Jia Xiuyue, Xu Jie, Gu Yang, Gu Xin, Li Weimin, Wang Yuping
Department of Obstetrics and Gynecology, Louisiana State University Health Sciences Center-Shreveport, Shreveport, Louisiana, USA.
Department of Cardiology, The First Affiliated Hospital Harbin Medical University, Harbin, China.
Biol Reprod. 2017 Jan 1;96(1):199-210. doi: 10.1095/biolreprod.116.142604.
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.
在包括先兆子痫在内的多种心血管疾病中，内皮微粒（MP）的释放都会增加。氧化应激是内皮功能障碍的有力诱导因素。在本研究中，我们旨在探究维生素D是否能保护内皮细胞（ECs）免受氧化应激诱导的MP释放。通过在低氧条件（2%O2）下培养细胞48小时来诱导内皮细胞（来自人脐静脉）氧化应激，在标准条件（21%O2）下培养的细胞作为对照。1,25(OH)2D3用作生物活性维生素D。使用膜联蛋白-V作为通过流式细胞术评估的释放MP的标志物，并通过细胞色素c还原测定法测量EC超氧化物的产生，我们发现当细胞在2%O2条件下培养时，MP释放和超氧化物产生显著增加，而1,25(OH)2D3可显著抑制这种增加。为了研究1,25(OH)2D3对ECs保护作用的潜在机制，测定了内皮型一氧化氮合酶（eNOS）、p-eNOSSer1177、p-eNOSThr495、小窝蛋白-1、细胞外信号调节激酶（ERK）、p-ERK、Akt、p-AktSer473、Rho相关卷曲螺旋蛋白激酶1（ROCK1）和维生素D受体在EC中的表达。还测定了MP中eNOS和小窝蛋白-1的表达。我们发现，在低氧条件下，1,25(OH)2D3可上调EC中eNOS、p-eNOSSer1177和p-AktSer473的表达，但抑制裂解的ROCK1表达。1,25(OH)2D3诱导的上调和抑制作用呈剂量依赖性。令人惊讶的是，我们还发现当培养中存在1,25(OH)2D3时，氧化应激诱导的MP中eNOS和小窝蛋白-1表达比例的降低可得到缓解。这些结果表明，上调EC中eNOSSer1177和AktSer473的磷酸化以及抑制ROCK1裂解，以及调节MP中eNOS和小窝蛋白-1的表达，可能是维生素D对ECs保护作用的合理机制。
