Environmental Stressors and their impact on Health and Disease MÜNZEL.pdf

Patients with established coronary artery disease and subsequent major adverse cardiovascular and cerebral events (MACCE), including cardiovascular death, myocardial infarction, and coronary revascularization by percutaneous coronary intervention or bypass surgery, hospitalization for unstable angina or decompensated heart failure, and nonhemorrhagic stroke, had a significantly lower FMD (+4.9%) compared with patients without MACCE (6.3% FMD). However, a pharmacological proof of this concept is still missing since so far no interventional study was published that showed normalization of FMD (e.g., by NO donors) improved the prognosis of patients, and even the prognostic value of FMD was questioned by several studies demonstrating that vascular function measurements without invasive interventions do not contribute much to the prognostic value of the European Society of Cardiology risk score (210) and was not independently correlated with cardiovascular events (240, 279). The pros and cons for FMD as a predictor of future cardiovascular events were discussed in detail previously (57). It is also of prognostic importance to what extent endothelial dysfunction is corrected by the acute administration of

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FIG. 9. Kaplan–Meier analysis of event-free survival refers to subgroups of patients categorized as being below and above the median values for mean FMD. Lower FMD was associated with higher cerebro-cardiovascular event incidence. FMD, flow-mediated dilation. Reprinted from Ostad et al. (182). Copyright (2014), with permission from IOS Press. The publication is available at IOS Press through http://dx.doi.org/10.3233/CH-131720 vitamin C, as this reflects the degree of oxidative stress within the vasculature (106). In patients with endothelial dysfunction and established coronary artery disease, Heitzer et al. established that endothelial dysfunction and higher burden of vascular oxidative stress represent useful predictors of future cardiovascular events (106). With these studies, the authors showed that endothelial dysfunction patients experiencing cardiovascular events, including cardiovascular mortality (e.g., myocardial infarction, ischemic stroke, coronary angioplasty, and coronary or peripheral bypass operation), had greater benefit from vitamin C than patients without any cardiovascular event (Fig. 10A, B) (106). Excessive ROS formation led to loss of progenitor repair capacity and contributes to the progression of atherosclerosis and adverse remodeling (193, 218), resulting in an imbalance between vascular damage and repair observed during the aging process (142, 146).

However, it should be noted that ROS do not only affect the health of patients but also of healthy subjects and that ROS are not only detrimental but, at lower concentrations, confer important redox signaling, involving stress adaptation processes (112, 242) and other essential cellular functions such as cell migration and differentiation (65, 159). This may also explain why so far most interventional studies based on antioxidant therapy failed to improve the prognosis of the treated subjects (92, 209).

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In addition, the effect of ascorbate infusion and age remained independent predictors of future cardiovascular events, even on adjustment for conventional risk factors that blunted endothelium-dependent vasodilation by acetylcholine, using a Cox proportional regression analysis (106). Thus, the degree of oxidative stress in the peripheral vasculature may go parallel with increased oxidative stress in the coronary arteries, thereby causing, for example, plaque rupture. Indeed, Sorescu et al. demonstrated that in atherosclerotic arteries, there was an intense area of superoxide in the plaque shoulder, which is rich in superoxide producing macrophages containing the gp91phox isoform of the NADPH oxidase (228). The authors concluded that increased intracellular oxidative stress in human coronary atherosclerosis may be involved in the genesis and progression of human coronary atherosclerotic disease (228). Interestingly, the strong superoxide signal in the shoulder of the plaque and the intense staining of macrophages in the same area may indicate that superoxide produced by inflammatory cells may be at least, in part, responsible for plaque rupture and therefore for the clinical events such as an acute coronary syndrome (228).

The most significant superoxide source within the vasculature causing endothelial dysfunction is vascular and phagocytic NADPH oxidase, uncoupled NO synthase, xanthine oxidase, and also the mitochondria (163). The direct effects of ROS on endothelial (vascular) function are shown in Figure 11. Based on this evidence of the prognostic importance of the endothelium and the crucial role of oxidative stress in modulating endothelial function, we decided to choose endothelial dysfunction as our primary endpoint in endothelial function studies in healthy subjects (208) and patients with established coronary artery disease (207), as well as in a novel animal model of aircraft noise exposure (161). B. Noise and translational studies in humans

In our recent field study, we found a correlation between the dose of nighttime aircraft noise exposure (in the subjects’ bedrooms) and endothelial function as measured by FMD of the arteria brachialis (Fig. 12) (208). The normalization of noise-induced endothelial dysfunction by acute administration of the antioxidant vitamin C pointed to an important role of oxidative stress in noisetriggered adverse vascular effects (Fig. 12). Vascular dysfunction was also associated with impaired sleeping quality and increased catecholamine production. There was also a ‘‘priming effect’’ of subjects who had previously exposed to noise, leading to a more pronounced adverse vascular effect of noise when exposed for the second time, suggesting sensitization of the vasculature to damage in response to repeated noise exposures. These findings provide a mechanistic explanation for a correlation between nighttime noise exposure and cardiovascular risk. More and more epidemiologic studies provide evidence that nighttime noise exposure has higher relevance for cardiovascular health than daytime noise exposure. The HYENA study observed no significant relationship for daytime noise and aircraft noise, but significantly increased BP with higher burden in nighttime noise (119). In support of these findings, it has been shown that road traffic noise exposure is more detrimental in people who

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FIG. 10. Impact of oxidative stress on endothelial function and event-free survival of patients. (A) Maximal ACh-induced vasodilation in patients with and without cardiovascular events during saline and vitamin C infusion. Vitamin C improved AChinduced vasodilation significantly larger in patients with events compared with patients without events. (B) Kaplan– Meier analysis demonstrating cumulative proportion of patients without cardiovascular events during follow-up. Effect of vitamin C on ACh-induced vasodilation is divided into values below and above the median, p < 0.05 versus group without vitamin C (blue bars). Adapted from Heitzer et al. (106). With permission of Wolters Kluwer Health, Inc. Copyright ª 2001, American Heart Association, Inc. sleep with open windows or have a bedroom facing the road (20). A sustained nocturnal decrease in BP (so-called dipping) is required for resetting the cardiovascular system and for long-term cardiovascular health (205). Repeated nighttime autonomic arousals likely interfere with BP dipping and

increase the risk for onset of hypertension in that part of the population with exposure to relevant levels of long-term environmental noise (102). A recent study in Switzerland demonstrated a more detrimental effect of railway noise on BP during nighttime

FIG. 11. Physiology and pathophysiology in the intact and damaged vasculature with direct effects of oxidative stress. NO, nitric oxide; ONOO-, peroxynitrite; XO, xanthine oxidase. Modified from Daiber and Münzel (54a). With permission of the publisher. Copyright ª 2006, Steinkopff Verlag Darmstadt.

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FIG. 12. Effects of simulated aircraft noise (noise 30 and 60 reflecting 30 or 60 playback aircraft noise events) on endothelial function (as measured by FMD) and (lower right) stress hormone levels of healthy volunteers. The administration of the antioxidant vitamin C (upper right) was associated with improved endothelial function, demonstrating a role of oxidative stress. Adapted from Schmidt et al. (208) with permission of the publisher. Copyright ª 2013, Oxford University Press.

exposure (62). The Night Noise Guidelines for Europe were published by the WHO in 2009 and contain an important correlation of four noise exposure ranges to negative health outcomes, ranging from ‘‘no substantial biological effects’’ to ‘‘increased risk of cardiovascular disease’’ (WHO night noise guidelines). According to these guidelines, noise levels of LAeq,outside 55 dB(A) (see Table 1 for definition) are defined as a goal that can be achieved rapidly to prevent negative health effects of noise, in cases where the more favorable value of 40 dB(A) cannot be reached. In summary, nighttime noise affects autonomic regulation [e.g., by increased heart rate, sympathetic activation and/or parasympathetic withdrawal (33, 102, 145), and increased BP (42)] as well as vascular function (208). Of note, endothelial dysfunction and reduced heart rate variability were associated with the prognosis of patients with peripheral artery disease, arterial hypertension, and acute coronary syndrome or chronic stable coronary artery disease (36, 140, 165). In a similar study, even more pronounced cardiovascular effects of noise were observed in patients with established coronary artery disease and the study had to be terminated earlier (207). With our field studies, we demonstrated sleep disturbance and increased BP as well as endothelial dysfunction in response to noise exposure (207) (Fig.

13). Of note, nighttime noise impaired endothelial function independently of noise sensitivity and annoyance, probably by subliminal processes (207). Our observations provide a mechanistic basis for induction of endothelial dysfunction in

patients with significant coronary artery disease by aircraft noise exposure, also providing an explanation for the epidemiological study results on more frequent arterial hypertension, chronic coronary artery disease, heart failure, stroke, and also cardiac arrhythmia in noisy, urbanized areas (166, 261). C. Noise and translational studies in animals

Noise in animal housing facilities is an inevitable environmental variable that can affect hearing, behavior, and physiology in mice (136). Noise levels in animal housing facilities can reach up to 80 dB(A), due to ventilation, and during human activity interference (e.g., changing the animal cages), peak SPL of more than 100 dB(A) can be reached. These facts should be considered when planning noise exposure experiments. According to a review article by Turner et al., the background noise level recorded from a representative animal housing unit (Southern Illinois University School of Medicine) was around 42 dB(A) with peak levels between 60 and 70 dB(A) (252). This review article also provides an excellent overview on noise susceptibility of different animal species and strains, their hearing frequencies, and common pathways of noise-induced adverse effects on health. Of great value are the examples for noise-induced cardiovascular, hormonal, biochemical, immunological, and other alterations (comparison of human and animal data). Most significant pathophysiological changes in response to

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FIG. 13. Impact of noise exposure during the sleep phase on different cardiovascular and hemodynamic parameters. Effects of nighttime aircraft noise on flow-dependent dilation (A), on systolic blood pressure (B) and on sleep quality as expressed by the VAS (A) and data are mean – SD in 60 patients with established coronary artery disease. Significance levels are *p = 0.001 for (A), *p = 0.03 for (B), and p < 0.001 for (C), respectively, adjusted for gender, age, night sequence, PSQI, overall noise sensitivity (NoiSeQ), sleep-related noise sensitivity, attitude toward aircraft noise, and the results of the Morning Evening Questionnaire. PSQI, Pittsburgh sleep quality index; SD, standard deviation; VAS, visual analog scale. Adapted from Schmidt et al. (207) with permission of the publisher/authors. Copyright: ª 2015, Schmidt et al. (open access). noise include increased BP, vascular dysfunction, stress hormones, immunomodulation, slower wound healing, weight loss, and impaired fertility and reproduction. 1. Direct pathway activation via ‡100 dB(A) noise exposure on the inner ear (hearing loss). The contribution of

noise and aging to hearing loss is distinct, yet interrelated (277). Noise-induced hearing loss (NIHL) is one of the leading causes of hearing loss worldwide. NIHL is usually characterized by an elevation in hearing threshold, and the area of damage is most pronounced one-half octave above the frequency of noise exposure. A significant body of evidence suggests that NIHL damage results from noise-induced free radical production after noise exposure (47, 80, 181, 219). Oxidative stress-dependent cochlear vascular dysfunction and inflammation were reported as a central player in NIHL (79, 246). Hearing loss could also be mimicked by administration of the redox cycler paraquat to chinchillas, which resulted in appreciable increases in cochlear superoxide formation and NIHL-typical threshold shifts (32). In another chinchilla model of NIHL, increased cochlear NADPH oxidase and NF-jB activity was observed on noise exposure (190). Wistar rats exposed to noise with an SPL of 100 or 110 dB(A) (8–12 kHz) for 24 h displayed increased cochlear expression of NOX1 and DUOX2 but decreased expression of NOX3 (262). The latter finding seems to be at variance with a genome-wide association study identifying Nox3 as a critical gene for susceptibility to NIHL (137).

The contribution of mitochondrial ROS to NIHL is supported by aggravation of cochlear complications in mice with heterozygous deficiency in manganese superoxide dismutase (SOD2) (251), which also holds true for age-related hearing loss (125). Lack of other antioxidant enzymes such as Cu,ZnSOD or glutathione peroxidase-1 also potentiates the complications of NIHL, further supporting a central role of oxidative stress in its pathogenesis (178, 179). Age is regarded as an independent risk factor for acquired hearing loss, although the specific effects leading to the pathophysiological phenotype may not be easy to separate from those of noise exposure in the clinical setting (194).

Despite the fact that the mechanisms underlying hearing loss in association with the aging process (presbycusis) are strongly influenced by the genetic susceptibility of a given subject, the pathophysiology of the aging process itself is highly interconnected with increased oxidative stress and low-grade inflammation (158, 239), reflecting major pathomechanisms in NIHL (194). Therefore, oxidative stress and inflammation may represent a link between noise- and agerelated hearing loss and accordingly a unifying target in both complications. This concept is further supported by observations that high-intensity noise [105 dB(A)] induced more pronounced cochlear damage by aggravated impairment of cochlear blood flow in diabetic mice and also less efficient recovery in the hyperglycemic animals (87). This is an important observation since oxidative stress also plays a central role for the pathogenesis of diabetes (109, 176, 267). Cochlear synaptic loss and hair cell death may play a role for noise- and age-related hearing impairment as observed on exposure to noise with an SPL of 100 dB(A) (8–16 kHz) for 2 h (132). When mice were exposed to that noise, there was a significant degree of synaptic loss and cochlear dysfunction starting at 16 weeks after exposure, which were not observed when mice were exposed to noise with an SPL of 91 dB(A) (8–16 kHz) for 2 h (77).

The hearing loss even was evident 1 h after noise exposure, when noise with an SPL of 100 dB(A) at frequencies >22 kHz was applied for 2 h, indicating that higher frequencies are even more detrimental. The authors concluded that one single exposure to noise with SPL ‡100 dB(A) can accelerate cochlear aging. When guinea pigs were exposed to noise with an SPL of 106 dB(A) (4–8 kHz) for 2 h, *30% of the auditory nerve synapses on inner hair cells were lost, leading to cochlear neuropathy and partial hearing loss (77). Application of higher SPL of up to 120 dB(A) also results in structural damage in the mouse cochlea (183). Reports on systemic effects of high-energy noise exposure were published for the prostaglandin E2-dependent hyperalgesia (increased inflammatory pain) of rats on exposure to a 105 dB tone with frequencies of 11–19 kHz (5 or 10 s per minute for 30 min per day) (124).

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Among the practical studies of induction of hearing loss, magnetic resonance imaging-induced acoustic noise was investigated, and it was found that SPL of 100 dB(A) and more can be reached that may cause inner ear damage not only to humans (patients and personnel) but also to companion and experimental animals (135). When hearing loss was induced in guinea pigs by exposure to noise with an SPL of 106 dB(A) for 30 min, red blood cell velocity was reduced gradually over 2 and 3.5 h as measured by in vivo fluorescence microscopy (12). This indicates that cochlear microcirculation plays an important role for the development of hearing loss. 2. Indirect, nonauditory vascular effects of £100 dB(A) noise exposure. There are only few experimental studies in

animals on the mechanisms of noise-dependent effects on endothelial function and cardiovascular risk. When rats were exposed to noise for periods of 2 and 4 weeks (100 dB(A), 4 h/day, 6 days/week) endothelium-dependent vasodilation (measured by acetylcholine in the thoracic aorta) was impaired, the sensitivity to the vasoconstrictor serotonin, but not phenylephrine or potassium chloride was increased, and systolic BP was elevated by 31 mmHg (272). In a similar study, the same authors demonstrated an increase in systolic BP after 3 weeks (25 mmHg) and 4 weeks (37 mmHg) of noise exposure and pronounced endothelial dysfunction in isolated mesenteric arterial rings (273). More recently, noise exposure of rats was reported to adversely affect the cardiovascular system by increased levels of circulating stress hormones (e.g., corticosterone, adrenaline, noradrenaline, endothelin-1 [ET-1]) and negatively affected oxidative stress markers, such as increased malondialdehyde and decreased SOD, all of which point toward endothelial dysfunction [octave band noise: 80–100 dB(A), 8–16 kHz, 8 h/ day for 20 days; 8 rats/group] (204). These elevated risk markers were associated with increased heart rate, mean arterial BP, and circulating nitrogen oxides, which could be a marker of NO coming from inducible NOS in inflammatory cells.

Of note, these authors also compared different noise exposure protocols, and the most pronounced adverse effects were observed in the chronic exposure group (8 h/day for 20 days), whereas the single exposure for 12 h induced moderate impairments that were comparable with those observed on chronic intermittent noise exposure (8 h/day for a total of 20 days with 3 days of exposure followed by 2 days of cessation) (204). Also, oxidative DNA damage (detected by the comet assay) was observed in the rat heart and adrenal gland on noise exposure ‡100 dB(A) (86, 139). Cardiovascular therapy using the cholesterol lowering substance rosuvastatin effectively prevented these adverse effects (70). Likewise, exposure of rats to white noise (90 dB, 15 min daily for 3 weeks) caused adverse effects on the morphology of the intestinal mucosa with separated or even detached mucosal epithelial cells and evidence of edema (24). Recovery of rats for another 3 weeks improved epithelial integrity and partially the function of mucosal cells. Using a similar protocol, the same authors showed that noise exposure induces mesenteric microvascular structural damage with increased number of leaks that was significantly reduced by anti-inflammatory and antioxidant cotreatment (23).

White noise exposure of 100 dB with frequencies of 0.4– 6.3 kHz (4 h per day for 30 days) caused a transient increase in blood glucose, markers of inflammation, triglycerides, and

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changes in the microbiome that returned to normal at 14 days after cessation of noise exposure (53). In 1981, a study reported on increases of mean BP by 30 mmHg in response to noise exposure [mean SPL of 85 dB(A), six episodes per day for 9 months, 97 dB(A) peak level] in two chronically exposed monkeys without significant effects on the auditory system (185). In another study from the year 1992, rats were exposed to noise with an SPL of 85 dB(A) for 12 h/day for 8 weeks and subsequently with an SPL of 95 dB(A) for 16 h/day for 4 weeks, and an increase of systolic and diastolic BP by 16 mmHg was observed along with magnesium deficiency and reduced lumen sizes of microvessels (7). Exposure of guinea pigs to noise [70 or 90 dB(A)] resulted in gradual loss of vascular endothelial growth factor (VEGF) in the cochlea. VEGF is a proangiogenic factor that regulates vascular permeability and also displays neuroprotective properties (217). Exposure of rats to moderate noise with an SPL of 70 or 85 dB(A) (8–16 kHz; 6 h/day for 3 months) led to neuroendocrine modulation with increased corticosterone levels and increased lipid peroxidation being more pronounced in the 85 dB(A) group, whereas upregulation of antioxidant enzyme catalase and SOD was similar in both exposure groups (89).

In addition, the authors reported on morphological changes in the heart [e.g., inflamed area of pericardium and dilated veins on 70 dB(A) exposure and more dilated veins in the periphery of pericardium on 85 dB(A) exposure] and analog changes in tissues of the thyroid and adrenal gland. 3. Indirect, nonauditory pathway activation with noise exposure £85 dB(A). Our recently established protocol of

noise exposure to study the effects of aircraft noise on vascular function consisted of repetitive playback of a 2-h-long noise pattern (69 aircraft noise events with a duration of 43 s) and a maximum SPL of 83 dB(A). The noise events were separated by irregularly distributed silent periods to prevent early adaptation. Downward facing loudspeakers were used for the playback of the noise pattern and were mounted *30 cm above the plastic mouse cages (Fig. 14). Playback of the noise pattern mp3 files was carried out using a Grundig MS 540 compact sound system with a total

FIG. 14. Setup of the noise exposure system used for the mouse studies.

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output of 65 W. Loudness and corresponding SPL were calibrated with a Class II sound level meter (Casella CEL-246) within one of the cages at initial setup. Actual SPLs during exposure were continuously recorded during the study period using the same device placed between cages with an upward facing microphone. The average SPL (equivalent to dB Leq) was 71.6 dB(A) with a usual intermediate background noise level of 50–55 dB(A) in the animal facility depending on the specific room. For investigation of the importance of specific noise patterns and characteristics, ‘‘white noise’’ was used as a control sound exposure with exactly the same average SPL and compared to the impact of continuous aircraft noise exposure for 1, 2, and 4 days on vascular function. ‘‘White noise’’ consists of a random noise with constant spectral density within the range of human hearing with frequencies of 20 Hz to 20 kHz. Already after 24 h of continuous exposure to aircraft noise, we observed increased stress hormone levels for catecholamines Ang II and ET-1 (161). Also, oxidative stress markers such as 3-nitrotyrosine- and malondialdehyde-positive proteins and endothelial (vascular) dysfunction, as well as BP, were increased on noise over the entire exposure period relative to pre-exposure levels (Fig. 15).

The increase in the NOX2 protein (phagocyte NADPH oxidase) content in the aorta of mice exposed to aircraft noise pointed toward increased infiltration of inflammatory cells into the vasculature as shown by FACS analyses (161). A major mechanism of the development of arterial hypertension consists of vascular infiltration of inflammatory cells leading to endothelial dysfunction and oxidative stress, previously demonstrated by genetic/pharmacological deletion of LysM-positive cells (265) and recently reviewed in detail (266). Endothelial nitric oxide synthase (eNOS) uncoupling was revealed in the aorta and hearts from animals exposed to aircraft noise (161). The presence of uncoupled eNOS was supported by increased levels of endothelial ROS formation, which was inhibited by the eNOS inhibitor L-NG-nitro-arginine [LNAME; using the dihydroethidium cryostaining method (55)]. As a surrogate parameter for the uncoupling reaction, eNOS S-glutathionylation was found elevated in the heart and aorta on noise exposure (summarized in Fig. 16) (44). GTP-cyclohydrolase-1 and dihydrofolate reductase are responsible for the synthesis and recycling of tetrahydrobiopterin (BH4), which is oxidized by high intracellular oxidative stress to the BH3 radical, thereby causing functional depletion of the eNOS cofactor (29, 134, 212).

The here observed increased expression of these enzymes in the noise-exposed groups points toward compensatory upregulation of these enzymes to overcome eNOS uncoupling and endothelial dysfunction. Accordingly, the NO bioavailability was decreased in aorta of noise-exposed mice (measured by electron paramagnetic resonance spectroscopy). The in-

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creased sensitivity to vasoconstrictors, such as norepinephrine, and the elevated vascular ET-1 immunohistochemical staining further supported the observed endothelial dysfunction in response to noise exposure (Fig. 15). Of note, exposure to ‘‘white noise’’ at similar mean SPL did not induce these adverse vascular effects in response to aircraft noise exposure, clearly indicating that not only the loudness (quantity) of a specific noise but also its characteristics (quality such as frequencies, pattern) determine its harmful effects (161). 4. Effects of aircraft noise on vascular gene regulation as established by next-generation sequencing. We were also

able to identify significant alterations in gene expression profiles by using next-generation sequencing (NGS) (summarized in Fig. 16) (161). Pathway analysis by gene ontology annotation from NGS data revealed major changes in the vascular smooth muscle cell (VSMC) contraction pathway and TGFb- and Smad signaling. Significant changes were detected in the NFjB-related pathway, adrenergic signal transduction, focal adhesion, cell cycle control, apoptosis, and kinase-mediated growth and proliferation signaling centered around the Forkhead-box-protein (Foxo) transcription factors herein (stress response and antioxidant defense) (161). The four strongest upregulated genes compared to controls were Zbtb44, Setad4, Ypel2, and Ihh and, similarly, a number of transcripts Sacs, Nbeal1, PTPN4, and NR4A3 were significantly reduced by noise (summarized in Fig. 16). These genes are quite novel in the context of cardiovascular physiology and are generally poorly understood. Therefore, their exact role in noise-induced pathophysiology warrants further investigation. Changes were also observed in expression of genes being involved in the regulation of the circadian rhythm, insulin, and calcineurin signaling pathways, although the relationship of these pathways to noise-induced vascular damage was not addressed in detail so far.

Zbtb44 codes for a highly conserved zinc finger domain DNA binding protein. It is involved in stem cell growth and responds to steroid and adrenergic stimulation (35, 271). The specific function in the cardiovascular vessels has not been determined. SERTAD4 is highly expressed in adult murine fibrous tissues. SERTAD4 resides predominantly in the nucleus throughout cell cycle progression and shows interaction with PP2A and PI3K (93, 186), which are crucial regulators in the pathways discussed here. Yipee-like 2 (YPEL2) is localized in the nucleus and contains several metal binding sites, it is known to interact with phosphatases and influences calcium signaling in cellular repolarization (11). Indian hedgehog (IHH) is involved in signaling and has been found among other pathways to participate in cartilage degeneration as well as in TGFb-driven chondrogenesis and ossification (99, 263). Sacsin is highly expressed in the central

‰ FIG. 15. Selected data of our recently published noise exposure mouse study. Exposure to aircraft noise led to elevated systolic blood pressure (red symbols) (A), impaired endothelial function (B), reduced vascular NO levels [measured with EPR as described (128)] (C), and enhanced sensitivity to vasoconstrictors (D). Exposure to aircraft noise led to increased staining of vascular 3-nitrotyrosine-positive proteins (E, F) and vascular autocrine endothelin-1 production (G, H). The staining reflects representative immunohistochemical images. Brown color indicates 3-nitrotyrosine-positive proteins or endothelin-1 expression and localization. EPR, electron paramagnetic resonance spectroscopy; ET-1, endothelin-1. Modified from Münzel et al. (161) with permission of the publisher/authors. Copyright ª 2017, Münzel et al. (open access). Published by Oxford University Press on behalf of the European Society of Cardiology.

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FIG. 16. Postulated mechanisms of noise-induced (cardio)vascular damage are based on the vascular functional and observational parameters as well as results from next-generation sequencing of our recent mouse study on the effects of aircraft noise exposure on the cardiovascular system. eNOS, endothelial nitric oxide synthase; Foxo, Forkhead-box-protein; Ihh, Indian hedgehog; NO, nitric oxide; Nox2, NADPH oxidase isoform 2, ROS, reactive oxygen species, Ypel2, Yipeelike 2.

nervous tissue and also shows abundant expression in fibrous tissue. It has chaperone-like function and is regulated by TGFb in epithelial–mesenchymal transition (241). NBEAL1 is known to be strongly expressed in the mouse aorta; its lysosomal import sequence may implicate a role in autophagy and hypoxia (45). PTPN4 belongs to a superfamily of protein phosphatases that is associated with cytoskeletal proteins. It has a role in cell growth and motility in various tissues and cell lines (281). Downregulation of the nuclear orphan receptor NR4A3/NOR1 is known to contribute to the regulation of matrix metalloprotease in vascular tissue and to the activation of VSMCs (197, 198). Conservative promoter database analysis of transcription factor binding sites of the corresponding gene products showed many binding sites with redox-active and cysteine-rich transcription factors (NF-jB, FOXO, and zinc-finger proteins) in the promoter regions of the eight most regulated genes (161). This might insinuate that noise-generated nitro-oxidative stress may directly influence transcription levels. Although these genes and their gene products are not well known in the cardiovascular context, through their interaction partners, they strongly contribute to the pathways discussed here. ROS play a key role in linking the different pathways in the present NGS analysis (summarized in Fig. 16) (161).

The IGF-1/insulin/PI3K/Akt pathway, for instance, can be activated via redox-sensitive mechanisms (10). Subsequently, Akt regulates the activity of FOXO transcription factors by phosphorylation, which has a transcriptional factor binding site on the Nr4a3, Sod1, and glutathione peroxidase 1 gene promoter explaining at least, in part, the observed changes in the transcription level of the NGS experiment (253). Oxidative stress also directly regulates transcription factors FOXO and NF-jB through reversible oxidation and reduction of cysteine residues (147), all of which may affect other transcription factors such as cysteine-rich zinc-finger pro-

teins such as ZBTB44. The changes in FOXO factor activity and other transcription factors may modulate cellular resistance capacity to oxidative stress by regulating key detoxification enzymes (e.g., SOD2 and catalase) (147). By a similar mechanism, TGF-b1 contributes to oxidative stress via decreasing the expression of antioxidant enzymes (e.g., SOD, catalase, glutathione peroxidase) (196). These events may result in increased vascular ROS production and reduced vascular NO-bioavailability in favor of the formation of the NO/superoxide reaction product peroxynitrite, leading to decreased cyclic guanosine monophosphate (cGMP) concentrations and sGC sensitivity along with an inhibition of the activity of the cGMP-dependent protein kinase I (cGK-I), which is able to induce major structural and transcriptional changes in smooth muscle cells. cGK-I may interact with the TGFb/Smad pathway, MAPK-signaling, as well as PI3K and FOXO pathways (76, 94, 144), which show relevant changes in the NGS transcription data. Some of the pathways modulated by nitrooxidative stress represent pathological mechanisms potentially mediating the detrimental effects of noise, while others are likely to reflect compensatory mechanisms in response to noise-induced injury.

Despite the presented evidence for potential redox regulation of vascular gene expression by noise exposure in multiple signaling pathways, we should keep in mind that the presented NGS data do not reflect posttranslational modifications of proteins (including kinases, phosphatases, and transcription factors), which undergo substantial changes in an oxidative stress milieu, largely affecting enzymatic function via sulfoxidation, S-nitros(yl)ation, S-glutathionylation, and phosphorylation. Accordingly, the overall impact of redox regulation on these multiple signaling pathways may be even more pronounced at the proteomic or metabolic level than expected by the established changes at the genomic level. These observations are also in good accordance with a

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recent overview on the impact of environmental stressors such as noise on redox-regulated epigenetic pathways published within this Forum (157). 5. Summary and conclusions of aircraft noise exposure in mice. Taken together, we propose the following patho-

physiological sequence of events causing vascular damage in response to aircraft noise exposure (Fig. 16). Aircraft noise exposure leads to an overactivation of the sympathetic system, resulting in elevated levels of noradrenalin (NA), adrenalin (A), angiotensin II (Ang II), and subsequently cortisol. Angiotensin II, in turn, activates endothelial NADPH oxidase causing oxidative stress, which may induce direct scavenging of NO and eNOS uncoupling through oxidation of BH4 and eNOS S-glutathionylation. ROS play a key role in linking different pathways, including PI3K/Akt signaling, the FOXO transcription factors, TGF-b1 and NFjB signaling, as well as the ET-1 system, increasing the circulating levels of IL-6, and the expression of vascular adhesion molecules. Superoxide and NO produced by infiltrating immune cells (neutrophils, natural killer cells, and monocytes/macrophages) promote the formation of 3-nitrotyrosine-, malondialdehyde-, and 4-hydroxynonenal-positive proteins and inflict additional cellular oxidative damage. The uncoupling of eNOS not only reduces NO production but also potentiates the pre-existing oxidative stress. Endothelial NO production is further reduced by glucocorticoids such as cortisol, leading to impaired vasodilation and increased BP.

The overproduction of noradrenalin, adrenalin, and ET-1 enhances contraction, which is further potentiated by glucocorticoids. All of these vascular alterations support the development of metabolic disorders as envisaged by increased blood glucose levels. ET-1 expression levels were also reported to contribute to the phenotype of Alport (Col4a3-/-) mice that display a particular susceptibility to toxic noise and represent a model of glomerular disease associated with hearing loss (151). IV. Adverse Effects of Simultaneous Noise and Air Pollution Exposure A. Adverse effects of noise and air pollution exposure share similar pathophysiological pathways

Recent data support the idea that air pollution and noise, similar to traditional risk factors, contribute to vascular (endothelial) dysfunction, hypertension, atherosclerosis, and other cardiovascular events such as myocardial infarction, stroke, and congestive heart failure. In general, four pathways have been introduced to better explain the combined effects of air pollution and noise (Fig. 17): (i) a dysregulated autonomic nervous system and/or an activated sympathetic system; (ii) generation of mediators of inflammation, modified lipids, or phospholipids, and recruitment of different immune cell populations; (iii) oxidative impairment of endothelial dysfunction; and (iv) higher activity of prothrombotic signaling pathways (25, 66, 96, 207, 208). These adverse pathways may overlap and act synergistically, and may be activated at different points in time to noise or air pollution exposures, and have probably varying impact on cardiovascular events. The immediate effects observed within seconds/ minutes/hours after noise or particulate matter (PM) expo-

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sures are most probably conferred via alterations in autonomic tone and/or activation of the sympathetic system, impairment of endothelial function (161), increased levels of procoagulant proteins leading to higher activity of procoagulant pathways, and more frequent thrombotic events [for review, see Münzel et al. (167) and Newby et al. (174)] (Fig. 17). Acute effects of air pollution and noise pollution are more likely in patients with higher susceptibility (e.g., with preexisting cardiovascular complications such as ‘‘vulnerable plaque,’’ ‘‘vulnerable myocardium’’ (arrhythmias), or ‘‘vulnerable circulation’’). Repeated exposures to environmental stressors mainly depend on the accumulation (duration of exposure), and accordingly, effects of noise and air pollution can be additive or synergistic. On chronic exposure also habituation/adaptation may occur, implying that most severe noise-induced physiologic effects are more likely within the first days of exposure. Likewise, the effects of noise observed in the field (where noise exposure of subjects for many years is frequently encountered) are usually less pronounced than those measured in laboratory conditions, where often exposures to unusual noise events are applied (25, 162, 187).

Although a weaker response to air pollution on repeated exposure challenges would be in line with biological adaption processes, this was never shown in detail in a controlled experimental setting. Under specific circumstances, however, contrary observations could be made meaning that the second exposure may lead to more pronounced effects than the initial exposure, a physiological and medical phenomenon that is well known as ‘‘priming’’ (208). B. Gaps in current knowledge concerning noise and air pollution

Only few studies investigate the additive or synergistic effects of simultaneous noise and air pollution exposure in humans (for animal research, we know not a single study addressing this issue). Studies in animals could answer important questions on the mechanisms of detrimental health effects by combined noise and air pollution exposures, providing the directions for future human studies. The questions comprise the extent and kinetics of response to coexposure, interactive effects of both factors on surrogate parameters (e.g., BP and metabolic risk), reversibility of effects, impact of low-grade background noise on air pollution exposure effects and vice versa, modulation of the circadian rhythm, and improvement by preventive measures and lifestyle changes (e.g., diet, stress, and exercise). Finally, new medical devices that allow to record health parameters as well as exposure levels to environmental pollutants may provide an important opportunity for research and understanding of the interactions of environmental with nonenvironmental risk factors (Fig. 18). V. Other Environmental Risk Factors, Oxidative Stress, and Cardiovascular Disease

Exposure to environmental pollutants is a major but significantly underestimated risk factor that contributes to the onset and progression of CVD (52). The cardiovascular system is susceptible to damage by various environmental risk factors such as air pollution (e.g., PM or reactive nitrogen/sulfur oxides) and heavy metals (e.g., arsenic, cadmium, or lead). These environmental risk factors may, similar to the

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FIG. 17. Proposed pathophysiological mechanisms of cardiovascular disease induced by environmental air pollution and noise. 7-KC, 7-ketocholesterol; ox-PAPC, oxidatively modified 1-palmitoyl-2-arachidonoyl-snphosphatidylcholine. Adapted from Münzel et al. (166) with permission of the publisher. Copyright ª 2016, Oxford University Press.

traditional risk factors (e.g., smoking, hypertension, and diabetes mellitus), increase the global burden of disease and mortality, also by adverse effects on regulatory pathways of vascular tone (leading to arterial hypertension), metabolism (leading to hyperlipidemia, obesity, and diabetes), and atherosclerosis. There is clear evidence that people living in highly polluted areas are at higher cardiovascular risk but more recently, there is also growing body of evidence that chronic exposure to these environmental risk factors, even at concentrations below the legal thresholds, leads to adverse effects on cardiovascular health. Since most of these environmental pollutants are omnipresent, even their minor impact on cardiovascular risk has

dramatic effects on public health (not only on mortality but also life years spent with severe illness and disability). Accordingly, as outlined above for the prevention of noiseinduced cardiovascular damage, mechanistic and epidemiological studies have been conducted or will address in the future how mitigation strategies can help to decrease the adverse health effects of air pollution and heavy metals. The list of environmental toxic compounds affecting cardiovascular health could be extended at will [e.g., pesticidedriven oxidative stress (63, 259) and CVDs (129, 215)], but in the present review with the focus on environmental noise effects on cardiovascular function, we only want to mention air pollution representing another major environmental

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FIG. 18. Hypothetical framework of investigations that combine technological innovation in biometric data with personalized exposure information in real time to study interactive effects of environmental risk factors on cardiovascular endpoints. ABP, ambulant blood pressure monitoring; BC, black carbon; PM, particulate matter. Adapted from Münzel et al. (167) with permission of the publisher. Copyright ª 2016, Oxford University Press.

cardiovascular risk factor (166, 167) that is mostly associated with noise exposure (e.g., traffic generates noise and air pollution and these stressors cannot be easily separated). Other environmental stressors are only briefly mentioned, mostly since they are discussed in detail in the reviews included in this Forum. A. Air pollution (PM/carbon black)

In contrast to the research on noise-induced cardiovascular risk, the cardiovascular effects of air pollution are well characterized demonstrating vascular (endothelial) dysfunction, vascular inflammation and, in the longer run, the de-

velopment of atherosclerosis (192). In addition, exposure to PM increases oxidative stress within the vasculature and increases the sensitivity to vasoconstrictors (280). Recent publications based on epidemiological and experimental studies suggest additive damage of noise on air pollutioninduced CVD (166, 234), although no study has ever investigated the functional and molecular mechanisms of the interaction of both environmental stressors for oxidative stress and the vasculature itself. In addition, the Heinz Nixdorf Recall study showed that environmental stress, for example, air pollution and traffic noise, is independently associated with increased aortic calcifications (121), another predictor of future cardiovascular events, which warrants detailed

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basic science studies to gain more mechanistic insight in the pathophysiology of these potential additive adverse effects at the vascular level. In a small cohort trial, 18 healthy individuals were subjected to diesel exhaust at 276 g/m3 from a passenger car or filtered air for 3 h, with coexposure to traffic noise at 48 or 75 dB(A) (107). Exposure to diesel exhaust had no effects on genotoxicity, oxidative stress, or inflammation in white blood cells isolated from the subjects, whereas exposure to noise caused oxidative DNA damage. In another study, 18 highway maintenance workers were monitored with respect to their exposure to PM and traffic noise, significantly associated with C-reactive protein, serum amyloid A, increased heart rate variability, or systolic and diastolic BP (152). The impact of PM and diesel exhaust on oxidative stress pathways and inflammation was reviewed in full detail in two articles within this Forum (191, 270). As outlined in a recent review article (52), around 7% of nonfatal myocardial infarctions (171) and 18% of sudden cardiac deaths (104) are potentially triggered by exposure to road traffic-dependent air pollution, representing comparable numbers to those published for the contribution of the major traditional and modifiable risk factors, from smoking, poor diet, or obesity to cardiovascular morbidity and mortality (104).

Despite the relatively low contribution of air pollution to the individual risk of a single human being, the cumulative cardiovascular risk conferred by chronic exposure of a large part of the population to ubiquitous air pollution (the cumulative global disease burden) even ranks above physical exertion, coffee, and alcohol in a comparative risk assessment of the major triggers of myocardial infarction (171). Only diet, high BP, and smoking represent more important cardiovascular risk factors for life years with severe illness and disability than air pollution (278). Strikingly, the short-term pollution-control activities (mainly reduction in traffic and industrial air pollution) applied during the 2008 Beijing Olympic Games led to a 13–60% reduction in the concentrations of air pollutants, which were perfectly mimicked by similar effects on biomarkers of inflammation, oxidative stress, and thrombosis in healthy adults (110, 126, 195, 203). Unfortunately, the beneficial effects quickly returned back to normal when the restrictions for air pollution were invalidated after the Olympic Games. An important feature of the ENNAH Network (see also the Introduction section) was the involvement of researchers mainly working on air pollution allowing joint considerations of the effects of both environmental risk factors, transportation noise and air pollution on public health (3).

The EU made efforts in supporting cohort studies on the effects of air pollution on public health by substantial funds. ENNAH provided an excellent platform for exploitation of existing European cohort data that provide reliable information on air pollution and enriching these cohort data on air pollution with noise components from existing noise maps. These joint studies may have direct impact on transportation and environmental guidelines with respect to future policies in air pollution and/or noise thresholds and the development of new mitigation strategies. Therefore, the ENNAH Network represents one of the first large initiatives to plan future research on the synergistic effects of air pollution and noise exposure on public health.

MÜNZEL ET AL. B. Other environmental stressors

Chronic mental stress in turn has been demonstrated to generate its own cardiovascular risk factors such as increased BP and dyslipidemia, increased blood viscosity and blood glucose, and activation of blood clotting factors (13). Findings from a large pan-European epidemiological study in 124,808 diabetes-free subjects indicate that job strain is a risk factor for type 2 diabetes in men and women independent of other lifestyle factors such as obesity and physical inactivity (175). A meta-analysis of nine case/control and cohort studies of good methodological quality showed positive associations between hypertension and job strain (22). The impact of mental stress on oxidative stress pathways and inflammation was reviewed in full detail by Siegrist and Sies (221) and in two articles within this Forum (155, 274). Environmental exposure to electromagnetic radiation (EMR) has been increasing with higher demand for advanced communication infrastructure (e.g., Wi-Fi) and devices (smart phones). The effects of EMR on oxidative stress, inflammation, and reproduction were previously reviewed (172). However, a recent critical review led to the conclusion that standardized protocols are required to obtain reliable data leading to better understanding of the underlying mechanisms (90).

As outlined in a recent review article (52), there is substantial evidence from epidemiological and experimental data suggesting that heavy metals [e.g., cadmium (248), a systematic review of 31 studies and lead, a systematic review of 12 studies (170)] and metalloids [e.g., arsenic, a systematic review of 12 studies (160)] can trigger CVDs. A modulation of BP, lipid metabolism, atherogenesis, and endothelial function was observed in response to these compounds (188). In the plasma, serum, and atherosclerotic lesions of mice treated with arsenic, increased levels of proinflammatory chemokines, cytokines, and markers of oxidative stress were observed (232). Likewise, cadmium leads to vascular damage, endothelial dysfunction, and atherosclerosis by oxidative mechanisms (e.g., by replacement of iron and copper in sulfur complexes, promoting Fenton reactions) (153, 154), interference with antioxidant responses (e.g., by disruption of zinc/sulfur complexes) (54), and inhibition of NO-mediated vasodilation (6). Of note, lead inhibits the NO/soluble guanylate cyclase signaling pathway, stimulates the renin–angiotensin– aldosterone system and the sympathetic nervous system, and activates protein kinase C activity, all of which resemble strikingly the adverse effects of noise exposure on the cardiovascular system (258).

The impact of environmental chemicals such as heavy metals on oxidative stress pathways and epigenetic gene regulation was reviewed in full detail in an article within this Forum (157). VI. Summary and Future Perspectives

Taken together, the present review summarizes important mechanisms for the development of cardiometabolic diseases in response to exposure to noise (and other environmental stressors). Noise leads to oxidative stress, vascular dysfunction, autonomic imbalance, and metabolic abnormalities, further increasing the adverse health effects of classical risk factors such as arterial hypertension, diabetes,

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FIG. 19. Overview on human and animal evidence of noise-induced cardiovascular complications. IL-6, interleukin-6; SOD, superoxide dismutase.

hypercholesterolemia, and smoking (e.g., accelerated progression of atherosclerosis and higher susceptibility to cardiovascular events). While there is substantial clinical symptomatic evidence but only minor mechanistic insight from human studies, there is appreciable experimental animal data available on underlying mechanisms but only few reports on the impact of chronic noise on disease progression in animal models (Fig. 19). Importantly, both noise and air pollution seem to cause vascular dysfunction by inducing oxidative stress involving partly similar enzymatic pathways, which warrants further preclinical studies addressing this topic. A note of caution with respect to the choice of suitable oxidative stress markers and their interpretation in the context of environmental stressors was published in this Forum (91). An effective reaction toward reducing environmental transportation noise exposures would require a paradigm shift in human activity and long-term costly mitigation strategies. Given the colocalization of noise and air pollution in big cities and the expected additive benefits of reducing both stressors, an alteration of the current approach, which considers the two exposures separately, may be required. Furthermore, even attempts that address several environ-

mental risk factors simultaneously, changing urban models completely, need to be considered and may lead to cobenefits such as greenhouse gas reduction and increased physical activity of the population. Given the increasing evidence for synergistic/additive effects of air and noise pollution as significant risk factors, their contribution to the pathogenesis of CVD needs to be considered. Accordingly, these environmental stressors should be acknowledged in current guidelines for cardiovascular prevention, acute coronary syndrome, and congestive heart failure. Acknowledgments

We thank Margot Neuser for graphical support. Our studies were continuously supported by the Center for Translational Vascular Biology (CTVB) and the Center for Thrombosis and Hemostasis (CTH, funded by the German Federal Ministry of Education and Research, BMBF 01EO1003) of Johannes Gutenberg University Medical Center (Mainz, Germany; A.D. und T.M.). Our experimental studies on noise effects in mice were supported by vascular biology research grants from the Foundation Heart of Mainz and the Boehringer Ingelheim Foundation for the

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collaborative research group ‘‘Novel and neglected cardiovascular risk factors: molecular mechanisms and therapeutic implications’’ (A.D. and T.M.). T.M. is Principle Investigator of the DZHK (German Center for Cardiovascular Research), Partner Site Rhine-Main (Mainz, Germany). References

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