Insulin-like growth factor binding protein 5 enhances survival of LX2 human hepatic stellate cells.

BACKGROUND: Expression of insulin-like growth factor binding protein 5 (IGFBP5) is strongly induced upon activation of hepatic stellate cells and their transdifferentiation into myofibroblasts in vitro. This was confirmed in vivo in an animal model of liver fibrosis. Since IGFBP5 has been shown to promote fibrosis in other tissues, the aim of this study was to investigate its role in the progression of liver fibrosis. METHODS: The effect of IGFBP5 was studied in LX2 cells, a model for partially activated hepatic stellate cells, and in human primary liver myofibroblasts. IGFBP5 signalling was modulated by the addition of recombinant protein, by lentiviral overexpression, and by siRNA mediated silencing. Furthermore, the addition of IGF1 and silencing of the IGF1R was used to investigate the role of the IGF-axis in IGFBP5 mediated effects. RESULTS: IGFBP5 enhanced the survival of LX2 cells and myofibroblasts via a >50% suppression of apoptosis. This effect of IGFBP5 was not modulated by the addition of IGF1, nor by silencing of the IGF1R. Additionally, IGFBP5 was able to enhance the expression of established pro-fibrotic markers, such as collagen Ialpha1, TIMP1 and MMP1. CONCLUSION: IGFBP5 enhances the survival of (partially) activated hepatic stellate cells and myofibroblasts by lowering apoptosis via an IGF1-independent mechanism, and enhances the expression of profibrotic genes. Its lowered expression may, therefore, reduce the progression of liver fibrosis.

Transcriptional profiling reveals novel markers of liver fibrogenesis: gremlin and insulin-like growth factor-binding proteins.

Activated hepatic stellate cells (HSC) that transdifferentiate to myofibroblasts in the injured liver are responsible for scar formation that leads to fibrosis and eventually cirrhosis. To investigate the gene expression profile during different stages of this process, we performed serial analysis of gene expression, representing a quantitative and qualitative description of all expressed genes. Stellate cells were isolated from human livers and cultured. Serial analysis of gene expression was performed on RNA isolated from quiescent, activated, and transdifferentiated HSC. Comparison of the three resulting transcriptomes showed that less than 5% of all genes changed significantly in expression. Established markers of liver fibrosis showed enhanced expression in accordance with the transdifferentiation process. In addition, induction was seen for several genes not yet recognized to be involved in liver fibrosis, such as insulin-like growth factor-binding proteins (IGFBP) and antagonists of bone morphogenic proteins: follistatin and gremlin. The induction of these genes was validated in vivo in mice developing liver fibrosis. The expression of IGFBPs and gremlin was measurable in the livers of these mice, whereas it was low or undetectable in control mice without liver fibrosis. Since gremlin modulates the activity of bone morphogenic growth factors, it may represent a novel pathway and a target for therapeutic intervention and together with IGFBPs it could be a specific marker of liver fibrosis. In conclusion, the comparison of the three transcriptomes of (activated) stellate cells reveals novel genes involved in fibrogenesis and provides an appreciation of the sequence and timing of the fibrotic process in liver.

Increased formation of pyridinoline cross-links due to higher telopeptide lysyl hydroxylase levels is a general fibrotic phenomenon.

Fibrosis is characterized by an excessive accumulation of collagen which contains increased levels of pyridinoline cross-links. The occurrence of pyridinolines in the matrix is an important criterion in assessing the irreversibility of fibrosis, which suggests that collagen containing pyridinoline cross-links significantly contributes to the unwanted collagen accumulation. Pyridinoline cross-links are derived from hydroxylated lysine residues located within the collagen telopeptides (hydroxyallysine pathway). Here, we have investigated whether the increase in hydroxyallysine-derived cross-links in fibrotic conditions can be ascribed to an increased expression of one of the lysyl hydroxylases (LH1, LH2 with its splice variants LH2a and LH2b, or LH3) and/or to an increased expression of lysyl oxidase (LOX). In fibroblast cultures of hypertrophic scars, keloid and palmar fascia of Dupuytren's patients, as well as in activated hepatic stellate cells, increased levels of LH2b mRNA expression were observed. Only minor amounts of LH2a were present. In addition, no consistent increase in the mRNA expression levels of LH1, LH3 and LOX could be detected, suggesting that LH2b is responsible for the overhydroxylation of the collagen telopeptides and the concomitant formation of pyridinolines as found in the collagen matrix deposited in long-term cultures by the same fibrotic cells. This is consistent with our previous observation that LH2b is a telopeptide lysyl hydroxylase. We conclude that the increased expression of LH2b, leading to the increased formation of pyridinoline cross-links, is present in a wide variety of fibrotic disorders and thus represents a general fibrotic phenomenon.