Biomarkers of hepatocellular synthesis in patients with decompensated cirrhosis

Using untargeted serum proteomics, in silico upstream regulator analysis and validation ELISAs alongside with hepatic gene expression analysis, we herein show that serum concentrations of proteins regulated by HNF-4α-and IL-6 are reduced in cirrhosis, associated with more advanced stages of cirrhosis, and indicate poor transplant-free survival. Low serum levels of apolipoprotein AI identified patients with very low likelihood of transplant-free survival in a large cohort of patients with acute decompensation of cirrhosis. The prognostic usefulness of apolipoprotein AI seen in our study further extends and corroborates previous findings [13, 14].

The prognosis in patients with advanced chronic liver disease is mainly determined by liver function, portal hypertension, the presence of extrahepatic organ failure, and systemic inflammation [15,16,17]. In advanced cirrhosis, inflammation is a major driver of complications and mortality, and pro-inflammatory cytokines, acute-phase proteins, and immune activation markers have been employed to improve risk prediction in decompensated liver disease [18,19,20,21]. The identified prognostic relevant hepatocellular proteins, albumin, apolipoprotein AI, transthyretin, and transferrin, are negative acute-phase proteins, and low concentrations of negative acute-phase proteins can serve as surrogates of reduced hepatic synthesis and impaired hepatocellular reserve but also indicate the presence of systemic inflammation. Upstream regulator analysis identified HNF-4α and IL-6 as the most important regulators underlying the observed serum proteome changes in patients with decompensated cirrhosis. Whereas the hepatic mRNA expression of the principal inducer of acute-phase response, IL6, was increased in cirrhotic livers, hepatic mRNA expression of HNF4A was reduced and positively correlated with the expression of ALB, TTR, TF and Apo-AI. The promoter regions of TTR and other negative APP contain HNF-4α binding sites, and this correlation has been observed in patients with alcoholic hepatitis [22,23,24].

Although BCHE expression did not correlate with HNF4A expression, its strong negative correlation with IL6 expression alongside the positive correlation of BCHE with transferrin and transthyretin on the protein level suggest a similar role as a negative APP and likely explains its marked decrease in patients with cirrhosis.

Despite a comparable hepatic regulation of the indicator proteins investigated in more detail, i.e., albumin, apolipoprotein AI, transthyretin, transferrin, and BCHE, their role as a prognostic biomarker somewhat differed. This may be due to different serum half-lives ranging from < 1 day (apolipoprotein AI), 2–4 days (transthyretin) across 8–12 days for transferrin and BCHE to 3 weeks (albumin). As higher levels of apolipoprotein AI identified a subgroup of patients with ACLF with better prognosis, apolipoprotein AI may be interpreted as the most dynamic marker of the hepatic reserve after liver injury and organ failure.

Hepatic HNF-4α-dependent gene expression is altered across the spectrum from fibrogenesis to decompensation and liver failure. Mechanistically, in early liver disease with fibrosis, liver matrix stiffness and cytoskeletal tension inhibit the hepatocellular HNF4-α transcriptional network [25]. In advanced liver disease, such as alcohol-related liver failure, hepatic activity of the liver-enriched transcription factor HNF-4α is severely inhibited, reducing hepatic expression of cytochrome P450 enzymes, apolipoproteins, and aldolases [26]. This process is driven by growth factors such as TGF-β, HGF, and EGF, cytokines such as TNF, IL-1β, IFN-γ, and inflammatory mediators such as PGE2 and LPS, and hepatocyte de-differentiation may play an additional role [26]. Because these upstream regulators play a prognostic role for complications of decompensated cirrhosis as well, the association of HNF-4α-regulated proteins with outcome in patients with acute decompensation of cirrhosis is plausible.

In a study on liver tissue from patients at different stages of decompensation, HNF-4α expression was downregulated and correlated with liver dysfunction, fibrosis stage, and prognostically relevant serum parameters bilirubin, albumin, and prothrombin time [27]. As a result, lower serum concentrations of transthyretin and BCHE correlated with the Child–Pugh stage and with the presence of complications such as hepatic encephalopathy in a study on patients with predominantly viral cirrhosis [28]. Plasma proteome of 459 patients with compensated alcohol-related chronic liver disease demonstrated that lower levels of plasma albumin, BCHE, and transthyretin were associated with the presence of significant liver fibrosis and hepatic inflammation (vs. no/minimal fibrosis/inflammation), but not with the degree of hepatic steatosis [29].

In this study, we demonstrate remarkable differences in serum levels of bona fide hepatocellular proteins as well as an association between lower apolipoprotein AI levels and transplant-free survival after adjusting for confounding factors such as age, MELD score, and the presence of hepatocellular carcinoma. Notably, the prognostic ability of apolipoprotein AI was comparable to the routinely used MELD score consisting of three different biomarkers. Moreover, apolipoprotein AI remained a significant predictor of mortality in two different multivariable models including MELD or ACLF.

These data suggest that the activity of the liver-enriched transcription factor HNF-4α as a surrogate for hepatocellular reserve, degree of fibrosis, hepatocyte differentiation, and inflammatory status may provide additional prognostic information in advanced stages of cirrhosis. However, several limitations of our work need to be considered. The observed analyses are based on a single-center cohort, and the clinical cohorts used for mRNA analyses differed from cohorts used for proteomic assessment since liver biopsies are rarely ethically justifiable in subjects with decompensated liver cirrhosis. Furthermore, the single biomarkers assessed in our study have been studied before and in this respect, our results are confirmative. The demonstrated heterogeneity in hepatocellular proteins in subjects with cirrhosis should spur large-scale proteomic studies addressing the prognostic usefulness of the individual protein patterns. Such studies might be able to identify and validate clinically useful prognostic biomarkers as it has been recently demonstrated for alcoholic liver disease [29].

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