TCF7 enhances pulmonary hypertension by boosting stressed natural killer cells and their interaction with pulmonary arterial smooth muscle cells

Figures

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  Fig. 1. The NK landscape of lung tissues from PAH patients and controls at single-cell resolution. A All cells in lung tissues from three PAH patients and three controls were clustered into 15 major cell types and visualized in UMAP plot. Each dot represents a cell, colored by cell type. B Cell distributions in lung tissues from either PAH patients or controls and were visualized in UMAP plot. C NK cells categorized into seven subclusters. D Distribution of lung NK cells from either PAH patients or controls. E Correlation between NK cells subsets and was visualized in heatmap. Red represents high correlation while blue represents low correlation. F NK cells clustered into four NK functional subtypes based on correlation. G Enriched GO biological process among different NK subtypes. Red bar represents lower P-value while blue bar represents higher P-value

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  Fig. 2. The NK landscape of lung tissues from MCT-induced PH rat model and controls at single-cell resolution. A All cells in lung tissues from six MCT-induced PH rats and six controls clustered into 16 major cell types and were visualized in UMAP plot. Each dot represents a cell, colored by cell type. B Cell distributions in lung tissues from either MCT-induced PH rats or controls. C NK cells clustered into four clusters. D Distribution of lung NK cells from either MCT-PH rats or controls. E Correlation between NK cells subsets and was visualized in heatmap. Red represents high correlation while blue represents low correlation. F NK cells clustered into three NK subtypes based on correlation. G GO biological process shows functional enrichment among NK subtypes. Red bar represents lower P-value while blue bar represents higher P-value

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  Fig. 3. The association of TCF7 and stress response in NK cells. A Stress score of lung NK cells from controls or PAH patients (n=3 each). B Stress score of each human NK cell subtypes in lung tissues. C Top 10 stress related genes differentiating low- and high-stress NK cells; red represents high expression while blue represents low expression. D Stress score of TCF7-deficient or TCF7-expressing NK cells. E Lung TCF7 expression level from controls or PAH patients (n=3 each). F TCF7 expression level of four NK cell subclusters from controls or PAH patients. Blue indicates elevated expression levels, and gray indicates reduced or absent expression. G Stress score of lung NK cells from control or MCT-PH rats (n=6 each). H Stress score of each rat NK cell subtypes in lung tissues. I Stress score of Tcf7-deficient or Tcf7-expressing NK cells. J Lung Tcf7 transcriptional levels from control group and MCT-PH group. K Lung Tcf7 protein levels from control group and MCT-PH group. L Lung Tcf7 expression level of three NK cell subclusters from control or MCT-PH rats. Data represent mean ± SEM. * P < 0.05 compared to corresponding controls, as analyzed by unpaired t test or Mann-Whitney test as…

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  Fig. 4. TCF7 might act as a moderator of NK cells responded to stress. A The expression of stress-related genes of lung NK cells from PAH patients or controls. Circle sizes represent the proportions of cells expressing genes of interest and filled colors from light gray to blue represent normalized expression levels from low to high. B The expression of stress-related genes of human lung TCF7- or TCF7+ NK cells. C Representative histogram of flow cytometry showing Tcf7 expression of isolated spleen NK cells from Tcf7WT or Tcf7KO mice. D Proportion of Tcf7+ NK cells among all NK cells from Tcf7WT or Tcf7KO mice (n=6 for each group). E The in vitro experimental schematic for gene validation in isolated splenic NK cells from Tcf7WT or Tcf7KO mice. F Hsp90aa1 mRNA expression in isolated spleen NK cells from Tcf7WT or Tcf7KO mice after hypoxia exposure or under normoxic condition for 24 h (n=3 mice/group for one experiment, repeat 3 experiments). G Hsp90ab1 mRNA expression in isolated spleen NK cells from Tcf7WT or Tcf7KO mice after hypoxia exposure or under normoxic condition for 24 h (n=3 mice/group for one experiment, repeat 3 experiments). H Proportion of Hsp90+ NK cells among all NK…

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  Fig. 5. TCF7 promotes proliferation and migration of PASMCs via upregulating secretion of SPP1 by NK cells. A The crosstalk between NK cells and SMCs in lung tissues of human samples from dataset GSE228644. B The ligand-receptor pairs in TCF7-deficient NK cells/SMCs and TCF7-expressing NK cells/SMCs. Communication probability refers to the likelihood of specific ligand-receptor interactions occurring between cell types. Red dots represent higher communication probabilities (stronger interactions); blue dots represent lower probabilities (weaker interactions). C SPP1 and TCF7 protein expression in LV-TCF7 or LV-Control infected NK92 cells after 48 h hypoxia exposure (n=3/group for one experiment, repeat 2 experiments). D The cell viability of hPASMCs after treatment with conditioned medium (CM) from NK92 cells infected with either LV-TCF7 or LV-Control along with 48 h hypoxia exposure (n=6/group for one experiment, repeat 4 experiments). E The representative images and quantification of the cell proliferation ability of hPASMCs after treatment with CM from NK92 cells infected with either LV-TCF7 or LV-Control along with 48 h hypoxia exposure (n=3/group for one experiment, repeat 3…

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  Fig. 6. TCF7 overexpression aggravates vascular remodeling in MCT-PH rat model. A An overview of the experimental design. B RVSP (n1=6, n2=6, n3=8, n4=9); (C) mPAP (n1=6, n2=6, n3=7, n4=6) and (D) Fulton index (n1=6, n2=6, n3=9, n4=11) from AAV6-hTCF7 treated or AAV6-Control rats at day 21 after MCT administration (60 mg/kg) or saline injection. E Representative images of EVG staining and (F) quantification of media thickness from AAV6-hTCF7 or AAV6-Control recipients at day 21 after MCT or saline injection (n1=6, n2=6, n3=9, n4=11); scale bar: 50 μm. G Representative image of Masson staining and (H) quantification of collagen abundance from AAV6-hTCF7 or AAV6-Control recipients at day 21 after MCT or saline injection (n1=5, n2=5, n3=9, n4=11); scale bar: 50 μm. I Representative images of double immunofluorescent staining and (J) quantification of α-SMA (for smooth muscle cells) and vWF (for endothelial cells) in lung tissues from AAV6-hTCF7 or AAV6-Control recipients at day 21 after MCT or saline injection (n1=6, n2=6, n3=10, n4=12); scale bar: 50 μm. K Representative images of multiplex immunofluorescent staining assays and (L) quantification of the proportion of Hsp90+NK cells among…

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  Fig. 7. Tcf7 knockout alleviates vascular remodeling in hypoxia-induced PH mouse model. A An overview of the experimental design. B RVSP (n1=7, n2=5, n3=7, n4=9) and (C) Fulton index (n1=8, n2=9, n3=7, n4=7) from Tcf7WT or Tcf7KO mice at day 28 after hypoxia or normoxia. D Representative images of EVG staining and (E) quantification of media thickness from Tcf7WT or Tcf7KO mice at day 28 after hypoxia or normoxia (n1=7, n2=7, n3=9, n4=9); scale bar: 50 μm. F Representative image of Masson staining and (G) quantification of collagen abundance from Tcf7WT or Tcf7KO mice at day 28 after hypoxia or normoxia (n1=7, n2=7, n3=9, n4=9); scale bar: 50 μm. H Representative images of double immunofluorescent staining and (I) quantification of α-SMA (for smooth muscle cells) and vWF (for endothelial cells) in lung tissues from Tcf7WT or Tcf7KO mice at day 28 after hypoxia or normoxia (n1=7, n2=7, n3=9, n4=9); scale bar: 50 μm. J Representative images of multiplex immunofluorescent staining assays and (K) quantification of the proportion of Hsp90+NK cells among all NK cells in lung tissues from Tcf7WT or Tcf7KO mice at day 28 after hypoxia or normoxia (n1=5, n2=5, n3=5, n4=5); scale bar: 20 μm. L…