Department of Endocrinology, The First Affiliated Hospital of Anhui Medical University, Hefei, China; Laboratory of Molecular Biology and Department of Biochemistry, Anhui Medical University, Hefei, China; Institute of Clinical Pharmacology, Anhui Medical University, Hefei, China.
Department of Geriatrics, The First Affiliated Hospital of Anhui Medical University, Hefei, China.
Biomed Pharmacother. 2019 May;113:108667. doi: 10.1016/j.biopha.2019.108667. Epub 2019 Mar 7.
Glucagon-like peptide-1 (GLP-1) showed protective effects on endothelium-dependent dilatation. Since endothelial barrier dysfunction also plays a pivotal role in atherosclerosis, this study was designed to investigate the effects of GLP-1 on endothelial barrier function in diabetic aortic endothelium and explore the underlying mechanism.
For in vivo studies, diabetic rats were established and subjected to 12- and 24-week treatment of exenatide. The morphological changes of aortic endothelium were observed with transmission electron microscope. A permeability assay of aortic endothelium was performed using the surface biotinylation technique. Protein expression was detected by immunohistochemical analysis and Western blots. For in vitro studies, human umbilical vein endothelial cells (HUVECs) were cultured in medium enriched with advanced glycation end products (AGEs) or AGEs plus GLP-1 and other reagents. The integrity of endothelium was evaluated by endothelial monolayer permeability assay and transendothelial resistance. The in vitro expressions of relevant proteins in signaling pathways were also detected by immunofluorescence and Western blots.
In vivo, the enhanced aortic endothelial permeability in diabetic aortas were attenuated by exenatide treatment. Additionally, myosin light chain (MLC) phosphorylation, related to actomyosin contractility, and activation of its upstream targets in diabetic aorta were inhibited after administration of exenatide. In vitro, the endothelial monolayer permeability and the assembly of stress fibers were reduced by GLP-1 intervention under diabetic condition. Meanwhile, AGE-induced MLC phosphorylation mediating ECs contractility was inhibited by GLP-1. Furthermore, GLP-1 down-regulated the upstream targets of MLC phosphorylation, including RAGE, Rho/ROCK and MAPK signaling pathways. Intriguingly, the effects of GLP-1 elicited on ECs contractility and barrier function in diabetes were blunted by inhibition of GLP-1R, cAMP or PKA and stimulation of Rho/ROCK and MAPK signaling pathways.
The findings of this study suggest that the stabilizing effect of GLP-1 on the endothelial barrier and contraction of AGE-treated ECs is caused by GLP-1R/cAMP/PKA activation and the subsequent inactivation of RAGE/Rho/ROCK as well as MAPK signaling pathways.
胰高血糖素样肽-1（GLP-1）对内皮依赖性舒张具有保护作用。由于内皮屏障功能障碍在动脉粥样硬化中也起着关键作用，因此本研究旨在探讨 GLP-1 对糖尿病主动脉内皮内皮屏障功能的影响，并探讨其潜在机制。
在体内研究中，建立糖尿病大鼠模型，并进行 12 周和 24 周的 exenatide 治疗。用透射电子显微镜观察主动脉内皮的形态变化。用表面生物素化技术进行主动脉内皮通透性测定。用免疫组化分析和 Western blot 检测蛋白表达。在体外研究中，将人脐静脉内皮细胞（HUVEC）在富含晚期糖基化终产物（AGEs）或 AGEs 加 GLP-1 和其他试剂的培养基中培养。用内皮单层通透性测定和跨内皮电阻来评估内皮的完整性。用免疫荧光和 Western blot 检测信号通路中相关蛋白的体外表达。
体内研究显示，exenatide 治疗可减轻糖尿病大鼠主动脉内皮通透性增强。此外，exenatide 给药后，糖尿病主动脉中肌球蛋白轻链（MLC）磷酸化，与肌球蛋白收缩性相关，其上游靶点被激活。在体外，在糖尿病条件下，GLP-1 干预可减少内皮单层通透性和应力纤维的组装。同时，GLP-1 抑制 AGE 诱导的调节 ECs 收缩性的 MLC 磷酸化。此外，GLP-1 下调 MLC 磷酸化的上游靶点，包括 RAGE、Rho/ROCK 和 MAPK 信号通路。有趣的是，GLP-1R/cAMP/PKA 抑制和 Rho/ROCK 和 MAPK 信号通路激活可削弱 GLP-1 对糖尿病状态下 ECs 收缩性和屏障功能的作用。
本研究结果表明，GLP-1 对内皮屏障的稳定作用和对 AGE 处理的 ECs 的收缩作用是由 GLP-1R/cAMP/PKA 激活以及随后的 RAGE/Rho/ROCK 和 MAPK 信号通路失活引起的。
