Study On The Phenotype And Mechanism Of Aortic Valve Disease

Aortic valve disease(AVD)is the most common heart valve disease.As the population ages,the proportion of degenerative aortic valve disease has increased year by year.At the same time,rheumatoid valvular disease still accounts for a large proportion of aortic valve disease in China.There is no effective medicine treatment for AVD patients.Aortic valve replacement surgery is the only effective choice,which has high cost,high risk and leads to heavy social burden.AVD involves a variety of cellular and biological processes.Although previous studies have explored its specific phenotype and mechanism to a certain extent,it has not systematically elucidated the key nodes of the disease.Therefore,the systematical describe of AVD phenotype is necessary.Based on the traditional molecular biology experimental research,the application of omics technology and AVD patient valve samples,we can comprehensively describe the regulation network of the disease via bioinformatics analysis.The interaction network is of great significance for the identification of key molecules and cell types in the pathogenesis,and it will also provide new targets for effective and precise intervention of diseases.The aortic valve anatomy includes normal tricuspid aortic valve,bicuspid aortic valve and its subtypes,and participates in the disease by affecting the aortic valve blood flow state.However,previous studies did not accurately distinguish the aortic valve anatomy,and the different anatomical structures corresponding to the AVD phenotype were not clear.Therefore,the first part of the study was based on accurate aortic valve anatomy classification,clinical features,aortic valve histopathological changes,hemodynamic status for patients with different types of AVD,including patients with different subtypes of bicuspid aortic valve.The disease phenotype was described and compared,which laid a foundation for systematic research on the phenotype and pathogenesis of aortic valve disease.The first part of the study clarified that aortic valve calcification is one of the major phenotypes of aortic valve disease.Valvular interstitial cell activation and osteogenic transdifferentiation are critical in aortic valve calcification.Biological processes associated with valve development are thought to be equally involved in the development of the disease.Meis2 is highly expressed in the valve area during the aortic valve development and is involved in the regulation of various valvular disease-related signaling pathways,but its role and mechanism in calcific aortic valve disease is not clear.Therefore,the second part of this study investigated the role and mechanism of Meis2 in the process of aortic valve calcification and vascular endothelial cell osteogenesis.Degenerative aortic valve disease includes aortic valve calcification and myxomatosis changes,and there are certain differences and connections between the two phenotypes.According to the results of the first part of the study,degenerative aortic valve disease has become the most important aortic valve disease phenotype.Degenerative changes are associated with environmental factors,and long noncoding RNAs(lncRNAs)can play a role in this process.Aortic valve mechanism studies have also found that some lncRNAs are involved in the regulation of disease.However,the overall differential expression of lncRNA in degenerative aortic valve disease and the differences between different pathological phenotypes are not clear.Therefore,the third part of this study used transcriptomics technology to study the differential expression of lncRNA in degenerative aortic valve disease,and analyzed its possible effects and mechanisms using bioinformatics methods.It provides a new basis for the comprehensive clarification of degenerative lesion-related IncRNA at the transcriptome level and the subsequent further investigation of pathogenesis.According to the first part of the study,aortic valve degeneration and rheumatism have covered the disease phenotype of almost all patients with AVD.The results of the second and third parts suggest that the process of cell activation,transdifferentiation and other processes are related to the disease,and these processes involve the biological functions of various cell types.However,changes in the type and composition of aortic valve cells under normal and disease states,as well as key cell types of the disease,are unclear.Therefore,the fourth part of this study used single-cell sequencing technology to further describe the microenvironment of aortic valve disease cells,and used bioinformatics methods to analyze key cell types and their functions.The specific four parts of the research on the phenotype and pathogenesis of aortic valve disease are summarized as follows:Part1:Phenotypes of Aortic Valves Disease according to Detailed Anatomical Classification of Patients underwent Aortic Valve Replacement SurgeryBackground:Anatomy of aortic valves plays an important role in the progression and treatment strategy of aortic valve disease(AVD).In this study,we described and compared differences of AVD phenotypes according to detailed anatomical classification.Methods:Patients underwent first time aortic valve replacement surgery(AVR)were enrolled.The anatomy of aortic valves diagnosed during operation was considered as the golden standard for detailed classification-Clinical characteristics,histopathological diagnosis,preoperative transthoracic echocardiography(TTE)and other available imaging examinations data were collected retrospectively.Phenotypes were described and compared according to the detailed classification.And the imaging diagnostic accuracy was evaluated.Results:For all 602 patients enrolled,74.1%were male and the average age was 55.51±12.94 years old.The number of patients diagnosed as tricuspid aortic valves(TAV),bicuspid aortic valves(BAV)and quadricuspid aortic valves(QAV)during operation were 370,228 and 4,respectively.TAV mainly experienced myxomatosis(48.4%),and fibrocalcification counted for 57.9%of BAV(P<0.001).96.2%TAV patients suffered aortic regurgitation(AR),while BAV patients showed high rate of aortic stenosis(AS,73.7%),and the severity of AS and AR varied among subtypes of BAV.The preoperative TTE diagnostic accuracy of aortic valve anatomy for the whole study population was 85.5%.In TAV and BAV group,the accuracy were 96.8%and 68.0%,respectively(P<0.001).Conclusions:Nearly 40%patients underwent AVR were BAV.Different phenotypes existed among patients with different detailed classification.More attention are needed for precise diagnosis of the anatomy of BAV in preoperative evaluation.Part2:Meis2 represses the osteoblastic transdifferentiation of aortic valve interstitial cells through the Notchl/Twistl pathwayBackground:Calcific aortic valve disease(CAVD)is the most common valvular disease worldwide.The osteoblastic transdifferentiation of aortic valve interstitial cells(VICs)is the essential process of CAVD,but the underlying mechanisms are poorly understood.Aortic VICs are generated from epithelial-to-mesenchymal transition(EMT)and migration of neural crest cells(NCCs).Meis2 has been associated with EMT and NCCs migration during development,but its role in CAVD is unknown.This study aims to elucidate the specific functions of Meis2 and its downstream targets in aortic valve calcification.Methods:Levels of Meis2 were examined in calcified(n=30)and normal(n=30)human aortic valve tissues,respectively.Meis2 was inhibited in porcine aortic VICs in vitro,and the effect on osteoblastic transdifferentiation and its downstream pathway were studied.Results:Meis2 gene and protein expression decreased significantly in calcified human aortic valve tissue compared with the normal ones.Inhibiting Meis2 by siRNAs reduced the gene and protein expression of Notchl and Twistl,and induced the osteoblastic transdifferentiation of the porcine aortic VICs in vitro.Conclusions:The present study indicated that Meis2 repress the osteoblastic transdifferentiation of aortic VICs through the Notchl/Twistl signaling pathway.The Results identify Meis2 as a potential intervention target for the prevention of CAVD.Part3:Landscape of Long noncoding RNA in Human Degenerative Aortic ValvesBackground:Long noncoding RNA(IncRNA)plays an important role in cardiovascular disease.However,the expression and specific function of lncRNA in degenerative aortic valve disease are still unclear.Methods:Aortic valve tissue of human degenerative lesions was collected and compared with normal aortic valve to analyze the differential expression of lncRNA.The bioinformatics method was used to analyze and predict the function and mechanism of the differential lncRNA.Results:A total of 32 aortic valve tissues were enrolled in the study,including 12 in the normal group,10 in the fibrosis group and 10 in the myxomatosis group.Compared with the normal group,93 differentially expressed lncRNA were found in the fibrosis group found,of which 46 were up-regulated and 47 were down-regulated;87 differentially expressed IncRNA were found in the myxomatosis group,of which 52 were up-regulated and 35 were down-regulated.The functional enrichment analysis of differential lncRNA co-expression mRNA suggests a correlation with pathophysiological processes such as cell proliferation,migration,and osteoblast differentiation.The IncRNA-miRNA-mRNA regulatory network was constructed to predict the mechanism of the function of lncRNA,and five key nodes lncRNA were obtained.Conclusion:This study found a series of differentially expressed lncRNAs in human fibrous calcification and mucin-like aortic valve tissues,and predicted the function and mechanism of differential IncRNA.Part4:Single-Cell Transcriptome Analysis of the Landscape of Human Aortic ValvesBackground:The process of aortic valve disease involves multiple cells,but the key cell types and pathophysiological processes are still unclear.This study intends to describe the cellular microenvironment of human aortic valve disease by single-cell sequencing,and analyze key cell types and their functions.Methods:The aortic valve tissue of patients undergoing cardiac surgery was collected and digested and isolated to obtain cells.The cell transcriptome was sequenced using single cell sequencing.Cell type and function were described and analyzed via bioinformatics analysis methods.Results:A total of 4,114 human aortic valve cells were sequenced,and 4,116 cell were analyzed.A total of 13 clusters of cells were obtained,including 1 type of endothelial cells,7 types of mesenchymal cells,4 types of immune cells and 1 type of cardiomyocytes.The proportion of endothelial cells was highest in normal aortic valves.Proliferation and differentiation of interstitial cells was the main process of aortic valve disease,and VIC-FOSB+and VIC-BMP2+were key cell types for aortic valve fibrosis.Atypical macrophages promoted the development of aortic valve disease.Conclusion:This study used single-cell sequencing technology to describe the cellular microenvironment of human aortic valve disease.VIC-FOSB+and VIC-BMP2+were found to be key cell types in the calcification of aortic valve fibers.It provided a target and basis for the comprehensive and in-depth mechanism research of subsequent aortic valve disease.