Plasma proteomic signature of fatty liver disease: The Rotterdam Study.

BACKGROUND AND AIMS: Fatty liver disease (FLD) is caused by excess fat in the liver, and its global prevalence exceeds 33%. The role of protein expression on the pathogenesis of FLD and accompanied fibrosis and its potential as a disease biomarker is currently not clear. Hence, we aimed to identify plasma proteomics associated with FLD and fibrosis using population-based data. APPROACH AND RESULTS: Blood samples were collected from 2578 participants from the population-based Rotterdam Study cohort. The proximity extension assay reliably measured plasma levels of 171 cardiometabolic and inflammatory-related proteins (Olink Proteomics). FLD was assessed by ultrasound, and fibrosis by transient elastography. Logistic regression models quantified the association of plasma proteomics with FLD and fibrosis. In addition, we aimed to validate our results in liver organoids. The cross-sectional analysis identified 27 proteins significantly associated with FLD surpassing the Bonferroni-corrected p <2.92×10 -4 . The strongest association was observed for FGF-21 (ß=0.45, p =1.07×10 -18 ) and carboxylesterase 1 (CES1) protein (ß=0.66, p =4.91×10 -40 ). Importantly, 15 of the 27 proteins significantly associated with FLD were also associated with liver fibrosis. Finally, consistent with plasma proteomic profiling, we found the expression levels of IL-18 receptor 1 (IL-18R1) and CES1 to be upregulated in an FLD model of 3-dimensional culture human liver organoids. CONCLUSIONS: Among the general population, several inflammatory and cardiometabolic plasma proteins were associated with FLD and fibrosis. Particularly, plasma levels of FGF-21, IL-18R1, and CES1 were largely dependent on the presence of FLD and fibrosis and may therefore be important in their pathogenesis.

Plasma MicroRNA Signature of Alcohol Consumption: The Rotterdam Study.

BACKGROUND: MicroRNAs (miRNAs) represent a class of noncoding RNAs that regulate gene expression and are implicated in the pathogenesis of different diseases. Alcohol consumption might affect the expression of miRNAs, which in turn could play a role in risk of diseases. OBJECTIVES: We investigated whether plasma concentrations of miRNAs are altered by alcohol consumption. Given the existing evidence showing the link between alcohol and liver diseases, we further explored the extent to which these associations are mediated by miRNAs. METHODS: Profiling of plasma miRNAs was conducted using the HTG EdgeSeq miRNA Whole Transcriptome Assay in 1933 participants of the Rotterdam Study. Linear regression was implemented to explore the link between alcohol consumption (glasses/d) and miRNA concentrations, adjusted for age, sex, cohort, BMI, and smoking. Sensitivity analysis for alcohol categories (nondrinkers, light drinkers, and heavy drinkers) was performed, where light drinkers corresponded to 0-2 glasses/d in men and 0-1 glasses/d in women, and heavy drinkers to >2 glasses/d in men and >1 glass/d in women. Moreover, we utilized the alcohol-associated miRNAs to explore their potential mediatory role between alcohol consumption and liver-related traits. Finally, we retrieved putative target genes of identified miRNAs to gain an understanding of the molecular pathways concerning alcohol consumption. RESULTS: Plasma concentrations of miR-193b-3p, miR-122-5p, miR-3937, and miR-4507 were significantly associated with alcohol consumption surpassing the Bonferroni-corrected P < 8.46 × 10-5. The top significant association was observed for miR-193b-3p (ß = 0.087, P = 2.90 × 10-5). Furthermore, a potential mediatory role of miR-3937 and miR-122-5p was observed between alcohol consumption and liver traits. Pathway analysis of putative target genes revealed involvement in biological regulation and cellular processes. CONCLUSIONS: This study indicates that alcohol consumption is associated with plasma concentrations of 4 miRNAs. We outline a potential mediatory role of 2 alcohol-associated miRNAs (miR-3937 and miR-122-5p), laying the groundwork for further exploration of miRNAs as potential mediators between lifestyle factors and disease development.

Plasma circulating microRNAs associated with obesity, body fat distribution, and fat mass: the Rotterdam Study.

BACKGROUND: MicroRNAs (miRNAs) represent a class of small non-coding RNAs that regulate gene expression post-transcriptionally and are implicated in the pathogenesis of different diseases. Limited studies have investigated the association of circulating miRNAs with obesity and body fat distribution and their link to obesity-related diseases using population-based data. METHODS: We conducted a genome-wide profile of circulating miRNAs in plasma, collected between 2002 and 2005, in 1208 participants from the population-based Rotterdam Study cohort. Obesity and body fat distribution were measured as body mass index (BMI), waist-to-hip ratio (WHR), android-fat to gynoid-fat ratio (AGR), and fat mass index (FMI) measured by anthropometrics and Dual X-ray Absorptiometry. Multivariable linear regression models were used to assess the association of 591 miRNAs well-expressed in plasma with these traits adjusted for potential covariates. We further sought for the association of identified miRNAs with cardiovascular and metabolic diseases in the Rotterdam study and previous publications. RESULTS: Plasma levels of 65 miRNAs were associated with BMI, 40 miRNAs with WHR, 65 miRNAs with FMI, and 15 miRNAs with AGR surpassing the Bonferroni-corrected P < 8.46 × 10-5. Of these, 12 miRNAs were significantly associated with all traits, while four miRNAs were associated only with WHR, three miRNAs only with FMI, and miR-378i was associated only with AGR. The most significant association among the overlapping miRNAs was with miR-193a-5p, which was shown to be associated with type 2 diabetes and hepatic steatosis in the Rotterdam Study. Moreover, five of the obesity-associated miRNAs and two of the body fat distribution miRNAs have been correlated previously to cardiovascular disease. CONCLUSIONS: This study indicates that plasma levels of several miRNAs are associated with obesity and body fat distribution which could help to better understand the underlying mechanisms and may have the biomarker potential for obesity-related diseases.

Dissecting the multifaceted impact of statin use on fatty liver disease: a multidimensional study.

BACKGROUND: Statin use could benefit patients with non-alcoholic fatty liver disease (NAFLD), but the evidence is segmented and inconclusive. This multidimensional study comprehensively investigated the potential benefits and mechanism-of-action of statins in NAFLD. METHODS: A cross-sectional investigation was performed within the Rotterdam Study (general population; n = 4.576) and the PERSONS cohort (biopsy-proven NAFLD patients; n = 569). Exclusion criteria were secondary causes for steatosis and insufficient data on alcohol, dyslipidemia or statin use. Associations of statin use with NAFLD (among entire general population), fibrosis and NASH (among NAFLD individuals and patients) were quantified. These results were pooled with available literature in meta-analysis. Last, we assessed statins' anti-lipid and anti-inflammatory effects in 3D cultured human liver organoids and THP-1 macrophages, respectively. FINDINGS: Statin use was inversely associated with NAFLD in the Rotterdam study compared to participants with untreated dyslipidemia. In the PERSONS cohort, statin use was inversely associated with NASH, but not with fibrosis. The meta-analysis included 7 studies and indicated a not significant inverse association for statin use with NAFLD (pooled-Odds Ratio: 0.69, 95% Confidence Interval: 0.46-1.01) and significant inverse associations with NASH (pooled-OR: 0.59, 95% CI: 0.44-0.79) and fibrosis (pooled-OR: 0.48, 95% CI: 0.33-0.70). In vitro, statins significantly reduced lipid droplet accumulation in human liver organoids and downregulated expression of pro-inflammatory cytokines in macrophages. INTERPRETATION: Pooled results demonstrated that statin use was associated with a lower prevalence of NASH and fibrosis and might prevent NAFLD. This may be partially attributed to the anti-lipid and anti-inflammatory characteristics of statins. Given their under-prescription, adequate prescription of statins may limit the disease burden of NAFLD. FUNDING: ZonMw, KWF, NWO, SLO, DGXII, RIDE, National and regional government, Erasmus MC and Erasmus University.

Circulatory microRNAs as potential biomarkers for fatty liver disease: the Rotterdam study.

BACKGROUND: Fatty liver disease (FLD) is the most common cause of liver dysfunction in developed countries. There is great interest in developing clinically valid and minimally invasive biomarkers to enhance early diagnosis of FLD. AIM: To investigate the potential of circulatory microRNAs (miRNAs) as biomarkers of FLD at the population level. METHODS: Plasma levels of 2083 miRNAs were measured by RNA sequencing in 1999 participants from the prospective population-based Rotterdam Study cohort. The Hounsfield Unit (HU) attenuation of liver was measured using non-enhanced computed tomography (CT) scan. Logistic and linear regression models adjusting for potential confounders were used to examine the association of circulatory miRNAs with liver enzymes (n = 1991) and CT-based FLD (n = 954). Moreover, the association of miRNAs with hepatic steatosis and liver fibrosis was assessed longitudinally in individuals who underwent abdominal ultrasound (n = 1211) and transient elastography (n = 777) after a median follow-up of >6 years. RESULTS: Cross-sectional analysis showed 61 miRNAs significantly associated with serum gamma-glutamyl transferase and/or alkaline phosphatase levels (Bonferroni-corrected P < 8.46 × 10-5 ). Moreover, 17 miRNAs were significantly associated with CT-based FLD (P < 8.46 × 10-5 ); 14 were among miRNAs associated with liver enzymes. Longitudinal analysis showed that 4 of these 14 miRNAs (miR-193a-5p, miR-122-5p, miR-378d and miR-187-3p) were significantly associated with hepatic steatosis (P < 3.57 × 10-3 ) and three (miR-193a-5p, miR-122-5p and miR-193b-3p) were nominally associated with liver fibrosis (P < 0.05). Nine of the 14 identified miRNAs were involved in pathways underlying liver diseases. CONCLUSIONS: Plasma levels of several miRNAs can be used as biomarkers of FLD, laying the groundwork for future clinical applications.