Dexpanthenol Promotes Cell Growth by Preventing Cell Senescence and Apoptosis in Cultured Human Hair Follicle Cells

Round 1

Reviewer 1 Report

Dexpanthenol is the pro-vitamin 5 which proved to treat human hair loss. The manuscript firstly showed that dexpanthenol reduced senescence and increased the growth factor in both DPCs and ORSCs. It may become the mechanism for clinic supports. However, here are some concerns that need to be addressed.

1. There are 2 papers highly relevant to the authors’ study which are worth inducing in the background. (Kutlu Ö, Metin A. Systemic dexpanthenol as a novel treatment for female pattern hair loss. J Cosmet Dermatol. 2021 Apr;20(4):1325-1330. doi: 10.1111/jocd.13729. Epub 2020 Oct 5. PMID: 32960484; Kutlu Ö. Dexpanthenol may be a novel treatment for male androgenetic alopecia: Analysis of nine cases. Dermatol Ther. 2020 May;33(3):e13381. doi: 10.1111/dth.13381. Epub 2020 Apr 24. PMID: 32255530.)
2. The concentration dosage of dexpanthenol showed inconsistency for all assays which is hard to convince.
3. Fig 1 B should provide the statistical result.
4. The authors showed dexpanthenol treatment protects DPCs from apoptosis and senescence. The apoptosis senescence markers are decreased. However, caspase 3, 9, p21, p16 only in mRNA level. Can authors check the protein level for these markers? However, in the skin hair follicle, there is no solid evidence that showed that DP cells apoptosis. Thus, the authors try to prove dexpanthenol reduced the apoptotic DPCs is not correct.
5. Fig 3, the authors showed ALP assay of DPCs. What is the passage of those DPCs? Because it is known that the HFDPC will lose alkaline phosphatase activity after being cultured and passaged gradually. The 561nm/594nm images are redundant, it is better to remove those images.
6. For all the experiments, how many repeats the authors did?
7. For the signaling pathway, in the anagen, there is a balance between Wnt, TGFb, BMP, and hedgehog. Did the authors check the hedgehog and BMP signaling pathways? The manuscript showed the TGFb1 decrease in RNA level. How about the protein level and its downstream Smad1/5/9? The b - catenin western blot quality is poor.
8. Fig 6, the authors use Minoxidil as a positive control for ORS cells. However, the P21 and P16 showed no change. The authors need to consider remove or change another positive control. Besides, the Bcl2, Bax, P21, P16 need to provide the protein results.
9. Fig 7, the VEGFA is highly expressed in DPCs, not ORSCs, why do authors detect VEGF in ORSCs?
10. It is better if the authors have the mouse model treated with dexpanthenol to facilitate the anagen in the hair cycle.
11. The discussion part needs to be more in-depth.

To summarize, it is a novelty that the authors showed dexpanthenol anti-senescence in vitro. However, they should focus on DP cells or ORS cells only, and test all the signaling to make the content clearer and cleaner.

Author Response

Dexpanthenol is the pro-vitamin 5 which proved to treat human hair loss. The manuscript firstly showed that dexpanthenol reduced senescence and increased the growth factor in both DPCs and ORSCs. It may become the mechanism for clinic supports. However, here are some concerns that need to be addressed.

1. There are 2 papers highly relevant to the authors’ study which are worth inducing in the background. (Kutlu Ö, Metin A. Systemic dexpanthenol as a novel treatment for female pattern hair loss. J Cosmet Dermatol. 2021 Apr;20(4):1325-1330. doi: 10.1111/jocd.13729. Epub 2020 Oct 5. PMID: 32960484; Kutlu Ö. Dexpanthenol may be a novel treatment for male androgenetic alopecia: Analysis of nine cases. Dermatol Ther. 2020 May;33(3):e13381. doi: 10.1111/dth.13381. Epub 2020 Apr 24. PMID: 32255530.)

We addressed those reports in introduction

2. The concentration dosage of dexpanthenol showed inconsistency for all assays which is hard to convince.

The treatment dosage in our experiments ranged essentially between 5μM and 5mM. We explored many kinds of effects of dexpanthenol and don’t think that all those effects are at the same concentrations. The effective concentrations will depend on what the effects are and also on the cell types. We know there are too many data points in some figures so we eliminated them.

3. Fig 1 B should provide the statistical result.

Corrected.

4. The authors showed dexpanthenol treatment protects DPCs from apoptosis and senescence. The apoptosis senescence markers are decreased. However, caspase 3, 9, p21, p16 only in mRNA level. Can authors check the protein level for these markers? However, in the skin hair follicle, there is no solid evidence that showed that DP cells apoptosis. Thus, the authors try to prove dexpanthenol reduced the apoptotic DPCs is not correct.

We added protein data for p21, p16. In case of caspase3, 9, further experiments will be conducted to elucidate post-translational modifications of caspase families.

Concerning the apoptosis of dermal papilla cells, Han et al. reported that minoxidil prevented cellular apoptosis in dermal papilla cells (J.H. Han, O.S. Kwon, J.H. Chung, K.H. Cho, H.C. Eun, K.H. Kim. Effect of minoxidil on proliferation and apoptosis in dermal papilla cells of human hair follicle J. Dermatol. Sci., 34 (2004), pp. 91-98).

Dermal papilla is considered to resist apoptosis associated with high levels of Bcl-2 and lack of death receptors during the entire hair cycle in mice and humans (Lindner et al., 1997. Analysis of apoptosis during hair follicle regression (catagen) Am J Pathol 151:1601–17; Matsuo et al., 1998. Apoptosis in murine hair follicles during catagen regression. Arch Dermatol Res 290:133–6; Soma et al., 1998. Analysis of apoptotic cell death in human hair follicles in vivo and in vitro. J Invest Dermatol 111:948–54).

However, they may be susceptible to apoptosis under certain experimental conditions (Ferraris et al., 1997. Induction of apoptosis through the PKC pathway in cultured dermal papilla fibroblasts. Exp Cell Res 234:37–46; Seiberg et al., 1997. Trypsin-induced follicular papilla apoptosis results in delayed hair growth and pigmentation. Dev Dyn 208:553–64).

Although it is not a common case, the apoptotic markers were reduced by dexpanthenol treatment in our experiments.

5. Fig 3, the authors showed ALP assay of DPCs. What is the passage of those DPCs? Because it is known that the HFDPC will lose alkaline phosphatase activity after being cultured and passaged gradually. The 561nm/594nm images are redundant, it is better to remove those images.

We used DPCs under passage 5, added in methods.

It is true DPCs lose ALP activities over culture time and passages. It doesn’t matter in our data since the control showed ALP activity.

We removed images except the representative one and quqntitated.

6. For all the experiments, how many repeats the authors did?

It has been already described in methods, statistical analysis, n≥3.

7. For the signaling pathway, in the anagen, there is a balance between Wnt, TGFb, BMP, and hedgehog. Did the authors check the hedgehog and BMP signaling pathways? The manuscript showed the TGFb1 decrease in RNA level. How about the protein level and its downstream Smad1/5/9? The b - catenin western blot quality is poor.

We did not check other proteins you recommended yet.

We substituted Western blot image with reduced data points which were too many.

8. Fig 6, the authors use Minoxidil as a positive control for ORS cells. However, the P21 and P16 showed no change. The authors need to consider remove or change another positive control. Besides, the Bcl2, Bax, P21, P16 need to provide the protein results.

We removed the minoxidil part.

9. Fig 7, the VEGFA is highly expressed in DPCs, not ORSCs, why do authors detect VEGF in ORSCs?

We tested several growth factors but only the expression of VEGF changed with significance.

10. It is better if the authors have the mouse model treated with dexpanthenol to facilitate the anagen in the hair cycle.

There are limitations in our study because we performed only in in vitro models but we could not do animal experiment because of the ethical problems and company policy.

As we mentioned dexpanthenol has clinical efficacy of hair growth stimulation, so we focused on the cellular mechanisms of dexpanthol.

11. The discussion part needs to be more in-depth.

We corrected discussion section.

To summarize, it is a novelty that the authors showed dexpanthenol anti-senescence in vitro. However, they should focus on DP cells or ORS cells only, and test all the signaling to make the content clearer and cleaner.

Reviewer 2 Report

This work by Shin et al. looks into the effects of D-Panthenol, a widely used ingredient in hair products, in two major hair follicle cell types: dermal papilla cells (hDPCs) and outer root sheath cells (hORSCs). They find that the increasing concentrations of D-Panthenol improve the cell viability, increase the expression levels of anagen markers and decrease the induction of senescence and apoptosis marker genes. The studies are in general well executed and their results do suggest a hair growth promoting effect of D-Panthenol. However, the study does have major limitations as it does not use any in vitro system mimicking the in vivo hair cycle like hair follicle cell culture and also no detailed mechanism of action of D-Panthenol is determined. The results do indicate that VEGF may be involved. An RNA-seq analysis of cells with and without treatment of D-panthenol might have provided a much clearer indication of global gene expression changes and the cellular pathways affected. Also, no flow cytometry analysis of the cells to check the effect of D-Panthenol on different cell cycle phases and DNA replication have been performed.

This study has important implications in the hair growth research and product development and as such the manuscript could benefit by significant improvements and further analysis.

Major changes:

1. In Figures 1A and 6A, the Y-axis is labeled as “Cell Proliferation” and I am not sure if that is correct. The CCK-8 assay output is the number of viable cells and is not a direct readout of cell proliferation. Is that correct? The authors should check this and modify the Y-axis accordingly.
2. It seems from the DAPI staining in Figure 1B, that most of the cells are not Ki-67 positive. Are they quiescent or senescent? To measure cell proliferation, the authors can do the flow cytometry analysis (such as Click-IT or similar method) as mentioned above. Also, all the immunofluorescence (IF) images can be quantified (% Ki-67 stained cells).
3. The effect on cell viability is seen at low concentrations of D-Panthenol (5 mM, Figure 1A) compared to apoptosis (3-4 mM) and senescence (4 mM) as shown in Figure 2. Can the authors explain that? How does this compared to the concentrations of D-Panthenol normally used in hair-related formulations (if known)?
4. Although p21 and p16 are used as senescence markers, p16 is expressed late during senescence and p21 could be induced upon DNA damage by non-senescent cells. The authors can look into the Table 1 of the paper by Kohli et al., Nature Protocols, 2021 for selecting senescence markers. Maybe they can try few more senescence markers like Lamin B1 (down with senescence).
5. In Figure 3, panel D is not labeled as “(D)”. The IF and western blots can be quantified.
6. The IF images in Figure 4B can be quantified.
7. Line 267 says that “several growth factors were evaluated”. What are these growth factors besides VEFFA and VEFGR? What are the findings?
8. The discussion mainly highlights the results again and is somewhat redundant. The authors could briefly discuss some genomic or mechanistic studies related to hair loss (if known) to indicate major players involved and how D-Panthenol may affect those players to put the results of this manuscript in a broader context. They could also highlight the importance and weaknesses of this study in a bit more detail.

Minor changes:

1. It may be helpful for the readers to have a simple schematic of the hair cycle stages with different cell types labeled. The introduction will be much clearer then.
2. Line 57: Is Caspase an activation marker? It is not clear. It would be good to make a distinction between proliferation and apoptosis markers.
3. In section 2.2 of the Materials and Methods, it would be good to mention the confluency of cells at the time of treatment. The authors can briefly mention how the D-Panthenol solution was prepared. Also, the incubation conditions and other important modifications (if different from the manufacturer’s protocol) could be added.
4. For the section 2.3, it could be mentioned what method was used to analyze RT-PCR data (delta-delta Ct method or something else).
5. For the sections 2.5 and 2.5, the steps in the analysis of the microscopy images could be briefly described.
6. Section 2.7 indicate that the standard deviation (SD) was used in the manuscript, but the figure legends say that SEM was used. Which one is it?
7. Some information about Minoxidil (positive control) such as manufacturer and solution preparation could be described in the Materials and Methods.
8. Change uM to μM everywhere in the manuscript.

Author Response

This work by Shin et al. looks into the effects of D-Panthenol, a widely used ingredient in hair products, in two major hair follicle cell types: dermal papilla cells (hDPCs) and outer root sheath cells (hORSCs). They find that the increasing concentrations of D-Panthenol improve the cell viability, increase the expression levels of anagen markers and decrease the induction of senescence and apoptosis marker genes. The studies are in general well executed and their results do suggest a hair growth promoting effect of D-Panthenol. However, the study does have major limitations as it does not use any in vitro system mimicking the in vivo hair cycle like hair follicle cell culture and also no detailed mechanism of action of D-Panthenol is determined. The results do indicate that VEGF may be involved. An RNA-seq analysis of cells with and without treatment of D-panthenol might have provided a much clearer indication of global gene expression changes and the cellular pathways affected. Also, no flow cytometry analysis of the cells to check the effect of D-Panthenol on different cell cycle phases and DNA replication have been performed.

This study has important implications in the hair growth research and product development and as such the manuscript could benefit by significant improvements and further analysis.

Major changes:

1. In Figures 1A and 6A, the Y-axis is labeled as “Cell Proliferation” and I am not sure if that is correct. The CCK-8 assay output is the number of viable cells and is not a direct readout of cell proliferation. Is that correct? The authors should check this and modify the Y-axis accordingly.

As you mentioned, CCK-8 assay results indicate the number of viable cells so the increase in viable cell number could represent the cell growth, proliferation.

2. It seems from the DAPI staining in Figure 1B, that most of the cells are not Ki-67 positive. Are they quiescent or senescent? To measure cell proliferation, the authors can do the flow cytometry analysis (such as Click-IT or similar method) as mentioned above. Also, all the immunofluorescence (IF) images can be quantified (% Ki-67 stained cells).

Ki-67 negative cells seem quiescent.

The original images show more Ki-67 positive cells seen in the manuscript figure where cells with medium to low fluorescent intensities were not detectable and Ki-67 positive cells were quantified.

3. The effect on cell viability is seen at low concentrations of D-Panthenol (5 mM, Figure 1A) compared to apoptosis (3-4 mM) and senescence (4 mM) as shown in Figure 2. Can the authors explain that? How does this compared to the concentrations of D-Panthenol normally used in hair-related formulations (if known)?

The treatment dosage in our experiments ranged essentially between 5μM and 5mM. We explored many kinds of effects of dexpanthenol and don’t think that all those effects are always at the same concentration. The effective concentrations will depend on what the effects are and also on the cell types.

4. Although p21 and p16 are used as senescence markers, p16 is expressed late during senescence and p21 could be induced upon DNA damage by non-senescent cells. The authors can look into the Table 1 of the paper by Kohli et al., Nature Protocols, 2021 for selecting senescence markers. Maybe they can try few more senescence markers like Lamin B1 (down with senescence).

I appreciate your suggestion but we could not do more experiments with senescent markers other than p21, and p16.

5. In Figure 3, panel D is not labeled as “(D)”. The IF and western blots can be quantified.

We corrected the figure and quantitated the data.

6. The IF images in Figure 4B can be quantified.

Quantitation done.

7. Line 267 says that “several growth factors were evaluated”. What are these growth factors besides VEFFA and VEFGR? What are the findings?

We tested several growth factors (VEGFA, EGF, KGF, bFGF, IGF1, TGFb1, PDGFA) but only the expression of VEGF changed with significance.

8. The discussion mainly highlights the results again and is somewhat redundant. The authors could briefly discuss some genomic or mechanistic studies related to hair loss (if known) to indicate major players involved and how D-Panthenol may affect those players to put the results of this manuscript in a broader context. They could also highlight the importance and weaknesses of this study in a bit more detail.

We revised the discussion section

Minor changes:

1. It may be helpful for the readers to have a simple schematic of the hair cycle stages with different cell types labeled. The introduction will be much clearer then.

We described about the hair cycle in introduction.

2. Line 57: Is Caspase an activation marker? It is not clear. It would be good to make a distinction between proliferation and apoptosis markers.

Caspase is not an activation marker so has been deleted.

3. In section 2.2 of the Materials and Methods, it would be good to mention the confluency of cells at the time of treatment. The authors can briefly mention how the D-Panthenol solution was prepared. Also, the incubation conditions and other important modifications (if different from the manufacturer’s protocol) could be added.

We specified the cell number seeded in culture plates/dishes.

Revised.

4. For the section 2.3, it could be mentioned what method was used to analyze RT-PCR data (delta-delta Ct method or something else).

Done

5. For the sections 2.5 and 2.5, the steps in the analysis of the microscopy images could be briefly described.

The instrument was operated according to the manual. I don’t think it is appropriate to describe an operation manual in the manuscript.

6. Section 2.7 indicate that the standard deviation (SD) was used in the manuscript, but the figure legends say that SEM was used. Which one is it?

SD is correct. Done

7. Some information about Minoxidil (positive control) such as manufacturer and solution preparation could be described in the Materials and Methods.

Done

8. Change uM to μM everywhere in the manuscript.

Corrected

Round 2

Reviewer 1 Report

The authors addressed some of my concerns but not all of them. For the dermal papilla apoptosis, the authors explain by using 90s paper, which is not convinced. My point is in vivo, we did not observe the apoptotic dermal papilla cells. Thus, the in vitro assay may not reflect the DP cell fate in the skin. The authors added the P21 and P16 western blot images, but not Caspase 3, 9 (Comment 4). The p21 and p16 indicate the senescence, caspase 3 and 9 are the direct markers for apoptosis. Authors should not ignore this. For comment 7, it is not an excuse because there are too much data. For comment 8, the authors did not provide the Bcl2, Bax, P21, P16 protein results. For comment 9 the authors only found VEGF changed in mRNA level. They should verify protein level and check the downstream of VEGF signaling pathway or rescue by using VEGF inhibitors.

Author Response

The authors addressed some of my concerns but not all of them. For the dermal papilla apoptosis, the authors explain by using 90s paper, which is not convinced. My point is in vivo, we did not observe the apoptotic dermal papilla cells. Thus, the in vitro assay may not reflect the DP cell fate in the skin. The authors added the P21 and P16 western blot images, but not Caspase 3, 9 (Comment 4). The p21 and p16 indicate the senescence, caspase 3 and 9 are the direct markers for apoptosis. Authors should not ignore this. For comment 7, it is not an excuse because there are too much data. For comment 8, the authors did not provide the Bcl2, Bax, P21, P16 protein results. For comment 9 the authors only found VEGF changed in mRNA level. They should verify protein level and check the downstream of VEGF signaling pathway or rescue by using VEGF inhibitors.

Reply:

As I mentioned, our work has major limitation that all the works were done in vitro, whose results might be deviated from those of the real, in vivo situation. Please appreciate that the purpose of our work is to find molecular and cellular clues of panthenol for its anti-hair loss efficacy.

We added the protein data of caspase 3 but could not for caspase 9 whose antibody we don’t have right now. I agree that it should be confirmed by protein level not only by mRNA level. I think the reduction in apoptosis could be demonstrated at least in part.

About comment 7, there should have been a miscommunication. We did not check hedgehog and BMPs not because there are too much data. I agree that these signaling pathways play important roles in anagen phase but our study focused on the prevention of catagen entry through apoptosis and senescence so we did not take those proteins into consideration. Those could be checked in our future works.

For comment 8 and 9, dermal papilla is the main regulator of hair cycle so most experiments were done in DPCs. Regarding the data in ORSCs, please appreciate we did some preliminary works, which should be elucidated in future works.

Reviewer 2 Report

The authors did make most of the changes as suggested. The Y-axis in Fig 1A and 6A can say "Viable cells (%)" instead of "Cell Proliferation (%)". The changes made to the discussion is OK, but more effort could have been made. The authors quantified all the data presented in the figures and that really helps in interpreting the figures better. Hope the authors can do more mechanistic and genomic studies in their future work as this work still leaves questions behind with regards to mechanism of action and pathways involved. 

Author Response

The authors did make most of the changes as suggested. The Y-axis in Fig 1A and 6A can say "Viable cells (%)" instead of "Cell Proliferation (%)". The changes made to the discussion is OK, but more effort could have been made. The authors quantified all the data presented in the figures and that really helps in interpreting the figures better. Hope the authors can do more mechanistic and genomic studies in their future work as this work still leaves questions behind with regards to mechanism of action and pathways involved. 

We changed the axis labels.

I appreciate your suggestions for future works.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.