Fibrosis is a common form of normal tissue damage after exposure to a wide variety of insults believed to involve oxidative stress. Plasminogen activator inhibitor-1 (PAI-1) is thought to play a major role in the development of progressive fibrosis via the inhibition of extracellular matrix degradation. Because radiation causes oxidative injury, which has been shown to trigger fibrogenic responses, the present study was designed to test the hypothesis that PAI-1 expression is redox-regulated after irradiation. Irradiating rat kidney tubule epithelial cells (NRK52E) with 1–20 Gy γ-rays led to dose-dependent increases in steady-state levels of PAI-1 mRNA and immunoreactive protein within 24 and 48 h, respectively. Enhancement of intracellular soluble thiol pools after incubation with N-acetylcysteine (2.5 mm), from 3.27 ± 0.27 nm/mg protein to 5.34 ± 0.52 nm/mg protein in cells incubated with N-acetylcysteine 30 min before and assessed 4 h after irradiation, abolished the radiation-induced up-regulation of PAI-1. In addition, overexpression of catalase inhibited radiation-induced increases in PAI-1 expression, suggesting a mechanistic role for hydrogen peroxide (H2O2) in regulating PAI-1 expression after oxidative insult. In support of this notion, incubating NRK52E cells with H2O2 (100 μm) also led to a nearly 3-fold increase in PAI-1 gene expression. These results demonstrate that PAI-1 is redox-regulated after exposure to ionizing radiation or H2O2 and suggest that H2O2 scavenging might represent a fundamental mechanism for modulating fibrogenic disease via inhibition of the induction of profibrogenic mediators after acute or chronic oxidative stress.

In vital organs such as the heart, kidney, lung, or liver, tissue injury caused by a range of insults and/or agents can result in the development of progressive fibrosis leading to ultimate organ failure (1). Indeed, the risk of radiation-induced fibrosis occurring in these normal tissues several months to years after radiotherapy remains a major factor in limiting the radiation dose that can be applied safely to cancer patients (2). Whereas the precise cell types, mediators, and pathogenic mechanisms involved in tissue fibrosis exhibit some organ specificity, a common feature is excessive accumulation of ECM.3 The latter results not only from increased ECM synthesis, primarily by myofibroblasts (3), but also from decreased ECM degradation. The PA/plasmin system is a key regulator of fibrinolysis and ECM degradation (4). Urokinase PA and tissue-type PA are arginine-specific serine proteases that convert the inactive zymogen plasminogen to the active broad-spectrum serine protease plasmin (5). Plasmin can degrade ECM both directly by its own proteolytic activity (6) and by activation of latent matrix metalloproteinases (7). Activity of the PA/plasmin system is regulated by a family of PA-inhibitors of which PAI-1, a member of the serine protease inhibitor (serpin) gene family, appears to be of primary importance (8).

PAI-1 is unique in its ability to efficiently inhibit urokinase PA as well as both chain forms of tissue-type PA (9). The apparent second-order inhibitory rate constants for the interaction of PAI-1 with PAs are all in the order of 107 M−1s−1, among the highest reported for any enzyme-inhibitor interaction (10). PAI-1 is synthesized and secreted by multiple cell types, including platelets, endothelial cells (11), vascular smooth muscle cells (12), hepatic stellate cells (13), mesangial cells (14), and proximal tubule epithelial cells (15). A role for PAI-1 in thrombosis has clearly been demonstrated. Increased PAI-1 levels are associated with thrombotic events (16), whereas a deficiency of PAI-1 results in hemorrhage (17). More recent data have shown an association with PAI-1 and fibrosis in the kidney (18, 19), liver (20), and lung (21). Additional support for a pathogenic role for PAI-1 in fibrosis comes from the use of transgenic mice that either lack or overexpress the PAI-1 gene. Using a bleomycin-induced model of pulmonary fibrosis, a direct correlation between the genetically determined level of PAI-1 expression and the extent of collagen accumulation was observed (22).

PAI-1 synthesis is believed to be regulated at the transcriptional level, with PAI-1 being rapidly secreted once synthesized (23); there is little evidence for any regulatory mechanism at the posttranscriptional level controlling PAI-1 synthesis or release. It has been shown in vitro that PAI-1 expression is regulated by glucocorticoids (24), thrombin (25), platelet-derived growth factor (26), and angiotensin II (27). The cytokines interleukin 1α, transforming growth factor β and tumor necrosis factor α also enhance PAI-1 secretion in vitro(28, 29, 30). Recent data have suggested a role for ROS in up-regulating PAI-1 expression. Interleukin 1-mediated up-regulation of PAI-1 expression in rat cardiac microvascular endothelial cells appears to be ROS-dependent (31). Furthermore, antioxidants have been shown to inhibit PAI-1 gene expression in endothelial cells (32). We have previously observed time- and dose-dependent increases in rat mesangial (33) and tubule epithelial cell PAI-1 gene expression (34) after in vitro irradiation. The biological effects of ionizing radiation are clearly largely attributable to energy deposition in irradiated cells and subsequent ROS generation (35). Thus, the present study was designed to test the hypothesis that PAI-1 expression is redox-regulated after exposure to ionizing radiation. We present data showing that increased ROS generation, and specifically H2O2, can indeed lead to increased PAI-1 expression in terms of both mRNA and immunoreactive protein in rat tubule epithelial cells. This up-regulation of PAI-1 could be blocked by the enhancement of intracellular reduced thiol pools or by increasing an enzyme that is specific for H2O2 scavenging (catalase) via a gene transfer approach. These findings could be of fundamental importance to understanding the underlying pathogenic mechanisms involved in a wide variety of fibrotic responses as well as providing possible strategies for antioxidant-based interventional approaches directed at the treatment of progressive fibrosis.

Culture of Rat Tubule Epithelial Cells.

A nontumorigenic rat renal epithelial cell line, NRK52E, derived from normal tubule epithelial cells (36), was obtained from American Type Culture Collection (Manassas, VA). Cells were routinely maintained in high-glucose DMEM containing 5% bovine calf serum, 2 mml-glutamine, 100 IU/ml penicillin, 100 μg/ml streptomycin, and 0.1 mm nonessential amino acids (all from Life Technologies, Inc., Gaithersburg, MD) at 37°C with 5% CO2 in air. Cells were trypsinized and reseeded at a 1:5 dilution every 3 days.

Irradiation.

NRK52E cells were grown to 90% confluence. To maintain the cells in a plateau phase, they were placed in serum-free medium for 24 h before irradiation. Cells were then irradiated with single doses (1–20 Gy) of γ irradiation using a 137Cs irradiator at a dose rate of 0.98 Gy/min. All irradiations were performed at room temperature; control cells received sham-irradiation. After irradiation, cells were returned to the incubator and maintained in the serum-free medium at 37°C in a 5% CO2/95% air mixture for up to 48 h.

Measurement of Intracellular ROS Generation.

ROS generation was measured in NRK52E cells using H2DCFDA (Molecular Probes, Inc., Eugene, OR) as described previously (37). Cells were incubated with 20 μm H2DCFDA (in PBS) for 30 min, washed, and then irradiated with single doses of 1–20 Gy γ-rays. After irradiation the cells were returned to the incubator for 1 h. The fluorescence intensity was then measured at excitation wavelength 485 nm and emission wavelength 530 nm using a Bio-Tek FL500 microplate fluorescence reader (Bio-Tek Instruments, Inc., Winooski, VT). Similar studies were done with an oxidation insensitive dye (C369; Molecular Probes, Inc.) to verify that uptake, ester cleavage, and efflux of the dye were not contributing to changes in fluorescence. This allows changes in fluorescence to be attributed to changes on oxidation of the dye and not to some other parameter.

RNA Extraction and Northern Blot Analysis.

Total RNA was isolated by the acid guanidinium thiocyanate-phenol-chloroform extraction method, using Tri-ZOL reagent (Life Technologies, Inc., Gaithersburg, MD); the concentration of RNA was determined spectrophotometrically at 260 nm. Northern blot analysis was performed as described previously (34). Dr. Thomas D. Gelehrter (University of Michigan, Ann Arbor, MI) generously provided the clone pSKPAI53 containing rat PAI-1 (2500 bp) inserted into the EcoRI site of pBluescript SK (−) (Stratagene). The clone containing rat GAPDH (316 bp) inserted into the PstI site of the pBR322 vector was obtained from Ambion (Austin, TX). The cDNA probes were labeled with [α-32P]dCTP (New England Nuclear) by the random primer extension method using a Random Primer DNA labeling kit (Boehringer Mannheim Biochimica, Germany). Autoradiographs were quantified by densitometry using an Alpha Imager 2000 (Alpha Innotech Corp., IL). To normalize the data, the densitometric signal for PAI-1 was divided by the densitometric signal of the GAPDH signal. PAI-1 induction was expressed in terms of the increase in mRNA signal measured in the irradiated tubule epithelial cells relative to control cells. Each experiment was performed a minimum of three times, and representative blots are shown.

Western Blot Analysis.

The media from irradiated or control rat tubule epithelial cell cultures were collected 48 h postirradiation and stored in aliquots at −70°C. This conditioned medium was concentrated using a Centricon concentrator (MW 10; Amicon, Beverly, MA). The amount of DNA/flask was measured fluorometrically (38). Western blot analysis was performed as described previously (34) using a rabbit polyclonal anti-PAI-1 (1:500 dilution; Calibiochem-Novabiochem Co., San Diego, CA) antibody; bands were visualized by chemiluminescence and exposure to film. Autoradiographs were quantified by densitometry using an Alpha Imager 2000. The protein content was expressed in terms of the increase in protein signal measured in the irradiated tubule epithelial cell conditioned media relative to that determined in the control cells. Each experiment was performed a minimum of three times, and representative blots are shown.

Measurement of Soluble Reduced Thiol Content.

Intracellular GSH, Cys, GGC, and NAC levels were assayed following previously published protocols (39, 40). Cell pellets were homogenized in 50 mm potassium phosphate buffer (pH 7.8) containing 1.34 mm diethylenetriaminepenta-acetic acid. Reduced thiol levels in cells were measured subsequent to derivatization with N-(1-pyrenyl) maleimide using a 25 cm C18 Reliasil column (Column Engineering, Ontario, CA) coupled with high-performance liquid chromatography with fluorescent detection (40). All biochemical determinations were normalized to the protein content of whole-cell homogenates using the method of Lowry et al.(41).

Cell Preparation for Antioxidant Enzyme Measurements.

At the end of the various experiments, tubule epithelial cells were rinsed twice with PBS and scraped into this buffer solution. After centrifugation, the supernatant solution was removed and the cell pellet stored at −80°C. The pellets were then thawed, sonicated on ice, and the protein content measured using the method of Bradford (42).

Immunoblotting for MnSOD and CAT.

Ten μg of total protein were separated by PAGE on a 12% gel by a modified version as described by Laemmli (43). Proteins were transferred to a nitrocellulose membrane for 1 h at 100 V, and then blocked with 5% milk in TBST. Membranes were incubated with CAT antibody (1:2500 dilution; Athens Biotech, Athens, GA.), or MnSOD antibody (1:5000 dilution; kindly provided by Dr. Larry Oberley, University of Iowa, Iowa City, IA) overnight, and then incubated with a horseradish peroxidase-conjugated secondary antibody (1:10000 dilution; Amersham Biotech). Bands were visualized by chemiluminescence and exposure to film.

Antioxidant Enzyme Activity.

Antioxidant enzyme activities were measured via native gel activity assays. For MnSOD, the native gel activity assay as described by Beauchamp and Fridovich (44) was used. CAT activity was visualized by staining as described previously (45).

Adenovirus Gene Transfer.

Adenovirus constructs containing the transgenes for human MnSOD, CAT, and the Escherichia coliLacZ reporter gene were obtained from Dr. B. Davidson (Director, Gene Therapy Vector Core, University of Iowa). To transiently transfect NRK52E cells, ∼1 × 106 cells were plated in a 100 mm Petri dish supplied with 10 ml complete media and allowed to attach for 24 h. Cells were then washed three times in serum- and antibiotic-free media, and the adenovirus constructs, suspended in 3% sucrose, were applied to cells in 5 ml of serum- and antibiotic-free media at 0–100 MOI units. Three h later, the serum- and antibiotic-free media were replaced with 10 ml of complete media. Transfection efficiency was determined using an 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside staining assay for β-galactosidase activity. Twenty-four h after incubation, AdLacZ-transduced cells were washed once in PBS and then fixed in a solution of 0.05% glutaraldehyde in PBS for 10 min at room temperature. After 2 more washes with PBS, the cells were then incubated in a solution containing 1 mg/ml 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (Sigma Chemical Co.), 5 mM K3 Fe(CN)6, 5 mM K4 Fe(CN)6 .3H2O and 1 mM MgCl2 .6H2O in PBS overnight at room temperature. Cells expressing the LacZ gene stained blue and were visualized under light microscopy; transfection efficiency was calculated as the ratio of blue-staining cells to total cell number using a hemocytometer.

Statistical Analysis.

Statistical analysis was carried out using a one-sample Student’s t test to compare differences between the irradiated tubule epithelial cells with control tubule epithelial cells. A P ≤ 0.05 was considered significant.

Effect of Radiation on PAI-1 Expression.

As we have demonstrated previously (34), irradiation of quiescent NRK52E cells with single doses of 1–10 Gy γ-rays led to a dose-dependent increase in steady-state PAI-1 mRNA levels 24 h postirradiation (data not shown). To determine whether this radiation-induced increase in PAI-1 gene expression was accompanied by increases in immunoreactive PAI-1 protein levels, the conditioned medium from irradiated and control sham-irradiated cells was collected and used for Western blot analysis. As shown in Fig. 1, radiation also led to a dose-dependent increase in the level of PAI-1 immunoreactive protein secreted into the conditioned medium at 48 h postirradiation; the maximum increase was found after 20 Gy γ-rays.

Radiation-induced Increase in ROS Production.

Fig. 2,A shows ROS production using the oxidation-sensitive fluorescent probe H2DCFDA. Irradiating NRK52E cells with 0–10 Gy γ-rays was associated with a dose-related increase in the fluorescence of the oxidation-sensitive probe, determined 1 h postirradiation. The mean level of fluorescence intensity in cells irradiated with either 1, 3, 5, or 10 Gy γ-rays was 1.5 ± 0.1, 2.2 ± 0.2, 2.2 ± 0.2, and 2.9 ± 0.4 (P < 0.05) times greater, respectively, than those observed in sham-irradiated controls. Fig. 2 B shows similarly treated cells labeled with the oxidation insensitive probe C369. The latter was used to control for alterations in fluorescence that might arise from irradiation-induced changes in probe uptake, ester cleavage, and efflux independent of oxidation. There was no apparent difference between the level of fluorescence observed in controls and cells irradiated with 10 Gy γ-rays, supporting the hypothesis that irradiation leads to dose-dependent increases in the generation of ROS in NRK52E cells.

Effect of H2O2 on PAI-1 Gene Expression.

The ability of ROS to up-regulate PAI-1 gene expression was tested further using H2O2. NRK52E cells were grown to 90% confluence and placed in serum-free medium for 24 h before incubation with 100 μm H2O2 for 3, 7, and 24 h. A marked increase in PAI-1 mRNA levels was observed 3 and 7 h after the addition of H2O2 (Fig. 3). Moreover, levels remained greater than those seen in control cells 24 h after incubation with H2O2. These results confirm that exogenous administration of H2O2 is capable of mediating the up-regulation of PAI-1 gene expression.

Effect of Antioxidants on ROS-induced Up-Regulation of PAI-1.

We investigated the ability of NAC to modulate radiation-induced changes in PAI-1 expression by incubating NRK52E cells with 2.5 mm NAC 30 min before and after irradiation. As shown in Fig. 4,A, incubating unirradiated cells with NAC led to a down-regulation of PAI-1 gene expression. Furthermore, NAC treatment abrogated the radiation-induced up-regulation of PAI-1 gene expression. Densitometric analysis revealed that the mean level of PAI-1 mRNA in cells treated with either 10 Gy, 2.5 mm NAC alone, or 10 Gy plus 2.5 mm NAC were 2.5 ± 0.2, 0.5 ± 0.2, and 0.4 ± 0.2, respectively, relative to the levels observed in control cells (Fig. 4 A).

The ability of NAC to abrogate the radiation-induced up-regulation of PAI-1 expression was also evident in terms of immunoreactive protein (Fig. 4,B). The amount of PAI-1 protein in the conditioned medium of NRK52E cells irradiated with a single dose of 20 Gy γ-rays was 2.8-fold greater than that determined in control cells. This increase was blocked in cells treated with 2.5 mm NAC. We measured the total soluble thiol content of the NRK52E cells incubated with 2.5 mm NAC 30 min before and 4 h after 10 Gy of radiation, including GSH, GGC, NAC, and Cys (Table 1). In control cells the mean total soluble thiol content was 3.27 ± 0.52 nm/mg protein, made up primarily of Cys (70%) and GSH (30%). Radiation-induced oxidative stress resulted in a significant decrease in GSH content (50%) associated with a concomitant increase in Cys levels (20%), such that the total soluble thiol content was unchanged. Incubating the cells with NAC before and 4 h after irradiation failed to abrogate the radiation-induced decrease in GSH. In contrast, Cys levels increased significantly 4 h postirradiation, reaching levels that were nearly 2-fold greater than those seen in control or irradiated cells that did not receive NAC; the mean total soluble thiol content increased to 5.34 ± 0.52 nm/mg protein. These findings indicate that Cys was the predominant small molecular weight-reducing thiol in the NRK52E cells, and that exposure to NAC led to a marked increase in intracellular NAC and Cys pools rather than in GSH content. Taken together, these findings provide additional support for the hypothesis that the radiation-induced up-regulation of PAI-1 expression is mediated, at least in part, via increased ROS production, because a thiol antioxidant that clearly increased intracellular reduced thiol pools was capable of suppressing the changes.

Effect of Overexpressing MnSOD and/or CAT on Radiation-induced Up-Regulation of PAI-1.

To directly test the involvement of superoxide (O2) and H2O2 in the radiation-induced up-regulation of PAI-1, an adenovirus-based gene transfer approach to directly modulate MnSOD and CAT levels was used. MnSOD, localized in the mitochondria, converts superoxide (O2) radical into H2O2, whereas CAT, found primarily in the peroxisomes, converts H2O2 to H2O and O2. Transduction efficiency was determined using the β-gal staining technique in cells transduced with AdLacZ. NRK52E cells transduced with 10 or more MOI adenovirus particles exhibited a transduction efficiency of >70% (data not shown). Transiently transducing NRK52E cells with AdMnSOD (0–100 MOI) led to a marked dose-dependent increase in MnSOD immunoreactive protein and activity determined 48 h postirradiation (Fig. 5,A). In contrast, no change in MnSOD gene expression or protein level was observed in cells transduced with 100 MOI of the AdLacZ control. Similar increases in CAT immunoreactive protein and activity were seen after infecting cells with AdCAT (Fig. 5 B).

NRK52E cells overexpressing either MnSOD or CAT (30 MOI) were used to evaluate further whether the radiation-induced up-regulation of PAI-1 is attributable, in part, to increased ROS generation. Transduced and control cells were irradiated using a single dose of 20 Gy γ-rays 48 h after infection and the conditioned medium collected at 48 h postirradiation. Densitometric analysis of Western blots revealed that overexpressing LacZ failed to alter the levels of PAI-1 secreted into the medium by unirradiated or irradiated cells (Fig. 6). Overexpressing MnSOD in NRK52E cells led to a small but statistically insignificant reduction in the radiation-induced increase in PAI-1 protein. In contrast, overexpressing CAT was associated with a nearly complete inhibition of the radiation-induced up-regulation of PAI-1 (Fig. 6). A similar inhibition of radiation-induced PAI-1 up-regulation was noted in cells transduced with both MnSOD and CAT (Fig. 6). These results demonstrate further that the radiation-induced up-regulation of PAI-1 is mediated, in part, via increased ROS production and indicate that H2O2 plays a major role in this induction.

These findings are the first to directly demonstrate that PAI-1 expression in kidney tubule epithelial cells is redox-regulated. Exposing cells to oxidative stress resulting from ionizing radiation or from direct incubation with H2O2 led to increased PAI-1 expression in terms of both mRNA and immunoreactive protein. These findings confirm previous observations demonstrating that PAI-1 expression increases in NRK52E cells (34) and in rat mesangial cells (33) after in vitro irradiation and in rat cardiac microvascular endothelial cells incubated with H2O2(32).

Additional evidence for a role of oxidative stress in mediating PAI-1 expression was obtained from studies in which the incubation of cells with the antioxidant NAC resulted in complete inhibition of the radiation-induced up-regulation of PAI-1 expression. As a thiol antioxidant, NAC can block oxidative stress directly through the scavenging of ROS and the inhibition of thiol oxidation reactions (46). Thus, the ability of NAC to prevent the radiation-induced up-regulation of PAI-1 expression suggests a direct role for ROS-mediated thiol oxidation/reduction reactions in regulating PAI-1 expression. However, interpreting these findings is complicated by the fact that NAC can also indirectly block oxidative stress by serving as a precursor for intracellular Cys pools and GSH synthesis (47). GSH, the most abundant cellular nonprotein thiol, plays a central role in maintaining cellular redox status and in protecting cells from sources of 32 oxidative injury, including H2O2(48). Elevation of intracellular GSH also occurs as an adaptive response to oxidative stress (48, 49). Radiation-induced oxidative stress resulted in a significant decrease in GSH content associated with a concomitant increase in Cys levels, such that the total soluble thiol content was unchanged (Table 1). Incubating the cells with NAC before and 4 h after irradiation failed to abrogate the radiation-induced decrease in GSH. In contrast, Cys levels in NAC-treated cells increased significantly 4 h postirradiation, reaching levels that were nearly 2-fold greater than those seen in control or irradiated cells that did not receive NAC. These findings indicate that Cys was the predominant small molecular weight-reducing thiol in the NRK52E cells, and that exposure to NAC led to a marked increase in intracellular NAC and Cys rather than in GSH content. The ability of a thiol antioxidant to block the radiation-induced up-regulation of PAI-1 expression suggests that some oxidant capable of perturbing thiol redox chemistry is involved in triggering the response. To identify the particular ROS involved, we used a gene transfer approach to overexpress specific antioxidant enzymes.

Superoxide dismutase and CAT, along with GPx, are the primary antioxidant enzymes in mammalian cells. SOD catalyzes the dismutation of O2 to H2O2 that is detoxified further by CAT and GPx (50). Three forms of SOD have been described in humans: (a) a homodimeric copper zinc containing SOD (CuZnSOD) found primarily in the cytoplasm (51); (b) a homotetrameric glycosylated CuZnSOD present in extracellular fluid (52); and (c) a homotetrameric MnSOD found in the mitochondrial matrix (52). Catalase is primarily located in the peroxisomes, whereas GPx is located in the cytosol and mitochondrial matrix. NRK52E cells were transiently transfected with adenovirus constructs containing the transgenes for human MnSOD and CAT. The AdMnSOD construct containing the human MnSOD gene (53) has previously been shown to be functional in transduced human lung epithelial cells (54). The AdCAT construct containing the human CAT gene has also been shown to be functional in vascular smooth muscle cells (55). Transient transduction of rat kidney epithelial cells with AdMnSOD and AdCAT led to significant increases in MnSOD and CAT mRNA, immunoreactive protein, and enzymatic activity. Subsequently, we determined that overexpression of CAT led to nearly complete inhibition of the radiation-induced up-regulation of PAI-1 expression. In contrast, overexpressing MnSOD did not appear to modulate the radiation-induced up-regulation of PAI-1 or enhance the effect of CAT. These findings strongly suggest that H2O2 rather than O2 may be the active moiety in this oxidative stress-mediated up-regulation of PAI-1.

These in vitro observations of radiation-induced redox up-regulation of PAI-1 expression are of interest in view of recent findings that suggest a potential role for PAI-1 in the pathogenesis of radiation-induced kidney fibrosis. Oikawa et al.(19) reported a 9-fold increase in PAI-1 mRNA in cortical tissue isolated from the kidneys of rats that had received a single dose of 12 Gy 60Co γ-rays to both kidneys 12 weeks before. In situ hybridization revealed significant association of PAI-1 expression with sites of glomerular lesions. More recently, a significant correlation between the degree of glomerulosclerosis and the level of PAI-1 immunostaining was observed within individual rats after kidney irradiation (56). Data on the possible role of PAI-1 in other late-responding normal tissues are limited. PAI-1 has been implicated in the pathogenesis of delayed radiation damage in the spinal cord (57). No PAI-1 was detected in normal spinal cord or in irradiated spinal cord up to 90 days after irradiation of the rat cervical cord with a single dose of 24 Gy. However, PAI-1 was detected using Western blot and ELISA assay 120 days after irradiation, and immunohistochemical studies revealed that PAI-1 was localized within and immediately adjacent to zones of necrosis at 145 days after irradiation; normal spinal cord was negative for immunoreactive protein. These limited data suggest a pathogenic role for PAI-1 in the expression of late radiation-induced normal tissue injury.

The finding that expression of PAI-1, an important profibrogenic mediator believed to play a major role in normal tissue fibrosis, is redox-modulated offers the potential to develop novel, rational, antioxidant-based approaches to treating fibrosis. Classically, fibrosis has been viewed as a chronic, progressive process in which normal tissue is replaced irreversibly with fixed fibrotic tissue. Recent data have lead to this view being challenged by a new paradigm in which fibrosis is viewed as a wound-healing response involving complex and dynamic interactions between several cell types (58). Such a model offers the opportunity of developing interventional therapies directed at modulating potential mediators to inhibit or indeed reverse the fibrotic process. A growing body of evidence supports a causative role of oxidative stress in fibrogenesis (59). Antioxidants, particularly SOD, have clearly proven to be effective both in terms of inhibiting and, indeed, in reversing established fibrosis (60, 61, 62). Of interest is the recent development of novel nonpeptidyl SOD mimics that offer the promise of improved clinical therapies for ROS-mediated injury (63). The current findings are supportive of this new paradigm and suggest that H2O2 scavenging might also offer potential for antioxidant-mediated modulation of fibrogenic disease via alterations in expression of the profibrogenic mediator PAI-1.

Fig. 1.

Irradiating rat tubule epithelial cells leads to a dose-dependent increase in PAI-1 protein level in rat tubule epithelial cell-conditioned medium. Quiescent tubule epithelial cells were irradiated with 0–20 Gy γ-rays, and conditioned medium was collected at 48 h postirradiation. Equal amounts of medium volume (based on the total DNA amount) were separated by SDS-PAGE. The protein level of PAI-1 was analyzed by Western immunoblotting using polyclonal rabbit anti-PAI-1 antibodies. A representative blot shows that irradiation induced increases in the PAI-1 immunoreactive protein level in a dose-dependent manner.

Fig. 1.

Irradiating rat tubule epithelial cells leads to a dose-dependent increase in PAI-1 protein level in rat tubule epithelial cell-conditioned medium. Quiescent tubule epithelial cells were irradiated with 0–20 Gy γ-rays, and conditioned medium was collected at 48 h postirradiation. Equal amounts of medium volume (based on the total DNA amount) were separated by SDS-PAGE. The protein level of PAI-1 was analyzed by Western immunoblotting using polyclonal rabbit anti-PAI-1 antibodies. A representative blot shows that irradiation induced increases in the PAI-1 immunoreactive protein level in a dose-dependent manner.

Close modal
Fig. 2.

Radiation stimulates ROS generation in rat tubule epithelial cells. Cells were preloaded for 30 min with the oxidation-sensitive fluorescence probe H2DCFDA, washed, irradiated with 0–10 Gy γ-rays, and the relative fluorescence measured at 1 h postirradiation. The oxidation-insensitive fluorescence probe C369 was used as control. Top, data generated using the oxidation-sensitive probe H2DCFDA; bottom, data generated using the oxidation-insensitive probe C369. Mean ± SE; n = 3 (three independent experiments); *P < 0.05.

Fig. 2.

Radiation stimulates ROS generation in rat tubule epithelial cells. Cells were preloaded for 30 min with the oxidation-sensitive fluorescence probe H2DCFDA, washed, irradiated with 0–10 Gy γ-rays, and the relative fluorescence measured at 1 h postirradiation. The oxidation-insensitive fluorescence probe C369 was used as control. Top, data generated using the oxidation-sensitive probe H2DCFDA; bottom, data generated using the oxidation-insensitive probe C369. Mean ± SE; n = 3 (three independent experiments); *P < 0.05.

Close modal
Fig. 3.

H2O2 increases PAI-1 mRNA levels in rat tubule epithelial cells in a time-dependent manner. Quiescent rat tubule epithelial cells were incubated with or without 100 μm H2O2, and total RNA samples were collected at 3, 7, and 24 h. Northern blot hybridization was used for PAI-1 mRNA analysis, and GAPDH was used as a loading control. A, Northern blot indicating that H2O2 increases steady-state PAI-1 mRNA levels in a time-dependent manner. B, the densitometric quantification of steady-state PAI-1 mRNA levels in cells treated with H2O2 relative to that seen in control cells.

Fig. 3.

H2O2 increases PAI-1 mRNA levels in rat tubule epithelial cells in a time-dependent manner. Quiescent rat tubule epithelial cells were incubated with or without 100 μm H2O2, and total RNA samples were collected at 3, 7, and 24 h. Northern blot hybridization was used for PAI-1 mRNA analysis, and GAPDH was used as a loading control. A, Northern blot indicating that H2O2 increases steady-state PAI-1 mRNA levels in a time-dependent manner. B, the densitometric quantification of steady-state PAI-1 mRNA levels in cells treated with H2O2 relative to that seen in control cells.

Close modal
Fig. 4.

Incubating rat tubule epithelial cells with NAC before irradiation abolishes the radiation-induced up-regulation of PAI-1 gene expression and protein level. Rat tubule epithelial cells were incubated with or without 2.5 mm NAC for 30 min before treatment and for 4 h after treatment with 10 Gy or 20 Gy γ-rays. Total RNA samples were collected 24 h postirradiation. Northern blot hybridization was used for PAI-1 mRNA analysis, and GAPDH was used as a loading control. Cell-conditioned medium was collected at 48 h postirradiation. The protein level of PAI-1 was analyzed by Western immunoblotting using polyclonal rabbit anti-PAI-1 antibodies. A, data in terms of the effects of NAC on PAI-1 mRNA subsequent to irradiation; B, data in terms of the effects of NAC on PAI-1 protein subsequent to irradiation. Mean ± SE; n = 3; ∗, P < 0.05.

Fig. 4.

Incubating rat tubule epithelial cells with NAC before irradiation abolishes the radiation-induced up-regulation of PAI-1 gene expression and protein level. Rat tubule epithelial cells were incubated with or without 2.5 mm NAC for 30 min before treatment and for 4 h after treatment with 10 Gy or 20 Gy γ-rays. Total RNA samples were collected 24 h postirradiation. Northern blot hybridization was used for PAI-1 mRNA analysis, and GAPDH was used as a loading control. Cell-conditioned medium was collected at 48 h postirradiation. The protein level of PAI-1 was analyzed by Western immunoblotting using polyclonal rabbit anti-PAI-1 antibodies. A, data in terms of the effects of NAC on PAI-1 mRNA subsequent to irradiation; B, data in terms of the effects of NAC on PAI-1 protein subsequent to irradiation. Mean ± SE; n = 3; ∗, P < 0.05.

Close modal
Fig. 5.

Immunoreactive protein and activity analysis of MnSOD and CAT in rat tubule epithelial cells transduced with AdMnSOD and AdCAT constructs. Tubule epithelial cells were transduced with 1–100 MOI AdMnSOD or AdCAT or 100 MOI AdLacZ. Total protein was collected 48 h after infection. Western blot and native gel activity assays were used to analyze protein and activity levels. Each panel shows the Western blot or native gel obtained after transduction of the cells with AdMnSOD or AdCAT and the densitometric quantification for antioxidant enzyme immunoreactive protein or enzymatic activity relative to that seen in control cells. These results show that the levels of MnSOD and CAT immunoreactive protein (a and c) and enzymatic activity (b and d) in cells transduced with MnSOD and CAT, respectively, increased in a dose-dependent manner.

Fig. 5.

Immunoreactive protein and activity analysis of MnSOD and CAT in rat tubule epithelial cells transduced with AdMnSOD and AdCAT constructs. Tubule epithelial cells were transduced with 1–100 MOI AdMnSOD or AdCAT or 100 MOI AdLacZ. Total protein was collected 48 h after infection. Western blot and native gel activity assays were used to analyze protein and activity levels. Each panel shows the Western blot or native gel obtained after transduction of the cells with AdMnSOD or AdCAT and the densitometric quantification for antioxidant enzyme immunoreactive protein or enzymatic activity relative to that seen in control cells. These results show that the levels of MnSOD and CAT immunoreactive protein (a and c) and enzymatic activity (b and d) in cells transduced with MnSOD and CAT, respectively, increased in a dose-dependent manner.

Close modal
Fig. 6.

Overexpression of CAT but not MnSOD led to inhibition of the radiation-induced increases in PAI-1 immunoreactive protein levels in rat tubule epithelial cells. Tubule epithelial cells were transduced with either AdLacZ (100 MOI), AdMnSOD, and/or AdCAT (30 MOI). Twenty-four h later, these cells and nontransduced cells were placed in serum-free medium for 24 h preceding irradiation with a single dose of 20 Gy γ-rays; control cells received sham-irradiation. Conditioned medium was collected at 48 h postirradiation. Equal amounts of medium volume (based on the total DNA amount) were separated by SDS-PAGE. The protein level of PAI-1 was analyzed by Western immunoblotting using polyclonal rabbit anti-PAI-1 antibodies. Densitometric quantification shows the effects of MnSOD and/or CAT gene transfection on PAI-1 protein induced by radiation in tubule epithelial cells. Mean ± SE; n = 3; ∗, P < 0.05 as compared with the protein level of PAI-1 observed in control cells.

Fig. 6.

Overexpression of CAT but not MnSOD led to inhibition of the radiation-induced increases in PAI-1 immunoreactive protein levels in rat tubule epithelial cells. Tubule epithelial cells were transduced with either AdLacZ (100 MOI), AdMnSOD, and/or AdCAT (30 MOI). Twenty-four h later, these cells and nontransduced cells were placed in serum-free medium for 24 h preceding irradiation with a single dose of 20 Gy γ-rays; control cells received sham-irradiation. Conditioned medium was collected at 48 h postirradiation. Equal amounts of medium volume (based on the total DNA amount) were separated by SDS-PAGE. The protein level of PAI-1 was analyzed by Western immunoblotting using polyclonal rabbit anti-PAI-1 antibodies. Densitometric quantification shows the effects of MnSOD and/or CAT gene transfection on PAI-1 protein induced by radiation in tubule epithelial cells. Mean ± SE; n = 3; ∗, P < 0.05 as compared with the protein level of PAI-1 observed in control cells.

Close modal

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by RO1 NIDDK DK-51612 (to M. E. C. R.), PO1 NIH CA-66081 (to L. W. O.), and RO1 NIH HL-51469 (to D. R. S.).

3

The abbreviations used are: ECM, extracellular matrix; PA, plasminogen activator; PAI-1, plasminogen activator inhibitor-1; ROS, reactive oxygen species; H2O2, hydrogen peroxide; H2DCFDA, 2′7′-dichlorodihydofluorescein diacetate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GSH, glutathione; Cys, cysteine; GGC, γ-glutamylcysteine; NAC, N-acetylcysteine; MOI, multiplicity of infection; GPx, glutathione peroxidase; MnSOD, manganese containing superoxide dismutase; CAT, catalase.

Table 1

Effect of radiation on the total soluble thiol content (nm/mg protein) of NRK52E cells incubated with or without 2.5 mM NACa

TreatmentNACGSHGGCCysTotal soluble thiol
Control ND 0.91 ± 0.11 0.03 ± 0.02 2.33 ± 0.15 3.27 ± 0.27 
10 Gy ND 0.46 ± 0.18b 0.02 ± 0.01 2.83 ± 0.18 3.31 ± 0.02 
NAC + 10 Gy 0.44 ± 0.19 0.25 ± 0.04b 0.03 ± 0.02 4.62 ± 0.52b 5.34 ± 0.52 
TreatmentNACGSHGGCCysTotal soluble thiol
Control ND 0.91 ± 0.11 0.03 ± 0.02 2.33 ± 0.15 3.27 ± 0.27 
10 Gy ND 0.46 ± 0.18b 0.02 ± 0.01 2.83 ± 0.18 3.31 ± 0.02 
NAC + 10 Gy 0.44 ± 0.19 0.25 ± 0.04b 0.03 ± 0.02 4.62 ± 0.52b 5.34 ± 0.52 
a

Data are mean ± SD; n = 3. ND, not detectable.

b

P < 0.05.

1
Franklin T. J. Therapeutic approaches to organ fibrosis.
Int. J. Biochem. Cell Biol.
,
29
:
79
-89,  
1997
.
2
Martin M., Lefaix J-L., Delanian S. TGF-β1 and radiation fibrosis: a master switch and a specific therapeutic target?.
Int. J. Radiat. Oncol. Biol. Phys.
,
47
:
277
-290,  
2000
.
3
Powell D. W., Mifflin R. C., Valentich J. D., Crowe S. E., Saada J. I., West A. B. Myofibroblasts. I. Paracrine cells important in health and disease.
Am. J. Physiol.
,
277
:
C1
-C19,  
2000
.
4
Vassalli J. D., Sappino A. P., Belin D. The plasminogen activator/plasmin system.
J. Clin. Investig.
,
88
:
1067
-1972,  
1991
.
5
Mayer M. Biomedical and biochemical aspects of the plasminogen activation system.
Clin. Biochem.
,
23
:
197
-211,  
1990
.
6
Werb Z., Banda M. J., Jones P. A. Degradation of connective tissue matrices by macrophages. I. Proteolysis of elastin, glycoproteins and collagen by proteinases isolated from macrophages.
J. Exp. Med.
,
152
:
1340
-1357,  
1980
.
7
Wong A. P., Cortez S. L., Baricos W. H. Role of plasmin and gelatinase in extracellular matrix degradation by cultured rat mesangial cells.
Am. J. Physiol.
,
263
:
F1112
-F1118,  
1992
.
8
Fearns C., Loskutoff D. J. Induction of plasminogen activator inhibitor 1 gene expression in murine liver by lipopolysaccharide. Cellular localization and role of endogenous tumor necrosis factor-α.
Am. J. Pathol.
,
150
:
579
-590,  
1997
.
9
Colucci M., Paramo J. A., Collen D. Inhibition of one-chain and two-chain forms of human tissue-type plasminogen activator by the fast acting inhibitor of plasminogen activator in vitro and in vivo.
J. Lab. Clin. Med.
,
108
:
53
-59,  
1986
.
10
Chmielewska J., Ranby M., Wiman B. Kinetics of the inhibition of plasminogen activator by the plasminogen activator inhibitor.
Biochem. J.
,
251
:
327
-332,  
1988
.
11
Erickson L. A., Hekman C. M., Loskutoff D. J. The primary plasminogen-activator inhibitors in endothelial cells, platelets, serum, and plasma are immunologically related.
Proc. Natl. Acad. Sci. USA
,
82
:
8710
-8714,  
1985
.
12
Knudsen B. S., Harpel P. C., Nachman R. L. Plasminogen activator inhibitor is associated with the extracellular matrix of cultured bovine smooth muscle cells in vivo.
J. Clin. Investig.
,
80
:
1082
-1089,  
1987
.
13
Leyland H., Gentry J., Arthur M. J., Benyon R. C. The plasminogen-activating system in hepatic stellate cells.
Hepatology
,
24
:
1172
-1178,  
1996
.
14
Peraldi M. N., Rondeau E., Medcalf R. L., Hagege J., Lacave R., Delarue F., Schleuning W. D., Sraer J. D. Cell-specific regulation of plasminogen activator inhibitor 1 and tissue type plasminogen activator release by human kidney mesangial cells.
Biochim. Biophys. Acta
,
1134
:
189
-196,  
1992
.
15
Kanalas J. J., Hopfer U. Effect of TGF-β1 and TNF-α on the plasminogen system of rat proximal tubular epithelial cells.
J. Am. Soc. Nephrol.
,
8
:
184
-192,  
1997
.
16
Wiman B., Hamsten A. The fibrinolytic enzyme system and its role in the etiology of thromboembolic disease.
Semin. Thromb. Hemost.
,
16
:
207
-216,  
1990
.
17
Schleef R. R., Higgins D. L., Pillemer E., Levitt L. J. Bleeding diathesis due to decreased functional activity of type 1 plasminogen activator inhibitor.
J. Clin. Investig.
,
83
:
1747
-1752,  
1989
.
18
Eddy A. A., Giachelli C. M. Renal expression of genes that promote interstitial inflammation and fibrosis in rats with protein-overload proteinuria.
Kidney Int.
,
47
:
1546
-1557,  
1995
.
19
Oikawa T., Freeman M., Lo W., Vaughan D. E., Fogo A. Modulation of plasminogen activator inhibitor-1 in vivo: a new mechanism for the anti-fibrotic effect of renin-angiotensin inhibition.
Kidney Int.
,
51
:
164
-172,  
1997
.
20
Zhang L. P., Takahara T., Yata Y., Furui K., Jin B., Kawada N., Watanabe A. Increased expression of plasminogen activator and plasminogen activator inhibitor during liver fibrogenesis of rats: role of stellate cells.
J. Hepatol.
,
31
:
703
-711,  
1999
.
21
Bertozzi P., Astedt B., Zenzius L., Lynch K., LeMaire F., Zapol W., Chapman H. A. J. Depressed bronchoalveolar urokinase activity in patients with adult respiratory distress syndrome.
N. Engl. J. Med.
,
322
:
890
-897,  
1990
.
22
Eltzman D. T., McCoy R. D., Zheng X., Fay W. F., Shen T., Ginsburg D., Simon R. H. Bleomycin-induced pulmonary fibrosis in transgenic mice that either lack or overexpress the murine plasminogen activator inhibitor-1 gene.
J. Clin. Investig.
,
97
:
232
-237,  
1996
.
23
Heaton J. H., Gelehrter T. D. Glucocorticoid induction of plasminogen activator and plasminogen activator inhibitor-1 messenger RNA in rat hepatoma cells.
Mol. Endocrinol.
,
3
:
349
-355,  
1989
.
24
Gelehrter T. D., Sznycer-Laszuk R., Zeheb R., Cwikel B. J. Dexamethasone inhibition of tissue-type plasminogen activator (tPA) activity: paradoxical induction of both tPA antigen and plasminogen activator inhibitor.
Mol. Endocrinol.
,
1
:
97
-101,  
1987
.
25
Heaton J. H., Dane M. K., Gelehrter T. D. Thrombin induction of plasminogen activator inhibitor mRNA in human umbilical vein endothelial cells in culture.
J. Lab. Clin. Med.
,
120
:
222
-228,  
1992
.
26
Reilly C. F., McFall R. C. Platelet-derived growth factor and transforming growth factor-β regulate plasminogen activator inhibitor-1 synthesis in vascular smooth muscle cells.
J. Biol. Chem.
,
266
:
9419
-9427,  
1991
.
27
Vaughan D. E., Lazos S. A., Tong K. Angiotensin II regulates the expression of plasminogen activator inhibitor-1 in cultured endothelial cells.
J. Clin. Investig.
,
95
:
995
-1001,  
1995
.
28
Hopkins W. E., Fujii S., Sobel B. E. Synergistic induction of plasminogen activator inhibitor type-1 in HEPG2 cells by thrombin and transforming growth factor-β.
Blood
,
79
:
75
-81,  
1992
.
29
Dawson S. J., Wiman B., Hamsten A., Green F., Humphries S., Henney A. M. The two allele sequences of a common polymorphism in the promoter of the plasminogen activator inhibitor-1 gene respond differently to interleukin in HepG2 cells.
J. Biol. Chem.
,
268
:
10739
-10745,  
1993
.
30
Van Hinsbergh V. W., Kooistra T., van den Berg E. A., Princen H. M., Fiers W., Emeis J. J. Tumor necrosis factor increases the production of plasminogen activator inhibitor in human endothelial cells in vitro and in rat in vivo.
Blood
,
72
:
1467
-1473,  
1988
.
31
Okada H., Woodcock-Mitchell J., Mitchell J., Sakamoto K., Marutsuka K., Sobel B. E., Fujii S. Induction of plasminogen activator inhibitor type 1 and type 1 collagen expression in rat cardiac microvascular endothelial cells by interleukin-1 and its dependence on oxygen-centered free radicals.
Circulation
,
97
:
2175
-2182,  
1998
.
32
Ceriello A., Curcio F., dello Russo P., Pegoraro I., Stel G., Amstad P., Cerutti P. The defense against free radicals protects endothelial cells from hyperglycaemia-induced plasminogen activator inhibitor 1 over-production.
Blood Coagul. Fibrinolysis
,
6
:
133
-137,  
1995
.
33
Zhao W., O’Malley Y., Robbins M. E. C. Irradiation of rat mesangial cells alters the expression of gene products associated with the development of renal fibrosis.
Radiat. Res.
,
152
:
160
-169,  
1999
.
34
Zhao W., O’Malley Y., Wei S., Robbins M. E. C. Irradiation of rat tubule epithelial cells alters the expression of gene products associated with the synthesis and degradation of extracellular matrix.
Int. J. Radiat. Biol.
,
76
:
391
-402,  
2000
.
35
Riley P. A. Free radicals in biology: oxidative stress and the effects of ionizing radiation.
Int. J. Radiat. Biol.
,
65
:
27
-33,  
1994
.
36
De Larco J. E., Todaro G. J. Epithelioid and fibroblastic rat kidney cell clones: epidermal growth factor (EGF) receptors and the effect of mouse sarcoma virus transformation.
J. Cell. Physiol.
,
94
:
335
-342,  
1978
.
37
Hempel S. L., Buettner G. R., O’Malley Y., Wessels D. A., Flaherty D. A. Dihydrofluorescein diacetate is superior for detecting intracellular oxidants: comparison to 2′7′ dichlorodihydrofluorescein diacetate, 5 (and 6)-carboxy-2′7′-dichlorodihydoflourescein diacetate, and dihydrorhodamine 123.
Free Radic. Biol. Med.
,
27
:
146
-159,  
1999
.
38
Kapuscinski J., Skoczylas B. Simple and rapid fluorometric method for DNA microassay.
Anal. Biochem.
,
83
:
252
-257,  
1977
.
39
Blackburn R. V., Spitz D. R., Liu X., Galoforo S. S., Sim J. E., Ridnour L. A., Chen J. C., Davis B. H., Corry P. M., Lee Y. J. Metabolic oxidative stress activates signal transduction and gene expression during glucose deprivation in human tumor cells.
Free Radic. Biol. Med.
,
26
:
419
-430,  
1999
.
40
Ridnour L. A., Winters R. A., Ercal N., Spitz D. R. Measurement of glutathione, glutathione disulfide, and other thiols in mammalian cell and tissue homogenates using high-performance liquid chromatography separation of N-(1-pyrenyl) maleimide derivatives.
Methods Enzymol.
,
299
:
258
-267,  
1999
.
41
Lowry O. H., Rosenbrough N. J., Randall R. J. Protein measurement using the folin phenol reagent.
J. Biol. Chem.
,
193
:
265
-275,  
1951
.
42
Bradford M. M. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal. Biochem.
,
72
:
248
-254,  
1976
.
43
Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature (Lond.)
,
227
:
680
-685,  
1970
.
44
Beauchamp C., Fridovich I. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels.
Anal. Biochem.
,
44
:
276
-287,  
1971
.
45
Sun Y., Elwell J. H., Oberley L. W. A simultaneous visualization of the antioxidant enzymes glutathione peroxidase and catalase on polyacrylamide gels.
Free Radic. Res. Commun.
,
5
:
67
-75,  
1988
.
46
Anbar M., Neta P. A compilation of specific bimolecular rate constants for the reactions of hydrated electrons, hydrogen atoms and hydroxyl radicals with inorganic and organic compounds in aqueous solution.
Int. J. Appl. Radiat. Isot.
,
18
:
495
-523,  
1967
.
47
Meister A., Anderson M. E. Glutathione.
Ann. Rev. Biochem.
,
52
:
711
-760,  
1983
.
48
Spitz D. R., Kinter M. T., Roberts R. J. Contribution of increased glutathione content to mechanisms of oxidative stress resistance in hydrogen peroxide resistant hamster fibroblasts.
J. Cell. Physiol.
,
165
:
600
-609,  
1995
.
49
Sagara Y., Dargusch R., Chambers D., Davis J., Schubert D., Maher P. Cellular mechanisms of resistance to chronic oxidative stress.
Free Radic. Biol. Med.
,
24
:
1375
-1389,  
1998
.
50
Fridovich I. The biology of oxygen radicals. The superoxide radical is an agent of oxygen toxicity: superoxide dismutases provide an important defense.
Science (Wash. DC)
,
201
:
875
-880,  
1978
.
51
McCord J. M., Fridovich I. Superoxide dismutase. An enzymatic function for erythrocuprein (hemocuprien).
J. Biol. Chem.
,
244
:
6049
-6055,  
1969
.
52
Marklund S. L. Human copper-containing superoxide dismutase of high molecular weight.
Proc. Natl. Acad. Sci. USA
,
79
:
7634
-7638,  
1982
.
53
Borgstahl G. E., Parge H. E., Hickey M. J., Boissinot M., Halliwell R. A., Lepock J. R., Cabelli D. E., Tainer J. A. Human mitochondrial manganese superoxide dismutase polymorphic variant Ile58Thr reduces activity by destabilizing the tetrameric interface.
Biochemistry
,
35
:
4287
-4297,  
1996
.
54
Zwacka R. M., Dudus L., Epperly M. W., Greenberger J. S., Engelhardt J. F. Redox gene therapy protects human IB-3 lung epithelial cells against ionizing radiation-induced apoptosis.
Hum. Gene Ther.
,
9
:
1381
-1386,  
1998
.
55
Brown M. R., Miller F. J., Jr., Li W. G., Ellingson A. N., Chatterje P., Engelhardt J. F., Zwacka R. M., Oberley L. W., Fang X., Spector A. A., Weintraub N. L. Overexpression of human catalase inhibits proliferation and promotes apoptosis in vascular smooth muscle cells.
Circ. Res.
,
85
:
524
-533,  
1999
.
56
Brown N. J., Nakamura S., Ma L., Nakamura I., Donnert E., Freeman M., Vaughan D. E., Fogo A. B. Aldosterone modulates plasminogen activator inhibitor-1 and glomerulosclerosis in vivo.
Kidney Int.
,
58
:
1219
-1227,  
2000
.
57
Sawaya R., Rayford A., Kono S., Ang K. K., Feng Y., Stephens L. C., Rao J. S. Plasminogen activator inhibitor-1 in the pathogenesis of delayed radiation damage in rat spinal cord in vivo.
J. Neurosurg.
,
81
:
381
-387,  
1994
.
58
Benyon R. C., Iredale J. P. Is liver fibrosis reversible?.
Gut
,
46
:
443
-446,  
2000
.
59
Poli G., Parola M. Oxidative damage and fibrinogenesis.
Free Radic. Biol. Med.
,
22
:
287
-305,  
1997
.
60
Delanian S., Baillet F., Huart J., Lefaix J. L., Maulard C., Housset M. Successful treatment of radiation-induced fibrosis using liposomal Cu/Zn superoxide dismutase: clinical trial.
Radiother. Oncol.
,
32
:
12
-20,  
1994
.
61
Lefaix J. L., Delanian S., Leplat J. J., Tricaud Y., Martin M., Nimrod A., Baillet F., Daburon F. Successful treatment of radiation-induced fibrosis using Cu/Zn-SOD and Mn-SOD: an experimental study.
Int. J. Radiat. Oncol. Biol. Phys.
,
35
:
305
-312,  
1996
.
62
Epperly M. W., Bray J., Kraeger S., Zwacka R., Engelhardt J. F., Travis E., Greenberger J. Prevention of late effects of irradiation lung damage by manganese superoxide dismutase gene therapy.
Gene Ther.
,
5
:
196
-208,  
1998
.
63
Salvemini D., Wang Z-Q., Zweier J. L., Samouilov A., Macarthur H., Misko T. P., Currie M. G., Cuzzocrea S., Sikorski J. A., Riley D. P. A nonpeptidyl mimic of superoxide dismutase with therapeutic activity in rats.
Science (Wash. DC)
,
286
:
304
-306,  
1999
.