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Academic Discipline of Paediatrics and Child Health, University of Queensland, Royal Children's Hospital, Herston, AustraliaPaediatric Intensive Care Unit, Royal Children's Hospital, Herston, AustraliaQueensland Children's Medical Research Institute, Royal Children's Hospital, Herston, AustraliaLeukaemia Foundation Laboratory, Queensland Institute of Medical Research, Herston, Australia
Queensland Children's Medical Research Institute, Royal Children's Hospital, Herston, AustraliaLeukaemia Foundation Laboratory, Queensland Institute of Medical Research, Herston, AustraliaAcademic Discipline of Medicine, University of Queensland, Royal Brisbane Women's Hospital, Herston, Australia
The Eph/ephrin receptor–ligand system plays an important role in embryogenesis and adult life, principally by influencing cell behavior through signaling pathways, resulting in modification of the cell cytoskeleton and cell adhesion. There are 10 EphA receptors, and six EphB receptors, distinguished on sequence difference and binding preferences, that interact with the six glycosylphosphatidylinositol-linked ephrin-A ligands and the three transmembrane ephrin-B ligands, respectively. The Eph/ephrin proteins, originally described as developmental regulators that are expressed at low levels postembryonically, are re-expressed after injury to the optic nerve, spinal cord, and brain in fish, amphibians, rodents, and humans. In rodent spinal cord injury, the up-regulation of EphA4 prevents recovery by inhibiting axons from crossing the injury site. Eph/ephrin proteins may be partly responsible for the phenotypic changes to the vascular endothelium in inflammation, which allows fluid and inflammatory cells to pass from the vascular space into the interstitial tissues. Specifically, EphA2/ephrin-A1 signaling in the lung may be responsible for pulmonary inflammation in acute lung injury. A role in T-cell maturation and chronic inflammation (heart failure, inflammatory bowel disease, and rheumatoid arthritis) is also reported. Although there remains much to learn about Eph/ephrin signaling in human disease, and specifically in injury and inflammation, this area of research raises the exciting prospect that novel therapies will be developed that precisely target these pathways.
Tissue injury or damage is followed by an orderly process that includes the induction of the following: i) an acute inflammatory process, ii) regeneration of cells, iii) migration and proliferation of both parenchymal and connective tissue cells, and iv) tissue remodeling.
The decreased adhesion between vascular endothelial cells allows the passage of plasma water and proteins into the interstitial space, and vascular endothelial cells become more adhesive to inflammatory cells, which leave the circulation and enter the injured tissue.
The Eph/ephrin receptor–ligand family (which subsequently will be referred to jointly as the Eph/ephrin proteins) is a group of cell surface proteins, and emerging evidence suggests that these proteins play an important role in injury
The direct implication of the Eph/ephrin proteins in inflammation remains relatively obscure; however, there is evidence to support their role in modulating vascular permeability during inflammation.
This review will summarize the extensive work on Eph/ephrin proteins in injury and present recent evidence to support the hypothesis that Eph/ephrin proteins play an essential role in inflammation mediated through the vascular endothelium. Finally, the review will anticipate future research that may lead to novel pharmacological approaches for treating injury and inflammation.
The Eph/Ephrin Proteins
Overview
The Eph receptor tyrosine kinases and their ephrin ligands are cell surface molecules with a wide range of biological functions that influence cell behavior during both embryogenesis and adult life.
These functions include roles in the following: i) the direction of cell positioning and migration; ii) axon guidance during development; iii) control of tissue morphogenesis and patterning; iv) defining tissue boundary formation during somatogenesis; v) the development of the vascular system; vi) neural plasticity; vii) tumor invasion and metastasis; viii) immune function, hematopoiesis, and blood clotting; ix) the biological characteristics of stem cells; and x) tissue repair and maintenance.
Cytoskeletal modification regulates the dynamics of cellular protrusions, affects cell-cell adhesion and attachment to the extracellular matrix, triggers cell segregation, and modulates migration.
Principle signaling cascades initiated by Eph/ephrin interactions converge on cytoskeletal targets, such as integrins and small Rho family GTPases, although emerging evidence reveals additional direct roles in modulating viability and proliferation, in particular stem and progenitor cells.
This is in contrast to other receptor tyrosine kinases, which were first identified as oncogenes, because they activated signaling pathways that target gene transcription and regulate cell proliferation and/or differentiation.
There are 10 EphA receptors, EphA1–EphA10 (pronounced eff-A), and six EphB receptors, EphB1–EphB4, and EphB6 in vertebrates and an additional EphB receptor, EphB5, which exists in chickens.
The ligands of the Eph receptors are known as ephrins (pronounced effrins), an abbreviation derived from Eph family receptor-interacting proteins. The initial distinction between EphA and EphB receptors was based on sequence differences within the extracellular ligand binding domain, but also corresponds to the binding preferences for the six glycosylphosphatidylinositol-linked ephrin-A ligands and the three transmembrane ephrin-B ligands, respectively
(Figure 1). The Eph/ephrin receptor–ligand interactions are promiscuous within each A or B class, with variations in binding affinities, although EphB4 only binds ephrin-B2.
Figure 1The structure of the Eph receptors and their ephrin ligands. The EphA and EphB receptors have a conserved domain structure. The ephrin-A ligands are attached to the cell membrane by a glycosylphosphatidylinositol anchor. The ephrin-B ligands are transmembrane proteins. PDZ, post-synaptic density protein-95; SAM, sterile alpha motif.
Figure 2Eph receptor and ephrin ligand binding preferences. Double-sided arrows, interactions between the specific Eph receptors and the respective interacting ephrins. GPI, glycosylphosphatidylinositol.
The interaction of an ephrin ligand with its cognate Eph receptor involves both forward (Eph-mediated) and/or reverse (ephrin-mediated) signaling, which can result in either cell–cell adhesion or de-adhesion.
Some reports imply that cis binding does not lead to active signaling but interferes with receptor activation by the ephrin-A presented on the surrounding cells.
The downstream effects of Eph/ephrin protein signaling are ultimately mediated through changes in cytoskeletal proteins (responsible for cell shape and motility) and cell surface receptors for extracellular matrix proteins (responsible for cell adhesion).
The specificity of the cellular response for both forward and reverse signaling events and the final outcome are determined by the type and abundance of Ephs and ephrins on the interacting cell surface and the sum of the Eph receptors on a cell surface, which are competing for available ephrin targets on the interacting cells.
Molecular Mechanisms of Eph/Ephrin Protein Signaling
The interaction of an Eph receptor with its ephrin ligand results in the formation of an Eph/ephrin tetramer and juxtaposition of two catalytically autoinhibited Eph receptor monomers in a ring-like complex, juxtaposing two Ephs for potential cross phosphorylation.
Once formed, the heterotetrameric Eph/ephrin complex promotes ephrin-independent Eph/Eph interactions between neighboring Ephs that promote the assembly of higher-order oligomers, which are required for Eph phosphorylation and activation of downstream signaling.
Recent findings suggest that this oligomerization via Eph/Eph protein interfaces in the globular (ephrin-binding) and cysteine-rich domains occurs independent of Eph signaling capacity and subclass specificity, so that the composition of the signaling cluster reflects the expression profile of Ephs on a cell surface.
Once activated, tyrosine phosphorylation–induced changes in the conformations of Eph/ephrin cytoplasmic domains, in particular release of the Eph kinase domain from the inhibitory interaction with the juxtamembrane domain, allow the specific binding of Src homology (domains) 2 and 3, phosphotyrosine domain, or post-synaptic density protein-95 domain–containing downstream signaling molecules and activate corresponding signaling pathways
Figure 3Eph/ephrin signaling pathways. Structurally and functionally significant domains within Eph and ephrin proteins and signaling pathways of activated Eph receptors and ephrins that modulate cell shape and attachment. Arrows, positive outcome; blue circle, phosphorylation; flat end lines, inhibition.
Eph/ephrin protein signaling results in the activation of several cytoplasmic downstream signaling pathways, including the following: i) Src family kinases, ii) mitogen-activated protein kinase, iii) p-21 activated kinase, iv) post-synaptic density protein-95–dependent pathways, v) chemokine pathways, vi) heterotrimeric G-protein pathways, and vii) integrin-mediated pathways.
The activity of each of these pathways is dependent on the activity of the Rho family GTPases, including RhoA, Ras-related C3 botulinum toxin substrate (Rac) 1, and cell division control protein 42 (Cdc42); the Rac–GTPase-activating protein (GAP); α-chimerin
; this may, in part, account for the different cell responses, either cell adhesion or cell–cell repulsion (de-adhesion), after Eph/ephrin receptor activation.
The pathological features that follow traumatic injury and tissue damage include formation of a platelet plug and coagulation of extravasated blood, which initiates a complex signaling cascade to recruit inflammatory cells, stimulate fibroblast and epithelial cell proliferation, direct cell migration, and induce angiogenesis to restore tissue integrity.
The classic histological observation that many of the features of normal wound healing are similar to the tumor microenvironment suggested that the tumor stroma is normal wound healing gone awry.
The fibroblast serum response was investigated with a cDNA microarray genome-wide survey and confirmed a gene expression signature similar to metastatic cancer.
However, although this approach of measuring gene expression has thus far not revealed Eph/ephrin proteins as candidates at the time points considered,
Tissue injury can also result from vascular disease in which the blood and nutrient supply is interrupted with severe consequences to major organs (eg, acute myocardial infarction and cerebrovascular stroke).
The ischemic damage is then followed by reperfusion injury when blood flow is restored, either as part of the natural history of the disease or as a result of therapeutic measures.
The ischemia-reperfusion injury is characterized by the following: i) an inflammatory response regulated by the pro-inflammatory cytokines, tumor necrosis factor (TNF)-α, IL-1, and IL-6; ii) up-regulation of endothelial adhesion molecules; and iii) recruitment of inflammatory cells to the damaged tissue.
A mouse skin flap model was used to determine the response of Eph/ephrin proteins to hypoxia. Partial cutaneous oxygen tension and tissue lactate/pyruvate measurements monitored by microdialysis confirmed tissue hypoxia, and quantitative PCR confirmed induction of hypoxia-inducible factor-1α and vascular endothelial growth factor (VEGF).
The expression levels of EphB4, ephrin-B2, EphA2, and ephrin-A1 were up-regulated in hypoxic skin, and the temporal expression pattern was determined, which supports the hypothesis that Eph/ephrin proteins are involved in revascularization after hypoxic injury.
Also, hypoxia-inducible factor-2α (but not hypoxia-inducible factor-1α) binds the hypoxia response element in the ephrin-A1 promoter and plays a role in tumor vascularization by inducing ephrin-A1 expression.
Furthermore, ephrin-B2 is required during angiogenesis and expressed specifically in arteries, which have a higher oxygen tension than veins. In fact, chromatin immunoprecipitation, mutagenesis, and small-interfering RNA knockdown experiments indicate that hypoxia drives arterial differentiation by increasing ephrin-B2 expression in endothelial cells through stimulating protein 1 activation.
Reciprocal gradients of Eph/ephrins were responsible for the precise projection of the retinal ganglion cells onto the optic tectum/superior colliculus.
In fish and amphibians, damage to the optic nerve or spinal cord is followed by infiltration of microglial cells and macrophages and subsequent axon regrowth and functional recovery.
In contrast, at the injury site in mammals, there is expression of chondroitin sulfate proteoglycan (lecticans and neuroglycan 2) in the extracellular matrix and inhibitory factors, including axon guidance molecules (semaphorins, ephrins, and netrins) and prototypic myelin inhibitors (Nogo, myelin-associated glycoprotein, and oligodendrocyte myelin glycoprotein), that actively inhibit axonal regeneration, resulting in poor functional recovery.
In fish, the neurons that undergo successful axonal regeneration have a similar, but not identical, molecular profile to neurons in the embryonic state.
In adult goldfish with optic nerve injury, immunohistochemical (IHC) studies indicated that there was transient up-regulation of EphA3 and EphA5 in the retinal ganglion cells (RGCs), coincident with up-regulated tectal ephrin-A2 expression, both of which were required for restoration of the normal retinotectal topographic map.
In mice with optic nerve de-afferentation, the graded expression patterns of ephrin-A2 and ephrin-A5 in the superior colliculus were similar to those found during development.
After optic nerve crush injury, macrophages expressing EphB3 accumulated at the injury site, and ephrin-B3 was expressed on RGC axons at the injury site.
This suggests a role for EphB3-expressing macrophages interacting with ephrin-B3–expressing RGC axons in the remodeling events that follow optic nerve injury.
Spinal cord injury in rats resulted in a marked increase in EphB3 mRNA at day 7 after injury, and was confirmed by immunolocalization of EphB3 expression in white matter astrocytes and gray matter neurons.
In another study using semiquantitative PCR of the injured adult rat spinal cord, EphA3, EphA4, and EphA7 mRNAs were up-regulated. Furthermore, EphA3, EphA4, EphA6, and EphA8 immunoreactivity was increased in the ventrolateral white matter. The EphA receptor expression localized in the white matter to glial cells, both astrocytes and oligodendrocytes, and localized to neurons in the gray matter. The expression of EphA3 mRNA and protein after spinal cord injury was elevated from day 2 to day 28, and EphA3 immunoreactivity was observed in reactive astrocytes.
EphB3 expression detected by in situ hybridization was up-regulated in rats subjected to complete thoracic spinal cord transection and was confirmed by IHC.
IHC data suggested that ephrin-B2 was expressed on reactive central nervous system astrocytes and that EphB2 was present on fibroblasts invading the injury site from the adjacent meninges.
After spinal cord hemisection, EphA4 was up-regulated in wild-type mice on astrocytes associated with the glial scar at the injury site, whereas EphA4-null mice showed markedly reduced astrocytic gliosis and scar formation.
The EphA4-null mice exhibited axonal regeneration, characterized by axons growing across the injury site, associated with significant functional recovery 1 to 3 months after the injury.
EphA4 up-regulation after spinal cord contusion injury in rats was blocked by infusing EphA4 antisense oligonucleotides; however, although this did not result in enhanced locomotor recovery, it did improve chronic pain scores.
A more complete understanding of the molecular basis of recovering axons in the fish, amphibian, and rodent central nervous systems will provide valuable insight into potential therapeutic advances after brain and spinal cord injury in humans.
The precise downstream intracellular signaling mechanisms that mediate the inhibitory effect of ephrins after spinal cord injury remain unknown. However, in the days after contusive spinal cord injury in rats, Western blot analysis studies identified an increased expression profile of the Rho guanine exchange factor, ephexin, in reactive astrocytes, activated macrophages, and neurons at the lesion site, which colocalized with EphA3, EphA4, and EphA7.
The interactions of EphB receptors with ephrin-B ligands modulate spinal cord synaptic efficiency in an N-methyl-D-aspartate receptor–dependent manner and contribute to neuropathic and inflammatory pain states mediated via a mitogen-activated protein kinase–dependent mechanism.
In this regard, single-nucleotide polymorphism and haplotype analyses suggest that the EFBN2 (ephrin-B2) locus is associated with schizophrenia in the Han Chinese population.
Furthermore, the Eph protein expression profiles in both active and inactive central nervous system lesions of multiple sclerosis, normal adjacent white matter, and control tissues have been characterized by IHC.
Inflammatory cells in active multiple sclerosis lesions expressed ephrin-A1 to A4 and EphA1, A3, A4, A6, A7, and not EphA2, A5, and A8. In axons adjacent to active multiple sclerosis lesions, EphA3, A4, and A7 and ephrin-A1 expression was increased.
The Role of the Eph/Ephrin Proteins in Inflammation
The Development of the Vascular Endothelium
The normal development of the cardiovascular and lymphatic system requires the coordinated function of several important transcription factors, receptor–ligand pairs, growth factors, and guidance molecules.
These molecules include the Eph/ephrin proteins, VEGF and receptors 1 and 2, angiopoietins (Ang-1 and Ang-2) and their receptors (Tie-1 and Tie-2), netrins, slits and their receptors (Robo), semaphorins and plexins (the receptors for the semaphorins), and neuropilins.
The expression of ephrin-B2 and its receptor, EphB4, in a complementary pattern on embryonic arteries and veins, respectively, suggested a reciprocal interaction in the vascular remodeling process.
The initial evidence for the role of Eph/ephrin proteins in vascular biology was the identification of ephrin-A1 (previously B61) as a TNF-α–responsive gene in endothelial cells highlighting a potential role in inflammatory responses.
The vascular endothelium controls the passage of fluid, proteins, and inflammatory cells from the blood into the interstitium via the paracellular spaces between endothelial cells.
The endothelial cell–cell junctional structures, which include the gap, adherens, and tight junctions (zona occludens), play an important role in determining and regulating this endothelial barrier function.
The endothelial cell–cell junctional structures are a complex of transmembrane proteins, and barrier function is influenced by several external factors acting through signaling pathways that regulate the paracellular space.
The adherens junctions are particularly important in the post-capillary venule, which also expresses the receptors for inflammatory mediators, including TNF-α, IL-1β, and VEGF.
The tight junctions (zona occludens-1) are located at the most apical part of the cell membrane, and the claudins, occludins, and JAM-A are major constituent proteins (Figure 4).
Figure 4Mechanism of EphA2 signaling in the endothelium. The passage of fluid and inflammatory cells across the endothelium is regulated by both the shape of the endothelial cell and the permeability of the endothelial gap junctions.
Increased low-molecular-weight phosphotyrosine phosphatase dephosphorylates the p190 RhoGAP that inhibits p190 Rho-GAP activity and up-regulates Rho-GTP, which destabilizes adherens junctions.
The cortical actin filaments are critical components of the cellular cytoskeleton and interact with myosin filaments through myosin light chain kinase, resulting in changes to endothelial cell shape.
The distortion of endothelial cell shape allows the development of gaps in the monolayer, permitting the passage of fluid, proteins, and inflammatory cells from the blood into the interstitial tissues.
The actin filaments are tethered to membrane proteins, including VE-cadherin, and are dynamically regulated by the Rho family guanosine triphosphatases (Rho-GTPases), specifically RhoA, Rac1, and Cdc42, which are known targets of Eph/ephrin signaling. In general, Rac1 and Cdc42 activation stabilizes actin, whereas RhoA activation, in response to inflammatory stimuli, including thrombin and VEGF, disrupts the actin cytoskeleton
When rats were administered LPS, there was altered regulation of several Eph/ephrin proteins in the LPS processing organs (liver and lung) determined by differential mRNA display and RT-PCR.
There was increased EphA2 expression in the hypothalamus, with no change in ephrin-A1; however, there was a counterchange in corresponding receptor–ligand pair expression in the liver and lung.
There was a biphasic change in ephrin-A1 expression with early (phase 2) down-regulation (threefold) and up-regulation of EphA2 (16-fold) in the liver (similar changes in the lung), followed by later (phase 3) up-regulation (fourfold) of ephrin-A1 and normalization of EphA2.
Furthermore, there was up-regulation of ephrin-B2 and down-regulation of EphB3 (liver and similar in lung), with up-regulation of ephrin-A1/ephrin-A3 and down-regulation of EphA1/EphA3 (EphA3 was 21-fold underexpressed in the lung during phase 3), and these findings were confirmed by immunoblotting.
The vasculature is central to the pathogenesis of inflammation, and the vascular endothelium plays a key role in orchestrating the response to injury or infection.
Changes in the phenotype of the endothelium allow the passage of fluid into the interstitium and enable inflammatory cells to localize and migrate into an injured or infected tissue.
The up-regulation of ephrin-A1 by TNF-α, IL-1β, and LPS in the endothelium is mediated by a p38 mitogen-activated protein kinase and a stress-activated protein kinase/c-Jun NH2-terminal kinase–dependent mechanism.
Tumor necrosis factor-alpha induction of endothelial ephrin A1 expression is mediated by a p38 MAPK- and SAPK/JNK-dependent but nuclear factor-kappa B-independent mechanism.
Ephrin-A1 is chemoattractive for migrating endothelial cells in vitro, induces tubule formation in assays with human umbilical vein endothelial cells, and has previously induced sprouting blood vessels in a corneal pocket assay.
EphA2/Ephrin-A1 Signaling in Vascular Endothelial Injury
Both EphA2 and ephrin-A1 are expressed in distal normal lung tissue, and in vitro studies of pulmonary vascular endothelial cells determined that ephrin-A1 increases monolayer permeability with evidence of tight and adherens junction disruption.
Intravenous injection of ephrin-A1 in rats induces leakage of labeled albumin, with histological evidence of endothelial disruption, and the EphA2 receptor was markedly up-regulated in the lungs of hypoxic infected rats.
Furthermore, when rats were exposed to viral infection, EphA2 was up-regulated by an endothelin-dependent effect in lung endothelial cells, and when EphA2 signaling was blocked with a soluble ligand competitor (ephrin-A1-Fc), there was markedly reduced extravasation of albumin and reduced lung edema formation.
The pattern of distribution of vascular EphA2 receptors in the lung is predominantly in the alveolar microvasculature, indicating localization to the capillary bed as the source of fluid and protein extravasation.
Surprisingly, EphA2-deficient mice treated with Mycoplasma pulmonis infection or sensitized to ovalbumin to cause airway inflammation displayed increased cytokine production and greater leukocyte infiltration at the site of inflammation,
EphA2/Ephrin-A1 Signaling in Retinovascular Disease
In retinopathy of prematurity, diabetic retinopathy, neovascular glaucoma, and age-related macular degeneration, vision loss results from abnormal retinal angiogenesis.
In a rat model of retinopathy of prematurity, an intraocular injection of a soluble EphA2-Fc receptor resulted in a significant reduction in abnormal retinal revascularization without affecting normal retinal vessels.
In a mouse model of proliferative retinopathy, an intraocular injection of ephrin-A1-Fc suppressed ischemic retinal revascularization in a dose-dependent manner by inhibiting VEGF-induced angiogenesis and vasopermeability.
The migration of T lymphocytes between the peripheral blood and organized lymphoid tissue is tightly regulated by an array of chemokines, and the migration of T cells to inflammatory sites is also chemokine dependent.
Similarly, the organization of the thymus and trafficking and maturation of T lymphocytes within thymic microenvironments is, in part, regulated by Eph/ephrin protein interactions, with the balance between Eph and ephrin-B signaling important for T-cell development, which is under the influence of thymic epithelial cells.
The blockade of Eph/ephrin signaling with EphA-Fc fusion proteins reduces CD4+CD8+ thymocytes. EphA4 knockout mice have a block in T-cell maturation because of changes in the nonlymphoid thymic microenvironment. Ephrin-B1 is critical for T-cell development, whereas EphB6 overexpression results in breakdown of the thymic cortex-medulla limits.
In chronic lymphocytic leukemia, EphA2/ephrin-A4 interactions mediate trafficking of malignant B lymphocytes into tissues through the high endothelial venules.
Recently, both ephrin-A2 and ephrin-B2 expression has been documented on human polymorphonuclear neutrophils, and gene microarray confirmed ephrin-A2 and ephrin-B2 expression in polymorphonuclear neutrophil–induced inflammation-mediated angiogenesis in a CD18-deficient mouse model.
The proangiogenic capacity of polymorphonuclear neutrophils delineated by microarray technique and by measurement of neovascularization in wounded skin of CD18-deficient mice.
However, the precise role of Eph/ephrins in human polymorphonuclear neutrophils remains obscure. Peripheral blood leukocytes stimulated with TNF-α led to an increased expression of ephrin-B2, which has the potential to activate the endothelium in inflammation.
There is evidence for the involvement of Eph/ephrin proteins in a range of chronic inflammatory diseases. The levels of circulating cytokines, TNF-α and IL-1β, are elevated in chronic heart failure, and differential display identified EphA3 as a cytokine-responsive gene in cultured rat cardiomyocytes; however, a link between EphA3 and cardiac failure has not been determined.
EphA2 (formerly Eck) and ephrin-A1 (formerly B61) have been described in the maintenance of the intestinal barrier, and EphB/ephrin-B expression gradients direct intestinal epithelial cell positioning within the crypts.
Real-time PCR and cDNA microarray analysis determined that EphA2, ephrin-A1, EphB2, and ephrin-B1/B2 expression was up-regulated in the intestinal epithelial cells of mucosal lesions in patients with inflammatory bowel disease.
Ephrin-B2 is differentially expressed in the intestinal epithelium in Crohn's disease and contributes to accelerated epithelial wound healing in vitro.
Stimulation of the ephrin-B2 reverse signaling pathway induced the expression of wound healing–associated genes in an intestinal epithelial cell line-6.
Ephrin-B2 is differentially expressed in the intestinal epithelium in Crohn's disease and contributes to accelerated epithelial wound healing in vitro.
Stimulation of ephrin-B1/2 with EphB1-Fc in intestinal epithelial cells induced pro-inflammatory genes (cyclooxygenase-2 and monocyte chemotactic protein-1) and genes involved in wound healing (FAK and ERK 1/2 mitogen-activated protein kinase pathway), resulting in faster wound healing.
Ephrin-B1 expression was significantly increased in patients with rheumatoid arthritis in the synovial fibroblast cells and invading CD3-positive lymphocytes compared with patients with osteoarthritis.
In an animal model of rheumatoid arthritis, animals treated with an ephrin-B1-Fc fusion protein that activates the EphB1 receptor resulted in an increase in TNF-α and IL-6 production and increased the number of peripheral blood lymphocytes migrating into the joint.
The Mechanism of Eph/Ephrin Receptor–Ligand Signaling and Vascular Leak
The principal effect of Eph/ephrin receptor–ligand interaction is cell repulsion or de-adhesion mediated through a complex signaling cascade converging on a final common pathway, which regulates the activity of the Rho family GTPases (RhoA, Rac1, and Cdc42), which mediate changes to cytoskeletal proteins
(Figure 3). Vascular leak in inflammation is well described, but the precise mechanism linking inflammatory mediators with increased endothelial paracellular permeability is still not well understood.
Ephrin-A1 is a TNF-α–responsive gene that potentially links systemic inflammation and the release of inflammatory mediators with EphA2-ephrin-A1 signaling events.
However, EphA2 stimulation by ephrin-A1-Fc in cultured bovine retinal endothelial cells resulted in suppression of VEGF receptor 2 phosphorylation and VEGF-mediated increased vasopermeability
Although the Eph/ephrin signaling system may be partly responsible for vascular leak, recently, other signaling systems (eg, the Slit-Robo system) have also been implicated.
In MDCK cells, EphA2 activation by ephrin-A1-Fc phosphorylates claudin-4 in tight junctions and attenuates claudin-4 association with zona occludens-1, increasing paracellular permeability.
In a brain microvascular endothelial cell line, EphA2 associated with the tight junction and stimulation by recombinant ephrin-A1-Fc increased monolayer permeability, whereas EphA2 inactivation by RNA interference or a kinase-inactive mutant promoted tight junction formation
(Figure 4). EphA2 co-associates with and regulates cadherin expression in the adherens junction, and activation of EphA2 suppresses cell proliferation and cell adhesion in a range of cells, including endothelial cells.
In human mammary epithelial cells (MCF10A), overexpression of EphA2 destabilizes the adherens junction by weakening E-cadherin–mediated cell–cell adhesion through activation of a Rho-GTPase signaling pathway, which involves Src-kinase–enhanced low-molecular-weight phosphotyrosine phosphatase activity and inhibition of Rho-GAP.
EphA2 mediates thrombin-induced up-regulation of intercellular adhesion molecule 1; therefore, EphA2 may be responsible for changes to the endothelial cell surface in addition to the change in permeability of the endothelial cell–cell junctions.
In addition, monocyte migration is partly mediated through interaction of monocyte-expressed EphB receptors with endothelial ephrin-B2, and endothelial cells overexpressing ephrin-B2 displayed stronger adhesions with monocytes than endothelial cells expressing truncated ephrin-B2 or no ligand.
The evidence suggests that EphA2/ephrin-A1 signaling on the vascular endothelial cell, possibly in response to inflammation-induced up-regulation of TNF-α, thrombin, and other inflammatory mediators, influences both the integrity of endothelial junctions and cytoskeleton structure, resulting in a vascular leak.
Both of these changes to the phenotype of vascular endothelium are fundamental to the response to injury and the pathogenesis of systemic inflammation (Figure 4).
The Future
The role of the Eph/ephrin proteins in human disease is an emerging field. Although much remains to be explored, the evidence raises hopes for the development of novel therapies that precisely modulate the molecular mechanisms of disease through the administration of specifically targeted molecules, such as Eph-Fc or ephrin-Fc, which disrupt Eph/ephrin signaling interactions.
Furthermore, in another mouse model of myocardial infarction, EphA2/ephrin-A1 signaling promoted cardiac stem cells to migrate into the injured tissue.
The ability to influence the migration of stem cells to restore tissue integrity after ischemia-reperfusion events, such as myocardial infarction and stroke, is a particularly exciting prospect.
In rodent models of spinal cord injury, the administration of EphA4 antagonists reduces astrocytic glial scarring and encourages spinal cord axons to regenerate across an area of spinal cord injury and promote functional recovery.
The postmortem analysis of human brains (n = 19) after traumatic brain injury revealed up-regulation of EphA4 expression, again suggesting that blocking EphA4 activation may represent a therapeutic approach to improving recovery after brain trauma.
The increased vascular permeability that results in fluid leakage plays a significant role in the pathogenesis of the circulatory failure (shock) that complicates sepsis/systemic inflammatory response syndrome
In fact, the restoration of endothelial barrier function by treatment with a recombinant Slit receptor fragment improved mortality in a mouse model of systemic inflammation, suggesting that endothelial barrier function is a potential therapeutic strategy.
A further understanding of the role of the Eph/ephrin proteins in the trafficking and maturation of lymphocytes may allow us to modify the natural history of chronic inflammatory and degenerative disorders.
Tumor necrosis factor-alpha induction of endothelial ephrin A1 expression is mediated by a p38 MAPK- and SAPK/JNK-dependent but nuclear factor-kappa B-independent mechanism.
The proangiogenic capacity of polymorphonuclear neutrophils delineated by microarray technique and by measurement of neovascularization in wounded skin of CD18-deficient mice.
Ephrin-B2 is differentially expressed in the intestinal epithelium in Crohn's disease and contributes to accelerated epithelial wound healing in vitro.
CME Disclosure: The authors of this article and the planning committee members and staff have no relevant relationships with commercial interest to disclose.
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