Conserved gene expression patterns in myofibroblastic murine and human hepatic stellate cells and nafld patients with liver fibrosis compatible with epithelial-to-mesenchymal like transition

Abstract

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) can progress to fibrosis and cirrhosis. The myofibroblastic activation of hepatic stellate cells (HSC) is critical for fibrosis progression in NAFLD. Previous studies showed that activation of cultured rat and human HSC occurs via an epithelial to mesenchymal transition (EMT)-like process. The aim of this study was to investigate whether culture activation of murine and human HSC reflects changes in liver gene expression that accompany fibrosis progression in NAFLD patients. METHODS: Liver gene expression patterns of activated murine and human HSC were compared to those of liver tissue from 72 patients with biopsyproven NAFLD (40 early fibrosis, F0-F1;32 advanced fibrosis, F3-F4). Using standard methods, primary HSC were isolated from residual healthy human liver that was used for split-liver transplantation and from healthy mice. Quiescent HSC were processed immediately after isolation or cultured to induce activation to myofibroblastic HSC (MF-HSC). Total RNA was extracted from 3mg liver tissue/patient and from quiescent and MF-HSC using Qiagen kits. cDNA was respectively hybridized to Affymetrix human genome U133A 2.0 array or mouse genome 430 2.0 array gene chips and analyzed via an empirical Bayes method after normalization in R/Bioconductor. Significance was determined by a False Discovery Rate of 5% after Benjamini-Hochberg correction. RESULTS: 1,143 genes were significantly differentially expressed in livers of NAFLD patients with advanced fibrosis vs. early fibrosis. The top upregulated genes included thrombospondin 2 (THBS2), EGF-containing fibulin-like extracellular matrix protein 1 (EFEMP1), lumican (LUM) and dickkopf homolog 3 (DKK3). 1,139 and 1,910 genes were differentially expressed during activation of human and mouse HSC to their respective MF-HSC phenotypes. 353 of the most differentially expressed genes were conserved in both species, and 62 of these were also differentially expressed in NAFLD livers with high vs. low fibrosis. Highly upregulated conserved genes included ACTA2, COL1A1, ENAH, LOX, FBN1, LBH, TGF-β2 and CDH11. 60% of the conserved differentially-expressed genes are involved in pathways modulated during EMT, such as cell motility, cell adhesion, morphogenesis and extracellular matrix interactions. CONCLUSIONS: Gene expression patterns from culture-activated human and mouse MF-HSC recapitulate some of the gene expression changes that accompany liver fibrosis progression in NAFLD patients. Gene expression patterns representative of EMT are amongst the most conserved gene expression changes, supporting a role for EMT in NAFLD-related fibrosis.