TET1s Deficiency Exacerbates Oscillatory Shear Flow-induced Atherosclerosis And The Mechanism

Cardiovascular diseases（CVD）are a group of medical conditions which related to the lesion of heart and blood vessels,mainly including coronary artery disease,cerebrovascular disease and peripheral artery disease.CVD are the leading cause of mortality globally.It brings long-term physical and psychological pain to patients,and a heavy burden to patients’families and society,because high incidence and difficulty to cure,which has become a global public health problem.Atherosclerosis（AS）is one of the main factors to lead CVD.Therefore,it is of great significance to further study on the pathogenesis of AS for the prevention,detection and treatment of CVD.Increasing evidence show that the focal lesion of AS is related to hemodynamics.Atherosclerotic plaques were more prone to occur in vessels that were stimulated by Oscillatory shear stress（OSS）,such vessel curve and branch,while plaques were less common in straight vessels stimulated by laminar shear stress（LSS）.AS starts with the endothelium is damaged.Endothelial cells（EC）are a single layer lining the lining of blood vessels which is essential for maintaining healthy arteries.The abnormal function of endothelial cells stimulated by OSS is the main reason for the occurrence and development of AS.Vascular endothelial cells directly perceive mechanical stimuli from blood flow,and conduct mechanical signals into the cell through a complex mechanical-biological signal transduction system,and make further biological responses.TET1（Tet methylcytosine dioxygenase 1）is a dioxygenase and it mediates DNA demethylation.In recent years,studies have reported that TET1 is involved in the AS occurrence and development by regulating the endothelial-to-mesenchymal transition（End MT）.The isoform of TET1（TET1 short,TET1s）was recently discovered that lacks the full-length amino-terminal 653 amino acids,but shares most of the protein sequence of canonical TET1.TET1s are more likely to be expressed in adult cells,whereas canonical TET1（TET1-FL）is less expressed in adult cells.Previous studies by our group have shown that TET1s are more highly expressed in ECs than TET1-FL,and their expressions are regulated by blood flow shear stress.Therefore,we speculate that TET1s is further involved in the AS occurrence and development by regulating the vascular endothelial cell function.In our research,we mainly used gene knockout mice TET1^-/-,TET1^cs/csApo E^-/-,Apo E^-/-TET1^-/-and Apo E^-/-TET1^CS/CS and primary human umbilical vein endothelial cells（p-HUVECs）as in vivo and in vitro model,combined with a mouse partial carotid artery ligation model and an in vitro parallel plate flow chamber model,to explore the effects and regulatory mechanisms of TET1s on the AS occurrence and development.The main research methods and results are as follows:1.Deletion of TET1s accelerates the development of OSS-induces AS.Two gene knockout mice,Apo E^-/-TET1^-/-and Apo E^-/-TET1^CS/CS,were constructed in the background of Apo E^-/-mice.TET1^-/-mice express neither TET1-FL nor TET1s,and TET1^CS/CS do not express TET1-FL,but TET1s.By comparing the overall plaque occurrence in the aorta of the two mice,we confirmed that the deletion of TET1s promoted the AS development.By comparing the plaques in different segments of the mouse aorta,and by partially ligating the carotid arteries of ApoE^-/-TET1^-/-and ApoE^-/-TET1^CS/CS mice,the blood flow environment of OSS was artificially constructed in mice.Comparing the plaque of ligated left（OSS）and right common carotid arteries（LSS）showed that TET1s deletion accelerated the development of OSS-induced AS.2.TET1s enhance endothelial barrier.To explore changes in vascular permeability in mice,we injected Evans blue and nanoscale erythrocyte membranes to TET1^-/-and TET1^CS/CS mice by tail vein injection,and calibrated vascular intimal permeability by analyzing the deposition of Evans blue and erythrocyte membranes on the vessel wall.The results showed that deletion of TET1s increased the deposition of Evans blue and erythrocyte membranes on the vessel wall,indicating that deletion of TET1s impairs the intimal barrier of vessels.At the same time,we analyzed the invasion of inflammatory cells and erythrocytes in the AS plaques of Apo E^-/-TET1^-/-and Apo E^-/-TET1^CS/CS mice by immunohistochemistry,and the results showed that TET1s deletion increased inflammatory cells and erythrocytes in the plaques invasion.The adenoviruses transfected p-HUVECs overexpressed TET1s,and the differences in gene expression of HUVECs between the TET1s overexpression group and the control group were compared,and the differential genes were found to include cell junction and transendothelial migration clusters,indicating that TET1s may be involved in regulating endothelial cell barrier function.Overexpressed TET1s did enhance endothelial barrier by Transwell-permeability assay.By immunofluorescence analysis of endothelial cytoskeleton F-actin and adherens junction VE-cadherin in overexpressed TET1s cells,it was found that overexpressed TET1s enhanced endothelial barrier may be related to cellular F-actin and VE-cadherin.3.TET1s overexpression enhances endothelial barrier by inhibiting histone deacetylase to promote CX40（Connexin 40）.By comparing the differences in the gene expression of p-HUVECs between the TET1s overexpression group and the control group,it was found that the expression of cell junction protein CX40 increased the most.Using plasmid transfection to knockdown CX40 and then overexpress TET1s,it was found that knockdown CX40 significantly reduced the TET1s overexpression-induced endothelial barrier enhancement.During this process,endothelial cytoskeleton F-actin strength and adhesion junction protein VE-cadherin stability were also significantly reduced.To further reveal how TET1s promotes CX40,overexpressed TET1s did not alter the methylation and hydroxymethylation levels of cellular genomic DNA,nor the methylation level of the CX40 promoter region,indicating that TET1s promotes CX40expression not through demethylation base.By analyzing the acetylation levels of histone H3K27 in the global histone and CX40 promoter region in ECs overexpressing TET1s,it was found that overexpressed TET1s increased cellular global H3K27acetylation and CX40 promoter region H3K27 acetylation.Further studies revealed that TET1s could interact with the Sin3a/HDAC complex and inhibit its histone deacetylation.Taken together,this study demonstrated that deletion of TET1s impaired endothelial barrier function and significantly increased OSS-induced AS in vivo.TET1s overexpression in vitro increases the interaction between TET1s and Sin3a/HDAC complex,inhibits histone deacetylation in the CX40 promoter region,and then promotes CX40 expression,which enhances the endothelial barrier.These results suggest that TET1s are an important protective factor of the vascular endothelial barrier and plays an important role in OSS-induced AS plaques.TET1s overexpression may be a core therapeutic strategy for OSS-induced AS.