Aims: 

cAMP signalling regulates several vascular functions including endothelial barrier and angiogenesis. Elevated intracellular levels of cAMP can activate a complex repertoir of signalling via its two well known effectors i.e. PKA and Epac. These signalling pathways interact at several points and control the vessel integrity and angiogenesis. We have previously shown that activation of the cAMP/Epac signalling attenuates the thrombin-induced hyperpermeability. Here the role of PI3K in cAMP/Epac-mediated endothelial barrier and angiogenesis was analysed.

Methods: 

Endothelial barrier function was analysed in cultured HUVEC. 8-CPT-cAMP (200 mM) was used to activate cAMP/Epac signalling. The cells were challenged by inflammatory mediator thrombin to induce hyperpermeability. Angiogenic response was analysed by proliferation and migration assays.

Results: 

Activation of cAMP/Epac signalling reduced the basal permeability of HUVEC monolayers and attenuated thrombin-induced hyperpermeability in a conc.-dependent manner. This barrier stabilisation effect was accompanied by an activation of PI3K/Akt, MEK/ERK, Rac1, and re-organisation of actin cytoskeleton and VE-cadherin at cell periphery. However, it did not affect RhoA/Rock activity. Inhibition of PI3K/Akt signalling by different PI3K isoform specific inhibitors or a specific Akt inhibitor did not abrogate its barrier protective effect against thrombin-induced hyperpermeability. On the other hand, inhibition of MEK/ERK pathway by a specific MEK inhibitor (UO126; 10mM) potentiated the barrier protective effect of cAMP/Epac signalling while inhibition of Rac1 by a specific inhibitor (NSC23766; 20 mM) abolished this effect. Similarly, activation of cAMP/Epac signalling induced endothelial cell proliferation and migration (wound assay) which was completely blocked by a specific PI3Ka and weakly by PI3Kb, PI3Kd, or PI3Kg inhibitors. The angiogenic response of cAMP/Epac signalling was also blocked by MEK/ERK inhibitors UO126 and PD59098.

Conclusion: 

cAMP/Epac stabilises the endothelial barrier via PI3K-independent activation of Rac1 and promotes angiogenesis via PI3Ka-dependent activation of MEK/ERK pathway.