The impact of cAMP signalling on the dynamics of endothelial tubule formation

Abstract

Signalling mediated by the ubiquitous cellular messenger cyclic adenosine monophosphate (cAMP) plays a central role in numerous cellular processes. Within vascular endothelial cells (VECs), cAMP signalling influences adhesion to extracellular matrix and formation of VE-cadherin cell-junctions, both part of the VECs angiogenic potential. The angiogenic signalling mechanism within VECs is not completely understood. Here we explored how the components of the cAMP signalling cascade, adenylyl cyclase (AC), protein kinase A (PKA), exchange protein activated by cAMP (EPAC), and phosphodiesterase enzymes (PDEs), influence VEC angiogenic potential.

Plating human aortic endothelial cells (HAECs; model VECs) on the pro-angiogenic Matrigel in vitro tubule forming assay, we show that treatment of VECs with adenylate cyclase activator forskolin leads to formation of a distinct phenotype. The structures formed are reminiscent of a monolayer and display some features of early tubule formation. This phenotype was not elicited by activating the two effector proteins (PKA and EPAC) individually. Inhibition of EPAC activity hindered VECs’ response to forskolin and resulted in formation of poor structures, occupying significantly less area and showing significantly less branching than controls.

Since the selectivity of cAMP-mediated responses is coordinated in part by actions of cAMP at individual VEC cAMP signalling complexes, we investigated the impact of PDEs on tubule formation. We recently showed that EPAC1-integration into a PDE3B-based VEC cAMP signalling complex allows PDE3 inhibitors to activate PI3Kγ and promote VEC adhesion to Matrigel, an early event in tubule formation. EPAC1 integrates into a PDE4D-containing, VEcadherin-based, cAMP signalling complex that allows EPAC1 to control VEC intercellular contacts, but not adhesions. We investigated how PDE4 inhibitors impacted VEC tubule formation and identified distinct roles for each PDE4B and PDE4D in the control of EPAC1-mediated effects in tubule formation and identify potential novel therapeutic strategies to control these angiogenesis-related events.