Endothelial actions of atrial natriuretic peptide prevent pulmonary hypertension in mice

Abstract

The cardiac hormone atrial natriuretic peptide (ANP) regulates systemic and pulmonary arterial blood pressure by activation of its cyclic GMP-producing guanylyl cyclase-A (GC-A) receptor. In the lung, these hypotensive effects were mainly attributed to smooth muscle-mediated vasodilatation. It is unknown whether pulmonary endothelial cells participate in the homeostatic actions of ANP. Therefore, we analyzed GC-A/cGMP signalling in lung endothelial cells and the cause and functional impact of lung endothelial GC-A dysfunction. Western blot and cGMP determinations showed that cultured human and murine pulmonary endothelial cells exhibit prominent GC-A expression and activity which were markedly blunted by hypoxia, a condition known to trigger pulmonary hypertension (PH). To elucidate the consequences of impaired endothelial ANP signalling, we studied mice with genetic endothelial cell-restricted ablation of the GC-A receptor (EC GC-A KO). Notably, EC GC-A KO mice exhibit PH already under resting, normoxic conditions, with enhanced muscularization of small arteries and perivascular infiltration of inflammatory cells. These alterations were aggravated on exposure of mice to chronic hypoxia. Lung endothelial GC-A dysfunction was associated with enhanced expression of angiotensin converting enzyme (ACE) and increased pulmonary levels of Angiotensin II. Angiotensin II/AT1-blockade with losartan reversed pulmonary vascular remodelling and perivascular inflammation of EC GC-A KO mice, and prevented their increment by chronic hypoxia. This experimental study indicates that endothelial effects of ANP are critical to prevent pulmonary vascular remodelling and PH. Chronic endothelial ANP/GC-A dysfunction, e.g. provoked by hypoxia, is associated with activation of the ACE–angiotensin pathway in the lung and PH.

Full text

ORIGINAL CONTRIBUTION
Endothelial actions of atrial natriuretic peptide prevent
pulmonary hypertension in mice
Franziska Werner
1
•Baktybek Kojonazarov
2,3
•Birgit Gaßner
1
•Marco Abeßer
1
•
Kai Schuh
1
•Katharina Vo
¨lker
1
•Hideo A. Baba
4
•Bhola K. Dahal
2,3
•
Ralph T. Schermuly
2,3
•Michaela Kuhn
1
Received: 30 June 2015 / Accepted: 16 February 2016 / Published online: 24 February 2016
ÓThe Author(s) 2016. This article is published with open access at Springerlink.com, corrected publication 2022
Abstract The cardiac hormone atrial natriuretic peptide
(ANP) regulates systemic and pulmonary arterial blood
pressure by activation of its cyclic GMP-producing
guanylyl cyclase-A (GC-A) receptor. In the lung, these
hypotensive effects were mainly attributed to smooth
muscle-mediated vasodilatation. It is unknown whether
pulmonary endothelial cells participate in the homeostatic
actions of ANP. Therefore, we analyzed GC-A/cGMP
signalling in lung endothelial cells and the cause and
functional impact of lung endothelial GC-A dysfunction.
Western blot and cGMP determinations showed that cul-
tured human and murine pulmonary endothelial cells
exhibit prominent GC-A expression and activity which
were markedly blunted by hypoxia, a condition known to
trigger pulmonary hypertension (PH). To elucidate the
consequences of impaired endothelial ANP signalling, we
studied mice with genetic endothelial cell-restricted abla-
tion of the GC-A receptor (EC GC-A KO). Notably, EC
GC-A KO mice exhibit PH already under resting, normoxic
conditions, with enhanced muscularization of small arteries
and perivascular infiltration of inflammatory cells. These
alterations were aggravated on exposure of mice to chronic
hypoxia. Lung endothelial GC-A dysfunction was
associated with enhanced expression of angiotensin con-
verting enzyme (ACE) and increased pulmonary levels of
Angiotensin II. Angiotensin II/AT
1
-blockade with losartan
reversed pulmonary vascular remodelling and perivascular
inflammation of EC GC-A KO mice, and prevented their
increment by chronic hypoxia. This experimental study
indicates that endothelial effects of ANP are critical to
prevent pulmonary vascular remodelling and PH. Chronic
endothelial ANP/GC-A dysfunction, e.g. provoked by
hypoxia, is associated with activation of the ACE–an-
giotensin pathway in the lung and PH.
Keywords Atrial natriuretic peptide Endothelium 
Guanylyl cyclase-A Cyclic GMP Pulmonary
hypertension
Introduction
Pulmonary hypertension (PH) is a complex and multifac-
torial disease which leads to overload of the right ventricle
(RV) and right heart failure. Pulmonary vasoconstriction,
endothelial cell (EC) dysfunction, vascular thickening,
inflammation and thrombosis contribute to disease pro-
gression in idiopathic and other forms of PH [3,21].
The cardiac hormone atrial natriuretic peptide (ANP),
via its cyclic GMP (cGMP)-synthesizing transmembrane
guanylyl cyclase A (GC-A) receptor, has critical functions
in the maintenance of systemic arterial blood pressure [6,
42] and also regulates pulmonary arterial blood pressure.
Hence, global inactivation of the genes encoding ANP or
GC-A increased resting pulmonary arterial pressure in
mice [28,29] or the susceptibility to hypoxia-induced PH
[58]. Conversely, infusion of synthetic ANP attenuated
hypoxia-induced experimental PH [56] and lowered
&Michaela Kuhn
michaela.kuhn@mail.uni-wuerzburg.de
1
Physiologisches Institut der Universita
¨tWu
¨rzburg,
Ro
¨ntgenring 9, 97070 Wu
¨rzburg, Germany
2
Department of Internal Medicine, University of Gießen and
Marburg Lung Center (UGMLC), Justus-Liebig University
Gießen, Giessen, Germany
3
German Center for Lung Research, Heidelberg, Germany
4
Institute of Pathology, University Hospital of Essen,
University of Duisburg-Essen, Essen, Germany
123
Basic Res Cardiol (2016) 111:22
DOI 10.1007/s00395-016-0541-x
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pulmonary pressure in patients with high-altitude disease
[32]. Together, these experimental and clinical studies
indicate that endogenous ANP plays a physiological role in
maintaining pulmonary arterial pressure homeostasis. And,
furthermore, that enhancement of endogenous ANP/GC-A/
cGMP signalling, for instance with drugs inhibiting ANP
or cGMP degradation, may have therapeutical implications
[5–7].
Pulmonary arterial remodelling in PH involves multiple
vascular (EC and smooth muscle cells (SMC), adventitial
fibroblasts) and nonvascular cell types (leucocytes, mast
cells, platelets) [3,21]. With the exception of platelets and
leucocytes, all these cell types express the GC-A receptor
[30]. Because synthetic ANP prevented acute hypoxia-in-
duced pulmonary vasoconstriction [26,58] and exerted
direct cGMP-mediated anti-proliferative effects in cultured
pulmonary arterial SMCs [24], the protective role of the
ANP/GC-A/cGMP pathway in the lung circulation has
mainly been attributed to its effects on pulmonary SMC.
However, as shown in the present study, the GC-A receptor
is also expressed at high levels in lung EC. Whereas
endothelial dysfunction is central to all forms of PH [3,21],
it is unknown whether this involves impaired ANP/GC-A/
cGMP signalling and how this could contribute to the
progression of this disease. Therefore, the goals of this
study were (1) to analyze the expression and activity of
GC-A in lung endothelial cells and the impact of hypoxia;
(2) to dissect the role of endothelial cells in mediating the
effect of ANP in the chronic regulation of pulmonary
arterial pressure by studying mice with selective disruption
of the GC-A-encoding gene (Npr1) in endothelial cells; and
(3) to elucidate the impact of endothelial ANP/GC-A
dysfunction on EC inflammatory activation as well as the
pulmonary levels of endothelin-1 (ET-1) and Angiotensin
II (Ang II). It is known that these hormones are activated
and contribute to cardiopulmonary remodelling in patients
with PH [21]. On the other hand, it was shown that ANP/
GC-A signalling diminishes endothelial ET-1 secretion
[55] and the (inter)actions of ET-1 and Ang II in the heart
and systemic circulation [19]. However, the relevance of
this functional antagonism between ANP and ET-1/Ang II
expression and action in the pulmonary circulation is
unknown.
Materials and methods
Genetic mouse models
Mice with global (GC-A
-/-
) or endothelial cell-restricted
deletion of the GC-A receptor (GC-A
fl/fl
;Tie2Cre
?/-
:EC
GC-A KO) and their respective control littermates (GC-A
?/?
or GC-A
fl/fl
, with unaltered GC-A expression levels) were
generated and genotyped as described [33,46]. The EC GC-
A KO mice have an unaltered median life span and do not
manifest clinically apparent, macroscopic changes
throughout life (mice were observed until the age of
15 months). All present studies were performed with 2- to
4-month-old mice. The experiments were conducted under
the guidelines on humane use and care of laboratory animals
for biomedical research published by NIH (No. 85-23,
revised 1996 [41]) and they were approved by the local
governmental animal care committee.
Hypoxia-induced pulmonary hypertension in mice
and losartan treatment
Experimental pulmonary hypertension (PH) was induced
by exposure to normobaric hypoxia. EC GC-A KO mice
and littermate controls were placed into a partially venti-
lated plexiglass chamber (Biospherix, New York, USA),
and exposed to chronic hypoxia (F
I
O
2
10 %, 90 % nitro-
gen) for 21 days under normobaric conditions [15]. Age-
matched mice of both genotypes were maintained in room
air and served as normoxic controls. For pharmacological
blockade of the Ang II AT
1
-receptor losartan was admin-
istered via the drinking water (10 mg/kg BW/day) during
3 weeks in normoxia or hypoxia. The concentration of the
drug in water was adjusted for body weight and daily water
intake.
Assessment of right ventricular pressures,
pulmonary vascular remodelling and perivascular
inflammation
Closed-chest right ventricular (RV) pressures were mea-
sured in anesthetized freely breathing mice (0.8–1 %
isoflurane) by insertion of a 1.4 F high-fidelity pressure
catheter (Millar Instruments, Houston, TX, USA) via the
external jugular vein. After these invasive hemodynamic
measurements, the lungs of isoflurane (1 %)-anesthetized
mice were fixed with a 1 % PFA solution through the
trachea at a constant pressure of 20 cmH
2
O. The trachea
was ligated, and the lungs and hearts were immersed in
fixative overnight. After paraffin embedding, 4 lm sections
were taken along the longitudinal lung axis (ten sections
per organ) and immunostained with antibodies against a-
smooth muscle actin (aSMA; Sigma, Munich, Germany;
dilution 1:900) and CD45 (Novus Biological, USA; dilu-
tion 1:20) to analyze the number and wall thickness of
muscularized distal arteries and perivascular leucocyte
infiltration [15,47]. Vessels of 20–70 lm external diameter
were classified as fully muscularized (actin staining [75 %
of the circumference), partially muscularized (actin stain-
ing 25–75 % of the circumference), or nonmuscularized
(\25 %). In each section, the percentage of fully or
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partially muscularized arteries was calculated [15].
Perivascular inflammation was assessed in the tissue sec-
tion after staining for CD45 [41]. Images were captured at
409using a Leica DM6000B microscope (Leica Instru-
ments, Nussloch, Germany) fitted with a Leica DFC310FX
digital camera. All blood vessels within lung section
ranging from 20 to 70 lm were analyzed using Leica
QWin software. Positively stained CD45 cells surrounding
the vessels were counted [47].
Morphometric analyses of cardiac hypertrophy
The heart was dissected to separate RV from LV plus sep-
tum (S). RV and LV ?S weights were normalized to tibia
lengths. Formaldehyde-fixed right (RV) and left ventricles
(LV) were embedded in paraffin, and 5 lmsectionswere
stained with hematoxylin eosin, periodic acid Schiff (PAS,
to discriminate cardiomyocyte cell borders) or picrosirius
red for quantification of interstitial collagen fractions. The
mean cross-sectional myocyte diameters were calculated by
measuring 50 (RV) to 100 (LV) longitudinally oriented
myocytes with a centrally located nucleus per specimen [18,
46]. Photomicrographs were evaluated using a computer-
assisted image analysis system (Olympus, Hamburg, Ger-
many), using the analySIS software (SIS), the investigator
being blinded to the genotypes [18,46].
Measurements of systemic arterial blood pressure
and left ventricular hemodynamics
Systemic arterial blood pressure was measured by tail cuff in
awake mice [33,46]. Left ventricular (LV) function was
evaluated in isoflurane-anesthetized by LV catheterization
[18]. A 1.4-F combined micromanometer-tipped conduc-
tance catheter (SPR-839, Millar) was retrogradely advanced
via the right carotid artery, and simultaneous recordings of
LV pressure and volume were performed [18].
Effects of ANP on cyclic GMP content
of microvascular lung endothelial cells (MLEC)
Human microvascular lung (ML) EC were purchased from
Promocell (Heidelberg, Germany). The cells were main-
tained in complete EC growth medium MV2 (Promocell)
and studied at passage 4 and 5. The isolation and culture of
murine MLEC has been described before [46]. Immuno-
cytochemistry with antibodies against the endothelial
marker VE-cadherin demonstrated that after the second
selection, more than 95 % of cultured cells were
endothelial. For the experiments the cells were seeded in
gelatine-coated 6-well (for western blot) or 24-well plates
(for cGMP determinations) and cultured for 48 h before
synchronization in medium containing reduced serum
(1 %) concentration for 24 h. The cells were thereafter
exposed to 24 h hypoxia in a humidified 37 °C chamber
(BioSpherix). The concentration of oxygen was reduced to
1 % by replacement with N
2
, keeping CO
2
constant at 5 %.
Control was defined as 95 % air and 5 % CO
2
. Thereafter
the cells were immediately used for the extraction of
membrane proteins (cell fractionation kit; Nanotools,
Teningen, Germany) and for determination of cGMP
responses to ANP. These steps were performed under
normoxic conditions. For cGMP determinations, MLEC
were pretreated with the phosphodiesterase inhibitor
3-isobutyl-1-methylxanthine (IBMX, 0.5 mmol/L, 15 min;
Sigma) and then incubated with ANP (0.1 nmol/L–1 lmol/
L; Bachem, Bubendorf, Switzerland) for additional 10 min.
The incubation media were rapidly removed and cellular
cGMP was extracted with ice-cold ethanol (70 %, v/v).
After centrifugation (30009g, 5 min, 4 °C), the super-
natants were dried in a speed vacuum concentrator,
resuspended in sodium acetate buffer (50 mmol/L, pH 6.0)
and acetylated, and the cGMP content was determined by
radioimmunoassay [31,48].
Determination of GC-A expression and activity
in murine lung cell membranes
ANP-dependent guanylyl cyclase activity in crude lung cell
membranes was determined as described [48]. Freshly
dissected lungs were homogenized using a Polytron
homogenizer in hepes buffer (HB) [25 mM HEPES (pH
7.4), 50 mM NaCl, 20 % glycerol and protease inhibitor
cocktail from Roche, Mannheim, Germany]. The suspen-
sions were pelleted by centrifugation at 45,000gfor 20 min
at 4 °C. Pellets were resuspended in HB and centrifuged
two more times. To initiate cyclase activity, 40 lg mem-
brane protein was incubated in assay buffer [25 mM/L
HEPES, 4 mM/L MgCl
2
, 1 mM/L IBMX, 2 mM/L ATP,
2 mM/L GTP, 30 mM/L phosphocreatine, 400 lg/mL
creatine phosphokinase (185 units/mg) and 0.5 mg/mL
BSA] at 37 °C, with or without ANP. At 10 min of incu-
bation, the reaction was stopped by addition of ice-cold
ethanol (final concentration 70 % v/v). cGMP content was
determined by radioimmunoassay as described above.
cGMP production was normalized to protein content
(40 lg/sample) and the increase in cGMP content in ANP-
treated samples was compared to parallel vehicle-treated
membrane preparations of the same lung.
Western blotting
Membrane proteins from whole lungs were extracted
(Thermo Scientific, Schwerte, Germany) and subjected to
SDS-PAGE and immunoblotting as described [18]. The
primary antibodies were against GC-A (generated in our
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laboratory [48]) and b-tubulin or GAPDH (for loading
control; Cell Signaling, Frankfurt/Main, Germany). The
blots were developed using the ECL detection system
(Biozym Scientific GmbH, Hessisch-Oldendorf, Germany)
and results were quantitated by densitometry
(ImageQuant).
Quantitative RT-PCR analysis of angiotensin
converting enzyme (ACE), endothelin-1,
intercellular cell adhesion molecule 1 (ICAM-1),
vascular cell adhesion protein 1 (VCAM-1) and E-
selectin mRNA expression levels
Extraction of mRNA from murine MLEC or peripheral lung
tissue and reverse-transcription were performed as described
using TRIzol reagent (Life Technologies GmbH, Darmstadt,
Germany) and Transcriptor First Strand cDNA synthesis kit
(Roche) [18]. Messenger RNA expression levels were ana-
lyzed by Real Time quantitative PCR with LightCycler
Technology (LC-96; Roche) and FastStart Essential Probes
Master with the following primers and probes (all from
Roche): for ACE, sense: 50-GTGGGTATCCCACTGAAAC
C-30; antisense: 50-CAGAAGGCTCCTGTGTCTGA-30;
and probe 121 (REF: 04693558001); for E Selectin, sense:
50-TCCTCTGGAGAGTGGAGTGC-30; antisense: 50-GGT
GGGTCAAAGCTTCACAT-30; and probe 19 (REF: 04686
926001); ET-1, sense: 50-CTGCTGTTCGTGACTTTCCA-
30, antisense: 50-TCTGCACTCCATTCTCAGCTC-30, and
probe 50 (REF: 04688112001); ICAM-1, sense: 50-CGAAG
CTTCTTTTGCTCTGC-30; antisense: 50-GTCCAGCCGA
GGACCATA-30; and probe 10 (REF: 04685091001);
VCAM-1: sense: 50-TGGTGAAATGGAATCTGAACC-30;
antisense: 50-CCCAGATGGTGGTTTCCTT-30; and probe
34 (REF: 04687671001). 12S ribosomal RNA served as
reference gene [sense: 30-GAAGCTGCCAAGGCCTTAG
A-30; antisense: 50-AACTGCAACCAACCACCTTC-30;
FastStart Essential DNA Green Master (Roche)].
Measurement of lung immunoreactive ET-1
Samples were assayed for ET-1 immunoreactivity with a
specific RIA (Bachem) as described by Aguirre et al. [1].
The peptide was extracted from lung tissue by boiling in
109(wt/vol) 1 mol/L acetic acid for 10 min. The samples
were then chilled and centrifuged at 5000gfor 10 min at
4°C. Aliquots (0.1 mL) of supernatant were applied to
Sep-PakC
18
columns (Waters Corporation, Milford, USA).
The columns were activated by 80 % acetonitrile in 0.1 %
TFA followed by 0.1 % TFA. After the column was slowly
washed with 10 % acetonitrile in 0.1 % TFA, samples were
eluted from the column with 80 % acetonitrile in 0.1 %
TFA into polypropylene tubes and evaporated to dryness in
a centrifugal concentrator. The samples were reconstituted
in RIA buffer and subjected to ET-1 radioimmunoassay
(Bachem) according to the manufacturer’s instructions.
Measurement of lung immunoreactive Angiotensin
II
Ang II from murine lungs was extracted and measured with
a commercial Ang II ELISA (Enzo Life Sciences GmbH,
Lo
¨rrach, Germany) according to the manufacturer’s
instructions.
Measurement of pulmonary bradykinin-9 levels
Bradykinin was measured with an EIA Kit (Phoenix Europe,
Karlsruhe, Germany). Tissue extractions and measurements
were performed according to the manufacturer’s protocol.
Freshly dissected lung samples were boiled in 75 % acetic
acid for 20 min (1 mL/100 mg tissue), homogenized with an
ULTRA-TURRAX, centrifuged (15,000g,30min,4°C)
and the supernatants were extracted with Sep-PakC
18
col-
umns. The eluates were dried, reconstituted in assay buffer
and subjected to Bradykinin EIA. Bradykinin levels were
normalized to protein content (BCA assay).
Statistics
Results are presented as mean ±SEM. Group comparisons
were performed using either unpaired ttest or two-way
ANOVA followed by the multiple-comparison Bonferroni
ttest to assess differences between groups. Pvalues of less
than 0.05 were considered statistically significant. The
individual sample sizes for each set of data (n) are provided
in the figure legends.
Results
GC-A is expressed in lung endothelial cells and is
downregulated by hypoxia
To assess the pulmonary endothelial role of GC-A, first
we tested the effects of ANP on cGMP levels of cultured
human and murine microvascular lung endothelial cells
(MLEC). Treatment with ANP (0.1 nmol/L–1 lmol/L,
10 min) provoked similar concentration-dependent
cGMP increases in both species (Fig. 1a). Accordingly,
western blot analyses revealed high lung GC-A levels in
control mice (Fig. 1b). As also shown, the immunoreac-
tive protein was not detected in lungs from mice with
global GC-A deletion (GC-A
-/-
), demonstrating the
specificity of our antibody [48]. Exposure of murine
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MLEC to hypoxia (1 % O
2
, 24 h) significantly attenuated
GC-A expression (Fig. 1c) and the cGMP-responses to
ANP (Fig. 1d). To study whether hypoxia-induced
downregulation of lung GC-A occurs in vivo, we exposed
mice to normobaric hypoxia (F
i
10 % O
2
) for 21 days
[15]. Figure 1e, f shows that pulmonary cell membrane
GC-A expression and activity were significantly impaired
by chronic hypoxia.
Endothelial cells are a main expression site of GC-A
in the lung
To dissect the role of endothelial cells in mediating the
homeostatic effect of ANP on pulmonary arterial pressure,
we studied mice with conditional, endothelial-restricted
disruption of GC-A (EC GC-A KO) and control littermates
[46]. As shown in Fig. 2a, in cultured MLEC isolated from
hypoxianormoxia
GC-A
GAPDH
normoxia hypoxia
0.0
0.2
0.4
0.6
0.8
1.0
1.2
lung GC-A / GAPDH
X-fold vs normoxia
*
F
E
ANP, nM
cGMP synthesis by lung
membranes, x-fold vs PBS
0
5
10
15
20
25
30 normoxia
hypoxia
*
*
D
intracellular cGMP
pmol / 100000 MLEC
normoxia
hypoxia
*
*
*
ANP, nM
0.0
0.2
0.4
0.6
0.8
1.0
A
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4 murine MLEC
intracellular cGMP
pmol/ 100000 MLEC
human MLEC
PBS 10 1000
PBS 0.1 1 10 100
PBS 0.1 1 10 100 1000
PBS 1 10 100
*
**
*
*
*
*
ANP, nM ANP, nM
CTR GC-A
-/-
HEK-293
lung membranes
GC-A
B
*
C
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
MLEC GC-A / -Tubulin
X-fold vs normoxia
GC-A
-Tubulin
hypoxianormoxia
normoxia hypoxia
β
β
Fig. 1 Pulmonary endothelial ANP/GC-A/cGMP signalling is atten-
uated by hypoxia. aEffect of ANP on cGMP content of cultured
human (6 wells per condition; 2 independent experiments) and murine
(15 dishes per condition; 5 experiments) microvascular lung endothe-
lial cells (MLEC, 10 min incubation). bRepresentative immunoblot:
strong GC-A expression (apparent MW is *130 kDa) in cell
membranes prepared from wildtype (CTR) lungs (loading 80 lg/
lane). The immunoreactive signal is abolished in membranes prepared
from mice with global GC-A deletion (GC-A
-/-
). Protein extracts
from GC-A-expressing HEK-293 cells were used as positive control.
c,dIn murine MLEC, hypoxia (1 % O
2
, 24 h downregulates GC-A
expression (c; western blots, 40 lg protein/lane) and ANP-induced
intracellular cGMP synthesis (d) (6 wells from 3 independent
experiments). e,f, In mice, chronic hypoxia (normobaric F
i
O
2
of
10 % during 3 weeks) downregulates pulmonary membrane GC-A
expression (e; western with 40 lg protein/lane) and ANP-stimulated
lung cell membrane GC-A/cGMP activity (n=6). *P\0.05 vs.
normoxia
Basic Res Cardiol (2016) 111:22 Page 5 of 16 22
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the KO mice GC-A expression and cGMP responses to
ANP were fully abolished, demonstrating efficient
endothelial GC-A deletion. Western blot analyses of
whole-lung protein extracts revealed &60 % reduction of
pulmonary GC-A protein levels in EC GC-A KO mice
(Fig. 2b). Even more, ANP-stimulated GC-A activity in
lung cell membranes was reduced by more than 80 %
(Fig. 2c). As already mentioned, different cell types in the
lung express the GC-A receptor. Considering that EC make
up *30 % of the lung cells [13], our studies of control and
EC GC-A KO mice indicate that endothelia are one main
expression site of GC-A in the lung.
Genetic deletion of endothelial GC-A in mice causes
PH and pulmonary vascular remodelling
To study the impact of endothelial GC-A dysfunction on
pulmonary arterial pressure we compared RV pressures in
anesthetized EC GC-A KO and control littermates. RV
catheterization revealed that EC GC-A KO mice have
increased RV systolic pressures (RVSP; Fig. 3a). This was
accompanied by RV hypertrophy, with enhanced RV
weight/tibia length ratios (Fig. 3b) and greater RV myocyte
diameters (Fig. 3c depicts the mean cross-sectional diam-
eters of RV myocytes with a centrally located nucleus).
Picrosirius red stainings did not reveal signs of RV inter-
stitial fibrosis (Fig. 3d). Together these observations indi-
cate that EC GC-A KO mice have mild but consistent PH
already under normoxic conditions. This phenotype was
independent of age (2- to 8-month-old mice were studied)
and gender. Notably, peak RVSP values in EC GC-A KO
mice nearly reached the levels of mice with global, sys-
temic GC-A deletion (GC-A
-/-
mice [33], see Fig. 3e),
whereas RV hypertrophy was more pronounced in the later
genotype (Fig. 3f). Exposure to chronic hypoxia induced
PH and RV hypertrophy in EC GC-A KO and control lit-
termates, again with greater RVSP and RV hypertrophy in
the former, without significant RV fibrosis (Fig. 3a–d).
However, the absolute increase in mean RVSP in response
to hypoxia was not greater in EC GC-A KO mice than that
in controls (?7.5 vs. 6.6 mmHg, respectively). Hypoxia-
induced hematocrite raises did not differ between geno-
types (controls 0.47 ±0.01 % (normoxia) vs.
0.6 ±0.01 %* (hypoxia); EC GC-A KO 0.46 ±0.01 vs.
0.57 ±0.08 %*; *P\0.05 vs. normoxia).
To investigate the effect of endothelial GC-A ablation
on pulmonary vascular remodelling, the degree of muscu-
larization of peripheral arterioles was analyzed by
immunostainings with anti-a-SMA antibodies [15]. Mor-
phometrical analyses showed an increase in the relative
number of fully and partially muscularized vessels and a
concomitant decrease of nonmuscularized vessels in EC
GC-A KO as compared with control lungs (Fig. 4a). In
addition, immunostainings with anti-CD45 antibodies [47]
revealed mild perivascular leucocyte infiltration (Fig. 4b).
Hypoxia provoked lung vascular remodelling and
perivascular inflammation in control and, significantly
more, in EC GC-A KO mice (Fig. 4a, b). Again, the rela-
tive changes (as compared to normoxia) were similar in
both genotypes.
Pulmonary hypertension in EC GC-A KO mice is
not secondary to left heart disease
In agreement with our previous report [40], EC GC-A KO
mice used in the present study had mild systemic hyper-
tension and subtle LV hypertrophy without fibrosis
(Table 1). The degree of LV hypertrophy was not changed
after hypoxia (Fig. 5). Pressure–volume relationships
(studied by LV catheterization) demonstrated that LV
contractile and relaxation functions of EC GC-A KO mice
were unaltered (Fig. 5). LV end-systolic pressures were
slightly greater, consistent with the mildly enhanced
afterload. As also shown in Fig. 5, hypoxia did not
Control mice
EC GC-A KO mice
cGMP synthesis
x-fold vs PBS
0
5
10
15
20
25
**
PBS 10 1000
ANP, nM
GC-A
β
β
-Tubulin
control KO
0.0
0.4
0.8
1.2
lung GC-A / -tubulin
X-fold vs CTR
*
CB
ANP, nM ANP, nM
A
0
0.4
0.8
1.2
1.6
2.0
intracellular cGMP
pmol/100000 MLEC
GC-A
CTR KO
GAPDH
PBS 0.1 1 10 100 1000 PBS 0.1 1 10 100 1000
*
*
*
*
Fig. 2 Inactivation of GC-A in lung endothelial cells of EC GC-A
KO mice. aEffects of ANP on intracellular cGMP content of MLEC
prepared from EC GC-A KO and control littermates (10 min
incubation; n=6 per genotype). Inset Representative western blot
of GC-A expression in MLEC. bImmunoblot analyses of GC-A
expression levels in whole lung protein extracts prepared from EC
GC-A KO and control mice (n=5). cGuanylyl cyclase activity
assays: ANP-dependent cGMP synthesis by lung cell membranes
prepared from EC GC-A KO and control mice (n=6). *P\0.05 vs.
controls
22 Page 6 of 16 Basic Res Cardiol (2016) 111:22
123
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Control mice
EC GC-A KO mice
A
0
10
20
30
40
RVSP
(mm Hg)
*
*
†
†
B
100 μm
Controls EC GC-A KO
normoxia
hypoxia
†
C
RV myocyte diameter
(m)
*
*†
normoxia hypoxia
0
4
8
12
16
RV weight / tibia length
(mg / cm)
0
0.4
0.8
1.2
1.6
normoxia hypoxia
**
†
†
FE
0
10
20
30
40 *
RVSP
(mm Hg)
GC-A-/-
CTR
RV weight / tibia length
(mg / cm)
*
GC-A-/-
CTR
0
0.4
0.8
1.2
1.6
2.0
D
0.00
RV collagen fraction (%)
normoxia hypoxia
0.02
0.04
0.06
0.08
0.10
50 μm
normoxia
hypoxia
Controls EC GC-A KO
normoxia hypoxia
μ
Fig. 3 Genetic deletion of the endothelial GC-A receptor in mice
causes pulmonary hypertension and right ventricular (RV) hypertro-
phy under normoxic conditions and, more, after chronic hypoxia
(F
i
O
2
10 % during 3 weeks). aElevated RV systolic pressures (SP) in
EC GC-A KO mice compared to respective controls under normoxia
and after hypoxia. b,cRatios of RV weight to tibia length and RV
myocyte diameters (indicated by white lines in longitudinal PAS
stained sections) were increased in EC GC-A KO mice. Hypoxia
further enhanced RV hypertrophy of EC GC-A KO mice. dPicrosirius
red stainings revealed that RV interstitial collagen fractions were not
different between genotypes and conditions (n=8 mice per group);
e,fIncreased RVSP and enhanced RVW/tibia length ratios in mice
with global, systemic GC-A deletion (GC-A
-/-
) compared to
respective controls (CTR) (n=6 mice per genotype studied under
normoxia). *P\0.01 vs. controls;

P\0.01 vs. normoxia
Basic Res Cardiol (2016) 111:22 Page 7 of 16 22
123
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influence LV function in control or EC GC-A KO mice. In
addition the lung wet-to-dry weight ratios were equal for
EC GC-A KO and controls (Table 1). Together, these data
indicate that the PH of EC GC-A KO mice is not secondary
to LV dysfunction.
Pulmonary levels of immunoreactive endothelin-1
are not altered in EC GC-A KO mice
To elucidate the mechanism(s) contributing to PH in EC
GC-A KO mice we studied specific ANP-modulated
pathways known to be altered in clinical PH. In particular,
ET-1 levels are upregulated in patients with PH and
endothelin receptor antagonists are used in its treatment
[45]. Synthetic ANP inhibits ET-1 release from cultured
human umbilical venous endothelial cells [55]. Therefrom,
we hypothesized that endothelial GC-A dysfunction leads
to increased lung ET-1 levels which, via the vasocon-
strictory and SMC proliferative actions of this peptide,
could contribute to PH in EC GC-A KO mice. However,
qRT-PCR did not reveal significant differences of the ET-1
mRNA levels in GC-A-deficient MLEC and in lungs from
EC GC-A KO mice in comparison to controls (Fig. 6a).
Even more, pulmonary ET-1 levels did not differ between
genotypes (Fig. 6b).
fully
partially
non
0
20
40
60
80
F P N F P N F P N F P N
Degree of muscularization (%)
Arterioles of 20-70 μm ø
CTR EC GC-A KO
normoxia
*
**
†
†
†
*
†
*
†
*
†
CTR EC GC-A KO
hypoxia
A
Controls KO Controls KO
normoxia hypoxia
100 μm
Control mice
EC GC-A KO mice
0.0
0.2
0.4
0.6
0.8
1.0
1.2
CD45 positive cells
perivascular (ø 20-70 m)
normoxia hypoxia
*
*
†
†
B
Controls KO Controls KO
normoxia hypoxia
100 m
μ
μ
Fig. 4 Genetic deletion of the endothelial GC-A receptor in mice
causes pulmonary vascular remodelling together with mild perivas-
cular inflammation under normoxic conditions and, more, after
chronic hypoxia. Lung sections were immunostained for SMC a-actin
or for lymphocyte common antigen (CD45). aQuantification of the
relative numbers of fully (F), partially (P) and non (N) muscularized
arterioles and bof perivascular CD45-positive cells per field
demonstrated enhanced pulmonary vascular remodelling and perivas-
cular inflammatory infiltration in EC GC-A KO mice under normoxia
and after hypoxia (n=8 mice per group). *P\0.01 vs. controls;

P\0.01 vs. normoxia
Table 1 EC GC-A KO mice have subtle systemic arterial hyper-
tension and mild left ventricular (LV) hypertrophy without fibrosis
Controls EC GC-A KO
SBP (mmHg) 118 ±2 133 ±3*
DBP (mmHg) 75 ±382±3*
HR (bpm) 589 ±18 564 ±13
Body weight (g) 25 ±1.5 25 ±1.2
Heart weight (mg) 118 ±4.5 145 ±6*
LV weight/tibia length (mg/cm) 4.78 ±0.19 5.9 ±0.2*
LV myocyte diameter (lm) 12 ±0.4 14.8 ±0.8*
Collagen fraction (%) 0.1 ±0.02 0.11 ±0.03
Lung wet/dry weight 4.4 ±0.04 4.5 ±0.04
Hematocrite (%) 43 ±1.7 42 ±2.4
Systemic systolic (SBP) and diastolic (DBP) arterial blood pressure,
heart rate (HR) (determined by tail cuff), hematocrite and LV mor-
phology (necropsy and histology) of EC GC-A KO and control mice.
n=8, * P\0.05 vs. control littermates
22 Page 8 of 16 Basic Res Cardiol (2016) 111:22
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Enhanced expression levels of ACE
and of endothelial adhesion molecules in EC GC-A
KO lungs
Experimental and clinical studies indicate that the renin–
angiotensin–aldosterone system (RAAS) is involved in
the pathophysiology of PAH [12,14,35,37,38,43].
Synthetic, exogenous ANP attenuates the expression of
angiotensin converting enzyme (ACE) and counterregu-
lates the cardiovascular effects of Ang II [17,19,27,52].
Thus, we evaluated whether the ACE/Ang II pathway
participates in PH of EC GC-A KO mice. Indeed, qRT-
PCR revealed increased ACE expression in GC-A-defi-
cient MLEC and lungs from EC GC-A KO mice (Fig. 6c).
As also shown, ACE mRNA expression was unaltered in
other tissues from the KO mice such as heart. The direct
effect of the dipeptidyl peptidase ACE is to increase
levels of Ang II and decrease levels of bradykinin. To
follow the hypothesis that increased Ang II together with
decreased local bradykinin levels contribute to PH of EC
GC-A KO mice we determined the lung levels of these
peptides. Indeed, levels of immunoreactive Ang II were
greater in EC GC-A KO lungs (Fig. 6d). Concomitantly,
the levels of bradykinin-9 were attenuated although, due
to high variability, the difference to control lungs did not
reach statistical significance (P=0.08; Fig. 6e). Lastly,
qRT-PCR revealed increased pulmonary expression of the
EC adhesion molecules VCAM-1 and ICAM-1 and mild
not-significant increases of E-selectin in EC GC-A KO
mice (Fig. 6f, g).
Enhanced ACE/Angiotensin II signalling contributes
to PH of EC GC-A KO mice
To study whether increased lung ACE/Ang II levels
contribute to the PH of EC GC-A KO mice, we compared
)nim/lm(tuptuocaidraC)mc/gm(htgnelaibit/thgiewVL
Stroke work (mmHg/μl)
End-systolic pressure (mmHg) End-diastolic pressure (mmHg)
Heart rate (beats/min)
tmax
Ejection fraction (%) dP/d (mmHg/s) -dP/dtmin (mmHg/s)
0
2
4
6
8
10
12
0
500
1000
1500
2000
0
2000
4000
6000
8000
10000
12000
14000
0
2500
5000
7500
10000
12500
15000
0
10
20
30
40
50
60
0
100
200
300
400
500
600
0
2
4
6
8
0
1
2
3
4
5
6
7
normoxia hypoxia
**
0
20
40
60
80
100
120
140
normoxia hypoxia normoxia hypoxia
normoxia hypoxianormoxia hypoxianormoxia hypoxia
normoxia hypoxia normoxia hypoxia
normoxia hypoxia
**
Control mice
EC GC-A KO mice
Fig. 5 Left ventricular (LV) weight and function of anesthetized
control and EC GC-A KO mice determined by pressure–volume
analyses after normoxia or chronic hypoxia. Ratios of LV weight to
tibia length and LV systolic pressures were similarly increased in EC
GC-A KO mice under normoxic and after hypoxic conditions. All
other parameters of LV contraction and relaxation were not different
between genotypes and conditions. n=6 mice per genotype and
condition; *P\0.05 vs. controls
Basic Res Cardiol (2016) 111:22 Page 9 of 16 22
123
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theeffectsofchronicblockadeoftheAngII/AT
1
-receptor
in both genotypes. Figure 7illustrates the impact of
losartan treatment (10 mg/kg/day, 3 weeks) on RVSP
(Fig. 7a) and on the ratios of RV weight/tibia length
(Fig. 7b) of mice maintained under normoxia or hypoxia.
As illustrated, losartan did not affect these parameters in
normoxic controls. The drug partly prevented the
increases in RVSP of control mice subjected to hypoxia
(Fig. 7a); however, this did not ameliorate either the
hypertrophy of the RV (Fig. 7b) or the thickening of the
distal pulmonary arteries. The percentage (%) of fully
muscularized distal arteries was: 0.96 ±0.44 in control
mice under normoxia; 6.84 ±1.6* in controls after
hypoxia; and 5.1 ±1.2* in controls treated with losartan
during hypoxia (n=6 mice per group; *P\0.05 vs.
normoxia).
Notably, while losartan had no appreciable effects in
normoxic control mice, it almost reversed the baseline
pulmonary hypertension of EC GC-A KO mice. This is
indicated by the decreases of RVSP (Fig. 7a), RV hyper-
trophy (Fig. 7b), pulmonary vascular remodelling and
perivascular inflammation (Fig. 7c, d). In addition,
administration of losartan during hypoxia partly but sig-
nificantly prevented the hypoxia-driven augmentation of
these cardiovascular changes (Fig. 7a–d). Lastly losartan
also reversed the mild (hypoxia-independent) LV hyper-
trophy of EC GC-A KO mice, as indicated by the following
LV weight/tibia length ratios (in mg/cm): 5.9 ±0.2 (KO,
ET-1 mRNA / S12
x-fold vs. controls
Lung
0
0.2
0.4
0.6
0.8
1.0
1.2
1.4 B
0
0.5
1.0
1.5
Endothelin-1
X-fold vs. controls
A
Control mice
EC GC-A KO mice
EDC
GF
mRNA /S12
x-fold vs. controls
*
VCAM-1 ICAM-1
*
0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
mRNA /S12s
x-fold vs. control
ET-1 mRNA /S12
x-fold vs. controls
MLEC
0
1.0
1.5
2.0
2.5
0.5
E-Selectin
1.8
0
1.0
1.2
1.4
1.6
0.8
0.2
0.4
0.6
Bradykinin
x-fold vs. controls
p = 0.0
6
0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
Angiotensin II
x-fold vs. controls
*
0
0.5
1.0
1.5
2.5
2.0
ACE mRNA /S12
x-fold vs. controls
tissues
RV LVLung
*
*
MLEC
0
1.0
2.0
2.5
3.0
0.5
1.5
*
Fig. 6 Unaltered endothelin-1 but altered levels of angiotensin
converting enzyme (ACE), Ang II, bradykinin and EC adhesion
molecules in cultured microvascular lung endothelial cells (MLEC)
and/or in lungs of EC GC-A KO mice. a,bReal-time RT-PCR and
radioimmunoassay (RIA) showed that the endothelial and pulmonary
mRNA and peptide levels of ET-1 were not significantly different
between genotypes. cACE mRNA expression was increased in GC-
A-deficient MLEC and in lungs from EC GC-A KO mice (n=5). d,
ePulmonary levels of immunoreactive Ang II were significantly
greater in EC GC-A KO mice whereas the pulmonary levels of
bradykinin were diminished (P=0.08). fVCAM-1, ICAM-1 and
E-Selectin mRNA levels were increased in lungs from EC GC-A KO
mice. The mRNA levels of all target genes were normalized to the
levels of 12S ribosomal RNA as reference gene. All data are
illustrated as x-fold changes in EC GC-A KO vs. control mice. n=8
per genotype; *P\0.05 vs. controls
22 Page 10 of 16 Basic Res Cardiol (2016) 111:22
123
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normoxia, vehicle); 3.9 ±0.11* (KO, normoxia, losartan);
5.8 ±0.14 (KO, hypoxia, vehicle); 3.8 ±0.27* (KO,
hypoxia, losartan) (n=9 mice per group; *P\0.05 vs.
vehicle). Together these observations indicate that AT
1
receptor signalling has a significant role in the cardiac and
pulmonary remodelling changes of EC GC-A KO mice.
Fig. 7 Blockade of the Ang II/AT
1
-receptor reversed the pulmonary
vascular changes in EC GC-A KO mice. aTreatment of control mice
with losartan (10 mg/kg BW/day during 3 weeks) had no effect on
baseline RVSP (normoxia) but attenuated the increment by chronic
hypoxia. In EC GC-A KO littermates losartan decreased elevated
RVSP under normoxic conditions and attenuated the increment by
chronic hypoxia. bIn control mice losartan did not prevent hypoxia-
induced RV enlargement. However, losartan reversed baseline RV
hypertrophy (under normoxia) in EC GC-A KO littermates and
prevented the increase by hypoxia. cThe number of fully muscular-
ized lung arterioles, and dsurrounding infiltration by CD45-positive
leucocytes in EC GC-A KO mice under normoxia and after hypoxia
were significantly decreased by losartan. n=6–9 mice per group;
*P\0.05 vs. vehicle;

P\0.05 vs. normoxia
Basic Res Cardiol (2016) 111:22 Page 11 of 16 22
123
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Discussion
Together with previous reports [32,56–58], our experi-
mental studies demonstrate that ANP, via its GC-A
receptor, plays an important physiological role in the
moderation of pulmonary arterial pressure and lung vas-
cular remodelling under normoxic and hypoxic conditions.
The major novel findings are (1) EC are a major expression
site of the GC-A receptor in the lung; (2) hypoxia impairs
pulmonary endothelial GC-A expression and signaling; (3)
genetic inactivation of the endothelial GC-A receptor in
mice (EC GC-A KO) provokes PH, pulmonary vascular
remodeling and subtle perivascular inflammatory infiltra-
tion already under normoxic conditions; (4) peak RVSP
values in EC GC-A KO mice were similar to the levels of
mice with deletion of GC-A in all cell types (GC-A
-/-
),
indicating that the endothelial effects of ANP are critically
involved in the chronic moderation of pulmonary arterial
pressure and vascular homeostasis by this hormone, at least
in the murine system; and (5) enhanced local ACE/Ang II
signaling contributes to the pulmonary vascular alterations
in mice with endothelial GC-A dysfunction.
The increases in RVSP and the extent of pulmonary
vascular remodeling in mice with global ANP or GC-A
inactivation [28,29], or endothelial-restricted GC-A abla-
tion are very consistent. In fact, less pronounced and more
variable changes were observed in other disease-relevant
genetic mouse models. For instance, wide ranges of RVSP
were observed in mice with endothelial deletion of the
BMPR2 gene (20.7–56.3 mmHg; median, 27 mmHg)
compared with control mice (19.9–26.7 mmHg; median
23 mmHg), and only a subset of BMPR2-deficient mice
with RVSP [30 mmHg exhibited RV hypertrophy and
pulmonary vascular remodeling [23]. Even more, exposure
of wild type rats or mice to chronic hypoxia (as accepted
experimental model of PH) increases RVSP by
7–10 mmHg [15,28,29]. Hence, in general the functional
and morphological pulmonary alterations in experimental
PH are much less pronounced as in the clinical setting,
emphasizing that patients have a multifactorial disease
whereas experimental studies attempt to dissect the con-
tribution of specific genes or mechanisms. The present
experimental study suggests that endothelial ANP/GC-A
dysfunction could be one aspect of the complex neurohu-
moral imbalance accompanying and aggravating PH, in
particular hypoxia-induced PH in chronic high-altitude
disease. Our observations may stimulate clinical studies to
follow this possibility.
Experimental and clinical studies showed that during
chronic hypoxia, right heart ANP and BNP synthesis and
circulating NP levels increase, possibly in response to the
RV pressure overload provoked by pulmonary
vasoconstriction [9–11,44]. Because synthetic ANP
counterregulates hypoxic pulmonary vasoconstriction [8,
22,26] and limits the interaction of endothelial and
inflammatory cells [25,39] and the proliferation of cultured
vascular SMC [24], it was proposed that enhanced
endogenous ANP/BNP release helps to mitigate the
development of hypoxic PH [9,10]. However, as shown
here, hypoxia can decrease lung GC-A levels and
endothelial GC-A/cGMP responses to ANP, which will
attenuate these protective ANP (and BNP) effects. The
inhibition of ANP/GC-A signaling by hypoxia has also
been observed in coronary EC [2] but the molecular
mechanism is presently unknown and requires further
study.
Endothelial GC-A dysfunction might cause PH in mice
by provoking chronic increases in pulmonary arteriolar
tone and/or vascular remodelling. Hence, we hypothesized
that ANP physiologically regulates the endothelial release
or (in) activation of factors locally modulating these pro-
cesses, such as ET-1, Ang II or bradykinin. And, con-
versely, that this effect of ANP is abolished in EC GC-A
KO mice. Interestingly, whereas ET-1 mRNA and protein
levels were unaltered, ACE mRNA levels were increased
in GC-A-deficient MLEC and in lungs from EC GC-A KO
mice. Concomitantly, pulmonary Ang II levels were
greater in the mutants whereas bradykinin levels tended to
be diminished. It is well known that Ang II, via AT
1
sig-
nalling, not only causes vasoconstriction, but also migra-
tion and proliferation of SMC as well as recruitment of
inflammatory cells [16,54]. Specifically, inhibition of ACE
decreased the cellular inflammatory response in experi-
mental models of lung inflammation [4]. Indeed, in the
present study AT
1
-receptor blockade with losartan largely
reversed PH, pulmonary vascular remodelling and inflam-
mation in normoxic EC GC-A KO mice. Even more,
losartan significantly attenuated the exacerbation of these
cardiovascular changes in response to hypoxia. Together
these observations indicate that enhanced ACE-dependent
local Ang II formation contributes to these phenotypical
alterations. In line with our results, several experimental
and clinical studies have implicated the involvement of the
RAAS in the pathogenesis of PH [35]. All components,
including renin, angiotensinogen, ACE and both subtypes
of Ang II receptors, are expressed in the lung [38,43].
Increased ACE expression and activity in the endothelium
of peripheral pulmonary arteries have been found in animal
models of PH and, importantly, in patients with various
forms of PAH [38,43]. However, the pathophysiological
mechanism(s) remain(s) unclear. Our studies add a novel
piece of information showing that pulmonary endothelial
ANP/GC-A/cGMP-dysfunction is associated with
enhanced ACE expression and activity. The inhibition of
22 Page 12 of 16 Basic Res Cardiol (2016) 111:22
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ACE levels by ANP was also observed by others [52] and
we will try to clarify the mechanism in our future
investigations.
Notably, in the present study losartan did not clearly
ameliorate hypoxic pulmonary hypertension in control mice.
The increase in RVSP was only partly prevented, whereas
RV hypertrophy and lung vascular remodelling were not at
all attenuated by the drug. Hence, mechanisms independent
of the AT
1
receptor seem to predominate. In line with our
observations, blockade of the AT
1
receptor by olmesartan
[49] or genetic deletion of ACE [53] also failed to amelio-
rate hypoxic PH and RV hypertrophy in mice. In contrast,
AT
1
antagonists (GR138950C, olmesartan) reversed
hypoxia-induced cardiopulmonary remodelling in rats [40,
57]. The discrepancy between these results remains unex-
plained; species differences might be involved.
Beside increased Ang II diminished bradykinin levels
may contribute to PH and lung perivascular inflammation
of EC GC-A KO mice. The small nine amino-acid
vasoactive peptide bradykinin has dual roles by exerting
pathophysiological as well as beneficial physiological
effects, mainly by stimulation of bradykinin B2 receptors.
Specifically in the lung, inhibition of bradykinin metabolic
breakdown by ACE inhibitors or exogenous administration
of B2 receptor agonists exerted protective effects, reducing
pulmonary arterial pressure in experimental hypertension
[50] and neutrophil recruitment by lipopolysaccharide [4].
These protective effects of bradykinin involve the
endothelial release of NO, prostacyclin and tissue-type
plasminogen activator [4]. Hence, we hypothesize that PH
and perivascular inflammation in EC GC-A KO mice is
mediated through both a local increase in Ang II and a
decrease in bradykinin mediated signalling.
In general, experimental and clinical studies emphasize
that a compromised endothelial barrier plays a central role
in the pathogenesis of PH [3,45]. In fact, both acute and
chronic hypoxia in mice and rats induce subtle but signif-
icant inflammation in the lung prior to the onset of struc-
tural changes in the vessel wall [34,36]. On the other hand,
numerous studies in vitro/in vivo indicated that ANP exerts
pulmonary endothelial barrier-protecting actions. Synthetic
ANP reduced hypoxia, TNF-a, thrombin, or bacterial
endotoxin (PepG)-induced paracellular hyperpermeability
of pulmonary microvascular and macrovascular endothelial
cells cultured on permeable supports and acute PepG-in-
duced lung injury in mice [30,51]. Conversely, enhanced
PepG-induced lung injury, ICAM-1/VCAM-1 expression
and vascular leak were observed in ANP
-/-
mice [51]. We
did not observe macroscopic signs of pulmonary edema in
EC GC-A KO mice under normoxic or hypoxic conditions.
However, we found increased pulmonary levels of the
endothelial adhesion molecules ICAM-1 and VCAM-1.
Together with the imbalance between Ang II and
bradykinin signalling these changes possibly contribute to
enhanced pulmonary neutrophil infiltration and PH in EC
GC-A KO mice.
Study limitations
One limitation of the EC GC-A KO mice is that the GC-A
receptor is absent not only in pulmonary but also in sys-
temic endothelia. Unfortunately, a selective disruption of
target genes within the pulmonary circulation is technically
impossible so far and therefore this limitation is shared by
other disease relevant genetic mouse models [20]. Hence,
because EC GC-A KO mice have mild systemic arterial
hypertension and subtle LV hypertrophy, it is possible that
PH was secondary to the systemic phenotype. However,
invasive haemodynamic studies clearly demonstrated that
cardiac output and LV function of EC GC-A KO mice are
unaltered, also after chronic hypoxia. In addition there are
no signs of pulmonary edema, corroborating that the PH of
these mice is not secondary to left ventricular failure. Even
more, we did not observe vascular thickening or inflam-
mation in other tissues of EC GC-A KO mice. Together
these observations indicate that the pulmonary vascular
alterations of EC GC-A KO mice are not secondary to
systemic changes.
Concordant to the mice with systemic ANP or GC-A
deletion ([28,29] and present study), mice with EC-re-
stricted GC-A ablation have mild PH already under base-
line, normoxic conditions, which was aggravated by
chronic hypoxia. However, the absolute increases in mean
RVSP and in vascular remodelling in response to CH were
similar in EC GC-A KO mice and in controls. Again this is
consistent with previous observations in mice with global
ANP or GC-A inactivation [28,29]. Hence, it remains
impossible to definitively determine whether ANP/GC-A
dysfunction aggravates hypoxic PH or merely produces
normoxic PH that is then amplified by hypoxia.
Conclusion
In summary, endothelial effects of ANP play a critical
physiological role in the chronic maintenance of pul-
monary vascular homeostasis. Our observations in vitro
and in EC GC-A KO mice suggest that ANP moderates the
endothelial expression (VCAM-1, ICAM-1) or formation
of local factors (ACE/Ang II, possibly bradykinin) regu-
lating SMC proliferation and the interaction of EC and
inflammatory cells. Our experimental observations in a
monogenetic mouse model suggest that chronic endothelial
ANP/GC-A dysfunction, e.g. provoked by hypoxia, might
contribute to lung endothelial barrier impairment and
vascular remodelling, and thereby to PH.
Basic Res Cardiol (2016) 111:22 Page 13 of 16 22
123
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Acknowledgments This study was supported by the Deutsche
Forschungsgemeinschaft (DFG KU 1037/6-1, to M.K.) and by the
Excellence Cluster Cardio Pulmonary System (ECCPS, to R.S.).
Compliance with ethical standards
Conflict of interest The authors declare that there are no conflicts
of interest.
Open Access This article is distributed under the terms of the
Creative Commons Attribution 4.0 International License (http://crea
tivecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided you give
appropriate credit to the original author(s) and the source, provide a
link to the Creative Commons license, and indicate if changes were
made.
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