Effects and Mechanism of Plasma-Activated Medium on Angiogenesis of Vascular Endothelial Cells

Round 1

Reviewer 1 Report

This is a large and very detailed study of the effects of LTP treatment where the authors described that plasma-activated medium has a dual effect on the angiogenesis of HUVECS.

However, there are some issues that should be checked;

Experiments are extensively and well described but there is important information missed;

• Sample size of each experiment should be described in materials and methods section
• Control of each experiment is not described throughout the text despite I can see the control group in the figures.
• Experiment 2.5 (Detection of angiogenesis) should be better describe; I cannot understand which is the PAM treatment, when are the cells treated with PAM?
• Regarding Elisa experiments, are samples measured in duplicate, please describe throughout the text.
• Parenthesis in the data should be removed in 3.1, 3.3, 3.4, and 3.5 sections.
• In Figures 4d and 5 differences between treatment and control are showed, are there also differences between groups?
• Figure legends should be better described and should be shown if data are represented as mean± SD or SEM.
• Magnification is missed in figure legends 2 and 3

Comments for author File: Comments.docx

Author Response

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Reviewer 2 Report

The manuscript is well written and interesting for the readers. It contains important information. The experimental section and research design is appropriate and well written, experimental results are nicely presented. I only suggest editing of English language and style.

Author Response

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Reviewer 3 Report

In this paper, the authors describe the effects of a plasma-activated medium on the angiogenesis of vascular endothelial cells. The manuscript is written moderately and experiments requires further analysis. 

In the introduction, the authors introduce their work at the very end and did not give any specific details about their own experiments. There is no information about the procedures that the authors are about to follow, experiments they will be doing to prove/provide a concept, what passages of HUVECs they used in the subsequent experiments, etc. The introduction should set the tone for the remaining paper. The authors have done a lot of work, but it is not properly explained in the introduction. Authors should re-write the introduction and highlight their procedures. 

Why plasma treatments were done for 15-75s and not given more time? There needs to be a specific explanation.

It would've been nice to see some cell-viability dye used in this work to characterize cell-viability rather than indirect calculation. 

In paragraph 2.6, detection of cell migration is best defined by cell speed/ velocity analysis which the authors didn't perform. Authors can use Particle Image Velocimetry (PIV) analysis to perform cell displacement analysis from consecutive phase images. The displacement from consecutive phase images then can be converted to cell velocity/speed. If the authors do not have access to PIV analysis codes, they can get them from this TFM paper (https://www.jove.com/v/60034/perturbing-endothelial-biomechanics-via-connexin-43-structural) which specifically worked with HUVECs. 

In paragraph 3.2 and in figure 3a), the authors report the decrease of nodes, junctions, mesh number, and vascular branches at different time points. From their figure, it is very unclear what is defined as nodes or mesh, junctions or, vascular branches. Authors should point out nodes, mesh, junctions from the control and from different time points, preferably with arrow points to show the difference/decrease.   

Many of the figures (2,3b,4d,5,6,7b,8,9) are missing their horizontal axis label (time). If you label the vertical axis, you should also label the horizontal axis. Also, figure 3b and 4d vertical axis just mention numbers. Is it a number of cells or what? Figure labels should be done more accurately and carefully. Also. figure description should include more words to describe the figures. 

Author Response

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Author Response File: Author Response.docx

Round 2

Reviewer 3 Report

The authors have done a good job addressing my concern about their work. However, I had expected them to perform PIV analysis and get the actual velocity results for cell migration. I do appreciate the author's response to acknowledge that PIV analysis is something they're unfamiliar with and they will probably try analysis like this next time. 

Looking at the results, I do have one more suggestion for the authors. Authors have reported a decrease in nodes, junctions, and meshes when the PAM time increases from 30-75 secs. Also, the HUVECs appear to be migrating slowly with increasing PAM time. There is no explanation given by the authors why HUVECs are slowing down or nodes/junctions are decreasing. One very possible explanation could be, PAM treatment could gradually decrease CX43 gap junction expression of HUVECs, which in turn lowers cell migration speed and decreases nodes/mesh. Authors should include this likely explanation in their discussion and can reference the works done by Islam et al. (https://link.springer.com/article/10.1007/s11340-018-00445-4, and https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7141411/) with HUVECs and CX43. In future work, authors should explore the impact of PAM on HUVEC gap junctions, adherens, and tight junctions and those experiments will likely provide some explanations to current results described in this paper. 

Author Response

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Author Response File: Author Response.docx