3D bioprinting of DPSCs with GelMA hydrogel of various concentrations for bone regeneration.

Bioprinting technology promotes innovation of fabricating tissue engineered constructs. Dental pulp stem cells (DPSCs) have significant advantages over classical bone mesenchymal stem cells (BMSCs) and are a promising seed cell candidate for bone engineering bioprinting. However, current reports about bioprinted DPSCs for bone regeneration are incomprehensive. The objective of this study was to investigate the osteogenic potential of DPSCs in methacrylate gelatin (GelMA) hydrogels bioprinted scaffolds in vitro and in vivo. Firstly, we successfully bioprinted GelMA with different concentrations embedded with or without DPSCs. Printability, physical features and biological properties of the bioprinted constructs were evaluated. Then, osteogenic differentiation levels of DPSCs in bioprinted constructs with various concentrated GelMA were compared. Finally, effects of bioprinted constructs on cranial bone regeneration were evaluated in vivo. The results of our study demonstrated that 10% GelMA had higher compression modulus, smaller pores, lower swelling and degradation rate than 3% GelMA. Twenty-eight days after printing, DPSCs in three groups of bioprinted structures still maintained high cell activities (>90%). Moreover, DPSCs in 10% GelMA showed an upregulated expression of osteogenic markers and a highly activated ephrinB2/EphB4 signaling, a signaling involved in bone homeostasis. In vivo experiments showed that DPSCs survived at a higher rate in 10% GelMA, and more new bones were observed in DPSC-laden 10% GelMA group, compared with GelMA of other concentrations. In conclusion, bioprinted DPSC-laden 10% GelMA might be more appropriate for bone regeneration application, in contrast to GelMA with other concentrations.

ED-71 Improves Bone Mass in Ovariectomized Rats by Inhibiting Osteoclastogenesis Through EphrinB2-EphB4-RANKL/OPG Axis.

Purpose: Estrogen deficiency is the main reason of postmenopausal osteoporosis. Eldecalcitol (ED-71) is a new active vitamin D analogue clinically used in the treatment of postmenopausal osteoporosis. We aimed to investigate whether EphrinB2-EphB4 and RANKL/RANK/OPG signaling cooperate in mediating the process of osteoporosis by ED-71. Methods: In vivo, the ovariectomized (OVX) rats were administered orally with 30 ng/kg ED-71 once a day for 8 weeks. HE staining, Masson staining and Immunofluorescence staining were used to evaluate bone mass, bone formation, osteoclastogenesis associated factors and the expression of EphrinB2, EphB4, RANKL and OPG. In vitro, H2O2 stimulation was used to simulate the cell environment in osteoporosis. Immunofluorescence, quantitative real time PCR (qRT-PCR), enzyme-linked immunosorbent assay (ELISA) and Western Blot were applied to detect the expression of EphrinB2, EphB4, RANKL and OPG. In osteoblasts, EphB4 was knocked down by EphB4 small-interfering RNA (siRNA) transfection. LY294002 (PI3K inhibitor) or ARQ092 (AKT inhibitor) was used to block PI3K/AKT pathway. An indirect co-culture system of osteoblasts and osteoclasts was established. The mRNA and protein expression of osteoclastogenes is associated factors were tested by qRT-PCR and Western Blot. Results: ED-71 increased bone mass and decreased the number of osteoclasts in OVX rats. Moreover, ED-71 promoted the expression of EphrinB2, EphB4, and decreased the RANKL/OPG ratio in osteoblasts. Osteoclastogenesis was restrained when osteoclasts were indirectly co-cultured with ED-71-treated osteoblasts. After silencing of EphB4 expression in osteoblasts, ED-71 inhibited the expression of P-PI3K and P-AKT and increased the ratio of RANKL/OPG. This reversed the inhibitory effect of ED-71 on osteoclastogenes. Therefore, in ED-71-inhibited osteoclastogenes, EphB4 is a key factor affecting the secretion of RANKL and OPG by osteoblasts. EphB4 suppressed the RANKL/OPG ratio through activating PI3K/AKT signaling in osteoblasts. Conclusion: ED-71 inhibits osteoclastogenesis through EphrinB2-EphB4-RANKL/OPG axis, improving bone mass in ovariectomized rats. PI3K/AKT pathway is involved this process.

Imbalanced EphB4/EphrinB2 Signaling Modulates Bone Resorption in Periodontitis Induced by Porphyromonas gingivalis.

Periodontitis, a chronic infectious disease in periodontal tissues, is characterized by an imbalance of alveolar bone resorption and remodeling, which eventually results in tooth loosening and even tooth loss. The etiology of periodontitis is polymicrobial synergy and dysbiosis, in which Porphyromonas gingivalis (P. gingivalis) is one of the primary pathogens responsible for periodontitis progression. The interplay of EphrinB2/EphB4 is crucial for osteoblast-osteoclast communication during bone remodeling and healing. This study investigates the mechanism of EphB4/EphrinB2 transduction modulating osteogenesis inhibition and bone resorption in periodontitis induced by P. gingivalis. An in vivo model of chronic periodontitis provoked by P. gingivalis was constructed, the inflammation and bone resorption were evaluated. The expression of EphB4 and EphrinB2 proteins in periodontal tissues was detected, which was also evaluated, respectively, in osteoblasts and osteoclasts infected with P. gingivalis in vitro. Then, a simulated coculture model of osteoblasts and osteoclasts was established to activate the forward and reverse pathways of EphB4/EphrinB2 with P. gingivalis infection. This study showed that P. gingivalis infection promoted alveolar bone resorption in rats and enhanced EphB4 and EphrinB2 expression in periodontal tissues. EphB4 and molecules associated with osteogenesis in osteoblasts infected with P. gingivalis were inhibited, while EphrinB2 and osteoclast differentiation-related markers in osteoclasts were activated. In conclusion, this study suggested that EphB4/EphrinB2 proteins were involved in alveolar bone remodeling in the process of periodontitis induced by P. gingivalis infection. Moreover, attenuated EphB4/EphrinB2 with P. gingivalis infection weakened osteoblast activity and enhanced osteoclast activity.

Low-intensity pulsed ultrasound regulates osteoblast-osteoclast crosstalk via EphrinB2/EphB4 signaling for orthodontic alveolar bone remodeling.

Background: The limited regenerative potential of periodontal tissue remains a challenge in orthodontic treatment, especially with respect to alveolar bone remodeling. The dynamic balance between the bone formation of osteoblasts and the bone resorption of osteoclasts controls bone homeostasis. The osteogenic effect of low-intensity pulsed ultrasound (LIPUS) is widely accepted, so LIPUS is expected to be a promising method for alveolar bone regeneration. Osteogenesis is regulated by the acoustic mechanical effect of LIPUS, while the cellular perception, transduction mode and response regulation mechanism of LIPUS stimuli are still unclear. This study aimed to explore the effects of LIPUS on osteogenesis by osteoblast-osteoclast crosstalk and the underlying regulation mechanism. Methods: The effects of LIPUS on orthodontic tooth movement (OTM) and alveolar bone remodeling were investigated via rat model by histomorphological analysis. Mouse bone marrow mesenchymal stem cells (BMSCs) and bone marrow monocytes (BMMs) were purified and used as BMSC-derived osteoblasts and BMM-derived osteoclasts, respectively. The osteoblast-osteoclast co-culture system was used to evaluate the effect of LIPUS on cell differentiation and intercellular crosstalk by Alkaline phosphatase (ALP), Alizarin Red S (ARS), tartrate-resistant acid phosphatase (TRAP) staining, real-time quantitative PCR, western blotting and immunofluorescence. Results: LIPUS was found to improve OTM and alveolar bone remodeling in vivo, promote differentiation and EphB4 expression in BMSC-derived osteoblasts in vitro, particularly when cells were directly co-cultured with BMM-derived osteoclasts. LIPUS enhanced EphrinB2/EphB4 interaction between osteoblasts and osteoclasts in alveolar bone, activated the EphB4 receptor on osteoblasts membrane, transduced LIPUS-related mechanical signals to the intracellular cytoskeleton, and gave rise to the nuclear translocation of YAP in Hippo signaling pathway, thus regulating cell migration and osteogenic differentiation. Conclusions: This study shows that LIPUS modulates bone homeostasis by osteoblast-osteoclast crosstalk via EphrinB2/EphB4 signaling, which benefits the balance between OTM and alveolar bone remodeling.

ED-71 inhibited osteoclastogenesis by enhancing EphrinB2-EphB4 signaling between osteoclasts and osteoblasts in osteoporosis.

BACKGROUND: Osteoporosis is a degenerative skeletal disease essentially caused by bone remodeling disorder. EphrinB2-EphB4 signaling play critical regulatory roles in bone remodeling via communication between osteoclasts and osteoblasts. Eldecalcitol (ED-71), a new vitamin D analog, is a high-potential drug for treating osteoporosis; however, its mechanism has yet to be determined. This study aims to investigate whether EphrinB2-EphB4 signal mediates the process of osteoporosis improved by ED-71. MATERIALS AND METHODS: An ovariectomized (OVX) rat model was constructed in vivo. ED-71 at 30 ng/kg was orally administered once daily for 8 weeks. Osteoclast activity and EphrinB2-EphB4 expression were evaluated by hematoxylin and eosin staining, tartrate-resistant acid phosphatase (TRAP) staining, and immunohistochemical staining. The mRNA levels of oxidation stress factors in the bone tissue were tested by reverse transcription polymerase chain reaction (RT-PCR). An H2O2-stimulated model in vitro was established to simulate the status of osteoporosis. Osteoclastogenesis and associated protein were detected by TRAP staining, F-actin ring formation assay, PCR, and Western blot analysis. EprhrinB2 and EphB4 levels were determined by immunofluorescence, PCR, and Western blot analysis. EprhrinB2 small-interfering RNA knocked down the EprhrinB2 in osteoclasts, and an EphB4 antibody blocked EphB4 in osteoblasts. RESULTS: ED-71 prevented bone loss and decreased the number of osteoclasts in vivo relative to the OVX group. In addition, the bone tissue of OVX rat displayed as an increased level of oxidation stress, which could be inhibited by ED-71. In vitro, in the simulation of osteoporosis with H2O2, ED-71 reversed the increase H2O2-induced oxidative stress. ED-71 then inhibited osteoclastogenesis and osteoclast function, accompanied by increased EphrinB2 expression in osteoclasts. Notably, EphrinB2 knockdown reversed the inhibitory effect of ED-71 on osteoclasts. ED-71 also enhanced EphB4 expression in osteoblasts in vivo and in vitro. Further research showed that ED-71 inhibited osteoclastogenesis in co-culture systems, which was weakened by blocking EphB4 in osteoblasts. CONCLUSIONS: ED-71 inhibited osteoclastogenesis by enhancing EphrinB2-EphB4 signaling between osteoclasts and osteoblasts, preventing osteoporosis. This theory explains the role of ED-71 in the treatment of osteoporosis.

The role of mural cells in hemorrhage of brain arteriovenous malformation.

Brain arteriovenous malformation (bAVM) is the most common cause of intracranial hemorrhage (ICH), particularly in young patients. However, the exact cause of bAVM bleeding and rupture is not yet fully understood. In bAVMs, blood bypasses the entire capillary bed and directly flows from arteries to veins. The vessel walls in bAVMs have structural defects, which impair vascular integrity. Mural cells are essential structural and functional components of blood vessels and play a critical role in maintaining vascular integrity. Changes in mural cell number and coverage have been implicated in bAVMs. In this review, we discussed the roles of mural cells in bAVM pathogenesis. We focused on 1) the recent advances in human and animal studies of bAVMs; 2) the importance of mural cells in vascular integrity; 3) the regulatory signaling pathways that regulate mural cell function. More specifically, the platelet-derived growth factor-B (PDGF-B)/PDGF receptor-ß (PDGFR-ß), EphrinB2/EphB4, and angiopoietins/tie2 signaling pathways that regulate mural cell-recruitment during vascular remodeling were discussed in detail.

The critical role of the interplays of EphrinB2/EphB4 and VEGF in the induction of angiogenesis.

The significant role of VEGF (vascular endothelial growth factor) as an angiogenesis inducer is well recognized. Besides VEGF, EphrinB2/EphB4 also plays essential roles in vascular development and postnatal angiogenesis. Compared with classical proangiogenic factors, not only does EphrinB2/EphB4 promote sprouting of new vessels, it is also involved in the vessel maturation. Given their involvement in many physiologic and pathological conditions, EphB4 and EphrinB2 are increasingly recognized as attractive therapeutic targets for angiogenesis-related diseases through modulating their expression and function. Previous works mainly focused on the individual role of VEGF and EphrinB2/EphB4 in angiogenesis, respectively, but the correlation between EphrinB2/EphB4 and VEGF in angiogenesis has not been fully disclosed. Here, we summarize the structure and bidirectional signaling of EphrinB2/EphB4, provide an overview on the relationship between EphrinB2/EphB4 signaling and VEGF pathway in angiogenesis and highlight the associated potential usefulness in anti-angiogenetic therapy.

Study of angiogenic signaling pathways in hemangioblastoma.

Hemangioblastoma (HB) is mainly located in the brain and the spinal cord. The tumor is composed of two major components, namely neoplastic stromal cells and abundant microvessels. Thus, hyper-vascularization is the hallmark of this tumor. Despite the identification of germline and/or epigenetic mutations of Von Hippel Lindau (VHL) gene as an important pathogenic mechanism of HB, little is known about the molecular signaling involved in this highly vascularized tumor. The present study investigated the key players of multiple angiogenic signaling pathways including VEGF/VEGFR2, EphB4/EphrinB2, SDF1α/CXCR4 and Notch/Dll4 pathways in surgical specimens of 22 HB. The expression of key angiogenic factors was detected by RT2 -PCR and Western blot. Immunofluorescent staining revealed the cellular localization of these proteins. We demonstrated a massive upregulation of mRNA levels of VEGF and VEGFR2, CXCR4 and SDF1α, EphB4 and EphrinB2, as well as the main components of Dll4-Notch signaling in HB. An increase in the protein expression of VEGF, CXCR4 and the core-components of Dll4-Notch signaling was associated with an activation of Akt and Erk1/2 and accompanied by an elevated expression of PCNA. Immuofluorescent staining revealed the expression of VEGF and CXCR4 in endothelial cells as well as in tumor cells. Dll4 protein was predominantly found in tumor cells, whereas EphB4 immunoreactivity was exclusively detected in endothelial cells. We conclude that multiple key angiogenic pathways were activated in HB, which may synergistically contribute to the abundant vascularization in this tumor. Identification of these aberrant pathways provides potential targets for a possible future application of anti-angiogenic therapy for this tumor, particularly when a total surgical resection becomes difficult due to the localization or multiplicity of the tumor.

Transgenic expression of ephrinB2 in periodontal ligament stem cells (PDLSCs) modulates osteogenic differentiation via signaling crosstalk between ephrinB2 and EphB4 in PDLSCs and between PDLSCs and pre-osteoblasts within co-culture.

BACKGROUND AND OBJECTIVE: The goal of periodontal therapy is to regenerate/reconstruct the damaged supporting tissues of diseased teeth and to facilitate recovery of their physiological functions. Combination of stem cell transplantation and gene therapy offers a viable method for accelerating periodontal repair and regeneration. In this study, the role of the ephrinB2/EphB4 signaling pathway in regulating osteogenic differentiation of periodontal ligament stem cells (PDLSCs) and crosstalk between PDLSCs and pre-osteoblasts within co-culture was investigated through ephrinB2 transgenic expression in PDLSCs. MATERIAL AND METHODS: PDLSCs isolated from premolar teeth of teenage patients undergoing orthodontic treatment were transfected with transgenic (hEfnB2-GFP-Bsd) vector or empty vector (GFP-Bsd). Vector-PDLSCs, EfnB2-PDLSCs, MC3T3-E1 and co-cultures of vector-PDLSCs with MC3T3-E1, and EfnB2-PDLSCs with MC3T3-E1 were subjected to osteogenic induction. The osteogenic differentiation of EfnB2-PDLSCs, vector-PDLSCs and co-cultures were assessed by reverse transcription-polymerase chain reaction, alkaline phosphatase (ALP) assay and Alizarin-red S staining. Protein expression levels of ephrinB2, EphB4, phosphorylated ephrinB2 and EphB4 were analyzed by western blot, immunoprecipitation and co-immunoprecipitation assays. RESULTS: ALP assay and Alizarin-red S staining demonstrated higher ALP activity and increased mineralization with EfnB2-PDLSCs vs. vector-PDLSCs and with co-culture of EfnB2-PDLSCs and MC3T3-E1 vs. vector-PDLSCs and MC3T3-E1. Reverse transcription-polymerase chain reaction revealed that the expression of human odonto/osteogenic markers were significantly enhanced in EfnB2-PDLSCs compared to vector-PDLSCs, and that the expression of mouse odonto/osteogenic markers were significantly higher in co-culture of EfnB2-PDLSCs with MC3T3-E1 vs. vector-PDLSCs with MC3T3-E1. The EphB4 receptor was activated through phosphorylation during osteogenic differentiation. CONCLUSION: Our data indicate that transgenic expression of ephrinB2 in PDLSCs could promote osteogenic differentiation via stimulation of the phosphorylation of ephrinB2 and EphB4, which regulates cell communication between PDLSCs and between PDLSCs and pre-osteoblasts within co-culture.

Positively charged polymers modulate the fate of human mesenchymal stromal cells via ephrinB2/EphB4 signaling.

Understanding the mechanisms by which mesenchymal stromal cells (MSCs) interact with the physical properties (e.g. topography, charge, ζ-potential, and contact angle) of polymeric surfaces is essential to design new biomaterials capable of regulating stem cell behavior. The present study investigated the ability of two polymers (pHM1 and pHM3) with different positive surface charge densities to modulate the differentiation of MSCs into osteoblast-like phenotype via cell-cell ephrinB2/EphB4 signaling. Although pHM1 promoted the phosphorylation of EphB4, leading to cell differentiation, pHM3, characterized by a high positive surface charge density, had no significant effect on EphB4 activation or MSCs differentiation. When the MSCs were cultured on pHM1 in the presence of a forward signaling blocking peptide, the osteoblast differentiation was compromised. Our results demonstrated that the ephrinB2/EphB4 interaction was required for MSCs differentiation into an osteoblast-like phenotype and that the presence of a high positive surface charge density altered this interaction.

Histochemical examination of the effects of high-dose 1,25(OH)2D3 on bone remodeling in young growing rats.

Vitamin D has an anabolic effect on bone developmental processes and is involved in maintaining skeletal integrity. In recent years, pediatric cases of vitamin D intoxication have attracted attention. Therefore, the aim of this study was to investigate the influence of long-term administration of physiologically-high-dose calcitriol (1,25(OH)2D3) on bone remodeling in young developing rats. Neonatal rats received once-daily subcutaneous injection of calcitriol (250 ng/kg body weight), or PBS only as a control, for 3 weeks. At 1, 2 and 4 weeks' post-administration, rats were sacrificed and fixed by transcardial perfusion with 4 % paraformaldehyde, following which tibiae were extracted for histochemical analysis. Compared with the control group, the number of tartrate-resistant acid phosphatase- and Cathepsin K-positive osteoclasts were significantly increased, and the expression of alkaline phosphatase in osteoblasts was decreased in trabecular bone of rats administered high-dose 1,25(OH)2D3, leading to decreased trabecular bone volume. In addition, the expression of receptor activator of nuclear factor kappa-B ligand (RANKL) was increased, while that of osteoprotegerin was weaker in osteoblasts in the experimental group compared with the control group. Moreover, there was weaker immunoreactivity for EphrinB2 in osteoclasts and EphB4 in osteoblasts of trabecular bone in the experimental group compared with the control group. These findings suggest that long-term use of physiologically-high dose calcitriol may result in bone loss through RANKL/RANK/osteoprotegerin and EphrinB2-EphB4 signaling pathways, and that these negative effects could continue after drug withdrawal. Therefore, optimal limits for vitamin D administration need to be established for children and adolescents.

Effect of EphB4/EphrinB2 reverse signal on angiogenesis induced by Xuefu Zhuyu Capsule () containing serum in human microvascular endothelial cell 1.

OBJECTIVE: To evaluate the effect of Xuefu Zhuyu Capsule ()-containing serum (XFZY-CS) on EphB4/ephrinB2 and its reverse signal in human microvascular endothelial cell-1 (HMEC-1). METHODS: XFZY-CS and the blank control serum were collected. HMEC-1 cells were randomly assigned to 6 groups including the concentration 1.25%, 2.5%, and 5% XFZY-CS groups and their blank serum control ones. The angiogenesis effect of XFZY-CS was tested with an in vitro tube formation assay and the best condition of pro-angiogenesis was determined. The effect of XFZY-CS on EphB4/ephrinB2 and the reverse signal were determined by Western blot and real-time quantitative polymerase chain reaction, respectively; we also confifirmed the results through activating and inhibiting the reverse signal by EphB4/fc and pyrophosphatase/ phosphodiesterase2 (PP2). RESULTS: XFZY-CS promoted angiogenesis at the concentration of 2.5% corresponding serum after being cultured for 48 h, while inhibited angiogenesis at the concentration of 5% after culturing for 48 and 72 h. Under the 2.5% serum concentration, XFZY up-regulated the expression of EphB4-mRNA at 12 h (P<0.05), and down-regulates its expression at 24 h (P<0.01). Protein expression of EphB4 was apparently up-regulated at 12 h and down-regulated at 24 h. The phosphorylation of ephrinB2 increased at 9 h (P<0.05). In addition, 2.5% XFZY-CS played a similar role as the reverse signaling activator EphB4/Fc ranging from 0.5 to 5 µg/mL (P>0.05). XFZY-CS also reduced the inhibitive effect of PP2 in limited periods. CONCLUSIONS: EphB4/ephrinB2 was the upstream signal in the process of angiogenesis and its reverse signaling was responsible for XFZY's effect on promoting angiogenesis.

EphrinB2/EphB4 pathway in postnatal angiogenesis: a potential therapeutic target for ischemic cardiovascular disease.

Ischemic cardiovascular disease remains one of the leading causes of morbidity and mortality in the world. Proangiogenic therapy appears to be a promising and feasible strategy for the patients with ischemic cardiovascular disease, but the results of preclinical and clinical trials are limited due to the complicated mechanisms of angiogenesis. Facilitating the formation of functional vessels is important in rescuing the ischemic cardiomyocytes. EphrinB2/EphB4, a novel pathway in angiogenesis, plays a critical role in both microvascular growth and neovascular maturation. Hence, investigating the mechanisms of EphrinB2/EphB4 pathway in angiogenesis may contribute to the development of novel therapeutics for ischemic cardiovascular disease. Previous reviews mainly focused on the role of EphrinB2/EphB4 pathway in embryo vascular development, but their role in postnatal angiogenesis in ischemic heart disease has not been fully illustrated. Here, we summarized the current knowledge of EphrinB2/EphB4 in angiogenesis and their interaction with other angiogenic pathways in ischemic cardiovascular disease.

Activation of multiple angiogenic signaling pathways in hemangiopericytoma.

Hemangiopericytoma (HPC) is a highly vascularized mesenchymal tumor. Local recurrence and distant metastasis are common features of HPC. Considering the remarkable hyper-vasculature phenotype of HPC, we assumed that dysregulated angiogenic signaling pathways were involved in HPC. The key components of angiogenic signaling pathways including VEGF-VEGF-R2, EphrinB2-EphB4 and DLL4-Notch were examined by real-time RT-PCR, Western blotting and immunostaining in 17 surgical specimens of HPC patients and in 6 controls. A significant upregulation of VEGF and VEGF-R2 associated with elevated levels of p-Akt and proliferating cell nuclear antigen (PCNA) was detected in HPC. Moreover, a dramatic increase in the mRNA and protein expression of EphB4 and its downstream factor p-Erk1/2 was found in HPC. A massive activation of core-components of DLL4-Notch signaling was detected in HPC. Double-immunofluorescent staining confirmed the expression of these upregulated key factors in the endothelial cells of tumor vessels. The present study identified the activation of multiple and crucial angiogenic signaling pathways, which could function individually and/or synergistically to stimulate angiogenesis in HPC and eventually contribute to tumor growth and progression. Our findings emphasize the importance to target multiple angiogenic signaling pathways when an anti-angiogenic therapy is considered for this highly vascularized tumor.

EphrinB2-EphB4 signals regulate formation and maintenance of funnel-shaped valves in corneal lymphatic capillaries.

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.