Abstract 607: cAMP-dependent Intimal Cushion Formation Of the Rat Ductus Arteriosus: The Role Of Epac
We have demonstrated that chronic stimulation of the prostaglandin E2-cAMP-dependent protein kinase A (PKA) signal pathway plays a critical role in intimal cushion formation (ICF) in perinatal ductus arteriosus (DA) through promoting synthesis of hyaluronic acd (HA) (Yokoyama et al. J Clin Invest 2006). Epac, exchange factor directly activated by cAMP, has been newly identified as a downstream target for cAMP and plays a distinct role from PKA. Since prostaglandin E2 increases cAMP in DA during late gestation, Epac is also likely to be activated. Here we examined the role of Epac in ICF in rat DA. Quantitative RT-PCR analysis revealed that the expression levels of Epac1 mRNA were significantly upregulated in rat DA during a perinatal period. Immunohistochemical analysis uncovered that Epac1 protein was expressed predominantly in the middle smooth muscle layer of fetal DA at term and in migrating SMCs in the central core of neonatal DA at birth. Acute stimulation (~ 4 hours) of Epac by 8-cCPT-2′-O-Me-cAMP, a cAMP analog selective to Epac, significantly promoted DA SMC migration in a dose-dependent manner (n=4, 220% increase at a concentration of 50 μM compared to control), and targeted disruption of Epac1 by siRNA negated this enhancement. However, acute stimulation of PKA by pCPT-cAMP (100 μM), a cAMP analog selective to PKA, significantly decreased DA SMC migration (n=4, 20% decrease compared to control). Furthermore, when Epac1 was overexpressed in DA SMCs by adenovirus-mediated gene transfer, the effect of 8-cCPT-2′-O-Me-cAMP on cell migration was further enhanced by 1.5 fold. Whereas PKA strongly produced HA by increasing the expression of HA synthase 2 mRNA, Epac did not increase HA production in DA SMCs. [3H]thymidine incorporation assay revealed that 8-cCPT-2′-O-Me-cAMP did not change SMC proliferation. Finally, adenovirus-mediated Epac1 gene transfer induced prominent ICF in rat DA explants. Intima-media ratio was significantly greater in Epac1-overexpressed DA by 1.7 fold when compared with green fluorescent protein-overexpressed DA (p<0.05, n=6). In conclusion, Epac plays a distinct role from PKA in promoting DA SMC migration, resulting in an increase in ICF in rat DA. Thus cAMP-downstream targets, Epac and PKA, synergistically promote ICF in DA.