Miroslava Kvandová

Sanela Rajlic

Paul Stamm

Isabella Schmal

Dominika Mihaliková

Marin Kuntic

Maria Teresa

Bayo Jimenez

Omar Hahad

Marta Kollárová

Henning Ubbens

Maria Teresa Bayo Jimenez

Lea Strohm

Katie Frenis

Georg Daniel Duerr

Marc Foretz

Benoit Viollet

Yue Ruan

Subao Jiang

Qi Tang

Hartmut Kleinert

Steffen Rapp

Adrian Gericke

Eberhard Schulz

Matthias Oelze

John F Keaney

Andreas 10

Daiber

Swenja Kröller-Schön

Thomas Jansen
email: t.jansen@kvb-klinik.de

Dr Thomas Münzel
email: tmuenzel@uni-mainz.de

activation: molecular proof of a protective key role of endothelial α1AMPK against environmental noise exposure

Keywords: aircraft noise exposure, noise mitigation, α1AMPK, exercise, fasting, AICAR, reactive oxygen species, endothelial dysfunction

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Environmental pollutants, such as noise exposure, increased substantially during the last decades due to modern lifestyle, industrialization, and urbanization. Epidemiological studies and the European Environment Agency and the World Health Organization (WHO) report that traffic noise is a rising environmental stressor with a substantial negative impact on the cardiovascular system and that more than 1/3 of the European population is exposed to noise levels exceeding the WHO guidelines (>55 dB(A) average over the entire day) (for review, see [START_REF] Münzel | Environmental risk factors and cardiovascular diseases: a comprehensive expert review[END_REF][START_REF] Munzel | Transportation noise pollution and cardiovascular disease[END_REF][START_REF] Munzel | Adverse Cardiovascular Effects of Traffic Noise with a Focus on Nighttime Noise and the New WHO Noise Guidelines[END_REF] ). It was shown that noise exposure, especially nocturnal aircraft noise, not only leads to annoyance and disturbed sleep [START_REF] Beutel | Noise Annoyance Is Associated with Depression and Anxiety in the General Population-The Contribution of Aircraft Noise[END_REF][START_REF] Hahad | Annoyance to different noise sources is associated with atrial fibrillation in the Gutenberg Health Study[END_REF] but also induces arterial hypertension, chronic coronary artery disease, heart failure, arrhythmia, and even triggers acute cardiovascular death [START_REF] Munzel | Transportation noise pollution and cardiovascular disease[END_REF][START_REF] Munzel | Adverse Cardiovascular Effects of Traffic Noise with a Focus on Nighttime Noise and the New WHO Noise Guidelines[END_REF][START_REF] Munzel | Environmental Noise and the Cardiovascular System[END_REF] , mainly related to increased stress hormone levels as a consequence of neurohormonal activation of the hypothalamic-pituitary-adrenal axis and the sympathetic nervous system [START_REF] Munzel | Cardiovascular effects of environmental noise exposure[END_REF][START_REF] Babisch | The Noise/Stress Concept, Risk Assessment and Research Needs[END_REF] . Even short-term noise exposure for one night causes endothelial dysfunction, a biomarker for early atherosclerosis, diastolic cardiac dysfunction, and increased oxidative stress. It induces a prothrombotic inflammatory phenotype in healthy volunteers [START_REF] Herzog | Acute exposure to nocturnal train noise induces endothelial dysfunction and pro-thromboinflammatory changes of the plasma proteome in healthy subjects[END_REF][START_REF] Schmidt | The impact of aircraft noise on vascular and cardiac function in relation to noise event number -a randomized trial[END_REF][START_REF] Schmidt | Effect of nighttime aircraft noise exposure on endothelial function and stress hormone release in healthy adults[END_REF] and patients with coronary artery disease [START_REF] Schmidt | Nighttime aircraft noise impairs endothelial function and increases blood pressure in patients with or at high risk for coronary artery disease[END_REF] .

In mice, noise-induced stress hormone release, activation of immune cells, and oxidative stress by NADPH oxidase (NOX-2) activation and circadian clock dysregulation were demonstrated to cause hypertension and endothelial dysfunction, an important biomarker for future cardiovascular events [START_REF] Munzel | Effects of noise on vascular function, oxidative stress, and inflammation: mechanistic insight from studies in mice[END_REF][START_REF] Kroller-Schon | Crucial role for Nox2 and sleep deprivation in aircraft noiseinduced vascular and cerebral oxidative stress, inflammation, and gene regulation[END_REF][START_REF] Frenis | Ablation of lysozyme M-positive cells prevents aircraft noise-induced vascular damage without improving cerebral side effects[END_REF] . Thus, it is mandatory to identify noise-induced molecular pathomechanisms to establish practical therapeutic tools to lower or prevent noise-induced cardio-and cerebrovascular injury. Non-pharmacological interventions such as regular physical activity, a balanced healthy diet, and weight reduction have been demonstrated to represent effective ways to prevent or to treat cardiovascular disease (CVD) (e.g., atherosclerosis, hypertension, and diabetes) [START_REF] Visseren | ESC Guidelines on cardiovascular disease prevention in clinical practice[END_REF] . Especially exercise has a substantial impact on CVD prevention and is currently discussed as a mitigation strategy against air pollutioninduced cardiovascular disease and mortality [START_REF] Munzel | Running in polluted air is a two-edged sword -physical exercise in low air pollution areas is cardioprotective but detrimental for the heart in high air pollution areas[END_REF][START_REF] Kim | Association of the combined effects of air pollution and changes in physical activity with cardiovascular disease in young adults[END_REF] .

In the past, adenosine monophosphate-activated protein kinase (AMPK) has been shown to play a central role in exercise-mediated vascular protection [START_REF] Kroller-Schon | alpha1AMPactivated protein kinase mediates vascular protective effects of exercise[END_REF] as well as suppression of inflammatory conditions by caloric restriction [START_REF] Lee | Fasting increases the phosphorylation of AMPK and expression of sirtuin1 in muscle of adult male northern elephant seals (Mirounga angustirostris)[END_REF][START_REF] Jordan | Dietary Intake Regulates the Circulating Inflammatory Monocyte Pool[END_REF] and beneficial health effects by intermittent fasting [START_REF] Tang | Combined intermittent fasting and ERK inhibition enhance the anti-tumor effects of chemotherapy via the GSK3beta-SIRT7 axis[END_REF] . AMPK acts as a fuel-sensing metabolic enzyme that phosphorylates several downstream targets like Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) [START_REF] Hardie | The AMP-activated/SNF1 protein kinase subfamily: metabolic sensors of the eukaryotic cell?[END_REF] , inhibiting energy consumption and promoting energy production to ensure cell survival. Especially the α1AMPK subunit, predominantly expressed in the vasculature, has gained much attention since it regulates vascular homeostasis by increasing eNOS expression and improving antioxidant mechanisms to limit vascular ROS production [START_REF] Jansen | The AMP-Activated Protein Kinase Plays a Role in Antioxidant Defense and Regulation of Vascular Inflammation[END_REF][START_REF] Kroller-Schon | Endothelial alpha1AMPK modulates angiotensin II-mediated vascular inflammation and dysfunction[END_REF] . Therefore, the present study aimed to test the hypothesis that noise-induced cardiovascular damage could be mitigated by lifestyle interventions based on caloric restriction, exercise training, and pharmacological activation of α1AMPK with AICAR. As a proof of concept, two approaches were also tested in endothelial-specific AMPK knock-out mice.

Materials & Methods

Animal models

All animal experiments within the present study were performed in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the U.S. National Institutes of Health and were approved by the ethics committee of the University Hospital Mainz (Az 23 177-07/ G17-1-066). For the purpose of the study, we created mice with endothelial-specific deletion of α1AMPK (referred to as α1AMPK EC KO) by breeding B6.Cg-Tg(Cdh5-cre)7Mlia/J mice on a C57BL/6J background (hereinafter referred to as wild type; WT) with α1AMPK fl/fl mice (Prkaa1 mutant mice possess loxP sites flanking exon 4 and 5) [START_REF] Boudaba | AMPK Re-Activation Suppresses Hepatic Steatosis but its Downregulation Does Not Promote Fatty Liver Development[END_REF] . Mice (only male, 10-14 weeks old) were exposed to aircraft noise (maximum sound pressure level of 85 dB(A), average sound pressure level of 72 dB(A)) as described previously [START_REF] Munzel | Effects of noise on vascular function, oxidative stress, and inflammation: mechanistic insight from studies in mice[END_REF][START_REF] Kroller-Schon | Crucial role for Nox2 and sleep deprivation in aircraft noiseinduced vascular and cerebral oxidative stress, inflammation, and gene regulation[END_REF] for the 4 last days of the treatment. α1AMPK EC KO and corresponding wild-type mice performed voluntary exercise for 7 weeks (start of the exercise at the age of 4-6 weeks) using cages with a running wheel as previously described [START_REF] Kroller-Schon | alpha1AMPactivated protein kinase mediates vascular protective effects of exercise[END_REF] , which may be considered an aerobic exercise model. Our previous data revealed no significant difference in the running distance between wildtype and AMPK knockout mice (4336 versus 4002 m/24h). Intermittent fasting was induced by exact daily food administration (2 weeksaverage food intake; 2 weeks -10 % reduction and 3 weeks -35 % reduction of calories); the protocol was adapted from [START_REF] Shinmura | Cardioprotective effects of short-term caloric restriction are mediated by adiponectin via activation of AMPactivated protein kinase[END_REF] . As previously published, the pharmacological activation of AMPK was induced by AICAR, administered by daily subcutaneous injections (200 mg/kg/day) for 7d [START_REF] Schulz | Suppression of the JNK pathway by induction of a metabolic stress response prevents vascular injury and dysfunction[END_REF] . For a detailed description of the treatment, see Figure 1A-C. At the end of the experiment, animals were sacrificed using ketamine anesthesia + xylazine analgesia, followed by cervical dislocation and sample collection (aorta, heart, brain, mesenteric arteries, eyes and plasma). In addition, blood samples were obtained by heart puncture using heparin and K 2 EDTA.

Blood pressure measurement

Non-invasive measurement of blood pressure (BP) was performed by plethysmography technique using the CODA system (Kent Scientific Corporation, Torrington, CT); measurements were provided after previous repeated training to minimize stress [START_REF] Munzel | Effects of noise on vascular function, oxidative stress, and inflammation: mechanistic insight from studies in mice[END_REF][START_REF] Kroller-Schon | Crucial role for Nox2 and sleep deprivation in aircraft noiseinduced vascular and cerebral oxidative stress, inflammation, and gene regulation[END_REF][START_REF] Frenis | Ablation of lysozyme M-positive cells prevents aircraft noise-induced vascular damage without improving cerebral side effects[END_REF] . BP was measured before, during and at the end of the treatment; the time schedule of BP measurements is illustrated in Figure 1A-C.

Endothelial function

Endothelial function studies were performed by isometric tension studies in large conductance or small resistance arteries (Aorta and mesenteric arteries) using an organ bath or wire myography. Endothelium-dependent relaxation of intact mouse aortic rings (3 mm, perivascular fat, and connective tissues free) was tested with acetylcholine (ACh 10 -9 -10 -5.5 M) upon pre-constriction with prostaglandin F2α [START_REF] Munzel | Effects of noise on vascular function, oxidative stress, and inflammation: mechanistic insight from studies in mice[END_REF][START_REF] Kroller-Schon | Crucial role for Nox2 and sleep deprivation in aircraft noiseinduced vascular and cerebral oxidative stress, inflammation, and gene regulation[END_REF] using Multi Wire Myograph system -620M (Danish Myo Technology, Aarhus, Denmark) as described [START_REF] Puzserova | Long-term social stress induces nitric oxideindependent endothelial dysfunction in normotensive rats[END_REF] . The isolated rings were pre-contracted with norepinephrine (10 -5 µM, final concentration), followed by the cumulative addition of acetylcholine (ACh 10 -9 -10 -5 M).

Retinal microvascular function

Microvascular function was measured in first-order arterioles of isolated retinas by using video microscopy as previously described [START_REF] Gericke | Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo[END_REF][START_REF] Gericke | Identification of the muscarinic acetylcholine receptor subtype mediating cholinergic vasodilation in murine retinal arterioles[END_REF] . Following dissection, the eyes were transferred into ice-cold Krebs Henseleit buffer, and the retina was isolated with the optic nerve and the ophthalmic artery attached. Next, the ophthalmic artery was cannulated and sutured to a micropipette with 10.0 nylon suture material and the retina was placed onto a transparent plastic platform. Subsequently, retinal arterioles were pressurized to 50 mmHg, visualized under brightfield conditions, and equilibrated for 30 min at 37°C. Subsequently, concentration-response curves for the thromboxane mimetic, U46619 (10 -11 to 10 -6 M; Cayman Chemical, Ann Arbor, MI, USA), were conducted. For measurement of vasodilation responses, vessels were preconstricted to 50-70% of the initial luminal diameter by titration of U46619, and responses to the endothelium-dependent vasodilator, acetylcholine (10 -9 to 10 -4 M; Sigma-Aldrich, Taufkirchen, Germany) and the endothelium-independent nitric oxide donor, sodium nitroprusside (SNP, 10 -9 to 10 -4 M, Sigma-Aldrich), were determined.

RNA sequencing and bioinformatical analysis

RNA sequencing was carried out at Novogene Bioinformatics Technology Co., Ltd., in Cambridge (UK). According to the manufacturer's protocol, aortic mRNA was isolated using the RNeasy Mini kit (Qiagen, Hilden, Germany). RNA sample quality control, mRNA library preparation (poly A enrichment), NovaSeq PE150 sequencing (9 G raw data per sample), data quality control and basic data analysis were performed by Novogene Bioinformatics Technology Co., Ltd., in Cambridge (UK). Volcano plots to envisage the number of changed genes by the three different therapeutic interventions as well as KEGG pathway analyses to envisage the biochemical processes that are changed by the three different therapeutic interventions were taken from the standard bioinformatical report provided by Novogene (suppl. Figure 4). Cluster analysis was performed by STRING (Search Tool for the Retrieval of Interacting Genes) version 11.0 32 . To identify interactive relationships among identified target proteins, the protein list was mapped to STRING.

Vascular oxidative stress

The vascular production of reactive oxygen species (ROS) in aortic tissue was measured by an HPLC-based dihydroethidium (DHE) assay as described previously [START_REF] Kroller-Schon | Crucial role for Nox2 and sleep deprivation in aircraft noiseinduced vascular and cerebral oxidative stress, inflammation, and gene regulation[END_REF][START_REF] Steven | Exacerbation of adverse cardiovascular effects of aircraft noise in an animal model of arterial hypertension[END_REF] . In addition, the mitoSOX/HPLC method [START_REF] Kalinovic | Comparison of Mitochondrial Superoxide Detection Ex Vivo/In Vivo by mitoSOX HPLC Method with Classical Assays in Three Different Animal Models of Oxidative Stress[END_REF] was used to determine mitochondria-specific production of ROS.

Mitochondria were isolated from cardiac tissues as described previously.

Protein and mRNA expression analysis

Western blot analysis was used to determine changes in protein expression. Aortic tissue was homogenized in cell lysis buffer (20 mM Tris-HCl; 250 mM saccharose; 3 mM EGTA; 20 mM EDTA; 0,5 mM PMSF; 1% Triton-X100; 0,5 mM Na-vanadate; 2,5 mM Na-fluoride; protease inhibitor cocktail % (P8340; Sigma-Aldrich, Taufkirchen, Germany); 1% phosphatase inhibitor cocktail (P2850; Sigma-Aldrich, Taufkirchen, Germany). Proteins (20 µg per lane) were separated using SDS-PAGE, followed by a blotting procedure onto nitrocellulose membranes. Immunostaining was performed as described previously [START_REF] Schulz | Suppression of the JNK pathway by induction of a metabolic stress response prevents vascular injury and dysfunction[END_REF] .

Determination of ADMA levels in plasma was performed by dot blot system (Schleicher & Schuell, 10484138CP) using 50 µg of proteins per dot; analysis was performed as stated previously [START_REF] Munzel | Effects of noise on vascular function, oxidative stress, and inflammation: mechanistic insight from studies in mice[END_REF][START_REF] Kroller-Schon | Crucial role for Nox2 and sleep deprivation in aircraft noiseinduced vascular and cerebral oxidative stress, inflammation, and gene regulation[END_REF][START_REF] Frenis | Ablation of lysozyme M-positive cells prevents aircraft noise-induced vascular damage without improving cerebral side effects[END_REF] . Polyclonal rabbit anti-α1AMPK (Merck-Millipore, Darmstadt, Germany; 1:1000), polyclonal rabbit anti-p-α1AMPK Thr172 (Cell Signaling, Boston, MA, USA; 1:500), monoclonal anti-α-Actinin (Sigma-Aldrich, Taufkirchen, Germany; 1:2000) and monoclonal rabbit anti-ADMA (Cell Signaling, Boston, MA, USA; 1:1000) were used for protein expression analysis. Gene expression changes were analyzed by quantitative real-time PC (qRT-PCR) using the QuantiTect™ Probe RT-PCR kit (Qiagen) used according to the manufacturer's protocol as described previously [START_REF] Livak | Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method[END_REF] . For analysis, we used TaqMan real-time PCR assays: Prmt1 (Mm00480135_g1), Ddah2 (Mm00516769_g1) and Foxo3 (Mm01185722_m1), TATA box binding protein was used as the housekeeping gene for normalization (TBP; MM00446973_m1). Gene expression was analyzed using the comparative ΔΔCt method and is expressed as the percentage of WT.

Statistical analysis

Prism for Windows, version 9, GraphPad Software Inc (GraphPad Software LLC, La Jolla, CA) was used for statistical analysis. Statistical comparisons were conducted using one-way ANOVA with Bonferroni correction to compare multiple means or two-way ANOVA with a repeated measurements approach with Tukey correction as appropriate. All presented results are expressed as the mean ± SEM, and the n for every independent experiment is indicated.

Results where p-values < 0.05 were accepted as statistically significant results.

Results

Effects of aircraft noise up to 4d on arterial blood pressure and endothelial function

All experimental animals were exposed to around-the-clock aircraft noise for 4d; this led to a significant elevation of systolic blood pressure in wild-type mice (Figure 1D-F). In addition, endothelial dysfunction was evident in the aorta (Figure 1G-I), mesenteric arteries (Figure 1J-L) and retinal arterioles (suppl. Figure 1) of all noise-treated animals. The impairment of these functional parameters by noise was also supported by RNA sequencing data and advanced pathway analysis pointing towards impaired redox homeostasis, changes in circadian clock genes, dysregulated AMPK and eNOS/NO/cGMP signaling, activation of the oxidative and general stress response as well as an altered inflammatory cascade (suppl. 1).

Effects of exercise training, caloric restriction, and AICAR treatment on noise-induced hypertension and endothelial dysfunction

We 3 and4). However, we could not establish such beneficial effects on the evaluated pathways for an exercise intervention in noise-exposed mice on the level of RNA sequencing data, despite the highly beneficial effects of exercise on the other functional and biochemical parameters (suppl. Figure 6 and suppl.

Table 2) and despite more than 3,000 genes showing a more than the log2-fold change in their expression, however without reaching significance (suppl. Figure 4).

Effects of exercise training, caloric restriction, and AICAR treatment on noise-induced increases in ADMA plasma levels

We next investigated whether aircraft noise may increase plasma levels of asymmetric dimethylarginine (ADMA), a potent endogenous competitive eNOS inhibitor shown to uncouple eNOS and cause endothelial dysfunction [START_REF] Karbach | eNOS uncoupling in cardiovascular diseases--the role of oxidative stress and inflammation[END_REF] . Noise pollution increased plasma levels of ADMA, which was completely prevented by exercise, fasting, and AICAR treatment (Figure 2A,B,C). ADMA is produced as a consequence of the methylation of L-arginine residues via the catalytic action of type 1 protein arginine N-methyltransferase (PRMT-1) and is degraded by dimethylarginine dimethylaminohydrolase 2 (DDAH-2) [START_REF] Chen | PRMT-1 and DDAHs-induced ADMA upregulation is involved in ROS-and RAS-mediated diabetic retinopathy[END_REF] . The elevated plasma levels of ADMA in noise-exposed mice were associated with a decreased expression of DDAH-2 mRNA levels, encoding for the most essential ADMA degrading enzyme (Figure 2D). They increased expression of PRMT-1 mRNA, encoding for a critical class of protein arginine N-methyltransferases that are responsible for ADMA formation (Figure 2E). The downregulated expression of DDAH-2 and upregulated expression of PRMT-1 was mostly reversed by exercise training, caloric restriction, and application of AICAR (at least by trend).

Effects of exercise training, caloric restriction and AICAR administration on noiseinduced oxidative stress in the cardiovascular system

The production of vascular superoxide formation was quantified using HPLC-based detection of oxidized dihydroethidium (DHE). Whereas noise exposure increased aortic superoxide levels, the mitigation measures exercise, fasting and AICAR treatment normalized superoxide formation (Figure 3A,B,C). Those data were also mirrored by the normalization of mitochondria-specific superoxide levels determined by mitoSOX/HPLC in isolated heart mitochondria by all 3 maneuvers (Figure 3D-F). Moreover, we can show that 4d of aircraft noise exposure increases the production of cerebral superoxide (suppl. Figure 1). Likewise, we observed a protective effect of exercise and fasting, resulting in reduced superoxide formation in response to noise. In addition, we observed the prevention of downregulation of the mRNA expression of cerebral Foxo3 (suppl. Figure 1) with two of the maneuvers, exercise, and fasting.

Effects of exercise training, caloric restriction, and AICAR administration on α1AMPK expression and activation

Noise exposure did not affect aortic α1AMPK protein expression nor its phosphorylation at threonine 172 (suppl. Figure 2). In contrast, voluntary exercise training and fasting significantly increased the protein expression of aortic α1AMPK and p-α1AMPK Thr172 (AICAR administration at least by trend), an activation marker of AMPK, compared to wildtype controls (suppl. Figure 2). Voluntary exercise training had the most substantial effect on α1AMPK protein expression and phosphorylation among the three investigated models.

Proof-of-concept studies by genetic deletion of endothelial-specific α1AMPK show abrogation of the beneficial vascular effects of exercise and fasting in noise-exposed mice

To prove the functional relevance of α1AMPK in preventing noise-mediated vascular damage, we used endothelial-specific α1AMPK knockout (α1AMPK EC KO) mice.

Endothelial-specific α1AMPK deletion resulted in a substantial reduction of α1AMPK and p-α1AMPK Thr172 expression, as shown at the level of aortic protein expression (suppl. Figure 2). In addition, α1AMPK EC KO mice developed arterial hypertension in response to noise exposure that was not prevented but even aggravated by exercise and fasting (Figure 4A,D).

Furthermore, α1AMPK EC KO mice developed endothelial dysfunction of the aorta in response to noise, which was not abolished by exercise and fasting (Figure 4B,E).

Importantly, the unexposed α1AMPK EC KO mice showed no significant impairment of endothelial function in comparison with the wildtype mice in accordance to our previous report [START_REF] Kroller-Schon | Endothelial alpha1AMPK modulates angiotensin II-mediated vascular inflammation and dysfunction[END_REF] (see also hatched lines in Figure 4). Endothelial dysfunction was also observed in resistance vessels of noise-exposed α1AMPK EC KO mice, as shown by the impaired vascular relaxation curve of mesenteric arteries (Figure 4C, F) as well as of retinal arterioles (suppl. Figure 3), all of which was also not prevented by exercise and fasting in the absence of endothelial α1AMPK. A key role of AMPK for AICAR-mediated protective effects was previously demonstrated in septic mice with global AMPK knockout [START_REF] Schulz | Suppression of the JNK pathway by induction of a metabolic stress response prevents vascular injury and dysfunction[END_REF] .

Effects of genetic deletion of endothelial-specific α1AMPK on plasma ADMA levels

We next examined the ADMA release in α1AMPK EC KO mice since ADMA plays an essential role in the impairment of NO production and subsequent endothelial dysfunction [START_REF] Kajimoto | Inhibition of eNOS phosphorylation mediates endothelial dysfunction in renal failure: new effect of asymmetric dimethylarginine[END_REF][START_REF] Smith | Effects of ADMA upon gene expression: an insight into the pathophysiological significance of raised plasma ADMA[END_REF] . In contrast to our findings mentioned above in wild-type animals (Figure 2), plasmatic ADMA levels were increased in response to noise exposure in our knock-out mice and were not decreased by exercise training and fasting, although for the latter a clear trend was noticed (Figure 5A). Furthermore, the expression of vascular DDAH-2 was almost absent in α1AMPK EC KO mice when compared to WT, which was also not improved by exercise or fasting (Figure 5B). Finally, aortic PRMT-1 expression displayed an opposite regulation, as it was dramatically increased in noise-exposed α1AMPK EC KO mice and rather further aggravated, at least by trend, by exercise, but was decreased mainly by fasting (Figure 5C). Endothelial α1AMPK deficiency did not change PRMT-1 expression when compared to WT.

The loss of antioxidant and vascular protective effects of AICAR in global AMPK knockout mice was previously demonstrated [START_REF] Schulz | Suppression of the JNK pathway by induction of a metabolic stress response prevents vascular injury and dysfunction[END_REF] .

Effects of deletion of endothelial-specific α1AMPK on antioxidant effects of exercise and fasting in noise-exposed mice

As measured by HPLC-based analysis in aortic tissue and cerebral cortex, we detected increased levels of superoxide in response to noise exposure in our knock-out animals, and mitigation by exercise and fasting failed in the knock-out mice (Figure 6A, C; suppl. Figure 3). Similar results were obtained by mitoSOX/HPLC analysis of isolated cardiac mitochondria, where noise exposure increased mitochondrial superoxide formation. Exercise and fasting had no protective effect on the exacerbation of oxidative stress in the knock-out mice (Figure 6B,D).

Discussion

The knowledge of environmental risk factors such as noise exposure and air pollution leading to cardiovascular complications and disease development has increased substantially during the last decades [START_REF] Münzel | Environmental risk factors and cardiovascular diseases: a comprehensive expert review[END_REF] . The alarming data stated by the WHO points to the fact that every year, up to 23% (12,6 million) of global premature deaths can be attributed to environmental risk factors [START_REF] Rojas-Rueda | Environmental Risk Factors and Health: An Umbrella Review of Meta-Analyses[END_REF] . Here, the most significant contributors (5 million deaths per year) are cardiovascular diseases such as stroke, myocardial infarction, coronary artery disease, arrhythmia, and heart failure. It is undeniable that traffic noise represents one of the most pronounced environmental risk factors in urbanized countries [START_REF] Munzel | Reduction of environmental pollutants for prevention of cardiovascular disease: it's time to act[END_REF] . A meta-analysis of the World Health Organization showed that road traffic noise elevates the incidence of coronary heart disease by 8% per 10 dB(A) increase starting at 50 dB(A) [START_REF] Kempen | WHO Environmental Noise Guidelines for the European Region: A Systematic Review on Environmental Noise and Cardiovascular and Metabolic Effects: A Summary[END_REF] . 10 dB(A) L den increase of traffic noise is also raising the risk of myocardial infarction incidence by 12% (starting at 42 dB(A)) [START_REF] Sorensen | Road traffic noise and incident myocardial infarction: a prospective cohort study[END_REF] and risk of stroke by 14% (starting at 55 dB(A)) [START_REF] Sorensen | Road traffic noise and stroke: a prospective cohort study[END_REF] , concluded by a Danish cohort studies. These findings were also supported by a nationwide study on noise effects on cardiovascular disease in Switzerland [START_REF] Heritier | A systematic analysis of mutual effects of transportation noise and air pollution exposure on myocardial infarction mortality: a nationwide cohort study in Switzerland[END_REF][START_REF] Vienneau | Transportation noise exposure and cardiovascular mortality: 15-years of follow-up in a nationwide prospective cohort in Switzerland[END_REF] . Although we studied the detrimental health effects of aircraft noise, it should be noted that road traffic noise is the dominant source of noise, followed by railway noise, since much more people in the EU-28 are exposed to nocturnal noise >50 dB(A) originating from roads (76 million) than railways (17.6 million) or airports (1.3 million) as reported under the EU Environmental Noise Directive (2002/49/EC). Therefore, it is highly important to identify the primordial and primary prevention mechanisms promoting cardiovascular health endangered by environmental risk factors in general and traffic noise exposure in particular. One-third of cardiovascular deaths could be prevented by a healthy lifestyle, as stated by The American Heart Association (AHA) [START_REF] Aha | ACC/AHA Guidance for Preventing Heart Disease[END_REF] .

Notably, up to 16% of the cardiovascular events and 9% of cardiovascular mortality could be reduced by calorie restriction and a healthy diet modification [START_REF] Collins | Blood pressure, stroke, and coronary heart disease. Part 2, Short-term reductions in blood pressure: overview of randomised drug trials in their epidemiological context[END_REF] .

It was shown that the prescription of physical activity represents a highly beneficial preventive/treatment maneuver against cardiometabolic diseases [START_REF] Colberg | Physical Activity/Exercise and Diabetes: A Position Statement of the American Diabetes Association[END_REF][START_REF] Andres | Evaluation of a cardiovascular Risk Reduction Program at a workplace medical clinic[END_REF][START_REF] Ashton | Effects of short-term, medium-term and long-term resistance exercise training on cardiometabolic health outcomes in adults: systematic review with meta-analysis[END_REF] and significantly improves the quality of life measures [START_REF] Jones | Promoting Physical Activity in a Primary Care Practice: Overcoming the Barriers[END_REF] . An inverse relation was observed between all-cause mortality and increased physical activity [START_REF] Munzel | Adverse Cardiovascular Effects of Traffic Noise with a Focus on Nighttime Noise and the New WHO Noise Guidelines[END_REF] . Dose-response (linear) dependence was observed between physical activity and the reduction of blood pressure dysregulation or endothelial dysfunction [START_REF] Ghadieh | Evidence for exercise training in the management of hypertension in adults[END_REF][START_REF] Huai | Physical activity and risk of hypertension: a meta-analysis of prospective cohort studies[END_REF] . Moreover, exercise improves the quality of life and physical and mental health in post-stroke patients [START_REF] Ali | Effect of Exercise Interventions on Health-Related Quality of Life After Stroke and Transient Ischemic Attack: A Systematic Review and Meta-Analysis[END_REF] . Another meta-analysis showed that regular physical activity positively affects mental health, especially in the elderly [START_REF] Yao | Effect of Aerobic Exercise on Mental Health in Older Adults: A Meta-Analysis of Randomized Controlled Trials[END_REF] . Likewise, intermittent fasting decreases cardiometabolic risk markers [START_REF] Yang | Effect of Epidemic Intermittent Fasting on Cardiometabolic Risk Factors: A Systematic Review and Meta-Analysis of Randomized Controlled Trials[END_REF][START_REF] Jahrami | Does four-week consecutive, dawn-to-sunset intermittent fasting during Ramadan affect cardiometabolic risk factors in healthy adults? A systematic review, meta-analysis, and meta-regression[END_REF] and caloric restriction decreases blood pressure and improves cardiovascular health [START_REF] Kirkham | The effect of caloric restriction on blood pressure and cardiovascular function: A systematic review and meta-analysis of randomized controlled trials[END_REF] . These health benefits are based on the activation of cellular stress response elements, improved autophagy, modification of apoptosis, and alteration in hormonal balance [START_REF] Golbidi | Health Benefits of Fasting and Caloric Restriction[END_REF] .

Several studies substantiated that the beneficial cardiovascular effects of calorie restriction and voluntary exercise depend on intact α1AMPK [START_REF] Kroller-Schon | alpha1AMPactivated protein kinase mediates vascular protective effects of exercise[END_REF][START_REF] Jordan | Dietary Intake Regulates the Circulating Inflammatory Monocyte Pool[END_REF][START_REF] Liu | TLR9 and beclin 1 crosstalk regulates muscle AMPK activation in exercise[END_REF] . However, corresponding mechanisms have not been fully elucidated, contrary to the growing understanding of the protective effect of exercise and calorie restriction mediated by α1AMPK activation. This knowledge is even more emphasized in conditions of environmental risk factors, where these cardioprotective strategies could be used in the first place. Understanding the pathways influenced by α1AMPK might yield a new and attractive mitigation strategy to protect the general population from noise-induced cardiovascular damage. Especially patients at high cardiovascular risk, with the pre-existing cardio-metabolic disease, or the elderly could benefit from supervised exercise training and/or calorie restriction to prevent additive cardio/cerebrovascular damage in response to transportation noise exposure.

With our previous study using the same aircraft noise exposure protocol, we were able to establish noise-mediated endothelial dysfunction, elevated blood pressure, cardiovascular and systemic oxidative stress and inflammation as well as substantial changes in gene networks [START_REF] Munzel | Effects of noise on vascular function, oxidative stress, and inflammation: mechanistic insight from studies in mice[END_REF] . In a subsequent study, we demonstrated a central role of the phagocytic NADPH oxidase (NOX-2) in the pathophysiological mechanisms of noise-mediated cardiovascular damage as Nox2 -/-mice were protected. They established cerebral oxidative stress and neuroinflammation as upstream mechanisms of cardiovascular complications [START_REF] Kroller-Schon | Crucial role for Nox2 and sleep deprivation in aircraft noiseinduced vascular and cerebral oxidative stress, inflammation, and gene regulation[END_REF] . Whereas most cardiovascular damage parameters became more pronounced during the exposure for 1, 2 or 4 days, endothelial dysfunction was already fully developed after 1 day of exposure [START_REF] Munzel | Effects of noise on vascular function, oxidative stress, and inflammation: mechanistic insight from studies in mice[END_REF] , in accordance with human data on impaired flow-mediated dilation after one night of aircraft noise exposure [START_REF] Schmidt | Effect of nighttime aircraft noise exposure on endothelial function and stress hormone release in healthy adults[END_REF] . We also conducted long-term noise exposure of mice for up to 28 days, proving that the mice neither became habituated to the noise stress nor developed hearing loss since the oxidative damage parameters, endothelial dysfunction and high blood pressure remained stable over the entire exposure time [START_REF] Frenis | Long-Term Effects of Aircraft Noise Exposure on Vascular Oxidative Stress, Endothelial Function and Blood Pressure: No Evidence for Adaptation or Tolerance Development[END_REF] . Although animal studies are still pending, there is evidence from human studies that a short-term reduction in aircraft noise exposure levels due to the coronavirus disease 2019 (COVID-19) lockdown may reverse the aircraft noise-induced negative effects on arterial stiffness and blood pressure [START_REF] Wojciechowska | Blood Pressure and Arterial Stiffness in Association With Aircraft Noise Exposure:Long-Term Observation and Potential Effect of COVID-19 Lockdown[END_REF][START_REF] Hahad | Reduced Aircraft Noise Pollution During COVID-19 Lockdown Is Beneficial to Public Cardiovascular Health: a Perspective on the Reduction of Transportation-Associated Pollution[END_REF] .

Therefore, with the present study, we sought to determine α1AMPK-dependent mechanisms of fasting and exercise to evaluate cardiovascular protective pathways against aircraft noise exposure-induced injury. AMPK is the "master" energy sensor, activated by several physiological stimuli responding to small AMP/ATP ratio changes, leading to increased ATP consumption and a concomitant rise in cellular AMP [START_REF] Jansen | The AMP-Activated Protein Kinase Plays a Role in Antioxidant Defense and Regulation of Vascular Inflammation[END_REF][START_REF] Hardie | Keeping the home fires burning: AMP-activated protein kinase[END_REF][START_REF] Li | Structural basis of AMPK regulation by adenine nucleotides and glycogen[END_REF] .

It is well-known that physical activity (voluntary exercise training) requires increased energy expenditure and vice versa; during fasting (calorie restriction), energy intake is reduced. Therefore, both interventions result in a relevant change in intracellular AMP/ATP ratio, resulting in AMPK activation [START_REF] Hardie | The AMP-activated/SNF1 protein kinase subfamily: metabolic sensors of the eukaryotic cell?[END_REF][START_REF] Hardie | AMP-activated protein kinase: the energy charge hypothesis revisited[END_REF] . Furthermore, in vivo studies showed that intact AMPK in skeletal muscle is essential for an adequate response to exercise [START_REF] Griffee | Skeletal muscle AMP-activated protein kinase is essential for the metabolic response to exercise in vivo[END_REF] .

Similarly, AMPK could be activated in the cardiovascular system. Chen et al. showed for the first time that AMPK could directly phosphorylate endothelial NO-Synthase (eNOS) in cardiac capillary endothelial cells and monocytes in vitro and during cardiac ischemia in rats [START_REF] Chen | AMP-activated protein kinase phosphorylation of endothelial NO synthase[END_REF] . That is mediated mainly by the α1AMPK isoform, predominantly expressed in the vascular endothelium and smooth muscle cells [START_REF] Fisslthaler | Activation and signaling by the AMP-activated protein kinase in endothelial cells[END_REF][START_REF] Rodriguez | AMPK, metabolism, and vascular function[END_REF] . Vascular α1AMPK is specifically responsible for maintaining vascular homeostasis by modulation of structure and function of endothelial, smooth muscle and immune cells [START_REF] Rodriguez | AMPK, metabolism, and vascular function[END_REF] . Vascular endothelium must constantly react to several physiological stimuli (shear stress) to modulate vascular tone and regulate tissuespecific metabolic demand. Laminar blood flow increases eNOS activation, phosphorylated by α1AMPK. Previously, we have shown that endothelium-specific α1AMPK deletion reduces expression and/or activation of eNOS (Ser1177 phosphorylation), resulting in increased oxidative stress [START_REF] Kroller-Schon | alpha1AMPactivated protein kinase mediates vascular protective effects of exercise[END_REF] .

Moreover, activating endothelial α1AMPK with AICAR is essential for attenuating oxidative endothelial injury caused by angiotensin II (AT-II) infusion [START_REF] Schulz | Suppression of the JNK pathway by induction of a metabolic stress response prevents vascular injury and dysfunction[END_REF] . We observed that endothelial α1AMPK plays a crucial role in vascular inflammation. The recruitment of inflammatory cells to the vascular wall is limited by endothelial α1AMPK expression and is closely related to the vascular redox status regulated by α1AMPK. During AT-II treatment, pro-inflammatory cytokines and oxidative stress lead to vascular dysfunction. Interestingly, this effect was aggravated in mice with endothelium-specific α1AMPK deletion compared to wild-type controls [START_REF] Kroller-Schon | Endothelial alpha1AMPK modulates angiotensin II-mediated vascular inflammation and dysfunction[END_REF] . Likewise, we know that noise causes inflammation [START_REF] Munzel | Effects of noise on vascular function, oxidative stress, and inflammation: mechanistic insight from studies in mice[END_REF][START_REF] Kroller-Schon | Crucial role for Nox2 and sleep deprivation in aircraft noiseinduced vascular and cerebral oxidative stress, inflammation, and gene regulation[END_REF] by exacerbated infiltration of LysM + -positive immune cells [START_REF] Frenis | Ablation of lysozyme M-positive cells prevents aircraft noise-induced vascular damage without improving cerebral side effects[END_REF] , where immune cell activation, endothelial adhesion, and pro-inflammatory phenotype of the plasma proteome also plays a central role in endothelial damage and microvascular dysfunction [START_REF] Eckrich | Aircraft noise exposure drives the activation of white blood cells and induces microvascular dysfunction in mice[END_REF] . Recently, array experiments have demonstrated changes in inflammatory clusters of cytokines and chemokines by noise exposure of mice [START_REF] Kuntic | Co-exposure to urban particulate matter and aircraft noise adversely impacts the cerebro-pulmonarycardiovascular axis in mice[END_REF] . Therefore the anti-inflammatory effects of exercise [START_REF] Sallam | Exercise Modulates Oxidative Stress and Inflammation in Aging and Cardiovascular Diseases[END_REF] , fasting [START_REF] Munzel | Environmental Noise and the Cardiovascular System[END_REF] , and AICAR [START_REF] Martin | 5-Amino-1-beta-D-Ribofuranosyl-Imidazole-4-Carboxamide (AICAR) Reduces Peripheral Inflammation by Macrophage Phenotype Shift[END_REF] may help to prevent noise-mediated cardiovascular damage as well as development and faster progression of atherosclerosis as driven by activated/infiltrated myelomonocytic cells.

Furthermore, we were able to highlight the vascular protective effects of α1AMPK in ROS-mediated vascular dysfunction and inflammation using tissue-specific knock-out models of α1AMPK. The deletion of α1AMPK resulted in aggravation of vascular ROS production at least by trend, which was by the known protective and anti-inflammatory effects of α1AMPK 25, 78, 79 . The AMP analog, 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR), was previously the most frequently used drug in animals to induce the pharmacological activation of α1AMPK. This results in reduced vascular oxidative stress and inflammation shown in an animal model of arterial hypertension [START_REF] Schulz | Suppression of the JNK pathway by induction of a metabolic stress response prevents vascular injury and dysfunction[END_REF] . Clinically, recent data suggest that metformin's antidiabetic drug mediates its beneficial cardioprotective effects, at least in part, via AMPK activation [START_REF] Xie | Phosphorylation of LKB1 at serine 428 by protein kinase C-zeta is required for metformin-enhanced activation of the AMPactivated protein kinase in endothelial cells[END_REF] . Thus, global, or cell-specific activation of α1AMPK could represent a novel preventive strategy against cardiovascular complications caused by environmental risk factors such as noise exposure. Although some of the reactive oxygen species-related beneficial effects of α1AMPK activation on the cardiovascular system are known, the exact mechanisms and their role during noise exposure have not been addressed.

The protective/beneficial role of α1AMPK is undeniable and its importance is even more highlighted during vascular oxidative stress. That suggests that activation of α1AMPK physiologically (by exercise or fasting) or pharmacologically (AICAR, shown previously in global AMPK knockout mice [START_REF] Schulz | Suppression of the JNK pathway by induction of a metabolic stress response prevents vascular injury and dysfunction[END_REF] ) could be used as a preventive strategy against aircraft noiseinduced injury. The crucial role of endothelial-specific α1AMPK during voluntary exercisemediated vascular protection was studied by our group as well. We demonstrated that the presence of the α1AMPK is essential during exercise, where its expression affects not only endothelial function (NO production, eNOS coupling) but also oxidative stress release.

Exercise training increased the expression of NOX-2 in endothelial-specific α1AMPK knock-out mice, resulting in increased vascular superoxide production. On the other hand, Nrf-2dependent antioxidant defense mechanisms were suppressed in mice lacking endothelial α1AMPK [START_REF] Jansen | Lack of Endothelial alpha1AMPK Reverses the Vascular Protective Effects of Exercise by Causing eNOS Uncoupling[END_REF] . In this regard, translational studies with measurements of individual α1AMPK in endothelial precursor cells might help to understand the variation in existing stress-and exercise tolerance among patients suffering from cardiovascular disease.

Our present data demonstrate that aircraft noise-mediated endothelial dysfunction and associated blood pressure elevation could be normalized and/or preserved by α1AMPK activation. Moreover, the beneficial effect of voluntary exercise training and calorie restriction was endothelial α1AMPK dependent. Undeniably, the endothelium's physiological function is closely linked to redox balance. Increased ROS production and/or insufficient antioxidant response impair endothelial function. Our previous study has shown that almost exclusively, nocturnal aircraft noise exposure triggers pathomechanisms (NO signaling disruption -eNOS uncoupling, oxidative stress, supersensitivity to vasoconstrictors, cardiovascular inflammation, etc.), causing vascular injury. Here, oxidative stress, predominantly driven by vascular NOX-2, played a crucial role [START_REF] Kroller-Schon | Crucial role for Nox2 and sleep deprivation in aircraft noiseinduced vascular and cerebral oxidative stress, inflammation, and gene regulation[END_REF] , since the deletion of Nox-2 almost completely prevented the aircraft noise-induced cardiovascular and cerebral side effects. The vital role of oxidative stress has been proven by vitamin C administration, which was able to re-establish the physiological function of the endothelium and reduce vascular damage caused by aircraft noise exposure [START_REF] Herzog | Acute exposure to nocturnal train noise induces endothelial dysfunction and pro-thromboinflammatory changes of the plasma proteome in healthy subjects[END_REF][START_REF] Schmidt | Effect of nighttime aircraft noise exposure on endothelial function and stress hormone release in healthy adults[END_REF][START_REF] Munzel | Effects of noise on vascular function, oxidative stress, and inflammation: mechanistic insight from studies in mice[END_REF] . The present studies demonstrated that aircraft noise initiates oxidative stress by NOX-2-derived increased ROS production. The presented data of our study suggest that oxidative stress development can be rescued by α1AMPK activation (AICAR, voluntary exercise, fasting). Whereas for exercise and fasting, we have shown in the present study that AMPK EC KO abrogates their beneficial effects on noise-mediated damage, the principle of action of AICAR via AMPK activation was previously demonstrated in a sepsis model with severe inflammation and oxidative stress [START_REF] Schulz | Suppression of the JNK pathway by induction of a metabolic stress response prevents vascular injury and dysfunction[END_REF] .

Intact α1AMPK, pharmacologically or physiologically activated by these manoeuvers, decreased cardiovascular ROS production and reduced expression of NOX-2, resulting in more negligible oxidative protein damage/modification (3-NT).

ADMA inhibits eNOS activity by competitive inhibition along with modulated phosphorylation and/or modified gene expression [START_REF] Kajimoto | Inhibition of eNOS phosphorylation mediates endothelial dysfunction in renal failure: new effect of asymmetric dimethylarginine[END_REF][START_REF] Smith | Effects of ADMA upon gene expression: an insight into the pathophysiological significance of raised plasma ADMA[END_REF] . Interestingly, the AMPK activator, metformin, has a similar structure to ADMA. It was proven that this structural analogue could reduce the ADMA level by DDAH-2 -dependent degradation, resulting in increased NO bioavailability in vivo [START_REF] Lai | Modulating DDAH/NOS Pathway to Discover Vasoprotective Insulin Sensitizers[END_REF] . In this study, we noticed that increased ADMA accumulation caused by noise-induced PRMT-1 catalyzed these α1AMPK-dependent mechanisms could decrease arginine methylation. However, crosstalk between α1AMPK and ADMA production and/or degradation is not adequately investigated. Despite the knowledge about PRMTs and DDAHs in skeletal muscle, their interplay with α1AMPK activation in the vasculature is not very well known.

Although our mouse model uses an average sound pressure level of 72 dB(A), it is close enough to the exposure levels of the population living nearby railways (often up to 65 dB(A)) or main roads (often up to 60 dB(A)) [START_REF]WHO Environmental Noise Guidelines for the European Region[END_REF] and far below the sound pressure levels used by most of the animal studies on noise health effects (usually >100 dB(A)) [START_REF] Munzel | The Adverse Effects of Environmental Noise Exposure on Oxidative Stress and Cardiovascular Risk[END_REF] . In addition, our mouse model of noise exposure is a short-term exposure protocol, which requires somewhat higher noise levels to see effects that in the population can accumulate over years. Also, a direct translation of the selected models into a human setting is not possible due to the complex interactions between noise exposure in the natural environment, which is often of chronic nature, and the characteristics of the subjects. Although there are studies showing that e.g. certain dietary patterns [START_REF] Lee | Association of dietary factors with noise-induced hearing loss in Korean population: A 3-year national cohort study[END_REF] and exercise [START_REF] Curhan | Body mass index, waist circumference, physical activity, and risk of hearing loss in women[END_REF] may be beneficial when it comes to the risk of noise-induced hearing loss, there are no manifest clinical guidelines quantifying the amount of calories restriction or exercise. However, this is an important point that should be addressed in future human experimental and observational studies.

Conclusions

The present study demonstrates for the first time that exercise, fasting (caloric restriction), and pharmacological stimulation of the α1AMPK subunit effectively mitigate aircraft noise-induced cardiovascular adverse side effects (see Figure 7 for summary). The detrimental health effects of noise and the protection against noise-mediated damage by fasting and AICAR therapy were also mirrored by RNA sequencing data and related bioinformatical analyses (for exercise, at least by trend). Moreover, genetic deletion of endothelial α1AMPK reversed these effects in certain treatment groups (exercise-mediated protection was almost completely lost, whereas fasting-dependent beneficial effects were partially lost), suggesting that the endothelial α1AMPK almost exclusively mediates these beneficial effects. Further, mainly prospective clinical trials need to prove now whether these strategies can effectively counteract the adverse cardiovascular and cerebral consequences of aircraft noise. According to previous data, the mode of activation of AMPK and subsequent signaling is comparable in human and experimental models. For example, activation of AMPK following exercise has been demonstrated in skeletal muscle from rodents and humans [START_REF] Blazev | Phosphoproteomics of three exercise modalities identifies canonical signaling and C18ORF25 as an AMPK substrate regulating skeletal muscle function[END_REF][START_REF] Lantier | AMPK controls exercise endurance, mitochondrial oxidative capacity, and skeletal muscle integrity[END_REF] . There have been also studies showing AMPK activation in response to various AMPK stimuli in both human and animal cell lines. For example, in primary human and mouse hepatocytes, AMPK is activated by direct and indirect activators to a similar extend and promotes the phosphorylation of conserved targets [START_REF] Boudaba | AMPK Re-Activation Suppresses Hepatic Steatosis but its Downregulation Does Not Promote Fatty Liver Development[END_REF][START_REF] Ford | Metformin and salicylate synergistically activate liver AMPK, inhibit lipogenesis and improve insulin sensitivity[END_REF] . This is also supported by the recent clinical trials testing AMPK activators in humans [START_REF] Fouqueray | Pharmacodynamic effects of direct AMP kinase activation in humans with insulin resistance and non-alcoholic fatty liver disease: A phase 1b study[END_REF] following promising results obtained in pathological mouse models [START_REF] Gluais-Dagorn | Direct AMPK Activation Corrects NASH in Rodents Through Metabolic Effects and Direct Action on Inflammation and Fibrogenesis[END_REF] . We, therefore, conclude that our present findings can be extrapolated to humans exposed to traffic noise.

Figure 5

 5 Figure 5 and suppl. Table1).

  used three different therapeutic interventions to prevent noise-induced cardiovascular damage, including exercise training, fasting, and AICAR-induced AMPK activation, as illustrated in Figure 1A-C. Voluntary exercise training, intermittent fasting, and AICAR treatment, all restored noise-induced hypertension to the level of unexposed control (Figure 1D-F). Fasting and AICAR treatment did not alter vascular function in wild-type controls, while all 3 interventions reversed endothelial dysfunction triggered by aircraft noise exposure. The beneficial effects of two therapeutic interventions on the noise-dependent impairment of these functional parameters were also supported by RNA sequencing data and advanced pathway analysis. The next-generation sequencing data revealed a striking improvement of antioxidant defense systems, activation of AMPK signaling and survival pathways, suppression of inflammation and largely improved eNOS/NO/cGMP signaling as well as a cellular response to oxidative stress in noise-exposed mice with fasting and AICAR interventions (suppl. Figures 7 and 8; suppl. Tables

Figures and Legends Fig. 1 .

 Legends1 Figures and Legends

Fig. 2 .

 2 Figure 1

  Figure 2

Fig. 3 .

 3 Fig. 3. Exercise training, caloric restriction, and AICAR administration abolish noiseinduced oxidative stress in the aorta. Effect of voluntary exercise training (A), fasting (B) and AICAR administration (C) on aortic superoxide production determined by HPLC-based analysis. Aircraft noise exposure-increased production of mitochondria-specific superoxide production measured by mitoSOX/HPLC method were prevented by exercise training (D), intermittent fasting (E) and AICAR administration (F). Representative chromatograms are shown beside the quantification data. Data are presented as mean ± SEM.

Fig. 4 .

 4 Fig. 4. Proof-of-concept studies: genetic deletion of endothelial-specific α1AMPK abrogates the beneficial vascular effects of exercise and fasting in noise-exposed mice. (A, D) Voluntary exercise training (n=4-12) and intermittent fasting (n=5-13) fail to prevent noise-induced blood pressure elevation in endothelial-specific α1AMPK knock-out mice. Voluntary exercise training (B [n=24-41] and C [n=6-7]) and intermittent fasting (E [n=11-29] and F [n=5-7]) did not ameliorate endothelial dysfunction in the aorta and mesenteric artery of endothelial-specific α1AMPK knock-out mice. Unexposed wildtype (WT) curves are shown for comparison and were reproduced from Figure 1 as the sum of all WT data for each parameter. The traces of untreated AMPK EC KO shown for exercise and fasting are the same. Data are presented as mean ± SEM. p-values < 0.05 were considered significant (*=AMPK EC KO vs. Noise; #=AMPK EC KO vs. Noise+Treatment; §=Treatment vs. Noise; &=Treatment vs. Noise + treatment; $=WT vs. AMPK EC KO).

Figure 4 Fig. 5 .

 45 Figure 4

  Figure 5

Fig. 6 .Fig. 7 .

 67 Fig. 6. Proof-of-concept studies: genetic deletion of endothelial-specific α1AMPK abrogates the beneficial antioxidant effects of exercise and fasting in noise-exposed mice. Voluntary exercise training failed to prevent noise-induced superoxide production (A) and mitochondrial superoxide formation (B). Also, intermittent fasting did not counteract noiseinduced superoxide in the aorta (C) and mitochondria-specific production of superoxide (D) as well in endothelial-specific α1AMPK knock-out mice. Besides the quantification data, representative HPLC chromatograms are shown. Data are presented as mean ± SEM.

0 200 400 600 PRMT-1 mRNA expression: Aorta [% of CTR ]

	A)	0.9595	0.0026	<0.0001	0.9829	0.9325
	ADMA-positive proteins:	Aorta [% of CTR ]	200 400 600 800	0.0181	<0.0001 0.0259	>0.9999 0.0716 <0.0001	0.9999 0.9999 0.1331 <0.0001
			0
			WT	a1AMPK	a1AMPK	a1AMPK	a1AMPK	a1AMPK
				EC KO	EC KO	EC KO	EC KO	EC KO
					+Noise	+Noise	+Fasting	+Noise
						+Exercise		+Fasting
	B)	<0.0001	<0.0001	0.0007	<0.0001	<0.0001
				0.9936	0.8398	>0.9999	>0.9999
					0.9800	0.9980	0.9922
						0.8874	0.8499
							>0.9999
	C)	0.9580	0.0074	<0.0001	0.9991	0.3942
				0.0029	<0.0001	0.9992	0.8871
					0.0343	0.0226	0.0003
						<0.0001	<0.0001
							0.7625
			WT	a1AMPK	a1AMPK	a1AMPK	a1AMPK	a1AMPK
				EC KO	EC KO	EC KO	EC KO	EC KO
					+Noise	+Noise	+Fasting	+Noise
						+Exercise		+Fasting

Acknowledgments

We thank Angelica Karpi, Nicole Glass, Alexandra Rosenberger, and Jörg Schreiner for excellent technical assistance. This manuscript contains parts of the doctoral thesis of Isabella Schmal at Johannes Gutenberg University Mainz and a PhD thesis on the retinal data of Yue Ruan at Mainz Research School of Translational Biomedicine of the University Medical Center Mainz. Sources of funding S.K.-S. and T.J. received funding from the German Research Foundation (DFG KR 4011/3-1). In addition, S.K., D.M., M.Ku., M.T.B.J. are/were supported by TransMed Ph.D. stipends funded by the Boehringer Ingelheim Foundation or in the case of H.U., L.S. by the Else-Kröner-Fresenius Foundation. The study was also funded by the Boehringer Ingelheim Foundation consortium on novel and neglected risk factors (A.D., T.J, T.M.). TM is a PI of the DZHK partner-site Rhine-Main, Mainz, Germany. The study was also supported by the Foundation Heart of Mainz.

Disclosures

There is no conflict of interest.