1 Introduction

The ductus arteriosus (DA) normally closes immediately after birth. Although the DA is a normal and essential fetal structure, it becomes abnormal if it remains patent after birth. DA closure occurs in two phases: functional closure of the lumen in the first hours after birth by smooth muscle constriction and anatomic occlusion of the lumen over the next several days due to extensive neointimal thickening in human DA [1–3]. There are several events that promote DA constriction immediately after birth. Increasing oxygen tension and a dramatic decrease in circulating PGE2 promote muscular constriction of the DA. In addition, DA remodeling is also necessary for its complete closure. Remodeling is characterized by (a) an area of subendothelial deposition of extracellular matrix [4], (b) the disassembly of the internal elastic lamina and loss of elastic fiber in the medial layer [5], and (c) migration into the subendothelial space of undifferentiated medial smooth muscle cells (SMCs). Some of these changes begin about halfway through gestation, and some occur after functional closure of the DA in the neonate [3, 6]. In addition to the well-known vasodilatory role of PGE2, our findings revealed the role of PGE2 in the anatomical closure of the DA.

2 The Molecular Mechanisms of Intimal Thickening of the Ductus Arteriosus

2.1 Hyaluronan-Mediated Intimal Thickening

PGE2 plays a primary role in maintaining the patency of the DA via its receptor EP4. However, previous studies have demonstrated that genetic disruption of the PGE receptor EP4 paradoxically results in fatal patent DA in mice [7, 8]. In addition, double mutant mice in which cyclooxygenase (COX)-1 and COX-2 are disrupted also exhibit patent DA [9]. We found that intimal thickening was completely absent in the DA of EP4-disrupted neonatal mice [3]. Moreover, a marked reduction in hyaluronan production was found in EP4-disrupted DA, whereas a thick layer of hyaluronan deposit was present in wild-type DA. PGE2-EP4-cyclic AMP (cAMP)-protein kinase A (PKA) signaling upregulates hyaluronan synthase type 2 mRNA, which increases hyaluronan production in the DA. Accumulation of hyaluronan then promotes SMC migration into the subendothelial layer to form intimal thickening [3].

EP4 is a Gs protein-coupled receptor that increases intracellular cAMP by adenylyl cyclases (ACs) consisting of nine different isoforms of membrane-bound forms of ACs (AC1 through AC9). We found that AC2 and AC6 are more highly expressed in rat DA than in the aorta during the perinatal period [10]. Our data using AC subtype-targeted siRNAs and AC6-deficient mice suggest that AC6 is responsible for hyaluronan-mediated intimal thickening of the DA, whereas AC2 inhibits AC6-induced hyaluronan production. The activation of both AC2 and AC6 induces vasodilation.

2.2 Epac-Mediated SMC Migration

In addition to PKA, a new target of cAMP that is an exchange protein activated by cAMP has recently been discovered; it is called Epac [11]. Epac is a guanine nucleotide exchange protein that regulates the activity of small G proteins and has been known to exhibit a distinct cAMP signaling pathway that is independent of PKA [12]. There are two variants: Epac1 is expressed in most tissues, including the heart and blood vessels, whereas Epac2 is expressed in the adrenal gland and the brain. Although both Epac1 and Epac2 are upregulated during the perinatal period, Epac1, but not Epac2, acutely promotes SMC migration and thus intimal thickening in the DA [13]. Since Epac stimulation does not increase hyaluronan production, the effect of Epac1 on SMC migration is independent of that of hyaluronan accumulation, which operates through a mechanism different from that underlying PKA stimulation.

2.3 Regulation of Elastogenesis

Elastic fiber formation begins in mid-gestation and increases dramatically during the last trimester in the great arteries. However, the DA exhibits lower levels of elastic fiber formation [5], which may contribute to vascular collapse and subsequent closure of the DA after birth. We found that EP4 significantly inhibited elastogenesis and decreased lysyl oxidase (LOX) protein, which catalyzes elastin cross-links in DA SMCs but not in aortic SMCs. In EP4-knockout mice, electron microscopic examination showed that the DA acquired an elastic phenotype that was similar to the neighboring aorta. More importantly, human DA and aorta tissues from seven patients showed a negative correlation between elastic fiber formation and EP4 expression, as well as between EP4 and LOX expression. Together with in vitro experiments, these data suggest that PGE2-EP4 signaling inhibits elastogenesis in the DA by degrading LOX protein. The EP4-cSrc-PLCγ-signaling pathway, a signaling pathway that has not previously been recognized, most likely promoted the lysosomal degradation of LOX [14, 15].

3 Future Direction and Clinical Implications

The persistently patent DA after birth is a major cause of morbidity and mortality, especially in premature infants, that can lead to severe complications, including pulmonary hypertension, right ventricular dysfunction, postnatal infections, and respiratory failure [16]. The incidence of DA patency has been estimated to be 1 in 500 in term newborns [17]. In preterm babies with birth weights <1,500 g, the incidence of patent DA exceeds 30 % [18]. Therefore, it is important to improve current pharmacological therapy through understanding the precise mechanisms of the regulation of the DA. Since both vascular contraction and remodeling are required for complete DA closure, pharmacological therapies that promote vasoconstriction and remodeling would be ideal for premature infants with persistently patent DAs. On the other hand, vasodilation and inhibition of intimal thickening are required for DA-dependent congenital heart diseases.

Our data suggest that PGE2-EP4-cAMP signaling promotes hyaluronan and Epac-mediated intimal thickening and that the EP4-PLC pathway attenuates elastogenesis in the DA. These cascades of events via different signaling pathways are thought to orchestrate the subsequent luminal DA reorganization (Fig. 35.1), leading to complete obliteration of the DA. In addition to its role in controlling vascular tone in the functional closure of the DA, the vascular remodeling of the DA is now attracting considerable attention as a target for novel therapeutic strategies for patients with persistently patent DA and DA-dependent cardiac anomalies.

Fig. 35.1
figure 1

The diverse EP4 signaling pathways. Both PKA and Epac synergistically promoted intimal cushion formation in the DA, but they work in two distinct ways. The cSrc-PLC pathway inhibited elastogenesis via degrading LOX proteins

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