Zhou Xiaoyan, Weng Jie, Xu Jing, Xu Qiulin, Wang Weiju, Zhang Weijin, Huang Qiaobing, Guo Xiaohua
Department of Pathophysiology, Key Laboratory for Shock and Microcirculation Research of Guangdong Province, Southern Medical University, Guangzhou, China.
Department of Pathology, Zhejiang Cancer Hospital, Zhejiang, China.
Cell Physiol Biochem. 2018;45(4):1717-1730. doi: 10.1159/000487780. Epub 2018 Feb 23.
BACKGROUND/AIMS: Disruption of endothelial barrier integrity in response to advanced glycation end products (AEGs) stimulation contributes to vasculopathy associated with diabetes mellitus. Mammalian diaphanous-related formin (mDia1) has been reported to bind to the cytoplasmic domain of the receptor for advanced glycation end products (RAGE), which induces a series of cellular processes. This study directly evaluated the participation of mDia1 in AGE-induced hyperpermeability and revealed the precise intracellular signal transductions of this pathological process.
Human umbilical vein endothelial cells (HUVECs) were used in the in vitro studies. Trans-endothelial electric resistance and permeability coefficient for dextran (Pd) were measured to analyze cell permeability. Western blotting, immunofluorescence staining and flow cytometry assay were performed to investigate the underlying mechanism. Dextran flux across the mesentery in mice was monitored to investigate in vivo microvascular permeability.
we found that AGEs evoked Nox4 membrane translocation, reactive oxygen species production, phosphorylation of Src and VE-cadherin, dissociation of adherens junctions and eventual endothelial hyperpermeability through RAGE-mDia1 binding. Cells overexpressing mDia1 by recombinant adenovirus infection showed stronger cellular responses induced by AGEs. Down-regulation of mDia1 by infection with an adenovirus encoding siRNA or blockade of RAGE-mDia1 binding by transfection with RAGE mutant plasmids into HUVECs abolished these AGE-induced effects. Furthermore, knockdown of mDia1 using an adenovirus or genetical knockout of RAGE in C57 mice rescued AGE-evoked microvascular hyperpermeability.
Our study revealed that mDia1 plays a critical role in AGE-induced microvascular hyperpermeability through binding to RAGE.
背景/目的：对晚期糖基化终末产物（AGEs）刺激的内皮屏障完整性破坏，促成了与糖尿病相关的血管病变。据报道，哺乳动物透明相关成膜蛋白（mDia1）与晚期糖基化终末产物受体（RAGE）的胞质结构域结合，从而诱导一系列细胞过程。本研究直接评估了mDia1在AGE诱导的高通透性中的作用，并揭示了这一病理过程精确的细胞内信号转导。
体外研究采用人脐静脉内皮细胞（HUVECs）。测量跨内皮电阻和葡聚糖的通透系数（Pd）以分析细胞通透性。进行蛋白质免疫印迹法、免疫荧光染色和流式细胞术检测以研究潜在机制。监测小鼠肠系膜的葡聚糖通量以研究体内微血管通透性。
我们发现，AGEs通过RAGE-mDia1结合引发Nox4膜转位、活性氧生成、Src和VE-钙黏蛋白磷酸化、黏附连接解离，最终导致内皮高通透性。通过重组腺病毒感染过表达mDia1的细胞，对AGEs诱导的细胞反应更强。用编码小干扰RNA的腺病毒感染下调mDia1，或在HUVECs中转染RAGE突变体质粒阻断RAGE-mDia1结合，消除了这些AGE诱导的效应。此外，在C57小鼠中用腺病毒敲低mDia1或基因敲除RAGE可挽救AGE引发的微血管高通透性。
我们的研究表明，mDia1通过与RAGE结合在AGE诱导的微血管高通透性中起关键作用。
