Cyclic AMP and Acidic Fibroblast Growth Factor Have Opposing Effects on Tight and Adherens Junctions in Microvascular Endothelial Cells in Vitro

Endothelial adherens junctions (AJ) and tight junctions (TJ) are important determinants of vascular permeability and cell morphology. Here, we investigate their regulation, in primary human placental microvascular endothelial cell (HPMEC) cultures, by either aFGF plus heparin (ECGS) or elevated cAMP. The proliferation of HPMEC was weakly stimulated by ECGS, while cAMP was inhibitory. ECGS had little effect on transendothelial resistance (TER), but increased macromolecular permeability, whereas cAMP induced a twofold increase in TER and reduced macromolecular permeability. Ultrastructurally, ECGS-treated HPMEC exhibited an “activated” phenotype typified by proliferating cells, with poorly organized cell–cell junctions, whereas cAMP-treated cells appeared quiescent and markedly flattened with extended paracellular junctions, resembling endothelium in situ. The expression and localization of junctional molecules, F-actin, and junctional phosphotyrosine were examined by confocal microscopy and immunoblotting. Junctional molecules in ECGS-treated cells were less organized at lateral membranes than in control cells, whereas in cAMP-treated cells, they were highly localized at continuous contacts. These differences correlated with the intensity of junctional phosphotyrosine, being lowest with cAMP treatment. In the AJ of ECGS-treated and control cells, β-catenin predominated but in cAMP-treated cells, γ-catenin/plakoglobin was enriched. In addition, cAMP upregulated junctional expression of VE-cadherin and PECAM-1 and increased the levels of the TJ molecules occludin and ZO−1. The expression levels of junctional components, and their tyrosine phosphorylation, play an important role in dynamic regulation of endothelial cell–cell junctions.