The Functions And Mechanisms Of Ascorbate Against Oxidant Stress In Gulo^(-/-) Mice And RAW264.7 Macrophages

Atherosclerosis, especially when considered with associated diseases such as diabetes and hypertension, remains the number one killer of both men and women in USA. It is the same situation in China since people's living standard has had a perceptible improvement. Accumulating in vitro and animal data continue to support a role for vitamin C and E, and especially for ascorbate, in countering multiple steps in the atherosclerotic process.The proposed studies will define the role of ascorbate in the function of the major cell types involved in early atherosclerosis both in cell culture and in animal models of atherosclerosis. Knowing whether and how ascorbate modifies early stages of the disease process in cells and animals will provide insight into how ascorbate and other antioxidants might best be used to prevent or mitigate atherosclerosis.To focus on these aspects of the role of ascorbate in atherosclerosis, we will use a mouse model which will lack the ability to synthesize the vitamin due to targeted deletion of the gene for gulonolactone oxidase(gulo(-/-)). In this mouse, we can modify the supply and total body content of the vitamin by dietary manipulations. The low dose of ascorbic acid group mice shows low ascorbate level in plasma. In mice with plasma ascorbic acid levels ranging from normal to subnormal levels, we find that plasma ascorbic acid levels in mice are inversely correlated with plasma cholesterol. To determine the effects of lesions on vascular permeability, wild-type and Gulo mice were injected with Evans blue dye before sacrifice. The abdominal aortas from ascorbate deficient mice showed leakage of macro-molecules into the vessel wall. These findings confirm that lower than normal levels of ascorbic acid are detrimental to the maintenance of the normal architectural integrity of the large vessels in the mutant mice.The macrophage is a crucial cell type in initiating the atherosclerotic process. Macrophages take up oxidized low density lipoprotein (oxLDL) to become a lipid-laden foam cells, secrete cytokines and other activators that enhance the inflammatory nature of this disease, and generate reactive oxygen species(ROS) that may damage both the macrophages and surrounding cells and tissue. LDL preparations were oxidized in the presence of Cu2+. Purity of the LDL preparation was assessed by electrophoresis using the TITAN Gel Electrophoresis System. We used the very minimally oxidized LDL.To assess whether oxLDL induces an oxidant stress in RAW264.7 macrophages, oxidation of intracellular dihydrofluorescein and lipid peroxidation such as the cell content of malondialdehyde were assessed. Both were markedly increased by culture of the cells with increasing concentrations of oxLDL. However, the ascorbate was without effect on the amount of oxLDL taken up by the cells.Since oxLDL caused an oxidant stress in RAW264.7 cells, it was important to determine whether this affected cellular function and survival. Four experiments (MTT,Typan Blue,Annexin V,Caspase3/7) had present. As expected, culture of RAW264.7 macrophages for 18 h with increasing amounts of oxLDL increased apoptosis. Although ascorbate treatment alone had no effect on macrophages apoptosis, ascorbate added did significantly decrease the extent of apoptosis induced by oxLDL. In previous studies using macrophages, ascorbate did not protect against oxLDL-induced apoptosis. Reasons for these discrepant findings regarding apoptosis likely relate to one or more differences in cell culture conditions, such as macrophage type, degree of LDL oxidation, and duration of culture with oxLDL.Ascorbate inhibits both NF-kB activation in monocytic cell lines and suppressed release of downstream products of NF-kB activation, including interleukin-6 and TNF-a in LPS-activated human monocytes. This may be due to scavenging of reactive oxygen species, which are well known to activate the NF-kB cascade in RAW264.7 cells.We found that specific inhibition of the NF-κB pathway completely prevented SVCT2 induction by oxLDL. The NF-κB pathway may indeed be fundamental to SVCT2 expression, since blockade of the NF-κB cascade significantly decreased basal SVCT2 expression. An oxLDL-induced increase in expression of the high affinity SVCT2, will increase ascorbate uptake of even low extracellular concentrations of ascorbate by the cells, which will help to alleviate intracellular oxidant stress. However, increased intracellular ascorbate will also affect SVCT2 synthesis. Thus, previous studies have shown that SVCT2 function or expression varies inversely with intracellular ascorbate in culture. Our current finding that ascorbate down-regulated SVCT2 protein expression both in the basal state and that induced by oxLDL in RAW264.7 macrophages could indicate that self-regulation of its transporter by ascorbate takes precedence over changes in SVCT2 expression induced by oxidant stress.