Department of Vascular Surgery, Qingpu Branch of Zhongshan Hospital, Fudan University, Shanghai, 9/F, Building 7, East Park Road No.1158, Qingpu District, Shanghai, 201700, People's Republic of China.
Glycoconj J. 2020 Apr;37(2):209-219. doi: 10.1007/s10719-020-09908-0. Epub 2020 Feb 3.
Diabetic vascular complications caused by endothelial dysfunction play an important role in the pathogenesis of diabetic foot. A well understanding of the role of endothelial dysfunction in diabetic foot vasculopathy will help to further reveal the pathogenesis of diabetic foot. This study aimed to assess whether the RhoA/ROCK signaling pathway is controlled by Rho GTPase-activating proteins (RhoGAP, ARHGAP) and advanced glycosylation end products (AGEs), and to clarify the roles of ARHGAP and AGEs in the RhoA/ROCK signaling pathway or the mechanism by which AGEs regulated RhoA. Real-time PCR was applied to detect gene expression. Manipulation of endothelial biological functions by ARHGAP18 and AGEs were studied via cell counting kit-8 (CCK-8), Western blot, transwell, FITC-Dextran and TEER permeability experiments. RhoA-specific inhibitor Y-27632 was used to silence the activity of RhoA. Dual Luciferase Reporter Assay, Western blot and ELISA assays were used to detect molecular mechanism of endothelial biological functions. In this study, we found that ARHGAP18 was negatively correlated with RhoA, and the expression of ARHGAP18 in human umbilical vein endothelial cells (HUVECs) was decreased with gradient-increased AGEs. Furthermore, AGEs and ARHGAP18 could orchestrate RhoA activity, then activate NF-κB signaling pathway, affect the structural and morphological of VE-cadherin and tight junction protein, and cause endothelial cell contraction, thereby increasing permeability of endothelial cells. In conclusion, AGEs and ARHGAP18 orchestrate cell proliferation, invasion and permeability by controlling the RhoA/ROCK signaling pathway, affecting NF-κB signaling pathway as well as the structure and morphology of VE-cadherin and tight junction protein, and regulating endothelial cell contraction.
糖尿病血管并发症由内皮功能障碍引起，在糖尿病足发病机制中起重要作用。深入了解内皮功能障碍在糖尿病足血管病变中的作用，有助于进一步揭示糖尿病足的发病机制。本研究旨在评估 RhoA/ROCK 信号通路是否受 Rho 鸟苷三磷酸酶激活蛋白（RhoGAP、ARHGAP）和晚期糖基化终产物（AGEs）的调控，并阐明 ARHGAP 和 AGEs 在 RhoA/ROCK 信号通路中的作用或 AGEs 调节 RhoA 的机制。应用实时 PCR 检测基因表达。通过细胞计数试剂盒-8（CCK-8）、Western blot、Transwell、FITC-Dextran 和 TEER 通透性实验研究 ARHGAP18 和 AGEs 对内皮生物功能的调控。采用 RhoA 特异性抑制剂 Y-27632 沉默 RhoA 的活性。双荧光素酶报告基因检测、Western blot 和 ELISA 检测用于检测内皮生物学功能的分子机制。在本研究中，我们发现 ARHGAP18 与 RhoA 呈负相关，人脐静脉内皮细胞（HUVECs）中 ARHGAP18 的表达随 AGEs 浓度梯度增加而降低。此外，AGEs 和 ARHGAP18 可以协调 RhoA 的活性，然后激活 NF-κB 信号通路，影响 VE-钙黏蛋白和紧密连接蛋白的结构和形态，引起内皮细胞收缩，从而增加内皮细胞的通透性。总之，AGEs 和 ARHGAP18 通过调控 RhoA/ROCK 信号通路，影响 NF-κB 信号通路以及 VE-钙黏蛋白和紧密连接蛋白的结构和形态，调节内皮细胞收缩，从而协调细胞增殖、侵袭和通透性。
