| 1.BackgroundMyocardial infarction is one of the most important causes of death in patients with coronary heart disease.After infarction,the tissues release various chemokines and cytokines,leading to aggregation of inflammatory cells and induction of inflammation,thereby causing myocardial tissue fibrosis and cardiac remodeling,thereby promoting the reduction of cardiac function and the occurrence of heart failure.Regulating the ECM remodeling after myocardial infarction can improve cardiac remodeling,enhance cardiac function and reduce mortality.The main components including fibroblasts in the cardiac muscle cells,endothelial cells and vascular smooth muscle cells.Other cells include lymphocytes,mast cells,and macrophages,which interact with major cells to affect heart function.Among these cells,fibroblasts are particularly important in maintaining normal heart function,maintaining homeostasis,and participating in scar repair.The major difference between fibroblasts and other cells is the loss of basement membrane,which makes them more likely to divide,migrate and transmit signals.This feature enables cardiac fibroblasts to play a key role in cardiac pathology and pathophysiology.After myocardial infarction,fibroblasts are activated to maintain the normal contractility of the heart by changing the electrophysiological characteristics of the heart muscle to accelerate the formation of blood vessels and secrete extracellular matrix such as collagen.Then it affects the process of ECM remodeling after myocardial infarction.The homeostasis of extracellular matrix is an important factor to maintain the cardiac stability after myocardial infarction.By breaking down the extracellular matrix,matrix metalloproteinase(MMP)regulates ventricular hypertrophy,dilation,and remodeling.The expression of MMP was strictly regulated at different levels.Different cytokines affect their transcription.Most MMPs are secreted in the form of non-active enzymes and require subsequent activation of active proteases.Finally,their activity is blocked by endogenous protein inhibitors,known as metalloproteinase tissue inhibitors(TIMP).Metalloproteinase 2(MMP2)is a member of the MMP family and is involved in tissue remodeling.Its proinflammatory effect can induce acute coronary syndrome and atrial fibrillation.MMP2 is involved in extracellular matrix remodeling by degradation of type IV collagen(the main structural component of basement membrane).The role of MMP2 in AF has been described in detail in the existing literature,which is mainly manifested in the remodeling of atrial structure.Sprouty(Spry)protein is an important regulator of cell growth and differentiation.Unlike mouse Spry3 located on x chromosome,human Spry3 gene is located in pseudoautosomal region 2.However,because of an unknown mechanism of epigenetic silence,the human y-allele does not express the position of sprouty.Human Spry3 is located in the pseudoautosomal region 2(PAR2)between the X-linked TMLHE gene and the par2-linked synaptobrevinl(SYBL1)gene,while the par2-linked synaptobrevinl(SYBL1)gene is conserved on the X chromosome of mice.PAR2 is not found in rodents or non-human primates,including chimpanzees,and so may be unique to humans,with several unusual genetic and epigenetic traits.At 320kb,it is much smaller than PAR1 and has a low frequency of pairing and recombination in male meiosis,which is not necessary for male reproduction.Spry protein is an important regulator of RTK/Ras/MAPK pathway.Recently,we discovered a new modulator family of RTK/Ras/MAPK pathway.The founding member of this family,the drosophila airways(dSpry),was originally described as a fgf-mediated negative feedback loop inhibitor of the drosophila tracheal branch,and mammals have four homologous genes spry1-4.These proteins contain highly conserved cysteine-rich estradiol-related regions at the carboxyl terminus,responsible for the intracellular localization of these proteins and conserved tyrosine residues near the amino terminus as binding sites for proteins containing the Src homologous 2 domain(SH2 domain).Spry3 is located in the pseudoautosomal region 2(PAR2)of X and Y chromosomes(Xq28 and Yq12),and its expression is believed to be limited to the brain and testicle of adults and fetal placental tissues.However,the role of Spry3 in cardiovascular disease and related diseases has not been reported.Therefore,this experiment intends to study the role of Spry3 in myocardial remodeling after myocardial infarction and its related mechanism by targeting myocardial fibroblasts.2.Objectives1.To detected the expression of Spry3 in MI-indued myocardial fibrosis;2.To study the effects of spry3 CFs’ migration;3.To investigate the effect of Spry3 in CFs’ MMP2 and ERK synthesis.3.Methods and Materials3.1.Tissue and cell cultureExperimental samples were collected within 48 hours after death.Human serum specimens were derived from autopsy cases.Human CFs(passages 1-20)was cultured in culture flasks.3.2.Haematoxylin-eosin(HE)stainingAn HE staining was performed to distinguish the normal tissue and the MI tissue.3.3.Masson stainingAn Masson staining was performed to distinguish the normal tissue and the MI tissue.3.4.Immunohistochemical stainingTo detect the expression and distribution of spry3,the slices of infarct tissues were immunohistochemically stained with the relevant antibodies,and the results were qualitative analysed statistically3.5.Cell transfectionSpry3 full length(pEX4-spry3)plasmid and spry3 small interfering RNA(siRNA-spry3)a non-targeting sequence(negative control,NC)were used to up-regulated and down-regulated the expressions of relative proteins.3.6.RT-qPCRRelative proteins mRNA expressions were determined using RT-qPCR.The relative expression quantity was calculated by 2-△△Ct.3.7.Cell migration assayHuman CF migration was detected by transwell assay.The cell number was measured within five randomly chosen fields at 200 x magnification,and the average number was calculated with ImageJ.3.8.Immunofluorescence stainingAn immunofluorescence assay was performed to detect the expression and distribution of MMP2(1:200).3.9.Western blot analysisRelative proteins expressions were determined using western blot.3.10.Statistical analysisThe results are expressed as the mean ± standard error of the mean.4.Results4.1.Sustained increase of spry3 expression in myocardial tissue induced by MI;4.2.After up-regulating and down-regulating spry3,respectively,the transfection4.3 The migration of human myocardial fibroblasts was enhanced by si-spry3,while the migration of human myocardial fibroblasts was weakened by pex4-spry3.4.4.The synthesis of MMP2 in human myocardial fibroblasts was enhanced by si-spry3,and the synthesis of MMP2 in human myocardial fibroblasts was weakened by pex4-spry4.5.The synthesis of phosph-ERK in human myocardial fibroblasts was enhanced by si-spry3,and the synthesis of phosph-ERK in human myocardial fibroblasts was weakened by pex4-spry5.Conclusion1.Spry3 can reduce the migration of myocardial fibroblasts and the synthesis of MMP2;2.Spry3 inhibits MMP2 synthesis through ERK pathway;... |
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