ONLINE REPOSITORY
Title: Overexpression of Pulmonary Extracellular Superoxide Dismutase Attenuates Endotoxin Induced Acute Lung Injury.
Authors: Dr. Patrick Hassett, MB, FCARCSI 1,2,
Dr. Gerard F. Curley, MB, FCARCSI 1,2,
Dr. Maya Contreras, MB, FCARCSI 1,2,
Ms Claire Masterson, BSc1,2
Dr. Brendan D. Higgins, PhD 1,2,
Prof. Timothy O’Brien, MD, PhD 3,
Dr. James Devaney, PhD 1,2,3,
Dr. Daniel O’Toole, PhD 1,2,3,
Prof. John G. Laffey, MD, FCARCSI 1,2.3.
Affiliation: 1Anaesthesia, School of Medicine, Clinical Sciences Institute, National University of Ireland, Galway; 2Lung Biology Group, National Centre for Biomedical Engineering Sciences, National University of Ireland, Galway, and 3Regenerative Medicine Institute, National University of Ireland, Galway, Ireland.
Materials and Methods
Preparation of AAV Vectors encoding EC-SOD and Null Transgenes
AAV vector production was carried out as previously described with several modifications [1]. Briefly, the EC-SOD transgene was first ligated into the multiple cloning site of a p-AAVMCS vector (Agilent Technologies Inc., Santa Clara, CA, USA), product size was confirmed by gel electrophoresis, and the plasmid was sequenced (Eurofins MWG Operon, Ebersberg, Germany) to validate insert sequence integrity. Commercially available plasmid-prep kits (QIAprep® Gigaprep kit; QIAGEN, West Sussex, UK) were used to generate the required amount of plasmid.
293T cells were used (passage 4-20) for the AAV particle synthesis.Plasmid DNA and adeno-associated virus envelope for AAV serotype 6 was transfected into 293T cells, using jetPEITM (Autogen Bioclear UK Ltd., Calne, UK) transfection reagent. Forty-eight hours after transfection, cells were harvested and pelleted. Cells were resuspended in lysis buffer (150 mM NaCl, 50 mM Tris, pH8.5) and subjected to three rounds of freeze/thaw followed by benzonase treatment (50 U/ml) for 30 min at 37 ◦C.
The cell lysate was centrifuged and the clear lysate fraction withdrawn and loaded on an iodixanol gradient, which was spun at 69,000 rpm for 1 h. The viral particles were further purified from the appropriate iodixanol layer by affinity column chromatography (Q-Sepharose affinity columns, GE Healthcare, Little Chalfont, UK). The particles were then concentrated and desalted by centrifuging through an Amicon Ultra centrifugal filter device (Millipore, Billerica, MA, USA) at 2000g for 20 minutes. Viral vector aliquots were stored at −70 ◦C. Viral vector particle titres (DNAse-resistant particles, drp) were determined by quantitative real-time polymerase chain reaction (qRT-PCR) using an ABI7000 Real-Time PCR machine (Applied Biosystems, Warrington, UK). Primers for the CMV promoter were used as target. Primers used were: sense: 5'-TGGAAATCCCCGTGAGTCAA-3', antisense: 5'-CATGGTGATGCGGTTTTGG-3'. Fluorescence was compared to a standard curve constructed using pAAVMCS-EC-SOD plasmid as template.
The process was then repeated for the synthesis of an empty AAV6 vector with no gene product ligated into the multiple cloning site. This viral vector is referred to subsequently as AAV-NULL.
Preparation of Vector for Instillation
The AAV particles were divided into aliquots of 125μl of frozen virus containing 3.0 x 108drp per l. As required, an aliquot was thawed and added to 125l of the porcine surfactant Curosurf® (120mg/ml) (Trinity-Chiesi Pharmaceuticals Limited, Cheadle, UK), to create a final instillate volume of 250l. For those animals receiving vehicle only, the instillate was 125l of Curosurf® mixed with 125ul of phosphate buffered saline (PBS).
Endotoxin Induced ALI Model
Specific-pathogen-free adult male Sprague Dawley rats (350 to 450g) were used. The experimental model was based on those previously reported, with several modifications [2-3]. All work was approved by the NUI Galway Research Ethics Committee and conducted under license from the Department of Health, Ireland.
Intra-pulmonary instillation Procedure: Animals were anesthetized by inhalational induction with isoflurane and an intra-peritoneal injection of 40 mg.kg-1 of ketamine (Pfizer, Kent, United Kingdom) [4-5], and the trachea intubated with a size 14 intravenous catheter (BD Insyte®; Becton Dickinson Ltd., Oxford, United Kingdom). Animals were randomized to intratracheal instillation of the surfactant/PBS mixture containing: (a) vehicle alone; (b) 3.5 x 1010 AAV-Null; or (c) 3.5 x 1010 AAV-EC-SOD. This dose of AAV-EC-SOD was determined to produce effective transgene expression in preliminary studies. The instillate was injected in two 125l aliquots over 2 minutes, with the animal turned on to the left and right sides respectively. The animals were subsequently extubated, allowed to recover from anesthesia, and placed in an individually ventilated recovery cages (Tecniplast Inc., Buguggiate, Italy). Subsequently, two additional groups were randomized to receive (a) vehicle or (b) AAV-EC-SOD instillation.
Endotoxin/Vehicle Instillation Protocol: Five days following vector/vehicle instillation, at which stage significant transgene expression had been demonstrated to develop in pilot studies, the animals were re-anesthetized as described above. 5 mg.kg-1 of Endotoxin derived from E. coli serotype 055:B5 (Fluka, Poole, U.K.) or a similar volume of vehicle was instilled intra-tracheally [2]. The animals were then allowed to recover from anesthesia and returned to their cages.
This strategy resulted in a 5 group design, with 2 sham injury and 3 endotoxin induced injury groups, as follows: Sham injury plus Vehicle treatment (Sham-Vehicle); Sham injury plus EC-SOD (Sham-EC-SOD); Endotoxin injury plus vehicle (LPS-Vehicle); Endotoxin injury plus null vector (LPS-Null); Endotoxin injury plus EC-SOD (LPS-EC-SOD).
Assessment of Lung Injury: Twenty four hours following endotoxin instillation, all animals were again anesthetized as described above, intravenous access was secured via the dorsal penile vein and anesthesia maintained with repeated intravenous boli of Saffan® (Alfaxadone 0.9% and alfadadolone acetate 0.3%; Schering Plough, Welwyn Garden City, UK) [4-5]. A tracheostomy tube (1mm internal diameter) was then inserted and intra-arterial access (22 or 24 gauge cannulae; Becton Dickinson, Franklin Lakes, NJ, USA) was sited in the carotid artery. Cis-atracurium besylate 0.5mg.kg-1 (GlaxoSmithKline, Dublin, Ireland) was administered intravenously and the lungs were mechanically ventilated (Model 683; Harvard Apparatus, Holliston, MA, USA) at a respiratory rate of 80.min-1, tidal volume 6 ml.kg-1 and positive end-expiratory pressure of 2cm H2O. To minimize lung derecruitment, a recruitment maneuver consisting of positive end-expiratory pressure 15cm H2O for 20 breaths was applied at the start of the protocol. All animals were ventilated with an inspired gas mixture of FiO2 = 0.3, and FiN2 = 0.7, for 20 min.Body temperature was maintained at 36–37°C using a thermostatically controlled blanket system (Harvard Apparatus) and confirmed with an indwelling rectal temperature probe. Systemic arterial pressure, peak airway pressures and temperature were continuously measured, and arterial blood samples drawn for assessment of systemic oxygenation, ventilation and acid-base status (ABL 710; Radiometer, Copenhagen, Denmark). Static inflation lung compliance was measured as previously described [2]. Heparin (400 IU.kg-1) was administered intravenously and the animals were then sacrificed by exsanguination under anesthesia [4-5].
Sampling and Assay Protocol
Bronchoalveolar Lavage Analysis:Immediately post-mortem, the heart-lung block was dissected from the thorax and bronchoalveolar lavage (BAL) collection performed as previously described [4-5]. Total cell numbers per ml in BAL fluid were counted and differential cell counts were performed following staining with Diff-Quik (Siemens Schweiz AG, Zürich, Switzerland). Samples of BAL fluid were centrifuged, snap frozen in liquid N2 and stored at -80C. The concentrations of Cytokine-Induced Neutrophil Chemoattractant-1 (CINC-1) and Interleukin-6 (IL-6) in the BAL were determined using a quantitative sandwich ELISA (R&D Systems Europe Ltd., Abingdon, U.K.) as previously described [6-7]. The Micro BCATM Protein assay kit (Pierce, Rockford, IL), was utilized to determine total BAL protein levels as previously described [8].
Histologic Analysis: The left lung was isolated and fixed [2, 9], and the extent of histologic lung damage determined using quantitative stereological techniques as previously described [10].
Assessment of Gene Transfer:SOD transgene expression was determined in lung homogenates by real-time rtPCR using transgene specific primers and Western Blotting as previously described [11-13]. Briefly, RNA was extracted from lung tissue following homogenization and, after treatment with DNAase (Ambion™ DNAase Treatment Kit; Applied Biosystems, Warrington, UK), each sample of RNA was then quantified by spectrophotometry (Thermo Scientific Nanodrop 2000; Loughborough, UK). Following quantification, cDNA synthesis from RNA was performed using the ImProm-II™ reverse transcription system (Promega, Madison, WI, USA) for use in quantitative real time PCR. Each cDNA sample was subjected to duplicate analysis. Quantitative PCR was performed for EC-SOD gene, normalised against a GAPDH control product. Primer sequences (from MWG-Biotech) used were as follows: GAPDH sense: 5'-TTGTGAAGCTCATTTCCTGG-3', antisense: 5'-CATGTAGGCCATGAGGTCCA-3'. EC-SOD sense: 5'-ATGCTGGCGCTACTGTGTTC-3', antisense: 5'-ACTCCGCCGAGTCAGAGTT-3'.
Tissue Western Blot Analysis for EC-SOD: Lung tissue western blot analysis for EC-SOD was carried out as previously described [14]. Briefly, total cell protein was extracted and protein concentration determined using a BCA protein assay kit (Pierce). Samples were electropheresed on an SDS-PAGE gel and transferred to nitrocellulose. Primary goat anti-human EC-SOD polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), was used, with anti-goat antibody conjugated to horseradish peroxidase (Cell Signaling Technology, Danvers, MA, USA) as the secondary antibody. The membrane was subsequently incubated with a chemiluminescent substrate (SuperSignal West Pico; Pierce) and visualized with a Kodak Image Station 4000MM Pro (Carestream Health, Inc., Rochester, NY, USA). EC-SOD protein was detected at size 32.5kDa.
SOD activity assays of lung homogenates: SOD activity was assessed utilizing a SOD Activity Assay Kit (Sigma) as per manufacturer instructions. 20µg of each lung homogenate sample was transferred in triplicate to a 96 well plate. 200µl of working solution (containing buffers and xanthine substrate) were then added to each well and enzyme solution (xanthine oxidase) was added. This is a colorimetric assay in which the degradation of a highly water soluble tetrazolium salt, WST-1(2(4-Iodophenyl)-3-(4-nitrophenyl)-5(2,4-disulfophenyl)- 2H tetrazolium monosodium salt produces a formazan dye on upon reduction with a superoxide anion. The rates of reduction with O2 are linearly related to the xanthine oxidase activity and are inhibited by superoxide dismutase activity. Absorbance was read at 450nm using a Wallac plate reader (Perkin Elmer Life Sciences, Waltham, MA, USA).
Immunohistochemistry:Lung tissue was flash-frozen in liquid nitrogen and subsequently cut to 4m thick sections. Slides were fixed in cold acetone, washed, and non-specific sites blocked with 10% (v/v) goat blocking serum in PBS. Sections were then incubated with EC-SOD primary antibody raised in goat (Santa Cruz), 1µg/mL with 1.5% (v/v) goat serum in PBS, for 60 minutes. After washing, slides were incubated in rhodamine conjugated donkey-anti-goat secondary antibody (Santa Cruz) at 1µg/mL in 1.5% (v/v) goat serum in PBS for 60 minutes and washed. Hardset Mounting Medium containing DAPI (Vector Laboratories Inc., Burlingame, CA, USA) was applied followed by a cover slip and allowed to set for 60 minutes and fluorescence subsequently imaged on an Olympus IX71 microscope using Cell^P® software (Olympus Europa GmbH, Germany).
Statistical Analysis
Results are expressed as mean ± (SD) for normally distributed data, and as median (interquartile range, IQR) if non-normally distributed. Data were analyzed by repeated measures two-way ANOVA or one-way ANOVA as appropriate, followed by Student-Newman-Keuls, t test or Kruskalis-Wallis followed by Mann-Whitney U test with the Bonferroni correction for multiple comparisons, as appropriate. A p value of <0.05 was considered statistically significant.
Figure Legends
Figure A
Panel A: Histogram representing mean (SD) expression of EC-SOD mRNA in lung homogenates from each group.
Panel B: Histogram representing mean (SD) SOD activity in lung homogenates from each group.
* Significantly different from Sham-Vehiclegroup (P<0.05, ANOVA).
Figure B
Photomicrographs of representative sections of lung tissue.
Panel A is an image from a normal uninjured lung, included for comparison purposes.
Panel B is an image from an endotoxin injured lung that received intra-tracheal vehicle, demonstrating increased wall thickness and inflammatory cell infiltrate.
Panel C is an image from an endotoxin injured lung that received intra-tracheal vector encoding a null transgene, demonstrating similar histologic changes to Panel B.
Panel D is an image from an endotoxin injured lung that received intra-tracheal vector encoding the EC-SOD transgene, demonstrating reduced injury and inflammatory cell infiltration
Note: The scale bar equals 200m.
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