PONE-D-21-14321

Glycolaldehyde induces vascular inflammation in TNF-α-stimulated vascular smooth muscle cells

PLOS ONE

Dear Dr. Sang Keun Ha

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 all points raised by the reviewers during the review process.

Especially, purity of nuclear and cytosol fractionation should be shown by including the specific nuclear and cytosolic markers in both fractions. Efffects of GA only should be included. Manuscripts have several mistakes and erros, which should be corrected.

From Editor: This study only uses mouse VSMC line, MOVAS-1, which may not reflect intact phenotype.Some key data should be repeated using primary VSMC. In additon, all the averaged graphs should be expressed by dot plots reflecting the individual raw value.

Please submit your revised manuscript by Sep 09 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're 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.

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If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

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We look forward to receiving your revised manuscript.

Kind regards,

Masuko Ushio-Fukai, PhD

Academic Editor

PLOS ONE

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"This research was supported by the Main Research Program (E 0210200) of the Korea Food Research Institute (KFRI) funded by the Ministry of Science and National Research Foundation of Korea (NRF) grant funded by the Korea government(MSIT) (NRF-2020R1A2C2012608)"

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Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: 

"This research was supported by the Main Research Program (E 0210200) of the Korea Food Research Institute (KFRI) funded by the Ministry of Science and National Research Foundation of Korea (NRF) grant funded by the Korea government(MSIT) (NRF-2020R1A2C2012608).

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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: No

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: No

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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

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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

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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: Lee et al found that GA further increased TNFα-induced inflammatory gene expression in mouse VSMC and concluded that GA may contribute atherosclerosis development. This study is not well presented and overstated statements with limited data.

There are several intermediates for AGE formation such as dicarbonyl compounds, such as methylglyoxal, glyoxal, 3-deoxyglucosone as well as GA. What is the rationale to use GA, not other intermediate, in this study?. How GA links with atherosclerosis development?

In introduction, authors described that previous studies reported that deposition of AGE is involved in atherosclerosis via increasing inflammation (Ref 15). Then it is highly expected that AGE precursor, GA involves in inflammation during atherosclerosis. Authors should explain what is new in this study in introduction section.

Reference is missing in last paragraph of introduction section (Line 77-86).

Give detail about source of GA and aminoguanidine, antibodies (especially AGE, RAGE etc), homogenization buffer etc in method section.

As TNF dose mentioned in figures, how authors made 10 nM concentration of TNF.

In Fig 3A, GA increased p-P65 translocation into nucleus but p-P65 in cytosolic fraction also increased. How authors interpret this data?

What is the procedure for nuclear and cytosolic fractionation?. Authors need to show successful fractionation using markers.

In Fig 4B, recombinant TNF treatment decreased protein expression of TNF by SP600125. What is the mechanism?

Provide replication of each experiment in figure legend.

There are several mistakes and missing reference throughout the manuscript.

Reviewer #2: According to previous literature the role of Advanced glycation end products (AGEs) which binds to the Receptor for advanced glycation end products (RAGE) to stimulate the signaling pathways involving in various cellular responses has been established. Indeed in investigating the mechanisms and effects of action of the AGE precursor glycolaldehyde (GA) in inflammation is interesting.

In abstract author claims that “This study investigated the effect of GA on the expression of adhesion molecules in the mouse VSMC line, MOVAS-1”. But throughout the figures author did not show any data related to the treatment of GA only. Instead, all data are reflected with the presence of TNF alpha stimulation. TNF alpha is a well known pro-inflammatory reagent to induce CAM proteins and MAPK signaling. Therefore, the effect of GA on the inflammation is still in question which is the aim of this study.

In addition, the effect of AG inhibitor is not convincing, mainly the Fig 3C, P65 nuclear translocations. Author should provide the good IF images with high resolution to confirm the role of GA on inflammation.

In Fig 1A, TNF-α treatment have no effect on VCAM1 expression which is not acceptable.

In Fig 3A author should confirm the purity of nuclear and cytosolic fractionation by their respective marker proteins. Actin is not accepted as the loading control for this blot.

In Fig 3B, why p-IkBα protein expression is high in basal level while total IkBα protein have no expression in basal.

In Fig 4B, there is a discrepancy in labelling, western blot labelled as TNFα while the respective quantification in bar graph represents IL-1b.

Overall, this manuscript does not fit for publication in PLoS One as it currently stands.

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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

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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 PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PONE-D-21-14321R1Glycolaldehyde induces synergistic effects on vascular inflammation in TNF-α-stimulated vascular smooth muscle cellsPLOS ONE

Dear Dr. Sang Keun Ha

Thank you for submitting your revised 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 by 2 reviewers during the review process. Especially, representative blots and cell fractionation assays should be revised.

Please submit your revised manuscript by Feb 18 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're 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.

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). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Masuko Ushio-Fukai, PhD

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

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2. 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: (No Response)

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: (No Response)

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4. 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: (No Response)

Reviewer #2: (No Response)

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5. 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: Yes

Reviewer #2: (No Response)

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6. 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: Authors revised manuscript based on reviewer’s comment, but several responses are not satisfactory.

For subcellular fraction (cytosolic and nuclear) assay, the procedure is not well described. What is meaning of buffer A, buffer B. Which kit used for this experiment. In figure 3A, what is the expression level of tubulin in NE and what is the level of Lamin A in CE. This will give the purity of fractions.

What software used for statistical analysis. Which test used for ANOVA.

In response to replication of experiments, Authors mentioned “quintuplicates” in figure legend. This will confuse the readers. Authors should follow the journal format (such as N=5).

Several representative blots are not good (Fig 2B; Fig 4A; Fig 6B), Replace with representative blots.

Reviewer #2: Thank you for your revised manuscript. The current manuscript has improved a lot. Still, I have some minor concerns on the following issues.

In the section of method and materials “Cytosol and nuclear extract preparation” protocol needs detailed information about the kit (like Company name and catalog number) or what is the composition of “buffer A” and “buffer B”.

Fig 2B immunoblot images are not acceptable for publication. Image’s qualities need to be improved.

In Fig 4B immunoblot, 3rd lane and 5th lane labelling are same, but the TNFα and VCAM1 expression in 3rd and 5th lane are completely different. Author should re-check the labelling and the quantification graph as well.

Overall, this manuscript does not fit for publication in PLoS One as it currently stands.

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7. 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

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 PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Glycolaldehyde induces synergistic effects on vascular inflammation in TNF-α-stimulated vascular smooth muscle cells

PONE-D-21-14321R2

Dear Dr. Sang Keun Ha

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. 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 help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- 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.

Kind regards,

Masuko Ushio-Fukai, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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2. 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: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. 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

**********

5. 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: Yes

Reviewer #2: (No Response)

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6. 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: All comments raised by reviewer has been addressed. No other comments

Reviewer #2: (No Response)

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7. 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

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