Epac1 Is Crucial for Maintenance of Endothelial Barrier Function through A Mechanism Partly Independent of Rac1

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.

Lack of adducin impairs the stability of endothelial adherens and tight junctions and may be required for cAMP-Rac1-mediated endothelial barrier stabilization

“…The relevance of cAMP for endothelial barrier function is well known 27 , 35 – 37 , however, whether Add is associated with cAMP-mediated barrier stabilization has not been investigated. Based on our data thus far, we speculated that in contrast to WT, Add-KO endothelium may have disturbed intracellular cAMP signaling.…”

“…The generation of immortalized mouse microvascular myocardial endothelial cells (MyEnd) was performed similary to previously described methods in the literature 27 , 28 . All endothelial cell isolations were carried out ex-vivo after mice sacrifice in a S2 cell culture laboratory in our facilities.…”

“…The function of the endothelial barrier was examined by using the ECIS Z Theta system (Applied Biophysics Inc, Troy, NYC, U.S.A.) as described previously 27 , 30 . In brief, MyEnd cells were seeded on 0.5% gelatine pre-coated 8W10E gold microarray electrodes (Ibidi, Martinsried, Germany).…”

cAMP Compartmentalization in Cerebrovascular Endothelial Cells: New Therapeutic Opportunities in Alzheimer’s Disease

“…They act as guanine exchange factors for Ras-like GTPases Rap1 and Rap2 [157,158]. Most studies have identified Epac1 as the principal isoform in vascular endothelial cells (see, i.e., [155,159]), although the existence of Epac2 has also been detected in some endothelial cell models and it was shown to be inducible in human microvascular endothelial cells [160].…”

“…In recent years, the role of the activation of GTPases by Epac in the control of endothelial permeability has been extensively studied (see, i.e., [160,[167][168][169]), although the review of these mechanisms exceeds the purpose of the present work. Furthermore, Epac/Rap signaling and the role of small GTPases in the regulation of BBB has recently been reviewed [165].…”