EphB4 Signaling Research Articles

Time-Course of Transcriptomic Change in the Lungs of F344 Rats Repeatedly Exposed to a Multiwalled Carbon Nanotube in a 2-Year Test.

Despite intensive toxicological studies of carbon nanotubes (CNTs) over the last two decades, only a few studies have demonstrated their pulmonary carcinogenicities in chronic animal experiments, and the underlying molecular mechanisms are still unclear. To obtain molecular insights into CNT-induced lung carcinogenicity, we performed a transcriptomic analysis using a set of lung tissues collected from rats in a 2-year study, in which lung tumors were induced by repeated intratracheal instillations of a multiwalled carbon nanotube, MWNT-7. The RNA-seq-based transcriptome identified a large number of significantly differentially expressed genes at Year 0.5, Year 1, and Year 2. Ingenuity Pathway Analysis revealed that macrophage-elicited signaling pathways such as phagocytosis, acute phase response, and Toll-like receptor signaling were activated throughout the experimental period. At Year 2, cancer-related pathways including ERBB signaling and some axonal guidance signaling pathways such as EphB4 signaling were perturbed. qRT-PCR and immunohistochemistry indicated that several key molecules such as Osteopontin/Spp1, Hmox1, Mmp12, and ERBB2 were markedly altered and/or localized in the preneoplastic lesions, suggesting their participation in the induction of lung cancer. Our findings support a scenario of inflammation-induced carcinogenesis and contribute to a better understanding of the molecular mechanism of MWCNT carcinogenicity.

Divergent Roles of Ephrin-B2/EphB4 Guidance System in Pulmonary Hypertension.

Smooth muscle cell (SMC) expansion is one key morphological hallmark of pathologically altered vasculature and a characteristic feature of pulmonary vascular remodeling in pulmonary hypertension. Normal embryonal vessel maturation requires successful coverage of endothelial tubes with SMC, which is dependent on ephrin-B2 and EphB4 ligand-receptor guidance system. In this study, we investigated the potential role of ephrin-B2 and EphB4 on neomuscularization in adult pulmonary vascular disease. Ephrin-B2 and EphB4 expression is preserved in smooth muscle and endothelial cells of remodeled pulmonary arteries. Chronic hypoxia-induced pulmonary hypertension was not ameliorated in mice with SMC-specific conditional ephrin-B2 knockout. In mice with global inducible ephrin-B2 knockout, pulmonary vascular remodeling and right ventricular hypertrophy upon chronic hypoxia exposure were significantly diminished compared to hypoxic controls, while right ventricular systolic pressure was unaffected. In contrast, EphB4 receptor kinase activity inhibition reduced right ventricular systolic pressure in hypoxia-induced pulmonary hypertension without affecting pulmonary vascular remodeling. Genetic deletion of ephrin-B2 in murine pulmonary artery SMC, and pharmacological inhibition of EphB4 in human pulmonary artery smooth muscle cells, blunted mitogen-induced cell proliferation. Loss of EphB4 signaling additionally reduced RhoA expression and weakened the interaction between human pulmonary artery smooth muscle cells and endothelial cells in a three-dimensional coculture model. In sum, pulmonary vascular remodeling was dependent on ephrin-B2-induced Eph receptor (erythropoietin-producing hepatocellular carcinoma receptor) forward signaling in SMC, while EphB4 receptor activity was necessary for RhoA expression in SMC, interaction with endothelial cells and vasoconstrictive components of pulmonary hypertension.

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

BackgroundOsteoporosis 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 methodsAn 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. ResultsED-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. ConclusionsED-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.

Osteoblast Jmjd3 regulates osteoclastogenesis via EphB4 and RANKL signalling.

Osteoblasts suppress osteoclastogenesis during the reversal phase of bone remodelling and the mechanism needs to be further investigated. Here, we investigated the role of histone demethylase Jumonji domain-containing 3 (Jmjd3) in osteoblasts on regulating osteoclastogenesis. Jmjd3 expression was silenced in osteoblasts. Osteoblasts and osteoclasts were co-cultured in direct or indirect contact ways, and osteoclastogenesis was determined by tartrate-resistant acid phosphatase (TRAP) staining and Western blotting. Additionally, Ephrin receptor B4 (EphB4) and receptor activator of nuclear factor-kappa Β ligand (RANKL) expression were quantified in osteoblasts via real-time PCR, Western blotting, and enzyme-linked immunosorbent assay. Subsequently, EphB4 was overexpressed in osteoblasts and RANKL expression and osteoclastogenesis was quantified. Osteoclastogenesis and marker protein expression levels was promoted when osteoclasts were co-cultured with Jmjd3-silenced osteoblasts. Silencing of Jmjd3 expression in osteoblasts decreased EphB4 expression, owing to suppression of demethylation of H3K27me3 on the promoter region of EphB4. Whereas RANKL expression was upregulated in Jmjd3-silenced osteoblasts. Overexpression of EphB4 in osteoblasts inhibited osteoclastogenesis and RANKL expression. Jmjd3 in osteoblasts is a crucial regulator of osteoblast-to-osteoclast communication through EphB4-EphrinB2, RANKL-RANK and EphB4-RANKL signalling axes, suggesting the pivotal role of Jmjd3 in bone remodelling process in bone destruction disease such as chronic apical periodontitis.

EphrinB2-EphB4 Signaling in Neurooncological Disease.

EphrinB2–EphB4 signaling is critical during embryogenesis for cardiovascular formation and neuronal guidance. Intriguingly, critical expression patterns have been discovered in cancer pathologies over the last two decades. Multiple connections to tumor migration, growth, angiogenesis, apoptosis, and metastasis have been identified in vitro and in vivo. However, the molecular signaling pathways are manifold and signaling of the EphB4 receptor or the ephrinB2 ligand is cancer type specific. Here we explore the impact of these signaling pathways in neurooncological disease, including glioma, brain metastasis, and spinal bone metastasis. We identify potential downstream pathways that mediate cancer suppression or progression and seek to understand it´s role in antiangiogenic therapy resistance in glioma. Despite the Janus-faced functions of ephrinB2–EphB4 signaling in cancer Eph signaling remains a promising clinical target.

Ligand-Dependent and Ligand-Independent Effects of Ephrin-B2-EphB4 Signaling in Melanoma Metastatic Spine Disease.

Tumor–endothelial cell interactions represent an essential mechanism in spinal metastasis. Ephrin-B2–EphB4 communication induces tumor cell repulsion from the endothelium in metastatic melanoma, reducing spinal bone metastasis formation. To shed further light on the Ephrin-B2–EphB4 signaling mechanism, we researched the effects of pharmacological EphB4 receptor stimulation and inhibition in a ligand-dependent/independent context. We chose a preventative and a post-diagnostic therapeutic window. EphB4 stimulation during tumor cell seeding led to an increase in spinal metastatic loci and number of disseminated melanoma cells, as well as earlier locomotion deficits in the presence of endothelial Ephrin-B2. In the absence of endothelial Ephrin-B2, reduction of metastatic loci with a later manifestation of locomotion deficits occurred. Thus, EphB4 receptor stimulation affects metastatic dissemination depending on the presence/absence of endothelial Ephrin-B2. After the manifestation of solid metastasis, EphB4 kinase inhibition resulted in significantly earlier manifestation of locomotion deficits in the presence of the ligand. No post-diagnostic treatment effect was found in the absence of endothelial Ephrin-B2. For solid metastasis treatment, EphB4 kinase inhibition induced prometastatic effects in the presence of endothelial Ephrin-B2. In the absence of endothelial Ephrin-B2, both therapies showed no effect on the growth of solid metastasis.

Lipopolysaccharides affect compressed periodontal ligament cells via Eph-ephrin signaling.

ObjectivesThe aim of this study is to investigate the underlying mechanism of the recovery of periodontal ligament cells (PDLCs) sequentially exposed to inflammation and mechanical loading.Materials and MethodsWe divided PDLCs into four groups: control; compressive force (CF) alone (2.0 g/cm2); lipopolysaccharides (LPS) pretreatment (0.1 μg/ml) followed by simultaneous LPS and CF stimulation, simulating uncontrolled periodontitis; and LPS pretreatment followed by CF exposure, simulating controlled periodontitis. The expression of EphB4‐ephrinB2 and EphA2‐ephrinA2, and the level of osteoclastogenesis and osteogenesis were evaluated.ResultsSimultaneous stimulation by LPS and CF, compared with CF alone and sequential LPS and CF exposure, significantly suppressed EphB4 and enhanced ephrinA2 expression. Similarly, the most intense osteoclastic differentiation was observed under simultaneous LPS and CF stimulation, while sequential exposure to LPS and CF only slightly increased osteoclastic cell numbers. Both the activation of EphB4 signaling and ephrinA2 silencing lowered osteoclastic differentiation, which had previously been upregulated by simultaneous LPS and CF stimulation. These treatments also increased osteogenic differentiation.ConclusionsSimultaneous LPS and CF stimulation critically enhances osteoclastogenesis in PDLCs through the suppression of EphB4 and the induction of ephrinA2 signaling. Sequential LPS and CF exposure partially abolishes the osteolytic effects of simultaneous stimulation.

The protective role of Ephrin-B2/EphB4 signaling in osteogenic differentiation under inflammatory environment

Inflammation and alveolar bone destruction constitute the main pathological process of periodontitis. However, the molecular mechanisms of bone destruction under the inflammation environment remain unclear. This study aims to explore the role of Ephrin-B2/EphB4 signaling in osteogenic differentiation under the inflammation environment. Mouse pre-osteoblasts MC3T3-E1 were pretreated with lipopolysaccharide of Porphyromonas gingivalis (Pg-LPS). The Ephrin-B2/EphB4 signaling was activated, and the osteogenic differentiation of cells was examined. The results showed that activation of Ephrin-B2/EphB4 signaling promoted the expression levels of osteogenic differentiation-related genes, and also relieved the inhibitory effect of Pg-LPS on osteogenesis. Noticeably, the effect of Ephrin-B2/EphB4 signaling might be related to the mitogen-activated protein kinase (MAPK) pathway. While applying Ephrin-B2-Fc and EphB4-Fc to periodontitis mice, we observed the reduction of alveolar crest destruction. The current study revealed the possible role of Ephrin-B2/EphB4 signaling in reducing bone destruction in periodontitis and suggested its potential values for further research.

Abstract 122: Targeting forward and reverse EphB4/EFNB2 signaling by a peptide with dual functions

Although chloroquine administration in vivo following haemorrhage in mice decreases tumour necrosis factor-alpha (TNF-alpha) release by macrophage (M phi), the mechanism remains unknown. To study this, peritoneal M phi (pM phi) from unmanipulated, sham-operated and post-haemorrhage mice were isolated, treated with 0.13 mg/ml chloroquine for 2 hr, and then stimulated with lipopolysaccharide (LPS) for 48 hr. Pretreatment of pM phi from various groups of mice with chloroquine resulted in 75-90% inhibition of TNF-alpha release, determined by bioassay. Total RNA was isolated from pM phi and murine M phi-derived cell lines (P388D1 and RAW 264.7), stimulated with LPS for 0.5 or 1 hr, respectively, and Northern blot analysis for TNF-alpha mRNA performed. Chloroquine inhibited TNF-alpha mRNA expression without interfering with mRNA stability, suggesting that this agent reduces M phi TNF-alpha release by disrupting TNF-alpha gene transcription.

Cantharidin treatment inhibits hepatocellular carcinoma development by regulating the JAK2/STAT3 and PI3K/Akt pathways in an EphB4-dependent manner

Hepatocellular carcinoma (HCC) is a lethal malignancy with limited treatment options. The tyrosine kinase receptor EphB4 promotes oncogenesis and tumor development and progression. Its inhibition is regarded as an effective strategy for the treatment of solid tumors. In the present study, we identified cantharidin as a novel EphB4 inhibitor for HCC treatment and evaluated the underlying molecular pharmacological mechanisms of action. We observed increased expression levels of EphB4 in HCC patients and a positive correlation between EphB4 and p-JAK2 levels in HCC patient samples. Knockdown of EphB4 using small interfering RNA decreased the expression levels of p-JAK2 and p-STAT3 in HCC cells, suggesting JAK2/STAT3 being a novel downstream signaling target of EphB4. Cell viability experiments revealed that the anti-cancer effect of cantharidin was positively correlated with EphB4 expression levels in HCC cell lines. We confirmed the potent antiproliferative activity of cantharidin on HepG2 cells with high expression of EphB4 and tumor xenograft. Molecular docking assay, immunoblotting assay and quantitative reverse transcription PCR assay indicated that cantharidin bound to EphB4, and thereby resulted in EphB4 suppression at mRNA and protein levels. Hep3B and SMMC-7721 cells were with low expression of EphB4. In EphB4−/HepG2, EphB4+/HepG2, and EphB4+/Hep3B cells, EphB4 knockdown alleviated the cantharidin-induced decrease in cell viability and colony formation ability and increase in apoptosis in HepG2 cells, while its overexpression exacerbated these effects in Hep3B cells and increased the apoptosis of HepG2 cells. In nude mouse models, cantharidin suppressed tumor growth more effectively in EphB4+/SMMC-7721 xenografts than in wild-type SMMC-7721 xenografts. Underlying mechanistic study showed that by targeting EphB4, cantharidin blocked a novel target, the downstream JAK2/STAT3 pathway, and the previously known target, the PI3K/Akt signaling, resulting in intrinsic apoptosis. These results indicated that cantharidin may be a potential candidate for HCC treatment by regulating the EphB4 signaling pathway.

Targeting connexin37 alters angiogenesis and arteriovenous differentiation in the developing mouse retina.

Connexin37 (Cx37) forms intercellular channels between endothelial cells (EC), and contributes to coordinate the motor tone of vessels. We investigated the contribution of this protein during physiological angiogenesis. We show that, compared to WT littermates, mice lacking Cx37 (Cx37-/- ) featured (i) a decreased extension of the superficial vascular plexus during the first 4days after birth; (ii) an increased vascular density at the angiogenic front at P6, due to an increase in the proliferative rate of EC and in the sprouting of the venous compartment, as well as to a somewhat displaced position of tip cells; (iii) a decreased coverage of newly formed arteries and veins by mural cells; (iv) altered ERK-dependent endothelial cells proliferation through the EphB4 signaling pathway, which is involved in the specification of veins and arteries. In vitro studies documented that, in the absence of Cx37, human venous EC (HUVEC) released less platelet-derived growth factor (PDGF) and more Angiopoietin-2, two molecules involved in the recruitment of mural cells. Treatment of mice with DAPT, an inhibitor of the Notch pathway, decreased the expression of Cx37, and partially mimicked in WT retinas, the alterations observed in Cx37-/- mice. Thus, Cx37 contributes to (i) the early angiogenesis of retina, by interacting with the Notch pathway; (ii) the growth and maturation of neo-vessels, by modulating tip, stalk, and mural cells; (iii) the regulation of arteriovenous specification, thus, representing a novel target for treatments of retina diseases.

Inhibition of erythropoietin-producing hepatoma receptor B4 (EphB4) signalling suppresses the vascularisation and growth of endometriotic lesions.

The development of endometriotic lesions is crucially dependent on the formation of new blood vessels. In the present study, we analysed whether this process is regulated by erythropoietin-producing hepatoma receptor B4 (EphB4) signalling. We first assessed the anti-angiogenic action of the EphB4 inhibitor NVP-BHG712 in different in vitro angiogenesis assays. Then, endometriotic lesions were surgically induced in the dorsal skinfold chamber and peritoneal cavity of NVP-BHG712- or vehicle-treated BALB/c mice. This allowed to study the effect of EphB4 inhibition on their vascularisation and growth by means of intravital fluorescence microscopy, high-resolution ultrasound imaging, histology and immunohistochemistry. Non-cytotoxic doses of NVP-BHG712 suppressed the migration, tube formation and sprouting activity of both human dermal microvascular endothelial cells (HDMEC) and mouse aortic rings. Accordingly, we also detected a lower blood vessel density in NVP-BHG712-treated endometriotic lesions. This was associated with a reduced lesion growth due to a significantly lower number of proliferating stromal cells when compared to vehicle-treated controls. Inhibition of EphB4 signalling suppresses the vascularisation and growth of endometriotic lesions. Hence, EphB4 represents a promising pharmacological target for the treatment of endometriosis.

Dysregulation of the EphrinB2\u2212EphB4 ratio in pediatric cerebral arteriovenous malformations is associated with endothelial cell dysfunction in vitro and functions as a novel noninvasive biomarker in patients

We investigated (1) EphrinB2 and EphB4 receptor expression in cerebral AVMs, (2) the impact of an altered EphrinB2:EphB4 ratio on brain endothelial cell function and (3) potential translational applications of these data. The following parameters were compared between AVM endothelial cells (AVMECs) and human brain microvascular endothelial cells (HBMVECs): quantified EphrinB2 and EphB4 expression, angiogenic potential, and responses to manipulation of the EphrinB2:EphB4 ratio via pharmacologic stimulation/inhibition. To investigate the clinical relevance of these in vitro data, Ephrin expression was assessed in AVM tissue (by immunohistochemistry) and urine (by ELISA) from pediatric patients with AVM (n = 30), other cerebrovascular disease (n = 14) and control patients (n = 29), and the data were subjected to univariate and multivariate statistical analyses. Compared to HBMVECs, AVMECs demonstrated increased invasion (p = 0.04) and migration (p = 0.08), impaired tube formation (p = 0.06) and increased EphrinB2:EphB4 ratios. Altering the EphrinB2:EphB4 ratio (by increasing EphrinB2 or blocking EphB4) in HBMVECs increased invasion (p = 0.03 and p < 0.05, respectively). EphrinB2 expression was increased in AVM tissue, which correlated with increased urinary EphrinB2 levels in AVM patients. Using the optimal urinary cutoff value (EphrinB2 > 25.7 pg/μg), AVMs were detected with high accuracy (80% vs. controls) and were distinguished from other cerebrovascular disease (75% accuracy). Post-treatment urinary EphrinB2 levels normalized in an index patient. In summary, AVMECs have an EphrinB2:EphB4 ratio that is increased compared to that of normal HBMVECs. Changing this ratio in HBMVECs induces AVMEC-like behavior. EphrinB2 is clinically relevant, and its levels are increased in AVM tissue and patient urine. This work suggests that dysregulation of the EphrinB2:EphB4 signaling cascade and increases in EphrinB2 may play a role in AVM development, with potential utility as a diagnostic and therapeutic target.

Inhibition of EphB4-Ephrin-B2 Signaling Reprograms the Tumor Immune Microenvironment in Head and Neck Cancers.

Identifying targets present in the tumor microenvironment that contribute to immune evasion has become an important area of research. In this study, we identified EphB4-ephrin-B2 signaling as a regulator of both innate and adaptive components of the immune system. EphB4 belongs to receptor tyrosine kinase family that interacts with ephrin-B2 ligand at sites of cell-cell contact, resulting in bidirectional signaling. We found that EphB4-ephrin-B2 inhibition alone or in combination with radiation (RT) reduced intratumoral regulatory T cells (Tregs) and increased activation of both CD8+ and CD4+Foxp3- T cells compared with the control group in an orthotopic head and neck squamous cell carcinoma (HNSCC) model. We also compared the effect of EphB4-ephrin-B2 inhibition combined with RT with combined anti-PDL1 and RT and observed similar tumor growth suppression, particularly at early time-points. A patient-derived xenograft model showed reduction of tumor-associated M2 macrophages and favored polarization towards an antitumoral M1 phenotype following EphB4-ephrin-B2 inhibition with RT. In vitro, EphB4 signaling inhibition decreased Ki67-expressing Tregs and Treg activation compared with the control group. Overall, our study is the first to implicate the role of EphB4-ephrin-B2 in tumor immune response. Moreover, our findings suggest that EphB4-ephrin-B2 inhibition combined with RT represents a potential alternative for patients with HNSCC and could be particularly beneficial for patients who are ineligible to receive or cannot tolerate anti-PDL1 therapy. SIGNIFICANCE: These findings present EphB4-ephrin-B2 inhibition as an alternative to anti-PDL1 therapeutics that can be used in combination with radiation to induce an effective antitumor immune response in patients with HNSCC.

Multivalent biomaterial platform to control the distinct arterial venous differentiation of pluripotent stem cells

Vascular endothelial cells (ECs) differentiated from pluripotent stem cells have enormous potential to be used in a variety of therapeutic areas such as tissue engineering of vascular grafts and re-vascularization of ischemic tissues. To date, various protocols have been developed to differentiate stem cells toward vascular ECs. However, current methods are still not sufficient to drive the distinct arterial venous differentiation. Therefore, developing refined method of arterial-venous differentiation is critically needed to address this gap. Here, we developed a biomaterial platform to mimic multivalent ephrin-B2/EphB4 signaling and investigated its role in the early arterial and venous specification of pluripotent stem cells. Our results show immobilized ephrinB2 or EphB4 on hydrogel substrates have a distinct effect on arterial venous differentiation by regulating several arterial venous markers. When in combination with Wnt pathway agonist or BMP4 signaling, the ephrin-B2/EphB4 biomaterial platform can create diverging EC progenitor populations, demonstrating differential gene expression pattern across a wide range of arterial and venous markers, as well as phenotypic markers such as anti-thrombotic, pro-atherogenic and osteogenic genes, that are consistent with the in vivo expression patterns of arterial and venous ECs. Importantly, this distinct EC progenitor population cannot be achieved by current methods of applying soluble factors or hemodynamic stimuli alone, illustrating that fine-tuning of developmental signals using the biomaterial platform offers a new approach to better control the arterial venous differentiation of stem cells.

Titanium particle‑mediated osteoclastogenesis may be attenuated via bidirectional ephrin‑B2/eph‑B4 signaling invitro.

The present study investigated the role of bidirectional ephrin-B2/erythropoietin-producing human hepatocellular receptor 4 (ephB4) signaling in the regulation of wear particle-mediated osteoclastogenesis in vitro. Mouse bone marrow macrophages (BMMs) were induced into osteoclasts by receptor activator of nuclear factor-κB ligand (RANKL, 50 ng/ml). EphB4-Fc, an osteoblast membrane surface receptor (4 µg/ml), was used to stimulate the ephrin-B2 ligand of osteoclasts in the presence and absence of titanium (Ti). Tartrate-resistant acid phosphatase (TRAP) staining was used to detect the number of osteoclasts, and phalloidin staining was used to examine the cytoskeletons of the osteoclasts. A bone pit absorption experiment was used to measure osteoclast function. Reverse transcription quantitative polymerase chain reaction and western blot analysis were used to examine osteoclastogenesis. ELISAs were used to detect the production of inflammatory factors. The data demonstrated that Ti significantly promoted the differentiation of BMMs into mature osteoclasts in the presence of RANKL and significantly promoted expression of the ephrin-B2, nuclear factor of activated T-cells 1 (NFATc1), TRAP, Fos proto-oncogene, AP-1 transcription factor subunit (C-FOS), and matrix metalloproteinase 9 (MMP9) genes. Phalloidin and TRAP staining revealed that following the addition of ephB4-Fc, the number, size and cytoskeletal elements of Key words: osteoclasts, osteoblasts, remodeling, ephrin-B2, osteoclastogenesis osteoclasts were significantly decreased compared with those in the titanium particle group without ephB4-Fc. Compared with the titanium particle group, the bone pit absorption experiment revealed significantly decreased absorption pit areas in the titanium particle+ephB4-Fc group. The expression of the NFATc1, TRAP, C-FOS and MMP9 genes was markedly decreased in the ephB4-Fc group; however, the expression of the ephrin-B2 gene was increased compared with the Ti particle group without ephB4-Fc after 5 days. Production of inflammatory cytokines was inhibited by Ti particles through bidirectional signals. Addition of ephB4-Fc inhibited the osteoclast-mediated formation of Ti particles via bidirectional ephrin-B2/ephB4 signaling. Activation of this bidirectional signaling pathway may be a potential clinical treatment for osteolysis surrounding prostheses.

Stromal cells of hemangioblastomas exhibit mesenchymal stem cell-derived vascular progenitor cell properties.

Hemangioblastoma is composed of neoplastic stromal cells and a prominent capillary network. To date, the identity of stromal cells remains unclear. Mesenchymal stem cells can give rise to committed vascular progenitor cells, and ephrin-B2/EphB4 and Notch signaling have crucial roles in these steps. The aim of our study was to elucidate that stromal cells of central nervous system hemangioblastomas have mesenchymal stem cell-derived vascular progenitor cell properties. Ten hemangioblastomas were investigated immunohistochemically. CD44, a mesenchymal stem cell marker, was detected in stromal cells of all cases, suggesting that stromal cells have mesenchymal stem cell-like properties. Neither CD31 nor α-SMA was expressed in stromal cells, suggesting that stromal cells have not acquired differentiated vascular cell properties. Both ephrin-B2 and EphB4, immature vascular cell markers, were detected in stromal cells of all cases. Jagged1, Notch1, and Hesr2/Hey2, which are known to be detected in both immature endothelial cells and mural cells, were expressed in stromal cells of all cases. Notch3, which is known to be detected in differentiating mural cells, was also expressed in all cases. These results suggest that stromal cells also have vascular progenitor cell properties. In conclusion, stromal cells of hemangioblastomas exhibit mesenchymal stem cell-derived vascular progenitor cell properties.

Pathogenic variant in EPHB4 results in central conducting lymphatic anomaly.

Central conducting lymphatic anomaly (CCLA) is one of the complex lymphatic anomalies characterized by dilated lymphatic channels, lymphatic channel dysmotility and distal obstruction affecting lymphatic drainage. We performed whole exome sequencing (WES) of DNA from a four-generation pedigree and examined the consequences of the variant by transfection of mammalian cells and morpholino and rescue studies in zebrafish. WES revealed a heterozygous mutation in EPHB4 (RefSeq NM_004444.4; c.2334 + 1G>C) and RNA-Seq demonstrated that the EPHB4 mutation destroys the normal donor site, which leads to the use of a cryptic splice donor that results in retention of the intervening 12-bp intron sequence. Transient co-expression of the wild-type and mutant EPHB4 proteins showed reduced phosphorylation of tyrosine, consistent with a loss-of-function effect. Zebrafish ephb4a morpholino resulted in vessel misbranching and deformities in the lymphatic vessel development, indicative of possible differentiation defects in lymphatic vessels, mimicking the lymphatic presentations of the patients. Immunoblot analysis using zebrafish lysates demonstrated over-activation of mTORC1 as a consequence of reduced EPHB4 signaling. Strikingly, drugs that inhibit mTOR signaling or RAS-MAPK signaling effectively rescued the misbranching phenotype in a comparable manner. Moreover, knock-in of EPHB4 mutation in HEK293T cells also induced mTORC1 activity. Our data demonstrate the pathogenicity of the identified EPHB4 mutation as a novel cause of CCLA and suggesting that ERK inhibitors may have therapeutic benefits in such patients with complex lymphatic anomalies.

PC236. Cav-1 Regulates EphB4-Mediated Arteriovenous Fistula Maturation

Arteriovenous fistula (AVF) continues to be the most common access created for hemodialysis. However, many AVFs fail to mature, suggesting a need to improve AVF maturation. Caveolin1 (Cav-1) is the major scaffolding protein of caveolae, a distinct microdomain that serves as a flow-activated mechanosensor at the membrane of endothelial cells; Eph-B4 is the embryonic venous determinant. We have previously shown that Cav-1 is a mechanism of Eph-B4-mediated vessel remodeling in the murine vein graft model. We have also shown that Eph-B4 expression increases during AVF maturation and Eph-B4 activity inhibits venous remodeling in the murine AVF model. Then we hypothesized that Cav-1 is a critical regulator of Eph-B4-mediated AVF maturation through inhibition of eNOS activity. We used a mouse aortocaval fistula model. The venous AVF limb and control (sham) inferior vena cava (IVC) of wild-type C57BL/6 (WT) were compared for Cav-1 expression. AVFs of WT mice, Cav-1 knockout (KO) mice, Cav-1 endothelial reconstituted (RC) mice, and Eph-B4 heterozygous (Eph-B4 het) mice were analyzed. A Cav-1 scaffolding domain peptide (cavtratin) was administrated to stimulate Cav-1 signaling; ephrinB2/Fc was used to stimulate Eph-B4 signaling. Vessel remodeling was assessed postoperatively by serial ultrasound measurements of the IVC/AVF diameter. AVFs were harvested at day 21 and examined with histology; IVC wall thickness was measured by computerized morphometry. Both Cav-1 mRNA and protein were increased in the fistula veins compared with control veins. Cav-1 KO mice showed enhanced venous remodeling with increased eNOS activity compared with WT mice. Inhibition of remodeling by ephrin-B2/Fc in WT mice was abolished in Cav-1 KO mice, but maintained in Cav-1 RC mice (Fig A). Administration of cavtratin decreased the fistula wall thickness in WT mice as well as in Eph-B4 het mice (Fig B). Endothelial Cav-1 is a critical regulator of Eph-B4-mediated AVF maturation. Manipulation of Cav-1 function may improve arteriovenous maturation within the fistula environment.

EphrinB2/EphB4 signaling regulates non‐sprouting angiogenesis by VEGF

Vascular endothelial growth factor (VEGF) is the master regulator of angiogenesis, whose best‐understood mechanism is sprouting. However, therapeutic VEGF delivery to ischemic muscle induces angiogenesis by the alternative process of intussusception, or vascular splitting, whose molecular regulation is essentially unknown. Here, we identify ephrinB2/EphB4 signaling as a key regulator of intussusceptive angiogenesis and its outcome under therapeutically relevant conditions. EphB4 signaling fine‐tunes the degree of endothelial proliferation induced by specific VEGF doses during the initial stage of circumferential enlargement of vessels, thereby limiting their size and subsequently enabling successful splitting into normal capillary networks. Mechanistically, EphB4 neither inhibits VEGF‐R2 activation by VEGF nor its internalization, but it modulates VEGF‐R2 downstream signaling through phospho‐ERK1/2. In vivo inhibitor experiments show that ERK1/2 activity is required for EphB4 regulation of VEGF‐induced intussusceptive angiogenesis. Lastly, after clinically relevant VEGF gene delivery with adenoviral vectors, pharmacological stimulation of EphB4 normalizes dysfunctional vascular growth in both normoxic and ischemic muscle. These results identify EphB4 as a druggable target to modulate the outcome of VEGF gene delivery and support further investigation of its therapeutic potential.