Activation of EphrinB2/EphB2 signaling in the spine cord alters glia-neuron interactions in mice with visceral hyperalgesia following maternal separation.

Background: Sress early in life has been linked to visceral hyperalgesia and associated functional gastrointestinal disorders. In a mouse model of visceral hyperalgesia, we investigated whether the EphB2 receptor and its EphrinB2 ligand in spinal cord contribute to dysregulation of glia-neuron interactions. Methods: An established mouse model of stress due to maternal separation (MS). Pups were separated from their mothers for 14 days during early development, then analyzed several weeks later in terms of visceral sensitivity based on the abdominal withdrawal reflex score and in terms of expression of c-fos, EphrinB2, EphB2, and phosphorylated MAP kinases (ERK, p38, JNK). Results: Visceral hyperalgesia due to MS upregulated EphB2, EphrinB2 and c-fos in the spinal cord, and c-fos levels positively correlated with those of EphB2 and EphrinB2. Spinal astrocytes, microglia, and neurons showed upregulation of EphB2, EphrinB2 and phosphorylated MAP kinases. Blocking EphrinB2/EphB2 signaling in MS mice reduced visceral sensitivity, activation of neurons and glia, and phosphorylation of NMDA receptor. Activating EphrinB2/EphB2 signaling in unstressed mice induced visceral hyperalgesia, upregulation of c-fos, and activation of NMDA receptor similar to maternal separation. Conclusion: The stress of MS during early development may lead to visceral hyperalgesia by upregulating EphrinB2/EphB2 in the spinal cord and thereby altering neuron-glia interactions.

EphrinB2-mediated chondrocyte autophagy induces post-traumatic arthritis via rupture of cartilage homeostasis.

EphrinB2, a member of the Ephrin family, has been linked to several orthopaedic conditions. Nevertheless, the correlation between ephrinB2 and post-traumatic arthritis (PTOA) remains unclear. Human PTOA cartilage from human and mouse knee joints was systematically analysed to investigate the relationship between EphrinB2 and PTOA using SO-FG and toluidine blue staining, micro-CT, histomorphometry, immunohistochemistry, immunofluorescence, lentiviral articular injection and in situ end labeling (TUNEL) assays. EphrinB2 expression was significantly downregulated in PTOA chondrocytes. Blocking EphrinB2 increased the breakdown of cartilage matrix in mice with PTOA via reducing the process of chondrocyte autophagy. The presence of severe cartilage damage was evident, as indicated by a considerable decrease in both cartilage thickness and area, accompanied by an increase in chondrocyte death. Altogether, EphrinB2 is required for the maintenance of cartilage homeostasis in post-traumatic arthritis, and EphrinB2 ablation is associated with accelerated chondrocyte matrix degeneration, finally causing damage to the articular cartilage.

Improved efficacy of pembrolizumab combined with soluble EphB4-albumin in HPV-negative EphrinB2 positive head neck squamous cell carcinoma.

OBJECTIVE: Patients with relapsed or metastatic head and neck squamous cell carcinoma (HNSCC) after primary local therapy have low response rates with cetuximab, systemic chemotherapy or check point inhibitor therapy. Novel combination therapies with the potential to improve outcomes for patients with HNSCC is an area of high unmet need. METHODS: This is a phase II single-arm clinical trial of locally advanced or metastatic HNSCC patients treated with a combination of soluble EphB4-human serum albumin (sEphB4-HSA) fusion protein and pembrolizumab after platinum-based chemotherapy with up to 2 prior lines of treatment. The primary endpoints were safety and tolerability and the primary efficacy endpoint was overall response rate (ORR). Secondary endpoints included progression free survival (PFS) and overall survival (OS). HPV status and EphrinB2 expression were evaluated for outcome. RESULTS: Twenty-five patients were enrolled. Median follow up was 40.4 months (range 9.8 - 40.4). There were 6 responders (ORR 24%). There were 5 responders in the 11 HPV-negative and EphrinB2 positive patients, (ORR 45%) with 2 of these patients achieving a complete response (CR). The median PFS in HPV-negative/EphrinB2 positive patients was 3.2 months (95% CI 1.1, 7.3). Median OS in HPV-negative/EphrinB2 positive patients was 10.9 months (95% CI 2.0, 13.7). Hypertension, transaminitis and fatigue were the most common toxicities. DISCUSSION: The combination of sEphB4-HSA and pembrolizumab has a favorable toxicity profile and favorable activity particularly among HPV-negative EphrinB2 positive patients with HNSCC.

Proangiogenic effect and underlying mechanism of holmium oxide nanoparticles: a new biomaterial for tissue engineering.

BACKGROUND: Early angiogenesis provides nutrient supply for bone tissue repair, and insufficient angiogenesis will lead tissue engineering failure. Lanthanide metal nanoparticles (LM NPs) are the preferred materials for tissue engineering and can effectively promote angiogenesis. Holmium oxide nanoparticles (HNPs) are LM NPs with the function of bone tissue "tracking" labelling. Preliminary studies have shown that HNPs has potential of promote angiogenesis, but the specific role and mechanism remain unclear. This limits the biological application of HNPs. RESULTS: In this study, we confirmed that HNPs promoted early vessel formation, especially that of H-type vessels in vivo, thereby accelerating bone tissue repair. Moreover, HNPs promoted angiogenesis by increasing cell migration, which was mediated by filopodia extension in vitro. At the molecular level, HNPs interact with the membrane protein EphrinB2 in human umbilical vein endothelial cells (HUVECs), and phosphorylated EphrinB2 can bind and activate VAV2, which is an activator of the filopodia regulatory protein CDC42. When these three molecules were inhibited separately, angiogenesis was reduced. CONCLUSION: Overall, our study confirmed that HNPs increased cell migration to promote angiogenesis for the first time, which is beneficial for bone repair. The EphrinB2/VAV2/CDC42 signalling pathway regulates cell migration, which is an important target of angiogenesis. Thus, HNPs are a new candidate biomaterial for tissue engineering, providing new insights into their biological application.

Predictive value of plasma ephrinB2 levels for amputation risk following endovascular revascularization in peripheral artery disease.

Background: The aim of this study is to investigate the expression levels of ephrinB2 in patients with lower extremity peripheral arterial disease (PAD) and explore its association with the severity of the disease and the risk of amputation after endovascular revascularization. Methods: During the period from March 2021 to March 2023, this study collected blood samples and clinical data from 133 patients diagnosed with lower extremity PAD and 51 healthy volunteer donors. The severity of lower extremity PAD patients was classified using the Rutherford categories. The expression of ephrin-B2 in plasma samples was detected using the Western Blotting. Results: Compared to the control group, the levels of serum ephrinB2 in patients were significantly elevated (p < 0.001). Moreover, the plasma EphrinB2 levels were positively correlated with white blood cell counts (r = 0.204, p = 0.018), neutrophil counts (r = 0.174, p = 0.045), and neutrophil-to-lymphocyte ratio (NLR) (r = 0.223, p = 0.009). Furthermore, the AUCs of plasma ephrinB2 level, NLR, and their combination as predictors for amputation events within 30 months after lower extremity PAD endovascular revascularization were 0.659, 0.730 and 0.811. In the high-ephrinB2 group, the incidence of amputation events within 30 months after endovascular revascularization was higher. Conclusions: Plasma EphrinB2 levels may be linked to lower extremity PAD development, inflammation, and postoperative amputation. Combining EphrinB2 and NLR can improve amputation prediction accuracy after endovascular revascularization in lower extremity PAD patients.

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.

Enzyme-Linked Immunosorbent Assay Using Henipavirus-Receptor EphrinB2 and Monoclonal Antibodies for Detecting Nipah and Hendra Viruses.

The Nipah virus (NiV) and the Hendra virus (HeV) are highly pathogenic zoonotic diseases that can cause fatal infections in humans and animals. Early detection is critical for the control of NiV and HeV infections. We present the development of two antigen-detection ELISAs (AgELISAs) using the henipavirus-receptor EphrinB2 and monoclonal antibodies (mAbs) to detect NiV and HeV. The NiV AgELISA detected only NiV, whereas the NiV/HeV AgELISA detected both NiV and HeV. The diagnostic specificities of the NiV AgELISA and the NiV/HeV AgELISA were 100% and 97.8%, respectively. Both assays were specific for henipaviruses and showed no cross-reactivity with other viruses. The AgELISAs detected NiV antigen in experimental pig nasal wash samples taken at 4 days post-infection. With the combination of both AgELISAs, NiV can be differentiated from HeV. Complementing other henipavirus detection methods, these two newly developed AgELISAs can rapidly detect NiV and HeV in a large number of samples and are suitable for use in remote areas where other tests are not available.

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.

EphB2 promotes enteric nitrergic hyperinnervation and neurogenic inflammation in DSS-induced chronic colitis in mice.

BACKGROUND: Enteric nervous system (ENS) has been closely associated with the neuro-immune response and is currently considered a reliable target for intestinal inflammation. Neuronal nitric oxide synthase (nNOS) nerves are involved in inflammatory diseases by releasing nitric oxide (NO). EphB2 expression and density of innervation of the mucosal layer are positively correlated with the severity of intestinal inflammatory responses. In this study, we hypothesized that a EphB2-mediated mechanism may regulate enteric immunity through modulation of nNOS nerves. METHODS: Firstly, the Western blot (WB) method was employed to quantify EphB2 expression in the intestinal mucosal layer of DSS mice and assess alterations in nerve fiber activation and density. Immunofluorescence (IF) double staining with nNOS and neuronal marker PGP9.5 was conducted to measure nNOS nerve fiber density within the intestinal mucosal layer of mice. Subsequently, in vivo experiments were performed to investigate the inhibitory or activatory effect of EphB2Fc or EphrinB2Fc on EphB2 expression and activation. Immunoprecipitation experiments confirmed the interaction between EphB2 and nNOS nerves. WB and IF experiments were carried out to evaluate both inflammatory conditions of mouse colonic mucosa following intervention with EphB2Fc/EphrinB2Fc as well as changes in nNOS nerve fibers expression. Finally, in vitro experiments, neurally-mediated inflammation was assessed in the organ bath system by activating intestinal mucosal innervation through Veratridine (VER) and electrical field stimulation (EFS) techniques for 3 h. The activation of nNOS nerves was inhibited by nitroindazole (7NI). WB was employed to detect changes in the expression of inflammatory factors in the intestinal mucosal layer in EphB2Fc/EphrinB2Fc treated mice and control group. KEY RESULTS: We found that the expression of EphB2 and density nNOS nerve fibers in the intestinal mucosa were positively correlated with the colitis response. Blocking (EphB2Fc)/activating (EphrinB2Fc) EphB2 in vivo significantly reduced/increased the density of nNOS nerve fibers and expression of inflammatory factors in colonic mucosa of DSS treated mice. In vitro, blocking nNOS nerves activation attenuated the inflammatory reaction induced by either EFS or EphB2. CONCLUSIONS: Our findings provided evidence that EphB2 mediated regulation of innate immunity-ENS crosstalk might represent an attractive target for novel therapeutic strategies in ulcerative colitis.

Discovery and Characterization of Ephrin B2 and EphB4 Dysregulation and Novel Mutations in Cerebral Cavernous Malformations: In Vitro and Patient-Derived Evidence of Ephrin-Mediated Endothelial Cell Pathophysiology.

Intracranial vascular malformations manifest on a continuum ranging from predominantly arterial to predominantly venous in pathology. Cerebral cavernous malformations (CCMs) are capillary malformations that exist at the midpoint of this continuum. The axon guidance factor Ephrin B2 and its receptor EphB4 are critical regulators of vasculogenesis in the developing central nervous system. Ephrin B2/EphB4 dysregulation has been implicated in the pathogenesis of arterial-derived arteriovenous malformations and vein-based vein of Galen malformations. Increasing evidence supports the hypothesis that aberrant Ephrin B2/EphB4 signaling may contribute to developing vascular malformations, but their role in CCMs remains largely uncharacterized. Evidence of Ephrin dysregulation in CCMs would be important to establish a common link in the pathogenic spectrum of EphrinB2/Ephb4 dysregulation. By studying patient-derived primary CCM endothelial cells (CCMECs), we established that CCMECs are functionally distinct from healthy endothelial cell controls; CCMECs demonstrated altered patterns of migration, motility, and impaired tube formation. In addition to the altered phenotype, the CCMECs also displayed an increased ratio of EphrinB2/EphB4 compared to the healthy endothelial control cells. Furthermore, whole exome sequencing identified mutations in both EphrinB2 and EphB4 in the CCMECs. These findings identify functional alterations in the EphrinB2/EphB4 ratio as a feature linking pathophysiology across the spectrum of arterial, capillary, and venous structural malformations in the central nervous system while revealing a putative therapeutic target.

A long non-coding RNA Leat1 mediates the hormone responsiveness of EfnB2 during male urogenital development.

The novel long non-coding RNA (lncRNA) Leat1 is extraordinarily conserved in both its location (syntenic with EfnB2, an essential gene in anogenital patterning) and sequence. Here we show that Leat1 is upregulated following the testosterone surge from the developing testis and directly interacts with EfnB2, positively regulating its expression. Leat1 expression is suppressed by estrogen, which in turn suppresses the expression of EfnB2. Moreover, the loss of Leat1 leads to reduced EfnB2, resulting in a severe hypospadias phenotype. The human LEAT1 gene is also co-expressed with EFNB2 in the developing human penis suggesting a conserved function for this gene in urethral closure. Together our data identify Leat1 as a novel molecular regulator of urethral closure and implicate it as a target of endocrine disruption in the etiology of hypospadias.

Bioprinted PDLSCs with high-concentration GelMA hydrogels exhibit enhanced osteogenic differentiation in vitro and promote bone regeneration in vivo.

OBJECTIVES: We aimed to explore the osteogenic potential of periodontal ligament stem cells (PDLSCs) in bioprinted methacrylate gelatine (GelMA) hydrogels in vitro and in vivo. MATERIALS AND METHODS: PDLSCs in GelMA hydrogels at various concentrations (3%, 5%, and 10%) were bioprinted. The mechanical properties (stiffness, nanostructure, swelling, and degradation properties) of bioprinted constructs and the biological properties (cell viability, proliferation, spreading, osteogenic differentiation, and cell survival in vivo) of PDLSCs in bioprinted constructs were evaluated. Then, the effect of bioprinted constructs on bone regeneration was investigated using a mouse cranial defect model. RESULTS: Ten percent GelMA printed constructs had a higher compression modulus, smaller porosity, lower swelling rate, and lower degradation rate than 3% GelMA. PDLSCs in bioprinted 10% GelMA bioprinted constructs showed lower cell viability, less cell spreading, upregulated osteogenic differentiation in vitro, and lower cell survival in vivo. Moreover, upregulated expression of ephrinB2 and EphB4 protein and their phosphorylated forms were found in PDLSCs in 10% GelMA bioprinted constructs, and inhibition of eprhinB2/EphB4 signalling reversed the enhanced osteogenic differentiation of PDLSCs in 10% GelMA. The in vivo experiment showed that 10% GelMA bioprinted constructs with PDLSCs contributed to more new bone formation than 10% GelMA constructs without PDLSCs and constructs with lower GelMA concentrations. CONCLUSIONS: Bioprinted PDLSCs with high-concentrated GelMA hydrogels exhibited enhanced osteogenic differentiation partially through upregulated ephrinB2/EphB4 signalling in vitro and promoted bone regeneration in vivo, which might be more appropriate for future bone regeneration applications. CLINICAL RELEVANCE: Bone defects are a common clinical oral problem. Our results provide a promising strategy for bone regeneration through bioprinting PDLSCs in GelMA hydrogels.

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.

EphrinB2 drives osteogenic fate of adult cardiac fibroblasts in a calcium influx dependent manner.

Cardiac calcification is a crucial but underrecognized pathological process, greatly increasing the risk of cardiovascular diseases. Little is known about how cardiac fibroblasts, as a central mediator, facilitate abnormal mineralization. Erythropoietin-producing hepatoma interactor B2 (EphrinB2), previously identified as an angiogenic regulator, is involved in fibroblast activation, while its role in the osteogenic differentiation of cardiac fibroblasts is unknown. Bioinformatics analysis was conducted to characterize the expression of the Ephrin family in human calcified aortic valves and calcific mouse hearts. The effects of EphrinB2 on cardiac fibroblasts to adopt osteogenic fate was determined by gain- and loss-of-function. EphrinB2 mRNA level was downregulated in calcified aortic valves and mouse hearts. Knockdown of EphrinB2 attenuated mineral deposits in adult cardiac fibroblasts, whereas overexpression of EphrinB2 promoted their osteogenic differentiation. RNA sequencing data implied that Ca2+-related S100/receptor for advanced glycation end products (RAGE) signaling may mediate EphrinB2-induced mineralization in cardiac fibroblasts. Moreover, L-type calcium channel blockers inhibited osteogenic differentiation of cardiac fibroblasts, implying a critical role in Ca2+ influx. In conclusion, our data illustrated an unrecognized role of EphrinB2, which functions as a novel osteogenic regulator in the heart through Ca2+ signaling and could be a potential therapeutic target in cardiovascular calcification.NEW & NOTEWORTHY In this study, we observed that adult cardiac fibroblasts but not neonatal cardiac fibroblasts exhibit the ability of osteogenic differentiation. EphrinB2 promoted osteogenic differentiation of cardiac fibroblasts through activating Ca2+-related S100/RAGE signaling. Inhibition of Ca2+ influx using L-type calcium channel blockers inhibited EphrinB2-mediated calcification of cardiac fibroblasts. Our data implied an unrecognized role of EphrinB2 in regulating cardiac calcification though Ca2+-related signaling, suggesting a potential therapeutic target of cardiovascular calcification.

The Therapeutic Effects of EFNB2-Fc in a Cell Model of Kawasaki Disease.

The EphrinB2/EphB4 signaling pathway involves the regulation of vascular morphogenesis and angiogenesis. However, little is known about EphrinB2/EphB4 in the pathogenesis of Kawasaki disease (KD) and coronary artery aneurysm formation. Hence, this study aimed to explore the role of EphrinB2/EphB4 and the potential therapeutic effect of EphrinB2-Fc in the coronary arterial endothelial injury of KD. The levels of EphB4 were compared between KD patients and healthy children. Human coronary artery endothelial cells (HCAECs) were stimulated with sera from acute KD patients to establish the KD cell model. The overexpression of EphB4 or treatment with EphrinB2-Fc was found to intervene in the cell model. The cell migration, angiogenesis, and proliferation ability were assessed, and the expression of inflammation-related factors was measured. Our study showed that EphB4 showed low expression in both KD patients and the cell model of KD. The EphB4 protein levels in the CECs of CAA+ KD patients were much lower than those in healthy children. EphrinB2-Fc treatment of KD sera-activated HCAECs suppressed cell proliferation, reduced the expression of inflammation-related factors (such as IL-6 and P-selectin), and elevated cell angiogenesis ability. The results reveal that EphrinB2-Fc has a protective function in endothelial cells and has promising clinical applications for protecting vascular endothelium in patients with KD.

Bioprinting EphrinB2-Modified Dental Pulp Stem Cells with Enhanced Osteogenic Capacity for Alveolar Bone Engineering.

Bioprinting, a technology that allows depositing living cells and biomaterials together into a complex tissue architecture with desired pattern, becomes a revolutionary technology for fabrication of engineered constructs. Previously, we have demonstrated that EphrinB2-modified dental pulp stem cells (DPSCs) are expected to be promising seed cells with enhanced osteogenic differentiation capability for alveolar bone regeneration. In this study, we aimed to bioprint EphrinB2-overexpressing DPSCs with low-concentrated Gelatin methacrylate (GelMA) hydrogels into three-dimensional (3D) constructs. The printability of GelMA (5% w/v) and the structural fidelity of bioprinted constructs were examined. Then, viability, proliferation, morphology, and osteogenic differentiation of DPSCs in bioprinted constructs were measured. Finally, the effect of EphrinB2 overexpression on osteogenic differentiation of DPSCs in bioprinted constructs was evaluated. Our results demonstrated that GelMA (5% w/v) in a physical gel form was successfully bioprinted into constructs with various shapes and patterns using optimized printing parameters. Embedded DPSCs showed round-like morphology, and had a high viability (91.93% ± 8.38%) and obvious proliferation (∼1.9-fold increase) 1 day after printing. They also showed excellent osteogenic potential in bioprinted constructs. In bioprinted 3D constructs, EphrinB2-overexpressing DPSCs expressed upregulated osteogenic markers, including ALP, BMP2, RUNX2, and SP7, and generated more mineralized nodules, as compared with Vector-DPSCs. Taken together, this study indicated that fabrication of bioprinted EphrinB2-DPSCs-laden constructs with enhanced osteogenic potential was possible, and 3D bioprinting strategy combined with EphrinB2 gene modification was a promising way to create bioengineered constructs for alveolar bone regeneration.

The role of ephrinB2-EphB4 signalling in bone remodelling during orthodontic tooth movement.

OBJECTIVE: The aim of this study was to investigate the role of ephrinB2-EphB4 signalling in alveolar bone remodelling on the tension side during orthodontic tooth movement (OTM). MATERIALS AND METHODS: An OTM model was established on sixty 8-week-old male Wistar rats. They were randomly divided into the experimental group and the control group. The animals in the experimental group were administrated with subcutaneous injection of EphB4 inhibitor NVP-BHG712 every other day, whereas the control group received only the vehicle. Samples containing the maxillary first molar and the surrounding bone were collected after 0, 3, 7, 14 and 21 days of tooth movement. RESULTS: EphrinB2-EphB4 signalling was actively expressed on the tension side during tooth movement. Micro-CT analysis showed the distance of tooth movement in the experimental group was significantly greater than that of the control group (P < .05) with significantly increased trabecular separation (Tb. Sp) and decreased trabecular number (Tb. N) from day 14 to day 21. The number of osteoclasts significantly increased in the experimental group compared with the control group after 3 and 7 days of tooth movement (P < .05). The expressions of alkaline phosphatase (ALP) and osteopontin (OPN) were significantly reduced by inhibition of EphB4 (P < .05). CONCLUSION: The inhibition of EphB4 suppressed bone formation and enhanced bone resorption activities on the tension side of tooth movement. The ephrinB2-EphB4 signalling might play an important role in alveolar bone remodelling during OTM.

EphrinB2 promotes the human aortic smooth muscle cell growth and migration via mediating F-actin remodeling.

OBJECTIVES: To evaluate the potential effect of EphrinB2 in human thoracic aortic dissection (TAD) and to illustrate the mechanisms governing the role of EphrinB2 in the growth of human aortic smooth muscle cells (HASMC). METHODS: In the study, EphrinB2 expression was investigated by qRT-PCR and immunohistochemistry in 12 pairs of TAD and adjacent human tissues. HASMCs were used for in vitro experiments. Next, EphrinB2 overexpression and depletion in HASMCs were established by EphrinB2-overexpressing vectors and small interfering RNA, respectively. The transfection efficiency was evaluated by qRT-PCR and Western blot. The effects of overexpression and depletion of EphrinB2 on cell proliferation, migration, and invasion were tested in vitro. Cell Counting Kit-8, flow cytometry and transwell migration/invasion, and wound healing assay were used to explore the function of EphrinB2 on HASMC cell lines. The relationship between EphrinB2 and F-actin was assessed by Western blot, immunofluorescence, and Co-IP. RESULTS: We found that EphrinB2 was a prognostic biomarker of TAD patients. Moreover, EphrinB2 expression negatively correlated to aortic dissection tissues, and disease incidence of males, suggesting that EphrinB2 might act as a TAD suppressor by promoting proliferation or decreasing apoptosis in HASMC. Next, over-expression of EphrinB2 in HASMC lines drove cell proliferation, migration, and invasion, and inhibited apoptosis while knockdown EphrinB2 showed the opposite phenomenon, respectively. Furthermore, the level of F-actin in mRNA, protein, and distribution in HASMC cell lines highly matched with the expression of EphrinB2, which indicated that EphrinB2 could mediate the HASMC cytoskeleton via inducing F-actin. CONCLUSIONS: In conclusion, our results first provided the pivotal role of EphrinB2 in HASMC proliferation initiated by mediating F-actin and demonstrated a prognostic biomarker and the potential targets for therapy to prevent thoracic aortic dissection.

EphA4/EphrinB2 signaling mediates pericyte-induced transient glia limitans formation as a secondary protective barrier after subarachnoid hemorrhage in mice.

BACKGROUND: Most patients with subarachnoid hemorrhage (SAH) do not exhibit brain parenchymal injury upon imaging but present significant blood-brain barrier (BBB) disruption and secondary neurological deficits. The aim of this study was to investigate whether stressed astrocytes act as a secondary barrier to exert a protective effect after SAH and to investigate the mechanism of glial limitan formation. METHODS: A total of 204 adult male C57BL/6 mice and an endovascular perforation SAH model were employed. The spatiotemporal characteristics of glial limitan formation after SAH were determined by immunofluorescence staining and transmission electron microscopy. The molecular mechanisms by which pericytes regulate glia limitans formation were analyzed using polymerase chain reaction, Western blotting, immunofluorescence staining and ELISA in a pericyte-astrocyte contact coculture system. The findings were validated ex vivo and in vivo using lentiviruses and inhibitors. Finally, pericytes were targeted to regulate glial limitan formation, and the effect of the glia limitans on secondary brain injury after SAH was evaluated by flow cytometry and analysis of neurological function. RESULTS: Stress-induced glial limitan formation occurred 1 day after SAH and markedly subsided 3 days after ictus. Pericytes regulated astrocyte glia limitan formation via EphA4/EphrinB2 signaling, inhibited inflammatory cell infiltration and altered neurological function. CONCLUSIONS: Astrocyte-derived glia limitans serve as a secondary protective barrier following BBB disruption after SAH in mice, and pericytes can regulate glial limitan formation and alter neurological function via EphA4/EphrinB2 signaling. Strategies for maintaining this secondary protective barrier may be novel treatment approaches for alleviating early brain injury after SAH.