Expression and role of VEGF--a in the ciliary body

Abstract

Purpose: The role of VEGF-A in the normal ciliary body is largely unexplored. The ciliary body is similar in many respects to the choroid plexus of the brain, and we demonstrated previously the importance of VEGF-A in maintenance of choroid plexus vasculature and ependymal cells. Therefore, the role of VEGF-A in ciliary body homeostasis was explored.

Methods: Swiss-Webster mice (VEGF-LacZ) were used to determine VEGF-A expression during ciliary body development and in the adult. VEGFR2 expression was determined in adult wild type C56BL/6J mice. Systemic VEGF-A neutralization in vivo was achieved with adenovirus-mediated overexpression of soluble VEGFR1 (sFlt1). Following VEGF-A neutralization, the ciliary epithelium was analyzed by light microscopy and transmission electron microscopy (TEM). The effect of VEGF-A blockade on ciliary body function also was assessed by measuring intraocular pressure.

Results: VEGF-A expression was detected at embryonic day 18.5 (E18.5), the onset of ciliary process formation. In the adult ciliary body, VEGF-A was expressed by the pigmented epithelium, whereas VEGFR2 was localized primarily to the capillary endothelium and nonpigmented epithelium. Systemic VEGF-A neutralization led to a thinning of the nonpigmented epithelium, vacuolization of the pigmented epithelium, loss of capillary fenestrations, and thrombosis. These changes were associated with impaired ciliary body function, as evidenced by decreased intraocular pressure in sFlt1-overexpressing animals (15.31 ± 2.06 mm Hg) relative to controls (18.69 ± 1.49 mm Hg).

Conclusions: VEGF-A has an important role in ciliary body homeostasis. Potential for undesired off-target effects should be considered with the chronic use of anti-VEGF-A therapies.

Figure 1.

VEGF-A expression during ciliary body development and in adult. Cryosections (10 μm) of nonpigmented (A, D) E18.5 and (B, E) adult VEGF-LacZ/+ eyes were stained for β-galactosidase (blue). (A, D) VEGF-A was detected in the primitive ciliary body at E18.5, the stage at which ciliary process infolding begins. (B, E) In the adult, VEGF-A expression (black arrows) was localized primarily to the presumptive pigmented epithelium. (C, F) TEM micrograph of adult ciliary body indicates location of pigmented epithelium, nonpigmented epithelium, and capillaries. Scale bar: 160 μm (A, D) and 20 μm (B, C, E, F). NPE, nonpigmented epithelium; PE, pigmented epithelium; FC, fenestrated capillary.

Figure 2.

VEGFR2 expression by the nonpigmented ciliary epithelium. VEGFR2 expression in the adult ciliary body was localized by immunofluorescent microscopy using a rabbit polyclonal anti-mouse VEGFR2 antibody. (A) Bright field microscopy demonstrates the extensive pigmentation of the pigmented epithelial layer, and the location of the nonpigmented layer (dotted line and arrow). (B) DAPI staining of nuclei along with immunofluorescent labeling reveals VEGFR2 primarily in the nonpigmented epithelium and capillaries (white arrowhead). (C, D) Merging of the bright field and fluorescent images confirms localization of VEGFR2 expression in nonpigmented epithelial cells. Scale bar: 20 μm.

Figure 3.

VEGF-A neutralization leads to ciliary capillary thrombosis. Richardson stain of epoxy embedded ultrathin sections of eyes from (A, B) Ad-null and (C, D) Ad-sFlt1–expressing mice at day 28 post-infection revealed numerous microthrombi (white arrowheads) in the capillaries of Ad-sFlt1–expressing mice, but not in the capillaries of Ad-null animals. Regions of the nonpigmented epithelium appeared markedly thinner (black arrow), indicating degeneration. Scale bar: 20 μm.

Figure 4.

Alterations in ciliary body ultrastructure following VEGF-A neutralization. Ultrastructural analysis of the ciliary body 14 days post-infection illustrates the normal ultrastructure of (A, B) Ad-null animals in comparison to the degeneration of the nonpigmented epithelial layer observed in (C, D) Ad-sFlt1–expressing mice. Arrow indicates the shrunken and nearly nonexistent cytoplasm of a nonpigmented epithelial cell. Scale bar: 2 μm.

Figure 5.

VEGF-A neutralization leads to loss of capillary fenestrations. TEM micrographs of the ciliary body from (A, B) Ad-null and (C, D) Ad-sFlt1–expressing mice at day 14 post-infection revealed a thickening of the capillary endothelial wall and associated loss of fenestrations (black arrowheads) in (D) Ad-sFlt1 eyes, whereas (B) Ad-null capillaries retained their normal appearance. The normal organization of the ciliary epithelia was observed in (A) Ad-null animals whereas (C) Ad-sFlt1–expressing animals displayed degenerated nonpigmented epithelium (black arrow) and vacuolization of the pigmented epithelium (black asterisks). Scale bar: 2 μm.

Figure 6.

VEGF-A neutralization leads to decreased IOP. The IOP of CD-1 mice was measured with a TonoLab tonometer before and at various time points following injection of the Ad-null or Ad-sFlt1. By day 11, there was a statistically significant reduction in IOP in the Ad-sFlt1–expressing animals. The values are expressed as the mean IOP ± SD of the animals in each group (n = 10). ***P < 0.001

Figure 7.

Schematic of VEGF-A and VEGFR2 localization, and aqueous humor secretion in ciliary epithelia. (A) A representation of a ciliary process depicting ciliary epithelial layers and fenestrated capillaries. VEGFR2 is expressed primarily by the nonpigmented epithelium and by capillary endothelium, whereas VEGF-A is expressed by the pigmented epithelium, the epithelium that lies in close proximity to the capillaries. Pigmented ciliary epithelium–derived VEGF-A is proposed to support the nonpigmented ciliary epithelium as well as maintain capillary fenestrations. (B) Aqueous humor secretion is driven by the transepithelial transport of ions, primarily Na⁺, Cl⁻, and to a lesser degree HCO₃⁻, across the ciliary epithelium, generating an osmotic gradient for water movement. Ions are taken up by the pigmented epithelial cells from the stroma by Na⁺, 2Cl⁻ , K⁺ symports, and parallel Na⁺/ H⁺ and Cl⁻ / HCO₃⁻ antiports. The ions then diffuse from the pigmented epithelium to the nonpigmented epithelium through gap junctions. Lastly, Na⁺ is released from the nonpigmented epithelium into the posterior chamber through Na⁺ K⁺-activated ATPase and Cl⁻ is released through Cl⁻ channels.