Spontaneous urinary bladder regeneration after subtotal cystectomy increases YAP/WWTR1 signaling and downstream BDNF expression: Implications for smooth muscle injury responses

Karen J. Aitken; Priyank Yadav; Martin Sidler; Thenuka Thanabalasingam; Tabina Ahmed; Prateek Aggarwal; Shing Tai Yip; Nefateri Jeffrey; Jia-Xin Jiang; Aliza Siebenaller; Chris Sotiropoulos; Ryan Huang; David Minh Quynh Le; Paul Delgado-Olguin; Darius Bagli

doi:10.1371/journal.pone.0287205

Peer Review History

Original SubmissionDecember 30, 2022
30 Mar 2023 Decision Letter - Panayiotis Maghsoudlou, Editor PONE-D-22-35632Spontaneous Urinary Bladder Regeneration After Subtotal Cystectomy (STC) Increases YAP/WWTR1 Signaling And Downstream BDNF Expression: Implications For Smooth Muscle Injury Responses.PLOS ONE Dear Dr. Aitken, 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 May 14 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. 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Reviewer #1: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: 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 ****** 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 ****** 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, the authors determined the role of the YAP/WWTR1 pathway and downstream BDNF, CTGF and Cyr61 in the regulation of bladder regeneration. It was found that scratch wound in vitro increased bladder smooth muscle cell migration and expression of BDNF, CTGF and CYR61 in a YAP/WWTR1-dependent manner. Furthermore, bladder regeneration is associated with YAP/WWTR1 signaling and BDNF expression. This manuscript is interesting and well written. I suggest to publish this manuscript after minor revision. 1. Please make sure to define all acronyms the first time they appear in the manuscript. There are some which are never defined, eg. FCS and CFSE. 2. The authors stated “bladder SMC migration is inhibited at the scratch wound edge (Fig. 1C)”. Images from a scratch assay at different time points should be added. 3. How do you explain “Mst1, which inhibits Yap/Wwtr1 activity, is transcriptionally downregulated during scratch wounding, but VP induced a further decrease”. 4. At 1 week, residual volumes in STC were lower than incised bladders, but why residual volumes in incised bladders at 7 weeks significantly decreased when compared to STC (Fig. 2C)? 5. Pan Bdnf was increased in STC vs sham after 1 week (1.68-fold+/-0.53) and 7 weeks (3.6-fold+/-1.1) (Fig. 3A). While the BDNF receptor, NTRK2, increased at 1 week, but decreased at 7 weeks. What's the explanation? 6. The statistical data charts in Figure 1D and E are too small and unclear, which is not convenient for readers to read. ****** 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 ******** [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.0287205.r001
Revision 1
19 May 2023 Author Response Response to Review for PONE-D-22-35632 We have now also included the annotated (highlighted in this case, as we utilized google docs to collaborate with authors) and non-annotated versions of the manuscript, as well as the figures that are revised as requested by the reviewer. 1. Please make sure to define all acronyms the first time they appear in the manuscript. There are some which are never defined, eg. FCS and CFSE. RESPONSE: We have revised acryonyms and abbreviations. Abbreviations are now described at their initial use, or are not included if only used once. However, we have left some abbreviations in the title, due to the length of the protein names for WWTR1 (Transcriptional co-activator with a PDZ-binding [WW] domain containing transcription regulator 1) and BDNF (Brain-derived neurotrophic factor). 2. The authors stated “bladder SMC migration is inhibited at the scratch wound edge (Fig. 1C)”. Images from a scratch assay at different time points should be added. RESPONSE: The original image was supposed to be embedded as a Quicktime movie. We had mentioned this during our initial submission, but unfortunately, the file could not contain a movie easily. We feel that the movies provide the information on the time points, but could revise this if required. Similar to figure S4, Figure 1C has two movies. We have added all of these movies in a zip file containing two pptx files with the movies for Figs.1C and S4, as we were unable to place the movies into pdfs. 3. How do you explain “Mst1, which inhibits Yap/Wwtr1 activity, is transcriptionally downregulated during scratch wounding, but VP induced a further decrease”. RESPONSE: During scratch wounding, we found that Mst1 was downregulated. This was consistent with the observed increased Yap/Wwtr1 activity during scratch wounding. VP is an inhibitor of Yap/Wwtr1 activity. It is possible that the decrease in their activity leads to a downregulation of Mst1 as a compensation to try to increase the Yap/Wwtr1 activity in the cell. It supports the potential for a negative feedback loop between Yap/Wwtr1 activity and Mst1 transcription, which is disrupted by VP’s inhibition of Yap/Wwtr1 activity. 4. At 1 week, residual volumes in STC were lower than incised bladders, but why residual volumes in incised bladders at 7 weeks significantly decreased when compared to STC (Fig. 2C)? RESPONSE: The incision at 7 weeks may have resolved and become more functional at this timepoint, while in STC ongoing responses may be preventing the reduction of residual volumes. The timecourse for resolution of the responses likely differs due to the nature and extent of the injury, which may also be associated with alterations in signaling and gene expression. This discussion point has been added into the manuscript: “...In contrast, incised bladders by week 7 exhibited significantly lower residual volumes in comparison to STC, which may be due to the nature and extent of the injury at the respective timepoints (Fig 2C). At 7 weeks, the incision may have resolved and led to increased functionality of the bladder, whereas STC responses may have prevented the reduction of residual volumes.” 5. Pan Bdnf was increased in STC vs sham after 1 week (1.68-fold+/-0.53) and 7 weeks (3.6-fold+/-1.1) (Fig. 3A). While the BDNF receptor, NTRK2, increased at 1 week, but decreased at 7 weeks. What's the explanation? RESPONSE: Yes, this is very interesting. It suggests that the particular NTRK2 and BDNF variants may be divergently regulated. Notably, NTRK2 var2 cannot respond to BDNF, as it lacks the kinase region. It appears that during long term STC BDNF expression, this BDNF variant increases as the NTRK non-kinase variant decreases, further increasing the potential for signaling in response to BDNF. We have adjusted the Primer pair designation and clarified this in the text as well. 6. The statistical data charts in Figure 1D and E are too small and unclear, which is not convenient for readers to read. RESPONSE: Figure 1D and 1E have been revised to increase size and clarity. Editorial Revisions: #1. Revisions of style: RESPONSE: Figure and supplemental figure names were adjusted to PLoSONE standards, as Fig # or S# Fig, respectively. Abbreviations and gene names were adjusted according to Plosone standards, which included HUGO names. The exception to this is that we did not change CTGF and CYR61 names to HUGO names CCN2 and CCN1, although both names are mentioned in the introduction. We decided to use the more commonly utilized gene names, CTGF and CYR61, for these two matricellular proteins, as they are utilized most often in the literature, which would make the manuscript more comprehensible. Indeed, a recent PLOS ONE paper utilizes CTGF instead of CCN2, for example: Al-U’datt, et al. (2023). Involvement and possible role of transglutaminases 1 and 2 in mediating fibrotic signalling, collagen cross-linking and cell proliferation in neonatal rat ventricular fibroblasts. PLOS ONE 18(2): e0281320 (https://doi.org/10.1371/journal.pone.0281320). Additionally, the standard nomenclature refers to human genes with capitals and rat names with the first letter as a capital, the rest lowercase. We have utilized this method. #2 and 3. Revisions of methodology “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, (2) methods of anesthesia and/or analgesia, and (3) efforts to alleviate suffering. 3. Please include your animal ethics statement in the Methods section of your manuscript. In the Methods section of your revised manuscript, please include the full name of the animal ethics committee that approved the protocol.” RESPONSE: Methodology details involving animals and the animal care committee are now updated. #4 and 6: RESPONSE: We have harmonized the granting information, but it is important to note that grant numbers are not available for CIHR awards – the PI or fellow name is the only referral. With regard to the funding statement, the funder did not have a direct role in the design, data collection, decision to publish or preparation. While the agency TPRM program approved KJA’s trainee application and trained KJA in regenerative medicine, this specific project was designed and carried out by the supervisors (DJB and PDO), the applicant (in this case KJA) and the accompanying authors. We have revised the statement to include the training aspect of the program: “The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript, and only played a role in the scientific training of the fellow KJA in regenerative medicine.” #5. RESPONSE: The acronym “CIHR” refers to the Canadian Institutes of Health Research. #7 and 9: RESPONSE: The corresponding author is no longer at the Institution where the work was originally performed, but the last author does not have an up-to-date ORCID ID. Both IDs are included here: ● KJA: 0000-0003-2995-0205 ● DB: 0000-0002-4521-3056 #8: RESPONSE: The minimal dataset is now being placed into a Github repository under the first author’s auspices: https://github.com/kjaitken/STC_repository. Additional code or instructions for VolocityTM (Perkin Elmer software that is useful for measuring intensities in micrographs) for plotting and analysis is being placed in this repository as well. ● The values behind the means, standard deviations and other measures reported; ● The values used to build graphs; ● The intensities and other measurements extracted from images for analysis. Examples of the fluorescent imaging data in support of reported results are provided in the paper itself. We are also in the process of submitting lab protocols on protocols.io as recommended by the editor. #10: RESPONSE: A full “REB” ethics statement might not be required, as this is an animal study which did not have any human component. However, we have updated the animal study information (See #2,3). #11: RESPONSE: The Table is now referenced and included immediately after the paragraph where it is first mentioned, as required in the PLOS ONE standards. #12: RESPONSE: We are not aware of any retracted articles in the reference list, but have noted that some of the references may have been flagged for various reasons: ● Reference 1: Xia et al posted an Author Correction to this article on 27 July 2018. ● Reference 2: had an erratum posted. ● References 6-10: these are old archival papers that are pertinent to human cystectomy which is rarely performed for non-cancer reasons now. Perhaps these articles appear to be unavailable on your search engine as they are quite old. ● Reference 18: This review is from the Journal of Tissue Science and Engineering. This journal’s articles appear to be available only through the publisher website after 2012, with most of the articles after 2012 not appearing on pubmed. The publisher website states: “Journal of Tissue Science and Engineering will support authors by posting the published version of articles by NIH grant-holders and European or UK-based biomedical or life sciences grant holders to PubMed Central immediately after publication.“ Though the article is not listed on pubmed, manuscripts submitted to this journal appear to undergo the usual peer review process of an academic journal. Finally, we would like to express our appreciation to the editors and reviewer for their review of our piece, and for providing the guidance needed to refine our work. As we believe that this work will benefit the research area of benign urologic disease and basic bladder cell and molecular biology, we want to thank your team for helping us showcase our efforts. Attachments Attachment Submitted filename: Response to Reviewers May 2023.docx https://doi.org/10.1371/journal.pone.0287205.r002
1 Jun 2023 Decision Letter - Panayiotis Maghsoudlou, Editor Spontaneous urinary bladder regeneration after subtotal cystectomy Increases YAP/WWTR1 signaling And downstream BDNF expression: implications for smooth muscle injury responses. PONE-D-22-35632R1 Dear Dr. Aitken, 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, Panayiotis Maghsoudlou Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0287205.r003
Formally Accepted
17 Jul 2023 Acceptance Letter - Panayiotis Maghsoudlou, Editor PONE-D-22-35632R1 Spontaneous urinary bladder regeneration after subtotal cystectomy increases YAP/WWTR1 signaling and downstream BDNF expression: implications for smooth muscle injury responses. Dear Dr. Aitken: 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. Panayiotis Maghsoudlou Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0287205.r004

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