Gene expression profile at single cell level of human pulmonary arteries (PA) of 2 healthy and 3 pulmonary hypertension associated with pulmonary fibrosis (PFPH) affected lungs

Dysfunction of pulmonary arterial endothelial cells (PAECs) is associated with the development and progression of vascular pathology. However, it remains unknown how pulmonary hypertension (PH) affects cellular composition and transcriptomic profile of pulmonary endothelium. Here, we have undertaken a single-cell, compartment specific approach to characterise alterations in PAECs associated with two different types of PH, i.e., pulmonary arterial hypertension (PAH) and pulmonary hypertension associated with pulmonary fibrosis (PHPF). Our unbiased analysis showed that endothelium of medium / small caliber pulmonary arteries is composed of three subsets of endothelial cells (ECs). The analysis of healthy and PH endothelium revealed that the three populations are persistently represented in remodelled arteries. Additionally, an exploratory analysis of human aorta (AO) and coronary arteries (CA) endothelium revealed that, although similar gene expression patterns were noticeable, PAECs subpopulations proportions differs significantly from pulmonary arteries (PA) endothelium. To address whether EC heterogeneity is a prime feature of human endothelium, we also performed a similar analysis in a murine model of hypoxia, revealing that similar EC populations were evident in this animal model. Comparative analysis of EC subpopulations in healthy and PH EC identified a common genetic deregulation accompanying vascular remodelling. Even though murine EC displayed some similarities with human EC subpopulations, the intense re-programming associated with hypoxia associated vascular remodelling displayed significant differences compared to the human disease. Finally, in depth comparative analysis of PAH and PHPF EC highlighted the development of disease-specific transcriptomic alterations in the three populations. Therefore, characterisation of transcriptomic differences in the endothelial bed of PAH and PHPF patients can facilitate identification of novel, disease-specific therapeutic targets. Using droplet-based single cell RNA sequencing we analyzed 3 pulmonary arteries (PA) from lungs affected with pulmonary hypertension associated with pulmonary fibrosis(PHPF_1, PHPF_2, PHPF_3) against PA from 2 healthy controls (Donor_4, Donor_5). Clustering, dimensionality reduction, trajectory inference and pseudotime, as well as transcription factor analyses were performed and select marker gene were examined for specific segregation of PA media layer using immunofluorescence in both the conditions.

肺动脉内皮细胞（pulmonary arterial endothelial cells, PAECs）功能异常与血管病变的发生及进展密切相关。然而目前尚不明确肺动脉高压（pulmonary hypertension, PH）如何影响肺内皮细胞的细胞组成与转录组谱。本研究采用单细胞、分区特异性分析方法，对两种不同类型肺动脉高压（即肺动脉高压（pulmonary arterial hypertension, PAH）与肺纤维化相关性肺动脉高压（pulmonary hypertension associated with pulmonary fibrosis, PHPF））相关的PAECs改变进行了表征。本研究的无偏分析显示，中/小口径肺动脉的内皮细胞由三类内皮细胞（endothelial cells, ECs）亚群构成。对健康肺内皮与PH肺内皮的分析表明，这三类细胞亚群在发生血管重构的动脉中持续存在。此外，对人主动脉（aorta, AO）与冠状动脉（coronary arteries, CA）内皮细胞的探索性分析显示，尽管二者存在相似的基因表达模式，但PAECs亚群的比例与肺动脉（pulmonary arteries, PA）内皮细胞存在显著差异。为探究内皮细胞异质性是否为人类肺内皮细胞的核心特征，本研究还在缺氧小鼠模型中开展了类似分析，结果显示该动物模型中同样存在相似的EC亚群。对健康与PH状态下的EC亚群进行比较分析后，本研究发现伴随血管重构存在一套共通的基因表达失调模式。尽管小鼠EC亚群与人类EC亚群存在部分相似性，但缺氧相关性血管重构所伴随的强烈细胞重编程过程，与人类疾病中的对应过程存在显著差异。最后，对PAH与PHPF患者的EC亚群进行深入比较分析后，本研究揭示了三类EC亚群中均存在疾病特异性的转录组改变。因此，对PAH与PHPF患者内皮细胞群体的转录组差异进行表征，将有助于识别新型疾病特异性治疗靶点。本研究采用基于液滴的单细胞RNA测序技术，对3例肺纤维化相关性肺动脉高压（PHPF_1、PHPF_2、PHPF_3）患者肺部的肺动脉，与2例健康对照者（Donor_4、Donor_5）的肺动脉进行了分析。本研究开展了细胞聚类、降维、轨迹推断与拟时间分析，以及转录因子分析，并通过免疫荧光染色在两种实验条件下检测了筛选出的标记基因（marker gene），以验证肺动脉中膜层的特异性分群情况。