PONE-D-22-01733Endothelial cell-specific Loss of eNOS Promotes ProliferationPLOS ONE

Dear Dr. Singh,

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.

The work is of interest since it proposes an original view of the potential role of eNOS on endothelial cells. However in the current form it seems yet preliminary and required many precisions and some experimental confirmations to be convincing. In particular it needs to take into account as underlined by one reviewer, in what context your hypothesis can take place and use the right tools for this aim (cell source, chemicals). Furthermore a point by point response to the concerns underlined by both reviewers is expected.

Please submit your revised manuscript by Apr 08 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.

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We look forward to receiving your revised manuscript.

Kind regards,

Alain-Pierre Gadeau, Ph.D

Academic Editor

PLOS ONE

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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: Yes

Reviewer #2: Yes

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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: No

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: Yes

Reviewer #2: No

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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: Title refer only to proliferation, but Fig 1K also migration.

The main question of the paper is quite unclear : Do the authors through proliferation and migration sought to modelize macrovascular endothelial cell healing, or do they sought to modelize angiogenesis ?

In addition, they refer to eNOS expression in SMC (ref 19) where the mechanisms are totaly different and which pathophysiological meaning is unclear.

FInally, they report rather preliminary results in nature, and acknowledge that at the end of the introduction « Our findings confirm that eNOS regulate endothelial function by directly controlling endothelial cell proliferation, and nonspecificity of L-NAME is responsible for inhibition of endothelial cell proliferation; both warranting further investigation.. ». Indeed, these results are very descriptive and the amount of limited to 2 Figures.

Thus, the apparent « discrepancy » between gene expression inhibition and eNOS actinity inhibition should be investigated more in deepth.

Major points :

1) INTRO :

a) « PKG increases extracellular concentration of calcium by facilitating the reuptake of cytosolic calcium into the sarcoplasmic reticulum and opening of calcium-activated potassium channels [5].

Ref 5 : Nitric oxide decreases [Ca2+]i in vascular smooth muscle by inhibition of the calcium current. Cell Calcium 1994. Is not appropriate as i) it demonstrate in SMC the opposite and ii) the roles of Calcium in SMC and in endothelial cells are very different and cannot be extrapolated.

2) METHODS :

a) Proliferation and Scratch Assay: the experimental conditions of cell culture should be better defined.

b) L-NAME is in vivo desestherified to L-NA, that is in fact the active coumpound. Thus,

Experiments should be done with L-NA and L-NMMA in order to minimize the pharmacological biais and to see if the results if these 3 complementary apporaches do concur. This would allow to attenuate the limitation indicated in the introduction : « non-specificity of eNOS pharmacologic inhibitor L-NAME « .

c) How many different sieNOS were used ? At least 3 should minimize the possible off-target effects.

Minor points :

Abstract : « On the contrary,.. » should be better explained : « at the level of the enzyme activity ».

Reviewer #2: The authors in their manuscript "Endothelial cell-specific Loss of eNOS Promotes Proliferation" attempted to describe function of eNOS as a negative regulator of cell proliferation. The study has a few major issues:

1. Authors should use correct term, authors claim that they are 'silencing' eNOS gene. Here, the word 'silencing' is used incorrectly, it should be replaced with 'siRNA mediated knockdown' or simply 'knockdown'. Genetic silencing, by definition, means traditional gene knockout using homologous recombination technology using the embryonic stem cells.

2. Can authors provide evidence that eNOS is a negative regulator of cell proliferation? For example, by over-expressing eNOS-cDNA in endothelial cells, which should increase the levels of cell cycle inhibitor such as p21, p27, p53 and down-regulation of Cyclin-D1. These can be done using cultured endothelial cells, at least two different primary endothelial cells must be used. The HUVECs at passage 4-6 are likely not quiescent, therefore, it would be more appropriate to use microvascular endothelial cells such as from lungs and heart. My fear is that the HUVECs may be displaying inappropriate adaptive response, in response to eNOS-knockdown.

3. Can authors normalize the activity of AKT (pAKT) in endothelial cells that received eNOS siRNA? Thereby rescue/restore the normal EC phenotype? Alternatively, can authors add back eNOS-cDNA to restore normal phenotype?

4. Figure 1L: Quantification of Western Blot is required.

5. The migration assay (wound healing) done over a period of 20 hours could include cell proliferation as well. Did author add BrdU during cell migration assay? This data is unclear.

6. In the Figure legend, can authors mention as to how many times experiments were repeated? Please explain N value clearly.

7. 'Tube formation' assay is misleading. Can authors indicate where the tubes are?

8. Methods are described inadequately.

Minor concerns:

1. 'matrigel' should be spelled Matrigel, where 'M' should be capitalized.

2. The manuscript could benefit from check on English grammar and clarity.

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Reviewer #1: Yes: JF ARNAL

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.]

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PONE-D-22-01733R1Endothelial Cell-Specific Loss of eNOS Differentially Affects Endothelial FunctionPLOS ONE

Dear Dr. Singh,

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. Please submit your revised manuscript by Sep 29 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,

Jeffrey S Isenberg, MD, MPH

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.

Additional Editor Comments (if provided):

The Reviewers felt that the revised manuscript was much improved, and the authors are thanked for this. Some concern still was found regarding terminology. See below and please address this. As well, a Reviewer requested an effort be made to improve the overall quality of the figures.

[However, the use of terminology remains confusing, e.g., tube formation. If authors claim that these are indeed tubes (Figure 4B, indicated by arrows), the Matrigel must be fixed with paraformaldehyde (4% PFA) and embedded in paraffin, make cross thin section, thereafter, stain with H&E and show lumen (vacuole/empty space). In other words, "lumen formation" is alternatively called "tube formation".]

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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

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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

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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: Yes

**********

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: Seems fine no additional comment Seems fine no additional comment Seems fine no additional comment Seems fine no additional comment

Reviewer #2: The authors have addressed most of my questions and concerns.

However, the use of terminology remains confusing, e.g., tube formation. If authors claim that these are indeed tubes (Figure 4B, indicated by arrows), the Matrigel must be fixed with paraformaldehyde (4% PFA) and embedded in paraffin, make cross thin section, thereafter stain with H&E and show lumen (vacuole/empty space). In other words, "lumen formation" is alternatively called "tube formation".

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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: Yes: Jean Francois ARNAL

Reviewer #2: No

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[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.]

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.

Endothelial Cell-Specific Loss of eNOS Differentially Affects Endothelial Function

PONE-D-22-01733R2

Dear Dr. Singh,

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.

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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,

Jeffrey S Isenberg, MD, MPH

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

The authors are thanked for the minor adjustment in terminology. The manuscript is acceptable for publication.

Reviewers' comments: