路特异性抑制剂Cyclopamine所抑制（p<0.01）。以上结果表明重组人SHH蛋白在激活SHH信号通路的下游信号蛋白表达过程中扮演重要的角。
6.急性心肌梗死患者冠脉侧枝循环较差组血浆SHH水平与对照组相比差异无统计学意义(p >0.05)；并且冠脉侧枝循环较好组中血浆SHH水平显著高于冠脉侧枝循环较差组以及对照组(p <0.01 )，提示在急性心肌梗死患者冠脉侧枝循环评分较高的患者中其血浆SHH含量也比较高(p <0.01)，并且其相关性独立于年龄、性别、BMI、高血压、糖尿病家族史、心肌梗死类型、吸烟史、饮酒史、空腹血糖水平、血肌酐、血尿素氮、总胆固醇、甘油三酯、HDL-C、BNP等危险因素单独存在。
结论：1.SHH对缺血再灌注损伤的心脏微血管内皮细胞具有保护作用；CMECs缺血再灌注损伤存在着SHH信号通路的激活；外源性加入重组人SHH蛋白可以增强细胞活力，减轻细胞凋亡，上调血管新生因子VEGF, FGF和Ang-1的表达，促进新生血管形成，提示SHH信号通路激活在CMECs缺血再灌注损伤后血管再生过程中发挥着举足轻重的作用，可能成为心肌缺血再灌注损伤的潜在靶点。
2.急性心肌梗死患者冠状动脉侧支循环建立与血浆SHH水平明显相关，且血浆SHH水平独立于年龄、性别、BMI、高血压、糖尿病家族史、心肌梗死类型、吸烟史、饮酒史、空腹血糖水平、血肌酐、血尿素氮、总胆固醇、甘油三酯、HDL-C、BNP等危险因素作用于急性心肌梗死患者缺血心肌区域侧枝循环的形成过程。提示可以将SHH血清水平作为急性心肌梗死患者冠状动脉侧枝循环形成状况好坏的预测因子。
博士研究生：国伟（心血管病）
指导教师：杨军（教授）
关键词：缺血再灌注损伤；血管再生；SHH；细胞凋亡；急性心肌梗死
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Roles of SHH signaling pathway in neovascularization
Abstract
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. mRNA 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
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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.mRNA 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 mRNA level of SHH .
2.Cell viability was significantly increased by SHH application compared to that in control, but this effect was significant1y suppressed with the application of SHH inhibitor of Cyclopamine(p<0.05), ind
icating 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 oxygen- glucose 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 1
6.7%, 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 mRNA 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 mRNA expressions of VEGF, FGF and Ang-1 compared to
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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 oxygen- glucose deprivation/reoxygenation condition.
5.SHH application significantly increased the mRNA 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 signific
antly 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-proBNP.
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
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plasma SHH levels can be used as a biomarker to predict coronary collateral formation in patients with acute myocardial infarction.
Graduate student: Guo Wei(Cardiology)
proliferationDirected by Prof. Yang Jun
Key words:Ischemic-reperfusion injury, vasculogenesis, SHH, cell apoptosis, acute myocardial infarction
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