Dataset Information

Effect of cAMP derivates on assembly and maintenance of tight junctions in human umbilical vein endothelial cells.

ABSTRACT:

Background

Endothelial 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.

Results

We 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.

Conclusions

pCPT-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.

SUBMITTER: Beese M

PROVIDER: S-EPMC2941681 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature

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Publications

Effect of cAMP derivates on assembly and maintenance of tight junctions in human umbilical vein endothelial cells.

Beese Michaela M Wyss Kristin K Haubitz Marion M Kirsch Torsten T

BMC cell biology 20100907

<h4>Background</h4>Endothelial 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 tig ...[more]

PMID: 20822540

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Project description:β-Cell functionality and survival are highly dependent on the cells' microenvironment and cell-cell interactions. Since the pancreas is a highly vascularized organ, the crosstalk between β-cells and endothelial cells (ECs) is vital to ensure proper function. To understand the interaction of pancreatic β-cells with vascular ECs, we sought to investigate the impact of the spatial distribution on the interaction of human cell line-based β-cells (EndoC-βH3) and human umbilical vein endothelial cells (HUVECs). We focused on the evaluation of three major spatial distributions, which can be found within human islets in vivo, in tissue-engineered heterotypic cell spheroids, so-called pseudo-islets, by controlling the aggregation process using magnetic levitation. We report that heterotypic spheroids formed by spontaneous aggregation cannot be maintained in culture due to HUVEC disassembly over time. In contrast, magnetic levitation allows the formation of stable heterotypic spheroids with defined spatial distribution and significantly facilitated HUVEC integration. To the best of our knowledge, this is the first study that introduces a human-only cell line-based in vitro test system composed of a coculture of β-cells and ECs with a successful stimulation of β-cell secretory function monitored by a glucose-stimulated insulin secretion assays. In addition, we systematically investigate the impact of the spatial distribution on cocultures of human β-cells and ECs, showing that the architecture of pseudo-islets significantly affects β-cell functionality. Impact statement Tissue engineering of coculture systems containing β-cells and endothelial cells (ECs) is a promising technique to stimulate β-cell functionality. In this study, we analyzed human pancreatic islet tissue and revealed three different native distributions of β-cells and ECs. We successfully recreated these distributions in vitro by employing magnetic levitation of human β-cells and ECs, forming controlled heterotypic pseudo-islets, which enabled us to identify a significant impact of the pseudo-islet architecture on insulin secretion.

Dataset Information

Effect of cAMP derivates on assembly and maintenance of tight junctions in human umbilical vein endothelial cells.

Background

Results

Conclusions

Publications

Effect of cAMP derivates on assembly and maintenance of tight junctions in human umbilical vein endothelial cells.

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