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Dear Authors, thank you for submission of the revised version of your manuscript. The submission was properly improved and can be now recommended for publication
no specific comments
no additional comments
no additional comments
The authors addressed all criticisms sufficiently and the manuscript is largely improved.
Dear Authors, thank you for submitting the revised version of your manuscript. It was evaluated by two reviewers and the editor. The submission has been significantly improved. However, one of the reviewers has indicated several point that have to be addressed in frames of minor revision, before your paper can be recommended for the publication. The specific point are listed in the comments of the reviewers
We are looking forward to receive the revised version of your paper
With best regards
They authors responded adequately to all questions and performed revisions so far as possible.
Finally the manuscript has been improved, but there are still a number of irregularities left and changes are necessary.
1. Introduction page 4, line 19: wild-type mice subjected to RAS – add the “to”
2. At page 6 line 2 the authors state that they performed RAS in 5-7 week old mice, later at page 11, line 23 they report obout 5-6 weeks – what is true?
3. Page 7 line 3: five aortas from db/db mice …. Are this db/db mice or db/dbRAS mice??
4. Page 9 line 6, - for assessment of hypertrophy the heart weight/tibia length or heart weigh/body weight ratio would be necessary. In db/db mice, RAS seems not to induce any hypertrophy, in wild-type TAS seems to induce heart hypertrophy but this was not tested for significance. So it is not clear whether RAS induce cardiac hypertrophy. Please verify this.
5. Figure 1 is not mentioned in results
6. Page 10, line 10. Instead of figure 2 – figure 2A-2D!
7. Page 10, line 11. Instead of figure 2 – figure 2E!
8. Page 10, line 12: actin is shown in figure 3 not 2 as stated in results
9. Aorta width is shown in figure 4 not 3 as stated at page 11, line 2
10. Page 11, line 6: replace “figure 4” with “figure 5”
11. Page 13, line 21: remove ”returns” in between “production” and “ is”
12. Table 1:
• mean and standard deviation should be indicated
• the absolute heart weight is no indicator for cardiac hypertrophy, heart weight tibia length or heart weight/ body weight ratio should also been shown. Taken from the presented data the db/db mice are not subjected to cardiac hypertrophy by RAS, however RAS seems to induce hypertrophic growth in WT. This would need to be statistically evaluated.
• A lot of single p-values are shown, would be enough to discriminate p< 0.05 and < 0.001. It should be mentioned which parameters are compared to each other.
No specific comments
The authors clarified details about experimental design in their revised manuscript which improved readability.
As far as I can judge the findings are valid.
The authors attempted to address all comments raised during initial review. They have performed additional analyses of which some have been included in the revised manuscript. Furthermore they improved clarity of the manuscript by revising the main text. In conclusion, the authors sufficiently addressed my previous comments.
Dear Dr. Grande,
Thank you for the submission of your manuscript „Aortic aneurysm in diabetic mice with renovascular hypertension “ for the consideration in PeerJ.
Your submission has been reviewed by the editor and 2 experts in the field. I regret to tell you that the manuscript in its present form is not suitable for publication. The concept of your study is interesting and original; however significant improvements are required prior the re-consideration of your submission for the publication. Please, find the specific criticism in the enclosed reports of the reviewers.
If you decide to resubmit the revised version, please summarise all the improvements made in the revised version and give answers to all critical points raised in the reviewers’ report in an accompanying letter.
1. Title: The title focus on the aortic aneurysm and do not reflect the investigations and findings in the cardiac tissue
2. Abstract: 50% of the abstract contain the description of the findings in a previous study – this is not adequate. The abbreviation “RAS” for renal aortic stenosis should be introduced for the first time in “abstract” line 3 and thereafter consistently used.
3. Methods: the authors should clearly indicate at which time points after RAS the mice were dissected and how many animals were studied in each of these groups
• At page 8 the authors extensively report about data obtained in a previous study. This part should be rather included in the discussion.
• Page 8 lower part – it is mentioned that the aortas were collected at different time points after RAS – it should be clarified at which time points and how much in each group.
• The figures 2 should show the aortic pathology score separately for the different time points after RAS and indicate the number of mice in each of these groups or it should be commented why it is not necessary.
• Figure 3: The number of analysed aortas and time points after RAS should be mentioned.
• Figure 1 and figure 2 should be combined
• Figure 4: again no information how many hearts were investigated in the different groups and at which time points after RAS.
In the manuscript, the authors do not provide any information about the relation between the severity of kidney disease in these db/db RAS mice and the aortic and cardiac damage. It seems that there were no correlation between aortic damage and heart fibrosis, this should be commented and discussed.
The present study address an important topic - the relation between diabetics and aortic aneurysm. This is specifically interesting in the light of recent case-control studies showing a reduced risk for aortic aneurisms in diabetic patients. The present study describes the occurrence of aortic aneurysm and cardiac fibrosis in diabetic db/db in contrast to wild-type mice subjected to renovascular hypertension due to renal artery stenosis (RAS). The authors performed this study in extension to a previous work in which they investigated the combined effects of hypertension and diabetes on renal disease progression and observed increased prevalence of sudden death associated with massive intrathoracic haemorrhage in db/db mice with RAS. The study is principally well performed and although interesting, the present data are still descriptive and rather preliminary. They do not provide a hint for the underlying mechanisms. The authors relate the findings to the combined effect of diabetes and renal hypertension. It is well known from other studies that angiotensin II in combination with hypercholesterolemia or disturbed fat metabolism stimulates the occurrence of aortic aneurysm. Db/db mice are obese and diabetic, so it is not clear whether hyperglycemia or rather obesity cause the observed findings. I miss information about clinical blood parameters such as triglyceride and cholesterol, about cardiac hypertrophy in these mice, blood pressure and body weight. These data at least should be mentioned in results. In contrast to other studies with angiotensin II related aortic aneurysm, in this study no increase in CD206 and iNOS as a sign of macrophage influx was observed. This indicates a different underlying mechanism and should be verified and studied in more detail.
This reviewer has some major concerns regarding the measurements performed i.e. selection of aorta and cardiac tissue used for analysis. Furthermore, it is not clear at what time points tissues were harvested and it appears that these are pooled data from a large number of animals sacrificed at different time points after surgery. This should be clarified. This has been explained in more detail in specific comments (section general comments to authors).
I have some serious methodological concerns as explained in more detail in specific comments (section general comments to authors).
In this article, Kashyap and colleagues describe a retrospective study performed on aorta and heart tissues that were obtained from wildtype and diabetic db/db mice with or without renal artery stenosis (RAS) induced by cuffing of the right renal artery. Results on a.o. kidney morphology, RAAS activation, and blood pressure from the same cohort were published last year (Hartono et al., BMC Nephrology 2014). Current study was instigated by the previous observation that RAS in db/db mice resulted in premature death of mice during follow-up (~23.5%), especially within the first 2 weeks after RAS induction. Massive intrathoracic hemorrhage reminiscent of aortic aneurysm was observed. In the current study the authors therefore determined morphological changes in aorta and heart of mice from the aforementioned cohort. The main observations of these analyses are 1) more severe aorta wall damage (cell loss and wall width) and 2) more severe cardiac fibrosis in db/db RAS mice when compared with wildtype RAS mice. No differences were observed with regard to macrophage subset influx.
The manuscript is clearly written. Data are descriptive and no functional analyses are being performed or at least discussed. I have some specific major and minor comments as detailed below.
1. The authors have analyzed aorta and cardiac tissue from a large number of mice. In their original publication they analyzed renal tissue harvested 2, 4 and 6 weeks after surgery. From the current study it is unclear at which time point after RAS or sham surgery the organs were harvested and used for analysis. These time points should be mentioned. I assume that analyses have been performed on the same time points, but this contrasts with the follow-up time indicated in Table 1. This needs to be clarified. Moreover, the severity of damage should be related to the time point of harvest.
2. They analyzed aorta pathology in a semi-quantitative manner as described in the Methods section. Aorta pathology is described as “small muscle dropout”. What do they mean? Should it read “smooth muscle dropout”? When analyzing SMC loss in the vascular wall it might be more appropriate to stain for these cells specifically using a well-established SMC markers e.g. alpha-smooth muscle actin, transgelin (SM22) etc.
3. In the Discussion section (lines 231-233) the authors state that they sampled abnormal regions of the aorta, and this might have led to an underestimation of the degree of histologic alterations given the focal nature of the lesions. However, from the Methods it is not clear at all how aorta tissue was selected(only thoracic, abdominal?). If they selected based on abnormal morphology, how can the authors explain that 16 out of 46 (35%) aorta’s (in db/db RAS group) have score 0 (i.e. normal aorta)? Same holds true for 11 out of 46 (24%) aorta’s with score 1 in the same group. Score 1 is virtually normal (based on figure 1B) and therefore unlikely to be visible during sacrifice. This needs explanation.
4. Related to comment 4: analyses should be performed at different levels within the aorta because of the focal nature, and not based on 1 HE stained section from 1 location. The same holds true for the cardiac fibrosis measurements.
5. The overall score of aorta pathology was 1.34 in db/db RAS and in WT RAS 0.41. Is the pathology sufficiently severe to speak about “aortic aneurysm” as stated in the title?
6. Apparently, there is quite a lot of variation in the severity of aorta pathology and cardiac fibrosis in the RAS groups. What is the explanation for this variation, and what is the relation between the development of aorta pathology and cardiac fibrosis? They are discussed as separate entities but they are possibly linked. The authors have the data to perform these correlations.
7. There is no rationale given for analyzing M1 and M2 macrophage subsets; moreover they do not show any data on the gene expression or IHC. They mention that no differences were observed between groups which is rather unexpected since fibrosis and vascular remodeling are processes associated with inflammation. What is their explanation? Why did they study this, what was the outcome (no inflammation at all, inflammation in all groups etc?). The way it is included and described in the manuscript does not add.
8. In the Discussion section their data are not sufficiently discussed; how does previously published work relate to their observations? The authors discuss e.g. the role of TGFb signaling, MMP activity in the development of aortic aneurysm. Additional analyses on these pathways would definitely add to the quality of their manuscript.
9. In legend Figure 4 they refer to dbc sham and dbc RAS; for clarity reasons rather use WT sham and WT RAS respectively.
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