Vitamin D suppresses oxidative stress-induced microparticle release by human umbilical vein endothelial cells

Abstract

Endothelial microparticle (MP) release was increased in numerous cardiovascular diseases including preeclampsia. Oxidative stress is a potent inducer of endothelial dysfunction. In this study, we aimed to investigate if vitamin D could protect endothelial cells (ECs) from MP release induced by oxidative stress. Endothelial cell (from human umbilical vein) oxidative stress was induced by cultivation of cells under lowered oxygen condition (2%O2) for 48 h and cells cultured under standard condition (21%O2) served as control. 1,25(OH)2D3 was used as bioactive vitamin D. Using annexin-V as a marker of released MP assessed by flow cytometry and cytochrome c reduction assay to measure EC superoxide generation, we found that MP release and superoxide generation were significantly increased when cells were cultured under 2%O2, which could be significantly inhibited by 1,25(OH)2D3. To study the potential mechanisms of 1,25(OH)2D3 protective effects on ECs, EC expression of endothelial nitric oxide synthase (eNOS), p-eNOSSer1177, p-eNOSThr495, caveolin-1, extracellular signal-regulated kinase (ERK), p-ERK, Akt, p-AktSer473, Rho-associated coiled-coil protein kinase 1 (ROCK1), and vitamin D receptor were determined. Microparticle expression of eNOS and caveolin-1 were also determined. We found that under lowered oxygen condition, 1,25(OH)2D3 could upregulate EC eNOS, p-eNOSSer1177, and p-AktSer473 expression, but inhibit cleaved ROCK1 expression. The upregulatory and inhibitory effects induced by 1,25(OH)2D3 were dose dependent. Strikingly, we also found that oxidative stress-induced decrease in ratio of eNOS and caveolin-1 expression in MP could be attenuated when 1,25(OH)2D3 was present in culture. These results suggest that upregulation of eNOSSer1177 and AktSer473 phosphorylation and inhibition of ROCK1 cleavage in EC and modulation of eNOS and caveolin-1 expression in MP could be plausible mechanisms of vitamin D protective effects on ECs.

Keywords: Akt; ROCK; eNOS; endothelial cells; microparticles; vitamin D.

Figure 1.

Effects of 1,25(OH)₂D₃ on MP release and superoxide generation by ECs cultured under 2%O₂ and 21%O₂. A) Annexin-V positive MP release from ECs was assessed by flow cytometry and the count was normalized by total cellular protein. Annexin-V positive MP release was significantly increased in cells cultured under 2%O₂ vs. 21%O₂, ^*P < 0.05, which could be suppressed when 1,25(OH)₂D₃ was present in culture, ^#P < 0.05. B) Superoxide generation by ECs was assessed by cytochrome c reduction assay and normalized by total cellular protein. Superoxide generation was significantly increased when cells were cultured under 2%O₂ vs. 21%O₂, ^**P < 0.01. Increased superoxide generation could be attenuated by 1,25(OH)₂D₃, ^#P < 0.05. ^*P < 0.05 and ^**P < 0.01: untreated cells cultured under 2%O₂ vs. untreated cells cultured under 21%O₂; ^#P < 0.05: 1,25(OH)₂D₃ treated cells cultured under 2%O₂ vs. untreated cells cultured under 2%O₂, respectively. Data are means ± SE from five to six independent experiments. C) A representative image of isolated EC MPs examined by TEM, showing that isolated MPs are intact and the size within the MP limitation between 0.1 and 1.0 μm in diameter, bar = 200 μm. D) Protein expression for eNOS and caveolin-1 in MP isolated from ECs cultured with 21%O₂.

Figure 2.

Protein expression for caveolin-1, eNOS, p-eNOS^Ser1177, p-eNOS^Thr495, and VDR in ECs cultured under 21%O₂ or 2%O₂ in the presence or absence of 1,25(OH)₂D₃. A) Representative western blots for caveolin-1, eNOS, p-eNOS^Ser1177, p-eNOS^Thr495, and VDR expression. β-Actin expression was determined and showed equal loading of total cellular protein for each sample. The bar graphs show relative protein expression after normalization with β-actin expression. Caveolin-1 expression was increased, but eNOS expression was reduced, in cells cultured under 2%O₂ vs. 21%O₂, ^*P < 0.05. The lowered oxygen induced downregulation of eNOS and upregulation of caveolin-1 could be inhibited by 1,25(OH)₂D₃, ^#P < 0.05. p-eNOS^Ser1177 expression was reduced, but p-eNOS^Thr495 expression was increased, in cells cultured under 2%O₂ vs. 21%O₂, ^*P < 0.05. 1,25(OH)₂D₃ could upregulate p-eNOS^Ser1177 expression in cells treated with 1,25(OH)₂D₃ in both 21%O₂ (^*P < 0.05) and 2%O₂ (^#P < 0.05) conditions. 1,25(OH)₂D₃ could upregulate VDR in both conditions when compared with the cells cultured under the same condition without 1,25(OH)₂D₃, ^**P < 0.01 and ^##P < 0.01, respectively. B) Western blots of endothelial caveolin-1, eNOS, p-eNOS^Ser1177, p-eNOS^Thr495, and VDR protein expression in cells cultured under 2%O₂ in the presence of different concentrations of 1,25(OH)₂D₃. The bar graphs show relative protein expression after normalization with β-actin expression. 1,25(OH)₂D₃ induced a dose-dependent decrease in caveolin-1 expression, ^#P < 0.05 and ^##P < 0.01, but a dose-dependent increase in eNOS expression, respectively. p-eNOS^Ser1177 expression was dose-dependently upregulated, but p-eNOS^Thr495 expression was dose-dependently downregulated in cells treated with 1,25(OH)₂D₃. Vitamin D receptor expression was also upregulated, ^##P < 0.01. Data are means ± SE from five independent experiments.

Figure 3.

Protein expression for Akt, p-Akt^Ser473, ERK, and p-ERK in ECs cultured under 2%O₂ or 21%O₂ in the presence or absence of 1,25(OH)₂D₃. A) Representative blots for Akt, p-Akt^Ser473, ERK, and p-ERK expression. β-Actin expression was also determined to show equal loading of total cellular protein for each sample. The bar graphs show relative protein expression for Akt, p-Akt^Ser473, ERK, and p-ERK expression after normalization with β-actin expression. p-Akt^Ser473 and p-ERK expression were significantly downregulated in cells cultured under 2%O₂ vs. 21%O₂, ^*P < 0.05 and ^**P < 0.01, respectively. p-Akt^Ser473 expression was upregulated in cells treated with 1,25(OH)₂D₃ in both 21%O₂ and 2%O₂ culture conditions. In contrast, 1,25(OH)₂D₃ had no effect on p-ERK expression. Both Akt and ERK expression were not affected in cells cultured under 21%O₂ vs. 2%O₂ with or without 1,25(OH)₂D₃ treatment. B) Akt, p-Akt^Ser473, ERK, and p-ERK expression in cells cultured under 2%O₂ treated with different concentrations of 1,25(OH)₂D₃. The bar graphs show relative protein expression after normalization with β-actin expression. p-Akt^Ser473 expression was dose-dependently upregulated by 1,25(OH)₂D₃ vs. untreated control cells, ^#P < 0.05, and ^##P < 0.01. Data are means ± SE from five to six independent experiments.

Figure 4.

Protein expression for ROCK1 and truncated ROCK1 in ECs cultured under 2%O₂ or 21%O₂ in the presence or absence of 1,25(OH)₂D₃. A) Western blot for ROCK1 and cleaved ROCK1 expression. β-Actin expression was determined to show equal loading of total cellular protein for each sample. The bar graphs show relative protein expression for ROCK1 and cleaved ROCK1 after normalization with β-actin expression. ROCK1 had no significant difference among the groups, the cleaved ROCK1 was increased in cells cultured under 2%O₂, ^*P < 0.05, whereas cleaved ROCK1 expression was reduced when 1,25(OH)₂D₃ was present in cultured, ^#P < 0.05. B) Western blots of native and cleaved ROCK1 expression in ECs cultured under 2%O₂ treated with different concentrations of 1,25(OH)₂D₃. The bar graphs show relative protein expression after normalization with β-actin expression. 1,25(OH)₂D₃ had no significant effect on native ROCK1 expression, but could reduce cleaved ROCK1 expression in a dose-dependent manner, ^##P < 0.01. Data are means ± SE from five independent experiments.

Figure 5.

Effect of 1,25(OH)₂D₃ on eNOS and caveolin-1 expression in MPs isolated from ECs. A) Representative western blot of eNOS and caveolin-1 expression in MPs isolated from ECs cultured under 2%O₂ and 21%O₂ in the presence or absence of 1,25(OH)₂D₃. B) Relative ratio of eNOS and caveolin-1 expression in endothelial MPs. The ratio of eNOS and caveolin-1 expression was dramatically decreased in MPs released from cells cultured under 2%O₂, ^**P < 0.01. 1,25(OH)₂D₃ had no significant effect on the ratio of MP eNOS and caveolin-1 expression in cells cultured under 21%O₂, but could increase the ratio of MP eNOS and caveolin-1 expression in cells cultured under 2%O₂, ^#P < 0.05. Data are means ± SE from five independent experiments.