Mechanism of arterial calcification: Role of matrix metalloproteinases

目的：目前糖尿病/透析患者的增加增加了治疗伴有钙化动脉的缺血性疾病的机会。本研究旨在探讨糖尿病/透析患者动脉钙化的机制和动物模型，以及基质金属蛋白酶在动脉钙化中的作用。材料和方法：在研究期间，12例糖尿病/透析患者在首次静脉搭桥术后3~10个月因肢体缺血而接受改良静脉移植狭窄手术的人静脉移植标本，以及来自40周龄的老年糖尿病大鼠(大冢Long Evans Tokushima Fatty大鼠；OLETF大鼠)的动物主动脉标本，保存在细胞库中供以后分析。对所有标本进行横断切片，用光学显微镜和扫描电子显微镜筛选钙化微点；当扫描电子显微镜鉴定出钙化微点时，用电子探针显微镜…进行分析同时行免疫组织化学染色观察钙化情况。免疫组织化学检测骨钙素和MMC-3、-9、-13的表达。结果：扫描电子显微镜显示人移植静脉和大鼠主动脉中膜有许多大小不一的微斑点，EPMA显示斑点内有磷和钙的存在，提示这些微斑点为钙化。在糖尿病/透析患者的人静脉移植物中，微钙化在植入后3个月内开始。免疫组织化学显示MMPs和骨钙素的表达，但与动脉钙化起始机制的详细关系尚不清楚。透射电子显微镜显示钙化斑点直径为70~300 nm，位于细胞外基质中。进一步的放大显示钙化是由直径为10 nm的纳米颗粒聚集而成。结论：动脉钙化在基质中形成细胞外基质，并形成直径为10 nm的颗粒聚集。钙化的发展与MMPs和骨钙素等基因表达之间的关系仍不清楚。较少

OBJECTIVES: Current increase of patients with diabetes/dialysis increases opportunities of management of ischemic diseases with calcified arteries. In the present study, we investigated the mechanism of arterial calcification in diabetic/dialysis patients as well as animal model, and a role of matrix metalloproteinases in arterial calcification.MATERIAL AND METHOD : During the study period, human vein graft specimens were obtained from 12 patients with diabetes/dialysis who underwent revised surgery for vein graft stenosis 3-10 months after the initial vein bypasses for limb ischemia, and animal aortic specimens were obtained from old diabetic rats (Otsuka Long Evans Tokushima Fatty rat; OLETF rat) with age of 40 weeks, and were stored in cell baner for later analysis. All of the specimens were cross-sectioned, and calcification microspots were screened by light microscopy and SEM; when calcified microspots were identified by SEM, the specimens were analyzed by the electroprobe microan … More alysis (EPMA) for confirmation of calcification, and were also subjected to immunohistochemistry. In immunohistochemistry, osteocalcin and MMC-3,-9, and-13 were examined. After the confirmation, the calcification spots was subjected to TEM to evaluate relation to the surrounding tissue.RESULTS : The SEM demonstrated many various sized microspots in the media in all human vein grafts as well as rat aortas, and EPMA demonstrated the presense of phosphorus and calcium in the spots, probing the microspots to be calcification. In human vein grafts in diabetic/ dialysis patients, microcalcification initiates within 3 months after implantation.Immunohistochemistry demonstrated the expression of MMPs as well as osteocalcin, but the detail relationship to the initiation mechanism of arterial calcification remained unclear. TEM showed calcification spots were with diameters of 70-300 nm, and located in extracellular matrix. Further magnification disclosed the calcification consisted of an aggreigation of nanoparticles with a diameter of 10nm.CONCLUSION : Arterial calcification develops extracellular matrix in media, and forms aggregation with a diameter of 10 nm particles. Relation between development of calcification and gene expressions such as MMPs and osteocalcin still remained unclear. Less