Antenatal inflammation reduces expression of caveolin-1 and influences multiple signaling pathways in preterm fetal lungs

Abstract

Bronchopulmonary dysplasia (BPD), associated with chorioamnionitis, results from the simultaneous effects of disrupted lung development, lung injury, and repair superimposed on the developing lung. Caveolins (Cavs) are implicated as major modulators of lung injury and remodeling by multiple signaling pathways, although Cavs have been minimally studied in the injured developing lung. We hypothesized that chorioamnionitis-associated antenatal lung inflammation would decrease the expression of Cav-1 in preterm fetal lungs. We tested whether changes occurred in the transcription factors Smad2/3, Smad1/5, Stat3, and Stat1, and we also studied the activation of acid-sphingomyelinase (a-SMase) with the generation of ceramide, along with changes in the expression of heme oxygenase-1 (HO-1) as indicators of possible Cav-1-mediated effects. Fetal sheep were exposed to 10 mg of intra-amniotic endotoxin or saline for 2, 7, or 2 + 7 days before preterm delivery at 124 days of gestation. The expression of Cav-1 and HO-1 and the phosphorylation of Smad and Stat were evaluated by real-time PCR, Western blotting, and/or immunohistochemistry. The activity of a-SMase and the concentrations of ceramide were measured. Intra-amniotic endotoxin decreased Cav-1 mRNA and protein expression in the lungs, with a maximum reduction of Cav-1 mRNA to 50% ± 7% of the control value (P < 0.05), and of Cav-1 protein expression to 20% ± 5% of the control value (P < 0.05). Decreased concentrations of Cav-1 were associated with the elevated phosphorylation of Smad2/3, Stat3, and Stat1, but not of Smad1/5. The expression of HO-1, a-SMase activity, and ceramide increased. Antenatal inflammation decreased the expression of Cav-1 in the preterm fetal lung. The decreased expression of Cav-1 was associated with the activation of the Smad2/3, Stat, and a-SMase/ceramide pathways, and with the increased expression of HO-1. The decreased concentrations of Cav-1 and changes in other signaling pathways may contribute to BPD.

Figure 1.

Study design. Six to seven animals per group received ultrasound-guided intraamniotic injections with 10 mg LPS or NaCl 0.9% (control), 2 days, 7 days, or 2 + 7 days before delivery at the same gestational age of 124 days. d, days.

Figure 2.

Intra-amniotic exposure to LPS decreases expression of caveolin-1 (Cav-1). (A) Real-time PCR measurements of Cav-1 mRNA expression in whole-lung homogenates. Mean fold change in lung mRNA expression of Cav-1 normalized for ovine ribosomal protein S15 by the ΔΔC_t method. (B) Western blot measurements of Cav-1 protein expression with an anti–Cav-1 antibody. The same membrane was analyzed with anti–β-actin antibody. (C) Concentrations of Cav-1 and β-actin protein were semiquantified by densitometry. Optical density of Cav-1 protein band was corrected to β-actin, and results are expressed as ratio (%) of endotoxin-exposed animals to control animals. (D–F) Immunohistochemical evaluation of Cav-1 expression in preterm lung tissue. Sections are representative of a control animal (D) and an animal exposed to LPS-induced chorioamnionitis for 2 days (E), stained for Cav-1. Magnification, ×50. (F) Immunostaining for Cav-1 was graded on a scale from 0–3. Values are means ± SE. *P < 0.05, versus control group.

Figure 3.

Effect of intra-amniotic exposure to LPS on Smad signaling. The phosphorylation of Smad2/3 (A–C) and Smad1/5 (D) was evaluated in lung tissue by immunohistochemistry. Sections are representative of a control animal (A) and an animal exposed to LPS-induced chorioamnionitis for 7 days (B), stained for phosphorylated Smad2/3. Magnification, ×50. Inset shows higher magnification, and arrow identifies nuclear staining for phosphorylated Smad2/3. (C) Immunohistochemical semiquantification of phosphorylated Smad2/3 in lung sections. (D) Immunohistochemical semiquantification of phosphorylated Smad1/5 in lung sections. Immunostaining for phosphorylated (P) Smad2/3 or Smad1/5 was graded on a scale from 0–3. Values represent means ± SE. *P < 0.05, versus control group.

Figure 4.

Effect of intra-amniotic exposure to LPS on Stat3 signaling. (A) Western blot measurements of Stat3 phosphorylation with an anti-phosphorylated Stat3 antibody. The same membrane was analyzed with anti–β-actin antibody. Phosphorylated Stat3 and β-actin protein concentrations were semiquantified by densitometry. (B) Optical density of phosphorylated Stat3 protein bands was corrected to β-actin, and results are expressed as the ratio (%) of LPS-exposed animals to control animals. (C–E) Evaluation of Stat3 phosphorylation in lung tissue by immunohistochemistry. Sections are representative of a control animal (C) and an animal exposed to LPS-induced chorioamnionitis for 7 days (D), stained for phosphorylated Stat3. Magnification, ×50. Inset shows higher magnification, and arrow identities nuclear staining for phosphorylated Stat3. (E) Immunohistochemical semiquantification of phosphorylated Stat3 in lung sections. Values represent means ± SE. *P < 0.05, versus control group.

Figure 5.

Effect of intra-amniotic exposure to LPS on Stat1 signaling. (A) Western blot measurements of Stat1 phosphorylation with an anti-phosphorylated Stat1 antibody. The same membrane was analyzed with anti–β-actin antibody. Concentrations of phosphorylated Stat1 and β-actin protein were semiquantified by densitometry. (B) Optical density of phosphorylated Stat1 protein band was corrected for β-actin, and results are expressed as the ratio (%) of LPS-exposed animals to control animals. Values represent means ± SE. *P < 0.05, versus control group. (C–E) Evaluation of Stat1 phosphorylation in lung tissue by immunohistochemistry. Representative sections from a control animal (C) and an animal exposed to LPS-induced chorioamnionitis for 2 days (D), stained for phosphorylated Stat1. Magnification, ×50. Inset shows higher magnification, and arrow indicates nuclear staining for phosphorylated Stat1. (E) Immunohistochemical semiquantification of phosphorylated Stat1 in lung sections. Values represent means ± SE. *P < 0.05, versus control group.

Figure 6.

Effects of intra-amniotic exposure to LPS on acid-sphingomyelinase (a-SMase) activity and ceramide. (A) Measurements of a-SMase activity in lung tissue by a modified micellar in vitro assay. (B) Measurements of ceramide concentrations by two-dimensional charring densitometry. Values represent means ± SE. *P < 0.05, versus control group.

Figure 7.

Intra-amniotic exposure to LPS induces expression of heme oxygenase–1 (HO-1). (A) Western blot measurements of HO-1 protein expression with an anti–HO-1 antibody. The same membrane was analyzed with anti–β-actin antibody. HO-1 and β-actin protein levels were semiquantified by densitometry. (B) Optical density of HO-1 protein bands was corrected for β-actin, and results are expressed as ratio (%) of endotoxin-exposed to control animals. (C–E) Evaluation of HO-1 expression in lung tissue by immunohistochemistry. Sections are representative of a control animal (C) and an animal exposed to endotoxin-induced chorioamnionitis for 7 days (D), stained for HO-1. Magnification, ×50. (E) Immunohistochemical semiquantification of HO-1 in lung sections, on a scale from 0–3. Values represent means ± SE. *P < 0.05, versus control group.