糖化apo(a) 促进血管内皮功能不全及机制研究

动脉内皮功能不全是糖尿病血管病变早期表型，脂蛋白糖化在内皮功能不全的发生中具关键作用。我们发现，在糖尿病患者脂蛋白(a)代谢中，载脂蛋白(a)[apo(a)]及其含kringle V序列降解肽段[apo(a)-k]能发生糖化，且糖化程度与冠状动脉病变严重性有关。体内外实验中，糖化apo(a)和糖化apo(a)-k均诱导内皮功能不全；且均促进小鼠动脉粥样硬化。mRNA芯片显示，此两种糖化蛋白显著上调抑制性核受体NR0B1。以siRNA下调NR0B1后，其致内皮功能不全效应减弱；敲低NR0B1则动脉粥样硬化显著降低。结合文献提示，此两种糖化蛋白经上调NR0B1而抑制保护性核受体诱导内皮功能不全。后续拟构建全身和内皮敲减的NR0B1小鼠及培养原代细胞，从体内外层面探讨外源性糖化apo(a)和糖化apo(a)-k、内源性糖化apo(a)及其降解肽段经NR0B1促进内皮功能不全效应及机制。

Arterial endothelial dysfunction is an early marker of diabetic vascular complications, and glycation of lipoproteins play a pivotal role in the development of endothelial dysfunction. In the present study, we found that apoprotein (a) [apo(a)] and its degradation-derived peptide of kringle V-like sequence [apo(a)-k] can be glycated during the metabolism of lipoprotein (a), and the degree of glycation was associated with atherosclerosis severity in type 2 diabetic patients with significant coronary artery disease. In vitro experiments demonstrated that glycated apo(a) and glycated apo(a)-k promoted inflammatory reaction and oxidative stress, increased vascular permeability, and impaired angiogenic function of endothelial cells. Intraperitoneal injection of these two glycated proteins induced atherosclerosis in apoE-/-mice. Moreover, cDNA microarray revealed that nuclear receptor NR0B1, the co-repressor of protective nuclear receptor NR4A1 and estrogen receptor (ER), was greatly up-regulated in glycated apo (a)- or glycated apo (a)-k-stimulated endothelial cells. In contrast, down-regulation of NR0B1 with siRNA decreased the effects of these two glycated proteins on endothelial dysfunction. Compared with control mice, NR0B1+/-mice developed less atherosclerosis in the aorta after feeding with high-fat food. Thus, our results, integrated with previous findings, collectively suggest that NB0B1 may be a key mediator in endothelial dysfunction caused by glycated apo(a) and glycated apo(a)-k. Our future studies include two parts. First, we project to determine the effects of glycated apo (a) and glycated apo (a)-k promoting vascular endothelial dysfunction with in vivo and in vitro experiments using global and endothelial specific NR0B1 deficient mice. Exogenous apo(a) and apo(a)-k and endogenous glycated apo (a) and its degraded peptide will be probed for their impact on endothelial dysfunction. Second, we search for the key glycation site of apo(a) related to endothelial dysfunction and atherosclerosis in type 2 diabetic patients by mass spectrometry, and the mechanism of its action and pathogenic pathway will be clarified.