The origins of the mesenchymal cells participating in tissue repair and pathological processes, notably tissue fibrosis, tumor invasiveness, and metastasis, are poorly understood. However, emerging evidence suggests that epithelial-mesenchymal transitions (EMTs) represent one important source of these cells. As we discuss here, processes similar to the EMTs associated with embryo implantation, embryogenesis, and organ development are appropriated and subverted by chronically inflamed tissues and neoplasias. The identification of the signaling pathways that lead to activation of EMT programs during these disease processes is providing new insights into the plasticity of cellular phenotypes and possible therapeutic interventions.

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The basics of epithelial-mesenchymal transition

Structure and function of the blood–brain barrier

Reactive oxygen species (ROS) are key signaling molecules that play an important role in the progression of inflammatory disorders. An enhanced ROS generation by polymorphonuclear neutrophils (PMNs) at the site of inflammation causes endothelial dysfunction and tissue injury. The vascular endothelium plays an important role in passage of macromolecules and inflammatory cells from the blood to tissue. Under the inflammatory conditions, oxidative stress produced by PMNs leads to the opening of inter-endothelial junctions and promotes the migration of inflammatory cells across the endothelial barrier. The migrated inflammatory cells not only help in the clearance of pathogens and foreign particles but also lead to tissue injury. The current review compiles the past and current research in the area of inflammation with particular emphasis on oxidative stress-mediated signaling mechanisms that are involved in inflammation and tissue injury.

Reactive Oxygen Species in Inflammation and Tissue Injury

The Blood-Brain Barrier in Health and Chronic Neurodegenerative Disorders

The blood-brain barrier (BBB) is the regulated interface between the peripheral circulation and the central nervous system (CNS). Although originally observed by Paul Ehrlich in 1885, the nature of the BBB was debated well into the 20th century. The anatomical substrate of the BBB is the cerebral microvascular endothelium, which, together with astrocytes, pericytes, neurons, and the extracellular matrix, constitute a "neurovascular unit" that is essential for the health and function of the CNS. Tight junctions (TJ) between endothelial cells of the BBB restrict paracellular diffusion of water-soluble substances from blood to brain. The TJ is an intricate complex of transmembrane (junctional adhesion molecule-1, occludin, and claudins) and cytoplasmic (zonula occludens-1 and -2, cingulin, AF-6, and 7H6) proteins linked to the actin cytoskeleton. The expression and subcellular localization of TJ proteins are modulated by several intrinsic signaling pathways, including those involving calcium, phosphorylation, and G-proteins. Disruption of BBB TJ by disease or drugs can lead to impaired BBB function and thus compromise the CNS. Therefore, understanding how BBB TJ might be affected by various factors holds significant promise for the prevention and treatment of neurological diseases.

The Blood-Brain Barrier/Neurovascular Unit in Health and Disease

Tight junctions are well-developed between adjacent endothelial cells of blood vessels in the central nervous system, and play a central role in establishing the blood-brain barrier (BBB). Claudin-5 is a major cell adhesion molecule of tight junctions in brain endothelial cells. To examine its possible involvement in the BBB, claudin-5–deficient mice were generated. In the brains of these mice, the development and morphology of blood vessels were not altered, showing no bleeding or edema. However, tracer experiments and magnetic resonance imaging revealed that in these mice, the BBB against small molecules (<800 D), but not larger molecules, was selectively affected. This unexpected finding (i.e., the size-selective loosening of the BBB) not only provides new insight into the basic molecular physiology of BBB but also opens a new way to deliver potential drugs across the BBB into the central nervous system.

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Size-selective loosening of the blood-brain barrier in claudin-5–deficient mice

Three integral membrane proteins, clau- din-1, -2, and occludin, are known to be components of tight junction (TJ) strands. To examine their ability to form TJ strands, their cDNAs were introduced into mouse L fibroblasts lacking TJs. Immunofluorescence microscopy revealed that both FLAG-tagged claudin-1 and -2 were highly concentrated at cell contact sites as planes through a homophilic interaction. In freeze-fracture replicas of these contact sites, well-developed networks of strands were identified that were similar to TJ strand networks in situ and were specifically labeled with anti-FLAG mAb. In glutaraldehyde-fixed samples, claudin-1–induced strands were largely associated with the protoplasmic (P) face as mostly continuous structures, whereas claudin-2–induced strands were discontinuous at the P face with complementary grooves at the extracellular (E) face which were occupied by chains of particles. Although occludin was also concentrated at cell contact sites as dots through its homophilic interaction, freeze-fracture replicas identified only a small number of short strands that were labeled with anti-occludin mAb. However, when occludin was cotransfected with claudin-1, it was concentrated at cell contact sites as planes to be incorporated into well- developed claudin-1–based strands. These findings suggested that claudin-1 and -2 are mainly responsible for TJ strand formation, and that occludin is an accessory protein in some function of TJ strands.

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A single gene product, claudin-1 or -2, reconstitutes tight junction strands and recruits occludin in fibroblasts.

Tight junctions (TJs) in endothelial cells are thought to determine vascular permeability. Recently, claudin-1 to -15 were identified as major components of TJ strands. Among these, claudin-5 (also called transmembrane protein deleted in velo-cardio-facial syndrome [TMVCF]) was expressed ubiquitously, even in organs lacking epithelial tissues, suggesting the possible involvement of this claudin species in endothelial TJs. We then obtained a claudin-6–specific polyclonal antibody and a polyclonal antibody that recognized both claudin-5/TMVCF and claudin-6. In the brain and lung, immunofluorescence microscopy with these polyclonal antibodies showed that claudin-5/TMVCF was exclusively concentrated at cell–cell borders of endothelial cells of all segments of blood vessels, but not at those of epithelial cells. Immunoreplica electron microscopy revealed that claudin-5/TMVCF was a component of TJ strands. In contrast, in the kidney, the claudin-5/TMVCF signal was restricted to endothelial cells of arteries, but was undetectable in those of veins and capillaries. In addition, in all other tissues we examined, claudin-5/TMVCF was specifically detected in endothelial cells of some segments of blood vessels, but not in epithelial cells. Furthermore, when claudin-5/TMVCF cDNA was introduced into mouse L fibroblasts, TJ strands were reconstituted that resembled those in endothelial cells in vivo, i.e., the extracellular face–associated TJs. These findings indicated that claudin-5/TMVCF is an endothelial cell–specific component of TJ strands.

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Endothelial Claudin: Claudin-5/Tmvcf Constitutes Tight Junction Strands in Endothelial Cells

In tight junctions (TJs), TJ strands are associated laterally with those of adjacent cells to form paired strands to eliminate the extracellular space. Claudin-1 and -2, integral membrane proteins of TJs, reconstitute paired TJ strands when transfected into L fibroblasts. Claudins comprise a multigene family and more than two distinct claudins are coexpressed in single cells, raising the questions of whether heterogeneous claudins form heteromeric TJ strands and whether claudins interact between each of the paired strands in a heterophilic manner. To answer these questions, we cotransfected two of claudin-1, -2, and -3 into L cells, and detected their coconcentration at cell–cell borders as elaborate networks. Immunoreplica EM confirmed that distinct claudins were coincorporated into individual TJ strands. Next, two L transfectants singly expressing claudin-1, -2, or -3 were cocultured and we found that claudin-3 strands laterally associated with claudin-1 and -2 strands to form paired strands, whereas claudin-1 strands did not interact with claudin-2 strands. We concluded that distinct species of claudins can interact within and between TJ strands, except in some combinations. This mode of assembly of claudins could increase the diversity of the structure and functions of TJ strands.

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Hiroyuki Sasaki

Papers

Size-selective loosening of the blood-brain barrier in claudin-5–deficient mice

A single gene product, claudin-1 or -2, reconstitutes tight junction strands and recruits occludin in fibroblasts.

Endothelial Claudin: Claudin-5/Tmvcf Constitutes Tight Junction Strands in Endothelial Cells

Manner of Interaction of Heterogeneous Claudin Species within and between Tight Junction Strands

ZO-1 and ZO-2 Independently Determine Where Claudins Are Polymerized in Tight-Junction Strand Formation