CD44 as a novel therapeutic target in pulmonary arterial hypertension: Insights from multi-omics integration and molecular docking

Wei Chen; Lingling Zhang; Haiyan Qi

doi:10.1371/journal.pone.0332817

Peer Review History

Original SubmissionFebruary 26, 2025
23 Jun 2025 Decision Letter - Naseem Ahamad, Editor Dear Dr. Qi, 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 Aug 07 2025 11:59PM.5. 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. 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'. 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Thank you for stating the following in the Acknowledgments Section of your manuscript: “This work was supported by grants from Scientific Research Foundation of the Education Department of Jiangxi Province, China (No. GJJ2200446 to H.Q.) and Jiangxi Provincial Graduate Innovation Special Foundation (No. YC2024-B178 to H.Q. and No. YC2023-B166 to W.C.).” We note that you have provided additional information within the Acknowledgements Section that is not currently declared in your Funding Statement. Please note that funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: “This work was supported by grants from Scientific Research Foundation of the Education Department of Jiangxi Province, China (No. GJJ2200446 to H.Q.) and Jiangxi Provincial Graduate Innovation Special Foundation (No. YC2024-B178 to H.Q. and No. YC2023-B166 to W.C.).” Please include your amended statements within your cover letter; we will change the online submission form on your behalf. Additional Editor Comments (if provided): Thank you for your submission. The reviewers have given important feedback that needs to be addressed before your work can be considered for publication. Please revise your manuscript to address their concerns and include a detailed response letter explaining how you have addressed each point raised by the reviewers. Their enthusiasm for your molecular docking approach indicates that with these revisions, your manuscript has strong potential for publication. [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? Reviewer #1: Yes Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #1: No Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available??> The PLOS Data policy Reviewer #1: No Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: No Reviewer #2: Yes ****** Reviewer #1: 1. No correction for batch effects or potential confounders. No ROC analysis or machine learning models to predict CD44’s clinical significance. 2. Gene expression and KEGG/GO data need to added in supplementary data. Reviewer #2: The way the authors represent their work is fabulous and I found all the work are prefect for the molecular docking level. based on these findings future research should focus on understanding CD44's regulatory mechanisms and developing targeted therapies in the wet lab. ****** 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: Prince Kumar Reviewer #2: Yes: Kannan Kanthaiah ******** [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. Attachments Attachment Submitted filename: Comments_R1.pdf https://doi.org/10.1371/journal.pone.0332817.r001
Revision 1
21 Aug 2025 Author Response Response to Reviewer Comments We sincerely thank the reviewers for their thoughtful and constructive feedback. We have carefully revised the manuscript according to the comments and provide detailed responses below. All modifications have been marked in the revised manuscript. 1. “The introduction lacks a critical discussion on prior CD44 studies in PAH (Ref 15 could be elaborated).” Response: Thank you for pointing this out. We have revised the Introduction section to include a more detailed discussion on previous studies involving CD44 in the context of PAH, particularly elaborating on Ref 15. This provides a more comprehensive background and better highlights the novelty of our study. 2. “The bulk RNA-seq analysis was conducted on samples from only 4 iPAH patients and 4 healthy controls. Similarly, the single-cell RNA-seq analysis used data from 3 iPAH patients and 3 controls. These are relatively small sample sizes, which can limit the statistical power of the study and the generalizability of the findings to the broader PAH patient population. This should acknowledged as a limitation.” Response: We agree that the small sample sizes are a limitation. We have added a clear statement acknowledging this in the Discussion section, emphasizing that our findings require validation in larger and independent clinical cohorts. 3. “Add supplementary files with complete DEG lists and enriched pathway tables.” Response: Done. We have now included the complete list of differentially expressed genes and enriched pathways (GO and KEGG) in Supplementary Tables S1 and S2, respectively. • Supplementary Table S1: Complete list of differentially expressed genes (DEGs) from the bulk RNA-seq analysis, including gene symbols, log2 fold changes, p-values, and adjusted p-values. • Supplementary Table S2: Full results of Gene Ontology (GO) and KEGG pathway enrichment analyses, including terms, gene counts, and FDR values. 4. “The Materials and Methods section and the Data Availability statement consistently refer to the single-cell RNA-seq dataset as GSE228644. However, the legend for Figure 3, which presents the scRNA-seq results, states the data is from GSE231993. This is a clear inconsistency that needs to be corrected.” Response: Thank you for noticing this. We have corrected the inconsistency. The correct dataset used for scRNA-seq is GSE228644, and this has been updated throughout the manuscript, including the Figure 3 legend. 5. “Enrichment tools and thresholds are standard, but exact GO/KEGG terms and p-values should be added in at least in Supplementary Data.” Response: We thank the reviewer for the comment. We used standard enrichment analysis parameters, specifically setting pvalueCutoff = 0.05 and qvalueCutoff = 0.2 to identify significantly enriched GO and KEGG terms. The full enrichment results, including term names and statistical values, have been provided in the Supplementary Data. Supplementary Table S2: Full results of Gene Ontology (GO) and KEGG pathway enrichment analyses, including terms, gene counts, and FDR values. 6. “PPI networks predict interactions but do not confirm functional effects experimentally. Lacks complementary co-expression analysis (from RNA-seq data) to strengthen findings.” Response: We appreciate the reviewer’s insightful suggestion regarding the integration of co-expression analysis to complement the PPI network findings. In response, we performed a Spearman correlation-based co-expression analysis using the bulk RNA-seq dataset. Among the top 30 genes most strongly co-expressed with CD44, we identified EFHD2 and MGLL, both of which also appeared in the PPI network constructed from inflammation-related upregulated DEGs. This overlap strengthens the functional relevance of these genes within the CD44-associated inflammatory module and supports the biological significance of our PPI network predictions. We have added the co-expression analysis results to the revised manuscript (Figure X). 7. “The molecular docking results suggesting a high-affinity interaction between progesterone and CD44 are based on computational predictions. While this provides a novel hypothesis for therapeutic intervention, these findings are preliminary and require experimental validation (e.g., in vitro binding assays, cellular or animal model studies) to confirm the interaction and its functional significance in a biological context.” Response: We agree. We have added a paragraph in the Discussion section clearly stating that the molecular docking results are computational predictions and must be validated experimentally through functional studies such as in vitro binding assays and animal models. 8. “Molecular docking analysis seems overexerted as the docking energy (−7.2 kcal/mol) is reasonable but not definitive. Moreover, lacks comparative analysis with known CD44 ligands (e.g., HA analogs) and no molecular dynamics simulation was performed to support binding stability. Therefore looking at aforesaid limitations, the identification of progesterone as a CD44 ligand is speculative, thus needs a more cautious tone or follow-up validation. Statements like “These results position progesterone as a potential CD44 antagonist” are premature without functional assays.” Response: Thank you for the insightful comment. We agree that molecular docking alone provides preliminary evidence and requires experimental validation. Accordingly, we adjust our statements. Furthermore, we performed a comparative docking analysis using hyaluronic acid (HA), a well-established CD44 ligand, and showed that progesterone exhibited a higher predicted binding affinity (−7.2 kcal/mol vs. −5.1 kcal/mol). These results are now included in the revised Figure S5 and discussed appropriately. We also acknowledged the absence of MD simulations as a limitation, and emphasized the need for further validation through in vitro binding assays and functional studies. 9. “Statement: “highlighting systemic immune suppression in advanced PAH” contradicts the theme that inflammation is increased and requires further clarification.” Response: We appreciate this insightful comment. We agree that the original statement “highlighting systemic immune suppression in advanced PAH” may seem inconsistent with the broader theme of inflammation. To address this, we have revised the sentence to clarify that the observed downregulation of certain inflammatory pathways reflects immune dysregulation or exhaustion, which is commonly seen in chronic inflammatory diseases like advanced PAH. 10. “Author should discuss about unintended consequences of CD44 inhibition.” Response: This is a valuable point. We have added a paragraph in the Discussion addressing potential unintended consequences of CD44 inhibition, such as impaired tissue repair or altered immune surveillance, which should be considered in therapeutic development. Minor Comments • “Fix typographical and grammatical errors throughout.” Response: We have carefully proofread the manuscript and corrected all identified typographical and grammatical errors. • “Improve figure quality and annotations.” Response: We have enhanced the resolution of all figures and improved labeling and annotation to ensure clarity and readability. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0332817.r002
5 Sep 2025 Decision Letter - Naseem Ahamad, Editor CD44 as a Novel Therapeutic Target in Pulmonary Arterial Hypertension: Insights from Multi-Omics Integration and Molecular Docking PONE-D-25-09501R1 Dear Dr. Qi, 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. For questions related to billing, please contact billing support . 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, Naseem Ahamad Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0332817.r003
Formally Accepted
Acceptance Letter - Naseem Ahamad, Editor PONE-D-25-09501R1 PLOS ONE Dear Dr. Qi, 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 You will receive further instructions from the production team, including instructions on how to review your proof when it is ready. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few days 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. You will receive an invoice from PLOS for your publication fee after your manuscript has reached the completed accept phase. If you receive an email requesting payment before acceptance or for any other service, this may be a phishing scheme. Learn how to identify phishing emails and protect your accounts at https://explore.plos.org/phishing. 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 Dr. Naseem Ahamad Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0332817.r004

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