Vitamin C matters: increased oxidative stress in cultured human aortic endothelial cells without supplemental ascorbic acid

Abstract

Because standard culture media for human aortic endothelial cells (HAEC) do not contain vitamin C, we hypothesized that HAEC may be under significant oxidative insult compared with the situation in vivo. To assess parameters of oxidative stress, intracellular vitamin C, glutathione (GSH), GSH/GSSG, and NAD(P)H/NAD(P)+ ratios, as well as oxidant appearance and oxidative damage, were measured in HAEC with or without vitamin C addition. The effect of vitamin C on eNOS activity was also determined. Results showed that HAEC without vitamin C treatment were essentially scorbutic. On addition of 100 mM vitamin C to the culture media, intracellular vitamin C levels increased and peaked at 6 h. A concomitant increase in the total GSH and the GSH/GSSG ratio was also observed; the NAD(P)H/NAD(P)+ ratio increased more slowly over the 24-h time course. Significantly lower (P <0.05) oxidant appearance and steady-state oxidative damage were also observed following vitamin C repletion. Vitamin C treatment increased eNOS activity by 600%. Thus, HAEC are scorbutic under normal culture conditions and exhibit higher oxidative stress than vitamin C repleted cells. Vitamin C supplementation should be considered when using cultured cells, especially when experimental endpoints are related to cellular redox status and eNOS activity.

Lipid Peroxidation and Antioxidants in Arterial Hypertension

“…It is hydrophilic and can directly scavenge ROS and lipid hydroperoxides. Vitamin C can also restore oxidized vitamin E and can spare selenium [131]. Carotenoids, such as -carotene are lipid soluble antioxidants that function as efficient scavengers of 1 O2 but may also quench ROO .…”

Early determinants of H2O2-induced endothelial dysfunction

“…Prior to experiments, cells were incubated in freshly made 60 μM BH 4 for 10 min to compensate for variable cell culture-induced BH 4 deficiencies (13)(14)(15). Experimental results in the absence of an initial BH 4 supplementation were qualitatively identical but with lower NO• production, suggesting that pretreatment was not likely to affect the interpretation of the results.…”

Simulated diving after heat stress potentiates the induction of heat shock protein 70 and elevates glutathione in human endothelial cells

“…In this context, antioxidants may play important roles, although the literature so far shows conflicting results. Ascorbic acid has been shown to increase both glutathione (GSH) and NOS activity in human endothelial cells (Smith et al 2002), and the effect seems to be due to stabilization of the BH4 molecule (Werner et al 2003). Glutathione is a major cellular redox determinant, but some investigators have indicated that glutathione does not seem to have significant impact on the NOS (Huang et al 2001), while others have indicated a correlation between GSH level and NOS activity (Laursen et al 2001;Smith et al 2002).…”

“…Ascorbic acid has been shown to increase both glutathione (GSH) and NOS activity in human endothelial cells (Smith et al 2002), and the effect seems to be due to stabilization of the BH4 molecule (Werner et al 2003). Glutathione is a major cellular redox determinant, but some investigators have indicated that glutathione does not seem to have significant impact on the NOS (Huang et al 2001), while others have indicated a correlation between GSH level and NOS activity (Laursen et al 2001;Smith et al 2002). Novel findings have suggested a regulatory role of glutathione in glutathiolating specific proteins at specific residues; the reversible glutathiolation may thus protect these otherwise reactive sites against irreversible oxidation (West et al 2006).…”