Constitutive hydrogen inhalation prevents vascular remodeling via reduction of oxidative stress

Takeshi Kiyoi; Shuang Liu; Erika Takemasa; Hirotomo Nakaoka; Naohito Hato; Masaki Mogi

doi:10.1371/journal.pone.0227582

Peer Review History

Original SubmissionAugust 26, 2019
1 Nov 2019 Decision Letter - Michael Bader, Editor PONE-D-19-24002 Constitutive hydrogen inhalation prevents vascular remodeling via reduction of oxidative stress PLOS ONE Dear Dr. Mogi, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. We would appreciate receiving your revised manuscript by Dec 16 2019 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. We look forward to receiving your revised manuscript. Kind regards, Michael Bader Academic Editor PLOS ONE Journal Requirements: 1. When submitting your revision, we need you to address these additional requirements. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. Please complete and submit a copy of the ARRIVE Guidelines checklist, a document that aims to improve experimental reporting and reproducibility of animal studies for purposes of post-publication data analysis and reproducibility: https://www.nc3rs.org.uk/arrive-guidelines. Please include your completed checklist as a Supporting Information file. Note that if your paper is accepted for publication, this checklist will be published as part of your article. Specifically, please ensure that you revise your methods section to include the method of euthanasia, as well as how frequently the condition of the animals was monitored. 3. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data. 4. Thank you for stating the following in the Financial Disclosure section: This study was supported by JSPS KAKENHI Grant Numbers 18K08389 (S.L.) and 25462220 (M.M.), and research grants from Panasonic Corporation (https://www.panasonic.com/jp/home.html). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. We note that you received funding from a commercial source: Panasonic Corporation Please provide an amended Competing Interests Statement that explicitly states this commercial funder, along with any other relevant declarations relating to employment, consultancy, patents, products in development, marketed products, etc. Within this Competing Interests Statement, please confirm that this does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests). If there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared. Please include your amended Competing Interests Statement within your cover letter. We will change the online submission form on your behalf. Please know it is PLOS ONE policy for corresponding authors to declare, on behalf of all authors, all potential competing interests for the purposes of transparency. PLOS defines a competing interest as anything that interferes with, or could reasonably be perceived as interfering with, the full and objective presentation, peer review, editorial decision-making, or publication of research or non-research articles submitted to one of the journals. Competing interests can be financial or non-financial, professional, or personal. Competing interests can arise in relationship to an organization or another person. Please follow this link to our website for more details on competing interests: http://journals.plos.org/plosone/s/competing-interests [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Partly Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: No Reviewer #2: I Don't Know ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: No Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: In this work, the authors investigated the effect of hydrogen gas inhalation to prevent vascular diseases. The authors have demonstrated that constitutive inhalation of hydrogen gas has a beneficial effect on vascular remodeling, partly due to its inhibitory effects on ROS generation, DNA damage and cell proliferation. I think this is a meaningful work drawing some interesting conclusions. But, The design of animal group and free radical signal detected is inappropriate. Reviewer #2: Specific comments Introduction Line 81, 88 - How the constitutive inhalation of H2 in real life can contribute to a healthy lifestyle (from a viewpoint of medical practice). Materials and Methods Line 98 - Compressed air containing 1.25% hydrogen gas cannot consist of O2 21% and N2 79% Results Please explain differences: Line 187 and 188 - We examined the effect of hydrogen gas on neointima formation, 14 days after polyethylene cuff placement around the femoral artery….but in Material and Method you write: Line 137 - Femoral arteries that had undergone cuff placement for 7 days… or line 147 group at 7 days after cuff...or line 154- Femoral arteries that had undergone performed cuff placement for 7 days …or line 222 media of the femoral artery 7 days after cuff placement… or line 230 quantitative RT-PCR in femoral artery 7 days after… or line 231 prepared from cuffed arteries 7 days after operation… and all other figs. have 7 days Discussion Explain: healthy daily living in rats Line 278: remodeling aimed at healthy daily living was examined. Line 279 What do you mean by: In this vascular injury model, NADPH oxidase activity and production of superoxide are known to be closely related, Line 302 - Molecular hydrogen alleviates oxidative stress by acting as a radical scavenger for OH- and ONOO- in vitro -please discuss why only in vitro. Is it functioning only in vitro or in vivo as well? Are there other possibilities of antioxidative action in vivo? Line 351 - Indeed, it is difficult to constitutively inhale hydrogen gas in daily living, because patients are not constantly at home. Please discuss if possible: Is it only reason or is it completely useless in everyday life? Is it better to drink HRW several times a day better? ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step. Attachments Attachment Submitted filename: Plos one comments.docx https://doi.org/10.1371/journal.pone.0227582.r001
Revision 1
14 Dec 2019 Author Response Response to Editor: (MS # PONE-D-19-24002) Thank you very much for your careful reading of our manuscript and helpful comments. In response to your comments, we have revised the manuscript carefully. Major changes are highlighted in the text. We believe this revised manuscript has been greatly improved by your constructive comments, and would be grateful if it could be re-considered for publication in PLOS ONE. Comments: 1. When submitting your revision, we need you to address these additional requirements. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. Response: We have checked and revised the entire manuscript accordingly. 2. Please complete and submit a copy of the ARRIVE Guidelines checklist, a document that aims to improve experimental reporting and reproducibility of animal studies for purposes of post-publication data analysis and reproducibility: Please include your completed checklist as a Supporting Information file. Note that if your paper is accepted for publication, this checklist will be published as part of your article. Specifically, please ensure that you revise your methods section to include the method of euthanasia, as well as how frequently the condition of the animals was monitored. Response: We have checked the ARRIVE Guidelines and completed the ARRIVE Guidelines checklist. The document has been submitted as a Supporting Information file. In addition, we have added the requested information to the “Animals and Treatment” section of the “Materials and Methods”. 3. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data. Response: We understand the PLOS ONE policy. We have omitted use of the phrase “data not shown”. The data are now shown in the Supporting Information file. 4. Thank you for stating the following in the Financial Disclosure section: We note that you received funding from a commercial source: Panasonic Corporation Please provide an amended Competing Interests Statement that explicitly states this commercial funder, along with any other relevant declarations relating to employment, consultancy, patents, products in development, marketed products, etc. If there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared. Please include your amended Competing Interests Statement within your cover letter. We will change the online submission form on your behalf. Response: The authors have no conflict of interest including employment, consultancy, patents, products in development, marketed products, etc. from Panasonic Corporation. We have added this statement in the revision. Moreover, we understand the PLOS ONE policy. There is no conflict of interest in this research, and this fact is stated in the cover letter. Response to Reviewer #1: (MS # PONE-D-19-24002) Thank you very much for your careful reading of our manuscript and helpful comments. In response to your comments, we have revised the manuscript carefully. Major changes are highlighted in the text. We believe this revised manuscript has been greatly improved by your constructive comments, and would be grateful if it could be re-considered for publication in PLOS ONE. Comments: In this work, the authors investigated the effect of hydrogen gas inhalation to prevent vascular diseases. The authors have demonstrated that constitutive inhalation of hydrogen gas has a beneficial effect on vascular remodeling, partly due to its inhibitory effects on ROS generation, DNA damage and cell proliferation. I think this is a meaningful work drawing some interesting conclusions. But, the design of animal group and free radical signal detected is inappropriate. Response: We appreciate your valuable comments. According to the comments, we have added more details on the design of animal groups and free radical signal detection in the “Materials and Methods” of the revised manuscript, as described in the response to the Editor’s comments. Response to Reviewer #2: (MS # PONE-D-19-24002) Thank you very much for your careful reading of our manuscript and helpful comments. In response to your comments, we have revised the manuscript carefully. Major changes are highlighted in the text. We believe this revised manuscript has been greatly improved by your constructive comments, and would be grateful if it could be re-considered for publication in PLOS ONE. Specific comments Introduction Line 81, 88 - How the constitutive inhalation of H2 in real life can contribute to a healthy lifestyle (from a viewpoint of medical practice). Response: We appreciate your constructive comment. We have revised the manuscript as follows: Introduction (Line 62) “It was suggested that molecular hydrogen prevents vascular remodeling in animal models such as ischemia and reperfusion (I/R) injury, vein grafting, carotid balloon injury and cerebral vasospasm in subarachnoid hemorrhage via reduction of oxidative stress (12-15).” Introduction (Line 74) “In this study, we have focused on the effects of hydrogen gas inhalation on CVD as a lifestyle intervention. CVD is induced by lifestyle-related disease with chronic/persistent oxidative stress; that is, the constitutive inhalation of molecular hydrogen in real life contributes to reducing chronic/persistent oxidative stress and has the potential to prevent CVD.” Materials and Methods Line 98 - Compressed air containing 1.25% hydrogen gas cannot consist of O2 21% and N2 79% Response: We appreciate your comment. We have revised the manuscript as follows: Materials and Methods (Line 85) “Compressed hydrogen gas (O2 21%, N2 77.7%, hydrogen 1.3%) or compressed air (O2 21%, N2 79%) flowed continuously at 0.4 L/min.” Results Please explain differences: Line 187 and 188 - We examined the effect of hydrogen gas on neointima formation, 14 days after polyethylene cuff placement around the femoral artery….but in Material and Method you write: Line 137 - Femoral arteries that had undergone cuff placement for 7 days… or line 147 group at 7 days after cuff...or line 154- Femoral arteries that had undergone performed cuff placement for 7 days …or line 222 media of the femoral artery 7 days after cuff placement… or line 230 quantitative RT-PCR in femoral artery 7 days after… or line 231 prepared from cuffed arteries 7 days after operation… and all other figs. have 7 days Response: We appreciate your comments. We previously reported that neointima is formed via increasing oxidative stress and NADPH oxidase. Neointima formation in the femoral artery 14 days after cuff placement was reflected by increased oxidative stress and mRNA levels of NADPH oxidase subunits at 7 days after cuff placement. Therefore, we conducted experiments of quantitative RT-PCR and detection of ROS at 7 days after cuff placement. Therefore, we assessed ROS production and mRNA expression by RT-PCR at 7 days after cuff placement. The following references have been helpful. 17. Chisaka T, Mogi M, Nakaoka H, Kan-No H, Tsukuda K, Wang XL, Bai HY, Shan BS, Kukida M, Iwanami J, Higaki T, Ishii E, Horiuchi M. Low-protein diet-induced fetal growth restriction leads to exaggerated proliferative response to vascular injury in postnatal life. Am J Hypertens. 2016;29(1):54-62. 18. Ohnishi A, Asayama R, Mogi M, Nakaoka H, Kan-No H, Tsukuda K, Chisaka T, Wang XL, Bai HY, Shan BS, Kukida M, Iwanami J, Horiuchi M. Drinking citrus fruit juice inhibits vascular remodeling in cuff-induced vascular injury mouse model. PLoS One. 2015;10(2):e0117616. Discussion Explain: healthy daily living in rats Line 278: remodeling aimed at healthy daily living was examined. Response: We appreciate your comment. We have revised this as follows: Discussion (Line 272) On the other hand, this study on the effects of hydrogen gas inhalation on CVD focused on lifestyle intervention. In the present study, considering the daily life of humans, the preventive effect of constitutive administration of hydrogen gas on vascular remodeling was examined in a mouse cuff injury model. Line 279 What do you mean by: In this vascular injury model, NADPH oxidase activity and production of superoxide are known to be closely related, Response: We appreciate your comment. We have revised this as follows: Discussion (Line 277) “In this vascular injury model, it is known that superoxide resulting from increased NADPH oxidase activity promotes VSMC proliferation and neointima formation (21).” Line 302 - Molecular hydrogen alleviates oxidative stress by acting as a radical scavenger for OH- and ONOO- in vitro -please discuss why only in vitro. Is it functioning only in vitro or in vivo as well? Are there other possibilities of antioxidative action in vivo? Response: We appreciate your comment. There are some reports that molecular hydrogen acts in vivo as well as in vitro. We have described two reports with respect to in vivo study. Discussion (Line 280) “Zhang et al. indicated that intraperitoneal injection of hydrogen-rich medium produced a decrease in expression of NADPH oxidase in an isoproterenol (ISO)-induced cardiac hypertrophy model rat (24). Qin et al. reported that injection of hydrogen-rich saline reduced superoxide and prevented VSMC proliferation and migration in a rat carotid balloon injury model (13). These reports suggest that molecular hydrogen can prevent CVD by downregulating NADPH activity and superoxide production. However, molecular hydrogen does not act as a radical scavenger for other ROS such as superoxide, hydrogen peroxide etc. in vitro (6). This is an unclear point regarding the mechanism of action of molecular hydrogen on biological activity. In the present results, inhalation of hydrogen gas downregulated the expression of NADPH oxidase, NOX1, but did not affect the expression levels of NADPH oxidase subunits such as p40phox and p47phox in the femoral artery 7 days after cuff placement. " Line 351 - Indeed, it is difficult to constitutively inhale hydrogen gas in daily living, because patients are not constantly at home. Please discuss if possible: Is it only reason or is it completely useless in everyday life? Is it better to drink HRW several times a day better? Response: We appreciate your comments. Recently, a hydrogen-producing machine has been used safely, and constitutive hydrogen inhalation has been suggested to provide benefit involuntarily in daily living. On the other hand, drinking HRW has a temporary effect and the patient needs to intend to drink HRW. Therefore, we consider that inhalation is the most effective and natural method for administration of hydrogen. We have revised the manuscript as follows: Discussion (Line 360) “Recently, a hydrogen-producing machine has been used safely, and constitutive hydrogen inhalation has been suggested to provide benefit involuntarily in daily living. On the other hand, drinking hydrogen-rich water has a temporary effect and the patient needs to intend to drink hydrogen-rich water. Therefore, we consider that inhalation is a more effective and natural method for administration of hydrogen. We can expect that hydrogen gas inhalation in the living environment could be useful for attenuating vascular diseases such as atherosclerosis.” Attachments Attachment Submitted filename: Response to Reviewers (Final).docx https://doi.org/10.1371/journal.pone.0227582.r002
23 Dec 2019 Decision Letter - Michael Bader, Editor Constitutive hydrogen inhalation prevents vascular remodeling via reduction of oxidative stress PONE-D-19-24002R1 Dear Dr. Mogi, We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements. Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication. Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. With kind regards, Michael Bader Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0227582.r003
Formally Accepted
7 Jan 2020 Acceptance Letter - Michael Bader, Editor PONE-D-19-24002R1 Constitutive hydrogen inhalation prevents vascular remodeling via reduction of oxidative stress Dear Dr. Mogi: I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. For any other questions or concerns, please email plosone@plos.org. Thank you for submitting your work to PLOS ONE. With kind regards, PLOS ONE Editorial Office Staff on behalf of Prof. Michael Bader Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0227582.r004

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .