The Human Alveolar Epithelial Studies On The Regulation Of Coagulation And Fibrinolysis In Acute Lung Injury Alveolar

Objectives: The protein C pathway plays important roles in the regulation of the coagulation system and inflammatory response. Recent studies have suggested that acute lung injury is associated with increased procoagulant activity in the alveolar compartment and the protein C pathway is significantly altered in ALL Thrombomodulin (TM) and endothelial cell protein C receptor (EPCR) are major receptors for protein C activation. Our study is to characterize that alveolar epithelium can express and release TM and EPCR in response to pro-inflammatory stimuli and to explore that the alveolar epithelium can modulate intra-alveolar coagulation through protein C pathway.Methods: 1) We measured soluble EPCR levels in pulmonary edema fluid and plasma which were collected from ALI/ARDS and hydrostatic pulmonary edema patients using ELISA method. We also compared levels of soluble thrombomodulin (previously published) and soluble EPCR in the pulmonary edema fluid from patients with ALI/ARDS. 2) A549 and primary isolated alveolar type II cells were cultured. We measured the levels of TM and EPCR antigen in conditioned mediums and cell lysates after stimulating the cells with different stimuli. Activation of protein C by alveolar epithelium was also measured simultaneously. 3) To explore the mechanism of EPCR and TM shedding from alveolar epithelium, we measured EPCR and TM levels in conditioned medium after treating A549 cells with or whithout PMA and different metalloproteinase inhibitors. 4) EPCR and TM mRNA expression on alveolar epithelial cells were studied by real-time PCR.Results: 1) Levels of EPCR in the undiluted pulmonary edema fluid were 2-fold higher in ALI/ARDS patients than in a control group of patients with hydrostatic edema (p = 0.04). Higher edema fluid levels of EPCR were associated with worse clinical outcomes. When simultaneous edema fluid and plasma samples were compared, levels in the pulmonary edema fluid were higher than the plasma in patients with ALI/ARDS (edema fluid to plasma ratio of 1.46 ± 1.28 in ALI/ARDS vs. 0.59 ± 0.37 in hydrostatic edema, p = 0.007), suggesting an intra-alveolar source inthe ALI/ARDS group. There was a strong correlation between soluble thrombomodulin and EPCR levels (r = 0.81, p < 0.001), suggesting alveolar epithelium maybe the common intra-alveolar source of EPCR and TM. 2) A549 and primary isolates of human alveolar type II cells activated protein C in the presence of thrombin, suggesting active TM and EPCR on the epithelial surface. Cytomix (TNF-α, IL-1β, IFN-γ) treatment enhanced TM and EPCR protein shedding into the conditioned medium in a time and dose dependent manner, resulting in a decrease in APC generation. The release of TM and EPCR from epithelial cells was promoted by a protein kinase C activator PMA, but blocked by the hydroxamic acid based metalloproteinase inhibitors, suggesting the release is a metalloproteolytic process. 3) EPCR shedding is probably mediated by TACE/ADAM-17 with LPS. In contrast to LPS, the shedding of EPCR that was stimulated by cytomix was much slower, suggesting that a different mechanism is involved. 4) Real-time PCR demonstrated TM and EPCR mRNA expression in A549 and human alveolar type II cells, but no changes in the mRNA expression after exposure to cytomix exposure. Conclusion: The alveolar epithelial cells can express and regulate the release of TM and EPCR to modulate activation of protein C on the cell surface. Thus, the alveolar epithelial cells play an important role in the regulation of intra-alveolar coagulation through protein C pathway.Part II: The Human Alveolar Epithelium is Regulating Intra-alveolar Fibrinolysis through expressing and releasing plasminogen activator inhibitor(PAI-1)Objectives: To characterize the ability of human alveolar epithelium to express and release PAI-1 when exposed to pro-inflammatory stimulation.Methods: Human alveolar epithelial -like cells (A549 cells) and primary isolated alveolar type II cells were grown in to be confluent monolayer. The medium was replaced with serum-free medium a mixture of pro-inflammatory cytokines (TNF-α,IL-1β and IFN-γ). PAI-1 activity was measured using a commercial availablechromogenic assay kit. One unit of PAI-1 activity is defined as the amount of PAI-1that inhibits one international uint of human single chain tPA as calibrated against theinternational standard of tPA. The quantities of PAI-1 protein in the conditionedmedium and cell lysates were measured using ELISA method. Total RNA wasextracted and PAI-1 mRNA was measured by Northern Blot.Results: Cytomix (TNF-α, IL-1β and IFN-γ) treatment stimulated PAI-1 release fromalveolar epithelium in a time and dose dependent manner. PAI-1 activity of A549 cellswas also increased in a dose dependent manner by cytomix. PAI-1 mRMA expressionin A549 cells was increased by cytomix treatment.Conclusion: Human alveolar epithelial cells express and secrete active PAI-1.Exposure of alveolar epithelial cells to pro-inflammatory cytokines stimulates activePAI-1 releasing, which may contribute to impaired fibrinolysis of alveolar epithelialcells.