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

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. 4 Maf-knockout samples vs. 4 control samples肝窦内皮细胞（Liver sinusoidal endothelial cells, LSEC）在肝血管微生态位中具有高度特化的功能，通过血管旁信号传导调控肝脏功能与疾病发病机制。本研究团队此前鉴定出GATA4是调控LSEC发育、抵御肝纤维化的关键转录因子。鉴于在Gata4缺陷型LSEC中，转录因子c-Maf的表达显著下调，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Maf敲除的Clec4g-iCre/Maf fl/fl小鼠（命名为Maf-LSEC-KO小鼠），并在小鼠3月龄时，通过组织学、免疫荧光及原位杂交技术对其肝组织进行分析；同时分离LSEC，开展RNA测序、转座酶可及性染色质测序（ATAC-seq）及单细胞RNA测序（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。研究发现，内皮细胞Maf敲除可引发窦周肝纤维化，且不影响肝脏代谢分区，同时伴随内皮细胞分化从窦状型向连续型的转变。此外，内皮细胞Maf敲除可导致肝内皮细胞增殖，并上调促纤维化血管旁信号因子的表达，包括Pdgfb、Igfbp5、Sparcl-1及Flrt2。scRNA-seq结果显示，原本具有分区特异性的LSEC亚群被促肝纤维化与血管生成的毛细血管化、增殖性、出芽型及分泌型内皮细胞亚群所替代。LSEC基因表达与分化的这一根本性失调，是由于Maf缺失后染色质开放状态发生改变，且启动子与增强子区域的转录因子网络出现重构所导致的。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可通过调控染色质开放状态，抵御代谢功能障碍相关脂肪性肝炎样肝纤维化，并调控内皮细胞分化与肝脏分区。本研究结果为开发靶向血管的肝脏疾病预防与肝修复策略提供了新的研究方向。本次研究共纳入4例Maf敲除样本与4例对照样本。