通过分析单细胞测序数据(single-cell RNA sequencing, scRNA-seq)，构建急性肺损伤(acute lung injury, ALI)中肺微血管内皮细胞(pulmonary microvascular endothelial cells, PMVECs)的单细胞动态变化图谱，解析损伤进程中细胞异质性及通讯特征。

从基因表达综合数据库(gene expression omnibus, GEO)获取单细胞RNA测序数据集(GSE148499)。利用Seurat软件包进行质量控制、降维聚类和细胞注释。通过基因本体(gene ontology, GO)和京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes, KEGG)进行差异基因富集分析。采用Monocle3包和CellChat包分别进行拟时序轨迹推断和细胞间通讯分析。利用GSE207651数据集及体外细胞实验进行验证。

PMVECs共鉴定出5种功能亚群，其中先天免疫-气细胞型微血管内皮细胞(innate immune aerocytes, IIAerocytes)和增殖型微血管内皮细胞(proliferative capillary, PCap)是关键调控亚群。ALI早期(6 h~24 h)，高表达Car4、Prx、Sox9和Ptgse的IIAerocytes迅速扩增，第1天达峰值(细胞占比13.4%)。该亚群共鉴定出338个差异基因(adj. P<0.05, |log2FC|>1)，显著富集于先天免疫激活与病原体应答通路，功能上可通过ESAM信号增强内皮间连接，调控炎症反应与血管通透性。ALI修复期(3 d)，高表达Gpihbp1、Tm4sf1、Cdk1、E2f1和Cdk4的PCap扩增至细胞占比32.7%。该亚群共鉴定出441个差异表达基因(adj. P<0.05, |log2FC|>1)，显著富集于细胞周期与DNA修复。拟时序分析显示IIAerocytes源自Aerocytes的分化轨迹，PCap起源于GCap。脓毒症模型中同样鉴定出类似IIAerocytes亚群(高表达Car4、Prx、Ltbp2及Higd1b)，功能集中于趋化因子信号、白细胞迁移调控及中性粒细胞募集。体外脂多糖刺激实验中，人PMVECs的Cdk4蛋白表达在刺激后第1天达峰值(P<0.0001)，E2f1在第2天达峰值(P<0.0001)。细胞免疫荧光染色结果与蛋白印迹分析趋势高度一致，Cdk4和E2f1细胞内荧光强度分别于刺激后第1天、第2天达到峰值(P<0.0001)，与PCap增殖标志物的时序表达特征相符。

基于scRNA-seq揭示了ALI下PMVECs功能特征由促炎向促血管生成转换，为ALI病理机制解析及靶向干预提供新见解。

To construct a single-cell dynamic atlas of pulmonary microvascular endothelial cells (PMVECs) in acute lung injury (ALI) by analyzing single-cell RNA sequencing (scRNA-seq) data, and to decipher cellular heterogeneity and communication characteristics during the injury progression.

The single-cell RNA sequencing dataset (GSE148499) was retrieved from the Gene Expression Omnibus (GEO) database. Quality control, dimensionality reduction, clustering, and cell annotation were performed using the Seurat package. Differential gene enrichment analysis was conducted through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Pseudotemporal trajectory inference and intercellular communication analysis were carried out using the Monocle3 and CellChat packages, respectively. Validation was performed using the GSE207651 dataset and in vitro cellular experiments.

PMVECs were classified into five functional subpopulations, among which innate immune aerocytes (IIAerocytes) and proliferative capillary(PCap) were identified as key regulatory subpopulations. During the early phase of ALI (6 h~24 h), IIAerocytes highly expressing Car4, Prx, Sox9, and Ptgse rapidly expanded, reaching peak abundance on day 1 (13.4% of total cells). This subpopulation exhibited 338 differentially expressed genes (adj. P<0.05, |log2FC|>1), significantly enriched in pathways associated with innate immune activation and pathogen response. Functionally, IIAerocytes may enhance endothelial junctions through ESAM signaling, thereby regulating inflammatory responses and vascular permeability. Entering the repair phase of ALI (3 d), PCap highly expressing Gpihbp1, Tm4sf1, Cdk1, E2f1, and Cdk4 dramatically expanded to 32.7% of total cells. This subpopulation exhibited 441 differentially expressed genes (adj. P<0.05, |log2FC|>1), significantly enriched in cell cycle and DNA repair pathways. Pseudotime analysis revealed that IIAerocytes originated from the differentiation trajectory of Aerocytes, whereas PCap derived from GCap. In a sepsis model, a similar IIAerocyte subpopulation (highly expressing Car4, Prx, Ltbp2, and Higd1b) was also identified; however, its functions were more concentrated on chemokine signaling, leukocyte migration regulation, and neutrophil recruitment. In vitro LPS stimulation experiments demonstrated that Cdk4 protein expression in human PMVECs peaked on day 1 post-stimulation (P<0.0001), while E2f1 peaked on day 2 (P<0.0001). Immunofluorescence staining results were highly consistent with Western blot analysis trends, with intracellular fluorescence intensities of Cdk4 and E2f1 reaching peaks on day 1 and day 2 post-stimulation, respectively (P<0.0001), consistent with the temporal expression characteristics of PCap proliferative markers.

Based on scRNA-seq, this study reveals that PMVECs undergo a functional transition from pro-inflammatory to pro-angiogenic phenotypes during ALI, providing novel insights into the pathological mechanisms and targeted therapeutic interventions for ALI.