ACSL4 Promotes Pulmonary Arterial Hypertension Development Via Akt/TXNRD1 Axis

Background:Pulmonary arterial hypertension(PAH)is a severe cardiopulmonary vascular disease characterized by progressive increase of pulmonary vascular resistance and pulmonary vascular remodeling,eventually leading to right heart failure and death.In recent years,although the application of targeted drugs has greatly improved the prognosis and survival of patients,the current pharmacological effects of these drugs only promote pulmonary artery vasodilation and do not directly target the process of pulmonary vascular remodeling.As the disease progresses,the therapeutic effect of vascular diastolic drugs will gradually decrease and cannot fundamentally delay disease progression.Therefore,exploring the molecular mechanism of pulmonary vascular remodeling is helpful to develop new targeted therapy strategies,which is of great significance for the diagnosis and treatment of PAH.Metabolic reprogramming has been recognized as a new feature of pulmonary vascular remodeling in recent years,however,studies on the role of fatty acid metabolism in the development of PAH are still insufficient.Long-chain fatty acyl-Co A synthase(ACSL4)is a key enzyme in fatty acid activation and is involved in various processes such as cell proliferation,survival,apoptosis,and metabolic rewiring.Whether it is involved in PAH progression has not yet been reported.Objectives:The purpose of this study was to explore:(i)the expression of ACSL4 in PAH datasets and animal models;(ii)the location of ACSL4 in pulmonary arteries;(iii)the effect of ACSL4 intervention on PAH animal models and cell phenotypes;(iv)the specific molecular mechanism of ACSL4 mediating cellular functional phenotypes.Methods:1.We firstly analyzed the expression of ACSL4 in the PAH dataset by differential expression analysis,then rats were intraperitoneally injected with monocrotaline(MCT)to construct the PAH animal model.The expression of ACSL4 in the animal model was verified by Western Blot and immunofluorescence of lung tissue sections.On this basis,intraperitoneal injection of the ACSL4 activity inhibitor PRGL493 was applied to assess the therapeutic effect of ACSL4 intervention in MCT rats.2.Primary rat pulmonary arterial smooth muscle cells(PASMCs)were isolated using collagenase digestion method.Then it was verified by immunofluorescence with smooth muscle cell specific marker alpha-sma.PASMCs were stimulated with PDGF-BB to construct cell model and cellular proteins were extracted to detect the expression of proliferation markers PCNA and ACSL4.ACSL4 expression was also confirmed by immunofluorescence.Then ACSL4 was knocked down by transfection of si RNA and the knockdown efficiency was verified by q PCR and WB.The effect of ACSL4 knockdown on PDGF-induced PASMCs proliferation was assessed by CCK8 assay,Ed U assay and PCNA expression detection.The effect of ACSL4 knockdown on apoptosis of PASMCs was assessed by Annexin V-PI staining,TUNNEL assay and apoptosis-related gene expression detection.The effect of ACSL4 knockdown on the oxidative stress state of PASMCs was observed by ROS detection,NADPH/NADP+ assay,and Mito SOX staining.3.We performed an integrated bioinformatics analysis to identify robust differentially expressed genes(DEGs)in IPAH.The gene expression profiles of GSE117261,GSE53408,GSE48149 and GSE15197 were downloaded from GEO database.After screening DEGs in each dataset,the RRA method was applied to identify robust DEGs.Then,we uploaded robust DEGs to the STRING database to construct global protein-protein interaction network.The module analysis was performed by the MCODE plug-in of Cytoscape based on the entire network and hub genes identification was carried out by the plug-in Cytohubba.Through verification of independent dataset and experimental validation,TXNRD1 was finally identified as a potential therapeutic target.to further explore the functions of TXNRD1 in PASMC,we knocked down the TXNRD1 in transcriptional level to evaluate if silencing TXNRD1 exacerbated PASMC malignant phenotype.Ed U assay and PCNA expression detection were used to observe the effect of TXNRD1 knockdown on the proliferation of PASMCs,and the apoptosis markers cleaved-PARP and BAX/Bcl-2 were detected by WB to observe the effect of TXNRD1 knockdown on the PDGF-induced apoptosis resistance of PASMCs.WB was used to identify the effect of ACSL4 knockdown on TXNRD1 expression and Akt phosphorylation.The Akt phosphorylation agonist SC79 was further added on the basis of ACSL4 knockdown to investigate whether ACSL4 could regulate the expression of TXNRD1 and its mediated phenotype via Akt activation.Results:1.The differential expression analysis of GSE117261 found that ACSL4 was significantly highly expressed in the lung tissue of PAH patients.Animal experiments confirmed that ACSL4 was highly expressed in the lung tissue of MCT rats and was mainly enriched in the middle layer of the pulmonary artery.Treatment of ACSL4 activity inhibitor PRGL493 can significantly improve the hemodynamics and pulmonary vascular and right heart remodeling in MCT rats.2.ACSL4 is highly expressed in PDGF-BB-stimulated PASMC,and ACSL4 knockdown can significantly regulate the over-proliferation and apoptosis resistance of PASMC while regulating oxidative stress state.3.By integrated bioinformatic analysis,169 robust differentially expressed genes were screened.Ten hub genes were found by PPI network construction and Cytohubba application,including ENO1,TALDO1,TXNRD1,SHMT2,IDH1,TKT,PGD,CXCL10,CXCL9 and CCL5.After validation through GSE113439 and experiments,TXNRD1 was proved to be inhibited in PAH animals and cell models.Further knockdown of TXNRD1 would exacerbate the hyperproliferation and apoptosis resistance of PASMCs.WB experiments confirmed that TXNRD1 is a downstream target regulated by ACSL4.Finally,rescue experiments confirmed that ACSL4 can promote Akt phosphorylation to regulate the expression of TXNRD1 and mediate the malignant phenotype of PASMCs.Conclusion:In summary,we found that ACSL4 may regulate the oxidative stress homeostasis to promote hyperproliferation and apopsosis resistance in PASMC,which is potentially mediated by inhibiting the expression of TXNRD1 in Akt-dependent manner.