Platonin, a Cyanine Photosensitizing Dye, Ameliorates Inflammatory Responses in Vascular Smooth Muscle Cells by Modulating Inflammatory Transcription Factors
Round 1
Reviewer 1 Report
Manuscript details:
Manuscript ID: applsci-1075618
Journal: Applied Sciences (ISSN 2076-3417)
Type of manuscript: Article
Title: Platonin, a Cyanine Photosensitizing Dye, Ameliorates Inflammatory Responses in Vascular Smooth Muscle Cells by Modulating Inflammatory Transcription Factors.
Authors: Chih-Wei Chiu , Chih-Hao Yang , Jie-Heng Tsai , Cheng-Ying Hsieh * , Shih-Yi Huang *
Revision:
The Article entitled: “Platonin, a Cyanine Photosensitizing Dye, Ameliorates Inflammatory Responses in Vascular Smooth Muscle Cells by Modulating Inflammatory Transcription Factors” is very interesting.
In this study, authors described as platonin, a cyanine photosensitizing dye with immunomodulating activity, suppressed LPS/IFN-γ or ox-LDL-induced inflammation in VSMCs. However, pretreatment with platonin significantly reduced the ox-LDL-induced accumulation of lipid drops in the VSMCs. Therefore, the inhibition of proinflammatory factors (and the subsequent inflammatory cascade) may be effective in atherosclerosis therapy. This suggests that platonin should be a potential therapeutic agent against vascular inflammatory diseases.
I think the work is well written, clear and well thorough and I think that the manuscript is suitable for its publication in Applied Science after minor revisions:
In the Materials and Methods section:
- Line 81: add the glutamine concentration
- A subparagraph should be added to explain the treatment times of VSMCs with LPS / IFN-γ and ox-LDL for the different parameters assayed. The treatment times are reported in the results section 24h, (line 115), 48h (line 152), 6h (line186)..... but they should be reported also in the Materials & Methods section or it should be emphasized that treatment times are specified below. The same applies to the pretreatment of cells with platonin.
In the Results section:
In line 152: the authors reported “48h”, but in the figure legend reported “24h” (line 178): clarify it.
In the References section:
In line 396: replace “37” with “637”
In line 406: replace “5” with “275”
In line 425, 432, 430….etc…… write the complete page numbers in full for the references.
Author Response
Response to Reviewer #1
We appreciate your comments for our study and paid careful attention to them as we were revising our manuscript. The following is our responses to the specific issues raised.
Comment 1.
In the Materials and Methods section:
Line 81: add the glutamine concentration
A subparagraph should be added to explain the treatment times of VSMCs with LPS / IFN-γ and ox-LDL for the different parameters assayed. The treatment times are reported in the results section 24h, (line 115), 48h (line 152), 6h (line186)..... but they should be reported also in the Materials & Methods section or it should be emphasized that treatment times are specified below. The same applies to the pretreatment of cells with platonin.
Response:
We are thankful for your reminder. The concentration of glutamine was added in line 81 and highlighted with red shading. We have also descripted this information of treatment times in the “Materials and Methods” section of newly revised manuscript and highlighted with red shading (p3; line 83-100).
Comment 2.
In the Results section:
In line 152: the authors reported “48h”, but in the figure legend reported “24h” (line 178): clarify it.
Response:
We are sorry for the wrong labeling. This mistake has been corrected in the figure legend of figure 2 and highlighted with red shading (p6; line 185).
Comment 3.
In the References section:
In line 396: replace “37” with “637”
In line 406: replace “5” with “275”
In line 425, 432, 430….etc…… write the complete page numbers in full for the references
Response:
We are really sorry about the inconsistency of reference typing. The references has been checked in the “References” section and highlighted with red shading in the newly revised manuscript.
We appreciate all your comments for this study, and hope the above changes meet with your agreement. Your comments and suggestions truly improve the study.
Author Response File: 
Author Response.docx
Reviewer 2 Report
Chiu et al. in the present original research article “Platonin, a Cyanine Photosensitizing Dye, Ameliorates Inflammatory Responses in Vascular Smooth Muscle Cells by Modulating Inflammatory Transcription Factors” investigated the inhibitory effects of platonin on LPS/IFNγ- and oxLDL-induced inflammation in murine VSMC. The authors found that platonin pretreated cells have significantly reduced the levels of iNOS, mIL1β, and phospho-p65 following LPS/IFNγ stimulation compared with control cells. Furthermore, platonin treatment suppressed oxLDL-induced MCP-1 production and lipid accumulation in VSMC. Overall, the manuscript is well-written. The introduction section is focused and Materials and Methods are adequately detailed. Data is clearly presented and easy to follow. However, I have the following minor concerns.
- There are some grammatical errors, which need to be corrected.
- In Fig. 1B and 1C, please put the representative Western blot images of mIL1β above α-tubulin images or provide image of other control protein used. The labeling of figures is quite confusing. The authors may simply label the panels like A, B, C, D, E…….. Furthermore, there is no 1C label.
- It is not clear from the figure legend of Fig. 2, what concentration of oxLDL was used in Fig. 2C? Please mention it. Did the authors check the apoptosis of VSMC after oxLDL treatment?
- The authors need to mention in the Fig 3 legend, what black and gray bars indicate in Fig. 3A (a-b)? In addition, they may consider writing Time (min) opposite to the X-axis of Fig. 3B (a). The figures should be self-explanatory.
- The authors need to show the levels of total p65 and total JNK1/2 in Fig. 3 and 4.
- Please provide colored representative images of Oil Red O (ORO) staining (Fig. 5A). What was the method used to quantify the number of ORO positive cells, how the cell numbers were normalized? Please include these information in the Materials and methods section.
Author Response
Response to Reviewer #2
We appreciate your comments for our study and paid careful attention to them as we were revising our manuscript. The following is our responses to the specific issues raised.
Comment 1. There are some grammatical errors, which need to be corrected.
Response:
We are sorry for the grammatical errors. In fact, the first manuscript had also been subjected to English editing. I have checked the manuscript again and corrected some apparent grammatical errors. These corrections have been highlighted with red shading (lines 30, 45, 47, 60, 66, 68, 191, 243, 249, 335, and 363).
Comment 2. In Fig. 1B and 1C, please put the representative Western blot images of mIL1β above α-tubulin images or provide image of other control protein used. The labeling of figures is quite confusing. The authors may simply label the panels like A, B, C, D, E…….. Furthermore, there is no 1C label.
Response:
I am so embarrassed about the wrong labeling of Fig. 1 and have corrected the mistake. However, the labeling of Aa, Ab, Ac… but not A, B, C, D, E is because that we want to indicate these results are interrelated. On the other hand, regarding the control of mIL-1b; in the present study, we actually determined the expression of mature form IL-1b which is secreted to the medium. There is no suitable protein control of conditioned medium. We thus used the cytosolic level of a-tubulin to normalize the expression of mIL-1b. The quantification of mIL-1b in medium was mentioned in figure legend and method section (lines 90, 155, 186). We also highlighted it in the newly revised manuscript.
Comment 3. It is not clear from the figure legend of Fig. 2, what concentration of oxLDL was used in Fig. 2C? Please mention it. Did the authors check the apoptosis of VSMC after oxLDL treatment?
Response:
We are sorry for the short of indication. This mistake has been corrected in the figure legend of Fig. 2 and highlighted with red shading (line 185). We did not check the apoptosis after ox-LDL treatment in VSMCs. Some previous studies indicated that ox-LDL could induce the phenomenon of apoptosis in VSMCs; however, ox-LDL was also found to stimulate proliferation and migration in VSMCs. The cell number issue may not affect the conclusion of anti-inflammatory effect of platonin in VSMCs.
Comment 4. The authors need to mention in the Fig 3 legend, what black and gray bars indicate in Fig. 3A (a-b)? In addition, they may consider writing Time (min) opposite to the X-axis of Fig. 3B (a). The figures should be self-explanatory.
Response:
We are sorry about the poor labeling of figure legend. The definition of black, grey and deep grey bars has been mentioned in the figure legend of Fig. 3 and Fig. 4 with red shading (lines 233,234, 281, 282). To make treatment time more clear, we have added “min” into the statistical graph of Fig. 3B(a) and Fig. 4B(a) in the newly revised manuscript.
Comment 5. The authors need to show the levels of total p65 and total JNK1/2 in Fig. 3 and 4.
Response:
Generally, total protein may not affected by the short term exposure of platonin treatment. However, the incidence may still exist. We thus have repeated the experiment by using total JNK1/2 as the internal control of p-JNK1/2 in Fig. 4, and it was not affected by the treatment of platonin. Nevertheless, we cannot provide the data of total p65 in the newly revised manuscript, because we cannot finish all the experiments within five days. If you feel that we must provide the data of total p65, we are still glad to repeat it.
Comment 6. Please provide colored representative images of Oil Red O (ORO) staining (Fig. 5A). What was the method used to quantify the number of ORO positive cells, how the cell numbers were normalized? Please include these information in the Materials and methods section.
Response:
We have replaced the representative images of ORO staining into colored images in Fig. 5A, and described the quantification of ORO positive cells in the section of “Methods and Materials” with red shading (lines 107-110).
We appreciate all your comments for this study, and hope the above changes meet with your agreement. Your comments and suggestions truly improve the study.
Author Response File: Author Response.docx
