Suter-Dick, Laura
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Laura
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Suter-Dick, Laura
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- PublikationMethotrexate-induced liver injury is associated with oxidative stress, impaired mitochondrial respiration, and endoplasmic reticulum stress in vitro(MDPI, 01.12.2022) Schmidt, Saskia; Messner, Catherine; Gaiser, Carine; Hämmerli, Carina; Suter-Dick, Laura [in: International Journal of Molecular Sciences]Low-dose methotrexate (MTX) is a standard therapy for rheumatoid arthritis due to its low cost and efficacy. Despite these benefits, MTX has been reported to cause chronic drug-induced liver injury, namely liver fibrosis. The hallmark of liver fibrosis is excessive scarring of liver tissue, triggered by hepatocellular injury and subsequent activation of hepatic stellate cells (HSCs). However, little is known about the precise mechanisms through which MTX causes hepatocellular damage and activates HSCs. Here, we investigated the mechanisms leading to hepatocyte injury in HepaRG and used immortalized stellate cells (hTERT-HSC) to elucidate the mechanisms leading to HSC activation by exposing mono- and co-cultures of HepaRG and hTERT-HSC to MTX. The results showed that at least two mechanisms are involved in MTX-induced toxicity in HepaRG: (i) oxidative stress through depletion of glutathione (GSH) and (ii) impairment of cellular respiration in a GSH-independent manner. Furthermore, we measured increased levels of endoplasmic reticulum (ER) stress in activated HSC following MTX treatment. In conclusion, we established a human-relevant in vitro model to gain mechanistical insights into MTX-induced hepatotoxicity, linked oxidative stress in HepaRG to a GSH-dependent and -independent pathway, and hypothesize that not only oxidative stress in hepatocytes but also ER stress in HSCs contribute to MTX-induced activation of HSCs.01A - Beitrag in wissenschaftlicher Zeitschrift
- PublikationSingle Cell Gene Expression analysis in a 3D microtissue liver model reveals cell type-specific responses to pro-fibrotic TGF-β1 stimulation(MDPI, 22.04.2021) Messner, Catherine; Babrak, Lmar; Titolo, Gaia; Caj, Michaela; Miho, Enkelejda; Suter-Dick, Laura [in: International Journal of Molecular Sciences]3D cell culture systems are widely used to study disease mechanisms and therapeutic interventions. Multicellular liver microtissues (MTs) comprising HepaRG, hTERT-HSC and THP-1 maintain multicellular interactions and physiological properties required to mimic liver fibrosis. However, the inherent complexity of multicellular 3D-systems often hinders the discrimination of cell type specific responses. Here, we aimed at applying single cell sequencing (scRNA-seq) to discern the molecular responses of cells involved in the development of fibrosis elicited by TGF-β1. To obtain single cell suspensions from the MTs, an enzymatic dissociation method was optimized. Isolated cells showed good viability, could be re-plated and cultured in 2D, and expressed specific markers determined by scRNA-seq, qRT-PCR, ELISA and immunostaining. The three cell populations were successfully clustered using supervised and unsupervised methods based on scRNA-seq data. TGF-β1 led to a fibrotic phenotype in the MTs, detected as decreased albumin and increased αSMA expression. Cell-type specific responses to the treatment were identified for each of the three cell types. They included HepaRG damage characterized by a decrease in cellular metabolism, prototypical inflammatory responses in THP-1s and extracellular matrix remodeling in hTERT-HSCs. Furthermore, we identified novel cell-specific putative fibrosis markers in hTERT-HSC (COL15A1), and THP-1 (ALOX5AP and LAPTM5).01A - Beitrag in wissenschaftlicher Zeitschrift
- PublikationIdentification of miR-199a-5p, miR-214-3p and miR-99b-5p as fibrosis-specific extracellular biomarkers and promoters of HSC activation(MDPI, 2021) Suter-Dick, Laura; Messner, Catherine; Özkul, Dilek; Gaiser, Carine; Schmidt, Saskia; Terraciano, Luigi; Krähenbühl, Stephan [in: International Journal of Molecular Sciences]Liver fibrosis is characterized by the accumulation of extracellular matrix (ECM) resulting in the formation of fibrous scars. In the clinic, liver biopsies are the standard diagnostic method despite the potential for clinical complications. miRNAs are single-stranded, non-coding RNAs that can be detected in tissues, body fluids and cultured cells. The regulation of many miRNAs has been linked to tissue damage, including liver fibrosis in patients, resulting in aberrant miRNA expression/release. Experimental evidence also suggests that miRNAs are regulated in a similar manner in vitro and could thus serve as translational in vitro–in vivo biomarkers. In this work, we set out to identify and characterize biomarkers for liver fibrosis that could be used in vitro and clinically for research and diagnostic purposes. We focused on miRNAs released from hepatic 3D cultures exposed to methotrexate (MTX), which causes fibrosis, and acetaminophen (APAP), an acute hepatotoxicant with no clinically relevant association to liver fibrosis. Using a 3D in vitro model, we corroborated compound-specific responses as we show MTX induced a fibrotic response, and APAP did not. Performing miRNA-seq of cell culture supernatants, we identified potential miRNA biomarkers (miR-199a-5p, miR-214-3p, niRNA-125a-5p and miR-99b-5p) that were associated with a fibrotic phenotype and not with hepatocellular damage alone. Moreover, transfection of HSC with miR-199a-5p led to decreased expression of caveolin-1 and increased α-SMA expression, suggesting its role in HSC activation. In conclusion, we propose that extracellular miR-214-3p, miR-99b-5p, miR-125a-5p and specifically miR-199a-5p could contribute towards a panel of miRNAs for identifying liver fibrosis and that miR-199a-5p, miR-214-3p and miR-99b-5p are promoters of HSC activation.01A - Beitrag in wissenschaftlicher Zeitschrift
- PublikationNrf2 protects stellate cells from Smad-dependent cell activation(Public Library of Science, 07/2018) Prestigiacomo, Vincenzo; Suter-Dick, Laura [in: PLOS ONE]Hepatic stellate cells (HSC) orchestrate the deposition of extracellular matrix (ECM) and are the primary effector of liver fibrosis. Several factors, including TGF-β1, PDGF and oxidative stress, have been shown to trigger HSC activation. However, the involvement of cellular defence mechanisms, such as the activation of antioxidant response by Nrf2/Keap1 in the modulation of HSC activation is not known. The aim of this work was to elucidate the role of Nrf2 pathway in HSC trans-differentiation involved in the development of fibrosis. To this end, we repressed Nrf2 and Keap1 expression in HSC with specific siRNAs. We then assessed activation markers, as well as proliferation and migration, in both primary and immortalised human HSCs exposed to Smad inhibitors (SB-431542 hydrate and SB-525334), TGF-β1 and/or PDGF. Our results indicate that knocking down Nrf2 induces HSC activation, as shown by an increase in αSMA-positive cells and by gene expression induction of ECM components (collagens and fibronectin). HSC with reduced Nrf2-levels also showed an increase in migration and a decrease in proliferation. We could also demonstrate that the activation of Nrf2-deficient HSC involves the TGF-β1/Smad pathway, as the activation was successfully inhibited with the two tested Smad inhibitors. Moreover, TGF-β1 elicited a stronger induction of HSC activation markers in Nrf2 deficient cells than in wild type cells. Thus, our data suggest that Nrf2 limits HSCs activation, through the inhibition of the TGF-β1/Smad pathway in HSCs.01A - Beitrag in wissenschaftlicher Zeitschrift