An exploratory study examining how nano-liquid chromatography–mass spectrometry and phosphoproteomics can differentiate patients with advanced fibrosis and higher percentage collagen in non-alcoholic fatty liver disease

Abstract

BackgroundNon-alcoholic steatohepatitis (NASH) is among the leading causes of liver disease worldwide. It is increasingly recognized that the phenotype of NASH may involve a number of different pathways, of which each could become important therapeutic targets. The aim of this study is to use high resolution mass spectrometry (MS) and phosphoproteomics techniques to assess the serum proteome and hepatic phosphoproteome in subjects with NASH-related fibrosis.MethodsSixty-seven biopsy-proven NAFLD subjects with frozen sera and liver tissue were included. Reverse phase protein microarray was used to quantify the phosphorylation of key signaling proteins in liver and nano-liquid chromatography (LC)-MS was used to sequence target biomarkers in the serum. An image analysis algorithm was used to quantify the percentage of collagen (% collagen) using computer-assisted morphometry. Using multiple regression models, serum proteomes and phosphorylated hepatic proteins that were independently (p ≤ 0.05) associated with advanced fibrosis (stage ≥ 2) and higher % collagen were assessed.ResultsPhosphorylated signaling pathways in the liver revealed that apoptosis signal-regulating kinase 1, mitogen-activated protein kinase (ASK1-MAPK pathway involving ASK1 S38 (p < 0.02) and p38 MAPK (p = 0.0002)) activated by the inflammatory cytokine interleukin (IL-10) (p < 0.001), were independently associated with higher % collagen. LC-MS data revealed that serum alpha-2 macroglobulin (α2M) (p = 0.0004) and coagulation factor V (p = 0.0127) were independently associated with higher % hepatic collagen.ConclusionsSimultaneous profiling of serum proteome and hepatic phosphoproteome reveals that the activation of ASK1 S38, p38 MAPK in the liver, and serum α2M and coagulation factor V are independently associated with hepatic collagen deposition in patients with NASH. These data suggest the role of these pathways in the pathogenesis of NASH-related fibrosis as a potential therapeutic target.Electronic supplementary materialThe online version of this article (10.1186/s12916-018-1136-1) contains supplementary material, which is available to authorized users.

Updates in the quantitative assessment of liver fibrosis for nonalcoholic fatty liver disease: Histological perspective

“…[31][32][33][34][35][36] Recently, there has been interest in gut microbiome to identify signatures for advanced fibrosis in NAFLD, 37,38 as well as proteomic or multihttp://www.e-cmh.org https://doi.org/10.3350/cmh.2020.0181 omic studies. 39,40 However, these methods are all indirect measures of fibrosis encumbered by confounders and lack of specificity. Some of the most pressing issues with noninvasive tests are difficulty in determining the optimum cut-off to differentiate intermediate stages of fibrosis; inability to reflect architectural changes/fibrosis stage that may not correspond with amount of collagen deposition; and the incapability to assess the dynamic nature of fibrosis progression or regression.…”

“…Composite scoring systems based on serum biomarkers, such as fibrosis-4 (FIB-4) index, NAFLD fibrosis score (NFS), and enhanced liver fibrosis (ELF) test, have also been developed as surrogate measures of fibrosis severity in NAFLD [ 31 - 36 ]. Recently, there has been interest in gut microbiome to identify signatures for advanced fibrosis in NAFLD [ 37 , 38 ], as well as proteomic or multiomic studies [ 39 , 40 ]. However, these methods are all indirect measures of fibrosis encumbered by confounders and lack of specificity.…”

The role of omics in the pathophysiology, diagnosis and treatment of non-alcoholic fatty liver disease

“…Several studies have investigated hepatic proteome alone or in combination with blood proteome, either in animal models or in humans with NAFLD, aiming to answer fundamental pathophysiological questions [ [127] , [128] , [129] ]. In one recent study, the levels and cellular distributions of 6000 liver proteins and 16,000 phosphopeptides have been assessed in the liver of mice developing hepatic steatosis due to high fat diet (HFD).…”

“…In a combined hepatic phosphoproteome and serum proteome analysis of 67 biopsy-proven NAFLD subjects, the ASK1-MAPK pathway that is activated by IL-10 was recognized as important for liver fibrosis, indicated by its strong association with higher % of hepatic collagen. In serum, alpha-2 macroglobulin and coagulation factor V were strongly associated with hepatic collagen [ 127 ]. It has thus been suggested that these pathways can potentially serve as therapeutic targets.…”

Mitogen Activated Protein Kinases in Steatotic and Non-Steatotic Livers Submitted to Ischemia-Reperfusion

“…It is well known that p38 MAPK participates in the regulation of inflammatory response [24]. The involvement of the apoptosis signal-regulating kinase 1 (ASK1)-MAPK pathway, the transducer activator transcription 3 and other survival pathways insulin receptor substrate 2 via phosphoinositide 3-kinase (PI3k) and its downstream effectors 3-phosphoinositide dependent protein kinase-1, ribosomal protein S6 kinase polypeptide 1 and v-protein kinase B (Akt) oncogene homolog) in NASH and NASH-related fibrosis has been also suggested [25,26].…”