GTMB 7 - Gene Therapy & Molecular Biology

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Epperly et al: Late injection of MnSOD-PL protects against pulmonary fibrosisof organizing alveolitis/fibrosis, an increase in TGF-β1 isalso detected (Epperly et al, 1999c). This late increase inTGF-β1 persists to day 120, the time at which TGF-β2expression also increases (Epperly et al, 1999c). Levels ofTGF-β2 remain elevated throughout the development oforganizing alveolitis/fibrosis (Epperly et al, 1999c).We have previously demonstrated that intratrachealinjections of MnSOD-PL complex or adenoviruscontaining the human MnSOD transgene 24 hours beforeirradiation protects the murine lung from irradiationinduceddamage (Epperly et al, 1998; 1999b; 2000a,2001b). Protection of the murine lung was measured as:(a) increased survival (Epperly et al, 1998, 1999b), (b)decreased pathologically quantifiable percent of lungshowing organizing alveolitis/fibrosis, (Epperly et al,1998; 1999b; 2000a, 2001b) and (c) decreased productionof inflammatory cytokine mRNA for IL-1, TNF-α, andTGF-β (Epperly et al, 1998, Epperly et al, 2001b). Theoptimal schedule for administration of MnSOD-PL is notknown. Injection prior to irradiation might be effective bypreventing ROS mediated DNA damage or protectingagainst mitochondrial mediated apoptosis (Epperly et al,1999a, 2002). However, injection following irradiation orat delayed time points when increases in TNF-α and TGFβmRNA are detected may reduce cytokine mediatedproduction of ROS and also protect against tissue injury.To determine the optimal time of MnSOD-PLadministration in the C57BL/6J mouse model, mice wereinjected with MnSOD-PL at 1, 80, 90, or 100 days after 20Gy whole lung irradiation and data were compared to thatwith mice treated before irradiation. The mice werefollowed for development of organizing alveolitis/fibrosisand the percent of lung displaying organizingalveolitis/fibrosis was determined. Since MnSOD is amitochondrial enzyme that dismutates superoxides only(Quinlan et al, 1994; Fridovich, 1995) the detection ofincreased survival in delayed injection groups of micemight indicate the presence of delayed increases insuperoxide production, and thus be interpreted to play arole in the development of pulmonary fibrosis. In thepresent studies, we sought to determine whether delayedelevation of MnSOD by transgene therapy protects lungsfrom irradiation-induced pulmonary fibrosis.II. Materials and methodsA. Injection of MnSOD-PLC57BL/6J were anesthetized using Nembutal and injectedintratracheally with MnSOD-PL complexes (500 µg plasmidDNA in a volume of 50 µl plus 28 µl of lipofectant) (Epperly etal, 1998; 1999b) Twenty-four hours later the MnSOD-PLinjectedmice plus control non-injected mice were irradiated to20 Gy to the pulmonary cavity. The mice were shielded so thatonly the pulmonary cavity was irradiated. A subgroup of thecontrol, irradiated mice was injected with MnSOD-PL 24 hoursafter irradiation. Other subgroups of each control irradiated orMnSOD-PL pre-irradiation injected mice were injectedintratracheally a second time at day 80, 90 or 100 followingirradiation. All mice were followed for development oforganizing alveolitis/fibrosis, at which time the mice weresacrificed.B. Determination of organizingalveolitis/fibrosisWhen 80% of the control, irradiated mice had beensacrificed due to moribund condition as indicator of pulmonaryorganizing alveolitis/fibrosis, a subgroup of mice from eachgroup was also sacrificed. The lungs were expanded withOptimum Cutting Temperature (OCT), removed, frozen in OCT,sectioned, and hematoxylin and eosin (H&E)-stained (Epperly etal, 1998; Epperly et al, 1999b). The sections were examinedmicroscopically and the percent of organizing alveolitis/fibrosiswas determined using an Optimus Image Analysis System(Epperly et al, 1998; Epperly et al, 1999b). In this system, thearea of organizing alveolitis/fibrosis was compared to the area ofthe entire lobe, and the percent of lung developing organizingalveolitis/fibrosis calculated.C. StatisticsThe irradiation survival curves of the different subgroupswere compared with control irradiated mice using a Log RankTest (Epperly et al, 1998; 1999b). The percent organizingalveolitis/fibrosis for the different subgroups of mice werecompared using a Student’s t-Test (Epperly et al, 1998; Epperlyet al, 1999b).D. Animal protocolsProtocols for animal usage were approved by theInstitutional Animal care and Use Committee of the Universityof Pittsburgh. Veterinary support was provided by the Divisionof Laboratory Animal Research of the University of Pittsburgh.III. ResultsA. Delayed injection of MnSOD-PL afterlung irradiation improves survivalTo determine whether intratracheal injection ofMnSOD-PL at delayed intervals following irradiationprotected the murine lung from irradiation-induceddamage, C57BL/6J mice were injected intracheally with500 µg of plasmid DNA containing the MnSOD transgeneat 1, 80, 90 or 100 days following 20 Gy irradiation to thepulmonary cavity. The mice were then followed for thedevelopment of organizing alveolitis/fibrosis and weresacrificed when moribund. Mice injected with MnSOD-PLat 80 or 100 days after irradiation showed a significantincrease in survival compared to 20 Gy irradiated noninjectedcontrol mice while mice injected with MnSOD-PL at day 1 or 90 after irradiation showed a detectable butnot significant increase in survival (Figure 1).B. Pre-irradiation injection of MnSOD-PL affords optimal protection and is notfurther enhanced by a second delayedtreatmentGroups of mice were next injected with MnSOD-PL24 hours before 20 Gy irradiation to the pulmonary cavityand then evaluated to determine whether a secondinjection of MnSOD-PL at 80, 90 or 100 days afterirradiation resulted in an additional increase in survivalcompared to single pre-irradiation therapy. In this study,62
Gene Therapy and Molecular Biology Vol 7, page 63subgroups of mice received a second injection of MnSOD-PL at 80, 90 or 100 days later. The mice were thenfollowed for the development of organizingalveolitis/fibrosis at which time they were sacrificed. Asshown in Figure 2, there was no significant improvementin survival following a second injection of MnSOD-PLcompared to the improvement seen with one preirradiationinjection. A second injection at day 90 resultedin a significantly decreased survival compared to preinjectiononly.A comparison of these injection groups is shown inFigure 3. All subgroups of mice injected with MnSOD-PL24 hours before irradiation had increased survivalcompared to mice that received no injection and only 20Gy irradiation. Furthermore, mice injected with MnSOD-PL 24 hours prior to irradiation showed the best survivalcompared to all other groups of mice including those thatreceived a second delayed injection.C. Decreased lung irradiation damagehistopathologically correlates to MnSOD-PLmediated increased survivalTo determine whether the differences in survival ofmice between groups correlated with histopathologicchanges in the lung, specifically the development oforganizing alveolitis/fibrosis, representatives of eachsubgroup of mice were euthanized at the time point when80% of the 20 Gy irradiated, control mice were sacrificeddue to moribund condition from developing organizingalveolitis/fibrosis. The lungs were expanded in OCT,removed, frozen in OCT, sectioned, and H&E-stained.The percent of lung displaying organizingalveolitis/fibrosis was calculated using an Optimus ImageAnalysis system as described in the Methods.Figure 2: Improved survival of mice injected with MnSOD-PL24 hours before pulmonary irradiation is not further enhanced bya second delayed injection. C57BL/6J mice were injected withMnSOD-PL 24 hours before 20 Gy irradiation to the pulmonarycavity. Subgroups were injected with a second dose of MnSOD-PL at 80, 90 or 100 days after the initial irradiation. There was nosignificant improvement in the overall survival by a secondinjection 80, 90 or 100 days after irradiation (p=0.547, 0.039, and0.309 respectively) compared to pre-irradiation administrationabove. A second injection at day 90 resulted in significantlydecreased survival compared to pre-injection only. Groupscontained ≥10 mice/group.Figure 1: Improved survival of pulmonary irradiated C57BL/6Jmice injected with MnSOD-PL at day 1, 80, 90 or 100 followingirradiation. C57BL/6J mice were irradiated to 20 Gy to thepulmonary cavity. Subgroups were subsequently injected withMnSOD-PL on day 1, 80, 90, or 100 following irradiation. Themice were followed for the development of organizingalveolitis/fibrosis, at which time they were sacrificed. Theseresults demonstrated that injection of MnSOD-PL at day 80 or100 following irradiation (or times when TNF-α and TGF-βproduction are increased) increases survival compared toirradiated, control mice (p = 0.0015 or 0.0005, respectively).Groups contained ≥10 mice/group.Figure 3: Pre-irradiation injection of MnSOD-PL providesoptimal protection from lung irradiation damage. C57BL/6J micewere injected with MnSOD-PL and irradiated 24 hours later to20 Gy to the lung, as were non-injected control mice. Subgroupsof mice were subsequently injected with MnSOD-PL at day 1(control, irradiated mice only), 80, 90 or 100 after irradiation.The mice were then followed for development of organizingalveolitis/fibrosis, and were sacrificed when moribund. All miceinjected with MnSOD-PL 24 hours before irradiation had asignificantly increased life span compared to control, irradiatedmice (p ≤ 0.0066). Groups contained ≥10 mice/group.63
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