Mechanisms for Ductus Arteriosus Closure

Flavio Coceani, MD, and Barbara Baragatti, BSc

Closure of the ductus arteriosus at birth is a complex phenomenon being conditioned by
antenatal events and progressing in preprogrammed steps. Functional at rst, narrowing of
the vessel is determined by 2 overlapping processesremoval of the prostaglandin E
2
-
based relaxation sustaining prenatal patency and activation of a constrictor mechanism by
the natural rise in blood oxygen tension. Two schemes have been proposed for oxygen
actionone involving a cytochrome P450 hemoprotein (sensor)/endothelin-1 (effector)
complex and the other a set of voltage-gated K

channels. These proposals, however, are

not mutually exclusive. Structural closure follows the constriction through a remodeling
process initiated antenatally with the development of intimal cushions and completed
postnatally by a host of humoral and mechanical stimuli. Research in this area has already
provided clinical applications. Nevertheless, management of premature infants with per-
sistent ductus remains troublesome and calls for an alternative approach to the prosta-
glandin E
2
inhibitors now in use. Studies in progress on the oxygen-sensing system may
lead to a denitive solution for this problem.
Semin Perinatol 36:92-97 2012 Elsevier Inc. All rights reserved.
KEYWORDS: ductus arteriosus, fetal physiology, neonatal physiology, oxygen sensing
T
hrough the years, the ductus has attracted a great deal of
attention for its forceful contraction to oxygen, the
abrupt closure at birth coinciding with the onset of lung
ventilation, and the importance of its demise for the well-
being of the neonate. Accordingly, research focused on the
mechanism for postnatal closure, assuming at rst that no
other active control would operate in the vessel. However,
the discovery that prenatal patency is also actively main-
tained introduced a new dimension in the approach to this
problem. In fact, the mechanism of closure acquired greater
complexity when it was realized that events originating ante-
natally could have an impact on postnatal adjustments. A
fascinating story then unfolded, with a host of factors being
implicated in the closure of the duct. Several questions still
remain outstanding, and while elaborating on them, our aim
will be to dene a framework for future research.
Conventionally, the process of ductus closure is divided in
2 sequential stepsfunctional and structuraland the same
distinction will be maintained here for better clarity. How-
ever, one will realize that these stages overlap when noting
that causative agents may be shared and certain transitional
adjustments progress without any obvious interruption.
Functional Closure
Under normal conditions, the ductus arteriosus constricts
immediately after birth, coincident with the physiological
rise in blood oxygen tension and the cessation of placental
function with attendant loss of a major source for prostaglan-
din E
2
(PGE
2
). PGE
2
is a potent relaxant of the vessel and a
prime determinant for its patency in utero.
1
It follows that 2
eventsdirect action of oxygen and withdrawal of PGE
2
-
induced relaxationcontribute synergistically to the con-
striction, hence functional closure, of the ductus, and they
will be considered separately.
Direct Action of Oxygen
The ductus muscle is peculiarly sensitive to oxygen and can
translate the stimulation from this agent into a sustained
contraction. The oxygen sensor recalls in its arrangement a
true receptor inasmuch as it (i) shows uneven distribution,
within and without the vasculature, with a few blood vessels
mimicking the ductus
2
; (ii) is developmentally regulated over
a timeframe not coinciding with the maturation of contractile
function
3,4
; (iii) may undergo desensitization on extended
exposure to the agent
5
; and (iv) is liable to selective activation
by certain compounds (eg, retinoic acid).
3,4
Institute of Life Sciences, Scuola Superiore SantAnna, Pisa, Italy.
Supported in part by Italian Ministry of Education and Research, Grant PRIN
2007E7Y7R.
Address reprint requests to Flavio Coceani, MD, Institute of Life Sciences,
Scuola Superiore SantAnna, Piazza Martiri della Libert 33, 56127 Pisa,
Italy. E-mail: coceani@sssup.it
92 0146-0005/12/$-see front matter 2012 Elsevier Inc. All rights reserved.
doi:10.1053/j.semperi.2011.09.018
Two main schemes have been proposed for the oxygen-
induced contraction of ductal muscle and, as discussed later,
they are not mutually exclusive. Scheme 1 originates from 2
outwardly distant lines of investigation involving, respec-
tively, the cytochrome P450 (CYP450) system and endothe-
lin-1 (ET-1), which dovetail into a novel operational model.
Supporting data are listed in Table 1. In brief, from the evi-
dence accrued it is postulated that a CYP450 of the 3A sub-
family, identied with CYP3A13 in the mouse, acts as the
main oxygen sensor and links with ET-1, serving as the ef-
fector for the sustained contraction.
4
ET-1, however, may
also become an accessory target for oxygen whenever, as it
happens in the mouse, the response includes phasic changes
in tone.
4
Hence, the model emerging from these studies en-
visages a partially different arrangement depending on the
character of the oxygen response. With the thin-walled duc-
tus of the mouse, where phasic discharges are usually super-
imposed on the tonic contraction, oxygen has a dual site of
action: a primary one on CYP450 (for tonic contraction) and
a secondary on ET-1 (for phasic activity). Conversely, in any
species not exhibiting a phasic component in the response
(eg, lamb), oxygen action is expectedly limited to the CYP450
target. In either case, however, the nature of the linkage be-
tween CYP450 and ET-1 remains to be dened, although
several data assign to the sensing hemoprotein a catalytic
function in a monooxygenase reaction (Table 1). Further-
more, in a recent development, convincing evidence has been
obtained pointing to a concerted interaction between 2 path-
ways for arachidonic acid metabolismCYP450-based ep-
oxygenase and 12(S)-lipoxygenasein the generation of this
hypothetical coupling agent.
20
Congruent with this possibil-
ity is also the marked upregulation of 12(S)-lipoxygenase in
the ductus at birth.
14
If conrmed, this arrangement would
(re)assert the central position of the eicosanoid system in
ductus regulation, with the relaxant PGE
2
prevailing prena-
tally, and an epoxygenase/lipoxygenase product taking over
postnatally.
In a separate line of investigation (Scheme 2), oxygen sens-
ing has been identied with a mitochondrial redox mecha-
nism, being located in ductus muscle and generating a reac-
tive oxygen species in a stimulus-dependent fashion. The
redox signal is thought to produce its effect by inhibiting a
distinct set of voltage-gated K

(K
v
) channels (ie, K
v
1.5 and
K
v
2.1) and causing, via the attendant depolarization, an ac-
tivation of voltage-dependent L-type and T-type Ca
2
chan-
nels.
21-24
In addition, oxygen is assigned a direct, accessory
action on the L-type Ca
2
channels.
23
Table 2 demonstrates
the evidence in support of this alternative oxygen-sensing
mechanism.
Potential weaknesses or apparent inconsistencies have
been agged with either proposal. For Scheme 1, a role of
ET-1 as oxygen mediator has been questioned because cer-
tain studies could not conrm either an increase in peptide
synthesis with oxygen tension or the curtailment of the oxy-
gen contraction with a peptide antagonist.
25,26
Leaving aside
the fact that this intermediary function for ET-1 has been
veried in several laboratories,
16-19
results purportedly con-
tradicting this concept are arguable on methodological
grounds.
4,11
By contrast, the redox scheme (Scheme 2), with
the attendant role being assigned to K
v
inhibition, is weak-
ened because oxygen is still effective in the presence of K

depolarizing solutions.
2,18
Furthermore, the mitochondrial
target may not be readily accessible to extracellular oxygen.
Despite the perceived difculties, the 2 proposals rest on a
solid body of evidence and, furthermore, have certain fea-
tures in common. Both mechanisms are liable to activation by
glucocorticoids and retinoic acid.
4,27
Glucocorticoids are par-
ticularly important for the natural maturation of oxygen sens-
ing and, in fact, their absence is regarded as a risk factor for
persistent ductus patency in the prematurely born infant.
27,28
Their facilitatory action, originally ascribed to interference
with PGE
2
,
29,30
may also involve the ET-1
31,32
and K
v
chan-
nel
27
systems. Another common element is found in the Rho/
Rho-kinase pathway, with the plasmalemmal RhoB protein
33
playing a key role in the oxygen response.
14,22
In the end, it is
plausible to think that the 2 mechanisms coexist, and in some
way interact, in the oxygen activation process. A possibility
would be that ET-1 exerts some of its effect through the
inhibition of K

channel function.
34
Alternatively, the 2
Table 1 Data Implicating a CYP450/ET-1 Complex in the Duc-
tus Contraction to OxygenScheme 1
CYP450
Any interference with CYP450 function curtails the
contraction to oxygen.
6
1-Aminobenzotriazole, a
suicide substrate for CYP450-based reactions, is also
effective in this respect
7
A CYP450 hemoprotein of the 3A subfamily, specically
CYP3A13 in the mouse, mediates the oxygen
response
4,8
A CYP3A hemoprotein is present in the plasma
membrane of ductus muscle cells and is, accordingly,
optimally positioned to sense oxygen changes in the
extracellular milieu
4,8,9
ET-1
ET-1 is a singularly potent ductus constrictor acting via
the ET
A
receptor subtype
10,11
ET-1 is present in ductal muscle and its synthesis
increases with oxygen tension
12,13
Ductal ET-1 is upregulated at birth
14,15
Oxygen contraction subsides when interfering with ET-1
function by pharmacologic means or through deletion
of the ET
A
receptor subtype
11,12,16-19
Table 2 Data Implicating a Redox-Linked Inhibition of K
v
Channels in the Ductus Contraction to OxygenScheme 2
Oxygen contraction subsides when interfering with redox
signaling and K

channel activity
2,5
Susceptibility to oxygen is identied with the operation of
distinct, voltage-gated K

channels (ie, K
v
1.5 and K
v
2.1)
21,23
Putative oxygen-sensing K
v
channels are developmentally
regulated.
21,23
Transfection of the same channels
promotes maturation of oxygen-sensing function
21,23
Mechanisms for ductus arteriosus closure 93
mechanisms could serve distinct purposes in a single se-
quence, wherein ET-1 activation precedes K
v
inhibition.
35
Whatever the answer, it is important to note that deletion of
the CYP3A complex promotes the appearance of an alterna-
tive oxygen-sensing mechanism not identiable with ET-1
(K
v
-based?).
4
The latter nding points to a potential for com-
pensation among allied mechanisms and, by inference, to the
feasibility of their arrangement changing in response to phys-
iological or pathophysiological challenges.
Withdrawal of PGE
2
Relaxation
Originally viewed only as a determinant of prenatal ductus
patency, PGE
2
has acquired increasing importance in the
process of closure. In fact, even before birth there are PGE
2
-
linked processes, both functional and structural, that prepare
the vessel for this transitional change. On the functional side,
there is an upsurge in blood PGE
2
toward the end of gestation
preceding an abrupt fall immediately after birth.
36
This sud-
den loss of exposure to a high level of PGE
2
, coinciding with
a reduced effectiveness of the compound,
37-40
represents a
singularly potent stimulus for ductus closure. In its absence,
as it happens with the deletion of mechanisms responsible for
PGE
2
metabolism,
41,42
the ductus fails to close, despite the
presence of a viable, oxygen-triggered constrictor system.
This peculiar lack of compensation to PGE
2
withdrawal
stands in contrast to the nding of a normal closure when the
duct is missing the ET
A
receptor subtype of ET-1 and, hence,
the capability to contract to oxygen.
11
Other Mechanisms for Ductus Closure
Any elaboration on additional constrictor agents for ductus
closure is necessarily speculative. Nevertheless, a special
place is occupied by angiotensin II (Ang II), its possible role
in this process has been heralded by the observation, made
some years ago, that a synthesis inhibitor reopens the closing
duct of the neonate.
43
Although of interest, that nding could
not rule out an unspecic response due to the rebound in-
crease in relaxant bradykinin overriding in its action the ex-
pected subsidence of constrictor Ang II.
43
Bradykinin would
certainly be effective in any such instance because it may
dilate the ductus by several means.
44
However, the idea of a
specic response to the inhibitor has gained more strength
with the demonstration, rst, that Ang II is potent as ductus
constrictor and, second, that the Ang II type 1 receptor un-
dergoes a selective upregulation at birth.
14
Signicantly, the
latter occurrence is not linked causally with oxygen stimula-
tion, and this feature distinguishes Ang II fromET-1, which is
instead closely dependent on oxygen for its action (Table
1).
14
Germane to ndings in the animal is the observation
that premature infants, with an enhanced potential for con-
striction to Ang II deriving from a particular polymorphism
of its type 1 receptor, are less prone to develop a persistent
duct.
45
The arachidonic acid -hydroxylation product, 20-hy-
droxyeicosatetraenoic acid, has been implicated as an inter-
mediary in the constriction of the systemic microvasculature
to oxygen.
46
The same mechanism, however, could not be
found in the ductus.
20
Ductus Closure Under Hypoxia
Outwardly at odds with established concepts, the ductus may
also close, albeit more slowly, in the absence of oxygen stim-
ulation. Exemplary in this respect are infants with obstructive
right heart malformations, and attendant cyanosis, in whom
the closing duct becomes an emergency condition and a
prime indication for the lifesaving use of PGE
2
(or PGE
1
).
47
An experimental correlate of the clinical situation is afforded
by newborn guinea pigs, reared in a low oxygen environ-
ment, whose duct does not remain open for more than 3-4
days.
48
A plausible cause for closure may be found among the
mechanisms so far discussed. The decrease in blood PGE
2
levels, taking place in the hypoxic as in the well-oxygenated
newborn, is undoubtedly a prime factor. However, Ang II
may also have a role because its expected greater effectiveness
after birth is not conditioned by oxygen.
14
The latter possi-
bility warrants further investigation.
Structural Closure
Remodeling of the ductus for its permanent closure is a com-
plex process, starting antenatally with a preset programming
and involving a host of humoral and mechanical stimuli.
Intertwined with functional closure, with which it shares
certain agents, structural closure may encompass up to 4
distinct mechanisms of varying impact depending on the
species. They are the following: (i) development of intimal
cushions, (ii) mechanical solicitation from turbulent blood
owalong the narrowing lumen, (iii) intramural hypoxia due
to the collapse of vasa vasorum in the constricting ductus,
and (iv) interaction of platelets with the vessel wall.
Intimal Cushions
Intimal ingrowth forming cushion-like structures is critical
for permanent closure of the ductus in certain species.
49
Starting prenatally during the last third of gestation, it is
completed after birth coincidentally with the constriction of
the vessel. PGE
2
plays a key role in 2 ways, both being cAMP-
dependent, that is, by eliciting the deposition of hyaluronic
acid within the intimal tissue via protein kinase A and by
stimulating the inward migration of smooth muscle cells via
the signaling protein Epac.
50,51
Hyaluronic acid, in turn,
magnies PGE
2
action by promoting cell migration.
50
Coin-
cidentally, in what appears to be a self-reinforcing loop, mus-
cle cells secrete the glycoprotein bronectin, promoting fur-
ther their drive for migration.
49
Two other factorsthe
glycoprotein laminin and growth hormonemay also inter-
vene in this process.
52,53
Intimal cushion growth persists
postnatally through the residual inuence of PGE
2
and,
moreover, through the oxygen-induced activation of T-type
Ca
2
channels.
24
An open question is whether ET-1, besides
its involvement in functional closure of the ductus, has also
an impact on tissue remodeling. Available knowledge of the
compound would favor this possibility.
94 F. Coceani and B. Baragatti
Shear Stress Stimulation
Mechanical solicitation secondary to alteration of blood ow
in the closing ductus promotes a host of factors for remodel-
ing.
14
Among them, particular importance is ascribed to one,
or more than one, integrin.
54,55
In addition, a contribution
may come from Ang II through its dual (constrictor and
trophic) action and the mechanosensitive properties of its
type 1 receptor.
14,56,57
Intramural Hypoxia
Oxygen presents a steep gradient across the wall of the duc-
tus.
58,59
Hence, when the wall exceeds a critical width,
60
the
presence of a viable vasa vasorum system becomes essential
for the maintenance of adequate tissue oxygenation. Postna-
tal constriction, with the attendant collapse of the intramural
vessels, disrupts this subtle equilibrium and promotes the
trophic action of hypoxia-inducible agents, such as the vas-
cular endothelial cell growth factor (VEGF) and transforming
growth factor-.
61,62
Together, VEGF and transforming
growth factor- stimulate endothelial cell proliferation and
the migration of muscle cells toward the intimal layer.
Platelet-Vessel Wall Interaction
A new mechanism for permanent closure of the ductus has
recently been introduced by documenting the importance of
a competent platelet population in initiating tissue remodel-
ing.
63
In the proposed scheme, the adhesion of platelets to the
luminal surface of the closing duct along with the local clus-
tering of monocytes/macrophages and, to a lesser degree, of T
lymphocytes is critical. Hence, a drive for closure results from
the concerted contribution of muscle contraction, tissue re-
modeling, and sealing of the narrowing lumen by aggregated
cells. Relevant to this process is the observation, made in an
earlier study, of an upregulation of platelet-endothelial cell
adhesion molecule-1 and platelet selectin taking place in the
neonatal ductus.
14
Redundancy of effector mechanisms for structural closure
of the ductus is mitigated by their uneven expression across
species. In the thin-walled vessel of rodents, intimal cushions
are replaced by a sheer thickening of the innermost layer, and
the vasa vasorum are also absent. It follows that the hypoxic
challenge is likely minimal, if not missing, and closure de-
pends primarily on shear stress stimulation and the interac-
tion of platelets with the luminal surface. Conversely, at the
other end of the scale, in the thick-walled ductus of a large
animal, such as the lamb, and by extension of humans, all
mechanisms come into play, with the possible exception of
the platelet-triggered process whose involvement in this case
is being questioned.
64,65
However, regardless of the relative
impact of individual mechanisms, the whole sequence un-
folds in a preset order.
1,66
Direct demonstration of this pro-
gramming comes also from experiments in which neonatal
oxygenation was reproduced in utero, and, despite the ensu-
ing ductus constriction, no transcript modication was noted
attesting to a tissue remodeling in progress.
14
As it happens with functional closure, distinct aspects of
ductus remodeling are also amenable to pharmacologic ma-
nipulation. In particular, retinoic acid promotes transcript
expression, prenatally for bronectin and hyaluronic acid
synthase and postnatally for certain integrins and VEGF.
15
Signicantly, the antenatal gene change is associated with an
accelerated formation of the structure (ie, intimal thickening)
replacing in rodents the intimal cushions of larger animals.
15
Clinical Implications
Advances in this eld open new perspectives for the clinic,
specically in the management of the premature infant with
persistent ductus. In fact, despite considerable effort through
the years, this condition remains troublesome, and its current
treatment, based on PGE
2
suppression by a cyclooxygenase
inhibitor (indomethacin or ibuprofen), is far from optimal
due to possible side effects and frequent failures.
67
Reasons
for the unsatisfactory outcome are multifold and relate to the
presence in the ductus of several relaxing mechanisms poten-
tially amenable to reciprocal compensation,
1,44
the possibility
of cyclooxygenase inhibition promoting nitric oxidebased
relaxation,
68
and the very nature of PGE
2
action comprising
opposing inuences on the natural evolution of the ves-
sel.
1,50,51
A better result is expected from the recently devel-
oped inhibitors of the main PGE synthase within the ductus
(ie, microsomal PGE synthase-1) because they are more se-
lective and are also free of any enhancing effect on the nitric
oxide system.
69
However, experimental data, obtained so far
with one such compound, are inconclusive.
69
Although the
therapeutic potential of microsomal PGE synthase-1 inhibi-
tors needs to be investigated further, a more direct approach
to the problem is also warranted, exploiting the growing
knowledge of the functional organization of the oxygen-sens-
ing system. In fact, the eld is ready for new lines of investi-
gation having as an ultimate aim the design of therapeutic
tools that favor the natural, oxygen-triggered process of clo-
sure. After all, the receptor-like arrangement being per-
ceived for oxygen sensing carries in itself the prerequisite for
a targeted intervention. For example, a better insight into the
mode of action of retinoic acid in promoting the susceptibil-
ity of the ductus to oxygen could provide some important
clue in this respect. Equally rewarding might be the charac-
terization of the putative messenger linking the CYP450-
based oxygen sensor with ET-1.
20
Closely intertwined with
any such approach would also be a reappraisal of glucocor-
ticoid action to fully dene its prime target in increasing the
drive for ductus constriction. In brief, several leads may be
followed in the search of a selective, mechanism-based treat-
ment of the persistent ductus complicating prematurity.
Without doubt, the stage is set for a new chapter in this
appealing story.
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