Roles Of SHH Signaling Pathway In Neovascularization

Objective:Recently,various evidences have demonstrated that SHH is involved in vasculogenesis among diversity ischemia models including cerebral ischemia,skeletal muscle ischemia and cornea and body model.However,few studies have mentioned the effects of SHH signaling in MIRI,hence the protective effects of SHH on cardiac microvascular endothelial cells injury induced by ischemia-reperfusion is not seen through the SHH-Patched-SMO-Gli signaling pathway-related literature reports.There is no evidence about the function of SHH in the promotion of angiogenesis in patients with acute myocardial infarction.1.This study aimed to analyze the effects of SHH signal proteins expression on CMECs apoptosis and proliferation,investigate the potential roles of SHH signaling pathway activation in vasculogenesis on cardiac microvascular endothelial cells(CMECs)injury induced by ischemia-reperfusion and its underlying mechanism.Our study may provide theoretical basis for the possibility application of SHH on MIRI protection in clinical.2.Investigate the correlation between the formation of coronary collateral circulation and plasma SHH levels,estimate the function of SHH in the promotion of angiogenesis in patients with acute myocardial infarction.Our study may provide a new therapeutic target for effective clinical prevention of acute myocardial infarction.Methods:Cardiac microvascular endothelial cells(CMECs)isolated from the SD rat hearts tissues were used to construct the OGD MIRI model,then SHH protein or Cyclopamine was injected.m RNA level of SHH in control cells and MIRI cells was detected using RT-PCR analysis.Furthermore,effects of SHH expression on CMECs viability and apoptosis were analyzed using MTT assay and Annexin-V-FITC kit respectively.The expression of angiogenetic factors were analyzed using ELISA and RT-PCR.Moreover,effects of SHH expression on the pathway signal proteins expression was analyzed using RT-PCR and western blotting.In the clinical study,298 patients who were admitted to the coronary care unit,Jining NO.1 People's Hospital,diagnosed with acute myocardial infarction and underwent primary percutaneous coronary intervention were stratified into three groups according to the Rentrop scoring system: the non-collateral group,poor collateral group and good collateral group.Collect the patients,clinical datas and laboratory outcomes,determine IRA and record the time of myocardial infarction.Detect the plasma SHH levels of the blood sample before percutaneous coronary intervention with ELISA methods.Analyze the data with the SPSS 20.0,estimate the correlation between the formation of coronary collateral circulation and plasma SHH levels.Results:1.m RNA level of SHH was significantly increased in the OGD group compared to the control group(p <0.05),indicating that oxygen-glucose deprivation reoxygenation injury can lead activation of SHH signaling pathway,and increase m RNA level of SHH.2.Cell viability was significantly increased by SHH application compared to that in control,but this effect was significant1 y suppressed with the application of SHH inhibitor of Cyclopamine(p<0.05),indicating that SHH may play certain contribute roles in enhancting cell viability under normal oxygen and glucose condition.On the contrary,when cells were treated with OGD,cell viability was significantly decreased(p<0.05),suggesting the harmful effects of OGD on cell viability.However,this effect was highly increased by SHH addition compared with the OGD group(p<0.05),indicating that SHH may play certain contribute roles in enhancing cell viability under oxygenglucose deprivation/reoxygenation condition.3.Although cell apoptotic percentage was slightly decreased 4.09% by SHH application compared to that in control,there was no significant difference of cell apoptosis among control,SHH and Cyclopamine group.When cells were added with Cyclopamine,apoptotic percentage was slightly increased but not significantly increased 6.43%.Besides,when cells were treated with OGD,apoptotic cells were more than that in the control group(12.4%,p <0.05),but this enhance effect was significantly suppressed by SHH application,which was decreased from 12.4% to 5.23%(p<0.05).However,when CMECs were treated with Cyclopamine,the percentage of apoptosis cells were again increased to 16.7%,indicating that SHH may play certain contribute roles in reducing cell apoptotic under oxygen-glucose deprivation/reoxygenation condition.4.SHH treatment significantly increased the plasma level and relative m RNA expressions of Vasculogenesis-related factors including VEGF,FGF and Ang-1 compared to that in control(p<0.05),but this effect was inhibited by Cyclopamine treatment(p<0.01),indicating that SHH may play certain contribute roles in vasculogenesis under normal oxygen and glucose condition.Similarly,SHH application significantly increased the plasma level and relative m RNA expressions of VEGF,FGF and Ang-1 compared to the OGD group(p<0.01),but this effect was reversed by Cyclopamine application(p<0.01),indicating that SHH may play certain contribute roles in vasculogenesis under oxygenglucose deprivation/reoxygenation condition.5.SHH application significantly increased the m RNA expressions of SHH,SMO,Pathced-1,Gli-1 and Gli-2 in cells compared to the OGD group(p<0.05).However,this enhance effect was significantly suppressed by the application of inhibitor of Cyclopamine(p<0.01).Similarly,SHH application significantly increased the relative density of protein level of SHH,SMO,Pathced-1,Gli-1 and Gli-2 in cells compared to the OGD group(p<0.05).However,this enhance effect was significantly suppressed by the application of inhibitor of Cyclopamine(p<0.01).These results showed that SHH treatment may play certain roles in activating the downstream signal proteins expression of SHH signaling pathway.6.There are no significant differences between plasma SHH level of poor collateral circulation group and control group(p >0.05),however good collateral group have higher plasma SHH level than poor collateral circulation group and control group(p <0.01).This study demonstrate those patients of acute myocardial infarction who have good collateral circulation may have a higher SHH plasma level.The plasma SHH is an independent risk factor beside age,gender,BMI,hypertension,diabetes,types of myocardial infarction,smoking,drinking,fasting blood glucose,serum creatinine,urea nitrogen,total cholesterol,HDL-C,NT-pro BNP.Conclusion:1.This study demonstrate that heart administration of SHH protein could protect heart from ischemic injury,enhance cell viability,reduce cell apoptotic,increase vasculogenesis-related factors including VEGF,FGF and Ang-1 and promote angiogenic repair.These data suggested that activating SHH signals pathway may play a pivotal contribute role in vasculogenesis on cardiac microvascular endothelial cells injury induced by ischemia-reperfusion.2.The plasma SHH level was strongly correlated with collateral circulation in acute myocardial infarction patients,and the correlation was independent of age,gender,BMI,hypertension,diabetes,types of myocardial infarction,smoking,drinking,fasting blood glucose,serum creatinine,urea nitrogen,total cholesterol,HDL-C,NT-pro BNP.Hence plasma SHH levels can be used as a biomarker to predict coronary collateral formation in patients with acute myocardial infarction.