Endothelial c-Maf protects against liver fibrosis by regulating chromatin accessibility to suppress pathogenic microvascular subpopulations [scRNA-seq]

Liver sinusoidal endothelial cells (LSEC) are highly specialized within the hepatic vascular niche, controlling liver function and disease pathogenesis by angiocrine signaling. Recently, we identified GATA4 as a major transcription factor controlling LSEC development and protecting against liver fibrosis. As the transcription factor c-Maf was strongly downregulated in Gata4-deficient LSEC, we hypothesized that c-Maf might be an important downstream effector of GATA4 in LSEC differentiation and liver fibrogenesis. Clec4g-iCre/Maf fl/fl mice (Maf-LSEC-KO) mice with LSEC-specific Maf deficiency were generated and liver tissue was analyzed by histology, immunofluorescence, and in situ-hybridization at the age of three months. LSECs were isolated for RNA-, ATAC-seq, and single-cell RNA-seq (scRNA-seq) analysis. The expression of MAF and its targets were analyzed in published human scRNA-seq data. Endothelial Maf deficiency resulted in perisinusoidal liver fibrosis without affecting metabolic liver zonation, accompanied by a switch from sinusoidal to continuous endothelial differentiation. Furthermore, endothelial Maf deficiency caused hepatic endothelial proliferation and expression of profibrotic angiocrine factors such as Pdgfb, Igfbp5, Sparcl-1, and Flrt2. scRNA-seq revealed replacement of zonated LSEC subpopulations by capillarized, proliferative, sprouting and secretory endothelial cell subset promoting liver fibrogenesis and angiogenesis. This fundamental dysregulation of LSEC gene expression and differentiation was caused by changes in chromatin accessibility and transcription factor network alterations at promoter and enhancer regions following loss of Maf. Notably, endothelial MAF expression was also significantly reduced in human liver cirrhosis patients. Hepatic endothelial c-Maf protects against metabolic dysfunction-associated steatohepatitis-like liver fibrosis and regulates endothelial differentiation and zonation by controlling chromatin opening. Notably, our findings may open up new avenues to develop angiotargeted strategies for hepatic disease prevention and liver repair. scRNA-seq experiment was performed to compare LSECs transcriptomic in WT and MAF KO mice肝窦内皮细胞（Liver sinusoidal endothelial cells, LSEC）是肝血管微环境中高度特化的细胞类群，通过血管分泌信号（angiocrine signaling）调控肝脏功能与疾病发生发展。近期本团队鉴定出GATA4是调控LSEC发育、抵御肝纤维化的关键转录因子。鉴于转录因子c-Maf在Gata4缺陷型LSEC中显著下调，我们推测c-Maf可能是GATA4调控LSEC分化与肝纤维化发生的重要下游效应分子。我们构建了LSEC特异性敲除Maf的Clec4g-iCre/Maf fl/fl小鼠（简称Maf-LSEC-KO小鼠），并于小鼠3月龄时通过组织学、免疫荧光及原位杂交（in situ-hybridization）技术分析其肝组织样本；同时分离LSEC进行RNA测序、转座酶可及性测序（ATAC-seq）及单细胞RNA测序（single-cell RNA-seq, scRNA-seq）分析。我们还在已发表的人类scRNA-seq数据中分析了MAF及其靶基因的表达情况。实验结果显示，内皮细胞Maf缺失会导致肝窦周纤维化，且不影响肝脏代谢分区，同时伴随内皮细胞分化从窦状表型向连续表型的转换。此外，内皮细胞Maf缺失会引发肝内皮细胞增殖，并上调PDGFB、IGFBP5、SPARCL-1及FLRT2等促纤维化血管分泌因子的表达。scRNA-seq分析显示，原本呈分区特征的LSEC亚群被毛细血管化、增殖性、出芽性及分泌性内皮细胞亚群替代，后者可促进肝纤维化与血管生成。LSEC基因表达与分化的这种显著失调，是由于Maf缺失后染色质开放状态改变以及启动子、增强子区域的转录因子网络发生紊乱所导致。值得注意的是，肝硬化患者的肝内皮细胞中MAF的表达水平同样显著降低。肝内皮细胞c-Maf可抵御代谢功能障碍相关脂肪性肝炎样肝纤维化，并通过调控染色质开放状态维持内皮细胞分化与肝脏分区。本研究发现可为开发肝脏疾病防治与肝修复的血管靶向策略提供全新思路。本研究通过scRNA-seq实验对比了野生型与MAF敲除小鼠LSEC的转录组差异。