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 O₂⁻ 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 O₂⁻ levels, MnSOD and eNOS expression and activity, intracellular hydrogen peroxide (H₂O₂) 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 O₂⁻ levels, and increased H₂O₂ 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.

the rapid decrease in pulmonary vascular resistance (PVR) that occurs at birth following initiation of respiration is primarily mediated by the release of endothelial-derived nitric oxide (NO) (1, 4, 43). Increased O₂ tension (Po₂), lung distension, and shear stress are known physiological signals that increase NO release by activating endothelial nitric oxide synthase (eNOS) in pulmonary circulation (1, 4, 21, 43). Impaired postnatal transition of pulmonary circulation results in persistent pulmonary hypertension of the newborn (PPHN) when PVR fails to decrease, and deoxygenated blood is shunted right to left across ductus arteriosus and/or foramen ovale (2, 32). PPHN is associated with increased superoxide levels (O₂⁻) in the pulmonary arteries, leading to depletion of NO (5). A successful postnatal transition, therefore, requires proper adaptive mechanisms to regulate the oxidant/antioxidant balance and release of NO.

Manganese superoxide dismutase (MnSOD), a metalloenzyme that catalyzes the dismutation of O₂⁻ to H₂O₂ and O₂, plays a critical role in regulating cellular oxidative stress by detoxifying excess mitochondrial O₂⁻ generated during respiration. MnSOD may be the first line of defense against reactive oxygen species (ROS) because of its localization in the mitochondrial matrix (12). The fetal lung prepares for the rapid increase in Po₂ at birth by an increase in MnSOD expression in late gestation (3, 48). Impairment of this adaptation is particularly important for infants being treated for PPHN, since they are exposed to a high fraction of inspired O₂ and mechanical ventilation. Hyperoxia increases mitochondrial O₂⁻ levels in pulmonary arteries and impairs pulmonary vasodilation (14, 26).

Recent studies in the fetal lamb model of PPHN induced by prenatal ductal ligation demonstrated that increased oxidative stress contributes to impaired pulmonary vasodilatation (5, 22). This may result from impaired scavenging of excess ROS by cellular antioxidants and increased ROS generation. Mitochondria are a major source of ROS, where an estimated 1–5% of total O₂ consumed for oxidative phosphorylation is reduced to O₂⁻ (6, 18). O₂⁻ can directly elicit vasoconstriction and also react with NO generated by eNOS three to five times faster than with superoxide dismutase (SOD) to form the radical peroxynitrite, which in turn can nitrate MnSOD and impair its antioxidant activity (29, 33, 37). Expression and activity of MnSOD have been previously shown to affect the responses of vascular endothelial cells to both acute and chronic oxidative stress (10). Endothelial expression of MnSOD is decreased in adult patients with established pulmonary hypertension (15). However, the contribution of decreased antioxidant defenses to vascular oxidative stress observed in PPHN remains unclear. Because MnSOD is critical to the regulation of mitochondrial O₂⁻ levels, we hypothesized that: 1) MnSOD expression and activity are decreased in the fetal lamb model of PPHN, resulting in increased mitochondrial O₂⁻ levels and impaired pulmonary vasodilation, and 2) enhancing MnSOD activity by adenoviral MnSOD gene transduction will decrease oxidative stress and improve eNOS expression and function and the nitric oxide synthase (NOS)-dependent relaxation response of PPHN pulmonary arteries. We performed these studies in PAEC and pulmonary arteries isolated from control (sham ligation with no pulmonary hypertension) and PPHN fetal lambs.

METHODS

Creation of PPHN lamb model.

This study was approved by the institutional Animal Care and Use Committee of the Medical College of Wisconsin and conformed to NIH guidelines for animal use. Ligation of fetal patent ductus arteriosus was performed at 128 ± 2 days of gestation (term = 144 days) as we described previously (22–23). Control fetal lambs had thoracotomy performed without ligation of the ductus arteriosus. After 8 days of ductal constriction, fetal lungs were harvested for the isolation of pulmonary arteries and pulmonary artery endothelial cells (PAEC). Studies were done in isolated cultured PAEC to investigate the expression and activity of MnSOD. Parallel experiments were done on pulmonary arteries and lung homogenates to study the in vivo changes in expression and activity of MnSOD and the relaxation response of pulmonary arteries in vitro.

Isolation of PAEC.

PAECs were isolated and characterized using techniques described previously (23–24). Endothelial cells were isolated with 0.25% collagenase type A (Roche Molecular Biochemicals, Indianapolis, IN), and cells were grown in Dulbecco's modified Eagle's medium (DMEM) with 20% FCS. Endothelial cell identity was verified by the presence of factor VIII antigen and acetylated low density lipoprotein uptake (23). PAEC isolated from at least six fetal lambs were used for each group. PAEC from control (NFL-PAEC) and ductal ligation (PPHN-PAEC) lambs between passages 3 and 6 were used at the same time in parallel for each experiment for appropriate comparison. PAECs were grown in round 60-mm tissue culture dishes (BD Falcon, BD Biosciences, San Jose, CA) to assess MnSOD expression and RNA extraction and six-well plates (BD Falcon) for MnSOD activity, intracellular H₂O₂ levels, and total nitrite + nitrate level quantification. Round 100-mm tissue culture dishes (BD Falcon) of 90% confluent cells were used for mitochondrial isolation and immunoprecipitation studies. Eight-well chamber glass slides (LabTek II; Nalgene, Naperville, IL) were used to measure mitochondrial O₂⁻ using mitochondrial targeted hydroethidine (mito-HE, MitoSOX; Invitrogen, Eugene, OR).

Transduction of PAEC with adenoviral MnSOD.

PAEC (1 × 10⁵) between passages 3 and 5 were grown to 50% confluence in DMEM containing 20% FCS and 1% antibiotic/antimycotic in round 60-mm tissue culture dishes. PAEC were transduced with 50, 100, or 200 multiplicity of infection (MOI) of adenoviral MnSOD (ad-MnSOD) or adenoviral green fluorescent protein (ad-GFP, control for adenoviral transduction effects) in 350 μl of antibiotic-free media. The MOI were chosen based on preliminary studies on transduction efficiency and survival of PAEC after transduction. After 1 h of incubation at 37°C, 2 ml of fresh complete DMEM were added. After transduction, MnSOD protein expression and activity, MnSOD nitration, eNOS expression and activity, catalase protein expression, and H₂O₂ levels were assessed.

Transduction of pulmonary artery rings with ad-MnSOD.

Five-millimeter segments of third- to fifth-generation pulmonary arteries were isolated from the lungs of PPHN lambs as described above. Pulmonary artery (PA) segments were washed in cold Hanks' balanced salt solution (HBSS) and incubated with 2.7 × 10⁷ viral particles/μl of ad-MnSOD or ad-GFP as control in 50 μl transduction media (DMEM and 20% FBS without antibiotics) in a 96-well plate at 37°C in 5% CO₂. After 4 h of incubation, the transduction media was removed, and 100 μl of growth medium were added. After 20 h of incubation, growth medium was removed; 1-mm-length PA rings were cut and suspended in 2-ml baths. Ring tension was measured by wire myography using standard techniques as we described previously (22, 23). MnSOD protein and activity in PA rings were also measured to assess the effect of ad-MnSOD transduction on these variables in the isolated PA as described above.

Mitochondrial isolation.

Mitochondria were isolated using the Pierce mitochondrial isolation kit (Thermo Scientific). Briefly, 2 × 10⁷ cells were pelleted by centrifuging the harvested cell suspension at 3,000 rpm for 2 min. The supernatant was carefully removed, and the cytosolic and mitochondrial fractions were isolated from the cell pellet using reagents and instructions from the kit. The cytosolic proteins were concentrated using centrifugal filters (Amicon ultra; Millipore). The mitochondrial pellet was lysed in radioimmunoprecipitation assay (RIPA) buffer, the protein contents in both fractions were analyzed using the bicinchoninic acid assay (BCA) method, and lysates were used to study expression and tyrosine nitration of MnSOD.

Western blot analysis for proteins.

PAEC were grown to 90% confluence and washed two times with ice-cold HBSS. The cells were lysed in modified RIPA buffer. The cell lysate was sonicated, and cell debris was removed by centrifugation at 13,000 rpm. Protein content of the lysate was determined by BCA. Separated proteins were transferred to nitrocellulose membranes and were blotted with specific polyclonal antibodies for MnSOD, eNOS, catalase, β-actin, and nitrotyrosine overnight at 4°C. The membranes were blotted with horseradish peroxidase (HRP)-conjugated anti-mouse IgG antibody (1:9,000; Bio-Rad) or HRP-conjugated anti-rabbit IgG antibody and exposed to CL-XPosure films (Pierce) after treatment with SuperSignal West Pico (Pierce). The signals were analyzed with Image J and normalized to the expression of the internal control β-actin for whole cell lysates and for cytosolic fractions and with porin, a structural protein of the mitochondrial outer membrane, for isolated mitochondria.

Lung tissue expression of MnSOD.

Peripheral lung tissue and third- to fifth-generation pulmonary arteries were flash-frozen in liquid nitrogen, pulverized, and placed in modified RIPA buffer to obtain homogenates as described above. An aliquot of protein (50 μg) was used for immunoblots for MnSOD as described above.

Quantification of mRNA abundance.

PAEC were plated and grown to 90% confluence. RNA was extracted via the TRIzol (Sigma) method, and the contaminated DNA was removed by the TURBO DNA-free Kit (Ambion) in a 37°C water bath for 60 min. cDNA was synthesized from the extracted RNA using the iScript cDNA synthesis kit (Bio-Rad). The PCR primers were designed using Primer3 as previously described (38) and are shown in Table 1. Real-time RT-PCR was performed using the iQ5 multicolor real-time PCR detection system (Bio-Rad). The PCR cycle was started at 95°C for 3 min followed by 40 cycles of 95°C for 10 s and then 58°C for 1 min. Melting temperatures were monitored for each pair of primers, and single-peak melting temperature was observed for all of the primer pairs. The number of the threshold cycle (C_t) for each target mRNA was corrected against the corresponding C_t of β-actin and 18S rRNA to obtain the C_t, and then 2^−ΔΔC_t was calculated against the corresponding control for mRNA abundance (28, 35).

Table 1.

Sequence of primers used for real-time RT-PCR analyses of mRNA for MnSOD, eNOS, 18S, and β-actin

Gene	Forward Primer	Reverse Primer
MnSOD	5′-ATT GCT GGA AGC CCA TCA AAC-3′	5′-AGC AGG GGG ATA AGA CCT GT-3′
eNOS	5′-CCT CAC CGC TAC AAC ATCC-3′	5′-GCA CAG CCA GGT TGA TCTC-3′
β-Actin	5′-GCG GGA AAT CGT GCG TGA CAG-3	5′-GAT GGA GTT GAA GGT AGT TTC GTG-3
18S rRNA	5′-CGG ACA CG GAC AGG ATT GAC AG-3′	5′-ATG CCA GAG TCT CGT TCG TTA TCG-3′

MnSOD, manganese superoxide dismutase; eNOS, endothelial nitric oxide synthase.

Measurement of MnSOD activity.

PAEC were grown in six-well plates to 90% confluence and washed with HBSS. Cells were trypsinized, and the cell pellet was collected by centrifugation at 3,500 rpm for 10 min at 4°C. The pellet was homogenized in cold 20 mM HEPES buffer containing 1 mM EGTA, 210 mM mannitol, and 70 mM sucrose, and cell debris was removed by centrifugation at 13,000 rpm at 4°C for 10 min. Potassium cyanide (1 mM) was added to the lysate during the assay to inhibit both cytosolic copper and zinc SOD (Cu/ZnSOD) and extracellular SOD and to detect copper-zinc SOD specific activity. SOD activity was measured by a colorimetric assay kit (Cayman) that detects the conversion of tetrazolium salt to formazan dye by superoxide generated by xanthine + xanthine oxidase, producing a color change. SOD activity was measured by the ability of the sample to scavenge O₂⁻ and prevent formation of formazan dye. Absorbance was read at 440–460 nm using a microplate reader (FLUOstar Omega by BMG LABTECH). One unit of SOD was defined as the amount of enzyme needed for 50% dismutation of the superoxide radical. The protein concentration in each well was estimated by the BCA method to calculate the MnSOD activity per milligram of protein.

Immunoprecipitation studies.

PAEC at 90% confluence in 100-mm round tissue culture dishes were treated with either HBSS or 10⁻⁵ M A-23187 (a calcium ionophore and eNOS agonist) in HBSS for 10 min. The supernatant was then aspirated, and cells were lysed in RIPA buffer. Samples were sonicated, and cell debris was removed by centrifugation. MnSOD was immunoprecipitated from cell lysate with specific antibody (assay Designs). The immunoprecipitated proteins were separated by SDS-PAGE (10%) and transblotted to nitrocellulose membrane. The membranes were blocked with 5% nonfat milk in TBS-Tween (0.1%) and then immunoblotted for MnSOD and nitrated MnSOD using nitrotyrosine antibody (Sigma). The bands were visualized as described above. The integrated optical density ratio of nitrated MnSOD to MnSOD was calculated for each immunoprecipitate.

Peripheral lung tissue and third- to fifth-generation pulmonary arteries were flash-frozen in liquid nitrogen, pulverized, and placed in RIPA buffer to obtain homogenates as described above. An aliquot of protein (500 μg) was used for immunoprecipitation for MnSOD as described above.

Measurement of mitochondrial O₂⁻.

PAEC from PPHN lambs were grown to 40–50% confluence in eight-well chamber glass slides. Cells in selected wells were transduced with the desired MOI of ad-MnSOD or ad-GFP. After 16–18 h incubation, cells were treated with HBSS alone or with 10⁻⁵ M A-23187 and with mito-HE (MitoSOX) reagent (10⁻⁵ M) to detect mitochondrial O₂⁻ levels as described previously (22, 42).

Assay for nitrite + nitrate.

PPHN-PAEC were grown to 40–50% confluence in six-well plates, and cells in some wells were transduced with ad-MnSOD or ad-GFP with the desired MOI. Activity of eNOS was assessed by accumulation of nitrite + nitrate in the supernatant, detected by the ozone-chemiluminescence method as we described previously (23, 36). Nitrite + nitrate levels were normalized to the protein concentration of each well.

Measurement of intracellular H₂O₂ level.

Freshly prepared carboxyl-2,7-dicholorofluorescein diacetate (carboxy-DCF) was used to measure intracellular H₂O₂ levels (9, 20). PPHN-PAEC were transduced with ad-MnSOD or ad-GFP, and nontransduced cells were also used for comparison. Cells were trypsinized and resuspended in prewarmed buffer (PBS) containing 1 mM carboxyl-DCF in a test tube. Before loading of the cells with DCF reagent, some wells were pretreated with polyethylene glycol (PEG)-catalase and N^G-nitro-l-arginine methyl ester (l-NAME) for 30 min, and all the cells were stimulated with A-23187 for 15 min. After incubation at 37°C for 45 min, the buffer was removed, and cells were returned to prewarmed growth medium and further incubated at 37°C for 10 min of recovery. H₂O₂ levels were quantified by flow cytometer. For flow cytometer, the forward and side scatter of cells was unchanged after dye loading and treatment. Signals were corrected against the fluorescence signals in cell-free mixtures of dye and buffer and untreated loaded cells that have been maintained in growth medium or simple buffer. We also compared the results with positive controls, consisting of cells treated with 100 μM H₂O₂.

Statistical analyses.

Data are shown as means ± SE. Student's t-test was used for normally distributed data, and Mann-Whitney U-test was used for data that did not pass the normality test for comparison between two groups. One-way ANOVA with Student-Newman-Keuls post hoc test was used when more than two groups were compared. Data were analyzed with MedCalc (www.medcalc.com). A P value of <0.05 was considered significant.

RESULTS

MnSOD expression.

MnSOD protein levels were 20% lower in the whole cell lysate of PPHN-PAEC compared with controls (P < 0.001, n = 7; Fig. 1A). MnSOD protein was decreased by twofold in mitochondria (Fig. 1B) and by approximately fourfold in the cytosol (Fig. 1C) of PPHN-PAEC compared with controls. Similarly, MnSOD protein levels in PA homogenates were decreased by approximately twofold in PPHN lambs (Fig. 1D), but no difference was detected in whole lung homogenates between PPHN lambs and the controls (Fig. 1E). MnSOD mRNA was 2.5-fold lower in PPHN-PAEC compared with controls (P < 0.001, n = 8; Fig. 1F). There was no difference in 18S rRNA and β-actin mean C_t (P > 0.3, n = 5) between NFL-PAEC and PPHN-PAEC.

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.

MnSOD activity in PPHN.

In whole cell lysates, we observed a twofold decrease in MnSOD activity in PPHN-PAEC compared with controls (Fig. 2A). MnSOD activity was also decreased by fourfold in isolated mitochondria from PPHN-PAEC compared with controls (Fig. 2B). However, MnSOD activity was barely detectable in the cytosol of either the controls or PPHN-PAEC. PA homogenates of PPHN lambs showed a similar threefold decrease in MnSOD activity, but no difference in the MnSOD activity was seen between whole lung homogenates of either the control or PPHN lambs (Fig. 2C).

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.

Tyrosine nitration of MnSOD.

After 15 min of incubation with calcium ionophore A-23187, nitration of MnSOD tyrosine residues increased by twofold in whole cell lysate of PPHN-PAEC compared with controls (Fig. 3A). Although tyrosine nitration of MnSOD protein increased by approximately threefold in mitochondria isolated from PPHN-PAEC (Fig. 3B), no difference was found in MnSOD nitration in the cytosol of either PPHN or control PAEC (Fig. 3C). PA homogenate of PPHN lambs showed an approximately twofold increase in tyrosine nitration of MnSOD at the basal level (Fig. 3D), but no significant difference in the MnSOD nitration was seen between lung homogenates of either the control or PPHN lambs (Fig. 3E).

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.

Effects of ad-MnSOD transduction on MnSOD mRNA, protein levels, and activity.

ad-MnSOD transduction of PPHN-PAEC increased MnSOD mRNA (P = 0.002, n = 6; Fig. 4A), protein levels (P < 0.05, n = 5; Fig. 4B), and activity (P < 0.05, n = 5; Fig. 4C), with increasing MOI compared with ad-GFP as controls. ad-MnSOD transduction of PPHN-PAEC decreased MnSOD nitration by 40–45% in a dose-dependent manner (Fig. 4D).

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).

Mitochondrial O₂⁻ levels.

The mito-HE fluorescence increased when PPHN-PAEC were stimulated with A-23187 (Fig. 5, A and B). Transduction with ad-GFP had no effect on basal or stimulated levels of mito-HE fluorescence (Fig. 5, C and D). ad-MnSOD transduction of PPHN-PAEC decreased mito-HE fluorescence signals by threefold at 100 (Fig. 5, E and F) and 200 (Fig. 5, G and H) MOI in PPHN-PAEC both at the basal level and in response to stimulation with A-23187 (Fig. 5, bar graph in I).

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 (#).

Effects of ad-MnSOD transduction on eNOS expression and function.

Increased oxidative stress in PPHN is known to be associated with decreased eNOS expression and function (15, 46). We observed that ad-MnSOD transduction leads to an increase in eNOS mRNA levels in PPHN-PAEC (Fig. 4A) and a six- to sevenfold increase in eNOS protein levels compared with ad-GFP-transduced PPHN-PAEC as controls (Fig. 6A). Total nitrite + nitrate levels were also increased in ad-MnSOD-transduced PPHN cells when stimulated with A-23187 (Fig. 6B). Preincubation with N-nitro-l-arginine (l-NNA), a competitive inhibitor of NOS, attenuated the rise in total nitrite + nitrate generated in ad-MnSOD-transduced PPHN-PAEC (Fig. 6B).

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.

Effect of H₂O₂ on eNOS protein expression.

Because increasing MnSOD levels can increase H₂O₂ levels in the cell, we investigated the effect of exogenous H₂O₂ on the eNOS mRNA levels. We observed that H₂O₂ increases eNOS mRNA in both nontransduced and ad-MnSOD-transduced cells, with a greater increase occurring when both are combined (Fig. 6C).

Effect of MnSOD on H₂O₂ levels.

Stimulation of ad-MnSOD-transduced PPHN-PAEC with A-23187 resulted in an increase in the carboxy-DCF signal with increasing MOI compared with ad-GFP-transduced PPHN-PAEC (Fig. 6D). Incubation of ad-MnSOD-transduced PPHN-PAEC with 100 U/ml PEG-catalase decreased the DCF signals in ad-MnSOD-transduced PPHN-PAEC.

Effect of ad-MnSOD transduction on catalase protein expression.

We investigated whether an increase in H₂O₂ levels with ad-MnSOD transduction leads to an increase in catalase expression. We observed that ad-MnSOD transduction increases catalase protein levels in PPHN-PAEC in a dose-dependent manner compared with controls (P < 0.05, n = 4; Fig. 7A).

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.

Effect of ad-MnSOD transduction on the relaxation response of PA rings.

The relaxation response to ATP, a NOS agonist, was significantly improved in PPHN lamb PA rings treated with adeno-MnSOD compared with ad-GFP-treated rings (Fig. 7B). The NOS inhibitor l-NNA attenuated the improved relaxation response to ATP in ad-MnSOD-transduced rings (Fig. 7B). We also observed that MnSOD expression and activity were increased in ad-MnSOD-transduced PA rings compared with ad-GFP controls (Fig. 7, C and D).

DISCUSSION

In this study, we observed that PPHN is associated with decreased MnSOD expression and activity in PAEC and pulmonary arteries; the downstream effects of these alterations were increased basal and stimulated mitochondrial O₂⁻ levels and impaired eNOS function. Enhancing MnSOD expression and activity via ad-MnSOD transduction attenuated the oxidative stress and improved eNOS function in this model of PPHN. In addition, MnSOD transduction improved the relaxation response of the pulmonary arteries from PPHN lambs. These data together suggest that impaired MnSOD function contributes to oxidative stress and impaired vasodilation in PPHN.

Our studies showed that MnSOD protein levels are decreased in PPHN. The decrease in MnSOD protein is associated with decreased mRNA levels in PAEC, suggesting alterations in nuclear transcription of MnSOD in PPHN. However, regulation of MnSOD is complex and is affected by the stability of mRNA as well as protein. Because a functionally active pool of MnSOD is localized to mitochondria, we investigated the protein levels and activity specifically in the mitochondrial pool. We observed greater changes in protein levels and activity in the mitochondrial fraction. Previous studies have shown decreased MnSOD expression in systemic and pulmonary hypertension (10, 15) and our laboratory previously reported decreased MnSOD levels in PPHN-PAEC (11). In contrast, Farrow et al. (14) previously reported that MnSOD expression is increased in cultured vascular smooth muscle cells from pulmonary arteries in PPHN lambs. The reason for observed differences in the expression of MnSOD between PAEC and vascular smooth muscle cells in PPHN remains unclear. Because endothelial cells express eNOS, which is uncoupled in PPHN (22), the mitochondria and MnSOD in PAEC may be uniquely susceptible to nitrosative stress. This possibility is supported by our observation of increased tyrosine nitration of MnSOD in PAEC. There appears to be a coordinated regulation of eNOS and MnSOD in PAEC, with a decrease in expression of both proteins in PPHN. Increasing the MnSOD expression was associated with an increase in eNOS protein. These data suggest that MnSOD may be regulated differently in PAEC compared with smooth muscle cells in PPHN. In addition, our studies on PA homogenates were done in freshly isolated vessels, which may show different alterations than cultured vascular smooth muscle cells.

MnSOD activity was also decreased in the mitochondria of PPHN-PAEC and PA homogenates of PPHN lambs. The activity in the cytosolic fraction was very low, as expected. MnSOD is normally synthesized in the cytosol and targeted with a leader polypeptide sequence to the mitochondria, where it is activated and functions as the antioxidant (12, 30, 46). The decrease in MnSOD activity was greater than the decrease in MnSOD expression, particularly in PA homogenates. We investigated tyrosine nitration of MnSOD, a posttranslational modification that can impair MnSOD function as a potential explanation for this observation. MnSOD has tyrosine residues at amino acids 34 and 166 near its active catalytic site. Nitration of MnSOD at tyrosine residue 34 to 3-nitrotyrosine can significantly impair its antioxidant activity (29, 30, 37, 41). Our studies demonstrated an increased tyrosine nitration of MnSOD in the mitochondria of PPHN-PAEC and in the PA homogenates of PPHN lambs. Restoring MnSOD protein levels via ad-MnSOD transduction decreased the relative levels of nitrated MnSOD in PPHN-PAEC. These data suggest that increasing MnSOD protein levels may result in increased dismutation of O₂⁻ to decrease its reaction with NO. The greater decrease in MnSOD activity we observed in PA homogenates compared with cell lysates of PAEC may be related to comparison of freshly isolated tissue with cultured cells. These data suggest that in vivo differences in MnSOD activity in PPHN may be more significant than in cultured cells.

The downstream effect of decreased MnSOD expression and activity is increased mitochondrial O₂⁻ levels in PPHN. We have previously reported an increase in the endothelial oxidative stress in pulmonary arteries of the ductal ligation model of PPHN (11, 22). Enhancing MnSOD activity in PPHN-PAEC via ad-MnSOD transduction decreased the O₂⁻ levels in a dose-dependent manner. This observation suggests that impaired MnSOD function contributes to increased mitochondrial O₂⁻ levels in PPHN.

The decreased MnSOD activity is associated with impaired eNOS function in PPHN, which may be secondary to increased oxidative stress. Farrow et al. (13) previously demonstrated that decreased total SOD activity and increased NADPH oxidase activity in PPHN lambs impairs eNOS expression and function. If the decreased activity of MnSOD affects the balance of mitochondrial O₂⁻ and NO, enhancing MnSOD activity is logically expected to improve endogenous eNOS protein expression and function. Our data demonstrated that enhancing MnSOD activity restores eNOS expression and function, suggesting that mitochondrial-derived O₂⁻ plays a significant role in regulating eNOS expression and function. Improvement in eNOS function may account for the improved relaxation response to ATP we observed in PA rings after MnSOD transduction. This observation may have therapeutic implications in the management of PPHN.

Increased eNOS expression and function after ad-MnSOD transduction may be due to the following mechanisms: 1) recoupling of eNOS as a result of decreased oxidative stress and 2) increased intracellular H₂O₂ levels, a biochemical effect of increased activity of MnSOD, which dismutates O₂⁻ to H₂O₂ and O₂. H₂O₂ has been shown in previous studies to increase eNOS expression and activity by a phospholipase C-dependent release of inositol trisphosphate (7, 8, 27).

Increased H₂O₂ levels from increased MnSOD activity can also constitute an oxidative stress if not effectively removed from the cells. We observed an increase in catalase protein levels that paralleled the increased intracellular H₂O₂ levels in ad-MnSOD-transduced PPHN-PAEC. These data suggest a compensatory response from PAEC to handle the excess H₂O₂ by the increased catalase protein expression, thus potentially limiting the oxidative injury when MnSOD activity is increased.

MnSOD is the first line of defense against mitochondrial O₂⁻ to maintain the balance of NO and O₂⁻ in the pulmonary circulation during birth-related transition (46). Previous studies demonstrated that PPHN is associated with decreased release of endothelium-derived NO by birth-related stimuli, particularly ATP and O₂ (13, 23, 40). We observed that ad-MnSOD transduction significantly improved the relaxation response of PPHN PA rings to ATP. These results suggest that enhancing MnSOD function can improve vasodilation in PPHN. This may be of therapeutic significance in the management of PPHN. Our results are consistent with previous studies that demonstrated a significant benefit with the intratracheal administration of recombinant Cu/ZnSOD to lambs with PPHN (13, 25). These data together suggest that antioxidants may be beneficial adjuncts to treatment of PPHN.

In conclusion, PPHN is associated with decreased MnSOD expression and function in PAEC and pulmonary arteries. The impaired function of MnSOD in PPHN may be both a contributing factor and result of oxidative stress in PPHN. Decreased MnSOD function in turn contributes to the impaired NOS-dependent vasodilation in PPHN. Whether improving MnSOD function with exogenous MnSOD restores postnatal transition in PPHN requires further investigation.

GRANTS

This work was supported by National Institutes of Health Grants RO1 HL-057268 and RO3 HD-065841 and by funding from the Advancing Healthier Wisconsin Foundation, Children's Research Institute, and Muma Endowed Chair in Neonatology (G. G. Konduri).

AUTHOR CONTRIBUTIONS

A.J.A., R.-J.T., and G.G.K. conception and design of research; A.J.A., A.E., R.-J.T., I.B., S.K., J.M.D., and G.G.K. performed experiments; A.J.A., R.-J.T., and G.G.K. analyzed data; A.J.A., R.-J.T., and G.G.K. interpreted results of experiments; A.J.A. and G.G.K. prepared figures; A.J.A. and G.G.K. drafted manuscript; J.M.D. and G.G.K. edited and revised manuscript; G.G.K. approved final version of manuscript.