Role For ephrin-B2 Research Articles

The roles of ephrinB2 in fear memory formation in excitatory neurons and astrocytes in the basolateral amygdala

The roles of ephrinB2 in fear memory formation in excitatory neurons and astrocytes in the basolateral amygdala

Ephrin-B2 paces neuronal production in the developing neocortex

BackgroundDuring mammalian cerebral cortex development, different types of projection neurons are produced in a precise temporal order and in stereotypical numbers. The mechanisms regulating timely generation of neocortex projection neurons and ensuring production in sufficient numbers of each neuronal identity are only partially understood.ResultsHere, we show that ephrin-B2, a member of the Eph:ephrin cell-to-cell communication pathway, sets the neurogenic tempo in the neocortex. Indeed, conditional mutant embryos for ephrin-B2 exhibit a transient delay in neurogenesis and acute stimulation of Eph signaling by in utero injection of synthetic ephrin-B2 led to a transient increase in neuronal production. Using genetic approaches we show that ephrin-B2 acts on neural progenitors to control their differentiation in a juxtacrine manner. Unexpectedly, we observed that perinatal neuron numbers recovered following both loss and gain of ephrin-B2, highlighting the ability of neural progenitors to adapt their behavior to the state of the system in order to produce stereotypical numbers of neurons.ConclusionsAltogether, our data uncover a role for ephrin-B2 in embryonic neurogenesis and emphasize the plasticity of neuronal production in the neocortex.

Roles of ephrin-B2+ and CD34+ cells in post-angioplasty pericardial patch repair

Roles of ephrin-B2+ and CD34+ cells in post-angioplasty pericardial patch repair

The role of Delta-like 4 ligand/Notch-ephrin-B2 cascade in the pathogenesis of preeclampsia by regulating functions of endothelial progenitor cell.

Preeclampsia is a hypertensive complication in pregnancy, closely related to endothelial dysfunction. Endothelial progenitor cells (EPCs) have the capacity for endothelial repair. Both Ephrin-B2 and Dll4/Notch pathway play critical roles in various steps of angiogenesis. In addition, there is an up-regulation of ephrin-B2 expression consequent to Dll4/Notch activation in endothelial cells (ECs). However, the roles of ephrin-B2 and Dll4/Notch signaling on EPCs, as well as the relationship between them, have not been completely characterized. We analyzed expression of ephrin-B2 in the EPCs and placenta from preeclampsia and normal pregnancy. Then up-regulation and down-regulation strategies were employed to detect the effects of ephrin-B2 on EPC proliferation, differentiation, migration and HUVEC-tube formation. The effects of Dll4/Notch signaling on EPCs' functions were determined by repeating the assays in the presence of Dll4 or DAPT as agonists or antagonists of Notch signaling, respectively. Ephrin-B2 expression increased notably in preeclampsia EPCs and placenta, compared with controls. Up-regulation of ephrin-B2 impaired EPCs' proliferation, differentiation, migration and HUVEC-tube formation capabilities. In contrast, down-regulation of ephrin-B2 in EPCs, resulted in the opposite effects. In addition, activation of Dll4/Notch signaling led to increased expression of ephrin-B2 and subsequent inhibition of EPCs activity. Ephrin-B2, a downstream of Dll4/Notch signaling pathway, might be act as an inhibitor of EPC-mediated vasculogenesis invitro, as well as a potential target in the effort to promote angiogenesis of patients with preeclampsia.

Abstract 511: Ephrin-B2 Is Essential for Ischemic Recovery following Femoral Artery Occlusion in Mice

The cell surface protein ephrin-B2 is expressed in arterial and not venous endothelial cells in adulthood mice. The role for ephrin-B2 in adult vascular physiology is currently unknown. We have recently reported that endothelial ephrin-B2 is essential in acute arterial vasodilation. Here we report that endothelial ephrin-B2 plays important protective roles against acute ischemia. We investigated the role of endothelial ephrin-B2 following experimental femoral artery occlusions in mice lacking ephrin-B2 specifically in the endothelium. Mutant mice exhibited lower foot blood flow by laser doppler perfusion imaging and developed gangrene not seen in controls, following femoral arterial occlusions. Histologically, neovascularization and muscle regeneration were impaired in the mutant hindlimb. S-nitrosoglutathione, a donor for nitric oxide, improved the foot blood flow and tissue survival in the mutants after the occlusion. Mechanistically, we demonstrated that acetylcholine-stimulated nitric oxide production was impaired in ephrin-B2 mutant, detected by both chemiluminescence that measures NO metabolites and electron spin resonance that measures bioavailable NO. Our data demonstrate that mice lacking ephrin-B2 in endothelial cells are defective in acute ischemic recovery after femoral artery occlusion and suggest that endothelial ephrin-B2 may interact with the nitric oxide pathway to mediate arterial dilation and ischemia protection.

Ephrin-B2/EphA4 forward signaling is required for regulation of radial migration of cortical neurons in the mouse

Postmitotic neurons in the neocortex migrate to appropriate positions and form layered structures of nascent cortex during brain development. The migration of these neurons requires precise control and coordination of a large number of molecules such as axon guidance cues. The Eph-ephrin signaling pathway plays important roles in the development of the nervous system in a wide variety of ways, including cell segregation, axon pathfinding, and neuron migration. However, the role of ephrin-B2/EphA4 signaling in cortical neuron migration remains elusive. Here we demonstrated that ephrin-B2 and its receptor EphA4 were expressed in complementary and overlapping patterns in the developing neocortex. Deletion of the EphA4 gene in the embryonic cerebral cortex resulted in faster migration of cortical neurons, whereas knockdown or overexpression of ephrin-B2 did not alter the normal process of migration. These results suggest that ephrin-B2 forward signaling through EphA4 is required for the precise control of cortical neuron migration.

Ephrin-B2 expression in the proprioceptive sensory system

Recent studies have shown that ephrin-B2 on sensory afferent fibers from the dorsal root ganglia (DRG) controls transmission of pain sensation to the spinal cord. We examined ephrin-B2 expression in mouse DRG and spinal cord using an ephrin-B2/ß-galactosidase chimeric allele. We found that ephrin-B2 is expressed exclusively in proprioceptive neurons and fibers in neonates, while expression in lamina III and IV of the adult spinal cord was observed in addition to that in the deeper laminae. We confirmed that ephrin-B2 protein causes co-clustering of EphB2 and glutamate receptors in spinal cord neurons. Our data are consistent with a role for ephrin-B2 in transmission of positional information to the CNS, and thus suggest a role in synaptic plasticity of spinal cord locomotor circuits that are known to be sensitive to proprioceptive sensory input after spinal cord injury.

Abstract 395: Host ephrinB2 regulates T-cells in tumor microenvironments

Abstract Background: The tumor microenvironment consists of immune, stromal, and vascular cells. Proteins known to play roles in vascular development during embryogenesis also have been implicated in the control of leukocyte migration. The vascular patterning ligand ephrinB2, for example, influences T-cell migration in vitro. We therefore hypothesized that host ephrinB2 signaling regulates tumor immune microenvironments. Methods: EphrinB2+/lacZ mice were implanted with B16 melanoma, KR158 glioma, LLC lung, and EL4 T-lymphoma tumor cells. Macroscopic tumors were harvested and ephrinB2 expression assayed using x-gal staining. Mx1-cre/efnb2fl/fl mice were generated which allow for the post-natal deletion of ephrinB2 after pIpC treatment. Mx1-cre/efnb2+/+ mice served as controls. 1 million B16 or KR158 tumor cells were implanted in hindlimbs and tumor volume measured (V= 0.52*L*W2) at least twice weekly. Tumor tissues were stained with anti-CD3, F4/80, anti-CD34, and anti-NG2 antibodies. Experiments were repeated following T-cell depletion with i.p. injections of anti-CD4 and anti-CD8 antibodies 3x week. Finally, C57BL6 mice were transplanted with Mx1-cre/efnb2fl/fl or Mx1-cre/efnb2+/+ bone marrow and ephrinB2 was deleted after engraftment but prior to tumor growth experiments. Results: EphrinB2 was expressed in vascular structures all tumors. In addition we observed upregulation of ephrinB2 in microvascular structures in adjacent muscle during tumor invasion. Inducible deletion of host ephrinB2 significantly reduced B16 tumor growth (control tumor volume 2846 mm3 versus deleted tumor 869 mm3, p<0.01) and KR158 tumor growth (2028 mm3 versus 1403 mm3). EphrinB2 deletion did not affect CD34+ microvessel density, NG2+ pericyte or F4/80 macrophage infiltration. Rather, tumors grown in ephrinB2 deficient microenvironments demonstrated a significant increase in infiltrating CD3+ T-cells. In vivo depletion of T-cells abrogated the effect of ephrinB2 deletion on tumor growth delay demonstrating a role for immune cells in impairing tumor growth. EphrinB2 deletion in transplanted hematopoietic cells did not lead to tumor growth delay demonstrating a requirement for intact ephrinB2 signaling in vascular cells for T-cell regulation. Conclusion: EphrinB2 is upregulated in tumor vasculature and at sites of tumor invasion. Post-natal ephrinB2 deletion did not affect tumor angiogenesis but led to tumor growth delay via a T-cell mediated mechanism. These findings demonstrate a novel role for ephrinB2 specifically and vascular cells generally in regulating immune microenvironments. Therefore this study identifies a novel therapeutic strategy to increase the accumulation of T-cells in tumor tissues with relevance to combination immunotherapeutic strategies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 395. doi:1538-7445.AM2012-395

Generation of transgenic mice overexpressing EfnB2 in endothelial cells

Genetic studies have shown that ephrin-B2 and its cognate EphB4 receptor are necessary for normal embryonic angiogenesis. Moreover, there is overwhelming evidence that ephrin-B2 is involved in tumor vascularization, yet its role in adult angiogenesis has been difficult to track genetically. Here, we report the generation of transgenic mice that over-express EfnB2 specifically in endothelial cells (ECs). We show that exogenous expression of EfnB2 under the control of the Tie2 promoter/enhancer regions in ECs does not affect viability or growth of the transgenic animals. We further show that targeted expression of EfnB2 in ECs is not sufficient to rescue severe cardiovascular defects at mid-gestation stages but rescues early embryonic lethality associated with loss-of-function mutation in EfnB2. This mouse model will be useful to study the role of ephrin-B2 in physiological and pathological angiogenesis.

Endothelial Cell EphrinB2-Dependent Activation of Monocytes in Arteriosclerosis

The expression of ephrinB2 in endothelial cells delineates their arterial phenotype and is a prerequisite for the development of the embryonic vasculature. Whereas the role of ephrinB2 in the microcirculation has been studied extensively, its expression and function in adult arteries is hardly understood. Our analyses showed that in mouse arteries, ephrinB2 is located on the luminal surface of endothelial cells and may physically interact with monocyte EphB receptors. Moreover, transdifferentiation of human monocytes into macrophages was associated with an increase in EphB2 expression, and exposing monocytes to immobilized ephrinB2 resulted in phosphorylation of the receptor followed by an increased expression of proinflammatory chemokines such as interleukin-8 and monocyte chemotactic protein-1/CCL2. Relating to the (patho)physiological relevance of these findings, immunofluorescence analyses revealed that ephrinB2 is most abundantly expressed in endothelial cells at arteriosclerosis predilection sites of the mouse aorta. Subsequent analyses indicated that monocyte adhesion to aortic segments abundantly expressing ephrinB2 is strongly enhanced and that endothelial cell ephrinB2 forward signaling is sufficient to upregulate cytokine expression in monocytes. These observations suggest a hitherto unknown link between vascular ephrinB2 expression and the proinflammatory activation of monocytes that may contribute to the pathogenesis of arteriosclerosis.

Role of ephrinB2 in nonproductive angiogenesis induced by Delta-like 4 blockade

AbstractDelta-like 4 (DLL4) is one of the Notch ligands and plays an important role in vascular development. DLL4 blockade inhibits tumor growth by promoting nonproductive angiogenesis, which is characterized by an increase in vascular density and decrease in tissue perfusion. However, a detailed mechanism remains unclear. In this study, newly developed neutralizing antibodies against mouse and human DLL4 were used to investigate the possible involvement of VEGF-DLL4-ephrinB2 cascade in nonproductive angiogenesis caused by DLL4 blockade. DLL4 blockade and soluble ephrinB2 treatment suppressed tumor growth and induced nonproductive angiogenesis. DLL4 was expressed in subcutaneous tumors, and DLL4 blockade suppressed ephrinB2 expression in the tumors. DLL4 blockade significantly promoted human umbilical vein endothelial cell (HUVEC) proliferation in vitro, and the effect was additive to that of VEGF. Both DLL4 blockade and VEGF significantly increased cord length and branch points in a tubular formation assay. Expression of ephrinB2 in HUVECs was enhanced by VEGF alone, and the enhancement was inhibited by DLL4 blockade. Moreover, when we studied the effect of ephrinB2 RNA interference on HUVEC tubular formation, knockdown of ephrinB2 mimicked the effect of DLL4. These results suggest that ephrinB2 plays a crucial role in nonproductive angiogenesis caused by DLL4 blockade.

Organ- and endotheliotropism of Nipah virus infections in vivo and in vitro

Nipah virus (NiV) is a highly pathogenic paramyxovirus that was first isolated in 1999 during an outbreak in Malaysia. In contrast to other paramyxoviruses NiV infects many mammalian species. Because of its zoonotic potential, the high pathogenicity and the lack of therapeutic treatment, NiV was classified as a biosafety level 4 pathogen. In humans NiV causes a severe acute encephalitis whereas in some animal hosts respiratory symptoms are predominantly observed. Despite the differences in the clinical outcome, microvascular endothelial cell damage predominantly underlies the pathological changes in NiV infections in all susceptible host species. NiV generally induces a pronounced vasculitis which is primarily characterised by endothelial cell necrosis and inflammatory cell infiltration. For future developments of specific antiviral therapies or vaccines, a detailed understanding of the molecular basis of NiV pathogenesis is required. This article reviews the current knowledge about natural and experimental infections in different mammals, focusing on the main organ and cell tropism in vivo, and summarises some recent studies in cell culture on the role of ephrin-B2 and -B3 receptors in NiV infection of endothelial cells.

Role for EphrinB2 in postnatal lung alveolar development and elastic matrix integrity

Role for EphrinB2 in postnatal lung alveolar development and elastic matrix integrity

Role for ephrinB2 in postnatal lung alveolar development and elastic matrix integrity

Alveoli are formed in the lung by the insertion of secondary tissue folds, termed septa, which are subsequently remodeled to form the mature alveolar wall. Secondary septation requires interplay between three cell types: endothelial cells forming capillaries, contractile interstitial myofibroblasts, and epithelial cells. Here, we report that postnatal lung alveolization critically requires ephrinB2, a ligand for Eph receptor tyrosine kinases expressed by the microvasculature. Mice homozygous for the hypomorphic knockin allele ephrinB2DeltaV/DeltaV, encoding mutant ephrinB2 with a disrupted C-terminal PDZ interaction motif, show severe postnatal lung defects including an almost complete absence of lung alveoli and abnormal and disorganized elastic matrix. Lung alveolar formation is not sensitive to loss of ephrinB2 cytoplasmic tyrosine phosphorylation sites. Postnatal day 1 mutant lungs show extracellular matrix alterations without differences in proportions of major distal cell populations. We conclude that lung alveolar formation relies on endothelial ephrinB2 function.

Ephrin-B2 Controls Cell Motility and Adhesion during Blood-Vessel-Wall Assembly

New blood vessels are initially formed through the assembly or sprouting of endothelial cells, but the recruitment of supporting pericytes and vascular smooth muscle cells (mural cells) ensures the formation of a mature and stable vascular network. Defective mural-cell coverage is associated with the poorly organized and leaky vasculature seen in tumors or other human diseases. Here we report that mural cells require ephrin-B2, a ligand for Eph receptor tyrosine kinases, for normal association with small-diameter blood vessels (microvessels). Tissue-specific mutant mice display perinatal lethality; vascular defects in skin, lung, gastrointestinal tract, and kidney glomeruli; and abnormal migration of smooth muscle cells to lymphatic capillaries. Cultured ephrin-B2-deficient smooth muscle cells are defective in spreading, focal-adhesion formation, and polarized migration and show increased motility. Our results indicate that the role of ephrin-B2 and EphB receptors in these processes involves Crk-p130(CAS) signaling and suggest that ephrin-B2 has some cell-cell-contact-independent functions.

Role of ephrin B2 in human retinal endothelial cell proliferation and migration

This study was designed to determine the presence of Eph B4 or ephrin B2 in human retinal endothelial cells (REC) and their signal transduction. Human retinal endothelial cells were stimulated with an Eph B4/Fc chimera and probed for phosphorylation of phosphatidylinositol-3-kinase (PI3K), Src, and mitogen-activated protein kinase (MAPK) pathways. Proliferation and migration were investigated after Eph B4/Fc stimulation in the presence of various pathway inhibitors. Human retinal endothelial cells express ephrin B2, with little expression of Eph B4. Treatment with EphB4/Fc chimera resulted in activation of PI3K, Src, and MAPK pathways. Eph B4-stimulated endothelial cell proliferation was mediated via PI3K, nitric oxide synthase, and extracellular signal-regulated kinase 1/2 (ERK1/2). Blockade of Src-PI3K pathways produced significant attenuation of Eph B4/Fc-stimulated migration. These results demonstrate for the first time that ephrin B2 is present in human retinal endothelial cells. Additionally, it appears that vascular growth may be modulated in the retina through activation of the PI3K pathway and its downstream components.

Ephrin-B2 Selectively Marks Arterial Vessels and Neovascularization Sites in the Adult, with Expression in Both Endothelial and Smooth-Muscle Cells

The Eph receptor tyrosine kinases and their membrane-tethered ephrin ligands provide critical guidance cues at points of cell-to-cell contact. It has recently been reported that the ephrin-B2 ligand is a molecular marker for the arterial endothelium at the earliest stages of embryonic angiogenesis, while its receptor EphB4 reciprocally marks the venous endothelium. These findings suggested that ephrin-B2 and EphB4 are involved in establishing arterial versus venous identity and perhaps in anastamosing arterial and venous vessels at their junctions. By using a genetically engineered mouse in which the lacZ coding region substitutes and reports for the ephrin-B2 coding region, we demonstrate that ephrin-B2 expression continues to selectively mark arteries during later embryonic development as well as in the adult. However, as development proceeds, we find that ephrin-B2 expression progressively extends from the arterial endothelium to surrounding smooth muscle cells and to pericytes, suggesting that ephrin-B2 may play an important role during formation of the arterial muscle wall. Furthermore, although ephrin-B2 expression patterns vary in different vascular beds, it can extend into capillaries about midway between terminal arterioles and postcapillary venules, challenging the classical conception that capillaries have neither arterial nor venous identity. In adult settings of angiogenesis, as in tumors or in the female reproductive system, the endothelium of a subset of new vessels strongly expresses ephrin-B2, once again contrary to earlier views that such new vessels lack arterial/venous characteristics and derive from postcapillary venules. While earlier studies had focused on a role for ephrin-B2 during the earliest embryonic stages of arterial/venous determination, our current findings using ephrin-B2 as an arterial marker in the adult challenge prevailing views of the arterial/venous identity of quiescent as well as remodeling adult microvessels and also highlight a possible role for ephrin-B2 in the formation of the arterial muscle wall.