Study On The Role Of Factor â…¦ Activating Protease In A Bleomycin-induced Lung Injury Rat Model

Acute lung injury (ALI) and its more severe form acute respiratory distress syndrome (ARDS) are common, devastating clinical syndromes which characterized by non-cardiogenic pulmonary edema, respiratory distress and hypoxemia (Gao & Barnes, 2009). Although the mortality rate associated with ARDS has improved in the last decade,40-70% of patients still die from this syndrome and survivors encounter significant physical and psychological impairments (Rubenfeld et al.,2005). ALI/ARDS are caused by a number of processes that direct or indirect injure to lung (Pietropaoli & Georas,2009). Traditional model of ARDS suggested that lung injury follows an orderly, sequential pattern in which damage to the alveolar capillary membrane accompanied by edema formation was followed by active clearance of edema fluid and subsequent repair of the alveolar-capillary membrane with a varying degree of fibrosis (Marshall et al.,1998). It is now recognized that these processes occur simultaneously in the lung of patient with ARDS (Chesnutt et al.,1997). Previous studies indicated that the changes of various cytokine such as platelet derived growth factor (PDGF) and transforming growth factor (TGF) are involved in these processes (Zagai et al.,2003, Budinger et al.,2005). Therefore, understanding the mechanisms of cytokine changes in these processes will provide possible opportunities for therapeutic intervention of ARDS.FactorⅦactivating protease (FSAP) is a plasma-derived protease structurally homologous to members of the haemostasis family (Romisch et al.,1999, Romisch, 2002). It serves to active pro-urokinase as well as factorⅦand might play a role in the regulation of both coagulation and fibrinolysis (Parahuleva et al.,2008). Recently, Wygrecka et al reported that FSAP protein level and activity were markedly increased in the plasma and BAL fluid of patients with ARDS, suggesting a role for FSAP in the development of ARDS (Wygrecka et al.,2007). Nonetheless, the precise role of FSAP in ARDS needs to be further elucidated. Accumulating evidences implicated that basic fibroblast growth factor (bFGF) and platelet-derived growth factor-BB (PDGF-BB) participate in the pathogenesis of ARDS by induction of endothelial cells activation, stimulation of fibroblasts proliferation as well as promotion pulmonary fibrosis (Henke et al.,1993, Madtes et al.,1994). Moreover, FSAP can inhibit hepatic stellate cells and vascular smooth muscle cells proliferation and migration by cleavage of PDGF-BB, thereby functions as a suppressor of fibrosis and inflammation response (Roderfeld et al.,2009). These findings led us to explore whether similar mechanisms of FSAP also play a role in the progression of ARDS. Additionally, since the pulmonary fibrosis is a long-term dynamic and progressive disease, the investigation of changing levels of the FSAP along the time course may have significance in understanding the real pathophysiology of the condition. To this end, in the present study we investigated the expression of FSAP in bleomycin-induced acute lung injury and fibrosis in rat model. The influence of FSAP on cultured fibroblasts was also further examined.Materials and Methods1.Animals modelsPathogen free male Sprague-Dawley rats (200±20g) were purchased from the Experimental Animal Center of China Medical University. Forty rats were randomly divided into control group (n=20) and bleomycin-administered experimental group (n=20). Each rat was anaesthetized with sodium pentobarbital (100mg/kg) and then subjected to a tracheostomy. The rats in the experimental groups received intratracheal instillation of 4% bleomycin (5mg/kg). Then upright spin was used to ensure a homogenous distribution of bleomycin to rat lungs. The control animals received intratracheal saline only. 2.Preparation of Lung SamPlesFive rats from experimental group and 5 from the control group were sacrificed at 3,7,14 and 28 days after bleomycin instillation. Left lung was inflation-fixed via tracheal cannula using 4%paraformaldehyde for morphology obverasion and immunohistochemistry study. Put the middle lobe of right lung inRNase-free Eppendorf tubes and stored at-80℃freezer for Quantitative Real-Time RT-PCR and Western blotting.3.Cell cultureHuman pulmonary fibroblasts (HPF) were obtained from Bioleaf Biotechnology Co., Ltd. (Shanghai, China). The cells were maintained in Dulbecco's modified Eagle medium (DMEM, GIBCO, Grand Island, NY, USA) supplemented with fetal bovine serum (FBS) and cultured at 37℃in a 5% CO2 incubator. The cells were routinely passaged and cells at logarithmic growth phase were used for experiments.4.Experimental methods(1) Morphology observation::histologicalstudy.(2) Immunohistochemistry measurement the expression levels of FSAP.(3)Quantitative Real-Time RT-PCR; measurement the expression levels of FSAP and collagenⅢmRNA.(4)Western blotting:measurement the expression levels of FSAP and collagenⅢprotein.(5) Bromodeoxyuridine (BrdU) incorporation.(6) Cell Migration Assay.(7) Phosphorylation of mitogen-activated protein kinase (MAPK) p42/p44.5. Statistical analysisNormally distributed data are expressed as the mean±SEM and were assessed for significance by Student's t test or ANOVA withPost-hoc continuity correction for multiple comparisons as indicated in the text. Non-normally distributed data were assessed for significance using the Wilcoxon rank sum test. Statistical calculations were Performed usingSPSS 13.0 software.Statistical difference was accepted at P<0.05.Results1.Lung morphology(1)Changes of lung pathology. Prominent inflammatory reaction was noted from the third day, showing congestion of small blood vessels, minor hemorrhage in interstitial and alveolar space, and effusion of neutrophils as well as occasional widening of alveolar septum. On the day 7, thickening of alveolar walls and intraalveolar edema became prominent. More neutrophils and macrophages were present and inflammatory cell infiltration spread to the interstitial and alveolar spaces. Diminishing of alveolar space and proliferation of blood capillary were strongly induced. Additionally, the number of fibroblasts was markedly increased.The number of infiltrating macrophages peaked on the day 14. On the other hand, marked widening of alveolar septum was observed and the collagen fibers became apparent and widen, accompanied by increasing of lymphocyte and fibroblasts. On the day 28, the level of fibrosis further progressed. Abundant collagen fibers deposition and collapse of alveolar spaces were observed, along with marked thickening of capillary walls. In the saline-treated control animals, such morphological changes were not observed at any time point examined.(2)Changes of FSAP expression.In the saline-treated control animals, positive signals of FSAP were observed weakly in a small number of alveolar macrophages. On the three days after instilled with bleomycin, positive signals for FSAP were observed prominently in alveolar epithelial cells and microvascular endothelial cells. At the later time points, no obvious changes of FSAP expression and localization was observed on day 7 and 14, whereas positive signals of FSAP were markedly diminished on day 28.2. Quantitative Real-Time RT-PCR. The mRNA expression of FSAP tended to increase at the beginning of bleomycin instillation, then peaked on day 7, and deceased thereafter. After incubated with PDGF-BB, the mRNA was markedly increased. FSAP and heparin alone have a slight inhibitory effect on the synthesis of collagenⅢ. Notably, when FSAP and PDGF-BB were incubated in the presence of heparin, PDGF-BB-mediated stimulus effect was also decreased by FSAP. In accordance with the above results, there is a loss of FSAP-mediated effect when in the presence of aprotinin.3.Western blottingThe changes in the Western blotting were in accordance with the findings in the quantitative Real-Time RT-PCR study. These results confirmed that the expression level of FSAP was markedly increased before the progression of pulmonary fibrosis, but decreased during pulmonary fibrosis.4. Bromodeoxyuridine (BrdU) incorporationCell proliferation was stimulated by both PDGF-BB and heparin. Although FSAP alone did not have a significant inhibitory effect on cell proliferation, when FSAP and PDGF-BB were incubated in the presence of heparin there was a loss of PDGF activity. In the presence of aprotinin, which inhibits the proteolytic activity of FSAP, there was no inhibition of PDGF-BB stimulation.5.Cell migrationFSAP has an inhibitory effect on the migration of HPF. Especially when in the present of heparin, a strong inhibitory effect of FSAP was observed and this effect can be reversed by the present of FSAP enzymatic activity blocking aprotinin.6.Effect of FSAP on PDGF-BB-stimulated phosphorylation in HPFPDGF-BB is a stimulator of MAPK-p42/44 phosphorylation in HPF. FSAP itself did not inhibit the effect of PDGF-BB on MAPK phosphorylation, but when in the presence of heparin, a strong inhibitory effect of FSAP was induced. Similarly, this effect also can be reversed by the presence of aprotinin. ConclusionIn summary, we demonstrated an inhibitory effect of FSAP on PDGF-stimulated proliferation and migration of HPF in vitro. In addition, the dynamic expression changes of FSAP in a bleomycin-induced pulmonary fibrosis rat model indicated that FSAP may modulate inflammation and exert a beneficial effect in ARDS, suggesting that exogenous administration of FSAP may serve as a potential strategy for therapeutic interventions of ARDS.