Zhejiang Chinese Medical University, Hangzhou, Zhejiang, China.
Rehabilitation Area of the Third Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou, Zhejiang, China.
Clin Hemorheol Microcirc. 2024;88(1):59-70. doi: 10.3233/CH-242171.
In the pathogenesis of atherosclerotic cardiovascular disorders, vascular endothelium is crucial. A critical step in the development of atherosclerosis is endothelial dysfunction. Furin may play a factor in vascular remodeling, inflammatory cell infiltration, regulation of plaque stability, and atherosclerosis by affecting the adhesion and migration of endothelial cells. It is yet unknown, though, how furin contributes to endothelial dysfunction.
We stimulated endothelial cells with oxidized modified lipoprotein (ox-LDL). Endothelial-to-mesenchymal transition (EndMT) was found using immunofluorescence (IF) and western blot (WB). Furin expression level and Hippo/YAP signal activation were found using reverse transcription-quantitative PCR (RT-qPCR) and WB, respectively. To achieve the goal of furin knockdown, we transfected siRNA using the RNA transmate reagent. Following furin knockdown, cell proliferation, and migration were assessed by the CCK-8, scratch assay, and transwell gold assay, respectively. WB and IF both picked up on EndMT. WB and RT-qPCR, respectively, were used to find furin's expression level. We chose the important micrornas that can regulate furin and we then confirmed them using RT-qPCR.
EndMT was created by ox-LDL, evidenced by the up-regulation of mesenchymal cell markers and the down-regulation of endothelial cell markers. Furin expression levels in both protein and mRNA were increased, and the Hippo/YAP signaling pathway was turned on. Furin knockdown dramatically reduced the aberrant migration and proliferation of endothelial cells by ox-LDL stimulation. Furin knockdown can also suppress ox-LDL-induced EndMT, up-regulate indicators of endothelial cells, and down-regulate markers of mesenchymal cells. After ox-LDL stimulation and siRNA transfection, furin's expression level was up-regulated and down-regulated.
Our study demonstrated that furin knockdown could affect ox-LDL-induced abnormal endothelial cell proliferation, migration, and EndMT. This implies that furin plays an important role in endothelial dysfunction.
在动脉粥样硬化性心血管疾病的发病机制中，血管内皮至关重要。动脉粥样硬化的一个关键步骤是内皮功能障碍。弗林可能通过影响内皮细胞的黏附和迁移，在血管重塑、炎症细胞浸润、斑块稳定性和动脉粥样硬化的调节中发挥作用。然而，弗林如何导致内皮功能障碍尚不清楚。
我们用氧化修饰的脂蛋白（ox-LDL）刺激内皮细胞。用免疫荧光（IF）和蛋白质印迹（WB）发现内皮-间充质转化（EndMT）。用逆转录定量聚合酶链反应（RT-qPCR）和 WB 分别检测弗林表达水平和 Hippo/YAP 信号激活。用 RNA transmate 试剂转染 siRNA 实现弗林敲低。用 CCK-8、划痕实验和 Transwell 金实验分别评估细胞增殖和迁移。WB 和 IF 都可以检测到 EndMT。WB 和 RT-qPCR 分别用于检测弗林的表达水平。我们选择了可以调节弗林的重要 microRNAs，并通过 RT-qPCR 进行了验证。
ox-LDL 诱导 EndMT，表现为间充质细胞标志物上调和内皮细胞标志物下调。蛋白质和 mRNA 中弗林的表达水平均增加，Hippo/YAP 信号通路被激活。ox-LDL 刺激引起的内皮细胞异常迁移和增殖在弗林敲低后明显减少。弗林敲低还可以抑制 ox-LDL 诱导的 EndMT，上调内皮细胞标志物，下调间充质细胞标志物。ox-LDL 刺激和 siRNA 转染后，弗林的表达水平上调和下调。
本研究表明，弗林敲低可影响 ox-LDL 诱导的内皮细胞异常增殖、迁移和 EndMT。这表明弗林在内皮功能障碍中起重要作用。
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