VE-Cadherin and Endothelial Adherens Junctions: Active Guardians of Vascular Integrity

Developmental Cell

Review VE-Cadherin and Endothelial Adherens Junctions: Active Guardians of Vascular Integrity Monica Giannotta,1 Marianna Trani,1 and Elisabetta Dejana1,2,* 1FIRC

Institute of Molecular Oncology, 20129 Milan, Italy of Biosciences, University of Milan, 20122 Milan, Italy *Correspondence: [email protected] http://dx.doi.org/10.1016/j.devcel.2013.08.020 2Department

VE-cadherin is a component of endothelial cell-to-cell adherens junctions, and it has a key role in the maintenance of vascular integrity. During embryo development, VE-cadherin is required for the organization of a stable vascular system, and in the adult it controls vascular permeability and inhibits unrestrained vascular growth. The mechanisms of action of VE-cadherin are complex and include reshaping and organization of the endothelial cell cytoskeleton and modulation of gene transcription. Here we review some of the most important pathways through which VE-cadherin modulates vascular homeostasis and discuss the emerging concepts in the overall biological role of this protein. Introduction Endothelial cells form monolayers that cover the internal surface of blood vessels and protect the tissues from noxious blood components and inflammatory cells. The endothelial integrity is maintained by a complex functional balance between multiple cell activities that modulate the inhibitory activity of the vessel wall to thrombosis or inflammatory reactions. Endothelial cell-to-cell junctions have crucial roles in these processes because they not only maintain intercellular adhesion but also transfer intracellular signals that modulate contact inhibition of cell growth, cell polarity, lumen formation, and interactions with pericytes and smooth muscle cells (for review, see Dejana et al., 2009). Therefore, conditions that disrupt endothelial junctions might not only increase vascular permeability by opening intercellular gaps but also change the endothelial cell responses to their environment and to the surrounding cells. Endothelial adherens junctions have been studied extensively by several groups (for reviews, see Dejana et al., 2009; Delva and Kowalczyk, 2009; Komarova and Malik, 2010; Vestweber et al., 2009). A schematic view of the multiple components that can associate to adherens junctions is shown in Figure 1. The ‘‘basic’’ organization of adherens junctions is provided by vascular endothelial cadherin (VE-cadherin), which is linked through its cytoplasmic domain to p120-catenin and b-catenin or plakoglobin (Figure 1) (Dejana and Vestweber, 2013). The identified intracellular partners of VE-cadherin so far are multiple, and it is likely that many more will be discovered in the future (Table 1). The cytoplasmic domain of VE-cadherin is highly homologous to that of classic cadherins such as N-cadherin or E-cadherin and shares with them some intracellular partners such as p120-catenin or b-catenin. An exception is plakoglobin, which is commonly associated with desmosomes in epithelial cells but can directly bind to VE-cadherin in endothelial cells, which lack desmosomes (for review, see Bazzoni and Dejana, 2004). A systematic study of the different partners of VE-cadherin in comparison with other members of the cadherin family is missing. Some partners are specific to endothelial cells, such

as VE-PTP, PECAM, or VEGFR2, and are, as a consequence, also specific for VE-cadherin. However, the majority of the molecules listed in Table 1 are expressed also in other cell types and have the potential to interact with other members of the cadherin family. Considering the large number of putative partners of VEcadherin, it is tempting to speculate that these proteins can assemble into different types of complexes, which would vary according to their composition and function, the different vessels and stages of development, and even within the same cell. This Review will mainly focus on VE-cadherin and adherens junction organization in endothelial cells to highlight the functional activity of these molecules and their roles in the control of vascular permeability and intracellular signaling. Some of the relevant members of the cadherin complex that have been identified and studied in other cell types will also be briefly mentioned. Cadherin Expression and Function in Endothelial Cells At adherens junctions, adhesion is mediated by the cadherins, which are calcium-dependent adhesion molecules. Endothelial cells express VE-cadherin and N-cadherin to comparable levels, although in most cases, only VE-cadherin is clustered at cell-tocell contacts, with N-cadherin excluded from these structures (Navarro et al., 1998). This particular behavior of N-cadherin is not endothelial-cell specific but is related to the functional and structural features of these two cadherins. Cotransfection of VE-cadherin with N-cadherin in Chinese hamster ovary (CHO) cells prevents N-cadherin localization to the adherens junctions (Navarro et al., 1998). A similar result was obtained upon transfection of a VE-cadherin mutant that was truncated in the cytoplasmic domain, which suggests that the extracellular region of VE-cadherin, its residual cytoplasmic tail, or its transmembrane domain is responsible for N-cadherin exclusion from the adherens junctions (Giampietro et al., 2012; Navarro et al., 1998). Regardless of the mechanism, these observations suggest that VE-cadherin and N-cadherin have different functions in endothelial cells. Because N-cadherin is expressed in several Developmental Cell 26, September 16, 2013 ª2013 Elsevier Inc. 441

Developmental Cell

Review Figure 1. Multiple Functions of Adherens Junctions in Endothelial Cells The organization of adherens junctions is provided by VE-cadherin, which is linked through its cytoplasmic domain to p120-catenin, b-catenin, and plakoglobin. These proteins assemble in diverse complexes and have different functions. The interactions of VE-cadherin with the growth factor receptors VEGFR2, FGF-R1, and the TGFb-R complex modulate their downstream signaling. The cytoskeletal remodeling controlled by VEcadherin is through its indirect interaction with various actin-binding proteins, such as a-catenin, vinculin, a-actinin, eplin, and others. In addition, the stability of the adherens junctions is regulated by the clustering of VE-cadherin and its indirect association with different partners, such as Tiam, the CCM complex, and vinculin. Some phosphatases such as VE-PTP, DEP-1, PTP-m, Csk, and SHP2 might also associate with VE-cadherin and, directly or indirectly, decrease its phosphorylation and increase endothelial barrier function. Conversely, phosphorylation of VE-cadherin by Src or FAK, as well as the VE-cadherin interaction with b-arrestin2, induces junction weakening. See the text for more details.

cell types, including mesenchymal and neural cells, an interesting possibility is that N-cadherin promotes endothelial cell adhesion and communication with other cells, such as pericytes or astrocytes (Takeichi, 1988). This indicates that such interactions are important in the elongation of vascular sprouts and in the formation of the so-called neurovascular unit in the brain. Indeed, direct contacts between endothelial cells and the underlying smooth muscle cells that define the myoendothelial junctions have been described using electron microscopy, which suggests that these myoendothelial junctions are required for coordinated responses of the vessel walls to various stimuli (Liebner et al., 2000). Specific loss of N-cadherin in endothelial cells leads to early embryonic death due to defects in vascular development. This occurs earlier than pericyte or smooth muscle cell engagement of the vasculature, which suggests that N-cadherin has a direct role in endothelial cell function (Luo and Radice, 2005). In a recent study, we showed that N-cadherin inhibits cell growth and apoptosis in a way comparable to VE-cadherin (Giampietro et al., 2012). However, while N-cadherin induced endothelial cell motility, VE-cadherin had the opposite effect of limiting cell movement. We also showed that VE-cadherin interacts with the fibroblast growth factor (FGF) receptor and reduces the receptor phosphorylation and signaling through coupling with density-enhanced phosphatase-1 (DEP-1). Conversely, N-cadherin maintains high FGF receptor activity in endothelial cells and induces a set of genes that are crucial for cell migration (Giampietro et al., 2012). Furthermore, we also showed that VEcadherin negatively controls both N-cadherin localization at the adherens junctions and N-cadherin expression by limiting p120-catenin availability and reducing b-catenin transcriptional activity (Giampietro et al., 2012). Overall, these findings suggest that any significant oscillation in VE-cadherin levels will increase N-cadherin expression in the endothelium. A tempting hypothesis is that these two cadherins contribute to angiogenesis by acting at different levels. N-cadherin promotes cell migration and contributes to vascular elongation, 442 Developmental Cell 26, September 16, 2013 ª2013 Elsevier Inc.

while VE-cadherin induces vascular quiescence by limiting growth factor receptor signaling. In some vessels, the endothelial cells express two other members of the cadherin family, P-cadherin and T-cadherin (Bazzoni and Dejana, 2004). T-cadherin is a glycosyl phosphatidylinositolanchored cell-surface glycoprotein, and it lacks the cytoplasmic domain. As well as promoting cell-to-cell adhesion, T-cadherin has a unique property: it can bind to the adipocyte-secreted hormone adiponectin, which, in turn, has a cardioprotective activity (Denzel et al., 2010) and promotes tumor angiogenesis (Denzel et al., 2009). VE-Cadherin and the Development of the Vascular System in the Embryo VE-cadherin is one of the first cell-specific markers to be expressed by endothelial cells during the development of the embryo vasculature and embryonic stem cell differentiation to endothelial cells in vitro (Breier et al., 1996; Vittet et al., 1996, 1997) (Figures 2A–2C). Using in situ hybridization in the mouse embryo, VE-cadherin transcripts have been detected at very early stages of vascular development (embryonic day 7.5 [E7.5]) in mesodermal cells of the yolk-sac mesenchyme (Figure 2A). At later embryonic stages, VE-cadherin transcripts were present in vascular structures of all of the organs examined, which included the ventricles of the heart, the inner cell lining of the atrium, the dorsal aorta, and the intersomitic vessels and capillaries entering the brain. This suggests that VE-cadherin is needed for the correct organization of the vasculature and for endothelial assembly (Breier et al., 1996). The early expression of VE-cadherin in endothelial progenitors in the embryo suggests that it contributes to endothelial differentiation. However, in VE-cadherin null mouse embryos, some early endothelial-cell-specific markers are still expressed, indicating that early endothelial cell differentiation may also occur in the absence of VE-cadherin (Breier et al., 1996; Crosby et al., 2005).  et al. (2009) showed that VE-cadherin participates in the Strilic establishment of correct endothelial cell polarity and lumen

Developmental Cell

Review Table 1. Functional Interactors of VE-Cadherin and Adherens Junctions Interactors

Function

References

b-catenin

junction architecture and signaling

Takeichi, 1988; Clevers, 2006; Dejana, 2010

p120

VE-cadherin stabilization and signaling

Reynolds and Carnahan, 2004; Xiao et al., 2007; McCrea et al., 2009

plakoglobin

junction architecture and signaling

Lampugnani et al., 1995; Nottebaum et al., 2008

a-catenin, a-actinin, eplin, N-WASP

cytoskeletal anchorage and organization

Hartsock and Nelson, 2008; Knudsen et al., 1995; Abe and Takeichi, 2008; Chervin-Pe´tinot et al., 2012; Rajput et al., 2013

Junction Architecture

Junction Stability and Cytoskeleton Organization Tiam

activation of Rac, junction stability

Lampugnani et al., 2002; Cain et al., 2010

Rap1, Raf1, MAGI

junction stability and maturation

Sakurai et al., 2006; Glading et al., 2007; Wimmer et al., 2012

VE-PTP

VE-cadherin dephosphorylation, permeability control, leukocyte diapedesis

Nottebaum et al., 2008; Nawroth et al., 2002; Broermann et al., 2011

PTP1B, PTP2A, SHP2, DEP-1, RPTP-m

permeability control

Grinnell et al., 2010, 2012; Ukropec et al., 2000; Ka´sa et al., 2013; Hatanaka et al., 2012; Lampugnani et al., 2006; Orsenigo et al., 2012, Sui et al., 2005

Csk

Src inactivation, inhibition of cell growth

Baumeister et al., 2005

PAR3/PAR6/aPKC

cell polarity, lumen formation

Lampugnani et al., 2010; McCaffrey and Macara, 2012; Iden et al., 2006; Koh et al., 2008

CCM1/Krit1

cell polarity, junction stability

Lampugnani et al., 2010; Glading et al., 2007

VEGFR2

contact inhibition of cell growth, permeability control

Nottebaum et al., 2008; Weis et al., 2004; Lampugnani et al., 2006; Gavard and Gutkind, 2006

FGF-R

cell motility and growth

Murakami et al., 2008; Giampietro et al., 2012

TGFb-R

inhibition of cell motility

Rudini et al., 2008; Rezaei et al., 2012

junction permeability

Gavard and Gutkind, 2006; Hebda et al., 2013; Kronstein et al., 2012

PECAM

mechanosensor

Conway and Schwartz, 2012; Tzima et al., 2005

PI3 kinase, Akt, Shc

signal transduction

Taddei et al., 2008; Zanetti et al., 2002

Src, FAK

increase in permeability

Weis et al., 2004; Wallez et al., 2007; Chen et al., 2012

Phosphatases Junction Stability

Cell Polarity

Growth Factor Receptors

Induction of Permeability b-arrestin1, b-arrestin2, Caveolin1 Other Functions

We report here some of the best-studied partners of VE-cadherin and adherens junctions. We apologize if, for space constraints, the table is not exhaustive.

formation in large vessels of the mouse embryo. During aorta development, endothelial cells adhere to each other, and the CD34-sialomucins—moesin, F-actin, and nonmuscle myosin II—localize at the endothelial cell-to-cell contacts to define the luminal cell surface. VE-cadherin is required for this CD34-sialomucin localization. This promotes the recruitment of moesin and F-actin, which results in changes in endothelial shape and the consequent lumen formation. Some observations of vascular development of isolated allantoises, which consist of all embryonic and extraembryonic tissues and simulate the first steps of vasculogenesis, have also underlined the crucial role of VE-cadherin in early vascular organization. In this system, in the absence of VE-cadherin or with a block of its extracellular domain by functional antibodies, endothelial cells cannot organize into a functional vasculature. A detailed analysis of vasculogenesis in this experimental sys-

tem has shown that VE-cadherin is not required for the early stages of de novo blood vessel formation. However, VEcadherin is needed to prevent disassembly of nascent blood vessels into disorganized endothelial aggregates (Figure 2D) (Crosby et al., 2005). In zebrafish, it has been possible to further dissect out the roles of VE-cadherin at different stages of vascular development (Montero-Balaguer et al., 2009). Although VE-cadherin does not regulate vascular patterning, it is implicated in vascular connections and inhibition of sprouting activity. These processes require stable cell-to-cell junctions, which are defective in the absence of VE-cadherin. Of note, an incomplete reduction of VE-cadherin expression also prevents the formation of a stable vasculature, suggesting that even a partial internalization or change in function of VEcadherin may strongly affect vascular stability (MonteroBalaguer et al., 2009). Developmental Cell 26, September 16, 2013 ª2013 Elsevier Inc. 443

Developmental Cell

Review

Figure 2. VE-Cadherin and the Development of the Vascular System in the Embryo (A) Expression of VE-cadherin at day E7.5 of the mouse embryo and in situ hybridization of frozen sections with 35S-labeled antisense VE-cadherin RNA. (a0 ) Parasagittal section of a day E7.5 mouse embryo in the maternal decidua. High levels of VE-cadherin transcripts were detected in the trophoblast, in blood vessels of the maternal decidua, and in the yolk sac mesenchyme. (b0 ) High-magnification field of the framed region (white rectangle) containing the embryo shown in (a0 ). C, chorion; MS, yolk sac mesenchyme; EC, embryonic ectoderm; EN, endoderm (outlined by arrowhead); D, decidua; E, embryo; EP, ectoplacental cavity; EX, exocoelom. (c0 ) Dark-field image of (b0 ). Arrows (in b0 and c0 ) denote VE-cadherin signals in yolk sac mesenchyme. Scale bars, 100 mm (a0 ) and 25 mm (b0 and c0 ). This research was originally published in Breier et al. (1996). Copyright the American Society of Hematology. (B) VE-cadherin null mutation impairs organized, vascular-like structures in embryonic stem-cell-derived 11-day-old embryoid bodies. PECAM whole-mount immunocytochemistry of representative wild-type (+/+, left), VE-cadherin mutant (+/, middle and /, right) embryoid bodies. Scale bar, 100 mm. This research was originally published in Vittet et al. (1997). Copyright the National Academy of Sciences. (C) Kinetics of endothelial marker expression in differentiating embryonic stem cells from days 3 to 7 by RT-PCR. Flk-l (VEGFR2) and PECAM gene expression are rapidly (day 4) upregulated during embryonic stem cell differentiation, whereas VE-cadherin and tie-1 transcripts appear 1 day later (day 5). After day 5, all transcript levels remained high and consistent. The negative control (no template) for each PCR reaction is also shown. This research was originally published in Vittet et al. (1996). Copyright the American Society of Hematology. (D) VE-cadherin null allantois explants fail to undergo normal vascular morphogenesis in culture. PECAM-immunolabeled allantois explants from 8.5 days postcoitum VE-cadherin+/+ and VE-cadherin/ embryos after 18 hr of culture. The interconnected networks of PECAM cells in wild-type explants (+/+) are absent in VE-cadherin null explants (/) and are replaced by isolated clusters of PECAM cells. Scale bars, 100 mm. This research was originally published in Crosby et al. (2005). Copyright the American Society of Hematology.

In a recent study, this concept was further supported by more extensive experimental data (Lenard et al., 2013). In a fish model wherein the vessels do not express functional VE-cadherin, the early sprouting vessels failed to anastomose correctly. However, VE-cadherin-deficient vascular sprouts can still recognize each other and form connections, possibly through other adhesive molecules that can promote homotypic adhesion. Interestingly, these connections do not last, and the filopodia of the endothelial tip cells of the sprouting vessels keep touching and retracting for a relatively long period of time before a functional anastomosis is created. Most importantly, in the normal vasculature, when tip cells connect with each other and create a successful anastomosis, they also stop forming filopodia. This important inhibitory 444 Developmental Cell 26, September 16, 2013 ª2013 Elsevier Inc.

process avoids uncontrolled sprouting and vascular branching. When the tips of endothelial cells lack VE-cadherin expression, they can still form anastomoses, as above, but they do not stop forming filopodia. In other words, in the absence of VEcadherin, the cells do not sense the cell-to-cell contact, but instead keep searching for other connections. These important in vivo observations underline the ability of VE-cadherin to transfer intracellular inhibitory signals to limit endothelial cell movement and cytoskeletal rearrangement (Lenard et al., 2013). Therefore, VE-cadherin appears to be an important factor in mediating vascular stability. Indeed, it is required to maintain the strength and duration of endothelial cell-to-cell interactions, which allows the fusion of the vascular sprouts and the creation

Developmental Cell

Review of a continuous lumen. In addition, VE-cadherin directly and indirectly transfers negative signals to switch the cells to a quiescent state. This concept was further supported by a study by Gaengel et al. (2012), who used genetic approaches in both zebrafish and mice. They showed that activation of sphingosine-1-phosphate receptor-1 (S1PR1), which is a receptor that recognizes the blood-borne lipid sphingosine-1-phosphate, limits unrestrained angiogenic sprouting and ectopic vessel branch formation. This effect is mediated by stabilization of VE-cadherin at adherens junctions, which, in turn, contributes to limit vascular endothelial growth factor receptor (VEGFR) signaling, and, as a consequence, aberrant angiogenesis (Gaengel et al., 2012). Furthermore, Jung et al. (2012) described the effect of laminar blood flow in transducing signals through S1PR1 in endothelial cells, which promotes vascular stability and suppresses ectopic sprout formation. Interestingly, sphingosine-1-phosphate colocalizes with VE-cadherin, which is a component of a mechanosensory complex together with platelet endothelial cell adhesion molecule (PECAM) and VEGFR2, as discussed below (Hahn and Schwartz, 2009; Tzima et al., 2005). It is therefore likely that VE-cadherin is a downstream effector of sphingosine-1-phosphate/S1PR1 signaling that acts by stabilizing the vasculature and preventing unrestrained vascular sprouting and angiogenesis. VE-Cadherin and Intracellular Signaling As previously described (for reviews, see Dejana and Giampietro, 2012; Dejana and Vestweber, 2013; Vestweber et al., 2009), in addition to promoting adhesion between endothelial cells, VE-cadherin and adherens junctions can transfer intracellular signals. This underlines the concept that adhesion is linked to intracellular signaling and that, when endothelial cells come into contact, they communicate with one another to build and maintain the vascular organization. The signaling network transduced by VE-cadherin is complex and varies under different functional conditions and in growing or resting vasculature (Dejana and Vestweber, 2013; Harris and Nelson, 2010). As a general concept, the natural state of endothelial cells in mature vessels is that of a confluent, resting monolayer, in which VE-cadherin is clustered at cell-to-cell contacts and adherens junctions are correctly organized. Under these conditions, signaling through VE-cadherin promotes vascular stability, such as contact inhibition of growth, protection from apoptosis, and control of endothelial permeability. However, as discussed above, VE-cadherin contributes to key and specific functional properties of endothelial cells as well during angiogenesis. These include establishment of cell polarity, formation of a correct lumen, vascular tubulogenesis (Lampug et al., 2009), rearrangement of the cytonani et al., 2010; Strilic skeleton (Komarova and Malik, 2010), tight junction remodeling, and gene transcription (Taddei et al., 2008). The complexity of VE-cadherin signaling is highlighted by the long list of signaling proteins that have been shown to be directly or indirectly associated to VE-cadherin or to adherens junctions in the endothelium (Figure 1 and Table1). VE-cadherin molecules are expressed at high numbers on endothelial cell membranes. Therefore, it is likely that VEcadherin also forms distinct complexes with different partners

on the same cell. These complexes might transfer different types of signals to the cells, depending on the functional conditions. Furthermore, it is conceivable that VE-cadherin association with one or another mediator is reversible and can be temporally and spatially regulated. Inhibition of Cell Growth Endothelial cells inhibit their growth and motility as soon as they come into contact and have established cell-to-cell junctions. This process is known as contact inhibition, and it is mediated by the action of different signaling pathways. Different molecules at adherens junctions contribute to this quiescent state. In particular, VE-cadherin can interact and cocluster with growth factor receptors and control their intracellular signals (Dejana and Vestweber, 2013). More specifically, VE-cadherin interacts with VEGFR2 and inhibits its downstream proliferation signal through the signaling of extracellular-regulated kinases 1 and 2 (ERK1 and ERK2) while maintaining or increasing antiapoptotic signaling through protein kinase B (PKB/Akt). The cytoplasmic domain of VE-cadherin and expression of b-catenin are required for association with the VEGFR2 (Lampugnani et al., 2002). When VEGFR2 is bound to VEcadherin, it is retained at the cell membrane, where it is quickly dephosphorylated by phosphatases on specific tyrosine residues and, in particular, by DEP-1 (Lampugnani et al., 2006). As described for other growth factor receptors (Le Roy and Wrana, 2005), internalization of VEGFR2 can strongly influence its signaling. VEGFR2 can be targeted to degradation, to recycling to the membrane, or to maintenance of signaling activity from the endosomal compartments. In confluent resting endothelial cells, VE-cadherin association to VEGFR2 is long lasting and maintains VEGFR2 dephosphorylation and the cells in a quiescent state (Hayashi et al., 2013). During angiogenesis, VEGF stimulates the sprouting of new vessels by inducing VEGFR2 activity in the tip cells, while in the stalk cells of the sprout, VEGFR2 activity is downregulated. It has been reported that VEGFR2 in stalk cells is dephosphorylated by VE-PTP at endothelial junctions. VE-PTP inactivation leads to excess VEGFR2 activity, increased tyrosine phosphorylation of VE-cadherin, and loss of cell polarity and lumen formation (Hayashi et al., 2013). Other molecules can also act in similar ways. A recent report showed that in vivo in retinal angiogenesis, Disabled 2 (Dab2) and the cell polarity factor partitioning defective protein 3 (PAR3) mediate VEGFR2 endocytosis by clathrin-coated vesicles. Atypical protein kinase C (aPKC) has been shown to inhibit VEGFR2 endocytosis by inhibition of Deb2 by reducing its interaction with VEGFR2 (Nakayama et al., 2013). VE-cadherin has been shown to codistribute and associate with PAR3, and the activation of aPKC is inhibited in the absence of VE-cadherin (Iden et al., 2006; Lampugnani et al., 2010). These indirect observations suggest that VE-cadherin might modulate VEGFR2 signaling and internalization, contributing to Dab2/PAR3 inhibition and to aPKC activation. Ephrin B2 is the transmembrane ligand of EphB receptors, and it can also promote VEGFR2 endocytosis and downstream signaling. Whether ephrin B2 may interact with VE-cadherin in this process is unknown (Nakayama et al., 2013; Wang et al., 2010). Similarly, synectin is involved in the trafficking of Developmental Cell 26, September 16, 2013 ª2013 Elsevier Inc. 445

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Review VEGFR2-containing endosomes. In the absence of synectin, VEGFR2-endosome movement is delayed and VEGFR2 is dephosphorylated by protein tyrosine phosphatase 1b (PTP1b) (Lanahan et al., 2010). Interestingly, PTP1b also stabilizes cellto-cell adhesions by reducing tyrosine phosphorylation of VEcadherin (Nakamura et al., 2008). Thus, the same phosphatase might contribute both to the reduction in VEGF signaling and to junction stability. A recent paper added more information to this picture. It was found that neuropilin-1 could associate to VEGFR2 and synectin via a PDZ-containing domain (Lanahan et al., 2013). Neuropilin-1 is required for a rapid internalization of the receptor in endosomes. In the absence of neuropilin-1 cytoplasmic domain, the receptor remains at the cell membrane and is rapidly dephosphorylated. As discussed above, a similar phenomenon occurs in absence of VE-cadherin (Lampugnani et al., 2006), but whether VE-cadherin can associate and influence the structure and function of neuropilin-1, synectin, and VEGFR2 complex is an attractive but still unknown possibility. Overall, as for other growth factor receptors, VEGFR2 internalization and trafficking depends on coreceptors. VE-cadherin, similarly to other cofactors such as neuropilin-1, synectin, ephrin-B2, and protein phosphatases, coordinates VEGFR2 activation and internalization, strongly influencing the intensity and duration of signaling. Other Pathways that Can Contribute to Inhibition of Cell Growth VE-cadherin was shown to be associated with c-Src and C-terminal Src kinase (Csk), a negative regulator of Src activation (Baumeister et al., 2005) and inhibitor of cell growth. The association with Csk requires phosphorylation of tyrosine 685 (Y685) in the VE-cadherin tail. Addition of VEGF to cultured endothelial cells stimulates Csk release from VE-cadherin by recruiting the SH2-domain-containing tyrosine phosphatase 2 (SHP2) to the VE-cadherin signaling complex, which leads to an increase in c-Src activation. Inhibition of VE-cadherin and SHP2 enhances ERK1/2 activation in response to VEGF (Ha et al., 2008). These findings reveal a novel role for VE-cadherin in the modulation of c-Src activation in VEGF signaling, thus providing new insights into the importance of VE-cadherin in VEGF signaling and vascular function (Ha et al., 2008). Transcriptional Signaling There is evidence that many VE-cadherin binding partners, such as b-catenin, p120-catenin, and plakoglobin, can also translocate to the nucleus to regulate transcription. This supports the idea that VE-cadherin can signal indirectly by recruiting these molecules to the membrane and thus inhibiting their nuclear signaling. The most-studied cadherin partner is probably b-catenin, which is one of the key players in canonical Wnt signaling (Clevers, 2006; Clevers and Nusse, 2012). It is constitutively bound to VE-cadherin. When b-catenin is free in the cytoplasm, it is quickly inactivated by phosphorylation and ubiquitination by a protein complex that includes axin and adenomatous polyposis coli (APC). However, when the amount of VE-cadherin expressed is reduced in endothelial cells, or when endothelial cells are activated by Wnt, b-catenin can 446 Developmental Cell 26, September 16, 2013 ª2013 Elsevier Inc.

translocate to the nucleus and modulate gene transcription (Dejana, 2010). Although nuclear b-catenin does not belong to the cadherinassociated pool in Drosophila and Xenopus (Funayama et al., 1995; Peifer et al., 1994), in other cell types, and in particular in the endothelium, cadherin expression and b-catenin signaling are interrelated (for reviews, see Jeanes et al., 2008; McEwen et al., 2012). As discussed above, VE-cadherin might act as a stoichiometric inhibitor of b-catenin by retaining the molecule at the cell membrane. Alternatively, cadherins might act by modulation of the rate of b-catenin destruction. This is supported by the observation that the phosphodestruction complex for b-catenin is localized at cell-to-cell contacts in nonendothelial cell lines (Maher et al., 2009). An interesting hypothesis is that the membrane-associated destruction complex is ‘‘tunable’’ by the cadherins, which may control, in this way, the amount of b-catenin degradation and nuclear signaling (Maher et al., 2009; McEwen et al., 2012) In the nucleus, b-catenin interacts with members of the lymphoid enhancer factor/T cell factor (LEF/TCF) protein family (Clevers and Nusse, 2012) and also with other transcription factors, such as Forkhead box protein O1 (FoxO1), hypoxia-inducible factor (HIF), Smads, and others (for review, see Dejana, 2010). As well as controlling b-catenin signaling, VE-cadherin might also bind other armadillo proteins that have transcriptional activity, such as p120-catenin and plakoglobin (Reynolds, 2007). At the transcriptional level, p120-catenin associates with the transcriptional repressor Kaiso, which it displaces from specific promoters, to allow activation of the corresponding genes (for review, see McCrea et al., 2009). The transcriptional activity of plakoglobin has not been studied as extensively as that of b-catenin (McCrea et al., 2009). Some reports have shown that plakoglobin can bind to TCF/LEF transcription factors and have transcriptional activity; this is, however, contradicted by other reports, which have shown that the plakoglobin-TCF complex cannot bind to DNA (McCrea et al., 2009; Zhurinsky et al., 2000). Another transcription factor modulated by E-cadherin and adherens junction organization is Yes-associated protein 1 (YAP). When this factor translocates to the nucleus and associates with the DNA-binding factors TEA domain family member-1 (TEAD) or transcriptional enhancer factor (TEF), this activates genes that promote cell proliferation. When the cells are confluent, YAP is phosphorylated and excluded from the nucleus, thus limiting cell proliferation. The mechanism through which the cadherin complex restrains YAP signaling remains poorly understood, although a-catenin appears to be indirectly or directly implicated (Kim et al., 2011; Schlegelmilch et al., 2011; Silvis et al., 2011). Taken together, the data discussed above imply that the cadherins, and VE-cadherin in particular, can either repress or stimulate gene transcription. An interesting question is which type of transcriptional program might be induced by VE-cadherin expression and clustering. Intuitively, it would be expected that VE-cadherin activates and maintains the functions related to endothelial cell quiescence and vascular stability. Gene profile analysis has shown that several genes implicated in the inhibition of cell proliferation and apoptosis are

Developmental Cell

Review upregulated by VE-cadherin expression (Spagnuolo et al., 2004). A number of these genes, and in particular the tight-junction adhesive protein claudin 5, are induced by a complex mechanism that involves VE-cadherin activation of phosphoinositide 3-kinase (PI3K) and Akt. Active Akt in turn phosphorylates and inhibits FoxO1. The FoxO1 transcription factor forms a complex with nuclear b-catenin, which represses claudin 5 (Taddei et al., 2008). In the presence of VE-cadherin, this repression is released and claudin 5 is upregulated, thus promoting junction tightening. Other vascular stability genes might follow the same mechanisms of regulation by VE-cadherin (Taddei et al., 2008). VE-Cadherin and Actin Cytoskeletal Remodeling The anchorage of junctional proteins to the cytoskeleton has a crucial role in the control of endothelial cell shape, movement, and permeability. VE-cadherin binds b-catenin and plakoglobin through its cytoplasmic tail, and these in turn can bind a-catenin, which regulates the actin cytoskeleton, and therefore cell shape and motility. How cadherin-catenin complexes interact with the actin cytoskeleton is still a matter of debate, as direct interaction of the cadherin–b-catenin–a-catenin complex with actin filaments has been questioned (Hartsock and Nelson, 2008). In its monomeric state, a-catenin binds b-catenin that is coupled to cadherin, but it does not bind actin (Yamada et al., 2005). However, homodimeric a-catenin binds actin, and not b-catenin, and promotes the bundling of actin filaments (Drees et al., 2005). Thus, the binding of a-catenin to the cadherin complex might negatively regulate the a-catenin action on actin polymerization (Hartsock and Nelson, 2008). Various actin-binding proteins, such as a-actinin (Knudsen et al., 1995; Nieset et al., 1997), vinculin (Watabe-Uchida et al., 1998; Weiss et al., 1998), and Eplin (Abe and Takeichi, 2008), can also bind a-catenin. Of these candidate linker molecules, Eplin has been shown to directly link the E-cadherin–b-catenin–a-catenin complex with actin (Abe and Takeichi, 2008; Chervin-Pe´tinot et al., 2012). A recent study described so-called focal adherens junctions between endothelial cells (Huveneers et al., 2012). These junctions were defined as attachment sites for radial F-actin bundles and sites of junction remodeling. They were formed upon stimulation with junction-challenging agents, such as thrombin. Vinculin associates with these structures in a way that is dependent on the actomyosin contractility and on the exposure of a specific vinculin-binding a-catenin domain. As a consequence of this force-dependent binding of vinculin, the mechanical stability of the focal adherens junctions was reinforced, and these endothelial junctions were prevented from opening (Huveneers et al., 2012). Endothelial cells appear to ‘‘sense’’ changes in the shear forces and respond by the reshaping of their cytoskeleton, which, in turn, alters the tension forces on the junction proteins. The VE-cadherin–catenins complex has a role in the transduction of the mechanical forces that shape endothelial cells exposed to blood pressure and shear stress. VE-cadherin can directly transduce the mechanical forces to form a mechanosensor complex through its coupling to the junctional protein PECAM and VEGFR2 (Conway et al., 2013; Tzima et al., 2005). This complex transfers signals that can modulate Src and other intracellular signals. These responses are needed for optimal

vascular homeostasis, to maintain the correct endothelial cell attachment to the matrix and to regulate the blood flow. Importantly, the mechanosensor complex does not require adhesion and clustering of VE-cadherin or PECAM at adherens junctions because it is also active in sparsely spread, nonconfluent, endothelial cells (Tzima et al., 2005). Furthermore, the signal is also transferred in the absence of VEGF, which suggests the activation of VEGFR2 independent of its ligand. This supports the concept that this particular mechanosensor complex can act independently of adhesion or its localization at adherens junctions. Thus, when VE-cadherin is associated with this particular complex, it has a specific functional role that has never been described previously for other members of the cadherin family. VE-Cadherin, the Vascular Barrier, and Small GTPases Despites its other functions, the best-studied role of VE-cadherin is to promote homotypic endothelial cell-to–cell adhesion, which contributes to the maintenance of vascular integrity. When junctions are disrupted, the permeability is increased and the vascular integrity is altered. This intuitive process, however, appears to be complex, and it is influenced by several signaling processes. In the present Review, we mostly focus on VEcadherin and vascular permeability to solutes, and we do not discuss in detail permeability to leukocytes because other excellent reviews are available on this topic (for reviews, see Engelhardt and Wolburg, 2004; Vestweber et al., 2009). It has been shown (for reviews, see Beckers et al., 2010; Goddard and Iruela-Arispe, 2013; Spindler et al., 2010) that different members of the Rho GTPase family, such as Ras homolog gene family, member A (RhoA), Ras-related C3 botulinum toxin substrate 1 (Rac1), cell division control protein 42 homolog (Cdc42), and Ras-related protein-1 small GTPase (Rap1), contribute to the fine-tuning of vascular permeability under both physiological and pathological conditions. Indeed, changes in their activities are associated with the regulation of endothelial barrier homeostasis, as well as with the disruption or reinforcement of the barrier integrity, according to the context in which they are activated (Komarova and Malik, 2010; Spindler et al., 2010; van Nieuw Amerongen et al., 2007). Rac1 and Cdc42 are generally thought to spatially control the formation of filopodia and lamellipodia, which favors barrier improvement, whereas RhoA is mainly involved in the organization of stress fibers during cell contraction and also impairs barrier integrity (Beckers et al., 2010; Spindler et al., 2010; Wojciak-Stothard and Ridley, 2002). However, recent studies have highlighted a more complex involvement of these small GTPases in positive and negative regulation of the endothelial barrier (Beckers et al., 2010). Under resting conditions in endothelial cells (Figure 3A), VEcadherin regulates adherens junction stability and Rho GTPase activation through interactions with p120-catenin and other molecules. It was demonstrated previously that VE-cadherin clustering increases the concentration of Tiam1, which is a guanine nucleotide exchange factor (GEF) for Rac1, and p21activated kinase (PAK), which is a Rac effector, in the membrane compartment, thus enhancing Rac1 activation (Lampugnani et al., 2002). Similarly, the levels of active Cdc42 are elevated in VE-cadherin-overexpressing endothelial cells (Kouklis et al., 2003). Moreover, p120-catenin binding to VE-cadherin activates Developmental Cell 26, September 16, 2013 ª2013 Elsevier Inc. 447

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Figure 3. Model for Regulation of Endothelial Barrier Function by Small GTPases (A) Resting conditions: VE-cadherin expression and clustering may activate the small GTPase Rac1 and Cdc42 through Tiam. Rac1 increases endothelial barrier function by stabilizing adherens junctions and inhibiting RhoA, which in turn decreases actomyosin contractility. Rap1 stabilizes cell-to-cell junctions and binds to adherens junctions indirectly through Krit1. (B) Permeability increasing agents: several barrier-destabilizing mediators, such as thrombin and VEGF, can induce transient and sustained increases in vascular permeability through modulation of the activity of different small GTPases. Thrombin leads to impaired cAMP signaling, which causes both Rap1 inactivation and activation of RhoA. VEGF increases permeability through different pathways (1) by Rac1 activation, ROS generation, and VE-cadherin endocytosis and (2) by activation of the RhoA/Rho kinase pathway. See text for details and further discussion.

Vav2, another specific GEF for Rac1 and Cdc42 (Abe et al., 2000; Noren et al., 2000; Schuebel et al., 1998). The activated state of these GTPases is maintained by IQ-motif-containing, GTPaseactivating protein 1 (IQGAP1), which inhibits their intrinsic GTPase activity (Hart et al., 1996). However, Rac1 activates different downstream targets, such as PAK and p190RhoGTPase activating protein (p190RhoGAP), which in turn inhibits RhoA (Herbrand and Ahmadian, 2006; Wildenberg et al., 2006). Therefore, under resting conditions, Rac1 activation stabilizes adherens junctions and promotes endothelial barrier properties. Conversely, in unperturbed endothelium, a basal Rho kinase activity is essential for endothelial cell junction maintenance and is necessary for the correct recruitment of VE-cadherin to adherens junctions (van Nieuw Amerongen et al., 2007). A more detailed analysis of endothelial dynamics has revealed that even under resting conditions, adherens junctions are continuously remodeled, which generates many tiny interendothelial gaps that appear and disappear spontaneously. In this context, RhoA activation occurs prior to gap closure, which suggests a novel role for RhoA in the maintenance of endothelial integrity (Szulcek et al., 2013). Although there have been extensive investigations into RhoA and Rac1 signaling in the control of basal endothelial functions, the role of Rap1 under resting conditions is still largely unknown. Both GAPs, such as RapGAP, and GEFs, such as exchange proteins activated by cyclic AMP (cAMP) (Epac1) and PDZ-GEF, have been proposed to regulate Rap1 in endothelial cells (Cullere et al., 2005; Kooistra et al., 2005). Recently, it was demonstrated (Pannekoek et al., 2011) that under resting conditions, Rap1 activity is mainly under the control of PDZ-GEF to maintain basal endothelial barrier functions. Although the triggers responsible for PDZ-GEF activation are still unclear, Epac1 activation is 448 Developmental Cell 26, September 16, 2013 ª2013 Elsevier Inc.

mediated by elevated levels of the second messenger cAMP (Pannekoek et al., 2011). In this context, Epac-Rap1 signaling stabilizes VE-cadherin at adherens junctions and induces actin cytoskeleton rearrangements. These changes include disruption of the radial stress fibers and induction of cortical actin bundles, with the consequent switch from motile and discontinuous to linear and stable junctions (Cullere et al., 2005; Fukuhara et al., 2005; Noda et al., 2010; Sakurai et al., 2006). Recently, the Rap1 effector Krev interaction trapped protein1/cerebral cavernous malformation 1 (Krit-1/CCM1) protein has been proposed to be involved in Rap1-mediated regulation of Rho signaling (Glading et al., 2007; Glading and Ginsberg, 2010; Stockton et al., 2010). Furthermore, through activation of the common binding partner Rho GTPase-activating protein 29 (ArhGAP29), two other Rap1 effectors, Ras-interacting protein 1 (Rasip1) and Ras-association and dilute domain-containing protein (Radil), cooperate to inhibit Rho-mediated stress fiber formation and induce junctional tightening by reducing the actomyosin-induced tension (Post et al., 2013). Several barrier-destabilizing mediators, such as thrombin and VEGF, can induce transient and sustained increases in vascular permeability through modulation of the activity of different small GTPases (Figure 3B). Several reports have shown fast and transient increases in RhoA activity upon thrombin stimulation (Birukova et al., 2004; van Nieuw Amerongen et al., 2008). One of the underlying mechanisms is Rho-kinase-mediated inactivation of myosin light chain (MLC) phosphatase, which leads to increased MLC phosphorylation and actin-myosin contractility (Essler et al., 1998; van Nieuw Amerongen et al., 2000). Thrombin increases RhoA activity along stress fibers and at cell margins both in vitro and in vivo, thus destabilizing vascular integrity (Szulcek et al., 2013). At the same time, thrombin-mediated

Developmental Cell

Review endothelial permeability involves Rac1 inactivation, mainly through a reduction in cAMP levels, which in turn results in inhibition of both the RapGEF Epac and Rap1 (Birukova et al., 2008). This weakens the junctional complex and results in enhanced vascular permeability. VEGF-mediated Rho GTPase activation appears more restricted to a direct effect on the disruption of adherens junctions. VEGF stimulation of endothelial cells activates Src, which stimulates Vav2, Rac1, and its downstream effector PAK1. In turn, PAK1 phosphorylates a serine residue in the cytoplasmic tail of VE-cadherin, thereby promoting its endocytosis and increasing the vascular permeability (Gavard and Gutkind, 2006). VEGF-induced Rac1 activation also promotes the generation of reactive oxygen species (ROS). ROS production is essential for Rac1/p190RhoGAP-induced downregulation of RhoA (Nimnual et al., 2003). Another level of regulation of small GTPase activity is the different localization of GAPs and GEFs. VEGF promotes displacement of the RhoA-specific GEF Syx from cell junctions, thereby promoting junction disassembly (Ngok et al., 2012). Conversely, treatment of endothelial cells with the vascular normalizing agent angiopoietin1 maintains Syx at the adherens junctions. This results in localized activation of RhoA and its effector mDia, which in turn induces adherens junction stabilization and prevents VEGF-induced permeability (Gavard et al., 2008; Ngok et al., 2012). Furthermore, other reports have shown that treatments of endothelial cells with vascular-protective agents, such as the phospholipid 1-palmitoyl-2-arachidonoylsn-glycero-3-phosphorylcholine (OxPAPC), promotes the recruitment of p190RhoGAP to adherens junctions and its binding to p120-catenin, which leads to enhancement of the endothelial barrier through the reduction of RhoA activity and the increase in Rac1 signaling (Birukova et al., 2011; Zebda et al., 2013). These findings suggest that precise spatial and temporal regulation of the activity of Rho family GTPases and related proteins is a strict requirement for the maintenance of endothelial barrier integrity. When all of these data are taken together, some of the reported findings in vitro and in vivo appear contradictory. However, there are different explanations that can reconcile these conflicting data. It is likely that the mechanisms that regulate endothelial permeability in vitro might differ from the in vivo setting. Most of the experimental evidence regarding the involvement of the small GTPases in the control of endothelial barrier function derives from cell-culture studies. Cultured endothelial cells show more pronounced features than their in vivo counterparts. These cultured cells have a more contractile phenotype and enhanced stress fiber formation, compared with intact microvessels, which reflects the situation of inflammatory conditions. In addition, it is possible that endothelial cells in culture are more susceptible to contractile stimuli, because they migrate and divide to generate a confluent monolayer that resembles the cellular organization of blood vessels. This makes the junctions of cultured cells relatively immature, and the resulting endothelial barrier is less stable than under in vivo conditions. In addition, endothelial cells are constantly exposed to shear stress and mechanical forces in vivo, whereas most in vitro studies are performed under static conditions. In conclusion, in vitro data of the small GTPases and endothelial barrier regulation need to be interpreted while taking into account both the

cellular background (e.g., microvascular-derived and macrovascular-derived endothelial cells) and the results obtained in vivo. In Vivo Modulation of Endothelial Barrier Function Under in vitro and in vivo conditions, it is important to bear in mind that adherens junctions are not static, but are dynamic structures that continuously lose and gain cadherins. The organization and disorganization of adherens junctions are regulated by a complex network of intracellular signaling pathways and by the cytoskeleton (as described above). The complexity and diversity of these pathways might reflect the multiple physiological needs of endothelial cells and the type of responses to permeability-increasing agents. A common concept is that permeability-increasing agents act by inducing VE-cadherin internalization and thereby reducing the amount of VE-cadherin at adherens junctions and disrupting the endothelial cell barrier. VE-cadherin phosphorylation in Ser665 has been implicated in the increase in permeability induced by VEGF. Phosphorylated VE-cadherin associates with b-arrestin2 and is internalized through clathrin-coated vesicles (Gavard and Gutkind, 2006). Src-induced VE-cadherin phosphorylation on tyrosine also appears to be required for an increase in permeability and VEcadherin internalization (Gavard et al., 2008; Orsenigo et al., 2012; Weis et al., 2004), and Src inhibitors are among the most potent inhibitors of permeability, both in vitro and in vivo. However, the precise mechanisms through which Src exerts its action are far from being completely clear. In vitro studies have shown that phosphorylation of VE-cadherin by Src is not enough to increase endothelial permeability (Adam et al., 2010). Accordingly, a recent study by our group (Orsenigo et al., 2012) showed that in vivo, under resting conditions, VE-cadherin is phosphorylated on tyrosine in the veins, but not in the arteries. VEcadherin phosphorylation is mediated by a specific pool of Src that is localized at intercellular junctions. Treatment of mice with permeability-increasing agents induces the specific internalization of phosphorylated VE-cadherin. Src-mediated VEcadherin phosphorylation acts as a ‘‘priming’’ mechanism, which creates a dynamic state of the protein, which then becomes more sensitive to internalization. Src therefore appears to be required, but not sufficient, for VE-cadherin internalization. P120-catenin dissociation from the VE-cadherin intracellular tail is also recognized as a way through which cadherin is destabilized at adherens junctions such that it is probably degraded (Oas et al., 2010). Recently, a specific motif was defined in the VE-cadherin tail that is required for VE-cadherin internalization and p120 binding (Nanes et al., 2012). Mutations in this region cause uncoupling of p120 and inhibition of internalization, which strongly and selectively affects cell migration (Nanes et al., 2012). However, the effect of the mutant on endothelial permeability was not reported. A knockin model of this mutant in vivo might help to understand the importance of this VE-cadherin tail motif on its different vascular functions. Another important mediator of endothelial permeability is focal adhesion kinase (FAK). This kinase was originally found to colocalize with integrins at focal contacts and to modulate cell adhesion and migration. Genetic and pharmacologic inhibition of FAK prevents VEGF-induced endothelial permeability. VEGF activation of endothelial cells leads to the rapid binding of FAK to Developmental Cell 26, September 16, 2013 ª2013 Elsevier Inc. 449

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Review VE-cadherin, which induces phosphorylation of b-catenin and dismantling of adherens junctions (Chen et al., 2012). As discussed above, another important aspect in the control of permeability by VE-cadherin is its association with the actin cytoskeleton. A VE-cadherin a-catenin fusion construct has been knocked in in mice (Schulte et al., 2011). This mutant protein interacts more effectively with actin, and the vasculature is resistant to the increase in permeability induced by histamine or VEGF or to leukocyte transmigration. This strongly suggests that a stable interaction with the actin cytoskeleton might stabilize endothelial cell junctions and reduce their dynamic opening. Recently, it was shown that VE-cadherin also modulates the microtubule cytoskeleton through the activation of End-binding protein 3 (EB3). This process has an important role not only in adherens junction assembly, but also in the modulation of vascular permeability (Komarova et al., 2012) Many other kinases and phosphatases have been shown to interact with and modulate phosphorylation of VE-cadherin and its partners and to modulate adherens junction strength (for review, see Dejana and Vestweber, 2013; for details, see Figure 1 and Table 1). Of particular importance is the association of VE-cadherin with the phosphatase VE-PTP. The dissociation of VE-PTP from VE-cadherin is triggered by endotoxin, VEGF, and the binding of leukocytes to endothelial cells in vitro and in vivo, suggesting that this dissociation is a prerequisite for the destabilization of endothelial cell contacts (Broermann et al., 2011; Vockel and Vestweber, 2013). Overall, the complexity and redundancy of the intracellular mechanisms that modulate adherens junction organization is such that it is difficult, if not impossible, to draw a unique picture. Although rapid internalization and turnover of VE-cadherin is associated with increases in endothelial permeability, this process might not explain all of the steps of adherens junction disassembly and barrier disruption. For instance, the amount of VE-cadherin internalized upon stimulation with permeabilityincreasing agents is relatively small, and, at least in vivo, this is not always accompanied by a diffuse disorganization of adherens junction proteins from cell-to-cell contacts (Orsenigo et al., 2012). It is possible that the permeability is also increased when VE-cadherin and adherens junctions undergo relatively subtle changes, which do not necessarily imply an obvious opening of gaps between the endothelial cells, but rather only subtle modifications of the junction architecture. It is also probable that disruption of VE-cadherin clusters leads to diffusion of VE-cadherin onto the cell membrane, which is not necessarily coupled to internalization. Moreover, junction reassembly is based on complex interactions between the VE-cadherin ectodomain, which promotes its clustering at intercellular contacts, and the reorganization of mature and stable junctions. This process coincides with actin and tubulin reorganization. Inactivation of actin polymerization or actomyosin contractility alters adherens junction formation. All of this evidence suggests that the cytoskeleton regulates VE-cadherin clustering and, conversely, that VE-cadherin reciprocally regulates cytoskeletal organization and barrier integrity. However, the molecular mechanisms through which all this occurs still remain elusive. 450 Developmental Cell 26, September 16, 2013 ª2013 Elsevier Inc.

Concluding Remarks As described here, the complexity of the organization of adherens junctions in the endothelium and the signaling pathways that they can trigger is extremely high. Through these structures, endothelial cells maintain continuous communication with each other and can ‘‘sense’’ the blood flow and adapt their cytoskeleton accordingly. Most of our knowledge about adherens junction and VEcadherin organization comes from studies performed on cultured cells that are frequently derived from large vessels. In contrast, endothelial cell responses in vivo vary profoundly in different types of vessels and organs. For instance, endothelial cells in postcapillary venules have poor control of the permeability, which allows the dynamic passage of fluids and circulating cells from the blood into the underlying tissue and vice versa. Conversely, endothelial cells in brain microcirculation or in large arteries apply tight control of permeability to protect the tissues from undesired infiltration of toxic agents and inflammatory cells. The most important challenge for the future in this field will be to develop suitable and sensitive imaging tools to study the organization of adherens junctions and their assembly and disassembly in vivo in the different vascular beds and under different pathophysiological conditions. ACKNOWLEDGMENTS This study was supported in part by grants from Fondation Leducq Transatlantic Network of Excellence, Associazione Italiana per la Ricerca sul Cancro (AIRC), ‘‘Special Program Molecular Clinical Oncology 5x1000’’ to AIRC-Gruppo Italiano Malattie Mieloproliferative, the European Community (ENDOSTEM-HEALTH-2009-241440, JUSTBRAIN-HEALTH-2009-241861, and ITN Vessels), the European Research Council (ERC), and CARIPLO Foundation. M.T. was a recipient of a fellowship from Structured International Post Doc Program. REFERENCES Abe, K., and Takeichi, M. (2008). EPLIN mediates linkage of the cadherin catenin complex to F-actin and stabilizes the circumferential actin belt. Proc. Natl. Acad. Sci. USA 105, 13–19. Abe, K., Rossman, K.L., Liu, B., Ritola, K.D., Chiang, D., Campbell, S.L., Burridge, K., and Der, C.J. (2000). Vav2 is an activator of Cdc42, Rac1, and RhoA. J. Biol. Chem. 275, 10141–10149. Adam, A.P., Sharenko, A.L., Pumiglia, K., and Vincent, P.A. (2010). Srcinduced tyrosine phosphorylation of VE-cadherin is not sufficient to decrease barrier function of endothelial monolayers. J. Biol. Chem. 285, 7045–7055. Baumeister, U., Funke, R., Ebnet, K., Vorschmitt, H., Koch, S., and Vestweber, D. (2005). Association of Csk to VE-cadherin and inhibition of cell proliferation. EMBO J. 24, 1686–1695. Bazzoni, G., and Dejana, E. (2004). Endothelial cell-to-cell junctions: molecular organization and role in vascular homeostasis. Physiol. Rev. 84, 869–901. Beckers, C.M., van Hinsbergh, V.W., and van Nieuw Amerongen, G.P. (2010). Driving Rho GTPase activity in endothelial cells regulates barrier integrity. Thromb. Haemost. 103, 40–55. Birukova, A.A., Smurova, K., Birukov, K.G., Kaibuchi, K., Garcia, J.G., and Verin, A.D. (2004). Role of Rho GTPases in thrombin-induced lung vascular endothelial cells barrier dysfunction. Microvasc. Res. 67, 64–77. Birukova, A.A., Zagranichnaya, T., Alekseeva, E., Bokoch, G.M., and Birukov, K.G. (2008). Epac/Rap and PKA are novel mechanisms of ANP-induced Racmediated pulmonary endothelial barrier protection. J. Cell. Physiol. 215, 715–724.

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