The Role And Mechanism Of Telomerase Reverse Transcriptase In Phenotypic Transformation Of Smooth Muscle Cells And Thoracic Aortic Aneurysm

BackgroundAortic aneurysm is a disease that the structural changes of the aortic wall caused by a variety of factors lead to the progressive expansion of the aortic lumen,which exceeds 50%of the normal diameter.Thoracic aortic aneurysm(TAA)is a kind of aneurysm occurring in the aortic sinus,ascending aorta,aortic arch or descending aorta.Compared with other common cardiovascular diseases,it has a hidden onset and is usually asymptomatic in the early stage,in addition,the incidence rate of TAA is low.With the progress of TAA,aortic emergency may occur and lead to lethal consequence.At present,there is no early diagnostic method and effective prevention and treatment drugs for TAA.Therefore,illustrating the pathogenesis of TAA has significant theoretical value for the study of its diagnostic markers and drug targets.The thoracic aortic wall is composed of the intima,the medial layer and the lateral layer.The medial layer is the main component to maintain the tension and strength of the aortic wall.Vascular smooth muscle cells(VSMCs)are the main cellular component of the middle layer of aorta.Phenotypic transformation of VSMCs is a vital role in the development of thoracic aortic aneurysms.There are two types in vascular smooth muscle cells,including contractile and synthetic phenotypes.In quiescent state,most of the VSMCs are contractile phenotypes,with long fusiform cell morphology,strong contractile ability but weak proliferation and migration ability.Smooth Muscle 22 Alpha(SM22α),α-smooth muscle actin(α-SMA),and other contractile related proteins are highly expressed.Of note,the synthetic phenotypes have great effects on vascular development and disease process.VSMCs with synthetic phenotypes present polygonal shape,accompanied with the enhanced secretion,proliferation and migration capabilities.Osteopontin(OPN),proliferating cell nuclear antigen(PCNA),reactive oxygen species(ROS)and matrix metalloproteinases(MMP)are up-regulated.As a result of factors such as MMP and ROS,extracellular elastic fibers are gradually degraded,which then weakens the tension of thoracic aortic wall and finally causes the occurrence of TAA.Telomerase is a kind of enzyme responsible for telomere elongation,which plays a vital role in maintaining telomere stability,genome integrity,cell long-term activity and potential continued proliferation ability.Telomerase reverse transcriptase(TERT)is a crucial component of telomerase.Maintaining telomere length after mitosis has long been considered to be the most important function of TERT.While a growing number of studies show that TERT also plays a significant role in cardiovascular diseases.TERT may participate in the pathogenesis of atherosclerosis by regulating the proliferation cycle of VSMCs.The change of proliferative ability is one of the essential characteristics of smooth muscle cell phenotypic transformation from contractile to synthetic phenotypes.In summary,we proposed a scientific hypothesis:TERT may participate in the pathogenesis of TAA by regulating the phenotypic transformation of VSMCs.Objectives1.To clarify the expression of TERT in human TAA tissue and mouse model of TAA.2.To investigate the regulatory effect of TERT on phenotypic transformation of VSMCs.3.To explore the mechanism by which TERT participates in the pathogenesis of TAA by regulating VSMCs phenotype.Methods1.Specimen collection and cell culture(1)Specimen collection:22 cases of TAA confirmed by ultrasound and CTA were included in the study after excluding genetic diseases such as Marfan syndrome,immunoinflammatory diseases such as great arteritis,and aortic aneurysms associated with bicuspid Aortic Valve.Meanwhile,tissues from 10 patients receiving heart transplantation were selected as normal controls.Immunohistochemistry and Western blot were used to detect the expression differences of TERT and VSMCs phenotypic markers between TAA group and control group.(2)Isolation and culture of VSMCs:Primary rat VSMCs were isolated and cultured by the method of aortic tissue attachment.Rats were sacrificed and thoracic cavity was opened under aseptic conditions,and the thoracic aorta tissue was separated into culture petri dishes under stereomicroscope.Then,the inner and outer membranes of the aorta were removed,and the remaining media layer was cut into 1mm~2 size and affixed to the 24-well plate with about 2-3 pieces of broken tissue in each hole.After adding about 300μl DMEM medium containing 20%fetal bovine serum(FBS)to each well,the primary VSMCs were cultured by adhesion culture until the VSMCs climbed out.(3)Identification of VSMCs:The expression ofα-SMA in primary cells obtained by the above procedure was determined by immunofluorescence assay.2.Establishment of a mice model of TAA and detection of TERT and VSMCs phenotypic markers(1)Establishment of mouse model of TAA:subcutaneous angiotensin II(AngⅡ)sustained-release pump was inserted in the scapular area of C57BL/6 mice for continuous intervention for 28 days to establish a mouse model of TAA.Normal saline sustained-release pump was set as control group.At 0,1,2,3 and 4 weeks of the experiment,ultrasound examination and tissue sampling of thoracic aorta were performed.(2)Detection of TERT and VSMCs phenotypic markers:Expression differences of TERT and VSMCs phenotypic markers in TAA group and control group were detected by immunohistochemistry.3.The function of TERT in phenotypic transformation of VSMCs(1)Cell model:VSMCs were stimulated with platelet-derived growth factor(PDGF-BB)at 10μg/m L to induce the synthetic phenotypic model.Meanwhile,VSMCs were cultured in DMEM medium containing 10%,5%and 1%FBS successively to construct the phenotypic transformation model of VSMCs.The expression of TERT and VSMCs phenotypic markers in the two cell models was detected.(2)Overexpression of TERT in VSMCs:The amount of virus was calculated based on the TERT-associated lentivirus titer and the appropriate Multiple of infection(MOI)value.The virus was added to the culture medium to infect VSMCs to overexpress TERT,and the expression of VSMCs phenotypic markers was subsequently detected.(3)Inhibition of TERT expression in VSMCs:lipofectomne 3000 liposomes were used to transfect an appropriate concentration of TERT specific si-RNA into VSMCs to inhibit the expression of TERT,and then the expression of VSMCs phenotype markers was detected.(4)CCK-8 assay:CCK-8 Kit was used to detect the proliferative activity of VSMCs.(5)Scratch test:scratch test was used to test the migration ability of VSMCs.4.Mechanism of TERT regulating phenotypic transformation of VSMCs(1)The expression of TERT and autophagy related proteins was detected in synthetic VSMCs.(2)Construction of VSMCs autophagy defect model:Synthetic VSMCs were stimulated with 3-methyladenine(3-MA)to induce the autophagy defect model,and the expression of TERT,phenotypic markers of VSMCs and autophagy-related proteins was detected.(3)Overexpression of TERT in VSMCs:TERT was overexpressed by transfection of TERT-associated lentiviruses into VSMCs,and the expression of TERT,autophagy related proteins and phenotypic markers of VSMCs was detected.(4)TERT was overexpressed by transfection of TERT-associated lentivirus into 3-MA treated VSMCs,and the expression of TERT,autophagy related proteins and phenotypic markers of VSMCs was detected.5.Statistics and analysis of experimental dataSPSS 20.0 software was used to process the experimental data.T test or Wilcoxon rank sum test was used to compare the two groups.P<0.05 meant that data could be used.Results1.TERT expression is up-regulated in TAA,and phenotypic transformation of VSMCs occurred in TAA.Hematoxylin-eosin(HE)staining and Victoria blue(VB)staining results showed structural changes in the membranes of the vascular wall of TAA,rupture of elastic fibers and disordered collagen fibers.Western blot and immunohistochemical results showed that the expression of TERT was increased in human TAA tissue and mouse TAA model,and the expression of OPN and PCNA of VSMC synthesis phenotypic marker was increased,On the contrary,the expression of SM22αwas decreased.The results of this part suggest that TERT expression is up-regulated in TAA,and phenotypic transformation of VSMCs occurred in TAA.It implies that there may be a correlation between TERT and the process of phenotypic transformation of VSMCs.2.TERT plays a regulatory role in phenotypic transformation of VSMCsWestern blot was used to detect the expression of TERT and VSMCs phenotypic markers in the synthetic VSMCs model and VSMCs phenotypic transformation model.The results showed that the expression of TERT,OPN and PCNA was increased and the expression of SM22αwas decreased in VSMCs stimulated by PDGF-BB at a concentration of 10μg/m L.The expression of TERT,OPN and PCNA was gradually down-regulated and the expression of SM22αwas gradually up-regulated in VSMCs cultured in DMEM medium with FBS concentration of 10%,5%and 1%successively.Western blot results showed that the expression of OPN and PCNA was increased and the expression of SM22αwas decreased after overexpression of TERT in VSMCs.Proliferation ability of VSMCs were enhanced after overexpression of TERT testified by CCK-8.While the results of scratch experiment showed that overexpression of TERT enhanced the horizontal migration of VSMCs.In contrast,aforementioned results were reversed by inhibiting expression of TERT.Taken together,TERT plays a regulatory role in the phenotypic transformation of VSMCs,and overexpression of TERT could induce the transformation of VSMCs from contractile to synthetic phenotype.3.TERT regulates phenotypic transformation of VSMCs through autophagyThe expression of TERT and autophagy-related proteins was detected in the synthetic VSMCs model induced by PDGF-BB.Western blot results showed that the expression of TERT and Beclin1 was up-regulated in synthetic VSMCs,and the ratio of LC3II/I was increased.The expression of autophagy-related proteins and VSMCs phenotypic markers was detected in VSMCs autophagy-deficient model induced by 3-MA.Results showed that the expression of Beclin1 was down-regulated,the ratio of LC3II/I was decreased,the expression of OPN and PCNA was decreased and the expression of SM22αwas increased,while there was no significant change in TERT expression.After overexpression of TERT in VSMCs,the expression of TERT,autophagy-related proteins and phenotypic markers of VSMCs was detected.Western blot results showed that the expression of Beclin1 was up-regulated,the ratio of LC3II/I was increased,the expression of TERT,OPN and PCNA was increased and the expression of SM22αwas decreased.The expression of autophagy related proteins,VSMCs phenotypic markers,and TERT was detected by Western blot after TERT was overexpressed in autophagy deficiency models.Results showed that overexpression of TERT could reverse phenotypic transformation of VSMCs caused by autophagy deficiency.The results in this part suggest that TERT may regulate phenotypic transformation of VSMCs through autophagy pathways.ConclusionOur study shows that TERT expression is up-regulated in TAA,accompanied by the transformation of VSMCs from contractile phenotype to synthetic phenotype.TERT plays a vital part in the pathogenesis of TAA by regulating phenotypic transformation of VSMCs through the autophagy pathway.