The EPAC inhibitor ESI-09 attenuates NF-κB signalling in vascular cells.

Vascular smooth muscle cells (VSMCs) and human coronary artery endothelial cells (HCAECs) are central mediators of vascular inflammation through NF-κB-dependent transcriptional responses, but the role of the cAMP effector exchange protein directly activated by cAMP 1 (EPAC1) remains unresolved. Here, we combined pharmacological modulation, EPAC1 knockdown, Rap1 activation assays, macrophage-containing vascular tri-culture RNA sequencing, cytokine profiling, and molecular docking to define the contribution of EPAC1 to inflammatory signalling in human vascular cells. In primary VSMCs, forskolin suppressed IL-1β- and TNF-α-induced NF-κB p65 phosphorylation and reduced EPAC1 abundance, confirming a strong anti-inflammatory effect of global cAMP elevation. The non-selective EPAC inhibitor ESI-09 also reduced cytokine-induced p65 Ser536 and Ser529 phosphorylation, whereas the EPAC1-selective inhibitor CE3F4 produced only modest effects on NF-κB signalling despite inhibiting Rap1 activation, confirming functional engagement of EPAC-dependent Rap1 signalling. Conversely, acute activation of EPAC1 using D-007, PWO577 or SY007 increased Rap1-GTP but failed to suppress p65 phosphorylation, and EPAC1 siRNA knockdown did not phenocopy forskolin or ESI-09. In a macrophage-driven tri-culture model, RNA sequencing revealed cell-type- and compound-specific transcriptional responses. ESI-09 produced the largest endothelial DEG burden and a prominent down-regulated VSMC signature, whereas CE3F4 exerted a comparatively modest endothelial effect but increased the VSMC up-regulated DEG pool. Molecular docking supported distinct EPAC ligand-binding modes. Collectively, these findings indicate that cAMP suppresses vascular NF-κB signalling predominantly through EPAC1-independent mechanisms and that ESI-09-associated anti-inflammatory effects should not be interpreted as selective EPAC1 biology alone.