PONE-D-19-17091

Factors influencing subclinical atherosclerosis in patients with biopsy-proven nonalcoholic fatty liver disease

PLOS ONE

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Reviewer #1: Arai T et al evaluated the factors influencing atherosclerosis in patients with biopsy-proven nonalcoholic fatty liver disease (NAFLD), and found advavalunced liver fibrosis was associated with arterial stiffness. The study is interesting, however, some issues need to be addressed.

Abstract:

1. Line 42-43: Please show the value of 95% CI.

2. Line 48: Please show the value of p.

Methods:

1. Line 119-123: Hyaluronic acid [28], type IV 120 collagen 7s domain [29, 30], and Wisteria floribunda agglutinin positive Mac-2-binding protein (WFA+-M2B) [30–32], all of which have been reported as useful liver fibrosis markers in NAFLD, were measured. In addition, the fibrosis scores such as the FIB-4 index [33] and NAFLD fibrosis scorer (NFS) [34] were calculated, as reported previously.--> please change to Hyaluronic acid [28], type IV collagen 7s domain [29, 30], Wisteria floribunda agglutinin positive Mac-2-binding protein (WFA+-M2B) [30–32], FIB-4 index [33] and NAFLD fibrosis scorer (NFS) were measured. It is not suitable to write comments in the Methods.

Results:

1. How about NFS score in analysis, including Table 1, Table 2 and Figure 1?

2. In this study, baPWV was used without considering ABI values. It has been widely recognized that baPWV value is meaning less to measure in the limb of ABI value <0.9. How are the results if you exclude baPWV data associated with ABI <0.9? Addition, how about the association between ABI and liver fibrosis stage?

3. Line 280-281: This paradox can be explained by the loss of hepatic fat in NAFLD patients with advanced fibrosis. This result may suggest that liver fibrosis affects the arterial stiffness more than the hepatic fat. This explanation is too simple to convince others.

4. Line 323: based on noninvasive fibrosis markers and scores may predict the risk of arteriosclerosis� please change to arterial stiffness. After all, the two nouns are different.

Reviewer #2: This is a well conducted study examining the association of the severity of NAFLD and subclinical atherosclerosis in 153 biops-proven NAFLD patients. The findings are interesting and important, while some points need to be further clarified.

1. Patients who underwent histological evaluation were eligible and included in this study. However, the reasons for histological evaluation are unclear. The authors need to show the reasons for histological examination of the study subjects.

2. This study shows an association between liver fibrosis and subclinical atherosclerosis, but not a causal association. Therefore, the statement "...liver fibrosis affects the arterial stiffness...." is incorrect.

3. The results in paragraphs "Prevalence of baPWV ≥1600 cm/s according to the clinical risk scores based on older age, hypertension, and advanced fibrosis diagnosed by liver biopsy" and "Prevalence of baPWV ≥1600 cm/s according to the clinical risk scores based on older age, hypertension, and advanced fibrosis as diagnosed by fibrosis markers and scores" are redudant and may be removed. Moreover, the authors need to exaplain why they chose a cut-off level of baPWV as 1600 cm/s.

4. What kinds of drugs that may induce fatty liver were examined and excluded in this study?

5. The authors need to exaplain why this study could not show the associations of metabolic factors/ and related disorders (sucah as daibetes mellitus and hyperlipidemia...) with liver fibrosis.

Reviewer #3: PONE-D-19-17091

Taeang Arai et al., Factors influencing subclinical atherosclerosis in patients with biopsy-proven nonalcoholic fatty liver disease

This study aimed to clarify the factors related to subclinical arteriosclerosis, including the histopathological severity of the disease and PNPLA3 gene 38 polymorphisms, in 153 biopsy-proven nonalcoholic fatty liver disease (NAFLD) patients. The authors found that older age (≥55 years), hypertension, and advanced fibrosis were independently linked to baPWV ≥1600 cm/s. The study is appropriately designed and executed. However, there are some concerns to be clarified:

1. The association between subclinical atherosclerosis and NAFLD, indeed, has been studied extensively in the past. Retrospective and prospective studies have provided evidences of a strong association between NAFLD and subclinical atherosclerosis, including increased intima-media thickness, endothelial dysfunction, arterial stiffness, impaired left ventricular function and coronary calcification. (Fargion S et al., World J Gastroenterol 2014;20:13306-24.). The relevant meta-analysis has also been published (Ampuero J et al., Rev Esp Enferm Dig;107:10-6.). A recent study has also addressed this issue (Gill C et al., Am J Cardiol 2017;119:1717-22.).

2. Also, the progression of histological liver fibrosis as an independent factor for a high PWV value in a study involving 100 biopsy-confirmed 276 NAFLD patients has been reported [Ref. 13 of the manuscript]. The authors also mentioned that the only difference of their study as compared with the previous studies is that theirs is a “relatively large Japanese cohort” (Line 1, Page 17, the manuscript), which made this study not novel enough.

3. Figure 1 shows that no correlation with baPWV was noted for histological findings of liver inflammation and ballooning. Multiple logistic regression analysis showed that the following the only histological variable independently linked to baPWV ≥1600 cm/s (Table 2) was advanced fibrosis. This raises the concern if baPWV is actually related only to liver fibrosis, but not necessary to steatosis (inflammation and ballooning). The previous study has indicated that the carotid intima thickness, a parameter of subclinical atherosclerosis, significantly increased in chronic hepatitis C virus patients especially in those with cirrhosis and closely correlated with each other (Barakat AAE et al., Egypt Heart J 2017;69:139-47.).

4. As discussed in the Paragraph 2, Discussion of the manuscript, the arterial stiffness-promoting mechanism of the presence and severity of NAFLD independent of conventional metabolic risk factors remains unclear. To strengthen the novelty of this study, the levels of parameters of chronic inflammation, oxidative stress, adiponectin and TGF-β can be checked.

5. Although single nucleotide polymorphisms (SNPs) in the patatin-like phospholipase domain containing 3 gene (PNPLA3) are associated with the development and severity of NAFLD [Ref. 16–18 of the manuscript], this study only mentioned the distribution of the PNPLA3 polymorphisms without showing the association among PNPLA3 polymorphisms and severity of NAFLD.

6. It would be interesting to have sub-group analyses to search for any further possible correlations. For instance, although the current study did not identify an association between baPWV and the PNPLA3 SNP genotype, sub-group analyses according to gender or age may exert different findings.

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Factors influencing subclinical atherosclerosis in patients with biopsy-proven nonalcoholic fatty liver disease

PONE-D-19-17091R1

Dear Dr. Atsukawa,

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With kind regards,

Jee-Fu Huang, M.D., Ph.D.

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Partly

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #3: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors had addressed all issues well. I think the manuscripts get better after the revise. Thank you for the efforts of the authors.

Reviewer #2: The authors have satisfactorily responded to all my questions and made the necessary changes to the revised manuscript.

Reviewer #3: The previously raised issues have been addressed by the authors appropriately and I have no further comments.

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