The Mechanism Of NOTCH-1in Bicuspid Aotic Valve Complicated With Ascending Aortic Dilation

Background and objective:Bicuspid aortic valve (BAV) disease is the mostcommon congenital heart defect, with a prevalence estimated between0.46%and1.37%.There is a male predominance of approximately3:1. For the reason that BAV patients canbe healthy in their whole life with no symptoms, they are difficult to be diagnosed. Most ofthem are made a definite diagnosis when visiting the hospital due to the other complication,such as valve dysfunction, infective endocarditis, and ascending aortic disease. Comparedwith another congenital heart disease, BAV patients face more difficulty for diagnosis andtreatment. When facing aortic lesions such as ascending aortic dilation, aortic aneurysm oraortic dissection, surgery risk and cost of treatment is a significant increase coupled withthe increasing of the incidence of postoperative complications and mortality. Therefore, themechanism of BAV complicated with AAD is not only to provide guidance on the earlydiagnosis and early treatment of disease, but also to provides a potential target for thebiological therapy of BAV complicated with AAD.It is widely believed that the dysfunction of vascular smooth muscle cells (VSMC) isrelevant to AAD. VSMC is the main component of the vascular media, which play animportant role in maintaining vascular integrity and function. In the other hand, VSMCsalso secrete the aortic wall extracellular matrix proteins, including collagen, elastin,laminin. But it is unknown that what make the VSMC dysfunction.The Notch pathway is an evolutionarily conserved signaling mechanism that playsessential roles both during development and in the maintenance of adult tissue homeostasisin metazoan. In the process of heart embryo development, NOTCH-1play an importantrole in the regulating endothelial cells to mesenchymal transformation (EMT), which is thekey pathway of the aortic valve development. Mutations in the NOTCH1result inabnormal aortic valve development (BAV) and later to de-repression of calcium deposition.But it is unknown whether is the NOTCH-1gene participate in aortopathy progress.The purpose of this experiment is to review our hospital database (from Jan1991to Dec2010) of BAV patient, summarize epidemiological characteristics, analysize of its complications' clinical feature, particularly the ascending aortic dilation. Based on this, weanalysize the risk factors of BAV complicated with AAD, and then provide the advice forclinical diagnosis and treatment in the future. Secondly, we analysize the pathologicalchange of the clinical tissue to find out the effect of NOTCH-1and VSMC function on thepathogenesis of BAV complicated with AAD. Thirdly, by means of cytological method weanalysize the relation between NOTCH1gene change and VSMC function.Methods and results:The present study was divided into six sections.1.Clinical retrospective analysis of406cases of bicuspid aortic valve disease.The20years' clinical data of surgical treatment of BAV patients was summarized,including patients gender, age, body surface area (BSA), preoperative complications,cardiac function level, and preoperative ultrasound examination index, BAV phenotype.Analysis the difference in clinical features and comorbidity according to gender and BAVphenotype. And on this basis, the binary Logistic regression analysis was use to search forthe risk factor of BAV complicated with AAD.Through analysis, we found that BAV patients with male-dominated major clinicalcomplications include valve dysfunction and infective endocarditis and ascending aorticdisease. The most common complication of congenital heart disease is coarctation of theaorta and ventricular septal defect. The most common aortic valve phenotype is theleft-right coronary valve fusion (L-R type), accounting for63.3%, followed by theright-noncoronary valve fusion type (R-N type), accounted for32.8%. The in-hospitalmortality of whole406patients was6.2%, which is higher than the other surgical aorticvalve disease in the same period. Preoperative infective endocarditis is one of the riskfactors of hospital mortality.Gender affect the clinical features of BAV patients, the age of male patients admitting tothe hospital was obviously lower than that of the female patients, and male patients weremore likely to complicated with SBE. The male patients showed worse cardiac function,and female patients tend to progressed aortic lesion. Aortic valve phenotype effect clinicalcharacteristics: type L-R is tend to involve aortic disease, otherwise type R-N is tend toinvolve valve dysfunction. Through the Logistic regression analysis, we found thatincreasing age and female is an independent risk factor of the the BAV complicated with AAD.2.Correlation study between NOTCH-1expression or VSMC function change andBAV complicated AAD.By analyzing the pathological change of BAV patients' aorta, we found out the histologicfeatures and extracellular matrix components of molecular biological change. Using theTUNEL method to compare the media VSMC apoptosis rate between BAV group andcontrol group. Immunohistochemical analysis of matrix metalloproteinase-2(MMP-2) andvascular smooth muscle α actin protein (α-SMA), and qRT-PCR detection expression ofNOTCH-1, apoptosis-related protein and VSMC differentiation migration marker proteinsin fresh specimens.Researches show that compared with normal control group the the BAV with AAD patientshave more damage about aortic wall structure, and the more loss of smooth muscle cells.Victoria blue (VB) staining show that red collagen fiber content increased and blue elasticfiber fracture in BAV aortic wall. Immunohistochemical results reveal that the MMP-2expression was significantly higher in the BAV and TAV complicated with AAD patientsthan the normal control group, which represents high α-SMA expression than in theBAV/TAV AAD group. TUNEL staining in the normal aortic wall show only a fewapoptotic cells, otherwise an increasing number apoptosis cell in the the the BAV/TAVAAD group, and the more severe degree of aortic dilatation, the more middle VSMCapoptosis. qRT-PCR detect that the expression of NOTCH-1genes in the BAV/N andBAV/AAD group was significantly lower than the control group (P <0.01), and inTAV/AAD group was lower than the control group (P <0.05). α-SMA,and SM22αexpression, which is on behalf of the contractile phenotype of VSMC, was significantlyreduced, and the MMP-2,9expression, which regulate the metabolism of extracellularmatrix components, is significantly up-regulated.3. The mechanism of NOTCH-1regulate smooth muscle cell apoptosis.To investigate the mechanism of NOTCH-1regulate VSMC apoptosis, firstly wesuccessfully culture VSMC in vitro which can be used for the cytological experiments, andthen chemically synthesize NOTCH-1siRNA to down-regulate its expression by lipofectintransfect. Base on those, we use Annexin-V/PI double labeling method to detect cell apoptosis rate, and use qRT-PCR and Western blot method to detect apoptosis-relatedprotein change in the mRNA and protein levels.VSMC with NOTCH-1siRNA have significantly increased apoptosis rate, correspondingwith apoptosis related proteins changing. Compared to the control group, the expression ofpro-apoptosis proteins Caspase-9and Bax in the siRNA group increased significantly,while the expression of anti-apoptosis proteins BCL-2and NF-Kappa B significantlyreduced; On the other hand, the expression of Caspase-8of of the have increased, but notas good as the degree of the other apoptotic proteins. Therefore, we draw the conclusionthat NOTCH-1regulate VSMC apoptosis mainly through mitochondrial pathway, but thedeath-receptor pathway is not excluded.4.The effect of NOTCH-1on VSMC migration and differentiation.Based on last experiment, we use qRT-PCR and Western blot to compare the expression ofα-SMA and SM22α, which is VSMC phenotype transformation marker gene, betweenNOTCH-1siRNA group and negative control group. Transwell migration experiment wasto assess the changes in VSMC migration ability, on the same time, the expression ofMMP-2,9were detected to analyze the effect of NOTCH-1on them.The researches reveals that in the siRNA group with the significant down-express ofNOTCH-1the VSMC transfer from contractile subtypes to the synthesis subtype,manifesting that the expression of α-SMA and SM22α reduced. Accompanied withNOTCH-1down-regulated the VSMC migration ability increased, but the MMP-2,9haveno significant rise. The reason mainly is that the VSMC migration ability is not effected bythe expression of MMPs but by the cell differentiation state. NOTCH-1do not regulate theexpression of MMP-2,9in direct effect.Conclusion:1.BAV patients with male to female ratio is approximately2.6:1, the most commoncomplication is valve dysfunction, infective endocarditis and ascending aortic dilatation.The most common congenital heart disease associated with BAV is coarctation of the aortaand ventricular septal defect. The most common aortic valve phenotype is the L-R fusion(63.3%), followed by R-N fusion (32.8%). 2.Male BAV patients are more susceptible to AI and infective endocarditis; and femalepatients are more likely to possess AS. Female patients tend to progress ascending aorticdilation. BAV patients with type L-R may predispose to the expansion of the ascendingaortic dilation and aortic sinus dilation, and are more vulnerable to the AI, while the typeR-N BAV patients are easier to develop with AS and AI.3.The independent risk factors of BAV patients with ascending aortic dilation includeincreasing age and women. Because of complicated with infective endocarditis, earlytreatment is to play a passive effect.4.The pathological changes of BAV/AAD patients is the cystic medial necrosis, includingsmooth muscle cell swelling, degeneration, lackage of elastin, and the severity ofpathological changes was correlated with the extent of aortic dilatation.5. The down-regulation of NOTCH-1, more apoptosis and phenotypic change of VSMC,the expression of MMP-2,9are relevant to the BAV complicated with AAD.6. The down-regulation of NOTCH-1is relevant to more apoptosis and phenotypic changeof VSMC.7.The down-regulation of NOTCH-1may promote VSMC apoptosis, the mitochondrialpathway of apoptosis played an important role, but does not exclude the role ofdeath-receptor pathway.8.The down-regulation of NOTCH-1could induce the conversion of VSMC fromcontractile subtype to synthetic subtype, with the down-regulation of α-SMA and SM22α.9.NOTCH-1do not regulate the expression of MMP-2,9in direct effect. When theexpression of NOTCH-1down-regulate, the expression of MMP-2was no significantchange.10.The down-regulation of NOTCH-1can promote VSMC migration through not the up-expression of MMP-2,9but the the cell differentiation state.