Single-cell transcriptomic profiling of lung endothelial cells identifies dynamic inflammatory and regenerative subpopulations
Lianghui Zhang, … , Asrar B. Malik, Jalees Rehman
Lianghui Zhang, … , Asrar B. Malik, Jalees Rehman
Published May 5, 2022
Citation Information: JCI Insight. 2022;7(11):e158079. https://doi.org/10.1172/jci.insight.158079.
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Research Article Infectious disease Pulmonology

Single-cell transcriptomic profiling of lung endothelial cells identifies dynamic inflammatory and regenerative subpopulations

Abstract

Studies have demonstrated the phenotypic heterogeneity of vascular endothelial cells (ECs) within a vascular bed; however, little is known about how distinct endothelial subpopulations in a particular organ respond to an inflammatory stimulus. We performed single-cell RNA-Seq of 35,973 lung ECs obtained during baseline as well as postinjury time points after inflammatory lung injury induced by LPS. Seurat clustering and gene expression pathway analysis identified 2 major subpopulations in the lung microvascular endothelium, a subpopulation enriched for expression of immune response genes such as MHC genes (immuneEC) and another defined by increased expression of vascular development genes such as Sox17 (devEC). The presence of immuneEC and devEC subpopulations was also observed in nonhuman primate lungs infected with SARS-CoV-2 and murine lungs infected with H1N1 influenza virus. After the peak of inflammatory injury, we observed the emergence of a proliferative lung EC subpopulation. Overexpression of Sox17 prevented inflammatory activation in ECs. Thus, there appeared to be a “division of labor” within the lung microvascular endothelium in which some ECs showed propensity for inflammatory signaling and others for endothelial regeneration. These results provide underpinnings for the development of targeted therapies to limit inflammatory lung injury and promote regeneration.

Authors

Lianghui Zhang, Shang Gao, Zachary White, Yang Dai, Asrar B. Malik, Jalees Rehman

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Figure 6

Regulation of central role of Sox17 in the inflammatory response of lung vascular ECs.

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Regulation of central role of Sox17 in the inflammatory response of lung...
(A and B) Activity of TFs and their regulatory targets estimated by SCENIC. Heatmap of estimated mean activities of TFs using SCENIC in each subpopulation at LPS treatment 6 hours (A). Enhanced activities of inflammatory TFs shown in top box and developmental TFs shown in lower box; Sox17 activity marked by an arrow. UMAP of estimated activities and predicted regulatory targets of Sox17 (B). (C–E) Human lung endothelial cells (HLECs) were infected with retroviruses expressing pMX-Sox17 to overexpress Sox17 and lentiviruses expressing GFP-Sox17-shRNA to knock down Sox17. The retroviruses expressing pMX vector and lentiviruses with GFP-scramble-shRNA were applied as control, respectively. Infected HLECs were treated with 20 ng/mL TNF-α for 2 hours. Representative Western blots (C) and quantification (D and E) shown. (F) Time course of lung transvascular permeability after LPS challenge in C57BL/6J mice with liposome delivery of Cdh5 vector or Cdh5-Sox17. Mice were injected with LPS 8 μg/kg 2 days later after liposome delivery of Sox17. Schematic diagram of Sox17 liposome delivery and LPS injection shown on top. n = 4 for each time point. (G) Western blots and quantification of fresh isolated lung ECs from C57BL/6J mice with liposome delivery of CDH5 vector or CDH5-Sox17 for 3 days. (H) Western blots and quantification of fresh isolated lung ECs from Sox17fl/fl mice or Sox17 EC cKO mice. n = 3 for each group. Mice were injected with LPS 8 μg/kg for 24 hours. n = 3 for each group. Data shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 compared with control by 2-way ANOVA (D and E) and unpaired 2-tailed t test (G and H).