Basic Research On The Cardiovascular Development And Abnormalities Correction

Cardiovascular disease is the leading cause of death worldwide,putting enormous burden on public health.Cardiovascular development requires precise tempo-spatial regulation.However,the molecular mechanisms are yet to be revealed.Identifying disease-causing genes,exploring their pathogenic mechanisms and seeking potential treatment strategies are main goals of basic medical research.This dissertation therefore focuses on two parts:1) to study the regulatory mechanism of the Wdpcp gene on coronary artery development;2) to show therapeutic effect of epigenetic drug tranylcypromine（TCP） on correction of cardiac outflow tract abnormalities.Wdpcp,a planar cell polarity（PCP） effector gene,was shown to play essential roles in embryonic organogenesis and developmental process through regulating collective cell movement and ciliogenesis.Mouse Wdpcp deficient mutants(Wdpcp^m/m)presented a spectrum of developmental anomolies including anophthalmia,polydactyly,intestinal cysts,polycystic kidney,pulmonary atresia,and atrioventricular septal defect.These defects resemble the phenotypic spectrum of human Bardet–Biedl and Meckel–Gruber syndromes.Here,we show that the Wdpcp gene is also required for the development of coronary arteries.Using the SM22α^LacZacZ allele to visualize smooth muscle cells of coronary vessels,we found Wdpcp mutants have reduced coronary artery branches and a significant reduction in the number of smooth muscle cells,whereas the development of coronary vein was hardly affected.PECAM immunohistochemical staining showed the formation of coronary plexus was not affected in Wdpcp mutants.Instead,the subepicardial coronary plexus encloses the heart in a faster speed in mutants than that in wild-type.Using Wt1^CreERT2-Rosa26^mTmG lineage-specific labeling system to trace epicardium-derived cells（EPDCs）,we found that the number of EPDCs was significantly reduced in Wdpcp mutants.Quantitative PCR analysis reveals that the expression of epithelial-mesenchymal transition（EMT）regulators,Snail2 and Twist1,and mesenchymal-specific markers Vinculin and Vimentin were also reduced.Three-dimentional collagen gel invasion assay demonstrated impaired migration of EPDCs in Wdpcp mutant.In addition,we recapitulated the coronary artery defects of Wdpcp^m/m mutant with epicardial-specific deletion of Wdpcp.These results demonstrated that Wdpcp play an essential role in coronary plexus remolding stage through promoting epithelial EMT and EPDCs migration.This is the first reported mouse model with accelerated primitive plexus formation but defective vascular remodeling.It will be a useful model to study the molecular mechanisms governing the primitive plexus remodeling process of coronary development.22q11.2 deletion syndrome（22q11.2DS）is the most common chromosomal microdeletion syndrome in mankind.Its common symptoms include:cardiovascular disease,craniofacial abnormalities,thymus and parathyroid dysplasia.The cardiovascular disease seen in 22q11.2DS is congenital heart diseases,such as tetralogy of Fallot,tetralogy of Fallot with pulmonary atresia,interrupted aortic arch,double outlet right ventricle and persistent truncus arteriosus（PTA）etc.Haploinsufficiency of the T-box transcription factor TBX1 is the major cause for the malformations in 22q11.2DS.Our collaborators found that Tbx1 regulates target gene expression by recruiting histone methyltransferases,which promotes monomethylation of histone 3 lysine 4（H3K4me1）.On the basis of this work,we hypothesize that TCP,an inhibitor of histone demethylase,can rescue the cardiovascular defects in the Tbx1 mutants(Tbx1^LacZ/+ and Tbx1^Neo2/LacZ).After intraperitoneal injection of TCP,ink injection was performed to visualize pharyngeal arch artery（PAA）in mouse E10.5 Tbx1^LacZ/+mutants.We found that the 4^thh pharyngeal arch artery defect was significantly rescued.PTA abnormalities were also partially rescued in E18.5 Tbx1^Neo2/lacZ embryos after TCP treatment.However,thymic phenotypes were not.Quantitative PCR analysis revealed that the expression of Tbx1 has no significant difference between the TCP treatment group and control group,suggesting the rescue is not due to increased expression of Tbx1.In situ hybridization showed Fgf8 expression in second heart field（SHF）was increased after TCP treatment.Moreover,the expression of cyclin D2 was also increased in the SHF,indicating an upregulation of cell proliferation.Immunostaining of phospho-histone H3 further validated this observation.Loss of SHF cell polarity was ameliorated after TCP treatment,as identified by redistribution of αPKCζ immun of luorescent signal to the apical suface.In conclusion,this study provided powerful in vivo evidence that TCP,a drug targeting epigenetic modification,can rescue Tbx1 gene haploinsufficiency.Our results shed new lights on a novel therapeutic strategy for clinical 22q11.2 microdeletion syndrome treatment.