Fig 2: Matrix Gla protein (MGP) inhibits bone morphogenic protein (BMP)-1, which regulates maturation of transforming growth factor (TGF)ß1 and in turn controls the differentiation of endothelial cell (EC)-like myofibroblasts. a) Binding of BMP-1 to MGP shown by immunoprecipitation. BMP-1 (200 ng) and conditioned medium containing N-FLAG-MGP (100 µL; ~200 ng) were combined as indicated in the top panel, and the presence of the respective protein was confirmed by immunoblotting (IB) (top two blots). Interactions between the proteins were analysed by immunoprecipitation (IP) followed by immunoblotting with antibodies as indicated and quantified by densitometry. b) Binding of BMP-1 to MGP shown by chemical crosslinking. BMP-1 (200 ng) and conditioned medium containing N-FLAG-MGP (100 µL; ~200 ng) were combined as indicated in lane 2 and 3. Interactions were analysed using chemical crosslinking followed by immunoblotting with antibodies as indicated and quantified by densitometry. c) Expression of Col3a1 and Fn1 in tdTomato+VE-cadherin+ cells isolated from the lungs of SM22acreRosatdTomato mice and treated with BMP-1 (200 ng·mL-1) with transfection of MGP siRNA or scrambled siRNA (SCR) (n=6). d) Time-course immunoblotting with densitometry of latent TGFß1 binding protein-1 (LTBP-1) in tdTomato+VE-cadherin+ cell lysates after treatment with BMP-1 (200 ng·mL-1) with or without conditioned medium containing N-FLAG-MGP (n=6). e) Levels of TGFß1 in culture media of tdTomato+VE-cadherin+ cell after treatment with BMP-1 with or without conditioned medium containing N-FLAG-MGP (n=3). f) Immunoblotting with densitometry of lung tissues of VE-cadherincreMgpflox/flox and Sm22acreMgpflox/flox and control mice (n=3). g) Masson's trichrome (MT) staining with quantification of the lungs of VE-cadherincreMgpflox/flox and Sm22acreMgpflox/flox mice and control mice after treatment with UK388367 (n=8). Scale bars=100 µm. h) A schematic representation. c, e and g) were analysed for statistical significance by ANOVA with post hoc Tukey's test. The bounds of the boxes are upper and lower quartiles with data points; in c and g) error bars are maximal and minimal values; in e) error bars are mean±sd. **: p<0.001, ***: p<0.0001.

Fig 3: Transforming growth factor (TGF)ß1 drives endothelial cell (EC)-like myofibroblasts towards myofibroblast differentiation. a) Fluorescence-activated cell sorting of whole lung cells of Sm22acreRosatdTomato mice. b) Gene expression of tdTomato-negative and VE-cadherin-positive (tdTomato-VE-cadherin+), tdTomato+VE-cadherin+ and tdTomato+VE-cadherin- cells isolated from lungs of Sm22acreRosatdTomato mice (n=6). c) Gene expression of tdTomato-VE-cadherin+, tdTomato+VE-cadherin+ and tdTomato+VE-cadherin- cells after matrix Gla protein (MGP) deletion with siRNA and treated with or without TGFß1 (1 ng·mL-1) (n=6). Data were analysed for statistical significance by ANOVA with post hoc Tukey's test. The bounds of the boxes are upper and lower quartiles with data points; the line in the box is median; error bars are maximal and minimal values. Fn1: fibronectin 1; SMMHC: smooth muscle myosin heavy chain; vWF: von Willebrand factor. ***: p<0.0001.

Fig 4: Endothelial cell (EC)-like myofibroblasts are present in healthy human lungs and contribute to human pulmonary fibrosis. a) Uniform manifold approximation and projection (UMAP) for cell populations subclustered from the whole population of CD34+CD45- pulmonary cells of healthy human lungs and human pulmonary fibrosis. b) Violin plots of the gene expression of the lineage markers. c) Pseudotemporal trajectories of the cell clusters. d) Alterations in cell compositions of different populations in healthy human lungs and human pulmonary fibrosis. Mgp: matrix Gla protein; vWF: von Willebrand factor; SMA: smooth muscle actin; Fn1: fibronectin 1.

Fig 5: Berbamine induces FoxA2 to regulate transforming growth factor (TGF)ß signalling and control endothelial cell (EC)-like myofibroblast differentiation. a) Expression of activin receptor-like kinase (ALK)5 in tdTomato+VE-cadherin+ cells isolated from the lungs of SM22acreRosatdTomato mice and treated with berbamine (20 µM). b) Gene expression in tdTomato+VE-cadherin+ cells transfected with matrix Gla protein (MGP) siRNA in combination with berbamine treatment with or without overexpression of ALK5 (n=5). c) Expression of FoxA2 in tdTomato+VE-cadherin+ cells isolated from the lungs of SM22acreRosatdTomato mice and treated with berbamine (20 µM). d) Immunoblotting with densitometry using tdTomato+VE-cadherin+ cell lysates treated with berbamine (Bbm) with or without overexpression of FoxA2 (cytomegalovirus promoter (CMV)-FoxA2) (n=3). e) Expression of endothelial markers in tdTomato+VE-cadherin+ cells treated with berbamine in combination with FoxA2 overexpression or FoxA2 knockdown (FoxA2 siRNA) (n=6). f) Immunoblotting with densitometry of the lungs of VE-cadherincreMgpflox/flox mice, Sm22acreMgpflox/flox mice and control mice after treatment of berbamine (Bbm) (n=6). g) Quantification of VE-cadherin-positive and platelet-derived growth factor receptor (PDGFR)a-positive (VE-cadherin+PDGFRa+) cells and VE-cadherin negative and PDGFRa positive (VE-cadherin-PDGFRa+) cells after immunostaining of the lungs of bleomycin-injected wild-type mice, where gene expression was examined by real-time PCR (n=8). h) Immunostaining of ALK5 and FoxA2 of human pulmonary fibrosis (n=3). Scale bars=100 µm. a) and c) were analysed for statistical significance by unpaired two-tailed t-test. b), e) and g) were analysed for statistical significance by ANOVA with post hoc Tukey's test. The bounds of the boxes are upper and lower quartiles with data points; the line in the box is median; error bars are maximal and minimal values. SCR: scrambled siRNA; Ctr: control; Fn1: fibronectin 1. **: p<0.001, ***: p<0.0001.