A novel assay to measure low-density lipoproteins binding to proteoglycans

Esmond N. Geh; Debi K. Swertfeger; Hannah Sexmith; Anna Heink; Pheruza Tarapore; John T. Melchior; W. Sean Davidson; Amy Sanghavi Shah

doi:10.1371/journal.pone.0291632

Peer Review History

Original SubmissionDecember 23, 2022
3 Mar 2023 Decision Letter - Boyan Grigorov, Editor PONE-D-22-35204A Novel assay to measure low-density lipoproteins binding to proteoglycansPLOS ONE Dear Dr. Geh, 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. One of the reviewers pointed a major problem concerning the cell model. Your immunofluorescence images should be improved. Close-ups are necessary and also if you speak about colocalzation, confocal microscopy should be performed. Please submit your revised manuscript by Apr 17 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. 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The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide 3. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section 4. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide [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: Partly Reviewer #2: No ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: I Don't Know Reviewer #2: No ****** 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 ****** 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 ****** 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: This study (Ms #: PONE-D-22-35204) sought to develop a novel, rapid, sensitive, and reproducible method for measuring low-density lipoproteins (LDLs) binding to proteoglycans (PGs) in combination of immunofluorescence microscopy with in-cell ELISA (ICE) technique (this technique is to determine LDL bound to PGs by quantifying ApoB). To test their method, mouse vascular smooth muscle (MOVAS) cells were used to produce PGs (extracted PGs and purified chondroitin sulfate or CS were also utilized), and LDLs were isolated from adults without (as a control) or with cardiovascular disease. By using the novel method, the authors confirmed the previously reported result that the LDLs from adults with cardiovascular disease have a higher affinity to PGs than those from healthy adults. LDL affinity to extracellular matrix (ECM; e.g., PGs) is a very important step in the initiation and development of atherosclerosis according to the retention hypothesis of atherogenesis. Therefore, any efforts to develop methods for detecting LDL-ECM/PGs affinity or interaction are encouraged and applausive. This manuscript can be considered for publication after addressing my following comments: 1. The background, applications (particularly its application in LDL and LDL-ECM/GP interaction if available), and advances of the ICE technique, should be described in the “Introduction” section. This description will help emphasize the novelty of this study. 2. Lines 83-85: The detailed information on the ultracentrifugation (e.g., speed, temperature, and time) should be provided. The abbreviation “UC-LDL” should be defined at the firm mention. Line 130: I suggest to remove the abbreviation UC and replaced with its full name (ultracentrifugation?). 3. In the “Materials and methods” section, the “Study participants” part should be moved to the location before or after the “lipoprotein isolation” part because the lipoproteins were isolated from the participants. 4. Line 125: There are two “overnight”. 5. Fig. 1: Based on the displayed 2-D fluorescence images, it is hard to determine whether the fluorescences (or molecules, e.g., ApoB and CS) are located pericellularly (or on the cell surface) or within the ECM or in cells. According to my experience, Fig. 1 (particularly the cells indicated by the white arrows) shows that the CS fluorescence is located mainly inside cells and that the ApoB fluorescence is located pericellularly (or on the cell surface). The 3-D fluorescence images should be provided to determine the location/co-localization of ApoB/CS. By the way, the legend to Fig. 1D,H is missing. 6. Besides CS, other types of PGs (e.g., heparin and dermatan sulfates) should also be detected by immunofluorescence imaging to determine CS is the major type of PGs produced by MOVAS cells and the major factor responsible for the LDL binding. If other types of PGs also contribute to the binding of LDL, the evidence from the experiments only testing LDL-CS interaction/co-localization is not solid enough to support the conclusions in this study. 7. Lines 208-209: Fig. 2A shows that LDL binds to MOVAS cells in a dose dependent manner which saturated at about 1 mg/mL. I cannot see the saturated concentration of LDL based on Fig. 1A (it seems that the bound LDL is still increasing at 2 mg/mL). 8. In this study, the authors used chondroitin sulfate antibody (alpha-CS) to block the CS-LDL interaction sites. However, it seems that the CS-LDL interaction sites (electrostatic interaction) are perhaps not overlapped with the CS-alpha-CS interaction sites (antigen-antibody interaction). Perhaps, a better interaction-blocking experiment is to block the negative charge of CS? 9. Fig. 4: The authors found that low pH enhances LDL binding to ECM/PGs/CS. However, a question may arise whether the influence of pH on the biomechanical properties of LDL particles also contributed to the changes in LDL binding to ECM/PGs/CS? This question should be addressed in the “Discussion” section. For example, it has been previously reported that low pH could not change the stickiness of LDL particles (Ref. 1) although the oxidation could increase the stickiness of LDL particles (Ref. 2). [Refences: (1) Wang K. etc. AFM detects the effects of acidic condition on the size and biomechanical properties of native/oxidized low-density lipoprotein. Colloids and Surfaces B: Biointerfaces. 2021. 208: 112053; (2) Wang K. et al. Dynamic AFM detection of the oxidation-induced changes in size, stiffness, and stickiness of low-density lipoprotein. Journal of Nanobiotechnology. 2020. 18: 167]. 10. Line 265: The abbreviation CV should be defined at the first mention. All abbreviations in Table 1 should be defined. 11. Legend to Fig. 5: (a) The abbreviations CBB and FPLC should be defined; (b) Fig. 5D-F is missing; (c) The numbers 1-5 (or I-IV) on each graph should also be defined. 12. It will be better to compare the current method with other existing methods in the “Discussion” section based on the experimental data. 13. The authors should carefully check the whole “Discussion” section to correct potential typos and gramma errors. Reviewer #2: This study by Geh and coworkers aims at designing a robust and reproducible assay to measure the binding of low-density lipoproteins (LDL) to proteoglycans, in view of a screening of blood samples from a population at risk of atherosclerosis. Although the idea is elegant, this study has a major flaw. Indeed, the core of proteoglycans harbour highly specific sugar molecules (GAG), chemically-modified at several places of the sugar ring (epimerisation, phosphorylation, sulfatation...). This high level of specificity is species-dependent (as pinpointed in Nikpour et al Glycobiology 2021, only to cite one). In consequence, using a mouse cell line to address such a question does not properly recapitulate the proteoglycan diversity in terms of GAG of a human organ/tissue. Experiments should then be performed on a human cell line. Minor point. With a p value < 0.05, the method should not be considered as "highly sensitive". ****** 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? 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Revision 1
25 Jul 2023 Author Response Dear Editor and Reviewers, We greatly appreciate the editor and reviewers’ comments and suggestions in reviewing our manuscript entitled. We have carefully considered the comments and recommendations and made corresponding revisions to the manuscript. Below are our responses to each specific points. Editor Comment. One of the reviewers pointed a major problem concerning the cell model. Your immunofluorescence images should be improved. Close-ups are necessary and also if you speak about colocalization, confocal microscopy should be performed. As suggested, we conducted an experiment using a human cell line (human ascending aorta smooth muscle) and we have included confocal microscopy images that show colocalization of the LDL with chondroitin and heparan sulfate in both mouse and human cell lines. Reviewer 1. 1. The background, applications (particularly its application in LDL and LDL-ECM/GP interaction if available), and advances of the ICE technique, should be described in the “Introduction” section. This description will help emphasize the novelty of this study” As recommended, this important bit of information has been added to the “Introduction” section of revised manuscript (lines 60-64). 2. Lines 83-85: The detailed information on the ultracentrifugation (e.g., speed, temperature, and time) should be provided. The abbreviation “UC-LDL” should be defined at the firm mention. Line 130: I suggest to remove the abbreviation UC and replaced with its full name (ultracentrifugation?). The revised manuscript now includes the information on speed, temperature, and time in the "Materials and Methods" section (lines 100). Additionally, we defined the abbreviation "UC-LDL" at its first mention and replaced UC in line 150. 3. In the “Materials and methods” section, the “Study participants” part should be moved to the location before or after the “lipoprotein isolation” part because the lipoproteins were isolated from the participants. As suggested, the "Study participants" part, has been moved just before “lipoprotein isolation” (see line 88), 4. Line 125: There are two “overnight”. We have corrected this duplication (see line 144). 5. Fig. 1: Based on the displayed 2-D fluorescence images, it is hard to determine whether the fluorescences (or molecules, e.g., ApoB and CS) are located pericellularly (or on the cell surface) or within the ECM or in cells. According to my experience, Fig. 1 (particularly the cells indicated by the white arrows) shows that the CS fluorescence is located mainly inside cells and that the ApoB fluorescence is located pericellularly (or on the cell surface). The 3-D fluorescence images should be provided to determine the location/co-localization of ApoB/CS. By the way, the legend to Fig. 1D,H is missing. To address this concern, we now include confocal microscopy images showing colocalization of APOB together with CS and HS in MOVAS (Fig 1), and human aortic vascular smooth muscle cells (supplemental S1 Fig) in the manuscript. The missing figure legends have also been included. 6. Besides CS, other types of PGs (e.g., heparin and dermatan sulfates) should also be detected by immunofluorescence imaging to determine CS is the major type of PGs produced by MOVAS cells and the major factor responsible for the LDL binding. If other types of PGs also contribute to the binding of LDL, the evidence from the experiments only testing LDL-CS interaction/co-localization is not solid enough to support the conclusions in this study. We now include confocal images of both heparin sulfate and chrondrotin sulfate. As CS has the highest affinity, in terms of binding to LDL than other proteoglycans, we have focused on CS but acknowledge other PGs may contribute to binding. (Reference: Mourão PA, Pillai S, Di Ferrante N. The binding of chondroitin 6-sulfate to plasma low density lipoprotein. Biochim Biophys Acta. 1981 May 5;674(2):178-87.) 7. Lines 208-209: Fig. 2A shows that LDL binds to MOVAS cells in a dose dependent manner which saturated at about 1 mg/mL. I cannot see the saturated concentration of LDL based on Fig. 1A (it seems that the bound LDL is still increasing at 2 mg/mL). Agree. We corrected this sentence by deleting the “which saturated at about 1 mg/mL” (lines 235). 8. In this study, the authors used chondroitin sulfate antibody (alpha-CS) to block the CS-LDL interaction sites. However, it seems that the CS-LDL interaction sites (electrostatic interaction) are perhaps not overlapped with the CS-alpha-CS interaction sites (antigen-antibody interaction). Perhaps, a better interaction-blocking experiment is to block the negative charge of CS? We agree. The CS-LDL interaction is an electrostatic interaction between positively charged LDL and negatively charged sulfate groups of CS. For this reason, in addition to antibody inhibition, we performed an endogenous inhibition of sulfation using sodium chlorate which is a 3′-phosphoadenosine 5′-phosphosulfate (PAPS) synthetase inhibitor (Fig 6D). Thus, reducing or indirectly blocking the amount of available sulfate groups not only on CS but also on other sulfate containing PGs. 9. Fig. 4: The authors found that low pH enhances LDL binding to ECM/PGs/CS. However, a question may arise whether the influence of pH on the biomechanical properties of LDL particles also contributed to the changes in LDL binding to ECM/PGs/CS? This question should be addressed in the “Discussion” section. For example, it has been previously reported that low pH could not change the stickiness of LDL particles (Ref. 1) although the oxidation could increase the stickiness of LDL particles (Ref. 2). [Refences: (1) Wang K. etc. AFM detects the effects of acidic condition on the size and biomechanical properties of native/oxidized low-density lipoprotein. Colloids and Surfaces B: Biointerfaces. 2021. 208: 112053; (2) Wang K. et al. Dynamic AFM detection of the oxidation-induced changes in size, stiffness, and stickiness of low-density lipoprotein. Journal of Nanobiotechnology. 2020. 18: 167]. We have made changes in the revised manuscript “Discussion” section to address this concern (see lines 398-402). In addition, we have added Ref 1 to the discussion section. 10. Line 265: The abbreviation CV should be defined at the first mention. All abbreviations in Table 1 should be defined. The abbreviations have been defined in line 296. 11. Legend to Fig. 5: (a) The abbreviations CBB and FPLC should be defined; (b) Fig. 5D-F is missing; (c) The numbers 1-5 (or I-IV) on each graph should also be defined. All the above changes have been made in the revised manuscript (see lines 320-321) 12. It will be better to compare the current method with other existing methods in the “Discussion” section based on the experimental data. We think that comparing methods based on experimental data could be challenging because of the variations in experimental protocols, specific antibodies and detection methods used. Additionally, most ELISA based techniques generally report lower limits of detection that vary 0.1 �g/mL to 20 �g/mL using a buffer that has a pH of about 5.5. In our case, we chose to use a physiologically relevant buffer, PBS at pH 7.4, and our limit of detection was about 0.5 �g/mL (Supplemental S4 Fig) so direct comparisons are challenging. We acknowledge existing methods with corresponding references. 13. The authors should carefully check the whole “Discussion” section to correct potential typos and gramma errors. The "Discussion" section has been revised to address grammatical errors and typos. Furthermore, new information has been incorporated into this section, which was not available at the time of submission. Some sentences that did not align with the discussion have been removed. Reviewer 2. 1. Although the idea is elegant, this study has a major flaw. Indeed, the core of proteoglycans harbour highly specific sugar molecules (GAG), chemically-modified at several places of the sugar ring (epimerisation, phosphorylation, sulfatation...). This high level of specificity is species-dependent (as pinpointed in Nikpour et al Glycobiology 2021, only to cite one). In consequence, using a mouse cell line to address such a question does not properly recapitulate the proteoglycan diversity in terms of GAG of a human organ/tissue. Experiments should then be performed on a human cell line. We appreciate your suggestion (see line 374). However, utilizing a human cell line expressing the LDL receptor presents challenges due to the binding of both human LDL and PGs and to the LDL receptor. We were unable to locate a human cell line that lacks the LDL receptor, particularly a vascular smooth muscle cell line. However, experiments were repeated in a human ascending aorta smooth muscle cells (HAAVAS) in parallel with the MOVAS cell line (supplemental S2 Fig). LDL binding in HAAVAS was over three times higher than in MOVAS, and we attributed a portion of this binding to the LDL receptor. Since the human LDL binds poorly to the mouse LDL receptor, most of the binding in the mouse cell line is attributed to PG binding. Maintenance cost of human cell lines is significantly higher, approximately 40 times more, making it financially impractical for widespread use and repeated experiments over time. Given the concerns with the human LDL receptor, high experimental costs, and data in human and mouse cell lines showing similar results, we believe that using the mouse cell line is a valid model to study LDL-PG interactions. 2. With a p value < 0.05, the method should not be considered as "highly sensitive". We have softened the language in the revised manuscript (see line 30). Attachments Attachment Submitted filename: Responses to Reviewers.docx https://doi.org/10.1371/journal.pone.0291632.r002
4 Sep 2023 Decision Letter - Boyan Grigorov, Editor A novel assay to measure low-density lipoproteins binding to proteoglycans PONE-D-22-35204R1 Dear Dr. Geh, 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, Boyan Grigorov 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 #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: I Don't Know 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: Most of my concerns have been addressed. Thank the authors for their contribution in this research field. 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.0291632.r003
Formally Accepted
15 Sep 2023 Acceptance Letter - Boyan Grigorov, Editor PONE-D-22-35204R1 A novel assay to measure low-density lipoproteins binding to proteoglycans Dear Dr. Geh: 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. Boyan Grigorov Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0291632.r004

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