Peer Review History
Original SubmissionSeptember 18, 2022 |
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PONE-D-22-25235Co-culture models of endothelial cells, macrophages, and vascular smooth muscle cells for the study if the natural history of atherosclerosisPLOS ONE Dear Dr. Chatzizisis, 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 proposed article describing cellular models of the different stages of atherosclerosis is of interest to the journal. However, the relevance of these models to describe the stages of atherosclerosis needs to be further developed and better argued. In addition, the reviewers have highlighted many points that need to be clarified and completed. Thank you for answering point by point to their questions and remarks. Please submit your revised manuscript by Nov 21 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:
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Kind regards, Alain-Pierre Gadeau, Ph.D Academic Editor PLOS ONE Journal requirements: When submitting your revision, we need you to address these additional 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 2. 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. 3. Thank you for stating the following in the Competing Interests section: “Yiannis S. Chatzizisis: Speaker honoraria, advisory board fees and research grant from Boston Scientific Inc., advisory board fees and research grant from Medtronic Inc., Co-founder of ComKardia Inc. All other authors have no relevant conflict of interests to disclose.” 4. 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. [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: Partly Reviewer #2: Partly ********** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: N/A ********** 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: In this manuscript by Liu et al, the authors have developed four different co-culture models in order to in vitro replicate different stages of early atherosclerosis: model 1 for normal arterial intima, model 2 for atherogenesis, model 3 for intima xanthomas (mimicking "fatty streaks"), and model 4 for pathological intima thickening. Each model was characterized by electron (SEM and TEM) and confocal microscopies together with measurements of oxidized LDL and expression of chemokines, cytokines and matrix proteinases. Model 4 was also subjected to pre-exposition to low shear stress in order to mimic what is encountered in human atherosclerosis. Even if the proposed co-culture models suffer from several limitations that are clearly mentioned by the authors at the end of the manuscript (pages 26-27), these models show improvements compared to those already published in the literature and are expected to help researchers working in the field. I recommend acceptation of this work after addressing the following comments. 1) Figure numbering must be corrected. Figure 1 should contain the graphical illustration of the co-culture models and study design but is missing. The current Figure 1 corresponds to the culture model 1 (indicated as Figure 2 in the manuscript). As a result, the numberings of the figures are shifted. 2) Figure relative to model 2: higher magnification/better resolution is recommended for confocal imaging to better show colocalization of LDL particles and endothelial cells. 3) Figure relative to model 2: what was the concentration of native LDL used? 0.1 and 1.0 mg/dL or 0.1 and 1.0 mg/mL (like for model 3)? This should be confirmed. 4) It would have been interesting to evaluate the permeability of the EC monolayer before and after adding LDL or monocytes? 5) If comparable concentrations of native LDL were added in the medium for model 2 and model 3 (see previous comment), does it means that adding monocytes in the model has no influence on the oxidization of native LDL? This should be discussed by the authors. 6) What is the percentage of monocytes that transmigrate through the EC monolayer for models 3 and 4? Is it comparable? 7) Figure relative to model 4: for the western blot profile (panel K), HCASMCs exhibit similar amount of SMA compared to HCASMCs in monoculture. Authors should check other marker proteins in order to better characterize the phenotype of the SMC (contractile versus synthetic). Is there production of ECM by SMC in this model? 8) Figure relative to model 4: to be homogeneous with figures relative to models 2 and 3, "LDL (µg/mL)" in the legend of panel L should be replaced by "native LDL added in the medium (µg/mL)". 9) Why authors did not used comparable concentrations of native LDL in the medium for model 4 (as for model 2 and 3)? This should be explained by the authors. 10) M&M section: - page 9: authors indicated that HCAECs and HCASMCs were used between passages 3 and 9. Have the authors checked the phenotype of the cells between passage 3 and 9? Unchanged? - page 9: preparation of type I collagen should be better explained. After extraction of collagen, any precipitation and/or dialysis of collagen? What about the presence of HCl? - page 12: authors should explain what does the LS column means? 11) page 19: Writing "the LSS effect on the concentration of oxidized LDL in both medium and matrix, as well as the expression of chemokines, cytokines, MMPs and cathepsins was directly proportional to the dose of added native LDL (Fig. 8)" is not correct. For instance, no variation was observed between 5 and 25 µg/mL LDL and between static and flow conditions for Il-1beta, Il-8, CathS, L, MMP1 and MMP9. The authors should modify the text accordingly. 12) Authors should comment on the differences observed for the concentrations measured in the medium+matrix (Fig. 6/7?) versus medium alone (Fig. 7/8?) for the different cytokines, chemokines, MMPs, … at 100µg/mL LDL and under low shear stress. For instance, 150-200 pg/mL MMP-1 in the medium (fig. 7/8?) versus 100.000-150.000 pg/mL in medium+matrix ; around 6.000 pg/mL IL-1b in the medium around 3.000 pg/mL for medium+ matrix. Reviewer #2: The aim of this work is to set up a cell co-culture system to represent the different stages of atherosclerosis. Although the idea of establishing in vitro cell culture system to mimic atherosclerosis is of interest, there are several limitations in this study. -in my opinion presenting the different models as representative stages of atherosclerosis development is irrelevant. For example, mixing endothelial cells with collagen is not representative of healthy vessel. They should focus only on their model 4 as in vitro co-culture system to “mimic atherosclerosis”. -the authors provided images but they should quantify them (e.g. monocyte transmigration, foam cells…) to have an idea of the frequency of the processes -quantifying oxLDL is useless, they should instead visualize oxLDL internalization into the macrophages -page 22, fig 5 results are missing -fig 3j , the quantification of oxldl is the same as in fig 2h. In this regard, why adding ldl at 0.1 mg/ml did not give the same result as in fig 2? Anyway, as said before this quantification is useless. ********** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No ********** [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. |
Revision 1 |
PONE-D-22-25235R1Co-culture models of endothelial cells, macrophages, and vascular smooth muscle cells for the study if the natural history of atherosclerosisPLOS ONE Dear Dr. Chatzizisis, 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 revised document is improved. However, there are still several points that need to be strengthened, in particular some that reviewer 2 had raised: 1) It is stated that the models represent “morphologically and functionally the different histopathologic stages of early atherosclerosis”. It would be more accurate to say that they represent “different cellular mechanisms of early atherosclerosis” as they do not take into account many other cell types and cellular and matrix organisation involved in vessel wall during the atherosclerosis process. 2) I understand the challenge of doing an absolute count of macrophages. However, could you provide some relative information on the aproximative frequency of observation of macrophages having crossed the endothelial layer in order to indicate to a future user of the models if this is a rare or easy to observe event? For example giving average macrophage number per confocal field you observed / size of observed confocal field. 3) You show that LDL oxidation is strongly increased in model 4 in the presence of SMC in no flow in comparison to model 3. If so, is there a role for SMC in LDL oxidation. Please submit your revised manuscript by Feb 02 2023 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:
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, Alain-Pierre Gadeau, Ph.D 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 #1: All comments have been addressed Reviewer #2: (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 #1: (No Response) Reviewer #2: Partly ********** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: (No Response) Reviewer #2: 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 #1: (No Response) Reviewer #2: (No Response) ********** 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: (No Response) 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: (No Response) Reviewer #2: The authors addressed some of my concerns but others remain as following: -comment 1: even that the authors claim that ECs were mixed with collagen in a “sophisticated way “, this does not mean that it is representative of an atherosclerosis stage. In my opinion, this part should be removed from the manuscript as it does not represent an atherosclerotic stage, neither a “healthy vessel”. - Comment 2: the authors provided images of monocyte transmigration.I have asked to provide a quantification of this phenomenon, however the authors estimated that this is too challenging. I do not understand the challenging part of this quantification. In case that the infiltration is rare, the authors should acknowledge it. ********** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No ********** [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. |
Revision 2 |
Co-culture models of endothelial cells, macrophages, and vascular smooth muscle cells for the study if the natural history of atherosclerosis PONE-D-22-25235R2 Dear Dr. Chatzizisis, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Alain-Pierre Gadeau, Ph.D Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: |
Formally Accepted |
PONE-D-22-25235R2 Co-culture models of endothelial cells, macrophages, and vascular smooth muscle cells for the study of the natural history of atherosclerosis Dear Dr. Chatzizisis: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. 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 plosone@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 Dr Alain-Pierre Gadeau Academic Editor PLOS ONE |
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