Round 1

Reviewer 1 Report

This observational study describe how high cholesterol diet increase the transcription of myocardial inflammatory, apoptotic, pro-fibrotic and damage markers in White New Zealand rabbits, and how statins moderate it.

Although a moderate novelty can be recognised, the paper is limited by the use of sole mRNA analyses. The lack of histopathological data doesn’t allow to speculate on the reason of marker expression increase. Are there inflammatory infiltrates? Is there coronary atherosclerosis? Is there cardiac fibrosis? Which cell type undergo apoptosis?

In addition, the paper is very difficult to follow, due to lack of clear figure legends and almost no explanations of the results.

Specifically:

Major issues

-The schematic figure of experimental protocol is not easy to follow. I suggest a graphical timeline representation for each of the 8 groups, detailed with denominations of each group and relative treatments (C120, C90… I believe these are samples?).

-Please detail the modality of intake (mixed with the diet, in the water, oral gavage, other tipe?) of daily -oral administration of 72 Rosuvastatin (0.7mg/kg BW) and Fluvastatin (2 mg/kg BW)

-These animals are “recycled” from other experiments, which is fine given the necessity of “reduction” in the 3R ethical rules. However, a power analyses should have been indicated.

-Please justify the statin dose choice, at least by providing references.

-In figure 1 I don’t understand the fold change respect to what. I don’t see any of the controls at 1

-What is reported in table 2? Fold change? Respect to what?

-Line 170: statistical correlation with what? I really don’t understand what figure 2 represent. Please explain

- the discussion of MMP is mostly based on their role in the vessels. however they have a role also in the myocardium. Please discuss both since no evidence is shown on the effect of HCD on the cardiac vessels only.

-please discuss the effect of HCD in other models, e.g. mouse, and compare with rabbits.

Minor issues

-Abstract: line 20: “C” should be corrected by “c”.

-a. b. and c. of lines 19-20 look different from a. and b. of line 23. If so please use other character to list the different groups and maintain it throughout the text.

-why you have a supplementary figure in the text? Can’t it be a normal figure (if improved?)

-Line 129: What is “a statistical increase”? maybe you meant “a statistically significant increase”?

Author Response

Dear Editor,

We would like to thank you for reviewing our manuscript entitled: “A high cholesterol diet increases TLRs and other harmful factors in the rabbit myocardium. The effect of statins.”

We are pleased to hear that our work was favorably reviewed and was found to be potentially acceptable for publication pending revisions.

We thank the Reviewers for their valuable insight and comments as these serve to further strengthen our manuscript.

Reviewer's comments:

Reviewer #1

This observational study describe how high cholesterol diet increases the transcription of myocardial inflammatory, apoptotic, pro-fibrotic and damage markers in White New Zealand rabbits, and how statins moderate it.

Although a moderate novelty can be recognized, the paper is limited by the use of sole mRNA analyses. The lack of histopathological data doesn’t allow to speculate on the reason of marker expression increase. Are there inflammatory infiltrates? Is there coronary atherosclerosis? Is there cardiac fibrosis? Which cell type undergo apoptosis?

1.1 Major Comments:

Comment 1.1.1:  The schematic figure of experimental protocol is not easy to follow. I suggest a graphical timeline representation for each of the 8 groups, detailed with denominations of each group and relative treatments (C120, C90… I believe these are samples?).

Authors’ reply: We thank the knowledgeable Reviewer for this important comment.  We have now changed the initial figure to time table in the Materials and Methods section (p.2, Supplementary Table S1).

Comment 1.1.2: Please detail the modality of intake (mixed with the diet, in the water, oral gavage, other type?) of daily -oral administration of 72 Rosuvastatin (0.7mg/kg BW) and Fluvastatin (2 mg/kg BW)

Authors’ reply: We thank the Reviewer for this comment. We added in the Methods section the following clarification (p.2):

“Daily oral gavage Reply to author 1.1.2 administration of Rosuvastatin (0.7mg/kg BW) and Fluvastatin (2 mg/kg BW)”.

Comment 1.1.3: These animals are “recycled” from other experiments, which is fine given the necessity of “reduction” in the 3R ethical rules. However, a power analyses should have been indicated.

Authors’ reply: We thank the Reviewer for this comment. We have now added the following sentence in the Statistical analysis section (p.3):

“According to the power analysis for independent samples, the required sample size per group was 6. (Power analysis=0.95, alpha=0.05, beta=0.05)”.

Comment 1.1.4: Please justify the statin dose choice, at least by providing references.

Authors’ reply: We thank the Reviewer for this comment. The dose of statins used in our experiments is justified according to the following publications:  

1. Tziakas D, Chalikias G, Kapelouzou A, Tentes I, Schäfer K, Karayannakos P, Kostakis A, Boudoulas H, Konstantinides S. Erythrocyte membrane cholesterol and lipid core growth in a rabbit model of atherosclerosis: modulatory effects of rosuvastatin. Int J Cardiol. 2013; 170(2):173-81.
2. Kapelouzou A, Giaglis S, Peroulis M, Katsimpoulas M, Moustardas P, Aravanis CV, Kostakis A, Karayannakos PE, Cokkinos DV. Overexpression of Toll-Like Receptors 2, 3, 4, and 8 Is Correlated to the Vascular Atherosclerotic Process in the Hyperlipidemic Rabbit Model: The Effect of Statin Treatment. J Vasc Res. 2017;54(3):156-169.

Comment 1.1.5: These animals are “recycled” from other experiments, which is fine given the necessity of “reduction” in the 3R ethical rules. However, a power analyses should have been indicated.

Authors’ reply: We thank the Reviewer for this comment. We have already answered this question (comment 1.1.3)

Comment 1.1.6: In figure 1 I don’t understand the fold change respect to what. I don’t see any of the controls at 1.

Authors’ reply: We thank the Reviewer for this comment. Fold change occurred between the groups and also between their Control groups C90 and C120. It is described with the following symbols  *,#,a,b,c,d,e. We added a new sentence according to your suggestion. The new sentence now reads as follows:

“Significant difference (p < 0.05) versus C90 (*); C120 (#); G30 (a); G60 (b); G90 (c); G120 (d); GF120 (e)”.

Comment 1.1.7: Line 170: statistical correlation with what? I really don’t understand what figure 2 represent. Please explain

Authors’ reply: We thank the Reviewer for this valuable comment. This figure shown the trend of biomarkers and not the correlation. We apologize for the discrepancy.

Comment 1.1.8: the discussion of MMP is mostly based on their role in the vessels. However they have a role also in the myocardium. Please discuss both since no evidence is shown on the effect of HCD on the cardiac vessels only.

Authors’ reply: We thank the Reviewer for this comment. We modified the Discussion section accordingly. The new sentences now read (p.8):

“MMP-2 structure and function is correlated with increased remodeling after an acute myocardial infarction [35].  MMP-9 is also correlated with remodeling and mortality after an infarct [36]. MMP-2 degrades troponin I in IRI ischemia reperfusion injury [37], while in aortic stenosis TIMP 1 and 2 are related to fibrosis [38].  TIMP 1 also promotes myocardial fibrosis in pressure overload [39]. In 669 patients in the Framingham Heart Study, MMP-9 was positively correlated to left ventricular mass and thickness and negatively to fractional shortening [40].  Moreover, serum MMP9 and TMP1 are also significant risk factors in population studies [41,42]”. 

Comment 1.1.9: please discuss the effect of HCD in other models, e.g. mouse, and compare with rabbits.

Authors’ reply: We thank the Reviewer for this comment. We have added the following sentence (p.9):

“From the above it can be seen that apart from our rabbit model, mice showed the same behavior [27,48]”.

1.2 Minor Comments:

Comment 1.2.1: Abstract: line 20: “C” should be corrected by “c”.

Authors’ reply: We thank the Reviewer for this comment. The revised manuscript now reads as follow:

“The aim of this study is to investigate the mRNA expression of TLRs and other noxious bi-omarkers expressing inflammation, fibrosis, apoptosis, and cardiac dysfunction on the rabbit myocardium during a. high cholesterol diet (HCD); b. normal diet resumption and c. fluvastatin or rosuvastatin treatment.” (p.1)

Comment 1.2.2: a. b. and c. of lines 19-20 look different from a. and b. of line 23. If so please use other character to list the different groups and maintain it throughout the text.

Authors’ reply: We thank the Reviewer for this comment. We have made the appropriate modifications. The revised manuscript now reads as follow (p.1):

“In the second experiment, 3 groups were fed with HCD for 3 months and followed by normal chow for one month; and administration of fluvastatin or rosuvastatin for one month”.

Comment 1.2.3: why you have a supplementary figure in the text? Can’t it be a normal figure (if improved?)

Authors’ reply: We thank the Reviewer for this comment. We have now changed the initial figure to time table in the Materials and Methods section (p.2, Supplementary Table S1).

Comment 1.2.4: Line 129: What is “a statistical increase”? maybe you meant “a statistically significant increase”?

Authors’ reply: We thank the Reviewer for this comment. The new sentence reads as follows (p4):

“A statistically significant increase was found in the mRNA expression and as re-gards TLRs 2,4 and 8 between C90, G30, G60, G90; TLR3 also increased, but the in-crease started at a later time interval (G60); (Table 2 and Figure 1)”.

Reviewer 2 Report

Kapelouzou et al. in their manuscript describe the effects of an high cholesterol diet and the beneficial role of two statins, Rosuvastatin being more effective than Fluvastatin. Ineffective was the return to normal diet.

The work is well organized but the results are limited to RT-PCR analysis.

Although the real-time experiments give important results, it can be useful to go inside by WB analysis at least for the most important genes. Some experiments can be of support if the authors have still stored material.

In Table 2 there is not described if the data are significantly different or not.

Title. I’m not favorable for an abbreviation in the title. Moreover it can be specified the kind of effect statin produce.

Also, it should be described in the discussion section that although the therapeutic value of statins, they can induce important side effects particularly in skeletal muscle (Camerino et al., Toxicol Appl Pharmacol. 2016 Sep 1;306:36-46. doi: 10.1016/j.taap.2016.06.032).

Author Response

Reviewer #2

Kapelouzou et al. in their manuscript describe the effects of an high cholesterol diet and the beneficial role of two statins, Rosuvastatin being more effective than Fluvastatin. Ineffective was the return to normal diet.

The work is well organized but the results are limited to RT-PCR analysis. 

Comment 2.1: Although the real-time experiments give important results, it can be useful to go inside by WB analysis at least for the most important genes. Some experiments can be of support if the authors have still stored material.

Authors’ reply: We thank the Reviewer for this valuable suggestion. Unfortunately, we have not stored material.

Comment 2.2: In Table 2 there is not described if the data are significantly different or not.

Authors’ reply: We thank the Reviewer for this comment. We added a new sentence regarding statistical significance. The new sentence now reads as follows:

“Significant difference (p < 0.05) versus C90 (*); C120 (#); G30 (a); G60 (b); G90 (c); G120 (d); GF120 (e)”.

Comment 2.3: Title. I’m not favorable for an abbreviation in the title. Moreover it can be specified the kind of effect statin produce.

Authors’ reply: We thank the Reviewer for this valuable comment. Based on that, the revise title reads as follow:

“A high cholesterol diet increases Toll like receptors and other harmful factors in the rabbit myocardium. The beneficial effect of statins”

Comment 2.4: Also, it should be described in the discussion section that although the therapeutic value of statins, they can induce important side effects particularly in skeletal muscle (Camerino et al., Toxicol Appl Pharmacol. 2016 Sep 1;306:36-46. doi: 10.1016/j.taap.2016.06.032).

Authors’ reply: We thank the Reviewer for this comment. We have now added the following sentences in the Discussion section (p.9):

“Statins can induce important important side effects particularly in skeletal muscle especially in aged rats [59]. However the effects of statins on the cardiomyocytes have not been specifically reported”.

Round 2

Reviewer 1 Report

Unfortunately, after the revision the manuscript did not show great improvements. The data are still not explained properly and thus it cannot be judged if they support the conclusions.

-The schematic table of experimental protocol is even more difficult to follow than the previous figure…

-The reference sample for the fold change (whose value needs to be 1) is still not shown in figure1 or table 1 nor mentioned, I guess is C30, but it is just a guess. What the authors now describe  “Significant difference (p < 0.05) versus C90 (*); C120 (#); G30 (a); G60 (b); G90 (c); G120 (d); GF120 (e)” is just the statistical comparison not the reference value respect to which the data are calculated (hopefully, otherwise it would make no sense).

-I still don’t understand figure2: mRNA fold respect to what?

If the author mean mRNA absolute expression is not measured in fold.

Increase respect to C30? No, because the values are not consistent with the graphs and the table shown above….

The authors had the chance to explain the results but unfortunately the opportunity was not taken.

Author Response

Please see the attachment. 

Author Response File: Author Response.pdf