|Current Perspectives on Pathobiology of the Ductus Arteriosus
|Jason Z. Stoller1*, Sara B. DeMauro1, John M. Dagle2 and Jeff Reese3
|1Department of Pediatrics, University of Pennsylvania School of Medicine, The Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA
|2Departments of Pediatrics and Biochemistry, University of Iowa, Iowa City, IA 52242, USA
|3Departments of Pediatrics, and Cell and Developmental Biology; Vanderbilt University, Nashville, TN, 37232, USA
||Jason Z. Stroller, MD
Division of Neonatology
Department of Pediatrics
University of Pennsylvania School of Medicine
Children’s Hospital of Philadelphia 3615 Civic Center Blvd
PA 19104, USA
|Received December 15, 2011; Accepted December 18, 2011; Published
December 22, 2011
|Citation: Stoller JZ, DeMauro SB, Dagle JM, Reese J (2011) Current Perspectives
on Pathobiology of the Ductus Arteriosus. J Clinic Experiment Cardiol S8:001.
|Copyright: © 2011 Stoller JZ, et al. This is an open-access article distributed under
the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and
source are credited.
|The ductus arteriosus shunts blood away from the lungs during fetal life, but at birth this shunt is no longer needed
and the vessel rapidly constricts. Postnatal persistence of the DA, patent ductus arteriosus (PDA), is predominantly
a detrimental condition for preterm infants but is simultaneously a condition required to maintain systemic blood
flow for infants born with certain severe congenital heart defects. Although PDA in preterm infants is associated
with significant morbidities, there is controversy regarding whether PDA is truly causative. Despite advances in our
understanding of the pathobiology of PDA, the optimal treatment strategy for PDA in preterm infants is unclear. Here
we review recent studies that have continued to elucidate the fundamental mechanisms of DA development and
|Patent ductus arteriosus; Vascular development
|During early embryogenesis, the paired dorsal aortae give rise to a
set of symmetric aortic arch arteries. As development progresses, these
arteries go through an elegant transformation to form the asymmetric
mature vessels of great arteries, head vessels and proximal aorta. Many
forms of congenital heart disease involve aberrant remodeling of these
arteries . The left sixth aortic arch artery persists to become the
ductus arteriosus (DA), a unique blood vessel linking the pulmonary
and systemic circulations in utero. The DA shunts blood away from the
lungs during fetal life, but at birth this shunt is no longer needed and
the vessel rapidly constricts. Postnatal persistence of the DA, patent
ductus arteriosus (PDA), is predominantly a detrimental condition for
preterm infants but is simultaneously a conditionrequired to maintain
systemic blood flow for infants born with certain severe congenital
heart defects such as hypoplastic left heart syndrome.
|Although PDA in preterm infants is associated with significant
morbidities including intraventricular hemorrhage (IVH), necrotizing
enterocolitis (NEC) and bronchopulmonary dysplasia (BPD) [2-4],
there is intense controversy regarding whether PDA is truly causative
. The use of prophylactic indomethacin is effective in reducing
symptomatic PDA and surgical PDA ligation but it does not improve
mortality or reduce the incidence of BPD or NEC . Prophylactic
indomethacin does lower the incidence and severity of IVH [7-9].
Studies have demonstrated improved neurodevelopment outcomes
at 4 years of age but not at 18 months or 12 years of age [7,10,11].
Despite advances in our understanding of the pathobiology of PDA,
the optimal treatment strategy for PDA in very low birth weight
preterm infants is unclear . Is it possible indomethacin and other
cyclooxygenase inhibitors are having detrimental effects on non-
DA tissues and thus confounding the beneficial effects of closing the
DA? Are there alternative pathways to target for treatment of PDA?
The hope for future targeted therapies and for the prevention of PDA
requires knowledge of the fundamental mechanisms controlling its
development and pathogenesis. Here we review recent studies that
expanded our understanding of the DA,building a foundation upon
which to base future investigations that address some of these questions.
|Genetic Considerations in PDA Pathobiology
|PDA in the mouse model system
|Elegant methods allowing targeted genetic manipulation have made the mouse an ideal system in which to investigate genes involved
in closure of the DA. Initial studies that focused on prostaglandin
pathway components were based upon both the important role of
prostaglandin E2 (PGE2) in maintaining patency of the DA in utero,
and the successful pharmacologic manipulation of postnatal PGE2
levels to either maintain patency or induce closure of the DA. Several
genes in the prostaglandin pathway have been investigated in knockout
mice, as shown in Table 1.
||Table 1: Summary of prostaglandin pathway mutant mouse models relevant to the ductus arteriosus.
|Prostaglandin-endoperoxide synthase 1 (Ptgs1) and 2 (Ptgs2)
encode bi-functional enzymes, with both cyclooxygenase (COX)
and peroxidase activities, that catalyze the rate limiting step in the
production of prostaglandins from arachidonic acid. Mice lacking both
isoforms of Ptgs have a normal DA in utero, suggesting the presence of
maternal and/or placental sources of PGE2 . Although the absence
of Ptgs1 does not affect neonatal closure of the DA, 35-57% of Ptgs2-/-
mice die shortly after birth as a result of a PDA [12,13]. Interestingly,
mortality and incidence of PDA was increased in Ptgs2-/- mice when
one copy of Ptgs1 was also inactivated, suggesting compensation by
Ptgs1 for loss of Ptgs2. Neonatal mice with combined deficiency of
Ptgs1 and Ptgs2 have PDA and substantial perinatal mortality [12,14].
A Ptgs2 mouse model has been generated in which cyclooxygenase
activity was inhibited without affecting peroxidase activity . These
mice showed normal DA closure after birth. The paradoxical persistent
ductal patency seen in knockout mice following elimination of PGE2
synthesis or signaling can be explained by the developmental role of
PGE2 in preparing the fetal DA for postnatal closure in response to
increasing oxygen tension .
|Genes encoding other components of the prostaglandin pathway have also been studied. Mice lacking Ptger4 (encoding the
prostaglandin E receptor 4) have PDAs resulting in neonatal death
[16-18]. In addition, stimulation of EP4 promotes DA closure by
enhancing intimal thickening . Thus, PGE2 binding to EP4 may
have two opposite effects on closure of the DA: vascular smooth
muscular relaxation and intimal thickening. Slco2a1-/- mice, in which
the prostaglandin transporter gene has been deleted, fail to close the
DA after birth and die prior to postnatal day 2 . Finally, deletion of
the Hpgd gene, which encodes hydroxyprostaglandin dehydrogenase
15-(NAD), attenuates the normal postnatal decrease in PGE levels,
resulting in PDA and neonatal death [21,22]
|Genes important in vascular smooth muscle development have
also been studied using knockout mice. Smooth muscle myosin heavy
chain (encoded by Myh11) contracts in responseto the postnatal rise
in oxygen levels. Myh11-/- mice have delayed closure of the DA, but
full closure does occur . Closure of the DA in these mice suggests
that DA smooth muscle cells may contract using non-muscle myosin
components. Myocardin is a transcriptional coactivator that is
important in both cardiovascular development and adaptation of the
cardiovascular system to hemodynamic stress. The selective deletion
of the myocardin gene in neural crestderived smooth muscle cells
populating the cardiac outflow tract and great arteries resulted in mice
that are born alive, but die before postnatal day 3 secondary to a PDA
. Selective deletion of Jag1 in smooth muscle cells resulted in mice
that are alive at birth but become cyanotic in the early neonatal period
. The mortality of these mice was 50% on day 1 and 100% by day
2. Histologic examination exhibited a significant defect in DA smooth
muscle cell differentiation and 95% of the mice had an identifiable PDA
on postmortem examination. Deletion of Brahma-related gene 1 (Brg1-
a component of the chromatin-remodeling complex) in smooth muscle
cells resulted in death from cardiovascular anomalies, including PDA
and ventricular septal defects, in one third of Brg1-/- offspring .
Finally, Tcfap2b encodes a transcription factor expressed in the neural
crest cells, from which the DA originates. The first study of Tcfap2b-/-
mice found that they were born alive, but died in the first 24 hours of life
from what was attributed to renal abnormalities . Ductal anatomy
was not examined in these mice. Interestingly, the morphology of
the fetal DA after mid-gestation is not altered in Tcfap2b-/- mice
compared to wild type mice . However, Tcfap2b-/- mice die within
the first day of life with a PDA and signs of pulmonary over-circulation
. Tcfap2b-/- mice exhibit decreased DA expression of calponin
and hypoxia-inducible factor 2α, which are markers of differentiated
smooth muscle cells. Thus, Tcfap2b in mice appears to play a role in
maturation of the muscular layer of the DA. In addition, transcription
factor AP-2 beta regulates the expression of both EPAS1 (also known as
hypoxia inducible factor 2 alpha), which is involved in oxygen sensing,
and endothelin-1, which is a potent vasoconstrictor of DA smooth
|These studies highlight two critical pathways involved in permanent
closure of the DA, smooth muscle formation and regulation of muscle
contraction. Many genes are involved in the development of the
various smooth muscle layers in the DA. The control of DA closure
also requires a complex set of regulators. Shortly after birth, when the
pulmonary vascular resistance drops dramatically, the DA constricts to
the point of complete luminal closure. Identifying and characterizing
the regulatory networks controlling this unique behavior remains an
area of active research.
|PDA in human infants
|PDA can be divided into 2 groups: 1) a relatively rare condition seen in term infants that can exist as part of a constellation of other
physical anomalies (syndromic PDA) or as an isolated finding (nonsyndromic
PDA); and 2) a common condition present in very preterm
infants in which the vast majority of PDA cases are non-syndromic and
have a significant developmental component, i.e., the PDA would likely
not be present if the preterm infant had been born at term.
|Several genetic studies have focused on PDA associated with
syndromes in small cohorts of subjects, often excluding preterm
infants. A syndrome of thoracic aortic aneurysm and PDA [30,31] has
been linked to a region of chromosome 16p12.2-13.13 . Mutations
in MYH11, a gene encoding smooth muscle myosin heavy chain
11 (located at 16p13.11) have been identified as one cause for this
syndrome . Missense mutations in the smooth muscle α-actin gene
(ACTA2) also present as a syndrome of thoracic aortic aneurysms and
PDA . This association between abnormal contractile proteins and
PDA is not surprising, given the interactions between actin and myosin
required to generate contraction in muscle cells. Finally, mutations in
the gene TFAP2B (Tcfap2b in the mouse) have been found to result in
Char syndrome, a rare disorder characterized by facial dysmorphism,
hand anomalies, and PDA [35,36]. TFAP2B mutations have also been
reported in familial non-syndromic cases of PDA, which likely reflects
phenotypic variability in Char syndrome [37,38]
|Two retrospective twin studies, investigating the concordance
rates of PDA in monozygotic compared to dizygotic preterm twins,
have suggested a familial component for PDA in preterm infants. The
first study included 70 monozygotic twin pairs and 89 dizygotic twin
pairs and reported a 93% heritability of PDA requiring indomethacin
therapy and 48% heritability for PDA requiring surgical ligation .
The second study included 99 monozygotic twin pairs and 333 dizygotic
twin pairs and found that genetic factors or a shared environmental
factor accounted for 76% of the variance in liability to PDA, with
only 12% being accounted for by genetic factors . Thus, although
these studies both identified a familial/heritable contribution to PDA,
there was disagreement with respect to the magnitude of the genetic
|In addition to twin studies that can quantify heritability (but not
risk from a specific locus), candidate gene studies have identified
specific gene polymorphisms associated with PDA in preterm infants.
For example, the p allele of the PvuII pP polymorphism (rs2234693) in
the estrogen receptor alpha gene (ESR1) is associated with a decreased
risk of PDA . A polymorphism in the interferon gamma gene
(+874) (rs2430561 T allele) was also associated with a decreased risk for
PDA . This result may help explain the clinical finding that bacterial
infection is associated with both reduced DA closure and increased
reopening following initial closure [43,44]. Finally, polymorphisms in
TFAP2B (rs987237, G allele,) and in TRAF1 (TNF receptor-associated
factor 1) (rs1056567, T allele) have been reported to be associated
with the presence of a PDA. An additional analysis considering
combinations of neighboring alleles identified PTGIS (prostaglandin
I2 synthase) as a gene containing polymorphisms (rs493694, G allele
and rs693649, A allele) associated with the absence of a PDA (i.e.,
protective). The association of sequence variants in TFAP2B with PDA
in preterm infants supports the concept that common variants in the
same genes that are responsible for syndromic forms of PDA may be
responsible for isolated, non-syndromic forms of PDA given the right
environmental or developmental context. TFAP2B genotype is also associated with altered levels of mRNA encoding three ion channels
that are expressed in the DA: CACNB2 (calcium L channel beta
subunit), CACNA1 (calcium Tchannel) and KCNA2 (KV1.2 potassium
channel) . These channels may be potential targets for therapeutic
|Gene expression profiles in the DA
|Technological advances and progress in bioinformatics analysis
over the past fifteen years have enabled genome-wide transcriptome
analysis. Several groups have recently leveraged this by utilizing
microarrays to examine DA development and function. There are four
published reports that take an unbiased approach to determine the gene
expression profiles of the DA [46-49]. The methodology and controls
for each study differed, in part, due to the disparate questions being
asked by each group of investigators. In addition to a broad survey of
DA gene expression, the goals of these studies included determining
the effect of maternal vitamin A exposure and the effects of oxygen
and birth on DA gene expression. The results of these studies have
revealed some common themes but also some differing and sometimes
|There has only been one microarray study analyzing the human
DA. Mueller and colleagues performed a comparative analysis of
several vessels including the DA and pulmonary artery harvested at
the time of surgery . These vessels were obtained from patients
ranging between 1 and 807 days of age and included vessels that were
stented for different indications. Placement of a stent undoubtedly
causes changes in gene expression that are unlikely to reflect aspects
of normal physiologic closure in either term or preterm infants. The
broad range of ages precluded the authors from being able to group the
samples as biologic replicates. Together, these factors make the study’s
findings of little relevance to the development of the DA or function of
the perinatal DA. The remaining three studies were performed using
vessels isolated from the rat. In 2006, Costa and colleagues published a
study examining the effects of oxygen and birth on DA gene expression
at E19 and at 3 hours of life . In 2007, Yokoyama and colleagues
administered maternal vitamin A to increase retinoic acid levels at late
gestation. They reported DA gene expression profiles at E19, E21, and
at 3 to 6 hours of life . In 2011, Jin and colleagues compared gene
expression profiles of the DA and aorta E19 and E21 . Rat gestation
is approximately 21 days. Thus, E19 represents a late preterm gestational
age and E21 represents term. The characteristics and platforms utilized
in each study are listed in Table 2. While the methodology was
different, important data can be obtained by grouping the differentially
expressed genes into functional categories.
||Table 2: Summary of the characteristics of published ductus arteriosus gene expression profiles.
|Changes in cytoskeletal gene expression during late gestation may
reflect a critical step in DA vascular smooth muscle differentiation
in preparation for the rapid changes at the time of birth. Some of
these genes include those encoding myofibrillar proteins and genes
associated with muscle differentiation. Contraction of vascular smooth
muscle is mediated by myosin II. Myosin II consists of both heavy
chains and light chains. The heavy chain classically associated with
smooth muscle is encoded by the gene Myh11 . There are two
groups of light chains – essential light chains (ELCs) and regulatory
light chains (RLCs). The RLCs are encoded by the Myl2 (a.k.a. MLC2v),
Myl5, Myh7 (a.k.a. MLC2a), and Myl9 genes and can be phosphorylated
by myosin light chain kinase (MLCK) [51,52]. Costa and colleagues
found that relative to the aorta, the late preterm E19 DA had higher
expression of myofibrillar genes such as Myl2 and Myh7. By 3 hours
of life, under normoxic conditions, these genes were no longer
differentially expressed in the DA compared with the aorta. Yokoyama
and colleagues took a slightly different experimental approach. They
analyzed changes in gene expression in the DA over time but did
not normalize the expression to that of any other tissue or vessel.
This makes it difficult to make direct comparisons, and based on the
Yokoyama data, it is difficult to determine whether these myofibrillar
genes are predominant in all E19 vessels. Yokoyama found that not
only Myh7 as in the Costa study, but also other myofibrillar genes such
Actc1, Myh7, and Tnnt2 were more highly expressed in the late preterm
E19 DA relative to the DA at later stages (E21 and 3-6 hours after birth).
Jin and colleagues found that the myo fibrillar genes Myh11, Myl6,
Actg2, Tpm1 were highly expressed in both the E19 and E21 (term) DA
relative to aortic expression. Myl6 encodes an ELC and thus is not a
MLCK target, but may have a phosphorylation independent role in the
DA . In contrast to the Costa study, this study showed that Myl2
and Myh7 were downregulated at both ages in the DA relative to the
aorta along with similar downregulation of other myofibrillar genes
such as Myh7, Actc1, Tnnt2, Myh6, and Tnni3. Although the specificity
of MLCK-mediated RLC phosphorylation is known for some tissues,
the RLC in the DA is unknown. These microarray studies provide a
clue that although Myl2 and Myl7 are sometimes considered cardiacspecific
RLCs, they may play a role in the DA. It is difficult to explain
this discrepancy in Myl2 and Myh7 between the Costa and Jin studies.
Coceani proposed that this may be due to rat strain differences .
It is plausible that genetic background may explain differences in the
magnitude of fold changes but seems unlikely to explain completely
contradictory findings. The exact mechanistic role of these proteins in
the DA has not been described with the exception of Myh1 [23,33].
|As discussed in more detail below, myosin light chain (MLC)-
mediated contraction in vascular smooth muscle is dependent on MLC
phosphorylation. This is modulated by MLCK and MLC phosphatase
(MLCP). Calcium sensitization occurs when MLCP activity is
inhibited thus increasing the sensitivity of MLCK to calcium. A
number of signaling pathways, including the Rho-Rho kinase system,
regulate MLCP activity. Rho signaling pathways, acting through Rhoassociated
coiled-coil containing kinases (ROCKs), decrease MLCP
activity and thus result in calcium sensitization [51,54]. Costa et al.
report upregulation of the Rho-Rho kinase system in the neonatal
DA. The gene encoding the RhoB GTPase (Rhob) was upregulated in
the newborn DA relative to the aorta and also upregulated compared
with the fetal DA. The gene encoding the Rho downstream effector
molecule, Rock2, was upregulated in both the neonatal DA and aorta
relative to the respective fetal vessel. Neither Yokoyama nor Jin detected
differential expression in Rho-Rho-kinase genes.
|Calcium and potassium ion channels are critical mediators of
DA closure (discussed below). The Costa, Yokoyama and Jin studies
revealed several differentially expressed potassium channel genes.
Kcnk3 encodes the TWIK-related acid-sensitive K1 potassium channel
(TASK 1) and interestingly, this potassium channel is phosphorylated
in a pulmonary artery smooth muscle ET-1 signaling pathway .
TASK-1 controls resting membrane potential and modulates sensitivity
to vasoactive factors. Costa found that Kcnk3 was upregulated in the
neonatal DA relative to the fetal DA. Yokoyama et al. identified the ATPsensitive
potassium channel component gene Abcc9 to be upregulated
in both the E21 and P0 DA relative to the E19 DA. Abcc9 encodes the
sulfonylurea receptor subunit Sur2. Sur2 and the inwardly rectifying
potassium channel subunit Kir6.1 (Kcnj8) can function as interacting
subunits to form a functional ATP-sensitive potassium channel .
Yokoyama et al. found that Kcnj8 was unchanged between E19 and E21
but subsequently upregulated by 3 to 6 hours after birth. Consistent
with the Yokoyama data, Kcnj8 was highly expressed in the E19 and
E21 DA relative to the aorta. Yokoyama et al. also reported another
inwardly rectifying potassium channel subunit, Kir1.1 (Kcnj1) that was
present in the DA but essentially unchanged between these three ages.
They also found that the potassium channel tetramerisation domain
containing 12 gene (Kctd12) increased progressively from E19 to E21
and to P0. The functional significance of this is unclear although recent
evidence shows that this protein may function as a GABAB receptor
subunit . This is particularly interesting as GABA receptors can
regulate both inwardly rectifying potassium channels and voltage gated calcium channels.
|Jin and colleagues discovered other membrane ion channels
that were highly expressed in the DA relative to the aorta. The genes
encoding the Na+/K+ ATPase pump beta subunit (Atp1b1) and an
auxiliary α2δ subunit of the L-type voltage sensitive calcium channel
(Cacna2d1) were highly expressed in the DA at both E19 and E21
relative to aortic expression. L-type calcium channels regulate vascular
smooth muscle contraction and are essential for DA constriction .
|During development, specification and differentiation occur as
the bilaterally symmetric aortic arch arteries undergo morphological
and functional changes. The left 6th aortic arch artery transforms into
the DA and acquires a very different contractile potential compared
to the other aortic arch artery derivatives such as the carotid arteries
and portions of the aortic arch. Endothelin (ET-1) signaling has been
implicated as a potential vasoconstrictive effector of oxygen in the
DA and othervessels[50,58,59] (discussed below). Although Jin and
colleagues found ET-1 (Edn1) to be highly expressed in both the E19
and E21 DA relative to aortic expression, neither Costa nor Yokoyama
detected significant expression of Edn1 in the DA. There are two
transmembrane G protein-coupled endothelin receptors, endothelinA
(ETA) and endothelinB (ETB). These two receptors are encoded by
the Ednra and Ednrb genes. The ETB receptor inhibits cell growth and
functions as a scavenger, clearing ET-1, and thus inhibiting endothelindependent
vasoconstriction . Costa et al. report downregulation
of Ednrb in the newborn DA relative to late preterm offspring.
Downregulation of ETB would be expected to result in increased levels
of ET-1 and thus have a vasoconstrictive effect. In the Yokoyama study,
neither Ednra nor Endnb were found to be in a dominant gene cluster
at any of the tested age groups although by qPCR, ET-1 and endothelin
converting enzyme (Ece1) were upregulated in the postnatal DA relative
to fetal levels. Gata2 may increase expression of ET-1 . Costa et al.
found Gata2 was upregulated in the newborn DA compared with the
late preterm E19 DA. Carboxypeptidase A3 (Cpa3) has been reported
to catalyze the degradation of ET-1. Costa et al. report that Cpa3
was upregulated in the neonatal DA compared with late preterm DA.
This seems to contradict the hypothesis that ET-1 contributes to DA
constriction after birth, as one would expect higher carboxypeptidase
A3 levels to result in less ET-1. This may be consistent if the relative
expression of ET-1 is higher than the carboxypeptidase A3mediated
|Costa and colleagues propose that angiotensin II may be a
vasoconstrictive effector inthe DA. They found a modest increase in
the expression of the angiotensin II type 1a receptor (Agtr1a) in the neonatal DA compared with late preterm DA. In follow up experiments
using alate preterm mouse DA explants, they showed that exposure
to angiotensin II resulted in a transient dose-dependent contraction.
Yokoyama and colleagues found that the angiotensin II type 2 receptor
(Agtr2) is upregulated at E21 compared with E19 and then decreases
to levels modestly higher than E19 levels by 3 to 6 hours after birth.
Similarly, Jin et al. found that Agtr2 expression in the DA increased from
E19 to E21 when normalized to aortic expression but interestingly was
expressed as an aortic-dominant transcript rather than a DA-dominant
one. None of these groups found a difference in the angiotensin II
precursor angiotensinogen (Agt). Waleh et al. have subsequently
identified polymorphisms in the angiotensin II type 1 receptor (Agtr1)
associated with PDA in preterm infants . Surprisingly, none of the
endothelin or angiotensin related genes mentioned above were highly
expressed in the late preterm or neonatal DA after normalization to
aortic expression, suggesting that they may be important for general
vascular development and not unique to the DA.
|Prostaglandins (PG) play a prominent role in maintaining DA
vasodilation. Arachidonicacid is metabolized by PGH2 synthase
(a.k.a. cyclooxygenase or COX) to prostaglandin H2. Prostaglandin
H2 can be metabolized by several enzymes but, most relevant to the
biology of the DA, it is metabolized by PGE synthase to prostaglandin
E2 . As discussed in more detail below, the prostaglandin
receptor EP4 is the most likely PG receptor relevant to DA smooth
muscle. Costa and colleagues did not report enrichment in any of
the prostaglandin receptor genes or in genes encoding the enzymes
involved in prostaglandin synthesis. Despite this, they did find
upregulation of Alox15 in the neonatal DA relative to the E19 DA.
Alox15 encodes arachidonate 15-lipoxygenase (previously known as
12S-lipoxygenase), an alternative metabolic pathway for PG precursor
arachidonic acid. This upregulation in Alox15 around the time of birth
may not be DA-specific as this upregulation was also seen in the aorta.
Neither Yokoyama nor Jin report differential expression of Alox15.
Yokoyama and colleagues reported the EP4 prostaglandin receptor
(Ptger4) in their term (E21) dominant cluster where it was upregulated
compared with the E19 expression levels. Expression levels of Ptger4
seemed to then modestly decrease by 3 to 6 hours after birth. Jin and
colleagues reported Ptger4 to be highly expressed in both the E19
and E21 DA relative to aortic expression. Yokoyama et al. report no
apparent change in COX-1 (Ptgs1), but show that COX-2 (Ptgs2) gene
expression increases between E19 and E21 and then remains fairly
constant in the first few postnatal hours. Neither Costa nor Jin report
differential expression of Ptgs1 or Ptgs2.
|The effects of prostaglandins, nitric oxide, and carbon monoxide
are mediated through the second messengers, cyclic guanosine
monophosphate (cGMP) and cyclic adenosine monophosphate
(cAMP) in DA endothelial and smooth muscle cells (discussed below).
Phosphodiesterases (PDEs) catalyze the degradation of both cGMP
and cAMP. The expression of several PDEs, including Pde1a, Pde1b,
Pde1c, Pde3a, Pde3b, Pde4d and Pde5a have been described in the
sheep and baboon [45,63]. A role for Pde5a mediating vasoconstriction
in DA smooth muscle cells has been proposed . Costa et al. found
that Pde4b was one of the most highly expressed genes in the postnatal
DA relative to the aorta and that its expression increased from E19 to
3 hours after birth. This high level of expression may not be limited
to the DA as they also found the same age-related pattern of Pde4b
expression in the aorta. Yokoyama et al. found that Pde1, Pde2, Pde3,
Pde4b, and Pde5a were expressed in the DA in at least one of the three
time points they analyzed. They found both Pde5a and Pde4b in the
group of genes most highly expressed at term (E21) dominant cluster. Subsequently, Pde5a and Pde4b expression decreases by 3 to 6 hours
after birth. Pde1 and Pde2 did not change significantly in the DA at the
three examined ages. Pde3 expression progressively increases from E19
to 3 to 6 hours postnatally. Neither the E19 nor the E21 gene expression
profiles reported by Jin et al. revealed any PDE genes that were highly
expressed relative to aortic expression.
|Many other differentially expressed genes were reported in these
three studies. Their biological significance remains to be determined.
Notably absent from these studies is Tcfap2b. This gene is expressed in
DA smooth muscle and has been implicated in human Char syndrome
and in a developmental DA transcriptional network [28,65]. It is
possible that Tcfap2b is only expressed earlier than the ages examined
by these three groups. Notably, several extracellular matrix genes
were also expressed. Yokoyama reported that lysyl oxidase (Lox),
is upregulated in the E21 and postnatal DA relative to the E19 DA.
Lysyl oxidase is strongly hypoxia induced and been associated with
vascular smooth muscledevelopment [66-68]. Costa et al. did not
report differential expression of Lox. Yokoyama et al. reported that
fibronectin (Fn1) was the most highly expressed gene comparing term
E21 to late preterm E19 expression and this high expression persisted
after birth. Data from the Costa study support this finding. Fibronectin
has been shown to play a role in intimal cushion formation [69,70].
Similar to Lox, Jin et al. found Fn1 to be an aortic dominant gene when
compared to DA expression. These findings for both Lox and Fn1 may
not be DA-specific as Jin et al. found both to be aortic dominant genes.
|There are several important caveats to these studies. All of the rat
studies used pooled samples. While pooling samples is convenient,
important information is lost. If there are outlier values, pooling the
mRNA may show a false positive finding of a differentially expressed
gene. After pooling the samples, there is no way to identify this outlier
thus masking the lack of a difference. The studies discussed above
may have overcome this by pooling dozens of vessels. When assaying
thousands of transcripts, statistical analysis is paramount. Statistical
power is lost when samples are pooled and thus these studies likely
report many false positive or false negative findings. Finally, the
platform used in these studies was an early rat microarray. Not only
was there incomplete probe coverage for every rat gene, but there was
also incomplete probe annotation at the time of publication. The three
rat studies have deposited their primary datasets (Table 2) into public
databases, which will enable reanalysis of the expression data with
the better annotation information available today. These microarray
studies looked at a mixed cell population. In the future, a more
refined approach may be to perform transcriptional profiling using
specific cell populations such as smooth muscle cells, endothelial cells
andfibroblasts  to gain insight into cell-specific mechanisms of DA
development and function.
|Molecular Considerations in PDA Pathobiology
|Oxygen and PDA
|The exquisite sensitivity of the DA to oxygen is one of its defining
characteristics. Functional closure by contraction of circular and
longitudinal smooth muscle cells was postulated by Virchow in
1856, but demonstration of the contractile effects of oxygen was not
established until the 1940s and 1950s [72,73]. In those studies, exposure
to increased oxygen tension or oxygen bubbles in the circulation
caused DA constriction in vivo and in vitro. Transient oxygen exposure
induced brief DA constriction that was reversible under low oxygen
|Despite the historical nature of these observations, decades of
subsequent studies have failed to provide consensus on the mechanism
for oxygen-induced DA constriction, possibly because multiple
interacting pathways are involved [5,76-78].
|Oxygen sensing by cytochrome P450 enzymes
|Recent efforts have focused on elucidating the sequence of steps
from the detection of oxygen levels to triggering a vascular response.
In one well-developed scheme, a member of the cytochrome P450
(CYP) enzyme system acts as the sensor for acute changes in oxygen
tension. Coceani and colleagues have extensively detailed the proposal
that a monooxygenase reaction by certain CYP enzymes serves as
the initial step for oxygen signaling in the DA . Prevention of
oxygen-induced DA constriction by carbon monoxide and various
CYP inhibitors, together with localization of CYP3A in DA smooth
muscle cells, support this concept. While the majority of CYP enzymes
are expressed in the liver, members of the CYP2 and CYP4 family are
also expressed in the cardiovascular system where they catalyze the
production of epoxyeicosatrienoic (EET) and hydroxyeicosatetraenoic
(HETE) acids, respectively, to modulate inflammation, angiogenesis,
and vascular tone .
|The mechanisms by which CYP enzymes might transduce
oxygen levels are not yet resolved. Baragatti et al. recently examined
a potential role for CYP enzymes as the source of an endotheliumderived
hyperpolarizing factor (EDHF)  to maintain relaxation
of the mouse fetal DA. A survey of CYP expression demonstrated
the presence of CYP4A, CYP4B, and CYP2J (but not CYP2C) family
members by conventional RT-PCR. CYP2J6 and CYP2J9 proteins
were immune localized in the muscular media of the DA wall, with
increased CYP2J6 expression in the intima and sub endothelial
region. DA explants metabolized arachidonic acid into EETs via the
epoxygenase activity of CYP2J but basal concentrations were low.
Although EET levels marginally increased in response to a shift in
oxygen concentration from 2.5 to 30% in the culture media, this aspect
of CYP function is unlikely to serve as a sensor for oxygeninduced
constriction since EETs are typically associated with a vasodilatory
response. An exception exists in the lung, where EETs are implicated
in pulmonary vasoconstriction . It is unknown whether EETs play
a contractile or vasodilatory role in the DA, as this has not been directly
tested. Likewise, the products of CYP4A (20-HETE and others) were
either absent or did not have a definitive role in the DA. However, 12-
HETE, the product of an active 12- lipoxygenase pathway or CYP4B
activity, was detected in abundance. The function of 12-HETE in the
DA was not determined. Although the goal of this study was to identify
the CYP enzymes responsible for EDHF in the DA, insightful data were
also provided on the spectrum of CYP activities that contribute to DA
regulation. In a recent follow-up study, inhibition of arachidonic acid
epoxygenation (presumably by CYP2J) completely blocked oxygeninduced
constriction of the isolated mouse DA, lending support to
the concept of CYP-mediated oxygen sensing by EETs or an unknown
intermediary . A similar response was noted following inhibition
of the 12-lipoxygenase pathway. Together, these data show that
monooxygenase and lipoxygenase metabolites of arachidonic acid that
are produced in the DA wall have the ability to respond to changes in
oxygen tension and influence DA tone.
|Direct transduction and execution of oxygen signals by a single
CYP enzyme metabolite may not be necessary. Instead, Coceani and
colleagues have long proposed that oxygeninduced DA constriction
occurs by a multistep process  that is mediated via separate
mechanisms for oxygen sensing (by CYP enzymes) and effectors of the oxygen response. Endothelin-1 (ET-1) is proposed as the effector,
based on its potent vasocontrictive effects, the local oxygen-stimulated
production of ET-1 in the DA wall, and inhibition of its actions on
the DA by receptor blockade (by BQ123 or others) or by inhibition of
ET-1 synthesis (by phosphoramidon) [79,83,84]. In mice, deletion of
the ETA receptor for ET-1 results in decreased oxygen-induced DA
constriction , further supporting its role as an effector. However,
postnatal closure of the ETA null DA occurs normally  and ETA
blockade in other species does not affect postnatal DA constriction ,
raising the possibility that CYP enzymes may lead to vasoconstriction
by acting through other pathways. Baragatti et al. have now extended
their original supposition for a CYP3A source of DA regulation
. In that paper, CYP3A13 was the only CYP3A family member
detected in the DA and was developmentally regulated, with declining
levels at term gestation. By confocal fluorescence imaging, CYP3A13
was localized in the endoplasmic reticulum and plasma membrane
throughout the DA wall. Mice with targeted deletion of the Cyp3a gene
had normal postnatal DA closure but its size was somewhat smaller
than wild type, and the luminal surface lacked the usual initimal growth
and endothelial undulations that are characteristic of the closing DA.
Myography studies showed that the isolated DA of Cyp3a null mice was
poorly responsive to increased oxygen tension in the bath. On closer
inspection, deletion of Cyp3a primarily affected the tonic response
to increased oxygen tension, but the periodic, phasic contractile
response was maintained. This finding is in contrast to deletion of the
ETA receptor, where disruption of both tonic and phasic effects was
observed . This again supports the notion that CYP enzymes may
modulate signaling pathways independent of ET-1. The Cyp3a null DA
also had diminished contractile response to ET-1 compared to wild
type. In both instances, the DA of animals treated with retinoic acid
(used to promote maturation and oxygen sensitivity) had restoration of
their contractile response to either oxygen or ET-1. Even the preterm
DA on day 17 of gestation developed contractile responses that were
similar to the term DA after retinoic acid exposure. Cultured smooth
muscle cells from Cyp3a null mice had delayed intracellular calcium
accumulation compared to wildtype.
|The findings described above support the proposed role of CYP3A
as an oxygen mediator in the DA. There are other data that do not
support this model, including the observation that Cyp3a null fetuses
developed greater DA constriction in response to maternal hyperoxia
and closed normally after birth. In addition, Cyp3a expression declined
with advancing gestation – opposite to what would be expected for an
oxygen sensor. However, there are at least eight CYP3A homologues
in the mouse  and it is possible that other CYP3A family members
are present in the murine DA (J.Reese; unpublished data). The identity
of the hypothetical monoxygenase product that serves as messenger
between the putative sensor (CYP3A13) and effector (ET-1) remains
unknown, as acknowledged by the authors [82,87]. An alternative
explanation might be found in the CYP epoxygenase products 11, 12-
and 14,15-EET, which can activate large conductance Ca2+-activated
potassium channels (BKCa) to stimulate vascular smooth muscle
. Another possibility is the interaction of ET-1 with voltage-gated
potassium channels , although these pathways might have parallel
functions in the closing DA [91,92]. Further investigation is required
to determine whether links exist between these signaling systems to
actively mediate oxygen-induced DA constriction.
|Oxygen sensing through redox state and ion channels
|Kovalcik summarized contemporary concepts on oxygen-induced
DA closure in the 1960s, stating, “The most obvious and most important metabolic effect of oxygen is in relation to the terminal steps
of the electron transport chain” . Depolarization of the DA cell
membrane by oxygen exposure was later reported in 1981 . Based on
observations in pulmonary arteries and other oxygen-sensitive tissues,
a role for potassium channels in the early phase of oxygen-induced DA
constriction was established using pharmacological inhibitors  and
electrophysiology techniques . A model emerged whereby exposure
to increased oxygen tension stimulates inhibition of the voltage-gated
(Kv) potassium channels that are involved in maintenance of resting
membrane potential, causing subsequent membrane depolarization.
This, in turn, leads to activation of voltage-dependent L-type calcium
channels and entry of calcium to initiate contraction . These in
vitro findings were ultimately confirmed in whole animal studies and
in human DA specimens . In contrast to the multistep proposal by
Coceani and colleagues, oxygen-induced DA closure might therefore
be accomplished through a different series of sensors (mitochondria),
mediators (peroxide), and effectors (Kv channels and L-type calcium
|A redox reaction in smooth muscle cells may represent the earliest
step in oxygen sensing by the DA , triggering downstream effects
on redox-sensitive potassium channels . Mitochondria are critical
to this scheme, where the activity of specific mitochondrial enzymes
(e.g., NADPH oxidase), mitochondrial energetics and the electron
transport chain (ETC), and the generation of reactive oxygen species
(ROS), including superoxide anion and hydrogen peroxide, determines
the cellular redox status . Inhibition of proximal (e.g.,rotenone, for
complex I), midpoint (e.g., antimycin A, for complex III), or distal (e.g.,
cyanide, for cytochrome oxidase) components of the mitochondrial
ETC alters DA tone in humans, mammals, and birds [100,101]. Recent
work by Dzialowsky and colleagues confirm this concept for oxygen
sensing in the DA of different avian species (chick and emu) and
predict its importance in reptiles and other vertebrates . In short,
the overall scheme purports that oxygen stimulates mitochondrial
production of ROS, and that inhibitors of different ETC complexes or
mitochondrial enzymes can block this step. Peroxide (or other ROS)
then inhibit redox-sensitive Kv channels in the plasma membrane,
causing depolarization. This results in opening of inositol triphosphate
(IP3)-sensitive sarcoplasmic reticulum (SR) calcium stores , L-type
calcium channels and store-operated channels (SOCs), allowing the
influx of calcium. Increased intracellular calcium binds calmodulin
(CaM), and the calcium-CaM complex activates myosin light-chain
(MLC) kinase (MLCK). Activated MLCK then phosphorylates the
MLC leading to actomyosin stimulation and muscle contraction. In
opposition to this cascade, MLC phosphatase (MLCP) dephosphorylates
MLC, allowing smooth muscle cell relaxation. Villamor and colleagues
recently extended these findings in the chick DA to show that there is
a parallel maturation of sensor, mediator, and effector functions .
Controversy exists regarding the nature and direction of the ROS
signal that is generated during oxygen stress, along with the timing,
methods, and target molecules to be assessed [78,104]. Although there
is no consensus yet on the actual mechanisms by which redox changes
alter vascular tone , this appears to be the most likely first step in
oxygen signaling for DA closure.
|Activation of Rho kinase, a family of small GTPases that act through
ROCKs, has recently gained attention as an important pathway in DA
regulation. The Rho kinase system affects vascular tone by modulating
the balance between MLC kinase and phosphatase activities. In
pulmonary vessels, hypoxia increases the GTP-bound (active) form of
the small Gprotein RhoA. This stimulates Rho kinase, which in turn,
inhibits MLC phosphatase, thereby prolonging MLC phosphorylation and increasing calcium sensitization and smooth muscle contraction.
Calcium sensitization yields sustained vasoconstriction independent
of changes in cytosolic calcium levels and is stimulated by common
vasoactive G-protein coupled receptor agonists. Inhibitors of Rho
kinase lead to pulmonary vascular relaxation and prevention of
pulmonary hypertension. The role of Rho kinase in hypoxic pulmonary
vasoconstriction prompted interest in its potential contribution to
oxygen-induced DA constriction, where it was expected to have
an opposite effect. Compared to the role of RhoA/Rho kinase in the
lungs, Costa and colleagues found upregulation of RhoB and Rock2
expression in the DA of newborn rats . Rho activation was
stimulated by increased oxygen tension rather than hypoxia, as in
the lung, and it was RhoB rather than RhoA that served as the initial
mediator. As predicted, inhibition of Rho kinase by fasudil or similar
agents blocked oxygen-induced DA constriction in rabbits . A
follow-up study identified RhoB and ROCK-1 as critical mediators of
oxygen sensing in the rabbit and human DA . RhoA, RhoB, ROCK-
1, and ROCK-2 were present in the human DA and upregulated by
oxygen. Approximately one-third of oxygen-mediated DA tone was
attributed to Rho-mediated calcium sensitization. In the term DA,
oxygen-stimulated Rho kinase effects were mimicked by the redox
mediator, peroxide, and blocked by mitochondrial ETC inhibitors.
Immaturity of the mitochondrial ROS system in the preterm rabbit
DA was compounded by failure to upregulate Rho kinase expression
in response to oxygen. Similar to hypoxic pulmonary vasoconstriction,
a signaling scheme was envisioned that incorporates Rho/Rho kinase
as an alternate pathway, independent of Kv and calcium channel
signaling, to initiate smooth muscle constriction via its effects on MLC
|Clyman et al. showed that RhoA, RhoB, and ROCK-1 are present
in the fetal lamb DA, with increasing expression of RhoA in the more
mature DA, but not aorta . Rho kinase inhibition relaxed the
isolated DA under normoxic and hypoxic conditions. Approximately
50% of normoxic tension was resistant to calcium depletion, suggesting
the importance of calcium sensitizing mechanisms and the role of Rho
kinases or tyrosine kinases for oxygen-induced DA constriction in
sheep. More recent studies in the rat  and chick DA confirm the
importance of Rho kinase actions on MLC phosphatase for calcium
sensitization and share the view that an alternative pathway for oxygeninduced
DA constriction is available and utilizes Rho signaling as an
effector [100,103]. Rho kinase inhibitors may therefore provide a useful
approach to maintain DA patency, although untoward side effects may
limit its applicability.
|Extracellular calcium entry is the principal mechanism for
increased [Ca2+]i and the final common pathway for DA smooth
muscle constriction. L-type calcium channels are the main source for
oxygen-stimulated increases in calcium. However, internal release
of calcium from IP3-sensitive SR stores has also been demonstrated
and may precede calcium influx by L-type calcium channels .
Conversely, release of calcium from ryanodine-sensitive SR stores does
not appear to be involved. Increased [Ca2+]i in the DA is also the result
of transient receptor potential channels (TRPCs) that are presumed to
form SOCs. Inhibition of TRPC transcription or SOC function causes
diminished oxygen-induced DA constriction [105,106]. Increased
[Ca2+]i is thus dependent on SR release and L-type channel stimulation,
with calcium repletion through SOCs. Calcium entry across the plasma
membrane via reverse-mode function of the Na+/Ca2+ exchanger is an
additional mechanism involved in DA smooth muscle response to
oxygen. In sheep, pharmacological perturbation of SR replenishment
or Na+/Ca2+ exchange function eliminated differences in tone between the immature and mature DA under both normoxic and hypoxic
conditions, suggesting a potential therapeutic strategy. In contrast,
efforts to pharmacologically manipulate L-type calcium channels
were not successful in the premature DA under hypoxic conditions
. In a more recent paper, Thébaud et al. show that L-type calcium
channels are themselves an oxygen-sensitive channel, similar to the
aforementioned subset of Kv channels. In this study, stimulation of
L-type channels did not have an effect on the term rabbit DA or human
DA cells, but caused the preterm DA to behave like the term DA,
including increased oxygen-induced constriction, increased whole-cell
calcium current, and increased [Ca2+]i. L-type channels were expressed
and physiologically capable of response in preterm tissues, but were
not activated by oxygen exposure alone . Previous studies showed
that reduced expression and function of the oxygen-sensitive Kv1.5
and Kv2.1 channels might explain failure of the premature DA to close.
Transfer of Kv channels into the preterm DA could partially restore
its response, to approximately 50% of term levels . In contrast,
pharmacological activation of the premature DA L-type channel
restored full oxygen responsiveness . This intrinsic sensitivity
of the L-type channel to oxygen adds another layer to the complex
mechanisms for oxygen-mediated DA regulation.
|A role for other calcium channels in oxygen-induced DA
constriction is becoming apparent. T-type calcium channels are also
members of the voltage-dependent family of calcium channels that
regulate calcium influx. There was initial disagreement regarding the
role of T-type calcium channels in the DA, but the presence of multiple
family members has been demonstrated in the rat, where the α1G
subunit was predominantly expressed . In a recent study, Akaike
et al. showed that the α1G subunit of the T-type channel is upregulated
in rat DA cells with increased oxygenation or in neonates compared
to term fetuses. Disruption of T-type channel function with a specific
pharmacological agent or α1G-specific siRNAs resulted in reduced
smooth muscle cell migration, decreased [Ca2+]I accumulation, and
impaired thickening of the intimal layers of cultured DA explants.
Pharmacological inhibition also partially inhibited oxygen-induced
constriction of isolated DA rings and delayed closure of the postnatal
DA in newborn offspring. Overexpression of α1G promoted smooth
muscle cell migration . The importance of these findings was
reinforced by a more recent study in humans that identified risk factors
for PDA based on predisposing polymorphisms in the TFAP2B gene,
since abnormalities in TFAP2B are associated with PDA in mice and
humans (Char syndrome) [28,29,65]. The purpose of this study was
to understand the mechanisms for failed PDA closure after treatment
with prostaglandin inhibitors . Genes that contribute to the
increased risk of persistent PDA were identified. Here, the presence of
the rs2817399 (A) allele of TFAP2B in human DA tissues was associated
with decreased expression of specific calcium- and potassium-channel
genes, including the Kv1.2 channel, the beta-2 isoform of the L-type
calcium channel, and the α1G isoform of the T-type calcium channel
. Together, these studies implicate T-type channels as important
members of the oxygen-induced events that regulate DA closure.
|Prostaglandins and PDA
|A role for prostaglandins in fetal DA regulation was initially
suspected when DA constriction occurred in utero in pregnant
women that were treated with salicylates or indomethacin [112,113].
Prostaglandins or their inhibitors were found to have potent effects
on DA tone in animal models [114-117]. In 1976, two clinical trials
subsequently established the effectiveness of indomethacin for PDA
closure in premature infants [118,119]. Ibuprofen was found to have similar effects on DA closure [120,121], but clinical trials in preterm
infants were not reported for another two decades [122-124].
|Prostaglandins and ductus arteriosus regulation
|Prostaglandins are synthesized by the cyclooxygenase enzymes
COX-1 (Ptgs1) and COX-2 (Ptgs2) and are critical mediators of DA
patency and closure. The COX products prostacyclin (PGI2), and
more importantly, PGE2, have well-established roles as vasodilators
of the DA . However, there are inconsistencies regarding the
predominance of COX-1 or COX-2 in DA regulation among different
species. In mice, targeted deletion of both COX isoforms (Ptgs1;Ptgs2
double knockout) causes uniform lethality in the immediate newborn
period with a large PDA despite high levels of inspired oxygen [12,14].
Targeted deletion of COX-1 alone has little or no effect on DA closure,
while COX-2 deletion makes an arguably stronger impact, resulting
in PDA in 35% of offspring . Trivedi et al. found increased COX-
2 expression with advancing gestation and after birth, and suggested
that reduced COX-2 expression in the DA of premature offspring
prevented its postnatal constriction . COX-2 may be coupled with
downstream microsomal PGE synthases that reinforce its preferential
role in prostaglandin production in the DA [62,126]. Chronic
pharmacological inhibition of COX-2 mimics COX-2 deletion and also
results in PDA [127,128]. Chronic inhibition of both COX isoforms
results in a large PDA, similar in caliber to the COX double knockout
mouse, but only if the inhibitors are given later in gestation, and not
during early DA development .
|The presence of a PDA in COX deficient or chronically COXinhibited
offspring is unexpected, since brief exposure to NSAIDs
causes DA constriction. There are several competing theories that
address this apparent contradiction. Some evidence suggests that
NO or other vasodilatory mediators are upregulated in the absence
of prostaglandins . However, inhibition of NO synthesis did not
rescue the PDA phenotype of knockout or chronically COX inhibited
mice . Alternatively, it is possible that prostaglandins play an
important role in a developmental vascular program that dictates
formation of the DA’ contractile apparatus [14,128]. At an earlier stage
in gestation, Srivastava and colleagues demonstrated the presence
of a similar transcriptional program, this time under the control of
TFAP2B, which modulates ET-1 and Hif2α in the DA , and which
may be important in the human DA . Based on this premise,
a recent study in sheep and mice with either chronic exposure to
PGE2 or chronic COX inhibition, respectively, showed that PGE has
a unique role in the development of DA contractility that is distinct
from its role as a vasodilator . In that study, chronic exposure
of the fetal DA to PGE2 in vitro increased the expression of L-type
calcium channels (CACNα1c, CACNα2) and the potassium channel
genes Kcnj8 (Kir6.1 or KATP8) and Kv1.5 (Kcna5) (which regulate
oxygen-induced constriction), without affecting the genes that regulate
Rho-kinase–mediated calcium sensitization. Conversely, chronic COX
inhibition (and PGE depletion) decreased the DA’ in vitro contractile
response to stimuli that use L-type calcium channels and potassium
channels, whereas the response to stimuli that act through Rhokinasemediated
pathways was not significantly affected. Chronic exposure
to COX inhibitors inutero decreased expression of these same L-type
calcium channels and K+-channel genes, without affecting Rho-kinase–
associated genes . Together, these observations implicate an
important subset of genes that act as downstream effectors of a putative
developmental program, where PGE2 plays an important role in the
expression of specific pathways that are necessary for the DA’ oxygeninduced
closure after delivery.
|The role of COX enzymes in the human DA is less clear since
suitable tissue specimens are difficult to obtain. Nevertheless, Koehne
and colleagues studied autopsy samples from fetuses of 11 - 38 weeks of
gestation and found an increase in COX-1 expression with advancing
maturity. COX-1 immunostaining was present in the endothelium,
intima, and media, and was developmentally regulated in all three
layers. COX-2 immunostaining was detected at much lower levels and
was not related to maturational stage . During pregnancy, the
nonselective COX inhibitor indomethacin crosses the placenta and
constricts the human DA in fetuses <32 weeks gestation [130,131].
The findings of Koehne, along with studies on the predominant role of
COX-2 in the human uterus during parturition, suggest that selective
COX-2 inhibition might be a promising approach to block uterine
contractions during preterm labor without the additional risks for
constriction of the fetal DA, where COX-1 would be predicted to have
an important role. Unfortunately, several studies show that the fetal DA
is affected by maternal COX-2 inhibition . Thus, pharmacological
studies suggest either that COX-2 is active in the human fetal DA or
that COX-2-mediated prostaglandin synthesis in peripheral tissues (or
circulating cells) is important for fetal DA patency. It will be difficult
to determine whether a PGE-mediated vascular program is active in
the human DA since COX inhibitors reduce vasa vasorum flow to the
thick muscular media of the DA in humans and large animals, causing
hypoperfusion and ischemic injury to the vessel wall . This finding
partially explains why infants born to some women that are treated
with COX inhibitors for tocolysis have increased incidence of PDA
[134,135]. The alternative possibility, that there is upregulation of other
vasodilators, or disruption of a fetal vascular transcriptional program,
awaits further investigation.
|Prostaglandin receptors and downstream signaling
|Prostaglandins exert their effects through a family of G-protein
coupled receptors. There are subtle differences in the expression and
function of each receptor in the DA of various species. Due to the
importance of PGE in DA regulation, the EP family of receptors has
been the focus of particular attention. The EP4 subtype appears to play
an essential role, since mice with targeted deletion of the gene encoding
EP4, Ptger4, die in the first few hours of life with a large PDA [16-18].
Deletion of EP4, which typically mediates a vasodilatory response,
would be expected to cause DA constriction. However, the PDA
phenotype in these mice may be due to vascular dysregulation, similar
to COX double knockout mice, suggesting a critical ligandreceptor
pathway for DA development. EP4 is also the predominant receptor in
rats, rabbits, and baboons, although not in sheep [136-138]. In humans,
Leonhardt et al. found significant mRNA and protein expression of the
PGE2 receptors, EP3 and EP4, along with FP, IP, and TP receptors,
for PGF2, PGI2, and thromboxane, respectively. Of these receptors,
EP4 and TP receptors were the most expressed and were primarily
localized to the medial layer of the DA . Rheinlaender et al.
also found a predominance of EP4 protein expression in the intima
and media of the human DA at the time of autopsy . EP4 levels
were increased in the later stages of DA maturation. More recently,
Fan et al. demonstrated that the isolated preterm human DA was less
responsive to oxygen in vitro, but that pharmacological inhibition of
the EP4 receptor caused potent constriction . A link between
EP4 signaling and Kv channels was suggested as an underlying cause
for the differential response between term and preterm human DA
samples. Polymorphisms in the human EP4 gene are associated with
susceptibility to aspirin-resistant asthma, Crohn’s disease and other
disorders [141,142], but there are no studies to date that indicate a
relationship to PDA.
|The downstream targets of EP4 are the subject of several ongoing
investigations. Stimulation of the EP4 receptor by PGE or other
agonists increases intracellular cAMP and activates cAMP dependent
kinase A (PKA), resulting in relaxation of vascular smooth muscle and
DA dilation. Recently, Yokoyama et al. hypothesized that PGE2/EP4
signaling that is important for vascular remodeling in the aorta and
other vessels might also play a role in promoting anatomical closure of
the rat and mouse DA. In addition to the potent vasodilatory effects of
PGE2, EP4 stimulation was postulated to prepare the DA in utero for
postnatal closure by promoting subendothelial hyaluronic acid (HA)
production and intimal cushion formation . As in other species,
EP4 was the predominantly expressed isoform. Prolonged exposure
of cultured DA smooth muscle cells to a selective EP4 agonist caused
cell migration that was dependent on HA synthesis; migration was
inhibited by HA removal or silencing of the HAS2 enzyme for HA
synthesis. Explants of immature rat DA did not respond to 48 hours
of stimulation with an EP4 agonist, while the mature DA explants had
increased HAS2 expression, increased HA deposition, and increased
cell proliferation. Transfection of HAS2 improved lumen closure
rates in immature DA explants. Moreover, the DA of Ptger4 null
mice had reduced HA deposition. HAS2 transfection also improved
lumen closure in Ptger4 null DA explants . Although these detailed
findings provide compelling new insights into PGE actions for DA
closure, some methodological and conceptual uncertainties remain.
First, it is surprising that HA accumulation was most pronounced
in the adventitia and outer layers of muscular media, rather than the
intima or subendothelial region of the closing DA. The proposed
interaction of EP4 and HAS2 would be critical in this region. Given its
proposed role in luminal closure, HA accumulation was remarkably
sparse in the subendothelium - it is unclear how HA deposition
in the outer wall would prepare the fetal DA for postnatal closure.
Information on EP4 and HAS2 localization might also be informative.
Second, there is confusion regarding the proposed role for EP4 and
HA in intimal cushion formation, since rats, mice, and other small
rodents do not form intimal cushions or neointimal mounds, as
classically described in humans and larger species [85,143-145].
Indeed, none of the studies shown depict formation of an intimal
cushion. Thus, intimal cushion formation may not be an appropriate
outcome measure in these models. Although intimal thickening was
also described and may be the actual difference of interest, it is difficult
to distinguish intimal thickening from endothelial cell crowding
that takes place as the muscular wall constricts and the lumen crosssectional
area is correspondingly reduced. Third, the contribution of
intimal thickening to DA closure in the PDA of Ptger4 null mice is
particularly difficult to estimate, since the vessel wall fails to constrict
and therefore does not experience the same forces that lift endothelial
cells from their anchorage to the underlying internal elastic lamina.
Endothelial upheaval and redundancy is regarded as part of the process
that creates increased subendothelial space. While these concerns do
not invalidate the hypothesis that PGE signaling is important for HA
deposition, additional information is required. Mice with conditional
deletion of HAS2 have an embryonic lethal phenotype . However,
Prx1-Cre;Has2flox/flox mice with conditional deletion of HAS2 under
the control of the Prx1 transcription factor (which is expressed in the
DA) survive postnatally but have severe skeletal anomalies . More
in-depth studies in mice with conditional HAS2 inactivation may be
informative and help to resolve the interactions of PGE and HAS2 for
|A recent follow-up study by Yokoyama et al. examined whether a
newly defined target of cAMP, exchange protein activated by cAMP (Epac), is an important downstream effector of PGE2-EP4 cAMP
signaling during postnatal DA constriction . Epac1 and Epac2
mRNA expression was upregulated at term gestation and after birth,
with immunolocalization in the media and endothelium of the closed
rat DA. EP4 stimulation activated both the cAMP-PKA and cAMPEpac
pathways. A selective agonist of the cAMP-Epac pathway
stimulated DA smooth muscle cell migration, whereas cAMP-PKA
stimulation was inhibitory. Adenoviral-mediated overexpression or
siRNA-mediated inhibition of each isoform suggested the importance
of Epac1 over Epac2 for cell migration. The selective agonist of
the cAMP-Epac pathway inhibited cell proliferation and did not
upregulate hyaluronin synthesis, while stimulation of the cAMP-PKA
pathway successfully stimulated HA accumulation, as seen in their
previous publication . Explants of immature rat DA had increased
intimal thickening after transfection with Epac1, but not Epac2 .
Reservations regarding the formation of intimal cushions as an
outcome measure also exist for this paper. It is unclear whether the
difference between acute (PKA) and chronic (Epac) activation of EP4
occurs in vivo. However, the overall data demonstrate a unique, second
pathway for the downstream mechanisms of PGE2-EP4-cAMP actions.
In contrast to PKA, Epac-promoted DA closure was independent of cell
proliferation and HA synthesis. Further study is required to determine
whether these mechanisms are active in the human DA and could be
|Platelets and PDA
|A relationship between circulating platelet counts and closure of
the DA in preterm infants has recently become the focus of considerable
research. Echtler et al. first reported this relationship in 2010 .
The authors demonstrated that activated platelets accumulated in and
adhered to the lumen of the constricted DA within minutes after birth in
newborn mice. Mice with defective platelet adhesion or biogenesis had
high rates of persistent PDA, even after treatment with indomethacin.
Echtler then performed a retrospective evaluation of the relationship
between thrombocytopenia, defined as platelet count <150,000/μl on
the first day of life, and DA closure, demonstrated by echocardiogram
on day of life 3-5, in a group of 123 human infants born at 24-30 weeks
gestation . Seventy-one percent of infants had a PDA on day 3-5.
In a logistic regression model, low platelet count was identified as an
independent predictor of hemodynamically significant PDA (OR 13.1,
p=0.0001). The authors did not present a similar model for all (both
hemodynamically significant and asymptomatic) PDAs. Based onthese
findings, it was concluded that formation of a platelet plug is a critical
step in closure of the DA, linking the initial reversible constriction and
final anatomic remodeling.
|Since the publication of Echtler’s mouse and human studies,
several additional studies in human subjects have been performed. In
a similar retrospective cohort, Fujioka reported that a platelet count
<150,000/μl on the first day of life was not related to the rate of DA
closure in 142 Japanese infants 24-30 weeks gestational age .
Median platelet counts in the two studies were similar, but overall rate
of PDA was significantly lower in the Japanese study. Interestingly,
the thrombocytopenic Japanese infants were overall smaller and
younger than those with higher platelet counts, so would have been
expected to have higher rates of PDA. A study presented at the 2011
meeting of the Pediatric Academic Societies evaluated the relationship
between platelet counts during the first 3 days of life and DA closure
in 148 extremely low birth weight infants. Rates of both spontaneous
and indomethacin-induced DA closure were lower in extremely low
birth weight infants with platelet counts <150,000/μl . However, this result is confounded by higher rates of small for gestational age
and maternal preeclampsia and lower average birth weight in the
|The largest published study evaluating the relationship between
platelets and DA patency in human subjects included 497 infants <28
weeks of gestation . The cohort was managed in a single center
with an aggressive protocol including prophylactic indomethacin,
echocardiographic screening, and the availability of additional
indomethacin treatment. Unlike previous studies, which only evaluated
platelet counts in the 1-3 days of life, this study examined platelet counts
over the first week of life, the time period in which initial constriction
is most likely to occur in human infants. Persistence of a PDA was not
related to platelet count at any time in the first week of life. Rather, high
platelet counts were found to promote initial DA constriction. Neither
high nor low counts influenced rates of final, permanent closure. This
finding conflicts with the Echtler model, which suggests that platelets
accumulate in and contribute to obliteration of the already constricted
DA lumen, promoting permanent anatomic closure. Unfortunately,
the true influence of platelet count on the duct in the absence of
indomethacin cannot be determined in this study because all infants
received prophylactic indomethacin.
|Older studies, performed before the Echtler publication, are
equally inconclusive. A prospective cohort study from Singapore
demonstrated that, when controlling for other factors, platelet count
was of borderline significance for predicting failed closure of PDA
with indomethacin (OR 0.987, p=0.045) . On the other hand, in
a randomized trial of transfusions to keep the platelet count >150,000/
μl in thrombocytopenic preterm infants, rates of PDA were nearly
identical in the two groups .
|These conflicting studies have led Sallmon and colleagues to suggest
an alternative hypothesis: immature platelet function, not platelet
count, plays a role in persistent patency of the DA in the preterm
human infant . The rationale for this theory includes evidence for
impaired platelet function in preterm infants compared to term infants
and adults and the important observation that term infants with severe
alloimmune or autoimmune thrombocytopenia do not have higher
than expected rates of PDA [156,157]. However, no definitive data in
humans or animals yet exists to support the theory that developmental
differences in platelet function contribute to persistent PDA in preterm
|Thus, despite multiple studies, the platelet-PDA relationship
remains unclear. Echtler presented compelling and novel murine data
about the role of platelets in successful DA closure. The few small, single
center studies that have been performed in preterm human subjects are
contradictory, but do not suggest that thrombocytopenia in the first
days of life consistently results in failure of DA closure. Physiological
differences in the mechanism of DA closure between mice and humans
or population differences between cohorts in the human studies
may account for these conflicting results. As Echtler and others have
suggested, it is likely that the role of platelets in DA closure cannot be
elucidated without large controlled clinical studies.
|While the DA is a vessel rarely dwelled upon after patients leave the
neonatal intensive care unit, it is critical for both fetal well-being and
the transition to newborn life. Recent research has provided a window
into the important molecular pathways regulating the development
and function of the DA. However, there continues to be a tremendous amount that is unknown. Many of the animal models have focused on
the late preterm or term DA, but there is a distinct possibility that the
DA of the ELBW is functionally dissimilar. Further analysis of these
pathways earlier in gestation is necessary. The extension of rodent and
non-human primate studies to human basic science and clinical studies
will likely reveal conserved pathways with potential therapeutic targets.
As detailed in this review, these targets may include modulation of
hematopoietic cells, specific ion channels, prostaglandins or signaling
pathways including angiotensin and endothelin. In the future,
improved mechanisms by which clinicians can modulate the patency
and closure of the human ductus arteriosus will undoubtedly improve
the lives of countless infants.
|Supported by NIH grants HL086633 (Stoller), HL109199, HD52953 (Dagle)
and HL77395, HL96967, HL109199 (Reese) and by AHA grant 11BGIA7370043
- Stoller JZ, Epstein JA (2005) Cardiac neural crest. Seminars in cell & developmental biology 16: 704-715
- Laughon MM, Simmons MA, Bose CL (2004) Patency of the ductus arteriosus in the premature infant: is it pathologic? Should it be treated? Current opinion in pediatrics 16: 146-151
- Clyman RI, Chorne N (2007) Patent ductus arteriosus: evidence for and against treatment. J Pediatr 150: 216-219.
- Benitz WE (2010) Treatment of persistent patent ductus arteriosus in preterm infants: time to accept the null hypothesis? J Perinatol 30: 241-252.
- Hamrick SE, Hansmann G (2010) Patent ductus arteriosus of the preterm infant. Pediatrics 125: 1020-1030.
- Fowlie PW, Davis PG, McGuire W (2010) Prophylactic intravenous indomethacin for preventing mortality and morbidity in preterm infants. Cochrane database Syst Rev: CD000174.
- Schmidt B, Davis P, Moddemann D, Ohlsson A, Roberts RS, et al. (2001) Long-term effects of indomethacin prophylaxis in extremely-low-birth-weight infants. N Engl J Med 344: 1966-1972.
- Ment LR, Oh W, Ehrenkranz RA, Phillip AG, Vohr B, et al. (1994) Low-dose indomethacin therapy and extension of intraventricular hemorrhage: a multicenter randomized trial. J Pediatr 124: 951-955.
- Ment LR, Oh W, Ehrenkranz RA, Philip AG, Vohr B, et al. (1994) Low-dose indomethacin and prevention of intraventricular hemorrhage: a multicenter randomized trial. Pediatrics 93: 543-550
- Luu TM, Ment LR, Schneider KC, Katz KH, Allan WC, et al. (2009) Lasting effects of preterm birth and neonatal brain hemorrhage at 12 years of age. Pediatrics 123: 1037-1044
- Ment LR, Vohr B, Allan W, Westerveld M, Sparrow SS, et al. (2000) Outcome of children in the indomethacin intraventricular hemorrhage prevention trial. Pediatrics 105: 485-491.
- Loftin CD, Trivedi DB, Tiano HF, Clark JA, Lee CA, et al. (2001) Failure of ductus arteriosus closure and remodeling in neonatal mice deficient in cyclooxygenase-1 and cyclooxygenase-2. Proc Natl Acad Sci U S A 98: 1059-1064.
- Yu Y, Funk CD (2007) A novel genetic model of selective COX-2 inhibition: comparison with COX-2 null mice. Prostaglandins Other Lipid Mediat 82: 77-84
- Reese J, Paria BC, Brown N, Zhao X, Morrow JD, et al. (2000) Coordinated regulation of fetal and maternal prostaglandins directs successful birth and postnatal adaptation in the mouse. Proc Natl Acad Sci U S A 97: 9759-9764.
- Reese J, Waleh N, Poole SD, Brown N, Roman C, et al. (2009) Chronic in utero cyclooxygenase inhibition alters PGE2-regulated ductus arteriosus contractile pathways and prevents postnatal closure. Pediatr Res 66: 155-161.
- Nguyen M, Camenisch T, Snouwaert JN, Hicks E, Coffman TM, et al. (1997) The prostaglandin receptor EP4 triggers remodelling of the cardiovascular system at birth. Nature 390: 78-81.
- Segi E, Sugimoto Y, Yamasaki A, Aze Y, Oida H, et al. (1998) Patent ductus arteriosus and neonatal death in prostaglandin receptor EP4-deficient mice. Biochem Biophys Res Commun 246: 7-12.
- Schneider A, Guan Y, Zhang Y, Magnuson MA, Pettepher C, et al. (2004) Generation of a conditional allele of the Mouse Prostaglandin EP4 Receptor. Genesis 40: 7-14.
- Yokoyama U, Minamisawa S, Quan H, Ghatak S, Akaike T, et al. (2006) Chronic activation of the prostaglandin receptor EP4 promotes hyaluronan-mediated neointimal formation in the ductus arteriosus. J Clin Invest 116: 3026-3034.
- Chang HY, Locker J, Lu R, Schuster VL (2010) Failure of postnatal ductus arteriosus closure in prostaglandin transporter-deficient mice. Circulation 121: 529-536.
- Coggins KG, Latour A, Nguyen MS, Audoly L, Coffman TM, et al. (2002) Metabolism of PGE2 by prostaglandin dehydrogenase is essential for remodeling the ductus arteriosus. Nat Med 8: 91-92
- Roizen JD, Asada M, Tong M, Tai HH, Muglia LJ, et al. (2008) Preterm birth without progesterone withdrawal in 15-hydroxyprostaglandin dehydrogenase hypomorphic mice. Mol Endocrinol 22: 105-112
- Morano I, Chai GX, Baltas LG, Lamounier-Zepter V, Lutsch G, et al. (2000) Smooth-muscle contraction without smooth-muscle myosin. Nat Cell Biol 2: 371-375.
- Huang J, Cheng L, Li J, Chen M, Zhou D, et al. (2008) Myocardin regulates expression of contractile genes in smooth muscle cells and is required for closure of the ductus arteriosus in mice. J Clin Invest 118: 515-525.
- Feng X, Krebs LT, Gridley T (2010) Patent ductus arteriosus in mice with smooth musclespecific Jag1 deletion. Development 137: 4191-4199.
- Zhang M, Chen M, Kim JR, Zhou J, Jones RE, et al. (2011) SWI/SNF complexes containing Brahma or Brahma-related gene 1 play distinct roles in smooth muscle development. Mol Cell Biol 31: 2618-2631.
- Moser M, Pscherer A, Roth C, Becker J, Mucher G, et al. (1997) Enhanced apoptotic cell death of renal epithelial cells in mice lacking transcription factor AP-2beta. Genes Dev 11: 1938-1948
- Ivey KN, Sutcliffe D, Richardson J, Clyman RI, Garcia JA, et al. (2008) Transcriptional regulation during development of the ductus arteriosus. Circ Res 103: 388-395.
- Zhao F, Bosserhoff AK, Buettner R, Moser M (2011) A heart-hand syndrome gene: Tfap2b plays a critical role in the development and remodeling of mouse ductus arteriosus and limb patterning. PLoS One 6: e22908.
- Glancy DL, Wegmann M, Dhurandhar RW (2001) Aortic dissection and patent ductus arteriosus in three generations. Am J Cardiol 87: 813-815.
- Khau Van Kien P, Wolf JE, Mathieu F, Zhu L, Salve N, et al. (2004) Familial thoracic aortic aneurysm/dissection with patent ductus arteriosus: genetic arguments for a particular pathophysiological entity. Eur J Hum Genet 12: 173-180.
- Khau Van Kien P, Mathieu F, Zhu L, Lalande A, Betard C, et al. (2005) Mapping of familial thoracic aortic aneurysm/dissection with patent ductus arteriosus to 16p12.2-p13.13. Circulation 112: 200-206.
- Zhu L, Vranckx R, Khau Van Kien P, Lalande A, Boisset N, et al. (2006) Mutations in myosin heavy chain 11 cause a syndrome associating thoracic aortic aneurysm/aortic dissection and patent ductus arteriosus. Nat Genet 38: 343-349.
- Guo DC, Pannu H, Tran-Fadulu V, Papke CL, Yu RK, et al. (2007) Mutations in smooth muscle alpha-actin (ACTA2) lead to thoracic aortic aneurysms and dissections. Nat Genet 39: 1488-1493.
- Mani A, Meraji SM, Houshyar R, Radhakrishnan J, Ahangar M, et al. (2002) Finding genetic contributions to sporadic disease: a recessive locus at 12q24 commonly contributes to patent ductus arteriosus. Proc Natl Acad Sci U S A 99: 15054-15059.
- Satoda M, Pierpont ME, Diaz GA, Bornemeier RA, Gelb BD (1999) Char syndrome, an inherited disorder with patent ductus arteriosus, maps to chromosome 6p12-p21. Circulation 99: 3036-3042
- Chen YW, Zhao W, Zhang ZF, Fu Q, Shen J, et al. (2011) Familial Nonsyndromic Patent Ductus Arteriosus Caused by Mutations in TFAP2B. Pediatr Cardiol 32: 958-965.
- Khetyar M, Syrris P, Tinworth L, Abushaban L, Carter N (2008) Novel TFAP2B mutation in nonsyndromic patent ductus arteriosus. Genet Test 12: 457-459.
- Lavoie PM, Pham C, Jang KL (2008) Heritability of bronchopulmonary dysplasia, defined according to the consensus statement of the national institutes of health. Pediatrics 122: 479-485.
- Bhandari V, Zhou G, Bizzarro MJ, Buhimschi C, Hussain N, et al. (2009) Genetic contribution to patent ductus arteriosus in the premature newborn. Pediatrics 123: 669-673.
- Derzbach L, Treszl A, Balogh A, Vasarhelyi B, Tulassay T, et al. (2005) Gender dependent association between perinatal morbidity and estrogen receptor-alpha Pvull polymorphism. J Perinat Med 33: 461-462.
- Bokodi G, Derzbach L, Banyasz I, Tulassay T, Vasarhelyi B (2007) Association of interferon gamma T+874A and interleukin 12 p40 promoter CTCTAA/GC polymorphism with the need for respiratory support and perinatal complications in low birthweight neonates. Arch Dis Child Fetal Neonatal Ed 92: F25-F29
- Gonzalez A, Sosenko IR, Chandar J, Hummler H, Claure N, et al. (1996) Influence of infection on patent ductus arteriosus and chronic lung disease in premature infants weighing 1000 grams or less. J Pediatr 128: 470-478.
- Rojas MA, Gonzalez A, Bancalari E, Claure N, Poole C, et al. (1995) Changing trends in the epidemiology and pathogenesis of neonatal chronic lung disease. J Pediatr 126: 605-610.
- Waleh N, Hodnick R, Jhaveri N, McConaghy S, Dagle J, et al. (2010) Patterns of gene expression in the ductus arteriosus are related to environmental and genetic risk factors for persistent ductus patency. Pediatr Res 68: 292-297.
- Costa M, Barogi S, Socci ND, Angeloni D, Maffei M, et al. (2006) Gene expression in ductus arteriosus and aorta: comparison of birth and oxygen effects. Physiol Genomics 25: 250- 262.
- Mueller PP, Drynda A, Goltz D, Hoehn R, Hauser H, et al. (2009) Common signatures for gene expression in postnatal patients with patent arterial ducts and stented arteries. Cordiol Young 19: 352-359.
- Jin MH, Yokoyama U, Sato Y, Shioda A, Jiao Q, et al. (2011) DNA microarray profiling identified a new role of growth hormone in vascular remodeling of rat ductus arteriosus. J Physiol Sci 61: 167-179
- Yokoyama U, Sato Y, Akaike T, Ishida S, Sawada J, et al. (2007) Maternal vitamin A alters gene profiles and structural maturation of the rat ductus arteriosus. Physiol Genomics 31: 139-157
- Bokenkamp R, DeRuiter MC, van Munsteren C, Gittenberger-de Groot AC (2010) Insights into the pathogenesis and genetic background of patency of the ductus arteriosus. Neonatology 98: 6-17.
- Somlyo AP, Somlyo AV (2003) Ca2+ sensitivity of smooth muscle and nonmuscle myosin II: modulated by G proteins, kinases, and myosin phosphatase. Physiol Reviews 83: 1325-1358
- Chen Z, Huang W, Dahme T, Rottbauer W, Ackerman MJ, et al. (2008) Depletion of zebrafish essential and regulatory myosin light chains reduces cardiac function through distinct mechanisms. Cardiovasc Res 79: 97-108.
- Coceani F, Scebba F, Angeloni D (2011) Gene profiling in ductus arteriosus and aorta: a question of consistency.J physiol Sci 61: 443-444
- Kajimoto H, Hashimoto K, Bonnet SN, Haromy A, Harry G, et al. (2007) Oxygen activates the Rho/Rho-kinase pathway and induces RhoB and ROCK-1 expression in human and rabbit ductus arteriosus by increasing mitochondria-derived reactive oxygen species: a newly recognized mechanism for sustaining ductal constriction. Circulation 115: 1777-1788.
- Tang B, Li Y, Nagaraj C, Morty RE, Gabor S, et al. (2009) Endothelin-1 inhibits background two-pore domain channel TASK-1 in primary human pulmonary artery smooth muscle cells. Am J Respir Cell Mol Biol 41: 476-483.
- Cui Y, Giblin JP, Clapp LH, Tinker A (2001) A mechanism for ATP-sensitive potassium channel diversity: Functional coassembly of two pore-forming subunits. Proc Natl Acad Sci U S A 16: 729-734.
- Metz M, Gassmann M, Fakler B, Schaeren-Wiemers N, Bettler B (2011) Distribution of the auxiliary GABAB receptor subunits KCTD8, 12, 12b, and 16 in the mouse brain. The J Comp Neurol 519 1435-1454.
- Kawanabe Y, Nauli SM (2011) Endothelin. Cell Mol Life Sci 68: 195-203
- Taniguchi T, Azuma H, Okada Y, Naiki H, Hollenberg MD, et al. (2001) Endothelin-1-endothelin receptor type A mediates closure of rat ductus arteriosus at birth. J Physiol 537: 579-585
- Stow LR, Jacobs ME, Wingo CS, Cain BD (2011) Endothelin-1 gene regulation. FASEB J 25: 16-28.
- Pejler G, Knight SD, Henningsson F, Wernersson S (2009) Novel insights into the biological function of mast cell carboxypeptidase A. Trends in immunology 30: 401-408.
- Baragatti B, Sodini D, Uematsu S, Coceani F (2008) Role of microsomal prostaglandin E synthase-1 (mPGES1)-derived PGE2 in patency of the ductus arteriosus in the mouse. Pediatr Res 64: 523-527.
- Liu H, Manganiello V, Waleh N, Clyman RI (2008) Expression, activity, and function of phosphodiesterases in the mature and immature ductus arteriosus. Pediatr Res 64: 477-481.
- Thebaud B, Michelakis E, Wu XC, Harry G, Hashimoto K, et al. (2002) Sildenafil reverses O2 constriction of the rabbit ductus arteriosus by inhibiting type 5 phosphodiesterase and activating BK(Ca) channels. Pediatric research 52: 19-24.
- Satoda M, Zhao F, Diaz GA, Burn J, Goodship J, et al. (2000) Mutations in TFAP2B cause Char syndrome, a familial form of patent ductus arteriosus. Nat Genet 25: 42-46.
- Nuthakki VK, Fleser PS, Malinzak LE, Seymour ML, Callahan RE, et al. (2004) Lysyl oxidase expression in a rat model of arterial balloon injury. J Vasc Surg: official publication, the Society for Vascular Surgery [and] International Society for Cardiovascular Surgery, North American Chapter 40: 123-129.
- Myllyharju J, Schipani E (2010) Extracellular matrix genes as hypoxia-inducible targets. Cell Tissue Res339: 19-29.
- Choudhary B, Zhou J, Li P, Thomas S, Kaartinen V, et al. (2009) Absence of TGFbeta signaling in embryonic vascular smooth muscle leads to reduced lysyl oxidase expression, impaired elastogenesis, and aneurysm. Genesis 47: 115-121.
- Mason CA, Bigras JL, O'Blenes SB, Zhou B, McIntyre B, et al. (1999) Gene transfer in utero biologically engineers a patent ductus arteriosus in lambs by arresting fibronectindependent neointimal formation. Nat med 5: 176-182.
- Mason CA, Chang P, Fallery C, Rabinovitch M (1999) Nitric oxide mediates LC-3-dependent regulation of fibronectin in ductus arteriosus intimal cushion formation. FASEB J 13: 1423-1434.
- Weber SC, Gratopp A, Akanbi S, Rheinlaender C, Sallmon H, et al. (2011) Isolation and culture of fibroblasts, vascular smooth muscle, and endothelial cells from the fetal rat ductus arteriosus. Pediatr Res 70: 236-241.
- Born GVR, Dawes GS, Mott JC, Rennick BR (1956) Constriction of the ductus arteriosus caused by oxygen and by asphyxia in newborn lambs. J Physiol 132: 304-342.
- Kennedy JA, Clark SL (1942) Observations on the physiological reactions of the ductus arteriosus. Am J Physiol 136: 140-147.
- Kovalcik V (1963) The response of the isolated ductus arteriosus to oxygen and anoxia. J Physiol 169: 185-197.
- Moss AJ, Emmanouilides GC, Adams FH, Chuang K (1964) Response of Ductus Arteriosus and Pulmonary and Systemic Arterial Pressure to Changes in Oxygen Environment in Newborn Infants. Pediatrics 33: 937-944.
- Fay FS (1971) Guinea pig ductus arteriosus. I. Cellular and metabolic basis for oxygen sensitivity. Am J Physiol 221: 470-479.
- Smith GC (1998) The pharmacology of the ductus arteriosus. Pharmacol Rev 50: 35-58.
- Ward JP (2008) Oxygen sensors in context. Biochim Biophys Acta 1777: 1-14.
- Coceani F (1999) Cytochrome P450 in the contractile tone of the ductus arteriosus: regulatory and effector mechanisms. In: Weir EK, Archer SL, Reeves JT, editors. The Fetal and Neonatal Pulmonary Circulations: Futura Publishing Co., Inc.; Armonk, NY. pp. 331-341.
- Fleming I (2008) Vascular cytochrome p450 enzymes: physiology and pathophysiology. Trends Cardiovasc Med 18: 20-25.
- Baragatti B, Schwartzman ML, Angeloni D, Scebba F, Ciofini E, et al. (2009) EDHF function in the ductus arteriosus: evidence against involvement of epoxyeicosatrienoic acids and 12S-hydroxyeicosatetraenoic acid. Am J Physiol Heart Circ Physiol 297: H2161-2168.
- Baragatti B, Coceani F (2011) Arachidonic acid epoxygenase and 12(S)-lipoxygenase: evidence of their concerted involvement in ductus arteriosus constriction to oxygen. Can J Physiol Pharmacol 89: 329-334.
- Takizawa T, Horikoshi E, Shen MH, Masaoka T, Takagi H, et al. (2000) Effects of TAK-044, a nonselective endothelin receptor antagonist, on the spontaneous and indomethacin- or methylene blue-induced constriction of the ductus arteriosus in rats. J Vet Med Sci 62: 505-509.
- Taniguchi T, Muramatsu I (2003) Pharmacological knockout of endothelin ET(A) receptors. Life Sci 74: 405 409.
- Coceani F, Liu Y, Seidlitz E, Kelsey L, Kuwaki T, et al. (1999) Endothelin A receptor is necessary for O(2) constriction but not closure of ductus arteriosus. Am J Physiol 277: H1521-1531
- Fineman JR, Takahashi Y, Roman C, Clyman RI (1998) Endothelin-receptor blockade does not alter closure of the ductus arteriosus. Am J Physiol 275: H1620-1626.
- Baragatti B, Ciofini E, Scebba F, Angeloni D, Sodini D, et al. (2011) Cytochrome P-450 3A13 and endothelin jointly mediate ductus arteriosus constriction to oxygen in mice. Am J Physiol Heart Circ Physiol 300: H892-901.
- Nelson DR, Zeldin DC, Hoffman SM, Maltais LJ, Wain HM, et al. (2004) Comparison of cytochrome P450 (CYP) genes from the mouse and human genomes, including nomenclature recommendations for genes, pseudogenes and alternative-splice variants. Pharmacogenetics 14: 1-18.
- Michaelis UR, Fleming I (2006) From endothelium-derived hyperpolarizing factor (EDHF) to angiogenesis: Epoxyeicosatrienoic acids (EETs) and cell signaling. Pharmacol Ther 111: 584-595.
- Whitman EM, Pisarcik S, Luke T, Fallon M, Wang J, et al. (2008) Endothelin-1 mediates hypoxia-induced inhibition of voltage-gated K+ channel expression in pulmonary arterial myocytes. Am J Physiol Lung Cell Mol Physiol 294: L309-318.
- Keck M, Resnik E, Linden B, Anderson F, Sukovich DJ, et al. (2005) Oxygen increases ductus arteriosus smooth muscle cytosolic calcium via release of calcium from inositol triphosphate-sensitive stores. Am J Physiol Lung Cell Mol Physiol 288: L917-923.
- Michelakis E, Rebeyka I, Bateson J, Olley P, Puttagunta L, et al. (2000) Voltage-gated potassium channels in human ductus arteriosus. Lancet 356: 134-137.
- Roulet MJ, Coburn RF (1981) Oxygen-induced contraction in the guinea pig neonatal ductus arteriosus. Circ Res 49: 997-1002.
- Nakanishi T, Gu H, Hagiwara N, Momma K (1993) Mechanisms of oxygen-induced contraction of ductus arteriosus isolated from the fetal rabbit. Circ Res 72: 1218-1228.
- Tristani-Firouzi M, Reeve HL, Tolarova S, Weir EK, Archer SL (1996) Oxygen-induced constriction of rabbit ductus arteriosus occurs via inhibition of a 4-aminopyridine-, voltage-sensitive potassium channel. J Clin Invest 98: 1959-1965.
- Weir EK, Lopez-Barneo J, Buckler KJ, Archer SL (2005) Acute oxygen-sensing mechanisms. N Engl J Med 353: 2042-2055.
- Archer SL, Wu XC, Thebaud B, Moudgil R, Hashimoto K, et al. (2004) O2 sensing in the human ductus arteriosus: redox-sensitive K+ channels are regulated by mitochondriaderived hydrogen peroxide. Biol Chem 385: 205-216.
- Reeve HL, Tolarova S, Nelson DP, Archer S, Weir EK (2001) Redox control of oxygen sensing in the rabbit ductus arteriosus. J Physiol 533: 253-261.
- Weir EK, Obreztchikova M, Vargese A, Cabrera JA, Peterson DA, et al. (2008) Mechanisms of oxygen sensing: a key to therapy of pulmonary hypertension and patent ductus arteriosus. Br J Pharmacol 155: 300-307.
- Greyner H, Dzialowski EM (2008) Mechanisms mediating the oxygen-induced vasoreactivity of the ductus arteriosus in the chicken embryo. Am J Physiol Regul Integr Comp Physiol 295: R1647-1659.
- Michelakis ED, Rebeyka I, Wu X, Nsair A, Thebaud B, et al. (2002) O2 sensing in the human ductus arteriosus: regulation of voltage-gated K+ channels in smooth muscle cells by a mitochondrial redox sensor. Circ Res 91: 478-486
- Dzialowski EM, Sirsat T, van der Sterren S, Villamor E (2011) Prenatal cardiovascular shunts in amniotic vertebrates. Respir Physiol Neurobiol 178: 66-74.
- Cogolludo AL, Moral-Sanz J, van der Sterren S, Frazziano G, van Cleef AN, et al. (2009) Maturation of O2 sensing and signaling in the chicken ductus arteriosus. Am J Physiol Lung Cell Mol Physiol 297: L619-L630.
- Weir EK, Archer SL (2010) The role of redox changes in oxygen sensing. Respir Physiol Neurobiol 174: 182-191
- Hong Z, Hong F, Olschewski A, Cabrera JA, Varghese A, et al. (2006) Role of storeoperated calcium channels and calcium sensitization in normoxic contraction of the ductus arteriosus. Circulation 114: 1372-1379.
- Clyman RI, Waleh N, Kajino H, Roman C, Mauray F (2007) Calcium-dependent and calcium-sensitizing pathways in the mature and immature ductus arteriosus. Am J Physiol Regul Integr Comp Physiol 293: R1650-R1656.
- Momma K, Toyoshima K, Sun F, Nakanishi T (2009) In vivo dilatation of the ductus arteriosus by Rho kinase inhibition in the rat. Neonatology 95: 324-331.
- Thebaud B, Wu XC, Kajimoto H, Bonnet S, Hashimoto K, et al. (2008) Developmental Absence of the O2 Sensitivity of L-Type Calcium Channels in Preterm Ductus Arteriosus Smooth Muscle Cells Impairs O2 Constriction Contributing to Patent Ductus Arteriosus. Pediatr Res 63: 176-181
- Thebaud B, Michelakis ED, Wu XC, Moudgil R, Kuzyk M, et al. (2004) Oxygen-sensitive Kv channel gene transfer confers oxygen responsiveness to preterm rabbit and remodeled human ductus arteriosus: implications for infants with patent ductus arteriosus. Circulation 110: 1372-1379.
- Yokoyama U, Minamisawa S, Adachi-Akahane S, Akaike T, Naguro I, et al. (2006) Multiple transcripts of Ca2+ channel alpha1-subunits and a novel spliced variant of the alpha1Csubunit in rat ductus arteriosus. Am J Physiol Heart Circ Physiol 290: H1660-H1670.
- Akaike T, Jin MH, Yokoyama U, Izumi-Nakaseko H, Jiao Q, et al. (2009) T-type Ca2+ channels promote oxygenation-induced closure of the rat ductus arteriosus not only by vasoconstriction but also by neointima formation. J Biol Chem 284: 24025-24034.
- Arcilla RA, Thilenius OG, Ranniger K (1969) Congestive heart failure from suspected ductal closure in utero. J Pediatr 75: 74-78.
- Zuckerman H, Reiss U, Rubinstein I (1974) Inhibition of human premature labor by indomethacin. Obstet Gynecol 44: 787-792.
- Coceani F, Olley PM (1973) The response of the ductus arteriosus to prostaglandins. Can J Physiol Pharmacol 51: 220-225.
- Coceani F, Olley PM, Bodach E (1975) Lamb ductus arteriosus: effect of prostaglandin synthesis inhibitors on the muscle tone and the response to prostaglandin E2. Prostaglandins 9: 299-308
- Sharpe GL, Thalme B, Larsson KS (1974) Studies on closure of the ductus arteriosus. XI. Ductal closure in utero by a prostaglandin synthetase inhibitor. Prostaglandins 8: 363-368.
- Starling MB, Elliott RB (1974) The effects of prostaglandins, prostaglandin inhibitors, and oxygen on the closure of the ductus arteriosus, pulmonary arteries and umbilical vessels in vitro. Prostaglandins 8: 187-203.
- Friedman WF, Hirschklau MJ, Printz MP, Pitlick PT, Kirkpatrick SE (1976) Pharmacologic closure of patent ductus arteriosus in the premature infant. N Engl J Med 295: 526-529.
- Heymann MA, Rudolph AM, Silverman NH (1976) Closure of the ductus arteriosus in premature infants by inhibition of prostaglandin synthesis. N Engl J Med 295: 530-533.
- Coceani F, Olley PM, Bishai I, Bodach E, Heaton J, et al. (1977) Prostaglandins and the control of muscle tone in the ductus arteriosus. Adv Exp Med Biol 78: 135-142.
- Coceani F, White E, Bodach E, Olley PM (1979) Age-dependent changes in the response of the lamb ductus arteriosus to oxygen and ibuprofen. Can J Physiol Pharmacol 57: 825-831
- Patel J, Marks KA, Roberts I, Azzopardi D, Edwards AD (1995) Ibuprofen treatment of patent ductus arteriosus. Lancet 346: 255.
- Van Overmeire B, Follens I, Hartmann S, Creten WL, Van Acker KJ (1997) Treatment of patent ductus arteriosus with ibuprofen. Arch Dis Child Fetal Neonatal Ed 76: F179-F184.
- Varvarigou A, Bardin CL, Beharry K, Chemtob S, Papageorgiou A, et al. (1996) Early ibuprofen administration to prevent patent ductus arteriosus in premature newborn infants. Jama 275: 539-544.
- Trivedi DB, Sugimoto Y, Loftin CD (2006) Attenuated cyclooxygenase-2 expression contributes to patent ductus arteriosus in preterm mice. Pediatr Res 60: 669-674. Epub 2006 Oct 26.
- Coceani F, Barogi S, Brizzi F, Ackerley C, Seidlitz E, et al. (2005) Cyclooxygenase isoenzymes and patency of ductus arteriosus. Prostaglandins Leukot Essent Fatty Acids 72: 71-77.
- Loftin CD, Trivedi DB, Langenbach R (2002) Cyclooxygenase-1-selective inhibition prolongs gestation in mice without adverse effects on the ductus arteriosus. J Clin Invest 110: 549-557
- Reese J, Anderson JD, Brown N, Roman C, Clyman RI (2006) Inhibition of cyclooxygenase isoforms in late- but not midgestation decreases contractility of the ductus arteriosus and prevents postnatal closure in mice. Am J Physiol Regul Integr Comp Physiol 29: R1717-R1723.
- Rheinlaender C, Weber SC, Sarioglu N, Strauss E, Obladen M (2006) Changing expression of cyclooxygenases and prostaglandin receptor EP4 during development of the human ductus arteriosus. Pediatr Res 60: 270-275.
- Moise KJ (1993) Effect of advancing gestational age on the frequency of fetal ductal constriction in association with maternal indomethacin use. Am J Obstet Gynecol 168: 1350-1353
- Vermillion ST, Scardo JA, Lashus AG, Wiles HB (1997) The effect of indomethacin tocolysis on fetal ductus arteriosus constriction with advancing gestational age. Am J Obstet Gynecol 177: 256-259.
- Groom KM, Shennan AH, Jones BA, Seed P, Bennett PR (2005) TOCOX--a randomised,
- double-blind, placebo-controlled trial of rofecoxib (a COX-2-specific prostaglandin inhibitor) for the prevention of preterm delivery in women at high risk. Bjog 112: 725-730.
- Clyman RI (2006) Mechanisms regulating the ductus arteriosus. Biol Neonate 89: 330-335. Epub 2006 Jun 1.
- Hammerman C, Glaser J, Kaplan M, Schimmel MS, Ferber B, et al. (1998) Indomethacin tocolysis increases postnatal patent ductus arteriosus severity. Pediatrics 102: E56.
- Norton ME, Merrill J, Cooper BA, Kuller JA, Clyman RI (1993) Neonatal complications after the administration of indomethacin for preterm labor. N Engl J Med 329: 1602-1607.
- Kajino H, Taniguchi T, Fujieda K, Ushikubi F, Muramatsu I (2004) An EP4 receptor agonist prevents indomethacin-induced closure of rat ductus arteriosus in vivo. Pediatr Res 56: 586-590.
- Smith GC, Wu WX, Nijland MJ, Koenen SV, Nathanielsz PW (2001) Effect of gestational age, corticosteroids, and birth on expression of prostanoid EP receptor genes in lamb and baboon ductus arteriosus. J Cardiovasc Pharmacol 37: 697-704.
- Waleh N, Kajino H, Marrache AM, Ginzinger D, Roman C, et al. (2004) Prostaglandin E2--mediated relaxation of the ductus arteriosus: effects of gestational age on g proteincoupled receptor expression, signaling, and vasomotor control. Circulation 110: 2326-2332.
- Leonhardt A, Glaser A, Wegmann M, Schranz D, Seyberth H, et al. (2003) Expression of prostanoid receptors in human ductus arteriosus. Br J Pharmacol 138: 655-659.
- Fan F, Ma A, Guan Y, Huo J, Ju Z, et al. (2011) Effect of PGE2 on DA tone by EP4 modulating Kv channels with different oxygen tension between preterm and term. Int J Cardiol 147: 58-65.
- Kim SH, Kim YK, Park HW, Jee YK, Kim SH, et al. (2007) Association between polymorphisms in prostanoid receptor genes and aspirin-intolerant asthma. Pharmacogenet Genomics 17: 295-304.
- Libioulle C, Louis E, Hansoul S, Sandor C, Farnir F, et al. (2007) Novel Crohn disease locus identified by genome-wide association maps to a gene desert on 5p13.1 and modulates expression of PTGER4. PLoS Genet 3: e58. Epub 2007 Mar 5.
- Hornblad PY (1967) Studies on closure of the ductus arteriosus. 3. Species differences in closure rate and morphology. Cardiologia 51: 262-282.
- Slomp J, van Munsteren JC, Poelmann RE, de Reeder EG, Bogers AJ, et al. (1992) Formation of intimal cushions in the ductus arteriosus as a model for vascular intimal thickening. An immunohistochemical study of changes in extracellular matrix components. Atherosclerosis 93: 25-39
- Tada T, Kishimoto H (1990) Ultrastructural and histological studies on closure of the mouse ductus arteriosus. Acta Anat (Basel) 139: 326-334.
- Camenisch TD, Spicer AP, Brehm-Gibson T, Biesterfeldt J, Augustine ML, et al. (2000) Disruption of hyaluronan synthase-2 abrogates normal cardiac morphogenesis and hyaluronan-mediated transformation of epithelium to mesenchyme. J Clin Invest 106: 349-360
- Matsumoto K, Li Y, Jakuba C, Sugiyama Y, Sayo T, et al. (2009) Conditional inactivation of Has2 reveals a crucial role for hyaluronan in skeletal growth, patterning, chondrocyte maturation and joint formation in the developing limb. Development 136: 2825-2835.
- Yokoyama U, Minamisawa S, Quan H, Akaike T, Suzuki S, et al. (2008) Prostaglandin E2-activated Epac promotes neointimal formation of the rat ductus arteriosus by a process distinct from that of cAMP-dependent protein kinase A. J Biol Chem 283: 28702-28709. Epub 2008 Aug 11.
- Echtler K, Stark K, Lorenz M, Kerstan S, Walch A, et al. (2010) Platelets contribute to postnatal occlusion of the ductus arteriosus. Nat Med 16: 75-82. Epub 2009 Dec 6.
- Dwarakanath K, Dereddy NR, Chabra D, Schabacker C, Calo J, et al. (2011) Spontaneous and pharmacological closure of PDAs in ELBW Infants Is influenced by thrombocytopenia E-PAS 21.
- Fujioka K, Morioka I, Miwa A, Morikawa S, Shibata A, et al. (2011) Does thrombocytopenia contribute to patent ductus arteriosus? Nat Med 17: 29-30; author reply 30-21.
- Shah NA, Hills NK, Waleh N, McCurnin D, Seidner S, et al. (2011) Relationship between circulating platelet counts and ductus arteriosus patency after indomethacin treatment. J Pediatr 158: 919-923 e911-912.
- Boo NY, Mohd-Amin I, Bilkis AA, Yong-Junina F (2006) Predictors of failed closure of patent ductus arteriosus with indomethacin. Singapore Med J 47: 763-768.
- Andrew M, Vegh P, Caco C, Kirpalani H, Jefferies A, et al. (1993) A randomized, controlled trial of platelet transfusions in thrombocytopenic premature infants. J Pediatr 123: 285-291.
- Sallmon H, Weber SC, von Gise A, Koehne P, Hansmann G (2011) Ductal closure in neonates: a developmental perspective on platelet-endothelial interactions. Blood Coagul Fibrinolysis 22: 242-244.
- Bussel JB, Zacharoulis S, Kramer K, McFarland JG, Pauliny J, et al. (2005) Clinical and diagnostic comparison of neonatal alloimmune thrombocytopenia to non-immune cases of thrombocytopenia. Pediatr Blood Cancer 45: 176-183