Anti-ATR001 monoclonal antibody ameliorates atherosclerosis through beta-arrestin2 pathway

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_q or G_i^2/i3) signal transduction events was also investigated.

McAb-ATR could specifically bind to the Phe¹⁸²-His¹⁸³-Tyr¹⁸⁴ 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_q or G_i^2/i3) and G_q-dependent intracellular Ca²⁺ release, nor cause RAS feedback activation.

Through regulating beta-arrestin2, McAb-ATR ameliorates atherosclerosis without affecting G_q or G_i^2/i3 pathways. Due to high selectivity for AT1R and biased interaction with beta-arrestin2, McAb-ATR could serve as a novel strategy for treating atherosclerosis.