Multi-Target Restoration of Dermal Elastic Fibers Through Elastin Upregulation, Elastase Suppression, and Scaffold Reinforcement
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe study proposed by Ye and collaborators aims at monitoring the effects of combined treatments on elastic fiber integrity following UV exposure of skin fibroblasts and ex vivo models. Although the topic is of high interest, several points have to be clarified or improved to be convincing.
Major points:
1- It is well known that UV exposure will induce elastic fiber degradation due to the release of proteases in response to oxidative stress. However, elastin gene expression is usually increased in cellular models, while Figure 1b shows a decrease in control UV when compared to unexposed cells. Is the decrease significant? Moreover, the problem of elastin expression following UV radiations is the unusual spliced form of elastin comprising the exon 26A that results in incorrect protein folding. Does the elastin mRNA induced by Copper Tripeptide-1 contain the exon 26A? If yes, the resulting elastic fibers would not function properly…
2- In figure 3a, all the tested genes appear upregulated. Is it specific for elastic-fibers related genes or a global effect? What about housekeeping genes? Are they stable? Can you provide a gene which expression is not increased by cedrol as negative control?
3- The images in figure 3b are not convincing because the fluorescent signal is intracellular while fibrillin-1 and MAGP-1 are expected to be extracellular proteins. The reason may be the fact that the study is conducted in sub-confluent proliferative cells. Usually, these proteins are observed in post-confluent cultures in which they will self-organized as fibrillar structures in the extracellular compartment. In consequence, the specificity of the signal is questionable on the images.
4- We know that elastic fibers are quite not homogenous in the dermis. What was the procedure to ensure that the comparisons were made in similar locations? How many images were quantified per sample? A more robust and quantitative method such as desmosin quantification would have been preferred.
5- The discussion section is particularly light. The authors remind the main results and explain that they did not explore the signaling pathways, nor the mechanical properties. Since the 3 compounds are already described in the literature, it would be quite easy to propose hypothetical mechanisms for each treatment.
Minor points:
6- Statistics are missing in Fig 1a and 2a.
7- Please indicate the “n” used to make statistics for each experiment, and if they are technical or biological replicates.
Author Response
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Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThis study reports that copper tripeptide-1, ethyl ferulate, and cedrol can promote elastin synthesis, inhibit elastase activity, and reinforce the microfibrillar scaffold, respectively, and that the complex composed of these three components can effectively repair UV-damaged dermal elastic fibers. I have the following questions and comments:
1.The rationale for the authors' selection of copper tripeptide-1, ethyl ferulate, and cedrol should be elaborated in the Introduction. In addition, the structural characteristics of the test substances, or corresponding structural diagrams, should be described in the Materials and Methods section—especially the distinct advantages of copper tripeptide-1.
2.A single UV irradiation was applied in the individual experiments of the test substances, whereas repeated UV irradiations were used in the combined experiment of the three components. What is the consideration for this difference in UV irradiation regimens? The UV irradiation protocol should be described in greater detail and accuracy.
3.Can copper tripeptide-1, ethyl ferulate, and cedrol penetrate the epidermis to reach dermal fibroblasts and exert their biological effects?
4.How should the "multi-target modulation" proposed by the authors be interpreted and defined?
5.This study involves almost no investigation into the underlying molecular mechanisms, resulting in a relatively superficial research level.
Author Response
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Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThe authors have addressed most of the comments made previously. Nevertheless, in my opinion, the intracellular labeling of matrix proteins remains a major weakness in this study, which would benefit from demonstrating the effect in overconfluent cultures (6-8 days). Intracellular labeling may be appropriate for quantitative flow cytometry studies following treatment with brefeldin A to block all secretion. However, in the present case, it is difficult to assign a qualitative or quantitative value to the result.
Author Response
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Author Response File: Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThe author carefully answered my questions and also proposed prospects and research plans for the problems that couldn't be solved temporarily.
Author Response
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Author Response File: Author Response.pdf
Round 3
Reviewer 1 Report
Comments and Suggestions for AuthorsNo further comments.
