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Cornea  |   June 2013
EphrinB2–EphB4 Signals Regulate Formation and Maintenance of Funnel-Shaped Valves in Corneal Lymphatic Capillaries
Author Affiliations & Notes
  • Hideto Katsuta
    Laboratory for Stem Cell Biology, RIKEN Center for Developmental Biology, Kobe, Japan
  • Yoko Fukushima
    Laboratory for Stem Cell Biology, RIKEN Center for Developmental Biology, Kobe, Japan
  • Kazuichi Maruyama
    Department of Ophthalmology, Tohoku University Graduate School of Medicine, Miyagi, Japan
  • Masanori Hirashima
    Division of Vascular Biology, Department of Physiology and Cell Biology, Kobe University Graduate School of Medicine, Kobe, Japan
  • Kohji Nishida
    Department of Ophthalmology, Osaka University Medical School, Osaka, Japan
  • Shin-Ichi Nishikawa
    Laboratory for Stem Cell Biology, RIKEN Center for Developmental Biology, Kobe, Japan
  • Akiyoshi Uemura
    Laboratory for Stem Cell Biology, RIKEN Center for Developmental Biology, Kobe, Japan
    Division of Vascular Biology, Department of Physiology and Cell Biology, Kobe University Graduate School of Medicine, Kobe, Japan
  • Correspondence: Akiyoshi Uemura, Division of Vascular Biology, Department of Physiology and Cell Biology, Kobe University Graduate School of Medicine, 7‐5‐1 Kusunoki-cho, Chuo-ku, Kobe 650‐0017, Japan; auemura@med.kobe-u.ac.jp
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 4102-4108. doi:10.1167/iovs.12-11436
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      Hideto Katsuta, Yoko Fukushima, Kazuichi Maruyama, Masanori Hirashima, Kohji Nishida, Shin-Ichi Nishikawa, Akiyoshi Uemura; EphrinB2–EphB4 Signals Regulate Formation and Maintenance of Funnel-Shaped Valves in Corneal Lymphatic Capillaries. Invest. Ophthalmol. Vis. Sci. 2013;54(6):4102-4108. doi: 10.1167/iovs.12-11436.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: To elucidate the role of signals mediated by EphB4 receptor tyrosine kinase and its transmembrane ephrinB2 ligand in corneal lymphatic capillaries.

Methods.: To detect expression of ephrinB2 and EphB4 in mouse corneas, immunohistochemistry of flat-mount corneas from 6- to 10-week-old wild-type, Efnb2-lacZ, and Ephb4-lacZ mice on a C57BL/6 background was performed. To induce formation of new blood vessels and lymphatic vessels, mouse corneal epithelia were swabbed with 0.1 M sodium hydroxide. To antagonize endogenous receptor–ligand interactions in corneal lymphatic vessels, recombinant EphB4/Fc proteins were injected into the subconjunctival spaces. To visualize the corneal lymphatic flow, FITC-dextran was injected subconjunctivally.

Results.: In lymphatic capillaries of adult mouse corneas, EphB4 was intensively expressed in lymphatic endothelial cells (LECs) of funnel-shaped valves, which were segregated from ephrinB2-expressing LECs. The number of corneal lymphatic valves was significantly decreased by Efnb2 haploinsufficiency, and subconjunctival EphB4/Fc injections resulted in the deformation of preexisting valves of corneal lymphatic capillaries. In alkali-burn corneas, ephrinB2 and EphB4 were highly expressed in LECs of valve-forming areas. Subconjunctival EphB4/Fc injections perturbed the morphologic maturation of new lymphatic valves, leading to reflux of FITC-dextran to peripheral lymphatic branches.

Conclusions.: The results demonstrate a pivotal role of ephrinB2–EphB4 signals in the formation and maintenance of funnel-shaped valves in corneal lymphatic capillaries, and further suggest the potential of ephrinB2–EphB4 signals as a target to therapeutically manipulate corneal lymphangiogenesis.

Introduction
In corneal limbus, blind-ended lymphatic capillaries maintain fluid homeostasis by absorbing water and macromolecules from the interstitium, and serve as a trafficking route for immune cells. 1,2 The quiescence of corneal lymphatic vessels is disrupted upon inflammation and wound healing associated with trauma, infection, and chemical burn, leading to the growth of new lymphatic vessels (lymphangiogenesis) as well as blood vessels (hemangiogenesis), which deteriorates corneal transparency. 1,2 Because lymphangiogenesis after corneal transplant surgery is a key determinant of graft rejection, it may be of clinical value to develop a modality that can manipulate corneal lymphangiogenesis. 1,2  
Based on anatomic and immunochemical features, lymphatic vasculature is generally subdivided into collecting vessels and capillaries. 35 In collecting vessels, lymphatic endothelial cells (LECs) are spindle-shaped, covered by smooth muscle cells (SMCs), and negative for lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), whereas capillary LECs are oak leaf–shaped, lack SMC coverage, and express LYVE-1. 35 Luminal valves are present only in collecting lymphatic vessels, but not in lymphatic capillaries. 36 However, this prevailing view was challenged by recent reports showing microvalves in corneal lymphatic capillaries. 7,8 Because luminal valves regulate the directions of lymphatic flow, 36 it is therefore important to fully understand the molecular mechanisms underlying the formation and maintenance of microvalves in corneal lymphatic capillaries. 
Among signaling molecules involved in lymphangiogenesis, the transmembrane ephrinB2 ligand regulates valve formation in developing lymphatic vasculature. 36,9 Binding of ephrinB2 to its cognate EphB4 receptor tyrosine kinase stimulates EphB4 autophosphorylation and activates downstream signaling cascades, which lead to repulsion of EphB4-expressing cells. 911 In addition to this “forward” signal, ephrinB2 itself mediates a “reverse” signal via its C-terminal PDZ-binding motif (PDZ is an acronym combining the first letters of three proteins: postsynaptic density protein [PSD95], Drosophila disc large tumor suppressor [Dlg1], and zonula occludens-1 [zo-1]), which is required for endothelial sprouting in newly formed blood vessels and lymphatic vessels. 12,13 In blood vessels, ephrinB2 and EphB4 are mainly expressed in arteries and veins, respectively, and the reciprocal ligand–receptor interactions are a prerequisite for the organized patterning of vascular networks. 1416 In collecting lymphatic vessels, ephrinB2 is highly expressed in valvular LECs, and the reverse ephrinB2 signal is necessary for valve formation, as demonstrated by the absence of lymphatic valves in mice lacking the ephrinB2 PDZ-binding motif. 17 In addition, inducible deletion of the Efnb2 gene in mature lymphatic vessels reduced the number and deformed the morphology of luminal valves in adult ear skins, indicating a continuous requirement of ephrinB2 signaling for the maintenance of valves in collecting lymphatic vessels. 18 However, the roles of ephrinB2–EphB4 signals remain unknown in corneal lymphatic capillaries. 
Here we demonstrate that in corneal lymphatic capillaries, LECs of funnel-shaped valves highly express EphB4, whereas the surrounding LECs diffusely express ephrinB2. We further demonstrate that perturbation of the ephrinB2–EphB4 signals disrupts the formation and maintenance of funnel-shaped valves in corneal lymphatic capillaries. Thus, the ephrinB2–EphB4 signals can be potential targets to manipulate corneal lymphangiogenesis, such as in the management of graft survival after transplant surgery. 
Materials and Methods
Animals
Under anesthesia after intraperitoneal (IP) injections of sodium pentobarbital (40 mg/kg), corneal epithelia of 6- to 10-week-old wild-type (WT), Efnb2-lacZ, 14 or Ephb4-lacZ 15 mice on a C57BL/6 background were swabbed with 0.1 M sodium hydroxide (NaOH) followed by washout with phosphate-buffered saline (PBS). After IP injections of sodium pentobarbital and cardiac perfusion with 4% paraformaldehyde (PFA) in PBS, eyeballs were enucleated and processed for immunochemical analyses. All experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee of RIKEN CDB, and were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Immunohistochemistry
After brief fixation of eyeballs in ice-cold 4% PFA/PBS, corneas were dissected under a stereo microscope (Stemi DV4; Carl Zeiss, Oberkochen, Germany) and were further fixed in 4% PFA/PBS overnight. The whole-mount corneas were washed with PBS, blocked in 1% bovine serum albumin and 0.1% Triton X-100 in PBS, and incubated overnight with the following primary antibodies (Abs): PECAM-1 (clone Mec13.3; BD Biosciences, San Jose, CA), LYVE-1 (AngioBio, Del Mar, CA), β-galactosidase (Merck Millipore, Billerica, MA), ephrinB2 (R&D Systems, Minneapolis, MN), integrin-α9 (R&D Systems), and Cy3-conjugated α-smooth muscle actin (clone 1A4; Sigma-Aldrich, St. Louis, MO). After washing with 0.1% Triton X-100 in PBS, the samples were further incubated with donkey anti-rat, -rabbit, and -goat secondary Abs conjugated with Alexa488 (Life Technologies, Carlsbad, CA), Cy3, or Cy5 (Jackson ImmunoResearch, West Grove, PA). Nuclei were labeled (TO-PRO-3; Life Technologies). Images were taken with a confocal microscope (LSM510 or LSM700; Carl Zeiss). 
Subconjunctival Injections
EphB4/Fc (R&D Systems), consisting of the extracellular domain of mouse EphB4 fused to the Fc portion of human IgG, or equivalent amounts (16 μg dissolved in 4 μL PBS) of control human IgG Fc (Jackson ImmunoResearch) proteins, were injected into subconjunctival spaces. To visualize the corneal lymphatic flow, high molecular weight FITC-dextran (MW = 2000 kDa, 8 mg/mL in PBS; Sigma-Aldrich) was injected subconjunctivally, and corneas were observed using a fluorescence stereomicroscope (Leica MZ FLIII; Leica Microsystems GmbH, Wetzler, Germany). 
Morphometric Analyses of Lymphatic Valves
The number of lymphatic valves was counted in whole-mount corneas immunostained with PECAM-1 and LYVE-1. The length of lymphatic valve invagination was measured in whole-mount corneas immunostained with integrin-α9, photographed with a ×63 objective using ZEN confocal image software (Carl Zeiss). 
Statistical Analysis
Statistical analysis was performed with commercial software (JMP software, version 8.0.2; SAS Institute, Inc., Cary, NC) using the Wilcoxon rank sum test or Kruskal–Wallis nonparametric analysis with Dunnett's post hoc test. Values of P < 0.05 were considered statistically significant. 
Results
Intense EphB4 Expression in Corneal Lymphatic Valves
To elucidate the expression pattern of ephrinB2 and EphB4 in quiescent lymphatic capillaries of adult corneas, we performed corneal whole-mount immunohistochemistry in mice heterozygous for the Efnb2 or Ephb4 gene, in which the endogenous ephrinB2 or EphB4 expression could be monitored by the lacZ reporter expression. In the corneal limbus, ephrinB2 was continuously expressed in arterial blood vessels, which expressed PECAM-1 but not LYVE-1, whereas capillary LECs that expressed both PECAM-1 and LYVE-1 diffusely expressed ephrinB2 (Fig. 1A). By contrast, intense EphB4 expression was periodically observed in LECs, most of which was located near the branching points of lymphatic capillaries and the traversing points of arterial blood vessels (Fig. 1B). Notably, the intense EphB4 expression was restricted to the LECs invaginating into the proximal, enlarged lymphatic vessels, which corresponded to the microvalves with high PECAM-1 and integrin-α9, but low LYVE-1 expression (Fig. 1C; Supplementary Data S1A).7,8 Unlike the bicuspid valves in collecting lymphatic vessels,6 the valvular LECs in corneal lymphatic capillaries were arranged circumferentially, forming a funnel-shaped structure (Supplementary Data S1B). In these valve areas, ephrinB2 was expressed diffusely in the outer LECs surrounding the invaginating LECs (Fig. 1D). Dual labeling of ephrinB2 and EphB4 further demonstrated that EphB4-expressing valvular LECs were segregated from the ephrinB2-expressing LECs (Figs. 1E, 1F; Supplementary Movie S1). Thus, the LECs of adult corneas continuously express ephrinB2 and EphB4, with distinct expression patterns in the funnel-shaped valves. 
Figure 1
 
Expression of ephrinB2 and EphB4 in corneas of adult Efnb2-lacZ or Ephb4-lacZ mouse. (A) EphrinB2 is expressed in PECAM-1(+) LYVE-1(−) arterial blood vessels (arrowheads), and in PECAM-1(+) LYVE-1(+) lymphatic capillaries (arrows). (B) EphB4 is periodically expressed in lymphatic capillaries near the branching points (arrow) and the crossing points with arterial vessels (arrowheads). (C) Intense EphB4 expression is restricted to the invaginating LECs of funnel-shaped valves. (D) EphrinB2 is diffusely expressed in outer LECs surrounding the valves. (E) EphB4-expressing valvular LECs are segregated from ephrinB2-expressing LECs. (F) A scheme representing the expression of ephrinB2 and EphB4 in corneal lymphatic valves. Scale bars: 100 μm (A, B); 10 μm (CE).
Figure 1
 
Expression of ephrinB2 and EphB4 in corneas of adult Efnb2-lacZ or Ephb4-lacZ mouse. (A) EphrinB2 is expressed in PECAM-1(+) LYVE-1(−) arterial blood vessels (arrowheads), and in PECAM-1(+) LYVE-1(+) lymphatic capillaries (arrows). (B) EphB4 is periodically expressed in lymphatic capillaries near the branching points (arrow) and the crossing points with arterial vessels (arrowheads). (C) Intense EphB4 expression is restricted to the invaginating LECs of funnel-shaped valves. (D) EphrinB2 is diffusely expressed in outer LECs surrounding the valves. (E) EphB4-expressing valvular LECs are segregated from ephrinB2-expressing LECs. (F) A scheme representing the expression of ephrinB2 and EphB4 in corneal lymphatic valves. Scale bars: 100 μm (A, B); 10 μm (CE).
Maintenance of Corneal Lymphatic Valves by EphrinB2–EphB4 Signals
In adult mice heterozygous for the Efnb2 gene, but not the Ephb4 gene, the number of corneal lymphatic valves marked as PECAM-1–positive and LYVE-1–negative was significantly decreased compared with WT mice (Fig. 2A), indicating that the reduced expression level of the ephrinB2 ligand affected the formation and/or maintenance of corneal lymphatic valves. To further evaluate the role of the ephrinB2–EphB4 signals in the maintenance of corneal lymphatic valves, we injected EphB4/Fc proteins, which antagonize endogenous ligand–receptor interactions, 19,20 into the subconjunctival spaces of adult mice. Surprisingly, at 3 days after daily EphB4/Fc injections, the preexisting valves of corneal lymphatic capillaries showed morphologic deformation (Fig. 2B), which was characterized by shortening of the invagination. Indeed, the invagination length measured at the center of the valves was significantly decreased in the EphB4/Fc-treated corneas (Fig. 2C). These results indicated that the ephrinB2–EphB4 signals constitutively contributed to the maintenance of corneal lymphatic valves. 
Figure 2
 
Defects in lymphatic valves by manipulating the ephrinB2–EphB4 signals in adult mouse corneas. (A) Quantification of lymphatic valve number in adult mouse corneas (n = 3, from three animals for each population). (B) Whole-mount integrin-α9 immunostaining at 3 days after daily subconjunctival injections of control Fc or EphB4/Fc proteins. Arrows indicate the invagination lengths at the center of the valves. Scale bar: 10 μm. (C) Quantification of valve invagination length in corneal lymphatic capillaries (n = 15, from three eyes [five valves per eye] of three animals for each population). Error bars represent SEM; ***P < 0.001. NS, not significant.
Figure 2
 
Defects in lymphatic valves by manipulating the ephrinB2–EphB4 signals in adult mouse corneas. (A) Quantification of lymphatic valve number in adult mouse corneas (n = 3, from three animals for each population). (B) Whole-mount integrin-α9 immunostaining at 3 days after daily subconjunctival injections of control Fc or EphB4/Fc proteins. Arrows indicate the invagination lengths at the center of the valves. Scale bar: 10 μm. (C) Quantification of valve invagination length in corneal lymphatic capillaries (n = 15, from three eyes [five valves per eye] of three animals for each population). Error bars represent SEM; ***P < 0.001. NS, not significant.
Expression of EphrinB2 and EphB4 in Alkali Burn Corneas
To address the role of ephrinB2–EphB4 signals in newly formed lymphatic vessels in adult mouse corneas, we examined expression of ephrinB2 and EphB4 in alkali burn corneas. Two days after the alkali treatment, both blood vessels and lymphatic vessels at the corneal limbus started to sprout toward the center of the cornea (Fig. 3A). The new blood vessels and lymphatic vessels continued to grow centripetally during the first week, then underwent morphogenetic remodeling during the second week (Fig. 3A). At the initial hemangiogenic and lymphangiogenic stages, ephrinB2 was expressed broadly in vascular endothelial cells (VECs) of arterial and venous vessels, but was almost undetectable in LECs (Fig. 3B). In contrast, EphB4 was expressed both in venous VECs and LECs (Fig. 3B). At the subsequent remodeling stages, ephrinB2 expression was periodically found in LECs (Fig. 3C), especially in enlarged lymphatic vessels, which seemed to form new valves (Fig. 3D). Likewise, the diffuse EphB4 expression in LECs became restricted to these immature valves (Figs. 3C, 3D). Thereafter, the invagination of new lymphatic valves further proceeded with high PECAM-1 and low LYVE-1 expression, thereby establishing the funnel-shaped structures (Fig. 3E). Thus, the expression of ephrinB2 and EphB4 dynamically changed in accordance with the formation of new lymphatic valves. 
Figure 3
 
Expression of ephrinB2 and EphB4 in alkali burn corneas. (A) New blood vessels and lymphatic vessels grow centripetally during the first week after the NaOH treatment, and undergo morphogenetic remodeling during the second week. (B) At the initial hemangiogenic and lymphangiogenic stages, ephrinB2 is expressed in arterial and venous VECs, but is barely detectable in LECs. EphB4 is expressed in LECs and venous VECs. (C) At the remodeling stages, ephrinB2 and EphB4 are periodically expressed both in VECs and LECs. (D) High-magnification images demonstrating intense expression of ephrinB2 and EphB4 with faint LYVE-1 expression in LECs of valve-forming areas. (E) Procedures of invagination of new lymphatic valves with high PECAM-1 and low LYVE-1 expression. Scale bars: 100 μm (AC); 10 μm (D, E).
Figure 3
 
Expression of ephrinB2 and EphB4 in alkali burn corneas. (A) New blood vessels and lymphatic vessels grow centripetally during the first week after the NaOH treatment, and undergo morphogenetic remodeling during the second week. (B) At the initial hemangiogenic and lymphangiogenic stages, ephrinB2 is expressed in arterial and venous VECs, but is barely detectable in LECs. EphB4 is expressed in LECs and venous VECs. (C) At the remodeling stages, ephrinB2 and EphB4 are periodically expressed both in VECs and LECs. (D) High-magnification images demonstrating intense expression of ephrinB2 and EphB4 with faint LYVE-1 expression in LECs of valve-forming areas. (E) Procedures of invagination of new lymphatic valves with high PECAM-1 and low LYVE-1 expression. Scale bars: 100 μm (AC); 10 μm (D, E).
EphrinB2–EphB4 Signals Facilitate Morphologic Maturation of New Valves in Corneal Lymphangiogenesis
To evaluate the roles of the ephrinB2–EphB4 signals in corneal lymphangiogenesis, we daily injected EphB4/Fc proteins into the subconjunctival spaces from day 7 after alkali treatment. At day 12 after alkali treatment, newly formed lymphatic vessels were enlarged with uneven protrusions in the EphB4/Fc-treated corneas (Fig. 4A). Given that mice lacking the ephrinB2 PDZ-binding motif displayed hyperplastic lymphatic capillaries with ectopic SMC association, 17 we suspected a possibility that the disorganized lymphatic network after EphB4/Fc injections was relevant to abnormal SMC coverage. However, lymphatic SMCs were completely absent in the EphB4/Fc-treated corneas, as in the control (Fig. 4A). Thus, the defects in lymphatic vessels after EphB4/Fc injections were likely to be attributable to lymphatic endothelial dysfunctions, especially the defective valve formation. Indeed, while new lymphatic valves in the control corneas were forming funnel-shaped structures, 75% of lymphatic valves in the EphB4/Fc-treated corneas appeared as vestigial ring shapes (Fig. 4B). Accordingly, the length of the valve invagination was significantly reduced in the EphB4/Fc-treated corneas (Fig. 4C). Because the defective valve formation results in retrograde fluid flow in collecting lymphatic vessels, 6 we further attempted to visualize the lymphatic flow by subconjunctival injections of high molecular weight FITC-dextran, which drains into lymphatic vessels but not blood vessels. 21,22 In the control corneas, the FITC-dextran was drained into the trunk lymphatic vessels surrounding the corneal limbus (Fig. 4D). In contrast, in the EphB4/Fc-treated corneas, peripheral lymphatic branches were filled with the FITC-dextran, indicating reflux of dye. Thus, the ephrinB2–EphB4 signals facilitated the maturation of funnel-shaped valves in newly formed lymphatic capillaries in alkali burn corneas. 
Figure 4
 
Defective valve formation in corneal lymphangiogenesis after EphB4/Fc injections. (A) Enlargement of lymphatic vessels with uneven protrusions after EphB4/Fc injections. Note the absence of SMC coverage in newly formed lymphatic vessels both of the control and EphB4/Fc–treated corneas. (B) Whole-mount immunostaining for integrin-α9. Lymphatic valves are forming funnel-shaped structures in control corneas and remain as vestigial ring shapes in EphB4/Fc–treated corneas. (C) Quantification of valve invagination length in corneal lymphatic vessels (n = 12, from three eyes [four valves per eye] of three animals for each population). Error bars represent SEM; **P < 0.01. (D) Subconjunctival injections of FITC-dextran. Note the reflux of dye (arrows) from the trunk lymphatic vessel to its peripheral branches in EphB4/Fc–treated corneas. Arrowheads indicate sites of FITC-dextran injection. Scale bars: 100 μm (A); 10 μm (B).
Figure 4
 
Defective valve formation in corneal lymphangiogenesis after EphB4/Fc injections. (A) Enlargement of lymphatic vessels with uneven protrusions after EphB4/Fc injections. Note the absence of SMC coverage in newly formed lymphatic vessels both of the control and EphB4/Fc–treated corneas. (B) Whole-mount immunostaining for integrin-α9. Lymphatic valves are forming funnel-shaped structures in control corneas and remain as vestigial ring shapes in EphB4/Fc–treated corneas. (C) Quantification of valve invagination length in corneal lymphatic vessels (n = 12, from three eyes [four valves per eye] of three animals for each population). Error bars represent SEM; **P < 0.01. (D) Subconjunctival injections of FITC-dextran. Note the reflux of dye (arrows) from the trunk lymphatic vessel to its peripheral branches in EphB4/Fc–treated corneas. Arrowheads indicate sites of FITC-dextran injection. Scale bars: 100 μm (A); 10 μm (B).
Discussion
In lymphatic capillaries of adult mouse corneas, we showed that ephrinB2–EphB4 signals play a pivotal role both in the maintenance of preexisting valves and in the formation of new valves. In many organs, lymphatic capillaries usually lack luminal valves, in which button-like junctions of monolayer LECs maintain unidirectional fluid entry from the interstitium into the lymphatics. 23 Although corneal LECs display anatomic and immunochemical features characteristic of capillary LECs such as oak leaf shapes and the absence of SMC coverage (Supplementary Data S1C, S1D), integrin-α9–positive and LYVE-1–negative microvalves were recently identified in corneal lymphatic capillaries, preferentially near the branching points. 7,8 Consistent with these observations, we demonstrated the funnel-shaped structure of corneal lymphatic valves, which is morphologically distinct from the bicuspid valves in collecting lymphatic vessels. 6 Nonetheless, in mouse corneas, it should be noted that the characters of lymphatic vessels as well as their valves are subject to change depending on the genetic backgrounds. 8,24,25 Therefore, further investigations will be required to ascertain whether the experimental results obtained from the present study using C57BL/6 mice can also be applied to various mouse strains. 
Although we showed that LECs of funnel-shaped valves intensively express EphB4 in corneal lymphatic capillaries, ephrinB2 is highly expressed in bicuspid valves of collecting lymphatic vessels. 17 This discrepancy implies differential roles of the ephrinB2–EphB4 signals in collecting lymphatic vessels and in corneal lymphatic capillaries. By exploiting mutant mice lacking the ephrinB2 PDZ-binding motif, it was shown that the reverse ephrinB2 signal is a prerequisite for valve formation in collecting lymphatic vessels. 17 Furthermore, inducible deletion of the Efnb2 gene has led to deformation of preexisting valves in collecting lymphatic vessels of adult ear skins. 18 In the present study, we showed that Efnb2 haploinsufficiency resulted in a decreased number of corneal lymphatic valves, which may partly be attributable to reduction of the reverse ephrinB2 signal. However, based on the intense EphB4 expression in valvular LECs, it seems likely that deficits in the forward EphB4 signal would also be responsible for the defective valves in corneal lymphatic capillaries. This possibility is consistent with the deformation of preexisting valves after subconjunctival injections of dimeric EphB4/Fc proteins, which block forward EphB4 signaling. 19,20 Nonetheless, it should also be noted that EphB4/Fc stimulates the reverse ephrinB2 signaling, which facilitates internalization and subsequent activation of vascular endothelial growth factor receptor 3 (VEGFR3) in LECs. 12 Although our study revealed the importance of the ephrinB2–EphB4 signals in corneal lymphatic valves, a full understanding of the distinct roles of the forward and reverse signals requires further elucidation, possibly utilizing monomeric EphB4 proteins that block both the forward and reverse signals. 20,26  
Based on their morphologic structure, the funnel-shaped valves may contribute to maintaining unidirectional fluid flow in corneal lymphatic capillaries, which is equivalent to the bicuspid valves in collecting lymphatic vessels. This notion is well corroborated with the frequent localization of EphB4-positive valves adjacent to the lymphatic–arterial crossing points, which may prevent backflow caused by arterial pulsate. Thus, retrograde lymphatic flow is an expectable outcome accompanying defective valve formation after EphB4/Fc injections. Indeed, we observed reflux of subconjunctivally injected FITC-dextran into the peripheral lymphatic branches in EphB4/Fc-treated corneas. In future studies, lymphatic dysfunctions caused by EphB4/Fc injections will further be clarified by alternative experimental approaches, such as by corneal transplantation. 
After corneal transplant surgery, the afferent lymphatic flow transports antigens and antigen-presenting cells to the draining lymph node, thereby inducing an adaptive immune response. 1,2 Indeed, surgical lymphadenectomy significantly improved the success rate of graft survival by suppressing immune rejection. 27 To date, various pharmacologic modalities targeting VEGF-A, VEGFR3, very late antigen 1, and integrin-α5, have been shown to suppress immune rejections after corneal transplantation by inhibiting lymphangiogenesis, dendritic cell trafficking, and leukocyte infiltration. 2831 Notably, it was recently shown that the LYVE-1–negative corneal LECs could serve not only as microvalves but also as immunologic hot spots that facilitate reentry of stromal macrophages. 8 Considering the involvement of the ephrinB2–EphB4 signals in leukocyte–endothelial interactions in blood vessels, 32 it is potentially expected that disruption of the ephrinB2–EphB4 signals would contribute to perturb the afferent arm of the immune reflex arc through affecting lymphatic valve formation as well as leukocyte–lymphatic interactions. Thus, it would be of possible clinical importance to evaluate the efficacy of pharmacologic reagents targeting the ephrinB2–EphB4 signals in posttransplantation management. 
Supplementary Materials
Acknowledgments
The authors thank David Anderson for providing Efnb2-lacZ and Ephb4-lacZ mice. 
Supported by Japan Society for the Promotion of Science Grant 22689046; the Global Center of Excellence from the Ministry of Education, Culture, Sports, Science and Technology (Japan); the Inamori Foundation (AU); the Takeda Science Foundation; and the Santen Foundation. 
Disclosure: H. Katsuta, None; Y. Fukushima, None; K. Maruyama, None; M. Hirashima, None; K. Nishida, None; S.-I. Nishikawa, None; A. Uemura, None 
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Footnotes
 HK and YF contributed equally to the work presented here and should therefore be regarded as equivalent authors.
Figure 1
 
Expression of ephrinB2 and EphB4 in corneas of adult Efnb2-lacZ or Ephb4-lacZ mouse. (A) EphrinB2 is expressed in PECAM-1(+) LYVE-1(−) arterial blood vessels (arrowheads), and in PECAM-1(+) LYVE-1(+) lymphatic capillaries (arrows). (B) EphB4 is periodically expressed in lymphatic capillaries near the branching points (arrow) and the crossing points with arterial vessels (arrowheads). (C) Intense EphB4 expression is restricted to the invaginating LECs of funnel-shaped valves. (D) EphrinB2 is diffusely expressed in outer LECs surrounding the valves. (E) EphB4-expressing valvular LECs are segregated from ephrinB2-expressing LECs. (F) A scheme representing the expression of ephrinB2 and EphB4 in corneal lymphatic valves. Scale bars: 100 μm (A, B); 10 μm (CE).
Figure 1
 
Expression of ephrinB2 and EphB4 in corneas of adult Efnb2-lacZ or Ephb4-lacZ mouse. (A) EphrinB2 is expressed in PECAM-1(+) LYVE-1(−) arterial blood vessels (arrowheads), and in PECAM-1(+) LYVE-1(+) lymphatic capillaries (arrows). (B) EphB4 is periodically expressed in lymphatic capillaries near the branching points (arrow) and the crossing points with arterial vessels (arrowheads). (C) Intense EphB4 expression is restricted to the invaginating LECs of funnel-shaped valves. (D) EphrinB2 is diffusely expressed in outer LECs surrounding the valves. (E) EphB4-expressing valvular LECs are segregated from ephrinB2-expressing LECs. (F) A scheme representing the expression of ephrinB2 and EphB4 in corneal lymphatic valves. Scale bars: 100 μm (A, B); 10 μm (CE).
Figure 2
 
Defects in lymphatic valves by manipulating the ephrinB2–EphB4 signals in adult mouse corneas. (A) Quantification of lymphatic valve number in adult mouse corneas (n = 3, from three animals for each population). (B) Whole-mount integrin-α9 immunostaining at 3 days after daily subconjunctival injections of control Fc or EphB4/Fc proteins. Arrows indicate the invagination lengths at the center of the valves. Scale bar: 10 μm. (C) Quantification of valve invagination length in corneal lymphatic capillaries (n = 15, from three eyes [five valves per eye] of three animals for each population). Error bars represent SEM; ***P < 0.001. NS, not significant.
Figure 2
 
Defects in lymphatic valves by manipulating the ephrinB2–EphB4 signals in adult mouse corneas. (A) Quantification of lymphatic valve number in adult mouse corneas (n = 3, from three animals for each population). (B) Whole-mount integrin-α9 immunostaining at 3 days after daily subconjunctival injections of control Fc or EphB4/Fc proteins. Arrows indicate the invagination lengths at the center of the valves. Scale bar: 10 μm. (C) Quantification of valve invagination length in corneal lymphatic capillaries (n = 15, from three eyes [five valves per eye] of three animals for each population). Error bars represent SEM; ***P < 0.001. NS, not significant.
Figure 3
 
Expression of ephrinB2 and EphB4 in alkali burn corneas. (A) New blood vessels and lymphatic vessels grow centripetally during the first week after the NaOH treatment, and undergo morphogenetic remodeling during the second week. (B) At the initial hemangiogenic and lymphangiogenic stages, ephrinB2 is expressed in arterial and venous VECs, but is barely detectable in LECs. EphB4 is expressed in LECs and venous VECs. (C) At the remodeling stages, ephrinB2 and EphB4 are periodically expressed both in VECs and LECs. (D) High-magnification images demonstrating intense expression of ephrinB2 and EphB4 with faint LYVE-1 expression in LECs of valve-forming areas. (E) Procedures of invagination of new lymphatic valves with high PECAM-1 and low LYVE-1 expression. Scale bars: 100 μm (AC); 10 μm (D, E).
Figure 3
 
Expression of ephrinB2 and EphB4 in alkali burn corneas. (A) New blood vessels and lymphatic vessels grow centripetally during the first week after the NaOH treatment, and undergo morphogenetic remodeling during the second week. (B) At the initial hemangiogenic and lymphangiogenic stages, ephrinB2 is expressed in arterial and venous VECs, but is barely detectable in LECs. EphB4 is expressed in LECs and venous VECs. (C) At the remodeling stages, ephrinB2 and EphB4 are periodically expressed both in VECs and LECs. (D) High-magnification images demonstrating intense expression of ephrinB2 and EphB4 with faint LYVE-1 expression in LECs of valve-forming areas. (E) Procedures of invagination of new lymphatic valves with high PECAM-1 and low LYVE-1 expression. Scale bars: 100 μm (AC); 10 μm (D, E).
Figure 4
 
Defective valve formation in corneal lymphangiogenesis after EphB4/Fc injections. (A) Enlargement of lymphatic vessels with uneven protrusions after EphB4/Fc injections. Note the absence of SMC coverage in newly formed lymphatic vessels both of the control and EphB4/Fc–treated corneas. (B) Whole-mount immunostaining for integrin-α9. Lymphatic valves are forming funnel-shaped structures in control corneas and remain as vestigial ring shapes in EphB4/Fc–treated corneas. (C) Quantification of valve invagination length in corneal lymphatic vessels (n = 12, from three eyes [four valves per eye] of three animals for each population). Error bars represent SEM; **P < 0.01. (D) Subconjunctival injections of FITC-dextran. Note the reflux of dye (arrows) from the trunk lymphatic vessel to its peripheral branches in EphB4/Fc–treated corneas. Arrowheads indicate sites of FITC-dextran injection. Scale bars: 100 μm (A); 10 μm (B).
Figure 4
 
Defective valve formation in corneal lymphangiogenesis after EphB4/Fc injections. (A) Enlargement of lymphatic vessels with uneven protrusions after EphB4/Fc injections. Note the absence of SMC coverage in newly formed lymphatic vessels both of the control and EphB4/Fc–treated corneas. (B) Whole-mount immunostaining for integrin-α9. Lymphatic valves are forming funnel-shaped structures in control corneas and remain as vestigial ring shapes in EphB4/Fc–treated corneas. (C) Quantification of valve invagination length in corneal lymphatic vessels (n = 12, from three eyes [four valves per eye] of three animals for each population). Error bars represent SEM; **P < 0.01. (D) Subconjunctival injections of FITC-dextran. Note the reflux of dye (arrows) from the trunk lymphatic vessel to its peripheral branches in EphB4/Fc–treated corneas. Arrowheads indicate sites of FITC-dextran injection. Scale bars: 100 μm (A); 10 μm (B).
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