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.