Effect of Atorvastatin on Angiogenesis-Related Genes VEGF-A, HGF and IGF-1 and the Modulation of PI3K/AKT/mTOR Transcripts in Bone-Marrow-Derived Mesenchymal Stem Cells

Round 1

Reviewer 1 Report

Authors have investigated the effect of the most widely prescribed statin, atorvastatin, on the characteristics and properties of bone marrow derived mesenchymal stem cells (BM-MSCs) cultured in vitro. This study concludes that t atorvastatin did not decrease the viability of BM-MSCs, nor did it change the expression of MSC markers. Atorvastatin upregulated the mRNA expression levels of VEGF-A and HGF, and reduced the mRNA expression levels of IGF-1. In addition, the PI3K/AKT signaling pathway was modulated by atorvastatin as indicated by the high mRNA expression levels of PI3K and AKT. There are some minor issues to be rectified;

1. The submitted manuscipt has some text with red color, which should be corrected. 

2. The method of cell treatment with Atorvastatin is not clearly illustrated, authors used 0.1% Atorvastatin in DMSO then how they treat the cells with this drug?

3. In Figure 5, authors have proposded the mechanism of atorvastatin-induced regulation of VEGF-A, HGF, IGF-1 and IGF-2 mRNA expression and the effects on PI3K/AKT/mTOR transcripts in BM-MSCs. RTKs, recepto tyrosine kinases. However, many factors/roles are unknown, It will be great to propose some future directions in accordance with the complete PI3K/AKT/mTOR pathway for stem cells accordingly.
In addition, there is a lietrature (10.1007/s13770-021-00362-z) about Atorvastatin Pretreatment Ameliorates Mesenchymal Stem Cell Migration through miR-146a/CXCR4 Signaling. It will be beneficial if authors can relate their proposed mechanism to this already published work. 

4. Some parts or sentences for example,
"Among statins, atorvastatin represents the most widely prescribed statin worldwide" repeated as it is  two times. 
Cells were treated for 20 minutes, 2, 24, 48 88 and 96 hours and treated with 10 µM Atorvastatin (ATO). not understandable

5. Please proof read the whole manuscript to eradicate typoor grammatical errors before resubmission.

6. There are some recent literatures to be included; 

• 10.2165/00003495-200161120-00012
• 10.1093/cvr/cvz139

Author Response

Reviewer 1

Authors have investigated the effect of the most widely prescribed statin, atorvastatin, on the characteristics and properties of bone marrow derived mesenchymal stem cells (BM-MSCs) cultured in vitro. This study concludes that t atorvastatin did not decrease the viability of BM-MSCs, nor did it change the expression of MSC markers. Atorvastatin upregulated the mRNA expression levels of VEGF-A and HGF, and reduced the mRNA expression levels of IGF-1. In addition, the PI3K/AKT signaling pathway was modulated by atorvastatin as indicated by the high mRNA expression levels of PI3K and AKT. There are some minor issues to be rectified;

1. The submitted manuscript has some text with red color, which should be corrected.
2. The method of cell treatment with Atorvastatin is not clearly illustrated, authors used 0.1% Atorvastatin in DMSO then how they treat the cells with this drug?
3. In Figure 5, authors have proposded the mechanism of atorvastatin-induced regulation of VEGF-A, HGF, IGF-1 and IGF-2 mRNA expression and the effects on PI3K/AKT/mTOR transcripts in BM-MSCs. RTKs, recepto tyrosine kinases. However, many factors/roles are unknown, It will be great to propose some future directions in accordance with the complete PI3K/AKT/mTOR pathway for stem cells accordingly.

In addition, there is a lietrature (10.1007/s13770-021-00362-z) about Atorvastatin Pretreatment Ameliorates Mesenchymal Stem Cell Migration through miR-146a/CXCR4 Signaling. It will be beneficial if authors can relate their proposed mechanism to this already published work.

4. Some parts or sentences for example,

"Among statins, atorvastatin represents the most widely prescribed statin worldwide" repeated as it is  two times.

Cells were treated for 20 minutes, 2, 24, 48 88 and 96 hours and treated with 10 µM Atorvastatin (ATO). not understandable

5. Please proof read the whole manuscript to eradicate typoor grammatical errors before resubmission.
6. There are some recent literatures to be included;

• 10.2165/00003495-200161120-00012
• 10.1093/cvr/cvz139

Response to reviewer 1:

Dear Reviewer #1, We thank you and the reviewer #2 for the valuable comments and suggestions. Accordingly, the manuscript has been rechecked for language and the necessary changes have been made throughout the manuscript. Point-by-point responses to the comments have been prepared and attached herewith. The revisions made in response to the comments are marked up using the “Track Changes” function in the revised manuscript. We look forward to working with you to move this manuscript closer to publication in the Current Issues in Molecular Biology. Thank you for your consideration.

1. We sincerely thank you for providing critical comments and useful suggestions, that have helped us improve our manuscript. We have corrected the text with red colour from our manuscript.
2. Thank you for your pertinent question. In our experiment, control cells were treated with cultivation medium (CTR) or 0.1% DMSO as a vehicle control (DMSO). Atorvastatin (Sigma, Germany) was dissolved in 0.1% DMSO to a final concentration of 10µM in cultivation medium (ATO). MSCs were pre-treated with 10µM atorvastatin for 20 minutes (20m ATO 10µM), 2 hours (2h ATO 10µM), 24 hours (24h ATO 10µM), 48 hours (48h ATO 10µM), or 96 hours (96h ATO 10µM). We modified the content of methods. Kindly see Page 2, Line 84-90 in revised manuscript.
3. We greatly appreciate your valuable suggestion. We modified the content and figure in the discussion according to the reviewer’s suggestions. The abovementioned reference has been added. Kindly see Page 9, Line 342-354 in revised manuscript.
4. We are very grateful for your suggestions and corrections. According to your instructions, we deleted repeating phrases in the introduction, Line 62-63. We have modified the method part of drug treatment. Kindly see Page 2, Line 88-90 in revised manuscript.
5. Thank you, the manuscript has been rechecked for language and grammatical errors.
6. We are very grateful for your suggestions. According to your instructions, the abovementioned references have been added. Kindly check Page 7, Line 248-259, Page 9, Line 355-366 in revised manuscript.

 

Author Response File: Author Response.docx

Reviewer 2 Report

The authors have completed a small study on the effects of Atorvastatin. While very short and compact the authors have shown the effect of atorvastatin in a time course and dose dependant way. overall the paper is well organized but requires and overall check of english.  This reviewer asks to explain better why the concentration at 10 uM was chosen. 

Author Response

Reviewer 2

The authors have completed a small study on the effects of Atorvastatin. While very short and compact the authors have shown the effect of atorvastatin in a time course and dose dependant way. overall the paper is well organized but requires and overall check of english.  This reviewer asks to explain better why the concentration at 10 uM was chosen.

Response to reviewer 2:

Dear Reviewer #2, We thank you and the reviewer #1 for the valuable comments and suggestions. Accordingly, the manuscript has been rechecked for language and the necessary changes have been made throughout the manuscript. Point-by-point responses to the comments have been prepared and attached herewith. The revisions made in response to the comments are marked up using the “Track Changes” function in the revised manuscript. We look forward to working with you to move this manuscript closer to publication in the Current Issues in Molecular Biology. Thank you for your consideration.

1. Thank you for your pertinent question. In our experiment the optimal concentration of atorvastatin (10µM) was chosen based on literature search identifying in vitro experiments where pleiotropic effects of statin are claimed (Reference: 15. Björkhem-Bergman, L.; Lindh, J.D.; Bergman, P. What Is a Relevant Statin Concentration in Cell Experiments Claiming Pleiotropic Effects?: Letter to the Editors. British Journal of Clinical Pharmacology 2011, 72, 164–165, doi:10.1111/j.1365-2125.2011.03907.x.

Reference 16. Youssef, S.; Radosevich, J.L.; Hur, E.M.; Bravo, M.; Mitchell, D.J.; Sobel, R.A.; Steinman, L.; Zamvil, S.S. The HMG-CoA Reductase Inhibitor, Atorvastatin, Promotes a Th2 Bias and Reverses Paralysis in Central Nervous System Autoimmune Disease. 2002, 420.)

We modified the content of methods with references. Kindly see Page 2, Line 88-90 in revised manuscript.

 

Author Response File: Author Response.docx