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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online May 8, 2002 as doi:10.1096/fj.01-0825fje. |
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Linus Pauling Institute and Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon, USA; and
* Department of Pharmacological Sciences, University of Milan, Italy
2Correspondence: Linus Pauling Institute/Oregon State University, 571 Weniger Hall, Corvallis OR 97331, USA. E-mail: tory.hagen{at}orst.edu
SPECIFIC AIMS
We sought to determine whether standard culture conditions result in vitamin C insufficiency in human aortic endothelial cells (HAEC) and the effects of vitamin C supplementation on parameters of oxidative stress. Other objectives were to determine the glutathione redox (GSH/GSSG) and pyridine nucleotide [NAD(P)H/NAD(P)+] redox ratios, mitochondrial and cellular oxidant production, oxidative nucleotide damage, and endothelial nitric oxide synthase (eNOS) activity before and after treatment of HAEC with vitamin C.
PRINCIPAL FINDINGS
1. HAEC grown under standard culture conditions are scorbutic but can be repleted with vitamin C supplementation
HAEC were grown to confluency using EGMTM medium. HPLC analysis of cellular vitamin C content showed no detectable vitamin C (detection limit
5 pmol). Fetal bovine serum and M199, another commonly used medium, was analyzed and contained no vitamin C.
To determine whether supplementing the culture medium could restore vitamin C status, cells were treated with 100 µM vitamin C in EGMTM. This regimen led to a rapid cellular appearance of vitamin C such that after 1 h, ascorbate content was 2.01 ± 0.21 nmol/mg protein. Maximum vitamin C levels were seen 
6 h (4.80±0.95 nmol/mg protein) after addition and fell to 0.47 ± 0.22 nmol/mg protein at 24 h.
2. Vitamin C treatment lowers oxidative stress in HAEC
Changes in GSH levels were followed over 24 h after vitamin C addition. HPLC analysis revealed that GSH and GSSG levels in scorbutic cells were 18.67 ± 3.42 and 0.334 ± 0.046 pmol/mg protein (n=4), respectively, which gives a GSH/GSSG ratio of 54.29 ± 6.26. Vitamin C repletion resulted in a significant doubling of total GSH levels after 24 h (38.41±3.58 pmol/mg protein; P<0.05; n=4) and an increase in the GSH/GSSG ratio, which almost doubled to 104.78 ± 10.66 (P<0.05; n=4). Most of the changes in GSH levels and GSH/GSSG ratio occurred during the first 6 h after vitamin C addition, which was also the period of greatest vitamin C accumulation. GSH and the GSH/GSSG ratio remained elevated over the 24 h time course, though ascorbate levels began declining after 6 h. This indicates that the scorbutic state leads to a compromised intracellular thiol redox status and that cultured HAEC without vitamin C repletion may be under increased oxidative stress.
The pyridine nucleotide redox ratio [NAD(P)H/NAD(P)+] of the cell was measured after vitamin C addition to the culture media. NAD(P)H/NAD(P)+ ratio for cells grown without vitamin C was 0.88 ± 0.27 (n=3). Again, this suggests that these cells exhibit an altered cellular redox state, considering this ratio is usually greater than 1.0 in most cell types measured. The pyridine nucleotide redox ratio improved significantly (P>0.05) to 2.08 ± 1.04 only after 24 h after vitamin C repletion. Thus, changes in pyridine redox state do not directly correlate with vitamin C repletion or GSH/GSSG ratio after vitamin C addition. However, indirect effects of vitamin C on NAD(P)H/NAD(P)+ cannot be ruled out. Thus, vitamin C status influences thiol and pyridine nucleotide redox status and strongly suggests that HAEC grown without vitamin C have distinctly lower overall redox status compared with vitamin C-repleted cells.
To determine whether the lack of vitamin C resulted in increased oxidant appearance, cells were incubated with 2',7'-dichlorodihydrofluorescein diacetate (DCF), a dye that fluoresces on oxidation by reactive oxygen and reactive nitrogen species (ROS/RNS). The rate of oxidant-induced fluorescence in scorbutic cells was significantly higher than that seen in vitamin C-repleted cells (Fig. 1
A; P<0.05). These results suggest that vitamin C markedly lowers general oxidant levels in cultured HAEC.
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Cells were also incubated with the eNOS inhibitor L-NAME to determine whether the enhanced oxidant appearance in vitamin C-deprived cells was partly due to eNOS-generated oxidants. Oxidant levels in L-NAME-treated cells were significantly lower than in scorbutic HAEC (Fig. 1A
; P<0.01), but were not completely inhibited, which suggests that cellular sources other than eNOS contribute to ROS/RNS production.
We also determined whether treatment with vitamin C affects mitochondrial oxidant production. Cells were incubated with dihydrorhodamine 123 (DHR), a dye that specifically associates with the mitochondria and, like DCF, fluoresces on oxidation by ROS/RNS. Scorbutic HAEC exhibited a significantly higher rate of fluorescence than vitamin C-repleted cells (Fig. 1B
, P<0.01). Similarly, the eNOS inhibitor L-NAME decreased DHR fluorescence (P<0.01), possibly indicating the presence of eNOS-derived RNS as well as ROS in the mitochondria. These observations were confirmed using scanning laser confocal microscopy. A markedly lower intensity of fluorescence was observed after treating HAEC with vitamin C (data not shown). Together, these results suggest that HAEC maintained under standard culture conditions are under increased oxidative stress compared with vitamin C-treated cells, in part due to heightened oxidant appearance from the mitochondria.
3. Scorbutic HAEC suffer increased oxidative damage compared with vitamin C-repleted cells
To determine whether altered antioxidant status and heightened oxidative stress lead to elevated steady-state oxidative damage, oxidized nucleotide levels were monitored immunocytochemically. We used a monoclonal antibody directed against 8-hydroxyguanine (8-oxo-dG), an oxidative adduct that arises primarily from hydroxyl radical or peroxynitrite attack on guanine nucleobases. Results showed intense staining in control cells, suggesting oxidative damage to nucleic acids. In contrast, daily addition of vitamin C (100 µM) to the culture media for 7 days resulted in markedly lower fluorescence intensity, indicating that vitamin C repletion lowers steady-state oxidative damage (data not shown).
Immunostaining in vitamin C-depleted and repleted cells was predominantly perinuclear in nature. Mitochondrial DNA damage did not account for a significant portion of the staining. To determine the extent that RNA damage contributed to perinuclear staining, fixed cells were incubated with RNase A before immunostaining with the 8-oxo-dG antibody. Pretreatment of fixed scorbutic cells with RNase A did not affect the relative signal intensity. In contrast, pretreatment of the vitamin C supplemented cells with RNase A abolished the fluorescence signal (data not shown). Thus, repletion of HAEC not only lowers the steady-state levels of oxidative damage, but alters the nature of perinuclear damage from non-RNA nucleotide pools to RNA.
4. Vitamin C treatment potentates NO production by endothelial NO synthase
Because eNOS activity can be modified by altered cellular redox status and is directly influenced by vitamin C levels, we postulated that scorbutic HAEC would have diminished eNOS activity relative to vitamin C-repleted cells. Endothelial NOS activity was monitored after stimulation with the calcium ionophore A23187. Vitamin C pretreatment significantly increased endothelial-derived NO (EDNO) production by 600% (Fig. 2
, P<0.01), a highly significant increase over scorbutic cells. This effect was blocked by the eNOS inhibitor L-NAME, indicating that vitamin C exerts its tonic effects directly on eNOS.
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CONCLUSIONS AND SIGNIFICANCE
Standard culture media for HAEC do not routinely include vitamin C because it is not stable in aqueous solution. We show that providing vitamin C results in uptake into cells and intracellular vitamin C levels are maintained for at least 6 h before declining. Thus, vitamin C supplementation results in a transient yet marked improvement in intracellular ascorbic acid levels despite the potential for oxidation or hydrolysis occurring in the media.
The scorbutic nature of HAEC in culture leads to major alterations in endothelial function that could directly affect interpretation of experimental results (Fig. 3
). For instance, lack of vitamin C leads to significant increases in ROS/RNS appearance, lower GSH/GSSG and NAD(P)H/NAD(P)+ ratios, and subsequent increased oxidative damage. These results suggest that in experiments without the addition of vitamin C, HAEC are in a nonphysiological, pro-oxidant environment.
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Whereas increased oxidative conditions may actually benefit HAEC growth in culture by causing higher rates of cell proliferation, the scorbutic intracellular environment may adversely affect parameters related to endothelial function, especially NO synthesis and availability (Fig. 3)
. NO is a radical that must diffuse to vascular smooth muscle cells to induce vasorelaxation. As a signal molecule, EDNO has a relatively short half-life and can be oxidized to RNS incapable of causing vascular relaxation. Therefore, lack of vitamin C could significantly enhance oxidative inactivation of EDNO and result in a culture model that does not mimic the physiological environment of the intact vessel.
Even though cells without vitamin C treatment had more intense antibody staining than cells repleted with vitamin C, both conditions showed perinuclear oxidative nucleic acid damage. It appears that most of the damage seen is to RNA. This is suggested because damage patterns did not overlap with mitochondria, and experiments using a free radical-generating system as a positive control showed that nuclear DNA damage was detected.
Aside from enhanced oxidative stress and resultant alterations in NO availability, the scorbutic nature of standard culture conditions could lead to altered EDNO production (Fig. 3)
. EDNO is produced by eNOS, a tetrahydrobiopterin-dependent heme protein. Tetrahydrobiopterin is required by eNOS as a reducing agent for its oxidase moiety and for enzyme dimerization. We show that vitamin C repletion of HAEC in culture leads to significantly higher eNOS activity, which is in agreement with previous data. Alternatively, it has been proposed that vitamin C can directly reduce critical thiol moieties on the enzyme and thereby up-regulate eNOS activity.
Finally, use of EC culture models has resulted in significant insights into endothelial cell function. In 2000 alone, 1999 papers were published using the EC culture model; at the time of this manuscript preparation, 1171 papers had appeared in 2001. The popularity of cultured EC as a research model is undeniable. We contend that the scorbutic nature of HAEC in culture can significantly alter parameters that directly pertain to the known function(s) of vitamin C in endothelial cells. Experimental data related to eNOS activity, oxidative stress, and any mechanism that can be influenced by redox status need to be examined in light of these findings. The results demonstrate that vitamin C supplementation to HAEC is critically important in order to establish a physiologically relevant culture model for HAEC function.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0825fje; to cite this article, use FASEB J. (May 8, 2002) 10.1096/fj.01-0825fje. 
This article has been cited by other articles:
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