Cobalt-induced oxidant stress in cultured endothelial cells: prevention by ascorbate in relation to HIF-1alpha

Abstract

Endothelial cells respond to hypoxia by decreased degradation of hypoxia-inducible factor 1alpha (HIF-1alpha), accumulation of which leads to increased transcription of numerous proteins involved in cell growth and survival. Ascorbic acid prevents HIF-1alpha stabilization in many cell types, but the physiologic relevance of such effects is uncertain. Given their relevance for angiogenesis, endothelial cells in culture were used to evaluate the effects of ascorbate on HIF-1alpha expression induced by hypoxia and the hypoxia mimic cobalt. Although EA.hy926 cells in culture under oxygenated conditions did not contain ascorbate, HIF-1alpha expression was very low, showing that the vitamin is not necessary to suppress HIF-1alpha. On the other hand, hypoxia- or cobalt-induced HIF-1alpha expression/stabilization was almost completely suppressed by what are likely physiologic intracellular ascorbate concentrations. Increased HIF-1alpha expression was not associated with significant changes in expression of the SVCT2, the major transporter for ascorbate in these cells. Cobalt at concentrations sufficient to stabilize HIF-1alpha both oxidized intracellular ascorbate and induced an oxidant stress in the cells that was prevented by ascorbate. Whereas the interaction of ascorbate and cobalt is complex, the presence of physiologic low millimolar concentrations of ascorbate in endothelial cells effectively decreases HIF-1alpha expression and protects against cobalt-induced oxidant stress.

Figure 1. HIF-1α immunoblotting

Panel A: Confluent EA.hy926 cells were treated with ascorbate or cobalt at the indicated concentrations and then cultured for 18 h under ambient atmospheric oxygen concentrations before rinsing the cells 3 times with KRH, followed by removal of the cells for immunoblotting as described under Experimental Procedures. Blots of HIF-1α are shown in the top row, with blots of β-actin shown in the second row as a control for gel protein loading. Results are representative of 4 such experiments performed. Panel B: EA.hy926 were cultured to confluence at ambient oxygen concentrations and then cultured with additions as noted for 18 h under a mixture of 1% oxygen, 5% carbon dioxide, and 94% nitrogen. Cells were rinsed 3 times with KRH and taken for immunoblotting of HIF-1α (top row) and β-actin (bottom row). Results are representative of 2 such experiments performed.

Figure 2. SVCT2 message and protein

Panel A: Confluent EA.hy926 cells in culture were treated for 18 h with ascorbate or cobalt as indicated, rinsed 3 times in buffer, and taken for determination of SVCT2 mRNA (Panel A, top row) or protein by immunoblotting (Panel B, top row), using glyceraldehyde 3-phosphate dehydrogenase (G3PDH) or β-actin as a control for loading of the gel lanes, respectively.

Figure 3. Cobalt-induced loss of ascorbate in overnight culture

Confluent EA.hy926 cells were treated for 15 min with 200 µM ascorbate, followed by addition of the indicated concentration of cobalt and culture for 18 h before sampling of medium aliquots for ascorbate (circles, right Y-axis) and removal of the cells for assay of intracellular ascorbate (squares, left Y-axis). Results are shown from 4 experiments, with an “*” indicating p < 0.05 compared to the sample not treated with cobalt.

Figure 4. Cobalt-induced decreases of intracellular ascorbate

Panel A: EA.hy926 cells were rinsed free of culture medium and incubated in KRH that contained 5 mM D-glucose and 250 µM DHA for 15 min before addition of 250 µM cobalt (circles) or no cobalt (squares), followed by culture for the times indicated before 3 cell rinses with KRH and removal of the cells for assay of intracellular ascorbate. Results are from 4 experiments, with an “*” indicating p < 0.05 compared to the sample at time zero. Panel B: EA.hy926 cells were incubated for 2 h in KRH that contained 5 mM D-glucose with the indicated concentration of cobalt, followed by 3 rinses and addition of 250 µM ascorbate. After an additional 30 min, the cells were rinsed 3 times in KRH and taken for assay of intracellular ascorbate. Results are from 4 experiments, with an “*” indicating p < 0.05 compared to the sample not treated with cobalt.

Figure 5. Cobalt-induced decreases in GSH

EA.hy926 cells were cultured for 18 h with the indicated concentration of cobalt, rinsed 3 times, and taken for assay of their GSH content. Results are shown from 7 experiments, with an “*” indicating p < 0.05 compared to the same not treated with cobalt.

Figure 6. Malondialdehyde formation in response to cobalt and ascorbate

Panel A: EA.hy926 cells were cultured for 18 h with the indicated concentration of cobalt, rinsed 3 times in KRH, and taken for assay of malondialdehyde. Results are shown from 5 experiments, with an “*” indicating p < 0.05 compared to cells not treated with cobalt. Panel B: EA.hy926 cells were treated with the indicated concentration of ascorbate for 15 min, and then cultured for 18 h without (square) or with (circles) 250 µM cobalt. Results are shown from 5 experiments, with an “*” indicating p < 0.05 compared to the sample treated with cobalt alone.