Single cell RNA-seq profiling of murine endothelial cells in response to pulmonary hypertension

(1) Rationale: Endothelial cell dysfunction plays a critical role in the development and pathogenesis of pulmonary arterial hypertension (PAH). (2) Objectives: We aimed to characterise the endothelial cell dynamics in PAH at a single cell resolution. (3) Methods: We carried out single-cell RNA sequencing of lung endothelial cells isolated from an endothelial cell lineage tracing mouse model in control and SU5416/Hypoxia-induced PAH conditions. (4) Measurements and Main Results: Endothelial cell populations corresponding to the different lung vessel types could be identified in both control and PAH mice. Differential gene expression analysis revealed novel global and vessel-type specific responses in endothelial cells due to PAH. Global changes included the up-regulation of the major histocompatibility complex class II pathway, supporting a role for endothelial cells in the inflammatory response in PAH. We also identified a PAH response specific to the second capillary EC population, with the up-regulation of genes involved in cell localization and angiogenesis. Comparison with human genetics and transcriptomics data revealed the regulation of four genes with variants associated to PAH and five genes up-regulated in endothelial cells in both human and mouse scRNA-seq. Among them, Aqp1 and Adam15 genes represent promising new candidates to target endothelial dysfunction. Finally, we identified zonation-dependent changes across the arteriovenous axis in PAH using an in-silico cell ordering approach and showed the up-regulation of the Serine/threonine-protein kinase Sgk1 at the junction between the macro- and micro-vasculature. (5) Conclusions: This study uncovers the murine endothelial cell transcriptomics changes in PAH at a high resolution, revealing novel candidates relevant to PAH. (1) Experiment1: To study ECs in murine lungs, we used a Cdh5-CreERT2-TdTomato mouse line, in which the EC-specific (Cdh5) expression of TdTomato is inducible with tamoxifen. After a two week wash out period, TdTomato-positive cells were obtained from the lung of two mice using flow cytometry and analysed using 10X Genomics scRNA-seq (ContA/ContB). (2) Experiment2: To examine EC changes due to pulmonary arterial hypertension (PAH), we used the SuHx model, exposing the Cdh5-CreERT2-TdTomato mice to chronic hypoxia for three weeks in combination with weekly subcutaneous injections of SU5416. ScRNA-seq was carried out for three SuHx mice (PAH1, PAH2, PAH3) and one control mouse that was maintained in normoxia (Cont1).

(1) 研究依据：内皮细胞功能异常在肺动脉高压（pulmonary arterial hypertension, PAH）的发生与发病机制中发挥关键作用。
(2) 研究目的：本研究旨在以单细胞分辨率解析肺动脉高压（PAH）中的内皮细胞动态变化。
(3) 研究方法：本研究对来自内皮细胞谱系示踪小鼠模型的肺内皮细胞进行了单细胞RNA测序（single-cell RNA sequencing），该模型分别设置了对照组与SU5416/低氧诱导的PAH模型组。
(4) 测量与主要结果：在对照组与PAH模型小鼠中，均可识别出对应不同肺血管类型的内皮细胞群体。差异基因表达分析揭示了PAH导致内皮细胞出现全新的全局及血管类型特异性应答反应。全局变化主要包括主要组织相容性复合体Ⅱ类通路的上调，这支持了内皮细胞在PAH炎症反应中发挥作用的观点。本研究还鉴定出仅在第二毛细血管内皮细胞（endothelial cell, EC）群体中出现的PAH特异性应答，涉及细胞定位与血管生成相关基因的上调。通过与人类遗传学及转录组学数据进行比对，发现有4个携带PAH相关变异的基因以及5个在人类与小鼠单细胞RNA测序的内皮细胞中均出现上调的基因。其中，Aqp1与Adam15基因是极具潜力的内皮细胞功能异常治疗新候选靶点。最后，本研究通过计算机细胞排序分析（in-silico cell ordering）方法，鉴定出PAH中动静脉轴的分区依赖性变化，并证实丝氨酸/苏氨酸蛋白激酶Sgk1（Serine/threonine-protein kinase Sgk1）在大血管与微血管的交界处出现上调。
(5) 研究结论：本研究以高分辨率解析了PAH模型小鼠的肺内皮细胞转录组变化，揭示了与PAH相关的全新候选靶点。
(1) 实验1：为研究小鼠肺组织中的内皮细胞（endothelial cell, EC），本研究使用了Cdh5-CreERT2-TdTomato小鼠品系，该品系中内皮细胞特异性（Cdh5）的TdTomato表达可通过他莫昔芬（tamoxifen）诱导。经过两周的洗脱期后，本研究通过流式细胞术（flow cytometry）从2只小鼠的肺组织中分离得到TdTomato阳性细胞，并采用10X Genomics平台进行单细胞RNA测序（scRNA-seq，ContA/ContB）。
(2) 实验2：为探究肺动脉高压（PAH）导致的内皮细胞变化，本研究采用SuHx模型，将Cdh5-CreERT2-TdTomato小鼠暴露于慢性低氧（chronic hypoxia）环境三周，并每周皮下注射SU5416。本研究对3只SuHx模型小鼠（PAH1、PAH2、PAH3）以及1只维持于常氧环境（normoxia）的对照小鼠（Cont1）进行了单细胞RNA测序（scRNA-seq）。