Inducible lung-specific urokinase expression reduces fibrosis and mortality after lung injury in mice

Thomas H. Sisson, Kerstin E. Hanson, Natalya Subbotina, Anjali Patwardhan, Noboru Hattori, and Richard H. Simon

Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Michigan School of Medicine, Ann Arbor, Michigan 48109


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Plasminogen activator inhibitor-1 (PAI-1)-deficient transgenic mice have improved survival and less fibrosis after intratracheal bleomycin instillation. We hypothesize that PAI-1 deficiency limits scarring through unopposed plasminogen activation. If this is indeed true, then we would expect increased urokinase-type plasminogen activator (uPA) expression to result in a similar reduction in scarring and improvement in mortality. To test our hypothesis, using the tetracycline gene regulatory system, we have generated a transgenic mouse model with the features of inducible, lung-specific uPA production. After doxycycline administration, these transgenic animals expressed increased levels of uPA in their bronchoalveolar lavage (BAL) fluid that accelerated intrapulmonary fibrin clearance. Importantly, this increased plasminogen activator production led to a reduction in both lung collagen accumulation and mortality after bleomycin-induced injury. These results suggest that PAI-1 deficiency does protect against the effects of bleomycin-induced lung injury through unopposed plasmin generation. By allowing the manipulation of plasminogen activation at different phases of the fibrotic process, this model will serve as a powerful tool in further investigations into the pathogenesis of pulmonary fibrosis.

plasminogen activator; plasmin; bleomycin; tetracycline


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

FIBROSIS OF THE LUNG OCCURS in a variety of diseases, including sarcoidosis and idiopathic pulmonary fibrosis, and in association with various collagen vascular diseases. Recently, in attempts to find new avenues for therapeutic intervention, researchers have made many insights into the pathophysiology of pulmonary fibrosis. The importance of the plasminogen activation system represents one of these key insights. Several studies have demonstrated impaired plasminogen activation in bronchoalveolar lavage (BAL) fluid of patients with various forms of pulmonary fibrosis (1, 6, 7, 13, 16). This abnormality in plasminogen activation results from increased expression of plasminogen activator inhibitor-1 (PAI-1). Identical alterations of plasminogen activation secondary to increased PAI-1 expression have been demonstrated in the bleomycin murine model of pulmonary fibrosis (22). Eitzman et al. (4) and Hattori et al. (9) demonstrated the critical role that PAI-1 plays in the pathogenesis of this fibrosis model. In these experiments, PAI-1-deficient mice were found to be relatively protected from collagen accumulation after intratracheal instillation of bleomycin. Neither of these studies elucidated the mechanism or the timing by which PAI-1 deficiency exerts its influence on fibrogenesis. It has been presumed that PAI-1 deficiency limits fibrosis by averting the bleomycin-induced impairment of plasminogen activation. This in turn leads to enhanced plasmin generation, and, through yet to be identified mechanisms, this latter protease mitigates fibrogenesis by decreasing collagen production, increasing collagen removal, or both. However, it remains possible that PAI-1 exacerbates fibrosis through an alternative pathway independent of its inhibition of plasminogen activation such as enhanced fibroblast migration or proliferation. If PAI-1 is exerting its profibrotic activity by blocking the activation of plasminogen to plasmin, then increased lung expression of a plasminogen activator (PA) in the setting of a fibrotic insult should be able to overcome the effects of this protease inhibitor and lead to protection. In fact, increased lung levels of PAs have been achieved through adenovirus-mediated gene transfer of the urokinase-type PA (uPA) gene (26). Although this approach did limit bleomycin-induced lung collagen accumulation, the improvement in fibrosis did not match that achieved through PAI-1 deficiency. A similar partial abrogation of lung collagen accumulation occurred in response to systemic administration of uPA in a dog model (14) and through intratracheal administration of recombinant uPA in a rat model of bleomycin-induced pulmonary fibrosis (8). The inability of these approaches to match the protection afforded by PAI-1 deficiency may be secondary to technical limitations. On the other hand, the results of these experiments may suggest that PAI-1 does indeed influence the pathogenesis of lung fibrosis through a pathway that is, at least in part, independent of its ability to inhibit uPA activity.

To further investigate this question as to whether or not PAI-1 is influencing fibrogenesis through the inhibition of plasminogen activation, it would be invaluable to have a mouse model that is capable of enhanced PA production throughout the lung's alveolar spaces. Also, if we confirm that increased uPA effectively reduces fibrosis, then further understanding the mechanism(s) by which this system interferes with the scarring process will be crucial in defining new therapeutic targets. Delineating when in the time course of fibrosis the PA system is exerting its influence is an important first step in this effort. Therefore, to begin addressing both the role of plasminogen activation in fibrogenesis and its mechanism of action, we set out to generate a transgenic mouse model with inducible, lung-specific, uPA production.

Following the lead of several recent reports, we have employed the doxycycline-inducible gene control system to construct this model (15, 17, 29). Several investigators have demonstrated a tight regulation of gene expression after doxycycline administration using this system. In our transgenic model, the tetracycline operator (tetO)-cytomegalovirus (CMV) minimal promoter drives exogenous uPA production when bound by the reverse tetracycline transactivator (rtTA) protein. The binding of rtTA to the tetO and the subsequent activation of uPA transcription can occur only in the presence of tetracycline or doxycycline. To restrict uPA production to the lung, rtTA expression is controlled by the rat Clara cell-specific protein (CCSP) promoter, which in this system induces transgene production predominantly in alveolar epithelial cells and conducting airways (29). Our report details the generation and characterization of this inducible, lung-specific, uPA-producing transgenic line. Specifically, this report illustrates the timing and quantity of lung uPA production in response to doxycycline exposure. This report also provides evidence that this transgenic model, after doxycycline administration, accelerates intra-alveolar fibrin removal, which has been associated with reduced fibrosis in PAI-1-deficient mice. Finally, this report demonstrates that increased production of uPA in the setting of a fibrotic injury reduces lung collagen accumulation and improves survival. Thus the capacity of this transgenic model to produce uPA in response to exogenous doxycycline administration, to enhance intrapulmonary fibrinolysis, and to protect against bleomycin-induced lung injury suggest that it will be a powerful tool in the further characterization of pulmonary fibrosis pathogenesis.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Generation of transgenic mice. The murine uPA (muPA) cDNA was excised from the PDB1519 plasmid with Xba I and Bgl II restriction enzymes, blunt ended, and cloned (using an EcoR V site) in the tetO expression cassette. This expression cassette consisted of seven tetO repeats, a CMV minimal promoter, and a bovine growth hormone polyadenylation sequence (a gift from Dr. J. Whitsett and Dr. J. Tischelaar, Cincinnati, OH). The resultant expression cassette was then excised from its plasmid backbone using the Asc I restriction enzyme and microinjected into (C57BL/6 × SJL) F2 mouse eggs. The microinjected eggs were implanted in pseudopregnant mothers. Resultant mice possessing the transgenic construct (founders) were then bred with 6- to 8-wk-old C57Bl/6 partners. The transgenic offspring (F1) from this cross were then bred with a second transgenic line containing the 2.3-kb rat CCSP promoter, the 1.0-kb rtTA coding sequence, and a 2.0-kb fragment from the human growth hormone containing introns and a polyadenylation sequence (a gift from Dr. J. Whitsett and Dr. J Tischelaar). Double-transgenic offspring possessing both the tetO-muPA and CCSP-rtTA were used in subsequent experiments. Littermates with either no or only a single transgenic construct were used as experimental controls.

Assessment of mouse genotypes. To determine the genotypes of offspring from various crossings, we purified DNA from mouse tail biopsies. To determine if the tetO-muPA construct was present in the genomic DNA of an individual mouse, we performed a PCR reaction with primers that distinguished this transgene from the native uPA gene. The tetO-muPA primer sequences are as follows: upper primer 5'-CTCTGCAACAGAGTCGT CAAATGGAGG-3', lower primer 5'-CGGGGGAGGGGCAAACAACAGATGGCTGGC-3'; product size 324 bp. We assessed the integrity of the purified DNA by simultaneously running a PCR reaction with primers specific for the beta -globin gene (upper primer 5'-CCAATCTGCTCACACAGGATAGAGAGGGCAGG-3', lower primer 5'-CCTTGAGGCTGTCCAAGTGATTCAGGCCATCG-3'; product size 494 bp). We also used a PCR technique to genotype offspring produced from the cross of tetO-muPA transgenic animals with CCSP-rtTA transgenic animals. The primer pair employed to identify the tetO-muPA transgene was identical to that described above. The primer pair sequences we employed to identify the presence of the CCSP-rtTA construct are as follows: upper primer 5'-ACTGCCCATTGCCCAAACAC-3', lower primer 5'-AAAATCTTGCCAGCTTTCCCC-3'; product size 525 bp. The PCR conditions were the same for each primer pair as follows: 90°C for 1 min followed by annealing at 62°C for 2 min followed by elongation at 72°C for 2 min. This temperature sequence was repeated for 30 cycles.

In vitro cotransfection experiments. Before being microinjected in fertilized eggs, the functional integrity of the tetO-muPA expression cassette was assessed by cotransfection of an A549 cell monolayer with the tetracycline-off plasmid [which contains the Tet-transactivator gene driven by the CMV promoter] in the presence and absence of doxycycline. A549 cells were grown in DMEM with 10% FCS serum to 80% confluence in a six-well plate. Following the manufacturer's guidelines, we used the Fugene reagent (Roche Molecular Biochemicals, Indianapolis, IN) to transfect the A549 monolayer with either the plasmid containing the tetO-muPA expression cassette alone (1.0 µg) or a 1.0 µg-1.0 µg mixture of this plasmid with the tetracycline-off plasmid. The cells were incubated with the Fugene-plasmid mixture for 24 h, after which the media was changed from DMEM with 10% FCS to 1.5 ml of DMEM without FCS containing 0, 1.0, or 4.0 µg/ml doxycycline. After another 24 h, conditioned media were collected and assayed for uPA activity (as detailed below).

Southern hybridization. Genomic DNA was digested with the Hind III restriction enzyme, separated on a 1.0% agarose gel, and blotted on a Zetabind membrane (Cuno, Meriden, CT). Next, following the manufacturer's protocol, we probed the membrane with a 1.0-kb DNA fragment generated from a Pst I restriction enzyme digest of the muPA cDNA that had been labeled with the Alk-Phos Direct labeling kit (AmershamPharmacia Biotech, Piscataway, NJ). After hybridization, the membrane was developed with the chemiluminescent detection reagent for 5 min and exposed to Kodak autoradiography film for 45 min.

Doxycycline treatment. Mice were exposed to doxycycline (Sigma Chemical, St. Louis, MO) by adding the antibiotic to their drinking water at a concentration of 0.5 mg/ml in 1% ethanol. The doxycycline solution was protected from light and was changed every other day during the course of each study.

PA activity assay. Double-transgenic (tetO-muPA:rtTA-CCSP) and littermate control mice were exposed to doxycycline for 1 or 2 wk. An additional group of double-transgenic (tetO-muPA:rtTA-CCSP) mice received no doxycycline to evaluate for non-doxycycline-induced gene expression. BAL fluid was collected by instilling 1.0 ml of sterile PBS intratracheally, aspirating the fluid after 10 s, and then centrifuging the samples for 10 min at 4,000 g. The supernatant was collected and then stored at -70°C until further analysis. PA activity in the BAL fluid samples was measured by an indirect colorimetric assay as described previously (5). Briefly, at the time of assay, the BAL fluid samples were mixed with or without human Glu-plasminogen (in a 96-well plate). After the plasminogen-sample mixtures were incubated for 30 min at 37°C, a color reagent consisting of 2.2 mM 5,5'-dithiobis-(2-nitrobenzoic acid), 0.2 M thiobenzyl benzyloxycarbonyl-L-lysinate, 1% Triton X-100, and PBS was added to each sample. The resulting color change was measured immediately, and the amount of PA activity in each sample was calculated from a standard curve generated from human uPA samples with known amounts of activity.

Casein zymography. Casein zymography was performed on BAL samples according to the method of Hattori et al. (10) with modifications as described previously. Samples were separated by SDS-PAGE on 10% gels containing alpha -casein (7 mg/ml) and human Glu-plasminogen (20 µg/ml). After electrophoretic separation, the gels were washed in 1% Tween 80 for 1 h at 37°C and then incubated in PBS containing 0.1% Tween 80 overnight at room temperature. Finally, the gels were stained with Coomassie blue dye and then destained in a solution of 10% acetic acid and 50% methanol. The molecular weights of the lytic bands were calculated by comparison to human uPA and human tissue-type plasminogen activator standards.

Imunohistochemistry. Double-transgenic (tetO-muPA:rtTA-CCSP) and littermate control mice were exposed to doxycycline for 1 wk. Lungs were inflation-fixed with 10% neutral-buffered formalin, and the trachea was ligated. Heart and lungs were removed en bloc, further fixed in 10% neutral-buffered formalin overnight, and paraffin embedded. Sections (4 µm) were deparaffinized and stained immunohistochemically with a polyclonal anti-murine uPA antibody (American Diagnostica, Greenwich, CT) diluted 1:25. Bound antibody was detected using a biotinylated goat anti-rabbit immunoglobulin and the Vectastain ABC kit (Vector Laboratories). Diaminobenzidine was used as a peroxidase substrate (Sigma Chemical), and the sections were counterstained with hematoxylin.

Measurement of fibrinolysis within lungs. Fibrinolysis in murine lungs was measured by a method described previously with modifications (11). After 1 wk of doxycycline exposure, mice were killed, and 800 µl of DMEM containing fibrinogen (1.5 mg/ml), fluorescein-labeled fibrinogen (0.1 mg/ml), plasminogen (60 µg/ml), thrombin (0.2 U/ml), and Texas red-conjugated BSA (0.25 mg/ml) were instilled intratracheally. The Texas red-conjugated BSA was included in the fibrinogen-plasminogen-thrombin mixture to allow for correction of dilutional changes that may have occurred with tissue processing. After instillation of the above mixture, we tied off the trachea of each animal, removed the lungs en bloc, and incubated them in a 50-ml tube at 37°C for 90 min. The lungs were then finely minced and centrifuged at 10,000 g for 10 min. Each supernatant (20 µl) was added to 80 µl of DMEM in a 96-well plate, and the fluorescence intensity was measured for fluorescein (excitation wavelength 485 nm, emission wavelength 515 nm) and Texas red (excitation wavelength 590 nm, emission wavelength 620 nm). To correct for the differences in dilution, the fluorescein fluorescence was divided by the Texas red fluorescence. During each experiment, we measured both 0 and 100% lysis. Zero percent lysis was determined by placing 500 µl of the instilled fibrinogen-plasminogen-thrombin mixture in a 1.5-ml tube and allowing the mixture to clot at 37°C. One hundred percent lysis was determined by placing the fibrinogen-plasminogen mixture in a 1.5-ml tube without thrombin such that no clotting took place. After centrifugation, we measured the fluorescein isothiocyanate (FITC)-to-Texas red fluorescence ratios of these control samples as described above. The percentage of fibrin degraded in the lung was calculated assuming a linear relationship between the values of the 0 and 100% lysis controls.

Survival curve. Six- to eight-week-old double-transgenic mice (tetO-muPA:CCSP-rtTA) and nontransgenic littermate controls were treated with doxycycline. After 1 wk of doxycycline exposure, groups of transgenic and control mice were treated with either an intratracheal instillation of bleomycin (0.0025 U/g body wt in 50 µl PBS) or PBS (50 µl) alone. After the intratracheal instillation of bleomycin or PBS, all mice were continued on doxycyline throughout the subsequent study period. Thereafter, the mice were observed daily, and any appearing moribund were killed humanely. At the time of death, BAL fluid was collected by the same technique described above, and these samples were subjected to casein zymography. Survival between groups was analyzed using a Kaplan-Meier curve.

Hydroxyproline assay. Six- to eight-week-old tetO-muPA:CCSP-rtTA mice and nontransgenic littermate controls were treated with doxycycline. Next, after 1 wk of doxycycline exposure, groups of transgenic and control mice were treated with either an intratracheal instillation of bleomycin (0.00225 U/g body wt in 50 µl PBS) or PBS (50 µl). Thereafter, the mice were continued on doxycyline until the time of death on day 21. Total lung hydroxyproline content was measured as previously described. Briefly, at the time of death, each lobe of the lung was excised individually with care taken to remove the extrinsic bronchi. The lungs were then homogenized in 1.0 ml PBS. To this resulting homogenate, 1.0 ml of 12 N HCl was added, and the samples were hydrolyzed at 120°C for 24 h. In a 96-well plate, 5 µl of each sample were combined with 5 µl citrate/acetate buffer (238 mM citric acid, 1.2% glacial acetic acid, 532 mM sodium acetate, and 85 mM sodium hydroxide). Thereafter, 100 µl of chloramine T solution (0.282 g chloramine T added to 16 ml citrate/acetate buffer, 2.0 ml of n-propanol, and 2.0 ml milli-Q water) were added, and the plate was incubated for 30 min at room temperature. After this incubation, 100 µl Ehrlich's reagent (2.5 g p-dimethylaminobenzaldehyde added to 9.3 ml of n-propanol and 3.9 ml of 70% perchloric acid) were added, and the samples were incubated at 65°C for 30 min. The absorbance of each sample was then measured at 550 nm. Standard curves for the experiment were generated using known concentrations of the reagent hydroxyproline (Sigma Chemical).

Histological analysis of fibrosis. Lungs were inflation-fixed with 10% neutral-buffered formalin, removed en bloc, further fixed in 10% neutral-buffered formalin overnight, and then paraffin embedded. Sections (8 µm) were stained with hematoxylin and eosin.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Generation of tetO-muPA transgenic line. After the muPA cDNA was cloned into the tetO expression cassette (Fig. 1), the functional integrity of this construct was assessed in vitro. For these experiments, we used the tet-off plasmid that encodes the tetracycline transactivator molecule. This transcription factor activates the tetO promoter in the absence of doxycycline, and the addition of this antibiotic shuts off expression. Cotransfecting A549 cells with both the tetO-muPA plasmid and the tet-off plasmid in the absence of doxycycline resulted in a marked increase in conditioned media PA activity (Fig. 2). Endogenous uPA production from the A549 cell monolayer was below the level of detection under the conditions of the activity assay. Transfecting A549 cells with the tetO-muPA plasmid alone resulted in no PA production. These results demonstrate that the tetO promoter required the tetracycline transactivator molecule to drive uPA expression. To demonstrate that this expression system was responsive to doxycycline, we added increasing amounts of this antibiotic to the cotransfected monolayers. As expected, because we had combined the tetO-muPA plasmid with the tet-off plasmid, the addition of doxycycline to the culture media led to a dose-dependent reduction in PA production. Upon confirmation of its functional integrity with this data, the tetO-muPA expression cassette was cleaved from its plasmid backbone and injected in 200 (C57BL/6 × SJL) F2 mouse eggs. These eggs were subsequently implanted into pseudopregnant mothers, and 118 pups were born after the 3-wk gestational period. Tail biopsies from the pups were used for genotyping, and, of these 118 mice, 11 were found to possess the tetO-muPA construct. To expand the colony size, these 11 transgenic founder mice were bred with C57Bl/6 partners. Six of these breeding pairs successfully generated litters. The F1 transgenic offspring from these successful crossings were subsequently bred with CCSP-rtTA transgenic animals to generate double-transgenic experimental mice.


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Fig. 1.   Constructs used to generate lung-specific inducible urokinase-type plasminogen activator (uPA) transgene. Murine uPA (muPA) was cloned into an expression cassette containing 7 copies of the tetracycline operator, a cytomegalovirus (CMV) minimal promoter, and the Simian virus 40 (SV40) polyadenylation sequence. This expression cassette was cleaved from its plasmid backbone using the Asc I restriction enzyme and microinjected in newly fertilized SJL × C57Bl/6 F2 mouse eggs. Resulting tetracycline operator (tetO)-muPA transgenic animals were bred with Clara cell-specific protine (CCSP)-reverse tetracycline transactivator (rtTA) animals to produce double-transgenic offspring. The CCSP-rtTA mice contain a 2.3-kb rat CCSP promoter, the 1.0-kb coding sequence for the rtTA, and a 2.0-kb fragment from the human growth hormone gene containing introns and a polyadenylation signal.



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Fig. 2.   In vitro assessment of the functional integrity of the tetO-muPA expression cassette. A549 monolayers were transfected with the tetO-muPA plasmid alone or with both the tetO-muPA plasmid and the tet-off plasmid. Doxycycline was added to the media of a subset of double-transfected monolayers at a concentration of 1.0 and 4.0 µg/ml. Conditioned media (24 h) were analyzed for uPA activity using a colorimetric assay. uPA activity is measured as mean milliplough units/µl ± SE, n = 4 experiments.

Transgene copy number and insertion sites. To assess both the copy number of tetO-muPA expression cassettes inserted and to assess the number of insertion sites, we performed Southern hybridization on Hind III-digested genomic DNA from the six breeding founders and several of their transgenic offspring. A 2.5-kb fragment, the size of the tetO-muPA expression cassette, was expected if two or more copies of the construct integrated. We assessed the presence of multiple insertion sites by comparing the founder's hybridization pattern with the hybridization pattern of several of their transgenic offspring. Each of the six successfully breeding founder mice possessed two or more copies of the tetO-muPA construct (Fig. 3). Compared with the 5-copy and 10-copy standards, the number of integrated cassettes ranged from ~2 (in line 848) to ~20 (in lines 756 and 823). Based on the identical hybridization patterns of the founders and their transgenic offspring (as demonstrated in Fig. 3 for lines 756, 825, and 829 but also true for the other founders), each transgenic line was determined to have a single insertion site. Importantly, the presence of a single insertion site meant we could assume each transgenic offspring from a particular founder to be genetically identical with regard to the tetO-muPA construct.


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Fig. 3.   Southern hybridization. Genomic DNA purified from a C57Bl/6 control animal and from tetO-muPA positive offspring of the 6 founder lines (756, 829, 858, 848, 825, and 823) was digested with the Hind III restriction enzyme and then separated on a 1% agarose gel. Transferred DNA was probed with a 1.0-kb probe generated from a Pst I digest of the muPA cDNA and alkaline phosphatase labeled. To assess transgene copy number, a known amount of tetO-muPA plasmid DNA (10× = 10 copies, 5× = 5 copies) was digested with Hind III and run on the same 1% agarose gel. Hind III cuts at a single site in the tetO-muPA plasmid and produces a 5.5-kb fragment. The tetO-muPA expression cassette measures 2.5 kb and has a single internal Hind III site. Thus, when the expression cassette has integrated at a frequency of greater than one copy, a 2.5-kb fragment will result after Hind III digestion.

BAL fluid PA activity from uninjured mice. After crossing founder lines 756, 823, 825, and 829 with CCSP-rtTA transgenic partners, we assessed the ability of the double-transgenic offspring to upregulate intrapulmonary PA activity in response to doxycycline. Mice possessing no transgenic construct and mice possessing only the tetO-muPA expression cassette served as controls. To determine whether transgenic uPA production occurred in the absence of antibiotic, we measured BAL fluid uPA activity in untreated double-transgenic mice. Without doxycycline exposure, the double-transgenic animals produced levels of BAL fluid PA activity similar to that of the nontransgenic controls. The amount of PA activity in the BAL fluid of doxycycline-treated mice possessing only the tetO-muPA expression cassette was also not significantly different from nontransgenic littermates. The administration of doxycycline for 7 and 14 days to the double-transgenic mice originating from founders 756 and 823 resulted in a marked increase in BAL fluid PA activity (Fig. 4). Day 3 expression of uPA was also increased significantly above baseline but had not yet peaked (data not shown). There was no further increase in PA activity between day 7 and day 14, suggesting the establishment of an equilibrium between uPA production, distribution, and metabolism sometime within the 1st wk after the initiation of doxycycline. Although the data are not shown, we observed similar results in the animals originating from the other two founder lines (825 and 829). Specifically, there was no significant difference in the magnitude of BAL fluid PA production at days 7 and 14 between any of the four different founder lines investigated. And thus, even though the number of integrated tetO-muPA constructs appears to be significantly greater in 756 and 823 lines compared with animals derived from the 825 and 829 founders, this does not appear to directly correlate with BAL fluid PA activity. In addition to measuring BAL fluid PA activity, we also performed casein zymography on these same BAL samples to ensure that the increased PA activity was indeed attributable to increased uPA production. After 7 and 14 days of doxycycline exposure, the BAL fluid from double-transgenic mice (founder line 756) generated appreciable lytic bands at 45 kDa (Fig. 5). The molecular weight of this band corresponds to that of muPA. No other lytic bands were present to account for the increased BAL fluid PA activity. In accordance with the PA activity measurements, the BAL fluid from the non-doxycycline-treated double-transgenic animals and from the doxycycline-treated controls generated a low amount of lysis at 45 kDa from endogenously produced uPA. The zymography results from the other three transgenic lines were similar to the results displayed for line 756 (data not shown).


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Fig. 4.   uPA activity of bronchoalveolar lavage (BAL) fluid. TetO-muPA:CCSP-rtTA double-transgenic, single-transgenic (tetO-muPA only), and nontransgenic littermate control mice from founder lines 756 (A) and 823 (B) were exposed to doxycycline for 7 and 14 days. BAL fluid was obtained and analyzed for uPA activity using an indirect colorimetric assay. In addition, BAL fluid from tetO-muPA:CCSP-rtTA double-transgenic mice not exposed to doxycycline was analyzed by the same technique. + and -, presence or absence of each transgenic construct. uPA activity is expressed in mean international units ± SE, n = 3 or 4.



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Fig. 5.   Casein zymography on BAL fluid from uninjured animals. TetO-muPA:CCSP-rtTA double-transgenic and nontransgenic littermate control mice from founder line 756 were exposed to doxycycline for 7 and 14 days. BAL fluid was obtained at these time points and was subjected to 10% SDS-PAGE containing alpha -casein. In addition, BAL fluid from tetO-muPA:CCSP-rtTA double-transgenic mice not exposed to doxycycline was subjected to this same technique. An huPA standard was run simultaneously as a positive control and as a size reference. +/+, Double-transgenic animals; -/-, nontransgenic littermate controls. huPA, human uPA.

Immunohistochemistry. The rat CCSP promoter has been reported to regulate gene transcription not only in the epithelial cells of small airways but also in alveolar epithelium. Because bleomycin induces fibrosis through injury to the alveolus, we wanted to insure that tetO-muPA:CCSP-rtTA mice expressed doxycycline-dependent uPA in this location. Using immunohistochemistry with an anti-rodent uPA antibody, double-transgenic mice treated with doxycycline for 7 days were found to have increased uPA production in both alveolar and small airway epithelial cells compared with identically treated littermate controls (Fig. 6). At this time point, the doxycycline-induced increase in intrapulmonary uPA expression did not produce any apparent alterations in lung architecture or cellular infiltration.


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Fig. 6.   Immunohistochemistry. A tetO-muPA:CCSP-rtTA double-transgenic mouse and a littermate control mouse from line 756 were exposed to doxycycline for 1 wk. Paraffin-embedded sections were stained with an anti-rodent uPA antibody or control rabbit serum. A: tetO-muPA:CCSP-rtTA mouse stained with anti-uPA antibody; B: nontransgenic animal stained with anti-uPA antibody; C: tetO-muPA:CCSP-rtTA stained with rabbit nonimmune serum; D: nontransgenic control animal stained with rabbit nonimmune serum.

Alveolar fibrinolysis. To determine if the increased alveolar uPA levels in the doxycycline-exposed double-transgenic animals could function in a biologically significant manner, we measured the ability of mouse lungs to degrade intra-alveolar fibrin matrixes. Within 5 min after instilling the FITC-labeled fibrinogen and thrombin mixture in the lungs of both transgenic and nontransgenic mice, the fluorescein-associated fluorescence became insoluble as the labeled fibrinogen was incorporated in a fibrin matrix (data not shown). After 90 min, the mean percentage of fibrin degradation in the double-transgenic mice from line 756 was 86 ± 12% and from line 823 was 64 ± 13% (Fig. 7). This percentage of fibrinolysis was increased markedly in the treatment mice from the two founder lines compared with control animals (37 ± 0.7 and 23 ± 5%, respectively P < 0.05).


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Fig. 7.   Ex vivo intrapulmonary fibrinolysis. TetO-muPA:CCSP-rtTA double-transgenic and nontransgenic littermate control mice from founder lines 756 (A) and 823 (B) were exposed to doxycycline for 7 days. Fluorescein isothiocyanate (FITC)-labeled fibrin matrixes were formed in the lungs of mice by intratracheal instillation of a mixture FITC-labeled fibrinogen, thrombin, and plasminogen. The amount of clot lysis was estimated by measuring the percentage of soluble fluorescent label at 90 min. Data are expressed as mean percent lysis ± SE; n = 5 mice/group.

BAL fluid PA activity from bleomycin-injured mice. To assess the influence of bleomycin injury on doxycycline-induced intrapulmonary uPA production, we performed casein zymography on BAL samples taken from double-transgenic and control mice at the time of death. The BAL fluid from doxycycline-exposed tetO-muPA:CCSP-rtTA mice contained increased levels of uPA despite bleomycin treatment (Fig. 8). The amount of uPA in the bleomycin-treated double-transgenic mice appeared slightly diminished compared with PBS-treated double-transgenic control mice. This slight decrease in uPA activity after injury is presumably secondary to bleomycin-induced PAI-1 expression and the formation of PAI-1-uPA complexes. Nontransgenic control animals treated with bleomycin demonstrated no casein lysis at 45 kDa. Again, the small amount of endogenously produced uPA seen in uninjured nontransgenic animals is presumably inhibited by the known increase in PAI-1 expression that occurs in the setting of fibrotic injuries.


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Fig. 8.   Casein zymography on BAL fluid from injured animals. After bleomycin injury, BAL fluid was collected at the time of death (when mice appeared moribund or at day 28) from doxycycline-treated tetO-muPA:CCSP-rtTA double-transgenic and nontransgenic littermate control mice. The BAL fluid was subjected to 10% SDS-PAGE containing alpha -casein. As a control, BAL fluid from PBS-treated tetO-muPA:CCSP-rtTA mice exposed to doxycycline for 28 days was subjected to this same technique.

Survival. To determine if a doxycycline-induced increase in uPA production protects animals from a fibrogenic pulmonary insult, we assessed the survival after intratracheal bleomycin administration of doxycycline-treated tetO-muPA:CCSP-rtTA mice compared with nontransgenic littermate controls. We observed mortality in the control mice at day 12, 4 days earlier than the first recorded death in the double-transgenic cohort of mice (Fig. 9). Ultimately, the mortality rate in the control group by day 28 was 50% compared with a mortality rate of <10% in the tetO-muPA:CCSP-rtTA group (P <=  0.05).


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Fig. 9.   Kaplan-Meier survival curve after bleomycin treatment. TetOET-muPA:CCSP-rtTA double-transgenic and nontransgenic littermate controls were exposed to doxycycline and after 7 days treated with intratracheal bleomycin (0.0025 U/g) or PBS. Mice were killed humanely at the time they became moribund. , PBS-treated tetO-muPA:CCSP-rtTA (n = 5) and nontransgenic mice (n = 5); black-triangle, bleomycin-treated tetO-muPA:CCSP-rtTA mice (n = 11); black-diamond , bleomycin-treated nontransgenic mice (n = 10).

Assessment of lung fibrosis. To investigate whether enhanced uPA expression also limited pulmonary fibrosis after bleomycin injury, we measured lung hydroxyproline content. For this experiment, we reduced the dose of bleomycin to increase the likelihood that control mice would survive to the targeted 3-wk time point. With a dose of 0.00225 U bleomycin/g mouse body wt, we observed no mortality in either the tetO-muPA:CCSP-rtTA or the control groups. The baseline lung collagen content for the double-transgenic and control mice was established by treating a subset of animals from each group with intratracheal PBS. As shown in Fig. 10, there was no difference between the groups in the hydroxyproline quantity per gram of body weight after PBS instillation (P = 0.85). On the other hand, treatment with intratracheal bleomycin resulted in a 75% increase (P < 0.0001) in lung collagen content per gram of body weight in the control group. Bleomycin treatment also resulted in an increased accumulation of lung collagen in the tetO-muPA:CCSP-rtTA mice, but the increase in hydroxyproline was 49% less in this group compared with control animals (P = 0.005). On histological evaluation, the appearance of the lung tissue from PBS-treated double-transgenic and control mice was normal (data not shown). The histological analysis of the lungs from bleomycin-treated tetO-muPA:CCSP-rtTA and control mice confirmed the differences in hydroxyproline measurement. Although areas of injury and collagen accumulation occurred in the double-transgenic animals, they were much less prominent than in the control group (Fig. 11). The tetO-muPA:CCSP-rtTA mice developed small patchy areas of injury in response to bleomycin in contrast to control mice, which developed large regions of confluent inflammation and fibrosis.


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Fig. 10.   Lung hydroxyproline content. TetO-muPA:CCSP-rtTA double-transgenic and nontransgenic littermate control mice were treated with doxycycline for 7 days and then were given either intratracheal bleomycin or PBS. The mice continued to receive doxycyline through day 21 at which time lung collagen content was assessed by measuring total hydroxyproline. +, Double-transgenic uPA overexpressing animals; -, nontransgenic littermate controls. Results are reported as µg/g body wt ± SE. ¶P = 0.005 for comparison of bleomycin-treated +/+ group with bleomycin-treated control group.



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Fig. 11.   Lung histology after bleomycin treatment. TetO-muPA:CCSP-rtTA double-transgenic and nontransgenic littermate control mice were treated with doxycycline for 7 days followed by intratracheal bleomycin. The mice then continued on doxycyline through day 21. Formalin-fixed, paraffin-embedded sections were stained with hematoxylin and eosin and photographed at ×10 magnification. A: tetO-muPA:CCSP-rtTA. B: littermate control.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Our report details the successful generation of a mouse model capable of producing intrapulmonary uPA in response to exogenously administrated doxycycline. Importantly, we also demonstrate the capability of doxycycline-induced uPA expression to accelerate intrapulmonary fibrin degradation and to limit the mortality and scarring associated with bleomycin-induced lung injury. This reduction in mortality and lung collagen accumulation mirrors the benefits afforded by PAI-1 deficiency after intratracheal bleomycin instillation.

We have shown that intrapulmonary uPA production can be markedly upregulated with the administration of doxycycline to double-transgenic mice. In the absence of doxycycline, these same mice produce uPA in amounts that are not different from nontransgenic littermate controls. The activity of uPA in the lungs reaches a maximum level sometime between days 3 and 7 after the initiation of the antibiotic and remains persistently elevated through the 2-wk time point. Our results are quite consistent with the transgenic expression of interluekin (IL)-11 using this same lung-specific tetracycline regulatory system (23). Ray et al. (23) demonstrated that CCSP-rtTA:tetO-IL-11 double-transgenic mice produced IL-11 in their BAL fluid after doxycycline exposure, with levels reaching maximum concentration by day 6. Thereafter the levels of IL-11 remained persistently elevated through day 20.

The overexpression of PAI-1 has been conclusively linked to the pathogenesis of pulmonary fibrosis. Current evidence suggests that PAI-1 exacerbates fibrosis by interfering with the activation of plasminogen to plasmin (28). However, the possibility exists that PAI-1 mitigates its effects on lung scarring through alternate activities such as fibroblast activation or migration. If PAI-1 is influencing fibrosis by blocking the activity of the PAs and preventing the conversion of plasminogen to plasmin, then this effect should be surmountable by the overexpression of a PA. Our results support this hypothesis. The marked transgenic overproduction of uPA in the lungs of mice does indeed provide protection from a fibrotic insult, as demonstrated by a significant improvement in survival and a reduction in lung collagen after intratracheal bleomycin instillation.

The mechanism by which enhanced plasminogen activation to plasmin limits fibrosis remains to be defined. Plasmin has many activities that could hypothetically reduce lung scarring. The activation of matrix metalloproteinases, the activation of growth factors, and the removal of fibrin (although not essential) could all lead to a reduction in fibrosis (2, 12, 18-21, 24, 25). To better understand which of these mechanisms may be playing a role, it would be helpful to define when in the time course of pathogenesis the PA system is exerting its influence. For example, the reduction of fibrosis after collagen has already accumulated in the lung would suggest that uPA overexpression is working through enhanced removal of extracellular matrix proteins. On the other hand, if increased plasminogen activation is found to protect against fibrosis early in the pathogenesis before collagen has been deposited, then this system must influence fibrosis through a pathway independent of collagen removal. The activation of a growth factor such as hepatocyte growth factor or the release of keratinocyte growth factor from the extracellular matrix with the subsequent restoration of the alveolar epithelium might also limit scarring. Exogenous administration of these growth factors has been found to limit lung fibrosis in rodent models of bleomycin-induced lung injury (3, 27, 31, 32). Our data suggest that the tetO-muPA:CCSP-rtTA double-transgenic mouse model, because it allows inducible uPA production, will be a powerful tool in our efforts to define this time course.

In summary, we have generated a lung-specific, inducible uPA-expressing transgenic mouse line that accelerates lung fibrin clearance. This transgenic animal is protected from the scarring and mortality associated with bleomycin-induced lung injury. Because uPA expression can be controlled, this transgenic model will be a powerful tool in our planned future studies to elucidate the mechanism(s) by which the PA system reduces pulmonary fibrosis. A better understanding of this mechanism will hopefully allow us to define new therapeutic targets.


    ACKNOWLEDGEMENTS

We thank Dr. Thomas Saunders and the Transgenic Core of the University of Michigan for advice in help generating the transgenic mouse lines.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grants K08-HL-04434 and P50-HL-56402.

Address for reprint requests and other correspondence: T. H. Sisson, 1150 W. Medical Center Dr., 6301 MSRB3, Ann Arbor, MI 48109-0642 (E-mail: tsisson{at}umich.edu).

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.

May 24, 2002;10.1152/ajplung.00049.2002

Received 4 February 2002; accepted in final form 28 July 2002.


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Am J Physiol Lung Cell Mol Physiol 283(5):L1023-L1032
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