Polymorphisms in CYP2C9 are associated with response to indomethacin among neonates with patent ductus arteriosus.

BackgroundPatent ductus arteriosus (PDA) is a common complication seen in preterm infants. Indomethacin is routinely used to treat PDA. Evidence suggests that the response of indomethacin is highly heritable. This study investigated the association between single-nucleotide polymorphisms (SNPs) in CYP2C9 and the closure of PDA in response to indomethacin.MethodsSix SNPs in CYP2C9 were analyzed for association with indomethacin response. A case-control analysis was performed among neonates who responded to indomethacin (responders) and among those who required surgical ligation (non-responders). Independent transmission disequilibrium tests were performed among parent-child trios of responders and non-responders.ResultsThe G allele of rs2153628 was associated with increased odds of response to indomethacin in the case-control analysis (odds ratios (OR): 1.918, 95% confidence interval (CI): 1.056, 3.483). Among indomethacin responders, the G allele of rs2153628 and the T allele of rs1799853 were overtransmitted from the parents to their child (OR: 2.667, 95% CI: 1.374, 5.177 and OR: 2.375, 95% CI: 1.040, 5.425, respectively), consistent with the case-control analysis.ConclusionWe identified an association between two SNPs in CYP2C9, rs2153628 and rs1799853, and indomethacin response for the treatment of PDA. These findings suggest that response to indomethacin in the closure of PDA may be influenced by polymorphisms associated with altered indomethacin metabolism.

Correlation between methyltetrahydrofolate reductase (MTHFR) polymorphisms and isolated patent ductus arteriosus in Taiwan.

BACKGROUND: Methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C gene polymorphisms are associated with the risk of patent ductus arteriosus (PDA) congenital heart defects. This study aimed to determine the association of these polymorphisms in patients with isolated PDA and in non-PDA patients group without congenital heart disease. METHODS: This retrospective case-controlled study was undertaken in 17 patients with isolated PDA and a control non-PDA group consisting of 34 subjects without congenital heart disease. MTHFR gene polymorphisms were analysed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). In addition, the genotype distribution of the MTHFR gene was compared among different ethnicities using the HapMap database. RESULTS: In contrast to the MTHFR C677T polymorphism, differences in the MTHFR A1298C genotype were observed between the two groups (P=0.002); a greater proportion of the PDA patients had the MTHFR 1298CC and 1298AA genotypes as compared to the non-PDA control group. After merging the data obtained from the Taiwanese participants with that from the HapMap database, genetic diversity of the MTHFR 1298AA genotype was observed. CONCLUSIONS: Thus, the MTHFR A1298C polymorphism is associated with isolated PDA in Taiwan. Larger studies are necessary to evaluate the prognostic value of determining MTHFR polymorphism in PDA.

Inhibition of phosphodiesterase type 3 dilates the rat ductus arteriosus without inducing intimal thickening.

BACKGROUND: Prostaglandin E(1) (PGE(1)), via cAMP, dilates the ductus arteriosus (DA). For patients with DA-dependent congenital heart disease (CHD), PGE(1) is the sole DA dilator that is used until surgery, but PGE(1) has a short duration of action, and frequently induces apnea. Most importantly, PGE(1) increases hyaluronan (HA) production, leading to intimal thickening (IT) and eventually DA stenosis after long-term use. The purpose of this study was therefore to investigate potential DA dilators, such as phosphodiesterase 3 (PDE3) inhibitors, as alternatives to PGE(1). METHODS AND RESULTS: Expression of PDE3a and PDE3b mRNAs in rat DA tissue was higher than in the pulmonary artery. I.p. milrinone (10 or 1mg/kg) or olprinone (5 or 0.5mg/kg) induced maximal dilatation of the DA lasting for up to 2h in rat neonates. In contrast, vasodilation induced by PGE(1) (10µg/kg) was diminished within 2h. No respiratory distress was observed with milrinone or olprinone. Most important, milrinone did not induce HA production, cell migration, or proliferation when applied to cultured rat DA smooth muscle cells. Further, high expression of both PDE3a and PDE3b was demonstrated in the human DA tissue of CHD patients. CONCLUSIONS: Because PDE3 inhibitors induced longer-lasting vasodilation without causing apnea or HA-mediated IT, they may be good alternatives to PGE(1) for patients with DA-dependent CHD.

Role of prostaglandin E and its receptors in the process of ductus arteriosus maturation and functional closure in the rabbit.

1. Patent ductus arteriosus (PDA) is a common congenital heart defect in premature infants. The present study was designed to determine the role of the prostaglandin (PG) E(2) pathway in the process of ductus arteriosus (DA) maturation and functional closure. 2. Changes in PGE(2) pathway-related enzymes and receptors in DA in preterm and term rabbits were examined at both the mRNA and protein levels. In addition, responses of DA rings to Po(2) and PGE(2) were determined. 3. Circulating PGE(2) levels remained high until 2 h after birth. High levels of the EP(4) receptor were found in preterm DA. These tissues were sensitive to PGE(2), which caused vessel dilation, but were insensitive to increased Po(2). In contrast, DA tissues from term rabbits exhibited an immediate contractile response to increased Po(2) and PGE(2) treatment resulted in vasoconstriction, which was associated with increased EP(3) and decreased EP(4) receptor expression in term DA. 4. In conclusion, the preterm PDA is maintained by high levels of PGE(2), which mainly binds to the EP(4) receptor under conditions of hypoxia. In contrast, in the term DA, in which levels of the EP(3) receptor are higher than in preterm DA, exposure to PGE(2) resulted in vasoconstriction under normoxic conditions. These findings suggest that blocking the EP(4) receptor may represent a more selective treatment for the preterm PDA, whereas activating the EP(3) receptor may be more suitable for the treatment of the term PDA.

Role of microsomal prostaglandin E synthase-1 (mPGES1)-derived PGE2 in patency of the ductus arteriosus in the mouse.

Prostaglandin E2 (PGE2) plays a key role in the ductus arteriosus, prenatally by maintaining patency and postnatally by promoting tissue remodeling for closure. Here, by using near-term mouse fetuses with (wild-type, WT) and without microsomal PGE synthase-1 (mPGES1-/-), we have examined the importance of this enzyme for PGE2 formation and function. mPGES1-/- ductus, unlike WT ductus, contracted little, or not all, to indomethacin in vitro. Coincidentally, as evident from responses to NG-nitro-L-arginine methyl ester and zinc photoporphyrin, the mutant showed no significant enhancement of nitric oxide (NO)- and carbon monoxide (CO)-based relaxation. mPGES1 suppression differs, therefore, from cyclooxygenase (COX) suppression, whether genetically or pharmacologically induced, where NO is markedly up-regulated. In vivo, the ductus was patent, albeit occasionally with a narrowed lumen, in all mPGES1-/- fetuses. Conversely, postnatal closure progressed regularly in mPGES1-/- animals thanks to residual PGE2 originating via mPGES2. We conclude that mPGES1 is critical for PGE2 formation in the ductus but its loss does not entail compensatory up-regulation of other relaxing mechanisms. Accordingly, an mPGES1 inhibitor stands out as a prospective better tool, compared with the currently used COX inhibitors, for the management of premature infants with persistent ductus.

Cyclooxygenase-1-selective inhibition prolongs gestation in mice without adverse effects on the ductus arteriosus.

Preterm delivery is the leading cause of neonatal mortality and contributes significantly to infant morbidity. Classical cyclooxygenase (COX) inhibitors, such as indomethacin, which inhibit both COX-1 and COX-2, are effective for delaying premature labor, but their use is limited by serious complications to the fetus and neonate, including adverse effects on the ductus arteriosus (DA). Using isoform-selective inhibitors, we characterized the roles of the COX isoforms in the initiation of labor and the regulation of fetal and neonatal DA closure in mice. Chronic inhibition of COX-2 during pregnancy (gestation days 15-18) significantly increased neonatal mortality by preventing closure of the DA after birth, whereas acute COX-2 inhibition near the end of term (gestation day 18) constricted the fetal DA. In contrast, the inhibition of COX-1 during pregnancy lacked these prenatal and postnatal adverse effects on the DA and effectively delayed the initiation of full-term labor and LPS-induced preterm labor. These findings suggest that premature fetal DA closure or neonatal patent DA observed following indomethacin tocolysis in women may result from the inhibition of COX-2. Therefore, COX-1-selective inhibitors may provide effective treatment to delay preterm labor with fewer adverse effects on fetal or neonatal health than nonselective or COX-2-selective inhibitors.

Constriction of the ductus arteriosus by selective inhibition of cyclooxygenase-1 and -2 in near-term and preterm fetal rats.

We studied the transplacental ductal constrictive effects of a selective cyclooxygenase (COX)-1 inhibitor (SC560), six selective COX-2 inhibitors including rofecoxib, and a non-selective COX inhibitor (indomethacin). Each drug was administered to the pregnant rats, and fetal ductus arteriosus (DA) was studied with a whole-body freezing method. The inner diameter ratio of the DA to the main pulmonary artery (DA/PA) was 1.02+/-0.03 (mean+/-S.E.M.) in controls. Every drug constricted the DA dose-dependently. In preterm rats on the 19th day of gestation, 10mg/kg of SC560, rofecoxib and indomethacin caused ductal constriction, with DA/PA reduced to 0.76+/-0.02, 0.80+/-0.03 and 0.75+/-0.02, respectively. In near-term on the 21st day, 10mg/kg of them caused ductal constriction, with DA/PA to 0.74+/-0.04, 0.26+/-0.02 and 0.33+/-0.05. In conclusion, both COX-1 and COX-2 selective inhibitors constrict fetal DA. They are not better alternatives for the fetus than non-selective COX inhibitors for tocolysis.

In utero remodeling of the fetal lamb ductus arteriosus: the role of antenatal indomethacin and avascular zone thickness on vasa vasorum proliferation, neointima formation, and cell death.

BACKGROUND: The ductus arteriosus (DA) of newborn infants exposed in utero to indomethacin is resistant to postnatal indomethacin; we hypothesized that this is due to ductus constriction in utero, with subsequent remodeling of the vessel. METHODS AND RESULTS: Infusion of fetal lambs with indomethacin for 48 hours constricted the DA and increased the thickness of the avascular zone of the DA, which in turn induced the expression of vascular endothelial growth factor, endothelial nitric oxide synthase (due to ingrowth of vasa vasorum), neointima formation, and loss of smooth muscle cells; moderate degrees of DA constriction in utero increased NO production, which inhibited DA contractility. Marked degrees of DA constriction decreased tissue distensibility and contractile capacity. CONCLUSIONS: DA patency is no longer controlled primarily by prostaglandins once it has been exposed to indomethacin in utero.

Nitric oxide synthase expression in lungs of pulmonary hypertensive patients with heart disease.

Since little is known about the contribution of endothelial nitric oxide synthase (e-NOS) to the mechanism of pulmonary vasospasm and the development of pulmonary vascular occlusive disease, we elucidate how e-NOS is expressed in lung biopsy specimens obtained from operative patients with pulmonary hypertension. Lung biopsy specimens were obtained from 17 patients who underwent open-heart operations for various heart diseases. A piece of normal lung specimen was also obtained from the resected lungs of three lung cancer patients as a control. e-NOS expression was visualized with a monoclonal antibody against e-NOS, and the level of expression was partially quantified. Significantly high levels of e-NOS expression were seen in adult patients, whose preoperative mean pulmonary arterial pressures were greater than 20 mm Hg. In contrast, e-NOS expression in pediatric patients with the same levels of mean pulmonary arterial pressure was the same as that in the controls and in low pulmonary arterial pressure. There was a statistically significant positive correlation between the level of e-NOS expression and Heath--Edwards grading. These data suggest that the e-NOS expression in lung tissue is induced when pulmonary vascular obstructive diseases progress.

Attenuated cyclooxygenase-2 expression contributes to patent ductus arteriosus in preterm mice.

Patent ductus arteriosus (DA) is the second most common congenital heart defect, the incidence of which is increased in premature infants, although mechanisms responsible are not clear. Our previous studies with genetic or pharmacological inactivation of cyclooxygenase-2 (COX-2) in mice, emphasized the importance of this enzyme in normal DA closure. The current study was designed to determine whether reduced COX-2 expression contributes to patent DA in preterm mice. Real-time PCR analysis indicated that COX-2 expression in the fetal mouse DA significantly increased with advancing gestational age. Furthermore, we observed a significant induction in COX-2 expression in the DA at 3 h after birth at full-term gestation. In contrast, COX-2 expression was significantly attenuated in the DA of preterm neonatal mice. DA closure was incomplete in preterm mice at 3 h postpartum, a time-point when the DA of full-term neonates was completely remodeled. Additionally, COX-2 expression was significantly attenuated in the DA of mice deficient in the prostanoid receptor EP4, which also show a patent DA phenotype, suggesting the importance of this receptor for the induction of COX-2 required for DA closure. Overall, these studies suggest that attenuated expression of COX-2 may contribute to increased patent DA at preterm gestation.

Studies of pulmonary prostaglandin biosynthetic and catabolic enzymes as factors in ductus arteriosus patency and closure. Evidence for a shift in products with gestational age.

The mechanisms underlying patency of the ductus arteriosus in utero and closure shortly after birth should permit optimal regulation of this important physiologic process. Although many studies indicate that vasodilatory prostaglandins contribute to patency, the contribution of the prostaglandin or prostanoid pathways to the closure mechanism has been less evident. The present studies tested a hypothesis which relates products of the cyclooxygenase pathway to modulation of ductal caliber. We began by defining the prostaglandin biosynthetic and catabolic activities of isolated fetal and neonatal lung as a function of gestational age. Inasmuch as biosynthetic enzymes compete for the same substrate, namely PGH2, the competitiveness of the enzymes was explored by varying endoperoxide concentration. The results indicate that the types and quantities of products formed by lung tissue are dependent markedly on both gestational age and substrate concentration. Cyclooxygenase activity was relatively constant through term, but increased sharply in neonatal lung tissue Although PHG2-PGE2 isomerase activity was high throughout gestation, prostacyclin synthase activity was only moderate and age-invariant after 130 d gestation. Lung thromboxane synthase activity increased markedly at 144 d gestation and in the neonatal lung. Catabolic activity (represented by 15-hydroxyprostaglandin dehydrogenase) was relatively high up to 130 d, became sharply minimal at term but increased in activity rapidly in the neonatal lung. These findings support the concept of a "balance" between dilatory and constrictor products of the cyclooxygenase pathway as one determinant of both patency of the ductus arteriosus and closure at birth or after administration of prostaglandin synthetase inhibitors.

Coordinated regulation of fetal and maternal prostaglandins directs successful birth and postnatal adaptation in the mouse.

Cyclooxygenase (COX)-derived prostaglandins (PGs) regulate numerous maternal-fetal interactions during pregnancy. PGs stimulate uterine contractions and prepare the cervix for parturition, whereas in the fetus, PGs maintain patency of the ductus arteriosus (DA), a vascular shunt that transmits oxygenated placental blood to the fetal systemic circulation. However, the origin and site of action of these PGs remain undefined. To address this, we analyzed mice lacking COX-1 (null mutation) or COX-2 (pharmacologic inhibition) or pups with a double null mutation. Our results show that COX-1 in the uterine epithelium is the major source of PGs during labor and that COX-1(-/-) females experience parturition failure that is reversible by exogenous PGs. Using embryo transfer experiments, we also show that successful delivery occurs in COX-1(-/-) recipient mothers carrying wild-type pups, establishing the sufficiency of fetal PGs for parturition. Although patency of the DA is PG dependent, neither COX-1 nor COX-2 expression was detected in the fetal or postnatal DA, and offspring with a double null mutation died shortly after birth with open DAs. These results suggest that DA patency depends on circulating PGs acting on specific PG receptors within the DA. Collectively, these findings demonstrate the coordinated regulation of fetal and maternal PGs at the time of birth but raise concern regarding the use of selective COX inhibitors for the management of preterm labor.