PONE-D-24-43059Tie-2 regulates endothelial morphological responses to shear stress by FoxO1-triggered autophagyPLOS ONE

Dear Dr. Shirakura,

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The subject of the paper is interesting, but needs revision to be acceptable.

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PONE-D-24-43059R1Tie-2 regulates endothelial morphological responses to shear stress by FoxO1-triggered autophagyPLOS ONE

Dear Dr. Shirakura,

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 Mar 30 2025 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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We look forward to receiving your revised manuscript.

Kind regards,

Saiedeh Razi-Soofiyani

Academic Editor

PLOS ONE

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Additional Editor Comments:

Please revise the paper based on reviewers comments.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

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Reviewer #1: Partly

Reviewer #2: Yes

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

Reviewer #1: I Don't Know

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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6. Review Comments to the Author

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Reviewer #1: This study investigated whether and how Tie2-FoxO1 axis is involved in flow-induced morphological changes in EC. There are several major concerns about the methodology used and the validity of conclusions. Moreover, the quality of some data need to be improved.

Major issues

Figures 1B, 3B, 5B lack statistical results; we cannot identify whether the effects are significant or not.

Similar to the data in Fig S1 and S2, the gene silencing efficacy by Tie2 siRNA should be presented.

Please clarify whether the data in FigS1 and S2 were from a single experiment only? If this is the case, these results should be clearly labeled as preliminary.

Data in Fig 4A to 4D: These fluorescent images of LC3B are not representative; it is difficult to identify the differences between groups, despite the numerical data showing statistical significances. High resolution images which highlight the differences in more detail are required to validate the numerical data. Moreover, autophagy is characterized by formation of LC3 puncta; it is not clear what “LC3 area” stands for, and how it is measured. These methodological details are critical and need to be added.

Figure 5A to 5C: it is obvious that Atg5 silencing had little effects on flow/Tie2-induced EC morphogenesis responses, indicating that autophagy induction is not mediating the actions. These data appear to be against the conclusion proposed by the authors.

Data analysis: A number of tests and post hoc detection methods were mentioned in the methods section. However, as indicated in the figure legends, only two methods (two-way ANOVA followed by Tukey’s multiple test and Mann–Whitney U test) were used. Please clarify.

Minor points

Page 9 line 178-181 (in the word document): Besides the question, whether FoxO1 is at all involved in the process, it was particularly interesting to know, whether it is the loss of the transcriptional activity of FoxO1 in the nucleus or whether it is a function of FoxO1 in the cytoplasm which involved in morphogenesis responses to flow. This sentence is hard to follow, please rephrase.

Page 9-10 line 188-190: This strongly suggests that it is not the inhibition of FoxO1 transcriptional activity in the is some function of FoxO1 in the cytoplasm which is needed for cellular morphological responses to shear stress. This sentence is hard to follow, please rephrase.

Figure 5: Results of validation of Atg5 silencing on autophagosome are to be presented first, well before its main actions on EC morphology.

Reviewer #2: This study identifies a novel Tie-2-FoxO1-autophagy pathway critical for endothelial alignment under shear stress. While the data are compelling, addressing the above points will enhance mechanistic clarity, validate key conclusions, and broaden the impact of the work. However, the paper still needs some improvements before acceptance for publication.

My detailed comments are as follows:

1. I don’t think “FoxO1” in the title is the standard name, FOXO1 is more commonly used in the literature, please check that.

2. Could the authors provide any evidence to expose HUVECs for 24 hrs to 15 dyn/cm2 shear?

3. Why does Atg5 silencing only partially inhibit alignment (e.g., test additional autophagy genes like ULK1 or Beclin-1)?

4. Clarify the rationale for Angpt1 concentration in 600 ng/ml, please cite previous studies or pre-experimental optimization.

5. This revised version still has some typos, such as “mechansims” should be “mechanisms”, “microgrpahs” should be “micrographs”.

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Reviewer #1: Yes:  Fan JIANG

Reviewer #2: No

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Tie-2 regulates endothelial morphological responses to shear stress by FOXO1-triggered autophagy

PONE-D-24-43059R2

Dear Dr. Keisuke Shirakura,

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.

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Kind regards,

Saiedeh Razi-Soofiyani

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

The paper was revised precisely based on reviewers comments. Congratulation.

Reviewers' comments: