Qin Xiao-Fei, Shan Ying-Guang, Dou Min, Li Feng-Xiang, Guo Yu-Xi
Department of Cardiology, The First Affiliated Hospital of Zhengzhou University, No.1, Jianshedong Rd, Zhengzhou, 450052, Henan, People's Republic of China.
Apoptosis. 2023 Feb;28(1-2):124-135. doi: 10.1007/s10495-022-01777-2. Epub 2022 Oct 14.
The Notch signaling pathway is related to endothelial dysfunction in coronary atherosclerosis. Our objective was to explore the role of Notch signaling in coronary microvascular dysfunction (CMD). CMD models were constructed by sodium laurate injection in vivo and homocysteine (Hcy) stimulation in vitro. The binding ability of Notch Intracellular Domain (NICD)/H3K9Ac/GCN5 (General Control Non-derepressible 5) to Neuregulin-1 (Nrg-1) promoter was examined by chromatin immunoprecipitation. Immunofluorescence staining was conducted to detect CD31 positive cells, NICD localization, and co-localization of NICD and GCN5. Flow cytometry and Tunel staining were conducted to identify the apoptosis. Hematoxylin and eosin staining, quantitative real-time PCR, western blot, immunohistochemical staining, co-immunoprecipitation, and double luciferase report analysis were also conducted. Notch signaling pathway-related protein levels were decreased, levels of Nrg-1 and the phosphorylation of ErbB2 and ErbB4 were enhanced in CMD models. Interference with Nrg-1 further increased the apoptosis in Hcy-induced cardiac microvascular endothelial cells (CMECs). Meanwhile, the activation of the Notch signaling pathway increased the levels of Nrg-1 and the phosphorylation of ErbB2 and ErbB4, as well as inhibited the apoptosis induced by Hcy. Furthermore, NICD and histone acetyltransferase enzyme GCN5 could regulate Nrg-1 promoter activity by affecting the expression of acetylation-modified protein H3K9Ac. In addition, NICD also interacted with GCN5. In vivo results also confirmed that the activation of the Notch signal alleviated CMD. Notch signaling pathway regulates Nrg-1 level through synergistic interaction with GCN5, thereby mitigating CMD.
Notch信号通路与冠状动脉粥样硬化中的内皮功能障碍有关。我们的目的是探讨Notch信号在冠状动脉微血管功能障碍(CMD)中的作用。通过在体内注射月桂酸钠和在体外进行同型半胱氨酸(Hcy)刺激来构建CMD模型。采用染色质免疫沉淀法检测Notch细胞内结构域(NICD)/组蛋白H3赖氨酸9乙酰化(H3K9Ac)/通用控制非抑制蛋白5(GCN5)与神经调节蛋白-1(Nrg-1)启动子的结合能力。进行免疫荧光染色以检测CD31阳性细胞、NICD定位以及NICD与GCN5的共定位。采用流式细胞术和Tunel染色来鉴定细胞凋亡。还进行了苏木精-伊红染色、定量实时聚合酶链反应、蛋白质免疫印迹、免疫组织化学染色、免疫共沉淀和双荧光素酶报告分析。在CMD模型中,Notch信号通路相关蛋白水平降低,Nrg-1水平以及ErbB2和ErbB4的磷酸化水平升高。干扰Nrg-1进一步增加了Hcy诱导的心脏微血管内皮细胞(CMECs)的凋亡。同时,Notch信号通路的激活增加了Nrg-1水平以及ErbB2和ErbB4的磷酸化水平,并抑制了Hcy诱导的细胞凋亡。此外,NICD和组蛋白乙酰转移酶GCN5可通过影响乙酰化修饰蛋白H3K9Ac的表达来调节Nrg-1启动子活性。此外,NICD还与GCN5相互作用。体内实验结果也证实,Notch信号的激活减轻了CMD。Notch信号通路通过与GCN5协同相互作用调节Nrg-1水平,从而减轻CMD。