The Repair Mechanism Of AEC2s After Acute Lung Injury

Acute lung injury is among the most common causes of acute hypoxemic respiratory failure in intensive care unit?ICU?patients.The adult lung is considered to be quiescent compared with other organs such as the liver and skin during homeostasis.However,recent research shows that the lung has facultative stem/progenitor cell populations with great reparative potential,especially after injury.Alveoli are gas-exchange sacs lined by squamous alveolar epithelial type 1 cells?AEC1s?and cuboidal,surfactant-secreting alveolar epithelial type 2 cells?AEC2s?.AEC2s are widely accepted as lung progenitor cells and contribute to lung repair and regeneration process.Recent studies show that,during development,AEC1s and AEC2s arise from a bipotent progenitor cell lineage,whereas after birth,AEC2s can undergo long-term self-renewal and give-rise to AEC1s during homeostasis.Barkauskas et al.suggest that SFTPC+AEC2s function as progenitor cells in the alveoli,proliferating and differentiating into AEC1s during homeostasis and regeneration.Therefore,AEC2s as a population are stem cells,proliferating in vivo and giving rise to AEC1s during ALI.Understanding the mechanism of the regenerative capacity of alveolar epithelial cells is essential for providing a theoretical basis the treatment of ALI.The aim of this study was to determine the proliferation and regeneration capacity of alveolar epithelium after acute lung injury and to further investigate the mechanism of alveolar epithelium regeneration.We established a LPS-induced acute lung injury model,and observed the changes of AEC2s at different time points after acute lung injury using pedigree tracer technology and immunostaining method.We found that AEC2s showed different proliferation and differentiation characteristics at different time points and in different injury areas.AEC2s showed the most obvious proliferation 3 days after injury,and the proliferation mainly occurred in the injured area.Through proteomics high-throughput screening,it was found that the changes of Hippo pathway after lung injury were highly consistent with the proliferation rules of AEC2s,and the key protein YAP1 of Hippo pathway was mainly expressed in AEC2s after lung injury.Cell experiments showed that inhibiting the expression of YAP1 would lead to the decreased proliferation of A549 cells.The results showed that Hippo-Yap1 signaling pathway was involved in the regulation of cell proliferation and played an important role in the regeneration after acute lung injury.Methods:Using a lung injury mouse model induced by hemorrhagic shock and LPS?O55:B5?,a protein mass spectrometry-based high-throughput screening and linage tracing technology to mark alveolar epithelial type 2 cells?AEC2s?,we analyzed the number of AEC2s in different lung injury days and regions to clarify the proliferation and migration of AEC2s after lung injury.Then,we used immunofluorescence staining,immunohistochemistry and in vitro tests to demonstrate the mechanism of alveolar epithelial cells proliferation.Results:1.The lung injury mouse model by intratracheal instillation indicated that a dose of10mg/kg LPS?O55:B5?and 50ul volume of drip for one mouse is suitable.Mice were sacrificed at 3,5,7 days after injury respectively.We found lung injury had been deteriorating after 3 days post injury?dpi?,then repaired to completely normal after 12 dpi.Different injury regions had different injury conditions.The damage was most severe near the hilus pulmonis and the lightest in the peripheral lung area.2.To lineage label mouse AEC2s,Sftpc-CreER^T2;Rosa26-RFP double heterozygous mice(Sftpc-CreER^T2;Rosa26-RFP)were given 4 doses of Tmx?0.1-0.15 mg/g body weight?and sacrificed at least 7 days after the final injection,which led to lineage labeling of approximately 89.2%of SFTPC+cells.Immunohistochemistry,location,and cell morphology analyses confirmed that the great majority?99.4%±7%?of these cells were AEC2s.3.The cell density of AEC2s showed the highest at 3 days after injury?205.20±46.14cells/HPF,P<0.05?,and the cell density at 5 and 7 days after injury was significantly higher than that in the control group?188.29±55.58/HPF,195.73±60.06/HPF,P<0.05?.Compared with the control group,the cell density of the damaged area and the injury-normal junction area increased significantly?P<0.05?,while the normal area showed no change?P<0.05?.4.We demonstrated that the expression of Hippo-YAP1 key proteins were highly consistent with the regularity of the proliferation of alveolar epithelial type 2 cells after ALI.Furthermore,the results showed that YAP1-positive cells in lung tissue after ALI were mainly Sftpc lineage-labeled AEC2s.An in vitro proliferation assay of AEC2s demonstrated that AEC2 proliferation was significantly inhibited by both YAP1 siRNA and Hippo inhibitor.Conclusion:In conclusion,our research revealed that the Hippo-YAP signaling pathway regulated pulmonary AEC2s proliferation after ALI.We observed that YAP mainly expressed on AEC2s through lineage-labeled AEC2s.Inhibiting the effect of YAP using a YAP-TAED inhibitor and small interfering RNA can suppress AEC2 proliferation.