Nestin and Notch3 collaboratively regulate angiogenesis, collagen production, and endothelial–mesenchymal transition in lung endothelial cells

Figures

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  Fig. 1. Characteristics of nestin-expressing cells in the lung. A Flowcytometric analysis of nestin-expressing cells in a nestin-GFP murine lung and CD45− lung cells. B The frequency of CD45−/nestin-expressing cells in 9–12-week-old male and female nestin-GFP murine lungs (n = 4–8 mice per group). C (left) Representative image of nestin-expressing cells in CD45− lung cells obtained from a 2-week-old nestin-GFP mouse. Effect of aging on (middle) frequency of CD45−/nestin-expressing cells and (right) Nes (nestin) mRNA expression (n = 4–8 mice per group). D Representative images of cell surface markers expressed on CD45−/nestin-expressing cells. E Fluorescent microscopic images of a nestin-GFP murine lung. Nuclei are shown in purple-blue with DAPI. Scale bars = 50 μm. F GFP immunostaining of wildtype (WT) and nestin-GFP murine lungs. Nestin signals are shown in brown with anti-GFP antibody. Scale bars = 100 μm. G Expression of Nes mRNA between CD31+ endothelial cells and CD31− non-endothelial cells obtained from murine lungs (n = 4 per group)

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  Fig. 2. Roles of nestin-expressing endothelial cells on angiogenesis. A and B Flowcytometric analysis of (A) VEGFR2 or (B) PDGFRβ expression in nestin-expressing and -nonexpressing endothelial cells of a murine lung (n = 5 per group). C (left) Workflow of 3-dimensional (3D) culture to examine the angiogenetic ability in nestin-expressing (Nes+) and -nonexpressing (Nes−) endothelial cells (EC) obtained from nestin-GFP murine lungs. (middle) Obtained Nes+ and Nes− ECs were cultured under 3D culture system supplemented with 50 ng/mL of VEGF or 50 ng/mL of PDGF. Representative results of 3D culture after 21 d. Scale bars = 100 μm. (right) Calculation of the vascular densities using imaging software as described in Method (n = 3 per group). D Nes mRNA expression in each murine cell line: 3T3 (fibroblasts), LA-4 (epithelial cells), and MS1 (endothelial cells) (n = 5 per group). E Effect of gene silencing using nestin siRNA on MS1 (n = 5 per group). F MS1 cells were cultured under 3D culture system supplemented with 50 ng/mL of VEGF or 50 ng/mL of PDGF. Representative results of MS1 treated with control or nestin siRNA after 6 d of 3D culture. Scale bars = 100 μm

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  Fig. 3. Roles of nestin-expressing endothelial cells during EndoMT. A Morphological changes of MS1 supplemented with TGFβ in 2D culture (at 24 or 72 h), and 3D culture (at day 8). Scale bars = 100 μm. B Expression of mesenchymal-related (Col1a1, collagen type 1; Fn1, fibronectin; and Acta2, αSMA), EndoMT-related (Snai1, snail1 and Snai2, snail2), and Nes mRNA in MS1 incubated for 10 h supplemented with or without TGFβ (n = 5 per group). C Expression of Nes and mesenchymal-related (Col1a1, Fn1, and Acta2) mRNA in MS1 at 5 h of incubation with or without TGFβ (n = 5–8 per group). D Effect of nestin gene silencing of normally cultured MS1 cells on Nes, Col1a1 and Fn1 mRNA expressions (n = 5–8 per group). E Effect of nestin gene silencing in MS1 undergoing TGFβ-induced EndoMT on Nes, mesenchymal-related (Col1a1, Fn1, and Acta2), and EndoMT-related (Snai1 and Snai2) mRNA expression (n = 8 per group). To induce EndoMT, MS1 was pre-treated with TGFβ for 10 h

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  Fig. 4. Notch3 expression is regulated by nestin in vitro. A Hierarchical clustered heatmap showing the gene expression patterns (fold change < − 2.0 or ≥ 2.0) in nestin-expressing (Nes+) and -nonexpressing (Nes−) endothelial cells (EC, n = 2 per group). Each row and line represent one sample and differentially expressed genes, respectively. Red and blue indicate upregulation and downregulation, respectively. B Dot plot showing the absolute value of fold change of differentially expressed genes between Nes+ and Nes− EC. Notch3 is presented at sixth place. C Pericyte markers (Notch3 and CD13) expressed on CD45−/nestin-expressing and -nonexpressing cells (n = 4 per group). D and E Effect of nestin (D, n = 8 per group) or Notch3 (E, n = 6 per group)-gene silencing in MS1 cells on mRNA expression. F Morphological changes in MS1 cells treated with control or Notch3 siRNA in 2D culture at 48 h. Scale bars = 100 μm

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  Fig. 5. Effect of nestin gene silencing in vivo. A (left) GFP immunostaining of nestin-GFP murine lungs administrated with control or nestin siRNA. Scale bars = 100 μm. (right) Calculation of the GFP/nestin-positive area using Image J (n = 9–13 per group). B Effect of in vivo nestin gene silencing on Notch3 expressing cells (n = 4 per group). C Effect of in vivo nestin gene silencing on mesenchymal-related mRNA expression in the lung (n = 6 per group). D Effect of in vivo nestin gene silencing on αSMA and Col1 expression in lung endothelial cells (n = 6 per group)

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  Fig. 6. Association between nestin-expressing cells and collagen production in bleomycin-induced pulmonary fibrosis. A Nes mRNA expression in murine lungs obtained at 2 d after administration with phosphate buffered saline (PBS, control group) or bleomycin (BLM, n = 4 per group). B Changes in CD45−/nestin-expressing cells during BLM-induced pulmonary fibrosis (n = 4–5 per group). C (left) GFP immunostaining of nestin-GFP murine lungs obtained at 14 d after administration with PBS or BLM. Scale bar = 100 μm. (right) Calculation of the GFP/nestin-positive area using Image J (n = 8 per group). D Flowcytometric analysis of the proportions of nestin-expressing cells in murine lungs 14 d after PBS or BLM treatment using anti-nestin antibody A and B. E The proportions of Notch3 and/or CD13-positive cells in the lungs obtained at 21 d after administration with PBS or BLM (n = 4 per group). F Correlations between the amount of hydroxyproline (indicating collagen content) and the number of CD45−/nestin-expressing cells in nestin-GFP murine lungs obtained at 7, 14, and 21 d after administration with BLM (n = 4–8 per group)