Antenatal endotoxin disrupts lung vitamin D receptor and 25-hydroxyvitamin D 1α-hydroxylase expression in the developing rat

Abstract

Vitamin D [vit D; 1,25-(OH)₂D] treatment improves survival and lung alveolar and vascular growth in an experimental model of bronchopulmonary dysplasia (BPD) after antenatal exposure to endotoxin (ETX). However, little is known about lung-specific 1,25-(OH)₂D₃ regulation during development, especially regarding maturational changes in lung-specific expression of the vitamin D receptor (VDR), 1α-hydroxylase (1α-OHase), and CYP24A1 during late gestation and the effects of antenatal ETX exposure on 1,25-(OH)₂D₃ metabolism in the lung. We hypothesized that vit D regulatory proteins undergo maturation regulation in the late fetal and early neonatal lung and that prenatal exposure to ETX impairs lung growth partly through abnormal endogenous vit D metabolism. Normal fetal rat lungs were harvested between embryonic day 15 and postnatal day 14. Lung homogenates were assayed for VDR, 1α-OHase, and CYP24A1 protein contents by Western blot analysis. Fetal rats were injected on embryonic day 20 with intra-amniotic ETX, ETX + 1,25-(OH)₂D₃, or saline and delivered 2 days later. Pulmonary artery endothelial cells (PAECs) from fetal sheep were assessed for VDR, 1α-OHase, and CYP24A1 expression after treatment with 25-(OH)D₃, 1,25-(OH)₂D₃, ETX, ETX + 25-(OH)D₃, or ETX + 1,25-(OH)₂D₃. We found that lung VDR, 1α-OHase, and CYP2741 protein expression dramatically increase immediately before birth (P < 0.01 vs. early fetal values). Antenatal ETX increases CYP24A1 expression (P < 0.05) and decreases VDR and 1α-OHase expression at birth (P < 0.001), but these changes are prevented with concurrent vit D treatment (P < 0.001). ETX-induced reduction of fetal PAEC growth and tube formation and lung 1α-OHase expression are prevented by vit D treatment (P < 0.001). We conclude that lung VDR, 1α-OHase, and CYP24A1 protein content markedly increase before birth and that antenatal ETX disrupts lung vit D metabolism through downregulation of VDR and increased vit D catabolic enzyme expression, including changes in developing endothelium. We speculate that endogenous vitamin D metabolism modulates normal fetal lung development and that prenatal disruption of vit D signaling may contribute to impaired postnatal lung growth at least partly through altered angiogenic signaling.

bronchopulmonary dysplasia (BPD), the chronic lung disease that follows preterm birth, continues to be the most common adverse outcome of prematurity despite ongoing advances in perinatal care (29). Surviving infants with BPD have sustained alterations in lung growth and structure, leading to abnormal lung function, recurrent respiratory exacerbation, exercise intolerance, and increased risk for pulmonary hypertension that persist beyond childhood (18–20). In addition to airway abnormalities, experimental models and lung pathology from severe BPD suggest that BPD is characterized by decreased distal alveolar and vascular development (22, 29, 51). Physiologically, Balinotti et al. (3) found decreased gas diffusion capacities but normal alveolar gas volumes in human infants with BPD, suggesting that decreased lung surface area with impaired alveolar development persists throughout infancy. However, mechanisms that lead to sustained abnormalities of lung function are poorly understood and effective preventive therapies are lacking.

Epidemiologic and experimental evidence suggests that inflammation induced by both prenatal (chorioamnionitis) and postnatal (infection, oxygen, and mechanical ventilation) events contribute to the pathogenesis of BPD (28, 32). Chorioamnionitis has been strongly associated with an imbalance in pro- and anti-inflammatory cytokines in tracheal fluid samples after birth (5, 47) and an increased risk for preterm infants to subsequently develop BPD (15, 24). However, clinical or histologic chorioamnionitis has not been uniformly confirmed in some studies, (1, 26, 33, 34, 46, 53, 54) suggesting that other factors may further modulate outcomes after exposure to prenatal inflammation. In animal models, antenatal exposure to endotoxin (ETX) can induce lung inflammation, cause significant neonatal mortality, and arrests subsequent alveolarization in infant rat lungs (36, 50, 52). However, the mechanisms through which antenatal inflammation leads to sustained disruption of lung growth in BPD or mechanisms that modulate the impact of inflammation on lung structure remain incompletely understood.

Recent studies have suggested an important role for vitamin D during lung development and support an emerging hypothesis that early maternal vitamin D deficiency may contribute to a high risk for subsequent asthma during infancy (6, 7, 9, 16, 58). In addition to data supporting the effect of vitamin D on modulating inflammation, past studies have recently shown that vitamin D directly regulates maturation of lung epithelium in the developing rat (39) and that vitamin D-deficient mice have decreased lung volume and alveolar number (58). Vitamin D further increases surfactant synthesis and secretion in alveolar type II cells (38) and inhibits airway smooth muscle proliferation (13). In addition, we have previously reported that antenatal vitamin D improved oxygenation and survival in newborn rat pups and enhanced late lung structure after exposure to intra-amniotic (IA) ETX in vivo (36). In addition to the maturational effects of vitamin D on the airway, our data further suggested that vitamin D has striking proangiogenic effects in the developing lung, which may be partly due to direct effects on endothelial growth(36).

Despite these observations, however, relatively little is known about lung-specific regulation of vitamin D metabolism, maturational changes in key signaling pathways involved with vitamin D production or responsiveness, and whether perinatal stress disrupts vitamin D metabolism in the fetal lung. Therefore, we hypothesized that vitamin D regulatory proteins are maturationally regulated in the late fetal and neonatal lung and that prenatal exposure to ETX may impair lung structure through disruption of endogenous vitamin D metabolism. We further hypothesized that abnormal vitamin D metabolism may contribute to the disruption of angiogenesis after ETX exposure in utero.

To begin to address these hypotheses, we sought to first examine maturational changes of key vitamin D regulatory enzymes, including 1α-hydroxylase (1α-OHase) or CYP27B1, the enzyme responsible for vitamin D activation, and CYP24A1, which is responsible for vitamin D catabolism, as well as changes in vitamin D receptor (VDR) expression in the fetal and early neonatal rat lung. We then studied whether antenatal ETX disrupts the normal maturational changes in vitamin D signaling in vivo and in fetal pulmonary artery endothelial cells (PAECs) in vitro.

MATERIALS AND METHODS

Animals

All procedures and protocols were approved by the Animal Care and Use Committee at the University of Colorado Heath Sciences Center. Timed pregnant Sprague-Dawley rats were purchased from Charles River Laboratories (Wilmington, MA) and maintained in room air at Denver's altitude (1,600 m; barometric pressure: 630 mmHg; inspired oxygen tension: 12 2 mmHg) for at least 1 wk before surgeries. Animals were fed ad libitum and exposed to day-night cycles alternatively every 12 h. The rats were fed a standard soy protein-free extruded rodent chow from Harlan Laboratories, which contains 1,500 IU/kg of vitamin D3. This chow is the standard chow fed to all rodents housed with in the vivarium facility at the University of Colorado. Rats were killed with an intraperitoneal injection of pentobarbital sodium (0.3 mg/g body wt; Fort Dodge Animal Health, Fort Dodge, IA).

Animal Model and Study Design

IA ETX administration.

For these studies, we used an established animal model of chorioamnionitis, as previously described (50). At 20 days of gestation (term: 22 days), pregnant rats were prepared for receiving IA injections. Five litters of rat pups received IA injections of either saline (1 litter-10 pups), ETX (2 litters-20 pups), or 1,25-(OH)₂D₃ + ETX as two separate injections (2 litters-20 pups). There were no significant differences in dam age or weight, and this was the first pregnancy for each dam. The average litter size for each dam is 10 pups, with a range of 9–12 pups/dam. After premedication with buprenorphine (0.01–0.05 mg/kg sc injection), laparotomy was performed under general anesthesia with 1–2% isoflurane inhalation via facemask (anesthesia machine: model VIP3000; Matrx by Midmark). Pregnant rats were randomly assigned to saline control (SA), ETX, or 1,25-(OH)₂D₃ group + ETX; the SA group received 50 μl of normal saline per amniotic sac, the ETX group received 10 μg of Escherichia coli 055:B55 ETX (Sigma Chemical, St. Louis, MO) diluted to 50 μl with normal saline per sac, and the 1,25-(OH)₂D₃ group received 50 pg diluted to 50 μl with normal saline. Under sterile preparation, a midline abdominal incision of 3–4 cm in length was made to expose the amniotic sacs for IA injections. The amniotic sac closest to the right ovary was first identified and injected, and then in a counterclockwise sequence each sac was identified and injected with a maximum of 10 sacs injected per dam. Injections were limited to 10 sacs to prevent maternal mortality due to systemic toxicities from accumulating doses of IA ETX.

The dose of ETX was established from previous studies in our laboratory that demonstrated ETX at lower doses (1–5 μg/sac) failed to induce abnormal lung structure at 14 days of age. The dose of vitamin D was established again from previous studies in our laboratory demonstrating vitamin D at higher doses (500 ng/g) produced subcutaneous calcium deposits noted in rat pups. The abdominal incision was closed with nylon sutures. Bupivacaine, 1–2 mg/kg im injection, used for postoperative pain control.

Cesarean section.

Two days after IA injections, cesarean section was performed on pregnant rats under general anesthesia with isoflurane inhalation, as described above. The fetus in the amniotic sac closest to the right ovary was first delivered, which was followed by delivery of the rest of the fetuses in a counterclockwise sequence, to identify fetuses exposed to IA injections. We performed cesarean sections instead of allowing vaginal deliveries to identify fetuses exposed to specific IA injections, based on the order of the amniotic sacs and their anatomic locations related to the ovaries. All of the rat pups in the injected amniotic sacs were delivered within 5 min after onset of anesthesia. Mother rats were then euthanized with pentobarbital sodium. Newborn rats were immediately dried and placed on a heating pad to avoid hypothermia. Pups received no supplemental oxygen or artificial ventilation at birth. Within 30 min after birth, pups were weighed and lungs were harvested for Western blot analysis.

Western Blot Analysis

Normal fetal rat lungs were harvested from timed pregnant Sprague-Dawley rats on gestation day 15 (n = 3), 18 (n = 3), 20 (n = 3), 22 (n = 3), and day of life 1 (n = 3), 7 (n = 4), and 14 (n = 4). Whole lung were also isolated from control (n = 5) and ETX (n = 5)-, ETX + 1,25-(OH)₂D₃ (n = 4)-, and 1,25-(OH)₂D₃ (n = 4)-exposed rats at birth and whole lung homogenates from all groups were for analyzed by Western blot analysis for 1α-OHase (CYP27B1; catalog no. AP9056b Abgent rabbit; 1:2,000 dilution), VDR (catalog no. SC1009 rabbit; Santa Cruz Biotechnology, Santa Cruz, CA 1:2,000 dilution), CYP24A1 (catalog no. SC66851 rabbit; 1:2,000 dilution; Santa Cruz Biotechnology), and β-actin (catalog no. A5316 mouse; Sigma, St. Louis, MO). Protein content was determined by the BCA assay (catalog no. 23225; Pierce Biotechnology, Rockford, IL), using bovine serum albumin as the standard. Whole lung homogenates were collected from four animals in each study group, and/or time point. A 20-mg protein sample per lane was resolved by SDS-PAGE, and proteins from the gel were transferred to PVDF membrane. After 1 h of blocking with 5% nonfat dehydrogenated milk, the blots were incubated with anti-CYP27B1, VDR, and CYP24A1 (as mentioned above) at 4°C overnight. Following overnight incubation blots were washed and subsequently incubated for 1 h at room temperature with goat anti-rabbit horseradish peroxidase-conjugate secondary antibody (catalog no. 170–6515; 1:10,000; Bio-Rad Technology). Bands of interest were visualized by enhanced chemiluminescence (ECL kit; Amersham Pharmacia Biotech, Buckinghamshire, UK) and identified by molecular weight as designated by the manufacturer for the protein of interest. All proteins of interest were normalized to β-actin (A2228 mouse; Sigma) as a loading control. Densitometry was performed using Image Lab (Bio-Rad Laboratories). Changes in protein expression were analyzed after normalizing for β-actin expression.

Fetal PAEC Isolation

Pulmonary PAECs were harvested from the proximal pulmonary arteries of late gestation control fetal sheep at day 135 (day 147 term), as previously described (23). Immunohistochemistry with standard endothelial markers confirmed the cell phenotype. Low-passage PAECs (p4-5) were then exposed to ETX, 25-(OH)D₃, 1,25-(OH)₂D₃, ETX + 25-(OH)D₃, or ETX + 1,25-(OH)₂D₃ in the experiments below.

Growth of PAECs during exposure to ETX and Vitamin D

Fetal PAECs were plated in triplicate at 50,000 cells/well in DMEM with 10% FBS into 12-well plates and allowed to adhere overnight in 21% oxygen. The following day (day 0) the cells were washed twice with PBS. DMEM with 2.5% FBS with VEGF, ETX, 25-(OH)D₃, 1,25-(OH)₂D₃, ETX + 1,25-(OH)₂D₃, or ETX + 25-(OH)D₃ was then added, and cells were incubated in 21% oxygen. Experimental media were changed daily and cells counted on day 3 after removing cells with 0.25% trypsin and counted with a cell counter (Beckman Coulter; Fullerton, CA). Growth studies with treatment were performed in DMEM with 2.5% FBS, based on previous studies that determined that this was the lowest serum concentration that supported fetal PAEC survival with some proliferation (23).

PAEC Tube Formation Assay

To assay in vitro angiogenesis, we cross-linked rat-tail collagen using 0.1% flavin mononucleotide and a UV Stratalinker 1800 (Stratagene). Then, 50,000 cells/well were added in serum free DMEM media supplemented with ETX, 25-(OH)D₃, 1,25-(OH)₂D₃, ETX + 1,25-(OH)₂D₃, or ETX + 25-(OH)D₃ and each condition was tested in triplicate for each animal. PAECs were then incubated for 12–18 h under 21% oxygen conditions based on previous studies (23). Branch-point counting was performed in blinded fashion under ×10 magnification from each of three wells with three to four field of view per well, as previously described. (23) Wells were imaged using an Olympus IX71 fluorescence microscope (Olympus).

Statistical Analysis

Data are presented as means ± SE. Statistical analysis was performed with the Prism software package (v. 5.0a; GraphPad). One-way ANOVA with Bonferroni posttest analysis were performed. P < 0.05 was considered significant.

RESULTS

Ontogeny of Regulators of Vitamin D Metabolism in the Lung

All results for the ontogeny of vitamin D metabolism in the lung are reported here as significant changes from embryonic day 15 (E15). The ontogeny for the vitamin D conversion enzyme (CYP27B1; 1α-OHase) in control fetal lungs demonstrates nondetectable expression on E15 with peak expression at term (E22; P < 0.01, E15 vs. E22; Fig. 1A) and postnatal day 1 (P < 0.01, E15 vs. ED1; Fig. 1A). Assessments of maturational changes in VDR expression demonstrate the presence of VDR protein in the late fetus (E15), with a 2.8-fold increase in expression on gestational day 18 (Fig. 1B; P < 0.05, E15 vs. E18; Fig. 1B). The vitamin D catabolism enzyme CYP24A1 demonstrates minimal expression on E15 and demonstrates a significant increase by E20 (P < 0.01) and E22 (P < 0.01; Fig. 1C). CYP24A1 decreases in expression before birth and undergoes an additional 75% decrease in expression on postnatal day 1 to values that are sustained through postnatal day 14.

Fig. 1.

A–C: developmental changes in vitamin D regulatory proteins in fetal and neonatal rat lungs. A: fetal expression of vitamin D conversion enzyme [CYP27B1; 1α-hydroxylase (1α-OHase)] increases late in fetal gestation with peak expression at birth and postnatal day 1 (P < 0.01). B: vitamin D receptor (VDR) demonstrates strong expression in late fetal life with peak expression on gestational day 18 (P < 0.01), with decreased expression on gestational day 20 (P < 0.01). C: vitamin D catabolism enzyme (CYP24A1) expression is present at day 18 with peak expression on gestation day 20 (P < 0.01) and decreases to fetal expression levels by postnatal day 1 (P < 0.01).

Effects of ETX on Vitamin D Metabolism in the Fetal Lung

Lung 1α-OHase expression increases nearly threefold in the newborn rat after intrauterine exposure to ETX + 1,25-(OH)₂D₃ compared with both control and ETX treatment groups (P < 0.001 for controls; P < 0.01 for ETX; Fig. 2A). Treatment with 1,25-(OH)₂D₃ increased 1α-OHase expression by 2.5-fold compared with both control and ETX groups (P < 0.01 for controls, P < 0.05 for ETX; Fig. 2A). VDR protein expression was decreased by 33% after ETX exposure (P < 0.01), which was prevented with cotreatment of 1,25-(OH)₂D₃ (P < 0.001). Fetal rats exposed to ETX + 1,25-(OH)₂D₃ demonstrated a 26% increase in lung VDR expression above that of controls (P < 0.01). In addition, 1,25-(OH)₂D₃ increased VDR protein by 40% when compared with rats exposed to antenatal ETX (P < 0.01; Fig. 2B). Lung expression of CYP24A1, the vitamin D catabolism enzyme, demonstrated a 2.2-fold increase after ETX exposure compared with controls (P < 0.01), ETX + 1,25-(OH)₂D₃, or 1,25-(OH)₂D₃ alone groups (P < 0.001; Fig. 2C).

Fig. 2.

A–C: effects of endotoxin (ETX) on vitamin D metabolism in the newborn rat lung. A: intra-amniotic (IA) ETX and vitamin D increases conversion enzyme expression (CYP27B1;1α-OHase; P < 0.001) compared with controls and IA ETX alone. B: IA ETX decreases lung VDR expression at birth (P < 0.01), which is prevented with vitamin D cotreatment (P < 0.001). C: IA ETX increases vitamin D catabolism enzyme expression compared with controls (P < 0.01), ETX alone, and vitamin D treatment alone (P < 0.001).

PAEC Growth Assay and Tube Formation

In control PAECs, 1,25-(OH)₂D₃ increased cell growth 25% above that of basal conditions (P < 0.001). Vascular endothelial growth factor (VEGF; 10 ng/ml) was used as a control for cell growth to illustrate the relative effects of 1,25-(OH)₂D₃ on cell growth, and as demonstrated 1,25-(OH)₂D₃ increased cell growth above that of control PAECs and also VEGF. 25-(OH)D₃-treated cells demonstrated a trend toward increasing cell growth, above that of controls, but did not reach significance. ETX exposure decreased PAEC growth by 53% in control cells (P < 0.001), while PAECs exposed to ETX who were also cotreated with either 25-(OH)D₃ or 1,25-(OH)₂D₃ demonstrated improved cell growth by 45% to levels similar to those of control cells [P < 0.001 for 25-(OH)D₃ + ETX and 1,25-(OH)₂D₃ + ETX compared with ETX; Fig. 3A]. 1,25-(OH)₂D₃-treated PAECs increased PAEC tube formation by 25% above that of control cells (P < 0.01), whereas 25-(OH)D₃ treatment did not affect PAEC tube formation. ETX exposure decreased tube formation by 55% (P < 0.001); this effect was attenuated by 83% with 25-(OH)D₃ coadministration and twofold increase in PAEC tube formation with 1,25-(OH)₂D₃ treatment (P < 0.001; Fig. 3B).

Fig. 3.

A and B: effects of 25-(OH)D₃ and 1,25-(OH)₂D₃ on PAEC growth (A) and network (tube) formation (B). In control PAECs, the precursor form of vitamin D [25-(OH)D₃] and 1,25-(OH)₂D₃ treatment increases PAEC growth above control values (P < 0.001) and to similar values achieved with VEGF treatment. PAEC growth is reduced by 53% during ETX treatment (P < 0.001), which is attenuated with 1,25-(OH)₂D₃ or 25-(OH)D₃ treatment (P < 0.001). 1,25-(OH)₂D₃ increases PAEC tube formation by 25% (P < 0.01; B). ETX exposure decreases tube formation (P < 0.001), and 1,25-(OH)₂D₃ or 25-(OH)D₃ treatment increases tube formation during ETX exposure (P < 0.001).

Effects of ETX on Vitamin D Metabolism in Fetal PAECs

Fetal PAECs exposed to ETX demonstrated a 50% decrease in 1α-OHase expression compared with control cells (P < 0.001). 1,25-(OH)₂D₃ treatment alone increased 1α-OHase expression slightly above that of controls (P = 0.05) and 2.3-fold increase compared with ETX alone (P < 0.01). Cells cotreated with 1,25-(OH)₂D₃ demonstrated expression that was restored to that of control levels compared with ETX alone (P < 0.01; Fig. 4A). VDR expression showed a 55% decrement in PAECs exposed to ETX compared with controls (P < 0.001). PAECs treated with 1,25-(OH)₂D₃ expressed VDR at equivalent levels to controls, which represented a 55% increase to those of PAECs exposed to ETX alone (P < 0.01). PAECs exposed to ETX + 1,25-(OH)₂D₃ demonstrated a 42% increase in expression of VDR compared with ETX alone. (P < 0.001; Fig. 4B). CYP24A1 expression decreased 66% in PAECs exposed to ETX compared with controls (P < 0.001). 1,25-(OH)₂D₃ treatment increased CYP24A1 expression 8-fold compared with ETX alone (P < 0.001) and 2.5-fold from control levels (P < 0.001). PAECs exposed to ETX + 1,25-(OH)₂D₃ demonstrated 8-fold increase in CYP24A1 expression compared with ETX alone (P < 0.001) and 2.6-fold increase compared with controls (P < 0.001; Fig. 4C).

Fig. 4.

A–C: effects of 1,25-(OH)₂D₃ and ETX on pulmonary artery endothelial cells (PAECs) vitamin D regulatory proteins. ETX decreases vitamin D conversion enzyme (CYP27B1;1α-OHase) expression by 50% (P < 0.001), which is prevented with cotreatment of 1,25-(OH)₂D₃. A: ETX decreases vitamin D receptor expression (P < 0.01). B: treatment with 1,25-(OH)₂D₃ during ETX exposure prevents the decrement in VDR expression (P < 0.01). C: ETX decreases vitamin D catabolism expression CYP24A1 (P < 0.001) whereas cotreatment with 1,25-(OH)₂D₃ increases CYP24A1 expression 8-fold (P < 0.001).

DISCUSSION

Recent studies have suggested that exogenous vitamin D modulates airway epithelial and vascular growth in utero, but lung-specific expression and maturational changes of key vitamin D regulatory proteins have not been previously studied. Similarly, whether prenatal stresses, such as intrauterine inflammation, alter fetal lung vitamin D metabolism remains unknown. In this study, we found that the key vitamin D regulatory enzymes that govern vitamin D activity, 1α-OHase, VDR, and CYP24A1, are strongly expressed in the late fetal lung and undergo striking developmental regulation immediately before birth. In addition, antenatal ETX exposure decreases VDR and increases CYP24A1 protein expression in the late fetal lung. These findings suggest that vitamin D potentially plays a role in normal lung development and that antenatal ETX-induced disruption of lung development may be partly due to disruption of vitamin D signaling. In addition, we found that ETX disrupts normal fetal PAEC growth and tube formation in vitro, which is associated with decreased VDR and increased CYP24A1 protein expression. These adverse effects on vitamin D regulatory protein expression with ETX exposure are prevented with 25-(OH)D₃ treatment.

Overall, these findings support the hypothesis that intrauterine inflammation may disrupt normal lung development at least partly through downregulation of key vitamin D regulatory proteins that favor decreased conversion to active vitamin D and decreased vitamin D receptor expression. These findings further demonstrate that normal fetal PAECs have site-specific vitamin D conversion enzyme activity, that the developing lung is capable of endothelial cell-specific activation of vitamin D, and that vitamin D may preserve angiogenesis after antenatal lung injury.

These data are the first to suggest a potential role of vitamin D during the late stages of fetal lung development and that antenatal ETX exposure alters expression of key regulatory vitamin D enzymes, which points to a potential mechanism for ETX-related abnormal lung development through vitamin D deregulation. While previous studies have demonstrated increasing concentrations of total fetal vitamin D content and major metabolites from gestational days 14–22 in fetal rats (12), we demonstrate lung-specific changes in vitamin D regulatory enzymes with advancing gestation in this present study. Our data further demonstrate that fetal PAECs are specifically capable of vitamin D activation, as 25-OHD improves growth and tube formation and rescues endothelial cell dysfunction during ETX exposure. Endothelial dysfunction is seen in a variety of conditions that adversely impact the cardiovascular system, including hypertension, diabetes mellitus, hypercholesterolemia, and chronic renal failure, conditions that are also known to be associated with vitamin D deficiency (11, 17, 25, 40, 43, 44, 56). These findings suggest a potential link between endothelial dysfunction and impaired vitamin D.

The role of vitamin D in normal lung development has gained support from epidemiologic studies of maternal vitamin D deficiency that suggest adverse outcomes including impaired lung function and increased childhood asthma (59), preterm birth and small for gestation newborns (55), preeclampsia (35, 48), and gestational diabetes (55). Importantly, each of these conditions has been associated with lung disease in premature and term newborns that may extend throughout childhood. Vitamin D has been previously shown to enhance maturation of alveolar type II cells in the fetal lung (37–39, 45). In addition, past studies have shown that vitamin D enhances infant lung growth and structure in a model of chorioamnionitis induced by antenatal ETX exposure and that these effects include preservation of lung vascular growth (36). Recently, Zosky et al. (58) found significantly higher airway resistance and lower lung volume of 2-wk-old, vitamin D-deficient mice and (58). Onwuneme et al. (42) reported a significant association between vitamin D status in 94 preterm births at birth with the need for early respiratory support and the need for assisted ventilation on admission to the neonatal intensive care unit.

Our results support a potential role for endogenous vitamin D activity during normal lung development, especially during late fetal life, as key vitamin D regulatory enzymes are upregulated just before birth in rats. We report that VDR and CYP27B1 are markedly increased in the normal fetal lung at gestational day 18 (for VDR) and day 20 and birth (for CYP27B1), correlating to the end of the cannalicular phase and the early saccular phase of lung development (41). We further demonstrate that IA ETX administration at gestational day 20 decreases VDR expression in the fetal rat lung and causes abnormal lung structure with alveolar simplification, decreased vessel density, and pulmonary hypertension at 2 wk of life (36, 50).

Mechanisms responsible for the disruption of lung growth seen with a single IA ETX injection may be partially mediated through disruption of vitamin D metabolism and signaling. As reported in this study, key vitamin D regulatory enzymes increase shortly before birth, and that ETX exposure during this window decreased expression of VDR and increases catabolism enzyme CYP24A1 expression. The CYP24A1 gene has very low basal expression but is upregulated by 1,25-(OH)₂D₃ through two vitamin response elements (VDRE) in the proximal promoter region (2, 10, 57). A potential mechanism for disruption of lung growth may be related increased metabolism of vitamin D by ETX-induced upregulation of the CYP24A1 gene potentially through binding of the two VDRE in the proximal promoter region. Reduced VDR expression may potentially decrease vitamin D activity and lower expression of downstream genes that are important in mediating lung growth. These include genes that are directly upregulated (CYP24A1, Rankl, and VEGF) or downregulated (PTH, CYP27B1) through VDR activation (4, 30, 31). Future studies are needed to more directly explore how disruption of vitamin D production may specifically impair fetal lung growth, especially in experimental models of BPD.

Potential limitations of the current study include the lack of vitamin D levels in fetal rats due to inaccessibility of blood samples for performing these assays. As we did not check vitamin D levels in our animals we cannot say they were in fact vitamin D sufficient. However, there is ample previous studies to support rodent vitamin D sufficiency-fed diets as low as 400-1,000 IU/kg (14, 21, 27), which is below the content of the chow used in these experiments, led to sufficient 25-(OH)D₃ levels. Additionally, our rats were maintained under conventional housing conditions, thus exposed to UVB light allowing for basal dermal synthesis of vitamin D. Given a vitamin D-sufficient diet and normal UVB exposure we assume our animals were vitamin D sufficient during these studies. In addition, although the use of ETX is an established animal model used to study the effects of chorioamnionitis on fetal lung development, it is unlikely that this model is sufficient to fully reflect the complexity of chorioamnionitis in human disease. Although our data provide further support regarding the direct effect of ETX on lung vascular growth in the absence of inflammatory cells, additional work is needed to better explore specific inflammatory and noninflammatory mechanisms that disrupt vitamin D regulation and the downstream consequences of decreased vitamin D. Finally, while we recognize the potential limitation of combining in vitro studies of fetal PAECs from sheep with in vivo rat experiments, fetal PAECs more closely reflect the developmental timing and lung-specific source that are particularly relevant to these study questions. Past studies have used high-performance liquid chromatography (HPLC) to assess conversion activity of cells transfected with CYP27B1 expression vector through detection of 1,25-(OH)₂D₃ in the incubated medium (49). Since VDR binds to 1,25-(OH)₂D₃ with 150 times the affinity as 25-(OH)D₃ (8), the effects of exogenous 25-(OH)D₃ in our studies mediated by its conversion to 1,25-(OH)₂D₃ by CYP27B1 in PAECs.

Thus we conclude that lung vitamin D regulatory protein expression increases during late fetal life and that antenatal ETX alters vitamin D regulatory protein expression in the developing lung, favoring less vitamin D conversion and VDR activation. Additionally, we found that fetal PAECs express vitamin D regulatory proteins and can locally activate vitamin D and that vitamin D treatment preserves CYP27B1 and VDR expression in fetal PAECs during ETX exposure. We speculate that vitamin D regulatory proteins contribute to normal development of the fetal rat lung and are impaired with antenatal injury due to endotoxin and the developing lung circulation is capable of endothelial cell-specific activations of vitamin D, which may preserve angiogenesis after perinatal lung injury.

GRANTS

This work was supported in part by National Heart, Lung, and Blood Institute Grants T32-HL-07670 and HL-68702, the Marshall-Klaus Grant from the American Academy of Pediatrics, Gilead Research Scholar Program, and Children's Hospital Colorado Research Institute.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

E.M., G.J.S., J.G., S.D.C., and S.H.A. conception and design of research; E.M., G.J.S., and S.L.R. performed experiments; E.M., S.L.R., and J.G. analyzed data; E.M., J.G., S.D.C., and S.H.A. interpreted results of experiments; E.M. and S.L.R. prepared figures; E.M. drafted manuscript; E.M., G.J.S., S.L.R., J.G., S.D.C., and S.H.A. edited and revised manuscript; E.M., G.J.S., S.L.R., J.G., S.D.C., and S.H.A. approved final version of manuscript.