ABSTRACT
Acute and chronic pulmonary diseases are characterized by impaired fibrinolytic activity within the lung. To determine the role of the fibrinolytic system in regulating the pathologies associated with lung injury, we examined the effect of bleomycin, an agent that induces the development of pulmonary fibrosis, in mice deficient for plasminogen (Pg(-)(/-)), urokinase (u-PA(-)(/-)), urokinase receptor (u-PAR(-)(/-)), or tissue plasminogen activator (t-PA(-)(/-)), and in control wild-type (WT) mice. Pg(-)(/-) and t-PA(-)(/-) mice demonstrated an enhanced increase in lung collagen content relative to that observed in WT mice. Levels in u-PA(-)(/-) and u-PAR(-)(/-) mice were similar to those in WT mice. Histological analysis 14 days after lung injury confirmed enhanced interstitial fibrosis in Pg(-)(/-), u-PA(-)(/-), and t-PA(-)(/-) mice relative to WT and u-PAR(-)(/-) mice. Areas of pulmonary hemorrhage were observed in bleomycin-treated WT mice and not in Pg(-)(/-), u-PA(-)(/-), and u-PAR(-)(/-) mice or saline controls. Instead, extensive areas of fibrosis were present throughout the lungs of bleomycin-treated Pg(-)(/-) and u-PA(-)(/-) mice. A mixed phenotype (hemorrhage and fibrosis) was observed in t-PA(-)(/-) and Pg(+/-) mice. Hemosiderin-laden macrophages were abundant in the lungs of mice exhibiting hemorrhage and these mice were prone to an early death. Enhanced macrophage levels in the lungs and activation of matrix metalloelastase (MMP-12) were found in mice with a hemorrhage phenotype. The results of these studies indicate a role for the fibrinolytic system in acute lung injury and suggests that intra-alveolar hemorrhage is the result of basement membrane degradation through cell-mediated u-PA activation of Pg with possible involvement of matrix metalloproteinases. Absence of these two components of the fibrinolytic system, either urokinase or plasminogen, results in accelerated fibrosis.
Subject(s)
Bleomycin/pharmacology , Blood Coagulation Factors/metabolism , Pulmonary Fibrosis/metabolism , Animals , Blood Coagulation Factors/genetics , Collagen/drug effects , Collagen/metabolism , Enzyme Activation/drug effects , Female , Fibrin/drug effects , Fibrin/metabolism , Genotype , Leukocyte Count/drug effects , Lung/drug effects , Lung/metabolism , Lung/pathology , Male , Matrix Metalloproteinase 12 , Metalloendopeptidases/drug effects , Metalloendopeptidases/metabolism , Mice , Mice, Inbred C57BL , Mice, Inbred Strains , Plasminogen/genetics , Plasminogen/metabolism , Pulmonary Fibrosis/chemically induced , Pulmonary Fibrosis/genetics , Receptors, Cell Surface/genetics , Receptors, Cell Surface/metabolism , Receptors, Urokinase Plasminogen Activator , Tissue Plasminogen Activator/genetics , Tissue Plasminogen Activator/metabolism , Urokinase-Type Plasminogen Activator/genetics , Urokinase-Type Plasminogen Activator/metabolismABSTRACT
OBJECTIVES: Our purpose was to investigate the relationship between expression of cyclooxygenase-2 and inducible nitric oxide synthase genes after labor induction with bacterial lipopolysaccharide in a murine model of preterm parturition. STUDY DESIGN: Pregnant C57B1/6 mice were given Escherichia coli lipopolysaccharide (20 micrograms per mouse) by intraperitoneal injection on day 16 of gestation, and the animals were followed up for signs of labor. Control mice received an equivalent volume of 0.9% saline solution. The latency from lipopolysaccharide injections until appearance of the first pup was recorded. Two separate groups of mice were given either aminoguanidine or indomethacin (5 mg/kg intragastric) 24 hours before induction of preterm labor. In a separate set of experiments mice were treated with lipopolysaccharide as described and were killed at intervals from 0.5 to 72 hours and intrauterine tissues (uterus, placenta, and fetal membranes) were removed and snap frozen in liquid nitrogen. Total protein and ribonucleic acid were extracted for Western and Northern blot analysis of cyclooxygenase-2 and inducible nitric oxide synthase protein and messenger ribonucleic acid, respectively. RESULTS: Northern blots from uterine, placental, and fetal membrane tissues of lipopolysaccharide- and saline solution-treated mice revealed that cyclooxygenase-2 and inducible nitric oxide synthase messenger ribonucleic acid transcripts were rapidly (within 0.5 to 2 hours) up-regulated after lipopolysaccharide administration but were unchanged in mice injected with saline solution. Immunoblot analysis with isoform-specific antibodies revealed that both enzymes were expressed in uterus, placenta, and fetal membranes in a coordinated fashion with peak expression seen at 6 to 8 hours. Although the steady-state accumulation of messenger ribonucleic acid transcripts encoding cyclooxygenase-2 and inducible nitric oxide synthase peaked at 6 hours and declined to baseline by 16 hours after injection with lipopolysaccharide, expression of cyclooxygenase-2 and inducible nitric oxide synthase was sustained through the period when premature delivery was observed. Nitric oxide-dependent cyclooxygenase-2 and inducible nitric oxide synthase expression was demonstrated by the elimination of accumulation of both messenger ribonucleic acid transcripts in mice pretreated with aminoguanidine before injection with lipopolysaccharide. CONCLUSIONS: These data indicate that nitric oxide synthesis may be a prerequisite for subsequent stimulation of cyclooxygenase-2 and inducible nitric oxide synthase gene expression. Taken together, the data suggest that cyclooxygenase-2 and inducible nitric oxide synthase are expressed in a coordinated manner in the uterus of endotoxin-challenged pregnant mice and that their enzymatic products may contribute to the signaling of uterine activity or cervical changes culminating in expulsion of the fetus.
