NLRP3 Inflammasome Activation Contributes To Mechanical Stretch-induced Endothelial-Mesenchymal Transition And Pulmonary Fibrosis

Objectives:Mechanical ventilation can induce lung fibrosis.Since mechanical stretch-induced lung endothelial injury is one of the key mechanisms involved in ventilator-associated lung injury,this study aimed to investigate whether ventilator-induced lung fibrosis was associated with endothelial—mesenchymal transition(EndMT)and if so,the underlying mechanisms of mechanical stress-induced EndMT formation.Design:Randomized,controlled animal study and cell culture study.Setting:University research laboratory.Subjects:Adult male ICR mice.Primary cultured mouse lung vascular endothelial cells(MLVECs).Interventions:ICR mice were subjected to mechanical ventilation(20ml/kg)for 2 h.Lung tissues were harvested at 3 days,7 days and 14 days after mechanical ventilation.MLVECs were subjected to cyclic stretch for 24 hours.Measurements and main Results:Mice subjected to mechanical stress exhibited significant increases in collagen deposition,hydroxyproline and type I collagen contents,and transforming growth factor(TGF)-β1 production in lung tissues on day 3 to day 14 after mechanical ventilation.Mechanical stress-induced lung fibrosis was associated with increased expression of mesenchymal markers(a smooth muscle actin[a-SMA]and vimentin),as well as decreased expression of epithelial marker(E-cadherin)and endothelial markers(VE-cadherin and CD31).Double immunofluorescence staining showed that staining for a-SMA was colocalized with CD31 in some lung vascular endothelial cells,indicating the mechanical stress-induced EndMT formation.Mechanical ventilation also induced NLRP3 inflammasome activation in lung tissues as evidenced by increased mRNA expression of NLRP3 and ASC,as well as increased levels of cleaved caspase-1 p10 and IL-1β.Double immunofluorescence staining demonstrated that increased NLRP3 expression was found in both macrophages and vascular endothelial cells.In vitro direct mechanical stretch of primary cultured MLVECs resulted in similar NLRP3 activation and EndMT formation,which were significantly reversed by NLRP3 knockdownConclusions:Mechanical stress induces NLRP3 inflammasome activation,which may contribute to pulmonary endothelial-mesenchymal transition and ventilator-induced lung fibrosis.