Reviewer ’s report  
Title: Non Alcoholic Fatty Liver disease and eNOS dysfunction in humans  
Version: 0 Date: 04 Nov 2016  
Reviewer: Yasuo Terauchi  
Reviewer's report:  
Although eNOS derangement has been demonstrated in experimental models, no studies ha ve 
directly shown that eNOS dysfunction is associated with NAFLD in humans. The aim of this 
study was to investigate eNOS function in NAFLD patients.  
Fifty four NAFLD patients underwent clinical and laboratory evaluation and liver biopsy. 
Patients were di vided into two groups by the presence of NAFL or NASH. Vascular reactivity 
demonstrated a reduced function induced from NAFLD platelets as compared to controls, 
associated with an impaired p -eNOS in both in platelets and in liver. NAFL showed a higher 
impa irment of eNOS phosphorylation as compared to NASH. In contrast to what observed in 
vitro, the vascular response by FMD was worse in NASH as compared to NAFL.  
The authors concluded that NAFLD patients showed a marked eNOS dysfunction, which may 
contribute  to a higher CV risk and that eNOS dysfunction observed in platelets and liver tissue 
didn't match with FMD.  
 
Criticisms  
1. Simple steatosis (NAFL), which represents the early stage of the disease, seems to be 
associated to a worse eNOS impairment compared  to steatohepatitis (NASH). On the other 
hand, clinical evaluation of endothelial dysfunction, measured via FMD, has shown to be 
worse in NASH than NAFL patients. Although the authors speculated the reasons for the 
discrepancy in Discussion section, it see ms to this reviewer that the authors should rather 
focus on the impacts of their findings on the higher risk of cardiovascular disease in NASH 
subjects.  
2. No statistically significant differences were found between the NAFL group and NASH 
group for age, sex, BMI, ALT, prevalence of hypertension, diabetes, dyslipidemia, obesity 
and metabolic syndrome (Table 1). However, there are marked differences were found 
between the control group and all NAFLD group for the prevalence of hypertension, 
diabetes, and dy slipidemia. The differences raise a concern that the results obtained from 
this control group is really adequate for the comparison with the NAFLD group. This 
reviewer asks the authors to comment on this issue.  
3. Six cirrhosis patients are included in the  NASH group. This reviewer has a concern that far 
advanced liver damage cirrhosis has a role on the clinical evaluation of endothelial 
dysfunction. Therefore, I would like to know the results of NASH subjects excluding 
cirrhosis (n = 33 -6 = 27).  
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
Yes 
 
Does the work include the necessary controls?  
If not, please spec ify which controls are required in your comments to the authors.  
 
Yes 
 
Are the conclusions drawn adequately supported by the data shown?  
If not, please explain in your comments to the authors.  
 
Yes 
 
Are you able to assess any statistics in the manuscript or wo uld you recommend an  
additional statistical review?  
If an additional statistical review is recommended, please specify what aspects require further  
assessment in your comments to the editors.  
 
I am able to assess the statistics  
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
Acceptable  
 
Declaration of competing interests  
Please complete a declaration of competing interests, considering the following questions:  
 
1. Have you in the past five years received reimbursements, fe es, funding, or salary from an  
organisation that may in any way gain or lose financially from the publication of this  
manuscript, either now or in the future?  
 
2. Do you hold any stocks or shares in an organisation that may in any way gain or lose  
financial ly from the publication of this manuscript, either now or in the future?  
 
3. Do you hold or are you currently applying for any patents relating to the content of the  
manuscript?  
 
4. Have you received reimbursements, fees, funding, or salary from an organization that 
holds  or has applied for patents relating to the content of the manuscript?  
 
5. Do you have any other financial competing interests?  
 
6. Do you have any non -financial com peting interests in relation to this paper?  
 
If you can answer no to all of the above, write 'I declare that I have no competing interests' 
below.  If your reply is yes to any, please give details below.  
 
I have no competing interests in relation to the paper  which I am reviewing.  
 
I agree to the open peer review policy of the journal. I understand that my name will be included  
on my report to the authors and, if the manuscript is accepted for publication, my named report  
including any attachments I upload will  be posted on the website along with the authors'  
responses. I agree for my report to be made available under an Open Access Creative Commons  
CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ). I understand that any comments  
which I do not wish to  be included in my named report can be included as confidential comments  
to the editors, which will not be published.  
 
I agree to the open peer review policy of the journal  
Reviewer ’s report  
Title: Non Alcoholic Fatty Liver disease and eNOS dysfunction in humans  
Version: 0 Date: 04 Nov 2016  
Reviewer: Yasuo Terauchi  
Reviewer's report:  
Although eNOS derangement has been demonstrated in experimental models, no studies ha ve 
directly shown that eNOS dysfunction is associated with NAFLD in humans. The aim of this 
study was to investigate eNOS function in NAFLD patients.  
Fifty four NAFLD patients underwent clinical and laboratory evaluation and liver biopsy. 
Patients were di vided into two groups by the presence of NAFL or NASH. Vascular reactivity 
demonstrated a reduced function induced from NAFLD platelets as compared to controls, 
associated with an impaired p -eNOS in both in platelets and in liver. NAFL showed a higher 
impa irment of eNOS phosphorylation as compared to NASH. In contrast to what observed in 
vitro, the vascular response by FMD was worse in NASH as compared to NAFL.  
The authors concluded that NAFLD patients showed a marked eNOS dysfunction, which may 
contribute  to a higher CV risk and that eNOS dysfunction observed in platelets and liver tissue 
didn't match with FMD.  
 
Criticisms  
1. Simple steatosis (NAFL), which represents the early stage of the disease, seems to be 
associated to a worse eNOS impairment compared  to steatohepatitis (NASH). On the other 
hand, clinical evaluation of endothelial dysfunction, measured via FMD, has shown to be 
worse in NASH than NAFL patients. Although the authors speculated the reasons for the 
discrepancy in Discussion section, it see ms to this reviewer that the authors should rather 
focus on the impacts of their findings on the higher risk of cardiovascular disease in NASH 
subjects.  
2. No statistically significant differences were found between the NAFL group and NASH 
group for age, sex, BMI, ALT, prevalence of hypertension, diabetes, dyslipidemia, obesity 
and metabolic syndrome (Table 1). However, there are marked differences were found 
between the control group and all NAFLD group for the prevalence of hypertension, 
diabetes, and dy slipidemia. The differences raise a concern that the results obtained from 
this control group is really adequate for the comparison with the NAFLD group. This 
reviewer asks the authors to comment on this issue.  
3. Six cirrhosis patients are included in the  NASH group. This reviewer has a concern that far 
advanced liver damage cirrhosis has a role on the clinical evaluation of endothelial 
dysfunction. Therefore, I would like to know the results of NASH subjects excluding 
cirrhosis (n = 33 -6 = 27).  
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
Yes 
 
Does the work include the necessary controls?  
If not, please spec ify which controls are required in your comments to the authors.  
 
Yes 
 
Are the conclusions drawn adequately supported by the data shown?  
If not, please explain in your comments to the authors.  
 
Yes 
 
Are you able to assess any statistics in the manuscript or wo uld you recommend an  
additional statistical review?  
If an additional statistical review is recommended, please specify what aspects require further  
assessment in your comments to the editors.  
 
I am able to assess the statistics  
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
Acceptable  
 
Declaration of competing interests  
Please complete a declaration of competing interests, considering the following questions:  
 
1. Have you in the past five years received reimbursements, fe es, funding, or salary from an  
organisation that may in any way gain or lose financially from the publication of this  
manuscript, either now or in the future?  
 
2. Do you hold any stocks or shares in an organisation that may in any way gain or lose  
financial ly from the publication of this manuscript, either now or in the future?  
 
3. Do you hold or are you currently applying for any patents relating to the content of the  
manuscript?  
 
4. Have you received reimbursements, fees, funding, or salary from an organization that 
holds  or has applied for patents relating to the content of the manuscript?  
 
5. Do you have any other financial competing interests?  
 
6. Do you have any non -financial com peting interests in relation to this paper?  
 
If you can answer no to all of the above, write 'I declare that I have no competing interests' 
below.  If your reply is yes to any, please give details below.  
 
I have no competing interests in relation to the paper  which I am reviewing.  
 
I agree to the open peer review policy of the journal. I understand that my name will be included  
on my report to the authors and, if the manuscript is accepted for publication, my named report  
including any attachments I upload will  be posted on the website along with the authors'  
responses. I agree for my report to be made available under an Open Access Creative Commons  
CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ). I understand that any comments  
which I do not wish to  be included in my named report can be included as confidential comments  
to the editors, which will not be published.  
 
I agree to the open peer review policy of the journal  
Please review this paper.
user
Reviewer ’s report  
Title: Non Alcoholic Fatty Liver disease and eNOS dysfunction in humans  
Version: 0 Date: 04 Nov 2016  
Reviewer: Yasuo Terauchi  
Reviewer's report:  
Although eNOS derangement has been demonstrated in experimental models, no studies ha ve 
directly shown that eNOS dysfunction is associated with NAFLD in humans. The aim of this 
study was to investigate eNOS function in NAFLD patients.  
Fifty four NAFLD patients underwent clinical and laboratory evaluation and liver biopsy. 
Patients were di vided into two groups by the presence of NAFL or NASH. Vascular reactivity 
demonstrated a reduced function induced from NAFLD platelets as compared to controls, 
associated with an impaired p -eNOS in both in platelets and in liver. NAFL showed a higher 
impa irment of eNOS phosphorylation as compared to NASH. In contrast to what observed in 
vitro, the vascular response by FMD was worse in NASH as compared to NAFL.  
The authors concluded that NAFLD patients showed a marked eNOS dysfunction, which may 
contribute  to a higher CV risk and that eNOS dysfunction observed in platelets and liver tissue 
didn't match with FMD.  
 
Criticisms  
1. Simple steatosis (NAFL), which represents the early stage of the disease, seems to be 
associated to a worse eNOS impairment compared  to steatohepatitis (NASH). On the other 
hand, clinical evaluation of endothelial dysfunction, measured via FMD, has shown to be 
worse in NASH than NAFL patients. Although the authors speculated the reasons for the 
discrepancy in Discussion section, it see ms to this reviewer that the authors should rather 
focus on the impacts of their findings on the higher risk of cardiovascular disease in NASH 
subjects.  
2. No statistically significant differences were found between the NAFL group and NASH 
group for age, sex, BMI, ALT, prevalence of hypertension, diabetes, dyslipidemia, obesity 
and metabolic syndrome (Table 1). However, there are marked differences were found 
between the control group and all NAFLD group for the prevalence of hypertension, 
diabetes, and dy slipidemia. The differences raise a concern that the results obtained from 
this control group is really adequate for the comparison with the NAFLD group. This 
reviewer asks the authors to comment on this issue.  
3. Six cirrhosis patients are included in the  NASH group. This reviewer has a concern that far 
advanced liver damage cirrhosis has a role on the clinical evaluation of endothelial 
dysfunction. Therefore, I would like to know the results of NASH subjects excluding 
cirrhosis (n = 33 -6 = 27).  
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
Yes 
 
Does the work include the necessary controls?  
If not, please spec ify which controls are required in your comments to the authors.  
 
Yes 
 
Are the conclusions drawn adequately supported by the data shown?  
If not, please explain in your comments to the authors.  
 
Yes 
 
Are you able to assess any statistics in the manuscript or wo uld you recommend an  
additional statistical review?  
If an additional statistical review is recommended, please specify what aspects require further  
assessment in your comments to the editors.  
 
I am able to assess the statistics  
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
Acceptable  
 
Declaration of competing interests  
Please complete a declaration of competing interests, considering the following questions:  
 
1. Have you in the past five years received reimbursements, fe es, funding, or salary from an  
organisation that may in any way gain or lose financially from the publication of this  
manuscript, either now or in the future?  
 
2. Do you hold any stocks or shares in an organisation that may in any way gain or lose  
financial ly from the publication of this manuscript, either now or in the future?  
 
3. Do you hold or are you currently applying for any patents relating to the content of the  
manuscript?  
 
4. Have you received reimbursements, fees, funding, or salary from an organization that 
holds  or has applied for patents relating to the content of the manuscript?  
 
5. Do you have any other financial competing interests?  
 
6. Do you have any non -financial com peting interests in relation to this paper?  
 
If you can answer no to all of the above, write 'I declare that I have no competing interests' 
below.  If your reply is yes to any, please give details below.  
 
I have no competing interests in relation to the paper  which I am reviewing.  
 
I agree to the open peer review policy of the journal. I understand that my name will be included  
on my report to the authors and, if the manuscript is accepted for publication, my named report  
including any attachments I upload will  be posted on the website along with the authors'  
responses. I agree for my report to be made available under an Open Access Creative Commons  
CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ). I understand that any comments  
which I do not wish to  be included in my named report can be included as confidential comments  
to the editors, which will not be published.  
 
I agree to the open peer review policy of the journal  
assistant