Background:

cAMP signalling regulates several endothelial functions such as endothelial barrier integrity, nitric oxide (NO) production, and proliferation and migration in response to growth factors. cAMP mediates these effects via activation of its two well known effector proteins i.e. PKA and Epac (exchange protein directly activated by cAMP). The signalling pathways activated by PKA and Epac might be overlapping, non-overlapping, or even antagonistic. The aim of the present study was to analyse the differential signalling response of PKA and Epac on endothelial barrier, cell migration and differentiation.

Methods:

The study was carried out on cultured human umbilical vein endothelial cells (HUVEC). cAMP analogues, 8-CPT-cAMP, 6-Bnz-cAMP and Forskolin (FSK) were used to activate Epac, PKA or adenylyl cyclase, respectively. Endothelial cell proliferation, migration, and sprouting were quantified by crystal violet staining, wound healing assay, and tube formation and spheroid sprouting assay, respectively. Endothelial barrier function was analysed by measuring the flux of trypan blue-labeled albumin.

Results:

Specific activation of either PKA or Epac induced barrier protective response in HUVEC monolayers which was additive when both PKA and Epac were activated together. Activation of Epac but not PKA caused Akt phosphorylation at S473 which was antagonised by inhibitors of alpha and beta but not by gamma and delta isoforms of PI3K. Inhibition of PKA with specific membrane permeable peptide inhibitor (PKI; 10 µM) enhanced Epac-mediated activation of PI3K/Akt signalling and eNOS phosphorylation which was abrogated by specific Akt inhibitor. Both Epac and PKA induced ERK phosphorylation accompanied by increased cell proliferation and migration which was abrogated by inhibition of MAPK pathway. Activation of PKA induced endothelial cell sprouting and tube formation and enhanced VEGF-mediated effect. Contrarily, activation of Epac antagonised VEGF-mediated sprouting and tube formation and endothelial cells tend to establish monolayer on matrigels rather than forming network like structures.

Conclusion:

The data of present study shows that endothelial cell functions are differentially regulated by PKA and Epac signalling. Both PKA and Epac activation stabilize endothelial barrier function, however, both have opposite effects on PI3K/eNOS signalling and angiogenesis. PKA activation promotes angiogenesis while Epac activation antagonises tube angiogenesis.