PGE, a potent vasodilator, plays a primary role in maintaining the patency of the ductus arteriosus (DA). Genetic disruption of the PGE-specific receptor EP4, however, paradoxically results in fatal patent DA (PDA) in mice. Here we demonstrate that EP4-mediated signals promote DA closure by hyaluronic acid-mediated (HA-mediated) intimal cushion formation (ICF). Chronic EP4 stimulation by ONO-AE1-329, a selective EP4 agonist, significantly enhanced migration and HA production in rat DA smooth muscle cells. When HA production was inhibited, EP4-mediated migration was negated. Activation of EP4, adenylyl cyclase, and PKA all increased HA production and the level of HA synthase 2 (HAS2) transcripts. In immature rat DA explants, ICF was promoted by EP4/PKA stimuli. Furthermore, adenovirus-mediated Has2 gene transfer was sufficient to induce ICF in EP4-disrupted DA explants in which the intimal cushion had not formed. Accordingly, signals through EP4 have 2 essential roles in DA development, namely, vascular dilation and ICF. The latter would lead to luminal narrowing, helping adhesive occlusion and permanent closure of the vascular lumen. Our results imply that HA induction serves as an alternative therapeutic strategy for the treatment of PDA to the current one, i.e., inhibition of PGE signaling by cyclooxygenase inhibitors, which might delay PGE-mediated ICF in immature infants.

Vascular remodeling after mechanoinjury largely depends on the migration of smooth muscle cells, an initial key step to wound healing. However, the role of the second messenger system, in particular, the cAMP signal, in regulating such remodeling remains controversial. Exchange protein activated by cAMP (Epac) has been identified as a new target molecule of the cAMP signal, which is independent from PKA. We thus examined whether Epac plays a distinct role from PKA in vascular remodeling. To examine the role of Epac and PKA in migration, we used primary culture smooth muscle cells from both the fetal and adult rat aorta. A cAMP analog selective to PKA, 8-(4-parachlorophenylthio)-cAMP (pCPT-cAMP), decreased cell migration, whereas an Epac-selective analog, 8-pCPT-2'-O-Me-cAMP, enhanced migration. Adenovirus-mediated gene transfer of PKA decreased cell migration, whereas that of Epac1 significantly enhanced cell migration. Striking morphological differences were observed between pCPT-cAMP- and 8-pCPT-2'-O-Me-cAMP-treated aortic smooth muscle cells. Furthermore, overexpression of Epac1 enhanced the development of neointimal formation in fetal rat aortic tissues in organ culture. When the mouse femoral artery was injured mechanically in vivo, we found that the expression of Epac1 was upregulated in vascular smooth muscle cells, whereas that of PKA was downregulated with the progress of neointimal thickening. Our findings suggest that Epac1, in opposition to PKA, increases vascular smooth muscle cell migration. Epac may thus play an important role in advancing vascular remodeling and restenosis upon vascular injury.

We have demonstrated that chronic stimulation of the prostaglandin E 2 -cAMP-dependent protein kinase A (PKA) signal pathway plays a critical role in intimal cushion formation in perinatal ductus arteriosus (DA) through promoting synthesis of hyaluronan. We hypothesized that Epac, a newly identified effector of cAMP, may play a role in intimal cushion formation (ICF) in the DA distinct from that of PKA. In the present study, we found that the levels of Epac1 and Epac2 mRNAs were significantly up-regulated in the rat DA during the perinatal period. A specific EP4 agonist, ONO-AE1-329, Prostaglandin E 2 (PGE 2 )3 is the most potent vasodilatory lipid mediator in the ductus arteriosus (DA), a fetal arterial connection between the pulmonary artery and the descending aorta (1). PGE 2 increases the intracellular concentration of cAMP, which activates cAMP-dependent protein kinase A (PKA), resulting in vasodilation in the DA (1, 2). In addition to its vasodilatory effect, our recent study has identified that chronic PGE 2 stimulation has another essential effect on DA development, namely intimal cushion formation (ICF) (3). Briefly, via EP4, a predominant PGE 2 receptor in the DA, the PGE 2 -cAMP-PKA stimulation up-regulates hyaluronic acid (HA) synthases, which increases HA production. Accumulation of HA then promotes smooth muscle cell (SMC) migration into the subendothelial layer to form intimal thickening. ICF then leads to luminal narrowing, helping adhesive occlusion of the vascular lumen and thus complete anatomical closure of the DA.A new target of cAMP, i.e. an exchange protein activated by cAMP, has recently been discovered; it is called Epac (4).

Retinoic acid (RA), a metabolite of vitamin A, has been proposed to regulate vascular remodeling and reactivity of the ductus arteriosus (DA). Using rat Affymetrix GeneChips, we found that a considerable number of genes in DA varied their expression levels in accordance with developmental mode: namely, preterm-, term-, and postnatal-dominant clusters. Among a total of 8,740 probe sets, maternal vitamin A administration (MVA) changed the expression levels of 91 genes (116 probe sets) >2.5-fold. About half of preterm- and term-dominant genes responded to MVA, whereas only 5% of postnatal-dominant genes responded to MVA, indicating that fetal-dominant genes were susceptible to RA signals. The expression levels of 51 genes in MVA-treated DA at preterm were similar to the expression levels in nontreated DA at term, indicating that the global gene profile at preterm resembled that of the control animal at term. We observed neointima formation in MVA-treated DA at preterm in accordance with upregulation of fibronectin and hyaluronic acid, whereas it was rarely observed in nontreated DA at preterm. Five fetal cardiac myofibrillar genes were also upregulated in MVA-treated in vivo DA, whereas they were developmentally downregulated in nontreated DA. The present study indicates that MVA-mediated alteration in gene profile was associated with early structural maturation of DA, although MVA-mediated maturation may differ from normal vascular remodeling of DA.