Timing matters in the use of renin-angiotensin system modulators and COVID-related cognitive and cerebrovascular dysfunction

Mackenzi Meier; Sara Becker; Erica Levine; Oriana DuFresne; Kaleigh Foster; Joshua Moore; Faith N. Burnett; Veronica C. Hermanns; Stan P. Heath; Mohammed Abdelsaid; Maha Coucha

doi:10.1371/journal.pone.0304135

Peer Review History

Original SubmissionFebruary 4, 2024
29 Feb 2024 Decision Letter - Michael Bader, Editor PONE-D-24-03599Timing Matters in the Use of Renin-Angiotensin System Modulators and COVID-Related Cognitive and Cerebrovascular DysfunctionPLOS ONE Dear Dr. Coucha, 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 Apr 14 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. 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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. [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: No Reviewer #2: Yes Reviewer #3: No ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: No Reviewer #2: Yes Reviewer #3: 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: No Reviewer #2: No Reviewer #3: No ****** 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 Reviewer #3: 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: 1. The authors 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 2. The figures lack legends that describe the statistical analyzes and sample sizes of each of the experimental groups. 3. The authors must validate that the preclinical model used emulates the recruited patients. In the preclinical model it is not specified whether they used only males or both males and females. The preclinical model used by the authors. Validation of the preclinical model is missing. 4. The patients received ACEI/ARB, however the mice received only ARB, losartan. The treatment with ACEi remains to be incorporated in the preclinical model. Reviewer #2: The paper is well written and addresses an important question of the timing of RAS modulators in COVID-19 related cognitive dysfunction. They show statistically better outcomes in patients receiving the medication post infection. I think the paper is acceptable in its current form. Reviewer #3: In this clinical/preclinical paper, the authors make an attempt to correlate RAS-inhibition before or post onset of Covid-19 with neurological dysfunction. While the preclinical part provides interesting results, appears to be well done and methodologically sound, I have major concerns with respect to the clinical part: In general, the observed increase in neurological dysfunction in patients on RAS inhibition my be simply due to the fact that these patients were more severely ill, had higher BP levels to start with or cardio-renal problems etc. which afforded additional treatment with RAS inhibitors. - The number of patients is relatively small. Meaningful statistics are difficult with such small numbers. - The proportion of black patients is too high to be representative for the US population, let alone the rest of the world. - Data on comorbidities ar lacking. - No information on BP control - No information on the methods to evaluate mental status - Coincidence of neurological problems: The number (91) with neurological dysfunction in RAS inhibitor- treated patients does not distinguish between i) one dysfunction in one patient or ii) several neurological problems in one patient. Minor problems: - line 318: Ang II has the same affinity to AT1R and AT2R. The difference comes rather from the number of receptors in a given tissue under given circumstances. - line 320: The AT2R-axis of the "protective RAS" needs to be referenced, e.g. Steckelings et al. Pharm. Reviews 2022. - line 323: The protective RAS is not only constituted by the Ang 1-7/Mas axis but also via the AT2R. Stimulation of the unopposed AT2R could be a mechanism of the protective effects of Losartan post SP in the animals. - line 348: Meta-analysis on Covid-19/ RAS inhibition by Gnanenthiran SR et al., J Am Heart Assoc. 2022 should be included. ****** 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: Yes: Abhinav Grover Reviewer #3: 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.] 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. https://doi.org/10.1371/journal.pone.0304135.r001
Revision 1
2 Apr 2024 Author Response Response to Journal Requirements: 1. 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 https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf The file naming was adjusted to follow PLOS ONE’s style requirements 2. To comply with PLOS ONE submissions requirements, in your Methods section, please provide additional information regarding the experiments involving animals and ensure you have included details on (1) methods of sacrifice, and (2) efforts to alleviate suffering. We have updated the method section as directed with the following: “Animals were sacrificed using carbon dioxide and cervical dislocation. Buprenorphine 0.1 mg/kg body weight was injected subcutaneously upon detecting any signs of animal distress.” 3. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section. We have updated the funding information. 4. Thank you for stating the following in the Competing Interests section: "Some data were presented as an abstract at the International Stroke Conference 2023 and the MIDYEAR 2023 Clinical Meeting & Exhibition. The authors have declared that no competing interests exist. Funding:This study was supported by American Heart Association 23AIREA1045073 to MA." 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 updated Competing Interests statement in your cover letter; we will change the online submission form on your behalf. “This does not alter our adherence to PLOS ONE policies on sharing data and materials.” was added to the manuscript and the cover letter as directed 5. In the online submission form you indicate that your data is not available for proprietary reasons and have provided a contact point for accessing this data. Please note that your current contact point is a co-author on this manuscript. According to our Data Policy, the contact point must not be an author on the manuscript and must be an institutional contact, ideally not an individual. Please revise your data statement to a non-author institutional point of contact, such as a data access or ethics committee, and send this to us via return email. Please also include contact information for the third party organization, and please include the full citation of where the data can be found. All relevant data are within the manuscript and its Supporting Information files. The online submission was updated accordingly 6. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions. The original uncropped images are submitted. 7. 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. The phrase was removed. Response to Reviewers Reviewer #1: 1. The authors 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 We thank the reviewer for their valuable comment. We assure the reviewer that we have adopted the NIH rigor policy and that we comply with transparent standards in our research. All our experiments have been rigorously designed. Our method section describes all experimental procedures in detail for transparency and reproducibility. Proper controls and animal randomization have been used to reduce bias. Our sample size was selected to ensure a power analysis at a significance level of P<0.05. Our animal studies used male and female h ACE-2 mice to avoid biological variables. Finally, all our antibodies and chemical reagent sources have been stated in our manuscript. These reagents have been authenticated by their vendor. 2. The figures lack legends that describe the statistical analyzes and sample sizes of each of the experimental groups. We apologize for this misunderstanding. According to the PLOS One manuscript author guide, all the figure legends are included in the manuscript body and they include sample size and statistical method, as directed. 3. The authors must validate that the preclinical model used emulates the recruited patients. In the preclinical model, it is not specified whether they used only males or both males and females. The preclinical model used by the authors. Validation of the preclinical model is missing. We apologize for the mistake and totally agree with the reviewer that the pre-clinical model must be validated. We and others have used hACE-2 mice in COVID-19 research. We have included the reference for studies using the animal model in our discussion. Both male and female hACE-2 mice have been used in our experimental design to avoid biological variables. We have updated our method section accordingly. (1) Munoz-Fontela C, Dowling WE, Funnell SGP, Gsell PS, Riveros-Balta AX, Albrecht RA, et al. Animal models for COVID-19. Nature. 2020;586(7830):509-15. (2) Burnett FN, Coucha M, Bolduc DR, Hermanns VC, Heath SP, Abdelghani M, et al. SARS-CoV-2 Spike Protein Intensifies Cerebrovascular Complications in Diabetic hACE2 Mice through RAAS and TLR Signaling Activation. Int J Mol Sci. 2023;24(22). 4. The patients received ACEI/ARB, however the mice received only ARB, losartan. The treatment with ACEi remains to be incorporated in the preclinical model. We thank the reviewer for their constructive feedback. While patients received ACEI or ARB therapy, we exclusively used ARB, Losartan, in the preclinical study. This choice was based on our recent research demonstrating Losartan's efficacy in restoring RAS balance and mitigating spike protein-induced cerebrovascular dysfunction (2). We acknowledge that including ACE inhibitor therapy in our preclinical model would offer a more comprehensive reflection of the clinical arm. However, incorporating ACE inhibitors would require a complete repetition of the whole preclinical study, which is unfeasible. The following statement was included in the manuscript to reflect this limitation. “Future studies should address the limitation of our preclinical model, which exclusively utilized ARB therapy, by incorporating ACEI to mimic the clinical arm where patients received either ARB or ACEI” Reviewer #2: The paper is well written and addresses an important question of the timing of RAS modulators in COVID-19 related cognitive dysfunction. They show statistically better outcomes in patients receiving the medication post infection. I think the paper is acceptable in its current form. Your feedback is greatly appreciated, and we're grateful for your time. Reviewer #3: In this clinical/preclinical paper, the authors make an attempt to correlate RAS-inhibition before or post onset of Covid-19 with neurological dysfunction. While the preclinical part provides interesting results, appears to be well done and methodologically sound, I have major concerns with respect to the clinical part: In general, the observed increase in neurological dysfunction in patients on RAS inhibition may be simply due to the fact that these patients were more severely ill, had higher BP levels to start with or cardio-renal problems etc. which afforded additional treatment with RAS inhibitors. - The number of patients is relatively small. Meaningful statistics are difficult with such small numbers. - The proportion of black patients is too high to be representative for the US population, let alone the rest of the world. - Data on comorbidities are lacking. – No information on BP control - No information on the methods to evaluate mental status - Coincidence of neurological problems: The number (91) with neurological dysfunction in RAS inhibitor- treated patients does not distinguish between i) one dysfunction in one patient or ii) several neurological problems in one patient. We would like to extend our sincere gratitude for the time and effort the reviewer dedicated to reviewing our manuscript. The reviewer’s feedback has been instrumental in strengthening our manuscript. Below is our response to the reviewer’s comments Firstly, we acknowledge that the clinical part of the study is a retrospective chart review, which inherently relies on electronic medical record documentation of subjective complaints. While we tried to identify objective measures of neurological dysfunction, such as CT scans or MRIs, not all patients underwent such evaluations due to various clinical reasons. In response to your concern regarding the potential influence of patients' overall clinical condition, including severity of illness, baseline blood pressure levels, or cardiorenal problems, we have implemented several measures to address this issue. Specifically, we have incorporated additional data on comorbidities and blood pressure control into our analysis. These findings indicate that the two groups exhibit similarities in these parameters, thereby indicating that the observed disparities in neurological dysfunction are less likely to be solely attributable to baseline clinical characteristics. We also acknowledge the high proportion of African American patients included in our study. As residents of the Southeast, where approximately half of the population is African American, it is not surprising that a significant number of African American patients were evaluated in our retrospective chart review. We appreciate your understanding of this demographic representation and assure you that it does not impact the validity of our findings. Minor problems: - line 318: Ang II has the same affinity to AT1R and AT2R. The difference comes rather from the number of receptors in a given tissue under given circumstances. We apologize for this mistake, and it was corrected in the manuscript to the following “Ang II binds with two main receptors, AT¬1R and AT2R, and triggers different biological responses.” - line 320: The AT2R-axis of the "protective RAS" needs to be referenced, e.g. Steckelings et al. Pharm. Reviews 2022. The review is being referenced in the manuscript. - line 323: The protective RAS is not only constituted by the Ang 1-7/Mas axis but also via the AT2R. Stimulation of the unopposed AT2R could be a mechanism of the protective effects of Losartan post SP in the animals. The following challenges were made to enhance the clarity of the manuscript: However, AT2R activation contributes to more vascular protective effects such as vasodilation and anti-inflammatory effects (3). Moreover, ACE-2 is an enzyme that catalyzes the degradation of Ang II to Ang-(1-7), and the resultant molecules activate the Mas receptor which results in protective actions (4-6). Therefore, Ang-(1-7)/Mas axis and the AT2R constitute the protective arm of the RAS. Additionally, unopposed AT2R stimulation could be a mechanism underlying the protective effect of Losartan demonstrated in the study. - line 348: Meta-analysis on Covid-19/ RAS inhibition by Gnanenthiran SR et al., J Am Heart Assoc. 2022 should be included. The Meta-analysis was included in the manuscript. In 2022, Gnanenthiran et al. published a meta-analysis to assess the safety and efficacy of RAS inhibiton in adults with COVID‐19. The authors found no significant difference in all-cause mortality between patients receiving RAS inhibition = therapy and those without it. However, there was a borderline decrease in acute myocardial infarction among patients on RASi. On the other hand, there was an increased risk of acute kidney injury (AKI) associated with RASi use, particularly in hospitalized patients. Despite this increased risk of AKI, there was no increase in the need for dialysis or other adverse outcomes such as congestive cardiac failure, stroke, or venous thromboembolism (7). 1. Munoz-Fontela C, Dowling WE, Funnell SGP, Gsell PS, Riveros-Balta AX, Albrecht RA, et al. Animal models for COVID-19. Nature. 2020;586(7830):509-15. 2. Burnett FN, Coucha M, Bolduc DR, Hermanns VC, Heath SP, Abdelghani M, et al. SARS-CoV-2 Spike Protein Intensifies Cerebrovascular Complications in Diabetic hACE2 Mice through RAAS and TLR Signaling Activation. Int J Mol Sci. 2023;24(22). 3. Steckelings UM, Widdop RE, Sturrock ED, Lubbe L, Hussain T, Kaschina E, et al. The Angiotensin AT(2) Receptor: From a Binding Site to a Novel Therapeutic Target. Pharmacol Rev. 2022;74(4):1051-135. 4. Raffai G, Durand MJ, Lombard JH. Acute and chronic angiotensin-(1-7) restores vasodilation and reduces oxidative stress in mesenteric arteries of salt-fed rats. Am J Physiol Heart Circ Physiol. 2011;301(4):H1341-52. 5. Santos RA, Campagnole-Santos MJ, Andrade SP. Angiotensin-(1-7): an update. Regul Pept. 2000;91(1-3):45-62. 6. Khajehpour S, Aghazadeh-Habashi A. Targeting the Protective Arm of the Renin-Angiotensin System: Focused on Angiotensin-(1-7). J Pharmacol Exp Ther. 2021;377(1):64-74. 7. Gnanenthiran SR, Borghi C, Burger D, Caramelli B, Charchar F, Chirinos JA, et al. Renin-Angiotensin System Inhibitors in Patients With COVID-19: A Meta-Analysis of Randomized Controlled Trials Led by the International Society of Hypertension. J Am Heart Assoc. 2022;11(17):e026143. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0304135.r002
26 Apr 2024 Decision Letter - Michael Bader, Editor PONE-D-24-03599R1Timing Matters in the Use of Renin-Angiotensin System Modulators and COVID-Related Cognitive and Cerebrovascular DysfunctionPLOS ONE Dear Dr. Coucha, 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 in which the clinical part is deleted. Please submit your revised manuscript by Jun 10 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, Michael Bader Academic Editor PLOS ONE [Note: HTML markup is below. Please do not edit.] 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 #3: (No Response) ******** 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 #3: Partly ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #3: N/A ****** 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 #3: 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 #3: 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 #3: Improvements regarding minor concerns acknowledged. Major concern still with respect to -small number of patients - high proportion of black patients with different responses to RAS inhibitors different from white and Asian populations - rigorous testing not in all patients ****** 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 #3: 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.] 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. https://doi.org/10.1371/journal.pone.0304135.r003
Revision 2
2 May 2024 Author Response The authors would like to thank all the reviewers (1, 2, and 3) for their invaluable feedback and constructive criticism of our manuscript titled "Timing Matters in the Use of Renin-Angiotensin System Modulators and COVID-Related Cognitive and Cerebrovascular Dysfunction." Their time and expertise are greatly appreciated. I am writing in response to Reviewer #3's feedback on our study. The authors appreciate Reviewer #3's time and effort in providing a thorough evaluation of our study. We understand Reviewer #3's concerns regarding the clinical part of our study and the potential limitations associated with it. However, we respectfully disagree with the suggestion to delete the clinical part of the study. We believe that the clinical data significantly contribute to the overall findings and provide valuable insights into the correlation between RAAS inhibition and neurological dysfunction in patients with COVID-19. The clinical data strengthens the scientific merit of our research. Here is a detailed response to Reviewer #3's concerns regarding the small sample size, the high proportion of African Americans, and the lack of rigorous testing in all patients. Small sample size: We understand the concerns raised by the reviewer regarding the sample size. However, we would like to emphasize that the goal of this study was to shed light on the potential correlation between RAAS inhibition and neurological dysfunction following COVID-19 exposure. In this study, we tried to identify areas for further investigation. While we acknowledge the importance of having a sufficient number of patients in the clinical study, the findings that we observed based on the 295 patients included in the study can serve as a foundation for future research projects with a larger sample of patients. Lack of rigorous testing in all patients: Firstly, we acknowledge that the clinical part of the study is a retrospective chart review, which inherently relies on electronic medical record documentation of subjective complaints. We acknowledge the concern raised regarding the absence of comprehensive neurological evaluations, such as CT scans or MRIs, in all patients within our retrospective chart review. It is important to note that the decision for these objective imaging tests was subject to the discretion of attending physicians based on clinical indications and resource availability. Given the retrospective nature of our study and the diversity of patient presentations, not all individuals underwent the same diagnostic procedures. While this may introduce variability in the data collection process, it does not compromise the integrity of our findings. We analyzed the available clinical data to identify trends and associations, ensuring transparency in our reporting of both objective and subjective measures of neurological dysfunction. Furthermore, our study's primary focus was on exploring potential correlations between RAAS inhibition and neurological outcomes in the context of COVID-19, rather than exclusively relying on imaging findings. We have acknowledged this limitation in our revised manuscript and provided interpretations of our results within the framework of available clinical data. Despite the variability in diagnostic evaluations, our study still provides valuable insights into the relationship between RAAS modulation and neurological dysfunction post-COVID-19 exposure, warranting further investigation. High proportion of African Americans: We acknowledge the high proportion of African American patients included in our study. The data was collected from St. Joseph’s/Candler Health System located in Savannah, Georgia. “In 2021, there were 1.41 times more Black or African American (non-Hispanic) residents (76k people) in Savannah, GA, than any other race or ethnicity. There were 54k White (non-Hispanic) and 4.01k White (Hispanic) residents, the second and third most common ethnic groups [1]”. Therefore, it is not surprising that a significant number of African American patients were evaluated in our retrospective chart review. We appreciate your understanding of this demographic representation and assure you that it does not impact the validity of our findings. To provide further clarity, we have included a table detailing the distribution of African American and White patients included in our study. As shown in the table, the majority of the patients were White. While we acknowledge the high proportion of African American patients included in our study; we believe that this demographic diversity enriches our findings and underscores the importance of considering racial disparities in healthcare outcomes. [1] https://datausa.io/profile/geo/savannah-ga#race_and_ethnicity Characteristic ACEI/ARB prior to admission (n=177) No ACEI/ARB prior to admission (n=118) Caucasian 97 (55%) 68 (58%) African American 78 (44% 44 (37%) Other 2 (1%) 6 (5%) We have revised the manuscript to clarify those issues, highlight any limitations within the study, and underscore the need for further studies to validate the findings. We believe that those changes will strengthen the quality and impact of our manuscript. We respectfully request that the journal reconsider its decision to remove the clinical part of our study. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0304135.r004
7 May 2024 Decision Letter - Michael Bader, Editor Timing Matters in the Use of Renin-Angiotensin System Modulators and COVID-Related Cognitive and Cerebrovascular Dysfunction PONE-D-24-03599R2 Dear Dr. Coucha, 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 will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, 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, Michael Bader Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0304135.r005
Formally Accepted
23 Jun 2024 Acceptance Letter - Michael Bader, Editor PONE-D-24-03599R2 PLOS ONE Dear Dr. Coucha, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, 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. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Prof. Michael Bader Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0304135.r006

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