The Mechanism Of TGF-? Mediating Aortic Smooth Muscle Cell Senescence In Marfan Syndrome

Objective:Adult aortic root expansion and aortic aneurysm formation are the most common cardiovascular manifestations and complications in the patients with Marfan syndrome(MFS).Dysfunction of vascular smooth muscle cells(VSMCs)in the medial wall of the aorta contributes to the disease.This study aimed to investigate the mechanisms of VSMC senescence in Marfan syndrome patients,and provides a new strategy and theoretical basis for the early intervention and treatment of Marfan syndrome aortic lesions in clinicMethods:Donor VSMCs(control-VSMCs)and Marfan syndrome VSMCs(MFS-VSMCs)were isolated from aortic tissues in organ donors and patients with MFS respectively.First,H&E staining,SA-?-gal staining,Ki67 proliferation assay,and immunofluorescence staining were used to compare the differences in the proliferation and senescence of VSMCs in aortic tissue between the two groups.Then we detected the concentrations of IL-6,IL-8,TNF-?,and INF-? in cell culture medium of control-VSMCs and MFS-VSMCs by ELISA,respectively.To investigate the effect of TGF-? in the senescence of aortic VSMCs,control-VSMCs were divided into the following three groups,and received TGF-?1(50ng/ml)and TGF-?1+TGF-?1-siRNA treatment for 48 h,and then the VSMC senescence of the three groups was measured.The levels of reactive oxygen species(ROS)in aortic tissue from control donors and MFS patients were detected by DHE staining,The mitochondrial ROS levels in control-VSMCs and MFS-VSMCs were detected by Mito-Sox staining.TGF-?1 and TGF-?1+Mito-Tempo(10uM)were added into the culture medium of control-VSMCs to investigate the effect of TGF-? induced mitochondrial ROS generation and the senescence of control-VSMCs.TGF-?1,TGF-?1+ Mito-Tempo and TGF-?1+SC-514(NF-?B Inhibitor)were added into control-VSMCs culture medium for 48 h to investigate the role of ROS/NF-?B signaling pathway in VSMC senescence.Results:The aortic tissue sections from patients with MFS displayed the typical characteristics of aneurysm,including increased dilation and degeneration of the medial layer of the aorta.Compared with control donors,levels of the cellular senescence markers p53 and p21 were significantly higher in aortic tissue from MFS patients.Immunofluorescence staining revealed that senescence mainly localized to medial VSMCs.Compared with control-VSMCs,MFS-VSMCs greatly enlarged and flattened,the proliferation ability decreased and higher SA-?-gal activity,increased SASP secretion and elevated levels of p53 and p21.The level of TGF-?1 was markedly upregulated in both the serum and aortic tissue from MFS patients than control donors.The addition of TGF-?1 can induce VSMCs senescence,while inhibition of TGF-?1 can reduce the senescence of VSMCs.High concentrations of TGF-?1 can stimulate ROS generation and lead to the activation of NF-?B.The activation of the ROS/NF-?B signaling pathway released SASP and induced the senescence of VSMCs under its combined effect.Conclusion:Highly expressed TGF-? in patients with Marfan syndrome stimulated excessive generation of ROS,which increased SASP secretion,decreased cell function,increased expression of senescence-related proteins and activated NF-?B.The activation of the ROS/NF-?B signaling pathway further promoted the generation of SASP,and together induced the senescence of VSMCs.