EphrinB2/ephB2 activation facilitates colonic synaptic potentiation and plasticity contributing to long-term visceral hypersensitivity in irritable bowel syndrome.

AIMS: Sustained visceral hypersensitivity is a hallmark of irritable bowel syndrome (IBS) could be partially explained by enteric neural remodeling. Particularly, synaptic plasticity in the enteric nervous system, a form of enteric "memory", has been speculated as a participant in the pain maintenance in IBS. This study aimed to elucidate the role of ephrinB2/ephB2 in enteric synaptic plasticity and visceral pain in IBS. MATERIALS AND METHODS: EphrinB2/ephB2 expression and synaptic plasticity were assessed in colonic tissues from IBS patients, and rats induced by Trichinella spiralis infection and those treated with ephB2-Fc (an ephB2 receptor blocker) or ifenprodil (a selective NR2B antagonist). Furthermore, abdominal withdrawal reflex scores to colorectal distention and mesenteric afferent firing were assessed. EphrinB2-Fc(an ephB2 receptor activator) induced enteric synaptic plasticity was further evaluated in longitudinal muscle-myenteric plexus(LMMP) cultures and primary cultured myenteric neurons. KEY FINDINGS: EphrinB2/ephB2 was specifically expressed in colonic nerves and upregulated in IBS patients and rats, which was correlated with pain severity. The functional synaptic plasticity, visceral sensitivity to colorectal distention and colonic mesenteric afferent activity to mechanical and chemical stimulus were enhanced in IBS rats, and were blocked by ephB2-Fc or ifenprodil treatment. EphrinB2-Fc promoted the phosphorylation of NR2B in IBS rats and LMMP cultures, and could mediate sustained neural activation via increased [Ca2+]i and raised expression of synaptic plasticity-related early immediate genes, including c-fos and arc. SIGNIFICANCE: EphrinB2/ephB2 facilitated NR2B-mediated synaptic potentiation in the enteric nervous system that may be a novel explanation and potential therapeutic target for sustained pain hypersensitivity in IBS.

Activation of EphrinB2 Signaling Promotes Adaptive Venous Remodeling in Murine Arteriovenous Fistulae.

BACKGROUND: Arteriovenous fistulae (AVF) are the preferred mode of vascular access for hemodialysis. Before use, AVF remodel by thickening and dilating to achieve a functional conduit via an adaptive process characterized by expression of molecular markers characteristic of both venous and arterial identity. Although signaling via EphB4, a determinant of venous identity, mediates AVF maturation, the role of its counterpart EphrinB2, a determinant of arterial identity, remains unclear. We hypothesize that EphrinB2 signaling is active during AVF maturation and may be a mechanism of venous remodeling. METHODS: Aortocaval fistulae were created or sham laparotomy was performed in C57Bl/6 mice, and specimens were examined on Days 7 or 21. EphrinB2 reverse signaling was activated with EphB4-Fc applied periadventitially in vivo and in endothelial cell culture medium in vitro. Downstream signaling was assessed using immunoblotting and immunofluorescence. RESULTS: Venous remodeling during AVF maturation was characterized by increased expression of EphrinB2 as well as Akt1, extracellular signal-regulated kinases 1/2 (ERK1/2), and p38. Activation of EphrinB2 with EphB4-Fc increased phosphorylation of EphrinB2, endothelial nitric oxide synthase, Akt1, ERK1/2, and p38 and was associated with increased diameter and wall thickness in the AVF. Both mouse and human endothelial cells treated with EphB4-Fc increased phosphorylation of EphrinB2, endothelial nitric oxide synthase, Akt1, ERK1/2, and p38 and increased endothelial cell tube formation and migration. CONCLUSIONS: Activation of EphrinB2 signaling by EphB4-Fc was associated with adaptive venous remodeling in vivo while activating endothelial cell function in vitro. Regulation of EphrinB2 signaling may be a new strategy to improve AVF maturation and patency.

EphrinB1 modulates glutamatergic inputs into POMC-expressing progenitors and controls glucose homeostasis.

Proopiomelanocortin (POMC) neurons are major regulators of energy balance and glucose homeostasis. In addition to being regulated by hormones and nutrients, POMC neurons are controlled by glutamatergic input originating from multiple brain regions. However, the factors involved in the formation of glutamatergic inputs and how they contribute to bodily functions remain largely unknown. Here, we show that during the development of glutamatergic inputs, POMC neurons exhibit enriched expression of the Efnb1 (EphrinB1) and Efnb2 (EphrinB2) genes, which are known to control excitatory synapse formation. In vivo loss of Efnb1 in POMC-expressing progenitors decreases the amount of glutamatergic inputs, associated with a reduced number of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptor subunits and excitability of these cells. We found that mice lacking Efnb1 in POMC-expressing progenitors display impaired glucose tolerance due to blunted vagus nerve activity and decreased insulin secretion. However, despite reduced excitatory inputs, mice lacking Efnb2 in POMC-expressing progenitors showed no deregulation of insulin secretion and only mild alterations in feeding behavior and gluconeogenesis. Collectively, our data demonstrate the role of ephrins in controlling excitatory input amount into POMC-expressing progenitors and show an isotype-specific role of ephrins on the regulation of glucose homeostasis and feeding.

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

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

The osteoprogenitor-specific loss of ephrinB1 results in an osteoporotic phenotype affecting the balance between bone formation and resorption.

The present study investigated the effects of conditional deletion of ephrinB1 in osteoprogenitor cells driven by the Osterix (Osx) promoter, on skeletal integrity in a murine model of ovariectomy-induced (OVX) osteoporosis. Histomorphometric and µCT analyses revealed that loss of ephrinB1 in sham Osx:cre-ephrinB1fl/fl mice caused a reduction in trabecular bone comparable to OVX Osx:Cre mice, which was associated with a significant reduction in bone formation rates and decrease in osteoblast numbers. Interestingly, these observations were not exacerbated in OVX Osx:cre-ephrinB1fl/fl mice. Furthermore, sham Osx:cre-ephrinB1fl/fl mice displayed significantly higher osteoclast numbers and circulating degraded collagen type 1 compared to OVX Osx:Cre mice. Confirmation studies found that cultured monocytes expressing EphB2 formed fewer TRAP+ multinucleated osteoclasts and exhibited lower resorption activity in the presence of soluble ephrinB1-Fc compared to IgG control. This inhibition of osteoclast formation and function induced by ephrinB1-Fc was reversed in the presence of an EphB2 chemical inhibitor. Collectively, these observations suggest that ephrinB1, expressed by osteoprogenitors, influences bone loss during the development of osteoporosis, by regulating both osteoblast and osteoclast formation and function, leading to a loss of skeletal integrity.

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

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

EphrinB2 Stabilizes Vascularlike Structures Generated by Endothelial Cells and Stem Cells from Apical Papilla.

INTRODUCTION: This study aimed to investigate the roles of ephrinB2 in stabilizing vascularlike structures generated by stem cells from apical papilla (SCAPs) and human umbilical vein endothelial cells (HUVECs). METHODS: HUVECs were seeded alone or with SCAPs concurrently or 12 hours later. Angiogenesis and ephrinB2 phosphorylation were assayed at different time points. Additionally, ephrinB2 expression in SCAPs and HUVECs was silenced with small interfering RNA, and vascularlike structure formation within coculture was assessed; 1 × 10(5) HUVECs were seeded in transwell inserts, and 6 × 10(5) SCAPs were plated in lower wells with or without ephrinB2-Fc. Migratory cells were stained and counted. Delayed addition of ephrinB2-Fc to the coculture of HUVECs and SCAPs was performed to evaluate the role of ephrinB2 on the stabilization of vascularlike structures. RESULTS: Concurrent coculture of SCAPs and HUVECs yielded significantly longer tubule lengths at 4, 8, and 12 hours (P < .05). Delayed addition of SCAPs to coculture with HUVECs resulted in vascularlike structures persisting longer than the HUVEC monoculture. Western blot confirmed that ephrinB2 phosphorylation was initiated at 0.5 hours of coculture and peaked at 1 hour. Silencing ephrinB2 expression in SCAPs and HUVECs resulted in the absence of vascularlike structures. Enhanced migration of HUVECs by SCAPs could be inhibited by ephrinB2-Fc. When ephrinB2-Fc was added at 3 hours of coculture, the vascularlike structures were stabilized for more than 12 hours as compared with 9 hours in the control group. CONCLUSIONS: EphrinB2 plays an important role in the stabilization of vascularlike structures generated by HUVECs and SCAPs.

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

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

Activation of multiple angiogenic signaling pathways in hemangiopericytoma.

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

Differential protein expression and oncogenic gene network link tyrosine kinase ephrin B4 receptor to aggressive gastric and gastroesophageal junction cancers.

Transmembrane tyrosine-kinase Ephrin receptors promote tumor progression and/or metastasis of several malignancies including leukemia, follicular lymphoma, glioma, malignant pleural mesothelioma, papillary thyroid carcinoma, sarcomas and ovarian, breast, bladder and non-small cell lung cancers. They also drive intestinal stem cell proliferation and positioning, control intestinal tissue boundaries and are involved in liver, pancreatic and colorectal cancers, indicating involvement in additional digestive system malignancies. We investigated the role of Ephrin-B4 receptor (EPHB4), and its ligand EFNB2, in gastric and gastroesophageal junction cancers in patient cohorts through computational, mathematical, molecular and immunohistochemical analyses. We show that EPHB4 is upregulated in preneoplastic gastroesophageal lesions and its expression further increased in gastroesophageal cancers in several independent cohorts. The closely related EPHB6 receptor, which also binds EFNB2, was downregulated in all tested cohorts, consistent with its tumor-suppressive properties in other cancers. EFNB2 expression is induced in esophageal cells by acidity, suggesting that gastroesophageal reflux disease (GERD) may constitute an early triggering event in activating EFNB2-EPHB4 signaling. Association of EPHB4 to both Barrett's esophagus and to advanced tumor stages, and its overexpression at the tumor invasion front and vascular endothelial cells intimate the notion that EPHB4 may be associated with multiple steps of gastroesophageal tumorigenesis. Analysis of oncogenomic signatures uncovered the first EPHB4-associated gene network (false discovery rate: 7 × 10(-90) ) composed of a five-transcription factor interconnected gene network that drives proliferation, angiogenesis and invasiveness. The EPHB4 oncogenomic network provides a molecular basis for its role in tumor progression and points to EPHB4 as a potential tumor aggressiveness biomarker and drug target in gastroesophageal cancers.

Role of EFNB2/EPHB4 signaling in spiral artery development during pregnancy: An appraisal.

EFNB2 and EPHB4, which belong to a large tyrosine kinase receptor superfamily, are molecular markers of arterial and venous blood vessels, respectively. EFNB2/EPHB4 signaling plays an important role in physiological and pathological angiogenesis, and its role in tumor vessel development has been extensively studied. Pregnancy and tumors share similar features, including continuous cell proliferation and increased demand for a blood supply. Our previous studies showed that Efnb2 and Ephb4 were expressed dynamically in the spiral arteries, uterine natural killer cells, and trophoblasts during mouse gestation Days 6.5-12.5. Moreover, uterine natural killer cells and trophoblasts are required for the modification of spiral arteries. Oxygen tension within the pregnant uterus, which contributes to the vascular development, also affects EFNB2 and EPHB4 expression. Considering the role of EFNB2/EPHB4 signaling in embryonic and tumor vascular development, and its dynamic expression in the decidua and placenta, we hypothesize that EFNB2 and EPHB4 are involved in the regulation of spiral artery remodeling. Investigating this hypothesis will help clarify the mechanisms of pathological pregnancy that may underlie abnormal spiral artery development.

Ephrin-Bs Drive Junctional Downregulation and Actin Stress Fiber Disassembly to Enable Wound Re-epithelialization.

For a skin wound to successfully heal, the cut epidermal-edge cells have to migrate forward at the interface between scab and healthy granulation tissue. Much is known about how lead-edge cells migrate, but very little is known about the mechanisms that enable active participation by cells further back. Here we show that ephrin-B1 and its receptor EphB2 are both upregulated in vivo, just for the duration of repair, in the first 70 or so rows of epidermal cells, and this signal leads to downregulation of the molecular components of adherens and tight (but not desmosomal) junctions, leading to loosening between neighbors and enabling shuffle room among epidermal cells. Additionally, this signaling leads to the shutdown of actomyosin stress fibers in these same epidermal cells, which may act to release tension within the wound monolayer. If this signaling axis is perturbed, then disrupted healing is a consequence in mouse and man.

Ephrin-B2 mediates trophoblast-dependent maternal spiral artery remodeling in first trimester.

INTRODUCTION: Maternal spiral artery remodeling after embryo implantation is a crucial process for successful pregnancy and rely on well-controlled trophoblast functions. Ephrin-B2 is found to be of great importance in various cell functions in both benign human tissue and tumors. However, its role in the regulation of trophoblast remains unknown. This study is conducted to investigate the role of ephrin-B2-induced trophoblast functions related to artery remodeling. METHODS: Trophoblast cell line HTR-8/SVneo was used to investigate the effects of ephrin-B2 inhibition on cell proliferation, apoptosis, migration, invasion and tube formation. Placental-decidual co-culture (PDC) system was conducted to verify ephrin-B2-induced trophoblast functions ex vivo. Factors involving in artery remodeling process, such as matrix metalloproteinases (MMPs), placental growth factors (PlGF) and vascular endothelial growth factor (VEGF) were tested at transcriptional level. RESULTS: Inhibition of ephrin-B2 suppressed cell proliferation and induced cell apoptosis in HTR-8/SVneo cells. Down-regulation of ephrin-B2 impaired migration/invasion capabilities of HTR-8/SVneo cells and significantly decreased gene expression of MMPs. Also, a worse tube formation and a decrease in gene expression of PlGF was observed after down-regulation of ephrin-B2. However gene expression of VEGF-A did not show significantly statistical difference. These effects were further confirmed by PDC system showing an inadequate trophoblast invasion and spiral artery remodeling. DISCUSSION: Ephrin-B2 might be act as a positive regulator in maternal artery remodeling via both trophoblast invasion and endovascular formation.

Role of EFNB1 and EFNB2 in Mouse Collagen-Induced Arthritis and Human Rheumatoid Arthritis.

OBJECTIVE: EFNB1 and EFNB2 are ligands for Eph receptor tyrosine kinases. This study was undertaken to investigate how the expression of Efnb1 and Efnb2 on murine T cells influences the pathogenesis of collagen-induced arthritis (CIA) and to assess correlations between the T cell expression of these 2 molecules and measures of disease activity in patients with rheumatoid arthritis (RA). METHODS: CIA was studied in mice with T cell-specific deletion (double gene knockout [dKO]) of both Efnb1 and Efnb2. Expression of EFNB1 and EFNB2 messenger RNA (mRNA) in peripheral blood T cells from patients with RA was determined by quantitative reverse transcription- polymerase chain reaction. RESULTS: In dKO mice, clinical scores of arthritis were reduced compared to those in wild-type (WT) control mice. Serum collagen-specific antibody titers in dKO mice were lower than those in WT mice. In analyses based on equal cell numbers, dKO mouse T cells, as compared to WT mouse T cells, provided vastly inferior help to B cells in the production of collagen-specific antibodies in vitro. T cells from dKO mice were compromised in their ability to migrate to the arthritic paws in vivo and in their ability to undergo chemotaxis toward CXCL12 in vitro. Deletion mutation of Efnb1 and Efnb2 intracellular tails revealed critical regions in controlling T cell chemotaxis. T cells from RA patients expressed higher EFNB1 mRNA levels, which correlated with RA symptoms and laboratory findings. CONCLUSION: Efnb1 and Efnb2 in T cells are essential for pathogenic antibody production and for T cell migration to the inflamed paws in mice with CIA. These findings suggest that the expression of EFNB1 in T cells might be a useful parameter for monitoring RA disease activity and treatment responses.

Endothelial ephrin-B2 is essential for arterial vasodilation in mice.

OBJECTIVE: The cell surface protein ephrin-B2 is expressed in arterial and not venous ECs throughout development and adulthood. Endothelial ephrin-B2 is required for vascular development and angiogenesis, but its role in established arteries is currently unknown. We investigated the physiological role of ephrin-B2 signaling in adult endothelium. METHODS: We generated adult conditional knockout mice lacking the Efnb2 gene specifically in ECs and evaluated the vasodilation responses to blood flow increase and ACh in the cremaster muscle preparation by intravital microscope and in carotid artery by in vivo ultrasound. RESULTS: We found that the Efnb2 conditional knockout mice were defective in acute arterial dilation. Vasodilation was impaired in cremaster arterioles in response to either increased flow or ACh, and in the carotid arteries in response to increased flow. Levels of cGMP, an effector of NO, were diminished in mutant arteries following ACh stimulation. GSNO, a donor for the vasodilator NO, alleviated the vasodilatory defects in the mutants. Immunostaining showed that a subset of ephrin-B2 proteins colocalized with caveolin-1, a negative regulator of eNOS. CONCLUSIONS: Our data suggest that endothelial ephrin-B2 is required for endothelial-dependent arterial dilation and NO signaling in adult endothelium.

[Ephrin B2 is involved in Porphyromonas gingivalis infection-enhanced adhesion of THP-1 to human umbilical vein endothelial cells].

OBJECTIVE: To investigate the mechanisms of Porphyromonas gingivalis (Pg) infection-mediated enhancement of adhesion between monocytes THP-1 and human umbilical vein endothelial cells (HUVEC) by detecting the effect of erythropoietin producing hepatomocellular receptor interacting protein B2 (Ephrin B2) and its receptors on the adhesion. METHODS: PgATCC33277 was cultured in an anaerobic jar, and THP-1 cells were infected with various concentrations of Pg at multiplicity of infection (MOI) of 1:100 for 8 and 24 h, respectively. The expression of Ephrin B2 receptor of THP-1 cells was detected. After removal of the free Pg, THP-1 cells were cocultured with HUVEC (overexpress of EphrinB2 or not) for 24 h to detect the expression of Ephrin B2 of HUVEC cells after additional cultivation for 23 h. RESULTS: The adhesion of THP-1 cells post infection by Pg to HUVEC was enhanced. The mRNA levels of Ephrin B2 receptors, including EphB3 (5.169±0.152, P = 0.005), EphB4 (11.040±1.195, P = 0.001), and EphA4 (4.976± 0.122, P = 0.001) expressed by THP-1, and Ephrin B2 (8.938±0.962, P = 0.008) expressed by HUVEC were significantly elevated 24 h post infection of Pg. Over expression of Ephrin B2 in HUVEC promoted the adhesion of THP-1 to HUVEC. CONCLUSIONS: Ephrin B2 and its receptors are involved in Pg infection mediated enhancement of the adhesion of THP-1 to HUVEC cells, suggesting that Ephrin B2 participates in the development of atherosclerosis.

[Effects of bidirectional EphB4-EphrinB2 signaling on bone remodeling].

Bidirectional Eph-Ephrin signaling as a focal point of research in cell-cell communications is critical for generation of nerves and vesssels as well as invation and metastasis of tumor cells. The roles for Ephrin-Eph bidirectional signaling in bone remodeling were important. EphrinB2 is expressed on osteoblasts and EphB4 is expressed on osteoclasts. Forward signaling through the EphB4 receptor into mesenchymal precursors promotes osteoblast differentiation, while reverse signaling through the EphrinB2 ligand into osteoclast suppresses differentiation. Signaling between the ligand EphrinB2 and the receptors EphB4 explains bidirectional signaling between osteoblasts and osteoclasts,bone absorption and remodeling, which may lay a theoretical foundation for identifying drug targeting and preventing and treating bone loss.

Direct inhibition of cell surface ephrin-B2 by recombinant ephrin-B2/FC.

First messengers and viral transfection are the two most common ways to stimulate cells for signal output, although their applications are limited. We investigated mechanisms of inducing neural stem cell differentiation using recombinant ephrin-B2/Fc and found that it acted as a ligand and inhibited endogenous ephrin-B2, which maintenance of the neural progenitor cell state, by direct interference. Our results showed the movement of ephrin-B2/Fc within the cell and indicated that it recycled to the plasma membrane surface, revealing a possible pattern of ephrin trafficking. Our results also serve as proof of concept for the reconstruction of the intracellular domain of ephrin using an artificial receptor to direct input signals in future studies.

Endothelial Shc regulates arteriogenesis through dual control of arterial specification and inflammation via the notch and nuclear factor-κ-light-chain-enhancer of activated B-cell pathways.

RATIONALE: Arteriogenesis, the shear stress-driven remodeling of collateral arteries, is critical in restoring blood flow to ischemic tissue after a vascular occlusion. Our previous work has shown that the adaptor protein Shc mediates endothelial responses to shear stress in vitro. OBJECTIVE: To examine the role of the adaptor protein Shc in arteriogenesis and endothelial-dependent responses to shear stress in vivo. METHODS AND RESULTS: Conditional knockout mice in which Shc is deleted from endothelial cells were subjected to femoral artery ligation. Hindlimb perfusion recovery was attenuated in Shc conditional knockout mice compared with littermate controls. Reduced perfusion was associated with blunted collateral remodeling and reduced capillary density. Bone marrow transplantation experiments revealed that endothelial Shc is required for perfusion recovery because loss of Shc in bone marrow-derived hematopoietic cells had no effect on recovery. Mechanistically, Shc deficiency resulted in impaired activation of the nuclear factor κ-light-chain-enhancer of activated B-cell-dependent inflammatory pathway and reduced CD45⁺ cell infiltration. Unexpectedly, Shc was required for arterial specification of the remodeling arteriole by mediating upregulation of the arterial endothelial cell marker ephrinB2 and activation of the Notch pathway. In vitro experiments confirmed that Shc was required for shear stress-induced activation of the Notch pathway and downstream arterial specification through a mechanism that involves upregulation of Notch ligands delta-like 1 and delta-like 4. CONCLUSIONS: Shc mediates activation of 2 key signaling pathways that are critical for inflammation and arterial specification; collectively, these pathways contribute to arteriogenesis and the recovery of blood perfusion.

Endothelial expression of guidance cues in vessel wall homeostasis dysregulation under proatherosclerotic conditions.

OBJECTIVE: Emerging evidence suggests that neuronal guidance cues, typically expressed during development, are involved in both physiological and pathological immune responses. We hypothesized that endothelial expression of such guidance cues may regulate leukocyte trafficking into the vascular wall during atherogenesis. APPROACH AND RESULTS: We demonstrate that members of the netrin, semaphorin, and ephrin family of guidance molecules are differentially regulated under conditions that promote or protect from atherosclerosis. Netrin-1 and semaphorin3A are expressed by coronary artery endothelial cells and potently inhibit chemokine-directed migration of human monocytes. Endothelial expression of these negative guidance cues is downregulated by proatherogenic factors, including oscillatory shear stress and proinflammatory cytokines associated with monocyte entry into the vessel wall. Furthermore, we show using intravital microscopy that inhibition of netrin-1 or semaphorin3A using blocking peptides increases leukocyte adhesion to the endothelium. Unlike netrin-1 and semaphorin3A, the guidance cue ephrinB2 is upregulated under proatherosclerotic flow conditions and functions as a chemoattractant, increasing leukocyte migration in the absence of additional chemokines. CONCLUSIONS: The concurrent regulation of negative and positive guidance cues may facilitate leukocyte infiltration of the endothelium through a balance between chemoattraction and chemorepulsion. These data indicate a previously unappreciated role for axonal guidance cues in maintaining the endothelial barrier and regulating leukocyte trafficking during atherogenesis.