Vitamin A and retinoic acid act synergistically to increase lung retinyl esters during normoxia and reduce hyperoxic lung injury in newborn mice

Abstract

We have shown that vitamin A (VA) and retinoic acid (RA) synergistically increase lung retinyl ester content in neonatal rats. To confirm whether this biochemical synergism attenuates early neonatal hyperoxic lung injury in mice, we exposed newborn C57BL/6 mice to 95% O2 or air from birth to 4 d. The agent [vehicle, VA, RA, or the combination vitamin A+retinoic acid (VARA)] was given orally daily. Lung and liver retinyl ester content was measured, and lung injury and development were evaluated. We observed that lung, but not liver, retinyl ester levels were increased more by VARA than by VA or RA alone. Hyperoxic lung injury was reduced by VA and RA, and more so by VARA. VARA attenuated the hyperoxia-induced increases in macrophage inflammatory protein (MIP)-2 mRNA and protein expression, but did not alter hyperoxia-induced effects on peptide growth factors (PDGF, VEGF, and TGF-beta1). The 4-d exposure to hyperoxia or retinoids did not lead to observable differences in lung development. We conclude that the VARA combination has synergistic effects on lung retinyl ester concentrations and on the attenuation of hyperoxia-induced lung injury in newborn mice, possibly by modulation of inflammatory mediators.

Figure 1. Lung and liver retinyl ester concentrations in neonatal mice exposed to room air or hyperoxia, while being treated with vehicle (Veh), vitamin A (VA) alone, retinoic acid (RA) alone, or vitamin A + retinoic acid (VARA)

Lung and liver RE (nmol/g) were analyzed on day 4. Panel A: Lung retinyl ester concentrations. [mean ± SEM; n per group shown at base of bar]. Panel B: Liver retinyl ester concentrations. [mean ± SEM of log₁₀ transformed data; n per group shown at base of bar] (*p<0.05 vs. corresponding Vehicle,† p<0.05 vs. corresponding VA, ‡ p<0.05 vs. corresponding RA, § p<0.05 vs. corresponding Air)

Figure 2. Photomicrographs of lungs from neonatal mice exposed to room air or hyperoxia while being treated with vehicle (Veh), vitamin A (VA) alone, retinoic acid (RA) alone, or vitamin A + retinoic acid (VARA)

(H&E stain; 400x; calibration bar = 50 μm; Br = Bronchus). Exposure to hyperoxia led to hemorrhage (erythrocytes within alveoli and in alveolar septae) and airway epithelial injury (denuded epithelial cells within Br) that were attenuated with VA, RA, and to a greater extent with VARA.

Figure 3. Cytokine gene expression and protein levels in lungs of neonatal mice treated with vehicle (Veh) or vitamin A + retinoic acid (VARA) while exposed to room air or hyperoxia

Gene expression and protein levels of cytokines in lung homogenates were analyzed on day 4 by competitive real-time PCR and by ELISA, respectively (Mean ±SEM, n= 6 animals per group; *p<0.05 vs. corresponding Vehicle, § p<0.05 vs. corresponding Air)

Figure 4. Peptide growth factor gene expression and protein concentration in lungs of neonatal mice treated with vehicle (Veh) or vitamin A + retinoic acid (VARA) while exposed to room air or hyperoxia

Gene expression and protein concentrations of PDGF, VEGF, and TGF-β1 in lung homogenates were analyzed on day 4 by competitive real-time PCR and by ELIS, respectively A. (Mean ±SEM, n= 6 animals per group; *p<0.05 vs. corresponding Vehicle, § p<0.05 vs. corresponding Air)