Mechanobiological Study On The Role Of TET1s In Vascular Endothelial Dysfunction Responses To Low And Oscillatory Shear Stress

At present,cardio-cerebral diseases are the leading cause of death in our country,and the disease burden is becoming more and more serious,which has become a major public hygiene issue.Atherosclerosis?AS?is the important pathological basis of ischemic cardiocerebral vascular disease which involves large and medium-sized arteries.Therefore,in-depth study of the pathogenesis of AS is of great significance for the prevention and treatment of cardio-cerebral vascular disease.Clinical studies have found that AS lesions occur in areas with irregular blood flow shear stress such as vascular stenosis,bending and bifurcation,suggesting that abnormal local stress stimulation is one of the important factors for AS formation.Vascular endothelial cells?VEC?are the most direct mechanoreceptors of blood vessels whose function changes are closely related to AS.Under low and oscillatory shear stress?OSS?,VEC are dysfunctional which can promote AS development,while under laminar shear stress?LSS?,VEC functions normally which can prevent AS formation.However,the molecular mechanism by which blood flow shear stress directs the biological function of the VEC has not been fully elucidated.TET1?10-11 translocation protein 1?is a key protein that catalyzes DNA demethylation and is highly expresses in early embryonic cells.Recent studies have identified TET1s,a truncated isoform of TET1,whose protein structure lacks the CXXC binding domain and is widely expressed in somatic tissues.Studies have shown that TET1s regulate gene expression through non-DNA demethylation mechanism.Our previous study found that OSS inhibited TET1 expression,and there was no significant change of DNA demethylation level in HUVEC.Based on these observation,we speculate that TET1s substantially responses to blood flow shear stress,and participates in the formation of AS by regulating the VEC function independent of the DNA demethylase activity.In this study,we investigate the role of TET1s in regulating the function of VEC in response to OSS and its underlying mechanical and biological mechanisms based on in vivo and in vitro models.The main research contents and conclusions are as follows:1.Mice carotid model was succesfully established,and it was found that OSS affects the normal endothelial structure and function in the early stage of AS.After partial ligation,ultrasonic was used to confirm that the mice left carotid artery?LCA?and right carotid arteries?RCA?exhibited OSS and LSS,respectively.The vascular tissue staining showed that OSS promoted AS lesions formation.The abnormal expression of adhesion factor ICAM-1 and VCAM-1 and the macrophage marker CD68were detected at 24h after ligation.Excessive proliferation of VEC was also observed at48h post-ligation.In vitro flow chamber mechanical loading of human umbilical vein endothelial cells?HUVEC?revealed that LSS maintained normal cell morphology and cytoskeleton arrangement,while OSS disrupted cell morphology and cytoskeleton.In addition,OSS also increased the amount of adhesion of monocyte THP-1 to HUVEC.2.It was revealed for the first time that TET1s is a shear stress sensitive protein,and it was clarfied that OSS promotes abnormal proliferation and inflammatory response of VEC by repressing TET1s expression.Through the experiments of qPCR,En face staining and western blot,it was found that TET1s was highly expressed in normal VEC.Subsequently,it was confirmed by mouse model that the expression of TET1s was significantly decreased in the OSS region?e.g.lesser curvature of aorta,LCA of ligation mice model?.In vitro mechanical loading experiments also confirmed that OSS inhibited the expression of TET1s.By using small interfering RNA,we found that knockdown of TET1s promotes HUVEC proliferation and inflammation.Infection of HUVEC with TET1s overexpressing adenovirus inhibited excessive proliferation induced by OSS.At the same time,overexpression of TET1s also reduced the endothelial inflammation induced by LPS.3.OSS down-regulates TET1s expression through inhibiting C/EBP beta.The potential mechanically sensitive transcription factor C/EBP beta was screened by bioinformatics analysis of the TET1s promoter sequence.We treated HUVEC with H-89,an inhibitor of C/EBP beta upstream signaling pathway PKA,and found that the expression of TET1s was significantly inhibited based on the inhibition of C/EBP beta and its phosphorylation level.4.There is physical interaction between TET1s and YAP,and TET1s mediates the nuclear activation of YAP by OSS.By using mechanical loading and TET1s small interference RNA treatment,it was confirmed that change in TET1s expression did not affect the DNA demethylation level of VEC.Immunofluorescence staining showed that overexpression of TET1s in vitro enhanced the cytoskeletal rearrangement of HUVEC.Subsequently,the physical interaction between TET1s and the mechanical effector YAP was confirmed by co-immunoprecipitation and immunofluorescence colocalization experiments.Both OSS treatment and knockdown of TET1s can promote the nuclear shuttling of YAP,while overexpression of TET1s inhibits the nuclear activation of YAP induced by OSS.OSS induces YAP nuclear import by inhibiting YAP?Ser127?site phosphorylation and promoting YAP?Tyr357?site phosphorylation.Knockdown of TET1s caused a decrease in p-YAP^S127 levels and an increase in p-YAP^Y357 levels,which was consistent with the observation under OSS loading.Overexpression of TET1s partially rescued changes in p-YAP^S127 and p-YAP^Y357 caused by OSS.Finally,qCR detection revealed that knockdown of TET1s expression led to up-regulation of YAP target genes expression,whereas inhibition of YAP activity with inhibitor verteporfin attenuated the abnormal up-regulation of genes caused by TET1s,suggesting that TET1s affects endothelial function by regulating YAP transcriptional activity.In summary,this study indicates that TET1s is an important mechanically sensitive protein that mediates the process of endothelial abnormal proliferation and inflammation induced by OSS.Besides,the effect of TET1s on YAP transcriptional activity in response to shear stress stimulation may provide the underlying mechanobiological mechanisms of OSS induced abnormal endothelial proliferation and inflammation.Therefore,OSS induced abnormal expression of TET1s may be an important inducement and a potential therapeutic target for AS.