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Identification of downstream
target genes regulated by the
nitric oxidesoluble
guanylate cyclasecyclic
guanosine monophosphate
signal pathway in pulmonary
hypertension
Abstract
Objective: To investigate the downstream target genes regulated by the nitric oxidesoluble
guanylate cyclasecyclic guanosine monophosphate (NO-sGC-cGMP) signal pathway and their
possible roles in the pathogenesis of pulmonary hypertension (PH).
Methods: Digital gene expression tag profiling was performed to identify genes that are
differentially expressed after activation of the NO-sGC-cGMP signal pathway in human pulmonary
artery smooth muscles cells using 8-bromo-cyclic guanosine monophosphate, BAY 41-2272 and
BAY 60-2770. Results were confirmed using real-time polymerase chain reaction. Gene ontology
and signal transduction network analyses were also performed.
Results: A number of genes were differentially expressed, including MMP1, SERPINB2, GREM1 and
IL8. A total of 68 gene ontology terms and seven pathways were found to be associated with these
genes. Most of these genes are involved in cell proliferation, cell migration and apoptosis, which
may contribute to the pathological pulmonary vascular remodelling in PH.
Conclusion: These results may provide new insights into the molecular mechanisms of PH.
1
Key Laboratory of Geriatrics, Beijing Hospital & Beijing
Institute of Geriatrics, Ministry of Health, Beijing, China
2
Department of Respiratory and Critical Care Medicine,
Beijing Hospital, Ministry of Health, Beijing, China
3
Department of Respiratory and Critical Care Medicine,
China-Japan Friendship Hospital, Ministry of Health,
Beijing, China
Creative Commons CC-BY-NC: This article is distributed under the terms of the Creative Commons Attribution-NonCommercial
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Zou et al.
509
Keywords
Pulmonary hypertension, NO-sGC-cGMP pathway, digital gene expression tag profiling array,
differentially expressed genes, gene ontology, signalling pathway
Date received: 17 November 2015; accepted: 9 February 2016
Introduction
Pulmonary hypertension (PH) is a serious
disease characterized by elevated blood
pressure in the pulmonary artery, pulmonary vein or pulmonary capillaries, leading to
shortness of breath, dizziness, weakness and
even symptoms of shock symptoms such as
fainting.1 Without treatment, the disorder
will progress rapidly to right heart failure
and death within 3 years of diagnosis.
The pathogenesis of PH is not completely
understood, but multiple studies have suggested that an imbalance of vasoconstriction/vasodilation and proliferation/
antiproliferation may be involved.2 Nitric
oxide (NO), which is produced by NO
synthase from L-arginine, is an important
vascular modulator in the development of
PH. It exerts its biological activity mainly by
activating soluble guanylate cyclase (sGC)
to synthesize the second messenger cyclic
guanosine monophosphate (cGMP),3 which
promotes vasodilation and inhibits smooth
muscle cell growth and proliferation, neuronal transmission, host defences, leukocyte
recruitment, platelet aggregation and vascular remodelling through a number of downstream targets.4
Impairment of the NO-sGC-cGMP pathway, associated with dysregulation of NO
production, sGC activity and cGMP degradation, has been shown to contribute to
the progression of PH.5 A number of sGC
stimulators and activators that can bind to
the b subunit of sGC and stimulate it in a
direct or indirect way have been identied,
including 3-(50 -hydroxymethyl-20 -furyl)-1benzyl indazole (YC-1), 3-(4-amino-5-cyclopropylpyrimidin-2-yl)-1-(2-uorobenzyl)1H-pyrazolo[3,4-b]pyridine (BAY 41-2272),
2-[1-[(2-uorophenyl)methyl]-1H-pyrazolo
[3,4-b]pyridin-3-yl]-5-(4-morpholinyl)-4,6pyrimidinediamine (BAY 41-8543) and riociguat (BAY 63-2521).6,7 They can act as
potent pulmonary vasodilators, decreasing
the mean pulmonary arterial pressure and
vascular resistance, and have been shown to
reverse structural lung vascular remodelling
and right heart hypertrophy in PH.8
Riociguat is the rst sGC stimulator to
undergo clinical study. In clinical trials
it signicantly improved the pulmonary
vascular haemodynamics in pulmonary
arterial hypertension and chronic thromboembolic PH with no serious adverse events.9
In contrast, a specic inhibitor of sGC,
1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one,
has been shown to block the rise in cGMP
and the antiproliferative action of the
NO-sGC-cGMP pathway.10
To date, studies suggest that once cGMP
is produced, its eects could be executed
though three main groups of cellular target
molecules: cGMP-dependent protein kinases (PKGs), cGMP-gated cation channels
and phosphodiesterases (PDEs).11 The PDE
inhibitor sildenal is used to treat idiopathic
PH.12,13 However, besides these three
groups of molecules, little is known about
the downstream target genes of the
NO-sGC-cGMP pathway and the signalling
events regulating gene expression. Using
genome-wide technologies that can measure
the expression of thousands of genes simultaneously, dierences in gene expression in
response to particular treatments can be
measured. In the present study, digital gene
expression tag proling was used to measure
dierences in gene expression in human
pulmonary artery smooth muscle cells
treated with two sGC agonists14 and a
510
Determination of differentially
expressed genes
To compare the dierential expression of
genes across samples, the raw data was
collected and ltered to remove low quality
tags, adaptor tags and tags with a single
copy number. The gene expression levels
Zou et al.
511
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Statistical analyses
All data were expressed as the mean SD.
Dierential gene expression levels were
based on log2 ratios among the dierent
groups. Statistical comparisons were performed using the Students t-test for analyzing two-group data. The threshold of
signicance was dened as a P-value < 0.05.
Results
Differentially expressed genes
Compared with controls, 539 upregulated
genes and 367 downregulated genes were
identied in cells treated with 8-Br-cGMP,
and 993 upregulated genes and 1002 downregulated genes were identied in cells
treated with BAY 41-2272 (100 mmol/l for
8 h) (Figure 1). In addition, 219 and 143
genes were signicantly upregulated and 353
and 351 genes were signicantly downregulated after treatment with BAY 41-2272
(10 mmol/l for 24 h) and BAY 60-2770
(10 mmol/l for 24 h), respectively. A total of
84 of the upregulated genes and 87 of the
downregulated genes showed similar variation trends in all four groups, suggesting
that activation of the NO-sGC-cGMP pathway might aect expression of a group of
important downstream factors. Nine genes
that were signicantly up- or downregulated
compared with controls when the NO-sGCcGMP pathway was activated are shown
in Table 1. Interestingly, these dierentially
expressed genes are involved in the
Figure 1. Differentially expressed genes (DEGs) in pulmonary artery smooth muscle cells from patients
with pulmonary hypertension treated with 8-bromo-cyclic guanosine monophosphate (8-Br-cGMP), BAY
41-2272 or BAY 60-2770 compared with control cells.
Zou et al.
513
Table 1. Significantly up- or downregulated genes in pulmonary artery smooth muscle cells from patients
with pulmonary hypertension treated with 8-bromo-cyclic guanosine monophosphate (8-Br-cGMP), BAY
41-2272 or BAY 60-2770 compared with control cells measured using digital gene expression tag profiling.
The values given are the number of unique reads, which is an indication of expression levels.
Gene
MMP1
SERPINB2
GREM1
0
2
609
310
1401
10168
508
2270
7528
35
163
1402
19
114
1456
IL8
ANGPTL4
COL5A3
PDGFRB
36
168
194
2697
1203
3064
60
865
1600
5275
12
559
29
1378
17
943
25
516
16
1104
TENC1
CXCL12
1591
3146
773
837
157
298
1221
2230
1209
2163
Extracellular matrix
Response to wounding
Epithelial to mesenchymal
transition
Cell adhesion
Regulation of angiogenesis
Collagen protein
G-protein coupled
receptor activity
Cell proliferation
Angiogenesis
GO analysis of differentially
expressed genes
Gene ontology analysis is widely used to
describe the molecular functions, cellular
components and biological processes of
dierentially expressed genes in a microarray. As the variation trends of most of
the dierentially expressed genes were
similar in the four treatment groups, GO
analysis of the biological process was
only performed in the group treated with
BAY 41-2272 (100 mmol/l for 8 h). A total
of 68 GO items associated with the dierentially expressed genes were identied
(Table 2). Regulation and negative regulation of cellular process, negative regulation of biological process and regulation
of cellular macromolecule biosynthetic
process were the most signicantly
enriched items (low P-value) (Table 2).
Moreover, other items associated with the
progression of PH such as cell proliferation,
cell migration and apoptosis were also
enriched.
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Figure 2. Expression of genes MMP1, SERPINB2, GREM1, IL8 and actin in human pulmonary artery smooth
muscle cells treated with BAY 41-2272 (100 mmol/l for 8 h) compared with controls treated with dimethyl
sulphoxide (DMSO) measured using quantitative real-time polymerase chain reaction. (A) Agarose gel
electrophoresis. (B) Ratio of gene expression compared with controls. Data presented as mean SD.
**P < 0.01, ***P < 0.001.
Discussion
Despite several advances in therapy, there is
currently no curative treatment for PH and
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2.27 1010
6.62 109
5.91 108
8.27 108
9.27 108
4.26 107
4.35 107
9.77 107
1.33 106
1.49 106
1.53 106
2.49 106
3.30 106
7.49 106
1.07 105
1.19 105
1.32 105
1.63 105
1.66 105
2.55 105
3.04 105
5
3.87 10
4.28 105
Table 2. Continued.
(continued)
Corrected
P-value
1.1 104
1.2 104
2.2 104
2.8 104
3.4 104
4.1 104
8.8 104
1.15 103
1.59 103
2.03 103
2.74 103
3.17 103
3.96 103
4.27 103
5.05 103
5.21 103
6.60 103
6.90 103
8.31 103
1.055 102
1.141 102
1.161 102
1.161 102
1.400 102
1.511 102
1.574 102
1.613 102
1.776 102
(continued)
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Table 2. Continued.
2.007 102
2.081 102
Pathway
Q-value
Pathways in cancer
p53 signalling pathway
NOD-like receptor
signalling pathway
Malaria
MAPK signalling pathway
Base excision repair
Basal cell carcinoma
1.760569 105
1.290269 104
2.943706 103
Corrected
P-value
2.258 102
2.339 102
2.551 102
2.620 102
3.041 102
3.041 102
3.133 102
3.607 102
3.803 102
4.082 102
4.321 102
4.540 102
4.572 102
4.639 102
4.956 102
8.883089 103
9.527016 103
1.002455 102
1.336772 102
domain;
MAPK,
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Funding
This work was supported by the National
High Technology Research and Development
Program (grant 2012AA02A511), the National
Department Public Benet Research Foundation
(no. 201302008), the National Key Technology
Research and Development Program (grant
2013BAI09B00) and the National Natural
Science Foundation of China (grant 81400036).
518
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