Effects of noise on vascular function, oxidative stress, and inflammation: mechanistic insight from studies in mice.

doi:10.1093/EURHEARTJ/EHX081

Effects of noise on vascular function, oxidative

stress, and inflammation: mechanistic insight

from studies in mice

Thomas Mu¨nzel

1,2

*, Andreas Daiber

1,2

, Sebastian Steven

1

, Lan P. Tran

1

,

Elisabeth Ullmann

1

, Sabine Kossmann

1

, Frank P. Schmidt

1

, Matthias Oelze

1

,

Ning Xia

3

, Huige Li

3

, Antonio Pinto

4

, Philipp Wild

2,4

, Kai Pies

5

, Erwin R. Schmidt

6

,

Steffen Rapp

6

, and Swenja Kro¨ ller-Scho¨n

1

Center for Cardiology, Cardiology I – Laboratory of Molecular Cardiology, University Medical Center at the Johannes Gutenberg University Mainz, Langenbeckstr. 1, 55131

Mainz, Germany;

2

German Center for Cardiovascular Research (DZHK), Partner Site Rhine-Main;

3

Department of Pharmacology;

4

Preventive Cardiology and Preventive

Medicine, Center for Cardiology, University Medical Center of the Johannes Gutenberg-University Mainz, Mainz, Germany;

5

Engineering Ofﬁce for Noise Protection, Mainz,

Germany; and

6

Institute for Molecular Genetics, Johannes Gutenberg University, Mainz, Germany

Received 24 August 2016; revised 25 October 2016; editorial decision 23 January 2017; accepted 6 February 2017; online publis h-ahead-of-print 17 February 2017

See page 2850 for the editorial comment on this article (doi: 10.1093/eurheartj/ehx217)

Aims Epidemiological studies indicate that trafﬁc noise increases the incidence of coronary artery disease, hypertension

and stroke. The underlying mechanisms remain largely unknown. Field studies with nighttime noise exposure dem-

onstrate that aircraft noise leads to vascular dysfunction, which is markedly improved by vitamin C, suggesting a

key role of oxidative stress in causing this phenomenon.

...................................................................................................................................................................................................

Methods

and results

We developed a novel animal model to study the vascular consequences of aircraft noise exposure. Peak sound

levels of 85 and mean sound level of 72 dBA applied by loudspeakers for 4 days caused an increase in systolic

blood pressure, plasma noradrenaline and angiotensin II levels and induced endothelial dysfunction. Noise increased

eNOS expression but reduced vascular NO levels because of eNOS uncoupling. Noise increased circulating levels

of nitrotyrosine, interleukine-6 and vascular expression of the NADPH oxidase subunit Nox2, nitrotyrosine-

positive proteins and of endothelin-1. FACS analysis demonstrated an increase in inﬁltrated natural killer-cells and

neutrophils into the vasculature. Equal mean sound pressure levels of white noise for 4 days did not induce these

changes. Comparative Illumina sequencing of transcriptomes of aortic tissues from aircraft noise-treated animals

displayed signiﬁcant changes of genes in part responsible for the regulation of vascular function, vascular remodel-

ling, and cell death.

...................................................................................................................................................................................................

Conclusion

We established a novel and unique aircraft noise stress model with increased blood pressure and vascular dysfunc-

tion associated with oxidative stress. This animal model enables future studies of molecular mechanisms, mitigation

strategies, and pharmacological interventions to protect from noise-induced vascular damage.

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Keywords

Environmental stressor

•

Noise exposure

•

Endothelial dysfunction

•

Oxidative stress

•

eNOS uncou-

pling

•

NADPH oxidase

•

Vascular inﬂammation

* Corresponding author. Tel: þ49 6131 17 7250, Fax: þ49 6131 17 6615, Email: tmuenzel@uni-mainz.de

V

C

The Author 2017. Published by Oxford University Press on behalf of the European Society of Cardiology.

This is an Ope n Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/),

which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact

journals.permissions@oup.com

European Heart Journal (2017) 38, 2838–2849

BASIC SCIENCE

doi:10.1093/eurheartj/ehx081

Hypertension

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Introduction

The health burden of environmental noise exposure has been in-

creasingly acknowledged. The World Health Organization (WHO)

estimates that each year almost 1 million disability adjusted life years

are lost due to noise exposure in the Western European population.

Epidemiological studies have demonstrated that traffic noise ex-

posure is associated with cardiovascular diseases such as hyperten-

sion, myocardial infarction, and stroke.

1–3

Noise is a non-specific

stressor that arouses the autonomic nervous system and endocrine

system. Chronic low levels of noise can cause disturbances of activity,

sleep, and communication leading to emotional responses such as an-

noyance and subsequent stress.

4,5

Chronic stress in turn has been

demonstrated to generate its own cardiovascular risk factors such as

increased blood pressure and dyslipidemia, increased blood viscosity

and blood glucose and activation of blood clotting factors in animal

models

6,7

and humans.

8–12

Persistent chronic noise exposure in-

creases the risk of cardiovascular and metabolic diseases such

as hypertension, coronary artery disease, diabetes, and stroke.

13–17

A recent meta-analysis reported on a linear relationship between

exposure to transportation noise and the incidence of ischaemic

heart diseases, warranting further studies to understand the mechan-

istic basis of this association.

18

We recently studied the effects of nighttime aircraft noise on vas-

cular (endothelial) function in healthy subjects

19

and demonstrated

that nighttime noise dose-dependently impairs endothelial function,

disrupts sleep quality and tends to increase blood pressure.

Importantly, in a subgroup of subjects, vitamin C significantly im-

proved flow-mediated dilation (FMD) suggesting that increased pro-

duction of reactive oxygen species (ROS) in response to noise

exposure contributes to vascular dysfunction.

19

In our subsequent

clinical study, we reported on synergistic adverse cardiovascular ef-

fects of noise exposure in patients with pre-established cardiovascu-

lar diseases, further supporting the role of oxidative stress.

20

Since previous animal models employed high dBA levels (up to

100 dBA),

21–23

which can lead to direct auditory damage, we de-

veloped a novel noise exposure model in mice (C57Bl/6j) with lower

peak sound levels (<85 dBA), lower mean sound pressure levels (72

dBA) and shorter exposure times (1–4 days), which has been shown

to be safe and to cause mainly non-auditory effects to animals such as

stress reactions.

24

To our knowledge, no study has so far investigated

directly the vascular consequences of noise exposure. In the present

studies we sought to characterize the mechanisms of aircraft noise-

induced vascular (endothelial) dysfunction, e.g. to identify the sources

of ROS production, to assess the degree of imbalance of vascular

NO/O

:

2

-

production and the extent of inflammation in the vascula-

ture of noise-exposed animals. We also tested the consequences of

noise on the gene expression profile in the vasculature by performing

Illumina transcriptomic next generation sequencing.

Methods

Noise exposure

All animals were treated in accordance with the Guide for the Care

and Use of Laboratory Animals as adopted by the U.S. National

Institutes of Health and approval was granted by the Ethics

Committee of the University Medical Center Mainz and the

Landesuntersuchungsamt Rheinland-Pfalz (Koblenz, Germany; permit

number: 23 177-07/G 12-1-021 E3 and 23 177-07/G 15-1-094).

Noise exposure consisted of repetitive playbacks of a 2 hour long

noise pattern of 69 aircraft noise events with a duration of 43 s and a

maximum sound pressure level of 85 dB(A) and a mean sound pres-

sure level of 72 dB(A), which does not lead to hearing loss.

24

Noise

events were separated by silent periods with irregular distribution to

prevent early adaptation. The noise pattern was played back from

downward facing loudspeakers mounted approximately 30 cm above

the mouse cages with a Grundig MS 540 compact sound system with

a total output of 65 W. Loudness and corresponding sound pressure

levels were calibrated with a Class II Sound level meter (Casella CEL-

246) within one the cages at initial setup. Actual SPLs during expos-

ure was continuously recorded during the study period with the

same device placed between cages with upward facing microphone.

The average SPL (Leq3) is 72 dB(A) at a usual background noise level

of 48 dB(A) in the animal facility. In control experiments, mice were

exposed to ‘white noise’ (this is a random noise with constant spec-

tral density within the range of human hearing from 20 Hz to 20 kHz)

using exactly the same average SPL as for aircraft noise. All SPL and

maximum sound pressure levels were measured within the mouse

cages.

For further information of the methodology employed for deter-

mination of blood pressure, stress hormones, vascular function, nitric

oxide quantification, oxidative stress parameters, inflammatory path-

ways, gene and protein expression, next generation sequencing see

the Supplementary material online.

Results

Effects of noise exposure on laboratory

values, blood pressure and stress

hormone levels

Noise exposure for 1–4 days caused no significant changes in heart/

body weight ratio (Supplementary material online, Figure 1S). Blood

glucose levels were increased compared with controls during the en-

tire noise exposure period, while triglyceride levels were not

changed (Supplementary material online, Figure 1S). Four days of

noise exposure also increased systolic and mean blood pressure

(Figure

1A, C) while diastolic blood pressure did not change signifi-

cantly (Figure

1B). Plasma catecholamine levels (noradrenaline, dopa-

mine significantly and adrenaline by trend) and angiotensin II levels

increased (Figure

1D–G). Cortisol levels in urine showed no significant

change (although a trend for increased values in response to noise

exposure) and were increased in kidney after 4 days of noise expos-

ure (Figure

1H, I).

Effects of noise exposure on vascular

function

Noise exposure for 1, 2, and 4 days caused a significant attenuation of

ACh-induced relaxation of mouse aorta (Figure

2A, Supplementary

material online, Table 2S). Responses to the endothelium independ-

ent vasodilator GTN were also significantly modified (Figure 2B,

Supplementary material online,Table2S). Noise exposure induced an

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increase in sensitivity of the aorta to norepinephrine- and ET-1 medi-

ated vasoconstriction (Figure

2C, D, Supplementary material online,

Table 3S).

Effects of noise exposure on eNOS

expression, eNOS-mediated NO produc-

tion and the functionality of the NO/

cGMP signaling pathway

Endothelial dysfunction was paralleled by a decrease in vascular NO

production assessed by electron paramagnetic resonance (EPR)

(Figure

2E, F). Paradoxically, eNOS was up-regulated in vascular tissue

on day 4 of noise exposure (Figure

3A) and phosphorylation of eNOS

at Ser1177 was enhanced over the entire time period of noise expos-

ure (Figure

3B, C). Since eNOS-mediated NO production was

decreased despite up-regulation of eNOS expression and Ser1177

phosphorylation, we tested whether eNOS was uncoupled. Using

DHE staining vascular superoxide production was increased in ves-

sels from noise exposed animals and treatment with the eNOS inhibi-

tor L-NAME consistently decreased endothelial superoxide

production, identifying eNOS as a significant superoxide source

(Supplementary material online, Figure 2S). Noise causes S-glutathio-

nylation of eNOS, an established mechanism of eNOS uncoupling in

the aorta (Figure

3D) and the heart (Figure 4H). The expression of

GCH-1 and DHFR, both responsible for providing the eNOS cofac-

tor tetrahydrobiopterin (BH4), were up-regulated, compatible with a

counter regulatory response (Figure

3E, F).

With respect to the NO/cGMP/cGK-I signaling pathway, we did

not observe any changes in the expression of the sGC subunits

(Supplementary material online, Figure 3S). However in parallel to the

decrease in NO production on d1 we observed a decrease in the ac-

tivity of cGK-I as assessed by the diminished phosphorylation of the

VAsodilator-Stimulated Phosphoprotein at serine 239 (P-VASP)

(Supplementary material online, Figure 3S).

Figure 1 Effects of noise for 1, 2, and 4 days on blood pressure and stress hormone release. Noise increased significantly systolic and mean arterial

(A, C) but not diastolic (B) blood pressure. Noise increased noradrenalin, dopamine and angiotensin II levels significantly and adrenalin by trend

(D–G). Cortisol levels in urine and kidney showed a weak trend for an increase under noise exposure, which was significant for kidney cortisol on day

4(H, I). Data are mean ± SD from n = 8–16 mice/day (A–C), 8–11 (D), 7–8 (E), 9–11 (F), 7–12 (G), 6–14 (H) and 7–12 (I) mice/group.

2840 T. Mu¨nzel et al.

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Effects of noise exposure on ROS

production in plasma, heart and the

vasculature

In plasma, noise exposure led to an increase in 3-nitrotyrosine-posi-

tive proteins (day 2–4), MDA-positive proteins (day 4) and interleu-

kin 6 (day 2–4) as established by Dot Blot measurements (Figure

4A–

C). In vascular tissue, noise exposure increased expression of the

NADPH oxidase subunit NOX-2 (day 1–4) and ET-1 (day 2), while

the NOX-1 subunit was not modified (Figure

4D–F). Likewise, we

observed an increase in NADPH oxidase activity and 3-nitrotyro-

sine-positive proteins in heart homogenates (Figure 4G, I).

Immunohistochemical analysis revealed that noise exposure

enhanced 3-nitrotyrosine staining throughout the vasculature (day 1–

4, Figure

5A), augmented 4-hydroxynonenal staining (day 1–4,

Supplementary material online, Figure 2S) and increased vascular ET-

1 levels on day 2, mainly within the endothelium (Figure

5B).

Effects of noise on gene expression in

MLEC

Noise exposure caused a significant increase in eNOS, HO-1, PGC-

1a and NOX-1 expression in freshly isolated mouse lung endothelial

cells (Supplementary material online, Figure 4S) from noise-exposed

animals. The changes in eNOS gene expression obtained in MLECsin

response to noise parallel changes in the aorta.

Figure 2 Effects of noise on vascular function, sensitivity to vasoconstrictors and vascular NO production. Relaxation by the endothelium-depend-

ent and -independent vasodilators acetylcholine (ACh, A) and nitroglycerin (NTG, B) were impaired by noise exposure. The sensitivity of the aorta

to vasoconstrictors like norepinephrine (C) and endothelin-1 (D) was increased upon noise exposure. Noise exposure for 1 and 4 days significantly

reduced the aortic NO production and bioavailability measured by EPR. (E) Quantification of the NO signal detected by Fe(DECT)

2

spin trapping. (F)

Representative NO traces of mice not exposed (Ctr.) and exposed to noise. For detailed statistical analysis see Supplementary material online, Tables

2S and 3S.Dataaremean±SDfromn =13–26(A, B), 8–22 (C, D), 8–22 (E) mice/group.

Effects of noise on vascular function, oxidative stress, and inflammation 2841

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Effects of noise on invasion of the

vasculature with inflammatory cells

FACS analysis of mouse aorta revealed that noise exposure led to a

significant increase of myelomonocytic cells and natural killer cells

within the vasculature on day 1 or 4 (Supplementary material online,

Figure 5S, panels A, B). No significant increase in infiltration was

observed for neutrophils (Supplementary material online, Figure 5S,

panel C) and at least one significantly increased day for leucocytes in

general as well as macrophages and monocytes (Supplementary ma

terial online, Figure 5S, panel D). No significant changes were observed

for T cells (Supplementary material online, Figure 5S, panel D). The

gating strategy for FACS analysis is shown in Supplementary material

online, Figure 6S.

Effects of white noise on parameters of

vascular function and oxidative stress

Exposure of mice to white noise did not change ACh-dependent re-

laxation (Supplementary material online, Figure 7S). Also other par-

ameters of NO/cGMP signaling such as eNOS phosphorylation of

eNOS at Ser1177 and of VASP at Ser239, BH4 generating enzymes

GCH-1 and DHFR, sGC, cGK-1 showed no major changes in the

white noise group (Supplementary material online, Figure 7S and 8S).

Plasma 3-nitrotyrosine positive proteins and IL-6 were not changed

(Supplementary material online, Figure 7S). Aortic NADPH oxidase

subunits NOX-1 and NOX-2 were not up-regulated (Supplementary

material online,Figure7S) and endothelial superoxide formation was

not changed with no indication of eNOS uncoupling as determined

by L-NAME incubation (Supplementary material online,Figure9S)

and as supported by unaltered S-glutathionylation of eNOS

(Supplementary material online,Figure8S). Aortic ET-1 protein ex-

pression showed no major changes (Supplementary material online,

Figure 7S).

Next generation seque ncing

Comparative analysis of the transcriptomes of aortic tissue from noise

treated animals vs. controls showed numerous differentially expressed

genes. If a threshold of P < 0.05 and Log2 fold change >_0.5 is applied,

224 genes were categorized as transcriptionally either up- or down-

regulated over the entire experimental period. The majority of these

genes are down-regulated. Depending on the length of noise treat-

ment, the level of gene expression of individual genes can fluctuate

(see Supplementary material onlin e, Figure 10S). In Supplementary

material online, Figure 11S, highly differentially expressed gene prod-

ucts from the NGS data are presented in box plots over day 1, 2, and

4 of noise treatment. The four strongest up-regulated genes

compared with controls were Zbtb44, Setad4, Ypel2,andIhh. Similarly,

the amount of transcripts of Sacs, Nbeal1, PTPN4,andNR4A3 were sig-

nificantly reduced by noise. In Supplementary material online, Tables

4S and 5S, the 30 most highly up- and down-regulated genes are listed.

Pathway analysis by gene ontology annotation from NGS data

Figure 3 Effects of noise for 1, 2, and 4 days on endothelial NO-synthase protein expression and activity. (A–C) Endothelial NO synthase protein

expression is increased after 4 days noise exposure and activating phosphorylation at Ser1177 is increased significantly at all time points measured.

(D) eNOS S-glutathionylation as a surrogate marker for uncoupling of the protein was increased significantly at all time points of noise exposure.

(E, F) Noise increased GTP-cyclohydrolase I (GCH-I) and dihydrofolate reductase (DHFR) expression leading to increased synthesis of the eNOS co-

factor tetrahydrobiopterin (BH

4

) responsible for the coupling of the enzyme. Data are mean ± SD from n = 6–8 samples (pooled from 2 to 3 mice

per sample) (A–C, E, F) and 7–10 samples (pooled from 2 mice per sample) (D).

2842 T. Mu¨nzel et al.

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Effects of noise on vascular function, oxidative stress, and inflammation: mechanistic insight from studies in mice.

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