Forkhead box transcription factor FoxC1 preserves corneal transparency by regulating vascular growth

Abstract

Normal vision requires the precise control of vascular growth to maintain corneal transparency. Here we provide evidence for a unique mechanism by which the Forkhead box transcription factor FoxC1 regulates corneal vascular development. Murine Foxc1 is essential for development of the ocular anterior segment, and in humans, mutations have been identified in Axenfeld-Rieger syndrome, a disorder characterized by anterior segment dysgenesis. We show that FOXC1 mutations also lead to corneal angiogenesis, and that mice homozygous for either a global (Foxc1(-/-)) or neural crest (NC)-specific (NC-Foxc1(-/-)) null mutation display excessive growth of corneal blood and lymphatic vessels. This is associated with disorganization of the extracellular matrix and increased expression of multiple matrix metalloproteinases. Heterozygous mutants (Foxc1(+/-) and NC-Foxc1(+/-)) exhibit milder phenotypes, such as disrupted limbal vasculature. Moreover, environmental exposure to corneal injury significantly increases growth of both blood and lymphatic vessels in both Foxc1(+/-) and NC-Foxc1(+/-) mice compared with controls. Notably, this amplification of the angiogenic response is abolished by inhibition of VEGF receptor 2. Collectively, these findings identify a role for FoxC1 in inhibiting corneal angiogenesis, thereby maintaining corneal transparency by regulating VEGF signaling.

Fig. 1.

FOXC1-attributable ARS is associated with a spectrum of corneal angiogenesis phenotypes. (A) In contrast to the normal anatomy (A), affected individuals from the deletion pedigree exhibit extensive iris (C and D) and corneal (E and F) anomalies. Of note, compared with the normal limbus (E, Inset), marked vascularization of the peripheral cornea (arrow in E) extends midway to the pupillary aperture (arrows in F). (G) The proband from the frameshift mutation pedigree (p.P274RfsX41) exhibits similar corneal vascularization (arrows). (H) Analogous but milder changes were observed in a pedigree with a 492-kb segmental duplication encompassing FOXC1 and FOXF2 with regional neovascularization extending across the superior cornea (arrowheads). (I and J) In a second member of this pedigree, in contrast to the normal circular limbus (I), there is squaring of the contour (J) implicating perturbed FOXC1 gene dosage in more extensive limbal anatomical changes.

Fig. 2.

NC-specific loss of Foxc1 in mice induces pupillary abnormalities and impaired collagen formation in the corneal stroma. (A) Compared with the eye of a control (Foxc1^F/F) mouse, the eye of a mouse heterozygous for the NC-Foxc1-null mutation (NC-Foxc1^+/−) had a displaced and misshapen pupil at age 11 wk. (B) Impaired collagen formation in NC-Foxc1^−/− embryos demonstrated by van Gieson staining of corneas at E15.5 and E17.5. NC-Foxc1^−/− embryos show a remarkable reduction of collagen formation (pinkish red) in the corneal stroma. Nuclei stained with hematoxylin are shown in brown. (C) The ultrastructure of E18.5 corneas. Note that the collagen fibers (arrow) are disorganized and surround in swirls the disrupted corneal stromal cells of NC-Foxc1^−/− embryos. st, corneal stromal cells. (Scale bars: 100 μm in B and 1 μm in C.)

Fig. 3.

NC-specific loss of Foxc1 induces angiogenesis and lymphangiogenesis in the mouse cornea during development. (A) Neovascularization was evaluated in CD31-stained corneas from control (Foxc1^F/F), NC-Foxc1^+/−, and NC-Foxc1^−/− mouse embryos (E14.5, E15.5, and E17.5) and newborn mice (P0), and from adult (7-wk-old and 43-wk-old) control (Foxc1^F/F) and NC-Foxc1^+/− mice. The corneas of control (Foxc1^F/F) embryos and mice were avascular with developing long ciliary arteries (long arrows) and limbal blood vessels (short arrows). The corneas of NC-Foxc1^+/− embryos and mice remained avascular through adulthood, but the long ciliary arteries (long arrows) and limbal blood vessels (Inset; short arrow) of embryos and newborn mice were disrupted. Increased sprouting of limbal blood vessels (arrowheads) was observed in adult NC-Foxc1^+/− mice. In NC-Foxc1^−/− embryos, corneal vascularization (red arrows) was observed by E14.5 and extended throughout the cornea at later time points; the disruption of limbal blood vessels was apparent on E17.5 and P0. (B) NC-specific loss of Foxc1 increases the growth of corneal lymphatic vessels in mice. Lymphangiogenesis was evaluated in corneas from control (Foxc1^F/F), NC-Foxc1^+/−, and NC-Foxc1^−/− embryos (E17.5) and newborn mice (P0), and also from adult (7-wk-old and 43-wk-old) Foxc1^F/F and NC-Foxc1^+/− mice. Embryonic corneas were stained with the lymphatic vessel marker Prox1, corneas from newborn mice were stained with Prox1 or the lymphatic vessel marker Lyve-1, and corneas from adult mice were stained with Lyve-1. Growth of lymphatic vessels from the limbus (red arrow) into the cornea was observed in NC-Foxc1^−/− embryo at E17.5 and in newborn mice (arrows). The corneas of adult NC-Foxc1^+/− mice remained free of lymphatic vessels, but showed increased sprouting of lymphatic vessels (arrowheads). (C) Quantification of blood and lymphatic vessel sprouting in 7-wk-old adult Foxc1^F/F and NC-Foxc1^+/− mice. The sprouting of both blood and lymphatic vessels was significantly greater in NC-Foxc1^+/− mice compared with control Foxc1^F/F mice. Values are mean ± SEM. Compared with control mice: blood vessel sprouting in NC-Foxc1^+/− mice, P = 0.0000006; lymphatic vessel sprouting in NC-Foxc1^+/− mice, P = 0.00003. *P < 0.05, Student t test. lca, long ciliary artery. (Scale bars: 100 μm.)

Fig. 4.

Up-regulation of MMP expression in the cornea of NC-Foxc1^−/− mice. (A) Gene expression was evaluated in the corneas of E15.5 control (Foxc1^F/F), NC-Foxc1^+/−, and NC-Foxc1^−/− embryos via real-time RT-PCR. Values are mean ± SEM. *P < 0.05, Student t test. Expression of FGF2, VEGF-A, sVEGFR-1, and MMP9 protein was evaluated in E15.5 control (Foxc1^F/F), NC-Foxc1^+/−, and NC-Foxc1^−/− embryos via immunofluorescent staining. MMP9 expression was observed in the corneal epithelium and superficial stroma of NC-Foxc1^+/− embryo and throughout the corneal stroma of NC-Foxc1^−/− embryo. (Scale bars: 100 μm.)

Fig. 5.

Foxc1 regulates corneal angiogenesis and lymphangiogenesis after corneal injury in adult mice. Corneal alkali burn injury was induced in 6-wk-old control (Foxc1^F/F) mice, NC-Foxc1^+/− mice, WT mice, and mice heterozygous for a global Foxc1-null mutation (Foxc1^+/− mice). (A) At 14 d after injury, angiogenesis and lymphangiogenesis were evaluated in corneas stained for CD31 (red) and Lyve-1 (green) expression, respectively. (B and C) Both the NC-Foxc1^+/− mutation and the Foxc1^+/− mutation enhanced the sprouting of blood and lymphatic vessels, and the blood and lymphatic vessel areas were significantly larger in NC-Foxc1^+/− mice than in control (Foxc1^F/F) mice (B) and in Foxc1^+/− mice than in WT mice (C). Values are mean ± SEM. *P < 0.05, Student t test. (Scale bars: 100 μm.)

Fig. 6.

Corneal angiogenesis after alkali burn injury in NC-Foxc1^−/− mice is blocked by inhibition of VEGF signaling. (A) Mice were treated with DMSO (control) or the VEGFR-2 blocker SU5416 for 7 consecutive days after corneal alkali burn injury, after which angiogenesis and lymphangiogenesis were evaluated in corneas stained for CD31 (red) and Lyve-1 (green) expression, respectively, on day 7 after corneal injury. The blockade of VEGFR-2 dramatically reduced the growth of corneal blood vessels but not that of lymphatic vessels. (B) The blood vessel area, but not the lymphatic vessel area, was significantly smaller after treatment with SU5416 than after DMSO treatment. Values are mean ± SEM. CI, corneal injury; NS, not significant. *P < 0.05, Student t test. (Scale bars: 100 μm.) (C) Model for FoxC1 function in corneal transparency. FoxC1 is essential for the establishment and maintenance of corneal avascularity during development and in postnatal life by regulating MMP expression in corneal stroma. Corneal avascularity is tightly controlled by angiogenic and antiangiogenic responses. In Foxc1-mutant corneas, elevated MMP levels degrade ECM and enhance VEGF bioavailability, thereby inducing corneal neovascularization, whereas sVEGFR1 suppresses activation of VEGF signaling by neutralizing VEGF.