The developmental origin, anatomical location, and other factors contribute to aortic heterogeneity in a physiological state. On this basis, vascular diseases occur at different ratios based on position specificity, even with the same risk factor. However, the continuous intersegmental aortic profile has been rarely reported at the single-cell level. To reveal aortic heterogeneity, we identified 15 cell subtypes from five continuous aortic segments by marker genes and functional definitions. The EC1 subtype highly expressed Vcam1 and Scarb2 genes in the aortic arch, which were reported to be associated with atherosclerosis. The newly identified Fbn1+ fibroblasts were found highly expressed in thoracic segments. More importantly, vascular smooth muscle cells (VSMCs) demonstrated a novel composition in which VSMC 4 marked with the gene Malat1 were mainly distributed in the abdominal segment. Malat1 knockout reduced MMPs and inflammatory factor production induced by Ang II in smooth muscle cells, and the Malat1 inhibitor exerted preventive, inhibitory, and reversing effects on AngII-induced abdominal aortic aneurysm (AAA) in vivo revealed by a series of animal experiments. Single-cell analysis of AngII-induced AAA tissues treated with or without the inhibitor further clarified the key role of Malat1+VSMC in the occurrence and progression of AAA. In summary, segmental gene expression and cell subtype features in normal aorta associated with different vascular diseases might provide potential therapeutic targets.

BACKGROUND: Atherosclerosis is a chronic inflammatory disease, in which macrophages determine the progression of atherosclerotic plaques. However, no studies have investigated how METTL3 (methyltransferase like 3) in macrophages affects atherosclerotic plaque formation in vivo. Additionally, whether Braf is modified by METTL3-dependent N6-methyladenosine (m6A) methylation remains unknown. METHODS: We analyzed single-cell sequencing data of atherosclerotic plaques in mice fed with a high fat diet for different periods. Mettl3 fl/fl Lyz 2 cre Apoe –/– mice and littermate control Mettl3 fl/fl Apoe –/– mice were generated and fed high fat diet for 14 weeks. In vitro, we stimulated peritoneal macrophages with ox-LDL (oxidized low-density lipoprotein) and tested the mRNA and protein expression levels of inflammatory factors and molecules regulating ERK (extracellular signal-regulated kinase) phosphorylation. To find METTL3 targets in macrophages, we performed m6A-methylated RNA immunoprecipitation sequencing and m6A-methylated RNA immunoprecipitation-qPCR. Further, point mutation experiments were used to explore m6A-methylated adenine. Using RNA immunoprecipitation assay, we explored m6A methylation-writing protein bound to Braf mRNA. RESULTS: In vivo, METTL3 expression in macrophages increased with the progression of atherosclerosis. Myeloid cell–specific METTL3 deletion negatively regulated atherosclerosis progression and the inflammatory response. In vitro, METTL3 knockdown or knockout in macrophages attenuated ox-LDL-mediated ERK phosphorylation rather than JNK (c-Jun N-terminal kinase) and p38 phosphorylation and reduced the level of inflammatory factors by affecting BRAF protein expression. The negative regulation of inflammation response caused by METTL3 knockout was rescued by overexpression of BRAF. In mechanism, METTL3 targeted adenine (39725126 in chromosome 6) on the Braf mRNA. Then, YTHDF1 could bind to m6A-methylated Braf mRNA and promote its translation. CONCLUSIONS: Myeloid cell–specific Mettl3 deficiency suppressed hyperlipidemia-induced atherosclerotic plaque formation and attenuated atherosclerotic inflammation. We identified Braf mRNA as a novel target of METTL3 in the activation of the ox-LDL-induced ERK pathway and inflammatory response in macrophages. METTL3 may represent a potential target for the treatment of atherosclerosis.