptake. In collecting lymphatics, adherens junctions were disrupted, and the vessels leaked upon ANG2 blockade or gene deletion. ANG2 inhibition also suppressed the onset of lymphatic valve formation and subsequent valve maturation. These data identify ANG2 as the first essential regulator of the functionally important interendothelial cell-cell junctions that form during lymphatic development.

The endothelial Tie1 receptor is ligand-less, but interacts with the Tie2 receptor for angiopoietins (Angpt). Angpt2 is expressed in tumor blood vessels, and its blockade inhibits tumor angiogenesis. Here we found that Tie1 deletion from the endothelium of adult mice inhibits tumor angiogenesis and growth by decreasing endothelial cell survival in tumor vessels, without affecting normal vasculature. Treatment with VEGF or VEGFR-2 blocking antibodies similarly reduced tumor angiogenesis and growth; however, no additive inhibition was obtained by targeting both Tie1 and VEGF/VEGFR-2. In contrast, treatment of Tie1-deficient mice with a soluble form of the extracellular domain of Tie2, which blocks Angpt activity, resulted in additive inhibition of tumor growth. Notably, Tie1 deletion decreased sprouting angiogenesis and increased Notch pathway activity in the postnatal retinal vasculature, while pharmacological Notch suppression in the absence of Tie1 promoted retinal hypervasularization. Moreover, substantial additive inhibition of the retinal vascular front migration was observed when Angpt2 blocking antibodies were administered to Tie1-deficient pups. Thus, Tie1 regulates tumor angiogenesis, postnatal sprouting angiogenesis, and endothelial cell survival, which are controlled by VEGF, Angpt, and Notch signals. Our results suggest that targeting Tie1 in combination with Angpt/Tie2 has the potential to improve antiangiogenic therapy.