Optimisation of cell and ex vivo culture conditions to study vascular calcification

Nathalie Gayrard; Karen Muyor; Cécile Notarnicola; Flore Duranton; Bernard Jover; Àngel Argilés

doi:10.1371/journal.pone.0230201

Peer Review History

Original SubmissionDecember 5, 2019
30 Dec 2019 Decision Letter - Harald Mischak, Editor PONE-D-19-33486 Optimisation of cell and ex vivo culture conditions to study vascular calcification PLOS ONE Dear Dr gayrard, 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. ============================== As you can see, all four reviewers were positive about the manuscript, but several minor changes were suggested, mostly requesting to provide more details in the methods, that could further strengthen the paper. ============================== We would appreciate receiving your revised manuscript by Feb 13 2020 11:59PM. 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Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. [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: Yes Reviewer #2: Yes Reviewer #3: Yes Reviewer #4: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes Reviewer #4: 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 Reviewer #3: Yes Reviewer #4: 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 Reviewer #3: Yes Reviewer #4: 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: COMMENTS FOR AUTHORS PlosONE MANUSCRIPT # PONE-D-19-33486 REVIEWER # 1 Nathalie Gayrard Optimization of cell and ex vivo culture 1 conditions to study vascular calcification The authors of this manuscript establish in vitro models mimicking in vivo vascular calcification and to select an optimal method to unravel the mechanisms involved in medial vascular calcification. The authors identified culture conditions, which allowed a reproducible and specific medial calcification of aortic explants as well as calcification of human aortic smooth muscle cells. The authors concluded these approaches will help to achieve reproducible results and allow result comparison in a consistent way. The data presented in this manuscript are interesting for terms of analyses of vascular calcification processes. However, the reviewer has some major remarks: Major points: 1. The introduction section has to be revised. The authors should focus on the main topic of the manuscript and should avoid an very general description of the methods available for the investigation of vascular calcification processes (eg.g page 3 , line 56-59). 2. The authors should prepare a table demonstrating the condition analyze within the study. 3. NG, KM, FD and AA are obviously employees of a company (RD-Nephrologie). A statement has to be added whether these authors have to declare a conflict of interest based on the employment. 4. Figure 3.B: A bar graph demonstrating the quantification of Phosphosmad1 should be added. 5. Not significant data has to be removed from the revised version of the manuscript (page 12, line 258 - 260) 6. The authors should discuss the overall aim of the study in the discussion section in more detail. 7. Some typos should be removed in the revised version of the manuscript, e.g. page 9, line 169 (3.8mM instead of 3.8 mM). Reviewer #2: This experiment allows overcoming of an ethical issue for using animal models in experiments of vascular calcification research as one of the most important outcome for experiencing major cardiovascular events in humans as a topic for research, although the authors well pointed out the extrapolation of the data should be taken carefully into account. Reviewer #3: The aim of this study is to analyze in vitro and ex vivo models of vascular calcification and to select factors that may affect the process of calcification. Th study is well performed and I cannot identify methodological defects However there are points that should be clarified -Fig1 shows that calcium deposits in HASMCs cultured with high phosphate (3.8 mM) are decreased by supplementing with 15% FCS. It is important to determine if ionized calcium and the concentration of phosphate are modified by 15% FCS. It is know that FCS contains large proteins “calciproteins” that may substract Ca and P from the solution. At least the authors should evaluate if for a given pH, FCS modifies the Ca x P product. -The total calcium content in the wells was significantly increased in the wells with cells cultured with the basic P donor only (P < 0.001) vs acidic and neutral conditions. It is likely that the high pH is increasing calcium deposit the opposed is expected to occur in acid pH. Again ionized Ca and pH should be measured minute after the addition of the different forms of phosphate. -It seems that FCS is required by alkaline phosphatase to increase activity on calcium deposition and that this enhancing activity may be independent of the presence of cells (Fig 2D). It will be important to analyze if FCS contains pyrophosphate; hydrolysis of pyrophosphate by alkaline phosphatase may affect calcification -The authors state the the optimal culture conditions to obtain medial calcification of aortic rings were 15% FCS supplemented medium without alkaline phosphatase. Thus data should be contrasted with results by KI 2008 73(9):1024 . Reviewer #4: The authors address a key problem in calcification research, which is the lack of reproducibility of in vitro calcification set ups. I have only minor comments. 1. What was the cell culture media pH for different phosphate donors used to induce calcification? 2. Please indicate the number of times that experiments were repeated and provide DS for graphs without DS 3. Specifically, for the optimal ACM, please indicate whether every time the experiment was repeated, calcification was obtained or whether there are still some uncontrolled factors that may prevent calcification in certain experiments 4. Please in the discussion, add the concentrations of calcium and phosphate used in mg/dl, which are commonly used units in many countries, so as readers can easily contextualize to the clinical situation. Since calcium binds albumin and albumin levels in 15% FCS are lower than in 100% human serum, please discuss what would be the free calcium and total calcium equivalent of the concentrations used in experiments. ****** 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: Yes: Joachim Jankowski Reviewer #2: No Reviewer #3: No Reviewer #4: 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 to be viewed.] 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 us at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0230201.r001
Revision 1
14 Feb 2020 Author Response Dear Editor, Please find enclosed the response to reviewers. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. We checked the style requirements 2. We note that you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. For information on unacceptable data access restrictions, please see http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. The complete data are available in supplemental data, S2_Table Data 3. 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. Blot/gel image data are in Supporting Information, S5_raw_images WB phosphosmad1 day 3 original images Figure 3B.pdf and S6_raw_images WB phosphosmad1 day 7 original images Figure 3B.pdf 4. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. Ok Reviewer #1: COMMENTS FOR AUTHORS PlosONE MANUSCRIPT # PONE-D-19-33486 REVIEWER # 1 Nathalie Gayrard Optimization of cell and ex vivo culture 1 conditions to study vascular calcification The authors of this manuscript establish in vitro models mimicking in vivo vascular calcification and to select an optimal method to unravel the mechanisms involved in medial vascular calcification. The authors identified culture conditions, which allowed a reproducible and specific medial calcification of aortic explants as well as calcification of human aortic smooth muscle cells. The authors concluded these approaches will help to achieve reproducible results and allow result comparison in a consistent way. The data presented in this manuscript are interesting for terms of analyses of vascular calcification processes. However, the reviewer has some major remarks: Major points: 1. The introduction section has to be revised. The authors should focus on the main topic of the manuscript and should avoid an very general description of the methods available for the investigation of vascular calcification processes (eg.g page 3 , line 56-59). The introduction has been revised following the suggestion of the reviewer and two references have been added. 2. The authors should prepare a table demonstrating the condition analyze within the study. A supplementary table including all the culture conditions as required by the reviewer has been added to the manuscript (See the S3_culture conditions in supplemental data). 3. NG, KM, FD and AA are obviously employees of a company (RD-Nephrologie). A statement has to be added whether these authors have to declare a conflict of interest based on the employment. NG, KM FD and AA are employees of RD – Néphrologie and have declared to have no conflict of interest. 4. Figure 3.B: A bar graph demonstrating the quantification of Phosphosmad1 should be added. The bar graph requested by the reviewer with the quantification of phosphosmad1 has been added to the figure 3B and the complete data have been included in the supplemental data file, S2_Table Data 5. Not significant data has to be removed from the revised version of the manuscript (page 12, line 258 - 260) The paragraph alluded to by the reviewer has been removed as requested 6. The authors should discuss the overall aim of the study in the discussion section in more detail. The overall aim of the manuscript has been now better presented in the discussion section of the new version of the manuscript. 7. Some typos should be removed in the revised version of the manuscript, e.g. page 9, line 169 (3.8mM instead of 3.8 mM). The typos have been corrected Reviewer #2: This experiment allows overcoming of an ethical issue for using animal models in experiments of vascular calcification research as one of the most important outcome for experiencing major cardiovascular events in humans as a topic for research, although the authors well pointed out the extrapolation of the data should be taken carefully into account. Reviewer #3: The aim of this study is to analyze in vitro and ex vivo models of vascular calcification and to select factors that may affect the process of calcification. Th study is well performed and I cannot identify methodological defects However there are points that should be clarified -Fig1 shows that calcium deposits in HASMCs cultured with high phosphate (3.8 mM) are decreased by supplementing with 15% FCS. It is important to determine if ionized calcium and the concentration of phosphate are modified by 15% FCS. It is know that FCS contains large proteins “calciproteins” that may substract Ca and P from the solution. At least the authors should evaluate if for a given pH, FCS modifies the Ca x P product. The remark of the reviewer is very pertinent. We checked, as suggested by the reviewer, the pH and the content of calcium and phosphate in both, serum free and serum supplemented media. The pH was 7.6 in both media showing that the DMEM medium used is highly buffered. Concerning the calcium content it was 1.9mM and 2.2mMy while the phosphate was 3mM and 3,25mM without and with 15% FCS respectively. The Ca x P product was 5.7 and 7.2 in 0% and 15% FCS medium respectively. Unfortunately, we were not able to measure the ionised calcium, as we do not have specific electrodes to do so. Therefore, the putative substraction of calcium and phosphate from the culture medium by the serum components added in 15% FCS conditions was not observed in our experiments. By the contrary, the slight increase in Ca x Phos product might be the result of the addition of the calcium and phosphate contained in the FCS. -The total calcium content in the wells was significantly increased in the wells with cells cultured with the basic P donor only (P < 0.001) vs acidic and neutral conditions. It is likely that the high pH is increasing calcium deposit the opposed is expected to occur in acid pH. Again ionized Ca and pH should be measured minute after the addition of the different forms of phosphate. The pH of culture medium was 7,6 for all the medium and was not modified by P donors. Unbound calcium was also measured and no difference was observed between the media with different P donors. The results are given in the S4_Medium calcium concentration of the open data. -It seems that FCS is required by alkaline phosphatase to increase activity on calcium deposition and that this enhancing activity may be independent of the presence of cells (Fig 2D). It will be important to analyze if FCS contains pyrophosphate; hydrolysis of pyrophosphate by alkaline phosphatase may affect calcification. The interaction between pyrophosphate and alkaline phosphatase is an interesting and probably very complex point. We included alkaline phosphatase effects on the tested media, both for cell culture systems and aortic explants on that basis. However, the surprising effects observed in both systems, with the decrease in the calcification of the aortic explants in the presence of alkaline phosphatase, led us to remove it from the proposed medium, as already stated in the discussion section (page 19). As underlined by reviewer #1 the main aim of the present paper was to find a calcifying system optimised to be proposed to a wider group of researchers interested in calcification processes. The interaction between pyrophosphate and alkaline phosphatase may be proposed as a separate topic to be explored in a completely different project. -The authors state the optimal culture conditions to obtain medial calcification of aortic rings were 15% FCS supplemented medium without alkaline phosphatase. Thus data should be contrasted with results by KI 2008 73(9):1024. The article of Lomashvili, has been commented (reference #28) and the results contrasted with our findings in the new version of the manuscript. Reviewer #4: The authors address a key problem in calcification research, which is the lack of reproducibility of in vitro calcification set ups. I have only minor comments. 1. What was the cell culture media pH for different phosphate donors used to induce calcification? The pH was 7.6 for all the culture media except for the culture media without FCS the pH was 7.9. 2. Please indicate the number of times that experiments were repeated and provide DS for graphs without DS The complete data of all the experiments performed are now available in the supplemental data (S2 of the present version). In it it can be found the number of repeats for all the experiments. 3. Specifically, for the optimal ACM, please indicate whether every time the experiment was repeated, calcification was obtained or whether there are still some uncontrolled factors that may prevent calcification in certain experiments. Calcification was obtained every time when using optimal ACM. However, the amount of calcium and calcium deposits may vary when changing the batch of FCS. 4. Please in the discussion, add the concentrations of calcium and phosphate used in mg/dl, which are commonly used units in many countries, so as readers can easily contextualize to the clinical situation. Since calcium binds albumin and albumin levels in 15% FCS are lower than in 100% human serum, please discuss what would be the free calcium and total calcium equivalent of the concentrations used in experiments. We added the equivalent concentration in the discussion in mg/dL in brackets. We understand the willingness of the reviewer in trying to make an easy parallelism between the proposed experimental systems and the clinical situation. However, we have to keep in mind that the in vitro and ex vivo settings are far from the in vivo and clinical situations and trying to make any equivalence is a hazardous approach that may be misleading and we would like to avoid if possible. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0230201.r002
25 Feb 2020 Decision Letter - Harald Mischak, Editor Optimisation of cell and ex vivo culture conditions to study vascular calcification PONE-D-19-33486R1 Dear Dr. gayrard, We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements. Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication. Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. 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 enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and 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. With kind regards, Harald Mischak Academic Editor PLOS ONE Additional Editor Comments (optional): 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: All comments have been addressed Reviewer #4: 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 #3: Yes Reviewer #4: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #3: Yes Reviewer #4: 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 #3: Yes Reviewer #4: 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 Reviewer #4: 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: All comments have been addressed. I do not think that additional experiments are required. The data definitely support the conclusion Reviewer #4: (No Response) ****** 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 Reviewer #4: No https://doi.org/10.1371/journal.pone.0230201.r003
Formally Accepted
27 Feb 2020 Acceptance Letter - Harald Mischak, Editor PONE-D-19-33486R1 Optimisation of cell and ex vivo culture conditions to study vascular calcification Dear Dr. gayrard: I am 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 notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, 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. For any other questions or concerns, please email plosone@plos.org. Thank you for submitting your work to PLOS ONE. With kind regards, PLOS ONE Editorial Office Staff on behalf of Prof. Harald Mischak Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0230201.r004

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