Application study of a novel apoptogenic protein for the pathogenesis of atherosclerotic diseases

新型凋亡蛋白在动脉粥样硬化疾病发病机制中的应用研究

基本信息

批准号：
18590810
负责人：
YASUDA Osamu
金额：
$ 2.49万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18590810/
关键词：
Atherosclerosis Apoptosis Mitochondria Smooth muscle cell

项目摘要

Apop is a protein originally isolated as a factor of unlkown function from cultured atherosclerotic smooth muscle cells. We found recently that expression of Apop induces apoptosis of vascular smooth muscle cells (SMC) in culture by releasing cytochrome c from mitochondria. Release of cytochrome c from the permeability transition pore (PTP) is believed to play an important role in ischemia/reeoxygenation-induced cardiomyocyte apoptosis, which contributes the development of heart failure following balloon angioplasty after myocardial infarction. This study was performed to investigate the role of Apop in hypoxia/reoxygenation-induced death of cardiomyocytes. Neonatal cardiomyccybas were prepared from ventricles of 2- to 3-day-old Sprague-Dawley rats and maintained in DMEM supplemented with 10% fetal bovine serum. Antisense plasmid to prevent Apop expression or control vector was introduced into cardiomyocybes using an electroporator. Cells were then incubated in hypoxic condition (1% O2 … More ) for 48 hours followed by 2 hours of reoxygenation. Cell viability was assessed by MTT assay. Anti-apoptotic bcl-2 family proteins, such as bcl-2 and bcl-xL were unable to prevent Apop-induced apoptosis, while it was prevented by inhibitors of cyclophilin D, an important component of PTP. These findings indicate that the apoptosis induced by Apop is not mediated by bcl-2 family-related channels, but may require PIP on mitochondrial membrane. Introduction of siRNA targeted toward Apop transcript into cultured cells almost completely abolished the green fluorescence of Apop-GFP fusion protein, indicating that siRNA prevents the expression of Apop. The inhibitory activity of siRNA was also confirmed by Western blot analysis. In the experiments of hypoxia/reoxygenation-induced cell death, introduction of siRNA inhibited the activation of caspase-cascade and the death of cultured cardiomyccytes, whereas introduction of control siRNA affected neither caspase activity nor viability; Inhibition of Apop expression prevented hypoxia/reoxygenation-induced death of cardiomyocytes, suggesting that Apop protein may play roles in the development of cardiac diseases following ischemia-reperfusion injury. Less

APOP是一种蛋白质，最初是从培养的动脉粥样硬化平滑肌细胞中分离出的不屈服功能的因素。最近，我们发现，通过从线粒体释放细胞色素C，凋亡在培养中诱导的血管平滑肌细胞（SMC）的凋亡表达。据信，从渗透性过渡孔（PTP）中释放细胞色素C在缺血/脱氧诱导的心肌细胞凋亡中起重要作用，在心肌梗死后气球血管成形术后，心力衰竭的发展有助于心力衰竭的发展。进行了这项研究，以研究APOP在缺氧/重氧诱导的心肌细胞死亡中的作用。从2至3天大的sprague-dawley大鼠的心室制备新生儿心脏肌cybas，并在补充10％胎牛血清的DMEM中保持。使用电氧化剂将防止APOP表达或对照载体的抗义质粒引入心肌围候。然后将细胞在低氧条件下（1％O2…更多）孵育48小时，然后进行2小时的重氧。通过MTT分析评估细胞活力。抗凋亡BCL-2家族蛋白（例如BCL-2和BCL-XL）无法预防凋亡诱导的凋亡，而Cyclophilin D的抑制剂则阻止了ptp的重要组成部分。这些发现表明，APOP诱导的凋亡不是由Bcl-2家族相关的通道介导的，而是在线粒体膜上需要PIP。将靶向APOP转录物靶向培养细胞的siRNA的引入几乎完全消除了APOP-GFP融合蛋白的绿色荧光，这表明siRNA阻止了APOP的表达。通过Western印迹分析也证实了siRNA的抑制活性。在缺氧/重氧诱导的细胞死亡的实验中，siRNA的引入抑制了caspase-cascade的激活和培养的心肌cy的死亡，而对控制siRNA的引入既不影响caspase活性，也没有影响生存能力。抑制APOP表达可防止缺氧诱导的心肌细胞死亡，这表明APOP蛋白可能在缺血 - 再灌注损伤后在心脏病的发展中起作用。较少的