Role of miRNA and posttranslational modifications as modulators of the functionality of high-density lipoprotein (HDL) in chronic kidney disease

慢性肾脏疾病(CKD)是血管事件发生的最大危险因素之一。其潜在的机制目前还不太清楚。然而，已知的是，内皮功能障碍、血管损伤再生障碍以及促炎微环境可能会促进肾功能受损患者的血管疾病。在我们之前的工作中，我们能够证明尿毒症将脂蛋白高密度脂蛋白和低密度脂蛋白转化为有害颗粒，从而导致内皮功能障碍。此外，我们发现这些脂蛋白的结构表型和功能之间存在很强的相关性。除了蛋白质组成的数量和质量变化外，翻译后蛋白质对高密度脂蛋白的修饰可能是其功能的关键调节因素。此外，高密度脂蛋白作为miRNA的载体，将它们输送到各种不同的细胞。然而，这种改变的高密度脂蛋白结构在特定疾病条件下的相关性还有待确定。本项目的目的是研究翻译后蛋白质修饰(第一部分)以及与高密度脂蛋白相关的miRNA(第二部分)如何调节高密度脂蛋白对血管再生和炎症细胞激活的影响。在翻译研究设计中，将使用从不同程度肾损害患者和健康受试者分离的高密度脂蛋白。在第一部分，我们将分析慢性肾脏病患者高密度脂蛋白相关蛋白的翻译后修饰。使用这些患者、健康受试者的高密度脂蛋白以及体外修饰的高密度脂蛋白，将研究这些高密度脂蛋白制剂对血管再生的影响。为此，我们将使用不同的体外实验(增殖、迁移、血管生成)和体内实验(小鼠颈动脉损伤模型/Matrigel Plug实验)。此外，我们的目的是通过激活天然免疫系统来确定高密度脂蛋白在促炎微环境中的作用。在第二部分中，我们将研究慢性肾脏病患者和健康受试者之间高密度脂蛋白相关miRNA的差异。然后我们将分析特定的高密度脂蛋白相关的miRNA如何干扰内皮细胞和单核细胞的功能特性来调节血管再生和炎症细胞的激活。总之，这些研究将有助于更好地理解促进血管疾病的生理和病理生理过程，特别是在CKD患者中。此外，我们的目标是将高密度脂蛋白相关的miRNA以及高密度脂蛋白的翻译后蛋白修饰确定为新的治疗靶点和/或生物标记物。

Chronic kidney disease (CKD) represents one of the strongest risk factor for the occurrence of vascular events. The underlying mechanisms have yet been poorly understood. However, it is known that endothelial dysfunction, impaired regeneration of vascular lesions as well as a proinflammatory microenvironment may promote vascular diseases in patients with impaired renal function. In our previous work, we were able to document that uremia transforms the lipoproteins HDL and LDL into noxious particles, which induce endothelial dysfunction. Moreover, we revealed a strong associated between the structural phenotype and the functionality of these lipoproteins. Besides quantitative and qualitative alterations of the protein composition, posttranslational proteins modifications of HDL might represent a crucial modulator of its functionality. Moreover, HDL serves as a carrier for miRNA and conveys them to a variety of distinct cells. However, the relevance of such an altered structure of HDL under specific disease conditions - particularly CKD - has to be determined.It is the aim of the present project to examine on how posttranslational protein modifications (part 1) as well as HDL-associated miRNA (part 2) modulate the effects of HDL on vascular regeneration and inflammatory cell activation. In a translational study design, HDL isolated from patients with different degrees of renal impairment as well as healthy subjects will be used.In part 1, we will analyze posttranslational modifications of HDL-associated proteins in patients with CKD. Using HDL from these patients, healthy subjects as well as ex vivo modified HDL, the effects of these HDL preparations on vascular regeneration will be studied. For this purpose, we will use distinct in vitro (proliferation, migration, angiogenesis) and in vivo assays (murine carotid injury model/matrigel plug assay). Moreover, we aim to determine on HDL contributes to a proinflammatory microenvironment by activating the innate immune system.In part 2, we will study differences in the profile of HDL-associated miRNA between patients with CKD and healthy subjects. We will then analyze on how specific HDL-associated miRNA interfere with the functional properties of endothelial cells and monocytes to regulate vascular regeneration and inflammatory cell activation.In summary, these studies should contribute to a better understanding of the physiological und pathophysiological processes promoting vascular disease in general and particularly in patients with CKD. Moreover, we aim to identify HDL-associated miRNA as well as posttranslational protein modifications of HDL as novel therapeutic targets and/or biomarkers.