Single-cell transcriptomic atlas of lung microvascular regeneration after targeted endothelial cell ablation
- Ottawa Hospital Research Institute, Canada
- Sinclair Centre for Regenerative Medicine, Canada
- Department of Cellular and Molecular Medicine, University of Ottawa, Canada
Figures
Figure 1 with 1 supplement
Establishment of the diphtheria toxin (DT)-induced endothelial cell (EC) ablation model.
(A) Transgenic mice harboring Cre recombinase cDNA downstream from a 2.5 kb fragment of the VE-Cadherin (Cdh5) mouse promoter (B6.FVB-Tg(Cdhn5-cre)7Milia) were crossed with mice with Cre-inducible …
Figure 1—figure supplement 1
Sublethal dose of diphtheria toxin (DT) induces pulmonary hypertension and selective increase in lung permeability.
(A) Single intra-tracheal (IT) administration of DT at doses below 20 ng was compatible with survival to 7 days post-treatment. (B) Doses above 1 ng resulted in a dose-dependent increase in right …
Figure 2
Diphtheria toxin (DT) administration results in an acute lung injury phenotype.
(A) Representative examples of Evans blue staining of lungs from binary transgenic mice. (B) Summary data showing a marked increase in Evans Blue lung content in binary transgenic mice treated with …
Figure 3 with 2 supplements
Multiplexed single-cell transcriptomic analysis after diphtheria toxin (DT)-induced lung endothelial cell (EC) ablation.
(A) Schematic of workflow showing the experimental design. Three separate cohorts (3, 5, and 7 days) of binary transgenic mice received DT (IT, 10 ng), and a fourth cohort of healthy animals served …
Figure 3—figure supplement 1
Quality control for single-cell RNA sequencing (scRNA-seq).
(A) All 12 barcodes were demultiplexed with a small number of cells with multiple barcodes (doublets) and a population lacking any barcode (negative). (B) Quality control was performed using the …
Figure 3—figure supplement 2
Major cell type classification.
Expression of selected genes characterizing the identity of the major lung cell clusters based on the Tabula Muris biological atlas, including (A) Cldn5 (endothelial), (B) Ptprc (immune), (C) Epcam …
Figure 4 with 1 supplement
Endothelial cell populations in control and diphtheria toxin (DT)-treated binary transgenic diphtheria toxin receptor (DTR) mice.
(A) Uniform manifold approximation and projection (UMAP) representation of the re-clustered endothelial populations for all cohorts produced eight distinct endothelial cell (EC) clusters. (B) The …
Figure 4—figure supplement 1
Aerocytes (aCap) and general capillary (gCap) genes in endothelial cells (EC) populations pre and post-injury.
Typical aCap ECs genes include: (A) Car4 (Carbonic Anhydrase 4); (B) Endrb (endothelin receptor type-B); (C) Fibin (Fin Bud Initiation Factor Homolog); and (D) Tbx2 (T-Box Transcription Factor 2). …
Figure 5
RNA velocity and trajectory analysis.
(A) This model showed a dynamic shift of endothelial cells (ECs) through Zones 2 and 3 of transitional Cluster 1 with strong vectors towards general capillary (gCap), arterial, and venous EC …
Figure 6 with 3 supplements
Apelin expression in a transitional general capillary (gCap) stem-like endothelial cell (EC) population at 3 days post-injury.
(A) Under basal conditions (day 0; blue color) Apln was only expressed by aCap ECs (aerocytes; Cluster 2), whereas Aplnr expression was seen in gCap ECs (Cd93 positive). 3 days after EC ablation …
Figure 6—figure supplement 1
Temporal evolution of expression of tip and stalk cell genes pre and post-endothelial cell (EC) ablation.
(A) Pdgfb (Platelet Derived Growth Factor Subunit B), (B) Npl1 (Neuropilin 1), (C) Angpt2 (Angiopoietin 2), (D) Notch1 (Notch Receptor 1), and (E) Kit (KIT Proto-Oncogene, Receptor Tyrosine Kinase). …
Figure 6—figure supplement 2
Unbiased inference of transcription factor (TF) activity based on known TF-gene set interactions (decoupleR) showing the top 25 significantly up- or down-regulated TFs.
Different endothelial cells (ECs) populations showed very distinct profiles with the gCap proliferative ECs (Cluster 7) exhibiting 13 activated TFs, including Tfdp1 (Transcription Factor Dp-1), E2f2 …
Figure 6—figure supplement 3
Serial single-cell transcriptomic analysis of a publicly available dataset endothelial cell (EC) gene expression in an endotoxin (LPS) model of acute lung injury (DOI: 10.1172/jci.insight.158079).
(A) Uniform manifold approximation and projection (UMAP) dimensionality reduction reveals 13 endothelial clusters. (B) The identity of these clusters is based on markers of general capillary (gCap), …
Figure 7 with 1 supplement
Differential gene expression and pathway enrichment analysis of endothelial sub-Clusters 1–7.
(A) Volcano plots showing the distribution of differentially expressed genes in Clusters 1–7 (C1 to C7) according to statistical significance (Benjamini-Hochberg (BH) adjusted p-value), magnitude …
Figure 7—figure supplement 1
Pathway enrichment analysis of endothelial Clusters 1–7 via Ingenuity Pathway Analysis.
Each panel shows a predefined functional category and specific canonical signaling pathways that were significantly enriched in genes altered in each endothelial Cluster 1–7 (x-axis). Circle size …
Figure 8 with 2 supplements
Differential expression and pathway enrichment analysis of time-dependent changes in Zones 1–4 of endothelial Cluster 1.
(A) Volcano plots showing the distribution of differentially expressed genes according to statistical significance, magnitude, and direction of fold change (FC) at different points following …
Figure 8—figure supplement 1
Pathway enrichment analysis of time-dependent changes in endothelial Cluster 1 at days 3, 5, and 7 versus day 0.
Each panel shows a predefined functional category and specific canonical signaling pathways (y-axis) that were significantly enriched in genes altered at each time point (days 3, 5, and 7 versus day …
Figure 8—figure supplement 2
Chord diagram showing the activation or inhibition of genes in three canonical pathways involved in apelin signaling (PI3/Akt, mTor, Elf4/p70S6K) during the emergence of the apelin/Procr expressing general capillary (gCap) endothelial cells (ECs) at day 3.
Figure 9
Cell Signaling inferences using NicheNet.
Circos plots showing color-coded sender populations (outer circle) indicating the 10 top genes signaling for each receiver population (shown in gray). (A) At day 3, only one population, general …
Figure 10
Impairment or inhibition of the apelin pathway leads to failure of recovery after endothelial cell (EC) ablation.
(A) Expression of apelin from a publicly available dataset (31) in young (3 months) versus old (24 months) mice (*=p<0.05, unpaired t-test). (B) Apelin expression assessed by qRT-PCR expressed as …
Figure 11
Schematic of endothelial cell (EC) populations contributing to microvascular repair.
At baseline (day 0), there are two main alveolar groups of capillary ECs: larger apelin-positive aerocytes (aCap) ECs, termed aerocytes, that play a key structural role in forming the air-blood …
Author response image 1
