Dyslipidemia as a risk factor for liver fibrosis progression in a multicentric population with non-alcoholic steatohepatitis

Background: Nonalcoholic fatty liver disease (NAFLD) is a serious worldwide health problem, with an estimated global prevalence of 24%; it has a notable relationship with other metabolic disorders, like obesity and type 2 diabetes mellitus (T2DM). Nonalcoholic steatohepatitis (NASH) is one of the most important clinical entities of NAFLD, which is associated with an increased risk of progression to liver cirrhosis and hepatocellular carcinoma (HCC). Mexico is one of the countries with the highest prevalence of metabolic diseases; therefore, we sought to investigate the impact that these clinical entities have in the progression to advanced fibrosis in Mexican patients with NASH. Methods: We performed a multicenter retrospective cross-sectional study, from January 2012 to December 2017. A total of 215 patients with biopsy-proven NASH and fibrosis were enrolled. NASH was diagnosed according NAS score and liver fibrosis was staged by the Kleiner scoring system. For comparing the risk of liver fibrosis progression, we divided our sample into two groups. Those patients with stage F0-F2 liver fibrosis were included in the group with non-significant liver fibrosis (n=178) and those individuals with F3-F4 fibrosis were included in the significant fibrosis group (n=37). We carried out a multivariate analysis to find risk factors associated with liver fibrosis progression. Results: From the 215 patients included, 37 had significant liver fibrosis (F3-4). After logistic regression analysis T2DM (p=0.044), systemic arterial hypertension (p=0.014), cholesterol (p=0.041) and triglycerides (p=0.015) were the main predictor of advanced liver fibrosis. Conclusions: In a Mexican population, dyslipidemia was the most important risk factor associated with advanced liver fibrosis and cirrhosis.

according NAS score and liver fibrosis was staged by the Kleiner scoring system. For comparing the risk of liver fibrosis progression, we divided our sample into two groups. Those patients with stage F0-F2 liver fibrosis were included in the group with non-significant liver fibrosis (n=178) and those individuals with F3-F4 fibrosis were included in the significant fibrosis group (n=37). We carried out a multivariate analysis to find risk factors associated with liver fibrosis progression. From the 215 patients included, 37 had significant liver fibrosis Results: (F3-4). After logistic regression analysis T2DM (p=0.044), systemic arterial hypertension (p=0.014), cholesterol (p=0.041) and triglycerides (p=0.015) were the main predictor of advanced liver fibrosis.
In a Mexican population, dyslipidemia was the most Conclusions: important risk factor associated with advanced liver fibrosis and cirrhosis.

Introduction
Nonalcoholic fatty liver disease (NAFLD) has a broad clinical spectrum, ranging from simple steatosis to cirrhosis, even developing in some cases with hepatocellular carcinoma (HCC) 1 . Nonalcoholic steatohepatitis (NASH) is one of the most important clinical entities of NAFLD, characterized by the histologic presence of liver steatosis, ballooning degeneration, and lobular inflammation, with or without fibrosis 2 . Once NASH is established, there is a significant increased risk of developing liver cirrhosis and HCC 3 .
Currently, NAFLD is the most common chronic liver disease in the world, with an important relationship with other metabolic disorders like obesity, type 2 diabetes mellitus (T2DM) and metabolic syndrome (MetS) 4-6 , being the second leading indication for liver transplantation in the United States 7 . A global prevalence of 24% is estimated, with the highest rates in South America and the Middle East, while the lowest prevalence has been reported in Africa 8 .
It is estimated that 30-40% of NAFLD patients will develop NASH 9,10 . Some studies have demonstrated that the risk of progression to liver cirrhosis in NAFLD patients is between 0-4%, while approximately 10-25% of NASH patients will develop cirrhosis 11-16 . This also depends on the ethnic origin of the patients, since Hispanic-Americans have been found to have a wide susceptibility to NAFLD and NASH development mainly from Mexican origin (33%) 8 .
Multiple risk factors for NASH progression have been identified, such as diet, MetS, T2DM, obesity, Hispanic ethnicity and polymorphisms in the patatin-like phospholipase domain-containing 3 (PNPLA3) gene [17][18][19] . However, the pathological mechanisms, by which some NAFLD patients progress to NASH are still not well understood 20 .
Mexico is one of the countries with the highest prevalence of metabolic diseases; 75.2% of the Mexican population are obese or overweight, 10.3% have T2DM and 19.5% have dyslipidemia 21 . We therefore sought to investigate the main metabolic factors involved in the progression to advanced fibrosis in Mexican patients with NASH.

Study design
We conducted a multicenter retrospective cross-sectional study from January 2012 to December 2017 in 7 tertiary referral centers from different parts of Mexico: Medica Sur Clinic and Foundation (Mexico City), General Hospital of Mexico "Dr. Eduardo Liceaga" (Mexico City), Civil Hospital of Guadalajara "Fray Antonio Alcalde" (Jalisco), Christus Muguerza "Super Speciality" Hospital (Nuevo Leon), Central Military Hospital (Mexico City), General Hospital of the Mexican Social Security Institute (Durango), and the General Regional Hospital, IMSS 1 (Morelos).
This study was reviewed and approved by the Ethics Committee of the Medica Sur Clinic and Foundation. Patients were not required to give informed consent to the study because the analysis used anonymous clinical data.

Patients and data collection
We included patients older than 20 years, of both genders, who had the diagnosis of biopsy-proven NASH. NASH was diagnosed according to NAS score 5,6 , and liver fibrosis was staged by the Kleinier scoring system 15 . We reviewed the medical records to obtain the baseline characteristics, biochemical parameters and comorbidities excluding all patients with other causes of liver disease, such as alcoholic liver disease, hepatitis B virus, hepatitis C virus, autoimmune liver disease, iron overload, drug-induced liver disease, and hereditary liver disease.
Several variables that have been commonly associated as risk factors for NASH progression were studied and included in the statistical analyses, such as MetS diagnosed with three or more of the following criteria established by the National Cholesterol Education Program (NCEP) Adult Treatment Panel III (ATP III) 22 : waist circumference over 102 cm (men) or 88 cm (women), blood pressure over 130/85 mmHg, fasting triglyceride (TG) level over 150 mg/dl, fasting high-density lipoprotein (HDL) cholesterol level less than 40 mg/dl (men) or 50 mg/dl (women) and fasting blood glucose over 100 mg/dl. To define overweight or obesity we use the body mass index (BMI) defined as weight (in kg) / height 2 (in m 2 ). Where overweight is defined above 25 kg / m 2 , and obesity as > 30 kg / m 2 . T2DM was diagnosed according to the history of diseases, fasting glucose (>126 mg/dL), and/or glucose tolerance test (>200 mg/dL two hours after the glucose load). Systemic arterial hypertension was diagnosed according to the history of diseases and/or intake of antihypertensive drugs. Cardiovascular disease (CVD) was defined as the presence of history such as systemic arterial hypertension, coronary heart disease, previous heart attack or stroke. Laboratory evaluation in all patients included serum liver tests (aspartate transaminase, alanine transaminase, total bilirubin, alkaline phosphatase, and albumin), hemoglobin, platelet count, cholesterol, low-density lipoprotein, HDL, TG, and blood glucose. The diagnosis of HCC was based on histopathological findings by liver biopsy.
For comparing the risk of liver fibrosis progression, we divided our sample into two groups. Those patients with stage F0-F2 liver fibrosis were included in the group with non-significant liver fibrosis (n=178) and the individuals with stage F3-F4 fibrosis were included in the significant fibrosis group (n=37).

Statistical analyses
We produced descriptive statistics for baseline patient characteristics. Univariate analysis of quantitative variables was conducted using Student's t-test, while for qualitative variables we used the Chi square test. Multivariate regression analyses were used to assess the independent association between significant fibrosis and possible risk factors. A p-value <0.05 was determined to indicate significance. All analyses were performed using STATA (v14.1, Stata Corp, College Station, TX, USA).

Results
We included 215 patients (Table 1). Non-significant fibrosis (F0-F2) was found in 83% of our sample; 7% had fibrosis at stage F-3, and 10% had fibrosis at stage F-4. Five patients were diagnosed with HCC (Table 2). De-identified individual-level results are available as Underlying data 23 .
Prediction of significant fibrosis stage and liver cirrhosis Compared to the group with non-significant liver fibrosis (stages F0, F1 and F2), those with significant fibrosis (stages F3 or F4) were more likely to be older and have lower levels of platelet count and albumin (Table 3 and Table 4). Moreover, those with significant fibrosis showed higher levels of blood glucose and proportions of dyslipidemia, T2DM, MetS, systemic arterial hypertension, and CVD.
Logistic multivariate regression analysis demonstrated that T2DM, systemic arterial hypertension, high cholesterol and hypertriglyceridemia significantly increased the risk for advanced liver fibrosis (Table 5), but only T2DM and hypertriglyceridemia significantly raised the risk of developing cirrhosis. (Table 6).

Discussion
In this study we found that T2DM, triglycerides and cholesterol were the main factors associated with advanced fibrosis stages and  cirrhosis. In this line, the association between T2DM and NASH progression has been widely demonstrated in a large number of studies from different geographical points 24-28 . Our findings support this association in patients of Mexican origin. On the other hand, in recent years some experimental and clinical studies have suggested an important role of lipid deregulation in the progression of fibrosis. Even though hepatic accumulation of triglycerides is the main feature of NAFLD, it has only been related with the development of simple steatosis due to its low lipotoxic characteristics. Contrarily, free cholesterol (FC) is an important lipotoxic species commonly found in NASH patients that has been linked to hepatic inflammation and fibrosis 29 .
The first cholesterol-induced NASH hypothesis was proposed more than 10 years ago by suggesting that mitochondrial FC loading changed the fluidity of the mitochondrial membranes, leading to the oxidation of mitochondrial glutathione, and sensitized hepatocytes to tumor necrosis factor and Fas-dependent death signaling via mitochondrial glutathione depletion, exacerbating NASH 30 . Furthermore, hyperinsulinemia (another characteristic usually found in NASH) and cholesterol accumulation was seen to upregulate sterol regulatory element-binding protein 2 (SREBP-2), leading to increased cholesterol synthesis and downregulation of mitochondrial β-oxidation, with the consequent accumulation of cholesterol and free fatty acids in the liver 31 . All  More recent evidence has found that when feeding C57BL/6 mice with a high cholesterol + methionine/choline-deficient diet for 12 weeks, there was an increase in the development of fibrosis due to the intrahepatic accumulation of FC in the hepatic stellate cells (HSCs) via TLR4 activation with the consequent downregulation of the transforming growth factor-beta (TGF-β) pseudoreceptor bone morphogenetic protein and activin membrane-bound inhibitor, leading to TGF-β-induced fibrosis 33 .
In addition, FC was found to alter the SREBP2-mediated feedback system, likely due to an increase in the expression of the SREBP cleavage-activating protein (SCAP) and the lack of expression of insulin induced gene 2 (Insig-2) in HSCs which favored the accumulation of cholesterol and the transdifferentiation of HSCs into myofibroblasts 33 .
In humans, a retrospective study conducted in cirrhotic patients found that cholesterol was a strong mortality predictor in those patients 34 . Also, in a more recent study, a group of NAFLD surgical specimens was studied observing an accumulation of FC and oxidized low-density lipoprotein in the portal vein and the hepatic sinusoidal parenchyma in the form of cholesterol crystals, related to the activation of NOD-like receptor family pyrin domain containing protein 3 (NLRP3) inflammasome promoting inflammation, HSCs activation and a characteristic chickenwire fibrosis 35 . Furthermore, basic research has demonstrated that cholesterol crystals (lipid droplets with a strong birefringence under polarized light) are only present in NASH-models (not in simple steatosis). Under these conditions, activated Kupffer cells (KCs) aggregate along cholesterol crystals forming "crown-like structures" intimately related to the development of foam cells and therefore atherosclerosis 36 .
The latter demonstrates the importance of CVD with NASH, being the first cause of death in this group of patients. Therefore, our findings demonstrate the important relation that metabolic diseases have with advanced stages of NASH. This will lead to the necessity of better preventive programs with a greater impact and coverage for general population around the world.
Finally, we must point out the limitations of this study such as the cross-sectional design which precluded the establishment of causal and temporal relationships between NASH progression and associated variables. Also, the small number of patients with significant fibrosis was important to mention.
In conclusion, our results show that dyslipidemia is one of the main risk factors associated with the development of significant liver fibrosis stages. Hispanics/Latinos and the Mexican population have one of the highest rates of metabolic diseases around the world; for this reason, it is expected that NAFLD and NASH will be the leading cause of cirrhosis in the near future. Therefore, it could be crucial to develop better screening programs for NAFLD patients with hypercholesterolemia and/or another component of MetS in order to prevent the development of more significant clinical complications. This is a multicentre retrospective cross-sectional study on NAFLD (2012-17) with 215 patients undergoing liver histology (NASH-fibrosis). Group F0-F2 fibrosis and Group F3-F4 were compare by logistic regression analysis. T2DM, systemic arterial hypertension, cholesterol and triglycerides were the main predictor of advanced liver fibrosis The paper is well presented and simple in the take-home message. The authors conclude that T2DM, serum triglycerides and cholesterol were the main factors associated with advanced fibrosis stages and cirrhosis.

Data availability
A general point is to discuss the limited n. of patients with fibrosis (N=37) and how this number could affect the overall results of the study. This aspect is only briefly touched at the end of discussion.
Also, can the authors provide any information about pre-T2DM, i.e. the presence of insulin resistance?
Abstract: please better describe the terms "cholesterol" and "triglycerides" (serum chol, TG) Intro: the authors should also mention the recent consensus paper in gastro re-classifying the metabolic NAFLD Eslam M, Sanyal AJ, George J, International Consensus Panel. MAFLD: A Consensus-Driven Proposed Nomenclature for Metabolic Associated Fatty Liver Disease .
When mentioning PNPLA3, please refer to a previous paper Krawczyk M, Portincasa P, Lammert F. PNPLA3-associated steatohepatitis: toward a gene-based classification of fatty liver disease . The authors declare that they have no conflict of interest.

Competing Interests:
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