Investigation of the role of Nrf2 in hepatic stellate cells during liver fibrosis using in vitro models

Liver fibrosis is a reversible wound-healing response characterized by fibroblast proliferation and extracellular matrix (ECM) remodelling. The development of fibrosis involves several intermediate steps, including hepatocyte injury and cell death, oxidative stress, activation of Kupffer cells (KC), activation of hepatic stellate cells (HSC), and chronic inflammation.
Currently, most liver fibrosis research is performed in vivo since suitable in vitro system are lacking. Here, we present the design and development of three-dimensional (3D) in vitro liver fibrosis models: a rat primary cell-based model and a human cell line based model. Both multicellular models responded to treatment with proinflammatory cytokine TGF-β1 and endotoxin lipopolysaccharide (LPS), displaying hepatocellular damage, HSC activation and deposition of ECM. In addition, the human model displayed a fibrotic phenotype after exposure to methotrexate (MTX) and thioacetamide (TAA), characterized by the activation of HSC, upregulation of genes involved in the development of fibrosis and the secretion and deposition of extracellular matrix. MTX and TAA also elicited the upregulation of Nrf2 and Keap1 suggesting oxidative defence pathway activation during early stages of fibrosis.
In addition, we have elucidated the role of key factors TGF-β1 and PDGF as well as the potential role of Nrf2 pathway in HSC activation. Simultaneous exposure of HSCs to both TGF-β1 and PDGF showed a synergistic effect leading to full cell activation, with increases in both proliferative and fibrogenic capacities. Moreover, activation markers expression, as well as proliferation and migration, were assessed in both human primary and hTERT-HSCs following Nrf2 or Keap1 repression by siRNAs. Knocking down Nrf2 induces αSMA production as well as induction of ECM components, clearly indicating an induction of HSC activation. This induced activation was depended on the TGF-β1/Smad pathway, as the two Smad inhibitors SB431542 hydrate and SB525334 successfully rescued the effect of the knockdown.
In summary, the studies discussed in this thesis deliver contributions addressing two of the limiting factors of liver fibrosis research: the lack of a suitable 3D model and the discovery of potential pathways on which act in order to revert HSC activation. Firstly, novel and well-suited 3D liver models, able to recapitulate the key fibrotic events, were developed. Secondly, the roles of TGF-β1 and PDGF were further clarified in both primary and hTERT-HSCs. Finally, this thesis describes a novel role for Nrf2 as a suppressor of HSC activation. The results underlines the importance of the Nrf2 defence pathway, which may play important roles in many cell types and may thus be involved in several liver diseases.