EphB4 Expression Research Articles

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.

A narrative review of the role of Eph receptors in head and neck squamous cell carcinoma.

Tyrosine kinase receptors (TKR) coordinate a variety of pathological processes in head and neck squamous cell carcinoma (HNSCC), and eventually play a role in patient outcomes. In this review, the role of Eph receptors in HNSCC progression and the possibility of targeting these receptors are illustrated. All relevant studies were identified through a comprehensive search of four electronic databases, including PubMed, Scopus, web of science, and Embase till August 2022. EphA2 and EphB4, along with ephrin-B2, were the most extensively studied proteins in this family. However, overexpression of EphB4 and its ligand ephrin-B2 were the only proteins that consistently showed association with a poor outcome, indicating that these proteins might serve as valuable prognostic markers in HNSCC. High expression of EphA3 and EphB4 was found to play a crucial role in radioresistance of HNSCC. EphB4 loss, in particular, was observed to induce an immunosuppression phenotypic HNSCC. Currently, ongoing clinical trials are investigating the benefits of EphB4-ephrin-B2 blockade in combination with standard of care treatment in HNSCC. Further efforts are needed to explore the biological role and behavioral complexity of this family of TKR in HNSCC with great attention to avoid heterogeneity of HNSCC subsites.

Fusobacterium nucleatum induced exosomal HOTTIP promotes gastric cancer progression through miR-885-3p/EphB2 axis.

Recent studies have demonstrated that Fusobacterium nucleatum (Fn) is associated with gastric cancer (GC). Cancer-derived exosomes contain key regulatory non-coding RNAs and are crucial medium of intercellular communication. However, the function and regulatory mechanism of exosomes (Fn-GCEx) secreted from Fn-infected GC cells remains unclear. In this study, Fn-GCEx enhanced the proliferation, migration and invasion capacity of GC cells in vitro, as well as tumor growth and metastasis in vivo. And, HOTTIP was upregulated in GC cells treated with Fn-GCEx. Moreover, knockdown of HOTTIP weakened the effects of Fn-GCEx in recipient GC cells. Mechanistically, HOTTIP promoted EphB2 expression by sponging miR-885-3p, thus activating the PI3K/AKT pathway in Fn-GCEx treated GC cells. Overall, Fn infection induced the upregulation of exosomal HOTTIP from GC cells that subsequently promoted GC progression via the miR-885-3p/EphB2/PI3K/AKT axis. Herein, we identify a potential molecular pathway and therapeutic target for GC.

Ten-m4 plays a unique role in the establishment of binocular visual circuits.

The patterning of binocular vision requires distinct molecular pathways for inputs arising from each side of the nervous system. Recent studies have demonstrated important roles for members of the Ten-m/Odz/teneurin family in the development of ipsilateral retinal projections. Here, we further highlight the significance of this gene family in visual development by identifying a role for Ten-m4 during the formation of the ipsilateral projection in the mouse. Ten-m4 was found to be expressed in the retina, dorsal lateral geniculate nucleus (dLGN), superior colliculus (SC), and primary visual cortex (V1) during development. Anterograde and retrograde tracing experiments in Ten-m4 knockout (KO) mice revealed a specific increase in ipsilateral retinal ganglion cells projecting to dLGN and SC. This increase was most prominent in regions corresponding to temporal retina. Consistent with this, EphB1 expression in the retina around the time of decussation was enhanced in this temporal region for KO mice, suggesting that the increased size of the ipsilateral population arises due to an increased number of retinal ganglion cells remaining ipsilaterally at the optic chiasm due to EphB1-mediated repulsion. The ectopic ipsilaterally targeted retinal ganglion cell projection observed in Ten-m4 KOs was associated with changes in response to ethologically-relevant visual stimuli. Together, these data demonstrate a requirement for Ten-m4 in the establishment of ipsilateral projections from the retina, which likely acts in combination with other Ten-m members (Ten-m2 and Ten-m3) to promote the formation of functional binocular circuits. This article is protected by copyright. All rights reserved.

1′-O-methyl-averantin isolated from the endolichenic fungus Jackrogersella sp. EL001672 suppresses colorectal cancer stemness via sonic Hedgehog and Notch signaling

Endolichenic fungi are host organisms that live on lichens and produce a wide variety of secondary metabolites. Colorectal cancer stem cells are capable of self-renewal and differentiation into cancer cells, which makes cancers difficult to eradicate. New alternative therapeutics are needed to inhibit the growth of tumor stem cells. This study examined the ability of an extract of Jackrogersella sp. EL001672 (derived from the lichen Cetraria sp.) and the isolated compound 1′-O-methyl-averantin to inhibit development of cancer stemness. The endolichenic fungus Jackrogersella sp. EL001672 (KACC 83021BP), derived from Cetraria sp., was grown in culture medium. The culture broth was extracted with acetone to obtain a crude extract. Column chromatography and reverse-phase HPLC were used to isolate an active compound. The anticancer activity of the extract and the isolated compound was evaluated by qRT-PCR and western blotting, and in cell viability, spheroid formation, and reporter assays. The acetone extract of EL001672 did not affect cell viability. However, 1′-O-methyl-averantin showed cytotoxic effects against cancer cell lines at 50 μg/mL and 25 μg/mL. Both the crude extract and 1′-O-methyl-averantin suppressed spheroid formation in CRC cell lines, and downregulated expression of stemness markers ALDH1, CD44, CD133, Lgr-5, Msi-1, and EphB1. To further characterize the mechanism underlying anti-stemness activity, we examined sonic Hedgehog and Notch signaling. The results showed that the crude extract and the 1′-O-methyl-averantin inhibited Gli1, Gli2, SMO, Bmi-1, Notch-1, Hes-1, and the CSL complex. Consequently, an acetone extract and 1′-O-methyl-averantin isolated from EL001672 suppresses colorectal cancer stemness by regulating the sonic Hedgehog and Notch signaling pathways.

Neddylation of EphB1 Regulates Its Activity and Associates with Liver Fibrosis

Liver fibrosis is a pathological process characterized by the excessive synthesis and accumulation of extracellular matrix proteins (ECMs) contributed mainly by the activated hepatic stellate cells (HSCs). Currently, no direct and effective anti-fibrotic agents have been approved for clinical use worldwide. Although the dysregulation of Eph receptor tyrosine kinase EphB2 has been reported to associate with the development of liver fibrosis, the involvement of other Eph family members in liver fibrosis remains underexplored. In this study, we found that the expression of EphB1 is significantly increased accompanying remarkable neddylation in activated HSCs. Mechanistically, this neddylation enhanced the kinase activity of EphB1 by the prevention of its degradation, thereby promoting the proliferation, migration, and activation of HSCs. Our findings revealed the involvement of EphB1 in the development of liver fibrosis through its neddylation, which provides new insights into the Eph receptor signaling and a potential target for the treatment of liver fibrosis.

MiR-218-5p-Modified Bone Marrow Mesenchymal Stem Cells Mediate the Healing Effect of EphrinB2-EphB4 Signals on Alveolar Bone Defect

Abnormally expressed miR-218-5p involves in alveolar bone defect. We intend to investigate whether miR-218-5p-modified bone marrow mesenchymal stem cells (BMSCs) mediates the healing effects of EphrinB2-EphB4 signals on the alveolar bone defect. Fifty germ-free rats (6-month-old) were utilized in this study. The grouping was set up as follows: blank group, model group, miR-218-5p group, EphrinB2-EphB4 antagonist group, and positive control group (10 rats in each group). HE staining was employed to quantify bone resorption lacunae number. And the following indicators were monitored: miR-218-5p expression, differentiation status of osteoblasts, concentrations of TNF-α/IL-10/ IL-8, and EphrinB2 and EphB4 expression. As shown in HE staining, massive infiltration of inflammatory cells was denoted at the alveolar bone defective sites in rats from model group. However, infiltration of inflammatory cells in lesions was moderate in rats from EphrinB2-EphB4 antagonist group and positive control group, which was accompanied by formation of small bone islands. Furthermore, lesser infiltration of inflammatory cells was denoted at the alveolar bone defective sites in rats from the miR-218-5p group, which also exhibited a larger number of newly formed bone trabeculae growing toward the center of lesions. On the 3rd day of culture, absorption lacunae were rare in the model group, while remaining undetectable in other groups. On the 7th day of culture, bone resorption lacunae number in samples from model group was significantly higher in comparison with that in other groups. Meanwhile, it was reduced significantly in miR-218-5p group. However, it was increased in EphrinB2-EphB4 antagonist group and positive control group (P <0.05). An elevation of the intracellular miR-218-5p level was denoted in the modified BMSCs in comparison with those unmodified BMSCs (P < 0.05). In comparison with blank group, other groups exhibited significantly elevated ALP levels, among which model group showed highest level. However, decline of ALP levels was denoted in positive control group, EphrinB2-EphB4 antagonist group and miR-218-5p group, with lowest ALP level in miR-218-5p group (P <0.05). Except blank group, rats in other groups exhibited a significant elevation of TNF-α, IL-10 and IL-8 in the serum, among which those in the model group displayed the most remarkable increase of these cytokines. Rats in miR-218-5p group, EphrinB2-EphB4 antagonist group and positive control group exhibited significantly reduced levels of IL-8, IL-10 and TNF-α in the serum, with miR-218-5p group showing lowest levels (P < 0.05). In comparison with the blank group, other groups showed significantly enhanced protein expression of EphrinB2 and EphB4, among which the model group displayed the most remarkable enrichment of these proteins. In comparison with the model group, samples from the miR-218-5p group, EphrinB2-EphB4 antagonist group and positive control group exhibited significantly weakened expression of EphrinB2 and EphB4, among which the miR-218-5p group displayed the most remarkable decrease of these proteins (P <0.05). miR-218-5p-modified BMSCs can modulate the EphrinB2-EphB4 signal transduction pathway to produce two-way transmission, which included their inhibition of the osteoclast generation and their enhancement of the osteoclast differentiation. In this way, they aided in alleviating inflammatory response in alveolar bone defective lesions, thereby accelerating the healing process of alveolar bone defect. The function of miR-218-5p-modified BMSCs is mainly achieved in the healing process of the alveolar bone defect.

M6PR- and EphB4-Rich Exosomes Secreted by Serglycin-Overexpressing Esophageal Cancer Cells Promote Cancer Progression.

Accumulating evidence shows that exosomes participate in cancer progression. However, the functions of cancer cell exosome-transmitted proteins are rarely studied. Previously, we reported that serglycin (SRGN) overexpression promotes invasion and metastasis of esophageal squamous cell carcinoma (ESCC) cells. Here, we investigated the paracrine effects of exosomes from SRGN-overexpressing ESCC cells (SRGN Exo) on ESCC cell invasion and tumor angiogenesis, and used mass spectrometry to identify exosomal proteins involved. Cation-dependent mannose-6-phosphate receptor (M6PR) and ephrin type-B receptor 4 (EphB4) were pronouncedly upregulated in SRGN Exo. Upregulated exosomal M6PR mediated the pro-angiogenic effects of SRGN Exo both in vitro and in vivo, while augmented exosomal EphB4 mediated the pro-invasive effect of SRGN Exo on ESCC cells in vitro. In addition, in vitro studies showed that manipulation of M6PR expression affected the viability and migration of ESCC cells. Both M6PR and EphB4 expression levels were positively correlated with that of SRGN in the serum of patients with ESCC. High level of serum M6PR was associated with poor overall survival rates. Taken together, this study presents the first proof that exosomal M6PR and EphB4 play essential roles in tumor angiogenesis and malignancy, and that serum M6PR is a novel prognostic marker for ESCC patients.

Extracellular matrix and dermal nerve growth factor dysregulation in prurigo nodularis compared to atopic dermatitis.

Prurigo nodularis (PN) is a chronic, pruritic, inflammatory skin disease characterized by hyperkeratotic nodules on the trunk and extremities. While there is growing research on the immunological basis of PN, the neuropathic and structural components of PN lesions are unknown. This study examines the inflammatory, neuropathic, and structural pathways in PN compared to atopic dermatitis (AD) using RNA-sequencing of the lesional and non-lesional skin tissue of PN and AD patients, as well as immunohistochemistry analysis of nerve growth factor (NGF), a neurotrophic factor that regulates nerve development. Transcriptomic analysis of skin biopsies revealed that compared to lesional AD skin, lesional PN skin had significantly increased expression of NGF, matrix metalloproteinases, OSM, MCEMP1, IL1α, IL1β, CXCL2, CXCL5, CXCL8, and insulin-like growth factors in PN compared to AD, and decreased expression of CCL13, CCL26, EPHB1, and collagens (COL4/6). Gene set enrichment analysis demonstrated higher enrichment of keratinization, cornified envelope, myelin sheath, TGF-beta signaling, extracellular matrix disassembly, metalloendopeptidase activity, and neurotrophin-TRK receptor signaling pathways in PN. On immunohistochemistry, PN lesions demonstrated higher dermal NGF expression compared to AD. We present novel findings demonstrating increased neurotrophic and extracellular matrix remodeling signatures in PN compared to AD, possibly explaining the morphological differences in their lesions. These signatures may therefore be important components of the PN pathogenesis and may serve as therapeutic targets.

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.

Isoflurane impairs GluN2B-containing NMDA receptors trafficking and cognition via decreasing histone acetylation and EphB2 expression in aged hippocampal neurons.

Perioperative neurocognitive disorders (PND) is a common complication that occurs among elderly patients in the perioperative course. Current clinical evidence has shown that isoflurane exposure could cause cognitive decline, but the exact molecular mechanisms remain unclear. As both NMDARs-dependent synaptic plasticity and histone acetylation play vital roles in processing learning and memory, we postulated that these alternations might occur in the isoflurane-associated PND. Here, we found that isoflurane impaired fear memory in aged mice, decreased GluN2B-containing NMDA receptors phosphorylation and trafficking, as well as the expression of EphB2, a key regulator of synaptic localization of NMDA receptors. We also identified that isoflurane could increase the expression of HDAC2, which was significantly enriched at the ephb2 gene promoter and regulated the transcription of ephb2. Furthermore, we showed that suberoylanilide hydroxamic acid (SAHA), a nonselective HDAC inhibitor or knocking-down HDAC2 rescued the cognitive dysfunction in isoflurane-treated aged mice via increasing acetylation of H3Ac, expression of EphB2 and promoting NMDA receptor trafficking. Collectively, our study highlighted the crucial role of histone posttranslational modifications for EphB2-GluN2B signals in isoflurane-associated PND, and modulating HDAC2 might be a new therapeutic strategy for isoflurane-associated PND.

Suppression of Multiple Myeloma Via Inhibition of EFNB2 Reverse Signaling

Multiple myeloma (MM) cells display a reciprocal relationship with neighboring niche cells in the bone marrow (BM), particularly vascular endothelial cells (BM ECs). This relationship drives disease initiation, progression, and therapy resistance, but the mechanisms underlying this interaction remain incompletely understood. Here, we show that silencing ephrin B2 (EFNB2) blocked MM cell proliferation and survival in vitro and abrogated MM engraftment and repopulation in vivo. EFNB2 expression is also increased in MM patients with adverse-risk cytogenetics and decreased survival, suggesting an important role for EFNB2 in MM disease progression. To identify novel regulators of MM pathogenesis, we performed transcriptome analyses of ECs from multiple human tissues and analyzed their capacity to promote MM growth. The EPH receptor, EPHB1, was overexpressed in human ECs that support MM expansion in culture. Silencing EPHB1 expression in ECs using siRNA or a lentiviral shRNA abrogated MM cell expansion compared to EC co-cultures treated with non-targeting shRNA and blocked the expansion of MM cells capable of in vivo repopulation in immune deficient mice. Additionally, transgenic Vk*MYC mice, which develop indolent murine MM through activation-induced deaminase-dependent MYC activation in germinal B cells, displayed increased expression of EPHB1 and EPHB4 in BM ECs compared to wild type BM ECs. We found that expression of EFNB2, an EPHB1/4 binding partner, is upregulated in 3 human MM cell lines and in primary MM cell populations from patients, whereas other EPHB1/4 binding partners were either not detected or expressed at a low level. shRNA-mediated inhibition of EFNB2 in MM cells blocked MM cell cycling, leading to a near 70-fold reduction in MM cell growth at day 12 of culture, and blunted MM cell repopulation when transplanted into NSG mice. In order to determine the therapeutic potential of targeting EFNB2 in MM in vivo, we transplanted irradiated NSG mice with MM cells and subsequently administered a single chain variable fragment (scFv) targeting EFNB2 on days +4, +6, +10 and +12. Mice treated with the EFNB2 scFv displayed significantly decreased engraftment of MM cells in vivo compared to control mice. MM reconstitution in vivo. Silencing EFNB2 expression in MM cells downregulated several pathways including those involved in cellular movement, cell death and survival, cellular assembly, cell cycle and DNA repair. Several STAT5 target genes were also downregulated in MM cells treated with shRNA-EFNB2, supporting the notion that STAT5 signaling is activated by ephrin B proteins (Bong et al. Proc Natl Acad Sci. 2007;104(44):17305-17310). Consistent with these findings, EFNB2 overexpression via dCas9-VPR increased MM cell survival, proliferation and STAT5 phosphorylation. Conversely, silencing EFNB2 decreased STAT5 activation, increased MM apoptosis, and decreased MM proliferation. Importantly, STAT5 inhibition in MM cells overexpressing EFNB2 blocked EFNB2-mediated MM cell proliferation, suggesting that EFNB2-mediated MM growth is dependent on STAT5. Overexpressing a mutant form of human EFNB2 with GFP replacing the intracellular cytoplasmic PDZ signaling domain did not result in cell growth comparable to overexpressing the full-length EFNB2. Silencing EPHB4, the main EFNB2 binding partner expressed on MM cells, resulted in significant impairment of growth in wild-type MM cells but not those with full-length EFNB2 overexpression. Updated downstream signaling and in vivo data of these will be presented at the meeting. Interrogation of MM clinical databases, including the Total Therapy studies (Zhan F. et al. Blood. 2006;108(6):2020-2028) and the CoMMpass data (Multiple Myeloma Research Foundation, https://research.themmrf.org/, accessed on 20 October 2021, n = 773 patients), revealed that EFNB2 expression is increased in MM patients with adverse risk cytogenetics, including t(4;14) and gain of chromosome 1q, while lower EFNB2 expression is associated with the more favorable, "standard risk" disease category, hyperdiploid MM. Increased EFNB2 expression is also associated with advanced stage disease and decreased survival in MM patients. Our analyses suggest that EFNB2 signaling represents an important new mechanistic target in MM. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Yi Shen Juan Bi Pill alleviates bone destruction in inflammatory arthritis under postmenopausal conditions by regulating ephrinB2 signaling.

Yi Shen Juan Bi Pill (YSJB) is a traditional Chinese medicine (TCM) formulation that has a therapeutic effect upon rheumatoid arthritis (RA), but how YSJB affects bone destruction in arthritis under postmenopausal conditions is not known. We evaluated the therapeutic role of YSJB in bone destruction in postmenopausal arthritis, We used collagen-induced arthritis (CIA) rats who had been ovariectomized (OVX) as models and explored the possible mechanism from the synovium and bone marrow (BM). Arthritis was generated after ovariectomy or sham surgery for 12 weeks. After 14 days of primary immunization, rats were administered YSJB or estradiol valerate (EV) for 28 days. YSJB could prevent bone destruction in the inflamed joints of rats in the OVX + CIA group. CIA promoted osteoclast differentiation significantly in the synovial membrane according to tartrate resistant acid phosphatase (TRACP) staining, and OVX tended to aggravate the inflammatory reaction of CIA rats according to hematoxylin-and-eosin staining. Immunohistochemistry revealed that the synovium did not have significant changes in erythropoietin-producing hepatocellular interactor (ephrin)B2 or erythropoietin-producing hepatocellular (eph) B4 expression after YSJB treatment, but YSJB treatment reduced nuclear factor of activated T cells (NFATc)1 expression. The BM of rats in the OVX + CIA exhibited remarkable increases in the number of osteoclasts and NFATc1 expression, as well as significantly reduced expression of ephrinB2 and ephB4 compared with the CIA group and sham group. YSJB treatment reduced NFATc1 expression significantly but also increased ephrinB2 expression in the BM markedly. These data suggest that YSJB exhibit a bone-protective effect, it may be a promising therapeutic strategy for alleviating bone destruction in arthritis under postmenopausal conditions, and one of the mechanisms is associated with the modulation of ephrinB2 signaling.

EPHB2 as a recurrence-related gene and a prognostic indicator in nasopharyngeal carcinoma: A bioinformatics screening and immunohistochemistry verification.

Recurrence and metastasis of nasopharyngeal carcinoma (NPC) after radical treatment is a major bottleneck in clinical treatment. Therefore, we aimed to find the genes related to metastasis after radical treatment in NPC patients. Public datasets in the Gene Expression Omnibus database were consulted and the differential expression genes (DEGs) were screened out. The possible roles of the DEGs were annotated by Gene Ontology, and pathway analysis. The hub genes/proteins were then filtered out through protein-protein interaction network construction. The key genes were sifted out from the hub genes, and their expressions were verified by qPCR and immunohistochemistry assays. A total of 28 DEGs were filtered out, which may be enriched in different signaling pathways. Of these DEGs, 11 hub genes were filtered out, among which EPHB2 was shown to be over-expressed in NPC tissues. Further experimental assays confirmed that EPHB2 was overexpressed in NPC cells, which might be associated with tumor recurrence, neck lymph node metastasis, and advanced clinical stages. Moreover, high EPHB2 expression predicted poor prognosis in NPC patients. EPHB2 might be a novel recurrence-related biomarker and a prognostic factor for NPC. Moreover, it might also be used as a potential treatment target for NPC.

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.

Endothelial EphrinB2 Regulates Sunitinib Therapy Response in Murine Glioma.

Vascular guidance is critical in developmental vasculogenesis and pathological angiogenesis. Brain tumors are strongly vascularized, and antiangiogenic therapy was anticipated to exhibit a strong anti-tumor effect in this tumor type. However, vascular endothelial growth factor A (VEGFA) specific inhibition had no significant impact in clinical practice of gliomas. More research is needed to understand the failure of this therapeutic approach. EphrinB2 has been found to directly interact with vascular endothelial growth factor receptor 2 (VEGFR2) and regulate its activity. Here we analyzed the expression of ephrinB2 and EphB4 in human glioma, we observed vascular localization of ephrinB2 in physiology and pathology and found a significant survival reduction in patients with elevated ephrinB2 tumor expression. Induced endothelial specific depletion of ephrinB2 in the adult mouse (efnb2i∆EC) had no effect on the quiescent vascular system of the brain. However, we found glioma growth and perfusion altered in efnb2i∆EC animals similar to the effects observed with antiangiogenic therapy. No additional anti-tumor effect was observed in efnb2i∆EC animals treated with antiangiogenic therapy. Our data indicate that ephrinB2 and VEGFR2 converge on the same pathway and intervention with either molecule results in a reduction in angiogenesis.

MicroRNA-122 inhibits hepatic stellate cell proliferation and activation in vitro and represses carbon tetrachloride-induced liver cirrhosis in mice

ObjectiveThis study aimed to determine the roles of microRNA (miR)-122 in the activation of hepatic stellate cells (HSCs) and liver cirrhosis. MethodsRat primary HSCs were incubated with transforming growth factor-beta (TGF-β), during which miR-122 and EphB2 expression was measured. miR-122 mimic and/or pcDNA3.1 EphB2 was transfected into TGF-β-induced HSCs. A mouse model of liver cirrhosis was established via an intraperitoneal injection of carbon tetrachloride (CCl4), followed by the injection of miR-122 agomir. Levels of serum alanine transaminase (ALT) and aspartate aminotransferase (AST) were measured. Fibronectin (FN), alpha smooth muscle actin (α-SMA), Collagen I, miR-122, and EphB2 expression was evaluated in liver tissues and HSCs. Cell proliferation was measured using CCK-8 assay. Interactions between miR-122 and EphB2 were assessed using dual luciferase reporter assay. ResultsmiR-122 (0.15-fold) was downregulated and EphB2 (mRNA: 5.06-fold; protein: 2.35-fold) was upregulated after TGF-β induction of HSCs. Overexpressed miR-122 decreased proliferation and EphB2 (mRNA: 0.46-fold; protein: 0.62-fold), FN (mRNA: 0.45-fold; protein: 0.64-fold), α-SMA (mRNA: 0.48-fold; protein: 0.51-fold), and Collagen I (mRNA: 0.44-fold; protein: 0.51-fold) expression in HSCs, which was abrogated by EphB2 upregulation. miR-122 expression was reduced by 0.21-fold and serum ALT and AST levels were enhanced in mice following 8-week CCl4 induction along with increased expression of FN, α-SMA, and Collagen I in liver tissues, which was blocked by miR-122 overexpression. Moreover, EphB2 was a target gene of miR-122. ConclusionmiR-122 curtails HSC proliferation and activation by targeting EphB2 and suppresses liver cirrhosis in mice.

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.

A phase II study of sEphB4-HSA in metastatic castration-resistant prostate cancer.

84 Background: Ephrin receptors and their membrane-localized ligands induce bidirectional signaling and facilitate tumor-stroma interactions. Expression of EphB4 is increased in prostate cancer tissue and cell lines and retained in castration resistant states, and can promote cell migration, invasion, and metastases. Blocking the EphB4-EphrinB2 pathway, which can be accomplished by soluble EphB4 conjugated to human serum albumin (sEphB4-HSA), has efficacy in preclinical models of aggressive prostate cancer. A phase I clinical trial of sEphB4-HSA led to response or stable disease in 56% of patients, with no grade 4 or 5 related adverse events, and combination pembrolizumab sEphB4-HSA led to a 52% response rate in EphrinB2 expressing urothelial cancer. We hypothesized that targeting the EphB4-EphrinB2 pathway may serve as a therapeutic target in the treatment of metastatic castration resistant prostate cancer (mCRPC). Methods: We conducted a single arm, phase II trial in patients with progressive mCRPC and treatment with at least one second generation androgen receptor (AR)-targeted therapy but no more than three prior therapies for mCRPC. On Day 1 of each cycle patients received sEphB4-HSA 1000 mg IV, with cycle length 14 days cycles 1-6 and cycle length 21 days for cycle 7 and beyond. The primary endpoint was confirmed prostate specific antigen (PSA) response rate (confirmed decrease in PSA by > 50%). We employed a Simon two stage Minimax design, requiring two or more responses among the first 15 patients to enroll an additional ten patients. Results: Fourteen eligible patients enrolled in the study. Median age was 73.5 years (range 52-83), patients had a median baseline PSA value of 65.11 ng/mL (range 7.77-2850 ng/mL) and received a median of three prior therapies (range 1-3) for mCRPC. Ten patients received prior taxane for mCRPC or hormone sensitive prostate cancer. The median length of treatment with sEphB4-HSA was 6.5 weeks (range 2-35 weeks). The potentially treatment-related adverse events (AEs) that occurred in more than 25% of patients were hypertension (10 patients) and fatigue (7 patients). Three patients experienced a serious adverse event potentially related to therapy, including one patient with a grade 5 event (cerebral vascular accident) possibly related to study drug. No patient had a confirmed PSA response, and the study was stopped for futility. Thirteen patients had PSA progression ( > 25% increase in PSA), and one patient withdrew due to toxicity prior to having an evaluable PSA response. The median time to PSA progression was 28 days (95% CI 28-64 days), and median time to radiologic progression was 55 days (95% CI 55 days-NR). Of three patients with measurable disease, two had stable disease and one had progressive disease. Conclusions: In patients with mCRPC who progressed on prior second generation AR-targeted therapy, sEphB4-HSA monotherapy had no discernable anti-tumor activity. Clinical trial information: NCT04033432.

Circular RNA-EPHB4 As A Potential Biotarget In Colorectal Cancer: A Preliminary Analysis

Colorectal cancer (CRC) is among the top causes of cancer-related deaths. With the advent of new biomedical technologies, new therapeutic targets are being actively discovered. Circular RNAs (circRNAs) are characterized by the absence of covalently closed-loop structures at the 3′ and 5′ ends. Recent studies have shown that circRNAs are abnormally expressed in tumor tissues. Certain circRNAs have been reported to have potential as cancer biomarkers. Among all the circRNAs previously discovered, circ-EPHB4 seemed promising. Therefore, this study is aimed to determine the function of circEPHB4 in CRC. By utilizing in-silico prediction tools, qPCR and cell-based assays, we were able to provide preliminary insights into the role of circEPHB4. The expression of both circEPHB4 and linear EPHB4 were upregulated in our Malaysian CRC tissues and commercial cell lines as compared to the non-cancerous counterparts. Reduction of both circEPHB4 and EPHB4 had effects on wound healing, drug response and clonogenic abilities of SW-480 and HCT-116 cells. Our initial findings suggest that circ-EPHB4 may be involved in the regulation of CRC though further in-depth studies are needed.