Abstract
BackgroundEndothelial tight and adherens junctions control a variety of physiological processes like adhesion, paracellular transport of solutes or trafficking of activated leukocytes. Formation and maintenance of endothelial junctions largely depend on the microenvironment of the specific vascular bed and on interactions of the endothelium with adjacent cell types. Consequently, primary cultures of endothelial cells often lose their specific junctional pattern and fail to establish tight monolayer in vitro. This is also true for endothelial cells isolated from the vein of human umbilical cords (HUVEC) which are widely used as model for endothelial cell-related studies.ResultsWe here compared the effect of cyclic 3'-5'-adenosine monophosphate (cAMP) and its derivates on formation and stabilization of tight junctions and on alterations in paracellular permeability in HUVEC. We demonstrated by light and confocal laser microscopy that for shorter time periods the sodium salt of 8-bromoadenosine-cAMP (8-Br-cAMP/Na) and for longer incubation periods 8-(4-chlorophenylthio)-cAMP (pCPT-cAMP) exerted the greatest effects of all compounds tested here on formation of continuous tight junction strands in HUVEC. We further demonstrated that although all compounds induced protein kinase A-dependent expression of the tight junction proteins claudin-5 and occludin only pCPT-cAMP slightly enhanced paracellular barrier functions. Moreover, we showed that pCPT-cAMP and 8-Br-cAMP/Na induced expression and membrane translocation of tricellulin.ConclusionspCPT-cAMP and, to a lesser extend, 8-Br-cAMP/Na improved formation of continuous tight junction strands and decreased paracellular permeability in primary HUVEC. We concluded that under these conditions HUVEC represent a feasible in vitro model to study formation and disassembly of endothelial tight junctions and to characterize tight junction-associated proteins
Highlights
Endothelial tight and adherens junctions control a variety of physiological processes like adhesion, paracellular transport of solutes or trafficking of activated leukocytes
human umbilical vein endothelial cells (HUVEC) treated with pCPT-cyclic 3’-5’-adenosine monophosphate (cAMP) or 8Br-cAMP/Na for 72 h showed no sign of cytotoxicity and the cell borders were clearly visible in phase contrast microscopy (Figure 1A)
Activation of caspases 3/7 did not significantly differ upon treatment with the cAMP derivates compared to untreated cells whereas stimulation with TNF-a and CPT strongly increased the signals for active caspases 3/7 (Figure 1D)
Summary
Endothelial tight and adherens junctions control a variety of physiological processes like adhesion, paracellular transport of solutes or trafficking of activated leukocytes. Endothelial cells line the surface of all vascular or lymphatic vessels and are connected by intercellular junctions consisting of tight junctions (zonula occludens), adherens junctions and gap junctions [1]. These junctional complexes control a variety of cellular mechanisms like adhesion, paracellular transport or signaling events [2]. In contrast to most epithelial cells that are characterized by a highly regulated organization of cellular junctions interendothelial junctions are far from being static structures Both adherens junctions and tight junctions seem to be intermingled throughout. A study published by Taddei and coworkers demonstrated the importance of adherens junctions in controlling proper tight junction formation in endothelial cells in vitro [11]
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