JCI - A lymphatic defect causes ocular hypertension and glaucoma in mice

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JCI - A lymphatic defect causes ocular hypertension and glaucoma in mice

J Clin Invest. doi:10.1172/JCI77162. 
Copyright © 2014, The American Society for Clinical Investigation.

Brief Report

A lymphatic defect causes ocular hypertension and glaucoma in mice

Benjamin R. Thomson1, Stefan Heinen2, Marie Jeansson3, Asish K. Ghosh1,Anees Fatima1, Hoon-Ki Sung2, Tuncer Onay1, Hui Chen4, Shinji Yamaguchi1,Aris N. Economides5, Ann Flenniken2, Nicholas W. Gale5, Young-Kwon Hong6,Amani Fawzi4, Xiaorong Liu4,7, Tsutomu Kume1 and Susan E. Quaggin1,2

1Feinberg Cardiovascular Research Institute, Northwestern University Feinberg School of Medicine, Chicago, Illinois, USA. 
2Lunenfeld-Tanenbaum Research Institute, Mt. Sinai Hospital, Toronto, Ontario, Canada. 
3Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden. 
4Department of Ophthalmology, Northwestern University, Chicago, Illinois, USA. 
5Regeneron Pharmaceuticals, Tarrytown, New York, USA. 
6Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, California, USA. 
7Department of Neurobiology, Northwestern University, Evanston, Illinois, USA.

Address correspondence to: Susan E. Quaggin, Northwestern University, Feinberg School of Medicine, 303 E. Superior Street, Lurie 10-105, Chicago, Illinois 60611, USA. Phone: 312.503.1534; E-mail: quaggin@northwestern.edu.

Published September 9, 2014
Submitted: May 20, 2014; Accepted: July 31, 2014.

Glaucoma is a leading cause of blindness, afflicting more than 60 million people worldwide. Increased intraocular pressure (IOP) due to impaired aqueous humor drainage is a major risk factor for the development of glaucoma. Here, we demonstrated that genetic disruption of the angiopoietin/TIE2 (ANGPT/TIE2) signaling pathway results in high IOP, buphthalmos, and classic features of glaucoma, including retinal ganglion degeneration and vision loss. Eyes from mice with induced deletion ofAngpt1 and Angpt2 (A1A2FloxWB mice) lacked drainage pathways in the corneal limbus, including Schlemm’s canal and lymphatic capillaries, which share expression of the PROX1, VEGFR3, and FOXC family of transcription factors. VEGFR3 and FOXCs have been linked to lymphatic disorders in patients, and FOXC1 has been linked to glaucoma. In contrast to blood endothelium, in which ANGPT2 is an antagonist of ANGPT1, we have shown that both ligands cooperate to regulate TIE2 in the lymphatic network of the eye. While A1A2FloxWB mice developed high IOP and glaucoma, expression of ANGPT1 or ANGPT2 alone was sufficient for ocular drainage. Furthermore, we demonstrated that loss of FOXC2 from lymphatics results in TIE2 downregulation, suggesting a mechanism for ocular defects in patients with FOXC mutations. These data reveal a pathogenetic and molecular basis for glaucoma and demonstrate the importance of angiopoietin ligand cooperation in the lymphatic endothelium.

Introduction

The angiopoietin/TIE2 (ANGPT/TIE2) signaling pathway is a major regulator of vascular development, and altered expression of ANGPT ligands or activity of the TIE2 receptor is linked to a variety of vascular diseases and adverse outcomes in patients (1–4). However, while the blood vascular role of the pathway has been extensively studied, the function of angiopoietins in lymphatic endothelium is uncertain. In vascular endothelium, ANGPT2 is reported to function as a competitive antagonist of ANGPT1/TIE2 signaling, inhibiting ANGPT1-mediated phosphorylation of TIE2 (5). ANGPT2 and the orphan receptor TIE1 are known to be involved in lymphatic development (6), but until now, the roles of TIE2 and its canonical ligand ANGPT1 have not been described.

Surprisingly, while Angpt1-KO mice die between E9.5 and E12.5 due to major cardiovascular defects, conditional deletion of Angpt1 after E13.5 produces no overt vascular phenotypes in adult mice (3). To determine whether there is unrecognized cooperation between ANGPT1 and ANGPT2, which provides compensation in mice lacking ANGPT1, we generated a conditional Angpt2 allele and produced mice lacking both major angiopoietin ligands (A1A2FloxWB). Strikingly, simultaneous deletion of both ligands at mid-gestation phenocopies deletion of Tie2, demonstrating cooperativity between ANGPT1 and ANGPT2. Whole-body deletion of the Tie2 receptor or both Angpt1 and Angpt2 at E12.5 leads to gross subcutaneous edema in embryos with patterning defects in dermal lymphatic vessels (S.E. Quaggin, unpublished observations). While Angpt2-KO mice exhibit lymphatic valve defects and mesenteric lymphatic abnormalities resulting in chylous ascites (2), they did not develop the embryonic edema observed in A1A2FloxWB or Tie2–conditional KO (cKO) mice, suggesting a compensatory role for ANGPT1 in lymphatic development.

Results and Discussion

To investigate the combined roles of ANGPT1 and ANGPT2 in adult mice, we deleted both ligands at E16.5. A1A2FloxWBΔE16.5 mice (Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI77162DS1) were born in Mendelian numbers and were indistinguishable from controls during the first 3 weeks of life. However, eyes of the mutant animals began to protrude 21–28 days after birth. Buphthalmos worsened with age, and by 8 weeks, mice had difficulty closing their eyelids (Figure 1, A–D). Gross examination revealed corneal enlargement and increased anterior chamber depth (Figure 1, F, G, I, and J). Eyes of TIE2COINWB-INVΔP0 mice (Supplemental Figure 2) exhibited an identical phenotype, confirming that the effect was mediated through the canonical ANGPT receptor TIE2 (Figure 1, E, H, and K). We measured the intraocular pressure (IOP) at 10 weeks using a rebound tonometer (7). While control animals were within the normal range of 12 to 16 mmHg, the IOP of mutant littermates was significantly elevated, ranging from 24 to 52 mmHg (Figure 1L). Using an optomotor response test (8), we found that A1A2FloxWBΔE16.5 mice had severely impaired vision, with visual acuity of less than 0.042 cycles per degree (Figure 1M). Histological analysis revealed optic nerve excavation (Figure 2, A and G) and other features of glaucomatous eye disease.

Figure 1

A1A2FloxWBΔE16.5- and TIE2COINWB-INVΔP0-cKO mice develop buphthalmos. While control eyes (A, C, F, and I) appeared normal, 8- to 9-week-old A1A2- (B, D, G, and J) and Tie2-cKO (E, H, and K) mice exhibited anterior chamber enlargement due to increased IOP (L). Optomotor response tests (M) showed impaired vision in mutant animals. Scale bars: 1 mm (F, G, and H) and 500 μm (I, J, and K). **P < 0.01 and ***P< 0.001 by Student’s 2-tailed t test. Error bars indicate SEM. No response (NR) indicates an optomotor response of less than 0.042 cycles per degree.