The Mechanism Of Cathepsin S In Regulating Caspase-4/11-mediated Pulmonary Artery Endothelial Cells Pyroptosis In Pulmonary Arterial Hypertension

Background:Endothelial dysfunction enhances vascular inflammation,triggers the pathogenesis of pulmonary arterial hypertension,and further induces vascular remodeling and right ventricular failure.Activation of the inflammatory Caspases is an important initial event at the onset of pyroptosis.Studies have shown that Caspase-1-mediated pyroptosis has played a crucial role in the pathogenesis of PAH.However,the role of Caspase-11,another inflammatory Caspase,remains to be elucidated.Preliminary studies in our laboratory have clarified the role of murine Caspase-11 and human Caspase-4 in pulmonary arterial hypertension in an MCT PAH rat model and in pulmonary artery endothelial cells induced by TNF-α.Application of Caspase-11 specific inhibitor Wedelolactone effectively alleviates the process of pulmonary arterial hypertension.Meanwhile,Caspase-4/GSDMD and Caspase-4/Caspase-3/GSDME-mediated pyroptosis pathways are involved in the mechanism of TNF-α-induced pulmonary artery endothelial injury.Purpose:this study firstly aimed to further clarify the key regulatory role of Caspase-11 in the occurrence and development of PAH by gene knockout mice.At the same time,based on the previous laboratory basics and bioinformatics analysis,we also explored the role of the key regulatory molecule Cathepsin S in pulmonary arterial hypertension and its regulation with the inflammatory Caspase-4-mediated pyroptosis pathway,providing an entry point for finding molecules of targeted diagnosis and treatment of pulmonary hypertension from inflammatory pathways.Methods:1.By using the GEO database to screen out suitable data sets related to the gene expression of pulmonary arterial hypertension population,and using bioinformatics analysis methods such as KEGG and GO to predict the key molecules and signaling pathways involved in the process of pulmonary arterial hypertension,and explore the mutual regulatory relationship between them.2.In vivo experiments:Su5416-Hypoxic（Su/Hx）pulmonary arterial hypertension mouse model was constructed by wild-type（WT）mice and Caspase-11^-/-mice,and randomly divided into four groups:1)WT mice normoxia group（WT+Norm）and Caspase-11^-/-mice normoxia group(Casp-11^-/-+Norm):the same amount of VEGFR inhibitor Su5416 diluted solvent was subcutaneously injected to the mice weekly for the first three weeks,and placed them in normoxia environment（21%）for 8 weeks;2)WT mice hypoxia group（WT+Su/Hx）and Caspase-11^-/-mice hypoxia group(Casp-11^-/-+Su/Hx):Su5416（20 mg/kg）was subcutaneously injected weekly for the first three weeks,and the mice were raised in a hypoxic chamber（10%O₂）for 3 weeks,and then they were placed in normoxia environment for 5 weeks.At the end of the modeling,right ventricular systolic blood pressure,mean pulmonary artery pressure and right ventricular hypertrophy index were detected in mice,and pulmonary vascular remodeling was detected by H&E and immunofluorescence staining;The expression levels of GSDMD and GSDMD-N in lung tissue were detected by Western blot.In vitro experiment:verify the previous results of our research group:under the stimulation of TNF-α（40 ng/ml）,the expression of Caspase-4 was intervened by si RNA silencing.The expression changes of pyroptosis related proteins such as Caspase-4,GSDMD,Caspase-3/GSDME pathway were detected by Western blot in the cell model.3.In vivo experiment:SD male rats were given single intraperitoneal injection of monocrotaline（MCT,60 mg/kg）to construct MCT-induced pulmonary arterial hypertension rat model.After 3 weeks of modeling,the right ventricular systolic blood pressure,mean pulmonary artery pressure and right ventricular hypertrophy index,H&E staining were used to detect pulmonary vascular remodeling;Western blot was used to detect the expression level of Cathepsin S and GSDME in lung tissue.At the same time,the expression levels of Cathepsin S and GSDME in the Su5416-hypoxia mouse model were detected by western blot.（1）Based on the previous laboratory basis,we stimulated HPAECs with TNF-α（40 ng/ml）,detected the changes of the commonly expressed cathepsins family in endothelial cells by q-PCR,and screened out the up-regulated Cathepsin S.HPAECs were intervened with different TNF-αconcentration（0,10,20,40 ng/ml）,and the expressions of Cathepsin S and the pyroptosis substrate proteins GSDMD and GSDME in HPAECs were detected by Western blot or q-PCR.The production of IL-1βwas detected by Western blot.（2）Under the stimulation of TNF-α（40 ng/ml）,si RNA silencing was used to interfere with the expression of Cathepsin S,and Western blot,q-PCR and LDH methods were used to detect the expression changes of Caspase-4 and its downstream pyroptosis-related protein,such as GSDMD,GSDME,and IL-1βin vitro.Results:1.Two related datasets of pulmonary arterail hypertension population were screened in GEO,and it was found that NOD-like receptor pathway was the main pathway enriched by KEGG,the biological processes found by GO enrichment analysis were mainly enriched in inflammatory pathways such as the cell response to interleukin-1（IL-1）,and the molecular functions were mainly enriched in cysteine-type peptidase activity.2.Compared with the normoxia control group,right ventricular systolic pressure,mean pulmonary arterial pressure,and right ventricular hypertrophy index in Su5416-hypoxia induced mouse pulmonary arterial hypertension model showed significantly higher;H&E staining showed pulmonary vascular remodeling in Su/Hx group mice,indicating that the model of pulmonary arterial hypertension was successful.At the same time,relative to WT+Su/Hx group,right ventricular systolic pressure,mean pulmonary artery pressure,right ventricular hypertrophy index was decreased in the group of Casp-11^-/-+Su/Hx;HE and immunofluorescence staining（CD31 labeled endothelial cells,α-SMA-labeled smooth muscle cells）showed marked improvement in pulmonary vascular remodeling.In addition,Western blot results showed that the expression of pyroptosis substrate protein GSDMD-N was significantly decreased in the Casp-11^-/-+Su/Hx group compared with WT+Su/Hx group,suggesting that Caspase knockout can inhibit the occurrence of pyroptosis pathway in the pulmonary arterial hypertension.Silencing of Caspase-4 by si RNA inhibited the activation of GSDMD and Caspase-3/GSDME pathway in HPAECs induced by TNF-α.3.（1）The right ventricular systolic blood pressure,mean pulmonary arterial pressure and right ventricular hypertrophy index of MCT-induced pulmonary arterial hypertension rats were significantly higher than those of the control group;H&E staining showed that the vascular remodeling of the MCT rats’group was obvious.Western blot shown that the expression level of Cathepsin S protein in the lung tissue of the MCT rats’group was significantly higher than that of the control group.In the Su5416-hypoxia induced mouse PAH model,the protein expression levels of Cathepsin S and GSDME-N were increased compared with the control group.（2）TNF-αcan promote the protein expression and activation of Cathepsin S,GSDMD,GSDME,and IL-1βunder the intervention of TNF-α;Silencing of Cathepsin S expression by si RNA inhibits TNF-α-induced activation of Caspase-4 in endothelial cells and the expression and activation of downstream proteins GSDMD,GSDME and IL-1β.Conclusion:Cathepsin S promotes pulmonary arterial endothelial cell pyroptosis to mediate pulmonary vascular remodeling in pulmonary arterial hypertension by promoting the Caspase-4/11-mediated GSDMD/E pathway.