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.

Figure 2 A1A2FloxWBΔE16.5-cKO mice develop glaucoma due to defects in ocular drainage. Compared with controls (A), the optic nerve head appeared abnormal in A1A2FloxWBΔE16.5 mice (G), with thinning of the nerve fiber layer (red arrowheads) and optic nerve excavation (asterisk). Semi-thin sections showed thinning of the nerve fiber, ganglion, and nuclear cell layers in the central retina (B and H). Loss of nerve fibers was confirmed by TuJ1 staining (C and I). Unlike their littermate controls (D), Schlemm’s canal was absent in A1A2-cKO mice (J). Anterior chamber drainage was further diminished by a loss of LYVE1-positive lymphatic capillaries in the corneal limbus (E and K). Lymphatic vasculature was present in nonocular tissues, but exhibited disturbed patterning as shown here in the dermis of the ear (F and L). Unlike double-KOs, mice lacking Angpt1 or Angpt2 individually developed lymphatics in the corneal limbus (M and N). Compared with controls, A1A2-cKO mice had fewer nuclei in the retinal ganglion cell layer (O). GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium; V, blood vessel; SC, Schlemm’s canal. Red arrowheads indicate thickness of the nerve fiber layer. Scale bars: 200 μm and 1 mm (F and L). **P < 0.01.

We observed reductions in the thickness of retinal cell layers in mutant eyes, including the ganglion and nuclear layers, as well as loss of the nerve fiber layer (Figure 2, B, C, H, I, and O, and Supplemental Figures 3 and 4). Unlike human glaucoma, which is rarely associated with photoreceptor loss, A1A2FloxWBΔE16.5 mice showed outer nuclear layer thinning that worsened toward the retinal periphery. This outer retina damage was similar to that described in laser-induced models of mouse glaucoma, suggesting the possibility of pressure-related or ischemic effects (9). While photoreceptor loss may be partially responsible for the vision loss we observed, this degree of atrophy was not sufficient to explain the dramatic decrease in visual acuity seen in A1A2FloxWBΔE16.5 mice. Instead, we hypothesize that the defect was due to loss of retinal ganglion cells and interneurons — which would confirm that A1A2FloxWB mice represent a new model of murine glaucoma.

To determine the cause of high IOP and glaucoma in A1A2FloxWBΔE16.5 mice, we studied the aqueous humor drainage system of the eye. While the trabecular meshwork and ciliary body were indistinguishable between KO and control mice, Schlemm’s canal was absent in 8 of 8 A1A2FloxWBΔE16.5 eyes examined (Figure 2, D and J). Schlemm’s canal was present in all control littermates. Although Schlemm’s canal is a major route of aqueous humor drainage, defects in Schlemm’s canal formation have not been reported to raise IOP in mice. Transgenic mice haploinsufficient for the transcription factor FOXC1 have been reported to exhibit a small or absent Schlemm’s canal, yet did not develop high IOP (10). Previous studies of aqueous humor dynamics in mice have suggested that only 20% of total fluid drainage is carried out via Schlemm’s canal, suggesting that alternate drainage routes, including uveoscleral and lymphatic routes, may be able to provide compensation for defects in Schlemm’s canal (11).

To better understand alternate drainage pathways of the anterior chamber, we studied lymphatic and vascular capillaries in the corneal limbus. We examined vessels by confocal microscopy, which revealed a complete absence of LYVE1-positive lymphatic endothelium in the limbus of A1A2FloxWBΔE16.5 mice (Figure 2, E and K). CD31-positive blood vasculature was present, but exhibited disturbed patterning with some capillary loops extending into the cornea. It is unclear whether this aberrant vascular morphology is a direct effect of Angpt1 and Angpt2 deletion, or a response to loss of lymphatic drainage and/or stretching of the cornea. Mice lacking ANGPT1 or ANGPT2 alone develop lymphatic vasculature in the corneal limbus (Figure 2, M and N), suggesting the presence of inter-ligand compensation in the lymphatic endothelium.

Corneal neovascularization and glaucoma have been described in patients with Axenfeld–Rieger syndrome due to mutations in FOXC1, and Foxc1 haploinsufficient mice exhibit defects in the anterior chamber and Schlemm’s canal, suggesting a link to this molecular pathway (10). In mice, a similar phenotype has been described for the related transcription factor FOXC2 (10, 12). Intriguingly, the downstream targets of FOXC1/2 responsible for these phenotypes have not been elucidated. Given the similar expression pattern of FOXC1/2 with TIE2 and ANGPT2 in lymphatics, as well as the overlapping phenotypes (13, 14), we hypothesized that FOXC1 or FOXC2 are responsible for regulating expression of TIE2 or angiopoietin ligands in the lymphatic endothelium. De Val and colleagues have reported the presence of a FOX:ETS transcriptional enhancer sequence in the Tie2 promoter region and demonstrated FOXC2-mediated enhancer activation using an in vitro reporter system, further suggesting a link between these pathways (15). We investigated the possibility of FOXC-mediated TIE2 regulation using complimentary in vivo and in vitro systems. siRNA targeting of FOXC2 in human dermal lymphatic cells caused a marked reduction in TIE2 protein expression compared with targeting of FOXC1 or scrambled siRNAs (Figure 3A). This result was replicated in vivo using a Foxc2 transgenic mouse line. Lymphatic endothelial cells were isolated from lymphatic-specific Foxc2-KO (Foxc2fl/fl Prox1-CreERT2; ref. 16) embryos at E15.5 using FACS. mRNA was isolated, and real-time PCR revealed a 74.5% reduction (n = 6 animals per group, P = 0.015) in Tie2 mRNA expression relative to that in controls (Figure 3B). These results suggest a mechanism for lymphatic phenotypes in patients with Foxc2 mutations or in Foxc2-KO mice and provide additional evidence of a connection between FOXC and ANGPT/TIE2 molecular pathways.

Figure 3 FOXC2 regulates TIE2 expression in the lymphatic endothelium. (A) Human dermal lymphatic endothelial cells were cultured in the presence of specific siRNAs targeting FOXC1, FOXC2, or a scrambled siRNA. Compared with scrambled siRNA (lane 1) or FOXC1 siRNA (lane 2), siRNA targeting FOXC2 (lane 3) caused a reduction in TIE2 protein expression. (B) Tie2 mRNA expression was measured by real-time PCR in lymphatic endothelial cells isolated from E15.5 Foxc2fl/fl (Ctrl) or Foxc2fl/fl Prox1-CreERT2 (Foxc2-cKO) mouse embryos. n = 6 control and 6 Foxc2-cKO embryos. Error bars indicate SEM. *P < 0.05.

Surprisingly, LYVE1-positive lymphatic vessels were present in nonocular tissues of A1A2FloxWBΔE16.5 mice, although patterning in some organs was abnormal. In the dermis of the ear, lymphatic vessels appeared sparse with variable vessel diameter and abnormal branching similar to those described in Angpt2-null mice (Figure 2, F and L, and ref. 2). It is possible that specialized lymphatic vessels of the anterior chamber are more dependent on ANGPT/TIE2 signaling than other lymphatic endothelia, suggesting that, like blood vascular endothelia, lymphatic capillaries are heterogeneous, with unique functions and regulatory mechanisms. Indeed, Schlemm’s canal has been described as a hybrid vessel, with features of both blood and lymphatic endothelium, expressing blood endothelial markers CD34 and endomucin and a subset of lymphatic markers, including PROX1 and VEGFR3, but not podoplanin (17).

The cardiovascular phenotypes reported in Angpt1- and Tie2-KO models highlight the importance of these molecules in cardiac development and angiogenesis. In older animals, our data suggest that ANGPT/TIE2 signaling is less critical in quiescent blood vasculature, but continues to play a major role in the lymphatic endothelium. At the outset, we hypothesized that A1A2FloxWB mice would develop severe vascular defects, revealing a compensatory role for ANGPT2 in the blood vasculature of mice lacking ANGPT1. Instead, we observed that the lymphatic defects reported in Angpt2-null mice were enhanced by the additional loss of ANGPT1, demonstrating cooperation between ligands in the lymphatic endothelium.

While the overall mechanism responsible for human glaucoma remains obscure, the most important risk factor is elevated IOP (18). IOP is determined by the relative rates of aqueous humor drainage and formation, and the majority of glaucoma treatment has focused on lowering IOP by targeting these systems (19). Aqueous humor is thought to drain through 2 major pathways: the trabecular meshwork leading to Schlemm’s canal and the uveoscleral pathway. In humans, studies have estimated that the uveoscleral pathway accounts for 46% to 54% of total outflow, with the remainder carried through Schlemm’s canal (20). The contribution of lymphatic vessels to each pathway has not been reported, but studies in sheep have suggested that they play an important role in allowing fluid to escape from the anterior chamber (21). Since the increased IOP we observed in A1A2FloxWBΔE16.5 mice was more severe than that of other models with an abnormal Schlemm’s canal, we hypothesize that lymphatic vessels are essential for maintaining aqueous humor flow through the uveoscleral route.

In summary, we show that A1A2FloxWBΔE16.5 mice lack both Schlemm’s canal and the lymphatic capillaries of the corneal limbus, leading to a dramatic increase in IOP and glaucoma. These data raise the intriguing possibility that promotion of lymphangiogenesis with therapies such as VEGFC or ANGPT/TIE2 agonists might represent novel and much-needed therapies for glaucoma.