Antenatal mesenchymal stromal cell extracellular vesicle treatment preserves lung development in a model of bronchopulmonary dysplasia due to chorioamnionitis

Abstract

Antenatal stressors such as chorioamnionitis (CA) increase the risk for bronchopulmonary dysplasia (BPD). Studies have shown that experimental BPD can be ameliorated by postnatal treatment with mesenchymal stromal cell-derived extracellular vesicles (MEx). However, the antenatal efficacy of MEx to prevent BPD is unknown. To determine whether antenatal MEx therapy attenuates intrauterine inflammation and preserves lung growth in a rat model of CA-induced BPD. At embryonic day (E)20, rat litters were treated with intra-amniotic injections of saline, endotoxin (ETX) to model chorioamnionitis, MEx, or ETX plus MEx followed by cesarean section delivery with placental harvest at E22. Placental and lung evaluations were conducted at day 0 and day 14, respectively. To assess the effects of ETX and MEx on lung growth in vitro, E15 lung explants were imaged for distal branching. Placental tissues from ETX-exposed pregnancies showed increased expression of inflammatory markers NLRP-3 and IL-1ß and altered spiral artery morphology. In addition, infant rats exposed to intrauterine ETX had reduced alveolarization and pulmonary vessel density (PVD), increased right ventricular hypertrophy (RVH), and decreased lung mechanics. Intrauterine MEx therapy of ETX-exposed pups reduced inflammatory cytokines, normalized spiral artery architecture, and preserved distal lung growth and mechanics. In vitro studies showed that MEx treatment enhanced distal lung branching and increased VEGF and SPC gene expression. Antenatal MEx treatment preserved distal lung growth and reduced intrauterine inflammation in a model of CA-induced BPD. We speculate that MEx may provide a novel therapeutic strategy to prevent BPD due to antenatal inflammation.

Keywords: bronchopulmonary dysplasia; chorioamnionitis; endotoxin; lung development; mesenchymal stromal cell-derived extracellular vesicles.

Figure 1.

Experimental model of chorioamnionitis (CA) caused by intra-amniotic injections of endotoxin (ETX). Timeline to illustrate the study design used for determining the antenatal effects of MEx treatment on lung structure and mechanics and placental modification in infant rats (n = 30 for each experimental group). E, embryonic day; MEx, mesenchymal stromal cell-derived extracellular vesicles; MLI, mean linear intercept; MWT, medial wall thickness; PVD, pulmonary vessel density; RAC, radial alveolar count.

Figure 2.

Effects of antenatal endotoxin (ETX) exposure and MEx treatment on placental spiral artery modification by cytokeratin positive trophoblast cells. Histological analysis of placental tissues at time of delivery (E22). A: low power view of spiral artery morphology Red: cytokeratin, blue: hematoxylin counter stain. Scale bars: 20 µm. B: high-power view of spiral artery morphology. Red: cytokeratin, blue: DAPI counterstain. Scale bars: 50 µm. C: quantification of percentage of transformed blood vessels (i.e., spiral arteries with cytokeratin positive trophoblast cells lining >50% of vessel lumen) per ×40 visual field. D: total blood vessels analyzed per ×40 visual field. BV, blood vessel. **P < 0.01; ***P < 0.001, n = 5 or 6 pregnant dams/experimental group, counts averaged from 3 placental tissues/pregnant dam. E, embryonic day; MEx, mesenchymal stromal cell-derived extracellular vesicles.

Figure 3.

Effects of antenatal ETX exposure and MEx treatment on placental inflammatory signaling. RT-PCR analysis of placental tissues at time of delivery (E22). A: NLRP-3 gene expression. B: IL-1β expression. **P < 0.01, *P < 0.05, n = 4 pregnant dams/experimental group, values averaged from 3 placental tissues/pregnant dam. ETX, endotoxin; MEx, mesenchymal stromal cell-derived extracellular vesicles.

Figure 4.

Effects of antenatal MEx treatment on distal lung structure in infant rats after antenatal intra-amniotic injection of endotoxin (ETX) in experimental chorioamnionitis (CA). A: antenatal exposure to ETX causes lung simplification as demonstrated by reduced alveolarization, while rats treated with ETX and MEx have preserved lung structure. B: quantification with mean linear intercept (MLI) and radial alveolar counts (RACs). Micrographs are representative and were obtained at the ×10 magnification (scale bars: 250 μm). *P < 0.05 vs. ETX, n = 12–20 pups/experimental group, values averaged from 5 or 6 lung tissue samples/pup. MEx, mesenchymal stromal cell-derived extracellular vesicles.

Figure 5.

Effects of antenatal MEx treatment on lung vascularization in infant rats after antenatal intra-amniotic injection of endotoxin (ETX) in experimental chorioamnionitis (CA). Antenatal exposure to ETX causes simplification of pulmonary vessel density (A) and medial wall thickness (B), which is improved with administration of MEx. C: quantification with pulmonary vessel density (PVD), medial wall thickness (MWT), and right ventricular hypertrophy (RVH). Micrographs are representative and were obtained at ×10 magnification (scale bars: 250 μm) for PVD. The ×40 objective (scale bars: 50 µm) was used for MWT. *P < 0.05 vs. ETX, n = 12–17 pups/experimental group for PVD and MWT and n = 30–45 pups/group for RVH, values averaged from 5 or 6 lung tissue samples/pup. MEx, mesenchymal stromal cell-derived extracellular vesicles.

Figure 6.

Effects of antenatal MEx treatment on lung mechanics after intra-amniotic exposure to ETX. Exposure to ETX decreased lung compliance and increased resistance in infant rats at 2 wk of age, while injections with MEx preserved lung mechanics. Antenatal injections of MEx decreased total respiratory system resistance (A) and increased compliance (B), to a level that did not differ significantly from saline controls. *P < 0.05 vs. ETX, n = 25–35 pups/experimental group, values averaged over at least 3–6 nonfailed attempts. ETX, endotoxin; MEx, mesenchymal stromal cell-derived extracellular vesicles.

Figure 7.

Effects of in vitro MEx treatment on distal alveolar branching after exposure to ETX. A: exposure to ETX decreased distal lung branching in in vitro lung explants at study end point day 3. Daily administration of MEx preserved distal branching to levels consistent with saline controls. B: quantification of distal branching for control, ETX, ETX + MEx, and MEx alone, n = at least 8 explants/experimental group. C: changes in fetal lung SPC and VEGF gene expression in fetal lung explants from control (CTL), MEx, ETX, and ETX + MEx groups. *P < 0.05 vs. ETX, n = 3 samples/experimental group. ETX, endotoxin; MEx, mesenchymal stromal cell-derived extracellular vesicles.