LDL isoforms in kidney-related atherosclerosisv

描述（由申请人提供）：伴有或不伴有尿毒症的慢性肾病（CKD）影响全球约10%的人口，并且由于动脉粥样硬化加速，患心血管疾病的风险增加至肾功能正常者的数倍。多项研究表明，动脉粥样硬化可由氧化低密度脂蛋白（oxLDL）诱导的内皮细胞损伤引发。最近在终末期肾病（ESRD）尿毒症患者中显示oxLDL水平升高，但尿毒症和oxLDL诱导的动脉粥样硬化之间的机制联系尚未建立。由罗克斯伯勒和杨发展的当前理论表明，尿素升高导致形成氨甲酰化LDL（cLDL），使LDL更容易氧化。我们的数据在不同层面上与这一理论相矛盾。一般来说，我们的体外实验表明oxLDL和cLDL对内皮细胞和血管平滑肌细胞的作用相似，但在人体中cLDL和oxLDL的水平显着不同。我们假设血浆cLDL是一种oxLDL非依赖性因子，与ESRD患者现有的动脉粥样硬化疾病及其进展相关，并且是体外内皮细胞损伤的原因。我们最近发展的一种定量cLDL的夹心ELISA方法表明，人血浆中cLDL的浓度比用类似方法测得的oxLDL高5-10倍。cLDL和oxLDL在尿毒症患者中均升高。然而，在尿毒症患者动脉粥样硬化内膜中层厚度（IMT）测量，cLDL升高约200%，而oxLDL没有显着增加相比，尿毒症患者没有增厚的IMT。使用人血浆的ELISA测定，未发现具有两种修饰类型的LDL的显著量。我们的初步数据表明，天然LDL的氨甲酰化部分抑制其氧化。在体外，cLDL产生的细胞损伤与动脉粥样硬化相似，几乎一样强烈的oxLDL。变化包括内皮细胞死亡、细胞粘附分子诱导、单核细胞吸引以及内皮和血管平滑肌细胞增殖诱导。通过体外化学修饰产生的cLDL比oxLDL聚集更多，并且cLDL在兔中比oxLDL更具免疫原性。总之，我们的数据表明，在尿毒症浓度下，cLDL可能比oxLDL对内皮细胞产生更多的致动脉粥样硬化损伤。我们的具体目标是：（1）在ESRD血液透析患者的横断面和前瞻性研究中，确定cLDL是否与动脉粥样硬化疾病指标相关，独立于oxLDL;（2）定量和研究由正常生理和尿毒症相关浓度的LDL亚型体外诱导的血浆cLDL和oxLDL介导的内皮细胞损伤机制。如果根据我们的预期，cLDL被证明与CKD相关的动脉粥样硬化相关，它可能在未来被用作CKD患者中可靠的和机械相关的心血管风险因素。

DESCRIPTION (provided by applicant): Chronic kidney disease (CKD) with or without uremia affects approximately 10% of worldwide population and increases the risk of developing cardiovascular diseases to several times that of persons with normal renal function because of accelerated atherosclerosis. Multiple studies have shown that atherosclerosis can be initiated by an endothelial cell injury induced by oxidized LDL (oxLDL). Increased levels oxLDL have been recently shown in uremic patients with end-stage renal disease (ESRD), however a mechanistic link between uremia and oxLDL-induced atherosclerosis has not been established. Current theory, developed by Roxborough and Young, suggests that elevated urea results in the formation of carbamylated LDL (cLDL) which makes LDL more susceptible to oxidation. Our data contradict this theory at different levels. In general, our in vitro experiments showed similarities between oxLDL and cLDL action on endothelial and vascular smooth muscle cells, but significantly different levels of cLDL and oxLDL in humans. We hypothesize that blood plasma cLDL is an oxLDL-independent factor that is associated with existing atherosclerotic disease and with its progression in ESRD patients, and is a cause of endothelial cell injury in vitro. A quantitative cLDL sandwich ELISA method recently developed by us showed that concentration of cLDL in human plasma is 5-10 times higher than oxLDL measured by a similar method. Both cLDL and oxLDL are elevated in uremic patients. However, in uremic patients with atherosclerosis measured by intima-media thickness (IMT), cLDL was elevated approximately 200% while oxLDL was not significantly increased compared to uremic patients without thickened IMT. No significant amount of LDL with both types of modifications was found using the ELISA assay of human plasma. Our preliminary data showed that carbamylation of native LDL partially inhibits its oxidation. In vitro, cLDL produced cell injury relevant to atherosclerosis similar to and almost as intense as oxLDL. Changes included endothelial cell death, induction of cell adhesion molecules, attraction of monocytes, and induction of endothelial and vascular smooth muscle cell proliferation. Produced by in vitro chemical modifications, cLDL aggregated more than oxLDL, and cLDL was much more immunogenic in rabbits than oxLDL. Taken together, our data suggest that, at uremic concentrations, cLDL is likely to produce more atherogenic injury to endothelial cells than oxLDL. Our Specific Aims are: (1) To determine whether cLDL is associated with atherosclerotic disease measures, independently of oxLDL, in cross-sectional and prospective studies of ESRD hemodialysis patients; and (2) To quantify and to study mechanisms of plasma cLDL- and oxLDL-mediated endothelial cell injury induced in vitro by normal physiological and uremia-associated concentrations of the LDL isoforms. If according to our expectation, cLDL is shown to be relevant to CKD-associated atherosclerosis, it may be used in the future as a reliable and mechanistically linked cardiovascular risk factor in CKD patients.