EphrinB2 in excitatory neurons and astrocytes in the basolateral amygdala controls long-term fear memory formation.

EphrinB2 regulates synaptic transmission and morphology however its role in memory formation is unknown. Here we show that deleting ephrinB2 from excitatory neurons in the basolateral amygdala (BLA) of male mice impairs long-term (LTM), but not short-term (STM), fear memory formation. Deleting ephrinB2 from astrocytes in the BLA impairs fear LTM but not STM. Removing ephrinB2 from astrocytes in the BLA reduces the level of the excitatory amino acid transporter 1 (EAAT1) in these cells. Inhibiting EAAT1 activity in the BLA during fear conditioning, by its specific inhibitor UCPH-101, impairs fear LTM showing that EAAT1 in the BLA is needed for fear LTM formation. The administration of ephrinB2 into the BLA during fear conditioning training enhances fear LTM. Moreover, ephrinB2 increases the ability of fear conditioning to activate cells in the BLA as detected by c-Fos labeling. EphrinB2 therefore determines the threshold for fear memory formation. In contrast to mature neurons, we show that ephrinB2 in neural stem cells (NSCs) is not needed for fear LTM. Our study shows that ephrinB2 in the BLA determines the strength of long-term memory consolidation.

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.

Development of Fusion-Based Assay as a Drug Screening Platform for Nipah Virus Utilizing Baculovirus Expression Vector System.

Nipah virus (NiV) is known to be a highly pathogenic zoonotic virus, which is included in the World Health Organization Research & Development Blueprint list of priority diseases with up to 70% mortality rate. Due to its high pathogenicity and outbreak potency, a therapeutic countermeasure against NiV is urgently needed. As NiV needs to be handled within a Biological Safety Level (BSL) 4 facility, we had developed a safe drug screening platform utilizing a baculovirus expression vector system (BEVS) based on a NiV-induced syncytium formation that could be handled within a BSL-1 facility. To reconstruct the NiV-induced syncytium formation in BEVS, two baculoviruses were generated to express recombinant proteins that are responsible for inducing the syncytium formation, including one baculovirus exhibiting co-expressed NiV fusion protein (NiV-F) and NiV attachment glycoprotein (NiV-G) and another exhibiting human EphrinB2 protein. Interestingly, syncytium formation was observed in infected insect cells when the medium was modified to have a lower pH level and supplemented with cholesterol. Fusion inhibitory properties of several compounds, such as phytochemicals and a polysulfonated naphthylamine compound, were evaluated using this platform. Among these compounds, suramin showed the highest fusion inhibitory activity against NiV-induced syncytium in the baculovirus expression system. Moreover, our in silico results provide a molecular-level glimpse of suramin's interaction with NiV-G's central hole and EphrinB2's G-H loop, which could be the possible reason for its fusion inhibitory activity.

Hypoxia-Induced Ephrin-B2 Facilitates Proliferation and Induces Glycolytic Metabolism in Cutaneous Squamous Cell Carcinoma by Modulating PKM2/HIF-1α Axis.

BACKGROUND: Cutaneous squamous cell carcinoma (cSCC) is a fatal disease characterized by metabolic dysregulation. The role of ephrin type-B receptor 2 (ephrin-B2), a crucial molecule in cancer cell biology, in regulating glycolysis and cell proliferation of cSCC is not well understood. This study aimed to investigate the biological pathways by which ephrin-B2 impacts the glycolysis and cell proliferation of cSCC. METHODS: Ephrin-B2 expression levels in cSCC were determined using quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and Western blotting. Ephrin-B2 expression in cSCC cells was manipulated using overexpression and knockdown approaches. A series of in vitro assays, such as cell counting kit-8 (CCK-8), Transwell assay, immunofluorescence assay, enzyme-linked immunosorbent assay (ELISA), qRT-PCR, and Western blotting, were employed to delineate the biological roles of ephrin-B2/pyruvate kinase muscle isoenzyme 2 (PKM2)/hypoxia-inducible factor 1 alpha (HIF-1α) in proliferation, migration, invasion, and glucose metabolism of cSCC. RESULTS: This study highlights an upregulation of ephrin-B2 expression in cSCC. Knockdown of ephrin-B2 significantly suppressed the proliferation, migration, invasion, and glucose metabolism of cSCC cells. Moreover, ephrin-B2 expression was upregulated under hypoxic conditions. At the molecular level, ephrin-B2 knockdown resulted in the downregulation of PKM2 and HIF-1α expression. Additionally, the overexpression of PKM2 or HIF-1α successfully rescued the diminished proliferation, migration, invasion and glucose metabolism induced by ephrin-B2 knockdown in cSCC cells. CONCLUSION: These findings suggest that ephrin-B2 suppression may hinder cSCC cell proliferation and glycolytic metabolism, potentially via the PKM2/HIF-1α axis modulation.

ephrin-B2 promotes nociceptive plasticity and hyperalgesic priming through EphB2-MNK-eIF4E signaling in both mice and humans.

Ephrin-B-EphB signaling can promote pain through ligand-receptor interactions between peripheral cells, like immune cells expressing ephrin-Bs, and EphB receptors expressed by DRG neurons. Previous studies have shown increased ephrin-B2 expression in peripheral tissues like synovium of rheumatoid and osteoarthritis patients, indicating the clinical significance of this signaling. The primary goal of this study was to understand how ephrin-B2 acts on mouse and human DRG neurons, which express EphB receptors, to promote pain and nociceptor plasticity. We hypothesized that ephrin-B2 would promote nociceptor plasticity and hyperalgesic priming through MNK-eIF4E signaling, a critical mechanism for nociceptive plasticity induced by growth factors, cytokines and nerve injury. Both male and female mice developed dose-dependent mechanical hypersensitivity in response to ephrin-B2, and both sexes showed hyperalgesic priming when challenged with PGE2 injection either to the paw or the cranial dura. Acute nociceptive behaviors and hyperalgesic priming were blocked in mice lacking MNK1 (Mknk1 knockout mice) and by eFT508, a specific MNK inhibitor. Sensory neuron-specific knockout of EphB2 using Pirt-Cre demonstrated that ephrin-B2 actions require this receptor. In Ca2+-imaging experiments on cultured DRG neurons, ephrin-B2 treatment enhanced Ca2+ transients in response to PGE2 and these effects were absent in DRG neurons from MNK1-/- and EphB2-PirtCre mice. In experiments on human DRG neurons, ephrin-B2 increased eIF4E phosphorylation and enhanced Ca2+ responses to PGE2 treatment, both blocked by eFT508. We conclude that ephrin-B2 acts directly on mouse and human sensory neurons to induce nociceptor plasticity via MNK-eIF4E signaling, offering new insight into how ephrin-B signaling promotes pain.

Enhancing precision targeting of ovarian cancer tumor cells in vivo through extracellular vesicle engineering.

Extracellular vesicles (EVs) function as natural mediators of intercellular communication, secreted by cells to facilitate cell-cell signaling. Due to their low toxicity, immunogenicity, biodegradability, and potential to encapsulate therapeutic drugs, EVs hold significant therapeutic promise. Nevertheless, their limited targeting ability often diminishes their therapeutic impact. Therefore, enhancing EVs by incorporating targeting units onto their membranes could bolster their targeting capabilities, enabling them to accumulate in specific cells and tissues. In this study, we engineered EVs to fuse ephrin-B2 with the EV membrane protein LAMP2b. This modification aimed to direct the engineered EVs toward the ephrin-B4 receptor expressed on the surface of ovarian cancer cells. The engineered EVs retained their inherent properties, including size, expression of EV membrane proteins, and morphology, upon isolation. In vitro experiments using real-time imaging revealed that EVs engineered with the ephrin-B2 ligand exhibited substantial internalization and uptake by ovarian cancer cells, in stark contrast to native EVs. In vivo, the engineered EVs carrying the ephrin-B2 ligand effectively targeted ovarian cancer cells, surpassing the targeting efficiency of control EVs. This innovative approach establishes a novel targeting system, enhancing the uptake of EVs by ovarian cancer cells. Our findings underscore the potential of using EVs to target cancer cells, thereby enhancing the effectiveness of anti-cancer therapies while minimizing off-target effects and toxicity in normal cells and organs.

Ephrin B2 (EFNB2) potentially protects against intervertebral disc degeneration through inhibiting nucleus pulposus cell apoptosis.

Nucleus pulposus (NP) cell apoptosis is a significant indication of accelerated intervertebral disc degeneration; however, the precise mechanism is unelucidated as of yet. Ephrin B2 (EFNB2), the only gene down-regulated in the three degraded intervertebral disc tissue microarray groups (GSE70362, GSE147383 and GSE56081), was screened for examination in this study. Subsequently, EFNB2 was verified to be down-regulated in degraded NP tissue samples. Interleukin-1 (IL-1ß) treatment of NP cells to simulate the IDD environment indicated that IL-1ß treatment decreased EFNB2 expression. In degenerative NP cells stimulated by IL-1ß, EFNB2 knockdown significantly increased the rate of apoptosis as well as the apoptosis-related molecules cleaved-caspase-3 and the Bax to Bcl-2 ratio. EFNB2 was found to promote AKT, PI3K, and mTOR phosphorylation; the PI3K/AKT signaling role was investigated using the PI3K inhibitor LY294002. EFNB2 overexpression significantly increased PI3K/AKT pathway activity in IL-1ß-stimulated NP cells than the normal control. Moreover, EFNB2 partially alleviated NP cell apoptosis induced by IL-1ß, reduced the cleaved-cas3 level, and decreased the Bax/Bcl-2 ratio after the addition of the inhibitor LY294002. Additionally, EFNB2 overexpression inhibited the ERK1/2 phosphorylation; the effects of EFNB2 overexpression on ERK1/2 phosphorylation, degenerative NP cell viability, and cell apoptosis were partially reversed by ERK signaling activator Ceramide C6. EFNB2 comprehensively inhibited the apoptosis of NP cells by activating the PI3K/AKT signaling and inhibiting the ERK signaling, obviating the exacerbation of IDD. EFNB2 could be a potential target to protect against degenerative disc changes.

Eph-ephrin signaling couples endothelial cell sorting and arterial specification.

Cell segregation allows the compartmentalization of cells with similar fates during morphogenesis, which can be enhanced by cell fate plasticity in response to local molecular and biomechanical cues. Endothelial tip cells in the growing retina, which lead vessel sprouts, give rise to arterial endothelial cells and thereby mediate arterial growth. Here, we have combined cell type-specific and inducible mouse genetics, flow experiments in vitro, single-cell RNA sequencing and biochemistry to show that the balance between ephrin-B2 and its receptor EphB4 is critical for arterial specification, cell sorting and arteriovenous patterning. At the molecular level, elevated ephrin-B2 function after loss of EphB4 enhances signaling responses by the Notch pathway, VEGF and the transcription factor Dach1, which is influenced by endothelial shear stress. Our findings reveal how Eph-ephrin interactions integrate cell segregation and arteriovenous specification in the vasculature, which has potential relevance for human vascular malformations caused by EPHB4 mutations.

EphrinB2 knockdown in cervical spinal cord preserves diaphragm innervation in a mutant SOD1 mouse model of ALS.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by motor neuron loss. Importantly, non-neuronal cell types such as astrocytes also play significant roles in disease pathogenesis. However, mechanisms of astrocyte contribution to ALS remain incompletely understood. Astrocyte involvement suggests that transcellular signaling may play a role in disease. We examined contribution of transmembrane signaling molecule ephrinB2 to ALS pathogenesis, in particular its role in driving motor neuron damage by spinal cord astrocytes. In symptomatic SOD1G93A mice (a well-established ALS model), ephrinB2 expression was dramatically increased in ventral horn astrocytes. Reducing ephrinB2 in the cervical spinal cord ventral horn via viral-mediated shRNA delivery reduced motor neuron loss and preserved respiratory function by maintaining phrenic motor neuron innervation of diaphragm. EphrinB2 expression was also elevated in human ALS spinal cord. These findings implicate ephrinB2 upregulation as both a transcellular signaling mechanism in mutant SOD1-associated ALS and a promising therapeutic target.

Inhibition of Ephrin B2 Reverse Signaling Abolishes Multiple Myeloma Pathogenesis.

Bone marrow vascular endothelial cells (BM EC) regulate multiple myeloma pathogenesis. Identification of the mechanisms underlying this interaction could lead to the development of improved strategies for treating multiple myeloma. Here, we performed a transcriptomic analysis of human ECs with high capacity to promote multiple myeloma growth, revealing overexpression of the receptor tyrosine kinases, EPHB1 and EPHB4, in multiple myeloma-supportive ECs. Expression of ephrin B2 (EFNB2), the binding partner for EPHB1 and EPHB4, was significantly increased in multiple myeloma cells. Silencing EPHB1 or EPHB4 in ECs suppressed multiple myeloma growth in coculture. Similarly, loss of EFNB2 in multiple myeloma cells blocked multiple myeloma proliferation and survival in vitro, abrogated multiple myeloma engraftment in immune-deficient mice, and increased multiple myeloma sensitivity to chemotherapy. Administration of an EFNB2-targeted single-chain variable fragment also suppressed multiple myeloma growth in vivo. In contrast, overexpression of EFNB2 in multiple myeloma cells increased STAT5 activation, increased multiple myeloma cell survival and proliferation, and decreased multiple myeloma sensitivity to chemotherapy. Conversely, expression of mutant EFNB2 lacking reverse signaling capacity in multiple myeloma cells increased multiple myeloma cell death and sensitivity to chemotherapy and abolished multiple myeloma growth in vivo. Complementary analysis of multiple myeloma patient data revealed that increased EFNB2 expression is associated with adverse-risk disease and decreased survival. This study suggests that EFNB2 reverse signaling controls multiple myeloma pathogenesis and can be therapeutically targeted to improve multiple myeloma outcomes. SIGNIFICANCE: Ephrin B2 reverse signaling mediated by endothelial cells directly regulates multiple myeloma progression and treatment resistance, which can be overcome through targeted inhibition of ephrin B2 to abolish myeloma.

In silico prediction of interaction between Nipah virus attachment glycoprotein and host cell receptors Ephrin-B2 and Ephrin-B3 in domestic and peridomestic mammals.

Nipah virus (NiV) is a lethal bat-borne zoonotic virus that causes mild to acute respiratory distress and neurological manifestations in humans with a high mortality rate. NiV transmission to humans occurs via consumption of bat-contaminated fruit and date palm sap (DPS), or through direct contact with infected individuals and livestock. Since NiV outbreaks were first reported in pigs from Malaysia and Singapore, non-neutralizing antibodies against NiV attachment Glycoprotein (G) have also been detected in a few domestic mammals. NiV infection is initiated after NiV G binds to the host cell receptors Ephrin-B2 and Ephrin-B3. In this study, we assessed the degree of NiV host tropism in domestic and peridomestic mammals commonly found in Bangladesh that may be crucial in the transmission of NiV by serving as intermediate hosts. We carried out a protein-protein docking analysis of NiV G complexes (n = 52) with Ephrin-B2 and B3 of 13 domestic and peridomestic species using bioinformatics tools. Protein models were generated by homology modelling and the structures were validated for model quality. The different protein-protein complexes in this study were stable, and their binding affinity (ΔG) scores ranged between -8.0 to -19.1 kcal/mol. NiV Bangladesh (NiV-B) strain displayed stronger binding to Ephrin receptors, especially with Ephrin-B3 than the NiV Malaysia (NiV-M) strain, correlating with the observed higher pathogenicity of NiV-B strains. From the docking result, we found that Ephrin receptors of domestic rat (R. norvegicus) had a higher binding affinity for NiV G, suggesting greater susceptibility to NiV infections compared to other study species. Investigations for NiV exposure to domestic/peridomestic animals will help us knowing more the possible role of rats and other animals as intermediate hosts of NiV and would improve future NiV outbreak control and prevention in humans and domestic animals.

Sequence basis for selectivity of ephrin-B2 ligand for Eph receptors and pathogenic henipavirus G glycoproteins.

IMPORTANCE: Ephrin-B2 (EFNB2) is a ligand for six Eph receptors in humans and regulates multiple cell developmental and signaling processes. It also functions as the cell entry receptor for Nipah virus and Hendra virus, zoonotic viruses that can cause respiratory and/or neurological symptoms in humans with high mortality. Here, we investigate the sequence basis of EFNB2 specificity for binding the Nipah virus attachment G glycoprotein over Eph receptors. We then use this information to engineer EFNB2 as a soluble decoy receptor that specifically binds the attachment glycoproteins of the Nipah virus and other related henipaviruses to neutralize infection. These findings further mechanistic understanding of protein selectivity and may facilitate the development of diagnostics or therapeutics against henipavirus infection.

Bioinformatic investigation of Nipah virus surface protein mutations: Molecular docking with Ephrin B2 receptor, molecular dynamics simulation, and structural impact analysis.

The SARS-CoV-2 outbreak resulted in significant challenges and loss of life. The Nipah virus, known for its high infectivity and severity, was designated an emergency concern by the World Health Organization. To understand its mutations, the Nipah virus proteins were analyzed extensively, with a focus on the essential G and F proteins responsible for viral entry into host cells. Our bioinformatics analysis unveiled multiple mutations, including simultaneous mutations within a single sequence. Notably, the G273S mutation in the F protein was identified as a potential cause of structural damage, which carries significant implications for vaccine development. Comparing the docking scores of G and F proteins with the Ephrin B2 receptor, it was found that the Y228H mutation in the G protein and the D252G mutation in the F protein likely affect virus entry into host cells. Moreover, our investigation into stability and deformability highlighted the impact of the Y228H mutation in the G protein complex. Molecular dynamics simulations revealed increased flexibility and conformational changes in the G protein complex with the Y228H mutation compared with the known complex. Furthermore, evaluating the root mean square deviation variation demonstrated greater dynamic behavior in the G protein complex and the Ephrin B2 receptor complex. This comprehensive study provides valuable insights into Nipah virus mutations, their significance for vaccine development, and the importance of understanding protein complex behavior in drug discovery. The identified mutations, especially G273S and Y228H, hold crucial implications for future research and potential interventions against the Nipah virus.

Computer-assisted identification of potential quinolone derivatives targeting Nipah virus glycoprotein attachment with human cell surface receptor ephrin-B2: Multistep virtual screening.

Nipah Virus (NiV) is a single-stranded, negative-sense, highly lethal RNA virus. Even though NiV has close to 70-80% of mortality in India and Bangladesh, still there is no available US FDA-approved drug or vaccine. NiV attachment glycoprotein (NiV-G) is critical for NiV to invade the human cell where ephrinB2 which is a crucial membrane-bound ligand that acts as a target of NiV. Most of the research has been performed targeting NiV or human ephrin-B to date. Quinolone derivatives are proven scaffolds for many approved drugs used to treat various bacterial, viral respiratory tract, and urinary tract infections, and rheumatologic disorders such as systemic lupus erythematosus, rheumatoid arthritis. Therefore, we have tried to find potential drug molecules employing quinolone scaffold-based derivatives from PubChem targeting both NiV-G and ephrin-B2 protein. A total of 1500+ quinolone derivatives were obtained from PubChem which were screened based on Drug Likeness followed by being subjected to XP docking employing Schrödinger software. The top ten best molecules were then chosen for their absorption, distribution, metabolism, excretion, and toxicity (ADMET) profiling based on the docking score ranking. Further, the top five molecules were selected for 200ns molecular dynamics (MD) simulation study with Desmond module followed by MM-GBSA study by Prime module of Schrödinger. The exhaustive analysis leads us to the top three probable lead drug molecules for NiV are PubChem CID 23646770, an analog of PubChem CID 67726448, and PubChem CID 10613168 which have predicted Ki values of 0.480 µm, 0.785 µm, and 0.380 µm, respectively. These proposed molecules can be the future drugs targeting NiV-G and human ephrin-B2 which requires further in vivo validation.

Overexpression of EphB6 and EphrinB2 controls soma spacing of cortical neurons in a mutual inhibitory way.

To establish functional circuitry, neurons settle down in a particular spatial domain by spacing their cell bodies, which requires proper positioning of the soma and establishing of a zone with unique connections. Deficits in this process are implicated in neurodevelopmental diseases. In this study, we examined the function of EphB6 in the development of cerebral cortex. Overexpression of EphB6 via in utero electroporation results in clumping of cortical neurons, while reducing its expression has no effect. In addition, overexpression of EphrinB2, a ligand of EphB6, also induces soma clumping in the cortex. Unexpectedly, the soma clumping phenotypes disappear when both of them are overexpressed in cortical neurons. The mutual inhibitory effect of EphB6/ EphrinB2 on preventing soma clumping is likely to be achieved via interaction of their specific domains. Thus, our results reveal a combinational role of EphrinB2/EphB6 overexpression in controlling soma spacing in cortical development.

Wnt4 and ephrinB2 instruct apical constriction via Dishevelled and non-canonical signaling.

Apical constriction is a cell shape change critical to vertebrate neural tube closure, and the contractile force required for this process is generated by actin-myosin networks. The signaling cue that instructs this process has remained elusive. Here, we identify Wnt4 and the transmembrane ephrinB2 protein as playing an instructive role in neural tube closure as members of a signaling complex we termed WERDS (Wnt4, EphrinB2, Ror2, Dishevelled (Dsh2), and Shroom3). Disruption of function or interaction among members of the WERDS complex results in defects of apical constriction and neural tube closure. The mechanism of action involves an interaction of ephrinB2 with the Dsh2 scaffold protein that enhances the formation of the WERDS complex, which in turn, activates Rho-associated kinase to induce apical constriction. Moreover, the ephrinB2/Dsh2 interaction promotes non-canonical Wnt signaling and shows how cross-talk between two major signal transduction pathways, Eph/ephrin and Wnt, coordinate morphogenesis of the neural tube.

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.

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.

Kindlin2 enables EphB/ephrinB bi-directional signaling to support vascular development.

Direct contact between cells expressing either ephrin ligands or Eph receptor tyrosine kinase produces diverse developmental responses. Transmembrane ephrinB ligands play active roles in transducing bi-directional signals downstream of EphB/ephrinB interaction. However, it has not been well understood how ephrinB relays transcellular signals to neighboring cells and what intracellular effectors are involved. Here, we report that kindlin2 can mediate bi-directional ephrinB signaling through binding to a highly conserved NIYY motif in the ephrinB2 cytoplasmic tail. We show this interaction is important for EphB/ephrinB-mediated integrin activation in mammalian cells and for blood vessel morphogenesis during zebrafish development. A mixed two-cell population study revealed that kindlin2 (in ephrinB2-expressing cells) modulates transcellular EphB4 activation by promoting ephrinB2 clustering. This mechanism is also operative for EphB2/ephrinB1, suggesting that kindlin2-mediated regulation is conserved for EphB/ephrinB signaling pathways. Together, these findings show that kindlin2 enables EphB4/ephrinB2 bi-directional signal transmission.

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

BACKGROUND: 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. METHODS AND RESULTS: 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. CONCLUSIONS: 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.