Activation of hepatic stellate cells under hypoxia and the inhibition of HSCs activation by TGF-b binding protein Decorin

Abstract

Liver fibrosis results from chronic damage to the liver in conjunction with the accumulation of extracellular matrix (ECM) proteins. The subsequent development of liver fibrosis would result in cirrhosis and even cancer. It is widely accepted that hepatic stellate cells (HSCs) are the main ECM-producing cells in the injured liver. In normal liver, HSCs are in the quiescent stage. Following chronic injury, HSCs are activated and transdifferentiate into myofibroblast-like cells, acquiring contractile, proinflammatory, and fibrogenic properties. Thus, the activation of HSCs plays the key role during fibrogenesis, and inhibition of HSCs activation offers a promising target for the development of antifibrotic agents. Hypoxia, a common physiological and environmental stress factor, is associated with various pathological conditions such as fibrogenesis. In the first part of thesis, the behavior of human HSCs, an LX-2 cell line in low oxygen tension (1% O2) was analyzed. Under hypoxic condition, the expression of hypoxia inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF) gene was induced, and the expression of α-smooth muscle actin (α-SMA) was increased. Hypoxia also elevated the expression of matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) genes, as well as the expression of the collagen type I protein in LX-2 cells. The analysis of transforming growth factor-β (TGF-β)/Smad signaling pathway showed that hypoxia potentiated the expression of TGF-β1 and the phosphorylation status of Smad2. In the second part of the thesis, the role of nuclear factor kappa B (NF-κB), an important transcription factor, and its signaling pathway in hypoxia-induced activation of HSCs were also investigated. The results showed that hypoxia induced the activation of NF-κB in HSCs, where the protein expression of nuclear p65 and the phosphorylation level of p65 were increased in hypoxic HSCs. Meanwhile, hypoxia reduced the expression of inhibitor subunit of nuclear factor kappa B (IκB-α) protein in HSCs, while elevating the phosphorylated IκB-α protein level. When the proteasome inhibitor MG132 was used to block NF-κB in HSCs, the hypoxia-induced expression of α-SMA and TGF-β protein was reduced, and the expression of MMP-2 gene was inhibited. The study showed that hypoxia activates NF-κB pathway in HSCs, which plays an important role in the activation of HSCs induced by low oxygen tension. Decorin is a small leucine-rich extracellular matrix proteoglycan involved in the regulation of formation and organization of collagen fibrils, and modulation of the activity of growth factors such as TGF-β. In the third part of the thesis, the core protein of human decorin was cloned and expressed in Escherichia coli. The purified recombinant human decorin (rhDecorin) was shown to significantly inhibit the proliferation of LX-2 cells stimulated by TGF-β1, a key stimulator of fibrosis. RT-PCR result showed that the expression of MMP-2 and TIMP-1 were reduced by rhDecorin in LX-2 cells stimulated by TGF-β1. Furthermore, the protein expression of α-SMA, collagen type III and phosphorylated Smad2 (p-Smad2) were significantly decreased in the presence of rhDecorin. rhDecorin also reduced fibrillogenesis of collagen type I in a dose-dependent manner. Gene expression profiles of LX-2 cells stimulated by TGF-β1 in the presence and the absence of rhDecorin were obtained by using cDNA microarray and differentially expressed genes were identified to provide further insight into the molecular action mechanism of decorin on LX-2 cells. In the fourth part of the thesis, the effect of decorin on liver fibrosis in vivo was studied using animal models. For carbon tetrachloride (CCl4)-induced chronic liver injury rats, the effect of rhDecorin on degrees of fibrosis and serological markers for liver fibrosis and liver function including hyaluronic acid (HA), type IV collagen (CIV), γ-glutamyl transferase (γ-GT), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined. α-SMA and proliferating cell nuclear antigen (PCNA) immunohistochemistry were performed. The treatment of rhDecorin on CCl4-induced chronic liver injury rats alleviated liver fibrosis and improved liver function. Serum HA, CIV, γ-GT, ALT and AST levels of the rhDecorin-treated group were significantly decreased. An increase in PCNA and decrease in α-SMA expression in the treated group were also observed. In addition, histological examination and serum markers were investigated for D-galactosamine (GalN)-induced acute damage of liver and the development of fibrosis in mice. Hepatic fibrosis and damage in acute liver injury mice induced by GalN was significantly alleviated by pre-treatment with rhDecorin. Pre-treatment with rhDecorin at high concentration also remarkably reduced the level of ALT and AST compared with GalN control group. It is concluded that rhDecorin shows inhibitory effects on liver fibrosis, both in vivo and in vitro, through inhibition of HSCs activation and protection of liver function.

Date of Award	15 Feb 2008
Original language	English
Awarding Institution	City University of Hong Kong
Supervisor	M YANG (Supervisor)