Expression And Regulation Mechanism Of Endothelial-to-Mesenchymal Transition In Calcified Aortic Valve Disease

With the aging of the population,the incidence of calcified aortic valve disease(CAVD)is increasing year by year,and it has become the most common heart valvular disease,which brings a huge burden to clinical treatment and social economy.Early studies have suggested that CAVD is a passive process related to valvular degeneration and calcium deposition.Recent studies have found that the occurrence of CAVD is an active process involving endothelial injury,cell differentiation,lipid infiltration,chronic inflammation,matrix remodeling,progressive calcification and neovascularization.Cell-to-cell transition and gene mutations also play an important role in the pathogenesis of CAVD.Endothelial-to-mesenchymal transition(EndoMT)plays a crucial role in the development of embryonic cardiovascular system,myocardial fibrosis,atherosclerosis and pulmonary hypertension,whereas more and more evidence show that aortic valve endothelial cell(VECs)also has a phenotypic and functional transition to valvular interstitial cell(VICs)in CAVD.However,there are several challenges in this field,including the lack of a description of the changing trend of EndoMT throughout the disease;the difficulty of a complete and accurate definition of EndoMT and the inability to identify cells that have developed complete EndoMT by traditional methods;and the obstacle to trace the cellular function and pathogenesis of EndoMT.Therefore,the expression of EndoMT in the pathogenesis of CAVD was described in detail by using human lesion specimens and CAVD animal model.To improve the traditional CAVD animal model which consums too long time.The changes of valvular endothelial cells during the disease were traced by lineage tracing technique,so as to further explore the function of partial and complete EndoMT cells.To Analyze the sequencing data of valvular sample from patients with CAVD and using traditional molecular biology experimental techniques to explore the key target genes that may affect EndoMT and CAVD.It is significant to clarify the role of EndoMT in CAVD and provide a new strategy for prevention and intervention.Previous studies only studied EndoMT process in tissue sample and in vitro cell experiments.There is no study using in vivo CAVD animal model to prove the expression and role of EndoMT in early and late stages of CAVD progression.As a result,we constructed the CAVD animal models of rabbits and mice in the first part of this study.The aortic valve samples were collected and observed at different stages of the disease process.Finally,the expression of EndoMT in CAVD was verified at the pathological and molecular level using model samples combined with human aortic valve calcification samples.In the first part,we constructed the traditional animal model and observed the expression of EndoMT in the course of the disease.However,the traditional CAVD mouse model has some problems including long modeling time and low success rate.This study combined the experience of hyperlipidemic mouse model and wire injury mouse model to develop a hybrid CAVD mouse model,which is more consistent with the clinical course of disease and has a short modeling cycle,high success rate and good consistency.The role of EndoMT in the process of CAVD is still controversial in academic circles.Lineage tracing technique is the most direct and powerful evidence to study the origin of cells,the cell transition in the process of disease.Therefore,this study established endothelial lineage tracing mice CAVD model to observe the fate and transition of VECs in the pathological process of lineage tracing mice CAVD model.There are no surgical methods to prevent or slower CAVD process due to the dimness of the mechanism The first two parts of the study found that EndoMT plays a crucial role in CAVD.The essence of EndoMT is the interaction and transition of VECs and VICs.We analyzed the human valvular sequencing data in the third part study and found two key genes—DUSP1 and PDK4.We investigated the function and related pathway of the two genes through in vitro experiments.It was found that DUSP1 may inhibit the calcification of VICs.Meanwhile,knocking down PDK4 inhibited the EndoMT process of VECs.The results provided new potential targets for the treatment of CAVD.Part ?:Expression of Endothelial-to-Mesenchymal Transition in Calcified Aortic Valve DiseaseBackground:Calcified aortic valve disease(CAVD)is is a serious threat to human health and a leading cause of mortality worldwide,and little is known about the mechanisms of pathogenesis.Endothelial-mesenchymal transition(EndoMT)plays a key role in the development of aortic valve in embryonic stage,and the occurrence and development of CAVD in adulthood may be related to the reactivation of EndoMT.EndoMT may be a valuable therapeutic target for the prevention and treatment of CAVD diseases.The purpose of this study was to investigate the expression of EndoMT and related transcription factors in calcified aortic valve.Methods:Calcified valve and normal valve were collected to verify the EndoMT phenomenon,the rabbit CAVD model was constructed,and the rabbit aortic valve tissues of the high-fat group and the control group were taken at 4,8 and 12 weeks,respectively,and the CAVD model of ApoE-/-mice fed with high-fat was established,and the aortic valves of mice were taken at 8,16,20,24,28,32,36 and 40 weeks respectively.To improve the decalcification method of calcified valve.H&E and Masson staining were used to observe valve morphology and structure;Alizarin red staining and Von Kossa staining were used to detect valve calcium deposits;the co-localization expression of endothelial markers CD31 and VE-cadherin with interstitial markers ? SMA,vimentin and EndoMT transcription factor SNAIL1/2 were detected by immunohistochemistry and immunofluorescence.The expression levels of EndoMT-related RNA in human and mouse aortic valves were detected at the molecular level.Results:It was found that endothelial and interstitial markers were co-expressed in the endothelial region of calcified aortic valve sections of human and rabbit,and EndoMT transcription factor was highly expressed in calcified group of mice.Compared with normal valves,there were significant differences in RNA expression levels of endothelial,interstitial markers and EndoMT-related transcription factors in human and mouse calcified valves.Conclusions:In this study,human aortic valve samples and CAVD animal model samples were used to verify the existence of EndoMT phenomenon in CAVD at the pathological and molecular level.The endothelial and interstitial markers of mice were detected at continuous time points.The trend reflects that EndoMT gradually worsens as the disease progresses.Part ?:Construction of modified CAVD Mouse Model and Endothelial Cell Lineage Tracing Mouse Model combined with Expression of Endothelial-to-Mesenchymal TransitionBackground:The process of traditional high-fat-fed CAVD mouse model is time consuming and not all animals develop hemodynamically significant stenosis.The wire injury CAVD mouse model is characterised by severe mechanical injury,difficult surgery and high mortality,but the pathophysiology of acute injury model is different from that of clinical chronic disease.A model accommodates both rapid calcific phenotype and similar clinical pathological process is needed.The role of EndoMT in CAVD is still lack of critical direct evidence.Endothelial-specific Cre-lox lineage tracking systems are a more rigorous approach for studying EndMT in vivo.Methods:Wire injury combined with high-fat-fed Apolipoprotein E(ApoE-/-knockout mouse model was compared with wire injury mouse model.Aortic velocity and aortic valve area were measured by echocardiogram and hearts with aortic valve were collected at 4,8,12 and 16 weeks after aortic valve injury.Valve morphology and structure were observed by H&E and Masson staining,and calcium deposition was detected by alizarin red staining.The co-localization expression of endothelial and interstitial markers was detected by immunofluorescence,and the modeling effects of different models were compared.A lineage tracing ApoE-/-mouse with endothelial specific expression of red fluorescent protein(RFP)was constructed.Calcified aortic valvular disease was induced by simple high-fat-fed method and high-fat combined with wire injury method.Results:Compared with high-fat-fed and wire injury mouse model,the aortic flow velocity of the ApoE-/-mouse CAVD model by wire injury combined with high-fat-fed was significantly faster,and calcification occurred earlier at 8 weeks.Endothelial cell lineage tracing ApoE-/-mouse was successfully constructed.?SMA and RFP co-localized regions were found after 8 weeks of high-fat-fed and 4 weeks after wire injury,which suggested EndoMT phenomenon.Conclusions:Wire injury combined with high-fat-fed ApoE-/-method is a modified CAVD mouse model with rapid calcification,good modeling effect and high consistency.It optimizes the previous CAVD mouse animal model and expands its application.It provides a suitable and reliable animal model for basic research of CAVD.This study used endothelial cell lineage tracing CAVD mouse model for the first time,providing new evidence for the role of EndoMT in pathogenesis of CAVD progression.Part ?:Expression of DUSP1&PDK4 and related mechanisms in CAVDBackground:The pathogenesis of CAVD is unknown.Surgery and intervention are the only means of treatment.The molecular mechanisms or targets of non-surgical treatment to prevent or delay the progress of CAVD is still difficult to find.It is necessary to use clinical samples and animal models to screen and verify potential intervention targets.Methods:mRNA sequencing and single cell transcriptome sequencing of calcified aortic valve and normal aortic valve were used to screen differential genes.Combined with external tissue verification and literature review,two down-regulated genes in calcified group--DUSP1 and PDK4,were identified.The CAVD mouse model tissue sections were used to explore the temporal and spatial distribution of DUSP1 on the aortic valve.Normal human aortic valves were collected and digested with collagenase to obtain primary aortic valve interstitial cells(VICs),and aortic valve endothelial cells(VECs)were isolated by magnetic beads method.The changes of DUSP1,PDK4 and related calcification markers were detected at different time points after VICs calcification culture.The osteogenic transformation of VICs was detected after suppressing the expression of DUSP1 with siRNA.The expression of PDK4 in VECs was overexpress and knockdown by adenovirus,then EndoMT process in VECs was induced by TGF ?.The changes of endothelial and mesenchymal markers and EndoMT transcription factors were detected after transfection.Results:Immunofluorescence staining indicated that DUSP1 was mainly distributed on the ventricular side of aortic valve.The trend of DUSP1 and PDK4 of VICs was observed for 7 consecutive days after calcification culture.It was found that DUSP1 increased significantly in the early stage and decreased in the later period.PDK4 decreased significantly in the early stage and then decreased continuously afterwards.The BMP2 level of cells was up-regulated after DUSP1 was inhibited by siRNA.After 3 days,Alizarin Red staining was darker than the control group,and the amount of Alizarin Red was significantly different.EndoMT process was promoted in VECs after PDK4 was overexpressed by adenovirus,while the TGF?-induced EndoMT process was inhibited after PDK4 was knocked down PDK4 in VECs.Conclusion:Knockdown of DUSP1 accelerates the calcification of VICs.Overexpression of PDK4 promotes EndoMT in VECs,while knockdown of PDK4 can inhibit the process of TGF ?-induced EndoMT in VECs.The vitro experiments indicate that DUSP1 has a protective effect on the calcification of VICs.Knockdown of PDK4 inhibits the EndoMT process of VECs,providing a target for future interventions for EndoMT and drug development for the prevention and treatment of CAVD.