Elisabetta Dejana

Endothelial cell-to-cell junctions.

The endothelium forme the main barrier to the passage of macromolecules and circulating cells from blood to tissues. Endothelial permeability is in large part regulated by intercellular junctions. These are complex structures formed by transmembrane adhesive molecules linked to a network of cytoplasmic/cytoskeletal proteins. At least four different types of endothelial junctions have been described: tight junctions, gap junctions, adherence junctions and syndesmos. lítese organelles have some features and components in common with epithelial cells but there are also some that are specific for the endothelium. The mechanisms that regulate the opening and closing of endothelial junctions are still obscure. It is conceivable that inflammatory agents increase permeabilty by binding to specific receptors generating intracellular signals, which in turn cause cytoskeletal reorganization and opening of interen‐ dothelial cell gaps. Endothelial junctions also control leukocyte extravasation. Once leukocytes have adhered to the endothelium, a coordinated opening of interendothelial cell junctions occurs. The mechanism by which this takes place is unknow, but it might present characteristics similar to that triggered by soluble mediators.—Dejana, E., Corada, M., Lampugnani, M. G. Endothelial cell‐to‐cell junctions. FASEBJ., 9, 910‐918 (1995)

Thrombin-Induced Increase in Endothelial Permeability Is Associated With Changes in Cell-to-Cell Junction Organization

Thrombin increases endothelial permeability in a rapid and reversible way. This effect requires the catalytic activity of the enzyme and thrombin receptor engagement. Endothelial cell permeability is mostly regulated by intercellular junction organization. In the present study, we investigated whether opening of intercellular gaps after thrombin treatment could be related to changes in adherence-junction molecular organization. By immunofluorescence analysis, we found that thrombin stimulation of endothelial cells caused a marked alteration of the distribution of vascular endothelial (VE)-cadherin and of the associated catenins. These molecules, which are strictly localized at intercellular boundaries in confluent resting cells, were absent in the areas of intercellular retraction. Immunoprecipitation analysis indicated that thrombin disrupted the VE-cadherin/catenin complex. This effect was reversible and correlated with the increase in endothelial permeability. The use of a protein kinase C inhibitor (calphostin C) blocked both thrombin-induced permeability and disassembly of adherence-junction components. We propose that thrombins effect on endothelial cell junction organization is an important determinant in the increase in endothelial permeability induced by this agent.

Functional Properties of Human Vascular Endothelial Cadherin (7B4/Cadherin-5), an Endothelium-Specific Cadherin

Human vascular endothelial cadherin (VE-cadherin, 7B4/cadherin-5) is an endothelial-specific cadherin localized at the intercellular junctions. To directly investigate the functional role of this molecule we cloned the full-length cDNA from human endothelial cells and transfected its coding region into Chinese hamster ovary cells. The product of the transfected cDNA had the same molecular weight as the natural VE-cadherin in human endothelial cells, and reacted with several VE-cadherin mouse monoclonal antibodies. Furthermore, it selectively concentrated at intercellular junctions, where it codistributed with alpha-catenin. VE-cadherin conferred adhesive properties to transfected cells. It mediated homophilic, calcium-dependent aggregation and cell-to-cell adhesion. In addition, it decreased intercellular permeability to high-molecular weight molecules and reduced cell migration rate across a wounded area. Thus, VE-cadherin may exert a relevant role in endothelial cell biology through control of the cohesion and organization of the intercellular junctions.

INHIBITION OF CULTURED CELL GROWTH BY VASCULAR ENDOTHELIAL CADHERIN (CADHERIN-5/VE-CADHERIN)

Endothelial cell proliferation is inhibited by the establishment of cell to cell contacts. Adhesive molecules at junctions could therefore play a role in transferring negative growth signals. The transmembrane protein VE-cadherin (vascular endothelial cadherin/cadherin-S) is selectively expressed at intercellular clefts in the endothelium. The intracellular domain interacts with cytoplasmic proteins called catenins that transmit the adhesion signal and contribute to the anchorage of the protein to the actin cytoskeleton. Transfection of VE-cadherin in both Chinese hamster ovary (CHO) and L929 cells confers inhibition of cell growth. Truncation of VE-cadherin cytoplasmic region, responsible for linking catenins, does not affect VE-cadherin adhesive properties but abolishes its effect on cell growth. Seeding human umbilical vein endothelial cells or VE-cadherin transfectants on a recombinant VE-cadherin amino-terminal fragment inhibited their proliferation. These data show that VE-cadherin homotypic engagement at junctions participates in density dependent inhibition of cell growth. This effect requires both the extracellular adhesive domain and the intracellular catenin binding region of the molecule.

Molecular Organization of Endothelial Cell to Cell Junctions

The endothelial monolayer forms the main barrier to macromolecular passage from blood to tissues and regulates the extravasation of circulating cells such as leukocytes. When endothelial permeability is altered such as in inflammatory conditions, edema takes place. Inflammatory edema is a significant component of many diseases e.g. adult respiratory syndrome, septicemia and hypoxia/reperfusion syndrome. This is frequently the result of both pressure-induced filtration of plasma proteins and changes in the permeability properties of the endothelial lining of the vascular wall (Majno et al., 1961).

Effect of Pentoxifylline and its Analogues on Cytokine Activation of Endothelial Cells

AbstractThe capacity of pentoxifylline and some of its analogues to inhibit endothelial cell activation by tumor necrosis factor (TNF) was investigated. The drugs were added to endothelial cells during incubation with the cytokine. Thromboplastin activity, polymorphonuclear cell and two different carcinoma cell line adhesion were measured. As expected, these parameters were strongly affected by TNF, but the drugs tested did not change TNF activation of the endothelium. The drugs were also inactive on endotoxin induced interleukin-6 production by endothelial cells. These data suggest that the beneficial effect of pentoxifylline observed in a series of experimental models of inflammation is not due to a change in the endothelial cell prothrombotic and proinflammatory properties.

Endothelial Cell Permeability Assays in Culture

One of the most specific functions of the endothelial layer is to act as a selective barrier between blood/lymph and tissues. A standardized assay able to measure permeability of in vitro cultured endothelial cells is therefore highly desirable. In principle such an assay measures the passage of an easily detectable tracer between two compartments separated by an endothelial layer. The assay of trans-monolayer electrical resistance, which measures the passage of ions through the cell layer, while very effective for testing barrier properties of epithelial cells which form very tight layers in culture (Madara 1998), is less suitable for endothelial cells. With the exception of arterial or brain microvascular endothelium, most types of endothelial cells in vitro form layers that present low electrical resistance, which makes it difficult to pick up a further decrease.

Adhesive Molecules at Luminal Surface and at Intercellular Junctions of the Endothelium in the Regulation of Leukocyte Transendothelial Traffic

Circulating leukocytes migrate from the vessels and enter tissues under both normal and pathological situations. Whereas monocytes, lymphocytes and natural killer cells exhibit a significant spontaneous migration through resting endothelium neutrophils and eosinophils require chemotactic stimuli and/or endothelial cell activation. Cell passage across endothelial monolayers involves leukocyte adherence to the endothelium, crawling on the endothelial surface and transmigration, most probably through endothelial clefts.

Adhesion Molecules at Endothelial Cell to Cell Junctions

Endothelial cells express a variety of adhesive receptors that regulate their adhesion to extracellular matrix and the organization of cell-cell junctions. Most of the endothelial cell receptors for matrix proteins belong to the inteh=grin superfamily. Endothelial cells, as many other cell types, have many different integrin on their surface. This indicates that the interaction with matrix protiens is a complex phenomenon that requires multiple recognition mechanisms. We have only a very limited knowledge of the molecules present at endothelial cell-cell junctions. Integrins have been found to be localized in these structures. This suggests that they can play a role also in this homoytpic type of cell inteaction. Other molecules, however, structurally and fuctionally distinct from integrins (including cadherins and immunoglobulins) have been found in endothelial junctions. The reciprocal role of these proteins remains tobe fully defined.