LincRNAp-21 Induces Abdominal Aortic Aneurysm By Promoting Synthetic And Pro-inflammatory Smooth Muscle Cell Phenotype

Background:Abdominal aortic aneurysm(AAA),defined as abdominal aorta with diameters>1.5 times their normal size,is a severe fatal disease with a mortality rate of up to 80%after rupture.However,there is still no effective drug therapy for AAA except surgical treatments,such as open repair and endovascular aneurysm repair,which is mainly attributed to the lack understanding of the mechanism AAA.It is generally believed that the transformation of vascular smooth muscle cells(VSMCs)from contractile phenotype to synthetic phenotype is an early stage of AAA development and plays an important role in the AAA formation.Our research team has previously found that long non-code RNA participates in the development of AAA by regulating VSMCs inflammation and apoptosis,but whether lncRNA mediates VSMCs phenotypic transformation and participates in AAA formation is unknown.We previously found that SM22?,a phenotype transformation regulatory protein of VSMCs,mediates the occurrence of AAA through ROS.Meanwhile,previous studies have found that ROS partpant in cardiovascular disease by modifing the activity of transcription factor p53 in different ways,and lincRNA-p21 was found to be a direct transcription target of p53.Therefore,we speculate that lincRNA-p21 may participate in the phenotype transformation of VSMC.Previous studies have found that lincRNA-p21 participates in the development of atherosclerosis by regulating the proliferation and apoptosis of VSMCs.However,the role of lincRNA-p21 in the phenotypic transformation of VSMCs and whether it mediates the development of AAA through the phenotypic transformation of VSMC have not been reported.The purpose of this study is aim to investigate the role of lincRNA-p21 in VSMC phenotype transformation and AAA formation in vitro and in vivo.Methods and Results:In situ hybridization and RTq-PCR showed that theexpression of lincRNA-p21 was significantly different betweenhuman normal abdominal aorta and AAA tissue,as well as in mouse normal abdominal aorta and mouse AAA tissue.In vitro,overexpression of lincRNA-p21 promote the expression of synthetic markers such as Opn and myh10,extracellular matrix metalloproteinase markers such as MMP1,MMP2,MMP3 and MMP9,and inflammatory markers such as IL-1b,IL-6 and Vcam1.Furthermore,the fluorescenceintensity changes of Opn,MMP2 and Vcam1,the respective markers of synthesis,matrix metalloproteinase and inflammatory phenotype,were observed by knocking down and overexpression of lincRNA-p21 on the basis of hydrogen peroxide(H2O2)stimulation in vitro,which promote VSMCs transforming to synthetic phenotype.VSMCs culture dishes were randomly divided into 6 groups:OE-lincRNA-p21 group,control group without any treatment,H2O2+SCR-siRNA group,H2O2+lincRNA-p21-siRNA group,H2O2+Vector group,and H2O2+O OE-lincRNA-p21 group.It was found that the fluorescence intensity of the three proteins increased in the OE-lincRNA-p21 group and H2O2 stimulation group,with the lowest in the untreated control group,and the highest in H2O2+OE-lincRNA-p21 group.Moreover,on the basis of H2O2 stimulation,the fluorescence intensity of these proteins was decreased when lincRNA-p21 was knocked down.In vivo,knocking down lincRNA-p21,as compared with compared with the control group,in Ang?-induced AAA model significantly reduce the rupture of vascular elastic fibers,the diameter of abdominal aorta,and the rate of aneurysm formation and improve the survival of mice.Compared with control group,overexpression of lincrnap21 in C57 mice stimulated by Ang? significantlyincrease the rupture of vascular elastic fibers,the diameter of abdominal aorta,and the rate of aneurysm formation and the mortality of mice.Furthermore,it was found that the decreased rate of AAA induced by Ang? in ApoE-/-mice after knockdown of lincRNA-p21 was achieved by reducing the expression of Opn,MMP2 and Vcam-1 in VSMCs,while the increased rate of AAA induced by Ang? in C57 mice after overexpression of lincRNA-p21 was achieved by increasing Opn,MMP2 and Vcam-1 in VSMCs.In mechanism,we found that lincrnap21 functions mainly by regulating Akt pathway.Conclusion:LincRNA-p21 induces abdominal aortic aneurysm by promoting synthetic and pro-inflammatory smooth muscle cell phenotype,which therefore may be a potential new target for clinical treatment of AAA.