Assessment of ICAM-1 N-glycoforms in mouse and human models of endothelial dysfunction

Kellie Regal-McDonald; Maheshika Somarathna; Timmy Lee; Silvio H. Litovsky; Jarrod Barnes; J. M. Peretik; J. G. Traylor Jr.; A. Wayne Orr; Rakesh P. Patel

doi:10.1371/journal.pone.0230358

Peer Review History

Original SubmissionJuly 23, 2019
29 Aug 2019 Decision Letter - Tohru Fukai, Editor PONE-D-19-20764 Assessment of ICAM-1 N-Glycoforms in Mouse and Human Models of Endothelial Dysfunction PLOS ONE Dear Dr. Rakesh Patel, 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 will gather from the reviews, the referees identified several methodological problems and questionable functional significance of the current findings. The editor concurs. Major issues include low resolution of images and lack of support data for PLA (e.g. western analysis). Other concerns pointed by reviewers should also be addressed. We would appreciate receiving your revised manuscript within three months. 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Please follow this link to our website for more details on competing interests: http://journals.plos.org/plosone/s/competing-interests 5. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions 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 study by Regal-McDonald et al used PLA to investigate co-localization of specific N-glycan structures and ICAM-1 in mouse and human atherosclerotic arteries and human AVF of chronic kidney disease patients (models of endothelial dysfunction). The results demonstrated that hypoglycosylated and α-2,6-sialylated ICAM-1 N-glycoforms are present in atherosclerotic murine and human arteries and AVF patients. The results also indicated that HM-ICAM-1 levels positively correlated with macrophage burden in both murine and human atherosclerotic tissue. The study is timely but lacks functional information on the role of ICAM-1 N-glycoforms in atherosclerosis or AVF. 1. The figures of the manuscript are very low resolution. 2. It would strengthen the findings of the manuscript if the authors confirm the results obtained using the innominate arteries by looking at total, HM / hybrid, a-2,6-sialylated, and a-2,3-sialylated ICAM-1 in the atherosclerotic mouse aorta. 3. How were the lesion and non-lesion segments identified? 4. Fig. 2 and 3 legends state that n=5 experiments were performed for CD68 immunostaining. The corresponding bar graphs show only 3 experiments. 5. What is the correlation coefficient for Fig. 4 studies? It seems there would be no correlation if only the closed symbols are taken into consideration. It is recommended to perform more experiments for the correlation studies. In the high fat diet model (Fig. 2; no ligation), there was atherosclerosis in the brachiocephalic artery and HM/hybrid, HM, a-2,6-sialylated and a-2,3-sialylated ICAM-1 were increased in lesion vs. non-lesion segments. If there is atherosclerosis in these mice but no correlation between CD68 and ConA or HHL puncta, the mechanisms regulating ICAM N-glycosylation may not be important for monocyte adhesion and macrophage accumulation. 6. Type 2 human atherosclerotic lesions show no ICAM-1 expression. The type 2 lesions are characterized by the presence of lipid-laden macrophages according to Stary et al (ATVB, 1995). These findings suggest that ICAM-1 (and its N-glycoforms) does not play a role at the early phase of monocyte transmigration in human atherosclerotic vessels. 7. Fig. 6B legends n=10 is misleading. It seems only n=5 samples per group was involved in the analysis. The authors need to carefully check the legends for accuracy. 8. This is an observational study and no functional experiments have been included to investigate the role of different ICAM-1 N-Glycoforms in the development of atherosclerosis or pathogenesis of AVF maturation failure. The manuscript would be strengthened by the addition of mechanistic data. 9. The rational for including the AVF data in the manuscript is not entirely clear. The mechanisms, leading to endothelial dysfunction in AVF failure vs atherosclerosis is different. Reviewer #2: This is a novel approach to assessment of ICAM-1 N-Glycoforms in mouse and human models of endothelial dysfunction. In this paper using the proximity ligation assay (PLA), authors assessed the relative formation of high mannose, hybrid and complex α-2,6-sialyated N-glycoforms of ICAM-1 in human and mouse models of atherosclerosis, as well as in arteriovenous fistulas (AVF) of patients on hemodialysis. To establish the distinct ICAM1-N-glycoforms in inflammatory disease in ivvivo authors solely used PLA assay. Indeed this assay has some limitation, however the PLA positive red puncta was not clear in all images throughout the manuscript. As this data represents the first study to demonstrating ICAM1-N-glycoforms invivo authors should provide convincing images with good quality pictures for PLA assay. There was no description for Figure 2F and 2G in result section. Author should mention about these two figures. In result section, Figure 3A author described that total ICAM-1, HM / hybrid N-glycoforms were increased in the ligated left carotid artery (LC) compared to the control right carotid artery (RC). But this statement was not matches with the Figure 3A. It looks like total ICAM1 and α-2,6-sialylated ICAM1 expressed more than HM/hybride. Fig 3C quantification was not reflects the Fig 3A images. Author should be careful about this discrepancy. In Fig 4 and Fig 6 show correlations between CD68 staining and HM / hybrid, HM and α-2,6- sialylated, ICAM-1 respectively. It was not clear how author made this correlation graph. Author should provide some direct evidence to prove this statement that CD68 expression higher in HM/hybrid not other ICAM1 N-glycoformes. Partial ligation and HFD are well establish model for atherosclerosis. Authors used only male mice without justification for the exclusion of female mice. Author should comment on this in discussion. Overall, this manuscript does not fit for publication in PLoS One as it currently stands. Reviewer #3: The current study showed correlation of high mannose (HM)-ICAM1 N glycoforms and a-2,6 sialylated ICAM1 N-glycoforms with macrophage burden in the lesions in human and mouse atherosclerosis, and in AVF maturation failure in hemodialysis patients. HM-ICAM1 N glycofoms and sialylated ICAM1 N-glycoforms were assessed by proximity ligation assay, while macrophage burden were assessed by CD68 staining. This is an extension of author’s previous studies. Findings are novel and important. There are several issues that should be addressed to make manuscript stronger. 1) Overall, image quality except Figure 7A is poor. High resolution image will be needed, since this is a crux of current study. 2) To support HM-ICAM1 N glycoforms and a-2,6 sialylated ICAM1 N-glycoforms in lesions, they need to be shown by western analysis as well. 3) In panel 2F, 3D, 6A, please show macrophage staining by immunohistochemistry, instead of immunofluorescence. [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.0230358.r001
Revision 1
5 Feb 2020 Author Response Dear Dr. Fukai, Thank you for forwarding reviewer comments on our article “Assessment of ICAM-1 N-glycoforms in Mouse and Human Models of Endothelial Dysfunction”. We appreciate the reviewer’s constructive comments. In the revised manuscript, we have addressed the comments raised. Specific responses are provided below with edits in the text indicated by red font color. Thank you Sincerely Rakesh Patel, PhD Reviewer #1: The study by Regal-McDonald et al used PLA to investigate co-localization of specific N-glycan stuctures and ICAM-1 in mouse and human atherosclerotic arteries and human AVF of chronic kidney disease patients (models of endothelial dysfunction). The results demonstrated that hypoglycosylated and α-2,6-sialylated ICAM-1 N-glycoforms are present in atherosclerotic murine and human arteries and AVF patients. The results also indicated that HM-ICAM-1 levels positively correlated with macrophage burden in both murine and human atherosclerotic tissue. The study is timely but lacks functional information on the role of ICAM-1 N-glycoforms in atherosclerosis or AVF. 1. The figures of the manuscript are very low resolution. Author response: Image resolution has been increased. Al images are at 600dpi resolution per journal guidelines. 2. It would strengthen the findings of the manuscript if the authors confirm the results obtained using the innominate arteries by looking at total, HM / hybrid, a-2,6-sialylated, and a-2,3-sialylated ICAM-1 in the atherosclerotic mouse aorta. Author response: We did not have access to mouse aortic tissue, but have now included new data from the left carotid arteries from ApoE-/- mouse fed high fa diet. These vessels are commonly used to interrogate atherosclerosis disease mechanisms; see revised Fig 2. 3. How were the lesion and non-lesion segments identified? Author response: Human lesions were characterized by a cardiovascular pathologist using the defining characteristics of fibrosis, calcification, and fatty deposits. Mouse lesion segments were identified by the presence of increased neointimal hyperplasia compared to non-lesion segments. 4. Fig. 2 and 3 legends state that n=5 experiments were performed for CD68 immunostaining. The corresponding bar graphs show only 3 experiments. Author response: We apologize for this oversight. The manuscript has been updated with additional experiments and n numbers corrected accordingly. 5. What is the correlation coefficient for Fig. 4 studies? It seems there would be no correlation if only the closed symbols are taken into consideration. It is recommended to perform more experiments for the correlation studies. Author response: Additional replicates have been added and correlation analyses separated out into HFD-fed experiments and partial carotid ligation to promote clarity. 6. In the high fat diet model (Fig. 2; no ligation), there was atherosclerosis in the brachiocephalic artery and HM/hybrid, HM, a-2,6-sialylated and a-2,3-sialylated ICAM-1 were increased in lesion vs. non-lesion segments. If there is atherosclerosis in these mice but no correlation between CD68 and ConA or HHL puncta, the mechanisms regulating ICAM N-glycosylation may not be important for monocyte adhesion and macrophage accumulation. Author response: As explained above, additional experiments were performed. With these additional replicates and the separation of models in the correlation analysis, ConA and HHL had a significant positive correlation with CD68 staining in HFD-mice and partial carotid ligation. 7. Type 2 human atherosclerotic lesions show no ICAM-1 expression. The type 2 lesions are characterized by the presence of lipid-laden macrophages according to Stary et al (ATVB, 1995). These findings suggest that ICAM-1 (and its N-glycoforms) does not play a role at the early phase of monocyte transmigration in human atherosclerotic vessels. Author Response: We do not make the claim that ICAM-1 N-glycoforms play a role in early monocyte adhesion. However, the lack of ICAM-1 staining may also be due in part to detection limitations in our assay. We have included discussion of this point in the revised manuscript. 8. Fig. 6B legends n=10 is misleading. It seems only n=5 samples per group was involved in the analysis. The authors need to carefully check the legends for accuracy Author response: Additional data was added to manuscript and legends adjusted accordingly. 9. This is an observational study and no functional experiments have been included to investigate the role of different ICAM-1 N-Glycoforms in the development of atherosclerosis or pathogenesis of AVF maturation failure. The manuscript would be strengthened by the addition of mechanistic data. Author response: Recently published work out of our lab demonstrates that different ICAM-1 N-glycoforms selectively recruit monocyte subsets under flow. Please see citation # 18 in the manuscript. 10. The rational for including the AVF data in the manuscript is not entirely clear. The mechanisms, leading to endothelial dysfunction in AVF failure vs atherosclerosis is different. Author response: Mechanisms leading to AVF do indeed differ from atherosclerosis, but endothelial dysfunction as a result of turbulent blood flow is an underlying component of both diseases states. We wanted to test distinct ICAM-1 N-glycoforms were also present in other disease settings where vascular inflammation and turbulent blood flow are underlying features. This is also further discussed in the manuscript. Reviewer #2: This is a novel approach to assessment of ICAM-1 N-Glycoforms in mouse and human models of endothelial dysfunction. In this paper using the proximity ligation assay (PLA), authors assessed the relative formation of high mannose, hybrid and complex α-2,6-sialyated N-glycoforms of ICAM-1 in human and mouse models of atherosclerosis, as well as in arteriovenous fistulas (AVF) of patients on hemodialysis. 1. To establish the distinct ICAM1-N-glycoforms in inflammatory disease in ivvivo authors solely used PLA assay. Indeed this assay has some limitation, however the PLA positive red puncta was not clear in all images throughout the manuscript. As this data represents the first study to demonstrating ICAM1-N-glycoforms invivo authors should provide convincing images with good quality pictures for PLA assay. Author response: Image resolution has been increased. 2. There was no description for Figure 2F and 2G in result section. Author should mention about these two figures. Author response: We apologize for this oversight. The manuscript has been edited to include these figures in the results section. 3. In result section, Figure 3A author described that total ICAM-1, HM / hybrid N-glycoforms were increased in the ligated left carotid artery (LC) compared to the control right carotid artery (RC). But this statement was not matches with the Figure 3A. It looks like total ICAM1 and α-2,6-sialylated ICAM1 expressed more than HM/hybride. Fig 3C quantification was not reflects the Fig 3A images. Author should be careful about this discrepancy. Author response: Analysis of PLA puncta is between paired LC and RC sections, not across lectins (i.e., α-2,6-sialylated ICAM-1 vs. HM/hybrid ICAM-1). Direct comparison between lectins is not possible as each have different binding affinities to their target epitopes- similar to how two different antibodies may bind with distinct affinities. The presented data is to show an increase in the ICAM-1 N-glycoforms in the ligated vessel compared to the non-ligated vessel (LC vs RC). We have clarified this point in the text. 4. In Fig 4 and Fig 6 show correlations between CD68 staining and HM / hybrid, HM and α-2,6- sialylated, ICAM-1 respectively. It was not clear how author made this correlation graph. Author should provide some direct evidence to prove this statement that CD68 expression higher in HM/hybrid not other ICAM1 N-glycoformes. Author response: Correlation studies were performed by measuring CD68 staining in the same (serial) sections on which PLA staining was performed. In the case of HM and hybrid ICAM-1, CD68 staining increased as puncta increased, indicating a positive association between the two. Statistical analyses were performed using Pearson correlation. Reviewer #3: The current study showed correlation of high mannose (HM)-ICAM1 N glycoforms and a-2,6 sialylated ICAM1 N-glycoforms with macrophage burden in the lesions in human and mouse atherosclerosis, and in AVF maturation failure in hemodialysis patients. HM-ICAM1 N glycofoms and sialylated ICAM1 N-glycoforms were assessed by proximity ligation assay, while macrophage burden were assessed by CD68 staining. This is an extension of author’s previous studies. Findings are novel and important. There are several issues that should be addressed to make manuscript stronger. 1) Overall, image quality except Figure 7A is poor. High resolution image will be needed, since this is a crux of current study. Author response: Image resolution has been increased. 2) To support HM-ICAM1 N glycoforms and a-2,6 sialylated ICAM1 N-glycoforms in lesions, they need to be shown by western analysis as well. Author Response: By western blot analyses, we have shown previously that ICAM-1 immunoprecipitated from atherosclerotic lesions has a MWt close to 75KDa suggesting it is a HM-glycoform. Our published studies show that ICMA-1 with complex N-linked sugars has a MWt closer to 100KDa. This information is included in the revised discussion. 3) In panel 2F, 3D, 6A, please show macrophage staining by immunohistochemistry, instead of immunofluorescence. Author Response: Immunofluorescence has been used extensively to assess CD68 staining in lesions, and per our experience there are no pros/cons in IF vs IHC for this marker. We have added an IgG staining control to Figure 6 to further verify specificity of the antibodies used for IF-based detection of CD68. Attachments Attachment Submitted filename: PLoS response final.docx https://doi.org/10.1371/journal.pone.0230358.r002
28 Feb 2020 Decision Letter - Tohru Fukai, Editor Assessment of ICAM-1 N-Glycoforms in Mouse and Human Models of Endothelial Dysfunction PONE-D-19-20764R1 Dear Dr. Rakesh Patel, 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, Tohru Fukai 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 #1: All comments have been addressed Reviewer #3: 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 #3: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #3: 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 #3: 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 #3: 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 #3: Revised manuscript has been improved, since image quality has been improved and necessary information was added. No further comments. ****** 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 #3: No https://doi.org/10.1371/journal.pone.0230358.r003
Formally Accepted
3 Mar 2020 Acceptance Letter - Tohru Fukai, Editor PONE-D-19-20764R1 Assessment of ICAM-1 N-Glycoforms in Mouse and Human Models of Endothelial Dysfunction Dear Dr. Patel: 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 Dr. Tohru Fukai Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0230358.r004

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