Regulation of Angiogenesis by Eph–Ephrin Interactions

REVIEW ARTICLES Regulation of Angiogenesis by Eph–Ephrin Interactions Sanne Kuijper, Christopher J. Turner, and Ralf H. Adams⁎

contact-mediated EC–EC, endothelial– mural, and vessel–tissue interactions are critical for vascular morphogenesis and are in part controlled by the Eph– ephrin signaling system.


The large families of Eph receptor tyrosine kinases and their ephrin ligands transduce signals in a cell-cell contact-dependent fashion and thereby coordinate the growth, differentiation, and patterning of almost every organ and tissue. Eph–ephrin interactions can trigger a wide array of cellular responses, including cell adhesion, boundary formation, and repulsion. The exact mechanisms leading to this diversity of responses are unclear but appear to involve differential signaling, proteolytic cleavage of ephrins, and endocytosis of the ligand–receptor complex. In the developing cardiovascular system, Eph and ephrin molecules control the angiogenic remodeling of blood vessels and lymphatic vessels and play essential roles in endothelial cells as well as in supporting pericytes and vascular smooth muscle cells. Recent evidence suggests that Ephs and ephrins may also be involved in pathological angiogenesis, in particular, the neovascularization of tumors. Consequently, the expression, interactions, or signaling of Eph–ephrin molecules might be targets for future therapeutic approaches. (Trends Cardiovasc Med 2007;17:145–151) n 2007, Elsevier Inc.


Introduction

Blood vessels form a highly branched hierarchical network of arteries, capillary beds, and veins in the vertebrate body to ensure that all tissues and organs have sufficient access to the blood circulation. The lymphatic vasculature forms a similarly extensive but unidirectional tree-like system of blind-ended capillaries (terminal lymphatics) that drain excessive interstitial liquid from tissues Sanne Kuijper, Christopher J. Turner and Ralf H. Adams are at the Vascular Development Laboratory, Cancer Research UK London Research Institute, London, UK. ⁎ Address correspondence to: Ralf H. Adams, Vascular Development Laboratory, Cancer Research UK London Research Institute, London, UK. Tel.: (+44) 207-269-3323; fax: (+44) 207-269-3581.; e-mail: [email protected]. © 2007, Elsevier Inc. All rights reserved. 1050-1738/07/$-see front matter

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and channel it as protein-rich lymph through collecting lymphatics, and the thoracic duct into the venous circulation (Carmeliet 2003, Jain 2003, Tammela et al. 2005). Despite their different functional roles, the endothelial cells (ECs) of the two vascular networks need to cope with analogous challenges during their growth into extensive tubular networks. Both systems maintain their function during their expansion, which requires the tight coordination of proliferation, migration, differentiation, branching, and patterning processes. The assembly of blood vessels also involves the recruitment of supporting mural cells, that is, pericytes (PCs), which cover blood vessel capillaries and postcapillary venules, as well as vascular smooth muscle cells (vSMCs), which are associated with arteries and larger veins. Smooth muscle cells are also found on collecting lymphatics and ducts, whereas terminal lymphatics remain uncovered. Direct

Eph–Ephrin Interactions and Signaling

Eph receptor tyrosine kinases (RTKs) form a large family of transmembrane proteins with a single cytoplasmic kinase domain, which is activated in response to binding of ephrin ligands to the globular domain in the extracellular region. Ephrins are presented on the cell surface either via a glycosylphosphatidylinisotol anchor, as in the case of the ephrin-A subclass, or by a transmembrane region present in the second subclass, the ephrin-B ligands (Figure 1, [1] and [2]). Based on their binding preference to one or the other class of ephrins, Ephs have been subdivided into EphA and EphB receptors (Figure 1, [3] and [4]) (Kullander and Klein 2002, Pasquale 2005, Poliakov et al. 2004). All these subclasses include a considerable number of molecules. No fewer than 10 EphA receptors (EphA1–A10), 6 EphB RTKs (EphB1–EphB6), 6 ephrinAs (ephrin-A1–A6), and 3 ephrin-B ligands (ephrin-B1–B3) are known in chick and mammals. Owing to the duplication of chromosomes during evolution, an even larger number of Eph–ephrin molecules exist in zebrafish, which has become an established model for the analysis of developmental blood vessel morphogenesis in recent years. Ephs and ephrins are not only numerous but their relationship is also complex. Receptor–ligand binding is highly promiscuous within each subclass, and there are several examples of interactions between members of different subclasses. In addition to their role as conventional ligands for their RTK counterparts, frequently referred to as ‘forward signaling’ (Figure 1, [5]), ephrins have the receptor-like ability to transduce signals into the cell that presents these molecules on its surface,

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Figure 1. Regulation of cell–cell interaction by Eph–ephrin signaling. Schematic drawing (dashed circle) of EphA and EphB receptors [3,4] showing the N-terminal, globular ligandbinding domain, two extracellular fibronectin type III-like regions, cytoplasmic kinase domain, conserved tyrosine phosphorylation sites, and a C-terminal PDZ binding motif. The corresponding ligands contain an N-terminal ephrin domain mediating receptor binding and are attached to the membrane (or membrane rafts, gray bars) via a glycosylphosphatidylinisotol anchor (ephrinAs, [1]) or a transmembrane region (ephrin-Bs, [2]). The latter contain several highly conserved tyrosine phosphorylation sites and PDZ binding motif at their C-terminus. Eph–ephrin interactions lead to bidirectional signal transduction both into the receptor (forward signaling, [5]) and ligand-expressing cells (reverse signaling, [6]). Receptor–ligand binding leads to the recruitment of more molecules into larger clusters within the cell membrane [7] and triggers signaling through ligand-associated membrane rafts (gray bars, [8]). Cis interactions between EphA and ephrin-A molecules block activation of the former [14]. However, some evidence suggests that ephrin-Bs are capable of signaling in cis [15]. Metalloproteinase cleavage of ephrins in trans releases their extracellular domain, permits endocytosis of the bound Eph receptor [10], and thereby converts adhesive into repulsive interactions [9]. Cytoplasmic cleavage fragments of B-class ligands may directly affect signaling through SFKs [11]. Repulsion has also been associated with the endocytosis of the intact (unprocessed) receptor–ligand complex [12].

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so-called ‘reverse signaling’ (Figure 1, [6]) (Kullander and Klein 2002, Pasquale 2005, Poliakov et al. 2004). Reverse signal transduction is better understood for the B-class ephrins, which can recruit cytoplasmic signaling and adaptor molecules to a number of highly conserved tyrosine phosphorylation sites in their intracellular part or a carboxyterminal PDZ binding motif. The latter allows the binding of several proteins containing PDZ-type protein–protein interaction domains. Some of these interactors, such as Grip1, Grip2, syntenin, Par3, PICK1, or Dvl2, are adaptors that may recruit additional proteins into larger complexes. By contrast, other intracellular binding partners, like FAP-1/PTP-BL, Tiam1, and PDZRGS3, can directly control signal transduction by dephosphorylating protein substrates, activating the small GTPase Rac1, or terminating the signaling downstream of G protein-coupled receptors, respectively (Kullander and Klein 2002, Pasquale 2005, Poliakov et al. 2004). Phosphorylation of the conserved tyrosine residues in ephrin-B ligands and signal transduction downstream of both ephrin subclasses involves the activity of Src family kinases (SFKs) (Figure 1, [7]). In the case of ephrin-As, unknown partner molecules, perhaps transmembrane proteins, might be required for reverse signaling. Alternatively, Eph binding might induce the clustering of ephrins within lipid rafts, microdomains within the cell membrane with distinct biophysical properties, and thereby trigger the activity of raft-associated SFKs (Figure 1, [8]). PDZ-mediated binding of the protein tyrosine phosphatase FAP-1/PTP-BL may counteract the effect of SFKs and block the recruitment of the adaptor protein Grb4/Nckβ (and perhaps other yet unknown binding partners) to the phosphotyrosine motifs in the ephrin-B cytoplasmic region. Eph receptor signaling is even more complex and involves the downstream activation of Abelson kinase (Abl) and SFKs. The network of Eph receptor interactors further includes phosphotyrosine-binding adaptors (Nck, Crk, Grb2, Grb10, SLAP), PDZ domain proteins (syntenin, Pick1, Grip1, AF-6), the 85-kDa subunit of phosphatidylinositol 3′-kinase, modulators of Ras and Rho family small GTPases (SHEP1, RasGAP, and ephexin), and the phosphatase

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LMW-PTP (Kullander and Klein 2002, Pasquale 2005, Poliakov et al. 2004).


Regulation of Repulsion, Adhesion, and Motility

The complexity of the signaling network downstream of Ephs and ephrins is mirrored by a similar array of cellular effects in response to the activation of these molecules. In many settings and cell types, forward signaling leads to repulsion of the Eph receptor-expressing cell. For example, activation of Eph RTKs on migrating neural crest cells or axonal growth cones (the navigation structure at the distal end of growing nerve fibers) triggers retraction away from tissues expressing the corresponding ephrins (Figure 1, [9]) (Klein 2004, Pasquale 2005, Poliakov et al. 2004). As a result, the Eph-positive subset of cells or nerve fibers may stall, turn, and change direction, whereas others are unaffected by the presence of ephrins because they lack receptor expression. A less pronounced repulsive signal, perhaps because of lower expression of Eph– ephrin molecules, may also explain why receptor-expressing cells are separated from an adjacent ephrin-positive tissue population (Kullander and Klein 2002, Poliakov et al. 2004). Given that repulsive signals can explain many of the biological functions of Ephs and ephrins, it may appear paradoxical that the same ligand and receptor molecules promote adhesion in certain settings. For example, high concentrations of ephrin-A2 trigger a repulsive response, whereas low concentrations promote the growth of cultured nerve cells (Hansen et al. 2004). In this context, it is noteworthy that Ephs and ephrins are sometimes presented in spatial gradients, so that cells get to choose between repellent or growth-promoting environments defined by a high or low level of Eph– ephrin interactions, respectively (Klein 2004, Poliakov et al. 2004). Signaling downstream of Eph receptors may play a key role in this decision because kinase-deficient versions of EphA7 promote adhesion in cultured cells and not repulsion, as it is the case for the full-length RTK (Holmberg et al. 2000). Cleavage of ephrins by the membranebound disintegrin metalloprotease ADAM10 provides another molecular

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switch between adhesion and repulsion (Figure 1, [10]) (Hattori et al. 2000). It has been shown that binding of EphA3 to ephrin-A5 exposes a metalloproteinase recognition motif and triggers the clipping of the ephrin by ADAM10. This release of the ephrin from the cell surface permits the withdrawal of the Ephexpressing cell, whereas blocking of cleavage leads to an extended (adhesive) interaction with the ligand-presenting cell (Figure 1, [11]) (Hattori et al. 2000). ADAM10 is constitutively associated with EphA3 (and perhaps other Ephs) so that cleavage of ephrin-A5 occurs in trans (Janes et al. 2005). Conservation of the ADAM recognition motif in ephrins of both subclasses suggests that this mechanism could be of wider relevance (Hattori et al. 2000). Removal of the Eph–ephrin complex from the cell surface by endocytosis is another mechanism mediating the conversion of adhesive interactions into a repulsive signal. In this setting, cell detachment requires both Eph activation and (‘forward’) internalization of the signaling complex into the receptorexpressing cell (Figure 1, [12]), and into some extend into the ligand-expressing cells (Figure 1, [13]) (Marston et al. 2003, Zimmer et al. 2003). Recent findings challenge the currently established view that Eph–ephrin interactions are confined to the contact zone between two adjacent cells. Whereas conventional interactions in trans involve the conserved ephrin domain and the corresponding ligandbinding domain located at the aminoterminal ends of ephrins and Eph receptors, respectively, ephrin-A5 can also bind in cis to the extracellular fibronectin type III-like domains in EphA3. The latter blocks receptor activation and thereby counteracts forward signaling in response to cell–cell contact (Figure 1, [14]) (Carvalho et al. 2006). The B-class ligand ephrin-B2 also appears to control cell motility and adhesion in cultured vSMCs even in the absence of cell–cell contact (Figure 1, [15]) (Foo et al. 2006).


Regulation of Vascular Morphogenesis by Ephs and Ephrins

Several Eph–ephrin molecules of both subclasses are expressed in the cardiovascular system, but EphB4 and its

ligand ephrin-B2 have attracted the most interest. Ephrin-B2 is predominantly expressed in arterial ECs, whereas EphB4 is mostly venous-specific (Figure 2). Although the functional relevance of these arteriovenous-specific expression patterns remains unclear, targeted inactivation of the genes encoding ephrin-B2 (Efnb2) and EphB4 (Ephb4) in mice has demonstrated that both are essential for angiogenic remodeling and embryonic survival (Adams et al. 1999, Gerety et al. 1999, Wang et al. 1998). Endothelial-cellspecific Efnb2 mutant recapitulates the severely pathological phenotype of the global knockout and proves that ephrinB2 has essential functions in the endothelial lining of blood vessels (Gerety and Anderson 2002). Knowledge is emerging about signaling pathways that interact with EphB4/ ephrin-B2 to establish arterial–venous specification. Up-regulation of ephrinB2 and simultaneous suppression of EphB4 expression in the arterial endothelium are controlled by the Notch pathway as well as by hemodynamic factors (Figure 2) (Heroult et al. 2006). Ephrin-B2 expression is also increased during physiological and pathological neoangiogenesis in the adult, which most likely reflects its regulation by vascular endothelial growth factor (VEGF) (Gale et al. 2001, Shin et al. 2001). Interestingly, arterial and venous ECs lose their characteristic asymmetrical ephrin-B2 expression pattern during in vitro culture, suggesting that ephrin-B2 and EphB4 expression is not hardwired but controlled by the local microenvironment within the vascular tree (Korff et al. 2006). Despite strong genetic evidence that ephrin-B2 and EphB4 play essential roles in growing blood vessels, their mechanistic role during angiogenic remodeling is largely unclear. Stimulation of cultured ECs with soluble dimeric forms of ephrin-B ligands and EphB receptors indicates that reverse and forward signaling can promote sprouting angiogenesis (Adams et al. 1999, Palmer et al. 2002). Activation of EphB4 induces EC migration and proliferation, which is at least in part mediated by PI3-kinase and the serin/threonine-specific kinase Akt/ PKB (Maekawa et al. 2003, Steinle et al. 2002). However, it has also been reported that forward EphB4 signaling inhibits EC migration, adhesion, and proliferation and suppresses a signal transduction

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of the lymphatic vasculature. It was shown that ephrin-B2 is strongly expressed in ECs of collecting lymphatic vessels, whereas EphB4 is found throughout the lymphatic vasculature (Makinen et al. 2005). The generation of mutant mice lacking the C-terminal PDZ interaction domain of ephrin-B2 has uncovered an important role of the ligand in lymphangiogenesis. Loss of the PDZ interaction motif disturbs the sprouting of lymphatic ECs and thereby compromises the formation of distal capillary lymphatic beds. Mutant collecting lymphatics are hyperplastic and devoid of valves. As a consequence of these malformations, mutants accumulate chylous fluid in their thorax and die within the first weeks of postnatal life. Although several of the PDZ proteins interacting with ephrin-B2 are expressed in lymphatic ECs, it is not yet known which of those are required for lymphangiogenesis (Makinen et al. 2005). It also appears that the PDZ motif in ephrin-B2 is not essential for developmental blood vessel morphogenesis, suggesting that ephrin-B2 controls the growth of blood vessels and lymphatics through different signaling mechanisms.


Figure 2. Ephrin-B2 and EphB4 in the regulation of arterial–venous identity. Schematic drawing of blood vessels (artery is shown in red, vein in blue, capillaries in white) with associated mural cells, that is, vSMCs and PCs. The unidirectional network of lymphatic capillaries and a collecting duct (with associated SMCs) is shown in green. Arterial differentiation and endothelial ephrin-B2 expression are controlled by VEGF signaling and activation of the Notch pathway as well as by hemodynamic factors (arterial blood flow). Expression of Notch pathway molecules on veins is suppressed by COUP-TFII. EphB4 is present in the venous endothelium and throughout the lymphatic vasculature. Ephrin-B2 and EphB4 are coexpressed in lymphatic collecting ducts.

cascade involving the small GTPase Ras and mitogen-activated protein kinase, which thereby interferes negatively with VEGF and angiopoietin-1 signaling (Fuller et al. 2003, Hamada et al. 2003, Kim et al. 2002). The reasons for these conflicting results remain unclear, but possible explanations include experimental design or differences in the concentration or clustering of recombinant Eph–ephrin fusion proteins. Ephs and ephrins are able to affect adhesion to extracellular matrix and cell migration by modulating integrin activity (Meyer et al. 2005, Miao et al. 2005). For instance, activation of endothelial ephrin-B1 enhances integrin-mediated

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migration, attachment, and promotes neovascularization in a mouse cornea micropocket assay (Huynh-Do et al. 2002). Reverse signaling by B-class ephrins to integrins involves the docking proteins Crk and p130Cas as well as activation of the small GTPase Rac1 (Foo et al. 2006, Nagashima et al. 2002). Unfortunately, there are currently very few data addressing the role of specific signaling cascades downstream of Eph– ephrin molecules in vivo, so that the biological relevance of many of the findings above remains unclear. In addition to a role in blood vessel development, EphB4 and ephrin-B2 have recently been linked to the development

Recruitment of Mural Cells

The importance of the association of PCs and vSMCs with the blood vessel endothelium is evident from a number of human pathologies. In diabetic retinopathy, loss (‘‘dropout’’) of PCs leads to microaneurysms, aberrant proliferation of immature vessels, macular edema, and eventually blindness (Adamis et al. 1999, Betsholtz et al. 2005). Similarly, low abundance or loose attachment of PCs might be in part responsible for the disorganized and leaky vasculature of tumors (Morikawa et al. 2002). Smooth muscle cells are involved in the pathogenesis of various vascular diseases such as atherosclerosis, aneurysms, varicose veins, or hypertension. Genetic studies in mice have demonstrated that four major receptor–ligand systems regulate critical steps of mural cell differentiation and recruitment: (1) platelet-derived growth factor-B and its receptor platelet-derived growth factor receptor-β; (2) transforming growth factor β (TGFβ) and its binding partners transforming growth factor β receptor II, Alk1, Alk5, and endoglin; (3) sphingosine

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1-phosphate and its receptor endothelial differentiation gene-1; and (4) angiopoietins 1 and 2 and their receptor Tie2/Tek (Armulik et al. 2005, Betsholtz et al. 2005). More recently, it has been shown that expression of Eph–ephrin molecules in mural cells is also critical for assembly of the vessel wall. To date, only the function of ephrin-B2 has been directly addressed, whereas the role of specific Eph receptors in PCs/vSMCs remains unclear (Foo et al. 2006, Gale et al. 2001, Shin et al. 2001). Mural cellspecific inactivation of the Efnb2 gene results in perinatal lethality, edema, and extensive hemorrhaging in the skin (Foo et al. 2006). Ephrin-B2-deficient PCs are only loosely attached, make insufficient contacts with ECs, and fail to envelope the endothelial monolayer. Likewise, mutant vSMCs show attachment defects, cover microvessels in a discontinuous fashion, and ectopically occupy the lymphatic capillaries of the skin. It is notable that the number of mural cells remains unchanged in these mice despite their improper incorporation into the vascular wall. This implies that mural cells or their precursors do not require expression of ephrin-B2 for differentiation, proliferation, or survival processes but rather for the assembly of the vessel walls during vascular maturation (Foo et al. 2006). Accordingly, expression of the Efnb2 gene in vSMCs commences at embryonic stage E12.5, that is, several days later than expression in the endothelium (Shin et al. 2001). Interestingly, the first detectable expression of ephrin-B2 in vSMCs is in the layer immediately adjacent to the endothelium, suggesting that an inductive signal from ECs may induce mural expression of ephrin-B2 (Shin et al. 2001). In vitro, ephrin-B2-deficient vSMCs are defective in spreading, focal adhesion formation, and show increased but unpolarized motility (Foo et al. 2006). Surprisingly, some of these defects appear to be cellcontact-independent, suggesting a cellautonomous role for ephrin-B2. Control of focal adhesion formation and integrin-mediated cell spreading of mural cells also involve activation of EphA receptors. Stimulation of vSMCs with ephrin-A1 fusion protein inhibits cell spreading and lamellipodia formation through inhibition of the small GTPase Rac1 and the kinase Pak1, a key regulator of the actin cytoskeleton, cell adhesion,

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and motility (Deroanne et al. 2003). Interestingly, a smooth muscle cell-specific guanine nucleotide exchange factor for the GTPase RhoA (Vsm-RhoGEF) has been found to associate with EphA4 (Ogita et al. 2003). Upon stimulation with ephrin-A1, EphA4 phosphorylation enhances Vsm-RhoGEF activity for RhoA and promotes the assembly of actin stress fibers. This suggests that interactions between ephrin-A1 and EphA4 control vSMC contractility, vascular tone, and blood pressure via Vsm-RhoGEF and RhoA (Ogita et al. 2003). Other downstream interactors of Eph receptors also stimulate Rho activity such as the guanine nucleotide exchange factors Vav, ephexin, and dishevelled, but it is currently unclear whether these molecules are relevant in the vasculature.


Regulation of Tumour Angiogenesis

Although the roles of Ephs and ephrins have mostly been studied during embryonic development, expression patterns suggest additional roles in the regulation of physiological and pathological angiogenesis in the adult (Dodelet and Pasquale 2000, Gale et al. 2001, Shin et al. 2001). Several Eph–ephrin molecules are up-regulated in cancer and may affect tumor growth and neovascularization. For example, elevated expression of EphA2 and its ligand ephrin-A1 has been linked to cancers of the breast, colon, prostate, lung, and to melanoma (Ogawa et al. 2000, Walker-Daniels et al. 2003). Blocking the activation of EphA receptors with soluble recombinant fusion proteins or inactivation of the Epha2 gene in mice reduces the size and vascularization of experimental tumors (Brantley-Sieders et al. 2004, Fang et al. 2005). EphA receptors appear to play important roles in tumor cells, in the endothelium, and at the tumor–endothelial interface, which makes these molecules potential targets for future therapies (Brantley-Sieders et al. 2004). The situation appears more complex for B-class molecules. Overexpression of ephrin-B ligands in cancer cells correlates with increased invasion and high vascularization of human tumors leading to a poor prognosis (Castellvi et al. 2006, Meyer et al. 2005). Although B-class receptors such as EphB4 appears to act as a tumor suppressor in colon and breast cancer, the receptor also enhances tumor

growth, cancer cell motility, and metastasis in the prostate, bladder, and in melanoma (Batlle et al. 2005, Noren et al. 2006, Xia et al. 2005, Xia et al. 2006). It has also been reported that tumor cell expression of EphB4 promotes angiogenesis through interactions with ephrin-B2 presented on ECs (Noren et al. 2006). By contrast, another recent study showed that EphB4 acts as a negative regulator of branching angiogenesis by promoting circumferential growth of blood vessels and suppressing endothelial sprouting. This process involves reverse signaling by ephrin-B2, which enhances mural cell association to tumor blood vessels through the activation of the angiopoietin-1/Tie2 pathway (Erber et al. 2006). Deregulated expression of ephrin-B2 and EphB4 in ECs has also been implicated in the abnormal remodeling of tumor blood vessels and loss of arterial– dvenous identity in Kaposi sarcoma (Masood et al. 2005). Similarly, hepatocarcinoma leads to an up-regulation of Notch pathway components and ephrinB2 in sinusoidal ECs, which normally display a venous identity and express EphB4 (Hainaud et al. 2006). Consistent with an important role of EphB4/ephrinB2 interactions in tumors, soluble fusion proteins containing the extracellular domain of EphB4 (sEphB4) reduce the growth and vascularization of tumors by antagonizing endogenous receptor–ligand interactions (Kertesz et al. 2006, MartinyBaron et al. 2004). These data indicate that targeting the activity of ephrin-B2 and/or EphB4 might be therapeutically beneficial in the context of cancer. Despite these first promising results, the exact biological role of ephrin-B2, EphB4, and Eph–ephrin molecules in tumors and other pathological processes remains unclear. Future work will have to address the exact relevance of Eph–ephrin interactions between endothelial, mural, and cancer cells. Here, it will be critical to understand how the activity of Ephs and ephrins controls cellular behavior in specific cell types and tissues. Tissuespecific mutant mice or engineered tumor cell lines expressing mutated versions of Eph–ephrin molecules may provide valuable insight in this direction. Moreover, evidence for tumor suppressor activity of Eph receptors raises the concern that inhibition of their activity may have detrimental effects at least in certain cancers. Detailed analysis

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of the downstream signaling pathways may explain how the same receptors can suppress certain cancers but promote the growth, vascularization, and metastasis of others. Given the versatile functions of Ephs and ephrins and their expression in almost every normal and diseased cell type and tissue, this effort should be worthwhile and might give access to a wide range of therapeutic opportunities.


Acknowledgments

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Cdc42 Regulates the Restoration of Endothelial Adherens Junctions and Permeability Michael T. Broman, Dolly Mehta, and Asrar B. Malik ⁎

The endothelial adherens junction (AJ) complex consisting of VEcadherin and its associated catenins is a major determinant of fluid, solute, and plasma protein permeability of the vessel wall endothelial barrier. Impairment of endothelial barrier function contributes to cardiovascular diseases such as vascular inflammation and atherosclerosis. Adherens junctions disassemble in response to proinflammatory mediators, producing an increase in endothelial permeability; however, AJs also have the capacity to reassemble, leading to restoration of endothelial barrier function. Activation of Cdc42, a member of the Rho family of monomeric GTPases, is an essential signal regulating reannealing of AJs and reversal of the increase in endothelial permeability. The possibility of activating Cdc42 therapeutically represents a novel approach to prevent inflammatory diseases resulting from breakdown of the endothelial barrier. This review summarizes recent findings concerning the role of Cdc42 in restoring endothelial barrier integrity. (Trends Cardiovasc Med 2007;17:151–156) n 2007, Elsevier Inc. The endothelial cell monolayer lining the intima of blood and lymphatic vessels (which comprises a vast surface area) forms a selective semipermeable barrier that performs the vital tasks of regulating tissue fluid homeostasis and migrating of blood cells across the vessel wall (Mehta et al. 2004, Rao et al. 2005). Impairment of endothelial barrier function is a crucial factor in the pathogenesis of several diseases, including atherosclerosis, diabetes-associated vascular disease, and acute lung injury. In acute lung

Michael T. Broman, Dolly Mehta, and Asrar B. Malik are at the Department of Pharmacology and the Center for Lung and Vascular Biology, University of Illinois College of Medicine, Chicago, IL 60612, USA. ⁎ Address correspondence to: Asrar B. Malik, Department of Pharmacology (M/C 868), University of Illinois, 835 South Wolcott Avenue, Chicago, IL 60612, USA. Tel.: (+1) 312-996-7635; fax: (+1) 312-996-1225; e-mail: [email protected]. © 2007, Elsevier Inc. All rights reserved. 1050-1738/07/$-see front matter

injury, increased microvessel endothelial permeability leads to protein-rich alveolar edema and impaired oxygenation. Increased endothelial permeability is also a critical determinant of migration of cancer cells, leading to metastasis (Weis et al. 2004). The endothelial adherens junction (AJ) complex consisting of VE-cadherin and its associated catenins dynamically controls the permeability of the vessel wall endothelial barrier. Thus, it is important to define the organization of the AJ complex of endothelial cells and how AJs, in response to external stimuli, regulate endothelial barrier function. The release of humoral factors during localized infection and activation of inflammatory cells triggers signals leading to disassembly of AJs and increased endothelial permeability (SiflingerBirnboim and Johnson 2003, Tinsley et al. 2005). Disruption of AJs can, in turn, also lead to the activation of other signals that promote the reassembly of AJs to restore endothelial barrier function. A comprehensive recent review

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