Renal Division, Department of Medicine, Peking University, First Hospital, Peking University Institute of Nephrology, Beijing, China.
Key Laboratory of Renal Disease, Ministry of Health of China, Beijing, China.
Front Immunol. 2019 Feb 15;10:237. doi: 10.3389/fimmu.2019.00237. eCollection 2019.
Activation of coagulation system plays an important role in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) pathogenesis. Thrombin, generated during coagulation could disrupt endothelial barrier integrity through protease-activated receptor 1 (PAR1). Our previous study found that sphingosine-1-phosphate (S1P) contributed to myeloperoxidase (MPO)-ANCA-positive IgG-induced glomerular endothelial cell (GEnC) activation through a S1P receptor (S1PR)-dependent route. In recent years, S1P signaling was reported to be involved in thrombin effects on endothelial cells. This current study investigated whether the interaction between thrombin-PAR and S1P-S1PR signaling contributed to MPO-ANCA-positive IgG-induced GEnC dysfunction. The effect of thrombin on GEnC activation was analyzed from three aspects. First, morphological alteration of GEnCs was observed. Second, permeability assay was performed to determine GEnC monolayer activation quantitatively. Third, endothelin-1 (ET-1) levels were measured. Expression levels of sphingosine kinases (SphKs) and S1PRs were detected. In addition, antagonists of PAR1 and S1PR3 were employed to determine their roles. Eventually, PAR1 and tissue factor (TF) expression levels as well as TF procoagulant activity were analyzed. Thrombin induced further damage of tight junction, increase in endothelial monolayer permeability as well as upregulation of ET-1 levels in GEnCs stimulated with MPO-ANCA-positive IgG. Blocking PAR1 downregulated ET-1 levels in the supernatants of GEnCs treated by thrombin plus MPO-ANCA-positive IgG. Expression levels of SphK1, S1PR3 increased significantly in GEnCs treated with thrombin plus MPO-ANCA-positive IgG. S1P upregulated PAR1 and TF expression, and enhanced procoagulant activity of TF in MPO-ANCA-positive IgG-stimulated GEnCs. Thrombin synergized with SphK1-S1P-S1PR3 signaling pathway to enhance MPO-ANCA-positive IgG-mediated GEnC activation.
凝血系统的激活在抗中性粒细胞胞浆抗体（ANCA）相关性血管炎（AAV）发病机制中起着重要作用。凝血过程中产生的凝血酶可以通过蛋白酶激活受体 1（PAR1）破坏内皮屏障完整性。我们之前的研究发现，鞘氨醇-1-磷酸（S1P）通过 S1P 受体（S1PR）依赖途径有助于髓过氧化物酶（MPO）-ANCA 阳性 IgG 诱导的肾小球内皮细胞（GEnC）激活。近年来，S1P 信号转导被报道参与了凝血酶对内皮细胞的作用。本研究探讨了凝血酶-PAR 和 S1P-S1PR 信号之间的相互作用是否有助于 MPO-ANCA 阳性 IgG 诱导的 GEnC 功能障碍。从三个方面分析了凝血酶对 GEnC 激活的影响。首先，观察 GEnC 的形态改变。其次，进行通透性测定以定量确定 GEnC 单层激活。第三，测量内皮素-1（ET-1）水平。检测鞘氨醇激酶（SphKs）和 S1PRs 的表达水平。此外，还使用了 PAR1 和 S1PR3 的拮抗剂来确定它们的作用。最后，分析了 PAR1 和组织因子（TF）的表达水平以及 TF 的促凝活性。凝血酶诱导了 MPO-ANCA 阳性 IgG 刺激的 GEnC 中紧密连接进一步受损，内皮单层通透性增加以及 ET-1 水平上调。阻断 PAR1 可降低凝血酶加 MPO-ANCA 阳性 IgG 处理的 GEnC 上清液中的 ET-1 水平。用凝血酶加 MPO-ANCA 阳性 IgG 处理的 GEnC 中 SphK1、S1PR3 的表达水平显著增加。S1P 上调了 PAR1 和 TF 的表达，并增强了 MPO-ANCA 阳性 IgG 刺激的 GEnC 中 TF 的促凝活性。凝血酶与 SphK1-S1P-S1PR3 信号通路协同作用，增强了 MPO-ANCA 阳性 IgG 介导的 GEnC 激活。
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