Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The overall topic of the manuscript has high value for molecular mechanism research, focusing on core signaling pathways and disease/biological processes, and is well aligned with the scope of IJMS. The research concept is relatively complete, with the experimental design largely centered on the core hypothesis, and each major conclusion supported by corresponding experimental data. The overall structure of the manuscript is clear, with a logical flow from background to methods, results, and discussion. The number of figures is appropriate and well matched to the main text, allowing the key experimental findings to be presented intuitively. The Discussion interprets the results in the context of existing literature, demonstrating that the authors have a reasonably systematic understanding of the relevant research field.

1. In the Methods section, some key experimental conditions (such as treatment duration, number of replicates, or sources of critical reagents) are not described with sufficient precision; it is recommended to supplement these essential details to ensure experimental reproducibility.

2. The description of statistical methods is relatively general, and some comparisons do not clearly specify the statistical tests used; it is recommended to provide a one-to-one correspondence between analyses and statistical tests, and to clarify whether normality testing or multiple-comparison corrections were performed.

3. If multiple-group comparisons were conducted without specifying the use of P adjustment or other correction methods, it is recommended to clearly explain the statistical strategy to avoid overestimation of significance.

4. The Results section mainly emphasizes statistical significance, with insufficient discussion of effect size and biological relevance; it is suggested to supplement fold changes, percentage changes, or effect size descriptions without adding new experiments.

5. In some figures, axis units, abbreviations, or symbols are not clearly explained in the legends; it is recommended to provide complete explanations in the figure legends to improve figure self-consistency.

6. Most results are presented as mean ± SD/SEM without showing individual data points; it is suggested to overlay scatter plots based on the existing data to enhance data transparency.

7. Some results that are not fully consistent with expectations or show relatively small changes are insufficiently discussed; it is recommended to provide reasonable explanations based on experimental conditions or differences from published literature to strengthen the completeness of the Discussion.

8. The manuscript does not explicitly define study limitations; it is recommended to add a short paragraph discussing limitations such as sample size, model constraints, or technical limitations to enhance scientific rigor.

9. There are minor issues with grammar and word choice throughout the manuscript; professional English language editing is recommended prior to acceptance.

10. The fluorescence images are somewhat unclear, and higher-magnification images should be added.

Author Response

Reviewer_comments:  

 

We have highlighted all modifications in the main body of the manuscript in YELLOW.

 

Reviewer #1:

 

1. In the Methods section, some key experimental conditions (such as treatment duration, number of replicates, or sources of critical reagents) are not described with sufficient precision; it is recommended to supplement these essential details to ensure experimental reproducibility.

Answer: Thank you so much for your comment. All key experimental conditions are described in the manuscript, although they are not consolidated into a single dedicated Methods subsection. Specifically, treatment conditions and durations are reported within the corresponding Results sections (e.g., SFN treatment at 5 µM for 16 h in murine brain endothelial bEnd.3 cells). The number of biological replicates and statistical analyses are provided in each figure legend (e.g., densitometric quantification of NRF2, HO-1, TIE2, and CLDN5 protein levels normalized to VCL or GAPDH, expressed as mean ± SD; n = 3; *p < 0.05, ***p < 0.001 versus vehicle, Student’s t-test). In addition, detailed information on critical reagents, including reagent type, catalog number, supplier, and country of origin, is provided in Section 4.1 (Cell Culture and Reagents) of the Materials and Methods.

 

2. The description of statistical methods is relatively general, and some comparisons do not clearly specify the statistical tests used; it is recommended to provide a one-to-one correspondence between analyses and statistical tests, and to clarify whether normality testing or multiple-comparison corrections were performed.

Answer: Thank you so much for your comment. In addition, Section 4.11 (Image Analysis and Statistics) provides a summary of the statistical approaches used throughout the manuscript. Detailed descriptions of the specific analyses and statistical tests applied to each experiment, including normalization methods, number of replicates, and significance thresholds, are reported in the corresponding figure legends. Thus, all statistical procedures are fully described and transparently reported within the manuscript.

For instance, in figure 2, (B) Densitometric quantification of NRF2, HO1, TIE2, and CLDN5 protein levels from representative immunoblots of A expressed as a ratio of VCL and GAPDH, respectively. Data are mean ± S.D. (n = 3). *p < 0.05 and ***p < 0.001 vs. empty vector according to Student´s t-test. (C) Transcript levels of Hmox1, Nqo1 and Slc7a11 and (D) of Tek, Cldn5, Cdh5, Ocln, and Tjp1 from bEnd.3 cells transduced with NRF2∆ETGE or empty vector maintained under low-serum conditions (16 h, 1% FBS), were determined by quantitative real-time PCR (qRT-PCR) and normalized to the geometric mean of the levels of Gapdh, Tbp, and Actb. Data are mean ± S.D. (n = 3). **p < 0.01 and ***p < 0.001 vs. empty vector according to Student´s t-test, or (F) Densitometric quantification of NRF2, TIE2, and CLDN5 protein levels from representative immunoblots of E expressed as a ratio of VCL and GAPDH, respectively. Data are mean ± S.D. (n = 3). *p < 0.05 and ***p < 0.001 vs. time 0 according to a one-way ANOVA followed by a Bonferroni post-hoc test.

 

3. If multiple-group comparisons were conducted without specifying the use of P adjustment or other correction methods, it is recommended to clearly explain the statistical strategy to avoid overestimation of significance.

Answer: Thank you so much for your comment. The specific statistical methods used for multiple-group comparisons are provided in the figure legends of Fig. 2F and Supplementary Fig. 3B, where one-way ANOVA followed by Bonferroni’s post hoc test was applied. In addition, the legend of Fig. 4 has been updated to clarify the statistical analysis performed, including a brief description of the comparison between vehicle- and SFN-treated groups, by employing two-way ANOVA followed by a Bonferroni post-hoc test.

 

4. The Results section mainly emphasizes statistical significance, with insufficient discussion of effect size and biological relevance; it is suggested to supplement fold changes, percentage changes, or effect size descriptions without adding new experiments.

Answer: Thank you very much for your appreciation. We have added brief paragraphs at the end of each Results subsection (highlighted in yellow) to succinctly discuss the obtained findings and their potential biological relevance. Nevertheless, the full discussion and overall conclusions of the study are presented in detail in the Discussion section.

 

5. In some figures, axis units, abbreviations, or symbols are not clearly explained in the legends; it is recommended to provide complete explanations in the figure legends to improve figure self-consistency.

Answer: Thank you very much for your comment. In the figure legends, we did not explicitly describe axis units because these are indicated within the figures themselves and are intended to be self-explanatory (e.g., Hmox1 mRNA levels or NRF2 protein levels expressed as fold change). All abbreviations are defined in the appropriate section at the end of the manuscript. Statistical significance is denoted by symbols (***p < 0.001, **p < 0.01, *p < 0.05; ###p < 0.001, ##p < 0.01, #p < 0.05). Nevertheless, we appreciate the reviewer’s comment and are happy to further clarify or revise the figure legends if deemed necessary.

6. Most results are presented as mean ± SD/SEM without showing individual data points; it is suggested to overlay scatter plots based on the existing data to enhance data transparency.

Answer: We thank the reviewer for this insightful comment and agree that displaying individual data points can enhance data transparency and facilitate the assessment of variability. In the present manuscript, however, we have chosen to present the results as mean ± standard deviation, which is a widely accepted practice in our field and consistent with previously published studies using similar experimental designs.

The standard deviation bars included in the figures adequately represent data dispersion and allow for a clear interpretation of differences between experimental conditions without compromising figure readability. Given the number of groups and comparisons shown, overlaying individual data points could result in overly crowded figures, potentially hindering visual clarity and the overall interpretation of the results.

Moreover, we believe that the current data presentation is sufficient to support the conclusions of the study, as all statistical analyses were performed using the full set of experimental replicates and are transparently described in the Methods section. Nevertheless, we appreciate the reviewer’s suggestion and will take it into consideration for future studies and figure designs when the format and number of comparisons allow.

 

7. Some results that are not fully consistent with expectations or show relatively small changes are insufficiently discussed; it is recommended to provide reasonable explanations based on experimental conditions or differences from published literature to strengthen the completeness of the Discussion.

Answer: We thank the reviewer for this insightful comment. We agree that several observations—particularly the modest or context-dependent changes in TIE2/Tek and tight junction–related genes upon NRF2 activation—required a more in-depth discussion.

Accordingly, we have substantially expanded the Discussion section to address these points. The revised text now provides mechanistic and contextual explanations for: (i) why NRF2 activation results in TIE2/Tek downregulation despite the generally protective roles attributed to both pathways; (ii) why some tight-junction components show modest or selective changes; and (iii) how differences in experimental conditions (acute vs. sustained NRF2 activation, in vitro endothelial models, redox context) may account for discrepancies with previously published literature reporting NRF2-mediated BBB protection.

These additions strengthen the biological interpretation of the data and clarify how our findings complement—rather than contradict—existing literature. We believe that the revised Discussion now more comprehensively addresses the reviewer’s concern.

 

8. The manuscript does not explicitly define study limitations; it is recommended to add a short paragraph discussing limitations such as sample size, model constraints, or technical limitations to enhance scientific rigor.

Answer: Thank you so much for your proposal. We have added a brief paragraph to the end of the discussion section, discussing the most relevant limitation of this study.

 

9. There are minor issues with grammar and word choice throughout the manuscript; professional English language editing is recommended prior to acceptance.

Answer: The English style has been revised throughout the entire manuscript.

 

10. The fluorescence images are somewhat unclear, and higher-magnification images should be added.

Answer: We provide a low-magnification view of the merged image from the randomized field, in Fig. 1E

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The paper entitled Modulation of the receptor tyrosine kinase TIE2/Tek pathway by NRF2 activation in neurovascular endothelial cells is presented. The authors showed  a role of the NRF2-mediated receptor tyrosine kinase TIE2/Tek pathway in neurovascular endothelial cells. They suggested an NRF2-dependent mechanism affecting TIE2/Tek levels and potentially influencing angiogenic regulation.  The paper is interesting.  The methods used were appropriate and the conclusions were reasonable.

 

There are some possible issues

Figure 4. NRF2 does not modify Tek mRNA stability and does not bind its promoter. Can you state the sample sizes with P value in all panels in the figure legends ?

In all other figures,  can you state the sample sizes with P value in all panels in the figure legends ?

Figure 2. Overexpression of NRF2 alters TIE2/Tek levels.  Do you have a KO of NRF2 as a comparison or control ?

Figure 1. Reduced TIE2/Tek levels with SFN are dependent on NRF2 activity. Do you have in vivo result or a disease model? Please at least discuss these aspects. Neurovascular endothelial cell pathways are involved in the regulation of blood vessels, endothelial cells, and pericytes  (for example, PMID: 35296645, PMID: 39757951  ).  It would be informative to discuss how Neurovascular endothelial cells interplay with pericytes?

Author Response

Reviewer_comments:  

 

We have highlighted all modifications in the main body of the manuscript in YELLOW.

 

Reviewer #2:

 

1. Figure 4. NRF2 does not modify Tek mRNA stability and does not bind its promoter. Can you state the sample sizes with P value in all panels in the figure legends ?

Answer: Thank you for your appreciation. We have added a brief comment to the legend of the corresponding Figure 4 describing the statistical methods employed, as well as the sample size (n) and the P values considered.

 

2. In all other figures, can you state the sample sizes with P value in all panels in the figure legends ?

Answer: Thank you for your comment. In all figure legends, we have meticulously described the statistical analyses employed, the sample size (n), and the P values considered statistically significant for each experiment.

 

3. Figure 2. Overexpression of NRF2 alters TIE2/Tek levels. Do you have a KO of NRF2 as a comparison or control ?

Answer: Yes, we compared this effect with ANGPT1 treatment under the same experimental conditions using shNRF2 (Supplementary Fig. S3). Activation of TIE2 signaling by ANGPT1 under conditions of NRF2 genetic ablation did not result in any significant change in CLDN5 protein levels.

 

4. Figure 1. Reduced TIE2/Tek levels with SFN are dependent on NRF2 activity. Do you have in vivo result or a disease model? Please at least discuss these aspects. Neurovascular endothelial cell pathways are involved in the regulation of blood vessels, endothelial cells, and pericytes (for example, PMID: 35296645, PMID: 39757951 ).  It would be informative to discuss how Neurovascular endothelial cells interplay with pericytes?

Answer: We thank the reviewer for this important comment. At present, our study does not include in vivo data or disease models, as it was designed to mechanistically dissect the endothelial-autonomous relationship between NRF2 activation and TIE2/Tek regulation under controlled conditions. We agree, however, that placing our findings in an in vivo and pathophysiological context is essential.

Accordingly, we have expanded the Discussion to explicitly address this limitation and to contextualize our results within the framework of neurovascular unit biology. The revised text now discusses how NRF2-dependent modulation of endothelial TIE2/Tek signaling could indirectly affect pericyte recruitment, survival, and endothelial–pericyte communication, given the well-established role of the ANGPT/TIE2 axis in maintaining pericyte coverage and vascular stability.

In addition, we now reference recent studies (including PMID: 35296645 and PMID: 39757951) highlighting the importance of endothelial–pericyte crosstalk in regulating blood–brain barrier integrity and vascular homeostasis. We propose that NRF2-mediated repression of TIE2/Tek may influence neurovascular dynamics in vivo by altering endothelial signaling cues required for pericyte function, particularly under chronic or pathological conditions.

We believe that these additions strengthen the physiological relevance of our findings and clearly outline future directions aimed at validating this NRF2–TIE2 regulatory axis in animal models and disease settings.

 

 

Author Response File: Author Response.pdf