8 The Journal of Clinical Investigation http://www.jci.org
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the PSQ 96 Gold Reagent Kit (Biotage AB) with the following oligonucle-
otide: ACE-S: CCTCCACACTCCAATTATAACTTTC-3′. The sequence ana-
lyzed by pyrosequencing was 85-bp long, encompassing 10 CpGs.
NO plasma concentration. NO was measured in plasma samples obtained
by cardiac punctuation by chemiluminescence with a NO analyzer (Sievers
280 NOA) after reduction of NO
to NO with VCl
Survival. Eighty male FVB mice generated by ART and 80 control mice
born and raised in our animal facility were observed in these studies. Three
to four animals were placed in a cage in the clean conventional mouse facil-
ity. Six ART and seven control mice had to be sacrificed because of inju-
ries related to fighting. Throughout the study, the mice were housed with
lights on from 7:00 am to 7:00 pm, and access to food and water was ad
libitum. Thirty-two ART and twenty control mice were fed normal chow,
42 ART mice and 53 control mice were fed a HFD (49.5% fat, 31.5% pro-
tein, and 0% carbohydrates; Safe). One week each month, mice in the HFD
groups were fed a standard chow diet (pellets) to avoid dental problems.
Kaplan-Meier survival curves were constructed using known birth and
death dates, and the log-rank test was used to evaluate statistical differ-
ences between groups.
Statistics. Statistical analyses were made using JMP v. 7.0 software (SAS
Institute). Bivariate analyses were made using the unpaired 2-tailed Stu-
dent’s t test and 1- or 2-factor ANOVA. Post hoc comparisons were made
using the Tukey HSD test and Dunnett’s multiple comparison test. A
P value of less than 0.05 was considered to indicate statistical significance.
Unless otherwise indicated, data are given as mean ± SD.
Study approval. All animal protocols were approved by the CHUV Institu-
tional Animal Care Committee.
We are indebted to Françoise Urner for help with the initial studies
and to Caroline Mathieu, Christelle Stouder, Pierre Dessen, and
Arnaud Bichat for invaluable technical assistance. This work was
supported by the Swiss National Science Foundation, the Eagle
Foundation, the Leenaards Foundation, the FABER Foundation,
the Swiss Society of Hypertension, the Prof. Dr. Max Cloetta Foun-
dation, and the Placide Nicod Foundation.
Received for publication January 22, 2013, and accepted in revised
form September 4, 2013.
Address correspondence to: Urs Scherrer, Swiss Cardiovascular Center
Bern, University Hospital, Bern, Switzerland. Phone: 41.31.632.01.02;
Fax: 41.31.632.42.11; E-mail: Urs.Scherrer2@insel.ch.
ratories) in 5% milk/TBST overnight and were detected using peroxidase-
conjugated secondary antibody (1:10000) in 5% milk/TBST for 1 hour.
Products were visualized by chemiluminescence (GE Healthcare), and
band intensity was measured using ImageQuant 5.0 software (Molecu-
lar Dynamics). Equal protein loading was confirmed by β-actin (1:10000;
Thermo Scientific Pierce Products) hybridization on the same membrane.
RNA preparation and gene expression analysis. Total RNA from aorta was
extracted using the TRIzol RNA Isolation Reagents according to the manu-
facturer’s protocol (Life Technologies Europe B.V.). From 500 to 1000 ng
total RNA were reverse transcribed using 400 units of Moloney Murine
Leukemia Virus Reverse Transcriptase (Invitrogen) in the presence of 1
U/μl RNasin (Promega Corp.), 0.2 μg of random primers (oligo[dN]6)
(Promega Corp.), 2 mM dNTP, and 20 μM of DTT (Invitrogen). The expres-
sion of the cDNAs for mouse eNOS was determined by quantitative real-
time PCR using an ABI StepOne Plus Sequence Detection System (Applera
Europe) and normalized using the housekeeping gene Rps29. PCR prod-
ucts were quantified using Master SYBR Green Mix (Applera Europe),
and results are expressed in AU relative to the control group mean value.
Primers were designed using Primer Express software (Applera Europe)
and chosen when possible on both sides of an intron to avoid amplifica-
tion of eventual contaminating genomic DNA. Oligos were used at 217
nM each (Microsynth). The sequence of the primers used were as follows:
eNOS, forward: 5′-AAGGCAGCGGTGGAAATTAA-3′; eNOS, reverse:
Methylation of the endothelin-1 gene promoter in the aorta. Methylation anal-
ysis was performed in the Edn-1 (GenBank NC_000079) promoter in a
region where DNA methylation was shown to control gene expression
(41, 42). The following oligonucleotides were designed with the Pyro-
Mark Assay Design 2.0 program (Qiagen): Edn-F: 5′-GTGATTTTTTA-
AGGAGTTTTAGAAATAGG-3′; Edn-R: 5′-biotin-7AACCCTACAACCCTA-
AACACACTTATT-3′. The biotinylated PCR products were purified using
streptavidin-sepharose beads (Amersham) and sequenced using the PSQ 96
Gold Reagent Kit (Biotage AB) with the following oligonucleotide: Edn-S:
5′-GAGTTTTAGAAATAGGTAGG-3′. The sequence analyzed by pyro-
sequencing was 239-bp long, encompassing 9 CpGs.
Methylation of the ACE gene promoter in the aorta. Methylation analysis was
performed in the ACE promoter, in a CpG island region where methyla-
tion was reported to be modified in the mouse fetus after maternal dietary
changes during pregnancy (19, 40, 43). The following oligonucleotides were
designed with the PyroMark Assay Design 2.0 program (Qiagen): ACE-F:
5′-biotin-7GGTGGTGGTTGGGTTTTATA-3′; ACE-R: 5′-CCCAACTAAC-
CACCATACTCTAAAACAT-3′. The biotinylated PCR products were puri-
fied using streptavidin-sepharose beads (Amersham) and sequenced using
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