PONE-D-23-22453Reactive Oxygen Species in Endothelial Signaling with COVID-19: Role of novel peptide PIP-2 in ProtectionPLOS ONE

Dear Dr. Chatterjee,

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Eliseo A Eugenin, Ph.D.

Academic Editor

PLOS ONE

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4. Thank you for stating the following financial disclosure: 

"YES

SC was supported by NIH R56 HL139559 and R41HL164161. ABF is a shareholder in Peroxitech LLC, a company that is developing PIP-2 as a therapeutic agent. We thank Nuala J. Meyer (PI), Caroline A.G. Ittner, Ariel R. Weisman, Roseline S. Agyekum, Oluwatosin Oniyide (coordinators) and the Penn COVID processing unit for access to COVID-19 samples. These were part of their Molecular Epidemiology of SepsiS in the ICU (MESSI-COVID) study where COVID-19 subjects were enrolled, screened, and samples collected."

  

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5. Thank you for stating the following in the Acknowledgments Section of your manuscript: 

"SC was supported by NIH R56 HL139559 and R41HL164161. ABF is a shareholder in Peroxitech LLC, a

company that is developing PIP-2 as a therapeutic agent. We thank Nuala J. Meyer (PI), Caroline A.G. Ittner,

Ariel R. Weisman, Roseline S. Agyekum, Oluwatosin Oniyide (coordinators) and the Penn COVID processing

unit for access to COVID-19 samples. These were part of their Molecular Epidemiology of SepsiS in the ICU

(MESSI-COVID) study where COVID-19 subjects were enrolled, screened, and samples collected."

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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: 

"YES

SC was supported by NIH R56 HL139559 and R41HL164161. ABF is a shareholder in Peroxitech LLC, a company that is developing PIP-2 as a therapeutic agent. We thank Nuala J. Meyer (PI), Caroline A.G. Ittner, Ariel R. Weisman, Roseline S. Agyekum, Oluwatosin Oniyide (coordinators) and the Penn COVID processing unit for access to COVID-19 samples. These were part of their Molecular Epidemiology of SepsiS in the ICU (MESSI-COVID) study where COVID-19 subjects were enrolled, screened, and samples collected."

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O.P., S.C. and A.B.F. have filed a U.S. Provisional Patent Application No. 63/346,663 for PIP-2 as a protective agent against endothelial damage with COVID-19."

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Additional Editor Comments:

Dear Dr. Chatterjee

thank you for submitting your manuscript to PLOSone, you manuscript was reviewer by two expert in the field and both have some suggestions and considerations. However, one reviewer and myself indicated additional concerns, listed below. Please, add all the information required and a point by point answer

1. How the staining for Fig. 1 and 2 was quantified? Negative controls with isotype are necessary

2. How were selected and catalog the blood vessels analyzed? veins, capillaries, or arteries.

3. Fig, 3, look like having a lot of autofluorescence, in the red and blue channel, as well as the green. That distribution of staining is typical of elastin. Negative controls are required.

4. NLRP3 is a protein and complex in the cytoplasm? Why most of the staining is in the nuclei. negative controls are required

5 P-selecting staining also needs controls

6. the apoptosis markers indicate that everything is going to apoptose? is this correct. How many cells are in the plate??

7 ROS Staining is wrong. ROS are mostly produced in the mitochondria or cytoplasm. Most staining is nuclear. Negative and positive controls are required

8. Fig. 7 and 8, needs controls, the pictures are overexposed. How was quantified

9. The figure legends need a better description.

Please also, check the text for a better description of the data and its interpretation

Thank you

Eliseo Eugenin

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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: Yes

Reviewer #2: Yes

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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: 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: Yes

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: In the introduction in the phrase "there is emerging evidence that endothelial damage occurs with COVID-19 and it impacts the outcome" is poorly worded and can be merged with the next sentence to make less"wordy".

In the results section when discussing Endothelial ICAM-1, NLRP3, P-Selectin in an in vitro model of COVID-19, you mention that P-Selectin tends to increase in COVID-19, but should probably mention that it is statistically not significant

In figure 9 Healthy serum treated cells with PIP-2 have a increase in caspase-1 that is significantly higher than the healthy, somewhat contradictory since you show that PIP-2 can decrease Caspace-1 in the COVID-19 serum treated cells.

Finally there is a type O, in the results section in the paragraph "NOX2-ROS un the regulation of the NLRP3 inflammasome, in which you refer to Covid-19 as Covid-10, and as much as I love virology i am not ready for Covid-10.....

Great work, and interesting paper.

Reviewer #2: The submitted paper by Oindrila Paul et al. gives new and interesting information about the role of endothelial cells in COVID-19 pathogenesis. Authors suggest that the cytokine storm induces the activation of NOX2 in endothelial cells via aiPLA2, inducing the expression of pro-inflammatory markers and cell death. The hypothesis was evaluated using an in vitro model (HPMVEC) exposed to serum from severe COVID-19 patients. The authors found that COVID-19 serum induced an increase in ROS, inflammatory markers, and cell death in HPMVEC cell cultures. PIP-2, a peptide that blocks NOX2 activation, prevented the COVID-19 serum effect on HPMVEC cells.

Although, in general, most results provide good data, several points need to be corrected and improved in a revised version of the manuscript.

Format

General

1. The manuscript was duplicated in the file.

2. Figures. I suggest that authors add the figures in their final format so that they can examine the context and sizes of fonts and graphics. These figures should be self-explanatory and with appropriate font sizes.

Results

1. For the three first subtitles, I suggest adding an opening line explaining the aim or why the particular analysis is being done and a concluding line for each subheading.

2. Subtitle: ICAM-1 expression along the vascular wall, Figure 2A. Check the order in the lower panel.

3. Subtitle: Endothelial ROS production in an in vitro model of COVID-19. I suggest the authors put together the results of ROS (induced by COVID-19 serum vs Healthy), CellROXTM green or Dihydroethidium (DHE) in a single figure and a second Figure showing the effect of PIP-2 on ROS production (COVID-19 vs COVID-19+PIP-2) with same format o make easy to understand for the reader.

4. About all the figures, I suggest they be ordered in the same way—for example, control vs treatment. Authors should review the format of all their graphics so that they are consistent in order, shape, and font size appropriate to the final size of the figure. In others, immunofluorescence photographs are not well aligned. Correct this.

Comments.

1. Is the effect of COVID-19 serum specific for human pulmonary microvascular endothelial cells? Or is it a general mechanism for endothelial cells?

2. Upstream, have the authors evaluated neutralizing antibodies against TNF- alpha?

3. In the manuscript, the authors show the preventive effect of PIP-2 on the inflammatory effect of CVID-19 serum. However, it could give more information about the therapeutic potential of PIP-2 if HPMVECs are simultaneously incubated with the COVID-19 serum or after its exposure to COVID-19 serum.

Minor comments

4. In the discussion section authors say, “We observed an increase in P-selectin expression in endothelial cells in the COVID-19 model as compared to healthy controls, but this increase was not significant”. The increase was not statistically significant. Therefore, there is no increase, but the authors could say they observed a tendency to increase.

This paper gives new and interesting in vitro data about the role of endothelial cells in COVID-19 pathogenesis. Most experiments are well executed, and conclusions are well presented and supported by the data. However, several results and figures described here must be improved.

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Reviewer #1: Yes: Karl M Valerdi

Reviewer #2: No

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PONE-D-23-22453R1Reactive Oxygen Species in Endothelial Signaling in COVID-19: Protective role of the novel peptide PIP-2 in ProtectionPLOS ONE

Dear Dr. Chatterjee,

Thank you very much for submitting your manuscript to PLoS ONE. After careful consideration, we have concluded that your manuscript has the potential to be published, although some minor aspects will need to be addressed. We therefore ask that you revise your manuscript in this direction.

Please submit your revised manuscript by May 04 2024 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,

Gianfranco Pintus, MSc, PhD.

Academic Editor

PLOS ONE

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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.

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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 #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 #2: Yes

**********

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

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 #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 #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 #2: The authors have addressed all suggestions made by this reviewer.

The quality of the text improved compared to the previous manuscript.

Minor comments.

In immunofluorescence figures, some photos are not the same size or do not align well.

No further comments

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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 #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.

Reactive Oxygen Species in Endothelial Signaling in COVID-19: Protective role of the novel peptide PIP-2 in Protection

PONE-D-23-22453R2

Dear Dr. Chatterjee,

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.

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Kind regards,

Gianfranco Pintus, MSc, PhD.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: