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      Association of single nucleotide polymorphisms in the NRF2 promoter with vascular stiffness with aging

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          Abstract

          Purpose

          Pulse wave velocity (PWV), an indicator of vascular stiffness, increases with age and is increasingly recognized as an independent risk factor for cardiovascular disease (CVD). Although many mechanical and chemical factors underlie the stiffness of the elastic artery, genetic risk factors related to age-dependent increases in PWV in apparently healthy people are largely unknown. The transcription factor nuclear factor E2 (NF-E2)-related factor 2 (Nrf2), which is activated by unidirectional vascular pulsatile shear stress or oxidative stress, regulates vascular redox homeostasis. Previous reports have shown that a SNP in the NRF2 gene regulatory region (−617C>A; hereafter called SNP−617) affects NRF2 gene expression such that the minor A allele confers lower gene expression compared to the C allele, and it is associated with various diseases, including CVD. We aimed to investigate whether SNP−617 affects vascular stiffness with aging in apparently healthy people.

          Methods

          Analyzing wide-ranging data obtained from a public health survey performed in Japan, we evaluated whether SNP−617 affected brachial-ankle PWV (baPWV) in never-smoking healthy subjects (n = 642). We also evaluated the effects of SNP−617 on other cardiovascular and blood test measurements.

          Results

          We have shown that not only AA carriers (n = 55) but also CA carriers (n = 247) show arterial stiffness compared to CC carriers (n = 340). Furthermore, SNP−617 also affected blood pressure indexes such as systolic blood pressure and mean arterial pressure but not the ankle brachial pressure index, an indicator of atherosclerosis. Multivariate analysis showed that SNP−617 accelerates the incremental ratio of baPWV with age.

          Conclusions

          This study is the first to show that SNP−617 affects the age-dependent increase in vascular stiffness. Our results indicate that low NRF2 activity induces premature vascular aging and could be targeted for the prevention of cardiovascular diseases associated with aging.

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          Most cited references42

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          Arterial and cardiac aging: major shareholders in cardiovascular disease enterprises: Part I: aging arteries: a "set up" for vascular disease.

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            Rare variant discovery by deep whole-genome sequencing of 1,070 Japanese individuals

            The Tohoku Medical Megabank Organization reports the whole-genome sequences of 1,070 healthy Japanese individuals and construction of a Japanese population reference panel (1KJPN). Here we identify through this high-coverage sequencing (32.4 × on average), 21.2 million, including 12 million novel, single-nucleotide variants (SNVs) at an estimated false discovery rate of <1.0%. This detailed analysis detected signatures for purifying selection on regulatory elements as well as coding regions. We also catalogue structural variants, including 3.4 million insertions and deletions, and 25,923 genic copy-number variants. The 1KJPN was effective for imputing genotypes of the Japanese population genome wide. These data demonstrate the value of high-coverage sequencing for constructing population-specific variant panels, which covers 99.0% SNVs of minor allele frequency ≥0.1%, and its value for identifying causal rare variants of complex human disease phenotypes in genetic association studies.
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              Decline in transcriptional activity of Nrf2 causes age-related loss of glutathione synthesis, which is reversible with lipoic acid.

              Glutathione (GSH) significantly declines in the aging rat liver. Because GSH levels are partly a reflection of its synthetic capacity, we measured the levels and activity of gamma-glutamylcysteine ligase (GCL), the rate-controlling enzyme in GSH synthesis. With age, both the catalytic (GCLC) and modulatory (GCLM) subunits of GCL decreased by 47% and 52%, respectively (P < 0.005). Concomitant with lower subunit levels, GCL activity also declined by 53% (P < 0.05). Because nuclear factor erythroid2-related factor 2 (Nrf2) governs basal and inducible GCLC and GCLM expression by means of the antioxidant response element (ARE), we hypothesized that aging results in dysregulation of Nrf2-mediated GCL expression. We observed an approximately 50% age-related loss in total (P < 0.001) and nuclear (P < 0.0001) Nrf2 levels, which suggests attenuation in Nrf2-dependent gene transcription. By using gel-shift and supershift assays, a marked reduction in Nrf2/ARE binding in old vs. young rats was noted. To determine whether the constitutive loss of Nrf2 transcriptional activity also affects the inducible nature of Nrf2 nuclear translocation, old rats were treated with (R)-alpha-lipoic acid (LA; 40 mg/kg i.p. up to 48 h), a disulfide compound shown to induce Nrf2 activation in vitro and improve GSH levels in vivo. LA administration increased nuclear Nrf2 levels in old rats after 12 h. LA also induced Nrf2 binding to the ARE, and, consequently, higher GCLC levels and GCL activity were observed 24 h after LA injection. Thus, the age-related loss in GSH synthesis may be caused by dysregulation of ARE-mediated gene expression, but chemoprotective agents, like LA, can attenuate this loss.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: Project administrationRole: SupervisionRole: ValidationRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: Investigation
                Role: Formal analysis
                Role: Formal analysis
                Role: Formal analysisRole: MethodologyRole: Project administration
                Role: SupervisionRole: Writing – review & editing
                Role: InvestigationRole: Writing – review & editing
                Role: Project administrationRole: Writing – review & editing
                Role: Project administration
                Role: Supervision
                Role: Writing – review & editing
                Role: Formal analysis
                Role: ConceptualizationRole: Funding acquisitionRole: Supervision
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: Project administrationRole: SupervisionRole: ValidationRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                11 August 2020
                2020
                : 15
                : 8
                : e0236834
                Affiliations
                [1 ] Department of Stress Response Science, Center for Advanced Medical Research, Hirosaki University Graduate School of Medicine, Hirosaki, Japan
                [2 ] Department of Vegetable Life Science, Hirosaki University Graduate School of Medicine, Hirosaki, Japan
                [3 ] Department of Nature & Wellness Research, Innovation Division, Kagome Co., Ltd. Nasushiobara, Tochigi, Japan
                [4 ] Health Intelligence Center, The University of Tokyo, Minato-ku, Tokyo, Japan
                [5 ] Human Genome Center, The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo, Japan
                [6 ] Department of Cardiology and Nephrology, Hirosaki University Graduate School of Medicine, Hirosaki, Japan
                [7 ] Tohoku Medical Megabank Organization, Tohoku University, Sendai, Japan
                [8 ] Department of Social Medicine, Hirosaki University Graduate School of Medicine, Hirosaki, Japan
                Showa University School of Pharmacy, JAPAN
                Author notes

                Competing Interests: The authors have declared that no competing interests exist. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

                [¤]

                Current address: Department of Hematology-Oncology, Institute of Biomedical Research and Innovation, Foundation for Biomedical Research and Innovation at Kobe, Kobe, Japan

                Author information
                http://orcid.org/0000-0002-8183-4677
                http://orcid.org/0000-0002-5518-0729
                Article
                PONE-D-20-12583
                10.1371/journal.pone.0236834
                7418968
                32780748
                174f306e-b849-4222-a8a5-e8b7e649a940
                © 2020 Shimizu et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 30 April 2020
                : 14 July 2020
                Page count
                Figures: 3, Tables: 3, Pages: 17
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/501100009033, Center of Innovation Program;
                Award ID: JPMJCE1302
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100009033, Center of Innovation Program;
                Award ID: JPMJCE1302
                Award Recipient :
                This work was supported by "Center for Innovation Program" of the Japan Science and Technology Agency Grant Number JPMJCE1302 ( https://projectdb.jst.go.jp/grant/JST-PROJECT-13423993/) and Joint Research Costs for the Department of Vegetable Life Science from Kagome Co., Ltd. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The Kagome Co., Ltd. provided support in the form of salaries for authors [SS, YU and HS] and the research costs as the Joint Research Costs, but did not have any additional roles in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section. SS: Sunao Shimizu, YU: Yusuke Ushida, HS: Hiroyuki Suganuma.
                Categories
                Research Article
                Physical Sciences
                Materials Science
                Material Properties
                Mechanical Properties
                Stiffness
                Medicine and Health Sciences
                Vascular Medicine
                Blood Pressure
                Biology and Life Sciences
                Biochemistry
                Lipids
                Cholesterol
                Medicine and Health Sciences
                Medical Conditions
                Cardiovascular Diseases
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                Cardiology
                Cardiovascular Medicine
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                Vascular Medicine
                Atherosclerosis
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                Medicine and Health Sciences
                Cardiology
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                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Artificial Gene Amplification and Extension
                Polymerase Chain Reaction
                Research and Analysis Methods
                Molecular Biology Techniques
                Artificial Gene Amplification and Extension
                Polymerase Chain Reaction
                Biology and Life Sciences
                Biochemistry
                Biomarkers
                Creatinine
                Custom metadata
                Data cannot be shared publicly because of the ethical concerns. Data are available from the Hirosaki University COI Program Institutional Data Access / Ethics Committee (contact via e-mail: coi@ 123456hirosaki-u.ac.jp ) for researchers who meet the criteria for access to the data. Researchers need to be approved by research ethics review board at the organization of their affiliation.

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