A novel role for PGE2-EP4 in the developmental programming of the mouse ductus arteriosus: consequences for vessel maturation and function.

The ductus arteriosus (DA) is a vascular shunt that allows oxygenated blood to bypass the developing lungs in utero. Fetal DA patency requires vasodilatory signaling via the prostaglandin E2 (PGE2) receptor EP4. However, in humans and mice, disrupted PGE2-EP4 signaling in utero causes unexpected patency of the DA (PDA) after birth, suggesting another role for EP4 during development. We used EP4-knockout (KO) mice and acute versus chronic pharmacological approaches to investigate EP4 signaling in DA development and function. Expression analyses identified EP4 as the primary EP receptor in the DA from midgestation to term; inhibitor studies verified EP4 as the primary dilator during this period. Chronic antagonism recapitulated the EP4 KO phenotype and revealed a narrow developmental window when EP4 stimulation is required for postnatal DA closure. Myography studies indicate that despite reduced contractile properties, the EP4 KO DA maintains an intact oxygen response. In newborns, hyperoxia constricted the EP4 KO DA but survival was not improved, and permanent remodeling was disrupted. Vasomotion and increased nitric oxide (NO) sensitivity in the EP4 KO DA suggest incomplete DA development. Analysis of DA maturity markers confirmed a partially immature EP4 KO DA phenotype. Together, our data suggest that EP4 signaling in late gestation plays a key developmental role in establishing a functional term DA. When disrupted in EP4 KO mice, the postnatal DA exhibits signaling and contractile properties characteristic of an immature DA, including impairments in the first, muscular phase of DA closure, in addition to known abnormalities in the second permanent remodeling phase.NEW & NOTEWORTHY EP4 is the primary EP receptor in the ductus arteriosus (DA) and is critical during late gestation for its development and eventual closure. The "paradoxical" patent DA (PDA) phenotype of EP4-knockout mice arises from a combination of impaired contractile potential, altered signaling properties, and a failure to remodel associated with an underdeveloped immature vessel. These findings provide new mechanistic insights into women who receive NSAIDs to treat preterm labor, whose infants have unexplained PDA.

Effects of antenatal betamethasone on preterm human and mouse ductus arteriosus: comparison with baboon data.

BACKGROUND: Although studies involving preterm infants ≤34 weeks gestation report a decreased incidence of patent ductus arteriosus after antenatal betamethasone, studies involving younger gestation infants report conflicting results. METHODS: We used preterm baboons, mice, and humans (≤276/7 weeks gestation) to examine betamethasone's effects on ductus gene expression and constriction both in vitro and in vivo. RESULTS: In mice, betamethasone increased the sensitivity of the premature ductus to the contractile effects of oxygen without altering the effects of other contractile or vasodilatory stimuli. Betamethasone's effects on oxygen sensitivity could be eliminated by inhibiting endogenous prostaglandin/nitric oxide signaling. In mice and baboons, betamethasone increased the expression of several developmentally regulated genes that mediate oxygen-induced constriction (K+ channels) and inhibit vasodilator signaling (phosphodiesterases). In human infants, betamethasone increased the rate of ductus constriction at all gestational ages. However, in infants born ≤256/7 weeks gestation, betamethasone's contractile effects were only apparent when prostaglandin signaling was inhibited, whereas at 26-27 weeks gestation, betamethasone's contractile effects were apparent even in the absence of prostaglandin inhibitors. CONCLUSIONS: We speculate that betamethasone's contractile effects may be mediated through genes that are developmentally regulated. This could explain why betamethasone's effects vary according to the infant's developmental age at birth.

Selective serotonin reuptake inhibitor exposure constricts the mouse ductus arteriosus in utero.

Use of selective serotonin reuptake inhibitors (SSRIs) is common during pregnancy. Fetal exposure to SSRIs is associated with persistent pulmonary hypertension of the newborn (PPHN); however, a direct link between the two has yet to be established. Conversely, it is well known that PPHN can be caused by premature constriction of the ductus arteriosus (DA), a fetal vessel connecting the pulmonary and systemic circulations. We hypothesized that SSRIs could induce in utero DA constriction. Using isolated vessels and whole-animal models, we sought to determine the effects of two commonly prescribed SSRIs, fluoxetine and sertraline, on the fetal mouse DA. Cannulated vessel myography studies demonstrated that SSRIs caused concentration-dependent DA constriction and made vessels less sensitive to prostaglandin-induced dilation. Moreover, in vivo studies showed that SSRI-exposed mice had inappropriate DA constriction in utero. Taken together, these findings establish that SSRIs promote fetal DA constriction and provide a potential mechanism by which SSRIs could contribute to PPHN.

Aminoglycoside-mediated relaxation of the ductus arteriosus in sepsis-associated PDA.

Sepsis is strongly associated with patency of the ductus arteriosus (PDA) in critically ill newborns. Inflammation and the aminoglycoside antibiotics used to treat neonatal sepsis cause smooth muscle relaxation, but their contribution to PDA is unknown. We examined whether: 1) lipopolysaccharide (LPS) or inflammatory cytokines cause relaxation of the ex vivo mouse DA; 2) the aminoglycosides gentamicin, tobramycin, or amikacin causes DA relaxation; and 3) newborn infants treated with aminoglycosides have an increased risk of symptomatic PDA (sPDA). Changes in fetal mouse DA tone were measured by pressure myography in response to LPS, TNF-α, IFN-γ, macrophage-inflammatory protein 2, IL-15, IL-13, CXC chemokine ligand 12, or three aminoglycosides. A clinical database of inborn patients of all gestations was analyzed for association between sPDA and aminoglycoside treatment. Contrary to expectation, neither LPS nor any of the inflammatory mediators caused DA relaxation. However, each of the aminoglycosides caused concentration-dependent vasodilation in term and preterm mouse DAs. Pretreatment with indomethacin and N-(G)-nitro-L-arginine methyl ester did not prevent gentamicin-induced DA relaxation. Gentamicin-exposed DAs developed less oxygen-induced constriction than unexposed DAs. Among 488,349 infants who met the study criteria, 40,472 (8.3%) had sPDA. Confounder-adjusted odds of sPDA were higher in gentamicin-exposed infants, <25 wk and >32 wk. Together, these findings suggest that factors other than inflammation contribute to PDA. Aminoglycoside-induced vasorelaxation and inhibition of oxygen-induced DA constriction support the paradox that antibiotic treatment of sepsis may contribute to DA relaxation. This association was also found in newborn infants, suggesting that antibiotic selection may be an important consideration in efforts to reduce sepsis-associated PDA.

Efficacy of paracetamol on patent ductus arteriosus closure may be dose dependent: evidence from human and murine studies.

BACKGROUND: We evaluated the clinical effectiveness of variable courses of paracetamol on patent ductus arteriosus (PDA) closure and examined its effect on the in vitro term and preterm murine ductus arteriosus (DA). METHODS: Neonates received one of the following three paracetamol regimens: short course of oral paracetamol (SCOP), long course of oral paracetamol (LCOP), and intravenous paracetamol (IVP) for 2-6 d. Pressure myography was used to examine changes in vasomotor tone of the preterm and term mouse DA in response to paracetamol or indomethacin. Their effect on prostaglandin synthesis by DA explants was measured by mass spectroscopy. RESULTS: Twenty-one preterm infants were included. No changes in PDA hemodynamics were seen in SCOP infants (n = 5). The PDA became less significant and eventually closed in six LCOP infants (n = 7). PDA closure was achieved in eight IVP infants (n = 9). On pressure myograph, paracetamol induced a concentration-dependent constriction of the term mouse DA, up to 30% of baseline (P < 0.01), but required >1 µmol/l. Indomethacin induced greater DA constriction and suppression of prostaglandin synthesis (P < 0.05). CONCLUSION: The clinical efficacy of paracetamol on PDA closure may depend on the duration of treatment and the mode of administration. Paracetamol is less potent than indomethacin for constriction of the mouse DA in vitro.

Embryonic domains of the aorta derived from diverse origins exhibit distinct properties that converge into a common phenotype in the adult.

Vascular smooth muscle cells (VSMCs) are derived from distinct embryonic origins. Vessels originating from differing smooth muscle cell populations have distinct vascular and pathological properties involving calcification, atherosclerosis, and structural defects such as aneurysm and coarctation. We hypothesized that domains within a single vessel, such as the aorta, vary in phenotype based on embryonic origin. Gene profiling and myographic analyses demonstrated that embryonic ascending and descending aortic domains exhibited distinct phenotypes. In vitro analyses demonstrated that VSMCs from each region were dissimilar in terms of cytoskeletal and migratory properties, and retention of different gene expression patterns. Using the same analysis, we found that these same two domains are indistinguishable in the adult vessel. Our data demonstrate that VSMCs from different embryonic origins are functionally distinct in the embryonic mouse, but converge to assume a common phenotype in the aorta of healthy adults. These findings have fundamental implications for aortic development, function and disease progression.

Cimetidine-associated patent ductus arteriosus is mediated via a cytochrome P450 mechanism independent of H2 receptor antagonism.

Persistent patency of the ductus arteriosus (PDA) is a common problem in preterm infants. The antacid cimetidine is a potent antagonist of the H2 histamine receptor but it also inhibits certain cytochrome P450 enzymes (CYPs), which may affect DA patency. We examined whether cimetidine contributes to PDA and is mediated by CYP inhibition rather than H2 blockade. Analysis of a clinical trial to prevent lung injury in premature infants revealed a significant association between cimetidine treatment and PDA. Cimetidine and ranitidine, both CYP inhibitors as well as H2 blockers, caused relaxation of the term and preterm mouse DA. CYP enzymes that are inhibited by cimetidine were expressed in DA subendothelial smooth muscle. The selective CYP3A inhibitor ketoconazole induced greater DA relaxation than cimetidine, whereas famotidine and other H2 antagonists with less CYP inhibitory effects caused less dilation. Histamine receptors were developmentally regulated and localized in DA smooth muscle. However, cimetidine caused DA relaxation in histamine-deficient mice, consistent with CYP inhibition, not H2 antagonism, as the mechanism for PDA. Oxygen-induced DA constriction was inhibited by both cimetidine and famotidine. These studies show that antacids and other compounds with CYP inhibitory properties pose a significant and previously unrecognized risk for PDA in critically ill newborn infants.

Association of amino acids with common complications of prematurity.

BACKGROUND: Tandem mass spectrometry has been proposed as a method of diagnosing or predicting the development of common complex neonatal diseases. Our objective was to identify metabolites associated with common complications of prematurity. METHODS: We performed a retrospective analysis of medical data and metabolite measurements from routine neonatal screening on 689 preterm (<37 wk of gestational age) neonates. RESULTS: We observed higher levels of phenylalanine (PHE) in infants with respiratory distress syndrome (RDS; P = 1.7 × 10(-5)), the only association that was significant after correction for multiple testing. We found suggestive significance (P < 0.001) of higher essential amino acids in infants with patent ductus arteriosus (PDA). Functionality of these findings was explored in the ductus arteriosus (DA) isolated from term and preterm mouse pups. None of the amino acids had a direct vasodilatory effect on the isolated DA. CONCLUSION: We found that newborns with RDS had higher levels of PHE that may be a result of impaired PHE hydroxylase activity. We also detected marginally higher levels of all measured essential amino acids in infants with PDA. We did not find dilation of the mouse ductus for these metabolites, indicating that instead of potentially causing PDA, they are probably serving as markers of catabolism.

Cardio-respiratory response to moderate chloral hydrate sedation in young lambs.

AIM: Chloral hydrate (CH) is the most commonly used sedative for medical procedures and lung function tests in infancy. The aim was to determine whether moderate CH sedation affects airway function, lung volume and ventilation. METHODS: Thirteen chronically instrumented 7- to 8-week-old lambs were studied both before and after CH sedation (50 mg/kg as intravenous bolus followed by 25 mg/kg/hour as continuous infusion). Nitrogen washout technique and lung mechanics analysis were used to assess functional residual capacity (FRC) and airway function. Moment analysis and lung clearance index were calculated as measures of gas mixing efficiency in distal airways. Respiratory rate, tidal volume, minute ventilation and indices of inspiratory drive were determined together with heart rate, blood pressure and oxygenation. RESULTS: No significant CH-induced changes were found for gas mixing efficiency, FRC or lung mechanics. Minute ventilation decreased slightly, but significantly, while indices of inspiratory drive remained unchanged. Heart rate increased significantly, but mean arterial blood pressure was unaffected. CONCLUSION: Moderate CH sedation did not significantly affect airway function or FRC. Although indices of inspiratory drive were not affected, minute ventilation decreased slightly. These findings indicate that reliable results can be obtained from lung function testing when CH is used for sedation.

Omental grafting: a cell-based therapy for blood vessel repair.

Clinicians regularly transplant omental pedicles to repair a wide variety of injured tissues, but the basic mechanism underlying this efficacious procedure is not understood. One possibility that has not been addressed is the ability of omentum to directly contribute regenerative cells to injured tissues. We hypothesized that if omental progenitor cells could be mobilized to incorporate into damaged tissue, the power of this therapy would be greatly expanded. Labelled omental grafts were transplanted into a murine carotid artery injury model. Selected grafts were treated with thymosin ß4 (Tß4) prior to transplantation to investigate the effects of chemical potentiation on healing. We found treatment of grafts with Tß4-induced progenitor cells to fully integrate into the wall of injured vessels and differentiate into vascular smooth muscle. Myographic studies determined that arteries receiving Tß4-stimulated grafts were functionally indistinguishable from uninjured controls. Concurrent in vitro analyses showed that Tß4 promoted proliferation, migration and trans-differentiation of cells via AKT signalling. This study is the first to demonstrate that omentum can provide progenitor cells for repair, thus revealing a novel and naturally occurring source of vascular smooth muscle for use in cell-based therapies. Furthermore, our data show that this system can be optimized with inducing factors, highlighting a more powerful therapeutic potential than that of its current clinical application. This is a paradigm-setting concept that lays the foundation for the use of chemical genetics to enhance therapeutic outcomes in a myriad of fields.

Isoprostanes as physiological mediators of transition to newborn life: novel mechanisms regulating patency of the term and preterm ductus arteriosus.

BACKGROUND: Increased oxygen tension at birth regulates physiologic events that are essential to postnatal survival, but the accompanying oxidative stress may also generate isoprostanes. We hypothesized that isoprostanes regulate ductus arteriosus (DA) function during postnatal vascular transition. METHODS: Isoprostanes were measured by gas chromatography-mass spectrometry. DA tone was assessed by pressure myography. Gene expression was measured by quantitative PCR. RESULTS: Oxygen exposure was associated with increased 8-iso-prostaglandin (PG)F2α in newborn mouse lungs. Both 8-iso-PGE2 and 8-iso-PGF2α induced concentration-dependent constriction of the isolated term DA, which was reversed by the thromboxane A2 (TxA2) receptor antagonist SQ29548. SQ29548 pretreatment unmasked an isoprostane-induced DA dilation mediated by the EP4 PG receptor. Exposure of the preterm DA to 8-iso-PGE2 caused unexpected DA relaxation that was reversed by EP4 antagonism. In contrast, exposure to 8-iso-PGF2α caused preterm DA constriction via TxA2 receptor activation. Further investigation revealed the predominance of the TxA2 receptor at term, whereas the EP4 receptor was expressed and functionally active from mid-gestation onward. CONCLUSION: This study identifies a novel physiological role for isoprostanes during postnatal vascular transition and provide evidence that oxidative stress may act on membrane lipids to produce vasoactive mediators that stimulate physiological DA closure at birth or induce pathological patency of the preterm DA.

Spontaneous Rhythmic Contractions (Vasomotion) of the Isolated, Pressurized Ductus Arteriosus of Preterm, but Not Term, Fetal Mice.

The mechanisms that regulate relaxation of the fetal ductus arteriosus (DA) and its postnatal constriction are the subject of ongoing studies. Using pressure myography, a pattern of rhythmic oscillatory contractions termed vasomotion was observed in the isolated DA of preterm (day 15) fetal mice. Vasomotion was enhanced by oxygen-induced DA constriction and other contractile agents, and diminished by vasodilatory stimuli or inhibition of chloride channels. The DA of late preterm (day 17) or term (day 19) gestation fetal mice did not exhibit vasomotion. These studies establish the stage-specific presence of vasomotion in the DA of fetal mice and suggest that complex events contribute to intrinsic mechanisms for control of fetal DA tone.

Fetal nicotine exposure increases airway responsiveness and alters airway wall composition in young lambs.

To test the hypotheses that fetal nicotine exposure alters airway wall composition and enhances the airway response to inhaled methacholine (MCh), lambs were exposed during the last fetal trimester to (1) a low dose (LN) (n=13, 0.5mg/kg/d (maternal weight) of free base nicotine, (2) a moderate dose (MN) (n=10, 1.5mg/kg/d) or (3) saline (n=14). Studies were performed at postnatal days 12, 26 and 52. Prenatal nicotine exposure induced a dose- and age-related hyper-responsiveness to MCh in the proximal airways. Moment analysis of nitrogen decay curves showed no nicotine or MCh effects on ventilation homogeneity or gas-mixing efficiency in the distal airways during MCh inhalations suggesting a bimodal response. Fetal nicotine exposure increased epithelial mucosubstance volume in central (LN, MN) and distal bronchi (LN), increased smooth muscle volume in distal bronchi and bronchioles (LN) and decreased bronchiolar diameter (MN). In conclusion, third trimester nicotine exposure causes hyperreactive proximal airways and alters proximal airway wall composition associated with airflow limitation.

Chronic in utero cyclooxygenase inhibition alters PGE2-regulated ductus arteriosus contractile pathways and prevents postnatal closure.

Although prostaglandin E2 (PGE2) vasodilates the ductus arteriosus, tocolysis with cyclooxygenase (COX) inhibitors delays postnatal ductus arteriosus closure. We used fetal mice and sheep to determine whether PGE2 has a role in the development of ductus contractility that is distinct from its function as a vasodilator. Prolonged exposure of fetal ductus to PGE2 in vitro increased the expression of CaL- and K+-channel genes (CaLalpha1c, CaLbeta2, Kir6.1, and Kv1.5, which regulate oxygen-induced constriction) without affecting the genes that regulate Rho-kinase-mediated calcium sensitization. Conversely, chronic exposure to COX inhibitors in utero decreased expression of CaL- and K+-channel genes, without affecting Rho-kinase-associated genes. Chronic COX inhibition in utero decreased the ductus' in vitro contractile response to stimuli that use CaL- and K+-channels (like O2 and K+), whereas the response to stimuli that act through Rho-kinase-mediated pathways (like U46619) was not significantly affected. Phosphodiesterase expression, which decreases the ductus' sensitivity to cAMP- or cGMP-dependent vasodilators, was increased by PGE2 exposure and decreased by COX inhibition, respectively. These studies identify potential downstream effectors of a PGE2-mediated, developmental program, regulating oxygen-induced ductus closure. Alterations in these effectors may explain the increased risk of patent ductus arteriosus (PDA) after in utero COX inhibition.

Regulation of the fetal mouse ductus arteriosus is dependent on interaction of nitric oxide and COX enzymes in the ductal wall.

Nitric oxide (NO) and cyclooxygenase (COX)-derived prostaglandins are critical regulators of the fetal ductus arteriosus. To examine the interaction of these pathways within the ductus wall, the ductus arteriosus of term and preterm fetal mice was evaluated by pressurized myography. The isolated preterm ductus was more sensitive to NOS inhibition than at term. Sequential NOS and COX inhibition caused 36% constriction of the preterm ductus regardless of drug order. In contrast, constriction of the term ductus was dependent on the sequence of inhibition; NOS inhibition prior to COX inhibition produced greater constriction than when inhibitors were given in reverse order (36+/-6% versus 23+/-5%). Selective COX-1 or COX-2 inhibition prior to N(G)-nitro-l-arginine methyl ester (l-NAME) induced the expected degree of constriction. However, NOS inhibition followed by selective COX-2 inhibition caused unexpected ductal dilation. These findings are consistent with NO-induced activation of COX in the ductus arteriosus wall and the production of a COX-2-derived constrictor prostanoid that contributes to the balance of vasoactive forces that maintain fetal ductus arteriosus tone.

Prenatal nicotine exposure transiently alters the lung mechanical response to hypoxia in young lambs.

To test the hypothesis that fetal nicotine exposure alters the lung mechanical response to hypoxia (10% O(2)) 10 lambs were exposed during the last fetal trimester to a low dose nicotine (LN) and 10 to a moderate dose (MN) (maternal dose 0.5 and 1.5mg/(kgday) free base, respectively). There were 10 controls (C). At 12 days, minute ventilation increased significantly less in MN compared with LN but not with C. In contrast to C and LN, MN did not show anticipated increases in dynamic compliance, specific compliance and FRC or decrease in lung resistance but had signs of airway hyperreactivity during hypoxia. Nicotine exposure did not alter the cardiovascular response. These adverse effects decreased with advancing age. In summary, prenatal nicotine exposure alters the lung mechanical response to hypoxia. We speculate that prenatal nicotine-induced alterations of lung mechanics during hypoxia may contribute to an increased vulnerability to hypoxic stress during infancy.

Altered lung development after prenatal nicotine exposure in young lambs.

There is compelling evidence that prenatal nicotine exposure permanently alters lung development and airway function. The aim of this study was to determine how prenatal nicotine exposure alters proximal and distal airway function. Thirteen lambs were continuously exposed during the last fetal trimester to low-dose nicotine (LN) and 12 to a moderate dose (MN) (maternal s.c. dose: 0.5 and 1.5 mg/kg/d, respectively). Ten lambs served as controls (C). Proximal airway function was measured by lung mechanics. A multiple-breath N2 washout technique was used to measure lung volume (functional residual capacity) and efficiency of gas mixing in distal airways, i.e. terminal respiratory units (moment ratio and nitrogen clearance). In comparison with C, both LN and MN had significantly reduced specific airway conductance to the same extent at a median study age of 12, 25, and 51 d, indicating signs of proximal airway obstruction. Distal airway function showed significant improvement in LN. Ventilation and functional residual capacity were unaffected. In summary, prenatal nicotine exposure induced airway obstruction in proximal airways and improved gas mixing in distal airways, possibly reflecting restriction in proximal airway growth and accelerated maturation of the acinar part of the lung, respectively. We speculate that prenatal nicotine exposure has a disparate impact on airway development and function. The effect on the distal airways seemed to be inversely related to dose, which was not the case in the large airways. The altered airway function persisted during the study period, indicating that the effects of prenatal nicotine exposure might be permanent.