Changes in vasoactive pathways in congenital diaphragmatic hernia associated pulmonary hypertension explain unresponsiveness to pharmacotherapy
Mous et al. Respiratory Research
Changes in vasoactive pathways in congenital diaphragmatic hernia associated pulmonary hypertension explain unresponsiveness to pharmacotherapy
Daphne S. Mous 1
Marjon J. Buscop-van Kempen 1
Rene M. H. Wijnen 1
Dick Tibboel 1
Robbert J. Rottier 0 1
0 Department of Cell Biology, Erasmus Medical Center , Rotterdam , The Netherlands
1 Department of Pediatric Surgery, Erasmus Medical Center, Sophia Children's Hospital , Wytemaweg 80, 3015 CN, PO Box 2040, Rotterdam , The Netherlands
Background: Patients with congenital diaphragmatic hernia (CDH) have structural and functional different pulmonary vessels, leading to pulmonary hypertension. They often fail to respond to standard vasodilator therapy targeting the major vasoactive pathways, causing a high morbidity and mortality. We analyzed whether the expression of crucial members of these vasoactive pathways could explain the lack of responsiveness to therapy in CDH patients. Methods: The expression of direct targets of current vasodilator therapy in the endothelin and prostacyclin pathway was analyzed in human lung specimens of control and CDH patients. Results: CDH lungs showed increased expression of both ETA and ETB endothelin receptors and the rate-limiting Endothelin Converting Enzyme (ECE-1), and a decreased expression of the prostaglandin-I2 receptor (PTGIR). These data were supported by increased expression of both endothelin receptors and ECE-1, endothelial nitric oxide synthase and PTGIR in the well-established nitrofen-CDH rodent model. Conclusions: Together, these data demonstrate aberrant expression of targeted receptors in the endothelin and prostacyclin pathway in CDH already early during development. The analysis of this unique patient material may explain why a significant number of patients do not respond to vasodilator therapy. This knowledge could have important implications for the choice of drugs and the design of future clinical trials internationally.
Nitric oxide; Endothelin; Prostacyclin; Development; Lung; Vasculature; Vasodilation
Pulmonary hypertension (PH) is the leading cause of
morbidity and mortality in patients with congenital
diaphragmatic hernia (CDH) [
]. The altered development
of the pulmonary vasculature and the disordered
pulmonary vascular remodeling [
] in combination with the
imbalance of vasoactive mediators caused by endothelial
dysfunction result in the arrest of pulmonary vascular
growth in these patients. Current treatment of CDH
patients is not evidence based [
] and is derived from
studies in adults, leading mainly to off-label and
unlicensed use of drugs. Current knowledge is based on
compassionate use and case reports, while some patients
with CDH were included in trials that were
underpowered for definitive conclusions. Even international
therapy guidelines are based on consensus only (level 3
]. In 2012, experts evaluated the current
antenatal and postnatal management of CDH and
emphasized the importance of optimal management of PH
in these patients [
]. Worldwide, PH treatment is mainly
directed against the receptors of the endothelin (ET)
and prostacyclin (PGI2) pathways or the conversion of
cyclic guanosine monophosphate (cGMP) in the nitric
oxide (NO) pathway (Fig. 1a). In spite of these targeted
treatments, it is still largely unknown how the different
Fig. 1 Three major pathways involved in vasodilation and vasoconstriction. a Schematic overview of the major pathways and the key proteins
involved in vasodilation and vasoconstriction. b Aberrant expression of key factors in the three pathways in both human and rat congenital
diaphragmatic hernia (CDH). Solid arrows represent up- or downregulation in human CDH, dashed arrows represent up- or downregulation in rat
CDH. ECE-1 = endothelin converting enzyme 1, ETA = endothelin A, ETB = endothelin B, eNOS = endothelial nitric oxide synthase, sGC = soluble
guanylate cyclase, COX = cyclooxygenase, PGIS = prostaglandin synthase, PGI2 = prostaglandin I2, AC = adenylate cyclase, TBXAS1 = thromboxane
synthase, TXA2 = thromboxane
components of these pathways are expressed in lungs of
unaffected individuals and CDH patients.
Previous studies reported increased levels of both the
endothelin A (ETA) and B (ETB) receptors in human
CDH as well as in the nitrofen rat model [
Endothelin-1 (ET-1) is a potent vasoconstrictor [
is increased in lung tissue of patients with pulmonary
hypertension. Moreover, high plasma levels of circulating
ET-1 associated with the severity of PH in human CDH
]. NO reduces the affinity of the ETA receptor for
ET1 and may therefore terminate the ET-1 mediated
]. NO is synthesized by different NO synthases
(NOS): endothelial NOS (eNOS), inducible NOS (iNOS)
and neuronal NOS (nNOS), which are all expressed in
the lung. However, only eNOS and, to some extent,
iNOS, are expressed in the pulmonary vasculature and
modulate pulmonary vascular tone [
human and rat CDH studies showed a decrease in eNOS
]. However, we and others showed either no
differences, or even an increased expression of eNOS in
both human and rat CDH [
]. PGI2 is an important
mediator of vasodilation, acting through the
prostaglandin-I2 receptor (PTGIR) [
prostacyclin receptor agonists have been used in the treatment
of persistent pulmonary hypertension of the newborn
with variable effects [
]. Limited data are available
about the use of these drugs in patients with CDH, but
the few available case reports show contrasting results
]. An overview of the current data for human and
the rat model is provided (Tables 1 and 2).
Since CDH patients respond poorly to current
treatment strategies, we hypothesized that these effects might
be due to an aberrant expression of important vasoactive
factors. Here, we are the first to analyze the expression
of the direct targets of the most commonly used
vasodilator drugs, as well as some of the important members
of the three major vasoactive pathways. Using unique
patient lung material, we show an increased expression
of both endothelin receptors and the rate-limiting
endothelin converting enzyme (ECE-1), as well as a
decreased expression of the prostaglandin-I2 receptor in
human CDH. Moreover, we found changes in the
expression of these and other important factors of the
pathways in rat CDH (Fig. 1b).
Human lung samples
Human lung samples were retrieved from the archives
of the Department of Pathology of the Erasmus Medical
Center, Rotterdam. In our high-volume, leading center
of the EURO consortium [
], approximately 15 to 20
CDH patients a year are born, which ensures a large
experience in the treatment of this disease.
Paraffinembedded lung samples, lacking severe hemorrhage or
necrosis, were selected of controls, of CDH patients and
of patients with lung hypoplasia or pulmonary
hypertension unrelated to CDH. Only lung material of patients
with a severe left-sided CDH and a survival of less than
7 h were selected to prevent secondary sequelae. Patient
characteristics are described in Table 3.
The well-established animal model was used, where in
short pregnant Sprague-Dawley rats received either
100 mg nitrofen dissolved in 1 ml olive oil or just 1 ml
olive oil by gavage on gestational age day E9.5 [
Nitrofen induces left-sided CDH in approximately 70%
of the offspring, while all pups have pulmonary
hypertension. At day E21 pups were delivered by
caesarean section and euthanized by lethal injection of
Immunohistochemistry (IHC) was performed on 5 μm
thick paraffin sections of lungs of both rats and
humans according to standard protocols, using the
Envision™ detection system (Dako Cytomatic,
Glostrup, Denmark) [
]. Briefly, sections were
deparaffinized with xylene and rehydrated in gradual
series of ethanol, after which antigen retrieval was
performed by boiling samples in 10 mM Tris
(pH 9.0), 1 mM EDTA. Primary and Horse Redox
Peroxidase conjugated secondary antibodies were
diluted in antibody dilution buffer (DAKO) with 0,5%
Tween-20, and the peroxidase was detected with
diamino-benzidine tetrahydrochloride (Fluka, Buchs,
Switzerland). Validated primary antibodies used for
IHC were Endothelin receptor A (ETA; 1:5000 (rat)
1:100 (human); Alamone, Jerusalem, Israel),
Endothelin receptor B (ETB; 1:2500 (rat) 1:500 (human);
Alamone), Endothelin Converting Enzyme (ECE-1;
1:500 (human); Abcam, Cambridge, MA, USA),
endothelial nitric oxide synthase (eNOS; 1:400 (rat);
Thermo Fisher Scientific, Waltham, MA, USA) and
prostaglandin-I2 receptor (Ptgir; 1:1000 (rat) 1:500
(human); Cayman Chemical, Ann Arbor, Michigan,
USA). Negative controls were performed by omitting
the primary antibody.
Quantitative real-time polymerase chain reaction (qPCR)
RNA isolation of whole lungs of rat pups, cDNA
synthesis and subsequent qPCR analysis was performed as
previously described [
]. The gene-specific primers used
are listed in Table 4. Actb was used as reference gene.
Data are presented as means (SD) for normally
distributed variables. Univariate analyses were performed
using independent samples t-tests for normally
distributed variables. The analyses were performed using SPSS
21.0 for Windows (Armonk, NY, USA: IBM Corp.). All
statistical tests were two-sided and used a significance
level of 0.05.
We analyzed the expression of receptors which are
targeted during treatment, as well as other critical factors
of the different vasoactive pathways, in order to unravel
the unresponsiveness of CDH patients to current
vasodilator therapies. Therefore, a unique set of lung
material of CDH patients was used and the data were verified
using the more dynamical nitrofen rat model.
Besides the use of inhaled NO (iNO), current treatment
is based on targeting the receptors in both the
prostacyclin and endothelin pathway. Therefore, we analyzed
the expression of the critical proteins of both pathways
in human lung samples of control and CDH patients by
immunohistochemistry. Human control lung samples
showed little expression of the main target of the
prostacyclin therapy, the important prostacyclin receptor
PTGIR, in the fetal period. This sharply increased later
during gestation at the preterm and term age. However
this significant increase was absent in CDH (Fig. 2). The
ETA receptor, which induces vasoconstriction and cell
proliferation, was expressed in the small (25–50 μm)
and larger (>50 μm) vessels as well as in the very small
capillaries (<25 μm) in CDH. In contrast, the
distribution of the ETA receptor in control lungs was limited to
the small and larger vessels only (Arrowheads in Fig. 3a).
The ETB receptor, involved in vasodilation through the
release of NO and PGI2 (Fig. 1), was expressed both in
the bronchial epithelium and in some of the larger
vessels (>50 μm) in CDH (Arrowheads in Fig. 3a). However,
the expression of ETB in control lungs was found only
in the bronchial epithelium (Fig. 3a). Next, we analyzed
the expression of ECE-1, a membrane-bound
metalloprotease that converts big-endothelin into the
biologically active compound ET1 and it is a
ratelimiting factor in the ET pathway. Early during gestation,
in the fetal period, ECE-1 is minimally expressed in the
vessels of the human control lung samples, with an
increase at preterm and term age. Increased expression
of this enzyme at both fetal, preterm and term age was
observed in CDH (Fig. 3b), indicating a potential
increased bio-availability of active ET-1 already at the
fetal stage of development.
To exclude that the differences in expression patterns of
the crucial prostacyclin – and endothelin receptors and
the rate-limiting factor ECE-1 was solely an effect of lung
hypoplasia (LH) or PH, we performed
immunohistochemistry on lungs of patients with LH and PH with other
cause than CDH. The PTGIR receptor expression was
reduced in both LH and PH (Fig. 4a). Increased expression
of ETA was detected in the smallest vessels in lungs of
both LH and PH (Arrowheads in Fig. 4b), whereas
increased expression of ETB was only observed in both
small and very small vessels of lungs of PH patients
(Arrowheads in Fig. 4c). ECE-1 was not expressed
differently in both LH and PH lung samples (Fig. 4d).
In order to validate these interesting human data, we
evaluated the expression patterns of the proteins of these
three pathways in the nitrofen rat model. This was
supplemented with RNA and protein expression analysis of
related factors. Real-time qPCR showed that the mRNA
expression of both the Eta and Etb receptors was
significantly higher in lungs of E21 pups with CDH compared
to those of control pups. We also analyzed the
expression of the ETA and ETB ligand, Et-1, but no significant
differences were found between the groups. However,
the mRNA encoding the rate-limiting factor Ece-1 was
significantly increased in CDH compared to control,
confirming the human data (Fig. 5a). Next, we analyzed
the protein expression pattern of the ET receptors with
immunohistochemistry. The ETA receptor was
expressed in the small capillaries of both groups at E15
until E21 with a stronger expression level in CDH. At
E21 only CDH lungs showed expression of the ETA
receptor in the larger vessels (>50 μm) (Fig. 5b). The
ETB receptor was expressed in the bronchial epithelium
of all lungs without significant differences between
control and CDH at all ages (Fig. 5c). There was a
significant higher mRNA expression of eNos in CDH rats
compared to control in relation to all cells as well as in
relation to only the smooth muscles cells (Fig. 6a) or
endothelial cells (data not shown). This increased
expression was clearly detectable with immunostaining
in the larger and smaller (<50 μm) vessels at E21.
However, no obvious differences were noted earlier during
development (E15 till E19) (Fig. 6b). Although there was
no difference in expression of prostaglandin-I2 synthase
(Ptgis) between control and CDH rat pups, there was a
slight increase in the expression of Ptgir and the
prostaglandin-E1 receptor (Ptger1) in CDH at the mRNA
level in both the whole lung as well as compared to the
number of smooth muscle cells. In contrast, the
expression of thromboxane synthase (Tbxas1), the enzyme
converting prostaglandin H2 into thromboxane A2,
which is in turn critical for vasoconstriction, was clearly
reduced in CDH, whereas the thromboxane receptor
(Tbxa2r) did not show significant differences (Fig. 7a).
However, immunostaining showed no clear differences
in expression of PTGIR in the vessels between both
groups (Arrowheads in Fig. 7b).
This is the first combined study showing the aberrant
expression of different important factors in the
endothelin, NO and PGI2 pathways in CDH patients (Fig. 1b)
and human patients with LH or PH unrelated to CDH,
possibly explaining why a large number of patients do
not respond to the current vasodilator therapy. We
focused our research on direct targets of the most
frequent used drugs to investigate the effectiveness of the
current approach and combined this with the analysis of
some key factors of the different pathways.
Since our unique human CDH material is scarce
and a limiting factor, since only specimens of
newborns who lived for a short period were analyzed to
prevent secondary morphological changes,
supplemental analyses were done on lung tissue of the nitrofen
In line with previous studies in both human and rat,
we found a significant increased expression of the ETA
and ETB receptor, important targets of vasodilator
therapy, in human CDH patients and the rat model [
]. However, we are the first to show an increased
expression of the crucial ECE-1 enzyme in both human
pulmonary vessels of CDH patients and whole lung
homogenates of nitrofen treated rat pups. ECE-1
converts big ET-1 into the active form of ET-1 and is
the rate-limiting step in the production of ET-1 [
Although there was no apparent difference in total ET-1
in CDH pups, the higher expression of ECE-1 in lungs
of CDH pups may lead to an increase in the active form
Previously, we and others showed no apparent
differences in the NO pathway [
]. In contrast to other
13, 14, 19, 29
], we found an increased
expression of eNOS in CDH rats in this study. This may be
explained by the decreased NO availability, or by the
process of eNOS uncoupling. In case of decreased
bioavailability of the cofactor tetrahydrobiopterin (BH4),
eNOS produces superoxide instead of NO [
superoxide leads to oxidative stress, which has been
observed in vessels of patients with PH [
enhanced activation of the ETA receptor, as mentioned
before, might lead to the increase in superoxide
production through the induction of reactive oxygen species
(ROS) and can thereby induce SMC proliferation and
vasoconstriction. Thus, eNOS uncoupling leads to a
reduction in NO bioavailability without a necessary
change in the amount of eNOS [
]. Furthermore, we
Fig. 7 Prostacyclin expression in rat pups. a Relative gene expression of prostaglandin I synthase (Ptgis), thromboxane synthase (Tbxas1),
prostaglandin-I2 receptor (Ptgir) and prostaglandin-E1 receptor (Ptger1) in lungs of control and CDH rat pups. b Representative images showing
the protein expression of PTGIR in the pulmonary vessels in control and CDH lungs.**p < 0.01, ***p < 0.001. Error bars represent SD. Arrows
indicate vessels, A indicates airways. Scale bars represent 100 μm (low power) and 20 μm (high power)
have shown a slight increase in expression of the
cGMPspecific phosphodiesterase 5 (Pde5) in the NO pathway
in nitrofen treated rat pups previously. However, no
differences were found in its phosphorylation or its
downstream targets, protein kinase G1 (Prkg1) and
The increased expression of PTGIR in control lungs
during gestation could result from the gradual increase
of placental PGI2 toward term [
]. The decreased
expression in CDH may be a sign of reduced activation
of this pathway. In contrast to our human results, we
found no differences in the expression of Ptgir in CDH
rat pups and an increase of this receptor on mRNA
level. Since PGI2 is a potent vasodilator and
thromboxane A2 (TXA2) a potent vasoconstrictor, the increased
expression of Ptgir and decreased expression of Tbxas1
was unexpected. However, this aberrant balance between
PGI2 and TXA2 in CDH was already previously
described by our group [
]. We showed an increased
level of 6-keto-PGF1α, the stable metabolite of PGI2, and
an increased ratio of 6-keto-PGF1α and TXA2 in both
lung homogenates and broncho-alveolar lavage (BAL)
fluid of nitrofen treated rat pups. The discrepancy
between the increased mRNA expression of Ptgir in CDH
lungs and the absence of differences at the protein level
is most likely caused by the difference in sensitivity
between qPCRs and IHC. Furthermore, the different
results in human and rat could be explained by the fact
that we only used the most severe cases of human CDH
where the rat model covers all cases.
Current treatment of CDH patients with PH is not
evidence based [
] and most patients respond poorly to the
used medication. Inhaled NO (iNO) is most commonly
used as a first line drug, but its use varies significantly
among different centers internationally [
]. In contrast
to the promising results of iNO in patients with
persistent pulmonary hypertension of the newborn [
in CDH have failed to show its efficacy [
], as no
trials have been performed to evaluate the potential role
of iNO specifically in CDH patients. Apart from iNO
therapy there are some case reports on the use of
sildenafil and prostacyclins in CDH patients with variable
25, 26, 38, 39
]. However, administration of
enteral sildenafil in neonates leads to highly variable
plasma concentrations because of variable gut
absorption and/or limited hepatic clearance [
]. The recent
availability of intravenous sildenafil may change its
], but solid pharmacokinetic data on optimal
dosage are still to be published. Treatment with
endothelin receptor antagonists is even a bigger problem
since these drugs are only available in oral form, while
data of its use in newborns are virtually absent
concerning dosage absorption and safety. The fact that the
current therapy should be considered mainly as” trial
and error” and is effective in the minority of patients
with CDH strengthens our results that there are possibly
more pathways affected. Furthermore, the severity of PH
in CDH patients has been known as an important
predictor of the outcome and further evaluation of current
therapies has been recommended by experts in the field
]. Future treatment should become more personalized
in this group of patients using pathway directed clinical
trials and risk stratification [
Although the nitrofen rat model is well established,
there are still differences in embryonic maturation
between rat and human, which may have affected the
results. Furthermore, ideally, we would like to be able to
directly correlate the findings of aberrant expression of
the different vasoactive pathways with the individual
response of patients to specific vasoactive drugs.
However, given the overall limitations of these types of
studies and the lack of material of patients who did
respond to one of the three therapies, this remains
impossible as repeated lung biopsies would be needed to
In conclusion, our study shows the aberrant expression
of specific vasodilator drug targets and crucial,
ratelimiting factors in human CDH and the nitrofen rat
model in both the endothelin, NO and PGI2 pathway
already early during development. Since PH is still a
major problem and the most important cause of
morbidity and mortality in CDH patients nowadays while
current treatment strategies are disappointing, a good
insight in these pathways is needed for specific and
patient directed targeting of pharmacotherapy.
CDH: Congenital diaphragmatic hernia; cGMP: Cyclic guanosine
monophosphate; ECE-1: Endothelin converting enzyme 1; eNOS: Endothelial
nitric oxide synthase; ET: Endothelin; ET-1: Endothelin 1; ETA: Endothelin
receptor A; ETB: Endothelin receptor B; IHC: Immunohistochemistry; LH: Lung
hypoplasia; NO: Nitric oxide; PDE5: Phosphodiesterase 5; PGI2: Prostacyclin;
PH: Pulmonary hypertension; PRKG1: Protein kinase G1; PRKG2: Protein kinase
G2; PTGER1: Prostaglandin-E receptor; PTGIR: Prostaglandin-I2 receptor;
PTGIS: Prostaglandin-I2 synthase; qPCR: Quantitative real-time polymerase
chain reaction; TBXA2r: Thromboxane receptor; TBXAS1: Thromboxane
synthase; TXA2: Thromboxane
Concept and design of study, D.S.M., D.T. and R.J.R. Acquisition, analysis and
interpretation of data, D.S.M., M.J.B., D.T. and R.J.R. Drafting of manuscript,
D.S.M., D.T. and R.J.R. Review of manuscript for important intellectual content,
D.S.M., R.M.H.W., D.T. and R.J.R. Final approval of manuscript, D.S.M., M.J.B.,
R.M.H.W., D.T. and R.J.R. Study supervision, D.T. and R.J.R. All authors read and
approved the final manuscript.
This study was supported in part by the Sophia Foundation for Medical
Research grant number 678.
Availability of data and materials
The datasets used and/or analysed during the current study are available
from the corresponding author on reasonable request.
Ethics approval and consent to participate
Human lung samples were retrieved from the archives of the Department of
Pathology of the Erasmus Medical Center, Rotterdam, following approval by
the Erasmus MC Medical Ethical Committee. According to Dutch law
following consent to perform autopsy, no separate consent is needed from
parents to perform additional staining of tissues.
All animal experiments were approved by an independent animal ethical
committee and according to national guidelines.
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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