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Exploring The Impact of Genetic Variations on Human Hepatic Stellate Cells Using Extracellular Matrix 3D Models

Caon, Elisabetta; (2023) Exploring The Impact of Genetic Variations on Human Hepatic Stellate Cells Using Extracellular Matrix 3D Models. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Background and aims: Liver fibrosis is a crucial pathophysiological step in chronic liver diseases (CLD). Activated hepatic stellate cells (HSCs) are the key mediators of fibrogenesis and therefore, research has been focusing on identifying and addressing pro-fibrogenic mechanisms in HSCs for the development of new drug candidates to treat fibrogenic liver diseases. This process has been hampered by the lack of appropriate models. In addition, only recently attention has been given to the importance of genetic polymorphisms for the onset of CLD, with numerous new variants being discovered, although their molecular role is still largely unclear. In this project, the impact of the PNPLA3 I148M and TM6SF2 E156K variants on the pro-fibrogenic behaviour of HSCs has been investigated utilizing as a platform 3D scaffolds and hydrogels obtained from the decellularized extracellular matrix (ECM) of healthy and diseased human livers. Methods: Healthy or cirrhotic decellularized human liver ECM 3D scaffolds were either utilized for the culture of primary human HSCs wild type (WT) or mutant for the genetic variant under investigation or lyophilized and solubilized to yield liver ECM solution. The ECM solution was mixed with two different gelling agents to form hydrogels for the culture of HSCs WT or mutant for the genetic variant under investigation. RNA sequencing was performed on 2D-cultured primary human HSCs and liver biopsies of obese individuals, genotyped for the PNPLA3 I148M variant. Cell behaviour of HSCs cultured in ECM hydrogels or scaffolds and treated with/without TGFB1/Endothelin-1/CytosporoneB (Csn-B) was evaluated. Results: Comparison between transcriptomic analysis of liver biopsies and primary human HSCs highlighted shared PNPLA3 I148M-driven dysregulated pathways related to mitochondrial function, antioxidant response, ECM remodelling and TGFB1 signalling. Analogous pathways were dysregulated in HSCs WT/I148M-PNPLA3 cultured in 3D liver scaffolds. Mitochondrial dysfunction in I148M-PNPLA3 cells was linked to respiratory chain complex IV insufficiency. Antioxidant capacity was lower in I148M-PNPLA3 HSCs, while ROS secretion was increased in I148M-PNPLA3 HSCs and higher in bioengineered cirrhotic scaffolds versus healthy scaffolds. TGFB1 signalling pathway followed the same trend. In I148M-PNPLA3 cells, TGFB1-endogenous inhibitor NR4A1 expression and activation were decreased: treatment with the Csn-B agonist increased total NR4A1 in HSCs cultured in healthy but not in cirrhotic 3D scaffolds. This led to a decreased TGFB1 signalling and increased NRF2 in Csn-B treated cells. Hydrogels made with human liver ECM proved to be stable and support viability and responsiveness to pro-fibrogenic treatment of HSCs. Furthermore, HSCs showed similar behaviour when cultured in ECM scaffolds or ECM hydrogels and featured a less activated phenotype compared to 2D plastic culture. HSCs carrying the PNPLA3 I148M and TM6SF2 E156K variants showed to have increased basal contraction and activation induced by the presence of the cirrhotic ECM compared to WT HSCs. Conclusions: This study illustrates how the usage of novel 3D models based on human derived healthy and cirrhotic decellularized liver ECM combined with omics and molecular biology techniques can help unravel the pro-fibrogenic behaviour of HSCs in presence of different genetic variants and their role in the progression of CLD.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Exploring The Impact of Genetic Variations on Human Hepatic Stellate Cells Using Extracellular Matrix 3D Models
Language: English
Additional information: Copyright © The Author 2023. Original content in this thesis is licensed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) Licence (https://creativecommons.org/licenses/by-nc-nd/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request.
UCL classification: UCL
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences > Div of Medicine
URI: https://discovery.ucl.ac.uk/id/eprint/10172389
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