Sustained-release prostacyclin analog ONO-1301 ameliorates tubulointerstitial alterations in a mouse obstructive nephropathy model

Tubulointerstitial injuries are crucial histological alterations that predict the deterioration of renal function in chronic kidney disease. ONO-1301, a novel sustained-release prostacyclin analog, accompanied by thromboxane synthase activity, exerts therapeutic effects on experimental pulmonary hypertension, lung fibrosis, cardiomyopathy, and myocardial ischemia, partly associated with the induction of hepatocyte growth factor (HGF). In the present study, we examined the therapeutic efficacies of ONO-1301 on tubulointerstitial alterations induced by unilateral ureteral obstruction (UUO). After inducing unilateral ureteral obstruction in C57/BL6J mice, a single injection of sustained-release ONO-1301 polymerized with poly (D,L-lactic-co-glycolic acid) sustained-release ONO-1301 (SR-ONO) significantly suppressed interstitial fibrosis, accumulation of types I and III collagen, increase in the number of interstitial fibroblast-specific protein-1 (FSP-1)(+) cells, and interstitial infiltration of monocytes/macrophages (F4/80(+)) in the obstructed kidneys (OBK; day 7). Treatment with SR-ONO significantly suppressed the increase of the renal levels of profibrotic factor TGF-β and phosphorylation of Smad2/3, and elevated the renal levels of HGF in the OBK. In cultured mouse proximal tubular epithelial cells (mProx24), ONO-1301 significantly ameliorated the expression of fibroblast-specific protein-1 and α-smooth muscle actin as well as phosphorylation of Smad3 and increased the expression of zonula occludens-1 and E-cadherin in the presence of TGF-β1 as detected by immunoblot and immunocytochemistry, partly dependent on PGI(2) receptor-mediated signaling. Administration of rabbit anti-HGF antibodies, but not the control IgG, partly reversed the suppressive effects of SR-ONO on tubulointerstitial injuries in the OBK. Taken together, our findings suggest the potential therapeutic efficacies of ONO-1301 in suppressing tubulointerstitial alterations partly mediated via inducing HGF, an antifibrotic factor counteracting TGF-β.     

The absence of reactive oxygen species production protects mice against bleomycin-induced pulmonary fibrosis

Background

Reactive oxygen species and tissue remodeling regulators, such as metalloproteinases (MMPs) and their inhibitors (TIMPs), are thought to be involved in the development of pulmonary fibrosis. We investigated these factors in the fibrotic response to bleomycin of p47^phox -/- (KO) mice, deficient for ROS production through the NADPH-oxidase pathway.

Methods

Mice are administered by intranasal instillation of 0.1 mg bleomycin. Either 24 h or 14 days after, mice were anesthetized and underwent either bronchoalveolar lavage (BAL) or lung removal.

Results

BAL cells from bleomycin treated WT mice showed enhanced ROS production after PMA stimulation, whereas no change was observed with BAL cells from p47^phox -/- mice. At day 1, the bleomycin-induced acute inflammatory response (increased neutrophil count and MMP-9 activity in the BAL fluid) was strikingly greater in KO than wild-type (WT) mice, while IL-6 levels increased significantly more in the latter. Hydroxyproline assays in the lung tissue 14 days after bleomycin administration revealed the absence of collagen deposition in the lungs of the KO mice, which had significantly lower hydroxyproline levels than the WT mice. The MMP-9/TIMP-1 ratio did not change at day 1 after bleomycin administration in WT mice, but increased significantly in the KO mice. By day 14, the ratio fell significantly from baseline in both strains, but more in the WT than KO strains.

Conclusions

These results suggest that NADPH-oxidase-derived ROS are essential to the development of pulmonary fibrosis. The absence of collagen deposition in KO mice seems to be associated with an elevated MMP-9/TIMP-1 ratio in the lungs. This finding highlights the importance of metalloproteinases and protease/anti-protease imbalances in pulmonary fibrosis.     

Plasmin overcomes resistance to prostaglandin E2 in fibrotic lung fibroblasts by reorganizing protein kinase A signaling

Collagen deposition by fibroblasts contributes to scarring in fibrotic diseases. Activation of protein kinase A (PKA) by cAMP represents a pivotal brake on fibroblast activation, and the lipid mediator prostaglandin E(2) (PGE(2)) exerts its well known anti-fibrotic actions through cAMP signaling. However, fibrotic fibroblasts from the lungs of patients with idiopathic pulmonary fibrosis, or of mice with bleomycin-induced fibrosis, are resistant to the normal collagen-inhibiting action of PGE(2). In this study, we demonstrate that plasminogen activation to plasmin restores PGE(2) sensitivity in fibrotic lung fibroblasts from human and mouse. This involves amplified PKA signaling resulting from the promotion of new interactions between AKAP9 and PKA regulatory subunit II in the perinuclear region as well as from the inhibition of protein phosphatase 2A. This is the first report to show that an extracellular mediator can dramatically reorganize and amplify the intracellular PKA-A-kinase anchoring protein signaling network and suggests a new strategy to control collagen deposition by fibrotic fibroblasts.     

TGFβ signaling in lung epithelium regulates bleomycin-induced alveolar injury and fibroblast recruitment

The response of alveolar epithelial cells (AECs) to lung injury plays a central role in the pathogenesis of pulmonary fibrosis, but the mechanisms by which AECs regulate fibrotic processes are not well defined. We aimed to elucidate how transforming growth factor-β (TGFβ) signaling in lung epithelium impacts lung fibrosis in the intratracheal bleomycin model. Mice with selective deficiency of TGFβ receptor 2 (TGFβR2) in lung epithelium were generated and crossed to cell fate reporter mice that express β-galactosidase (β-gal) in cells of lung epithelial lineage. Mice were given intratracheal bleomycin (0.08 U), and the following parameters were assessed: AEC death by terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling assay, inflammation by total and differential cell counts from bronchoalveolar lavage, fibrosis by scoring of trichrome-stained lung sections, and total lung collagen content. Mice with lung epithelial deficiency of TGFβR2 had improved AEC survival, despite greater lung inflammation, after bleomycin administration. At 3 wk after bleomycin administration, mice with epithelial TGFβR2 deficiency showed a significantly attenuated fibrotic response in the lungs, as determined by semiquantitatve scoring and total collagen content. The reduction in lung fibrosis in these mice was associated with a marked decrease in the lung fibroblast population, both total lung fibroblasts and epithelial-to-mesenchymal transition-derived (S100A4(+)/β-gal(+)) fibroblasts. Attenuation of TGFβ signaling in lung epithelium provides protection from bleomycin-induced fibrosis, indicating a critical role for the epithelium in transducing the profibrotic effects of this cytokine.     

CC-chemokine receptor 2 required for bleomycin-induced pulmonary fibrosis

MCP-1, which signals via the CC chemokine receptor 2 (CCR2), is induced in lung fibrosis that is accompanied by mononuclear cell recruitment and activation of lung fibroblasts. To evaluate the role of CCR2 in lung fibrosis, CCR2 knockout (ko) mice were used in a model of bleomycin-induced lung fibrosis. Wild type (wt) and ko mice were injected endotracheally with bleomycin to induce lung injury and fibrosis, and then analyzed for degree of lung fibrosis and cytokine expression. The results showed significantly reduced fibrosis in ko mice as evidenced by decreased lung type I collagen gene expression and hydroxyproline content relative to those in wt mice. Lung TNF-alpha and TGF-beta1 expression was significantly lower in ko vs. wt mice, while MCP-1 expression was unaffected. Interestingly, lung alpha-smooth muscle actin (alpha-SMA) expression, a marker for myofibroblast differentiation, was also decreased in ko mice, which was confirmed by analysis of isolated lung fibroblasts. Fibroblasts from ko mice exhibited decreased responsiveness to TGF-beta1 induced alpha-SMA expression, which was associated with reduced expression of TGF-beta receptor II (TbetaRII) and Smad3. These findings suggest that CCR2 signaling plays a key role in bleomycin-induced pulmonary fibrosis by regulating fibrogenic cytokine expression and fibroblast responsiveness to TGF-beta.     

Dual roles of IL-4 in lung injury and fibrosis

Increased lung IL-4 expression in pulmonary fibrosis suggests a potential pathogenetic role for this cytokine. To dissect this role, bleomycin-induced pulmonary inflammation and fibrosis were analyzed and compared in wild type (IL-4(+/+)) vs IL-4-deficient (IL-4(-/-)) mice. Lethal pulmonary injury after bleomycin treatment was higher in IL-4(-/-) vs IL-4(+/+) mice. By administration of anti-CD3 Abs, we demonstrated that this early response was linked to the marked T lymphocyte lung infiltration and to the overproduction of the proinflammatory mediators such as TNF-alpha, IFN-gamma, and NO in IL-4(-/-) mice. In contrast to this early anti-inflammatory/immunosuppressive role, during later stages of fibrosis, IL-4 played a profibrotic role since IL-4(-/-) mice developed significantly less pulmonary fibrosis relative to IL-4(+/+) mice. However, IL-4 failed to directly stimulate proliferation, alpha-smooth muscle actin, and type I collagen expression in lung fibroblasts isolated from the wild-type mice. Upon appropriate stimulation with other known fibrogenic cytokines, fibroblasts from IL-4(-/-) mice were relatively deficient in the studied parameters in comparison to fibroblasts isolated from IL-4(+/+) mice. Taken together, these data suggest dual effects of IL-4 in this model of lung fibrosis: 1) limiting early recruitment of T lymphocytes, and 2) stimulation of fibrosis chronically.     

Cadherin-11 contributes to pulmonary fibrosis: potential role in TGF-β production and epithelial to mesenchymal transition

Pulmonary fibrosis, characterized by excess deposition of extracellular matrix by myofibroblasts, is a serious component of chronic lung diseases. Cadherin-11 (CDH11) is increased in wound healing and fibrotic skin. We hypothesized that CDH11 is increased in pulmonary fibrosis and contributes its development. CDH11 expression was assessed in lung tissue from idiopathic pulmonary fibrosis patients. The role of CDH11 in lung fibrosis was determined using the bleomycin model of pulmonary fibrosis, and in vitro analyses were performed on A549 cells during the process of epithelial to mesenchymal transition (EMT). Immunohistochemical studies demonstrated CDH11 expression on fibroblasts, epithelial cells, and alveolar macrophages of patients with pulmonary fibrosis and mice given bleomycin. Interestingly, CDH11-deficient mice had decreased fibrotic endpoints in the bleomycin model of pulmonary fibrosis compared to wild-type mice. Furthermore, anti-CDH11-neutralizing monoclonal antibodies successfully treated established pulmonary fibrosis induced by bleomycin. TGF-β levels were reduced in bronchoalveolar lavage (BAL) fluid, BAL cells, and primary alveolar macrophages from CDH11-deficient mice. Mechanistic studies demonstrated that TGF-β up-regulated CDH11 expression on A549 cells, and inhibition of CDH11 expression using siRNA reduced TGF-β-induced EMT. Together, these results identify CDH11 as a novel therapeutic target for pulmonary fibrosis.     

Role of extracellular superoxide dismutase in bleomycin-induced pulmonary fibrosis

Bleomycin administration results in well-described intracellular oxidative stress that can lead to pulmonary fibrosis. The role of alveolar interstitial antioxidants in this model is unknown. Extracellular superoxide dismutase (EC-SOD) is the primary endogenous extracellular antioxidant enzyme and is abundant in the lung. We hypothesized that EC-SOD plays an important role in attenuating bleomycin-induced lung injury. Two weeks after intratracheal bleomycin administration, we found that wild-type mice induced a 106 +/- 25% increase in lung EC-SOD. Immunohistochemical staining revealed that a large increase in EC-SOD occurred in injured lung. Using mice that overexpress EC-SOD specifically in the lung, we found a 53 +/- 14% reduction in bleomycin-induced lung injury assessed histologically and a 17 +/- 6% reduction in lung collagen content 2 wk after bleomycin administration. We conclude that EC-SOD plays an important role in reducing the magnitude of lung injury from extracellular free radicals after bleomycin administration.     

Sustained-Release Delivery of Prostacyclin Analogue Enhances Bone Marrow-Cell Recruitment and Yields Functional Benefits for Acute Myocardial Infarction in Mice

Background

A prostacyclin analogue, ONO-1301, is reported to upregulate beneficial proteins, including stromal cell derived factor-1 (SDF-1). We hypothesized that the sustained-release delivery of ONO-1301 would enhance SDF-1 expression in the acute myocardial infarction (MI) heart and induce bone marrow cells (BMCs) to home to the myocardium, leading to improved cardiac function in mice.

Methods and Results

ONO-1301 significantly upregulated SDF-1 secretion by fibroblasts. BMC migration was greater to ONO-1301-stimulated than unstimulated conditioned medium. This increase was diminished by treating the BMCs with a CXCR4-neutralizing antibody or CXCR4 antagonist (AMD3100). Atelocollagen sheets containing a sustained-release form of ONO-1301 (n = 33) or ONO-1301-free vehicle (n = 48) were implanted on the left ventricular (LV) anterior wall immediately after permanent left-anterior descending artery occlusion in C57BL6/N mice (male, 8-weeks-old). The SDF-1 expression in the infarct border zone was significantly elevated for 1 month in the ONO-1301-treated group. BMC accumulation in the infarcted hearts, detected by in vivo imaging after intravenous injection of labeled BMCs, was enhanced in the ONO-1301-treated hearts. This increase was inhibited by AMD3100. The accumulated BMCs differentiated into capillary structures. The survival rates and cardiac function were significantly improved in the ONO-1301-treated group (fractional area change 23±1%; n = 22) compared to the vehicle group (19±1%; n = 20; P = 0.004). LV anterior wall thinning, expansion of infarction, and fibrosis were lower in the ONO-1301-treated group.

Conclusions

Sustained-release delivery of ONO-1301 promoted BMC recruitment to the acute MI heart via SDF-1/CXCR4 signaling and restored cardiac performance, suggesting a novel mechanism for ONO-1301-mediated acute-MI heart repair.     

Protective effect of orally administered carnosine on bleomycin-induced lung injury

Carnosine is an endogenously synthesized dipeptide composed of beta-alanine and L-histidine. It acts as a free radical scavenger and possesses antioxidant properties. Carnosine reduces proinflammatory and profibrotic cytokines such as transforming growth factor-beta (TGF-beta), IL-1, and TNF-alpha in different experimental settings. In the present study, we investigated the efficacy of carnosine on the animal model of bleomycin-induced lung injury. Mice were subjected to intratracheal administration of bleomycin and were assigned to receive carnosine daily by an oral bolus of 150 mg/kg. One week after fibrosis induction, bronchoalveolar lavage (BAL) cell counts and TGF-beta levels, lung histology, and immunohistochemical analyses for myeloperoxidase, TGF-beta, inducible nitric oxide synthase, nitrotyrosine, and poly(ADP-ribose) polymerase were performed. Finally, apoptosis was quantified by terminal deoxynucleotidyltransferase-mediated UTP end-labeling assay. After bleomycin administration, carnosine-treated mice exhibited a reduced degree of lung damage and inflammation compared with wild-type mice, as shown by the reduction of 1) body weight, 2) mortality rate, 3) lung infiltration by neutrophils (myeloperoxidase activity and BAL total and differential cell counts), 4) lung edema, 5) histological evidence of lung injury and collagen deposition, 6) lung myeloperoxidase, TGF-beta, inducible nitric oxide synthase, nitrotyrosine, and poly(ADP-ribose) polymerase immunostaining, 7) BAL TGF-beta levels, and 8) apoptosis. Our results indicate that orally administered carnosine is able to prevent bleomycin-induced lung injury likely through its direct antioxidant properties. Carnosine is already available for human use. It might prove useful as an add-on therapy for the treatment of fibrotic disorders of the lung where oxidative stress plays a role, such as for idiopathic pulmonary fibrosis, a disease that still represents a major challenge to medical treatment.     

Deficiency of developmental endothelial locus-1 (Del-1) aggravates bleomycin-induced pulmonary fibrosis in mice

Pulmonary fibrosis is a lung disease wherein lung parenchyma is gradually and irreversibly replaced with collagen. The molecular pathogenesis of pulmonary fibrosis is not fully understood and the only effective treatment available is lung transplantation. To test if Del-1, an endogenous anti-inflammatory molecule, may be implicated in the development of pulmonary fibrosis, we induced pulmonary fibrosis in wild type (WT) and Del-1^−/− mice by intratracheal administration of bleomycin. Del-1 expression in the lung was decreased in the WT mice treated with bleomycin compared to control mice. In addition, bleomycin-induced pulmonary fibrosis increased collagen deposition and TGF-β production in the lung of Del-1^−/− mice. Finally, Del-1^−/− mice treated with bleomycin displayed higher weight loss and greater mortality than did WT mice identically treated. These findings suggest that Del-1 may negatively regulate development of pulmonary fibrosis. Further delineation of a role for Del-1 in the development of pulmonary fibrosis will broaden our understanding of the molecular pathogenesis of this disease and hopefully help develop potential therapeutics.     

Intermittent administration of a sustained-release prostacyclin analog ONO-1301 ameliorates renal alterations in a rat type 1 diabetes model

Diabetic nephropathy is the most common pathological disorder predisposing end-stage renal disease. ONO-1301 is a novel sustained-release prostacyclin analog possessing thromboxane (TX) synthase inhibitory activity. Here, we aimed to investigate the therapeutic efficacies of ONO-1301 in a rat type 1 diabetic nephropathy model. Streptozotocin (STZ)-induced diabetic rats received injections of slow-release form of ONO-1301 (SR-ONO) every 3 weeks. Animals were sacrificed at Week 14. SR-ONO significantly suppressed albuminuria, glomerular hypertrophy, mesangial matrix accumulation, glomerular accumulation of monocyte/macrophage, increase in glomerular levels of pro-fibrotic factor transforming growth factor (TGF)-beta1 and the number of glomerular alpha-smooth muscle actin (SMA)(+) cells in diabetic animals. The glomerular levels of hepatocyte growth factor (HGF) were significantly increased in SR-ONO-treated diabetic animals. Taken together, these results suggest the potential therapeutic efficacy of intermittent administration of SR-ONO in treating diabetic nephropathy potentially via inducing HGF, thus counteracting the pro-fibrotic effects of TGF-beta1.     

Synthetic liposomes are protective from bleomycin-induced lung toxicity

Idiopathic pulmonary fibrosis is a devastating disease characterized by a progressive, irreversible, and ultimately lethal form of lung fibrosis. Except for lung transplantation, no effective treatment options currently exist. The bleomycin animal model is one of the best studied models of lung injury and fibrosis. A previous study using mouse tumor models observed that liposome-encapsulated bleomycin exhibited reduced lung toxicity. Therefore, we hypothesized that airway delivery of synthetic phosphatidylcholine-containing liposomes alone would protect mice from bleomycin-induced lung toxicity. C57BL/6 mice were administered uncharged multilamellar liposomes (100 μl) or PBS vehicle on day 0 by airway delivery. Bleomycin (3.33 U/kg) or saline vehicle was then given intratracheally on day 1 followed by four additional separate doses of liposomes on days 4, 8, 12, and 16. Fluorescent images of liposomes labeled with 1,1'-dioctadecyl-3,3,3',3' tetramethylindocarbocyanine perchlorate confirmed effective and widespread delivery of liposomes to the lower respiratory tract as well as uptake primarily by alveolar macrophages and to a lesser extent by type II alveolar epithelial cells. Results at day 22, 3 wk after bleomycin treatment, showed that airway delivery of liposomes before and after intratracheal administration of bleomycin significantly reduced bleomycin-induced lung toxicity as evidenced by less body weight loss, chronic lung inflammation, and fibrosis as well as improved lung compliance compared with controls. These data indicate that airway-delivered synthetic liposomes represent a novel treatment strategy to reduce the lung toxicity associated with bleomycin in a mouse model.     

Treatment with alpha-galactosylceramide attenuates the development of bleomycin-induced pulmonary fibrosis

Pulmonary fibrosis is an end-stage disorder for which efficacious therapeutic options are not readily available. Although its pathogenesis is poorly understood, pulmonary fibrosis occurs as a result of various inflammations. NKT cells modulate inflammation because of their ability to produce large amounts of cytokines by stimulation with their glycolipid ligand. In the present study, we investigated the effects of alpha-galactosylceramide (alpha-GalCer), a selective NKT cell ligand, on the development of bleomycin-induced pulmonary fibrosis. Treatment of mice with alpha-GalCer prolonged their survival under bleomycin administration by attenuating the development of pulmonary fibrosis. The protective effects of alpha-GalCer were associated with an increase in the pulmonary level of IFN-gamma and a decrease in the pulmonary level of fibrogenic cytokines such as TGF-beta and connective tissue growth factor. The initial pulmonary inflammation caused by bleomycin was also attenuated by alpha-GalCer with the reduction of the macrophage inflammatory protein-2 level. The protective effects of alpha-GalCer were markedly reduced in mice lacking NKT cells or as a result of treatment with anti-IFN-gamma Ab. These results suggest that alpha-GalCer suppresses bleomycin-induced acute pulmonary inflammation and thus attenuates the development of pulmonary fibrosis possibly by regulating several cytokine levels.     

Dual effect of AMD3100, a CXCR4 antagonist, on bleomycin-induced lung inflammation

The chemokine receptor CXCR4, which binds the chemokine stromal cell-derived factor 1, has been reported to be involved in the chemotaxis of inflammatory cells. In addition, AMD3100, an antagonist of CXCR4, has been reported to be an attractive drug candidate for therapeutic intervention in several disorders in which CXCR4 is critically involved. However, little is known about the therapeutic value of AMD3100 in the treatment of pulmonary fibrosis. In this study, we examined the effects of AMD3100 on a murine bleomycin-induced pulmonary fibrosis model. Concurrent administration of AMD3100 and bleomycin apparently attenuated bleomycin-induced pulmonary inflammation. In this process, an inhibition of neutrophil recruitment at early stage followed by the decrease of other inflammatory cell recruitment in the lung were observed. In addition, it also inhibited the expression of cytokines, including MCP-1, MIP-2, MIP-1alpha, and TGF-beta. In contrast, when AMD3100 was administered following bleomycin treatment, the bleomycin-induced lung inflammation progressed and resulted in severe pulmonary fibrosis. In this process, an increase of inflammatory cell recruitment, an up-regulation of lung MCP-1 and TGF-beta, and a remarkable activation of p44/42 MAPK in neutrophils were observed. U0126, an inhibitor of p44/42 MAPK, significantly abolished these effects. Thus, AMD3100 has dual effect on bleomycin-induced pulmonary fibrosis. Difference of inflammatory cell recruitment and activation might be associated with the dual effect of AMD3100 on bleomycin-induced pulmonary fibrosis.     

Overexpression of cathepsin K in mice decreases collagen deposition and lung resistance in response to bleomycin-induced pulmonary fibrosis

Background

Lung fibrosis is a devastating pulmonary disorder characterized by alveolar epithelial injury, extracellular matrix deposition and scar tissue formation. Due to its potent collagenolytic activity, cathepsin K, a lysosomal cysteine protease is an interesting target molecule with therapeutic potential to attenuate bleomycin-induced pulmonary fibrosis in mice. We here tested the hypothesis that over-expression of cathepsin K in the lungs of mice is protective in bleomycin-induced pulmonary fibrosis.

Methods

Wild-type and cathepsin K overexpressing (cathepsin K transgenic; cath K tg) mice were challenged intratracheally with bleomycin and sacrificed at 1, 2, 3 and 4 weeks post-treatment followed by determination of lung fibrosis by estimating lung collagen content, lung histopathology, leukocytic infiltrates and lung function. In addition, changes in cathepsin K protein levels in the lung were determined by immunohistochemistry, real time RT-PCR and western blotting.

Results

Cathepsin K protein levels were strongly increased in alveolar macrophages and lung parenchymal tissue of mock-treated cathepsin K transgenic (cath K tg) mice relative to wild-type mice and further increased particularly in cath K tg but also wild-type mice in response to bleomycin. Moreover, cath K tg mice responded with a lower collagen deposition in their lungs, which was accompanied by a significantly lower lung resistance (R_L) compared to bleomycin-treated wild-type mice. In addition, cath K tg mice responded with a lower degree of lung fibrosis than wild-type mice, a process that was found to be independent of inflammatory leukocyte mobilization in response to bleomycin challenge.

Conclusion

Over-expression of cathepsin K reduced lung collagen deposition and improved lung function parameters in the lungs of transgenic mice, thereby providing at least partial protection against bleomycin-induced lung fibrosis.     

Prostaglandin E₂ protects murine lungs from bleomycin-induced pulmonary fibrosis and lung dysfunction

Prostaglandin E(2) (PGE(2)) is a lipid mediator that is produced via the metabolism of arachidonic acid by cyclooxygenase enzymes. In the lung, PGE(2) acts as an anti-inflammatory factor and plays an important role in tissue repair processes. Although several studies have examined the role of PGE(2) in the pathogenesis of pulmonary fibrosis in rodents, results have generally been conflicting, and few studies have examined the therapeutic effects of PGE(2) on the accompanying lung dysfunction. In this study, an established model of pulmonary fibrosis was used in which 10-12-wk-old male C57BL/6 mice were administered a single dose (1.0 mg/kg) of bleomycin via oropharyngeal aspiration. To test the role of prostaglandins in this model, mice were dosed, via surgically implanted minipumps, with either vehicle, PGE(2) (1.32 μg/h), or the prostacyclin analog iloprost (0.33 μg/h) beginning 7 days before or 14 days after bleomycin administration. Endpoints assessed at 7 days after bleomycin administration included proinflammatory cytokine levels and measurement of cellular infiltration into the lung. Endpoints assessed at 21 days after bleomycin administration included lung function assessment via invasive (FlexiVent) analysis, cellular infiltration, lung collagen content, and semiquantitative histological analysis of the degree of lung fibrosis (Ashcroft method). Seven days after bleomycin administration, lymphocyte numbers and chemokine C-C motif ligand 2 expression were significantly lower in PGE(2)- and iloprost-treated animals compared with vehicle-treated controls (P < 0.05). When administered 7 days before bleomycin challenge, PGE(2) also protected against the decline in lung static compliance, lung fibrosis, and collagen production that is associated with 3 wk of bleomycin exposure. However, PGE(2) had no therapeutic effect on these parameters when administered 14 days after bleomycin challenge. In summary, PGE(2) prevented the decline in lung static compliance and protected against lung fibrosis when it was administered before bleomycin challenge but had no therapeutic effect when administered after bleomycin challenge.     

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

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.