The Senescence Mechanism Of Vascular Progenitor Cells In Patients With Marfan Syndrome Via TGF-?/AMPK Pathway

Objective:Cardiovascular diseases are the main cause of morbidity and mortality in patients with Marfan syndrome(MFS),and vascular progenitor cells(VPCs)present in the adventitia of the vessel wall play a critical role in the regulation of vascular repair following injury.This study aimed to investigate the senescence mechanism of VPCs in MFS patients,reveal the cause of vascular dysfunction,pave the way to restoring the function of VPCs and provide a novel strategy for treatment of MFS.Methods:c-kit~+VPCs were isolated from aortic tissues in organ donors and patients with MFS respectively.The proliferation,migration ability and senescence degree of the two VPCs were detected by Ki67 proliferation assay,Transwell assay,SA-?-gal staining and the expression level of senescence associated protein p53/p21.RNA-seq analysis was performed to define the differentially expressed genes between control-VPCs and MFS-VPCs.To Explore the role of TGF-?in VPCs senescence,TGF-?1(50ng/ml)was applied into the medium of donor VPCs(control-VPCs)for 48h.And the TGF-?1 protein synthesis of VPCs from MFS(MFS-VPCs)were reduced by TGF-?1-siRNA,then the senescence degree of the two cells was detected by SA-?-gal staining.TGF-?1 and TGF-?1+Mito-Tempo were added into the culture medium of control-VPCs,and Mito-Tempo(10uM)was added to MFS-VPCs for 48h to explore the role of Mito-SOX in cell senescence.TGF-?1and Mitofusion2-siRNA were added into control-VPCs medium,and Mitofusion2-siRNA was added into MFS-VPCs medium for 48 h to investigate the effect of mitochondrial function in the senescence of MFS-VPCs.TGF-?1 and TGF-?1+AICAR(AMPK activator)were added into control-VPCs medium for 48 h,and AICAR(1 mM)was added into MFS-VPCs to investigate the role of AMPK signaling pathway in cellular senescence.Results:Compared with control-VPCs,the proliferation and migration ability of MFS-VPCs decreased and exhibited cellular senescence with increased cell size,higher SA-?-gal activity and elevated levels of p53 and p21.RNA-sequencing showed that several cellular process-related pathways including cell cycle and cellular senescence were significantly enriched in MFP-VPCs.Moreover,MFS-VPCs expressed high level of TGF-?,mitochondrial fusion increased,highly expressed of Mito-SOX,and p-AMPK expression is decreased.TGF-?induces mitochondrial ROS production and mitochondrial fusion of control-VPCs,and the addition of ROS removers,as well as inhibition of mitochondrial fusion protein expression,can alleviate cellular senescence.Moreover,TGF-?can inhibit the AMPK signaling pathway,and activation of this pathway can ameliorate TGF-?-induced cellular senescence.Conclusion:The high expression level of TGF-?in patients with Marfan syndrome lead to mitochondrial dynamics change and increased mitochondrial ROS production which caused VPCs senescence and dysfunction by inhibiting AMPK signaling pathway.