The angiopoietin receptor Tie2 is atheroprotective in arterial endothelium

Figures

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  Fig. 1. Arterial Tie2 deletion promotes atheroma formation. a, Detailed schematic of the gene deletions. Numbers indicate nucleotide positions of the loxP sites relative to the transcription start site and names of the genotyping primers (Supplementary Table 1). b, Outline and time schedule (in weeks, w) of the experimental setup. c, Control and Tie2-deleted aortas stained with Sudan IV (n = 7 independent Tie2WT and n = 8 independent Tie2iΔAEC mice; scale bar, 2 mm). d, Quantification of aortic lesion areas (total, P = 0.0004; aortic arch, P = 0.099; thoracic aorta, P = 0.0005; abdominal aorta, P = 0.0003). Values show mean ± s.d. Statistical significance was determined using Student’s t-test (two-tailed, unpaired). Source data

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  Fig. 2. scRNA-seq analysis of cells in the atheromatous aortas. a, Schematic of the analysis. b, UMAP plots of aligned gene expression data from aortic cells isolated and pooled from 2–3 individual Tie2WT (n = 7,903 in the aortic arch, n = 6,310 in the thoracic plus abdominal aorta isolated and pooled from 2–3 mice) and Tie2iΔAEC (n = 6,578 and n = 6,178, respectively) mice after 20 weeks of Western diet, partitioned into 13 distinct clusters. Trem2hi macrophage (red arrow), T cell (black arrow) and B cell (green arrow) clusters showed the most obvious changes after endothelial cell Tie2 deletion. c, Proportions of immune cell clusters in the analyzed aortic regions. d, Quantification of CD68 macrophages (adventitia, P = 0.0096; intimal area, P = 0.0019) and CD3e T cells (P = 0.0010) in the atherosclerotic arotas (n = 3 independent mice per group). Values show mean ± s.d. Statistical significance was determined using Student’s t-test (two-tailed, unpaired). Source data

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  Fig. 3. Tie2 deletion increases inflammatory cells in atherosclerotic lesions. a, Representative images of aortas stained with Sudan IV from the Tie2WT and Tie2iΔAEC (n = 4 independent Tie2WT and n = 3 independent Tie2iΔAEC mice) mice after 4 weeks of Western diet (scale bar, 2 mm). b, Quantification of aortic lesion areas. Values show mean ± s.d. c, Quantification of CD45+ immune cells (P = 0.012), CD11b+F4/80+ macrophages (P = 0.038) and CD3e+ T cells (P = 0.034) in the aortas (n = 3 independent mice per group) using flow cytometry. Values show mean ± s.d. Statistical significance was determined using Student’s t-test (two-tailed, unpaired). d, Outline and time schedule (in weeks) of the experimental setup in the intravital microscopy analysis. e,f, Images of CD11b+ myeloid cells adherent to the carotid artery wall in the bifurcation area (f). Scale bar, 100 µm. Quantification of CD11b+ myeloid cells in the external carotid artery and its bifurcation (n = 5 independent Tie2WT and n = 6 Tie2iΔAEC mice) (e). Each data point represents one mouse. Values show mean ± s.d. Statistical significance was determined using two-way ANOVA with Sidak post hoc test. Source data

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  Fig. 4. Expression of leukocyte adhesion receptors and transcripts associated with lymphocyte activation in the Tie2WT and Tie2iΔAEc mice. a, Representative immunofluorescence images of aortic arch from the Tie2WT and Tie2iΔAEC mice 8 weeks after Western diet and Pcsk9 overexpression stained for platelet and endothelial cell adhesion molecule 1 (PECAM1; green) and VCAM1 (red) (left). Quantification of the VCAM1/PECAM1 ratio (n = 4 independent Tie2WT mice and n = 5 independent Tie2iΔAEC mice) (right). Scale bar, 100 μm. Values show mean ± s.d. Statistical significance was determined using Student’s t-test (two-tailed, unpaired). b, Violin plots of Vcam1, Clu and Fbln5 expression in AECs. Note that after Tie2 deletion, Vcam1 and Clu are upregulated in both parts of the aorta, whereas Fbln5 is significantly upregulated only in the thoracic and abdominal parts. Scale, log-transformed gene expression. Statistical significance was determined using a Wilcoxon rank-sum test. P value adjustment was performed using Bonferroni correction based on the total number of genes in the dataset. c, Representative immunofluorescence images of aortic sections 20 weeks after Western diet and Pcsk9…

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  Fig. 5. Tie2 restricts inflammatory signaling in the Tie2+ fibroblasts. a, UMAP plots of aortic cells from adult Tie2WT mice that received normal diet and Tie2WT mice after 20 weeks of Western diet. b, Dot plots showing top changed genes in the top regulated pathways in the Tie2+ fibroblasts. c, Heatmap showing upregulated genes related to inflammatory cell migration, immune response, inflammatory response, and chemokine and chemotaxis pathways upon Tie2 silencing. d, Gene ontology analysis on the GO terms that were upregulated in the Tie2-silenced Tie2+ fibroblasts. Bonferroni correction (multiple comparison test) was applied, followed by Benjamin–Hochberg adjusted P value or FDR values to plot the gene ontology. e, Quantification of IL-6 secreted in the Tie2+ fibroblasts after Tie2 silencing (n = 3 independent samples in SCR, n = 2 independent samples in shTie2-A and n = 3 independent samples in shTie2-D; 3 h, P = 0.0281, 6 h, P = 0.000102; 12 h, P = 3.21 × 10−8; 18 h, P = 2.16 × 10−13; 24 h, P = 3.5 × 10−14). Values show mean ± s.d. Statistical significance was determined using two-way ANOVA with Greenhouse–Geisser correction and Tukey’s multiple comparisons test. Source data

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  Fig. 6. Tie2 function dominates over Tie1 function in atherosclerosis development. a, Representative images of Tie1WTTie2WT and Tie1;Tie2iΔAEC (n = 7 independent Tie1WTTie2WT mice and n = 10 independent Tie1;Tie2iΔAEC mice) aortas stained with Sudan IV (scale bar, 2 mm). b, Quantification of lesion areas in the Tie1WTTie2WT and Tie1;Tie2iΔAEC aortas (n = 7 independent Tie1WTTie2WT mice and n = 10 independent Tie1;Tie2iΔAEC mice over two independent experiments; total, P = 0.0132; thoracic aorta, P = 0.0012; abdominal aorta, P = 0.0063). Values show mean ± s.d. Statistical significance was determined using Student’s t-test (two-tailed, unpaired). c, Quantification of CD68+ area (n = 5 independent Tie1WTTie2WT mice and n = 4 independent Tie1;Tie2iΔAEC mice; P = 0.0081). Values show mean ± s.d. Statistical significance was determined using Student’s t-test (two-tailed, unpaired). d, Representative immunofluorescence images of the Tie1WTTie2WT and Tie1;Tie2iΔAEC aorta sections stained for PECAM1 (green), DAPI (blue) and VCAM1 (red). e, Quantifications of VCAM1 in the Tie1WTTie2WT and Tie1;Tie2iΔAEC aortas (n = 5 independent Tie1WTTie2WT mice and n = 4 independent Tie1;Tie2iΔAEC mice). Values…

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  Fig. 7. Tie2 deletion promotes nuclear localization of Foxo1. a, Representative en face images of Tie2WT and Tie2iΔAEC (n = 3 per group) aortas stained for Foxo1 (red) and DAPI (blue) (top). Scale bar, 20 μm. Quantification of nuclear versus cytoplasmic Foxo1 expression in the endothelial cells (dashed lines) (n = 3 independent mice per group; P = 0.0044) (bottom). Values show mean ± s.d. Statistical significance was determined using Student’s t-test (two-tailed, unpaired). b, Schematic showing a summary of the main results. GWAS analysis indicates that Tie2 activity is involved in human CAD via disruption of Tie–Akt signaling axis and via inhibition of Foxo1 nuclear localization and transcriptional activity. When Tie2 alone or the whole Tie1;Tie2 receptor complex is deleted from the aortic endothelium, VCAM1 expression is induced via Foxo1 transcriptional transactivation and blood leukocytes adhere to the aortic endothelium, which promotes atheroma formation. ECs, endothelial cells. Source data

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  Extended Data Fig. 1. GWAS identify TIE2 (TEK) as a risk locus for CAD. a. A LocusZoom plot with eQTL association in GTEX v8 Tibial Artery (top) and CAD GWAS association (middle) and LD matrix (bottom) generated using ezQTL tool. The red triangle indicates the rs1322052 SNP which demonstrates strong colocalization between eQTL and GWAS signals within the LD block (HyPrColoc posterior probability, PP: 0.8789). The insert in top figure indicates the direction of eQTL association with the risk variant T being associated with lower TIE2 expression. b. IGV80 genome browser screenshots of the TIE2 locus that demonstrates ATAC-Seq, H3K27ac ChIP-Seq and Hi-C data from teloHAECs treated with 0–24 TNFα stimulus65. The rs1322052 localizes to cis-regulatory enhancer element that is TNFα-responsive and loops to the TIE2 promoter. c. CRISPR deletion (CRISPRΔ) and CRISPR inhibition (CRISPRi) experiments were used to study the effect of rs1322052 variant site on TIE2 expression in teloHAECs. Two pairs of gRNAs were used to make the deletion of 945 bps and 1120 bps centered around rs1322052 variant (schematic). Quantification of DNA band intensities on the gel image demonstrate ~80% deletion of the intronic rs1322052…

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  Extended Data Fig. 2. Expression of Bmx gene and confirmation of Tie1 and Tie2 gene deletions in the aorta. a. Representative images of β-gal staining in the thoracic aorta, b. aortic arch and c. aortic root from WT (Bmx+/+) mice and in mice expressing the LacZ gene targeted into the Bmx locus. Scale bar, 1 mm (A-B) and 50 μm (C). d. Fold differences of Tie1 and Tie2 transcripts in the Tie1iΔAEC (n = 3 independent mice per group, Tie1: P = 0.0025), Tie2iΔAEC (n = 4 independent mice per group, Tie2: P = 0.0003), and Tie1;Tie2iΔAEC mice (n = 5 independent Tie1;Tie2WT mice and n = 4 independent Tie1;Tie2iΔAEC mice, Tie1: P = 0.0003,Tie2: P = 0.0015) in comparison with their littermate controls. Statistical significance was determined using Student’s t-test (two tailed, unpaired). e. Immunofluorescence of Tie2 and PECAM1 in the aortic root, aortic arch and thoracic aorta of the Tie2iΔAEC and Tie2WT mice (n = 3 independent mice per group). Scale bar, 50 μm. f. Pcsk9-D377Y concentration in serum samples and body weight measurements at the indicated time points in the Tie2iΔAEC and Tie2WT mice (n = 6 independent mice per group). g. Quantification of cholesterol (CHOL) and triglyceride (TG) concentrations in…

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  Extended Data Fig. 3. Tie2iΔAEC is associated with alteration of T cell and macrophage subsets during atherosclerosis. a. Feature plot showing the expression of Bmx (AECs), Cytl1 (AECs), Gpihbp1 (nAECs), Pdgfra (fibroblasts), Tagln (SMCs) and Ptprc (immune cells). b. Tie1 and Tie2 expression shown in the feature plot. Note that unlike Tie2, Tie1 was not expressed in the Tie2+ FB cluster. c. Representative images of immunohistochemical (chromogenic) staining of CD68 macrophages and CD3e T cells in the atherosclerotic aortas (n = 3 independent mice per group). Scale bar, 50 μm. d. UMAP plots of (12) immune cell clusters from the atheromatous aortic arch and thoracic plus abdominal aortas of the Tie2WT and Tie2iΔAEC mice. e. UMAP plots of immune cell annotation referenced to the published datasets using SingleR. f. Pie plots show the relative proportions of the main immune cell types (macrophage/monocyte, T cells, DCs, B cells, NK and mast cells) (upper), macrophages (middle), and T cells (lower). g. Gene enrichment analysis showing pathways that are related the upregulated genes in the Cxcl2hi macrophages in the Tie2iΔAEC mice. P value adjustment is performed using a Benjamini-Hochberg (BH)-procedure…

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  Extended Data Fig. 4. No difference in bone marrow leukocyte production between Tie2WT and Tie2iδAEC mice. a. Peripheral blood cell counts indices in the Tie2WT and Tie2iΔAEC mice four weeks after Western diet. b-c. Quantification of BM cellularity and spleen weight/bodyweight in the Tie2WT and Tie2iΔAEC mice (n = 3 independent mice per group). d-e. Quantification of BM stem and progenitor cell numbers in the Tie2WT and Tie2iΔAEC mice (n = 3 independent mice per group). f. Quantification of CD3e+ T cells, B220 + B cells and CD11b + F4/80+ macrophages in the BM from the Tie2WT and Tie2iΔAEC mice (n = 3 independent mice per group). g. UMAP plots of (15) immune cell clusters from red blood cell-depleted total bone marrow of the Tie2WT and Tie2iΔAEC mice four weeks after Western diet with Immgen annotation. h. Quantification of immune cell clusters from the experiment. Values show mean ± s.d. Statistical significance was determined using Student’s t-test (two tailed, unpaired). i. Gene enrichment analysis showing pathways that are related the upregulated genes in the BM neutrophils and monocytes in the Tie2iΔAEC mice. P value adjustment is performed using a Benjamini-Hochberg (BH)-procedure (Multiple…

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  Extended Data Fig. 5. scRNA analysis of Tie2WT and Tie2iΔAEC mice on normal diet or after 4 weeks of Western diet. a. UMAP plots of total aortic cells from adult Tie2WT and Tie2iΔAEC mice after 4 weeks of Western diet. b. Representative images of cells labelled with fluorescent microbeads in the carotid arteries of the Tie2WT and Tie2iΔAEC mice (n = 5 Tie2WT and n = 6 Tie2iΔAEC). Scale bar, 100 µm. c. Schematic of the experimental workflow. d. Proportions of the distinct clusters and total immune cells among the total aortic cells. e. UMAP plots of total aortic cells (n = 5936 and n = 5615, respectively, that partition into 11 distinct clusters) from adult Tie2WT and Tie2iΔAEC mice on normal diet, four weeks after Tie2 deletion. f. Tie2 (p.adj=2.14×10-99) and Icam1 (p.adj=0.0258) expression in aortic AECs shown in the violin plot. Statistical significance was determined using a Wilcoxon rank-sum test. P value adjustment was performed using Bonferroni correction based on the total number of genes in the dataset as indicated in the Seurat package. g. Gene ontology annotation analysis of the differentially expressed genes (DEGs) in the AECs in the Tie2iΔAEC aortas. P value adjustment is performed using a…