Jagged/Notch proteins promote endothelial‐mesenchymal transition‐mediated pulmonary arterial hypertension via upregulation of the expression of GATAs

Figures

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  FIGURE 1. Jagged2 regulated the protein expression of GATAs. (A) Protein expression of GATA3, * versus †, p < 0.01. (B) Protein expression of GATA4, * versus †, p < 0.01. (C) Protein expression of GATA6, * versus †, p < 0.01. Statistical analysis by unpaired Student t‐test (n = 3 in each group). HUVECs, human umbilical vein endothelial cells; siRNA‐JAG2, silenced the JAG2 gene.

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  FIGURE 2. GATAs regulated expressions of the endMT markers. (A) Protein expression of Snail, * versus †, p < 0.01. (B) Protein expression of N‐cadherin, * versus †, p < 0.01. (C) Protein expression of CD31, * versus †, p < 0.01. (D) Protein expression of Zeb1, * versus †, p < 0.01. (E) Protein expression of vimentin, * versus †, p < 0.01. (F) Protein expression of fibronectin, * versus †, p < 0.01. (G) Protein expression of alpha smooth actin (α‐SMA), * versus †, p < 0.01. (H) Protein expression of phosphorylated (p)‐Smad2, * versus †, p < 0.01. (I) Protein expression of E‐cadherin, * versus †, p < 0.01. Statistical analysis by unpaired Student t‐test (n = 3 in each group). endMT, endothelial‐mesenchymal transition; HUVECs, human umbilical vein endothelial cells; siRNA‐GATA3, silenced the GATA3 gene.

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  FIGURE 3. Th capacity of HUVECs migratory assay in condition of siRNA‐GATA3. (A–F) Illustrating the microscopic finding of Giemsa stain in low‐power field (i.e. 40×) (A–C) and higher‐power filed (i.e. 200×, scale bars in right lower corners represent 50 μm) (D–F) for identification of the losing migratory ability of HUVECs, that is a reflection of endothelial to mesenchymal processing (i.e. reendothelialization). (G) Analytical result of number of migratory cells, * versus †, p < 0.001. Statistical analysis by unpaired Student t‐test (n = 5 in each group). (H) Relative gene expression of miRNA‐200b‐3p, * versus other groups with different symbols (†, ‡), p < 0.01. n = 3 for each group. (I) Relative gene expression of miRNA‐200c‐3p, * versus other groups with different symbols (†, ‡), p < 0.01. n = 3 for each group. (J) Cell viability of Jagged2 at 24 h, * versus †, p < 0.001. Statistical analysis by unpaired Student t‐test (n = 6 in each group). (K) Cell viability of Jagged2 at 48 h, * versus †, p < 0.001. Statistical analysis by unpaired Student t‐test (n = 6 in each group). (L) Cell viability of Jagged2 at 72 h, * versus †, p < 0.001. Statistical analysis by unpaired Student t‐test (n =…

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  FIGURE 4. Effect of TGF‐ß1, MCT and PTU on regulating the expression of endMT markers in in vitro study. (A) Protein expression of Jagged1, * versus other groups with different symbols (†, ‡), p < 0.001. (B) Protein expression of Jagged2, * versus other groups with different symbols (†, ‡), p < 0.001. (C) Protein expression of Zeb1, * versus other groups with different symbols (†, ‡), p < 0.001. (D) Protein expression of GATA3, * versus other groups with different symbols (†, ‡), p < 0.001. (E) Protein expression of GATA4, * versus other groups with different symbols (†, ‡, §), p < 0.001. (F) Protein expression of GATA6, * versus other groups with different symbols (†, ‡), p < 0.001. (G) Protein expression of Notch1, * versus other groups with different symbols (†, ‡), p < 0.001. (H) Protein expression of Notch2, * versus other groups with different symbols (†, ‡), p < 0.001. (I) Protein expression of Snail, * versus other groups with different symbols (†, ‡), p < 0.001. (J) Protein expression of fibronectin, * versus other groups with different symbols (†, ‡), p < 0.001. (K) Protein expression of Vimentin, * versus other groups with different symbols (†, ‡), p < 0.001. (L) Protein…

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  FIGURE 5. Exo vivo study of pulmonary arterial vasorelaxation and vasoconstriction and nitric oxide (NO) release by Day 42 after MCT treatment. (A) Phenylephrine (PE)‐induced vasoconstriction was significantly increased in MCT group than in other groups. Analytical result of vasoconstriction (%), * versus other groups with different symbols (†, ‡), p < 0.0001. (B) Sodium nitroprusside (SNP)‐induced vasorelaxation did not differ among the three groups, suggesting that SNP only acted on the capillaries, that is a direct NO provider. Analytical result of vasorelaxation, p > 0.5. (C) Acetylcholine (ACh)‐induced vasorelaxation (i.e. dilatation response) was significantly reduced in MCT group than in the other two groups. Analytical result of vasorelaxation, * versus †, p < 0.0001. (D) The baseline NO release from endothelial cells of PA detected by indirect measurement was significantly lower in NCT group than in the other two groups. Analytical result of NO release (%) among the three groups, * versus †, p < 0.001. All statistical analyses were performed by one‐way anova, followed by Bonferroni multiple comparison post hoc test (n = 8 for each group). Symbols (*, †, ‡) indicate significance…

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  FIGURE 6. Arterial oxygen saturation (SaO2), right ventricular systolic pressure (RVSP), LVSBP, femoral arterial systolic blood pressure (FASBP) and the ratio of RV weight, whole wet lung weight (WWLW) and septum + LV weight to tibial length by Day 42 after MCT treatment. (A) The level of SaO2, * versus other groups with different symbols (†, ‡), p < 0.001. (B) The value of RVSBP, * versus other groups with different symbols (†, ‡), p < 0.0001. (C) The FASBP, * versus †, p < 0.001. (D) The left ventricular systolic blood pressure (LVSBP), p > 0.5. (E) Ratio of RV weight to tibial length, * versus other groups with different symbols (†, ‡), p < 0.0001. (F) The ratio of WWLW to tibial length, * versus other groups with different symbols (†, ‡), p < 0.001. (G) The ratio of septal + left LV weight to tibial length, p > 0.1. All statistical analyses were performed by one‐way anova, followed by Bonferroni multiple comparison post hoc test (n = 6 for each group). Symbols (*, †, ‡) indicate significance (at 0.05 level). MCT, monocrotaline; PTU, propylthiouracil.

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  FIGURE 7. Lung injury score, lung consolidation area and inflammatory cell infiltrating score by Day 42 after MCT treatment. (A–C) Histopathological findings (i.e. H.E. staining) of lung parenchyma under microscopic finding (200×) among the three groups. Small squares of a1, b1 and c1 were magnified to larger squares of a2, b2 and c2, respectively. (D) The number of alveolar sacs among three groups. * versus other groups with different symbols (†, ‡), p < 0.0001. (E) Crowded scores of lung parenchyma. * versus other groups with different symbols (†, ‡), p < 0.0001. (F–H) Illustrating the microscopic finding (200×) for identification of lung consolidation area (yellow‐dotted line). (I) Analytical result of consolidation area, * versus other groups with different symbols (†, ‡), p < 0.0001. (J) Analytical result of inflammatory cell infiltration (grey colour) score, * versus other groups with different symbols (†, ‡), p < 0.0001. The scale bars in right lower corners represent 50 μm. (K–M) Illustrating the microscopic finding (400×) of H.E., stain for identification of thickness of pulmonary arterial wall (yellow arrow). The scale bars in right lower corner represent 20 μm. (N)…

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  FIGURE 8. Fibrotic area and antioxidant biomarkers in lung parenchyma by Day 42 after MCT treatment. (A–C) Illustrating the microscopic finding (100×) of Masson's trichome stain for identification of fibrosis in lung fibrosis (blue colour). (D) Analytical result of fibrotic area, * versus other groups with different symbols (†, ‡), p < 0.0001. The scale bars in right lower corner represent 100 μm. (E–G) Illustrating the microscopic finding (200×) for identification of the intensity of glutathione peroxidase (GPx) expression (grey colour). (H) Analytical result of intensity of GPx expression, * versus other groups with different symbols (†, ‡), p < 0.0001. (I–K) Illustrating the microscopic finding (200×) for identification of the intensity of glutathione reductase (GR) expression (grey colour). (L) Analytical result of intensity of GR expression, * versus other groups with different symbols (†, ‡), p < 0.0001. The scale bars in right lower corner represent 50 μm. All statistical analyses were performed by one‐way anova, followed by Bonferroni multiple comparison post hoc test (n = 8 for each group). Symbols (*, †, ‡) indicate significance (at 0.05 level). MCT, monocrotaline; PTU,…

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  FIGURE 9. Immunofluorescent stain for identification of inflammatory cells in the lung parenchyma by Day 42 after MCT treatment. (A–C) Illustrating the immunofluorescent (IF) microscopic finding (400×) for identification of CD11b cell infiltration in the lung parenchyma (green colour). (D) Analytical result of number of CD11+ cells, * versus other groups with different symbols (†, ‡), p < 0.0001. (E–G) Illustrating the immunofluorescent (IF) microscopic finding (400×) for identification of CD68 cell infiltration in the lung parenchyma (green colour). (H) Analytical result of number of CD68+ cells, * versus other groups with different symbols (†, ‡), p < 0.0001. The scale bars in right lower corner represent 20 μm. All statistical analyses were performed by one‐way anova, followed by Bonferroni multiple comparison post hoc test (n = 8 for each group). Symbols (*, †, ‡) indicate significance (at 0.05 level). MCT, monocrotaline; PTU, propylthiouracil.

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  FIGURE 10. Protein expressions of pressure overload/cardiac hypertrophic, oxidative stress, DNA‐damaged and cellular jap junction biomarkers in RV myocardium by Day 42 after MCT treatment. (A) Protein expression of beta‐myosin heavy chain (ß‐MHC), * versus other groups with different symbols (†, ‡), p < 0.0001. (B) Protein expression of brain natriuretic peptide (BNP), * versus other groups with different symbols (†, ‡), p < 0.0001. (C) Protein expression of α‐MHC, * versus other groups with different symbols (†, ‡), p < 0.0001. (D) Protein expression of phosphorylated connexin43 (p‐Cx43), * versus other groups with different symbols (†, ‡), p < 0.0001. (E) Protein expression of NOX‐1, * versus other groups with different symbols (†, ‡), p < 0.0001. (F) Protein expression of NOX‐2. (G) Protein expression of phosphorylated (p) γ‐H2AX, * versus other groups with different symbols (†, ‡), p < 0.0001. All statistical analyses were performed by one‐way anova, followed by Bonferroni multiple comparison post hoc test (n = 6 for each group). Symbols (*, †, ‡) indicate significance (at 0.05 level). MCT, monocrotaline; PTU, propylthiouracil.

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  FIGURE 11. Protein expressions of promoters and transcriptional regulators of endMT and gene expression of miR‐200 in lung parenchyma by Day 42 after MCT treatment. (A) Protein expression of Jagged1, * versus other groups with different symbols (†, ‡), p < 0.0001. (B) Protein expression of Jagged2, * versus other groups with different symbols (†, ‡), p < 0.0001. (C) Protein expression of Notch1, * versus other groups with different symbols (†, ‡), p < 0.0001. (D) Protein expression of Notch2, * versus other groups with different symbols (†, ‡), p < 0.0001. (E) Protein expression of GATA3, * versus other groups with different symbols (†, ‡), p < 0.0001. (F) Protein expression of GATA4, * versus other groups with different symbols (†, ‡), p < 0.0001. (G) Protein expression of GATA6, * versus other groups with different symbols (†, ‡), p < 0.001. (H) Relative gene expression of miRNA‐200b‐3p, * versus other groups with different symbols (†, ‡), p < 0.001. (I) Relative gene expression of miRNA‐200c‐3p, * versus other groups with different symbols (†, ‡), p < 0.0001. All statistical analyses were performed by one‐way anova, followed by Bonferroni multiple comparison post hoc test (n = 6 for each…

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  FIGURE 12. Protein expressions of endMT markers in lung parenchyma by Day 42 after MCT treatment. (A) Protein expression of Snail, * versus other groups with different symbols (†, ‡), p < 0.0001. (B) Protein expression of Zeb1, * versus other groups with different symbols (†, ‡), p < 0.0001. (C) Protein expression of Fibronectin, * versus other groups with different symbols (†, ‡), p < 0.0001. (D) Protein expression of Vimentin, * versus other groups with different symbols (†, ‡), p < 0.0001. (E) Protein expression of transforming growth factor (TGF)‐ß, * versus other groups with different symbols (†, ‡), p < 0.0001. (F) Protein expression of phosphorylated (p)‐Smad2, * versus other groups with different symbols (†, ‡), p < 0.0001. (G) Protein expression of p‐Smad3, * versus other groups with different symbols (†, ‡), p < 0.0001. All statistical analyses were performed by one‐way anova, followed by Bonferroni multiple comparison post hoc test (n = 6 for each group). Symbols (*, †, ‡) indicate significance (at 0.05 level). endEMT, endothelial‐mesenchymal transition; MCT, monocrotaline; PTU, propylthiouracil.