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. 2022 May 17;77(12):1176–1186. doi: 10.1136/thoraxjnl-2021-218083

Figure 2.

Figure 2

Significant reduction in BMP signalling at the earliest stage of lung vascular pathology is accompanied by microvessel loss in postnatal lung injury. (A) Preclinical mouse model of bronchopulmonary dysplasia (BPD) with induction of lung injury by exposure of neonatal mice to mechanical ventilation (MV) and/or hyperoxia (FiO2=0.4) for 8 hours or 24 hours. (B) Immunoblot analysis reveals a significant decrease in pSMAD 1-5-9 and Id-1 protein levels in WT mice in the early course of postnatal lung injury provoked by short-term MV-O2 and O2 exposure. (C) Representative images of immunofluorescence (IF) staining for cleaved caspase-3 (green) and VE-cadherin positive cells (red) in lung tissue sections (4 µM, 400X, upper panel; nuclei (DAPI, blue) from 5 to 8 day old pups show a significant increase in EC apoptosis, confirmed by quantitative analysis (lower-left panel). Histological analysis reveals a significant decrease in the number of micro vessels (20–100 µm diameter) in the lungs of mice undergoing only 8 hours of O2 and/or MV when compared with room air (RA) controls (lower-right panel). (D) In BMPR2 deficient mice, immunoblot analysis demonstrates significantly lower VE-cadherin protein expression in the neonatal lung at baseline (RA) when compared with WT pups (left panel). Postnatal exposure to moderate O2 further decreases VE-cadherin protein expression in BMPR2 deficient mice (right panel). (E) Representative images of immunofluorescence (IF) staining for BMPR2 (green) and VE-cadherin positive cells (red) in lung tissue sections (4 µM, 400X, upper panel; nuclei (DAPI, blue) following 24 hours MV show a prolonged and significant decrease in BMPR2 expression, confirmed by quantitative analysis (right panel). (F) ISH demonstrates a persistent decrease in BMPR2 transcription in neonatal mouse PCLS exposed to O2 for 24 hours when compared with RA control samples. Data are mean±SD *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n=3–4 mice/group compared with RA controls. Quantification of if in 10 fields of view (FOV) per section in two sections per animal, normalisation of positive cells to 100 nuclei; arrows point to positive cells. ISH quantification in 212.55 µm x 212.55 µm FOV. BMP, bone morphogenetic protein; BMPR2, BMP receptor 2; ISH, RNA in situ hybridization; PCLS, precision cut lung slices; VE, vascular endothelial; WT, wild type.