METTL3-METTL14 complex induces necroptosis and inflammation of vascular smooth muscle cells via promoting N6 methyladenosine mRNA methylation of receptor-interacting protein 3 in abdominal aortic aneurysms

Abstract

Abdominal aortic aneurysms (AAA) have the highest incidence and rupture rate of all aortic aneurysms. The N6 methyladenosine (m6A) modification is closely associated with angiotensin (Ang II)-induced aortic diseases. This study aimed to identify whether the m6A writer METTL3/METTL4 regulates rip3 mRNA expression in AAA. To induce the mouse AAA model, apolipoprotein E-deficient (ApoE-/-) mice were subcutaneously infused with Ang II, and C57BL/6 mice were infused with type I elastase. Vascular smooth muscle cells (VSMCs) were induced with Ang II. Necroptosis was detected using an Annexin V-FITC/PI apoptosis detection kit, and ELISA assays measured inflammatory cytokines. The RNA immunoprecipitation-qPCR determined the methylated rip3 mRNA level. The increased expressions of inflammatory factors, aortic adventitia injury, degradation of elastin, and CD68-positive cells suggested the successful establishment of mouse AAA models. In AAA aorta wall tissues, the m6A modification level and the expression of METTL3/METTL14 were elevated. In Ang II-induced VSMCs, necroptosis and inflammatory cytokines in the supernatants were increased. RNA immunoprecipitation and co-immunoprecipitation assays confirmed the binding between the METTL3-METTL14 complex and rip3 mRNA, the interaction between YTHDF3 and rip3 mRNA, and between the METTL3-METTL14 complex and SMAD2/3. Interference with METTL3/METTL14 attenuated VSMC necroptosis, inflammatory response, and the AAA pathological process in vivo. The METTL3-METTL14 complex, which was increased by the activation of the SMAD2/3, elevated the m6A modification of rip3 mRNA by promoting the binding between YTHDF3 and rip3 mRNA, thus contributing to the progression of AAA. The activation of SMAD2/3 in VSMCs of abdominal aortic wall tissues is stimulated by Ang II. Subsequently, it promotes METTL3 METTL14 complex mediated m6A modification of rip3 mRNA. Meanwhile, the level of rip3 mRNA becomes more stable under the m6A reader of YTHDF3, which increases the protein level of RIP3 and further induces VSMC necroptosis. In addition, cell debris induces inflammatory factors in neighboring VSMCs and recruit monocytes/macrophages to the lesion.

Keywords: Abdominal aortic aneurysm; N6 methyladenosine; Necroptosis; RIP3; m6A modification.