LDL isoforms in kidney-related atherosclerosisv

描述(申请人提供)：慢性肾脏病(CKD)合并或不合并尿毒症影响全球约10%的人口，由于动脉粥样硬化加速，发生心血管疾病的风险增加到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型低密度脂蛋白(OxLDL)引起的内皮细胞损伤启动。最近有研究显示尿毒症终末期肾病(ESRD)患者体内oxLDL水平升高，但尿毒症与oxLDL诱导的动脉粥样硬化之间的机制联系尚未建立。目前由Roxborough和Young发展的理论认为，尿素升高会导致氨基甲酰化低密度脂蛋白(CLDL)的形成，从而使低密度脂蛋白更容易被氧化。我们的数据在不同程度上与这一理论相矛盾。总的来说，我们的体外实验显示oxLDL和cLDL对内皮细胞和血管平滑肌细胞的作用相似，但在人类中cLDL和oxLDL的水平显著不同。我们假设血浆cLDL是一种oxLDL非依赖的因子，与ESRD患者现有的动脉粥样硬化性疾病及其进展有关，并且是体外培养内皮细胞损伤的原因。我们最近建立的cLDL夹心ELISA定量方法显示，人血浆中cLDL的浓度是用类似方法测得的oxLDL的5-10倍。尿毒症患者cLDL和oxLDL均升高。然而，与无IMT增厚的尿毒症患者相比，有动脉粥样硬化(IMT)的尿毒症患者的cLDL升高约200%，而oxLDL升高不明显。用人血浆的酶联免疫吸附试验没有发现有两种类型的修饰的显著低密度脂蛋白。我们的初步数据显示，天然低密度脂蛋白的氨甲酰化部分抑制了它的氧化。在体外，cLDL产生与动脉粥样硬化相关的细胞损伤，与oxLDL相似，而且几乎同样严重。改变包括内皮细胞死亡，细胞黏附分子的诱导，单核细胞的吸引，以及诱导内皮细胞和血管平滑肌细胞的增殖。通过体外化学修饰产生的cLDL比oxLDL聚集更多，并且在兔体内比oxLDL具有更强的免疫原性。综上所述，我们的数据表明，在尿毒症浓度下，cLDL对内皮细胞的致动脉粥样硬化损伤可能比oxLDL更严重。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的措施有关，独立于oxLDL；以及(2)量化和研究正常生理和尿毒症相关浓度的LDL亚型在体外诱导血浆cLDL和oxLDL介导的内皮细胞损伤的机制。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患者的一个可靠的和机械联系的心血管危险因素。

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.