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-eNOS^Ser1177, p-eNOS^Thr495, caveolin-1, extracellular signal-regulated kinase (ERK), p-ERK, Akt, p-Akt^Ser473, 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-eNOS^Ser1177, and p-Akt^Ser473 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 eNOS^Ser1177 and Akt^Ser473 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.

Summary Sentence

1,25(OH)₂D₃ induced upregulation of eNOS^Ser1177 and Akt^Ser473 phosphorylation and inhibition of ROCK1 cleavage could be plausible mechanisms of vitamin D protective effects to suppress MP release by ECs.