Context-dependent functions of angiopoietin 2 are determined by the endothelial phosphatase VEPTP Tomokazu Soumaa,b,1, Benjamin R. Thomsona,b,1, Stefan Heinenc,1, Isabel Anna Carotaa,b, Shinji Yamaguchia,b, Tuncer Onaya,b, Pan Liua,b, Asish K. Ghosha, Chengjin Lid, Vera Ereminad, Young-Kwon Honge, Aris N. Economidesf, Dietmar Vestweberg, Kevin G. Petersh, Jing Jina,b, and Susan E. Quaggina,b,2 aFeinberg Cardiovascular Research Institute, Northwestern University Feinberg School of Medicine, Chicago, IL 60611; bDivision of Nephrology/ Hypertension, Northwestern University Feinberg School of Medicine, Chicago, IL 60611; cHurvitz Brain Sciences Program, Sunnybrook Research Institute, Toronto, ON MN4 3M5, Canada; dLunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON M5G 1X5, Canada; eKeck School of Medicine, University of Southern California, Los Angeles, CA 90033; fRegeneron Pharmaceuticals, Tarrytown, NY 10591; gMax Planck Institute of Molecular Biomedicine, 48149 Münster, Germany; and hAerpio Therapeutics, Cincinnati, OH 45242 Edited by Kari Alitalo, Wihuri Research Institute and University of Helsinki, Helsinki, Finland, and approved December 20, 2017 (received for review August 20, 2017) The angiopoietin (ANGPT)–TIE2/TEK signaling pathway is essential for outcomes (2, 19). Developmental mouse models provide further blood and lymphatic vascular homeostasis. ANGPT1 is a potent TIE2 support for ANGPT2-mediated antagonism of ANGPT1–TIE2 ac- activator, whereas ANGPT2 functions as a context-dependent agonist/ tivation in blood vessels (1, 3, 4). Deletion of either Angpt1 or Tie2 antagonist. In disease, ANGPT2-mediated inhibition of TIE2 in blood results in embryonic lethality at embryonic day (E) 10.5 due to severe vessels is linked to vascular leak, inflammation, and metastasis. Using defects in cardiovascular development (7–11). This phenotype is conditional knockout studies in mice, we show TIE2 is predominantly reproduced by endothelial overexpression of Angpt2, supporting an activated by ANGPT1 in the cardiovascular system and by ANGPT2 in antagonistic role for ANGPT2 in the blood endothelium (12). the lymphatic vasculature. Mechanisms underlying opposing actions In contrast to its antagonistic role in the blood vasculature, of ANGPT2 in blood vs. lymphatic endothelium are poorly understood. ANGPT2 functions as a TIE2 agonist in the lymphatic endo- Here we show the endothelial-specific phosphatase VEPTP (vascular thelium (1, 3, 21). Angpt2 knockout mice display lymphatic de- endothelial protein tyrosine phosphatase) determines TIE2 response fects, including chylous ascites, as well as a sprouting defect in CELL BIOLOGY to ANGPT2. VEPTP is absent from lymphatic endothelium in mouse the retinal blood vascular capillaries (22–24). Intriguingly, only in vivo, permitting ANGPT2/TIE2-mediated lymphangiogenesis. Inhibi- the lymphatic phenotypes were rescued by the obligate TIE2 tion of VEPTP converts ANGPT2 into a potent TIE2 activator in blood agonist ANGPT1, supporting an agonistic role for ANGPT2 endothelium. Our data support a model whereby VEPTP functions as a specific to the lymphatic endothelium (22–24). rheostat to modulate ANGPT2 ligand effect on TIE2. Two recent papers have suggested that TIE2 is not required for lymphatic function in vivo, raising questions about the mechanism angiopoietin–TIE2 pathway | VEPTP | angiogenesis | lymphangiogenesis | tyrosine kinase of ANGPT2-mediated lymphangiogenesis (25, 26). However, here we report that LEC-specific loss of TIE2 phenocopies the lym- phatic defects observed in Angpt2 knockout mice, confirming that he angiopoietin–TIE2 (tyrosine kinase with Ig and EGF TIE2 is required for lymphatic development. Based on these data, Thomology domains, also known as TEK) receptor tyrosine kinase pathway regulates vascular homeostasis, maturation, and remodeling, and has been described as the “gatekeeper” of vascular Significance quiescence (1–4). TIE2 phosphorylation enhances vascular stability by promoting endothelial cell survival, reducing responsiveness to Reducing vascular leakage and stabilizing the endothelium inflammatory stimuli, and strengthening cellular junctions in ma- through activation of the angiopoietin (ANGPT)–TIE2 receptor ture vessels (5–10). The primary TIE2 agonist, angiopoietin 1 tyrosine kinase pathway is a promising therapeutic strategy for (ANGPT1), is secreted from perivascular cells, acting in a paracrine vascular diseases. ANGPT2 is one of two major ligands for the manner (7, 11). A second ligand, ANGPT2, has been described as a TIE2 receptor. Uniquely, ANGPT2 possesses an agonistic role in context-dependent agonist/antagonist, despite having similar re- lymphatic endothelium, but acts as a competitive antagonist in ceptor affinity as the agonistic ligand ANGPT1 (12–14). Unlike blood endothelium. The molecular basis for the opposing actions ANGPT1, ANGPT2 is secreted by endothelial cells and acts on the of ANGPT2 in these two vascular beds is poorly understood. Here TIE2 receptor in an autocrine manner (15). we demonstrate that the absence of VEPTP expression in the The ANGPT–TIE2 pathway has attracted attention due to lymphatic endothelium confers an agonist function of ANGPT2 on strong associations and causal links with human diseases, in- TIE2 receptor, but VEPTP expression in blood endothelium abro- cluding rare genetic disorders, such as hereditary vascular mal- gates its activity. Our findings provide mechanistic insights formations and primary congenital glaucoma (16–18), as well as needed to advance therapeutic targeting of this pathway. common diseases, such as sepsis, cancer, diabetes, and cardio- vascular disease (7, 19, 20). Vigorous efforts have been made to Author contributions: T.S., B.R.T., S.H., J.J., and S.E.Q. designed research; T.S., B.R.T., S.H., I.A.C., S.Y., T.O., P.L., A.K.G., C.L., and V.E. performed research; Y.-K.H., A.N.E., D.V., understand and translate this pathway to the clinic. However, a K.G.P., and J.J. contributed new reagents/analytic tools; T.S., B.R.T., S.H., J.J., and S.E.Q. major question remains: why does ANGPT2 display opposing analyzed data; and T.S., B.R.T., S.H., J.J., and S.E.Q. wrote the paper. context-dependent roles in different vascular beds (14)? In blood Conflict of interest statement: A.N.E. is an employee of Regeneron Pharmaceuticals. endothelial cells (BECs), ANGPT2 is described as an antagonist K.G.P. is an employee of Aerpio Therapeutics. of ANGPT1-mediated TIE2 activation (1, 3, 12, 14), while the This article is a PNAS Direct Submission. situation is reversed in lymphatic endothelial cells (LECs), where This open access article is distributed under Creative Commons Attribution-NonCommercial- ANGPT2 serves as the primary TIE2 agonist (21). NoDerivatives License 4.0 (CC BY-NC-ND). In the blood endothelium, where TIE2 signaling plays an im- 1T.S., B.R.T., and S.H. contributed equally to this work. portant role in vascular stability, elevated levels of circulating 2To whom correspondence should be addressed. Email: [email protected]. ANGPT2 in vascular diseases, such as sepsis, result in TIE2 in- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. hibition, leading to increased capillary leakiness and poor clinical 1073/pnas.1714446115/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1714446115 PNAS Early Edition | 1of6 Downloaded by guest on September 25, 2021 we hypothesize that the context-dependent agonist/antagonist function of ANGPT2 and its opposing effects on TIE2 in different vascular beds (i.e., LEC vs. BEC) might be explained by differ- ential expression of molecular components of the pathway, in- cluding negative regulators, such as the endothelial specific phosphatase, vascular endothelial protein tyrosine phosphatase (VEPTP) (27–30). To elucidate the molecular basis of opposing functions of ANGPT2 in LECs vs. BECs, we generated a series of gene- modified mouse lines and determined a critical cell-autonomous role for TIE2 signaling in lymphangiogenesis. We found that VEPTP is absent from LECs but abundant in BECs, and then used cell-biologic and proteomic-based approaches to explore the effect of VEPTP on ANGPT2–TIE2 activity. Our results show that VEPTP functions as a molecular “rheostat,” modulating re- ceptor sensitivity to enable discrimination between ANGPT li- gands, and provide a molecular mechanism to explain the opposing roles of ANGPT2 in blood and lymphatic vasculature. Results ANGPT2–TIE2 Signaling Is Essential for Embryonic Lymphangiogenesis. To identify the molecular basis of the differential functions of ANGPT2 in LECs and BECs, we characterized the role of ANGPT2–TIE2 signaling in lymphatic development, where ANGPT2 has a well-defined agonistic role (22–24). As expected, whole-body Tie2 deletion from conception using the Rosa26rtTA; tetOCre bitransgenic system in mice harboring a Tie2 conditional Δ by inversion (COIN) allele (Tie2WB E0) resulted in embryonic lethality between E9.5 and E10.5 (17, 18). However, embryos in- duced at E12.5 were found to survive until late gestation, allowing analysis of the lymphatic vasculature. At E16.5, all embryos were found alive. However, subcutaneous edema was observed in Fig. 1. ANGPT2–TIE2 signaling is essential for dermal lymphatic develop- Δ Tie2WB E12.5 knockout embryos, which was never observed in Tie2 ment. (A and B) Subcutaneous edema was observed following genetic de- wild-type or heterozygous controls (Fig. 1A). This result was re- letion of Tie2. Gross view of