FOSL2 binding to enhancer elements regulates endothelial cell state in systemic sclerosis pulmonary vascular disease

Systemic Sclerosis (SSc) is characterized by fibrosis and vasculopathy affecting the skin and internal organs, leading to multi-organ dysfunction. Injury of microvascular ECs in SSc impairs blood flow and causes tissue ischemia, leading to vascular complications such as Raynaud's, digital ulcers, and pulmonary hypertension (PH). PH in SSc presents as group 1 pulmonary arterial hypertension (PAH) or as group 3 PH related to hypoxia and interstitial lung disease (ILD), both major causes of mortality. Analysis of multiome data from SSc ILD-PH lungs inferred transcription factors regulating EC phenotype, including FOSL2. Overexpression of FOSL2 in transgenic mice (Fosl2tg) leads to vascular changes mirroring human SSc-PH, such as intimal thickening and fibrosis. ScRNA-seq analysis of altered EC gene expression in the Fosl2tg mice showed strong overlap with altered EC gene in SSc-ILD-PH. Overlapping as well as discrete EC gene expression in SuGene/hxpoxia- and hypoxia-treated mice suggested that FOSL2 regulates both hypoxia-dependent and independent pathways in Fosl2tg mice and SSc-ILD-PH. The deep learning model, ChromBPNet, inferred increased AP-1 binding at base pair resolution in SSc-ILDPH ECs and binding to the same motifs was found upon FOSL2 overexpression in primary vascular ECs, highlighting FOSL2's key role in driving the pathological changes seen in SSc-ILDPH. Overall design: 12 mouse lung samples were run using 10X Genomics FLEX assay. Conditions include 'WT_2', 'FRA2_1','FRA2_2','RAGko','FRA_RAGko1','FRA_RAGko2','WT_3','hypPVP','hypT5224','hypSugenePVP1','hypSugenePVP2', and 'hypSugeneT5224'

系统性硬化症（Systemic Sclerosis, SSc）以累及皮肤与内脏器官的纤维化及血管病变为特征，可引发多器官功能障碍。硬皮病患者的微血管内皮细胞（endothelial cells, ECs）损伤会损害血流并诱发组织缺血，进而导致雷诺现象、指端溃疡及肺动脉高压（pulmonary hypertension, PH）等血管并发症。硬皮病相关PH可表现为1型肺动脉高压（pulmonary arterial hypertension, PAH），或与缺氧及间质性肺疾病（interstitial lung disease, ILD）相关的3型PH，二者均为硬皮病的主要致死原因。对硬皮病合并ILD-PH患者肺部的多组学（multiome）数据进行分析后，鉴定出调控内皮细胞表型的转录因子，其中包括FOSL2。在转基因小鼠（Fosl2tg）中过表达FOSL2，可引发与人类硬皮病-PH相似的血管病变，包括内膜增厚与纤维化。对Fosl2tg小鼠内皮细胞的差异表达基因进行单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）分析，结果显示其与硬皮病-ILD-PH患者内皮细胞的差异表达基因存在高度重合。对SuGene/缺氧处理及缺氧处理小鼠的内皮细胞基因表达分析显示，其既有重合的差异表达基因，也存在特异性表达的基因，这表明FOSL2在Fosl2tg小鼠及硬皮病-ILD-PH中，可同时调控缺氧依赖与非缺氧依赖的通路。深度学习模型ChromBPNet在碱基分辨率下鉴定出，硬皮病-ILD-PH患者的内皮细胞中AP-1结合位点显著增加；而在原代血管内皮细胞中过表达FOSL2后，也可检测到相同基序的结合，这进一步凸显了FOSL2在介导硬皮病-ILD-PH病理变化中的核心作用。实验整体设计：采用10X Genomics FLEX检测技术对12份小鼠肺部样本进行测序，实验分组包括：'WT_2'、'FRA2_1'、'FRA2_2'、'RAGko'、'FRA_RAGko1'、'FRA_RAGko2'、'WT_3'、'hypPVP'、'hypT5224'、'hypSugenePVP1'、'hypSugenePVP2'以及'hypSugeneT5224'