NATIVE LDL, CHOLESTEROL AND IMPAIRED VASORELAXATION

血管松弛丧失与血浆天然低密度脂蛋白（LDL）胆固醇浓度升高有关。低密度脂蛋白胆固醇损害松弛的机制尚不清楚。先前，我们观察到天然LDL使培养内皮细胞的胆固醇含量增加了近2倍。更重要的是，超氧化物的释放量增加了4倍以上。当超氧化物以接近扩散速率与一氧化氮反应时，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起核心作用。我们假设，长时间暴露于致动脉粥样硬化浓度的天然低密度脂蛋白会使内皮改变一氧化氮和超氧化物的平衡，从而损害松弛反应。尽管存在大量间接证据表明高胆固醇血症会增加超氧化物，但尚未进行确定和量化血管内皮产生的活性氧的明确测量。天然低密度脂蛋白、胆固醇与血管内皮细胞生成超氧化物和一氧化氮之间的直接联系尚未确定。本应用研究了胆固醇改变一氧化氮和超氧化物平衡的机制，以及这种改变对内皮依赖和非内皮依赖、受体依赖和非内皮依赖的离体动脉舒张的功能后果。最先进的电子自旋共振（ESR）和自旋捕获将用于鉴定和量化培养的内皮细胞和冠状动脉环的自由基种类。动脉环将与天然低密度脂蛋白或富含胆固醇的脂质体预先孵育，以改变一氧化氮和超氧化物的平衡。动脉环将被悬挂在含有自旋陷阱的组织浴中。记录对P或A23187物质的松弛反应。自旋捕获自由基的ESR将提供弛豫反应过程中活性氧生成的第一个直接测量。LDL和胆固醇诱导eNOS产生超氧化物的机制将在四氢蝶呤代谢和翻译后修饰方面进行研究，这些修饰与eNOS、caveolin和CaM之间相互作用的干扰有关。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化机制。

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.