Background and aims

Metabolic dysfunction-associated steatotic liver disease (MASLD) ranges from Metabolic dysfunction-associated steatotic liver (MASL) to Metabolic dysfunction-associated steatohepatitis (MASH) with fibrosis. Activation of Hepatic Stellate Cells (HSCs) into fibrogenic myofibroblasts plays a critical role in the pathogenesis of MASH liver fibrosis. We compared transcriptome and chromatin accessibility of human HSCs from NORMAL, MASL, and MASH livers at single cell resolution. We aimed to identify genes that are upregulated in activated HSCs and to determine which of these genes are key in the pathogenesis of MASH fibrosis.

Methods

18 human livers were profiled using single-nucleus (sn)RNA-seq and snATAC-seq. High priority targets were identified, then tested in 2D human HSC cultures, 3D human liver spheroids, and HSC-specific gene knockout mice.

Results

MASH-enriched activated (A) HSC subclusters are the major source of extracellular matrix proteins. We identified a set of concurrently upregulated and more accessible core genes (GAS7, SPON1, SERPINE1, LTBP2, KLF9, EFEMP1) that drive activation of (A) HSC subclusters. Expression of these genes was regulated via crosstalk between lineage-specific (JUNB/AP1), cluster-specific (RUNX1/2) and signal-specific (FOXA1/2) transcription factors. The pathological relevance of the selected targets, such as SERPINE1 (PAI-1), was demonstrated using dsiRNA-based HSC-specific gene knockdown or pharmacological inhibition of PAI-1 in 3D human MASH liver spheroids, and HSC-specific Serpine1 knockout mice.

Conclusion

This study identified novel gene targets and regulatory mechanisms underlying activation of MASH fibrogenic HSCs and demonstrated that genetic or pharmacological inhibition of select genes suppressed liver fibrosis.

Impact and implications

Here we present snRNA-seq and snATAC-seq analysis of human HSCs from NORMAL, MASL, and MASH livers. We identified additional subclusters that were not detected by previous studies and characterized the mechanism by which HSCs activate in the MASH livers, including the transcriptional machinery that activates HSCs into myofibroblasts. For the first time, we described the pathogenic role of activated HSC-derived PAI-1 (a product of SERPINE1 gene) in the development of MASH liver fibrosis. Targeting of RUNX1/2-SERPINE1 axis may provide a novel strategy for treatment of liver fibrosis in patients.

中文翻译：

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人肝星状细胞的多模式分析确定了代谢功能障碍相关脂肪性肝病的新治疗靶点

 背景和目标

代谢功能障碍相关脂肪性肝病 （MASLD） 的范围从代谢功能障碍相关脂肪性肝 （MASL） 到伴有纤维化的代谢功能障碍相关脂肪性肝炎 （MASH）。肝星状细胞 （HSC） 活化为纤维化肌成纤维细胞在 MASH 肝纤维化的发病机制中起关键作用。我们在单细胞分辨率下比较了来自 NORMAL 、 MASL 和 MASH 肝脏的人类 HSC 的转录组和染色质可及性。我们旨在鉴定在活化的 HSC 中上调的基因，并确定这些基因中哪些是 MASH 纤维化发病机制的关键。

 方法

使用单核 （sn）RNA-seq 和 snATAC-seq 分析 18 例人肝脏。确定了高优先级靶标，然后在 2D 人 HSC 培养物、3D 人肝球和 HSC 特异性基因敲除小鼠中进行了测试。

 结果

富含 MASH 的活化 （A） HSC 亚簇是细胞外基质蛋白的主要来源。我们确定了一组同时上调且更容易获得的核心基因 （GAS7 、 SPON1 、 SERPINE1 、 LTBP2 、 KLF9 、 EFEMP1 ），这些基因驱动 （A） HSC 亚集群的激活。这些基因的表达通过谱系特异性 （JUNB/AP1） 、簇特异性 （RUNX1/2） 和信号特异性 （FOXA1/2） 转录因子之间的串扰进行调节。使用基于 dsiRNA 的 HSC 特异性基因敲低或对 3D 人 MASH 肝球体和 HSC 特异性 Serpine1 敲除小鼠中 PAI-1 的药理学抑制来证明所选靶标的病理相关性，例如 SERPINE1 （PAI-1）。

 结论

本研究确定了 MASH 纤维化 HSC 激活的新基因靶点和调控机制，并证明选定基因的遗传或药理学抑制抑制了肝纤维化。

 影响和影响

在这里，我们介绍了来自 NORMAL、MASL 和 MASH 肝脏的人 HSC 的 snRNA-seq 和 snATAC-seq 分析。我们确定了先前研究未检测到的其他亚集群，并表征了 HSC 在 MASH 肝脏中激活的机制，包括将 HSC 激活成肌成纤维细胞的转录机制。我们首次描述了激活的 HSC 衍生的 PAI-1 （SERPINE1 基因的产物） 在 MASH 肝纤维化发展中的致病作用。靶向 RUNX1/2-SERPINE1 轴可能为治疗患者肝纤维化提供一种新的策略。