The Expression And Significance Of Matrix Metallproteinases And Their Tissue Inhibitors In Rheumatic Mitral Valve

Rheumatic heart disease(RHD) is a total carditis involving the endocardium, myocardium,epicardium and conductive system caused by rheumatic inflammation, however permanent damage is often confined to the heart valves,in which simply mitral valve disease is most common,accounting for 95%to 98%.Matrix Metalloproteinases(MMPs) is a zinc-dependent protein hydrolysis endopeptidase,can specially degrade Extracellular Matrix(ECM),mainly involved in ECM reconstruction process.Tissue inhibitor of matrix metalloproteinases(TIMPs) is endogenous inhibitors of MMPs in in our body,closely related to the regulation of MMPs' activities.The imbalance of expression and regulation of MMPs can cause a variety of diseases such as cardiovascular diseases,lung diseases,arthritis,cancer,etc.MMPs and the relationship between the various kinds of diseases have become research hotspots at home and abroad.This study is divided into two parts,first observes the level of MMPs and its endogenous inhibitor TIMPs in RHD mitral valve tissue,then explores the relationship between the expression of MMPs and TIMPs in rheumatic mitral valve disease and the pathological types,clinical classification of cardiac function and atrial fibrillation,and further discusses the mechanism of ECM reconstruction of rheumatic mitral valve.Part 1 The study of expression of MMPs and their inhibitors TIMPs in rheumatic mitral valveObjective1.To observe morphology and ultrastructure of the mitral valve with RHD;2.To explore the expression and significance of MMPs and their tissue inhibitors TIMPs in rheumatic mitral valve.Methods1.Experiment group:From June to November of 2007,experimental group composed of 16 mitral valves which were collected from the adult patients accepted mitral valve replacement surgery in Wuhan General Hospital of Guangzhou Military Command.Controls were from 7 adults who were died accidentally without cardiovascular system diseases.2.Histopathological examination:All specimens were stained with Hematoxylin and eosin(HE) method to observe the morphological changes of experimental group and control group.Typical mitral valves lesions of RHD patients were made into electron microscopy specimens,using transmission electron microscope(TEM) to observe changes of collagen fibers and mitochondria in rheumatic mitral valve.3.Semi-quantitative reverse transcriptase - polymerase chain reaction(RT-PCR) was used to detected mRNA content of MMP-2,MMP-13 and TIMP-1 and TIMP-2 between the experimental group and control group.After gel electrophoresis images of PCR products scanned,the ratio of electrophoresis band gray integration between target genes andβ-actin gene meant the target genes' relative mRNA content. 4.By immunohistochemical staining(SABC method) MMP-2,MMP-13, TIMP-1 and TIMP-2 of mitral valves between the experimental group and control group were dyed.After the completion of light microscope observations,image acquisition by HPAIS 1000 image analysis system,using Image - Pro Plus version 6.0 software on each slice of expression levels of MMP-2,MMP-13,integral optical density(IOD) were determinated,average data were chosen as a quantitative indicators compared the two groups of MMP-2,MMP-13 protein expression levels.Results1.Compared with the control group,HE staining revealed that the valve structures of experimental group were unclear,with fibrous tissue hyperplasia, mucoid degeneration,collagenous fibers of glass-like degeneration and calcification. Transmission electron microscopy showed degenerative lesions including collagenous fibers proliferated,disorder arranged or ruptured;mitochondria swollen, membrane structure damaged,vasiculated,crista dissolved,condensed,ruptured or disappeared.2.Compared with the control group,RT-PCR results showed that mRNA levels of MMP-2,13 and TIMP-1,2 in rheumatic mitral valve were significantly increased (t=3.109,P=0.005:t=2.862,P=0.009;t=2.357,P=0.028;t=2.844,P=0.010).3.Compared with the control group,immunohistochemistry results indicated that tissue structures of mitral valves in the experimental group were unclear.We can also see more neovascularization.The expressions of MMP-2,MMP-13 were increased in the cytoplasm of the valve interstitial cells,endothelial cells and inflammatory cells.The expression of MMP-2,MMP-13 in experimental group were significantly higher than in controls(Z= -2.633,P=0.008:Z= -2.936,P=0.003).Conclusion1.Obvious pathologic changes of ECM had been seen in microstructure and ultrastructure of rheumatic mitral valve,i.e.collagenous fibers hyperplasia,mucoid degeneration,collagenous fiber disorder,glass-like degeneration and calcification.2.The gene and protein expressions of MMP-2,13 and TIMP-1,2 in rheumatic mitral valve tissue were significantly increased,suggesting that MMPs / TIMPs up-regulation may be an important molecular mechanism of ECM remodeling in rheumatic mitral valve,and our study lays a theoretical basis for further researches on preventing ECM reconstruction of rheumatic mitral valve.Part 2 The relationship between the expression of MMPs/TIMPs in rheumatic mitral valve and different pathological types,clinical classification of cardiac function and atrial fibrillationObjective1.To compare changes of major ultrasonic indexes in different types of mitral valve lesions,clinical classification of cardiac function and atrial fibrillation;2.To study the influence of different pathological types,clinical classification of cardiac function and atrial fibrillation to the gene levels of MMPs / TIMPs in rheumatic mitral valve tissues.Methods1.Experiment group:From June to November of 2007,select 16 adult patients accepted mitral valve replacement surgery in Wuhan General Hospital of Guangzhou Military Command,including 3 males and 13 females with mean age 42.06 years old. According to the different pathological types,16 patients were divided into mainly mitral stenosis(MS) group(mitral valve orifice area＜1.2cm~2,reflux≤2+) and mainly mitral insufficiency(MR) group(mitral valve orifice area＞1.2 cm~2,reflux 3+ to 4+).In accordance with NYHA functional classification standards patients were divided into NYHA ClassⅢorⅣ(A) group and NYHA ClassⅡ(B) group. According to with or without atrial fibrillation,patients were also divided into atrial fibrillation(Ⅰ) group and sinus rhythm(Ⅱ) group.2.Detection:clinical data were collected;Doppler ultrasound diagnostic apparatus of Agilent SONOS 5500 was used,and patients were undergone traditional echocardiography before operation.Let the patients lie left position,by parasternal long axis section from Echo measured LVDd,LAd,LVEF and LVFS. Semi-quantitative reverse transcriptase - polymerase chain reaction(RT-PCR) was used to detect mRNA content of MMP-2,MMP-13 and TIMP-1 and TIMP-2 of adult patients with rheumatic heart disease mitral valve.After gel electrophoresis images of PCR products scanned,the ratio of electrophoresis band gray integration between target genes andβ-actin gene meant the target genes' relative mRNA content.Results1.Ultrasonic indexes analysis:Compared MS group with MR group,there were significant differences in LVDd(t= -3.429,P=0.004).Compared group A with group B,there were no significant differences in all the indexes.Compared groupⅠwith groupⅡ,there were significant differences in LAd(t= 2.209,P=0.044).2.Compared MS group with MR group and group A with group B,there were no signifcant differences in mRNA levels of MMP-2,13 and TIMP-1,2.Compared groupⅠwith groupⅡ,mRNA levels of MMP-2 and MMP-13 were significant(t= 2.681,P=0.018;t=2.392,P=0.031).Conclusion1.Compared with MS group,LVDd in MR group was significantly enlarged;it shows that the pathological types and left ventricular geometric patterns may have relationship.Compared with sinus rhythm group,LAd in atrial fibrillation group was significantly enlarged;this indicated atrial fibrillation plays an important role in the configuration of the left atrium and development.2.The expression of MMPs and TIMPs had no significant differences between different pathological types,clinical classification of cardiac function in rheumatic mitral valve.The appearance and existence of atrial fibrillation follow the up-regulation of MMP-2 and MMP- 13.