Lymphatic regulator PROX1 determines Schlemm’s canal integrity and identity

Schlemm’s canal (SC) is a specialized vascular structure in the eye that functions to drain aqueous humor from the intraocular chamber into systemic circulation. Dysfunction of SC has been proposed to underlie increased aqueous humor outflow (AHO) resistance, which leads to elevated ocular pressure, a factor for glaucoma development in humans. Here, using lymphatic and blood vasculature reporter mice, we determined that SC, which originates from blood vessels during the postnatal period, acquires lymphatic identity through upregulation of prospero homeobox protein 1 (PROX1), the master regulator of lymphatic development. SC expressed lymphatic valve markers FOXC2 and integrin α₉ and exhibited continuous vascular endothelial–cadherin (VE-cadherin) junctions and basement membrane, similar to collecting lymphatics. SC notably lacked luminal valves and expression of the lymphatic endothelial cell markers podoplanin and lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1). Using an ocular puncture model, we determined that reduced AHO altered the fate of SC both during development and under pathologic conditions; however, alteration of VEGF-C/VEGFR3 signaling did not modulate SC integrity and identity. Intriguingly, PROX1 expression levels linearly correlated with SC functionality. For example, PROX1 expression was reduced or undetectable under pathogenic conditions and in deteriorated SCs. Collectively, our data indicate that PROX1 is an accurate and reliable biosensor of SC integrity and identity.

Schlemm’s canal (SC) is an endothelium-lined vascular channel that encircles the margin of the cornea and drains aqueous humor, which is produced in the ciliary body and supplies the cornea and lens with nutrients through collector channels into the circulation (1, 2). SC is a conventional aqueous humor outflow (AHO) pathway accounting for the majority of AHO and is responsible for appropriate resistance against it to maintain a balance between the secretion and drainage of aqueous humor, thereby controlling intraocular pressure (IOP) (3, 4). Uveoscleral tract, which includes the extracellular spaces within the iris, the ciliary muscle (CM), and sclera, is an unconventional AHO pathway and covers the egression of remaining aqueous humor (~20% in human, ~80% in mice) (5, 6). In physiologic conditions, conventional AHO is largely proportional to pressure, although unconventional AHO is relatively pressure independent (5). On the other hand, when the resistance of SC increases with age and disease reduces conventional AHO, IOP is elevated, thereby sometimes leading to irreversible ocular nerve damage and glaucoma (7).

Lymphatic vessels (LVs) are lined by a single layer of lymphatic endothelial cells (LECs) and are responsible for draining tissue fluid into the circulation (8, 9). LECs are differentiated from blood endothelial cells (BECs) during early development, and the homeodomain transcription factor prospero homeobox protein 1 (Prox1) controls the BEC-to-LEC differentiation process. Notably, endothelial cell (EC) fate is found to be astonishingly plastic; ectopic expression of Prox1 in BECs induces lymphatic reprograming, while Prox1 deletion in LECs results in regaining of BEC fate in both embryonic and postnatal settings, labeling Prox1 as the master regulator for lymphatic cell-fate specification (8–12).

SC displays some important features found in LVs. Although originating from and directly connected to the blood vascular system, normal SC is devoid of blood-borne cells, such as red blood cells (13). SC controls the ocular tissue fluid homeostasis and tissue pressure by functioning as a major draining channel. Furthermore, the junction between SC ECs and trabecular meshwork (TM) is composed of a specialized tethering structure, which is responsible for providing an appropriate pressure gradient across the inner wall of an SC to maintain the integrities of SC ECs, thereby providing a tissue pressure-sensing/responding mechanism, such as anchoring filaments for LVs (14–16).

Despite SC’s critical functions, its organogenesis and cell-fate–specification mechanism remain unclear. Here, we report that SC ECs, initially BECs, are postnatally respecified to acquire lymphatic phenotypes by the upregulation of Prox1. We uncovered that AHO, but not VEGF-C/VEGFR3 signaling, is essential for the development and maintenance of SC integrity. Moreover, our pathology mimicking models with disrupted AHO manifested impairments in SC development as well as losses of its lymphatic characteristics. Above all, we detected synchronous fluctuations of Prox1 expression in SC with the changes of SC integrity and lymphatic characteristics, indicating Prox1 as a biosensor of SC integrity and identity.

SC expresses the lymphatic-specific transcriptional factor Prox1. We first examined whether SC is detectable with lymphatic and blood vascular markers in the whole-mounted and cross-sectioned corneas of Prox1-GFP reporter mice (17). We confirmed that Prox1 was highly expressed in the CD31⁺ SC and limbal LVs and moderately expressed in aqueous veins (AVs), which act as collecting channels between SC and episcleral blood vessels (BVs) (Figure 1, A–D, and Supplemental Video 1; supplemental material available online with this article; doi:10.1172/JCI75392DS1). The Prox1⁺ SC was readily distinguishable from the PDGFRβ⁺ TM and α-SMA⁺ CM (Figure 1E). A bead passage assay revealed that the AHO could be traced within the Prox1⁺ SC (Figure 1F). The SC also expressed VEGFR3 and endomucin, a venous EC marker (18), but lacked the expression of differentiated LEC markers, podoplanin and lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) (19–21), whereas limbal LVs highly expressed podoplanin and LYVE-1 (Figure 1, G–K). In addition, SC showed segmental heterogeneities: the portion of SC adjacent to the bifurcation of long posterior ciliary arteries (LPCAs) displayed lower expressions of Prox1 and VEGFR3 and weaker drainage capacity (49%, 42%, and 84%, respectively), but stronger expression of vWF (3.6-fold), and was lacking α-SMA⁺ CM compared with the SC located in other regions (Supplemental Figure 1, A–D). Importantly, SC displayed 2 structural features of collecting lymphatics: zipper-like continuous vascular endothelial–cadherin (VE-cadherin) junctions (22) and collagen IV⁺ basement membrane (ref. 23 and Figure 1, K and L). In contrast, the limbal LVs (capillaries) demonstrated button-like discontinuous VE-cadherin junctions without basement membrane investment (Figure 1, K and L). Interestingly, however, the ECs of SC expressed the lymphatic valve markers Foxc2 (Supplemental Figure 2, A and B, and ref. 24) and integrin α₉ (Itga9) (Figure 1H and ref. 25), even though SC did not have luminal valves in its lumen according to our observations of the blood regurgitation from BVs to SC following an ocular puncture (Supplemental Figure 3, A and B). Thus, SC is a unique vascular structure composed of collecting LV-like assembly of ECs, which can be detected with Prox1.

Figure 1

SC drains out aqueous humor into BVs and exhibits lymphatic markers but does not perfectly match the features of terminally differentiated LVs. (A) Schematic diagram of cornea showing SC (light green), AV (dark green), limbal LVs (light blue), and BVs (red). The square portion, immunostained and highly magnified, is shown in B. (B and C) Image and schematic diagram showing Prox1-GFP⁺ SC, AV, and CD31⁺ BV networks. Dashed lines demarcate limbal LV and AV, while dashed arrows indicate the AHO from SC into BVs. (D) H&E staining of corneal limbus in section. Arrow indicates SC. (E) Cross-sectioned image showing Prox1-GFP⁺ SC, PDGFR-β⁺ TM, and α-SMA⁺ CM in corneal limbus. (F) Perfused red fluorescent microbeads (diameter, 1 μm) in Prox1-GFP⁺ SC. Prox1-GFP mice were given 0.5 μl of the microbeads by intraocular injection; sample was harvested after 1 hour. (G) Image showing VEGFR3 expression in CD31⁺ SC. (H) Cross-sectioned image showing Itga9 (arrowhead), CD31⁺ SC, and α-SMA⁺ CM in corneal limbus. Magnification of the square portion is shown in the top right corner. (I) Comparison of expression and distribution of endomucin between SC (left panel) and limbal LVs (right panel). Arrows indicate limbal veins, while arrowheads indicate limbal artery. (J–L) Comparisons of expressions and distributions of podoplanin, VE-cadherin, Prox1-GFP, LYVE-1, and collagen IV⁺ basement membrane between SC (upper panels) and limbal LVs (lower panels). Dashed lines demarcate SC or limbal LVs (I–L).The square portions are magnified on the right. Scale bars: 100 μm; 20 μm (enlarged squares).

SC acquires lymphatic signatures during postnatal development. To elucidate how SC is formed, we examined the morphological changes of primitive SCs and their expression patterns of angiogenic (or lymphangiogenic) growth factor receptors during postnatal development. Initial endothelial buds of SC derived from choroidal veins (CVs) were detected at P1 (Figure 2A). The CVs expressed VEGFR2, VEGFR3, and Tie2, and these expressions were retained in the ECs of primitive SC at P1 (Figure 2, A and B). At P4, the ECs of elongated primitive SC became fused and separated from the CVs with the start of Prox1 expression (Figure 2A). However, Sox18, an upstream inducer of Prox1 in the cardinal vein (Supplemental Figure 4A and ref. 26), was not detected in the ECs of SC at P1 and P4 (Supplemental Figure 4B). To trace the development of SC, we performed a serial intravital imaging of the SC in Prox1-GFP mice. Active sprouting from preexisting SC was still detected at P7–P8 (Supplemental Figure 5), and the SC area grew gradually until 2 months of age (Figure 2, A and B). Moreover, while Prox1 expression rapidly and concomitantly increased with VEGFR3 in SC from P4 to adulthood, the expressions of BV markers VEGFR2 and Tie2 gradually decreased (Figure 2, A and B). Additionally, we evaluated the temporal change of Foxc2 expression in SC compared with that in limbal LVs. In the limbal LVs, Foxc2 was expressed in the developing LECs at P7 and in the lymphatic valves after P14 (Supplemental Figure 2A). Although the initial endothelial bud of SC lacked Foxc2 expression, Foxc2 expression emerged around P7 (Supplemental Figure 2B). Intriguingly, some Prox1⁺ SC ECs were devoid of Foxc2 expression until adulthood, but for ECs that expressed Foxc2, it was mainly colocated with the expression of Prox1 (Supplemental Figure 2B). Collectively, primitive SC acquires lymphatic phenotype through lymphatic reprogramming, which we speculate to be activated by a certain stimulus during postnatal development.

Figure 2

Acquisition of lymphatic characteristics in SC dependent on AHO during postnatal development. Unless otherwise noted, for the bar graphs, the quantification of the control group was normalized to 100%, from which the quantifications of other groups were calculated. (A and B) Temporal changes and quantifications of SC area and expression of Prox1, VEGFR2, VEGFR3, and Tie2 in SC during postnatal development. Dashed lines indicate the buds of SC derived from the CVs. Arrowheads demarcate the remnant communication between SC and CVs. Mean ± SD are shown (n = 4). Scale bars: 50 μm. For the quantifications, the group with the highest value was normalized to 100%, from which the relative values of other groups are shown. (C) Schematic diagram showing generation of the ocular puncture. Upper panel: a puncture on the sclera through 31-gauge syringe needle; lower panel: reduction of AHO achieved by leakage through the puncture. (D–G) Images and comparisons of the SC between the punctured eyes (puncture) and the sham-operated control eyes. Eyes were punctured from P5 to P6, and the corneas were harvested at P7. (D) Comparison of IOP. Each group, n = 4–5. (E) Images showing Prox1⁺, Prox1-GFP⁺, or VEGFR3⁺ cells in CD31⁺ SC. Arrowheads indicate the remnant communication between SC and CVs. Scale bars: 100 μm. (F and G) Comparisons of relative area and expressions of Prox1, Prox1-GFP, and VEGFR3 in SC. Each group, n = 4–5. *P < 0.05 versus control.

AHO regulates morphogenesis and lymphatic fate of SC during development. We hypothesized that the initiation (and increase) of AHO through SC might be a major factor for its growth and Prox1 expression during development. To address the effect of AHO on SC development, we utilized an ocular puncture model (Figure 2C). To reduce the AHO through SC, eyes were punctured daily from P5, causing an artificial leakage of aqueous humor through the puncture (Figure 2C). After the puncture of the eye, IOP dropped by 34% compared with the contralateral sham-operated eye (hereafter called “control eye”) (Figure 2D). As a result, the SC area, the protein and mRNA levels of PROX1, and the VEGFGR3 protein level in SC at P7 decreased by 61%, 69%, 74%, and 68%, respectively, in the punctured eye compared with the control eye (Figure 2, E–G). In addition, the reduction of AHO by a single puncture decreased the PH3⁺ proliferating ECs by 57%, while it increased the caspase-3⁺ apoptotic ECs by 5.2-fold at P6 in the growing SC of the punctured eye compared with that of control eye (Figure 3, A–C). Furthermore, a long-term reduction of AHO and subsequently decreased IOP induced by punctures from P4 to P14 also reduced the SC area (58%) and Prox1 expression (70%) (Supplemental Figure 6, A–D). In addition, we applied Cosopt from P5–P7, a drug that is conventionally used for lowering IOP in glaucoma patients by inhibiting aqueous humor production in the ciliary body (27) (a combined agent of β-adrenergic receptor inhibitor and carbonic anhydrase inhibitor), as an alternative method to decrease IOP (16% at P7) to confirm our findings with the ocular puncture model. Similar to the findings of the ocular puncture model, the SC area and protein and mRNA levels of PROX1 were reduced by 21%, 54%, and 47%, respectively, at P7 compared with the control eye (Figure 3, F–I). Conversely, to increase AHO during SC development, eyes were injected with donor aqueous humor once at P6. We found that the IOP was significantly elevated by 1 hour after the injection compared with the contralateral sham-operated eye (control eye), but returned to baseline IOP after 24 hours (Figure 3J). Consequently, the SC area was increased by 11% at P7 in the injected eye compared with the control eye (Figure 3, K and L). Also, the SC in the injected eye tended to have increased PROX1 and VEGFR3 protein levels, but without any significance (Figure 3, K and M). These findings support the hypothesis that incremental AHO positively regulates the development of SC.

Figure 3

AHO determines the expansion of SC ECs during postnatal development. Unless otherwise noted, the relative SC area and expression in control was normalized to 100%, from which the relative expressions of other groups were calculated. n = 4, each group. (A–E) Images and comparisons of the SC between punctured eyes and control eyes. Eyes were punctured at P6 and harvested at P7. (A and D) Images showing the expression of PH3, caspase-3, Klf4, and Prox1 in SC. (B, C, and E) Comparison of numbers of PH3⁺ cells and caspase-3⁺ cells, and relative expressions of Klf4 and Prox1 in SC. (F–I) Eyes of WT and Prox1-GFP mice were treated with control buffer (control) or Cosopt (B&C) daily from P5 to P7, and corneas were harvested at P7. (F) Comparison of IOP. (G) Images showing expression of Prox1-GFP and Prox1 in SC. (H and I) Comparison of relative SC area and expression of Prox1-GFP and Prox1. (J–M) Images and comparisons of the SC between injected eyes (injection) and sham-operated eyes (control). Eyes were injected with donor aqueous humor at P6 and harvested at P7. (J) Comparison of IOP. (K) Images showing Prox1 and VEGFR3 expression in SC. (L and M) Comparison of relative SC area and expression of Prox1 and VEGFR3 in SC. Scale bars: 50 μm. *P < 0.05 versus control.

Analysis of the molecular basis of AHO-mediated lymphatic reprograming in SC. We therefore investigated the molecular mechanisms underlying the flow-mediated lymphatic reprograming in SC ECs. Among a number of genes regulated by flow (Supplemental Table 1), Klf4, which was previously identified as a key shear stress–responsive transcription factor (28), attracted our attention as it was also highly upregulated in our RNA-Seq analyses. Accordingly, we confirmed the Klf4 expression in the nuclei of corneal epithelium (29) and ECs of SC, limbal BVs, and limbal LVs at P5, and, importantly, Klf4 expression was found to precede Prox1 expression in SC (Supplemental Figure 6E), indicating a possible existence of flow before the completion of lymphatic differentiation of SC. This relationship between Klf4 and Prox1 was confirmed with an in vivo ocular puncture model that showed that the expressions of Klf4 and Prox1 concomitantly decreased by 73% and 72%, respectively, in the punctured eye compared with the control eye (Figure 3, D and E). Moreover, the reduction of AHO caused by punctures in the earlier postnatal period (P3 to P5) retarded SC development, resulting in a 53% reduction in the SC area and 64% and 62% decreased expressions of Prox1 and Klf4, respectively, in the punctured eye compared with the control eye (Supplemental Figure 6, F–H).

We questioned whether the shear stress–responsive transcription factor Klf4 may play a role in the AHO-mediated Prox1 upregulation. We first confirmed that laminar flow (~2 dyne/cm²) efficiently upregulated Klf4 expression in cultured postnatal human dermal BECs and LECs within 12 hours (Supplemental Figure 7A). However, this specific in vitro experimental condition neither activated Prox1 expression in postnatal BECs, nor altered the expression level of Prox1 in postnatal LECs (Supplemental Figure 7A). Consequently, because 2 previous genome-wide ChIP studies (30, 31) have suggested a possible binding of Klf4 to the Prox1 gene, we next questioned whether KLF4 protein could bind to putative Prox1 enhancer regions. Indeed, our ChIP-PCR analyses revealed that 12-hour–long laminar flow could physically associate KLF4 protein with a putative enhancer area (chr1:212,229,489–212,229,614) in the first intron of the Prox1 gene of BECs (Supplemental Figure 7B), indicating that KLF4 protein is recruited to the Prox1 regulatory region in BECs under the shear stress condition. Despite this protein/DNA interaction, however, adenoviral overexpression of Klf4 did not turn on the expression of Prox1 in BECs (Supplemental Figure 7C). Moreover, we cloned the Klf4-binding area of the Prox1 gene in the pGL3-promoter vector to assess the capability of Klf4 to activate the luciferase reporter through its binding region. However, Klf4 could not activate the expression of the luciferase reporter (Supplemental Figure 7D). These data indicate that although Klf4 acquires a binding affinity to the Prox1 regulatory region in BECs in response to shear stress, the employed experimental condition was not sufficient for Klf4 to switch on the otherwise silenced Prox1 expression in BECs.

Next, we investigated the role of integrin β₁ during SC development because integrin β₁–mediated VEGFR3 phosphorylation in response to fluid acquisition has been known to trigger LV expansion (32). After confirming the presence of integrin β₁ in SC ECs, limbal BVs, and CM (Supplemental Figure 8A), mice were i.p. treated with anti–integrin β₁ functional blocking antibody (25 mg/kg) from P1 to P6 and harvested at P7. We confirmed the antiangiogenic effect (33) caused by the suppressed growth of retinal BVs as shown by decreased radial length of BVs and number of filopodia (10% and 25%, respectively) compared with control mice (Supplemental Figure 8, B–D). Meanwhile, the analysis showed no differences in the SC area or expression of Prox1 and VEGFR3 (Supplemental Figure 8, E–G), indicating that the blockade of integrin β₁ was not sufficient to affect the growth and lymphatic identity of SC.

VEGF-C/VEGFR3 system plays a substantial role in the early development of SC. To explore the significance of VEGF-C/VEGFR3 signaling in SC development, we checked the expression pattern of VEGF-C by using VEGF-C–β-gal knockin reporter mice (Vegfc^+/LacZ) (34). We observed that VEGF-C was highly expressed in the superficial layer of the cornea and iris at P1, whereas its expression was detected in the iris and the junctional tissue between SC and TM during adulthood (Figure 4, A–C). In addition, compared with control mice (Vegfc^+/+), Vegfc^+/LacZ mice displayed delayed buddings of SC ECs from the CVs, retarded tubular fusion, smaller SC area (38% at P1 and 38% at P7), reduced Prox1 expression (87% at P7), and markedly diminished limbal LVs (99%) during postnatal development (Figure 4, D–F, and Supplemental Figure 9, A and B). In line with these results, we confirmed that daily i.p. injection of soluble VEGFR3-Fc (sVEGFR3-Fc) (25 mg/kg) from P1 to P6 markedly reduced the area of SC and limbal LVs (68% and 69%, respectively) at P7 (Figure 4, G and H, and Supplemental Figure 9, C and D). Regardless of the severe outcomes of VEGF-C/VEGFR3 signaling disruption during SC development, the SC of Vegfc^+/LacZ had well-organized morphology, normalized SC area, and recovered Prox1 expression by P14 compared with Vegfc^+/+ (Figure 4, D–F), whereas the limbal LVs were still affected (Supplemental Figure 9, E and F).

Figure 4

VEGF-C/VEGFR3 system plays a crucial role in the early development of SC. (A–C) β-Gal staining of corneas in VEGF-C–β-gal knockin reporter mice (Vegfc^+/LacZ) at P1 and 1 month. Robust VEGF-C expression in superficial layer of cornea (white arrowhead) and iris (black arrowhead) at P1 is shown, while rarer VEGF-C expression in TM (black arrow) and iris (black arrowhead) at 1 month is shown. (D) Prox1 and CD31 staining of SC ECs in Vegfc^+/LacZ and Vegfc^+/+ mice during postnatal development. Arrowheads indicate buddings of SC ECs. Arrows indicate holes in SC, which denote defects of tubular fusion. (E and F) Comparisons of relative area and Prox1 expression of SC. The area and Prox1 expression of Vegfc^+/+ at 1 month was set as 100%, respectively. n = 4, each group. *P < 0.05 versus Vegfc^+/+. (G and H) Mice were i.p. given sVEGFR3 (25 mg/kg) daily from P1 to P6 and designated as control Fc and sVEGFR3; corneas were harvested at P7. (G) Image showing CD31⁺ SC ECs. (H) Comparison of relative SC area. The quantification of control Fc group was normalized to 100%, from which the quantifications of other groups were calculated. n = 4, each group. *P < 0.05 versus control Fc. Scale bars: 100 μm.

Reduced AHO induces a loss of lymphatic characteristics in SC, leading to EndMT. As AHO often decreases due to aging and pathological insults (2), we next investigated the requirement of AHO in the maintenance of the lymphatic identity of SC in adults using the murine eye puncture model. Puncture was made in the sclera of adult mice daily for 7 days to decrease their IOP (Figure 5A). Compared with the SC of the control eye, the SC of the punctured eye exhibited reduced expressions of Prox1 (43%), VEGFR3 (43%), and Klf4 (56%), whereas they displayed increased expressions of vWF (1.8-fold), α-SMA (8.7-fold), fibroblast-specific protein 1 (FSP-1) (2.1-fold), vimentin (2.8-fold), and desmin (6.3-fold) (Figure 5, B–E). We also noted that α-SMA expression was colocalized with the CD31⁺ area of SC ECs in the punctured eye (Figure 5D).

Figure 5

Reduced AHO induces loss of lymphatic feature in SC ECs, leading to EndMT. (A–E) Images and comparisons of SC between punctured eyes and sham-operated control eyes. Eyes were punctured daily for 7 days, and corneas were harvested at day 7. (A) Comparison of IOP. n = 4–5, each group. (B and C) Images and comparisons of expressions of Prox1, VEGFR3, vWF, and Klf4 in the CD31⁺ ECs of SC. n = 4–5, each group. The quantification of control group was normalized to 100%, from which the quantifications of other groups were calculated. (D and E) Images and comparisons of mesenchymal markers α-SMA, FSP-1, vimentin, and desmin in CD31⁺ SC ECs. Solid-line square is magnified on the right, which reveals α-SMA expression in the CD31⁺ EC of SC (white arrows). Arrowheads indicate expression of FSP-1, vimentin, and desmin in CD31⁺ SC ECs, respectively. n = 4–5, each group. (F) Lineage-tracing study in which Prox1-Cre^ERT2 R26mTmG mice were treated with tamoxifen 3 times from 8 weeks. Then eyes were punctured daily for 7 days, and corneas were harvested at day 7. Solid-line square is magnified in the bottom right corner. Arrows indicate coexpression of FSP-1 in GFP⁺ SC ECs. *P < 0.05 versus control. Scale bars: 100 μm; 20 μm (enlarged squares).

To trace the fate of ECs in SC, we performed a lineage-tracing study with Prox1-Cre^ERT2 Rosa26mTmG mice, in which Prox1-expressing cells retained permanent membrane GFP expression after tamoxifen injection. Following the daily puncture of eyes over 7 days, the GFP⁺ SC ECs partly exhibited FSP-1 expression, whereas no FSP-1 expression was observed in that of control eye (Figure 5F). These findings indicate that the ECs of SC undergo endothelial-mesenchymal transition (EndMT) (35–37) when the AHO through SC is reduced. To support our data, which suggest that AHO is critical for the maintenance of lymphatic characteristics in SC, we created an experimental glaucoma model using beads (6 μm in diameter) that blocks the AHO and elevates the IOP (38). One day after the intraocular injection of the beads, an accumulation of these beads was notable around the margin of the cornea (Figure 6A). Compared with the PBS-injected eyes, the IOP of the bead-injected eyes remained elevated (Figure 6B), and the Prox1 expression was reduced by 57%, while the α-SMA expression increased by 6.8-fold in their SC (Figure 6, C–E).

Figure 6

Analysis of lymphatic identity in SC with defective AHO within bead-induced glaucoma model. (A–E) Eyes were injected with 2 μl of microbeads (bead diameter, 6 μm) or 2 μl of PBS, and corneas were harvested after 7 days. (A) Appearance of a bead-injected eye. Arrows indicate deposited beads adjacent to SC. (B) Comparison of IOP. n = 4, each group. (C) IHC of SC stained for Prox1 and α-SMA in CD31⁺ SC. Scale bars: 100 μm. (D and E) Comparisons of relative Prox1 expression and of the coverage of α-SMA⁺ cells by measuring α-SMA⁺ area divided by CD31⁺ SC area, expressed as a percentage. The relative expression of PBS was normalized to 100%, from which the relative expressions of other groups were calculated. n = 4, each group. *P < 0.05 versus PBS.

Aged SC displays the features of senescent endothelium and reduced lymphatic markers as well as increased mesenchymal markers. SCs are constantly exposed to external stress over a lifetime (39), which suggests that they may be undergoing the aging process faster than any other organs. In fact, in the SC of 1-year-old mice, the activities of senescence-associated β-gal (SA–β-gal) and ROS were markedly upregulated in the SC, by 4.8- and 20.3-fold, respectively, whereas the expressions of MMP-2 and MMP-9 in the junctional tissue between SC and TM were downregulated by 65% and 71%, respectively, and the AHO was reduced by 86% along with the 57% and 67% suppressed expressions of Klf4 and eNOS, respectively, compared with the SC of 2-month-old mice (Figure 7, A–D). Nevertheless, no significant differences in the IOP between the young and old mice were detected (Figure 7E), in agreement with previously reported knowledge (40). We assumed that this is because uveoscleral AHO has a greater role in mice, accounting for approximately 80% of the total AHO, thereby compensating for the dysfunctional drainage through SC, unlike in humans (6).

Figure 7

Aged SC exhibits features of senescent endothelium and reduced AHO. (A–D) Images and comparisons of expressions in SA β-gal, ROS, MMP-2, MMP-9, Klf4, eNOS, and perfused microbead (bead) in SC of 2-month-old and 1-year-old mice. Arrows and arrowheads indicate expressions of MMP-2 and MMP-9, respectively, in CD31⁺ SC. The relative expression of SC at 2 months was normalized to 100%, from which the relative expressions of other groups were calculated. n = 5, each group. *P < 0.05 versus 2 months. (E) Comparisons of IOP at 2, 6, and 12 months. n = 5, each group. Scale bars: 100 μm.

In terms of LEC markers, compared with the SC of 2-month-old mice, the SC of 1-year-old mice exhibited 59%, 65%, 55%, and 67% less expression of Prox1, Prox1-GFP, VEGFR3, and Itga9, respectively, while vWF expression was 2.1-fold higher (Figure 8, A and B). Consistent with this, we confirmed a 62% decreased Prox1 mRNA level in the corneal limbus, as shown by RT-PCR (Figure 8C). These findings indicate that the lymphatic identity of the SC is suppressed in aged mice. Furthermore, the SC of 1-year-old mice exhibited an 11.0-fold increase in α-SMA expression compared with that of 2-month-old mice (Figure 8, A and D). Most of the α-SMA⁺ cells appeared to be star shaped and mimicked fibroblasts. Furthermore, the analyses in the SC of 1-year-old mice showed 3.0-fold and 13.6-fold increased expressions of FSP-1 and desmin, respectively, both of which were partially correlated with α-SMA expression (Figure 8, A and D). Collectively, senescent SC showed analogous phenotypes to SC in punctured eyes, which might be manifested by the long-term suppression of AHO (Figure 9).

Figure 8

Aged SC displays reduced lymphatic markers and increased mesenchymal markers. (A, B, and D) Images and comparisons of expression in Prox1, Prox1-GFP, VEGFR3, Itga9, vWF, FSP-1, α-SMA, and desmin in CD31⁺ SC of 2-month-old and 1-year-old mice. Arrows indicate Itga9 expression in CD31⁺ SC. Arrowheads denote CD31⁺ limbal BVs with desmin coverage. The relative expression of SC at 2 months was normalized to 100%, from which the relative expressions of other groups were calculated. n = 5, each group. *P < 0.05 versus 2 months. (C) Comparisons of Prox1 mRNA expressions in corneal limbus. n = 5, each group. *P < 0.05 versus 2 months. Scale bars: 100 μm.

Figure 9

Schematic diagram depicting the dynamic changes of lymphatic identities of SC during development, adulthood, and senescence. SC ECs are postnatally transdifferentiated from BECs (VEGFR2⁺, Tie2⁺, and Prox1^–) to acquire lymphatic phenotypes by the upregulation of Prox1 and increased AHO. With ageing, senescent SC ECs, which by then have reduced AHO, start to lose lymphatic markers (Prox1, VEGFR3, and Itga9), but gain blood vessel markers (vWF) as well as mesenchymal markers (α-SMA, FSP-1, vimentin, and desmin), leading to EndMT.

Heterogeneity and plasticity are the two most important features of ECs (41). ECs often respond to physiological stimuli and pathological insults by altering their cell fates. This astonishing cell-fate plasticity is believed to underlie the remarkable heterogeneity of ECs. ECs of SC in the eye have long been suspected as being distinct from BECs of the capillaries or postcapillary venules, mainly because SC drains and carries tissue fluids (aqueous humor), not blood, although directly connected to BVs. Here, we report that SC ECs originate from the CVs and undergo a partial lymphatic reprogramming during postnatal development, giving rise to a novel class of ECs that display both BEC and LEC phenotypes. SC ECs express a list of key lymphatic-signature genes, such as Prox1, Vegfr3, and Itga9 (10), along with the venous marker endomucin (18), but, most notably, lack expressions of podoplanin and LYVE-1 (Figure 9). In fact, it is known that podoplanin, but not LYVE-1, is regulated by Prox1 (41, 42). However, the nonexistent expressions of both genes in SC ECs, despite the presence of Prox1 expression, is quite unusual and striking, since podoplanin and LYVE-1 are predominantly expressed in capillary LECs (43, 44), raising a possibility that SC ECs may not be terminally differentiated from LECs and their lymphatic phenotype may be intermediate. This possibility is further supported by our findings that Prox1 expression levels in SC ECs, but not in the limbal LVs, are dynamically regulated by AHO and aging. Additional unusual features of SC compared with normal LVs are that it displays structural features of collecting lymphatics (zipper-like continuous VE-cadherin junctions, ref. 22; and collagen IV⁺ basement membrane, ref. 23) without luminal valves, a hallmark of the collectors (25). All these data cohesively suggest that SC is a specialized vascular structure that displays some lymphatic phenotypes and carries out lymphatic function, but is distinct from normal LVs (Figure 9).

Interesting similarities can be found between embryonic lymphatic development and postnatal lymphatic differentiation of SC in terms of their formation. Both processes involve progenitor cell migration from the veins and subsequent acquisition of lymphatic phenotypes (45). Also, Prox1 appears to play a key role in both cell-reprograming events. The differentiated progenitor cells assemble to form new tubular structures, which later dissociate from the veins. Our current study found that AHO is essential for all stages of SC development as well as maintenance of its lymphatic phenotypes, which is also the case for lymphatic development (32). However, one major difference is that while Prox1 is upregulated in cardinal vein ECs before they migrate out during embryogenesis, Prox1 expression begins in the primitive SC structure, not in the CVs, at P4 and continues to increase until mice are 1 month old. In particular, Sox18 expression was absent in SC during early postnatal development, but present in the cardinal vein during embryonic lymphangiogenesis (26). It remains to be defined whether and how these differences affect the cell-fate specification as well as expression of lymphatic markers such as podoplanin and LYVE-1 (41).

Importantly, Prox1 expression in SC ECs was found to be highly sensitive to AHO; reduction in AHO induced by our surgical (ocular puncture model) or noninvasive (pharmacological) approach consistently downregulated Prox1. Although the strategy to increase AHO with incremental IOP over a long duration (>24 hours) was not successful, attributed to the small volume of the eyes of pups limiting the injection of adequate amount of aqueous humor, transiently elevated IOP tended to upregulate Prox1 and VEGFR3. Moreover, segmental heterogeneity in Prox1 expression was also found in the normal cornea; a weaker Prox1 expression was found in disorganized sections in SC with decreased AHO due to lack of CM (13), which were frequently found adjacent to the bifurcation portion of LPCAs. Furthermore, senescent SCs, which are similar to those of aged humans (46), showed a deficiency of lymphatic markers and increased density of mesenchymal cells, similar to the aftermath of artificially reduced AHO models. Previous studies reported that the inner wall of SC is the principal site of flow resistance against AHO (14). Furthermore, AHO resistance between SC and TM has been found to increase with age in humans (2). Indeed, senescent ECs in old mice involve disturbed AHO, coinciding with the dysfunction of MMP activities, which are responsible for ECM remodeling of the junction between SC and TM to maintain the appropriate AHO resistance (47). Consequently, the impairment of AHO through SC with aging leads to suppressed lymphatic identity and loss of EC integrity in SC. Overall, we propose that deficient AHO negatively affects the lymphatic fate of SC ECs.

Hemodynamic forces have been postulated to maintain the integrities of BVs (48). The importance of mechanotransduction in response to gain of fluid was highlighted in LEC proliferation via VEGFR3 activation (32). Previous studies comprehensively predicted the existence of a fluid-induced mechanistic factor in LECs (24, 49, 50). Furthermore, lymphatic vascular integrity can be compromised by a dose-dependent Prox1 depletion in vivo and in vitro (10, 11, 51, 52). However, the upstream pathways of Prox1 in LECs are still poorly understood, except for the Sox18 transcription factor and RAF1/ERK signaling (26, 53). In this study, we confirmed that Klf4 is upregulated in flow-exposed dermal BECs and LECs in vitro and in developing SC ECs in vivo (28, 54). Moreover, previous studies indicated that even if the current of aqueous humor is low, the presence of AHO itself is enough to actively modulate the behaviors of SC ECs, which is in agreement with our data (15). We demonstrated that Prox1 and Klf4 expressions in SC are simultaneously modulated in a temporal manner and are associated with AHO levels during developmental stage and adulthood. Accordingly, we addressed that the defective integrities of SC could be explained by the plasticity of Prox1, which was dependent on the level of AHO. However, our experimental conditions could not turn on the Prox1 expression in BECs. The failure of Prox1 upregulation in BECs by the shear stress could be due to multiple reasons: postnatal dermal BECs may not properly represent the molecular and cellular features of the primitive BECs that differentiate to SC ECs; the magnitude or nature of the shear stress generated in vitro may significantly differ from that of in vivo AHO, or AHO alone, although necessary, may not be sufficient for Prox1 upregulation and lymphatic differentiation of the primitive BECs. Further studies are needed to address the molecular basis of AHO-induced Prox1 upregulation in SC.

We found that Foxc2 in SC emerged from at least P7 and was retained through adulthood. Foxc2 expression is triggered by oscillatory shear stress in LECs (24). Because of the absence of a valve between SC and AV, it is postulated that the oscillatory pulse wave of aqueous humor from AV to SC, induced by the ocular pulse, blinking, and eye movements of normal eyes (55), might be associated with Foxc2 expression in SC. However, the heterogeneous Foxc2 expression that is only partly visible in Prox1⁺ SC is contrasted with the distinct expression of that in lymphatic valve (24), thereby supporting the hypothesis of intermediate lymphatic phenotype in SC.

The senescence of SC in aged mice is quite similar to that in aged humans (46). However, there are only few works in the literature depicting the integrity of SC ECs, except decreased densities of transendothelial pores in SC ECs, depending on AHO with aging (56). We reveal that the impairment of the integrity of SC ECs manifests increased numbers of mesenchymal cells, including fibroblasts and myofibroblasts, on the outer wall of SC in aged mice or those in pathological conditions. This phenomenon can be correlated with the higher accumulated plaque-like material (presumed to be elastic fibers produced from fibroblasts) around SC in aged humans (57). Furthermore, the transition of SC ECs to mesenchymal cells can be interpreted as a temporary action taken to avoid apoptosis against the sublethal cellular stress resulting from deficient AHO (7). VEGF-C/VEGFR3 signaling is closely related to the development of SC, where the disturbance of this signaling induces growth retardation of SC, accompanied by the same phenotype in limbal LVs. However, the rescued morphology and lymphatic identity of SC in VEGF-C heterozygous mice at a later postnatal period indicates that VEGF-C is not the main growth factor for the development of SC.

In summary, our evidence strongly points to AHO as the main element to maintain SC integrity and identity and above all presents Prox1 as an accurate biosensor for SC. Considering our data, which support the reliability of using Prox1 to reflect the functionality of SC, further research to detect Prox1 expression in the SC of humans will allow for the early detection and prevention of IOP-induced pathologies such as glaucoma.

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We are grateful to Soo-Il Chang, Sujin Seo, Eun Soon Lee, and Tae-Chang Yang for their technical assistance. This study was supported by grants from the National Research Foundation funded by the Ministry of Science, ICT, & Future Planning, Korea (NRF-2011-0019268, to G.Y. Koh), the Korea Healthcare Technology R&D Project, Ministry of Health & Welfare (A110076, to G.Y. Koh), and the National Heart, Lung and Blood Institute/NIH (HL121036, HD059762, to Y.K. Hong).

Address correspondence to: Gou Young Koh, Graduate School of Medical Science and Engineering, KAIST, 373-1, Guseong-dong, Daejeon, 305-701, Korea. Phone: 82.42.350.2638; E-mail: gykoh@kaist.ac.kr. Or to: Young Kwon Hong, Departments of Surgery and of Biochemistry & Molecular Biology, Keck School of Medicine, University of Southern California, 1450 Biggy St., Los Angeles, California 90033, USA. Phone: 323.442.7825; E-mail: young.hong@usc.edu.

Conflict of interest: The authors have declared that no conflict of interest exists.

Reference information: J Clin Invest. 2014;124(9):3960–3974. doi:10.1172/JCI75392.

See the related articles at The Schlemm’s canal is a VEGF-C/VEGFR-3–responsive lymphatic-like vessel and Schlemm’s canal: more than meets the eye, lymphatics in disguise.

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