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

Oltre il Segno/OltreMare

La realizzazione di un volume contenente le incisioni scelte all’interno della Scuola di Grafica d’Arte dell’Accademia di Belle Arti di Palermo, coordinata dai Proff. Giovanni D’Alessandro e Riccardo Mazzarino rappresenta motivo di orgoglio e di soddisfazione per la nostra Istituzione che costruisce i percorsi didattici dei propri corsi a partire dall’esperienza laboratoriale. L’incisione grafica è tra le tecniche artistiche più antiche ma nel contempo più contemporanee. La gestualità intrinseca al segno, che si manifesta nella carta, svela universi della visione inaspettati.(Mario Zito - Direttore dell’Accademia di Belle Arti di Palermo) Il segno è il risultato di un gesto a volte deciso, a volte contorto, a volte leggero, i cui risultati spesso sono inattesi e sorprendenti. Il volume contiene esemplari di incisioni fortemente caratterizzanti della scuola di Grafica d’Arte che vanta all’interno del proprio corso di studi docenti-artisti che consapevoli della ricchezza del loro bagaglio esperienziale offrono agli studenti gli strumenti necessari per far sì che l’arte del saper fare artigianale, si trasformi in mera poetica artistica

East Asia’s new developmentalism: state capacity, climate change and low-carbon development

This paper argues that to understand the relevance of developmental states in East Asia and elsewhere, we need to focus on the changing development agenda in the early twenty-first century, especially how this connects with the global challenge of climate change and thereby sustainable, low-carbon development. It combines theories on state capacity and ecological modernisation to form the ‘new developmentalism’ concept. This is applied to study revitalised and refocused forms of state capacity aimed at realising the transformative economic objectives associated with sustainable development. New developmentalism helps us understand not only current state capacity practice in a climate challenged world but also how we have moved beyond original conceptions of developmental statism. It may be understood in the wider context of the sustainable development agenda and climate interventionism. As is argued, new developmentalism is most clearly evident in East Asia but can be applied in a wider geographic sense where strong forms of developmental state capacity are exercised towards meeting transformative sustainable development goals

Nrf2 intervention into the TGF-β1/Smad signalling pathway.

<p>Panel A, basal condition: HSCs express high levels of both Nrf2 and its target genes (e.g. Nqo1), thereby controlling reactive oxygen species (ROS) level. Nrf2 also inhibits Smad pathway by binding directly to Smad protein or through the action of phosphatases [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0201044#pone.0201044.ref041" target="_blank">41</a>]. In these conditions, the TGF-β1/Smad pathway has a low activity, resulting in low level of αSMA, collagens and TGF-β1. Thus, HSC cells exhibit a quiescent phenotype. Panel B, Nrf2 knockdown: we have found out that Nrf2 knockdown, with a consequent decrease of its target genes, induces stellate cells activation. Decrease in Nrf2 was in fact associated with an increase in the levels of Extracellular matrix (ECM) components as well as αSMA and TGF-β1. TGF-β1 further induces the expression of ATF3, which acts as functional repressor of Nrf2. We found out that this siNrf2-induced stellate cell activation may be regulated by the Smad inhibitors SB-431542 hydrate (SB43) and SB-525334 (SB52), confirming the role of Nrf2 in relation to the Smad pathway.</p

Effect of siRNAs, TGF-β1 and/or PDGF-AB on HSC proliferation.

<p>(A-B) hTERT-HSCs (A) and primary HSCs (B) were transfected for 72 hours with siNrf2, siKeap1 or siCON. Cells were then detached and plated for EdU staining as described in Materials and methods section. The pictures were taken with 10X magnification by using confocal microscopy and nuclei were counted with Image J software. siCON: scrambled siRNA; siNrf2: Nrf2 knockdown; siKeap1: Keap1 knockdown. *, P ≤ 0.05; **, P ≤ 0.01; *** P ≤ 0.001 vs control or siCON. Values are expressed as rate of proliferation (mean ± SD); N = 4 independent experiment with 5 replicates each for hTERT-HSC, and N = 6 different batches with 5 replicates each. (C-D) Proliferation rate of transfected hTERT-HSC (C) and primary HSC (D) after exposure to 0.5–1 ng/mL TGF-β1 and/or 1–5 ng/mL PDGF-AB for 48 hours. EdU was added during the last 30 hours of the experiment. Values are expressed as percentage of proliferation over each control sample. *, P ≤ 0.05; **, P ≤ 0.01; *** P ≤ 0.001 vs Control (mean ± SD, N = 4 replicates for hTERT-HSC and N = 3 from different batches with 4 replicates each for primary HSC).</p

TGF-β1 suppresses mRNA expression of Nrf2 in HSCs.

<p>(A) hTERT-HSCs were exposed to 1–5 ng/mL TGF-β1 for 48 hours. mRNA was extracted using TRIzol conventional procedure and fold changes were calculated as 2^(-ΔΔCT) for each sample and control and expressed as mean fold change ± SD (N = 3). Beta-2-microglobulin (B2M) was used as reference gene for each sample. The results show a significant downregulation of Nrf2, Keap1 and Nqo1 after exposure to TGF-β1. (B) Primary HSCs were exposed to 1 ng/mL TGF-β1, 1 μM SB-525334 (SB52) and/or 5 ng/mL PDGF-AB for 48 hours. mRNA was extracted using TRIzol conventional procedure and fold changes were calculated as 2^(-ΔΔCT) for each sample and control and expressed as mean fold change ± SD (N = 3 different batches). Beta-2-microglobulin (B2M) was used as reference gene for each sample. The results show a significant downregulation of Nrf2, and Nqo1 following exposure to TGF-β1. SB52 induces upregulation of Nrf2 and Nqo1, efficiently inhibiting the TGF-β1 effect. (C-D) The protein levels of Nrf2, Nqo1 and actin beta were analysed by Western blot analysis after exposure to 0–10 ng/mL TGF-β1 in hTERT-HSC (C) and primary HSCs (D) for 24 and 48 hours. (E-F) The protein levels of Keap1 and Tubulin were analysed by Western blot analysis after exposure to 0–10 ng/mL TGF-β1 in hTERT-HSC (E) and primary HSCs (F) for 24 and 48 hours.</p

TaqMan probes used for the research.

<p>TaqMan probes used for the research.</p

Pro-fibrotic compounds induce stellate cell activation, ECM-remodelling and Nrf2 activation in a human 3D-multicellular model of liver fibrosis.

Currently most liver fibrosis research is performed in vivo, since suitable alternative in vitro systems which are able to recapitulate the cellular events leading to liver fibrosis are lacking. Here we aimed at generating a system containing cells representing the three key players of liver fibrosis (hepatocyte, Kupffer cells and stellate cells) and assess their response to pro-fibrotic compounds such as TGF-β1, methotrexate (MTX) and thioacetamide (TAA).Human cell lines representing hepatocytes (HepaRG), Kupffer cell (THP-1 macrophages) and stellate cells (hTERT-HSC) were co-cultured using the InSphero hanging drop technology to generate scaffold-free 3D microtissues, that were treated with pro-fibrotic compounds (TGF-β1, MTX, TAA) for up to 14 days. The response of the microtissues was evaluated by determining the expression of cytokines (TNF-α, TGF-β1 and IL6), the deposition and secretion of ECM proteins and induction of gene expression of fibrosis biomarkers (e.g. αSMA). Induction of Nrf2 and Keap1, as key player of defence mechanism, was also evaluated.We could demonstrate that the multicellular 3D microtissue cultures could be maintained in a non-activated status, based on the low expression levels of activation markers. Macrophages were activated by stimulation with LPS and hTERT-HSC showed activation by TGF-β1. In addition, MTX and TAA elicited a fibrotic phenotype, as assessed by gene-expression and protein-deposition of ECM proteins such as collagens and fibronectin. An involvement of the antioxidant pathway upon stimulation with pro-fibrotic compounds was also observed.Here, for the first time, we demonstrate the in vitro recapitulation of key molecular and cellular events leading to liver fibrosis: hepatocellular injury, antioxidant defence response, activation of Kupffer cells and activation of HSC leading to deposition of ECM