Deficiency of Lung Antioxidants in Idiopathic Pulmonary Arterial Hypertension

Abstract

Idiopathic pulmonary arterial hypertension (IPAH) is associated with lower levels of the pulmonary vasodilator nitric oxide (NO) and its biochemical reaction products (nitrite [NO₂ ⁻], nitrate [NO₃ ⁻]), in part, due to the reduction in pulmonary endothelial NO synthesis. However, NO levels are also determined by consumptive reactions, such as with superoxide to form peroxynitrite, which subsequently may generate stable products of nitrotyrosine (Tyr‐NO₂) and/or NO₃ ⁻. In this context, superoxide dismutase (SOD) preserves NO in vivo by scavenging superoxide and preventing the consumptive reactions. Here, we hypothesized that reactive oxygen species (ROS) consumption of NO may contribute to the low NO level and development of pulmonary hypertension. To test this, nitrotyrosine and antioxidants glutathione (GSH), glutathione peroxidase (GPx), catalase, and SOD were evaluated in IPAH patients and healthy controls. SOD and GPx activities were decreased in IPAH lungs (all p < 0.05), while catalase and GSH activities were similar among the groups (all p > 0.2). SOD activity was directly related to exhaled NO (eNO) (R ²= 0.72, p= 0.002), and inversely related to bronchoalveolar lavage (BAL) NO₃ ⁻ (R ²=–0.73, p= 0.04). Pulmonary artery pressure (PAP) could be predicted by a regression model incorporating SOD, GPx, and NO₃ values (R ²= 0.96, p= 0.01). These findings suggest that SOD and GPx are associated with alterations in NO and PAP in IPAH.

Introduction

Idiopathic pulmonary arterial hypertension (IPAH) is a fatal disease characterized by progressive increase in pulmonary artery pressure (PAP) and vascular resistance. ¹ , ² Abnormalities in vasodilator substances, and specifically, nitric oxide (NO) have been implicated in the pathogenesis of IPAH. ³ , ⁴ , ⁵ , ⁶ , ⁷ , ⁸ , ⁹ , ¹⁰ NO, endogenously produced in normal lungs by nitric oxide synthases (NOS), ¹¹ accumulates in the human lungs as a first‐order process resulting in steady‐state NO levels, indicating a constant rate of production balanced by a constant rate of consumption or scavenging of NO. One major consumptive pathway is the rapid reaction of NO with superoxide anion (O₂ ⁻) to form peroxynitrite (ONOO⁻), a potent oxidant that may mediate free radical‐induced injury. For example, peroxynitrite contributes to nitrotyrosine formation, allowing nitrotyrosine to be used as a marker of reactive oxygen and nitrogen species. ¹² , ¹³ , ¹⁴ In oxygenated environments, an interconversion among NO species also occurs, with end products of NO₂ ⁻ and NO₃ ⁻. The consumption of NO by oxidant reactions in vitro is regulated by antioxidants, most important of which may be the superoxide dismutases (SOD) that remove superoxide by conversion to hydrogen peroxide (H₂O₂), ¹⁵ which can, in turn, be removed by catalase or glutathione peroxidase (GPx) reactions. ¹⁶ These enzymes act cooperatively in protecting cells against oxidative stress. ¹⁷ However, NO also reacts with glutathione (GSH), which is present in abundant levels in the lung epithelial lining fluid, to form S‐nitrosothiols. ¹² , ¹³ In this context, the oxidants and antioxidants modulate the consumption of NO by chemical reactions and thus control the bioavailability of free NO in the lungs. The finding of NO₂ ⁻, NO₃ ⁻, and S‐nitrosothiols in the epithelial lining fluid confirms that a significant portion of the NO produced in the lungs is consumed by chemical reactions with oxidants and antioxidants. Our work and of others show that NO and NO biochemical reaction products (NO₂ ⁻, NO₃ ⁻) are lower in individuals with IPAH than in healthy controls, in part, due to lower levels of NO synthesis. ³ , ⁸ , ⁹ , ¹⁰ , ¹⁸ In this study, we investigate the potential consumptive mechanisms for the low levels of NO in IPAH. We hypothesize that an increased oxidative consumption may contribute to the lower levels of NO in IPAH, and subsequently contribute, to pulmonary arterial hypertension. To investigate this hypothesis, SOD, catalase, GSH, GPx, and nitrotyrosine were quantitated in the lungs of patients with IPAH as compared to healthy controls. The expression of SOD proteins was evaluated in lysates from bronchial tissues obtained at explantation of the lungs from IPAH patients undergoing transplantation, or from donor lungs not used in transplant. The results show that SOD and GPx activity are reduced in IPAH lungs, and that both are related to PAPs, indicating that redox events contribute to the abnormal vasoregulation in the IPAH lungs.

Materials and Methods

Study population

Healthy nonsmoking controls and patients with IPAH were studied. Individuals with IPAH diagnosed class 1.1 were diagnosed according to the standard criteria for pulmonary hypertension provided by the National Registry Criteria ¹⁹ , ²⁰ and were classifed according to the World Health Organization criteria as IPAH class 1.1. The study was approved by the Cleveland Clinic Institutional Review Board, and written informed consent was obtained from all individuals.

Isolation of bronchial epithelial cells and bronchus tissues

The volunteers underwent bronchoscopy with a fexible fiberoptic bronchoscope with cytology brushings to obtain samples of bronchial epithelial cells from second‐ and third‐order bronchi with a 1‐mm cytology brush (Microvasive, Inc., Watertown, MA, USA). Blood pressure, pulse oximetry, and electrocardiogram (EKG) were continuously monitored during and following the procedure. The brushing sample was immediately placed in RPMI 1640 (Gibco BRL, Carlsbad, CA, USA), and an aliquot was taken for cell count and differential determination, with the remaining cells used for the study of nitrotyrosine and antioxidants. The bronchus samples were obtained from the lungs explanted at the time of transplantation and were immediately snap‐frozen in liquid nitrogen until further evaluation for SOD expression and activity.

Evaluation of cellular morphology

Freshly isolated human bronchial epithelial cells were sedimented (Cytospin, Shandon Instruments, Waltham, MA, USA), stained with Diff‐Quick (American Scientific Products, Stone Mountain, GA, USA), and evaluated for cell differential and morphology by light microscopy at ×500 (×40 objective, NA 0.95) magnification in a blinded fashion by a pulmonary pathologist. For each individual sample, 400 intact cells were analyzed to determine differentials for epithelial and inflammatory cells (macrophages, neutrophils, eosinophils, basophils, and lymphocytes). The epithelial cells were classified into four categories (ciliated, secretory, basal, and unclassifiable) on the basis of the previously described criteria. ²¹

Antioxidant activity

SOD activity was determined in the cell lysate by measuring the rate of reduction of cytochrome C, as previously described. ²¹ One unit of activity is defined as the amount of SOD required to inhibit the rate of cytochrome C reduction by 50%.

Total GPx activity was determined spectrophotometrically in the cell lysate through an indirect coupled assay. ²² The cell lysate was incubated in the presence of 0.1 mM sodium azide, 1 U/mL glutathione reductase, 0.1 mM GSH and 0.12 mM βNADPH, 0.016 mM dithiothreitol, 0.38 mM EDTA, and 50 mM sodium phosphate (pH 7.0) for 2 minutes at 37°C. The reaction was initiated by the addition of 0.2 mM H₂O₂. The decrease in the absorbance at 340 nm over 3 minutes as NADPH is converted to NADP is proportional to the GPx activity. One unit of activity is defined as the activity that catalyzes the oxidation of 1 nM of NADPH per minute using an extinction molar coefficient of 6.22 × 10⁶/M/cm for NADPH.

Catalase activity was quantified by a method in which H₂O₂ is reacted with the components present in the cell lysate. ²¹ The initial rate of disappearance of H₂O₂ (0–60 seconds) is recorded spectrophotometrically at a wavelength of 240 nm; one unit of catalase activity is defined as the rate constant of the first‐order reaction.

GSH in the cell lysate was measured by the standard methods, as previously described. ²³ In brief, total GSH was determined by mixing equal amounts of the cell lysate with 10 mM 5,5′‐dithiobis‐2‐nitrobenzoic acid (DTNB) in 100 mM potassium phosphate, pH 7.5, which contained 17.5 μM EDTA. An aliquot of the solution was added to a cuvette containing 0.5 U of glutathione disulfde reductase (Sigma type III; Sigma Chemical, St. Louis, MO, USA) in 100 mM potassium phosphate and 5 mM EDTA, pH 7.5. Afer 1 minute, the reaction was initiated with 220 nM of NADPH in a final reaction volume of 1 mL. The rate of reduction of DTNB was recorded continuously at 412 nm by a spectrophotometer with a kinetics/time feature (Beckman DU‐640; Beckman Instruments, Inc., Fullerton, CA, USA). The GSH present in the cell lysate was based on the standard curves generated from known concentrations of GSH in phosphate‐buffered saline.

Nitrite and nitrate quantitation

The biochemical reaction products of NO (NO₃ ⁻, N O ₂ ⁻) present in bronchoalveolar lavage (BAL) fluid were detected by conversion to NO with a saturated solution of VCl₃ in 0.8 M HCl, and the NO was subsequently measured through a gas‐phase chemiluminescent reaction between NO and ozone, using the NOA 280 analyzer, as previously described. ³ Nitrite levels were determined with a solution of KI (1% wt/vol) in glacial acetic acid to convert NO₂ to NO, and NO detected by chemiluminescence. ³

SODs and Nitrotyrosine Western analyses

The cell lysates were prepared and electrophoresed on sodiumdodecyl sulfate (SDS)‐polyacrylamide gels and transferred onto a nitrocellulose membrane overnight. The membranes were blocked with 1% bovine serum albumin containing 0.1% Tween overnight at 4°C. The membranes were probed with a monoclonal antibody (Ab) against nitrotyrosine (1:4,000; Upstate Biotechnology, Lake Placid, NY, USA), followed by a goat anti‐mouse horseradish peroxidase conjugate (1:5,000; Amersham Biosciences, England, UK). Immunopositive spots were visualized using ECL‐Plus (Amersham Biosciences). The bronchus samples were homogenized in a lysis buffer containing 50 mM Tris (pH 7.9), 50 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.5% Nonidet P‐40, 10% glycerol, 1 mM PMSF, 5 μg/mL leupeptin, 10 μg/mL pepstatin A, 200 μM NaOV, and 20 μg/mL aprotinin, followed by incubation on ice for 30 minutes and centrifugation. The lysates were separated on a gradient gel (4–15%) SDS‐polyacrylamide gels, and transferred to a polyvinylidene difuoride (PVDF) membrane. Afer blocking in 5% milk, the membranes were incubated overnight with primary Ab at 4°C, then with a peroxidase‐conjugated secondary Ab, and a signal was detected by the enhanced chemiluminescent system (ECL; Amersham Biosciences). The primary Abs were polyclonal anti‐copper‐zinc SOD (CuZnSOD; Oxis, Foster City, CA, USA), polyclonal anti‐manganese SOD (MnSOD; Oxis), polyclonal anti‐extracellular SOD (EC‐SOD; Stressgen, Victoria, British Columbia, Canada), and polyclonal anti‐β‐actin Ab (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Proteomic analysis of nitrated proteins

Proteomic analysis was performed as describe by Aulak et al. ²⁴ Briefly, human lung tissues and BAL fluid were extracted in a lysis buffer (8 M urea, 4% Chaps, 1% DDT, 2% IPG‐ampholytes), followed by homogenization and sonication. Two‐dimensional (2D) polyacrylamide gel electrophoresis was performed on a Bio‐Rad system using pH 5–8 immobilized pH gradients (IPG) strips and 12.5% gels (Bio‐Rad, Hercules, CA, USA). Afer completion of the run, the proteins were partially transferred to a PVDF membrane (Millipore Corporation, Bedford, MA, USA) using semidry transfer. The gels were stained with colloidal Coomassie blue (Gel code blue, Pierce, Rockfod, IL, USA), and Western blot analysis performed on the PVDF membrane. The membranes were probed with a monoclonal Ab against nitrotyrosine (1:4,000; clone 1A6; Upstate Biotechnology) followed by a goat anti‐mouse horseradish peroxidase conjugate (1:2,000, Amersham Biosciences). Immunopositive spots were visualized by using ECL‐Plus.

Protein identification

The proteins were identified either by matrix‐assisted laser desorption ionization/time‐of‐flight (MALDI‐ToF) mass spectrometric or by LCQ‐Deca ion trap mass spectrometer system, as described by Kinter et al. ²⁵ Briefly, immunopositive nitrated protein spots were digested with trypsin, and the generated peptides extracted and analyzed on a Finnigan LCQ‐Deca ion trap mass spectrometer system with a Protana microelectrospray ion source interface using a self‐packed 10 cm × 75 μm i.d. (LCQ‐Deca, Waltham, MA, USA). Phenomenex Jupiter C18 reversed‐phase capillary chromatography column (Phenomenex Jupiter, Torrance, CA, USA). Data were then analyzed using all collision‐induced dissociation spectra (CID) spectra collected in the experiment to search the National Center for Biotechnology Information (NCBI) nonredundant database with the search program Mascot (http://www.matrixscience.com, Matrix Science Inc., Boston, MA, USA). A matching spectrum for each reported identification was verified by manual interpretation. Some of the samples were also analyzed by MALDI‐ToF. Here, the spectra were internally calibrated using trypsin autolysis peptides and analyzed using mass accuracies less than 25 ppm.

Statistical analyses

Continuous variables were summarized by group as sample size, mean and standard error of the mean (SEM). All statistical comparisons were performed using Student's t‐test for parametric data, and a median test for nonparametric data. Linear correlation coeficients, logarithmic regression fit, and stepwise regression model of the data were performed using the statistical discovery software JMP (version 5; SAS Institute, Cary, NC, USA).

Results

Clinical characteristics

The controls and IPAH individuals participating in the study were similar in terms of age, sex, and race (age [years]: control 35 ± 2, IPAH 35 ± 3; sex [female/male]: control 6/2, IPAH 7/1; race [Caucasian/African, American/Hispanic]: control 6/2/0, IPAH, 6/1/1). Pulmonary hypertension was present for a mean time of 2.3 ± 0.5 years and was diagnosed in all IPAH individuals by right heart catheterization (PAPs [mmHg]: systolic 97 ± 12, diastolic 43 ± 7, and mean 64 ± 9). None of the participants in the study were current users of tobacco products. IPAH individuals were receiving vasodilators, anticoagulants, diuretics, digitalis, and/or oxygen. Bronchial brushings and BAL were performed in 8 healthy and 8 IPAH individuals without complication. Predominant cells obtained by bronchial brushing were epithelial cells (>90%), with ciliated cells comprising the majority of the epithelial cell type (56%) ( Table 1 ). Due to the limitation of samples, not all assays could be performed in all individuals' samples. The numbers evaluated are noted in the text. In addition to bronchoscopically obtained samples, bronchial tissues for the studies were obtained from the lungs of IPAH patients (2 women/1 man) at explantation and from 5 donor lungs not used for transplantation (2 women; age at explantation [years]: control 57 ± 3, IPAH 64 ± 10; p= 0.6).

Table 1.

Cell differentials obtained by bronchial brushing.

	Control	IPAH
Epithelial cells
Ciliated	53 ± 2.7	56 ± 3
Secretory	2.3 ± 0.6	2.3 ± 0.2
Basal	7.7 ± 1.2	9.3 ± 1.1
Unclassifiable	23.5 ± 2.6	20 ± 1.8
Neutrophils	1.1 ± 0.3	1.0 ± 0.19
Eosinophils	0.025 ± 0.025	0
Lymphocytes	0.85 ± 0.23	2.4 ± 1.2
Macrophages	4.16 ± 1.9	4.2 ± 1.9
Indeterminate	6.3 ± 1.6	3.5 ± 0.7

Values are mean ± SE; all comparisons p > 0.05.

Antioxidants in airway epithelium

SOD and GPx activities were decreased in IPAH airway epithelial cells as compared to healthy controls (SOD U/mg protein: controls 167 ± 44 [n= 8], IPAH 88 ± 20 [n= 8]; p= 0.01; GPx U/mg: controls 0.89 ± 0.21 [n= 8], IPAH 0.51 ± 0.05 [n= 8]; p= 0.02; Figure 1 ). In contrast, catalase activity and GSH in the epithelial cells were not different between the groups ( Figure 1 ; catalase mU/mg protein: controls 138 ± 26 [n= 8], IPAH 221 ± 72 [n= 8], p= 0.3; GSH nM/mg protein: controls 28 ± 10 [n= 8], IPAH 24 ± 9 [n= 8], p= 0.7). Intracellular GSH was directly related to GPx activity in healthy control and IPAH epithelium (R ²= 0.71, n= 16, p= 0.003).

Figure 1.

Antioxidants in IPAH airway epithelial cells as compared to healthy controls. SOD and GPx activities are decreased in IPAH airway epithelial cells as compared to healthy controls. In contrast, catalase activity and glutathione in epithelial cells are not different between the groups (all p > 0.2).

SOD activity was also decreased in the lysates of bronchial tissues that were obtained at explantation of IPAH lungs at transplantation as compared to control bronchial tissues obtained from donor lungs not used in transplantation (SOD U/mg protein: control 51 ± 1 [n= 5]; IPAH 37 ± 3 [n= 3]; p= 0.039; Figure 2 ). The decrease in SOD activity in the bronchus was associated with a lower expression of MnSOD and EC‐SOD isoforms on Western analyses (expression relative to β‐actin: CuZnSOD: control 1.5 ± 0.5, IPAH 0.61 ± 0.05, p= 0.16; MnSOD: control 0.61 ± 0.07, IPAH 0.31 ± 0.07, p= 0.01; EC‐SOD: control 1.4 ± 0.2, IPAH 0.5 ± 0.2, p= 0.02; Figure 2 ), SOD activity was related to the levels of NO in the gas phase of controls and IPAH individuals ([NO]= 5.52 log [SOD]– 20.5, R ²= 0.72, p= 0.002; Figure 3A ). On the other hand, SOD activity in BAL of IPAH individuals was inversely associated with nitrate ([NO₃ ⁻]= 2.1 – 1.3 [SOD], R ²=–0.73, p= 0.04; Figure 3B ), but was not related in healthy controls (p= 5 0.5). SOD activity tended to inversely correlate with pulmonary systolic pressure (R ²=−0.572, p= 0.1). The inverse relationship of SOD to NO suggests that SOD regulates free NO bioavailability in the lungs by scavenging superoxide and preventing NO–superoxide consumptive reactions. Furthermore, these results suggest that the overall low NO in IPAH may be related to oxidant consumptive reactions.

Figure 2.

SOD activity and proteins in bronchial tissues from IPAH and control lungs. (A) SOD activities are decreased in the lysates of bronchial tissues of IPAH as compared to controls. (B) Western analyses of the lysates of bronchial tissues for CuZnSOD, MnSOD, and EC‐SOD. (C) Quantitation of expression of SOD proteins relative to β‐actin protein. The MnSOD and EC‐SOD expression in IPAH were less than in controls (all p < 0.05). Mean ± SEM values are shown.

Figure 3.

Correlation between SOD activity and exhaled NO. (A) SOD activity in freshly obtained airway cells correlates to levels of NO measured in the gas phase of the airways of controls and IPAH individuals. (B) SOD activity in BAL of IPAH individuals is inversely associated with nitrate.

Prediction model for systolic pulmonary arterial pressure

A stepwise regression model was used to determine factors that would create a prediction model for PAP. We evaluated all the variables, including all antioxidants (SOD, GPx, GSH, and catalase), eNO, and NO products (NO₂ ⁻, NO₃ ⁻), in the model. On the basis of the stepwise regression model, only SOD, GPx, and NO₃ ⁻ contributed significantly to the determination of systolic PAP:

Systolic PAP = 201 – 46 [NO₃ ⁻]– 0.21 [SOD]– 96.6 [GPx]; R ²= 0.96, p= 0.01.

SOD, GPx, and NO₃ ⁻ were inversely related to the levels of systolic PAP in IPAH. The equation indicates that all the three variables decrease systolic PAP significantly.

Nitrotyrosine in airway epithelial cells, BAL, and tissue of IPAH individuals

NO–superoxide reactions were directly evaluated by quantitating nitrotyrosine by Western analyses using a monoclonal nitrotyrosine Ab. As previously reported, in lung adenocarcinoma cells (A549) stimulated for 3 days with interferon (IFN)‐γ, interleukin (IL)‐1β, and tumor necrosis factor (TNF)‐α to induce NOS II and high‐level NO synthesis, multiple nitrotyrosine‐containing proteins were detectable in contrast to unstimulated cells with low levels of detectable nitrated proteins ( Figure 4I ), validating the method for the detection of nitrotyrosine. Fresh lysates of IPAH and control airway epithelium obtained at bronchial brushing had a similar amount of nitrotyrosine on 1D gel electrophoresis (nitrotyrosine/β‐actin: control 1.02 ± 0.03 [n= 3], IPAH 1.1 ± 0.1 [n= 3]; p= 0.5). However, differences in the pattern of nitrated proteins were found on 2D gels, suggesting an altered metabolism of NO ( Figure 4I ).

Figure 4.

Proteomic analyses of nitrotyrosine expression in human lung tissue and BAL of IPAH and controls. (I) IPAH and control airway epithelium obtained at bronchial brushing have similar nitrotyrosine, but have differences in the pattern of nitrated proteins (lanes 3 and 4). Lung adenocarcinoma cells (A549) were used to validate the method for nitrotyrosine detection. A549 cells stimulated for 3 days with cytokine mix have multiple nitrotyrosine‐containing proteins in contrast to unstimulated cells with no detectable nitrated proteins (lanes 1 and 2). (II) Proteomic analysis of BAL of IPAH and controls. Samples were solubilized in the denaturing buffer and 400 μg subjected to 2D electrophoresis. The gels were then partially transferred to a PVDF membrane and subjected to Western blot analysis using an anti‐nitrotyrosine antibody. Coomassie blue‐stained polyacrylamide gels of control‐BAL (A) and IPAH‐BAL (B) are shown with the corresponding Western blots (right panel, C and D). The proteins spots that corresponded to the immunoreactive proteins observed in the Western blot were excised and subjected to mass spectrometry and identified. The proteins on the Western blot and the corresponding spots on Coomassie blue‐stained gel are identified. The nitrotyrosine Westerns indicate NO‐superoxide reactions occur to a similar degree in healthy and IPAH individuals, suggesting that while oxidative consumption occurs, this is not quantitatively greater than in controls. However, the comparisons between control and IPAH show different protein nitration profiles, suggesting alterations and/or different localization of nitration/oxidation processes.

Identification of nitrotyrosine‐containing proteins

A proteomic approach was used to identify and analyze for changes in the expression of nitrotyrosine‐containing proteins. The protein lysates from BAL of controls (n= 3) and IPAH individuals (n= 3) were run on 2D gels using isoelectrofocusing in the first dimension and SDS‐page in the second dimension ( Figure 4II , left panel). The gels were partially transferred onto membranes and probed with an anti‐nitrotyrosine Ab ( Figure 4II , right panel). Immunopositive proteins were isolated from the original polyacrylamide gels and analyzed by mass spectrometry ( Table 2 ). Nitration of transferrin and vitamin D‐binding proteins were seen in BAL of IPAH and controls, whereas nitration of fibrinogen gamma‐β was present only in IPAH samples.

Table 2.

Nitrated proteins in IPAH.

Proteins	pI	Mol Wt. (KD)	Accession Number	Function
Cell death and proliferation
Annexin I1	6.6	38.7	4502101	Regulates activity of Ca2+‐phospholipid.
Annexin II1	7.6	38.6	113950	Ca2+‐ regulated membrane‐binding Protein.
Annexin VII1	5.5	52.7	55584155	Ca2+‐ and phospholipid‐binding protein.
Dnase‐I deoxyribonuclase3	5.1	29.1	494869	Binds to G‐actin and blocks actin polymerization.
Tyrosine phosphatase70kd sHP‐I3	8	70.6	74761982	Regulate cell proliferation and differentiation.
Kinesin family member 3A2	6.2	80	46852174	Involved in chromosome movement.
Metabolic Proteins
GaPDH1	8.57	36.2	7669492	Glycolytic enzyme.
Alpha enolase1 Tryptophan‐tRNA ligase1	7	47.3	119339	Glycolytic enzyme.
Transferrin 2 , 3	5.8	53.5	107990	Mitochondrial tryptophanyl‐tRNA synthetase.
Vitamin D‐binding protein 2 , 3	6.8	77.1	4557871	Plasma protein and carrier for iron.
Structure Proteins	5.3	52.9	32483410	Vitamin transport.
Alpha 1 actin 1 , 2	5.2	42.4	4501883	Structural protein.
Albumin 1 , 2 , 3	5.9	69.4	4502027	Structural protein.
Myosin light chain alkali3	4.6	16.9	17986258	Regulate muscle contraction.
Antioxidants Proteins
Selenium binding protein 12	5.9	52.9	55663924	Participates in intra‐golgi protein transport.
Others
Surfactant A22,3	4.8	32	20470503	Respiratory gaseous exchange; host defense.
Fibrinogen gamma –βchain1,2	5.4	51.5	70906439	Involved in clot formation.
Arsenite‐resistant protein ASR22	5.5	89.6	74715925	Tumor suppresser gene.

Nitrated proteins in IPAH‐tissue¹, IPAH‐BALF² and controls‐BALF³.

Discussion

NO is a critical determinant of pulmonary vascular tone, as has been confirmed in many species, including humans, and from different experimental model systems, including isolated cells, arterials rings, and intact animals genetically deficient in NO synthesis. ²⁶ , ²⁷ , ²⁸ The loss of this critical vasodilator appears to be fundamental to the pathophysiology of IPAH. IPAH patients have reduced NO and its biochemical reaction products (NO₂ ⁻, NO₃ ⁻) in the epithelial lining fluid of IPAH lungs. ³ , ¹⁸ The low levels of NO in IPAH are related to substrate L‐arginine limitation and subsequent decrease in NO synthesis. ²⁹ Here, we provide evidence for oxidant consumption of NO as another contributor to the reduction in NO in IPAH. An alteration in NO metabolism was demonstrated by identification of lower levels of SOD and GPx, alteration in proteins modified by tyrosine nitration in IPAH, and the close correlation of SOD to levels of NO and its reaction product nitrate. The enzymatic inactivation of ROS is achieved mainly by SODs, GPx, and catalase. ³⁰ These enzymes act in a cooperative fashion. ³¹ SODs represent the first line of cellular defense against O₂ ⁻ and formation of peroxynitrite ³² , ³³ by converting superoxide to H₂O₂, which can, in turn, be removed by GPx or catalase reactions ¹⁶ ( Figure 5 ). The eukaryotic SODs exist in two distinct intracellular forms and one extracellular form (EC‐SOD). Cu/ZnSOD is located in the cytosol and peroxisomes, while MnSOD is located in the mitochondria. EC‐SOD is highly expressed in the blood vessels, particularly the arterial walls, ³⁴ , ³⁵ and has been shown to play a critical role in the regulation of the vascular system. ³⁶ Studies indicate that superoxide production is increased in systemic arterial diseases such as in atherosclerosis, ³⁷ , ³⁸ and that increased superoxide levels interfere with NO‐mediated regulation of the vascular tone. ³⁹ The expression of EC‐SOD is substantially reduced in patients with coronary artery disease, and the reduced SOD activity is associated with endothelial dysfunction in patients with this disease. ⁴⁰ The precise mechanisms by which SODs protect against vascular disease are still poorly understood. Previous studies support the idea that SOD enhances the bioavailability of NO by scavenging superoxide, resulting in a prolongation of its beneficial effect. In support of this notion, the administration of recombinant human SOD to the lungs of piglets reduces pulmonary inflammation from extended exposure to hyperoxia and high concentrations of NO (100 ppm). ⁴¹ Studies implicate that NO and SOD are coordinately regulated and are the interacting determinants of vascular endothelial function. ⁴² For example, the inducible NOS (iNOS) and EC‐SOD are both elevated in human and rabbit atherosclerotic lesions. ⁴³ Similarly, EC‐SOD expression in eNOS‐deficient mice is markedly reduced as compared to wild‐type mice under normal physiological conditions. ⁴⁴ GPx and catalase serve as the primary enzymatic pathways for clearance of H₂O₂. ⁴⁵ There are at least fve GPx isoenzymes in mammals. ⁴⁶ In mice, the lack of GPx1 appears to be associated with an abnormal vascular and cardiac function and structure. ⁴⁷ A previous study identifies that the decrease or absence of GPx1 activity in carotid atherosclerotic plaques/lesions is associated with the development of more severe lesions in humans. ⁴⁸ In patients with coronary artery disease, a low level of red cell GPx1 activity is an independent risk factor of cardiovascular events. ⁴⁶ Although not measured in this study, previous reports have shown that the S‐nitrosated (GSNO) form of GSH is an equivalently effective substrate for the GPx enzymes ⁴⁹ , ⁵⁰ ( Figure 5 ). In fact, GSNO concentration decreases in the presence of GPx, indicating that GPx increases the availability of NO by its release from GSNO. This suggests that reduced levels of GPx may contribute to the reduced level of NO in IPAH and provides a potential mechanism for the inverse association of GPx to PAP discovered in the stepwise regression prediction model. ⁴⁹ , ⁵⁰ This study implicates the alterations of antioxidant enzymes in the pathophysiology of IPAH. The logarithmic relation of NO and SOD indicates that at low levels of SOD, NO concentration increases in direct proportion with increasing SOD activity. However, this relation also predicts a plateau NO level that indicates that even in the situation of infinite SOD to prevent superoxide consumption, other factors limit NO levels within the lungs. Together with the inverse association of SOD to NO₃ ⁻, these findings support the hypothesis that SOD levels are a determinant of NO levels through effects on oxidant consumption pathways. Importantly, Browers and colleagues previously identified that the amount and the activity of Mn‐SOD were reduced in the smooth muscle cells of the vessel walls in lung tissue from patients with IPAH as compared to normal lung tissue. ⁵¹ Furthermore, the overexpression of human EC‐SOD in monocrotalin (MCT)‐induced rats significantly reduces pulmonary hypertension development. ⁴⁹ Previously, the reports have demonstrated an increase in nitrotyrosine immunostaining in IPAH lungs. ⁵¹ This study shows that the amount of nitrotyrosine present in IPAH BAL was not greater than in controls. However, the protein nitration profile was altered, supporting the existence of a nitrative stress in IPAH. The nitration of proteins with or without concomitant oxidation affects protein functions. MnSOD is one of the prominent enzymes that have been identified as a target for nitration. ⁵² Although our analyses did not detect MnSOD nitration, protein modifications may result in a more rapid turnover of protein, or be at a level not detectable by proteomic techniques. Furthermore, previous studies show that MnSOD and EC‐SOD expression are regulated by NO synthesis. ⁴⁴ , ⁵³ For example, endothelial production of NO induces EC‐SOD expression through cGMP‐mediated pathways. ⁴⁴ In mice genetically deficient in endothelial NOS, EC‐SOD levels are more than 2‐fold lower than wild‐type mice. ⁴⁴ Consistent with these previous studies, MnSOD and EC‐SOD expression were both reduced in the low NO environment of IPAH. Thus, although the SODs prevent oxidant consumption and thus augment and prolong NO bioactivity, MnSOD and/or EC‐SOD are also reciprocally upregulated by NO. On the other hand, although the loss of SOD activity is related to the loss of EC‐SOD and MnSOD expression in IPAH, this does not exclude the other possible causes for SOD activity loss, such as oxidative inactivation of the proteins. ⁵⁰ , ⁵¹ , ⁵² , ⁵³ Nevertheless, these findings suggest that the reduction of the SOD and GPx antioxidants, in particular SOD, is involved in the pathophysiology of IPAH, most likely through effects on NO availability for vasoregulation. In addition to vasoregulatory effects via cGMP, NO and/or other reactive nitrogen species regulate cyclooxygenases production of prostacyclin, a potent vasodilator. ⁵⁴ , ⁵⁵ It is interesting to speculate that antioxidant loss and lower‐than‐normal NO levels may combine to exacerbate the vasoconstrictive process in IPAH through adverse effects on prostacyclin synthesis. Future studies evaluating SOD supplementation to augment endogenous NO effects, including effects on endogenous prostacyclin synthesis, will allow the determination of any benefit for the reduction in PAPs and improvement in clinical outcomes of patients.

Figure 5.

Schematic diagram illustrating the consumptive pathway of NO. Increased ROS and reduction in SOD lead to an increased reaction of superoxide with NO and the formation of peroxynitrite, which nitrates tyrosine residues of proteins. NO can also be consumed by its reaction with oxygen, hemoglobin, and heme‐containing proteins. The loss of NO likely contributes to the lower MnSOD and EC‐SOD expression in IPAH (see text). Dysregulation of NO and antioxidants SOD and GPx contribute to pulmonary hypertension. PAP = pulmonary artery pressure; NO = nitric oxide; eNO = exhaled nitric oxide; NOS = nitric oxide synthases; NO₂ ⁻= nitrite; NO₃ ⁻= nitrate; ONOO⁻= peroxynitrite; MPO = myleoperoxidase; SOD = superoxide dismutases; GPx = glutathione peroxidases; GSH = glutathione; O₂ ⁻. = superoxide; H₂O₂= hydrogen peroxide; GSSG = glutathione disulfde.