GTMB 7 - Gene Therapy & Molecular Biology

Epperly et al: Late injection of MnSOD-PL protects against pulmonary fibrosisTherefore, MnSOD-PL action on ROS produced byinflammatory cells such as macrophages at 80 days doesnot appear to explain the present data implyingsuperoxides might have been produced by macrophagesand neutralized by injections of MnSOD-PL at 80 or 100days after irradiation (Epperly et al, 2003).We previously demonstrated that at 80 days afterirradiation of the mouse lung there is an increase in TNF-αmRNA expression which decreases to background level byday 120 (Epperly et al, 1999b). This increase isaccompanied by increased expression of mRNA for TGFβat day 100. Initially, there is an increase in TGF-β1isoform until day 120 at which time TGF-β1 expressiondecreases, and TGF-β2 expression increases and stayselevated until development of organizing alveolitis/fibrosis(Epperly et al, 1999b). The detectable protection byMnSOD-PL injection at day 80 might have been attributedto an effect on the TNF-α elevation at that time point.ROS production might increase TNF-α expression at day80 leading to further increased ROS production (Haddad,2002). Treatment of alveolar epithelial cells with a ROSgenerating system results in increased TNF-α expressionand a depletion of glutathione (Haddad, 2002). TNF-αtreatment inhibits myogenesis by causing a decrease inglutathione levels and elevation of ROS (Langen et al,2002). Pre-treatment with the anti-oxidant N-acetyl-1-cysteine (NAC) restored the formation of multi-nucleatedmyotubes, indicating that myogenesis inhibition wasattributable to ROS expression (Langen et al, 2002). Pretreatmentof HELA cells with gammaglutamylcysteinylglycineinhibits TRAIL-inducedapoptosis (Lee et al, 2002). TNF-α expression mayincrease the production of ROS and result in a furtherincrease in TNF-α expression. ROS response to andinduction of TNF-α expression may be a cyclicmechanism in the lung at day 80, and MnSOD-PLtreatment at this time point may have interrupted the cycle.Further studies will be required to explain the protectionby injections of MnSOD-PL at 80 days after irradiation.Pulmonary increases in TGF-β1 and TGF-β2 mRNAat 100 to 120 days after irradiation have been detected(Epperly et al, 1999b). It has been demonstrated that ROScan also increase TGF-β expression (Bellocq et al, 1999)The treatment of human alveolar lung cell line A549 withxanthine and xanthine oxidase or nitric oxide generator S-nitroso-N-acetyl-penicillamine (SNAP) leads to release ofTGF-β1 (Bellocq et al, 1999). The xanthine-xanthineoxidase induced release of TGF-β1 can be inhibited by theaddition of catalase but not superoxide dismutase,implicating the involvement of hydrogen peroxide(Bellocq et al, 1999) TGF-β1 has been demonstrated toinduce production of extracellular hydrogen peroxide inhuman fibroblasts that mediate oxidative dityrosinedependentcross-linking of ECM (Larios et al, 2001).TGF-β and hydrogen peroxide have been observed toinduce connective tissue factor (CTGF) that then inducescollagen type 1 and fibronectin, a deposition leading tofibrosis (Park et al, 2001).The mechanism of action of TGF-β in cells of thelung may involve the mitochondria since TGF-β1 can leadto downregulation of Bcl-2 and Bcl-xl, which normallyprevent apoptosis (Lafon et al, 1996; Herrera et al, 2001a).Overexpression of Bcl-2 suppresses the effects of TGF-β(Huang and Chou, 1998). Following exposure to TGF-βthere is also a loss of mitochondrial membrane potential,release of cytochrome-C, and activation of caspase-3(Herrera et al, 2001a). TGF-β1 activates caspase- 3, 8 and9, which precede the loss of mitochondrial membranepotential (Herrera et al, 2001b). Activation of caspase-8results in cleavage of Bid and Bcl-xl, which may lead toan amplification loop resulting in the mitochondrialmediated apoptosis (Zha et al, 2000). Irradiation of murinebone marrow stromal cell line D2XRII in vitro inducesrelease of TGF-β into the culture medium (Greenberger etal, 1996). Co-cultivation of 32D cl 3 cells or subclones1F2 or 2C6 overexpressing MnSOD with irradiated bonemarrow stromal cells resulted in higher levels ofintracellular ROS in the non-irradiated 32D cl 3, 1F2 or2C6 cells compared to cells co-cultivated with nonirradiatedstromal cell lines (Greenberger et al, 1996). TheMnSOD overexpressing subclonal line 1F2 or 2C6 formedmore cobblestone islands on the irradiated stromal cellsthan 32D cl 3 cells (Greenberger et al, 1996). IncreasedMnSOD activity in 1F2 or 2C6 cells may have resulted ina decrease in ROS, thus allowing for greater attachment ofthe MnSOD overexpressing cell lines to the irradiatedstromal cells. Therefore, injections of MnSOD-PL into thelung at day 100 when TGF-β levels are beginning toincrease may inhibit ROS production, and/or stabilize thebronchoalveolar cell or endothelial cell mitochondria,preventing some (but not all) of the late effects ofirradiation damage to the lung. We are currently exploringthis possible mechanism.The present report indicates that a singleadministration of MnSOD-PL 24 hours prior to 20 Gylung irradiation is significantly more effective thanadministration at any of four post-irradiation time pointsranging from 1-100 days after irradiation. We did notevaluate time points between 1 and 80 days as there wasno histopathologic or other evidence to suggest thatinitiation steps in the late organizing alveolitis/fibrosisresponse began prior to 80 days. Our results may helpexplain the dynamics of late irradiation pulmonary injury.One interpretation of the results is that it representsevidence of a pleiotropic effect of ionizing irradiation onseveral cellular and physiologic targets within the lung.Initiation events at the time of irradiation may lead to amultiplicity of effectuating events beginning at around day100 and leading to rapid organizing alveolitis/fibrosis.Prevention of some of the initiating events by MnSOD-PLadministration prior to irradiation may have a significantlygreater effect at reducing the overall outcome compared tomodulation of some of the late effectuating events byMnSOD-PL administration at that time. For example,neutralization of free radical moieties induced byirradiation at day 0 by overexpression of MnSOD at thattime may be a significant early event which impacts onmultiple downstream/delayed effectuating targets(macrophage migration, fibroblast migration into the lung,66