Review Reports - Extracellular Adenosine Contributes to the Hydrogen Peroxide-Induced Calcification of Cultured Tendon Cells

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Review for cimb-4096521

In this original research article entitled “The Extracellular Adenosine Contributes to the Hydrogen Peroxide-Induced Calcification of Cultured Tendon Cells”, the author (Sakuma et al.,) studied the extracellular ATP (ex-ATP) and its metabolites by osteogenic culturing the TT-D6 cells and primary mouse tendon cells. The authors used 2 weeks of osteogenic culture associated intermittent H2O2 followed by metabolomic and gene expression explorations. They reported that osteogenic calcification of tendon cells associated increased extracellular nucleotide metabolites, particularly adenosine.

Overall, the manuscript is acceptable but requires some minor improvements. Hereafter some comments revealed after reviewing its current version.

The major lack of the study is related to the potential involvement of growth factors, which are commonly implicated and might highly be mediated by hydrogen peroxide. Such potential effects are overlooked.
The methodological approach is consistent, appropriate and technically sound.
Regarding the used primers (whatever forward or reverse), it is recommended to keep the same way of writing the sequences; separation of each 3 nucleotides or not.
The change in the period of study (in days) for the culture should be explained. The readers would find it unclear why sometimes 4, 8, 12 and 16 days and sometimes 6, 12 and 15 days. Particularly for the longest period 14, 15 or 16. Why?
The sentences “Thus, metastasis –the biological …related to angiogenesis” and “Oxidative stress also …tumor progression.” can be supported by the following recent and relevant citation; doi: 10.3390/life15101564.
The conclusions are consistent with the evidence and arguments that the authors presented.
English language is overall acceptable just a minor editing and checking is required.
Minor comment: Use capital letter to the unit of liter “L” such as in “1 mg/ml” and “2 mg/ml”.

Author Response

Please see the attached Word file.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Heterotopic ossification is a mechanistically and clinically significant problem with some understanding for stress-associated induction of ectopic calcification. However, limited insight into compromised parameters of biological control are currently available. The authors examined the consequences of hydrogen peroxide-induced mineralization of mouse tendon cells, addressing the hypothesis that secreted extracellular ATP and its metabolites enhance and accelerate osteogenic mineralization. While additional metabolites have the potential to influence stress induced mineralization in the model utilized in this study and under diverse and clinically relevant conditions, the clarity of experimental design and interpretation of findings from the experimental protocol pursued is justifiable and provides important information.

Strengths of results include: genomic, histological and metabolic-based experimental strategies that are reinforced by mass spectrometric analysis. The evidence for contributions by extracellular ATP metabolites to molecular mechanisms that mediate heterotopic calcification advance understanding for a cellular pathology that has been characterized to a limited extent.

The results and presented and interpreted in an accessible manner for biologically and clinically oriented investigators

Author Response

Thank you very much for evaluating our manuscript.

We revised our manuscript in response to the other reviewers and believe our points were strengthened.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript of Ezura and coworkers aims to investigate the role of extracellular adenosine in a model of cultured tendon cells during the calcification process induced by hydrogen oxide supplementation.

The role of both adenosine and hydrogen peroxide have been investigated in several models, although with some conflicting results, thus making the analysis of results potentially challenging and highly dependent on culture conditions.

The manuscript and the experimental design lack of clarity in several parts and conclusions are not adequately supported by “significant” results.

Authors should consider the following aspects:

Lines 71-73: it is not clear why ABCC6 is link to GACI patients, since this gene/transporter is typically associated with PXE (pseudoxanthoma elasticum) and only a negligible percentage of GACI patients instead of ENPP1 present aBCC6 mutations

Line 146: Primer sequences are provided in Table 1 and not in Suppl Table 1. Moreover, regarding RT-PCR experiments, data are evaluated using Gapdh as the housekeeping gene. Authors should provide evidence for the stability of this gene in this context and therefore for its suitability for correctly evaluate RT-PCR results

Line 208: Authors in this part of the results as well as in many other parts (i.e., line 250) mentioned the occurrence of cell death although at levels that were acceptable to continue the osteogenic experiments. Since both adenosine and hydrogen peroxide can modulate cell death (depending on concentrations, cell type and culture conditions) it would be very informative to evaluate and quantify cell death (necrosis and/or apoptosis)

Line 218: Authors reported the addition of hydrogen peroxide at the concentration of 0.3 mM. However, in figure 2A two different concentrations (0,1 and 0,3) are reported.

Lines 225-226: Since data are not present in figure 2B Authors should add “data not reported”

Figure 2B: it is not clear to which condition the lines correspond in graphs. Moreover, Authors should explain the difference in the response of the two different concentrations of H2O2.

Line 230: same comment as before for mentioning only 0.3 mM concentration

Lines 247-248: The response is different depending on the two treatments. Text does not correspond to data reported in figure and should be better explained especially when they want to compare results reported in figure 2. The behavior in the two figures is not the same. Moreover, it is not clear why Authors refer in the text to enhanced calcification, when in the legend of figure 3 state that results were statistically not significant. If differences in calcification are not significant which is the meaning or the relationship with differences in osteogenic markers reported in panel C. Authors should better explain and clarify.

Figure 4: since Authors are frequently using two different concentrations of H2O2, they should clearly specify (if it is the case) that all data reported in figure 4 refer to a 0,3 mM concentration (this is mentioned only for panel F). Authors should also explain why the time points of figure 4F are different from those in panels B-E. In addition, Authors reported that data were statistically analyzed only for the addition of adenosine. As also mentioned in line 332 and 349 statistical analysis was not always done because of the small sample size. If experiments are always done at least three time in triplicate this might an adequate size, or, if results are heterogeneous, authors should perform more experiments. This can be easily done, since they are using a cell line. Significance of results is very important to support consistent conclusions.

Line 307: it seems that Authors should refer to figure 5 instead of figure 6

Figure 5: Explanation of lines is missing in the third panel. Are differences reported for Adora2b significant?

Line 320: The indication of figure 3 is not correct. It should be figure 4

Figure 6: Statistical analysis should be applied, especially because differences are not clear according to the images. Which are the differences at 20 days between vehicle and adenosine (50 uM) or the differences between all images in panel B?

Line 341: Authors mentioned results of Figure S1. Why are they using a concentration (200 uM) different from that of all other experiments?

Conclusions: considering the results conclusions are too speculative or at least very preliminary

Figure S1: Authors report three different concentrations, but in the figure the concentrations are four.

Figure S2: Authors report that high dose adenosine enhanced the calcification. There are absolutely no differences between vehicle and Adenosine supplementation

Comments on the Quality of English Language

English should be revised to remove typos and to increase the clarity of sentences

Author Response

Please find the attached Word file.

Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

The revised manuscript presents the following drawbacks:

Line 75. It is still not clear why Authors refers, as an example, to GACI. In GACI calcification is due to the lack of the inhibitor PPi and not to the possible excess of ATP.
Line 146: reference to Figure S1 is not correct
Line 158: Authors should explain why they are using a protocol for the Alizarin staining that is different from the traditional one in terms of concentration of the stain, pH of the stain and incubation time. Although the protocol used in this manuscript has been reported in one paper from the same laboratory, this is not sufficient to justify all the differences introduced in the traditional and widely used staining protocol, especially if we consider the results of the poor staining reported in most figures. A clear and detailed explanation is required.
Line 309: significance among which samples?
Line 321-323: Description is not coherent with the figures in terms of increased or suppressed expression
Line 329: Figure panel 8, changes in the densitometric analyses are not significant. Authors should explain why there are no significant differences in the amount of calcification. If there no changes, speculation on other results are without any consistent meaning. Or the staining is not performed appropriately.
Line 346: significance among which samples?
Line 372: Figure 4. Significance is missing in panels from B to E.
Line 419: significance among which samples?
Line 527. Significance is required. The sample size should be the same as in the other experiments where the significance is reported. I wonder if differences are not significant and therefore comments are only on a speculative basis. If the sample size is so crucial (as Authors seems to suggest in lines 542-545) they should increase the samples to drown more consistent and convincing conclusions
Figure S1. This is not the way to assess the stability of housekeeping genes. Authors must show the Ct values and the analysis of gene stability performed with computational programs such as RefFinder. If the stability of the housekeeping gene is not accurately evaluated all results are not reliable
Figure S2. In the legend is mentioned the adenosine treatment at 200 micromolar, but figures report 50 and 100 micromolar
Figure S3. Staining is barely detectable in all conditions and in few cases present only at the border of the plate. I wonder if the monolayer is homogeneous or if the few stained spots are an artifact. Images are not comparable to those in figure S5 or S6.
Figure S4. Similar comments as before for the staining. Moreover, a control (0 adenosine and 0 dypyridamole) is missing

Comments on the Quality of English Language

English should be revised to remove typos and to increase the clarity of sentences

Author Response

Thank you very much for your valuable comment on our manuscript.

Please find the attached Word file.

We used an English editing software "Grammarly Pro" to improve the English presentation.