The BBB is a multicellular vascular structure separating the central nervous system from peripheral blood circulation (Obermeier et al., 2013). It is composed of cerebrovascular endothelial cells (EC) forming brain vessels, astrocytes and extracellular matrix (ECM) components providing structural support (Abbott et al., 2006). Pericytes are also relevant to form the BBB and their functional roles have been fully described elsewhere (Cabezas et al., 2014; Figure 1). Together, all these elements exert their functions as a selective physical (Abbott et al., 2006), transport (Begley and Brightman, 2003) and metabolic (Pardridge, 2003, 2016) barrier, tightly controlling the passage of molecules in and out of brain parenchyma and preventing the penetration of toxins or pathogens (Obermeier et al., 2013).

Cerebral EC have a unique characteristic in comparison to peripheral EC: they are interconnected by continuous intracellular multiprotein complexes called tight junctions (TJs), which lack fenestrations and undergo extremely low rates of transcytosis (Figure 1). This limits paracellular passage of substances and directs molecular trafficking to take a rigorously controlled transcellular route across the BBB (Abbott et al., 2006). Such a strong physical barrier allows only small gaseous and lipophilic molecules to diffuse freely in and out of the brain, whereas bigger molecules need to be actively transferred via transporter/carrier systems, such as the glucose transporter-1 (GLUT-1) or the large neutral amino acid transporter-1 (LAT-1) located on the luminal (blood facing) or abluminal (brain facing) EC sites (Borst and Schinkel, 2013). Potentially harmful compounds like glutamate are actively cleared from the brain even against a concentration gradient requiring ATP as energy source (e.g., via excitatory amino acid transporter 1/2; EAAT1/2 (Hawkins and Viña, 2016). Generally, large hydrophilic molecules cannot be transferred across the BBB unless by specific receptor- or adsorptive-mediated transcytosis (Pardridge, 2003, 2016; Strazielle and Ghersi-Egea, 2016).

The TJ are key regulators of paracellular permeability and transendothelial electrical resistance. Major constituents of the TJ are transmembrane molecules like occludin (Yu et al., 2005), which links to the cytoskeleton via the accessory proteins zonula occludens (ZO-1/2) and claudins (Piehl et al., 2010), and junctional adhesion molecules (JAM-A, -B, -C, Mandel et al., 2012). During early embryogenesis, pre-existing vessels sprout and undergo angiogenesis (Obermeier et al., 2013). Sealing properties, including refinement of the protein complexes, establishment of efflux transporters and limitation of transcytosis, seems to only mature when sprouting vessels come in close contact with pericytes and astrocytes (Daneman et al., 2010; Obermeier et al., 2013). However, the role of astrocytes in this process is still a matter of controversy (see below). Afterwards, matured TJ are fixed and need to be maintained throughout life.

Astrocytes regulate features of the BBB through the tips of their processes, called astrocytic endfeet, which surround and contact brain micro-vessels (Kettenmann and Verkhratsky, 2008). Among other functions, they regulate the ion balance around the BBB and secrete and recycle neurotrophic factors necessary to control TJ (Gee and Keller, 2005). A very elegant example of how astrocytes maintain the ionic homeostasis is represented by their synchronized spatial K⁺ buffering at synaptic and BBB locations mediated by their perivascular and perisynaptic endfeet (Olsen and Sontheimer, 2008). This controls ion concentrations during normal brain activity and can thereby link and adapt responses of blood vessels to synaptic neuronal activity to guarantee the appropriate supply of oxygen and nutrients (Wolburg et al., 2011). Additionally, astrocytes secret several molecules such as the glia cell-derived neurotrophic factor (GDNF; Igarashi et al., 1999), transforming growth factor β (TGF-β; Dobolyi et al., 2012), angiopoietin 1 (ANG1; Easton, 2012), fibroblast growth factor 2 (FGF2; Reuss et al., 2003) and vascular endothelial growth factor (VEGF; Rosenstein et al., 2010) which act on EC to either promote TJ formation and/or regulate BBB permeability (Figure 1).

At present, it is still controversial whether astrocytes are necessary for the induction of TJ, because of the temporal shift between EC differentiation/maturation and astrocyte development. Recent work suggests that astrocytes are dispensable for the induction of TJ (Saunders et al., 2016), but are necessary for their further strengthening and maintenance throughout life (Alvarez et al., 2011, 2013). However, meningeal blood vessels which lack contacts with astrocytes display higher vascular permeability than EC-BBB, supporting indeed the necessity of astrocytes to induce BBB properties (Lécuyer et al., 2016).

The non-cellular component of the NVU is the basement membrane, which is composed of structural proteins such as collagen-IV, laminin and fibronectin, among others (Cardoso et al., 2010; Figure 1). The main function of the basement membrane is to provide stability to the other members of the NVU and regulate their crosstalk enabled by matrix transmembrane receptors like integrins and dystroglycans (Baeten and Akassoglou, 2011).

Besides the well-known bidirectional signaling activated upon cell-cell interactions, which is described in detail in some excellent reviews (Pasquale, 2008; Murai and Pasquale, 2011; Klein, 2012; Lisabeth et al., 2013), several alternative signaling mechanisms have been proposed for the EphR/ephrin system.

Upon receptor/ligand interaction, several downstream signaling cascades are activated to mediate cell adhesion or repulsion, depending on the type and abundance of ligands and receptors present on cell surfaces (Janes et al., 2012). These signaling pathways include, among others, the Src kinase family, mitogen-activated protein kinase, and integrin mediated pathways (Lackmann and Boyd, 2008; Pasquale, 2008; Pitulescu and Adams, 2010). Their activity is dependent on Rho family GTPases, including RhoA, Rac1, Cdc42 and a variety of guanine nucleotide exchange factors (GEF), like ephexins (Cowan et al., 2005; Pasquale, 2008). After the initial receptor/ligand interaction, intact EphR/ephrin complexes together with potentially associated cytoplasmic proteins and the surrounding membrane are internalized in either cell. This Rac1-dependent mechanism is termed trans-endocytosis and provides a mechanism to switch between cell adhesion and retraction fates and to terminate receptor signaling activity (Lisabeth et al., 2013).

Besides trans-endocytosis, the activation of enzymes which initiate proteolytic cleavage represents another alternative signaling mechanism (Atapattu et al., 2014). Among such enzymes, A disintegrin and metalloproteases (ADAM) and matrix metalloproteases (MMP) are implicated in signal termination (Atapattu et al., 2014).

In mammalian tissues, members of the ADAMs family are transmembrane metalloproteases able to process and shed ectodomains of membrane bound receptors (Klein and Bischoff, 2011). They play crucial roles in pathological conditions such as inflammation or stress-mediated angiogenic responses (Weber and Saftig, 2012). Several EphRs/ephrins of both A and B subclasses can associate with ADAMs resulting in their own cleavage. Cleavage of the ligand-bound receptor leads to a breakdown of the molecular tethers between interacting cells, thereby favoring the internalization of receptor/ligand complexes, as exemplified by ADAM10 initiated cleavage of the EphA3/ephrinA2 complex during axon detachment (Hattori et al., 2000; Mancia and Shapiro, 2005) or of the EphA2/ephrinA1 complex (Salaita et al., 2010).

MMPs cleave proteins located either on membranes or in extracellular spaces (Miller et al., 2008). Their main function is to degrade structural components of the ECM to facilitate cell migration (Streuli, 1999), especially during angiogenesis and inflammatory processes (Kessenbrock et al., 2010; Palmisano and Itoh, 2010). Recently, it has been shown that MMPs cleave ephrinA1 and ephrinA2 from their GPI-anchor, leading to the release of functional soluble monomers which can act on distant Eph receptors (Beauchamp and Debinski, 2012). Followed by an initial shedding step mediated by ADAMs or MMPs, EphRs/ephrins can further be processed by intramembrane cleaving proteases such as γ-secretase (Bergmans and De Strooper, 2010) or neuropsin (Attwood et al., 2011; Morohashi and Tomita, 2013). This events generates cytoplasmic active fragments (Litterst et al., 2007; Xu and Henkemeyer, 2009) which may i.e., regulate behavioral responses such as anxiety (Attwood et al., 2011).

The signaling cluster propagation represents another noteworthy alternative signaling mechanism to be mentioned. This type of signaling, originally initiated by receptor/ligand interactions in trans, causes the formation of lateral clusters through receptor-receptor interactions in cis. These receptor clusters do no longer rely on ephrin interaction to get activated, enabling the strong amplification of an originally small signal generated by a first short cell-cell contact (e.g., EphA3/ephrinA5; Wimmer-Kleikamp et al., 2004).

Such signaling effectors of the Eph/ephrin system might become relevant in brain disorders to identify alternative targets for drug discovery.

The interaction of specific cell types to properly develop the vascular system and a functional BBB is an essential process which requires the appropriate temporally- and spatially-regulated expression of distinct guidance cues. Among them, the EphR/ephrin system represents an ideal candidate to exert those functions.

During vasculogenesis, VEGF induces ephrinA1 expression which activates EphA2 on neighboring EC, thus exerting angiogenic effects—in vitro and in vivo (Cheng et al., 2002; Brantley-Sieders et al., 2004). Despite the previously mentioned controversy, astrocytes release VEGF during embryonic development and might therefore contribute to the early TJ formation. Later on in development, however, for the further differentiation of EC and formation of an efficient BBB, the inhibition of EphA2 activity in human brain micro-vessel EC (HBMEC) is instrumental to promote TJ strengthening (Zhou et al., 2011; Figure 1). These different functions mediated by the tightly controlled expression levels of EphA2 suggest that the regulation of EphA2 dosages may underlie the “switch” between early/angiogenic and late/barriergenic effects of EphA2 in EC. Moreover, they suggest that putative interactions between EphA2-expressing EC with ephrinA1-expressing perivascular astrocytes or pericytes may also control TJ formation in physiological conditions or their disruption during pathogenic processes (Lécuyer et al., 2016). In a different system, the pulmonary system, stimulation of arterial EC with ephrinA1 also increases their permeability (Larson et al., 2008), further supporting that the overexpression of certain EphR/ephrin interactions might influence barrier integrity, ultimately impacting brain homeostasis. Astrocytes express several other members of the EphR/ephrin system (Nestor et al., 2007) which may be relevant during both vasculogenesis and/or barriergenesis. For example, the proper interaction between EphA4/ephrinA5 located on EC and astrocyte endfeet, respectively, is necessary for the development of a normal vascular system in the hippocampus of adult mice (Hara et al., 2010). Additionally, radial glia cells provide a physical scaffold and chemical signals to support the very early stages of angiogenesis (Cheslow and Alvarez, 2016). Among such signals, EphA4 expressed on EC has been indicated to guide the invasion of the developing brain by newly forming micro-vessels in response to glial-dependent stimulation (Goldshmit et al., 2006).

With regard to the “B” types, ephrinB2 controls VEGF receptor (VEGFR)-2 internalization, which is necessary for receptor activation and VEGF-induced filopodial extension in EC during angiogenesis (Bochenek et al., 2010; Sawamiphak et al., 2010; Pitulescu and Adams, 2014). During these events, the role of the EphB2/ephrinB2 interaction is essential for blood vessel assembly (Foo et al., 2006). During cardiovascular development, EphB4/ephrinB2 signaling in EC is additionally activated to properly specify arterial vs. venous identity (Wang et al., 1998; Adams et al., 1999; Gerety et al., 1999; Gale et al., 2001; Augustin and Reiss, 2003).