Hyperoxic Lung Injury in Newborn Mice is Abrogated by Neutralizing mAb 2A11 to Gastrin‐Releasing Peptide (GRP)

Abstract

RationaleGastrin‐releasing peptide (GRP) promotes fetal lung development in mice and baboons. Postnatally, GRP levels in lung normally drop, but rise in newborns who later develop bronchopulmonary dysplasia (BPD), chronic lung disease of newborns, with interstital fibrosis and arrested alveolarization. In premature baboons, blocking anti‐GRP mAb2A11 abrogated BPD. Rodent BPD models could help to clarify mechanisms by which GRP mediates BPD. Mice are born at the canalicular stage of lung development, ~24–28 wks human gestation, when BPD peaks.MethodsNewborn (NB) timed‐pregnant Swiss Webster mice were treated with continuous 75% O2 and twice weekly injections of mAb MOPC21 (negative control IgG1) or mAb2A11. On day 18, right lungs were inflation‐fixed and paraffin embedded. Myofibroblasts were immunostained with alkaline‐phosphatase‐conjugated mAb1A4 (Sigma‐Aldrich), developed with Vector Red. Eight‐ten random lung 20x images were captured per mouse. Using ImageJ, the area‐percent of SMA+ alveolar tissue was determined as: (area of SMA+ alveolae)/(alveolar tissue) ×100%. Mean linear intercept (MLI) was used to determine alveolar size with a fixed grid with lines totaling 1‐mm: 1/(#air‐tissue interfaces)×1000 = mean distance between two air‐tissue interfaces in microns. Data are shown as mean ±SEM, with significance at p < 0.05 (one‐way t‐test).ResultsNormoxic (NO) mice ± MOPC or 2A11, had area‐percent SMA+ alveoli unchanged (5.1–6.3 ±0.5–1.4). Hyperoxia+MOPC led to 3‐fold increased SMA (15.6±2%). Hyperoxia+2A11 reduced SMA to 10.3±1.2%; P<0.01 comparing hyperoxia+2A11 vs. hyperoxia+MOPC. 2A11 had no effect on MLI with either normoxia or hyperoxia, possibly due to insufficient time. We performed Affymetrix cDNA analysis of lung RNA from hyperoxic mice +2A11 or MOPC for 18d. HO+2A11 vs. HO+MOPC altered expression of >1000 genes (FC>10, P<0.05), many potentially protective vs. lung injury/fibrosis. Increased gene expression included: nucleoredoxin, an antioxidant related to thioredoxin (FC x78); surfactant protein A (FC x140); Tom20, mitochondrial transport protein that reduces lung injury (x74), and multiple other mitochondrial genes (>20x). Reduced gene expression included: Adam10, profibrotic; amphiregulin, elevated in BPD; amyloid beta A4, toxic fragment; laminin B1, which usually declines by birth; Tgfb1i4, profibrotic; and Stat1, profibrotic.ConclusionsAfter 18d exposure to 75% O2, newborn mice have >3‐fold more interstitial myofibroblasts that are significantly reduced by mAb2A11. Many pulmonary genes are up‐ or down‐regulated by mAb2A11 consistent with a multigenic downstream effect on reducing interstitial fibrosis via interacting signaling pathways. Mice can provide a useful model for carrying out preclinical investigations.Support or Funding InformationThis work was supported by NIH grants #R01‐HL44984 (M.E.S.), U01‐HL52638 (M.E.S.), and a Scientific Research Bequest from the Jenny Lillian Semans Koortbojian Trust Fund