Protective role of Pannexin1 in lymphatic endothelial cells in the progression of atherosclerosis in female mice

Avigail Ehrlich; Graziano Pelli; Bernard Foglia; Filippo Molica; Brenda R. Kwak

doi:10.1371/journal.pone.0315511

Peer Review History

Original SubmissionSeptember 12, 2024
4 Oct 2024 Decision Letter - Michael Bader, Editor PONE-D-24-40523Atheroprotective role of Panx1 deletion in lymphatic endothelial cells in female micePLOS ONE Dear Dr. Kwak, 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 Nov 18 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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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 ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The authors investigated the function of Panx1 in lymphatic endothelial cells concerning atherosclerotic lesions and revealed that this molecule specifically regulates lesions in female mice. They concluded that this phenomenon is due to changes in the ability of lymphatic endothelial cells to transport T cells. The results are clear, without significant contradictions, and the novelty is evident, but there appears to be a lack of experimental data regarding immune cells to support the above conclusions. Specifically, the possibility that lymphatic endothelial cells induce qualitative and quantitative changes in immune cells has been overlooked. Comments The authors conclude that the promotion of atherosclerosis in female Prox1-CreERT2+Panx1 fl/flApoe-/- mice affects T cell accumulation in the lesions, yet this study does not assess T cell accumulation in the lesions or the populations of T cells in the blood. Since T cells are roughly classified into proinflammatory and anti-inflammatory subsets, both quantitative and qualitative evaluations are essential. Previous reports (PMID: 36519468) have indicated that dysfunction of lymphatic endothelial cells reduces the stability of anti-inflammatory regulatory T cells systemically, increasing susceptibility to atherosclerosis, suggesting that impaired cellular transport via lymphatics may not always be the primary cause. In the author’s context, evaluating lymphatic endothelial cell functions, such as adhesion molecules, is warranted. The authors should discuss why the observed sex differences occur, in terms of Panx1. The current draft provides extensive explanations regarding gender differences in dyslipidemia; however, as these do not lead to a definitive conclusion, they should be condensed. Instead, the discussion should focus on why there are gender differences in Panx1's contribution to atherosclerosis. It would be beneficial to examine the differences in Panx1 expression between males and females. Lipoprotein cholesterol and triglycerides should be fractionated into at least VLDL, LDL, and HDL for evaluation. Merely assessing changes in total cholesterol is insufficient to rule out the involvement of dyslipidemia. The two experimental strategies (initiation and progression) are not clearly linked to the figures, even in the legends. Please revise this for clarity. Reviewer #2: The goal of this manuscript is to examine the impact of lymphatic endothelial Pannexin 1 (LEC Panx1) on the development and stability of atherosclerotic plaques. Despite being an intriguing subject, the data provided does not indicate a role for LEC Panx1 in atherosclerosis. New findings reveal variations in serum lipid levels, plaque size, and plaque vulnerability based on sex. However, these differences are not linked to the presence or absence of Panx1. Given the data presented, the significance of the results remains uncertain. ****** 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. 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Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0315511.r001
Revision 1
18 Nov 2024 Author Response Reviewer 1 We thank the Reviewer for his/her critical comments that helped us to improve our manuscript. We are happy to note that the Reviewer considers our results as clear without contradictions and that the novelty of our study was evident. We have responded to each of his/her remarks in detail below. Specific comments: 1) The authors conclude that the promotion of atherosclerosis in female Prox1-CreERT2+Panx1 fl/flApoe-/- mice affects T cell accumulation in the lesions, yet this study does not assess T cell accumulation in the lesions or the populations of T cells in the blood. Since T cells are roughly classified into proinflammatory and anti-inflammatory subsets, both quantitative and qualitative evaluations are essential. Previous reports (PMID: 36519468) have indicated that dysfunction of lymphatic endothelial cells reduces the stability of anti-inflammatory regulatory T cells systemically, increasing susceptibility to atherosclerosis, suggesting that impaired cellular transport via lymphatics may not always be the primary cause. In the author’s context, evaluating lymphatic endothelial cell functions, such as adhesion molecules, is warranted. Response: We thank the Reviewer for this insightful suggestion, which helped us to further understand the potential mechanism underlying the sex-specific increased progression of atherosclerosis in female Panx1LECdelApoe-/- mice. Following the suggestion of the Reviewer we show in the revised manuscript that 1) as compared to Panx1fl/flApoe-/- controls, the number of T lymphocytes is increased in advanced plaques of female Panx1LECdelApoe-/- mice but not in male mice of the same genotype (Figures 8E and 10E), and 2) the expression level of VCAM-1 was lower in Panx1-deficient LECs than in Panx1-expressing LECs isolated from lymph nodes of female mice (Figure 10F). Although we appreciate the suggestion to classify the populations of T lymphocytes in the blood, this requires fresh samples and thus new studies with 10 weeks high cholesterol diet on all groups of mice (40 mice), which was not possible in the 6 weeks given for the revision of our manuscript. 2) The authors should discuss why the observed sex differences occur, in terms of Panx1. The current draft provides extensive explanations regarding gender differences in dyslipidemia; however, as these do not lead to a definitive conclusion, they should be condensed. Instead, the discussion should focus on why there are gender differences in Panx1's contribution to atherosclerosis. It would be beneficial to examine the differences in Panx1 expression between males and females. Response: The new results mentioned under the above point 1 are extensively discussed in the revised manuscript (page 20, lines 1-20), resulting in a better balance between the discussion on sex-differences in early atherosclerosis that are independent of Panx1 and the sex-differences in the progression of atherosclerosis, which involves Panx1 in LECs. As requested, we mention in the revised manuscript that Panx1 expression is higher in LECs of males (page 17, line 1), an observation that was already described in detail in our earlier study (Ehrlich et al., Physiol Rep 2024; reference 18 in this manuscript). 3) Lipoprotein cholesterol and triglycerides should be fractionated into at least VLDL, LDL, and HDL for evaluation. Merely assessing changes in total cholesterol is insufficient to rule out the involvement of dyslipidemia. Response: As requested by the Reviewer, we have performed additional measurements and show now the serum LDL and HDL values (Figures 3C-D, 7C-D and 9C-D). Unfortunately, VLDL measurements are not possible on the Cobas C111 analyser available to us. 4) The two experimental strategies (initiation and progression) are not clearly linked to the figures, even in the legends. Please revise this for clarity. Response: We thank the Reviewer for this suggestion. We revised the legends and the two experimental strategies are now clearly mentioned for all Figures (pages 23-25). In addition, we have critically revised the text of the manuscript and the title of the manuscript to better link results and strategies. Reviewer 2 We thank the Reviewer for his/her comments on our manuscript. The goal of this manuscript is to examine the impact of lymphatic endothelial Pannexin 1 (LEC Panx1) on the development and stability of atherosclerotic plaques. Despite being an intriguing subject, the data provided does not indicate a role for LEC Panx1 in atherosclerosis. New findings reveal variations in serum lipid levels, plaque size, and plaque vulnerability based on sex. However, these differences are not linked to the presence or absence of Panx1. Given the data presented, the significance of the results remains uncertain. Response: We are happy to note that the Reviewer considers the subject of our study intriguing. Following the suggestions of Reviewer 1, we performed additional experiments to better understand the potential mechanism underlying the sex-specific increased progression of atherosclerosis female Panx1LECdelApoe-/- mice. We show in the revised manuscript that 1) as compared to Panx1fl/flApoe-/- controls, the number of T lymphocytes is increased in advanced plaques of female Panx1LECdelApoe-/- mice but not in male mice of the same genotype (Figures 8E and 10E), and 2) the expression level of VCAM-1 was lower in Panx1-deficient LECs than in Panx1-expressing LECs isolated from lymph nodes of female mice (Figure 10F). These new results mentioned are extensively discussed in the revised manuscript (page 20, lines 1-20), resulting in a better balance between the discussion on sex-differences in early atherosclerosis that are independent of Panx1 and the sex-differences in the progression of atherosclerosis, which involves Panx1 in LECs. Journal Requirements Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file? Response: The manuscript has been formatted according to the template. 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, (2) methods of anesthesia and/or analgesia, and (3) efforts to alleviate suffering. Response: All required details are in the methods section (page 7). When completing the data availability statement of the submission form, you indicated that you will make your data available on acceptance. We strongly recommend all authors decide on a data sharing plan before acceptance, as the process can be lengthy and hold up publication timelines. Please note that, though access restrictions are acceptable now, your entire data will need to be made freely accessible if your manuscript is accepted for publication. This policy applies to all data except where public deposition would breach compliance with the protocol approved by your research ethics board. If you are unable to adhere to our open data policy, please kindly revise your statement to explain your reasoning and we will seek the editor's input on an exemption. Please be assured that, once you have provided your new statement, the assessment of your exemption will not hold up the peer review process. Response: Once YARETA DOI has been finalized to make it publicly available, no further changes can be made. It is therefore advised to only do this for accepted manuscripts. We will do so immediately after acceptance of the manuscript and provide the DOI in the proofs of the manuscript. 4. Thank you for stating the following financial disclosure: This study was supported by grants from the Swiss National Science Foundation (grant number 310030_182573 to BRK) and the Gabbiani fund (to AE). Please state what role the funders took in the study. If the funders had no role, please state: ""The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."" Response: This statement has been included in the revised manuscript (page 22). 5. Thank you for stating the following in your Competing Interests section: NO authors have competing interests. Please complete your Competing Interests on the online submission form to state any Competing Interests. If you have no competing interests, please state ""The authors have declared that no competing interests exist."", as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now This information should be included in your cover letter; we will change the online submission form on your behalf. Response: The above requested changes have been made (page 22). Attachments Attachment Submitted filename: Response to reviewers.pdf https://doi.org/10.1371/journal.pone.0315511.r002
27 Nov 2024 Decision Letter - Eliseo A Eugenin, Editor Protective role of Pannexin1 in lymphatic endothelial cells in the progression of atherosclerosis in female mice PONE-D-24-40523R1 Dear Dr. Kwak, 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. 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The manuscript was evaluated by 2 experts in the field with excellent comments. Thank you for addressing all the comments. Best Regards Eliseo Eugenin 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 ******** 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: I Don't Know ****** 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: 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: The authors have adequately addressed the concerns raised by this reviewer. There are no additional comments. Reviewer #2: The authors have attempted to address my comments. The manuscript is written in an easy to understand manner. ****** 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: Takuro Miyazaki Reviewer #2: No ******** https://doi.org/10.1371/journal.pone.0315511.r003
Formally Accepted
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