Basic research
Involvement of reactive oxygen species in angiotensin II-induced endothelin-1 gene expression in rat cardiac fibroblasts

Presented at the International Young Investigator Prize Symposium at the XIVthWorld Congress of Cardiology.
https://doi.org/10.1016/j.jacc.2003.06.010Get rights and content
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Abstract

Objectives

The aim of this study was to investigate the effects of angiotensin II (Ang II) on fibroblast proliferation and endothelin-1 (ET-1) gene induction, focusing especially on reactive oxygen species (ROS)-mediated signaling in cardiac fibroblasts.

Background

Angiotensin II increases ET-1 expression, which plays an important role in Ang II-induced fibroblast proliferation. Angiotensin II also stimulates ROS generation in cardiac fibroblasts. However, whether ROS are involved in Ang II-induced proliferation and ET-1 expression remains unknown.

Methods

Cultured neonatal rat cardiac fibroblasts were stimulated with Ang II, and then [3H]thymidine incorporation and the ET-1 gene expression were examined. We also examined the effects of antioxidants on Ang II-induced proliferation and mitogen-activated protein kinase (MAPK) phosphorylation to elucidate the redox-sensitive pathway in fibroblast proliferation and ET-1 gene expression.

Results

Both AT1receptor antagonist (losartan) and ETAreceptor antagonist (BQ485) inhibited Ang II-increased DNA synthesis. Endothelin-1 gene was induced with Ang II as revealed by Northern blotting and promoter activity assay. Angiotensin II increased intracellular ROS levels, which were inhibited with losartan and antioxidants. Antioxidants further suppressed Ang II-induced ET-1 gene expression, DNA synthesis, and MAPK phosphorylation. PD98059, but not SB203580, fully inhibited Ang II-induced ET-1 expression. Truncation and mutational analysis of the ET-1 gene promoter showed that AP-1 binding site was an important cis-element in Ang II-induced ET-1 gene expression.

Conclusions

Our data suggest that ROS are involved in Ang II-induced proliferation and ET-1 gene expression. Our findings imply that the combination of ATIand ETAreceptor antagonists plus antioxidants may be beneficial in preventing the formation of excessive cardiac fibrosis.

Abbreviations

Ang II
angiotensin II
AP-1
activator protein-1
AT1
angiotensin II type 1 receptor
CAT
chloramphenicol acetyltransferase
DCF-DA
dichlorofluorescin diacetate
DPI
diphenyleneiodonium
ECM
extracellular matrix
ERK
extracellular signal-regulated kinase
ETA
ET-1 type A receptor
ET-1
endothelin-1
GPCRs
G-protein coupled receptors
JNK
c-Jun N-terminal kinase
MAPK
mitogen-activated protein kinase
MEK
MAPK/ERK kinase
mRNA
messenger RNA
NAC
N-acetylcysteine
redox
cellular oxidation reduction
ROS
reactive oxygen species

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Supported by National Science Council Grant, Taipei, Taiwan, and NHRI Postdoctoral Fellowship Award 91N002 (to T.-H. Cheng).