The Role of the Aryl Hydrocarbon Receptor in Vascular Factors Related to Preeclampsia in a Smoking Mouse Model

Round 1

Reviewer 1 Report (New Reviewer)

Comments and Suggestions for Authors

 

The authors investigated the response of several cellular signaling elements involved in preeclampsia to smoking in an experimental mouse model. The investigation aims to elucidate the mechanism behind the previously observed protective effect of smoking on preeclampsia. The authors demonstrate, that the anti-angiogenic factor sFlt-1 was significantly decreased in mice exposed to cigarette smoke and increased after introduction of an AhR-antagonist. The authors conclude that the beneficial effects of smoking on preeclampsia are due to sFlt-1 modulation via AhR-receptor.

This is a very interesting study that not only provides a valuable contribution to the question related to beneficial effects of smoking in preeclampsia, but also provides some insights to an important pathophysiology that is still not understood completely.

The English language is overall quite good, but grammar and wording are sometimes slightly weird due to non-native writing.

Examples:

line 28: “This study provides a possible role…”

line 55: Inhibition of cytokine production and antioxidant property of nicotine lead

beneficial effect of smoking evidenced by both in vitro and in vivo

There are also several spaces missing (line 22,23: 2-cigarette/p-value…)

Visual demonstration of immunohistochemistry results is not highly convincing, but we are aware that it is generally quite difficult to show differences in tissue based fluorescence.

Figure 3,6: Provide explanation for all three colors/slides in the legend (assume it is HOECHST, VEGF-GFP and overlay). Please also provide, how the images were evaluated for fluorescence intensity.

Please also provide complete (uncut) western blots to original images set of images.

Table 1, Figure 1,2,4,5,7: Provide the number of N (repeats) for all statistical evaluations in the legends. Please note that a minimal number of 3-6 repeats should be provided for a meaningful statistical evaluation (3 independent blots, six independent stainings).

Fig.5, Western Blot: Please indicate size in kD on left side of blots.

Comments on the Quality of English Language

 

 

The English language is overall quite good, but grammar and wording are sometimes slightly weird due to non-native writing.

Examples:

line 28: “This study provides a possible role…”

line 55: Inhibition of cytokine production and antioxidant property of nicotine lead beneficial effect of smoking evidenced by both in vitro and in vivo

There are also several spaces missing (line 22,23: 2-cigarette/p-value…)

Author Response

The authors investigated the response of several cellular signaling elements involved in preeclampsia to smoking in an experimental mouse model. The investigation aims to elucidate the mechanism behind the previously observed protective effect of smoking on preeclampsia. The authors demonstrate, that the anti-angiogenic factor sFlt-1 was significantly decreased in mice exposed to cigarette smoke and increased after introduction of an AhR-antagonist. The authors conclude that the beneficial effects of smoking on preeclampsia are due to sFlt-1 modulation via AhR-receptor.

 

This is a very interesting study that not only provides a valuable contribution to the question related to beneficial effects of smoking in preeclampsia, but also provides some insights to an important pathophysiology that is still not understood completely.

 

The English language is overall quite good, but grammar and wording are sometimes slightly weird due to non-native writing.

 

Examples:

 

line 28: “This study provides a possible role…”

• We rewrote this sentence “This study offers insights into a potential role~”

 

line 55: Inhibition of cytokine production and antioxidant property of nicotine lead beneficial effect of smoking evidenced by both in vitro and in vivo

 

• We rewrote this sentence “Inhibition of cytokine production and activation of antioxidant property by nicotine lead beneficial effect of smoking evidenced by both in vitro and in vivo”

 

There are also several spaces missing (line 22,23: 2-cigarette/p-value…)

• We corrected spaces missing in abstract.

Visual demonstration of immunohistochemistry results is not highly convincing, but we are aware that it is generally quite difficult to show differences in tissue based fluorescence.

• Quantification was done accordingly.

Figure 3,6: Provide explanation for all three colors/slides in the legend (assume it is HOECHST, VEGF-GFP and overlay). Please also provide, how the images were evaluated for fluorescence intensity.

• We provided all three colors in the legend and intensity of fluorescence was quantified accordingly.

Please also provide complete (uncut) western blots to original images set of images.

• We provided complete (uncut) western blots.

Table 1, Figure 1,2,4,5,7: Provide the number of N (repeats) for all statistical evaluations in the legends. Please note that a minimal number of 3-6 repeats should be provided for a meaningful statistical evaluation (3 independent blots, six independent stainings).

• For ELISA assays, duplication was done and for western blots, 6 independent blots and staining of sflt-1 and 3 blots and staining of VEGF were done. We provided the statement in manuscript.

Fig.5, Western Blot: Please indicate size in kD on left side of blots.

• We added kD on right side of blots.

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

The study titled "The role of aryl hydrocarbon receptor in vascular factors related to preeclampsia in smoking mouse model" investigates the effect of cigarette smoke on the expression of soluble fms-like tyrosine kinase-1 (sFlt-1), vascular endothelial growth factor (VEGF), and endoglin (sEng) in the context of preeclampsia and the involvement of aryl hydrocarbon receptor (AhR) in this process. This study utilized a smoking mouse model, where pregnant mice were exposed to cigarette smoke and the effects on the aforementioned factors were analyzed.

 

**Negative Points and Areas for Improvement:**

1. **Insufficient Sample Size:** The study involved a limited number of animals in each group, which might not adequately represent the broader population. Increasing the sample size could enhance the statistical power and validity of the findings.

 

2. **Lack of Long-Term Follow-up:** The study does not mention long-term follow-up of the mice post-exposure to cigarette smoke. Long-term effects are crucial, especially to understand the implications of prenatal exposure to cigarette smoke.

 

3. **Single Model of Exposure:** The study exclusively uses a smoking model. Including other models of AhR activation could provide a broader understanding of the AhR's role in preeclampsia.

 

4. **Control of Confounding Variables:** The study should ensure strict control of confounding variables such as the nutritional status and health condition of the mice to ensure the observed effects are solely due to the cigarette smoke exposure.

 

5. **Mechanistic Insights:** The study primarily focuses on observational findings. More detailed mechanistic insights, perhaps through molecular biology techniques, would be beneficial in understanding how cigarette smoke and AhR activation influence the expression of sFlt-1, VEGF, and sEng.

 

6. **Human Relevance:** While the study provides valuable insights, the relevance to human pregnancy and preeclampsia needs to be more explicitly addressed. Translational research or comparative studies involving human subjects might be required for comprehensive understanding.

 

7. **Ethical Considerations:** Given the known adverse effects of cigarette smoke, ethical considerations should be thoroughly addressed, particularly in the context of potentially harmful exposure during pregnancy, even in an animal model.

 

**Recommendation for Additional Reference:**

The manuscript could be strengthened by adding the following reference, which investigates the effects of Chinese green tea consumption on oxidative stress, inflammation, and tissue damage in smoke-exposed rats:

 

- Al-Awaida W, Akash M, Aburubaiha Z, Talib WH, Shehadeh H. "Chinese green tea consumption reduces oxidative stress, inflammation and tissues damage in smoke exposed rats." Iranian journal of basic medical sciences. 2014 Oct;17(10):740.

 

This reference is pertinent as it provides insight into the impact of an antioxidant-rich diet (green tea) on mitigating the adverse effects of smoke exposure. It could offer a contrasting perspective to the current study by demonstrating a potential protective strategy against the harmful effects of cigarette smoke.

Comments on the Quality of English Language

Minor editing of English language required

Author Response

The study titled "The role of aryl hydrocarbon receptor in vascular factors related to preeclampsia in smoking mouse model" investigates the effect of cigarette smoke on the expression of soluble fms-like tyrosine kinase-1 (sFlt-1), vascular endothelial growth factor (VEGF), and endoglin (sEng) in the context of preeclampsia and the involvement of aryl hydrocarbon receptor (AhR) in this process. This study utilized a smoking mouse model, where pregnant mice were exposed to cigarette smoke and the effects on the aforementioned factors were analyzed.

 

 **Negative Points and Areas for Improvement:**

 

1. **Insufficient Sample Size:** The study involved a limited number of animals in each group, which might not adequately represent the broader population. Increasing the sample size could enhance the statistical power and validity of the findings.

à We appreciate for these constraints. However, our study was conducted with a total of 35 mice, and we believe that this is not a small number for an animal study and influence of exposure to toxic material as smoking should be considered ethically in performing experiments suggested below. We fully agree that having a larger sample size would have likely yielded more statistically significant results.

 

2. **Lack of Long-Term Follow-up:** The study does not mention long-term follow-up of the mice post-exposure to cigarette smoke. Long-term effects are crucial, especially to understand the implications of prenatal exposure to cigarette smoke.

à This study lacks long-term follow-up unfortunately and added this limitation in discussion. We had to sacrifice all the mice before delivery to have all the placenta therefore follow up of mice after delivery was not possible. Nevertheless long term follow up of prenatal exposure to smoking might be another theme of research and we would definitely plan do the long term follow up research in the future.

 

3. **Single Model of Exposure:** The study exclusively uses a smoking model. Including other models of AhR activation could provide a broader understanding of the AhR's role in preeclampsia.

à We appreciate these contraints. We only focused on single model of exposure and added this limitation in discussion. In the future, we would like to plan for further research on different models of AhR activation.

 

 

4. **Control of Confounding Variables:** The study should ensure strict control of confounding variables such as the nutritional status and health condition of the mice to ensure the observed effects are solely due to the cigarette smoke exposure.

àAlthough we did not conduct objective tests to assess the nutritional and health status, our study focused on mice raised under identical conditions. As outlined in the methods section, we ordered the same strain of mice from a single supplier and maintained them in a consistent environment with identical water, food, humidity and temperature conditions. Therefore, we believe that this standardization helps compensate for the lack of direct nutritional and health assessments.

 

5. **Mechanistic Insights:** The study primarily focuses on observational findings. More detailed mechanistic insights, perhaps through molecular biology techniques, would be beneficial in understanding how cigarette smoke and AhR activation influence the expression of sFlt-1, VEGF, and sEng.

à We appreciate these contraints. Our study lacks molecular biology techniques so we wrote this lack of finding in limitation. In the future, we would like to plan for further research using molecular biology techniques of AhR activation.

 

6. **Human Relevance:** While the study provides valuable insights, the relevance to human pregnancy and preeclampsia needs to be more explicitly addressed. Translational research or comparative studies involving human subjects might be required for comprehensive understanding.

à We added several references on studies involving human subjects in introduction to explain the human relevance. (Line 86-96)  Reference 23, 24, 28

 

7. **Ethical Considerations:** Given the known adverse effects of cigarette smoke, ethical considerations should be thoroughly addressed, particularly in the context of potentially harmful exposure during pregnancy, even in an animal model.

à Above all we followed rules for animal research according to the institutional review board of Animal Ethics Committee (KUIACUC-2012-121). Before research, alternative methods was considered however no other way to examine the effect of smoking was possible, our research was carried out by professionals with ethical treatment and scientific knowledge and experience in animal experiments and the minimum number of mice were used. To alleviate pain, we used appropriate measures such as analgesia, sedation and anesthesia. Lastly we promptly examined the animals after the experiment was completed and when pain or permanent damage was recognized, we handled in a manner that avoids causing pain as soon as possible.

 

 

**Recommendation for Additional Reference:**

 

The manuscript could be strengthened by adding the following reference, which investigates the effects of Chinese green tea consumption on oxidative stress, inflammation, and tissue damage in smoke-exposed rats:

- Al-Awaida W, Akash M, Aburubaiha Z, Talib WH, Shehadeh H. "Chinese green tea consumption reduces oxidative stress, inflammation and tissues damage in smoke exposed rats." Iranian journal of basic medical sciences. 2014 Oct;17(10):740.

 

 This reference is pertinent as it provides insight into the impact of an antioxidant-rich diet (green tea) on mitigating the adverse effects of smoke exposure. It could offer a contrasting perspective to the current study by demonstrating a potential protective strategy against the harmful effects of cigarette smoke.

• We added comments on a research of green tea on smoking as a future perspective in discussion section.

Round 2

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

No comments

 

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In this Manuscript the authors study the putative  effect of cigarette smoke in modulating the presence of preeclampsia factors using a  pregnant mouse model and both exposure to controlled cigarette smoke and modulators of the aryl hydrocarbon receptor (AhR) . Overall the goal paper is interesting and clear.

Unfortunately the data is not convincing and in no way makes the case for the authors argument in the Abstract, as I see no differences that warrant what the authors write. Most of the time there are no clear or any differences (fig 2 to 4), when they exist they are small and, while statistically significant, are doubtful to have any physiological relevance (such as the AhR agonist/antagonist experiments); finally in other cases they are nor logical in terms of what the authors describe.

The Western blot data is particularly poor and unconvincing, and thus I cannot be more positive in my recommendation

Minor issues:

-The legends of Figures are at the top, they should be at the bottom. The authors should show the number of experiments in each figure and Table legend as well as the different data points in the figures, not just the average.

-The  western blots should be fully shown, as they seem unconvincing. Figure 4 is particularly bad and unfit for publication, and suggests (as does the rest of the data) a large variability and an inability to make strong conclusions.

Comments on the Quality of English Language

There are minor issues that have to do with sentence organization and grammar.

Author Response

In this Manuscript the authors study the putative effect of cigarette smoke in modulating the presence of preeclampsia factors using a pregnant mouse model and both exposure to controlled cigarette smoke and modulators of the aryl hydrocarbon receptor (AhR) . Overall the goal paper is interesting and clear.

 

Unfortunately the data is not convincing and in no way makes the case for the authors argument in the Abstract, as I see no differences that warrant what the authors write. Most of the time there are no clear or any differences (fig 2 to 4), when they exist they are small and, while statistically significant, are doubtful to have any physiological relevance (such as the AhR agonist/antagonist experiments); finally in other cases they are nor logical in terms of what the authors describe.

 

The Western blot data is particularly poor and unconvincing, and thus I cannot be more positive in my recommendation

• We updated our figures in western blotting and added immunocytochemistry to improve our results.
• We appreciate your thoughtful suggestion. Our research is inspired by the observation that smoking is associated with a lower incidence of preeclampsia in humans, and it serves as an experimental study. Therefore, among numerous factors related to smoking, we aimed to investigate the causality regarding the association with Aryl hydrocarbon and angiogenic factors. The results of this study confirmed that sFlt-1 was not inhibited when examining changes in sFlt-1 in the context of smoking in an environment where AhR antagonists were administered, in addition to simply observing the results of sFlt-1 based on smoking in the blood. Although the results from Western blotting, which assesses local effects in the placenta, did not show significant findings, it suggests that AhR may be involved in reducing sFlt-1, thereby potentially lowering the incidence of preeclampsia.

 

Minor issues:

 

-The legends of Figures are at the top, they should be at the bottom. The authors should show the number of experiments in each figure and Table legend as well as the different data points in the figures, not just the average.

à We placed figure legends at the bottom.

-The western blots should be fully shown, as they seem unconvincing. Figure 4 is particularly bad and unfit for publication, and suggests (as does the rest of the data) a large variability and an inability to make strong conclusions.

à We had mistaken the western picture matched with graph so we corrected figure 1 and 4.

 

Comments on the Quality of English Language

There are minor issues that have to do with sentence organization and grammar.

• We thoroughly reviewed our manuscript and corrected.

Reviewer 2 Report

Comments and Suggestions for Authors

The Authors deal with an interesting aim where pregnant mice were exposed to cigarette smoke concomitantly being treated with aryl hydrocarbon receptor (AhR) agonist and antagonist. This is a very interesting model showing one of the mechanisms leading to preclampsia however not much-obtained data is presented and therefore article (results and discussion are poor). Only after the enrichment of the article with the above, it can be encouraged for further processing.

1. Lines 53-55 provide reference and information what are other mechanisms of action of cigarette smoke on the human body, what the content of cigarette smoke

2. The aim of the study should be extended and more details need to be added

3. Provide histological analysis of obtained placenta

4. Describe in detail haw wos treatment with AhR agonist and antagonist was done (how many days, provide injection place etc.)

5. There is no WB for VEGF

 

2.

 

Author Response

The Authors deal with an interesting aim where pregnant mice were exposed to cigarette smoke concomitantly being treated with aryl hydrocarbon receptor (AhR) agonist and antagonist. This is a very interesting model showing one of the mechanisms leading to preclampsia however not much-obtained data is presented and therefore article (results and discussion are poor). Only after the enrichment of the article with the above, it can be encouraged for further processing.

 

1. Lines 53-55 provide reference and information what are other mechanisms of action of cigarette smoke on the human body, what the content of cigarette smoke

-> As suggested, we added references explaining AhR and its mechanism of action (Line 64-66)

2. The aim of the study should be extended and more details need to be added

-> We added more references and evidences to support our aim of this study. (Line 52-63)

3. Provide histological analysis of obtained placenta

-> We ran immunofluorescent staining with placenta and provided our results.  (Figure 3 and 6)

4. Describe in detail haw wos treatment with AhR agonist and antagonist was done (how many days, provide injection place etc.)

-> We wrote in detail about treatment with AhR agonist and antagonist. (Line 126-132)

5. There is no WB for VEGF

-> We attached WB for VEGF in the manuscript.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Unfortunately I am not convinced by the authors  revison, and consider that the problems remain.

Reviewer 2 Report

Comments and Suggestions for Authors

My comments were incorporated in the ms text