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Role of elastin degradation in the process of vascular calcification in uremic milieu

弹性蛋白降解在尿毒症环境血管钙化过程中的作用

基本信息

批准号：
22790806
负责人：
MIZOBUCHI Masahide
金额：
$ 2.5万
依托单位：
Showa University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Young Scientists (B)
财政年份：
2010
资助国家：
日本
起止时间：
2010 至 2012
项目状态：
已结题

项目摘要

Vascular calcification is the most important cause of cardiovascular disease in patients with chronic kidney disease (CKD). Medial layer vascular calcification, which is recognized to be an active process (i.e., the transformation of vascular smooth muscle cells into osteoblast-like cells), is common in CKD patients. We have recently reported the possibility of an interaction between elastin degradation and medial layer vascular calcification. Matrix metalloproteinase-2 (MMP-2), which induces the degradation of elastin, has been implicated in the elastic calcification in arteries of dialysis patients. However, the precise mechanisms by which elastin degradation interacts with the development of vascular calcification remain to be studied. To clarify the mechanisms by which elastin degradation is involved in the development of medial layer vascular calcification in the uremic milieu, we induced aortic medial layer calcification in 5/6 nephrectomized uremic rats (male Sprague-Dawley rats … More ) fed a diet containing high phosphate (1.2%) and lactate (20%). After 10 weeks, the rats were euthanized for the measurement of serum chemistry profiles and histological analyses. The uremic rats showed significant increases in blood pressure, serum creatinine, phosphate, and parathyroid hormone levels compared with normal rats. von Kossa staining showed medial layer aortic calcification in the uremic rats. In calcified lesions, thin elastic lamellae were observed by elastin staining, indicating that elastin degradation could occur in the area. Furthermore, MMP-2 expression determined by immunohistochemistry was also observed in the same area. Elastin degradation accompanied by MMP-2 expression might be involved in the development of medial layer vascular calcification in uremic rats. In the next step we focused on anti-inflammatory effect of vitamin D receptor activators (VDRAs) on vascular smooth muscle cell (VSMC) mineralization induced by phosphate and TNF-α since inflammatory cytokines induce MMP-2 which contributes to elastin degradation.Human VSMCs were treated with either vehicle, maxacalcitol (10^<-9> M to 10^<-7> M), or calcitriol (10^<-9> M to 10^<-7> M) in 2.5 mM of phosphate media with TNF-α (1 ng/ml) for 9 days. VSMC mineralization was determined and expression of genes associated with the osteogenic process was examined by real-time RT-PCR. Expression of matrix metalloproteinase-2 (MMP-2) mRNA in VSMCs and MMP-2 protein in media were also analyzed. Vehicle-treated VSMCs exhibited massive mineralization, which was inhibited by maxacalcitol in a concentration-dependent manner. Calcitriol also inhibited the mineralization. While vehicle-treated VSMCs exhibited increased mRNA expression of genes associated with the osteogenic process (Cbfa1/Runx2 and osteocalcin) compared with VSMCs grown in normal media without TNF-α (control), maxacalcitol and calcitriol suppressed the increase in mRNA species. Furthermore, vehicle-treated VSMCs exhibited increased MMP-2 mRNA and protein in the media that were suppressed notably by maxacalcitol. Both of the VDRAs abrogated the acceleration of the osteogenic process induced by phosphate and TNF-α in VSMCs, which was linked to inhibition of mineralization in VSMCs. MMP-2 blockade by VDRAs may contribute to an inhibitory effect on vascular calcification. Less

血管钙化是慢性肾脏病（CKD）患者心血管疾病的最重要原因。中层血管钙化，这被认为是一个活跃的过程（即，血管平滑肌细胞转化成成骨细胞样细胞），在CKD患者中很常见。我们最近报道了弹性蛋白降解和中层血管钙化之间相互作用的可能性。基质金属蛋白酶-2（MMP-2）诱导弹性蛋白降解，与透析患者动脉弹性钙化有关。然而，弹性蛋白降解与血管钙化发展相互作用的确切机制仍有待研究。为了阐明弹性蛋白降解参与尿毒症环境中中层血管钙化发展的机制，我们在5/6肾切除的尿毒症大鼠（雄性Sprague-Dawley大鼠）中诱导主动脉中层钙化 ...更多信息饲喂含高磷酸盐（1.2%）和乳酸盐（20%）的饲料。10周后，对大鼠实施安乐死，以测量血清化学特征和组织学分析。与正常大鼠相比，尿毒症大鼠的血压、血清肌酐、磷酸盐和甲状旁腺激素水平显著升高。von Kossa染色显示尿毒症大鼠主动脉中层钙化。在钙化病变中，弹性蛋白染色观察到薄的弹性膜，表明该区域可发生弹性蛋白降解。免疫组化法检测MMP-2的表达。弹性蛋白降解伴随MMP-2表达可能参与尿毒症大鼠血管中层钙化的发生。在下一步中，我们集中于维生素D受体激活剂（VDRA）对由磷酸盐和TNF-α诱导的血管平滑肌细胞（VSMC）矿化的抗炎作用，因为炎性细胞因子诱导MMP-2，其有助于弹性蛋白降解。人VSMC用赋形剂、马沙骨化醇（10 μ <-9>M至10 μ <-7>M）或骨化三醇（10 μ <-9>M至10 μ <-7>M）在含有TNF-α（1 ng/ml）的2.5 mM磷酸盐培养基中处理9天。测定VSMC矿化，并通过实时RT-PCR检测与成骨过程相关的基因表达。检测VSMCs中基质金属蛋白酶-2（MMP-2）mRNA的表达及培养上清中MMP-2蛋白的表达。溶剂处理的VSMCs表现出大量矿化，这是由maxacalcitol以浓度依赖性方式抑制。骨化三醇也抑制矿化。虽然与在无TNF-α的正常培养基中生长的VSMC（对照）相比，溶剂处理的VSMC显示与成骨过程相关的基因（Cbfa 1/Runx 2和骨钙素）的mRNA表达增加，但maxacalcitol和骨化三醇抑制mRNA种类的增加。此外，媒介物处理的VSMCs在介质中表现出MMP-2 mRNA和蛋白质的增加，这被maxacalcitol显著抑制。两种VDRA均消除了VSMC中磷酸盐和TNF-α诱导的成骨过程加速，这与VSMC中矿化的抑制有关。VDRA阻断MMP-2可能有助于抑制血管钙化。少