PONE-D-24-46813Inhibition of PHB1/PHB2 suppresses atherosclerotic plaque formation by interrupting PI3K/AKT/mTOR signalingPLOS ONE

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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: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Partly

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: 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

Reviewer #3: Yes

Reviewer #4: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes

Reviewer #4: 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: In the present manuscript, Li and colleagues showed that PHB1/PHB2 levels were elevated in serum of patients with hyperlipidemia. They further explored the effect of PHB1/PHB2 inhibition in the ApoE-/- fed with HFD murine model. They demonstrated that inhibition of PHB1 or PHB2 reduces the atherosclerosis progression. Mechanistically, they found that downregulating PHB1/PHB2 expression under inflammatory stimulation reduced the adhesion, proliferation, migration, and autophagy of ECs and VSMCs by inhibiting the PI3K/Akt/mTOR pathway activation. They have concluded that PHB1/PHB2 may play an important role in the promotion of endothelial and vascular smooth muscle cell injury. Thus, they could serve as a therapeutic target for atherosclerosis.

This finding is novel. Data are presented with qualitative and quantitative analysis; which is convincing. The whole manuscript could have been written in a more concise manner. Many methods/approaches/observations providing the same information repetitively appear in different sections throughout the manuscript. Extensive edition on the manuscript writing is warranted.

Here are two suggestions to scientifically improve the manuscript.

1. PHB1 and PHB2 are physically associated and functionally related proteins. It would be interesting to see the effect of double knockdown PHB1 and PHB2 in the AS progression. Are they functionally redundant?

2. Could you please provide a deeper discussion on the significance of the finding? Including The current knowledge on PHB1/PHB2 on VSMCs and ECs, future directions beyond this manuscript.

Minor points:

1. In figure 4B, the representative images and quantitative data do not indicate that overexpression of PHB1 increases THP-1 cell adhesion in the presence of il-8, as stated in Line 416

2. The order of Fig 4A and 4B is reversed, as the findings of VCAM-1 and ICAM-1 come later in the manuscript. (line 419)

Reviewer #2: This study reports that PHB1/PHB2 are up-regulated in AS tissues and cell lines. The knockdown of PHB1/PHB2 inhibits the adhesion of monocytes to ECs as well as proliferation, migration, and autophagy of IL-8-induced VSMCs, which might be mediated by the PI3K/Akt signaling pathway.The authors’ work represents PHB1/PHB2 as a potential therapeutic target for the treatment of AS in the clinical setting. The experiments are informative and sand the results are persuasive. However, there are still several comments that should be addressed:

1.In Figure 2D, hematoxylin and eosin staining and Masson’s staining of aortic plaques cannot reflect activity, please modify.

2.The confocal microscopy for PHB in Figure 3D appears to be located in organelles. They are not diffused in the cytosol and more likely punctalized.

3.The protein expression level in Figure 4A is not significant, please reassign the picture.

4.THP-1 is not strictly a monocyte.It should be induced to differentiate into monocytes to enhance the persuasiveness of experiments.

5.Why choose MMP9 over other members of MMPs? Moreover, Gelatin Zymography method should also be used to detect the activity of MMP9 for the reason that Western blotting cannot demonstrate that PHB1 and PHB2 inhibit IL-8-induced VSMC migration.

6.When the Akt downstream signaling pathway was activated, phosphorylated Akt levels were increased and both PHB1 and PHB2 were up-regulated compared to IL-8-treated A7r5 cells. If the AKT signaling pathway is down-regulated, will the levels of both PHB1 and PHB2 also decrease?

Minor:

1.It will be more convincing if the clinical cases can be more.

2.In Figure S3 B,why is there no picture that show the protein level of PHB2 in AoSMC ?

3.In Figure 4, there is no statistical significance in OE-PHB1.In addition, why is there no PHB2?

Reviewer #3: Dear Editor/Authors,

I am writing to inform you that I have completed my review of the manuscript titled " Inhibition of PHB1/PHB2 suppresses atherosclerotic plaque formation by interrupting PI3K/AKT/mTOR signaling” and I am pleased to accept it for publication in its current form. After careful consideration, I have found the manuscript to be well-written, well-structured, and of high quality. The authors have presented their research in a clear and concise manner, and the manuscript meets all the journal's requirements.

I have recommended minor revisions mentioned in PDF file as comment, and I believe the manuscript should be accepted for publication. I have checked the manuscript for its originality, relevance, and impact, and I am confident that it will make a significant contribution to the field.

Please let me know if you need any further information from me. I appreciate the opportunity to review this manuscript and look forward to seeing it in print.

Best regards,

Muhammad Asmat Ullah Saleem

Reviewer #4: Major Comments:

Please cite the relevant figures for lines 353-355, 362-365, and 380-382 (show data supporting PHB localization in the cytoplasm).

Please comment on why PHB1/PHB2 levels are lower at 100 ng/mL in Fig S3—is this caused by cell death?

Does Figure 3D correspond to stimulated or unstimulated conditions? How does the localization of PHB1 and PHB2 vary under both conditions?

Please discuss the text in the order that the results are displayed—figure 3D is discussed before 3C.

The legend for figures 3C and 3D are swapped.

How is pex-PHB1 overexpressed? Please elaborate in the methods section.

Figure 4 and its legend are confusing. Significant improvement in clarity is needed for figure 4.

1) What are the two controls in 4A? The legends for 4A and 4B are flipped.

2) No obvious change in ICAM1 and VCAM1. Confirmation using alternative methods such as immunofluorescence will further boost confidence.

3) Does overexpression of PHB1 increase ICAM1 and VCAM1 expression levels using western blot in figure 4? Please

show.

For autophagy experiments, it is important to confirm the flux of autophagy to make sure the increase in autophagosomes is a result of autophagy activation and not due to blockage of clearance. Please see doi: 10.1080/15548627.2020.1797280 on how to measure flux.

Need more text elaborating on the findings in figures 8H, I, J, K, and L.

Activation of the AKT-mTOR axis as shown in figures 8A, B, C, and D should inhibit autophagy, but the results claim otherwise. Please address this discrepancy.

Activation of AKT using the AKT agonist SC79 should inhibit autophagy (see DOI: 10.1007/s11064-023-04057-w). The results appear contradictory; please address this.

Minor Comments:

In lines 102-103, should it be “IL-8 inducing AS” and not the other way around?

Color code aSMA, PHB1, and DAPI for better clarity in figure S1B.

Please point out the specific changes with arrows to guide the audience in S1.

Please include a scale bar on the last panel of figure S1B.

Please cite literature for lines 374-375, 458-459, and 469.

Lines 359-361 can be structured better for clarity. Both uninfected and infected mice were fed with a normal diet, correct? The main difference is whether they are infected or not.

Please mention each abbreviation used in the figure in the legend for all figures.

Figures 7C and 7D: the y-axis label could be more accurate. Does it indicate expression of the protein relative to alpha-tubulin?

For figure 8B, are GAPDH levels measured? Please check the y-axis.

Please mention where SC79 is sourced from in the materials/methods section. Please check grammar and improve clarity.

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Reviewer #1: Yes:  Ying Wang

Reviewer #2: Yes:  Wuyang Wang

Reviewer #3: Yes:  Muhammad Asmt Ullah Saleem

Reviewer #4: No

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Submitted filename: PONE-D-24-46813 Reviewd file.pdf

Inhibition of PHB1/PHB2 suppresses atherosclerotic plaque formation by interrupting PI3K/AKT/mTOR signaling

PONE-D-24-46813R1

Dear Dr. Gao,

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,

Alexis G. Murillo Carrasco

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

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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

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

Reviewer #1: I Don't Know

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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

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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: No

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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 quality of revised manuscript has been significantly improved. Minor English editing is needed to make it more concise and informative.

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