An H3K18la-ALKBH5-Runx2-LDHA positive feedback loop promotes vascular calcification in the osteogenic phenotypic transition of vascular smooth muscle cells.

Osteogenic phenotypic transition of vascular smooth muscle cells (VSMCs) is the core pathological event underlying the initiation and progression of vascular calcification (VC). Runt-related transcription factor 2 (Runx2), a master osteogenic transcription factor, induces the transdifferentiation of VSMCs toward an osteoblast-like phenotype, which subsequently facilitates calcium salt deposition and calcified plaque formation in the vascular wall. However, its upstream and downstream regulatory networks remain incompletely defined. This study found that lactate levels were increased in calcified aortic tissues and osteogenic VSMCs. Additionally, it demonstrated that global protein lactylation and histone H3 lysine 18 lactylation (H3K18la) were concurrently elevated. In VSMCS, lactate consistently improved H3K18la modification and the osteogenic phenotypic transition, while the glycolytic inhibitor 2-deoxy-d-glucose (2-DG) reversed these phenotypic alterations. Consistent with the above phenotypic changes, we identified AlkB homolog 5 (ALKBH5) as a potential transcriptional target of H3K18la through CUT&Tag combined with proteomic screening and analysis. H3K18la is enriched at the ALKBH5 promoter, which is required to activate its transcription. Mechanistic investigations revealed that the Runx2 serves as a downstream target of ALKBH5-mediated m6A demethylation. ALKBH5 positively regulates Runx2 expression by erasing m6A modifications. Conversely, ALKBH5 knockdown leads to excessive m6A accumulation and subsequently accelerates Runx2 mRNA degradation. Additionally, Runx2 directly transcriptionally regulates lactate dehydrogenase A (LDHA), thereby increasing lactate production and H3K18la levels and establishing the H3K18la-ALKBH5-Runx2-LDHA positive feedback loop that persistently drives osteogenic phenotypic transition. This study identifies a novel synergistic regulatory mechanism between histone lactylation and RNA m6A methylation modifications in VC pathogenesis.