Hydrocortisone normalizes oxygenation and cGMP regulation in lambs with persistent pulmonary hypertension of the newborn

Abstract

In the pulmonary vasculature, cGMP levels are regulated by soluble guanylate cyclase (sGC) and phosphodiesterase 5 (PDE5). We previously reported that lambs with persistent pulmonary hypertension of the newborn (PPHN) demonstrate increased reactive oxygen species (ROS) and altered sGC and PDE5 activity, with resultant decreased cGMP. The objective of this study was to evaluate the effects of hydrocortisone on pulmonary vascular function, ROS, and cGMP in the ovine ductal ligation model of PPHN. PPHN lambs were ventilated with 100% O(2) for 24 h. Six lambs received 5 mg/kg hydrocortisone every 8 h times three doses (PPHN-hiHC), five lambs received 3 mg/kg hydrocortisone followed by 1 mg·kg(-1)·dose(-1) times two doses (PPHN-loHC), and six lambs were ventilated with O(2) alone (PPHN). All groups were compared with healthy 1-day spontaneously breathing lambs (1DSB). O(2) ventilation of PPHN lambs decreased sGC activity, increased PDE5 activity, and increased ROS vs. 1DSB lambs. Both hydrocortisone doses significantly improved arterial-to-alveolar ratios relative to PPHN lambs, decreased PDE5 activity, and increased cGMP relative to PPHN lambs. High-dose hydrocortisone also increased sGC activity, decreased PDE5 expression, decreased ROS, and increased total vascular SOD activity vs. PPHN lambs. These data suggest that hydrocortisone treatment in clinically relevant doses improves oxygenation and decreases hyperoxia-induced changes in sGC and PDE5 activity, increasing cGMP levels. Hydrocortisone reduces ROS levels in part by increasing SOD activity in PPHN lambs ventilated with 100% O(2.) We speculate that hydrocortisone increases cGMP by direct effects on sGC and PDE5 expression and by attenuating abnormalities induced by oxidant stress.

Fig. 1.

Hydrocortisone improves the arterial to alveolar (a/A) ratio in persistent pulmonary hypertension of the newborn (PPHN) lambs. a/A ratios were measured in three groups of lambs: PPHN lambs (n = 6; white circles), lambs that received 3 mg/kg hydrocortisone followed by 1 mg·kg⁻¹·dose⁻¹ times two doses (PPHN-loHC lambs; n = 5; grey triangles), and lambs that received 5 mg/kg hydrocortisone every 8 h times three doses (PPHN-hiHC; n = 6; black diamonds). There was 1 death in each of the 3 groups (shown by arrows). Data are shown as means ± SE. *P < 0.05 vs. PPHN lambs.

Fig. 2.

Mean systemic blood pressure in PPHN lambs ventilated with 100% oxygen with and without hydrocortisone. Mean systemic blood pressure in PPHN lambs (n = 6; white circles), PPHN-loHC lambs (n = 5; grey triangles), and PPHN-hiHC lambs (n = 6; black diamonds). There was no significant difference in mean systemic blood pressure between PPHN lambs ventilated with 100% oxygen alone and PPHN lambs ventilated with 100% oxygen receiving hydrocortisone.

Fig. 3.

Ventilator settings and blood gas variables in PPHN lambs ventilated with 100% oxygen with and without hydrocortisone. Changes in mean airway pressure (MAP; A), ventilator rate (B), pH (C), and Pco₂ (D) in PPHN lambs (white circles), PPHN-loHC lambs (grey triangles), and PPHN-hiHC lambs (black diamonds) during the study period. Data are shown as means ± SE. *P < 0.05 vs. PPHN lambs by ANOVA over time.

Fig. 4.

Hydrocortisone increases cGMP in pulmonary arteries from PPHN lambs. cGMP concentrations were measured in resistance pulmonary arteries from 1-day spontaneously breathing lambs (1DSB) lambs (n = 4), PPHN lambs (n = 6), PPHN-loHC lambs (n = 5), and PPHN-hiHC lambs (n = 6). cGMP concentration was assayed by enzyme-linked immunoassay and normalized for milligrams of total protein. Data are shown as means ± SE. *P < 0.05 vs. PPHN lambs.

Fig. 5.

High-dose hydrocortisone increases sGC activity but does not affect soluble guanylate cyclase-α (sGCα) protein expression in lambs with PPHN. sGCα protein expression (A) was measured in resistance pulmonary arteries from 1DSB lambs (n = 4), PPHN lambs (n = 6), PPHN-loHC lambs (n = 5), and PPHN-hiHC lambs (n = 6) via Western blot and normalized to β-actin. Representative blot is shown for sGCα and β-actin (B). sGC activity (C) was measured in lung tissue by EIA and normalized for milligrams of total protein. Data are shown as means ± SE relative to fetal controls for sGCα expression and as means ± SE for sGC activity. #P < 0.05 vs. 1DSB; *P < 0.05 vs. PPHN.

Fig. 6.

High-dose hydrocortisone decreases phosphodiesterase 5 (PDE5) protein expression in resistance pulmonary arteries from PPHN lambs. PDE5 mRNA and protein expression were measured in resistance pulmonary arteries from 1DSB lambs (n = 4), PPHN lambs (n = 6), PPHN-loHC lambs (n = 5), and PPHN-hiHC lambs (n = 6). A: PDE5 mRNA expression was measured by real-time PCR and normalized to β-actin. B: PDE5 protein expression was analyzed via Western blot, normalized to β-actin. Data are shown as means ± SE. C: representative Western blot is shown for PDE5 and β-actin. D: phase-contrast images (20×) of frozen lamb lung sections (top) with the corresponding sections stained for PDE5 shown in red (bottom). #P < 0.05 vs. 1DSB; *P < 0.05 vs. PPHN.

Fig. 7.

Hydrocortisone decreases PDE5 activity in pulmonary arteries from PPHN lambs. PDE5 activity was measured in resistance pulmonary arteries from 1DSB lambs (n = 4), PPHN lambs (n = 6), PPHN-loHC lambs (n = 5), and PPHN-hiHC lambs (n = 6). PDE5-specific activity was measured as the sildenafil-inhibitable fraction of total cGMP hydrolysis, normalized for total milligrams of protein. Data are shown as mean ± SE. #P < 0.05 vs. 1DSB; *P < 0.05 vs. PPHN.

Fig. 8.

Hydrocortisone decreases oxidative stress in lambs with PPHN. Dihydroethidium (DHE) fluorescence (A) and total SOD activity (B) were measured in lung tissue from 1DSB lambs (n = 4), PPHN lambs (n = 6), PPHN-loHC lambs (n = 5), and PPHN-hiHC lambs (n = 6). A: frozen lung sections were incubated with DHE and imaged using fluorescence microscopy. Conversion of DHE by superoxide to ethidium results in red nuclear fluorescence. Values are means ± SE (5–7 vessels quantified per animal imaged) relative to 1DSB lambs. Representative images of DHE staining are shown at bottom. B: total SOD activity was measured using a commercially available colorimetric assay. Activity was normalized to protein content and expressed relative to control samples. #P < 0.05 vs. 1DSB. *P < 0.05 vs. PPHN.

Fig. 9.

High-dose hydrocortisone decreases 3-NT levels. 3-NT levels were measured in resistance pulmonary arteries from 1DSB lambs (n = 4), PPHN lambs (n = 4), PPHN-loHC lambs (n = 3), and PPHN-hiHC lambs (n = 4). 3-Nitrotyrosine (3-NT) expression was quantified by immunohistochemistry. Data are shown as means ± SE relative to fetal controls. Representative fluorescent images of pulmonary arteries with corresponding light microscopy images from various groups of lambs are shown at bottom. #P < 0.05 vs. 1DSB. *P < 0.05 vs. PPHN.