Abstract
Epac1 (exchange protein activated by cAMP) stabilizes the endothelial barrier, but detailed studies are limited by the side effects of pharmacological Epac1 modulators and transient transfections. Here, we compare the key properties of barriers between endothelial cells derived from wild-type (WT) and Epac1-knockout (KO) mice myocardium. We found that KO cell layers, unlike WT layers, had low and cAMP-insensitive trans-endothelial resistance (TER). They also had fragmented VE-cadherin staining despite having augmented cAMP levels and increased protein expression of Rap1, Rac1, RhoA, and VE-cadherin. The simultaneous direct activation of Rac1 and RhoA by CN04 compensated Epac1 loss, since TER was increased. In KO-cells, inhibition of Rac1 activity had no additional effect on TER, suggesting that other mechanisms compensate the inhibition of the Rac1 function to preserve barrier properties. In summary, Epac1 is crucial for baseline and cAMP-mediated barrier stabilization through mechanisms that are at least partially independent of Rac1.
Highlights
The aim of the present study was to further investigate the cAMP-dependent mechanisms stabilizing the intercellular junctions between vascular endothelium
Our current study describes the establishment of Epac1-deficient endothelial cells and their use to elucidate in more detail the role of Epac1 in cAMP-mediated regulation of Rho GTPases and endothelial barrier homeostasis
To judge the importance of Epac1 for cell monolayer integrity in vitro, the transendothelial electrical resistance (TER) was determined in the myocardial endothelial cell (MyEnd) cell lines derived from WT and Epac1-null
Summary
The aim of the present study was to further investigate the cAMP-dependent mechanisms stabilizing the intercellular junctions between vascular endothelium. The specific focus was the role of the exchange protein activated by cAMP-(Epac1)-dependent pathways These investigations were designed as follow-up research in Epac1−/− (Epac1-knockout (KO)) mice that increased the basal permeability to high and low molecular weight tracers in different microvascular beds such as in the skin, small and large intestine, and adipose tissue. Their baseline microvascular permeability failed to drop further when exposed to atrial natriuretic peptide or histamine, and the permeability reduction in response to the phosphodiesterase 4 (PDE4) inhibitor Rolipram was blunted [3,4]. No increased heart permeability [3]
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