What allows communication and passage of small molecules between adjacent cells?

Cell-to-Cell Junctions and Vascular Permeability

Fluid homeostasis and capillary permeability are regulated by the complex intercellular communications within the cells of the neurovascular unit which comprises endothelial cells, pericytes and closely associated macro- and microglia and neurons. This cellular unit responds dynamically to complex circulatory and neural cues to control blood flow and regulate the intercellular junctions of the inner BRB. Intercellular junctions are complex structures formed by the assembly of transmembrane and cytoplasmic/cytoskeletal protein components. At least four different types of endothelial junctions have been described: tight junctions, gap junctions, adherence junctions, and syndesmos. Tight junctions are the most apical component of the intercellular cleft and are most relevant for the BRB (Fig. 30.5 online

Fig. 30.5. Intercellular junctions in endothelial cells: Endothelial cells are connected and communicate with each other by tight junctions and adherens junctions. Tight junctions resemble a major part of the inner blood–retinal barrier. They are built by different proteins including occludin, ZO-1, and the claudin family.

Fluid moves from the retina to the choroid largely due to the osmotic pressure exerted by the proteins in the choroidal stroma and disruption of this normal flow can lead to significant edema. Especially in the context of ischemia and diabetic retinopathy, there is evidence that the RPE becomes dysfunctional and that leakage from the choriocapillaris occurs in unison with impaired fluid clearance contributing to retinal edema. When the RPE shows stress responses resulting from oxidative or nitrosative damage, this can result in significant loss of fluid control and damage to junctional integrity. Similar to the breakdown of the inner BRB, the breakdown of the outer BRB is associated with a significant depletion of the occludin in the RPE of ischemic and diabetic rodents.

Intercellular junctions

Desmosomes are the major intercellular adhesion complexes in the epidermis. They anchor keratin intermediate filaments to the cell membrane and link adjacent keratinocytes (Fig. 1.45). Desmosomes are found in the epidermis, myocardium, meninges and cortex of lymph nodes. Ultrastructurally, desmosomes contain plaques of electron-dense material running along the cytoplasm parallel to the junctional region, in which three bands can be distinguished: an electron-dense band next to the plasma membrane, a less dense band, and then a fibrillar area (Fig. 1.46).1 Identical components are present on opposing cells which are separated by an intercellular space of 30 nm within which there is an electron-dense midline. There are three main protein components of desmosomes in the epidermis: the desmosomal cadherins, the armadillo family of nuclear and junctional proteins, and the plakins (Fig. 1.47).2 The transmembranous cadherins comprise mostly heterophilic associations of desmogleins and desmocollins. There are four main epidermis-specific desmogleins (Dsg1–4) and three desmocollins (Dsc1–3). These show differentiation-specific expression. For example, Dsg1 and Dsc1 are found predominantly in the superficial layers of the epidermis whereas Dsg3 and Dsc3 show greater expression in basal keratinocytes. The intracellular parts of the cadherins interact with the keratin filament network via the desmosomal plaque proteins, mainly desmoplakin, plakoglobin and plakophilin.1

Clues to the biologic function of these desmosomal components have arisen from various inherited and acquired human diseases.3,4 Naturally occurring human mutations have been reported in ten different desmosome genes with variable skin, hair and heart abnormalities and several desmosomal proteins serve as autoantigens in immunobullous blistering skin diseases such as pemphigus (Figs 1.48and1.49).5 Antibodies to multiple desmosomal proteins may develop in diseases such as paraneoplastic pemphigus through the phenomenon of epitope spreading.6 Cleavage of the extracellular domain of Dsg1 has also been demonstrated as the basis of staphylococcal scalded skin syndrome and bullous impetigo.7

Adherens junctions are recognized ultrastructurally as electron-dense transmembrane structures, with two opposing membranes separated by approximately 20 nm, that form links with the actin skeleton.8 They are 0.2–0.5 µm in diameter and can be found as isolated cell junctions or in association with tight junctions and desmosomes. Adherens junctions are expressed early in skin development and contribute to epithelial assembly, adhesion, barrier formation, cell motility and changes in cell shape. They may also spatially coordinate signaling molecules and polarity cues as well as serving as docking sites for vesicle release. Adherens junctions contain two basic adhesive units: the nectin-afadin complex and the classical cadherin complex.9,10 The nectins form a structural link to the actin cytoskeleton via afadin (also known as AF-6) and may be important in the initial formation of adherens junctions. The cadherins form a complex with the catenins (α-, β-, and p120 catenin) and help mediate adhesion and signaling. Cell signaling via β-catenin can activate several pathways linked to morphogenesis and cell fate determination.

Destabilization of EC–EC Junctions

Intercellular junctions between ECs, mediated by adherens junctions and tight junctions, stabilize the vascular endothelial monolayer. These junctions need to be destabilized to allow sprouting ECs to migrate from existing microvessels in response to angiogenic inducers. ECs uniquely express the adherens junction transmembrane protein vascular endothelial-cadherin (VE-cadherin), which contains five extracellular Ig-like domains, a single-pass transmembrane helical sequence and C-terminal intracellular region. VE-cadherins from adjacent ECs are thought to form a zipper-like structure between them. Although the VE-cadherin crystal structure shows overlap between the N-terminal Ig-like domains on VE-cadherins from opposing directions, they are also thought to be able to generate narrower adherens junctions by overlapping multiple Ig-like domains as illustrated in Figure 7. VE-cadherin binds near the membrane to p120 that influences VE-cadherin retention and spatial organization at the cell surface. The C-terminal tail of VE-cadherin binds β-catenin that in turn binds α-catenin, which attaches to actin thereby linking cell surface intercellular junctions to intracellular cytoskeleton microfilaments (Bravi et al., 2014).

EC tight junctions, also illustrated in Figure 7, are interspersed with adherens junctions but are thought to be more prevalent near the apical (luminal) surface. They limit passage of molecules through the EC–EC contact interface to approximately 800 Da. Tight junctions are composed of linear strands consisting of multiple proteins including occludins, EC-specific claudin-5, junctional adhesion molecules (JAMs), and zona occludens-1 (ZO-1), which links it to the actin cytoskeleton. VE-cadherin/β-catenin, acting through the PI3K/Akt pathway, expels a claudin-5 transcriptional repressor from the nucleus, effectively upregulating claudin-5 transcription and facilitating EC tight junction formation (Bravi et al., 2014).

The stability of the intercellular junctions is controlled by phosphorylation. VEGFR2, acting through endothelial nitric oxide synthase (eNOS) generation of nitric oxide, can nitrosylate β-catenin promoting its dissociation from VE-cadherin. VEGFR2-activated signaling pathways also phosphorylate VE-cadherin tyrosines in the binding sites for p120 and β-catenin, inhibiting adherens junction complex formation and promoting the internalization and degradation of VE-cadherin. Either the same or a different pool of β-catenin migrates to the nucleus and interacts with additional transcription factors to downregulate expression of the tight junction protein claudin-5. VEGF also induces serine phosphorylation of occludin, which increases ubiquitination that marks it for proteasome degradation. In addition to destabilizing EC intercellular junctions, growth factor-induced Rho GTPase activity promotes actin cytoskeleton contraction-mediated opening of the gaps between ECs. In contrast, Ang1-activated Tie2 signaling can inhibit VEGF-mediated EC junction destabilization, a process that can be functionally inhibited by the Tie2 weak agonist Ang2 (Goddard and Iruela-Arispe, 2013; Bravi et al., 2014). Therefore, as denoted in Figure 7, VEGF-induced destabilization of both adherens and tight junctions not only increases vascular permeability but also inhibits intercellular adhesion facilitating EC migration.

Basic Principles in Epithelial Repair: Cell Spreading and Migration, Cell Proliferation, and Junctional Resealing

Repair of the epithelium is crucial for clinical recovery, including reestablishment of AT1 cells and resolution of alveolar edema fluid. AT1 cells are flat cells that normally cover the majority of the gas exchange surface and exhibit active ion and fluid transport properties, whereas AT2 cells are cuboidal and synthesize surfactants to prevent the alveoli from collapsing.256,257 After death of AT1 cells, surviving AT2 cells supposedly spread and migrate onto the denuded basement membrane as an immediate process to reestablish the cellular barrier. Although this has not been directly observed in the lung in vivo, cell spreading and migration is likely to be the initial mechanism for resealing the leaky epithelium189 based on in vitro observations258–260 and on the importance of these phenomena in wound repair of other organs.261 Cell migration depends on a tightly regulated assembly of the cytoskeleton leading to protrusion of “lamellipodia” and “filopodia” from the leading edge, followed by contractile forces that drive release of the rear edge from the extracellular matrix, processes that depend on the Rho GTPases.262,263 In the lung epithelium, cell spreading and migration after injury are triggered by soluble factors such askeratinocyte growth factor (KGF), also known asfibroblast growth factor 7 (FGF7),260 TGF-α,259 TGF-β,264interleukin-1β (IL-1β),265,266 and CXC motif chemokine 3.267 Cell migration is controlled by signaling pathways involving Rac1/Tiam1,268phosphatase and tensin homolog (PTEN),269 β-catenin,270–273 syndecan-1,274,275adenosine triphosphate (ATP) and dual oxidase 1,276,277 and vimentin,264 as reviewed elsewhere.278 Integrins, which are up-regulated in the alveolar epithelium after injury,279 contribute to cell migration via interactions with both the actin cytoskeleton280 and theextracellular matrix (ECM).258,281–283 The production of ECM and MMPs is essential for cell migration.141,275 MMPs enhance wound healing by cleavage ofcell-cell and cell-ECM adhesion molecules, proteolytic activation of chemokines and growth factors, and degradation of the provisional matrix.142 Of note, cyclic stretch, imposed by mechanical ventilation during the acute and the recovery phase in ARDS, impedes cytoskeletal reorganization during cell spreading.284

Intercellular Junctions

Intercellular junctions join epithelial cells to one another and to adjacent tissue; some are named by their type and some by their shape. Protein components of intercellular junctions include cell adhesion molecules, transmembrane proteins (occludin, claudin), junctional adhesion molecules, and associated cytoplasmic proteins.7 Junctions between cells or with connective tissue can have additional functions other than adhesion. Physical changes, such as pressure and biochemical or pharmaceutical factors, can modulate junctions and alter the junctional proteins. This allows information about changes in the extracellular environment to be relayed to the cell interior affecting intracellular processes.

In a tight (occluding) junction, the outer leaflet of the cell membrane of one cell comes into direct contact with its neighbor. Ridgelike elevations on the surface of the cell membrance fuse with complementary ridges on the surface of a neighboring cell.8 As the paired strands meet, the neighboring cell membranes are fused.9 The fibers of tight junctions are connected to the cytoskeleton within the cell.

A tight junction that forms a zone or belt around the entire cell, joining it with each of the adjacent cells is called a zonula occludens (ZO) (Figure 1-5). In these zones, row on row of interwining ridges effectively occlude the intercellular space. A substance cannot pass through a sheet of epithelium whose cells are joined by ZO by passing between the cells, but must pass through the cell.In stratified epithelia, whose surface layer is constantly being sloughed and replaced from below, ZO, if present, will be located in the surface layer. The components of the tight junction are found in increasing numbers as a cell moves from its origin in the basal layer until, finally when the cell reaches the surface, its occluding junction is complete.10 The complex formed by the junctional proteins in the ZO can be affected in some diseases, causing in a breakdown in the barrier function, allowing a pathway to open through the network. Currently, researchers are developing pharmaceuticals that will cause a temporary disruption of the barrier, and that would allow other drugs or substances to pass through the intercellular route. In some instances, ridges in a tight junction are fewer and discontinuous, resulting in a “leaky epithelium.”8

A zonula adherens (ZA), an intermediate junction, is a similarly-shaped zone. However, the adjacent plasma membranes are separated, leaving a narrow intercellular space that contains a glycoporotein material causing cell adhesion but allowing intercellular passage.12 ZA junctions produce relatively firm adhesions. Adjacent to the adhering junction are fine microfilaments that extend from a plaque just inside the membrane to filaments of the cytoskeleton, contributing to cell stability.8 A terminal bar consists of a zonula occludens and a zonula adherens side by side, with the tight junction lying nearest the cell apex.1,8

Round, buttonlike intercellular junctions are either macula occludens (MO) or macula adherens (MA), depending on the type of adhesion.

A desmosome is a strong, spotlike attachment between cells (see Figure 1-5). A dense disc or plaque is present within the cytoplasm adajcent to the plasma membrane at the site of the adherence. Hairpin loops of cytoplasmic filaments called tonofilaments extend from the disc into the cytoplasm and link to keratin filaments in the cytoskelton, contributing to cell stability. Other filaments, transmembrane linkers, cadherins, extend from the plaque across the intercellular space, holding the cell membranes together and forming a strong bond.12 The intercellular space contains an acid-rich mucoprotein that acts as a strong adhesive.8

A hemidesmosome provides a strong connection between the cell and its basement membrane and underlying connective tissue. It contains similar intracellular components; the protein complex extends through the cell membrane to attach to keratin in the basement membrane. Bundles of filaments join the intracellular plaque to underlying connective tissue matrix, often attaching to a plaque embedded in the connective tissue.10

Gap junctions are formed by a group of (usually six)proteins, called connexins, that span the cell membrane and unite with connexins of a neighboring cell, forming a channel called a connexon (see Figure 5-1).13 These narrow channels allow rapid cell-to-cell communication, i.e., passage of small molecules and ions from one cell to another. A group of cells with such connection act like a syncytium, that is, a single cell with multiple nuclei.

Junctional Interendothelial Adhesion Molecules in Afferent LECs

The intercellular junctions in the initial lymphatics lack tight junctions at the tip but instead are anchored to the neighboring cells by discontinuous button-like junctions, which differ from conventional continuous zipper-like junctions, which are present in collecting lymphatics and blood vasculature (Figure 1). Despite different morphology, both of these junctions have the same molecules, such as vascular endothelial cadherin (VE-cadherin), the classical tight junction protein claudin-5, the tight junction protein zonula occludens-1 (ZO-1) and the transmembrane proteins, endothelial cell selective molecule (ESAM) and junctional adhesion molecule-A (JAM-A). Most likely, the lymph enters the lymphatic vessels via the buttons, whereas most leukocytes come in to the afferent lymphatics more proximally (Baluk et al., 2007) by penetrating into the lymphatic vessels via the preexisting pores (Pflicke and Sixt, 2009).

Transport Across Endothelial Cells

The presence of intercellular junctions is responsible for the control of paracellular pathway, which includes the passage of small and water-soluble molecules between adjacent ECs. In fact, this pathway is used by ions and solutes that cross the BBB accordingly to their concentration gradient (review by Petty & Lo, 2002). Since this type of transport is regulated by TJs and AJs, alterations of endothelial intercellular junctions will interfere with this route. Additionally, transcellular transport through the ECs can occur by passive diffusion of lipophilic compounds, or mediated by vesicles or selective transporters (reviewed by Abbott, Ronnback, & Hansson, 2006), such as the glucose transporter-1 (GLUT-1) and adenosine triphosphate-binding cassette transporters. Noteworthy, brain ECs present a low pinocytic activity (Claudio, Kress, Norton, & Brosnan, 1989) that can highly increase under pathological conditions. Indeed, endocytic vesicles play an important role in the transport into the brain, and the most studied are the caveolae-derived vesicles. Importantly, caveolin-1 is the primary coat protein of caveolae and regulates cellular proliferation, as well as both nitric oxide and calcium signaling (reviewed by Minshall, Sessa, Stan, Anderson, & Malik, 2003). Moreover, this vesicular protein modulates TJs proteins, since its silencing could prevent the downregulation of occludin, ZO-1, and ZO-2, induced by human immunodeficiency virus (HIV) proteins (Zhong et al., 2008).

In addition to the abovementioned routes of transport, migration of cells through the BBB is tightly regulated. Transmigration occurs with immune system cells, such as leukocytes that are recruited into the brain parenchyma under some pathological conditions. This phenomenon, known as diapedesis and rare under physiological condition, can occur through transcellular or paracellular pathways with the involvement of several adhesion molecules (AMs). Importantly, the paracellular diapedesis also involves alterations of intercellular junctions (reviewed by Engelhardt & Ransohoff, 2005).

Abstract

Desmosomes are adhesive intercellular junctions that mechanically integrate adjacent cells by coupling adhesive interactions mediated by desmosomal cadherins to the intermediate filament cytoskeletal network. Desmosomal cadherins are connected to intermediate filaments by densely clustered cytoplasmic plaque proteins comprising members of the armadillo gene family, including plakoglobin and plakophilins, and members of the plakin family of cytolinkers, such as desmoplakin. The importance of desmosomes in tissue integrity is highlighted by human diseases caused by mutations in desmosomal genes, autoantibody attack of desmosomal cadherins, and bacterial toxins that selectively target desmosomal cadherins. In addition to reviewing the well-known roles of desmosomal proteins in tissue integrity, this chapter also highlights the growing appreciation for how desmosomal proteins are integrated with cell signaling pathways to contribute to vertebrate tissue organization and differentiation.

Intercellular adhesion complexes

Two types of intercellular junctions are established at the level of the lateral surface of epithelial cells: tight junctions (TJs) and adherens junctions (AJs). Although made up of different proteins, both junctional complexes associate with actin, and formation and maturation of cell–cell contacts involves reorganization of the actin cytoskeleton. Both TJs and AJs are composed of transmembrane proteins, responsible for the adhesion of adjacent cells, and of intracellular scaffolding proteins for anchorage to the cytoskeleton. In mammalian epithelia TJs localize apically to AJs at the border of the apical and basolateral domain (Figure 1). In Drosophila epithelia the septate junction (SJ), the structure analogous to TJs in invertebrates, sits basal to AJs, while C. elegans epithelial cells have a single apical junction that acts as both AJs and TJs. TJs form a belt-like structure and provide both a fence function, to limit the diffusion of proteins and lipids between apical and basolateral membrane domains, and a barrier function, which restricts the passage of molecules between cells. TJs consist of a number of integral proteins, such as claudin, occludin, and junctional adhesion molecules (JAMs), and associated cytoplasmic proteins such as zonula occludens-1 (ZO-1) and its homologs ZO-2 and ZO-3, which in turn interact with F-actin (Tsukita et al., 2009; Figure 1). AJs provide the main mechanical link between neighboring epithelial cells. AJs consist of two basic adhesive units, the cadherin–catenin and nectin–afadin complexes (Niessen and Gottardi, 2008). Classical cadherins mediate strong cell–cell adhesion by a homophilic calcium-dependent binding of their extracellular domain. Intracellularly, they bind through β-catenin to α-catenin, which in turn interacts with actin-binding proteins (actin-BP) (Kobielak and Fuchs, 2004; Figure 1). Nectins link AJs to the actin cytoskeleton through the scaffolding protein afadin (AF6) (Takai and Nakanishi, 2003; Figure 1). In addition, nectins bind to ZO-1 and α-catenin, thus providing a link between AJs and TJs.