Hepatic stellate cells (HSCs) are the key drivers of fibrosis in metabolic dysfunction-associated steatohepatitis (MASH), the fastest growing cause of hepatocellular carcinoma (HCC) worldwide. HSCs are heterogenous, and a senescent subset of HSCs is implicated in hepatic fibrosis and HCC. Administration of anti-uPAR (urokinase-type plasminogen activator receptor) CAR T cells has been shown to deplete senescent HSCs and attenuate fibrosis in murine models. However, the comprehensive features of senescent HSCs in MASH, as well as their cellular ontogeny have not been characterized; hence, we aimed to comprehensively characterize and define the origin of HSCs in human and murine MASH. To comprehensively characterize the phenotype and ontogeny of senescent HSCs in human and murine MASH, we integrated senescence-associated beta galactosidase activity with immunostaining, flow cytometry and single-nucleus RNA sequencing (snRNAseq). We integrated the immunohistochemical profile with a senescence score applied to snRNAseq data to characterize senescent HSCs and mapped the evolution of uPAR expression in MASH. Using pseudotime trajectory analysis, we establish that senescent HSCs arise from activated HSCs. While uPAR is expressed in MASH, the magnitude and cell-specificity of its expression evolve with disease stage. In early disease, uPAR is more specific to activated and senescent HSCs, while it is also expressed by myeloid-lineage cells, including Trem2 macrophages and myeloid-derived suppressor cells, in late disease. Furthermore, we identify novel surface proteins expressed on senescent HSCs in human and murine MASH that could be exploited as therapeutic targets. These data define features of HSC senescence in human and murine MASH, establishing an important blueprint to target these cells as part of future antifibrotic therapies. Hepatic stellate cells (HSCs) are the primary drivers of scarring in chronic liver diseases. As injury develops, a subset of HSCs become senescent; these cells are non-proliferative and pro-inflammatory, thereby contributing to worsening liver injury. Here we show that senescent HSCs are expanded in MASH (metabolic dysfunction-associated steatohepatitis) in humans and mice, and we trace their cellular origin from the activated HSC subset. We further characterize expression of uPAR (urokinase plasminogen activated receptor), a protein that marks senescent HSCs, and report that uPAR is also expressed by activated HSCs in early injury, and in immune cells as liver injury advances. We have integrated high-resolution single-nucleus RNA sequencing with immunostaining and flow cytometry to identify five other novel proteins expressed by senescent HSCs, including mannose receptor CD206, which will facilitate future therapeutic development.

中文翻译：

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代谢功能障碍相关脂肪性肝炎中衰老肝星状细胞的表型和个体发育


肝星状细胞 (HSC) 是代谢功能障碍相关脂肪性肝炎 (MASH) 纤维化的关键驱动因素，而脂肪性肝炎是全球肝细胞癌 (HCC) 增长最快的原因。 HSC 是异质的，HSC 的衰老亚群与肝纤维化和 HCC 有关。在小鼠模型中，施用抗 uPAR（尿激酶型纤溶酶原激活剂受体）CAR T 细胞已被证明可以消耗衰老的 HSC 并减轻纤维化。然而，MASH 中衰老 HSC 的综合特征及其细胞个体发育尚未得到表征；因此，我们的目标是全面表征和定义人类和小鼠 MASH 中 HSC 的起源。为了全面表征人和小鼠 MASH 中衰老 HSC 的表型和个体发育，我们将衰老相关的 β 半乳糖苷酶活性与免疫染色、流式细胞术和单核 RNA 测序 (snRNAseq) 相结合。我们将免疫组织化学谱与应用于 snRNAseq 数据的衰老评分相结合，以表征衰老的 HSC，并绘制 MASH 中 uPAR 表达的演变图。通过伪时间轨迹分析，我们确定衰老的 HSC 是由激活的 HSC 产生的。虽然 uPAR 在 MASH 中表达，但其表达的程度和细胞特异性随着疾病阶段而变化。在疾病早期，uPAR 对活化和衰老的 HSC 更具特异性，而在疾病晚期，它也由髓系细胞表达，包括 Trem2 巨噬细胞和髓源性抑制细胞。此外，我们还发现了人类和小鼠 MASH 中衰老 HSC 上表达的新型表面蛋白，可将其用作治疗靶点。 这些数据定义了人类和小鼠 MASH 中 HSC 衰老的特征，为靶向这些细胞作为未来抗纤维化治疗的一部分建立了重要的蓝图。肝星状细胞（HSC）是慢性肝病疤痕形成的主要驱动因素。随着损伤的发展，HSC 的一部分会衰老；这些细胞具有非增殖性和促炎性，从而导致肝损伤恶化。在这里，我们展示了衰老的 HSC 在人和小鼠的 MASH（代谢功能障碍相关脂肪性肝炎）中扩增，并且我们从激活的 HSC 亚群中追踪它们的细胞起源。我们进一步表征了 uPAR（尿激酶纤溶酶原激活受体）的表达，uPAR 是一种标记衰老 HSC 的蛋白质，并报告说，在早期损伤中，激活的 HSC 也表达 uPAR，并且随着肝损伤进展，在免疫细胞中也会表达 uPAR。我们将高分辨率单核 RNA 测序与免疫染色和流式细胞术相结合，鉴定了衰老 HSC 表达的其他五种新蛋白，包括甘露糖受体 CD206，这将有助于未来的治疗开发。