OBJECTIVE To quantify pulmonary vascular endothelial subpopulations during bleomycin-induced pulmonary fibrosis in mice.METHODS Sixty male C57BL/6 mice were randomly divided into five groups(n=12 per group)corresponding to distinct observation timepoints:0,1,2,3,and 4 weeks.A model was established via intratracheal instillation of bleomycin(3 mg·kg-1).Lung tissues were harvested at 0,1,2,3 and 4 weeks post-bleomycin induction.Pathological staining was performed to assess lung histoarchitecture and collagen fiber deposition.Single-cell suspensions were analyzed by flow cytometry to quantify temporal changes in pulmonary vascular endothelial subpopulations,including pulmonary macrovascular endothelial cells,general capillaries,and aerocyte capillaries.Immunofluo-rescence staining was performed to validate the expressions of endothelial markers(CD31,APLN,APLNR,CD93).Single-cell transcriptomic data from the Tabula Muris database was analyzed to evalu-ate gene expression profiles of vascular endothelial subpopulations.RESULTS Pathological staining revealed progressive destruction of lung histoarchitecture and collagen deposition during bleomycin-induced pulmonary fibrosis.Flow cytometry demonstrated three-phase dynamics in vascular endothelial cells(CD45-CD31+CD90.2-):a significant decrease during the acute inflammatory phase,stabilization in the fibrotic phase,and partial recovery during the resolution phase.The proportion of von Willebrand factor-positive(VWF+)vascular endothelial cells significantly decreased during the resolution phase,whereas VWF-vascular endothelial cells increased.Single-cell transcriptomics identified Cd93 asa specific gene for general capillary endothelial cells,with a negative correlation with"aerocyte"genes enriched in gas-exchange alveolar capillary endothelial cells.Immunofluorescence confirmed CD93 localization to general capillary endothelial cells.A flow sorting strategy based on CD45-CD31+CD90.2-VWF-CD93-effectively enriched alveolar capillary endothelial cells.This subpopulation trended upward in pulmonary vascular endothelial composition during bleomycin induction.CONCLUSION During bleo-mycin-induced pulmonary fibrosis in mice,pulmonary vascular endothelial subpopulations exhibit dynamic compositional heterogeneity across fibrotic injury and repair phases.

OBJECTIVE To quantify pulmonary vascular endothelial subpopulations during bleomycin-induced pulmonary fibrosis in mice.METHODS Sixty male C57BL/6 mice were randomly divided into five groups(n=12 per group)corresponding to distinct observation timepoints:0,1,2,3,and 4 weeks.A model was established via intratracheal instillation of bleomycin(3 mg·kg-1).Lung tissues were harvested at 0,1,2,3 and 4 weeks post-bleomycin induction.Pathological staining was performed to assess lung histoarchitecture and collagen fiber deposition.Single-cell suspensions were analyzed by flow cytometry to quantify temporal changes in pulmonary vascular endothelial subpopulations,including pulmonary macrovascular endothelial cells,general capillaries,and aerocyte capillaries.Immunofluo-rescence staining was performed to validate the expressions of endothelial markers(CD31,APLN,APLNR,CD93).Single-cell transcriptomic data from the Tabula Muris database was analyzed to evalu-ate gene expression profiles of vascular endothelial subpopulations.RESULTS Pathological staining revealed progressive destruction of lung histoarchitecture and collagen deposition during bleomycin-induced pulmonary fibrosis.Flow cytometry demonstrated three-phase dynamics in vascular endothelial cells(CD45-CD31+CD90.2-):a significant decrease during the acute inflammatory phase,stabilization in the fibrotic phase,and partial recovery during the resolution phase.The proportion of von Willebrand factor-positive(VWF+)vascular endothelial cells significantly decreased during the resolution phase,whereas VWF-vascular endothelial cells increased.Single-cell transcriptomics identified Cd93 asa specific gene for general capillary endothelial cells,with a negative correlation with"aerocyte"genes enriched in gas-exchange alveolar capillary endothelial cells.Immunofluorescence confirmed CD93 localization to general capillary endothelial cells.A flow sorting strategy based on CD45-CD31+CD90.2-VWF-CD93-effectively enriched alveolar capillary endothelial cells.This subpopulation trended upward in pulmonary vascular endothelial composition during bleomycin induction.CONCLUSION During bleo-mycin-induced pulmonary fibrosis in mice,pulmonary vascular endothelial subpopulations exhibit dynamic compositional heterogeneity across fibrotic injury and repair phases.