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 (TGFß1) 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 TGFß1, on fibrotic phenotype and in vivo disease recapitulation in primary human hepatic stellate cells (HSCs). Methods: Human primary HSCs were cultured, stimulated (TGFß1 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 TGFß1 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. Overall design: Human primary HSCs (Hepatic Stellate Cells) were cultured -/+ stimulation with compound combinations (in total 8 treatments): Control without additions (C), 4 ng/ml TGFß1 (T), 4 ng/ml TGFß1 + 5 µM Alk5 (TA), 4 ng/ml TGFß1 + 0.3 µM IOX2 + 5 µM Alk5 (TI.3A), 0.3 µM IOX2 (I.3), 1 µM IOX2 (I1), 4 ng/ml TGFß1 + 0.3 µM IOX2 (TI.3), 4 ng/ml TGFß1 + 1 µM IOX2(TI1). TGFß1 = Transforming growth factor beta 1. Alk5 = ALK5 kinase inhibitor (ALK5 = TGFß type I receptor kinase). IOX2 = a selective Inhibitor of the Hypoxia Inducible Factor (HIF) Prolyl-Hydroxylases.

引言：当前用于纤维化研究的临床前模型仅能部分模拟人类疾病进程。具体而言，传统转化生长因子β1（transforming growth factor beta 1, TGF-β1）诱导的星状细胞模型无法激活缺氧诱导通路，而该通路在肝病患者中具有重要相关性。本研究探讨了两种缺氧模拟化合物（DMOG与IOX2）联合TGF-β1对原代人肝星状细胞（hepatic stellate cells, HSCs）的纤维化表型及体内疾病特征体外再现能力的协同效应。

方法：培养原代人HSCs，采用TGF-β1与缺氧模拟化合物的组合进行刺激并收集细胞，以筛选可实现最优纤维化表型、细胞活力与毒性水平的刺激条件。为评估纤维化表型，我们检测了纤维化标志物（胶原蛋白、金属蛋白酶组织抑制剂1（tissue inhibitor of metalloproteinases 1, TIMP-1）、纤连蛋白（fibronectin））的蛋白水平。提取RNA并通过下一代测序（next generation sequencing, NGS）进行测序。分别使用差异表达分析工具Deseq2（differential expression analysis）与Ingenuity通路分析（Ingenuity Pathway Analysis, IPA）鉴定差异表达基因（differentially expressed genes, DEGs）并将其映射至生物学功能通路。

结果：本研究构建的HSC模型显示，在TGF-β1存在的条件下，使用0.3 μM与1 μM的IOX2进行刺激可显著提升纤维化标志物（总胶原蛋白、TIMP-1及纤连蛋白）的表达水平。低浓度且无毒性的DMOG未产生明显效果。当以浓度依赖方式添加IOX2时，差异表达基因的生物学功能分析凸显了多种纤维化相关通路、缺氧相关基因及相关交联标志物。此外，与人类纤维化与非纤维化组织的差异表达基因进行对比分析后发现，添加IOX2的HSC模型可更好地再现疾病特征。

结论：上述研究结果表明，IOX2可加重纤维化疾病进程，并提升体外模型的体内疾病特征再现能力。诸如IOX2这类缺氧模拟化合物有望增强体外纤维化模型的构建效果，为阐明纤维化发病机制、缺氧相关基因及潜在治疗策略提供有价值的研究思路。

整体实验设计：培养原代人肝星状细胞（HSCs），分别设置8种化合物组合刺激处理组：无添加对照组（C）、4 ng/ml TGF-β1组（T）、4 ng/ml TGF-β1 + 5 μM ALK5抑制剂组（TA，ALK5即转化生长因子βⅠ型受体激酶）、4 ng/ml TGF-β1 + 0.3 μM IOX2 +5 μM ALK5组（TI.3A）、0.3 μM IOX2组（I.3）、1 μM IOX2组（I1）、4 ng/ml TGF-β1 +0.3 μM IOX2组（TI.3）、4 ng/ml TGF-β1 +1 μM IOX2组（TI1）。

补充说明：TGF-β1即转化生长因子β1；ALK5为ALK5激酶抑制剂（ALK5即转化生长因子βⅠ型受体激酶）；IOX2为缺氧诱导因子（hypoxia inducible factor, HIF）脯氨酰羟化酶的选择性抑制剂。