Orion Institute for Translational Medicine, Temple, TX, USA.
Emergent Biotechnologies LLC, Temple, TX, USA.
Mol Cell Biochem. 2021 Aug;476(8):3207-3213. doi: 10.1007/s11010-021-04147-9. Epub 2021 Apr 18.
Edema is common in preeclampsia (preE), a hypertensive disorder of pregnancy. Cardiotonic steroids (CTSs) such as marinobufagenin (MBG) are involved in the pathogenesis of preE. To assess whether CTSs are involved in the leakage of lymphatic endothelial cell (LEC), we evaluated their effect on monolayer permeability of LECs (MPLEC) in culture. A rat mesenteric LECs were treated with DMSO (vehicle), and CTSs (MBG, CINO, OUB) at concentrations of 1, 10, and 100 nM. Some LECs were pretreated with 1 μM L-NAME (N-Nitro-L-Arginine Methyl Ester) before adding 100 nM MBG or cinobufotalin (CINO). Expression of β-catenin and vascular endothelial (VE)-cadherin in CTS-treated LECs was measured by immunofluorescence and MPLEC was quantified using a fluorescence plate reader. Western blot was performed to measure β-catenin and VE-cadherin protein levels and myosin light chain 20 (MLC20) phosphorylation. MBG (≥ 1 nM) and CINO (≥ 10 nM) caused an increase (p < 0.05) in the MPLEC compared to DMSO while ouabain (OUB) had no effect. Pretreatment of LECs with 1 μM L-NAME attenuated (p < 0.05) the MPLEC. The β-catenin expression in LECs was downregulated (p < 0.05) by MBG and CINO. However, there was no effect on the LECs tight junctions for the CINO group. VE-cadherin expression was downregulated (p < 0.05) by CINO, and MLC20 phosphorylation was upregulated (p < 0.05) by MBG. We demonstrated that MBG and CINO caused an increase in the MPLEC, which were attenuated by L-NAME pretreatment. The data suggest that CTSs exert their effect via nitric-oxide-dependent signaling pathway and may be involved in vascular leak syndrome of LEC lining in preE.
水肿是子痫前期（preE）的一种常见病症，是一种妊娠高血压疾病。强心甾类化合物（CTS），如蟾蜍灵（MBG），参与了 preE 的发病机制。为了评估 CTS 是否参与了淋巴管内皮细胞（LEC）的渗漏，我们评估了它们对培养的 LEC 单层通透性（MPLEC）的影响。用 DMSO（载体）和浓度为 1、10 和 100 nM 的 CTS（MBG、CINO、OUB）处理大鼠肠系膜 LEC。一些 LEC 在加入 100 nM MBG 或华蟾毒配基（CINO）之前用 1 μM L-NAME（N-硝基-L-精氨酸甲酯）预处理。用免疫荧光法测量 CTS 处理的 LEC 中β-连环蛋白和血管内皮（VE）-钙粘蛋白的表达，并使用荧光板读数器定量 MPLEC。进行 Western blot 以测量β-连环蛋白和 VE-钙粘蛋白蛋白水平以及肌球蛋白轻链 20（MLC20）磷酸化。与 DMSO 相比，MBG（≥1 nM）和 CINO（≥10 nM）引起 MPLEC 增加（p<0.05），而哇巴因（OUB）没有作用。LEC 用 1 μM L-NAME 预处理可减弱（p<0.05）MPLEC。MBG 和 CINO 下调了 LEC 中的β-连环蛋白表达（p<0.05）。然而，CINO 组对 LEC 紧密连接没有影响。VE-钙粘蛋白表达被 CINO 下调（p<0.05），而 MBG 上调 MLC20 磷酸化（p<0.05）。我们证明 MBG 和 CINO 引起 MPLEC 增加，L-NAME 预处理可减弱其作用。数据表明 CTS 通过一氧化氮依赖性信号通路发挥作用，可能参与 preE 中 LEC 衬里的血管渗漏综合征。
