Journal: EMBO Molecular Medicine
Article Title: Attenuated PDGF signaling drives alveolar and microvascular defects in neonatal chronic lung disease
Figure Lengend Snippet: Elevated TGF ‐β levels causally relate with reduced PDGF ‐Rα expression in nCLD patients and mice undergoing MV ‐O 2 , reducing downstream signaling and migration in pulmonary myofibroblasts Representative immunofluorescence images showing reduced expression of PDGF‐Rα (red) in the lungs of patients ( n = 7) developing nCLD (lower left panel, red stain; white arrows) together with increased pSMAD‐2 expression (lower middle panel, green stain; white arrows) as compared to lung sections from a non‐nCLD patient ( n = 1) (upper panel) (200×). Negative correlation between PDGF‐Rα and TGF‐β1 in transcriptome analysis 72 h after birth in preterms with nCLD ( n = 11) in contrast to non‐nCLD ( n = 9); z test of the difference of the Fisher's z transformed correlations divided by the standard error of the difference ( P = 0.048); scatter plots log2‐gene expression; linear regression (blue), with 95% CI (gray). MV‐O 2 reduced lung PDGF‐Rα (main: red stain; white arrows) and increased pSMAD‐2 levels (insert: red stain; white arrows) in ventilated neonatal WT (lower panel) when compared to control WT mice (upper panel); ( n = 4 mice/group; 10 images/mouse; 200×). Luciferase assay of CCL‐206 cells transfected with pGL4.14 containing PDGF‐Rα promoter revealing reduced promoter activity upon TGF‐β application (normalized to control) ( n = 3 experiments). Immunoblot analysis showing reduced PDGF‐Rα (E), VEGF‐A (F), and pERK/EKR (G) protein levels upon TGF‐β application alone in primary pulmonary myofibroblasts from 5–7‐day‐old WT mice ( n = 6–9 mice/group). Reduced migration of myofibroblasts (MFBs) from neonatal WT mice upon TGF‐β application alone ( n = 5 mice/group, 3 technical replicates). Translation of the results in fibroblasts isolated from tracheal aspirates of ventilated preterm infants (hMFBs) displayed reduced PDGF‐Rα levels (I) and migration assessed by Boyden chamber assay (J) upon TGF‐β application ( n = 3–5 patients/group). Representative phase contrast images (100×) of scratch migration assays in human lung fibroblasts (hMFBs) after 48 h of TGF‐β incubation indicating decreased wound closure (K) quantified by reduced velocity (L) and distance travelled (M) ( n = 3 patients/group). Data information: In (D–J) and (L, M), data are presented as mean ± SD and normalized to control. Statistical test used is two‐tailed unpaired Student's t ‐test or Mann–Whitney test ( P = 0.0002–0.039). *** P
Article Snippet: After measurement of protein concentrations (BCA, #23227, Pierce Scientific), immunoblots were performed using a Bis‐Tris or a Tris‐Acetate gel (#NP0321BOX, #EA0375BOX, Life Technologies) using the following antibodies: PDGF‐Rα (C‐20, Santa Cruz Biotechnology #338), VEGF‐A (147, Santa Cruz Biotechnology #507), VEGF‐R2 (Abcam, Cambridge, USA #Ab2349), VE‐cadherin (H‐72, Santa Cruz Biotechnology #28644), cleaved caspase‐3 (Cell Signaling Technology #9661), cleaved caspase‐9 (Cell Signaling Technologies #7237), eNOS (Cell Signaling Technologies #5880), phospho‐ERK (Cell Signaling Technologies #4370), total ERK (Cell Signaling Technologies #4695), RAS (Cell Signaling Technologies #8955), PI3K (Cell Signaling Technologies #13666), JAK‐2 (Cell Signaling Technologies #3230), STAT‐3 (Cell Signaling Technologies #9139).
Techniques: Expressing, Mouse Assay, Migration, Immunofluorescence, Staining, Transformation Assay, Luciferase, Transfection, Activity Assay, Isolation, Boyden Chamber Assay, Incubation, Two Tailed Test, MANN-WHITNEY