Shiho Iwasaki

Chronic activation of the prostaglandin receptor EP4 promotes hyaluronan-mediated neointimal formation in the ductus arteriosus

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.

Prostaglandin E2-activated Epac Promotes Neointimal Formation of the Rat Ductus Arteriosus by a Process Distinct from That of cAMP-dependent Protein Kinase A

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 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, increased Rap1 activity in the presence of a PKA inhibitor, PKI-(14-22)-amide, in DA smooth muscle cells. 8-pCPT-2′-O-Me-cAMP (O-Me-cAMP), a cAMP analog selective to Epac activator, promoted migration of DA smooth muscle cells (SMC) in a dose-dependent manner. Adenovirus-mediated Epac1 or Epac2 gene transfer further enhanced O-Me-cAMP-induced cell migration, although the effect of Epac1 overexpression on cell migration was stronger than that of Epac2. In addition, transfection of small interfering RNAs for Epac1, but not Epac2, significantly inhibited serum-mediated migration of DA SMCs. In the presence of O-Me-cAMP, actin stress fibers were well organized with enhanced focal adhesion, and cell shape was widely expanded. Adenovirus-mediated Epac1, but not Epac2 gene transfer, induced prominent ICF in the rat DA explants when compared with those with green fluorescent protein gene transfer. The thickness of intimal cushion became significantly greater (1.98-fold) in Epac1-overexpressed DA. O-Me-cAMP did not change hyaluronan production, although it decreased proliferation of DA SMCs. The present study demonstrated that Epac, especially Epac1, plays an important role in promoting SMC migration and thereby ICF in the rat DA.

T-type Ca2+ channels promote oxygenation-induced closure of the rat ductus arteriosus not only by vasoconstriction but also by neointima formation

The ductus arteriosus (DA), an essential vascular shunt for fetal circulation, begins to close immediately after birth. Although Ca2+ influx through several membrane Ca2+ channels is known to regulate vasoconstriction of the DA, the role of the T-type voltage-dependent Ca2+ channel (VDCC) in DA closure remains unclear. Here we found that the expression of α1G, a T-type isoform that is known to exhibit a tissue-restricted expression pattern in the rat neonatal DA, was significantly up-regulated in oxygenated rat DA tissues and smooth muscle cells (SMCs). Immunohistological analysis revealed that α1G was localized predominantly in the central core of neonatal DA at birth. DA SMC migration was significantly increased by α1G overexpression. Moreover, it was decreased by adding α1G-specific small interfering RNAs or using R(−)-efonidipine, a highly selective T-type VDCC blocker. Furthermore, an oxygenation-mediated increase in an intracellular Ca2+ concentration of DA SMCs was significantly decreased by adding α1G-specific siRNAs or using R(−)-efonidipine. Although a prostaglandin E receptor EP4 agonist potently promoted intimal thickening of the DA explants, R(−)-efonidipine (10−6 m) significantly inhibited EP4-promoted intimal thickening by 40% using DA tissues at preterm in organ culture. Moreover, R(−)-efonidipine (10−6 m) significantly attenuated oxygenation-induced vasoconstriction by ∼27% using a vascular ring of fetal DA at term. Finally, R(−)-efonidipine significantly delayed the closure of in vivo DA in neonatal rats. These results indicate that T-type VDCC, especially α1G, which is predominantly expressed in neonatal DA, plays a unique role in DA closure, implying that T-type VDCC is an alternative therapeutic target to regulate the patency of DA.

Differential Regulation of Vascular Tone and Remodeling via Stimulation of Type 2 and Type 6 Adenylyl Cyclases in the Ductus Arteriosus

Rationale: Prostaglandin (PG)E2, which increases intracellular cAMP via activation of adenylyl cyclases (ACs), induces vasodilation and hyaluronan-mediated intimal thickening (IT) in the ductus arteriosus (DA) during late gestation. After birth, however, differential regulation of vasodilation and IT is preferable for treatment of patients with patent DA and DA-dependent congenital cardiac malformations. Objective: Our objectives were to examine whether AC isoforms play differential roles in DA vasodilation and IT. Methods and Results: AC2 and AC6 were more highly expressed in rat DA than in the aorta during the perinatal period. AC6-targeted siRNA counteracted PGE1-induced hyaluronan production in rat DA smooth muscle cells. Overexpression of AC6 enhanced PGE1-induced hyaluronan production and induced IT in DA explants. Furthermore, IT of the DA was less marked in mice lacking AC6 than in wild-type and AC5-deficient mice. Stimulation of AC2 attenuated AC6-induced hyaluronan production via inhibition of the p38 mitogen-activated protein kinase pathway and AC6-induced IT of the DA. An AC2/6 activator, 6-[N-(2-isothiocyanatoethyl) aminocarbonyl] forskolin (FD1), did not induce hyaluronan-mediated IT in DA explants, although an AC5/6 activator, 6-[3-(dimethylamino)propionyl]-14,15-dihydroforskolin (FD6) did. Moreover, FD1 induced longer vasodilation of the DA than did PGE1 without significant adverse effects in vivo. Conclusions: AC6 is responsible for hyaluronan-mediated IT of the DA and AC2 inhibited AC6-induced hyaluronan production. Stimulation of both AC2 and AC6 by FD1 induced longer vasodilation without hyaluronan-mediated IT in the DA in vivo. FD1 may be a novel alternative therapy to currently available PGE therapy for patients with DA-dependent congenital heart disease.

Urinary β2-microglobulin in premature infants with chorioamnionitis and chronic lung disease

Abstract The urinary β 2 -microglobulin (MG) concentration on day 0 to 2 was significantly higher in premature infants with chorioamnionitis (CAM) than in infants without CAM and in infants who developed chronic lung disease (CLD) than in those who did not. We propose that an elevated urinary β 2 -MG can indicate a fetal inflammatory response and identify neonates at risk for the development of CLD.

Interleukin-15 inhibits smooth muscle cell proliferation and hyaluronan production in rat ductus arteriosus.

Neointimal cushion formation (NCF) is an important vascular remodeling for anatomical closure of the ductus arteriosus (DA). Inflammatory responses to vascular injury or atherosclerosis are known to be associated with the pathogenesis of NCF. We found that the expression of interleukin (IL)-15 mRNA was significantly higher in rat DA than in the aorta. IL-15 immunoreactivity was detected predominantly in the internal elastic laminae (IEL) and to a lesser extent in smooth muscle cells (SMCs) in rat DA. Prostaglandin E (PGE) increased the expression of IL-15 mRNA in cultured DA SMCs. IL-15 significantly attenuated the platelet-derived growth factor (PDGF)-BB–mediated SMC proliferation, but did not change SMC migration. IL-15 significantly attenuated PGE1-induced hyaluronic acid (HA) production in a dose-dependent manner, which is a potent stimulator of NCF. Accordingly, IL-15 might have an inhibitory effect on the physiologic vascular remodeling processes in closing the DA.

Early discharge from a neonatal intensive care unit and rates of readmission

Background:  Increasing admissions to neonatal intensive care units (NICUs) demand early discharge from the units. Our hospital aims to early discharge patients who meet the following requirements: they are able to regulate body temperature; neither apnea nor bradycardia is observed; and bodyweight increases with lactation. We studied the real state of this strategy.

Blood Flow Velocities in the Anterior Cerebral Artery and Basilar Artery in Asphyxiated Infants

Objective. The aim of this series was to determine whether cerebral blood flow velocities (CBFVs) in the anterior cerebral artery (ACA) and basilar artery (BA) correlate with the severity of asphyxia in infants and whether these velocity measures can be useful for predicting early developmental prognosis. Methods. We measured CBFVs in the ACA and BA by using pulsed Doppler sonography in 29 healthy and 17 asphyxiated infants (11 with mild asphyxia [median gestational age, 38 weeks; median birth weight, 2856 g] and 6 with severe asphyxia [38.5 weeks; 2910 g]). Results. In the mildly asphyxiated infants, the median diastolic and systolic velocities in the ACA were 10.4 and 32.5 cm/s, respectively, and those in the BA were 10.5 and 33.1 cm/sec. In the severely asphyxiated infants, the median diastolic and systolic velocities in the ACA were 19.8 and 40.5 cm/s, and those in the BA were 30.2 and 60.5 cm/s. The BA:ACA ratios (CBFV in the BA/CBFV in the ACA) in both the diastolic and systolic periods were higher in the severely asphyxiated infants than in the mildly asphyxiated infants (0.94 versus 1.35; P < .01; 1.01 versus 1.38; P < .01). Conclusions. These preliminary results suggest that CBFVs in the BA correlate with the degree of neonatal asphyxia and may be useful to predict the neurodevelopmental outcome. We submit that the increased CBFV in the BA may represent the preferential blood flow to the brain stem region.

Decreased serum osmolality promotes ductus arteriosus constriction

AIMS At birth, dynamic changes occur in serum components and haemodynamics, such as closure of the ductus arteriosus (DA). A previous study demonstrated that, in full-term human neonates, serum osmolality decreased transiently after birth, but recovered over the next few days. However, the significance of this transient decrease in osmolality has never been addressed. The objective of the present study was to examine the role of changes in serum osmolality after birth in DA closure. METHODS AND RESULTS We found that rats exhibited a similar transient hypoosmolality after birth. Hypotonic stimulation induced constriction of DA rings and increased Ca(2+) transient in DA smooth muscle cells, but not in the aorta. The hypoosmotic sensor transient receptor potential melastatin 3 (TRPM3) was highly expressed in the rat DA, and TRPM3 silencing abolished the Ca(2+) response to hypoosmolality. Pregnenolone sulfate stimulation of TRPM3 induced rat DA constriction ex vivo and in vivo. Furthermore, hypertonic fluid injection impaired rat DA closure. In humans, neonatal serum hypoosmolality was observed in relatively mature preterm infants (≥28 weeks). In extremely preterm infants (<28 weeks), however, this hypoosmolality was absent. Instead, a rapid increase in osmolality occurred thereafter. Such an increase was greater, in particular, among patent DA (PDA) patients. CONCLUSIONS A transient decrease in serum osmolality may promote DA closure during the first few days of life. Adjusting serum osmolality to proper levels might help to prevent the onset of PDA, improving the therapeutic outcome in extremely preterm infants.

Soluble tumor necrosis factor receptor‐I in preterm infants with chorioamnionitis

Aim:  The aim of our study was (i) to determine whether chorioamnionitis (CAM) is associated with an elevated soluble tumor necrosis factor receptor I (sTNFR‐I) level and (ii) to examine the time course of the concentration of sTNFR‐I in preterm infants after birth.