Study On The Role And Molecular Mechanism Of Ferroptosis Of Aortic Smooth Muscle Cells In Aortic Dissection

Background and Objective:Aortic dissection(AD)is an acute and extremely dangerous vascular condition.AD is initiated by tearing of the intimal and medial layers of the aorta.The key pathological feature of AD is medial degeneration of the aorta,which is characterized by the loss of smooth muscle cells(SMCs)and the breakage of elastic fibers.Multiple types of programmed cell death have been reported to participate in the death of SMCs during the development of AD,such as autophagy,necroptosis and apoptosis.Ferroptosis is a newly form of programmed cell death,driven by the iron-dependent accumulation of lipid hydroperoxides.There is growing evidence that autophagy can promote ferroptosis by degrading ferritin.Autophagic cell death of aortic SMCs is crucial for the development of AD.However,whether ferroptosis contributes to SMCs loss during the formation of AD remains unknown.It has been reported that N6-methyladenosine(m6A)RNA methylation is involved in various cardiovascular diseases.Methyltransferase-like 3(METTL3),the major methyltransferase,installs m6A on RNA molecules.METTL3 has been reported to participate in the regulation of autophagy,and autophagy is closely related to ferroptosis and AD.However,whether METTL3 affects the development of AD through regulation of ferroptosis is unclear.This study found that ferroptosis was involved in the development of AD,and the RNA m6A mechanism that regulates ferroptosis in SMCs was further explored to provide theoretical basis and molecular strategies for delaying the pathological process of AD.Methods:Several types of programmed cell death have been shown to participate in the development of AD.Ferroptosis is a newly identified type of programmed cell death.To investigate whether ferroptosis participates in the occurrence of AD,a RNA-sequence dataset of AD samples in human was downloaded from gene expression omnibus(GEO)database for bioinformatics analysis.To further confirm ferroptosis involvement in the development of AD,we detected the expression levels of the ferroptosis-regulating molecules in human aortic tissues of non-AD and Stanford type A AD(TAAD)patients via immunohistochemical staining and western blot analysis.To investigate the effect of ferroptosis on AD,we treated BAPN-induced mice with the ferroptosis-specific inhibitor liproxstatin-1.Several studies have reported that RNA m6A methylation is closely related to aortic diseases.To analyse the expression patterns of RNA m6A methylation regulatory molecules in AD,we detected the protein levels of m6A modulators in non-AD and TAAD aortic tissues by using western blot analysis.A tissue microarray including aorta samples from 30 nonAD and 60 TAAD patients was performed to evaluate the medial degeneration of the aorta,elastin fiber fragmentation,and the expression patterns of m6A modulator by using HE,EVG and immunohistochemical staining.METTL3,the major methyltransferase,has a regulatory effect on autophagy,and autophagy is closely related to ferroptosis and AD.To elucidate the affection of METTL3 on ferroptosis and AD,we made correlation analysis between the protein levels of METTL3 and the ferroptosis key regulatory molecules,solute carrier family 7 member 11(SLC7A11),ferroptosis suppressor protein 1(FSP1)and glutathione peroxidase 4(GPX4)in human aorta samples.The expression levels of METTL3,SLC7A11,FSP1 and GPX4 were detected in the aortas of mice stimulated with BAPN and human aorta SMCs(HASMCs)to further investigate the correlation between them.Since studies have reported that m6A methylation of SLC7A11 mRNA can promote its degradation,we used Actinomycin D treatment to inhibit mRNA synthesis and detect the effect of METTL3 overexpression on the stability of SLC7A11 and FSP1 mRNA.To investigate the effect of METTL3 on ferroptosis of HASMCs,we subjected HASMCs to cystine deprivation or treated them with IKE to induce ferroptosis.Iron assay kit was used to detect the content of ferrous ions in HASMCs.Cell viability and injuried cells were analyzed by using Cell Counting Kit-8(CCK8)and lactate dehydrogenase(LDH)release assays.We further evaluated the impact of METTL3 on lipid peroxidation by using BODIPY-C11 fluorescent probe,MDA kit and 4-HNE staining.The protein levels of SLC7A11 and FSP1 were evaluated in HASMCs with overexpressed METTL3 or METTL3 knockdown under cystine deprivation treatment.SLC7A11 or FSP1 was overexpressed in HASMCs with METTL3 overexpression to reverse cystine deprivation-and IKE-induced ferroptosis.Cell viability,cell injury and lipid oxidation were detected to further investigate the molecular mechanisms that mediate the function of METTL3 in ferroptosis in HASMCs.Results:From bioinformatics analysis,ferroptosis was found the potential programmed cell death involved in the occurrence of AD.The protein levels of ferroptosis key regulatory molecules,SLC7A11,FSP1 and GPX4 were downregulated in aortic tissues of TAAD patients.Liproxstatin-1,a specific inhibitor of ferroptosis,remarkably reduced the incidence and mortality of AD induced by BAPN in mice,protected the mice from aortic dilatation,medial degeneration and breakage of elastic fibers in the aorta.The aortas of TAAD patients exhibited medial degeneration and elastin fiber fragmentation.The expression of METTL3,a major methyltransferase of RNA m6A modulators,was greatly enhanced in aortic tissues of TAAD patients,and mainly expressed in the nucleus.The expressions of SLC7A11 and FSP1 were significantly negatively regulated by METTL3.METTL3 overexpression accelerated the mRNA decay of SLC7A11 and FSP1 in HASMCs.Overexpression of METTL3 accelerated HA SMC death induced by cystine deprivation or IKE.However,METTL3 knockdown protected HASMCs from cystine deprivation-and IKE-induced ferroptosis.SLC7A11 or FSP1 significantly offset the reduced cell viability and increased cell injury and lipid oxidation caused by overexpression of METTL3 in HASMCs treated with cystine deprivation or IKE.Conclusions:The study has revealed that ferroptosis is a critical cause of AD in both humans and mice.The expression level of METTL3 was largely increased in aortic tissues of TAAD patients and was negatively correlated with the expression of the ferroptosis regulatory factors SLC7A11 and FSP1.Furthermore,METTL3 facilitated ferroptosis of SMCs by inhibiting the expression levels of SLC7A11 and FSP1,and suppressing ferroptosis greatly reversed BAPN-induced AD in mice.Thus,targeting ferroptosis or RNA m6A methylation is expected to become a new potential strategy for delaying AD.