Human Pulmonary Artery Endothelial Cells Increased Glycolysis and Decreased Nitric Oxide Synthase O-GlcNAcylation in Pulmonary Arterial Hypertension
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe study titled “Human pulmonary artery endothelial cells increased glycolysis while decreasing nitric oxide synthase O-GlcNAcylation in pulmonary arterial hypertension” by Sarah et al., is interesting. However, the study lacks precision due to the following issues.
1) The study should have first checked if the levels of eNOS is downregulated under PAH compared to healthy controls. This may be the fundamental cause of decrease in NO production rather than any changes in glycolysis or O-GlcNAcylation of eNOS.
2) Figure 3B is not acceptable as the total level of eNOS is not shown only the O-GlcNAcylated total proteins. The O-GlcNAcylation level changing without looking at the total eNOS levels is not giving any meaning conclusion. It may very well be that total level of eNOS are down in PAH. Therefore, it is necessary to check in the same blot what is the level of total eNOS. Ideally, one should pull-down total eNOs and check for the O-GlcNAcylation of eNOS with quantification of total eNOS and the modified eNOS. The ratio of modified to total eNOS level is what should be done here. This again confirms to the point that I raised in the first comment.
3) The trend in female and male as the author’s claim is not very significant in my opinion as the response is the same in male and female when comparing healthy and PAH. That is in both case the levels are decreasing in PAH compared to healthy ones. Only thing is that Females have higher amount to begin with. If the male and female respond differentially under PAH the point of projected sex-difference could have been a valid point.
4) The title of the study is also not a plausible one because there is no literature backup to suggest that increased glycolysis should always lead to increased O-GlcNAcylation of eNOS. Glycolysis can lead to several end points.
Author Response
Response to Reviewers
Thank you for your expert and insightful comments on the manuscript. We responded to and acted upon each of your suggestions and concerns. This paper is significantly improved as a result of the review, and we are grateful for the scrutiny and care with which it was previously considered. Below we include our response to each item in detail. We listed each concern raised verbatim and followed with an explanation of how we revised the manuscript through textual emendation as suggested.
Reviewer 1
Summary:
“The study titled “Human pulmonary artery endothelial cells increased glycolysis while decreasing nitric oxide synthase O-GlcNAcylation in pulmonary arterial hypertension” by Sarah et al., is interesting. However, the study lacks precision due to the following issues.”
Concern 1:
“The study should have first checked if the levels of eNOS is downregulated under PAH compared to healthy controls. This may be the fundamental cause of decrease in NO production rather than any changes in glycolysis or O-GlcNAcylation of eNOS.”
Response 1:
Thank you for bringing up this important point. We now include a Western blot of eNOS in Figure 3b. We did not observe any significant changes in eNOS in the healthy vs. pulmonary hypertension cells.
Concern 2:
“Figure 3B is not acceptable as the total level of eNOS is not shown only the O-GlcNAcylated total proteins. The O-GlcNAcylation level changing without looking at the total eNOS levels is not giving any meaning conclusion. It may very well be that total level of eNOS are down in PAH. Therefore, it is necessary to check in the same blot what is the level of total eNOS. Ideally, one should pull-down total eNOs and check for the O-GlcNAcylation of eNOS with quantification of total eNOS and the modified eNOS. The ratio of modified to total eNOS level is what should be done here. This again confirms to the point that I raised in the first comment.”
Response 2:
We checked in the same blot the level of eNOS. Unfortunately, we were not able to pull-down total eNOS and check for O-GlcNAcylation after that, as the protein yield was too small.
Concern 3:
“The trend in female and male as the author’s claim is not very significant in my opinion as the response is the same in male and female when comparing healthy and PAH. That is in both case the levels are decreasing in PAH compared to healthy ones. Only thing is that Females have higher amount to begin with. If the male and female respond differentially under PAH the point of projected sex-difference could have been a valid point.”
Response 3:
The reviewer is correct that these data may have been even more interesting if there had been a different trend between the male and female healthy vs. pulmonary hypertension cells. We believe it is important to keep these data in the paper, as the difference in male and female cells helps explain some of the variability in the human cell data.
Concern 4:
“The title of the study is also not a plausible one because there is no literature backup to suggest that increased glycolysis should always lead to increased O-GlcNAcylation of eNOS. Glycolysis can lead to several end points.”
Response 4:
The reviewer is correct that glycolysis leads to many different end points. We now clarify in the title that while we observed both increased glycolysis and decreased eNOS O-GlcNAcylation, we do not have data in this paper to show that increased glycolysis led to decreased eNOS O-GlcNAcylation.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsVery important and interesting topic considering basic research of the pathophysiology of pulmonary hypertension. This is in-vitro, experimental study on the pulmonary endothelial cells investigating the role of disturbed glycoses metabolisms and their connection to NO-synthase function in pulmonary hypertension.
The introduction is well written and introduce the reader to the all-important aspects of this work.
I have several questions on the methodology section.
HuPA endothelial cells were obtained from the failed donor pulmonary transplantation. In that case I suppose that pulmonary endothelial cell origin from the cadaver donors? In that case it is difficult to say that these cell origin from the healthy men and women. The procedure of harvesting cells and the donor’s history is needed to provide the accuracy of the statement that these cells derived from the healthy lungs.
On the other side, what is the origin of the PAH endothelial PA cells? It is important how long patients with PAH had the disease, in what risk they belong, if they are treated with anti PH drugs? What is the origin of material? Biopsy specimens? Why the biopsy performed? If this is type I PH patients, please precise the subclass, idiopathic, hereditary, associated to systemic connective tissue disorders, with congenital heart diseases, portal hypertension, HIV… We must know all about patients and control group to realize the significance of results and to estimate the role of confounding factors.
The average age of the control group is 44.5 y, and for the patient’s group it is 33.3 y, this is non-comparable and all the differences maybe from the these differences of age.
The number of specimens is too small for the conclusion about the differences in metabolism of NO-synthase and their O-GlcNAcylation between men and women.
Although this is very interesting investigation it is on the pilot level, hypothesis generated study.
Since I’m clinician dealing with PAH patients, I couldn’t comment in-vitro methodology, some lab. Scientists who know well cell culture methods are needed.
The publishing of this hypothesis generated studies depends on the Journal. According to my clinician mind several improvements I suggested could be done.
Author Response
Response to Reviewers
Thank you for your expert and insightful comments on the manuscript. We responded to and acted upon each of your suggestions and concerns. This paper is significantly improved as a result of the review, and we are grateful for the scrutiny and care with which it was previously considered. Below we include our response to each item in detail. We listed each concern raised verbatim and followed with an explanation of how we revised the manuscript through textual emendation as suggested.
Reviewer 2
Summary:
“Very important and interesting topic considering basic research of the pathophysiology of pulmonary hypertension. This is in-vitro, experimental study on the pulmonary endothelial cells investigating the role of disturbed glycoses metabolisms and their connection to NO-synthase function in pulmonary hypertension. The introduction is well written and introduce the reader to the all-important aspects of this work. I have several questions on the methodology section.”
Concern 1:
“HuPA endothelial cells were obtained from the failed donor pulmonary transplantation. In that case I suppose that pulmonary endothelial cell origin from the cadaver donors? In that case it is difficult to say that these cell origin from the healthy men and women. The procedure of harvesting cells and the donor’s history is needed to provide the accuracy of the statement that these cells derived from the healthy lungs.”
Response 1:
Thank you for raising this important point. We unfortunately do not have any additional information about the donors for the failed lung transplant except that they did not have PAH. We therefore changed the terminology in the paper to refer to these cells as non-PAH cells.
Concern 2:
“On the other side, what is the origin of the PAH endothelial PA cells? It is important how long patients with PAH had the disease, in what risk they belong, if they are treated with anti PH drugs? What is the origin of material? Biopsy specimens? Why the biopsy performed? If this is type I PH patients, please precise the subclass, idiopathic, hereditary, associated to systemic connective tissue disorders, with congenital heart diseases, portal hypertension, HIV… We must know all about patients and control group to realize the significance of results and to estimate the role of confounding factors.”
Response 2:
The reviewer is correct that the length of time that the patients had PAH as well as the treatment regimen are both important. Unfortunately, we do not have that information. We do know that the samples were obtained at the time of lung transplant for the PAH patients, and that they all had idiopathic PAH. We now include those details in the paper.
Concern 3:
“The average age of the control group is 44.5 y, and for the patient’s group it is 33.3 y, this is non-comparable and all the differences maybe from these differences of age.”
Response 3:
It is possible that the differences observed are due to the different patient ages. We were not able to control for this factor fully, since the number of male and female donors is small. We now include the age difference as a limitation of the study.
Concern 4:
“The number of specimens is too small for the conclusion about the differences in metabolism of NO-synthase and their O-GlcNAcylation between men and women.”
Response 4:
Unfortunately, the number of samples that we could use was limited by both sample availability and experimental factors. We emphasize that this paper should be considered a pilot study in the limitations section.
Concern 5:
“Although this is very interesting investigation it is on the pilot level, hypothesis generated study. Since I’m clinician dealing with PAH patients, I couldn’t comment in-vitro methodology, some lab. Scientists who know well cell culture methods are needed. The publishing of this hypothesis generated studies depends on the Journal. According to my clinician mind several improvements I suggested could be done.”
Response 5:
Thank you for raising your concerns. We have more than 20 years of experience in endothelial cell in vitro culture. We hope to follow up on this pilot, hypothesis driven study with additional experiments in animal models and human tissue.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThe response and the changes done are satisfactory.
Reviewer 2 Report
Comments and Suggestions for AuthorsThis is pilot, hypothesis generated study. All my concerns are sustained, see the cover letter.
