NATIVE LDL, CHOLESTEROL AND IMPAIRED VASORELAXATION

血管舒张的损失与天然低密度脂蛋白（LDL）胆固醇的血浆浓度增加有关。 天然LDL胆固醇损害舒张的机制尚不清楚。 先前，我们观察到天然LDL使培养的内皮细胞的胆固醇含量增加近2倍。 更重要的是，超氧化物的释放增加了4倍以上。 由于超氧化物与一氧化氮以接近扩散速率反应，因此内皮细胞超氧化物产生的增加可能在损害一氧化氮介导的血管舒张中发挥核心作用。 我们推测，长期暴露于致动脉粥样硬化浓度的天然LDL条件的内皮细胞转移平衡的一氧化氮和超氧化物，这反过来又损害舒张反应。 尽管存在大量间接证据证明高胆固醇血症会增加超氧化物，但尚未进行用于鉴定和定量血管内皮产生的活性氧的确定性测量。 天然低密度脂蛋白，胆固醇和血管内皮细胞产生的超氧化物和一氧化氮之间的直接联系尚未建立。 本申请研究胆固醇改变一氧化氮和超氧化物平衡的机制，以及这种改变对离体动脉的内皮依赖性和非内皮依赖性以及受体依赖性和非依赖性舒张的功能后果。 将采用最先进的电子自旋共振（ESR）和自旋捕获技术来鉴定和定量培养的内皮细胞和冠状动脉环中的自由基种类。 动脉环将与天然LDL或富含胆固醇的脂质体预孵育，以改变一氧化氮和超氧化物平衡。 动脉环将悬挂在含有自旋捕集器的组织浴中。 将记录对P物质或A23187的弛豫反应。 ESR自旋捕获自由基物种从浴将提供第一个直接测量的活性氧物种的产生在弛豫响应。 LDL和胆固醇诱导eNOS产生超氧化物的机制将检查与四氢蝶呤代谢和翻译后修饰有关的干扰eNOS，小窝蛋白和钙调素之间的相互作用。 这些研究将确定胆固醇与松弛反应受损的生理相关性。 该研究结合了细胞生物学和血管生理学的应用，以了解内皮细胞胆固醇富集损害血管舒张的生物物理和生物化学机制。

Losses in vascular relaxation are associated with increased plasma concentrations of native low-density lipoprotein (LDL) cholesterol. The mechanisms by which native LDL cholesterol impairs relaxation remain unclear. Previously, we observed that native LDL increased the cholesterol content of cultured endothelial cells nearly 2-fold. More importantly, the release of superoxide was increased more than 4-fold. As superoxide reacts with nitric oxide at near diffusion rates, increases in endothelial cell superoxide production may play a central role in impairing nitric oxide-mediated vasorelaxation. We hypothesize that prolonged exposure to atherogenic concentrations of native LDL conditions the endothelium to shift the balance of nitric oxide and superoxide, which, in turn impairs relaxation responses. Although ample indirect evidence exists demonstrating that hypercholesterolemia increases superoxide, definitive measurements for identifying and quantifying the reactive oxygen species generated by vascular endothelium have not been performed. Direct links between native LDL, cholesterol and the generation of superoxide and nitric oxide by vascular endothelial cells have not been established. This application examines the mechanisms by which cholesterol shifts the balance of nitric oxide and superoxide and the functional consequences of such shifts on endothelium- and non-endothelium-dependent and receptor-dependent and -independent relaxation of isolated arteries. State-of-the-art Electron Spin Resonance (ESR) and spin-trapping will be employed to identify and quantify radical species from cultured endothelial cells and coronary arterial rings. Arterial rings will be pre-incubated with native LDL or cholesterol-rich liposomes to alter nitric oxide and superoxide balance. Arterial rings will be hung in tissue baths containing spin-traps. Relaxation responses to substance P or A23187 will be recorded. ESR of spin-trapped radical species from the baths will provide the first direct measurements of reactive oxygen species generation during relaxation responses. The mechanisms by which LDL and cholesterol induce eNOS to generate superoxide will be examined with respect to tetrahydropterin metabolisms and post-translation modifications related to disturbances in the interactions between eNOS, caveolin and CaM. These studies will define the physiological relevance of cholesterol to impaired relaxation responses. The research combines the use of cell biology and vascular physiology to understand the biophysical and biochemical mechanisms by which cholesterol enrichment of the endothelium impairs vasorelaxation.