PONE-D-24-36911GATA2 participates in protection against hypoxia-induced pulmonary hypertensionPLOS ONE

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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: No

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: No

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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: No

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: Yes

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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: The current reserch describes the role of the transcription factor GATA2 in in the context of pulmonary hypertension and hypoxia-induced vascular damage. The authors found that GATA2 deficiency predisposes the pulmonary vasculature to damage and fibrosis in response to hypoxia, contributing to pulmonary hypertension. The study topic is of interest but the experiments are poorly designed and lack some key data to support the role of GATA2 in PH model.

1.The authors used Rosa26-CreERT to induce systematic GATA2-KO in mice model. It's better to also include cell specific GATA-KO data (such as using Cdh5-CreERT2, Myh11-CreERT2...) to study which cell type contribute to the GATA2 regulation of PH. Moreover, data showing diminished GATA2 expression in the KO mice tissue are needed to demostrate the knockout efficiency.

2. The study don't have the Right ventricular systolic pressure (RVSP) data to support the conclusion of the role of GATA2 in PH. The RVH measuement is flawed since they used formaldehyde-fixed dry hearts in stead of fresh tissues. The narrow histological difference shown in Figure 3 may come from gender or age difference of animals since the authors used 10 to 16-week-old Gata2 CKO and control mice in the hypoxia study.

3. The whole mice hypoxia study need normoxia controls to show the effects of hypoxia. The authors mentioned the housing of normoxic mice in the methods section but no data shown in the manuscript.

4. In figure 2, the authors show histological data of Gata2 GFP knock-in mice. However, there is no information about this strain provided in the Methods section. Do this mice strain show different PH phenotypes under hypoxia?

Reviewer #2: In this article, the authors used animal models, tissue immunohistology analyses, cell culture-based analyses, and bioinformatic analyses with public-available data bases to show the important roles that GATA2 may be involved in pulmonary arterial endothelial cells fate and pulmonary vascular remodeling. This research is a pioneer work in further understanding the progression of pulmonary vascular remodeling in pulmonary hypertension. Although the authors have provided some solid evidence to indicate the roles and functions of GATA2, there are major and minor issues that the authors should address. The authors can also refer to the attached file for revision suggestions.

Major revisions:

1. In Figure 2 and “GATA2 is abundantly expressed in endothelial cells of the pulmonary vasculature” section, the authors used “Gata2 GFP knock-in mice”. However, the development of the strain is not included in the “Methods” section, and it is recommended to include genotyping results in the supplemental figures. Please also do not forget to include the sequences that are used in the genotyping in Table 1.

2. The authors used the word “control” for many groups (Groups 2, 3 and 4) of mice, but the text did not clearly define which control was used for analysis. It is suggested that the authors just use Group name, instead of using “control”, when presenting the data. For example, what control is used in Line 339

3. In addition to Major revision #2 above, the authors claimed that mice were also kept in normoxic condition; however, these mice were not presented in data. These mice are essential controls to verify the success of hypoxia-induced vascular remodeling. The authors should show that G2f/f mice not bearing the CreERT allele and not treated with 4-OHT developed mild pulmonary vascular remodeling after hypoxia exposure, compared to these mice kept in normoxic condition. This data can be included in supplemental data.

4. In addition to Major revision #2 and #3 above, the authors claimed GATA2 is involved hypoxia-induced pulmonary hypertension; however, what the authors have performed only showed the importance of GATA2 in hypoxia-induced pulmonary vascular remodeling. In order to extend the argument to pulmonary hypertension, right ventricular pressure is required, which will be further supported with right ventricular hypertrophy analysis. The authors should either include the right ventricular pressure data, or correctly conclude their results to be limited to hypoxia-induced pulmonary vascular remodeling, but not pulmonary hypertension.

5. The authors showed that Bmp4 was decreased in G2-CKO lung endothelial cells, but to claim the trend “mirrored” siGATA2 knockdown in HMVEC is not entirely correct. The authors should notice that siGATA2-1 knockdown in HMVEC did very minimum effect on Bmp4 gene expression in HMVEC.

Minor revisions:

1. In Line 186 and Figure 5, there is no statistical analysis.

2. In Figure 7B

a. The spelling for change is not right

b. Use Log2(fold change) instead. Explain the meaning of fold change in figure legend.

3. From Line 211 to Line 219, the authors called for Figures 7B and 7C. However, it is not very reader-friendly to call 7C earlier than 7B. Please find a way to make the figure calling in order.

4. Is the induced GATA2 KO in mice global? If it is a global GATA2 KO, did the authors find any phenotype represent myelodysplastic syndromes? Have the authors ever thought of using heterozygous deletion to mimic the GATA2 haploinsufficiency?

5. The authors are suggested to use abbreviations with consistency. For example, Gata2 CKO in Line 349 should be called G2-CKO to avoid confusion.

6. In Line 376, the authors used VWT to represent pulmonary artery wall thickness; however, there is no full name for this abbreviation.

7. From Line 378 to 380, the authors explained to use Ed and Id to calculate wall thickness. In reality, pulmonary artery’s Ed and Id are not consistent. How did the authors average the parameters within one artery is not clear. Please specify.

8. In Line 383 and 384, the authors claimed that they used rabbit anti-GFP and rabbit anti-CD31, while Figure 2C,D showed co-staining of GFP and CD31. If the authors used a specific kit to facilitate this co-staining, please include.

9. From Line 399 to 405, the authors described a TUNEL assay for pulmonary smooth muscle cells. However, there is no data showed in the article.

10. Please also include a brief description of statistical analysis before “Data availability”.

11. Some captions were covered up in Figure 1A

12. Figure 2 legend mentioned panels E and F, but they were not included in the figure.

13. In Figure 3 legend, Line 483, the authors mentioned a “table”, but it is not Table 1. Please include this table after revised.

14. In Figure 4 legend, Line 489, the authors explained they used arrowheads, but there are no arrowheads in Figure 4A

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Reviewer #1: No

Reviewer #2: Yes: Yuanjun Shen

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Attachments

Attachment

Submitted filename: Major revision suggesion.docx

GATA2 participates in protection against hypoxia-induced pulmonary vascular remodeling

PONE-D-24-36911R1

Dear Dr. Moriguchi,

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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You-Yang Zhao

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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

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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 addressed the reviewers' comments and corrected all the minor issues in the manuscript.

Reviewer #2: With the modifications that the authors made, and additional discussions of their figures, I am overall satisfied.

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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

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