Phosphodiesterase 4 inhibition attenuates persistent heart and lung injury by neonatal hyperoxia in rats

Abstract

Phosphodiesterase (PDE) 4 inhibitors are potent anti-inflammatory drugs with antihypertensive properties, and their therapeutic role in bronchopulmonary dysplasia (BPD) is still controversial. We studied the role of PDE4 inhibition with piclamilast on normal lung development and its therapeutic value on pulmonary hypertension (PH) and right ventricular hypertrophy (RVH) in neonatal rats with hyperoxia-induced lung injury, a valuable model for premature infants with severe BPD. The cardiopulmonary effects of piclamilast treatment (5 mg·kg(-1)·day(-1)) were investigated in two models of experimental BPD: 1) daily treatment during continuous exposure to hyperoxia for 10 days; and 2) late treatment and injury-recovery in which pups were exposed to hyperoxia or room air for 9 days, followed by 9 or 42 days of recovery in room air combined with treatment started on day 6 of oxygen exposure until day 18. Prophylactic piclamilast treatment reduced pulmonary fibrin deposition, septum thickness, arteriolar wall thickness, arteriolar vascular smooth muscle cell proliferation and RVH, and prolonged survival. In the late treatment and injury-recovery model, hyperoxia caused persistent aberrant alveolar and vascular development, PH, and RVH. Treatment with piclamilast in both models reduced arteriolar wall thickness, attenuated RVH, and improved right ventricular function in the injury recovery model, but did not restore alveolarization or angiogenesis. Treatment with piclamilast did not show adverse cardiopulmonary effects in room air controls in both models. In conclusion, PDE4 inhibition attenuated and partially reversed PH and RVH, but did not advance alveolar development in neonatal rats with hyperoxic lung injury or affect normal lung and heart development.

Fig. 1.

A: in the early concurrent treatment model for experimental bronchopulmonary dysplasia (BPD), neonatal rat pups were exposed to 100% oxygen (O₂; solid bars) or room air (RA) directly after birth (day 1) until day 10. Treatment of RA and O₂ pups with piclamilast (pic; 5 mg·kg⁻¹·day⁻¹; shaded bars; RA-pic and O₂-pic, respectively) or 0.01% DMSO in 0.9% NaCl (open bars) was started on day 2 until day 10. †Lung and heart tissues were harvested on days 1, 3, 6, and 10. B: in the late treatment and recovery model for experimental BPD, neonatal rat pups were exposed to O₂ (solid bars) or RA directly after birth (day 1) until day 9. Hereafter, pups were allowed to recover in RA up to day 51. Treatment with pic (shaded bars) or DMSO in 0.9% NaCl (open bars) was started on day 6 until day 18. †Lung and heart tissues were harvested on days 9 (end-hyperoxic period), 18 (end-treatment period), and 51. Right ventricular (RV) function was determined on day 18.

Fig. 2.

Growth (A), body weight (B and D), and survival (C and E) at day 10 after early concurrent treatment (N = 12; A–C) and after late treatment and recovery (N = 8; D and E) on days 9, 18, and 51 in RA controls (open bars, ◊), RA pups treated with 5.0 mg·kg⁻¹·day⁻¹ pic (RA-pic; hatched bars, ♦), age-matched O₂-exposed controls (solid bars, ▵), and O₂ pups treated with 5.0 mg·kg⁻¹·day⁻¹ pic (O₂-pic; shaded bars, ▴). Growth and body weight are expressed as means ± SE. C: Kaplan-Meier survival curve of O₂-pic rat pups (▵), age-matched, O₂-exposed controls (▵), RA-exposed controls (◊), and RA-pic pups (♦) during the first 10 days after birth (N = 12). Survival data are expressed as percentage ± SE of pups surviving at the observed time point. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. age-matched O₂-exposed controls. ^Δ P < 0.05, ^ΔΔ P < 0.01, and ^ΔΔΔ P < 0.001 vs. RA-pic pups. ^φ P < 0.001 vs. recovery on postnatal day (pd) 18.

Fig. 3.

Lung sections stained for von Willebrand factor (A–D) and lung morphometry (E–G) of RA controls (A and open bars), RA-pic pups treated with 5.0 mg·kg⁻¹·day⁻¹ pic (B and hatched bars), age-matched O₂-exposed controls (C and solid bars), and O₂-pic pups treated with 5.0 mg·kg⁻¹·day⁻¹ pic (D and shaded bars) on day 10 (A–D, F, and G) or on days 1, 3, 6, and 10 (E) after early concurrent treatment. Pictures were taken at a ×200 magnification. Lung morphometry, including the quantifications of number of pulmonary vessels (E), septum thickness (F), and alveolar crest per tissue ratio (G), was determined on paraffin sections in RA and O₂ pups daily injected either with saline or pic. Values are means ± SE (N = 12). *P < 0.05 and **P < 0.001 vs. age-matched, O₂-exposed controls. ^Δ P < 0.05, ^ΔΔ P < 0.01, and ^ΔΔΔ P < 0.001 vs. RA-pic pups. ^φ P < 0.001 vs. pd6. ^γ P < 0.01 vs. pd3. ^δ P < 0.001 vs. pd1.

Fig. 4.

Serial lung sections stained for α-smooth muscle actin (ASMA; A–D) and for the proliferation marker Ki67 (E–H), and lung morphometry (I–K) of RA controls (A and E, open bars), RA-pic (B and F, hatched bars), age-matched, O₂-exposed controls (C and G, solid bars), and O₂-pic pups (D and H, shaded bars) on day 10 (A–H) or on days 1, 3, 6, and 10 (I–K) after early concurrent treatment. Pictures were taken at a ×1,000 magnification. Lung morphometry, including the quantifications of medial wall thickness (I) and number of Ki67-positive cells in the ASMA-positive layer of small arterioles relative to vessel diameter (J) or medial wall thickness (K), was determined on paraffin sections in RA and O₂ pups daily injected either with saline or pic. Values are means ± SE (N = 12, I; and N = 6, J and K). Arrows in G indicate Ki67-positive cells in the ASMA-positive layer of the arteriole. *P < 0.05, **P < 0.01 and ***P < 0.001 vs. age-matched, O₂-exposed controls. ^φ P < 0.001 vs. pd6.

Fig. 5.

Quantification of number of pulmonary vessels (A), alveolar crest per tissue ratio (B), and medial wall thickness (C) determined on paraffin sections after late treatment and recovery on days 9, 18, and 51 in RA controls (open bars), RA-pic pups treated with 5.0 mg·kg⁻¹·day⁻¹ pic (hatched bars), age-matched, O₂-exposed controls (solid bars), and O₂-pic pups treated with 5.0 mg·kg⁻¹·day⁻¹ pic (shaded bars). Values are means ± SE (N = 8). *P < 0.001 vs. age-matched, O₂-exposed controls. ^Δ P < 0.01 and ^ΔΔ P < 0.001 vs. RA-pic pups. ^δ P < 0.05, ^δδ P < 0.01, and ^δδδ P < 0.001 vs. recovery on pd9. ^φ P < 0.05, ^φφ P < 0.01, and ^φφφ P < 0.001 vs. recovery on pd18.

Fig. 6.

Western blot analysis of fibrin deposition (A) and quantification of cyclic AMP (cAMP; B) in lung homogenates of RA controls (open bars), RA-pic pups treated with 5.0 mg·kg⁻¹·day⁻¹ pic (hatched bars), age-matched, O₂-exposed controls (solid bars), and O₂-pic pups treated with 5.0 mg·kg⁻¹·day⁻¹ pic (shaded bars) on day 10 after early concurrent treatment. Values are means ± SE (N = 12). **P < 0.01 and ***P < 0.001 vs. age-matched, O₂-exposed controls. ^ΔΔΔ P < 0.001 vs. RA-exposed controls.

Fig. 7.

Relative mRNA expression in lungs, determined with RT-PCR, of genes related to inflammation [interleukin-6 (IL-6); A], coagulation [tissue factor (TF); B], alveolar growth [vascular endothelial growth factor A (VEGFA), C; and VEGF receptor 2 (VEGFR2); D], and the endothelin (ET) signaling pathway [ET-1, E; endothelin converting enzyme-1 (ECE-1), F; ET_A, G; and ET_B, H] on day 10 after early concurrent treatment. Experimental groups include RA controls (open bar), RA-pic rat pups treated with 5.0 mg·kg⁻¹·day⁻¹ pic (hatched bar), age-matched O₂-exposed controls (solid bar), and O₂-pic rat pups treated with 5.0 mg·kg⁻¹·day⁻¹ pic (shaded bar). Values are means ± SE (N = 12). *P < 0.05 and ***P < 0.001 vs. age-matched, O₂-exposed controls. ^Δ P < 0.05, ^ΔΔ P < 0.01, and ^ΔΔΔ P < 0.001 vs. RA-exposed controls.

Fig. 8.

Ventricular free wall thickness, indicated as the RV-to-left ventricle (LV) ratio (A) and RV hypertrophy depicted as the ratio RV/[LV + interventricular septum (IVS)] (C) in RA-exposed controls (open bars), RA-pic pups treated with pic (5.0 mg·kg⁻¹·day⁻¹; hatched bar), age-matched, O₂-exposed controls (solid bar), and O₂-pic pups treated with pic (5.0 mg·kg⁻¹·day⁻¹; shaded bar) on day 10 (C) or on days 1, 3, 6, and 10 (A and B) after early concurrent treatment. Values are means ± SE (N = 12). B: paraffin heart sections stained with hematoxylin and eosin at a × 40 magnification in RA controls, RA-pic, O₂ controls, and O₂-pic on days 1, 3, 6, and 10. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. age-matched, O₂-exposed controls. ^δ P < 0.01 vs. pd3. ^φ P < 0.01 vs. pd6.

Fig. 9.

A: RV hypertrophy depicted as the RV/LV ratio, after late treatment and recovery on days 9, 18, and 51 in RA controls (open bars), RA-pic pups treated with 5.0 mg·kg⁻¹·day⁻¹ pic (hatched bars), age-matched, O₂-exposed controls (solid bars), and O₂-pic pups treated with 5.0 mg·kg⁻¹·day⁻¹ pic (shaded bars). B: paraffin heart sections stained with hematoxylin and eosin at × 40 magnification in RA controls, RA-pic, O₂ controls, and O₂-pic on days 9, 18, and 51. Values are means ± SE (N = 8). *P < 0.05, **P < 0.01, and ***P < 0.001 vs. age-matched, O₂-exposed controls.

Fig. 10.

Relative mRNA expression in the RV free wall and LV, including the IVS (LV + IVS), determined with RT-PCR, of atrial natriuretic peptide (ANP; A) and brain natriuretic peptide (BNP; B) in RA-exposed controls (open bars), RA-pic pups treated with pic (5.0 mg·kg⁻¹·day⁻¹; hatched bar), age-matched, O₂-exposed controls (solid bar), and O₂-pic pups treated with pic (5.0 mg·kg⁻¹·day⁻¹; shaded bar) on day 10 after early concurrent treatment. Values are means ± SE (N = 12). *P < 0.05 and **P < 0.001 vs. age-matched, O₂-exposed controls. ^ΔP < 0.001 vs. RA-exposed controls.