Department of Cardiology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China; Key Lab of Molecular Biological Targeted Therapies of the Ministry of Education, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China.
International Research Center for Sensory Biology and Technology of MOST, Key Laboratory of Molecular Biophysics of MOE, School of Life Science and Technology, Huazhong University of Science and Technology, 430074, Wuhan, China.
Biochem Biophys Res Commun. 2021 Mar 12;544:1-7. doi: 10.1016/j.bbrc.2021.01.054. Epub 2021 Jan 28.
Our previous study developed ATRQβ-001 vaccine, which targets peptide ATR001 from angiotensin Ⅱ (Ang Ⅱ) receptor type 1 (AT1R). The ATRQβ-001 vaccine could induce the production of anti-ATR001 monoclonal antibody (McAb-ATR) and inhibit atherosclerosis without feedback activation of the renin-angiotensin system (RAS). This study aims at investigating the underexploited mechanisms of McAb-ATR in ameliorating atherosclerosis.
AT1R-KO HEK293T cell lines were constructed to identify the specificity of McAb-ATR and key sites of ATRQβ-001 vaccine. Beta-arrestin1 knock-out (Arrb1) mice, Beta-arrestin2 knock-out (Arrb2) mice, and low-density lipoprotein receptor knock-out (LDLr) mice were used to detect potential signaling pathways affected by McAb-ATR. The role of McAb-ATR in beta-arrestin and G proteins (G or G) signal transduction events was also investigated.
McAb-ATR could specifically bind to the Phe-His-Tyr site of AT1R second extracellular loop (ECL2). The anti-atherosclerotic effect of McAb-ATR disappeared in LDLr mice transplanted with Arrb2 mouse bone marrow (BM) and BM-derived macrophages (BMDMs) from Arrb2 mice. Furthermore, McAb-ATR inhibited beta-arrestin2-dependent extracellular signal regulated kinase1/2 (ERK1/2) phosphorylation, and promoted beta-arrestin2-mediated nuclear factor kappa B p65 (NFκB p65) inactivity. Compared with conventional AT1R blockers (ARBs), McAb-ATR did not inhibit Ang Ⅱ-induced uncoupling of heterotrimeric G proteins (G or G) and G-dependent intracellular Ca release, nor cause RAS feedback activation.
Through regulating beta-arrestin2, McAb-ATR ameliorates atherosclerosis without affecting G or G pathways. Due to high selectivity for AT1R and biased interaction with beta-arrestin2, McAb-ATR could serve as a novel strategy for treating atherosclerosis.
我们之前的研究开发了 ATRQβ-001 疫苗，该疫苗针对血管紧张素Ⅱ（Ang Ⅱ）受体 1（AT1R）的肽 ATR001。ATRQβ-001 疫苗可诱导产生抗 ATR001 单克隆抗体（McAb-ATR），并抑制动脉粥样硬化，而不会反馈激活肾素-血管紧张素系统（RAS）。本研究旨在探讨 McAb-ATR 改善动脉粥样硬化的未充分利用机制。
构建 AT1R-KO HEK293T 细胞系以鉴定 McAb-ATR 的特异性和 ATRQβ-001 疫苗的关键部位。使用β-arrestin1 敲除（Arrb1）小鼠、β-arrestin2 敲除（Arrb2）小鼠和低密度脂蛋白受体敲除（LDLr）小鼠来检测 McAb-ATR 可能影响的潜在信号通路。还研究了 McAb-ATR 在β-arrestin 和 G 蛋白（G 或 G）信号转导事件中的作用。
McAb-ATR 可特异性结合 AT1R 第二细胞外环（ECL2）的 Phe-His-Tyr 位点。在 LDLr 小鼠中，McAb-ATR 的抗动脉粥样硬化作用在接受 Arrb2 小鼠骨髓（BM）和源自 Arrb2 小鼠的 BM 衍生巨噬细胞（BMDMs）移植的小鼠中消失。此外，McAb-ATR 抑制了β-arrestin2 依赖性细胞外信号调节激酶 1/2（ERK1/2）磷酸化，并促进了β-arrestin2 介导的核因子 kappa B p65（NFκB p65）失活。与传统的 AT1R 阻滞剂（ARBs）相比，McAb-ATR 不抑制 Ang Ⅱ诱导的异三聚体 G 蛋白（G 或 G）解偶联和 G 依赖性细胞内 Ca 释放，也不会引起 RAS 反馈激活。
通过调节β-arrestin2，McAb-ATR 改善动脉粥样硬化而不影响 G 或 G 途径。由于对 AT1R 具有高选择性和与β-arrestin2 的偏向性相互作用，McAb-ATR 可作为治疗动脉粥样硬化的新策略。
