**2.2.3 PGE2 – EP4: A critical player in regulating intimal thickening**

PGE2, the most potent vasodilator affecting the DA, is produced in the placenta (Smith 1998) and in the DA itself (Clyman, 1978; Coceani, 1978). During gestation, PGE2 contributes to DA patency *in utero*. Stimulation of PGE2 receptors activates adenylyl cyclases (ACs). The resulting increased intracellular concentrations of cyclic AMP (cAMP) inhibit myosin light chain kinase, inducing DA relaxation (Smith 1998). The dilator effect of PGE2 on the mammalian DA is mediated mainly by the PGE2 receptor, EP4. After birth, the concentration of circulating PGE2 declines dramatically as the placenta is removed and PGE2 is rapidly catabolised through lung circulation. Furthermore, the expression levels of PGE2 receptors are decreased in the DA wall (Smith 1998).

Although PGE2 plays a primary role in maintaining the patency of the DA, previous studies have demonstrated that genetic disruption of EP4 paradoxically results in fatal PDA in mice (Nguyen, 1997; Segi, 1998). We have found that intimal thickening was completely absent in DA from EP4-disrupted neonatal mice (Yokoyama, 2006a). 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-cAMP-protein kinase A (PKA) signalling up-regulates hyaluronan synthase type 2 mRNA, which increases hyaluronan production in the DA. Accumulation of hyaluronan then promotes SMCs migration into the subendothelial layer to induce intimal thickening (Yokoyama, 2006a). Therefore, signalling through PGE2-EP4 plays two essential roles in DA development, namely, vascular dilation and intimal thickening.

#### **2.2.4 Specific adenylyl cyclases regulate intimal thickening of the DA**

Chronic PGE2-EP4-AC-cAMP-PKA signalling during gestation induces vascular remodelling of the DA and thereby promotes hyaluronan-mediated intimal thickening and structural closure of the vascular lumen. Both PGE1 and PGE2 also induce vasodilation in the DA. Since intracellular cAMP is synthesized by ACs, which are transmembrane enzymes activated by G protein-coupled receptors, including PGE receptors, ACs must play an important role in regulating vasodilation and remodelling in the DA. To date, nine different isoforms of membrane-bound forms of ACs (AC1 through AC9) have been identified in vertebrate tissues. Most tissues express several AC isoforms, which exhibit remarkable diversity in their biochemical properties (Sunahara & Taussig 2002). Since SMCs in the DA exhibit biological

Recent Advances Concerning the Molecular Mechanism of Patent Ductus Arteriosus 89

induces vascular endothelial cell growth factor (VEGF), which in turn stimulates neointimal proliferation and vasa vasorum ingrowth (Waleh, 2010; Clyman, 2002). They have also emphasized that hypoxia in the vessel wall plays a role in permanent DA closure. Yet before the blood flow through the DA is completely obstructed, the DA is exposed to blood containing higher oxygen content after birth compared to the blood passing through it during the fetal period. Recently we have found that oxygenation promotes migration of DA SMCs followed by intimal thickening of the rat DA (unpublished data). Accordingly, oxygenation, in addition to inducing contraction, plays an important role in completing DA closure through promoting intimal thickening after PGE2 removal. Further investigation is warranted to elucidate the molecular mechanism by which oxygenation promotes intimal

A growing body of evidence has demonstrated that voltage-dependent calcium channels (VDCCs), in addition to their role in determining the contractile state, play an important role in regulating differentiation, proliferation, migration, and gene expression in vascular SMCs. VDCCs are classified according to their distinct electrophysiological and pharmacological properties into low-voltage-activated (T-type) and high-voltage-activated (L-, N-, P-, Q-, and R-type) VDCCs. VDCCs consist of different combinations of α1 subunits and auxiliary subunits. Among the α1 subunits, α1C and α1G are the most predominant isoforms in the rat DA (Yokoyama, 2006b). In addition to a conventional α1C subunit, a novel alternatively spliced variant of the α1C isoform is highly expressed in the neointimal cushion of the rat DA, although the role of the spliced variant of the α1C isoform in neointimal cushion formation has not yet been investigated (Yokoyama, 2006b). Interestingly, α1G, a T-type VDCC, is significantly up-regulated in oxygenated rat DA tissue and in the region of intimal thickening in the DA (Akaike, 2009). α1G plays a role in migration of DA SMCs and neointimal cushion formation. *R(-)*-efonidipine, a T-type VDCCspecific antagonist, delays the closure of the rat DA through inhibiting the contraction and neointimal formation of the DA (Akaike, 2009). Therefore, isoform-specific inhibition of

VCDD may be an alternative therapeutic strategy to regulate the patency of the DA.

Inflammatory responses to vascular injury or atherosclerosis are known to be associated with the pathogenesis of neointimal thickening. Given that several proinflammatory cytokines are known to play an essential role during vascular remodelling, it is reasonable to assume that cytokines might contribute to physiological vascular remodelling processes as well, in particular, the permanent closure of the DA. Accordingly, it is likely that VEGF stimulates neointimal proliferation and vasa vasorum ingrowth during permanent DA closure, as described above (Waleh, 2010; Clyman, 2002). In addition, transforming growth factor-beta (TGF-β) 1 in endothelial cells and SMCs probably regulates vascular remodelling of the DA, though the function and expression of TGF-β 1 in the DA are controversial

Clyman's group has demonstrated that the expression levels of vascular cell adhesion molecule (VCAM)-1 (an important ligand for the mononuclear cell adhesion receptor VLA4), E-selectin, IL-8, macrophage colony stimulating factor-1, CD154, interferon-gamma, IL-6, and tumor necrosis factor-alpha are increased in the ductus wall. They have also found that VLA4+ monocytes/macrophages (CD68+ and CD14+) and, to a lesser extent, T-

thickening of the DA.

**2.2.7 Calcium channel** 

**2.2.8 Cytokines** 

(Tannenbaum, 1996; Zhou, 1998).

lymphocytes adhere to the postnatal DA (Waleh, 2005).

properties distinct from those of SMCs in other vessels such as the aorta, it is possible to identify specific AC isoforms that play a distinct role in the DA. Recent advances concerning AC isoform-selective activation or inhibition have allowed us to investigate the role of AC isoforms and the availability of AC isoform-selective activators in regulating DA vascular tone and remodelling. AC2 and AC6, for example, are more highly expressed in rat DA than in the aorta during the perinatal period (Yokoyama, 2010). AC6-targeted siRNA counteracts PGEinduced hyaluronan production in rat DA SMCs. Overexpression of AC6 enhances PGEinduced hyaluronan production and induces intimal thickening in DA explants. Furthermore, intimal thickening of the DA is less marked in mice lacking AC6 than in wild-type mice. Interestingly, stimulation of AC2 attenuates AC6-induced hyaluronan production via inhibition of the p38 mitogen-activated protein kinase pathway and AC6-induced intimal thickening of the DA. The AC2/6 activator 6-[N-(2-isothiocyanatoethyl) aminocarbonyl] forskolin (FD1) does not induce hyaluronan-mediated intimal thickening in DA explants, though the AC5/6 activator 6-[3-(dimethylamino)propionyl]-14 15-dihydroforskolin (FD6) does. Therefore AC6 must be responsible for hyaluronan-mediated intimal thickening of the DA, while AC2 inhibits AC6-induced hyaluronan production. It should be noted that the DA is dilated by stimulation with both FD1 and FD6, the effect of which is longer than that of stimulation with PGE**1** (Yokoyama, 2010). These data suggest that the activation of both AC2 and AC6 induces vasodilation, although the effectiveness of AC2 and AC6 activation has not yet been directly investigated. Administration of FD1 induces vasodilation without intimal thickening of the DA, suggesting that combinative stimulation with AC2 and AC6 or AC2 specific stimulation may be a novel alternative therapy to current PGE therapy for patients with DA-dependent congenital heart disease.

#### **2.2.5 Epac (Exchange Protein Activated by cAMP): A novel target of cAMP**

A new target of cAMP, i.e., a new exchange protein activated by cAMP, has recently been discovered and is called Epac. Epac has been known to utilize a distinct cAMP signalling pathway that is independent of PKA (Bos 2006). Epac is a guanine nucleotide exchange protein that regulates the activity of small G proteins. 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 up-regulated during the perinatal period, Epac1, but not Epac2, acutely promotes SMC migration and thus intimal thickening in the DA (Yokoyama, 2008). 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. Epac stimulation improves the organization of actin stress fibers and enhances focal adhesion of DA SMCs. Therefore, the EP4-cAMP signal pathway can induce intimal thickening in the DA via a PKA-dependent mechanism or an Epac-dependent mechanism.

#### **2.2.6 Oxygen and reactive oxygen species**

The DA is an oxygen-sensitive tissue, and oxygen is its most potent vasoconstrictor. The DA senses the change in oxygen tension through the change in redox status. Redox and reactive oxygen species are known to play an important role in vascular remodelling of pathological arteries. Therefore, the change in oxygen tension that occurs after birth should affect postnatal vascular remodelling of the DA. In this regard, Clyman's group has demonstrated that muscular constriction produces a region of ischemic hypoxia in the middle of the ductus muscle media and that intense hypoxia within the constricted vessel wall of the DA induces vascular endothelial cell growth factor (VEGF), which in turn stimulates neointimal proliferation and vasa vasorum ingrowth (Waleh, 2010; Clyman, 2002). They have also emphasized that hypoxia in the vessel wall plays a role in permanent DA closure. Yet before the blood flow through the DA is completely obstructed, the DA is exposed to blood containing higher oxygen content after birth compared to the blood passing through it during the fetal period. Recently we have found that oxygenation promotes migration of DA SMCs followed by intimal thickening of the rat DA (unpublished data). Accordingly, oxygenation, in addition to inducing contraction, plays an important role in completing DA closure through promoting intimal thickening after PGE2 removal. Further investigation is warranted to elucidate the molecular mechanism by which oxygenation promotes intimal thickening of the DA.

#### **2.2.7 Calcium channel**

88 Congenital Heart Disease – Selected Aspects

properties distinct from those of SMCs in other vessels such as the aorta, it is possible to identify specific AC isoforms that play a distinct role in the DA. Recent advances concerning AC isoform-selective activation or inhibition have allowed us to investigate the role of AC isoforms and the availability of AC isoform-selective activators in regulating DA vascular tone and remodelling. AC2 and AC6, for example, are more highly expressed in rat DA than in the aorta during the perinatal period (Yokoyama, 2010). AC6-targeted siRNA counteracts PGEinduced hyaluronan production in rat DA SMCs. Overexpression of AC6 enhances PGEinduced hyaluronan production and induces intimal thickening in DA explants. Furthermore, intimal thickening of the DA is less marked in mice lacking AC6 than in wild-type mice. Interestingly, stimulation of AC2 attenuates AC6-induced hyaluronan production via inhibition of the p38 mitogen-activated protein kinase pathway and AC6-induced intimal thickening of the DA. The AC2/6 activator 6-[N-(2-isothiocyanatoethyl) aminocarbonyl] forskolin (FD1) does not induce hyaluronan-mediated intimal thickening in DA explants, though the AC5/6 activator 6-[3-(dimethylamino)propionyl]-14 15-dihydroforskolin (FD6) does. Therefore AC6 must be responsible for hyaluronan-mediated intimal thickening of the DA, while AC2 inhibits AC6-induced hyaluronan production. It should be noted that the DA is dilated by stimulation with both FD1 and FD6, the effect of which is longer than that of stimulation with PGE**1** (Yokoyama, 2010). These data suggest that the activation of both AC2 and AC6 induces vasodilation, although the effectiveness of AC2 and AC6 activation has not yet been directly investigated. Administration of FD1 induces vasodilation without intimal thickening of the DA, suggesting that combinative stimulation with AC2 and AC6 or AC2 specific stimulation may be a novel alternative therapy to current PGE therapy for patients

with DA-dependent congenital heart disease.

**2.2.5 Epac (Exchange Protein Activated by cAMP): A novel target of cAMP** 

PKA-dependent mechanism or an Epac-dependent mechanism.

**2.2.6 Oxygen and reactive oxygen species** 

A new target of cAMP, i.e., a new exchange protein activated by cAMP, has recently been discovered and is called Epac. Epac has been known to utilize a distinct cAMP signalling pathway that is independent of PKA (Bos 2006). Epac is a guanine nucleotide exchange protein that regulates the activity of small G proteins. 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 up-regulated during the perinatal period, Epac1, but not Epac2, acutely promotes SMC migration and thus intimal thickening in the DA (Yokoyama, 2008). 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. Epac stimulation improves the organization of actin stress fibers and enhances focal adhesion of DA SMCs. Therefore, the EP4-cAMP signal pathway can induce intimal thickening in the DA via a

The DA is an oxygen-sensitive tissue, and oxygen is its most potent vasoconstrictor. The DA senses the change in oxygen tension through the change in redox status. Redox and reactive oxygen species are known to play an important role in vascular remodelling of pathological arteries. Therefore, the change in oxygen tension that occurs after birth should affect postnatal vascular remodelling of the DA. In this regard, Clyman's group has demonstrated that muscular constriction produces a region of ischemic hypoxia in the middle of the ductus muscle media and that intense hypoxia within the constricted vessel wall of the DA A growing body of evidence has demonstrated that voltage-dependent calcium channels (VDCCs), in addition to their role in determining the contractile state, play an important role in regulating differentiation, proliferation, migration, and gene expression in vascular SMCs. VDCCs are classified according to their distinct electrophysiological and pharmacological properties into low-voltage-activated (T-type) and high-voltage-activated (L-, N-, P-, Q-, and R-type) VDCCs. VDCCs consist of different combinations of α1 subunits and auxiliary subunits. Among the α1 subunits, α1C and α1G are the most predominant isoforms in the rat DA (Yokoyama, 2006b). In addition to a conventional α1C subunit, a novel alternatively spliced variant of the α1C isoform is highly expressed in the neointimal cushion of the rat DA, although the role of the spliced variant of the α1C isoform in neointimal cushion formation has not yet been investigated (Yokoyama, 2006b). Interestingly, α1G, a T-type VDCC, is significantly up-regulated in oxygenated rat DA tissue and in the region of intimal thickening in the DA (Akaike, 2009). α1G plays a role in migration of DA SMCs and neointimal cushion formation. *R(-)*-efonidipine, a T-type VDCCspecific antagonist, delays the closure of the rat DA through inhibiting the contraction and neointimal formation of the DA (Akaike, 2009). Therefore, isoform-specific inhibition of VCDD may be an alternative therapeutic strategy to regulate the patency of the DA.

#### **2.2.8 Cytokines**

Inflammatory responses to vascular injury or atherosclerosis are known to be associated with the pathogenesis of neointimal thickening. Given that several proinflammatory cytokines are known to play an essential role during vascular remodelling, it is reasonable to assume that cytokines might contribute to physiological vascular remodelling processes as well, in particular, the permanent closure of the DA. Accordingly, it is likely that VEGF stimulates neointimal proliferation and vasa vasorum ingrowth during permanent DA closure, as described above (Waleh, 2010; Clyman, 2002). In addition, transforming growth factor-beta (TGF-β) 1 in endothelial cells and SMCs probably regulates vascular remodelling of the DA, though the function and expression of TGF-β 1 in the DA are controversial (Tannenbaum, 1996; Zhou, 1998).

Clyman's group has demonstrated that the expression levels of vascular cell adhesion molecule (VCAM)-1 (an important ligand for the mononuclear cell adhesion receptor VLA4), E-selectin, IL-8, macrophage colony stimulating factor-1, CD154, interferon-gamma, IL-6, and tumor necrosis factor-alpha are increased in the ductus wall. They have also found that VLA4+ monocytes/macrophages (CD68+ and CD14+) and, to a lesser extent, Tlymphocytes adhere to the postnatal DA (Waleh, 2005).

Recent Advances Concerning the Molecular Mechanism of Patent Ductus Arteriosus 91

molecular mechanism of PDA. Char syndrome is a genetic syndrome consisting of PDA in association with facial anomalies and minor skeletal anomalies (Chen, 2011; Satoda, 2000; Khetyar, 2008). In addition, Ivey et al. has demonstrated that TFAP2B disruption affects the development of vascular SMCs in the DA (Ivey, 2008), although they did not observe morphological differences between TFAP2B–/– and wild-type mice at embryonic days 13.5, 15.5 and 18.5. It will be intriguing to examine in future studies whether TFAP2B plays a role

The evolutionarily conserved Notch signaling pathway plays a major role in vascular development in mammals and other vertebrates (Kurpinski, 2010; High, 2008). Mice with SMC-specific deletion of Jag1, which encodes a Notch ligand, die postnatally from PDA (Feng, 2010). These mice exhibit defects in contractile SMC differentiation in the medial vascular wall of the DA, which therefore fails to express contractile SMC proteins. However, the differentiation of contractile vascular SMCs is confined to the region adjacent to the endothelial cell layer in the DA. Therefore, propagation of the Jag1-Notch signal throughout the width of the vascular wall is required for contractile SMC differentiation of the DA.

Vascular cells are defined by the ways in which they regulate their extracellular matrix, and changes in the extracellular matrix, in turn, determine vascular cell phenotype, i.e. the ability to differentiate, proliferate, and migrate (Rabinovitch, 1996). As described in section 2.2.3, PGE-induced hyarulonan plays a critical role in the onset of intimal thickening of the DA (De Reeder, 1988; Yokoyama, 2006a). In addition, it has been reported that DA SMCs produce twice as much fibronectin as aortic SMCs do (Rabinovitch, 1996). Mason et al. have demonstrated that preventing fibronectin-dependent intimal thickening would be a feasible manipulation to cause PDA as a mode of treatment of congenital heart diseases (Mason, 1999). TGF β and nitric oxide induce extracellular matrix, including hyaluronan and fibronectin, in DA SMCs (Rabinovitch, 1996). Future studies will be needed to determine the other constituents of extracellular matrix that play a role in vascular remodelling of the DA.

**2.5 Disassembly of the internal elastic lamina and loss of elastic fiber in the medial** 

The disassembly and fragmentation of the internal elastic lamina and sparse elastic fibers in the middle layer of the DA is a hallmark of vascular remodelling in the DA. In the normally closing DA, impaired elastogenesis coincides with increased SMC migration and proliferation, contributing to physiological occlusion. The reduced elasticity of the DA wall may help its structure collapse easily as a prelude to postnatal permanent closure of the DA. In PDA, in contrast, an abundance of elastin lamellae in the intima, a subendothelial elastic lamina, and a failure of intimal SMC migration are found in humans and animal models (de Reeder, 1990; Hinek, 1991; Slomp, 1992). In humans and in animal models such as canine puppies and the inbred Brown-Norway (BN) rat (Bokenkamp, 2006), the subendothelial elastic lamina is thought to limit the passage of SMCs from the media to the intima. In PDA in BN rats, the media of elastin lamellae are absent, and the intima contains many elastic fibers. The abnormal distribution of elastin in the PDA of BN rats suggests that impaired elastin metabolism is related to the persistence of the DA and implicates a genetic factor that may link the PDA with aortic fragility. In this regard, recent studies have identified a new

**2.4 Extracellular matrix involved in vascular remodelling of the DA** 

in neointimal formation of the DA.

**2.3.3 Jag1** 

**layer of the DA** 

The expression of IL-15 mRNA is significantly higher in rat DA than in the aorta. IL-15 immunoreactivity is detected predominantly in the internal elastic laminae, and to a lesser extent in SMCs, in the rat DA. IL-15 attenuates PDGF-BB-mediated SMC proliferation and PGE1-induced hyaluronan production in a dose-dependent manner. Accordingly, IL-15 might have an inhibitory effect on the physiological vascular remodelling processes involved in closing the DA (Iwasaki, 2007).

Moreover, growth hormones promote the migration of DA SMCs, thus enhancing intimal cushion formation in the DA. Growth hormones also regulate the expression of cytoskeletal, genes in DA SMCs, which may retain a synthetic phenotype in the smooth muscle-specific cytoskeletal genes (Jin, 2011).

#### **2.3 Vascular smooth muscle differentiation**

SMCs retain the ability to reversibly alter their phenotype in response to various environmental and physiological changes (McDonald and Owens 2007). This property has been termed phenotypic switching or SMC plasticity. Contractile SMCs express high levels of contractile proteins involved in establishing and maintaining myofilament structure and function, including SM22α, SMA and SMMHC. In contrast, synthetic SMCs express lower levels of contractile muscle proteins and have higher rates of proliferation, migration and production of extracellular matrix components. The DA is a very specialized blood vessel, with a vascular wall composed of highly differentiated and contractile smooth muscle (Slomp, 1997). A unique transcriptional program is probably responsible for generating this particular artery during fetal development (Ivey, 2008). A growing body of evidence has demonstrated that maturation and differentiation of DA SMCs is essential for postnatal DA closure. Deletion of several factors that are required for DA SMCs to adopt a contractile phenotype results in PDA. It should be noted, however, that neointimal thickening is induced by migrating and proliferating vascular SMCs, which are characterized as a dedifferentiated (synthetic) phenotype. Therefore, the DA must consist of SMCs exhibiting phenotypic heterogeneity.

#### **2.3.1 Myocardin**

Myocardin is a remarkably potent transcriptional coactivator that regulates SMC contractile proteins. Myocardin-null mouse embryos exhibit a block in vascular SMC differentiation as well as defects in the yolk sac vasculature (Li, 2003). Importantly, mice generated after selective myocardin ablation of cardiac neural crest-derived vascular SMCs exhibit PDA and die at postnatal day 3 (Huang, 2008). In these mutant mice, the *myocardin-*deficient vascular SMCs populating the DA exhibit ultrastructural features generally associated with the synthetic, rather than the contractile, SMC phenotype. In addition, the architecture of the neointima and tunica media of the DA is markedly disturbed in association with a dramatic increase in extracellular matrix and a relative decrease in vascular SMC volume. These data demonstrate that myocardin regulates the expression of genes required to induce the contractile phenotype in neural crest–derived SMCs and provide new insights into the molecular and genetic systems that underlie the vascular remodeling of the DA.

#### **2.3.2 TFAP2B**

Identifying gene mutations in TFAP2B, a neural crest cell-specific transcription factor involved in Char syndrome, is one of the most important discoveries regarding the molecular mechanism of PDA. Char syndrome is a genetic syndrome consisting of PDA in association with facial anomalies and minor skeletal anomalies (Chen, 2011; Satoda, 2000; Khetyar, 2008). In addition, Ivey et al. has demonstrated that TFAP2B disruption affects the development of vascular SMCs in the DA (Ivey, 2008), although they did not observe morphological differences between TFAP2B–/– and wild-type mice at embryonic days 13.5, 15.5 and 18.5. It will be intriguing to examine in future studies whether TFAP2B plays a role in neointimal formation of the DA.
