Abstract Body (Do not enter title and authors here): Background: Understanding the intricate dynamics of lung microcirculation and capillary endothelial cells (ECs) is crucial for shedding light on both normal physiological processes and the pathogenesis of pulmonary diseases. Recent advancements in single-cell transcriptomics (scRNA-seq) have unveiled distinct lung EC populations, including aerocytes and general capillary cells (gCaps).
Hypothesis: We hypothesized that gCaps demonstrate more extensive diversity than previously understood, marked by distinct molecular and functional profiles.
Methods: We utilized scRNA-seq and computational analysis on enriched populations of lung ECs. The mitostress Seahorse assay was conducted to study metabolic function. Electric cell-substrate impedance sensing (ECIS) assessed cell growth, and the Matrigel assay evaluated angiogenic potential of isolated ECs.
Results: scRNA-seq analysis identified five novel gCap populations, each with unique molecular signatures and functions. Notably, two populations characterized by the expression of ion transporters Scn7a (Na+) and Clic4 (Cl−) are crucial for establishing arterial-to-vein zonation and enhancing the capillary barrier. We identified "root" cells (Flot1+), exhibiting the highest G2M and S scores, essential for the mitotic regeneration and repair of neighboring ECs. Depletion of Flot1+ cells significantly impaired EC proliferation, highlighting their critical role in cell repair. Furthermore, we discovered that gCaps aiding in the venous transition were characterized by the expression of Lingo2. Additionally, we identified a population of detached gCaps marked by an elevated expression of Fabp4, metabolically active genes, and tip-cell markers, suggesting a key role in regulating angiogenesis. The Matrigel sprouting assay confirmed that Fabp4+ gCaps significantly contribute to angiogenesis. Metabolic assays indicated that both Clic4+ and Fabp4+ cells exhibited increased mitochondrial function. Finally, western blot analysis across various EC junction and signaling proteins in isolated gCaps showed significant heterogeneity, further illustrating the complex nature of these cell populations.
Conclusion: Our study highlights the diverse roles and molecular signatures of lung capillary ECs, enhancing our understanding of their contributions to both normal lung function and pulmonary diseases. These insights also offer valuable direction for developing therapeutic strategies aimed at addressing cardiovascular disorders.