Decreases in manganese superoxide dismutase expression and activity contribute to oxidative stress in persistent pulmonary hypertension of the newborn

Abstract

A rapid increase in the synthesis and release of nitric oxide (NO) facilitates the pulmonary vasodilation that occurs during birth-related transition. Alteration of this transition in persistent pulmonary hypertension of the newborn (PPHN) is associated with impaired function of endothelial nitric oxide synthase (eNOS) and an increase in oxidative stress. We investigated the hypothesis that a decrease in expression and activity of mitochondrial localized manganese superoxide dismutase (MnSOD) in pulmonary artery endothelial cells (PAEC) increases oxidative stress and impairs eNOS function in PPHN. We isolated PAEC and pulmonary arteries from fetal lambs with PPHN induced by prenatal ductus arteriosus ligation or sham ligation (control). We investigated MnSOD expression and activity, tyrosine nitration of MnSOD, and mitochondrial O(2)(-) levels in PAEC from control and PPHN lambs. We introduced exogenous MnSOD via an adenoviral vector (ad-MnSOD) transduction into PAEC and pulmonary arteries of PPHN lambs. The effect of ad-MnSOD was investigated on: mitochondrial O(2)(-) levels, MnSOD and eNOS expression and activity, intracellular hydrogen peroxide (H(2)O(2)) levels, and catalase expression in PAEC. MnSOD mRNA and protein levels and activity were decreased and MnSOD tyrosine nitration was increased in PPHN-PAEC. ad-MnSOD transduction of PPHN-PAEC increased its activity two- to threefold, decreased mitochondrial O(2)(-) levels, and increased H(2)O(2) levels and catalase expression. ad-MnSOD transduction improved eNOS expression and function and the relaxation response of PPHN pulmonary arteries. Our observations suggest that decreased MnSOD expression and activity contribute to the endothelial dysfunction observed in PPHN.

Fig. 1.

Manganese superoxide dismutase (MnSOD) protein levels in pulmonary artery endothelial cells (PAEC), pulmonary artery, and lung homogenates. Representative Western blots and summarized data for 7 experiments for the whole cell lysate (A) and 4 experiments for mitochondrial and cytosolic fractions (B and C) are shown as a ratio of MnSOD to internal control, β-actin integrated optical density (IOD). MnSOD protein levels were decreased in PAEC and in pulmonary arteries (D) but not in whole lung homogenates (E) of lambs with persistent pulmonary hypertension of the newborn (PPHN). These data are shown as representative Western blots and summarized data for 4 samples each for controls and PPHN lambs. MnSOD mRNA levels were decreased in PPHN-PAEC compared with controls (F). The mRNA levels were estimated by QRT-PCR using β-actin and 18S rRNA as the housekeeping genes for 7 samples each from control and PPHN-PAEC. Data from the control PAEC were normalized to 1, and values are shown as fold change from the controls. *P < 0.05 from control.

Fig. 2.

MnSOD activity is shown as summarized data from 5 experiments for whole cell lysate (A), for mitochondrial and cytosolic fractions in control and PPHN-PAEC (B), and for pulmonary artery and lung homogenates (C). MnSOD activity was decreased in whole cell lysate and mitochondria of PPHN-PAEC and in the pulmonary arteries of PPHN lambs. *P < 0.05 from control.

Fig. 3.

Representative immunoblots and summarized data for tyrosine nitration of MnSOD proteins by immunoprecipitation. Data are shown as the ratio of nitrated MnSOD (nMnSOD) to MnSOD IOD in the control and PPHN-PAEC after stimulation of the cells with A-23187, a nitric oxide synthase (NOS) agonist, in the whole cell lysate (A) and in mitochondrial and cytosolic fractions (B and C). The signal for nitrated tyrosine is higher in PPHN in whole cell and mitochondrial lysate but not in the cytosolic fraction. Immunoprecipitation of MnSOD followed by immunoblotting for MnSOD and nitrotyrosine in pulmonary artery and lung tissue homogenates (D and E) shows increased MnSOD tyrosine nitration in PPHN in pulmonary arteries but not in whole lung homogenates. Summarized data below are shown as open bars for controls and filled bars for PPHN samples. *P < 0.05 from control.

Fig. 4.

Effects of transduction of PPHN-PAEC with adenoviral (ad)-MnSOD on the expression of MnSOD and endothelial nitric oxide synthase (eNOS). Transduction of PPHN-PAEC increased both MnSOD and eNOS mRNA levels (A). ad-MnSOD transduction with increasing multiplicity of infection (MOI) also increased MnSOD protein levels (B) and activity (C) and decreased nitrated MnSOD levels (D). P < 0.05 from corresponding controls (*) and from 50 MOI (#) (C).

Fig. 5.

Mitochondrial O₂⁻ levels assessed by mitochondrial-targeted hydroethidine (mitoSOX) fluorescence in PPHN-PAEC at the basal level and after stimulation with calcium ionophore A-23187. Representative photomicrographs of mitoSOX fluorescence from control nontransduced cells (A and B), ad-green fluorescent protein (GFP) transduced (C and D), and ad-MnSOD 100 MOI (E and F) and 200 MOI (G and H) are shown at basal (A, C, E, and G) and A-23187-stimulated (B, D, F, and H) conditions. Both basal and stimulated fluorescence signals were higher in untreated and ad-GFP-transduced PPHN-PAEC. ad-MnSOD transduction decreased both basal and stimulated fluorescence. Bar graph in I shows summarized data for 6 experiments each for control and PPHN-PAEC. P < 0.05 from corresponding basal levels of untreated and ad-GFP-treated cells (*) and from stimulated levels of the same groups (#).

Fig. 6.

Effects of MnSOD overexpression on eNOS expression and function. Representative Western blots and summarized data are shown for 4 experiments in PPHN cells transduced with ad-GFP or ad-MnSOD (A). eNOS protein levels increased in ad-MnSOD-transduced PPHN-PAEC in a dose-dependent manner with increasing MOI. Total nitrite + nitrate levels in transduced cells were measured after the cells were stimulated with A-23187 (B). Total nitrite + nitrate levels increased with ad-MnSOD transduction, which was inhibited by N-nitro-l-arginine (l-NNA). Endothelial NOS mRNA levels increased after treating nontransduced and ad-MnSOD-transduced PPHN-PAEC with 100 μM H₂O₂ (C). P < 0.05 from untreated cells (*) and from H₂O₂ alone and ad-MnSOD alone (#) groups. ad-MnSOD transduction also increased the intracellular H₂O₂ levels compared with ad-GFP-transduced PAEC (D). Preincubation with 100 U/ml of polyethylene glycol (PEG)-catalase for 15 min attenuated the H₂0₂ increase in ad-MnSOD-transduced PPHN-PAEC (D). carboxy-DCF-DA, carboxyl-2,7-dicholorofluorescein diacetate. *P < 0.05 from ad-GFP cells.

Fig. 7.

A: ad-MnSOD transduction increased the catalase expression in PPHN-PAEC. P < 0.05 from control (*) and from 100 MOI (#). B: effect of ad-GFP and ad-MnSOD transduction on the relaxation response of pulmonary artery rings to ATP. The basal tone after preconstriction with norepinephrine was normalized to 100%. Data are means ± SD for 6 rings each in both groups. C and D: representative immunoblots of MnSOD expression (C) and MnSOD activity in ad-GFP- and ad-MnSOD-transduced pulmonary artery ring homogenates (D). *P < 0.001 from ad-GFP-transduced rings.