Do T2DM and Hyperglycaemia Affect the Expression Levels of the Regulating Enzymes of Cellular O-GlcNAcylation in Human Saphenous Vein Smooth Muscle Cells?

Round 1

Reviewer 1 Report

The authors presented an interesting manuscript concerning T2DM and hyperglycaemia impact on the expression levels of the regulating enzymes of cellular O-GlcNAcylation in human saphenous vein smooth muscle cells. Here are some suggestions for modifications of the manuscript:

Abstract: Please, add methods that were used for the determination of expression levels of target enzymes.

Introduction: please double check for font theme (line 78).

Materials and methods: mL instead of ml

Table 1. Is there any information on the treatment type of T2DM patients?

Regarding statistical analysis- why mean ± standard error of mean were used? All data were with normal distribution?

In this section, authors should only comment on the results, without further explanations. There is a discussion section for that. There is no place for citation. So please, rewrite this section. For example, lines 301-307 are completely unnecessary. Same for all subsections in Materials and Methods.

What are the limitations of the study? Is there any other method that authors would use to confirm their hypothesis that neither T2DM nor hyperglycaemia caused any significant alteration in the expression levels of regulatory enzymes that control O-GlcNAcylation in HSVSMCs?

Author Response

Reviewer 1

The authors presented an interesting manuscript concerning T2DM and hyperglycaemia impact on the expression levels of the regulating enzymes of cellular O-GlcNAcylation in human saphenous vein smooth muscle cells. Here are some suggestions for modifications of the manuscript:

Abstract: Please, add methods that were used for the determination of expression levels of target enzymes.

Introduction: please double check for font theme (line 78).

Response: Thank you for this observation, this has now been corrected. See line 77-79 on the tracked version of the revised manuscript.

Materials and methods: mL instead of ml

Response: Thank you for the observation, these have now been corrected in the entire manuscript. See tracked version of the revised manuscript.

Table 1. Is there any information on the treatment type of T2DM patients?

Response: Thank you for this question. We have information on the treatment each patient either T2DM or non-T2DM received. We have now submitted this as a supplementary document.

Regarding statistical analysis- why mean ± standard error of mean were used? All data were with normal distribution?

Response: Thank you for your question. We used mean ± SEM because we want to accommodate as much as possible all uncertainties around the estimate of the mean measurement which is a more dependable approach in calculating the confidence interval.

In this section, authors should only comment on the results, without further explanations. There is a discussion section for that. There is no place for citation. So please, rewrite this section. For example, lines 301-307 are completely unnecessary. Same for all subsections in Materials and Methods.

Response: Thank you so much for the suggestion and recommendation. This section has now been revised as suggested. Detailed descriptions with references previously included in this section have now been moved to the discussion section. Also, the reference numbers have been adjusted appropriately to reflect these changes. See lines 450-452 and 457-461.

What are the limitations of the study? Is there any other method that authors would use to confirm their hypothesis that neither T2DM nor hyperglycaemia caused any significant alteration in the expression levels of regulatory enzymes that control O-GlcNAcylation in HSVSMCs?

Response: Thank you and we appreciate your technical question and insight. We recognise that there are some limitations to the study, some of which have been highlighted in some paragraphs of the discussion. For example, we have highlighted that our observation could be cell specific and not full characteristic of vascular cells and saphenous vein cells specifically (See lines 493-495). Also, we have highlighted that while there are no significant changes in the expression of these enzymes in HSVSMCs, this finding does not suggest that activities of these enzymes and their effect on other PTMs such as phosphorylation might not be significantly altered (See lines 518-520). Additionally, our group continues to investigate the possible involvement of HSVSMCs dysfunction in vascular pathologies and we intend to continue to optimise our approach as we expand this study.

Reviewer 2 Report

The study by Bolanle IO et al, titled “Do T2DM and hyperglycaemia affect the expression levels of the regulating enzymes of cellular O-GlcNAcylation in human saphenous vein smooth muscle cells?” They have compared protein of OGA, OGT, and GFAT in HSVSMCs of T2DM and non-T2DM patients and concluded that no changes between T2DM and non-T2DM patients. Although this is an interesting finding, I have some concerns about this article.

Specific comments

It would be nice to measure mRNA level of respective enzyme by q-PCR.

Author should estimate enzyme activity of respective OGA, OGT, and GFAT in HSVSMCs of T2DM and non-T2DM patients.

Page 5, line 142, para run like this “Permeabilised cells were also stained with 500 µl of 5 µg/m Hoechst to identify nuclei”- Is it 500 µl of 5 µg/mL Hoechst?

Author Response

Reviewer 2

The study by Bolanle IO et al, titled “Do T2DM and hyperglycaemia affect the expression levels of the regulating enzymes of cellular O-GlcNAcylation in human saphenous vein smooth muscle cells?” They have compared protein of OGA, OGT, and GFAT in HSVSMCs of T2DM and non-T2DM patients and concluded that no changes between T2DM and non-T2DM patients. Although this is an interesting finding, I have some concerns about this article.

Specific comments

It would be nice to measure mRNA level of respective enzyme by q-PCR.

Author should estimate enzyme activity of respective OGA, OGT, and GFAT in HSVSMCs of T2DM and non-T2DM patients

Response: Thank you for your insightful questions. We acknowledge that measuring the mRNA level of the enzymes are useful. This is an ongoing project, and it is our intention to continue to explore more biological tools and techniques such as q-PCR to investigate the role of hyperglycaemia/T2DM-driven vascular dysfunction in cardiac pathologies. Meanwhile, our findings in this current study highlight for the first time in HSVSMCs the impact of T2DM and hyperglycaemia on the expression levels of these O-GlcNAcylation regulating enzymes. However, we acknowledged possible limitations to this study in some paragraphs of the discussion which indicate future perspective on how to advance this study. For example, we have highlighted that our observation could be cell specific and not full characteristic of vascular cells and saphenous vein cells specifically (See lines 493-495). Also, we have highlighted that while there are no significant changes in the expression of these enzymes in HSVSMCs, this finding does not suggest that activities of these enzymes and their effect on other PTMs such as phosphorylation might not be significantly altered (See lines 518-520).

Page 5, line 142, para run like this “Permeabilised cells were also stained with 500 µl of 5 µg/m Hoechst to identify nuclei”- Is it 500 µl of 5 µg/mL Hoechst?

Response: Thank you for this observation. This is a typographical error and it has been corrected. See line 143 on the tracked version of the revised manuscript.

Reviewer 3 Report

This study by Bolanle et al. examines the protein expressions of enzymes of cellular O-GlcNAcylation, including glutamine 17 fructose-6P amidotransferase (GFAT), O-GlcNAc transferase (OGT), and O-GlcNAcase 18 (OGA), in human saphenous vein smooth muscle cells (HSVSMCs) isolated from human type 2 diabetes mellitus (T2DM) and non-T2DM patients. They found no significant difference in these protein expressions in HSVSMCs between D2DM and non-T2DM patients. They also found no significant effect of high glucose on these protein expressions in HSVSMCs isolated from non-T2DM patients. While the topic of the study is interesting and potentially important, the reviewer has considerable concerns about the experimental design, result interpretation, and conclusion.

Major comments:

1.       Protein expressions of GFAT, OGT, and OGA in isolated HSVSMCs are not equal to those in the saphenous vein. The procedure of VSMC explantation from the human saphenous vein may alter these protein expressions. It is more informative to examine protein expressions of GFAT, OGT, and OGA in the human saphenous veins by immunocytochemistry and immunoblot from T2DM and non-T2DM patients. Thus, the conclusion that D2DM does not affect the expression of these enzymes is not conclusive.

2.      Since GFAT, OGT, and OGA are enzymes, their enzymatic activity rather than protein expressions may be regulated by T2DM or glycemia in HSVSMCs. Thus, it is important to examine GFAT, OGT, and OGA enzymatic activity to support their conclusions.

3.      Treating HSVSMCs with high glucose for 48 hours may not be long enough to induce GFAT, OGT, and OGA protein expression. The time course of the high glucose treatment, especially a longer time treatment, needs to address whether hyperglycemia affects GFAT, OGT, and OGA protein expression in HSVSMCs. Also, a positive control to show that hyperglycemia affects some protein expressions should be included in the context of no effect of hyperglycemia on GFAT, OGT, and OGA protein expressions in HSVSMCs.

4.      It is unclear why the OGA and OGT protein expressions are normalized to the STAT3 protein expression, whereas the GFAT protein expressions are normalized to the GAPDH protein expressions. Since GAPDH is involved in glucose metabolism and diabetes, it may not be the appropriate loading control for the experiments.

Author Response

Reviewer 3

This study by Bolanle et al. examines the protein expressions of enzymes of cellular O-GlcNAcylation, including glutamine 17 fructose-6P amidotransferase (GFAT), O-GlcNAc transferase (OGT), and O-GlcNAcase 18 (OGA), in human saphenous vein smooth muscle cells (HSVSMCs) isolated from human type 2 diabetes mellitus (T2DM) and non-T2DM patients. They found no significant difference in these protein expressions in HSVSMCs between D2DM and non-T2DM patients. They also found no significant effect of high glucose on these protein expressions in HSVSMCs isolated from non-T2DM patients. While the topic of the study is interesting and potentially important, the reviewer has considerable concerns about the experimental design, result interpretation, and conclusion.

Major comments:

1. Protein expressions of GFAT, OGT, and OGA in isolated HSVSMCs are not equal to those in the saphenous vein. The procedure of VSMC explantation from the human saphenous vein may alter these protein expressions. It is more informative to examine protein expressions of GFAT, OGT, and OGA in the human saphenous veins by immunocytochemistry and immunoblot from T2DM and non-T2DM patients. Thus, the conclusion that D2DM does not affect the expression of these enzymes is not conclusive.

Response: We appreciate this insightful contribution and acknowledge that there are several methods to isolate SMCs from the saphenous vein including digestion of the entire vein. In our group, we have tried some of these techniques, but the method highlighted in this manuscript gave us the best success. This method has also been well documented in several other studies by other groups across the globe that currently work with HSVSMCs e.g. Madi et al., 2009; Riches et al., 2014. Furthermore, as shown in figures 1-3, we ensured that the isolated HSVSMCs represented a true SMCs population and ruled out endothelial cell contamination. In addition, a specialised growth medium specific for SMC growth medium, SmGM2 (supplemented with 5% (v/v) foetal bovine serum, 0.5 ng/mL epidermal growth factor, 2 ng/mL basic fibroblast growth factor, and 5 g/mL insulin; Promocell, Heidelberg, Germany, Cat. No: C-22162), was used to culture cells. These approaches helped optimised our successful isolation of HSVSMCs. Agreeably, further studies and optimisation of technique would enhance these findings. Once again, we appreciate the insights which we would bring on board in our further studies.

2. Since GFAT, OGT, and OGA are enzymes, their enzymatic activity rather than protein expressions may be regulated by T2DM or glycemia in HSVSMCs. Thus, it is important to examine GFAT, OGT, and OGA enzymatic activity to support their conclusions.

Response: We really appreciate this insightful comment. While there are documented studies in other cell types from different vascular tissues implicated in cardiac pathologies, our findings in this current study highlight for the first time in HSVSMCs the impact of T2DM and hyperglycaemia on the expression levels of these O-GlcNAcylation regulating enzymes. However, just as you have highlighted, we acknowledged possible limitations to this study in some paragraphs of the discussion which indicate future perspective on how to advance this study. For example, we have highlighted that our observation could be cell specific and not full characteristic of vascular cells and saphenous vein cells specifically (See lines 493-495). Also, we have highlighted that while there are no significant changes in the expression of these enzymes in HSVSMCs, this finding does not suggest that activities of these enzymes and their effect on other PTMs such as phosphorylation might not be significantly altered (See lines 518-520).

3. Treating HSVSMCs with high glucose for 48 hours may not be long enough to induce GFAT, OGT, and OGA protein expression. The time course of the high glucose treatment, especially a longer time treatment, needs to address whether hyperglycemia affects GFAT, OGT, and OGA protein expression in HSVSMCs. Also, a positive control to show that hyperglycemia affects some protein expressions should be included in the context of no effect of hyperglycemia on GFAT, OGT, and OGA protein expressions in HSVSMCs.

Response: We appreciate this insightful contribution and comment. In this case, we employed published protocol with similar research objectives to ours (See Masaki et al., 2020 and Riches et al., 2014). These references are well cited in this manuscript. However, we acknowledged that increasing the duration of treatment with high glucose concentrations may produce changes as you have suggested. This will be considered in future studies.

4. It is unclear why the OGA and OGT protein expressions are normalized to the STAT3 protein expression, whereas the GFAT protein expressions are normalized to the GAPDH protein expressions. Since GAPDH is involved in glucose metabolism and diabetes, it may not be the appropriate loading control for the experiments.

Response: Thank you for this observation and comment. The choice of GAPDH as the loading control is because of the size of GFAT (77 kDa) which is smaller than other enzymes (OGA-130 kDa and OGT-110kDa) investigated in this study. Hence, a smaller loading/housekeeping protein may be more suitable. Other studies where GFAT has been quantified have also adopted this approach (See Zhang et al., 2019; Phoomak et al., 2017). Also, these data are all normalised which technically reduce the level of variation.

References

1. Masaki N, Feng B, Bretón‐Romero R, Inagaki E, Weisbrod RM, Fetterman JL, Hamburg NM. O‐GlcNAcylation Mediates Glucose‐Induced Alterations in Endothelial Cell Phenotype in Human Diabetes Mellitus. Journal of the American Heart Association. 2020 Jun 16;9(12):e014046.
2. Riches K, Alshanwani AR, Warburton P, O’Regan DJ, Ball SG, Wood IC, Turner NA, Porter KE. Elevated expression levels of miR-143/5 in saphenous vein smooth muscle cells from patients with Type 2 diabetes drive persistent changes in phenotype and function. Journal of molecular and cellular cardiology. 2014 Sep 1;74:240-50.
3. Zhang N, Zhu T, Yu K, Shi M, Wang X, Wang L, Huang T, Li W, Liu Y, Zhang J. Elevation of O-GlcNAc and GFAT expression by nicotine exposure promotes epithelial‐mesenchymal transition and invasion in breast cancer cells. Cell death & disease. 2019 Apr 24;10(5):343.
4. Phoomak C, Vaeteewoottacharn K, Silsirivanit A, Saengboonmee C, Seubwai W, Sawanyawisuth K, Wongkham C, Wongkham S. High glucose levels boost the aggressiveness of highly metastatic cholangiocarcinoma cells via O-GlcNAcylation. Scientific reports. 2017 Mar 6;7(1):43842.

Round 2

Reviewer 2 Report

The manuscript is suitable for publication.

Author Response

We appreciate your comments and review.

Reviewer 3 Report

None.

Author Response

We appreciate your review, insights, and recommendations.