Aims Endothelial cell dysfunction drives the initiation and pathogenesis of pulmonary arterial hypertension (PAH). We aimed to characterise endothelial cell (EC) dynamics in PAH at single-cell resolution. Methods and Results We carried out single-cell RNA sequencing (scRNA-seq) of lung ECs isolated from an EC lineage-tracing mouse model in Control and SU5416/Hypoxia-induced PAH conditions. EC populations corresponding to distinct lung vessel types, including two discrete capillary populations, were identified in both Control and PAH mice. Differential gene expression analysis revealed global PAH-induced EC changes that were confirmed by bulk RNA-seq. This included upregulation of the major histocompatibility complex class II pathway, supporting a role for ECs in the inflammatory response in PAH. We also identified a PAH response specific to the second capillary EC population including upregulation of genes involved in cell death, cell motility and angiogenesis. Interestingly, four genes with genetic variants associated with PAH were dysregulated in mouse ECs in PAH. To compare relevance across PAH models and species, we performed a detailed analysis of EC heterogeneity and response to PAH in rats and humans through whole-lung PAH scRNA-seq datasets, revealing that 51% of up-regulated mouse genes were also up-regulated in rat or human PAH. We identified promising new candidates to target endothelial dysfunction including CD74, the knockdown of which regulates EC proliferation and barrier integrity in vitro. Finally, with an in silico cell ordering approach, we identified zonation-dependent changes across the arteriovenous axis in mouse PAH and showed upregulation of the Serine/threonine-protein kinase Sgk1 at the junction between the macro- and micro-vasculature. Conclusions This study uncovers PAH-induced EC transcriptomic changes at a high resolution, revealing novel targets for potential therapeutic candidate development.

中文翻译：

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小鼠内皮细胞响应肺动脉高压的单细胞 RNA-seq 分析

目的 内皮细胞功能障碍导致肺动脉高压 (PAH) 的发生和发病。我们的目的是在单细胞分辨率下表征 PAH 中的内皮细胞 (EC) 动力学。方法和结果我们对在对照和 SU5416/缺氧诱导的 PAH 条件下从 EC 谱系追踪小鼠模型中分离出的肺 EC 进行了单细胞 RNA 测序 (scRNA-seq)。在对照小鼠和 PAH 小鼠中均鉴定出与不同肺血管类型相对应的 EC 群体，包括两个离散的毛细血管群体。差异基因表达分析揭示了 PAH 引起的整体 EC 变化，并通过批量 RNA 测序证实。这包括主要组织相容性复合物 II 类通路的上调，支持 EC 在 PAH 炎症反应中的作用。我们还确定了第二毛细血管 EC 群体特有的 PAH 反应，包括细胞死亡、细胞运动和血管生成相关基因的上调。有趣的是，PAH 小鼠 EC 中四个具有与 PAH 相关遗传变异的基因失调。为了比较 PAH 模型和物种之间的相关性，我们通过全肺 PAH scRNA-seq 数据集对大鼠和人类的 EC 异质性和对 PAH 的反应进行了详细分析，发现 51% 上调的小鼠基因也上调在大鼠或人类 PAH 中。我们发现了针对内皮功能障碍的有前景的新候选药物，包括 CD74，其敲低可在体外调节 EC 增殖和屏障完整性。最后，通过计算机细胞排序方法，我们确定了小鼠 PAH 中动静脉轴的分区依赖性变化，并显示大脉管系统和微脉管系统之间连接处的丝氨酸/苏氨酸蛋白激酶 Sgk1 上调。结论 这项研究以高分辨率揭示了 PAH 诱导的 EC 转录组变化，揭示了潜在候选治疗药物开发的新靶点。