Enhanced relevance of hypoxia-induced in vitro models in mimicking liver fibrosis pathways

Introduction: Preclinical models that are currently used for fibrosis research only partially mimic human disease processes. Particularly, traditional transforming growth factor beta 1 (TGFβ1) induced stellate cell models are lacking in activation of hypoxia-induced pathways, which is a relevant process in patients with liver disease. Here we investigated the synergic effect of two hypoxia-mimicking compounds (DMOG and IOX2) and TGFβ1, on fibrotic phenotype and in vivo disease recapitulation in primary human hepatic stellate cells (HSCs). Methods: Human primary HSCs were cultured, stimulated (TGFβ1 and hypoxia-mimicking compounds combinations) and harvested to identify optimal stimulation conditions for fibrotic phenotype, cell viability and toxicity levels. To assess the fibrotic phenotype, protein levels was assayed for fibrosis markers (collagen, TIMP-1, Fibronectin) were evaluated. RNA was isolated and sequenced through next generation sequencing (NGS). Bioinformatic tools using differential expression analysis (Deseq2) and Ingenuity Pathway Analysis (IPA) were used to identify differentially expressed genes (DEGs) and map these to biological functionality, respectively. Results: Our HSC model showed that stimulation with IOX2 (0.3 and 1 µM) in the presence of TGFβ1 significantly increased fibrotic markers levels (total collagen, TIMP-1, and fibronectin). DMOG showed no effect using low, non-toxic concentrations. Biological functionality from DEGs highlighted various fibrosis-related pathways, hypoxia-related genes and relevant crosslinking markers when IOX2 was added in a concentration-dependent manner. Furthermore, comparative analysis with human DEGs (Fibrotic vs non-fibrotic) showed improved disease representation in our HSC model with IOX2 addition. Conclusion: These findings suggest that IOX2 may aggravate fibrotic disease and improve in vivo disease recapitulation in vitro. Hypoxia-mimicking compounds like IOX2 hold promise for enhancing fibrosis in in vitro models, providing valuable insights into fibrosis pathogenesis, hypoxia-related genes and potential therapeutic strategies.