Extraction of coronary thrombus-derived exosomes from patients with acute myocardial infarction and its effect on the function of adventitial cells

Youfu He; Bo Wang; Yu Qian; Debin Liu; Qiang Wu

doi:10.1371/journal.pone.0313582

Peer Review History

Original SubmissionAugust 2, 2024
3 Sep 2024 Decision Letter - Vinay Kumar, Editor PONE-D-24-31496Extraction of coronary thrombus-derived exosomes from patients with acute myocardial infarction and its effect on the function of adventitial cellsPLOS ONE Dear Dr. He, 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 Oct 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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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: No Reviewer #2: No ****** 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 manuscript "Extraction of coronary thrombus-derived exosomes from patients with acute myocardial infarction and its effect on the function of adventitial cells" describes a method to isolate exosomes from coronary thrombus. This study is important because Type I acute myocardial infarction (T1MI) has a very high morbidity and mortality rate across the world and the role of coronary thrombus derived exosomes is not well studied. In this manuscript the authors tested different concentrations and incubation time for collagenase and trypsin for the extraction of exosomes from coronary thrombus. They were able to isolate vesicles which could be exosomes after centrifugation. They then performed several cell biological experiments to test the effect of TEs on thrombus-adjacent cells. Finally they performed a microarray analysis for lncRNA's from thrombus isolated exosomes and compared them with HSEs and ASEs. They found several differentially expressed lncRNA's among the three exosomes. This is a well planned and executed study but I have a few concerns Major Concerns 1. Throughout the manuscript the authors have over analysed the findings. This becomes more problematic in the discussion section where the authors talk in great deal about the deferentially expressed lncRNA which they identified only by one microarray experiment and were not validated in an independent experiment like Real Time PCR. 2. The use of strong language and over interpretation of results can also be seen in Lines 505-506 and 514-515. 3. In line 514 the authors say "our findings confirmed". They should rather use a language such as our findings suggest. No one experiment can confirm anything in biology and a series of experiments preferentially done by different group of people are required before stating that something is confirmed. 4. Several parts of the manuscript needs to be rewritten especially the discussion. Minor Concerns 1. There are several grammatical and writing errors for example line 359, 488, 369 etc 2. A brief explanation of vesicles is needed on line 61. Authors jump from exosomes to vesicles making the reader wonder whether they are talking about the same thing or if they are different. Reviewer #2: In the present study, the authors investigate the role of thrombus-derived exosomes (TEs) in Type I acute myocardial infarction (T1MI), focusing on their effects on adjacent cells and their potential as therapeutic targets. The authors optimize the protocol for extracting TEs and examined their impact on cardiomyocytes and endothelial cells. The findings show that TEs promote necrosis, autophagy, and ferroptosis in human cardiomyocytes, while inhibiting the proliferation and migration of endothelial cells. Additionally, TEs stimulate smooth muscle cell proliferation and migration. Bioinformatics analysis highlighted five lncRNAs (AC068418.2, AC010186.3, AL031430.1, AC121333.1, and AL136526.1) that regulate autophagy and ferroptosis. However, the following issues must be addressed. 1. As noted in the introduction, exosomes contain a complex mixture of cellular components. The authors could have provided a more comprehensive analysis of the exosomal composition, such as through proteomics or genomics, rather than focusing solely on lncRNAs. The rationale for selecting only lncRNAs should be clearly explained in the manuscript. 2. Additionally, the authors could have performed GO term or KEGG pathway analyses to explore the broader functional implications of the identified lncRNAs. 3. The study is limited to cell lines, and the inclusion of experiments using isolated primary cardiomyocytes or endothelial cells would have strengthened the findings. This limitation should be acknowledged in the discussion. 4. It is unclear whether the exosomes used for cell line treatments were pooled from five AMI patients. The methods section lacks clarity and should be updated with detailed information on the exosome isolation and pooling procedures. 5. Figures 2F and 3F might be better presented in the supplementary material, perhaps in a table format. 6. Lines 369-373 contain repeated sentences and should be revised for clarity. The authors should also carefully review the manuscript for English language accuracy. 7. Finally, is there a possibility that the authors isolated exosomes from patients who underwent AMI treatment? If so, it would be insightful to assess the activity of these TEs on HCM cell lines. ****** 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: Yes: Suman Asalla ******** [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.0313582.r001
Revision 1
14 Oct 2024 Author Response List of Responses Dear Editors and Reviewers: Thank you for your letter and for the reviewers’ comments concerning our manuscript entitled “Extraction of coronary thrombus-derived exosomes from patients with acute myocardial infarction and its effect on the function of adventitial cells”. (ID: PONE-D-24-31496). Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our researches. We have studied reviewer’s comments carefully and have made revision which marked in red in the paper. We have tried our best to revise our manuscript according to the comments. Attached please find the revised version, which we would like to submit for your kind consideration. The main corrections in the paper and the responds to the reviewer’s comments are as flowing: Reviewer #1: The manuscript "Extraction of coronary thrombus-derived exosomes from patients with acute myocardial infarction and its effect on the function of adventitial cells" describes a method to isolate exosomes from coronary thrombus. This study is important because Type I acute myocardial infarction (T1MI) has a very high morbidity and mortality rate across the world and the role of coronary thrombus derived exosomes is not well studied.In this manuscript the authors tested different concentrations and incubation time for collagenase and trypsin for the extraction of exosomes from coronary thrombus. They were able to isolate vesicles which could be exosomes after centrifugation. They then performed several cell biological experiments to test the effect of TEs on thrombus-adjacent cells. Finally they performed a microarray analysis for lncRNA's from thrombus isolated exosomes and compared them with HSEs and ASEs. They found several differentially expressed lncRNA's among the three exosomes. This is a well planned and executed study but I have a few concerns. Major Concerns 1. Throughout the manuscript the authors have over analysed the findings. This becomes more problematic in the discussion section where the authors talk in great deal about the deferentially expressed lncRNA which they identified only by one microarray experiment and were not validated in an independent experiment like Real Time PCR. Response: Thank you very much to reviewer 1 for recognising our study. Regarding the validation of lncRNA, we have actually done a lot of qPCR validation. For example, in Figure A of this reply letter, it is the qPCR validation of the differentially expressed gene top20. Of course, the purpose of this study was mainly to confirm the existence of thrombus-derived exosomes (TE) and to explore their role on thrombus clinical cells. Therefore, after completing the RNA microarray analysis, we did not delve further into its specific mechanism. 2. The use of strong language and over interpretation of results can also be seen in Lines 505-506 and 514-515. Response: Many thanks to reviewer 1 for your patience and correction. We have re-examined this manuscript and corrected many places that were inappropriately described. 3. In line 514 the authors say "our findings confirmed". They should rather use a language such as our findings suggest. No one experiment can confirm anything in biology and a series of experiments preferentially done by different group of people are required before stating that something is confirmed. Response: Many thanks to reviewer 1 for your patience and correction. Regarding the word confirmed, we also found inappropriate use of the word elsewhere in the manuscript. It has now been corrected in the manuscript. 4. Several parts of the manuscript needs to be rewritten especially the discussion. Response: Thank you so much for your feedback. After receiving the review comments, our team members did find a lot of inappropriate things after checking and reviewing again. It has now been reworked for revision. Minor Concerns 1. There are several grammatical and writing errors for example line 359, 488, 369 etc Response: Indeed, as reviewer 1 said, in many places our wording is not very accurate and there is a suspicion of exaggerating the results. We fully accept the criticisms of the reviewers and editors and thank you for your comments. After careful consideration, many inappropriate places have been reworked. 2. A brief explanation of vesicles is needed on line 61. Authors jump from exosomes to vesicles making the reader wonder whether they are talking about the same thing or if they are different. Response: Many thanks to reviewer 1 for his patience and correction. Indeed we have overlooked these basic details. Considering that exosome itself also belongs to a kind of extracellular vesicle, so we added a part of explanation and definition for exosome. Reviewer #2: In the present study, the authors investigate the role of thrombus-derived exosomes (TEs) in Type I acute myocardial infarction (T1MI), focusing on their effects on adjacent cells and their potential as therapeutic targets. The authors optimize the protocol for extracting TEs and examined their impact on cardiomyocytes and endothelial cells. The findings show that TEs promote necrosis, autophagy, and ferroptosis in human cardiomyocytes, while inhibiting the proliferation and migration of endothelial cells. Additionally, TEs stimulate smooth muscle cell proliferation and migration. Bioinformatics analysis highlighted five lncRNAs (AC068418.2, AC010186.3, AL031430.1, AC121333.1, and AL136526.1) that regulate autophagy and ferroptosis. However, the following issues must be addressed. 1. As noted in the introduction, exosomes contain a complex mixture of cellular components. The authors could have provided a more comprehensive analysis of the exosomal composition, such as through proteomics or genomics, rather than focusing solely on lncRNAs. The rationale for selecting only lncRNAs should be clearly explained in the manuscript. Response: Thanks a lot for this professional review. Because of the complexity of the mechanism of lncRNA, that is why we chose to do the lncRNA microarray analysis separately. Also, the microarray analysis we did was not exclusively for lncRNAs, but also for many mRNAs, and we wrote a separate article to discuss this part. Of course, as reviewer 2 said, we did lack the explanation of this part. Therefore, we have made some additions in RESULT 3.7 of the manuscript. 2. Additionally, the authors could have performed GO term or KEGG pathway analyses to explore the broader functional implications of the identified lncRNAs. Response: Reviewer 2 is a very knowledgeable professor of academics, so please allow me to express my high regard to you. As we said earlier, our microarray sequencing analyses also included a lot of mRNA results. So combining these mRNA and lncRNA results, we did perform a lot of KEGG and Pathway analyses. A portion of the results can be seen in Figure B. Considering that the position of this article is to confirm that the substance TE does exist and can have a certain regulatory effect on thrombus adventitial cells. So our exploration in this part would like to be a bit more superficial. 3. The study is limited to cell lines, and the inclusion of experiments using isolated primary cardiomyocytes or endothelial cells would have strengthened the findings. This limitation should be acknowledged in the discussion. Response: Thanks again to reviewer 2 for your comments. Your rigour is something we should respect and learn from. We did use some common cell lines for our study. So this is an important shortcoming of our study. Combined with your comments, we have added in the limitation section comments. 4. It is unclear whether the exosomes used for cell line treatments were pooled from five AMI patients. The methods section lacks clarity and should be updated with detailed information on the exosome isolation and pooling procedures. Response: Please forgive us for our oversight. In this study, practically all exosomes used for cellular studies were obtained from these three T1MI patients. Taking your comments into account, we have refined the description in the Methods section. 5. Figures 2F and 3F might be better presented in the supplementary material, perhaps in a table format. Response: Please allow me to explain the rationale for this data. For exosome identification, we routinely need to use these three methods, i.e. western blot, electron microscopy and Nanoparticle tracking analysis (NTA). Figure 2F and Figure 3F are both essential NTA data in the exosome identification repertoire. In this field, we need to see the maximum peaks in addition to the distribution of particle sizes to understand the purity of the extracted exosomes. So we ultimately chose to keep this part of the data in order to demonstrate the credibility of our data as much as possible. 6. Lines 369-373 contain repeated sentences and should be revised for clarity. The authors should also carefully review the manuscript for English language accuracy. Response: We are very grateful to reviewer 2 for your careful correction. Please excuse our carelessness, the repetitive description of grouping in this section has been corrected. 7. Finally, is there a possibility that the authors isolated exosomes from patients who underwent AMI treatment? If so, it would be insightful to assess the activity of these TEs on HCM cell lines. Response: Indeed, the TEs as well as the ASEs we extracted were from patients undergoing emergency PCI in our hospital. As described in our methods, all thrombi and sera were extracted during emergency PCI. Considering that TE is really something that is overlooked by all of us researchers in the cardiovascular field, we take it very seriously. We also think it's a very valuable subject to study. We are currently doing everything we can to explore the association of TE with AMI as a disease. We hope that our research can provide a new line of treatment for AMI, especially T1MI as a disease. Therefore, we also very much hope that the reviewers and editors will give us this opportunity to present our study to the cardiovascular field researchers worldwide through this journal. We also hope that more researchers will join the TE study to provide more theoretical basis for the treatment of T1MI patients in the future. We tried our best to improve the manuscript and made some changes in the manuscript. These changes will not influence the content and framework of the paper. And here we did not list the changes but marked in red in revised paper. In addition, we will be happy to edit the paper further, based on helpful comments from the reviewers. We appreciate for Editors/Reviewers’ warm work earnestly, and hope that the correction will meet with approval. Once again, thank you very much for your comments and suggestions. Figure A. RT-PCR analysis of DElncRNA expression in each group of exosomes a, compared with the HE group, p < 0.05; b, compared with the SE group, p < 0.05 HE, serum exosomes from healthy individuals; SE, serum exosomes from patients with AMI; TE, coronary thrombus-derived exosomes from patients with AMI Figure B. Bioinformatics analysis of lncRNAs enriched in coronary thrombus-derived exosomes (a) Heat map showing the expression of DElncRNAs in TEs compared with those in healthy human serum exosomes. Each row in the heat map represents a single gene and each column represents a tissue sample. The color scale, ranging from blue (low expression) to red (high expression), represents the raw Z-score mRNA intensity values; (b) Volcano plot of DElncRNAs between TEs and healthy human serum exosomes. The red nodes represent up-regulated DEGs and the green nodes represent down-regulated DEGs; (c) Heat map showing the expression of DEmRNAs in TEs compared with those in healthy human serum exosomes; (d) Volcano plot of DEmRNAs between TEs and healthy human serum exosomes; (e,f) Circle chart and bubble diagram of gene ontology (GO) analysis of DEmRNAs showing enriched biological processes, cell components, and molecular functions; (g,h) Circle chart and bar chart of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of targeted genes showing enriched signaling pathways. HE, serum exosomes from healthy individuals; SE, serum exosomes from patients with AMI; TE, coronary thrombus-derived exosomes from patients with AMI Attachments Attachment Submitted filename: response to reviewer.docx https://doi.org/10.1371/journal.pone.0313582.r002
28 Oct 2024 Decision Letter - Vinay Kumar, Editor Extraction of coronary thrombus-derived exosomes from patients with acute myocardial infarction and its effect on the function of adventitial cells PONE-D-24-31496R1 Dear Dr. He, 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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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: 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 #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 addressed all the comments in a satisfactory manner. They have toned down the language in the discussion section according to the suggestion. Reviewer #2: (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 #1: No Reviewer #2: No ******** https://doi.org/10.1371/journal.pone.0313582.r003
Formally Accepted
7 Nov 2024 Acceptance Letter - Vinay Kumar, Editor PONE-D-24-31496R1 PLOS ONE Dear Dr. He, 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 If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks 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. 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. Vinay Kumar Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0313582.r004

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