1Department of Gene Therapy, National Heart and Lung Institute, and 2Anaesthetics and Intensive Care, Chelsea and Westminster Hospital, and 3Electron Microscopy Unit, National Heart and Lung Institute, Faculty of Medicine, Imperial College London, London; and 4Gene Medicine Research Group, Nuffield Department of Clinical Laboratory Sciences, University of Oxford, United Kingdom
Submitted 1 December 2004 ; accepted in final form 24 January 2005
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ABSTRACT |
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acute respiratory distress syndrome; alveolar epithelial damage; oleic acid; gene therapy
Human keratinocyte growth factor (KGF) was first cloned in 1989 from an embryonic fibroblast cell line by Finch et al. (19). KGF is a heparin-binding growth factor that is secreted by fibroblasts and is known to act via a receptor specific to epithelial cells (37). KGF induces potent proliferative activity in a variety of epithelial cells including keratinocytes, important to hair follicle development (8) and skin growth during wound healing (35). Additionally, KGF stimulates proliferation of mammary (62) and pancreatic ductal epithelia (75). Importantly, KGF also stimulates proliferation of ATII cells in vitro (47) and in vivo (18). Intratracheal administration of recombinant human KGF (rhKGF) to normal rat lungs results in ATII cell hyperplasia 2 days following administration (63). This process initially produces a "piled up" appearance to the alveolar epithelium, caused by the increased number of ATII cells. By day 3, however, the newly formed ATII cells appear to migrate outward to cover a larger area of the alveoli, and by day 6, the alveolar epithelium appears normal. However, not all newly formed ATII cells differentiate into ATI cells, and apoptosis has been shown to be involved in maintaining homeostasis between cell populations in the lung (18, 57).
Administration of exogenous rhKGF has been shown to ameliorate lung injury in a range of animal models. A number of studies have demonstrated that KGF pretreatment resulted in reduced mortality following intratracheal instillation of hydrochloric acid (43, 71), intratracheal bleomycin (55, 73), hyperoxia (5, 46) and Pseudomonas aeruginosa-induced lung injury (65). Amelioration of both morphological damage to the alveolar epithelium and inflammation has been demonstrated in the bleomycin (22) and hydrochloric acid models (43, 71), P. aeruginosa-induced lung injury (65), and hyperoxia-induced lung injury (5, 46). Exogenous KGF upregulates active ion transport by increasing the expression of sodium pumps, primarily the Na+-K+-ATPase 1-subunit (6). Thus in the
-naphthylthiourea model of acute permeability edema, KGF administration reduced lung wet-to-dry weight ratios and bronchoalveolar lavage (BAL) protein (21, 36). Both of these effects were also demonstrated in P. aeruginosa-induced (65) and ventilator-induced lung injury (67).
The protective effects of KGF observed in the above studies suggest that, in addition to ATII cell hyperplasia, KGF may have many other beneficial effects on ALI. These may include scavenging of reactive oxygen species and increased DNA repair (59, 69). Additionally, KGF has been implicated in the reduction of alveolar epithelial susceptibility to mechanical deformation in vitro (44), possibly related to changes in the cytoskeleton or the extracellular matrix. Furthermore, KGF accelerates the rate of wound closure during mechanical deformation in various cell lines and primary cells by a combination of increased cell migration rate and cell spreading (20, 66). Atabai et al. (2) demonstrated that KGF-treated cells were more adherent to the extracellular matrix. Enhanced epithelial cell adherence to the denuded basement membrane may provide more rapid restoration of the alveolar architecture following lung injury (49). KGF also directly affects the expression of surfactant (70) and has been shown to stimulate the synthesis of all surfactant components in rat fetal ATII cells, thereby promoting maturation of the lung epithelium (7).
These studies raise the possibility that exogenous recombinant KGF protein might be used to stimulate alveolar epithelial repair in ALI and thus become a future therapy for ARDS. However, the significant cost of large quantities of rhKGF would likely limit the possibilities of prophylactic studies, particularly in larger animals and humans. Gene therapy, in which transfer of KGF DNA to alveolar epithelial cells may result in prolonged protein expression, has been proposed as an alternative approach. Thus, topical or systemic application of KGF-encoding plasmid DNA, complexed with cationic liposomes, may result in sufficient expression to produce ATII cell proliferation. Recently, the feasibility of this approach was demonstrated by Jeschke et al. (28) using liposome-mediated KGF gene transfer to improve dermal and epidermal regeneration following thermal injury in rats.
Several experimental models of lung injury have been developed that demonstrate pathophysiological changes similar to those in ARDS (50). ALI induced by intravenous administration of oleic acid (OA) resembles ARDS in many morphological, histological, and physiological respects (reviewed in Ref. 53). OA-induced lung injury has been studied in many laboratory animal species and is consistently associated with severe respiratory distress characterized by hypoxemia and reduced lung compliance due to acute alveolar damage, intra-alveolar hemorrhage, and leakage of proteinaceous fluid into the air spaces (10, 58). Histological changes include intra-alveolar edema and hemorrhage, epithelial disruption, fibrin deposition, and formation of hyaline membranes (11, 29, 52). The model has, however, not been well characterized in mice. Gene therapy for ARDS remains a relatively unexplored possibility, and mice have been used extensively as models of gene transfer to the lungs. With a view to investigating the potential of KGF gene therapy for ARDS, we therefore characterized a murine model of OA-induced lung injury and subsequently assessed the potential of both recombinant KGF protein and liposome-mediated gene transfer to ameliorate alveolar damage. For the latter, we assessed both topical and systemic administration, since KGF is a secreted protein, which may produce its biological effects whether produced by alveolar epithelium or pulmonary endothelium.
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MATERIALS AND METHODS |
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The human KGF (hKGF) cDNA was kindly supplied by Stuart Aaronson (Derald H. Ruttenberg Cancer Center, Mount Sinai School of Medicine, New York, NY) as a 2.1-kb insert in a pCEV9 backbone. The hKGF insert was excised from the pCEV9 backbone by restriction enzyme digestion and ligated into the eukaryotic expression vector pCI (Promega). The resulting plasmid was termed pCIhKGF. Evaluation of in vivo transfection efficiency was carried out using a plasmid expressing the reporter gene chloramphenicol acetyltransferase (pCF1CAT), kindly supplied by Genzyme (Framingham, MA). Large-scale purification of plasmid DNA for in vivo gene transfer was carried out using Qiagen EndoFree Giga plasmid purification columns according to the manufacturer's recommendations (Qiagen, Crawley, UK).
Intratracheal Administration of rhKGF
Experiments were carried out under the guidelines of the Animals (Scientific procedures) Act 1986, United Kingdom. Male BALB/c mice (68 wk, 1925 g, Harlan) were anesthetized with Hypnorm/Midazolam/water (Hypnorm:MZ:H2O; 1:1:3, 0.1 ml/10 g body wt). The trachea was visualized by blunt dissection and KGF (Amgen, Thousand Oaks, CA), and 1, 5, 10, or 15 mg/kg body wt was injected directly into the trachea in a volume of no more than 60 µl. Control animals received an equal volume of KGF reconstitution solution (sterile H2O, 0.0055% Tween 20, Amgen). The incision was closed with two sutures using 6/0 silk braided suture (Johnson & Johnson, Edinburgh, UK), and the animals were left in a heated cage at 30°C until they fully recovered from the anesthetic. Analgesic (Vetagesic, 1 in 100 dilution, 6 µl/g body wt) was administered before surgery.
Immunohistochemical Quantification of ATII Cell Proliferation
At appropriate time points (24 h to 1 wk) following rhKGF administration, animals were killed by dorsal arteriosection under terminal anesthesia, the trachea was cannulated, and the lungs expanded with 800900 µl of 10% formalin (Sigma, Poole, UK). The lungs were removed in toto, immersed in 10% formalin, fixed overnight at room temperature, processed, and embedded in paraffin wax. Serial 4- to 5-µm sections were cut, and the first section was stained with Harris's hematoxylin and eosin (BDH, Lutterworth, UK) for overall morphology. ATII cells were stained with a rabbit anti-sheep surfactant protein B (SP-B) polyclonal antibody (Chemicon International, Southampton, UK). The SP-B antibody binds to the surfactant producing lamellar bodies within the ATII cell, which following binding to a secondary detection antibody can be visualized as a characteristic brown stain. The antibody also stains SP-B within Clara cells in the airways and any SP-B that has been engulfed by alveolar macrophages. However, airways were not included in the quantification process, and alveolar macrophages are morphologically and spatially distinct from ATII cells. Cross-reactivity was therefore not a confounding problem. Briefly, paraffin-embedded sections were dewaxed, and endogenous peroxidase activity was blocked with 3% H2O2. This was followed by 2-h incubation with normal goat serum (DAKO, Ely, UK). Primary antibody binding [rabbit anti-sheep SP-B (Chemicon)] was carried out for 1.5 days at 4°C, followed by binding of biotinylated goat anti-rabbit IgG secondary antibody for 1 h, incubation with streptavidin-horseradish peroxidase (DAKO) for 1 h, and incubation in 3,3'-diaminobenzidine tetrahydrochloride (DAKO) for 15 min. The slides were counterstained in Mayer's hematoxylin (BDH) for 12 min and then washed, dehydrated in graded alcohol, and mounted with DPX mountant (BDH). Immunoreactive ATII cells were identified as cells with brown staining within the cytoplasm and quantified by counting 10 randomly selected fields of view per slide (x200, 1 slide/animal). All slides were evaluated in a blinded fashion.
Topical Gene Transfer
Preparation of lipid67/KGF pDNA complexes. The cationic liposome GL67 (Genzyme) was rehydrated in sterile endotoxin-free water to a final concentration of 1.2 mM. Plasmid DNA was resuspended in sterile endotoxin-free water to a final concentration of 1.6 mg/ml. Equal volumes of DNA and lipid were incubated separately at 30°C for 5 min to equilibrate temperature. Subsequently, the lipid was gently added to the DNA. The mixture was left to complex at 30°C for 15 min.
Intranasal instillation of vector-DNA complexes. Animals were individually anesthetized with methoxyflurane (Metofane; Mallingckrodt Veterinary, Mundelein, IL) in a closed chamber and held vertically while pressure was applied to the lower mandible to immobilize the tongue and prevent swallowing. Lipid-DNA complexes (80 µg pCIhKGF complexed with 120 µg GL67 in a total volume of 100 µl) were applied dropwise onto the nostrils of the animal with a pipette, allowing the mixture to be inhaled naturally. Control animals received the same volume of lipid complexed with an irrelevant plasmid (pCF1CAT).
Systemic Gene Transfer
Preparation of lipid and DNA. Sequential injection of lipid and DNA was carried out as described by Tan et al. (60). Lipid [1,3-dioleoyl-3-trimethylammonium propane (DOTAP)/cholesterol in a 1:1 molar ratio, kindly supplied by Dr. Leaf Huang, Pittsburgh, PA] was diluted with 5% dextrose to contain 900 nmol of lipid in 100 µl. DNA (pCIhKGF or pCF1CAT) was prepared with sterile water to contain 50 µg in 100 µl.
Sequential injection of lipid and DNA. Animals were anesthetized with Avertin (2.5%, 0.1 ml/10 g), the tail was heated transiently with warm water, and 100 µl of lipid solution (900 nmol) were injected into the tail vein. Two minutes later, 100 µl of the DNA (50 µg, pCIhKGF or pCF1CAT) solution were injected in a similar manner, and the animal was left to recover from the anesthetic in a heated cage at 30°C.
TaqMan RT-PCR for Detection of Vector-Specific Expression
Whole lungs were submerged in RNAlater (Ambion, Huntingdon, Cambridgeshire, UK) and stored at 4°C until further analysis. Samples were homogenized in 4 ml of RLT (Qiagen) before extraction of total RNA using RNeasy mini protocol (Qiagen). Levels of plasmid-derived mRNA were quantified by real-time quantitative multiplex TaqMan RT-PCR using the ABI Prism 7700 Sequence Detection System and Sequence Detector version 1.6.3 software (Applied Biosystems, Warrington, Cheshire, UK). The oligonucleotide primer and fluorogenic probe sequences were designed using Primer Express Software version 1.0 (Applied Biosystems). Plasmid-specific mRNA from the pCIhKGF was quantified using forward primer (5'-GCTTCTGACACAACAGTCTCGAA-3'), reverse pCI primer (5'-GGAGTGGACACCTGCCCA-3'), and the fluorogenic pCI probe (5'-FAM-TGCCTCACGACCAACTTCTGCAGC-TAMRA-3'). 18S ribosomal RNA was quantified using Ribosomal RNA Control Reagents (Applied Biosystems).
RNA was heated to 75°C for 5 min and then reverse transcribed with TaqMan RT reagents (Applied Biosystems). The RT-reaction mix (5 µl) consisted of 1x TaqMan RT buffer, 5.5 mM MgCl2, 500 µM each dNTP, 0.4 U/µl RNase inhibitor, 1.25 U/µl MultiScribe reverse transcriptase, 0.4 µM pCI reverse primer, 0.4 µM reverse rRNA primer, and 5 ng total RNA. Reactions were incubated at 48°C for 30 min followed by 95°C for 5 min. Subsequently, triplicate 25-µl PCRs were performed for each sample. Each 25-µl reaction consisted of 1x TaqMan Universal PCR Mastermix (Applied Biosystems), 300 nM forward pCI primer, 300 nM reverse pCI primer, 100 nM pCI probe, 50 nM forward rRNA primer, 50 nM reverse rRNA primer, 50 nM rRNA probe, and 5 µl reverse-transcribed template. Reactions were incubated at 50°C for 2 min and then 95°C for 10 min followed by 40 cycles of 95°C for 15 s and 60°C for 1 min.
Controls included no template and no reverse transcriptase control in which total RNA or MultiScribe reverse transcriptase and RNase inhibitor were omitted from the reverse transcriptase reaction, respectively. Relative levels of plasmid-derived mRNA were determined using the CT method (as described in Ref. 1a). In this study, the amount of pCI plasmid was normalized to 18S rRNA (endogenous reference) and expressed relative to a calibrator that was used throughout the study. The calibrator was total RNA extracted from mouse lung treated with 100 µg of pCIkLux in 150 µl via intranasal instillation (as described above) and harvested 24 h postdose.
Detection of Transgene Expression
KGF expression in lung homogenate. KGF ELISA was carried out using ELISA development reagents according to the manufacturer's recommendations (R&D Systems, Abingdon, UK). Lung homogenates (100 µl) from KGF-transfected mice or rhKGF standards were tested. Absorbance was determined in a microplate reader at 450 nm (wavelength correction 540 and 570 nm). The KGF concentration was calculated from a standard curve using known amounts of hKGF (1,000 pg/ml to 15.6 pg/ml) correlated to amount of protein detected in the lung homogenate.
Induction of Lung Injury
Male BALB/c mice (68 wk, 1925 g; Harlan, Bicester, UK) were anesthetized with Avertin (2.5%, 0.1 ml/10 g). The trachea was visualized by blunt dissection, and the animal was intubated via the oral route with a 22-gauge cannula (3S Healthcare, London, UK). Mechanical ventilation (120 strokes/min, stroke volume 200 µl) was carried out using a MiniVent (type 845; Hugo Sachs Elektronik). OA (0.2 ml/kg or 0.4 ml/kg body wt; Sigma, Poole, UK) suspended in 50 µl of sterile PBS was administered through the tail vein with a 0.5-ml insulin syringe (3S Healthcare). Control animals received 50 µl of PBS. Animals were monitored for the 1-h duration of the experiment and then killed by anesthetic overdose.
Lung Wet-To-Dry Weight Ratios
Lungs were removed in toto at the end of the experiment. The trachea and esophagus were separated from the lungs by blunt dissection, and the wet weight of the latter was determined. Subsequently, the lungs were incubated at 55°C overnight to remove all moisture. The dry weight was then measured, and the ratio of wet-to-dry weight was calculated.
BAL
The animal was extubated, and a 22-gauge cannula (3S Healthcare) was inserted into the trachea. The lungs were lavaged with 500 µl of PBS three times (total volume 1.5 ml). Retrieval volume was maximized by compression of the thorax following the last lavage.
Total Cell Counts
BAL fluid (BALF) samples were centrifuged at 900 relative centrifugal force (gav) for 5 min at 4°C, the supernatant was removed, and the pellet was resuspended in 100 µl of PBS. Ten microliters of the cell suspension were stained with crystal violet stain (BDH), and nucleated cells were counted in a Neubauer hemocytometer. A total of 0.5 x 106 cells in a volume of 100 µl of PBS were centrifuged (Cytospin 3; Shandon, Astmoor, UK) onto slides (700 gav for 4 min) and stained for 5 min with May and Grunwald and Giemsa stains (BDH). The slides were quantified for macrophages, neutrophils, and lymphocytes by counting a total of 200 cells/slide at x25 magnification.
Macrophage Inflammatory Protein-2 in Lung Homogenates
Macrophage inflammatory protein-2 (MIP-2) levels were measured using a precoated murine MIP-2 colorimetric sandwich ELISA kit (R&D Systems) according to the manufacturer's recommendations. The MIP-2 concentration in the lung homogenate supernatant was calculated from a standard curve using known amounts of murine MIP-2 in a range from 7.8 pg/ml to 500 pg/ml.
BALF
Total protein and lactate dehydrogenase measurements. The total protein concentration in the BALF supernatant was measured by the Folin-Lowry method (48). Lactate dehydrogenase (LDH) levels were determined by the rate of pyruvate substrate conversion to lactate according to the manufacturer's recommendations (Sigma Diagnostics, Poole, UK).
Albumin estimation. To quantify the leak of albumin from the serum into the alveoli, the albumin concentration in the BAL supernatant was measured. This was carried out according to the manufacturer's recommendations (Sigma Diagnostics). The absorbance was measured spectrophotometrically at 628 nm (Unicam UV1; Thermo Electron Spectrometry, Cambridge, UK). The albumin concentration in the samples was calculated from a standard curve generated by using known amounts of murine albumin (Sigma) in the range of 5100 mg/ml.
Quantification of Lung Injury
Light microscopy. All slides were coded and evaluated in a blinded fashion to prevent bias. A point scoring system was used to quantify the extent of lung injury and was defined semiquantitatively as the presence of any one of: 1) capillary congestion; 2) alveolar/interstitial edema; 3) presence of fibrin; 4) alveolar/interstitial hemorrhage; 5) necrosis; or 6) alveolar/interstitial neutrophils. The mean % damage score for an animal was calculated by counting a total of 24 randomly selected fields/slide (x200 magnification) for one section. For quantification, each field of view was required to contain >50% alveolar tissue.
Transmission electron microscopy. The lungs were inflated with fixative (2.5% glutaraldehyde, 50 mM sodium cacodylate buffer) in situ to a constant pressure of 2530 cm and removed in toto into fixative and stored at 4°C for a minimum of 24 h. Samples of parenchyma of the left lobe and right upper lobe were postfixed in 1% osmium tetroxide in 50 mM sodium cacodylate buffer, dehydrated in graded alcohol and propylene oxide, and subsequently embedded in Araldite epoxy resin. Blocks were initially cut as semithin sections (1 µm) on a Reichart Ultracut E and stained with 1% toluidine blue (in 1% sodium tetraborate) for the purposes of orientation and initial morphological assessment. Ultrathin (80100 nm) sections were cut, contrasted with uranyl acetate and lead citrate, and examined with a Hitachi 7000 transmission electron microscope.
Physiological Measurements of Lung Function: Dosing Optimization
Administration of OA for the physiological measurements of lung function was undertaken via a catheter placed in the jugular vein, as tail vein administration proved difficult and unreliable due to the experimental set up needed for lung function measurements. More efficient OA delivery is likely through the jugular compared with the tail vein. In keeping with this, administration of 0.2 ml/kg through the jugular vein resulted in 100% mortality within 1 h, equivalent to that seen with 0.4 ml/kg through the tail vein. Reduction of the jugular dose to 0.1 ml/kg prevented premature deaths over the 1-h time period, as was the case for 0.2 ml/kg administration by tail vein injection. Thus 0.1 ml/kg was used for all studies of lung mechanics and blood gas analysis.
Physiological Measurements of Lung Function
Lung physiological measurements were carried out as previously described in detail by Wilson et al. (68). In brief, male BALB/c mice (810 wk, 2228 g, Harlan) were anesthetized by intraperitoneal injection of Hypnorm/Midazolam (Hypnorm:MZ:water 1:1:3, 0.1 ml/10 g) and placed in the supine position. An endotracheal cannula [0.76-mm inner diameter (ID), 1.22-mm outer diameter (OD)] was inserted via tracheotomy and secured with a suture. Animals were ventilated with a custom-made mouse jet ventilator system, as described by Ewart et al. (17). Airway pressure was monitored by a pressure transducer (MLT0380; ADInstruments, Chalgrove, UK), and airway flow was determined by a differential pressure transducer (PX137; OMEGA Engineering, Manchester, UK) connected to a miniature pneumotachogram in the ventilator circuit. A polyvinyl chloride (PVC) catheter (0.28-mm ID, 0.6-mm OD; Critchley Electrical Products, Silverwater, Australia) was introduced into the left carotid artery for monitoring arterial pressure using a pressure transducer (MLT844, ADInstruments) and measurement of blood gases. Rectal temperature was maintained between 36 and 37°C by the use of a heated pad. All data collected from the ventilator and blood pressure transducers during the experiment were recorded by PowerLab Data Recording System (ADInstruments).
After standardization of volume history of the lungs with a sustained inflation of 35 cm/H2O for 5 s, the animals were ventilated with a tidal volume of 910 ml/kg, a respiratory rate of 120 breaths/min, and inspiratory:expiratory ratio of 1:2 throughout the experiment. Administration of OA was carried out via a jugular vein cutdown and cannulation. Briefly, 0.1 ml/kg of OA was loaded into 0.28-mm single-lumen PVC tubing (Critchley Electrical Products) and connected to the jugular vein line with a 30-gauge needle. OA was infused at a controlled rate using a syringe pump (0.3 ml/h, KD Scientific). Control animals received an equal volume of sterile PBS. Respiratory system compliance and resistance were measured every 15 min by the end-inflation occlusion technique. Blood gas analyses were made on serial arterial blood samples (60 µl) collected via the carotid cannula before and at 30 min and 1 h after OA administration and analyzed by a fetal scalp blood gas analyzer (Chiron Rapidlab 248; Bayer Diagnostics, Newbury, UK).
Statistical Analysis
Data are cases represented in summary plots that are based on the median, quartiles, and extreme values. The box represents the interquartile range, which contains the 50% of values. The whiskers are lines that extend from the box to the highest and lowest values, excluding outliers, which are defined separately with circles. A line across the box indicates the median. Selected data (see Figs. 3A and 4) are represented as dot plots or as median ± first and third data quartile (see Fig. 9). Comparison of data between groups used the Kruskal-Wallis analysis of variance for multiple unpaired, nonparametric groups, followed (where permitted) by Mann-Whitney's U-test, with the Bonferroni correction for multiple comparisons. The null hypothesis was rejected at P < 0.05.
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RESULTS |
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Intratracheal instillation of rhKGF at 115 mg/kg body wt resulted in a significant (P < 0.01 for all groups) increase in SP-B-positive cells compared with animals receiving diluent alone 2 days following administration (Fig. 1A). The KGF-induced increase in ATII cell numbers was dose related with a peak of proliferation following 10 mg/kg of rhKGF [23 ± 2.7 compared with 8.2 ± 1 ATII cells/field of view (x200 magnification) following administration of diluent]. Representative images of SP-B immunohistochemical staining in animals receiving the diluent alone or rhKGF at 10 mg/kg body wt are shown in Fig. 2. To ensure SP-B antibody specificity, morphological counting of ATII cells using hematoxylin and eosin-stained sections was also carried out. Significantly (P < 0.01) increased numbers of ATII cells were seen at all KGF doses (1 mg/kg, 15.2 ± 3.4; 5 mg/kg, 21.5 ± 5.2; 10 mg/kg, 28.4 ± 3.6; 15 mg/kg, 28.3 ± 2.2 compared with 10.1 ± 1.4 in animals receiving the diluent). In agreement with SP-B immunohistochemistry, 10 mg/kg produced the peak effect.
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To exclude rhKGF-induced toxicity, hematoxylin and eosin-stained tissue sections from animals receiving 1, 5, 10, and 15 mg/kg of rhKGF obtained at day 2 were examined for the presence of edema, hemorrhage, and neutrophils. Approximately 10% of the total area from each lung section was found to include one or more of the above-mentioned parameters. However, no increase in lung damage compared with control animals was demonstrable in lung tissue following rhKGF administration (data not shown).
Topical Administration of Liposome-KGF DNA Complexes Produces KGF mRNA, Protein, and ATII Cell Proliferation
mRNA. To circumvent the problem of detection of contaminating plasmid DNA, pCI-specific mRNA was amplified by TaqMan PCR, and relative expression was calculated by comparison to expression levels following intranasal instillation of a known standard (100 µg pCIKLux). Significant (P < 0.01) relative expression of pCIhKGF-specific mRNA was demonstrated 24 and 48 h following intranasal administration of 80 µg of pCIhKGF complexed with GL67 compared with untransfected animals (Fig. 3A).
Protein. In lung homogenate, low levels of KGF were detected in pCIhKGF/GL67-transfected animals by ELISA (21.5 ± 5.7 pg/mg protein at 48 h and 21.5 ± 13.8 pg/mg protein at 72 h), with a threshold sensitivity level of the ELISA of 5 pg/ml.
ATII cell quantification. Intranasal instillation of pCIhKGF/GL67 (80 µg) did not result in significantly increased ATII cell numbers as quantified by SP-B immunohistochemistry (Fig. 3B). However, morphological ATII cell counts revealed a significant (P < 0.05) increase in ATII cell numbers 72 and 96 h following intranasal transfection [15.2 ± 4.4 and 15.3 ± 3.9 ATII cells/field of view (x200 magnification) compared with 8.1 ± 1.9 in animals receiving an irrelevant plasmid].
Systemic Administration of Liposome-KGF DNA Is Toxic But Able To Produce KGF mRNA and ATII Cell Proliferation
Lipid-mediated gene transfer resulted in high mortality (1540%) after intravenous injection in both pCIhKGF- and pCF1CAT-transfected animals. Thus group sizes were not consistently large enough to allow for statistical analysis.
mRNA. pCI-specific mRNA was amplified by TaqMan PCR, and relative expression was calculated by comparison to expression levels following intranasal instillation of a known standard (100 µg of pCIKLux). Significant (P < 0.01) relative expression of pCIhKGF-specific mRNA was demonstrated 8 and 24 h following intravenous administration of pCIhKGF (50 µg) and 2 min after injection of 900 nmol of DOTAP/cholesterol compared with untransfected animals (Fig. 4A).
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ATII cell quantification. Sequential intravenous gene transfer of pCIhKGF/lipid was associated with an increase in ATII cell numbers 48, 72, and 96 h after transfection as demonstrated by SP-B immunohistochemistry (Fig. 4B). However, due to the high mortality following intravenous gene transfer, the number of animals in each group was low, and statistical testing was not appropriate. A similar trend was observed following morphological counts [12.4 ± 1.4 at 24 h, 14.4 ± 2.7 at 48 h, 14.3 ± 1.9 at 72 h, 17 ± 0.3 at 96 h compared with 8.7 ± 1 ATII cells/field of view (x200 magnification) in animals receiving an irrelevant plasmid]. Pooling of data obtained from all time points (24, 48, 72, and 96 h) showed a significant (P < 0.01) increase in ATII cell numbers by SP-B immunohistochemistry (12.9 ± 3.7 ATII cells/field of view compared with 8.7 ± 1.6 in controls animals), which was supported by morphological assessment (14.3 ± 2.3 ATII cells/field of view compared with 8.6 ± 2.2 in control animals).
OA Produces A Dose-Related Increase in ALI
Macroscopic changes following OA administration. A dose-related difference in mortality was observed in animals treated with 0.2 and 0.4 ml/kg of OA, respectively. The higher dose was associated with 100% mortality, with death usually occurring 3040 min following OA administration. The animals appeared cyanotic, and bloody fluid spilled out of the trachea upon extubation. At postmortem, the lungs were grossly enlarged, and severe hemorrhage affected nearly all of the lung surface. In contrast, no deaths occurred within 1 h following administration of 0.2 ml/kg of OA. At this dose, the injury appeared patchy and less severe and was commonly found at the periphery of the lung lobes. The lungs were enlarged, and bloody fluid was often found in the trachea.
Wet-to-dry weight ratios. Intravenous administration of OA (0.2 and 0.4 ml/kg body wt) was associated with a significant (P < 0.01) increase in the lung wet-to-dry weight ratios (Fig. 5A) compared with ventilated control animals receiving only PBS. This effect was not dose related. Furthermore, compared with untreated nonventilated animals, control animals that were ventilated and received intravenous PBS also had significantly (P < 0.01) increased wet-to-dry weight ratios.
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BALF protein and LDH. Administration of OA was associated with a dose-related increase in total protein concentration measured in BALF supernatants (Fig. 5C). Thus compared with control animals receiving PBS alone, BALF total protein was significantly (P < 0.001 and 0.01, respectively) higher in animals receiving the 0.2 and 0.4 ml/kg of OA. Again, compared with baseline untreated animals, a small but significant (P < 0.01) increase in total BALF protein was detected in ventilated animals that received PBS alone. This was associated with an increase in BALF supernatant albumin (0.2 ml/kg OA, 0.25 ± 0.00 µg/µl; 0.4 ml/kg OA, 0.25 ± 0.00 µg/µl compared with undetectable levels in control animals). BALF supernatant LDH activity, a marker of local cell damage, was significantly (P < 0.01) increased in a dose-related manner in animals injected with OA (Fig. 5D) compared with ventilated control animals given PBS alone.
Lung histology. Compared with untreated animals and ventilated PBS controls, OA (0.4 ml/kg) induced a significant (P < 0.01) increase in histological lung damage (Fig. 6). The principal histological changes seen included the presence of fibrin in the alveolar spaces, hemorrhage, and necrosis of alveolar tissues (Fig. 6B). Whereas alveolar fibrin, hemorrhage, and necrosis were barely detectable in sections examined from the lungs of untreated or PBS control animals (Fig. 6A), all three features were significantly (P < 0.05) increased in lung sections from the OA-treated animals (Fig. 6C).
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BALF cell counts. Pretreatment with rhKGF (10 mg/kg) did not significantly affect the total cell numbers found in the BALF (KGF, 2 x 106 ± 2.4/ml of BALF, diluent, 2.6 x 106 ± 1.8/ml of BALF) or MIP-2 levels (KGF, 15.1 ± 5.5 pg/mg of protein, diluent, 15.4 ± 3.5 pg/mg of protein).
BALF protein and LDH. No significant differences were observed in the BALF protein concentration (KGF, 11.5 ± 4.3 µg/µl; diluent, 11.6 ± 5.3 µg/µl), albumin (KGF, 9.3 ± 3.1 µg/µl; diluent, 7.1 ± 4.2 µg/µl), and LDH activity (KGF, 790 ± 522 U/l; diluent, 1,138 ± 581 U/l) in animals pretreated with rhKGF 48 h before OA.
Lung histology. No improvements were observed in histological damage in KGF-pretreated OA-injured animals (KGF, 60 ± 12% lung damage; diluent, 43 ± 14% lung damage).
Blood gases and lung compliance. Pretreatment of mice with rhKGF (10 mg/kg) 48 h before intra-jugular OA (0.1 ml/kg) challenge resulted in a significant (P < 0.001 and P < 0.05, respectively) improvement in arterial PO2 30 and 60 min after OA administration compared with control mice receiving the diluent only (Fig. 9A). Additionally, rhKGF pretreatment was associated with significantly (P < 0.01) decreased arterial PCO2 30 and 60 min after OA administration (Fig. 9B) compared with diluent only. Lung compliance was significantly (P < 0.01) improved in animals pretreated with rhKGF (Fig. 9C).
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DISCUSSION |
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The mouse remains an important model system with transgenic technology making it possible to study effects of a single gene on the whole organism. Additionally, gene delivery methods and gene transfer efficiency have been well characterized in the mouse. This study has for the first time characterized the KGF response in this species in detail. Intratracheal instillation of 5 mg/kg body wt has been used most frequently in experimental ALI studies in both mice and rats (55, 65, 71, 73, 74). We confirmed that this dose is associated with significant ATII cell proliferation 2 days after administration. However, administration of 10 mg/kg appeared to result in higher and less variable ATII cell numbers. Studies in the rat have demonstrated ATII proliferation to be focal and thus resulting in piles of ATII cells within the alveoli (63). However, significant evidence of focal proliferation was not observed in this study, and cell proliferation in the lung sections from KGF-treated animals appeared to be diffuse. Furthermore, we demonstrated that ATII cell numbers were still increased at day 7 after administration, in contrast to Ulich et al. (63), who showed resolution of proliferation in rats. The administered dose was 5 mg/kg of rhKGF, and thus it is possible that the higher KGF dose used in this study extends the ATII cell proliferative response.
Novel interventions for ALI are needed as mortality remains high, even in well-equipped intensive care units. A number of studies have recently demonstrated the possibility of gene therapy as a potential intervention, particularly addressing the inflammatory aspects of ALI, using IL-10 (38, 41), elafin (54), neutrophil inhibitory factor (77), and heme oxygenase-1 (25, 45). However, no studies to date have used gene transfer to address alveolar repair following ALI.
Nonviral KGF Gene Delivery Results in Modest ATII Cell Proliferation
GL67 was first described in 1996 by Lee et al. (31), who demonstrated a 100-fold increase in reporter gene expression following intranasal transfection in mice compared with other first-generation cationic liposomes. Expression was found to peak 48 h following intranasal delivery of 80 µg of DNA. Although high levels of CAT reporter gene activity were demonstrated in lung homogenate 2472 h following intranasal administration of pCF1CAT/GL67 complexes, significant KGF levels could not be detected in parallel studies using pCIKGF/GL67 despite evidence for gene transfer. In keeping with this, no increase in SP-B-positive cells was seen, with only a small increase in the numbers of ATII cells assessed by routine histology. Systemic lipid-mediated gene transfer has been demonstrated to transfect primarily pulmonary endothelial cells (32). However, KGF produced by capillary endothelial cells as a result of systemic gene transfer may reach across the alveolar endothelial-epithelial barrier, thus stimulating ATII cell proliferation. Lipid (DOTAP/cholesterol)-protamine sulfate-DNA (LPD) has previously been shown to be effective for intravenous lung delivery (32) but also has toxic effects. The toxicity is caused by a strong inflammatory response to the unmethylated CpG motifs in the DNA following uptake of LPD by circulating immune cells, primarily resulting in high levels of TNF- and IFN-
(34). Recently, reduced toxicity and increased gene transfer efficiency have been demonstrated by sequential delivery into the tail vein of cationic liposomes followed by DNA (60). Administration of the lipid before the DNA is believed to increase the retention time of plasmid DNA within the lung capillaries (33). However, the toxicity caused by systemic gene transfer was confirmed in this study, as mortality was high. In surviving animals, quantitative RT-PCR confirmed the expression of pCI-specific mRNA 8 and 24 h following systemic transfection. However, KGF could not be detected in lung homogenate or serum following intravenous pCIhKGF/DOTAP/cholesterol gene transfer at any time points. Although a clear trend toward an increase in ATII cell numbers was observed following sequential intravenous gene transfer, significance was not tested due to the low numbers in each group. However, by grouping the data from the different time points, a highly significant increase in ATII cell numbers could be demonstrated. As mentioned above regarding topical gene delivery, the resulting ATII cell numbers achieved by systemic gene transfer were lower than those resulting from delivery of KGF protein. Additionally, in the light of the high mortality observed in these experiments, systemic KGF gene transfer is currently not an appropriate strategy for evaluating the protective effect in of KGF gene transfer in the OA model of ALI. We conclude that at present, nonviral lung gene transfer by topical and systemic administration cannot match the efficacy of recombinant protein. Virus-mediated gene transfer, using vectors such as adenovirus, would likely have resulted in more efficient gene transfer. However, the significant inflammatory response associated with viral delivery would be detrimental in the ALI setting.
Intravenous OA in Mouse Results in Severe Alveolar Injury
The oleic acid model is regarded as relevant for ARDS and has been extensively characterized in many larger animal species, including sheep, pigs, dogs, rats, and rabbits. Intravenous OA infusion causes severe pulmonary edema in a number of species due to an increase in the pulmonary alveolar barrier permeability. The arrest of small OA microemboli in the pulmonary capillaries results in severe congestion and hemorrhage (10). This microinfarction results in extensive necrosis of the alveolar epithelial type I cells (39) and also affects the permeability of endothelial cells (26). Neutrophil infiltration, fibrin deposition, and hyaline membrane formation are also common features (15, 40). These pathological events lead to hypoxemia and severely compromised lung compliance (9).
There is only one previously reported study of OA administration in mice (56). Although poorly characterized, the pathophysiology described was similar to that seen in other animal species. In our study, intravenous OA administration in mice resulted in severe pulmonary edema and a dose-related increase in BALF total protein concentration, likely representative of a combination of serum protein leakage (24), inflammatory cells, and necrotic alveolar epithelial cells (10). Further measurement of BALF supernatant albumin levels, however, suggested that leaked serum protein contributed only 10% of the total BALF protein levels with the majority likely to originate from protein debris released from necrotic alveolar cells. This is supported by the significant increases in BALF total cell counts and by LDH levels in BALF supernatant as well as the finding of marked alveolar epithelial cell necrosis on TEM analysis. TEM analysis also demonstrated the presence of some endothelial injury and necrosis, which would further contribute to the overwhelming pulmonary edema and the albumin found in the BALF supernatant.
Acute OA Does Not Result in Inflammatory Cell Infiltration
Surprisingly, differential cell counts demonstrated only a small (albeit significant) increase in neutrophil numbers at 1 h following 0.2 ml/kg of OA. This finding contrasted with other studies in which OA was shown to cause a marked influx of neutrophils into the alveoli 4 h after OA administration (53). It is possible that transepithelial neutrophil migration does not occur within the initial hour after OA administration, and had we measured later time points, increased BAL neutrophils may have been detected (15). This possibility was supported by a significant increase in MIP-2 following OA (0.2 ml/kg) compared with untreated baselines in which no MIP-2 was detected. MIP-2 has potent neutrophil chemotactic activity and is secreted by epithelial cells as a key mediator of neutrophil recruitment in response to tissue injury (14). Thus the significant increase demonstrated in total BAL cell counts is likely to be a result of the presence of sloughed necrotic epithelial cells.
Mechanism of OA Injury Differs from ARDS
The OA mouse model has a number of dissimilating features to human ARDS. First, the initiating events leading to OA-induced lung injury do not resemble the clinical situation. ARDS generally results from sepsis or multiple injuries and evolves secondary to an overwhelming inflammatory cascade. OA-induced lung damage is likely to be caused by OA microemboli being trapped in the capillary bed, causing subsequent local necrosis followed by inflammation. Second, the time course of OA-induced injury development and progression is much shorter than that of human ARDS. The acute phase of human ARDS develops over 17 days and is also characterized by extensive damage to the alveolar epithelium, hemorrhage, and pulmonary edema, leading to severely decreased lung compliance and hypoxemia (3, 61). However, with a view to evaluating KGF as a potential therapy for alveolar epithelial repair, this model consistently reproduces the histopathological features of ARDS.
Mechanical Ventilation Causes Some Degree of Lung Injury
Compared with untreated baseline animals, a significant increase in wet-to-dry weight ratios was also observed in ventilated control animals given intravenous PBS. This suggests that mechanical ventilation is associated with the development of at least some degree of the pulmonary edema observed following OA administration and confirms previous observations that mechanical ventilation causes lung injury (42, 64), including increased microvascular permeability and leakage of fluid into alveolar spaces (23). Ventilator-induced lung injury may be partly attributed to the effects of excessive airway pressure and overdistension of the alveoli during ventilation (13). It is, however, important to distinguish the above studies of lung injury caused by deliberate hyperventilation from this study, where the injury is most likely caused by dry gas ventilation and atelectasis due to low positive end-expiratory pressure (PEEP) and inadequate alveolar recruitment procedures. In these experiments, no PEEP was applied, which may affect lung expansion and exacerbate edema formation during mechanical ventilation (12). The addition of PEEP in the ventilatory strategy prevents complete alveolar collapse and reopening repeatedly during the breathing cycle and thereby reduces the shear stress forces generated.
rhKGF Improves Gas Exchange and Lung Compliance in OA-Injured Mice Without Evidence of Increased Epithelial Repair
Pretreatment of mice with intratracheal rhKGF (10 mg/kg) 48 h before intrajugular administration of OA (0.1 ml/kg) resulted in a significant improvement in PaO2 30 and 60 min after OA administration. The improvement in PaCO2 was significant 30 and 60 min after OA administration. Lung compliance was also significantly improved in animals pretreated with rhKGF and was observed as early as 15 min after OA administration. However, by comparison of the lung physiological results from rhKGF-pretreated animals to ventilated animals not receiving OA, it becomes clear that the KGF-induced improvements are relatively small. It is possible that the existing pool of ATII cells generated by the rhKGF pretreatment reduces the extent of epithelial damage caused by OA, thereby increasing gas exchange and improving the mechanics of the lung. Additionally, intratracheal rhKGF pretreatment may increase cell migration rate, cell spreading, and adherence, as described by Waters and Savla (66) and Atabai et al. (2), to repair areas of denuded basement membrane. However, whether this is likely to occur within the short time course of OA injury in this model is not known. Some evidence exists demonstrating that tracheal epithelial cells can migrate minutes after wound induction in vivo (16), but whether this occurs in alveolar epithelial cells is not known.
Pretreatment of the animals with rhKGF did not appear to ameliorate the extent of protein exudation (BALF protein and albumin), epithelial cell damage (LDH activity), or cellular infiltrate (BALF cell counts) induced by OA. Additionally, KGF pretreatment did not protect against the histological damage induced by OA, and no differences in fibrin, hemorrhage, or necrosis were detected. These findings were surprising in light of the physiological improvements demonstrated. Intrajugular administration of 0.1 ml/kg of OA in mice produces very acute and severe lung injury, resulting in damage to >40% of the lung tissue. The injury may have been too severe to detect any changes in the pathological end points, and further titration of the OA dose to result in a less acute and overwhelming lung injury may reveal an effect of rhKGF pretreatment on the less sensitive pathological end points. Furthermore, there may have been alternative mechanisms underlying the physiological improvements seen. Some studies have demonstrated increased surfactant protein production following KGF administration (72), most likely due to the hyperplasia of ATII cells. This increase in surfactant production may have helped to improve gas exchange and lung compliance by preventing alveolar collapse in the early stages of OA-induced lung injury development but would not alter the pathological and histological features of OA-induced lung injury. Additionally, KGF may have increased the number of Na+K+-ATPase pumps, as has previously been demonstrated (6), thus reducing fluid accumulation within the alveoli. Nevertheless, the results raise questions regarding the relevance of the improvements seen in the mice to the clinical picture of ALI and ARDS. It may be possible that a small, but significant, improvement in gas exchange may not be accompanied by a measurable reduction in damage to the alveolar epithelial membrane but may be sufficient to prevent morbidity/mortality by reducing the amount of time spent on mechanical ventilation. Additionally, prolonging the time KGF is present in the lung tissue may enhance the protective effect observed. Whereas we have established that a single dose of intratracheal administration of 10 mg/kg of rhKGF appears to produce the maximum ATII proliferative effect, multiple rhKGF administrations may further improve the protective effect. Finally, it is possible that prolonged expression achieved by gene transfer with a vector expressing high levels of KGF may result in enhanced protective effects.
The potential of ameliorating ALI by KGF pretreatment has been confirmed in this study using a murine model of very severe ALI. Whereas KGF did not appear to directly affect alveolar epithelial repair and integrity, measurements of blood gases and lung compliance demonstrated significant improvements in gas exchange, possibly linked to increased presence of surfactant proteins secreted from proliferating ATII cells. Further optimization of the OA model with respect to time course and dosing may reveal histological and pathological improvements.
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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