背景:肝星状细胞(HSC)是代谢功能障碍相关脂肪变性肝病(MASLD)纤维化的关键驱动因素,全球肝细胞癌增长最快的原因。HSC是异质性的,HSC的衰老亚群与肝纤维化和HCC有关。施用抗uPAR(尿激酶型纤溶酶原激活物受体)CART细胞消耗衰老的HSC并减轻小鼠肝损伤模型中的纤维化,包括MASLD。然而,MASLD中衰老HSC的综合特征,以及它们的细胞个体发育尚未被表征。
目的:我们的目的是通过整合衰老相关的β-半乳糖苷酶活性和免疫染色来全面表征和定义人和鼠MASLD中衰老HSC的起源,流式细胞术和单核RNA测序(snRNAseq)。我们将免疫组织化学谱与应用于snRNAseq数据的衰老评分进行整合,以表征衰老HSC,并绘制了uPAR在MASLD中表达的演变图。
结果:使用伪时间轨迹分析,我们确定衰老的HSC来自活化的HSC。虽然uPAR在MASLD中表达,其表达的大小和细胞特异性随疾病阶段而演变,在疾病早期,uPAR对活化和衰老的HSCs更具特异性,在晚期疾病中,uPAR也由骨髓谱系细胞表达,包括Trem2+巨噬细胞和骨髓来源的抑制细胞。此外,我们鉴定了在人和鼠MASLD中的衰老HSC上表达的新型表面蛋白,这些蛋白可以用作治疗靶标。
结论:这些数据定义了人和鼠MASLD中HSC衰老的特征,建立一个重要的蓝图,靶向这些细胞作为未来抗纤维化治疗的一部分。
背景:肝星状细胞(HSC)是慢性肝脏疾病中瘢痕形成的主要驱动因素。随着损伤的发展,一组HSC衰老;这些细胞是非增殖和促炎的,从而导致肝损伤恶化。在这里,我们表明,衰老的HSC在人类和小鼠的代谢功能障碍相关的脂肪变性肝病(MASLD)中扩增,我们从激活的HSC子集追踪它们的细胞起源。我们进一步表征uPAR(尿激酶纤溶酶原激活受体)的表达,一种标记衰老HSC的蛋白质,并报道uPAR在早期损伤中也由活化的HSC表达,和免疫细胞随着肝损伤的发展。我们已经将高分辨率单核测序与免疫染色和流式细胞术结合起来,以鉴定衰老HSC表达的其他五种新蛋白。包括甘露糖受体CD206,这将有助于未来努力清除衰老的HSC以治疗纤维化。
OBJECTIVE: 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.
METHODS: 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.
RESULTS: 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.
CONCLUSIONS: 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.
UNASSIGNED: 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.