PONE-D-21-05884

Effect of umbilical cord blood stem cell transplantation on restenosis after endovascular interventional therapy for diabetic hindlimb vascular disease

PLOS ONE

Dear Dr Wang

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Xianwu Cheng, M.D., Ph.D., FAHA

Academic Editor

PLOS ONE

Additional Editor Comments:

As the authors gather from the reviews, the referee identified substantive methodological and statistical problems as well as several data presentation and the conclusions are very confusing and not clear.

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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: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: Yes

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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

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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: No

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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: 1- Introduction- sentence structure is awkward (...due to its advantages such as small trauma and quick effect.)- consider revising

2-Introduction- I agree that percutaneous intervention has become indispensible for treating PAD; however, increasingly providers are using less indwelling stents and/or are using drug-eluting technologies to ameliorate the risk of neointimal hyperplasia- can the authors provide more context for this evolving practice pattern in their introduction?

3-Introduction- regarding the sentence: "After interventional treatment, local injection of cord blood stem cells...between intimal area and medium film area..." please clarify.

4-Introduction- can the authors provide clarification for why they targeted RCAN1 specifically for in-stent restenosis? There are a variety of other pathways that are implicated in neointimal hyplerplasia proliferation. I understand that there are multi-step regulatory check points that this targets but why this and not another molecule?

5-Methods- I appreciate that the authors have catalogued the materials used in the experiments; however, it is difficult to interpret without its appropriate context- is there a way to integrate the information offered in the 1st paragraph into subsequent aspects of the methods so this information doesn't sit unsupported or without specific context to an experiment?

6-Methods- STZ model, can the authors provide a reference for the 11.0mmol/L threshold to corroborate the diabetic state diagnosis?

7-Methods- rabbit model- if I am reading the manuscript correctly, rabbits underwent 'gentle ligation' of the femoral artery using 7-0 suture; however, the subsequent paragraph discusses 'successful remodeling' and then subsequent angioplasty -- what was being angioplastied if the femoral artery was surgically ligated? Also, in-stent restenosis was discussed in the introduction but angioplasty alone was used, was there a group of animals that received an intravascular stent?

8-Methods- cell phenotype identification- several sentences use non-scientific/peer reviewed publication appropriate nomenclature to define what occurred- would consider revision (e.g. 'sucked into the centrifuge', etc.).

9-Methods- Interventional therapy and HUCBSC- can the authors provide a study table and/or flow chart that outlines how the experiments were conducted?

10-Methods- there is a description of placing the balloon in the femoral artery but also proximal and distal occlusion with vascular clamps- how can the clamps occlude the vessel proximally and distally while the balloon is in place, wont the artery have the shaft of the balloon in it so one of the vessels cannot be fully occluded?

11- Discussion- the authors highlight stents in the discussion; however, they only present experiments for plain balloon angioplasty- please clarify the overlap since mechanisms for in-stent restenosis and neointimal hyperplasia at balloon angioplasty sites are somewhat different.

Reviewer #2: In this manuscript, the authors provided evidence showing that the mechanism of human umbilical cord blood stem cell (HUCBSC) transplantation on the restenosis of percutaneous transluminal angioplasty (PTA) in the diabetic hindlimb vascular disease via RCAN1, Can and JAK/STAT3 pathway. In PTA+HUCBSC group, the expression levels of RCAN1 and CnA were significantly lower than those in PTA group and the HUCBSC inhibited the migration and proliferation of HA-VSMC and suppressed the levels of JAK and STAT3.

The following shortcomings need to be addressed to strengthen the study.

1. Why use the paclitaxel (an anti-cancer agent) as a control group in the cell experiments and how many doses were used in the HA-VSMC cell lines.

2. Dil-labeling kit was used to detect the HUCBSC, detailed information of procedure should be provided.

3. The authors did not provide the methods of HUCBSC treatment in vitro, does it have co-culture of HUCBSC and HA-VSMC cells?

4. In Fig 1 and Fig3 legends fluorescence electron microscope was employed, but there are no electron microscope images in the paper, please check it.

5. In Fig 4, please provide more detailed information of methods for better understanding.

6. No. 3, 4, 5, 6, 13, and 22 of references are in Chinese, please address.

7. No. 11 of reference is showed “INVALID CITATION”, please check it.

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Reviewer #1: No

Reviewer #2: No

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Effect of umbilical cord blood stem cell transplantation on restenosis after endovascular interventional therapy for diabetic hindlimb vascular disease

PONE-D-21-05884R1

Dear Dr Wang

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.

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Kind regards,

Xianwu Cheng, M.D., Ph.D., FAHA

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

All original concerns have been addressed by the authors.

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

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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

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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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

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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

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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 provided satisfactory responses to all my questions and comments. I have no further questions.

Reviewer #2: Methods: the samples ..…..observed under a laser confocal microscope.

Fig 1 Third line: Fluorescence electron microscopy showed…...

Fig 3 First line: …...under fluorescence electron microscope.

but there are no electron microscope images in the Figs, please check it.

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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