| Part One Platelet Microparticles: A Tool to assess Infarction Area in RatsPlatelet microparticles(PMPs)are small membrane vesicles budding from the cell surface,relieved from platelet following its activation and apoptosis.With the in-depth study,PMPs possess a multiple proinflammatory effect and play an important role in the atherosclerosis,especially acute coronary syndrome process.They are recognized as an important intercellular messenger contributing complex pathological and physiological effect.There are increasing evidences that PMPs involve in the processes of the formation and development of acute coronary syndrome,especially acute myocardial infarction.Studies has shown PMPs are related to extent of myocardial damage in acute myocardial infarction,not just an independent risk predictor.However,the value of PMPs in acute myocardial infarction and the mechanisms involved in PMPs regulation need the further research and quests still.Objective: Firstly,to observe the correlation among PMPs and myocardial infarction area,and evaluate the clinical value of PMPs in acute myocardial infarction.Try to find if there is a quantitative relationship between PMPs and myocardial infarction area.Secondly,to observe the influence of antiplatelet drugs(aspirin,clopidogrel and ticagrelor)on the expression of PMPs and infarction area,try to illuminate the intervention mechanism of antiplatelet drugs on PMPs.Methods:1.30 eight-week-old male Wistar rats were used for all murine experiments and divided into five groups(the sham-operated group,the myocardial infarction group,the aspirin intervention group,the aspirin combined with clopidogrel intervention group,and the aspirin combined with ticagrelor intervention group).Myocardial infarction was induced surgically by a permanent ligation of the left anterior descending coronary artery.Pharmaceutical interventions(aspirin 10.42 mg/kg,clopidogrel 18.7 mg/kg and ticagrelor 7.81 mg/kg)were taken by oral gavage daily,one week before and after the operation.2.Animals were humanely sacrificed one week after the operation.Infarction size was determined by TTC staining method.Stained myocardial samples were photographed from both sides.The infarct area was compared to the total left ventricular area using Image J software(NIH)and was calculated as the percentage of infarct area of the total cross-sectional area of the heart in each section for comparison;that is,infarct area/(infarct area plus non infarct area)×100%.ELISA was performed to measure PMPs using antibodies to PMPs according to the manufacturer's protocol.3.All data were analyzed by statistical software SPSS 13.0 editions.The difference of measurement data was detected by analysis of one-way analysis of variance.Two-two comparison among all means was done by Student-Newman-Keuls test and LSD pairwise comparison methods.The correlation between PMPs and infarction areas was analyzed by Pearson correlation analysis.Linear regression analysis was applied to further asses the correlation between PMPs and infarction areas.Results: 1.Significant differences(p < 0.01)were revealed among all the groups in PMPs and infarction area.PMPs and infarction areas in sham-operated group were decreased most than other groups followed by dual antiplatelet intervention groups and then aspirin alone intervention group.There were no differences in PMPs level between any two groups of sham-operated group,aspirin combined with clopidogrel intervention group and aspirin combined with ticagrelor intervention group.2.No difference was found in infarction area between two dual antiplatelet intervention groups(P=0.22).3.PMPs was significantly correlated with infarction area.(r=0.85,P<0.01).5.The linear regression analysis model of infarction(Infarction Area = 4.61 +0.28 ~*PMPs)was achieved.Conclusions:1.PMPs was significantly correlated with infarction area.PMPs can be used as a tool to assess infarction area based on the regression model of Infarction Area=4.61+0.28~*PMPs.We provide a novel method to assess infarction area with PMPs,which is more precise and convenient.2.Antiplatelet drugs can downregulate PMPs expression by inhibiting platelet activation.Our study has established PMPs is valuable in assessing severity and extent of myocardial infarction more than an independent biomarker and a risk factor of acute coronary syndrome.PMPs is novel and potential targets for intervention of myocardial infarction.All of these conclusions result from fundamental study based on rats MI models.We will testify them with clinical research in our follow studies.Part Two Calpain10 Regular Platelet Microparticles and Myocardial InfarctionCalpains are calcium dependent intracellular cysteine proteases,which respond to Ca2+ signals by removing limited portions of protein substrates.Activation of calpains are involved in a multitude of physiological and pathological events.And,there are increasing evidences that calpain take part in ischemia/reperfusion damage and the process of atherosclerosis.Besides,Research shows calpains are related to platelet microparticles up-regulation.However,their functions in myocardial infarction and the regulatory mechanism of platelet microparticles are still poorly understood and need the further research.Objective: Firstly,to clarify the correlation between myocardial infarction area and Calpain10,and evaluate the clinical value of Calpain10 in myocardial infarction.Try to establish the quantitative correlation between calpain10 and myocardial infarction area.Secondly,to illuminate the mechanism of Calpain10 involved in PMPs regulation.Finally,by analyzing the influence of antiplatelet drugs(aspirin,clopidogrel and ticagrelor)on the expression of Calpain10 and infarction area,try to illuminate the intervention mechanism of antiplatelet drugs on Calpain10.Methods: 1.30 eight-week-old male Wistar rats were used for the murine experiments and randomly divided into five groups(the sham-operated group,the myocardial infarction group,the aspirin intervention group,the aspirin combined with clopidogrel intervention group,and the aspirin combined with ticagrelor intervention group.Myocardial infarction was induced surgically by a permanent ligation of the left anterior descending coronary artery.Pharmaceutical interventions(aspirin 10.42 mg/kg,clopidogrel 18.7 mg/kg and ticagrelor 7.81 mg/kg)were taken by oral gavage daily,one week before and after the operation.2.All rats were humanely sacrificed one week after operation.Infarction size was determined by TTC staining method.Stained myocardial samples were photographed from both sides.The infarct area was compared to the total left ventricular area using Image J software(NIH)and was calculated as the percentage of infarct area of the total cross-sectional area of the heart in each section for comparison.ELISA was performed to measure PMPs and Calpain 10 level using antibodies to PMPs and Calpain 10 according to the manufacturer's protocol.3.All data were analyzed by statistical software SPSS 13.0 editions.The difference of measurement data was detected by analysis of variance.Two-two comparison among all means was done by Student-Newman-Keuls test and LSD pairwise comparison methods.Pearson Correlation analysis was applied to analyze the correlation among Calpain10,infarction areas and PMPs.Linear regression analysis was applied to further asses the correlation between calpain10 and infarction areas.Results: 1.Significant differences(P < 0.01)were revealed among all the groups in calpain 10 and infarction area.Calpain10 and infarction areas in sham-operated group were decreased most than other groups followed by dual antiplatelet intervention groups and then aspirin alone intervention group.No difference was detected in calpain 10 levels between sham-operated group and aspirin combined with clopidogrel intervention group(P =0.42).Calpain10 and infarction areas were all decreased in drug intervention groups,significantly in dual antiplatelet intervention groups.2.No difference was found in infarction area between dual antiplatelet intervention groups(P =0.22).3.Calpain10 were significantly correlated with infarction area.(r =0.84,P< 0.01).4.The linear regression analysis model of infarction area= 3.350+ 0.342~*Calpain10 was achieved.Conclusions: 1.Firstly,Calpain10 was significantly correlated with infarction area.Calpain10 could be applied to assess infarction area with the regression model of Infarction Area=3.350+ 0.342~*calpain10.2.Calpain10 is involved in regulation of PMPs expression and could increase PMPs expression by promote platelet secretion,aggregation,and spreading.3.Antiplatelet drugs can decrease Calpain 10 expression,and the mechanism may be related to inhibition of calcium influx.4.Antiplatelet drugs can decrease PMPs expression through downregulating Calpain10.Our study has established Calpain10 are valuable in assessing the extent of myocardial infarction.Calpain 10 could regular PMPs expression and take part in the development of myocardial infarction.Calpain 10 is novel and potential target for intervention of myocardial infarction..Part Three Platelet Microparticles is Relevant to Left Ventricular Ejection Fraction,as a cardiac remodeling markerPlatelet microparticles(PMPs)result from platelet activation and apoptosis and play an important role in the coronary heart disease,hypertension,pulmonary artery hypertension and so on.However,the value of PMPs in cardiomyopathy and the correlation between PMPs and cardiac remodeling need further research.Objective: Firstly,to observe the correlation between PMPs and cardiac remodeling in mice model of transverse aortic constriction(TAC).Secondly,to evaluate the clinical value of PMPs in the hypertrophy cardiomyopathy.Try to find if there are correlations among PMPs,left ventricular mass(LV Mass)and left ventricular ejection fraction(LVEF).Finally,to observe the influence of losartan on the expression of PMPs and LV Mass,try to illuminate the intervention mechanism of losartan on PMPs.Methods: 1.18 eight-week-old male C57BL/6J mice were used for all murine experiments and divided into three groups(the sham-operated group,the TAC group,the losartan intervention group).Transverse aortic constriction was induced surgically by a permanent ligation of the aortic arch.Pharmaceutical interventions(losartan 5 mg/kg)were taken by oral gavage daily,one week before and after the operation.2.Animals were humanely sacrificed one week after the operation.The various indicators of cardiac structure and function under cardiac ultrasound were evaluated before sacrifice.ELISA was performed to measure PMPs using antibodies to PMPs according to the manufacturer's protocol.3.All data were analyzed by statistical software SPSS 13.0 editions.The difference of measurement data was detected by analysis of variance.Two- two comparison among all means was done by LSD pairwise comparison methods.Kruskal Wallis and Wilcoxon test were applied to analyze non-normally distributed or unequal variances data.The correlations among PMPs,LV Mass and LVEF were analyzed by Pearson Correlation analysis.Results: 1.Significant differences(P < 0.01)were revealed among all the groups in PMPs and LV Mass.PMPs and LV Mass in sham-operated group were decreased most than the other groups.The highest levels of PMPs and LV Mass were detected in TAC group.There were no differences in PMPs and LV Mass between the losartan intervention group and sham-operated group.2.No difference was found in LVEF among the three groups.3.PMPs was significantly positive correlated with LV Mass(r=0.908,P<0.01).4.PMPs was significantly negative correlated with LVEF(r=-0.5,P<0.05).Conclusions: 1.PMPs was significantly positive correlated with LV Mass.It can be used as a biomarker to assess cardiac remodeling.2.Losartan can downregulate PMPs expression by inhibiting platelet AT1 receptor.Our study has established PMPs is valuable in assessing cardiac remodeling and cardiac function.We will testify them with clinical research in our follow studies. |
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