Maternal Vitamin D Deficiency Causes Sustained Impairment of Lung Structure and Function and Increases Susceptibility to Hyperoxia-induced Lung Injury in Infant Rats

Abstract

Vitamin D deficiency (VDD) during pregnancy is associated with increased respiratory morbidities and risk for chronic lung disease after preterm birth. However, the direct effects of maternal VDD on perinatal lung structure and function and whether maternal VDD increases the susceptibility of lung injury due to hyperoxia are uncertain. In the present study, we sought to determine whether maternal VDD is sufficient to impair lung structure and function and whether VDD increases the impact of hyperoxia on the developing rat lung. Four-week-old rats were fed VDD chow and housed in a room shielded from ultraviolet A/B light to achieve 25-hydroxyvitamin D concentrations <10 ng/ml at mating and throughout lactation. Lung structure was assessed at 2 weeks for radial alveolar count, mean linear intercept, pulmonary vessel density, and lung function (lung compliance and resistance). The effects of hyperoxia for 2 weeks after birth were assessed after exposure to fraction of inspired oxygen of 0.95. At 2 weeks, VDD offspring had decreased alveolar and vascular growth and abnormal airway reactivity and lung function. Impaired lung structure and function in VDD offspring were similar to those observed in control rats exposed to postnatal hyperoxia alone. Maternal VDD causes sustained abnormalities of distal lung growth, increases in airway hyperreactivity, and abnormal lung mechanics during infancy. These changes in VDD pups were as severe as those measured after exposure to postnatal hyperoxia alone. We speculate that antenatal disruption of vitamin D signaling increases the risk for late-childhood respiratory disease.

Keywords: hyperoxia; lung development; maternal vitamin D deficiency; vitamin D deficiency.

Figure 1.

Vitamin D deficiency model characteristics. Serum 25(OH)D levels were significantly lower in the vitamin D deficiency (VDD) dams as compared with control dams (P < 0.01). Offspring from VDD dams had serum 25(OH)D levels significantly lower than control pups at 14 days of life (P < 0.01). Birth weights between VDD offspring and control newborn pups were not different (P = ns). At 14 days of life VDD offspring had lower body weights as compared with control pups (P < 0.01). Values are mean ± SEM; one-way ANOVA used for all comparisons. n = 9–15 animals for each group. *P < 0.0001. Error bars are 100 μm. 25-OHD = 25-hydroxyvitamin D; CTL = control; ns = not significant.

Figure 2.

Serum profile of calcium axis. Serum calcium levels in VDD dams and their offspring were not different as compared with control animals (P = ns). Serum phosphorous levels were not statistically different between VDD and control dams (P = ns). Serum phosphorous levels in VDD offspring were higher as compared with controls (P < 0.05). Serum parathyroid levels were not significantly different between VDD offspring and controls (P = ns). Values are mean ± SEM. Two-way ANOVA used for all comparisons. n = 4 samples for each group. Error bars are 100 μm. *P < 0.05.

Figure 3.

Effects of maternal VDD and postnatal hyperoxia on distal lung structure at Day 14. (A) Lung histology from CTL-RA and VDD-RA are in the top panels, and CTL-HX and VDD-HX are in the bottom panels. (B) Quantification of the lung structure of vitamin D deficient offspring at Day 14. VDD-RA pups had lower RAC as compared with CTL-RA (P < 0.01). VDD-HX had decreased RAC compared with both VDD-RA and CTL-HX (P < 0.01). MLI was increased in VDD-RA offspring as compared with CTL-RA (P < 0.01), and MLI was increased in VDD-HX compared with CTL-HX and also VDD-RA (P < 0.01). Values are mean ± SEM. Two-way ANOVA used for all comparisons. n = 8–12 samples for each group. *P < 0.01 versus CTL-RA, ^#P < 0.01 versus VDD-RA, and ^‡P < 0.01 versus CTL-HX. Scale bars: 100 μm. Fi_O2 = fraction of inspired oxygen; HX = postnatal hyperoxia exposure; MLI = mean linear intercept; RA = room air; RAC = radial alveolar counts.

Figure 4.

Distal pulmonary vessel density in lung of VDD offspring and postnatal hyperoxia. As compared with CTL-RA pups, we found decreased pulmonary vessel density (PVD) in VDD-RA pups, CTL-HX pups, and VDD-HX pups (P < 0.001 for each group). In addition, there was decreased PVD in VDD-HX pups as compared with both VDD-RA and CTL-HX pups (P < 0.001 for each). Values are mean ± SEM; one-way ANOVA was used for all comparisons. n = 8–15 animals for each group. *P < 0.001 versus CTL, ^#P < 0.001 versus VDD-RA, and ^‡P < 0.001 versus CTL-HX. HPF = High power field. Scale bars: 100 μm.

Figure 5.

Vessel wall thickness in VDD offspring. VDD-RA pups had increased vessel wall thickness compared with CTL-RA pups (P < 0.01). CTL-HX pups had increased vessel wall thickness compared with CTL-RA and VDD-RA pups (P < 0.01 and P < 0.05, respectively). Values are mean ± SEM; one-way ANOVA used for all comparisons. n = 8-15 animals for each group. *P < 0.01 versus CTL and ^#P < 05 versus VDD. Scale bars: 50 μm.

Figure 6.

(A) Effects of maternal VDD on lung function at Day 14. VDD-RA pups had increased resistance as compared with CTL-RA pups (P < 0.05). Both CTL-HX and VDD-HX pups had increased resistance compared with CTL-RA pups (P < 0.05). VDD-RA pups had decreased compliance compared with CTL-RA pups (P < 0.05). Both CTL-HX and VDD-HX pups had decreased compliance compared with CTL-RA pups (P < 0.05). Values are mean ± SEM; one-way ANOVA used for all comparisons. n = 10–15 animals for each group. *P < 0.05 versus CTL. (B) Effects of maternal VDD on lung function at Day 14. VDD-RA, CTL-HX, and VDD-HX pups had increased resistance in response to MCH challenge compared with CTL-RA pups (P < 0.01). VDD-RA, CTL-HX, and VDD-HX pups had decreased compliance in response to MCH challenge compared with CTL-RA pups (P < 0.01). Values are mean ± SEM; one-way ANOVA used for all comparisons. n = 10–15 animals for each group. *P < 0.01 versus CTL. MCh = methacholine.

Figure 7.

(A) Effects of maternal VDD and postnatal hyperoxia on mRNA expression of VEGF, KDR, HIF-1α, and VDR in whole lung homogenates. VEGF: VDD-RA pups had decreased VEGF mRNA as compared with CTL-RA pups (P < 0.05). CTL-HX and VDD-HX pups had decreased VEGF mRNA fold change as compared with CTL-RA pups (P < 0.05). KDR: Pups from CTL-HX and VDD-HX groups had a decrease in KDR mRNA compared with both CTL-RA and VDD-RA pups (P < 0.05). HIF-1α: VDD-RA pups had a small but nonsignificant decrease in HIF-1α mRNA expression compared with CTL-RA pups (P = ns). CTL-HX and VDD-HX pups had decreased HIF-1α mRNA compared with CTL-RA pups (P < 0.05). VDR: Pups from CTL-HX and VDD-HX groups had decreased VDR mRNA expression as compared with CTL-RA and VDD-RA pups (P < 0.05). Values are mean ± SEM; one-way ANOVA used for all comparisons. n = 4 animals for each group. ^#P < 0.05 versus CTL and *P < 0.05 versus VDD. (B) Effects of maternal VDD and postnatal hyperoxia on protein expression of VEGF, KDR, HIF-1α, and VDR in whole lung homogenates. VEGF: VDD-RA pups did not have a change in VEGF protein expression compared with CTL-RA pups. CTL-HX and VDD-HX pups had a decrease in VEGF protein expression as compared with CTL-RA pups (P < 0.01 for each). KDR: Pups from CTL-HX and VDD-HX had decreased KDR expression compared to CTL-RA pups (P < 0.001 for each). As compared with VDD-RA pups, both CTL-HX and VDD-HX pups had decreased KDR expression (P < 0.05 for each). HIF-1α: VDD-RA pups had decreased HIF-1α expression as compared with CTL-RA pups (P < 0.05). In addition, VDD-HX pups also had decreased HIF-1α protein expression as compared with CTL-RA pups (P < 0.05). VDR: As compared with CTL-RA, CTL-HX and VDD-HX pups had decreased VDR expression (P < 0.05). Values are mean ± SEM; one-way ANOVA used for all comparisons. n = 4 animals for each group. ^#P < 0.01 versus CTL. HIF-1α = hypoxia-inducible factor-1 α; KDR = vascular endothelial growth factor 2; VEGF = vascular endothelial growth factor; VDR = vitamin D receptor.