Conceived and designed the experiments: EN-G. Performed the experiments: TH RB SV MM LV. Analyzed the data: EN-G TH RB SV SB KS. Contributed reagents/materials/analysis tools: EN-G KS. Wrote the paper: EN-G TH SV LV. Assisted with editing: SV SB KS RB MM.
The authors have declared that no competing interests exist.
Exposure of newborn calves to chronic hypoxia causes pulmonary artery (PA) hypertension and remodeling. Previous studies showed that the redox-sensitive transcription factor, early growth response-1 (Egr-1), is upregulated in the PA of chronically hypoxic calves and regulates cell proliferation. Furthermore, we established in mice a correlation between hypoxic induction of Egr-1 and reduced activity of extracellular superoxide dismutase (EC-SOD), an antioxidant that scavenges extracellular superoxide. We now hypothesize that loss of EC-SOD in chronically hypoxic calves leads to extracellular superoxide-mediated upregulation of Egr-1. To validate our hypothesis and identify the signaling pathways involved, we utilized PA tissue from normoxic and chronically hypoxic calves and cultured calf and human PA smooth muscle cells (PASMC). Total SOD activity was low in the PA tissue, and only the extracellular SOD component decreased with hypoxia. PA tissue of hypoxic calves showed increased oxidative stress and increased Egr-1 mRNA. To mimic the
Infants, children and adults with chronic lung diseases complicated by alveolar hypoxia are at risk for developing pulmonary hypertension, which is associated with a high morbidity and mortality
The contribution of EC-SOD to the pathogenesis of neonatal pulmonary hypertension has not been substantially investigated. Broadly it has been recognized that the neonatal lung is susceptible to oxidative stress due to the developmental regulation of antioxidant defenses
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All animal studies were approved by the Institutional Animal Use and Care Committees (Colorado State University School of Veterinary Medicine, Fort Collins, CO (Protocol 10-1927A) or University of Colorado School of Medicine, Aurora, CO (Protocol # 71108(03)1E). Chronic exposure to hypoxia is a well-established model of pulmonary hypertension, characterized by elevated pulmonary artery pressures and pulmonary artery remodeling. In this study, 1-day-old male Holstein calves (Laluna Dairy Farm, Fort Collins) were placed in a hypobaric hypoxia chamber at PB 445 mm Hg (simulating 15,000 ft or 12% FIO2) for 14 days along with age-matched calves maintained in ambient Denver altitude (PB 640 mm Hg). Tissue from chronically hypoxic calves was harvested within the chamber under hypobaric hypoxic conditions while tissue from normoxic calves was obtained in normobaric atmosphere. Calves were euthanized with an overdose of pentobarbital sodium (160 mg/kg body weight) and lung tissue and intraparenchymal proximal pulmonary artery tissue containing all three layers of the vessel wall were rapidly dissected and flash frozen. Another pulmonary artery segment was placed in cold media for preparation of primary vascular cell lines. Tissue was flash frozen for analysis.
To test how extracellular O2.− generated in the pulmonary artery wall can upregulate the redox sensitive transcription factor, Egr-1, experiments were done with smooth muscle cells (SMC) isolated from the pulmonary artery of normoxic or chronically hypoxic calves using an explant technique as previously described
Cells were treated with XO because it is an enzyme known to be upregulated in the pulmonary circulation in models of pulmonary hypertension and it is a reproducible method to generate extracellular O2.− in order to study the impact of O2.− released in this particular cell compartment. Cells were treated with XO dissolved in phosphate buffered saline (PBS) (Sigma, St Louis, MO) and hypoxanthine (HX) (Sigma) dissolved in 0.1 M sodium hydroxide (NaOH). The optimal dose of XO and HX was established with a dose and time response curve. Based on pilot results, all remaining studies were carried out using cells treated for 1 hour with 8 mU/mL XO and 0.5 mM HX (XO/HX). Control cells for XO/HX treatment received the vehicles alone (PBS and 0.1 M NaOH). For studies with extracellular antioxidant treatments, cells were pretreated for 30 minutes with 500 U/mL SOD (Sigma) and/or catalase 600 U/mL (Boehringer Mannheim, Mannheim, Germany). Catalase was first dialyzed against PBS for 24 hours in PBS and activity determined as previously described
An siRNA targeting EC-SOD and a non-targeting siRNA were transfected into human PASMC (Lonza Walkersville, Walkersville, MD) using the siPORT NeoFX transfection reagent (Ambion). Cells (12×105) in a 6-well plate, were transfected with a final concentration of 5 nM of non-targeting (negative control, siNEG)) or siRNA targeting EC-SOD, (siEC-SOD). The manufacturer's suggested protocol of a reverse transfection was followed. Transfected cells were then harvested after 48 h for mRNA or protein analysis. After every transfection, the EC-SOD knockdown was confirmed by qPCR.
For pulmonary artery segments, the fibrous tissue was first pulverized using a mortar and pestle on liquid nitrogen to facilitate homogenization. Cells, 4×104 per well, were seeded in a 6 well plate (Corning, Lowell MA) and allowed to grow for 48 hours prior to treatment. RNA was isolated from tissue using TRIzol (Invitrogen, Carlsbad, CA) and from cells using the RNeasy Plus kit (Qiagen, Germantown, MD) according to manufacturers' instructions. The concentration and purity of each total RNA sample was determined by the NanoDrop® ND-1000 spectrophotometer (NanoDrop, Wilmington, DE). The integrity of total RNA samples was examined by 2100 Bioanalyzer and the RNA 6000 Nano Kit (Agilent Technologies).
RNA (1 µg per reaction) was reverse transcribed with the Maxima First Strand cDNA synthesis kit (Fermentas International Inc, Glen Burnie, MD). PCR was performed on the MyiQ Detection System (Bio-Rad, Hercules, CA) with the RT2 Real-Time SYBR Green/Fluorescein PCR master mix (SABiosciences, Frederick, MD). Reactions were run in triplicate and results analyzed by the 2 (−delta delta CT) Method, normalizing the gene copy numbers to hypoxanthine-guanine phosphoribosyltransferase (HPRT). Data are expressed in figures as either actual copies of Egr-1/HPRT or, to more easily visualize fold change in certain experiments, Egr-1/HPRT normalized to the mean expression of the control group. Primers for calf tissue were designed with NCBI Primer-BLAST software. Primers used were Egr-1 forward:
Pulverized lung or pulmonary artery tissue was homogenized in RIPA buffer (Sigma) containing protease (Sigma) and phosphatase (Thermo Scientific, Rockford, IL) inhibitors and centrifuged to remove cellular debris. For total cell lysates from cultured cells, 5.0×105 cells were seeded in 100 mm dishes (Corning) and allowed to grow for 48 hours prior to treatment. After appropriate treatment, media was removed and plates placed on ice, cells washed 2× PBS and then lysed in RIPA buffer (Sigma) containing protease (Sigma) and phosphatase (Thermo Scientific) inhibitors. Cells were scraped and placed in −80°C overnight followed by sonification and centrifugation at 4°C degrees to collect the supernatant. Protein concentration was determined using the Pierce 660 nm protein assay reagent (Thermo Scientific). Nuclear protein was extracted from calf PASMC (1×106 cells per condition) using NE-PER Nuclear and Cytoplasmic Extraction Reagents according to product instructions (Pierce Biotechnology, Rockford, IL).
For Western blot analysis with the phosphoERK1/2 and total ERK1/2 antibodies, 20 µg human or calf total PASMC protein was loaded on a 4–12% Bis-Tris Gel (Invitrogen, Carlsbad, CA) and protein separated by gel electrophoresis. Proteins were transferred to PDVF membranes using the semi-dry method (Invitrogen). Blots were blocked with 5% milk in Tris Buffered Saline with 0.05% Tween 20 (TBST) and probed with the primary monoclonal mouse phospho-p44/42 MAPK antibody (Cell Signaling, 1∶1000) overnight at 4°C followed by an anti-mouse HRP-conjugated secondary antibody (Millepore, Billerica, MA). Blots were developed with Enhanced Chemiluminescence (ECL) plus (Thermo Scientific) and then stripped with Restore Plus Western Blot Stripping Buffer according to kit instructions (Thermo Scientific) and reprobed with rabbit monoclonal Total ERK antibody (Cell Signaling, 1∶1000), for normalization: Bands were quantified by densitometry and expressed as the ratio of phosphoERK/totalERK. Blots from human PASMC total cell lysates were also probed with polyclonal rabbit anti-Egr-1 (Cell Signaling, 1∶500), cyclin D1 (Cell Signaling , 1∶1,000) and ß-actin as a loading control. For calf PASMC, to reduce non-specific binding by the Egr-1 antibody, likely due to poor cross-reactivity with the anti-human Egr-1 antibody, 25 µg nuclear extracts were used instead for Western blot. Equal nuclear protein loading was confirmed by Ponceau S staining (Millipore). For the Western blot with protein from pulmonary artery or lung tissue, 20–100 mg of protein homogenate was loaded on the gel as well as 1 mg purified bovine EC-SOD protein as a positive control. The blot was probed overnight at 4°C with 1∶1,000 rabbit anti-human EC-SOD ab in 5% milk, which recognizes bovine EC-SOD (purified EC-SOD and EC-SOD antibody kindly provided by Tim Oury, MD, PhD, University of Pittsburgh). Western blots for PA protein were also probed with antibodies against Cu,Zn SOD (Abcam, 1∶2,000) Mn SOD (Millipore, 1∶1,000), and ß-actin in protein homogenates from normoxic and chronically hypoxic calves.
2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide, disodium salt (XTT) (Sigma) was used to detect extracellular O2.− generated in response to XO/HX or hypoxia in cultured calf PASMC. SMC (3×104), were plated in triplicate in a 96 well plate in phenol free medium containing the supplements as described above. Cells were grown for 48 hours and prior to treatment, cells were washed with 1× PBS and replaced with fresh phenol-free medium. To measure the change in extracellular O2.− generated by XO/HX, XTT (100 µm) was added to the cells along with either XO/HX or vehicle. The change in absorbance at 470 nm was read by a Biorad 680 microplate reader (Biorad, Hercules, CA) at 1 hr following XO/HX. One set of cells were pre-treated for 30 minutes with 500 U/mL SOD (Sigma) prior to the addition of XO/HX. The experiment was repeated three times. Using the extinction coefficient of 21600 M−1 cm−1 (XTT), the superoxide flux was calculated and data expressed as rate of superoxide flux (M•s−1). Hydrogen peroxide was measured in calf PASMC treated with 1 hour of XO/HX using a fluorometric assay as described by Hyslop
Pulverized lung and pulmonary artery tissue (300 mg) were homogenized in 10 volumes of ice-cold buffer (50 mM potassium phosphate, pH 7.4, with 0.3 M KBr, 0.05 mM phenylmethylsulfonyl flouride, and 3 mM diethylene-triaminepentaacetic acid) and centrifuged to remove cellular debris. For the pulmonary artery homogenates, EC-SOD was separated from intracellular SOD (Cu,Zn SOD and Mn SOD) using a concanavalin A column (Pierce,Rockford,Illinois) as previously described
The GSH/GSSG ratio was measured as a marker of oxidative stress in PA tissue following chronic hypoxia as previously described
These assays were carried out using human PASMC. Cell proliferation was measured by MTS assay using CellTiter 96 AQueous One Solution (Promega, Madison, WI). Twenty-dour hours after transfection with siRNA, ∼2000 cells were plated in a 96-well plate and twenty microliters of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) was added to the wells on consecutive dates. MTS is bio-reduced by cells into a colored formazan product that reduces absorbance at 490 nm. Plates were read at 24 and 48 hours using a BioTek
Data are expressed as means ± SE. Unpaired t-test analysis or one-way Anova with Bonferroni post-hoc analysis was performed using Prism software (GraphPad, San Diego, CA, USA). Statistical significance was defined as
We speculated that chronic hypoxia would lead to a loss of EC-SOD activity in the pulmonary artery of the chronically hypoxic neonatal calf, similar to our observation in the lungs of weanling mice exposed to hypoxia
In pulmonary artery tissue homogenates from two-week old chronically hypoxic (Hypo) and age-matched normoxic (Norm) control calves, extracellular SOD was separated from intracellular SODs (Cu,Zn SOD or SOD1 and Mn SOD or SOD2) using a concavalin A sepharose column, based on EC-SOD's predominantly glycosylated state that enables it to bind to the column and thus separate intracellular SODs (nonbound) from extracellular SOD (bound, then eluted) fraction. SOD activity was measured in each fraction using the SOD assay kit-WST.
Since EC-SOD activity is markedly decreased in the tissues of chronic hypoxic calves, it could trigger oxidative stress. Oxidative stress in the pulmonary artery was evaluated by determining the ratio of reduced to oxidized glutathione (GSH/GSSG) in the calf PA following 2 weeks of hypoxia. During periods of increased oxidative stress, GSSG will accumulate and the ratio of GSH to GSSG will decrease. Therefore, the determination of the GSH/GSSG ratio is a general indicator of oxidative stress in cells and tissues. We observed that the ratio of GSH/GSSG in calf PA decreased by 50% with hypoxia (
We have previously reported that the level of EC-SOD activity in the lung modulates the hypoxic upregulation of Egr-1 in mice
Egr-1 mRNA expression was measured by qPCR in the pulmonary artery of 2 week old chronically hypoxic calves compared to age-matched normoxic control calves . Data are expressed as copies of Egr-1 per copy HPRT, mean ± SEM. *p<0.05 vs. Norm; n = 4–5.
To evaluate whether the increased Egr-1 expression detected in the pulmonary artery of chronically hypoxic calves could be directly mediated by extracellular ROS, we performed a series of experiments in SMC treated with XO+HX as an exogenous source of ROS. A dose response curve with a concentration of XO between 1–16 mU/mL and a time course between 30 minutes to 4 hours was initially performed to select the experimental conditions (
(
XO generates extracellular O2.−, which rapidly dismutates to H2O2, though XO can also generate H2O2 directly, depending on conditions such as pH or oxygen concentration
MAPK/ERK signaling pathways have been shown to regulate Egr-1, and many MAPK enzymes are redox sensitive. To determine if extracellular O2.−, or H2O2 arising from the dismutation of O2.−, induced the upregulation of Egr-1 via MAPK, we first evaluated whether XO/HX treatment resulted in phosphorylation of ERK1/2. Treatment with XO/HX led to phosphorylation of ERK1/2 (
Calf PASMC were treated with XO (8 mU/mL) and hypoxanthine (0.5 mM) for 1 hour (XO) and protein was isolated for Western blot analysis.
Upon completing studies that tested the impact of extracellular O2.− on Egr-1 expression, we performed a final series of experiments in PASMC isolated from normoxic and chronically hypoxic calves and in human PASMC following siRNA silencing of EC-SOD. These experiments were designed to confirm the impact of the level of EC-SOD expression on Egr-1 expression in the
We then performed additional experiments with human PASMC to confirm the impact of EC-SOD on Egr-1 expression. We selected human PASMC because these cells express high levels of EC-SOD at baseline, thus knock-down of EC-SOD would have a more significant impact than in the calf cells with low EC-SOD expression levels at baseline. Human PASMC were transfected with human EC-SOD siRNA (siEC-SOD) or the negative control siRNA vector (siNEG), and EC-SOD mRNA and protein was measured. We first confirmed that we significantly knocked down EC-SOD mRNA (
Human PASMC were transfected with human EC-SOD siRNA (siEC-SOD) or the negative control siRNA vector (siNEG) and experiments were performed 48 hours later.
Pulmonary vascular cells isolated from humans with PAH or animals with experimental PH exhibit increased cell proliferation and decreased cell apoptosis. To evaluate the impact of EC-SOD on PASMC growth characteristics, we measured cell proliferation and apoptotic index following knock down of EC-SOD. After siRNA treatment to knock down EC-SOD, there was significant increase in cell proliferation (
Understanding the molecular mechanism that induces pulmonary hypertension in neonates is important for the development of improved therapeutics. In this current study, we used the chronically hypoxic calf model of pulmonary hypertension to evaluate the activity of EC-SOD in a severe neonatal disease model and to better understand the role for extracellular O2.− in the upregulation of Egr-1, a redox sensitive transcription factor implicated in vascular remodeling. We tested the hypothesis that loss of EC-SOD in chronically hypoxic calves leads to extracellular O2.− -mediated upregulation of Egr-1 via activation of MAPK pathways. We tested tissue from chronically hypoxic calves to demonstrate the decrease in EC-SOD in the pulmonary artery
We previously reported that EC-SOD activity is impaired following exposure to chronic hypoxia, and overexpression of lung EC-SOD in two murine models of pulmonary hypertension protected against pulmonary vascular remodeling and prevented the early upregulation of the redox sensitive transcription factor, Egr-1
To test the impact of extracellular O2.−on Egr-1 expression in PASMC, we treated cells with XO, as an enzymatic source of O2.−. This exogenous model has potential relevance to the
Published studies have shown enhanced induction of Egr-1 with hypoxia
There is extensive data implicating the MAPK/ERK1/2 pathway in the regulation of Egr-1 and in the pathogenesis of chronic hypoxic pulmonary hypertension
The regulation of Egr-1 has been implicated in vascular remodeling in a number of models including the calf model of chronic hypoxic pulmonary hypertension. For example, upregulation of Egr-1 contributes to cyclin D1 expression and hypoxia-induced cell proliferation in fetal lung fibroblasts and stimulates insulin-like growth factor-1 receptor, resulting in vascular remodeling of vein grafts
In summary, we report that the neonatal calf has low SOD activity levels in the pulmonary artery and EC-SOD activity decreases further in the calf with pulmonary hypertension secondary to chronic hypoxia. The loss of EC-SOD is associated with an increase in Egr-1 mRNA in the pulmonary artery. We thus tested the impact of extracellular O2.− and its product, H2O2, on Egr-1 expression in vascular smooth muscle and found that exogenous production of these ROS, via xanthine oxidase, upregulated Egr-1. Hypoxia itself had a minimal effect on either Egr-1 expression or O2.− production. Extracellular ROS together with activation of ERK1/2 by phosphorylation regulated the increase in Egr-1. Therefore, targeting Egr-1 by controlling extracellular ROS generation and thereby balancing the cellular redox status could be a potential therapeutic pathway for pulmonary artery remodeling. Our data provided new insight into the role of extracellular O2.− and EC-SOD in the pathogenesis of pulmonary hypertension.
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