The Schlemm's canal is a VEGF-C/VEGFR-3-responsive lymphatic-like vessel

Abstract

In glaucoma, aqueous outflow into the Schlemm's canal (SC) is obstructed. Despite striking structural and functional similarities with the lymphatic vascular system, it is unknown whether the SC is a blood or lymphatic vessel. Here, we demonstrated the expression of lymphatic endothelial cell markers by the SC in murine and zebrafish models as well as in human eye tissue. The initial stages of SC development involved induction of the transcription factor PROX1 and the lymphangiogenic receptor tyrosine kinase VEGFR-3 in venous endothelial cells in postnatal mice. Using gene deletion and function-blocking antibodies in mice, we determined that the lymphangiogenic growth factor VEGF-C and its receptor, VEGFR-3, are essential for SC development. Delivery of VEGF-C into the adult eye resulted in sprouting, proliferation, and growth of SC endothelial cells, whereas VEGF-A obliterated the aqueous outflow system. Furthermore, a single injection of recombinant VEGF-C induced SC growth and was associated with trend toward a sustained decrease in intraocular pressure in adult mice. These results reveal the evolutionary conservation of the lymphatic-like phenotype of the SC, implicate VEGF-C and VEGFR-3 as critical regulators of SC lymphangiogenesis, and provide a basis for further studies on therapeutic manipulation of the SC with VEGF-C in glaucoma treatment.

Figure 7. A single injection of recombinant VEGF-C is associated with a sustained decrease in IOP in adult mice.

(A–C) IOP after injection of rVEGF-C or rMSA into the anterior chamber of aged (A, 43 weeks; B, 41 weeks) or young (C, 7 weeks) mice. 9.6 μg protein was injected in 4 μl. A substantial proportion of the injected fluid was backflushed. Age-controlled female NMRI mice were used. (D) Meta-analysis of all IOP experiments performed (see A–C and Supplemental Figure 11 for individual experiments). All postinjection IOP measurements from 1 eye were averaged. Postinjection t test: rVEGF-C, 8.153 ± 0.1711 mmHg, n = 84; rMSA, 9.239 ± 0.1341 mmHg, n = 81; difference, 1.086 ± 0.2184; 95% CI, 0.6543 to 1.517; R² = 0.1316. *P < 0.05; **P < 0.01; ***P < 0.001; ^#P < 0.0001.

Figure 6. A single injection of rVEGF-C induces enlargement of the SC without corneal neovascularization in adult mice.

Mice received a single injection of 3.8 μg rVEGF-C or rMSA. (A) Immunofluorescence staining of the SC on day 14 after injection. Dashed outlines denote the SC. rVEGF-C administration showed downregulation of VEGFR-3 by SC ECs (arrowheads). (B) Representative macroscopic images of eyes on day 9 after injection, after IOP measurement. (C) Corneal and ES vasculature on day 14 after injection. (D) Representative images of H&E-stained paraffin-embedded eye sections on day 14. Scale bars: 100 μm (A and C), 400 μm (D).

Figure 5. VEGF-C overexpression induces directional sprouting, proliferation, and migration of SC ECs in adults.

(A–E) AdVEGF-C, AdVEGF, or an AdControl was injected into the anterior chamber, as indicated. (A) Immunofluorescence staining of the SC with antibodies against PECAM-1 (green), BrdU (red), and PROX1 (blue) at days 4 and 14 after injection. Mice were injected with 100 mg/kg BrdU 2 hours prior to sacrifice. Sprouts of SC ECs (asterisks) are indicated. Changes in limbal vascular anatomy on day 14 are illustrated. C, cornea; S, sclera; AC, anterior chamber; PC, posterior chamber. (B and C) Quantitative analysis of SC area (B) and SC sprouts extending toward the cornea or sclera (C). Each symbol represents data from 1 eye. (D and E) IOP before and 3 (D) and 14 (E) days after injection. (F and G) AAV–VEGF-C or AAV-HSA was injected into the anterior chamber. (F) Immunofluorescence staining of the SC with indicated antibodies at week 6 after transduction, and illustration of changes in limbal vascular anatomy. (G) IOP before and after injection. (H) X-gal staining in mice injected with AdLacZ. (I) Immunofluorescence in mice injected with reporter AAV vectors encoding EGFP and staining with antibodies against GFP (red) and PECAM-1 (blue). Scale bars: 200 μm (A, F, and I). *P < 0.05; **P < 0.01; ***P < 0.001; ^#P < 0.0001.

Figure 4. The lymphangiogenic receptor VEGFR-3 is essential for SC development.

(A and B) SC morphology (A) and area (B; data from 1 litter) after injection of rat anti–VEGFR-3 antibodies (n = 4), rat anti–VEGFR-2 antibodies (n = 4), their combination (n = 3), or control rat IgG (n = 3) once daily during P0–P7 into littermate mice. (C and D) SC morphology (C) and mean area (D; data from 1 litter) in Vegfr3^iΔLEC and littermate control Vegfr3^fl/fl mice (n = 3 per group) at P7 after induction of Cre activity. Vegfr3 deletion was confirmed using antibodies against VEGFR-3. (E and F) SC morphology (E) and mean area (F; data pooled from 2 litters) in Vegfr2^iΔLEC and littermate control Vegfr2^fl/fl mice (n = 4 per group) mice at P7. Vegfr2 deletion was confirmed using antibodies against VEGFR-2. Arrowhead indicates residual VEGFR-2. Scale bars: 100 μm (A, C, and E). *P < 0.05; **P < 0.01.

Figure 3. The lymphangiogenic growth factor VEGF-C is critical for SC development.

(A and B) SC morphology (A) and mean area (B; data from 1 litter) in transgenic K14-VEGFR-3_1–3-Ig mice (n = 3), their WT littermate controls (n = 3), and K14-VEGFR-3_4–7-Ig mice (n = 4) at P7. (C and D) SC morphology (C) and mean area (D; data from 2 litters) in Vegfc^iΔR26 (n = 4) and control Vegfc^fl/fl littermate (n = 5) mice at P7, after induction of Cre activity from P1 to P5 with daily 4-OHT injections. (E and F) SC morphology (E) and mean area (F; data from 1 litter) in Vegfd^–/– (n = 6) and littermate WT (n = 3) mice. (G and H) SC morphology (G) and mean area (H; data from 3 litters, and data from F included for comparison) in Vegfc^iΔR26 Vegfd^–/– (n = 8) and control littermate Vegfc^fl/fl Vegfd^–/– (n = 5) mice at P7 after induction of Cre activity. Scale bars: 100 μm (A, C, E, and G). *P < 0.05; **P < 0.01; ^#P < 0.0001.

Figure 2. The SC develops postnatally from transscleral veins.

(A–O) Immunofluorescence staining with antibodies against PECAM-1 (green), PROX1 (red), and VEGFR-3 (blue) were used to visualize SC development. (P–T) SC developmental stages. Dashed lines denote the subset of confocal z stacks selected for SC visualization. Blue cells, PROX1^– BECs; green cells, PROX1⁺ LEC-like cells. EV, ES vein. For 3D volume renderings of entire confocal z stacks with CCs and ES veins, see Supplemental Videos 1–5. (A–C and P) At P0, lateral sprouting (asterisks and inset in C) of transcleral veins toward adjacent transcleral veins was observed. (D–F and Q) At P1, connection of adjacent transcleral veins by strings of future SC ECs was apparent. (G–I and R) At P2, maturation and induction of PROX1 expression (arrowheads) was observed. (J–L and S) P4 revealed lumenization and expression of PROX1, induction of VEGFR-3 (arrowheads), regression of connections to CCs (hashtags), and lateral sprouting (asterisks). (M–O and Y) Mature SC at P7. Note that the AVs (arrow) did not regress. Scale bars: 50 μm (A–O), 12.5 μm (C, inset).

Figure 1. SC ECs display molecular features of lymphatic endothelium.

(A–M) Whole-mount immunofluorescence staining of the adult murine eye using antibodies against PECAM-1, PROX1, and VEGFR-3. The entire thickness of the limbus was visualized by confocal imaging into 1 z stack. Subsets with the SC (A–D), the AV (E–H), and the ES vasculature (I–L) are shown. The joining point of the AV into the SC (arrow) and the joining point of the AV into the ES vein (arrowhead) are indicated. Dashed outlines denote the SC and ES lymphatic vessels. c, capillary; pcv, postcapillary venule; v, vein; a, artery. (M) AH drainage route in a 90° y-axis projection of the confocal stack in A–L. xyz axes are shown for orientation between A–L and M. (N) The SC (dashed outline) and ES lymphatic vessels (asterisks) in vivo in Prox1-CreER^T2 LSL-tdTomato lineage tracer mice after 4-OHT administration. (O and P) Immunohistochemical PROX1 staining and negative control staining of the SC in a human eye. Arrows indicate PROX1 expression in SC ECs. (Q) Visualization of zebrafish SC by staining with antibodies against human and mouse PROX1. (R–T) Immunofluorescence staining of murine SC and ES lymphatic vessels using antibodies against CCL21 (R) and LYVE-1 (S and T). Scale bars: 100 μm (A–L, S, and T); 50 μm (M); 200 μm (N–R).