Adenylate kinase 4 (AK4) deficiency prevents vascular smooth muscle cell phenotypic switching by regulating mitochondrial dysfunction through AMPKα inactivation.

Background and aims: Mitochondrial dynamics are key mechanism regulating the conversion of vascular smooth muscle cells (VSMCs) from a contractile to a synthetic phenotype, which is involved in neointima formation and restenosis. However, the underlying mechanisms leading to mitochondrial dysfunction are not fully understood.

Methods: Western blot was used to detect the expression of relevant molecules at the protein level. CCK-8, EdU assays, and transwell were used to test cell proliferation and migration capacity. Flow cytometry was used to assess cell cycle and ROS.

Results: AK4 was upregulated in 10 % fetal bovine serum (FBS)- and 20 ng/mL platelet-derived growth factor-BB (PDGF-BB)-induced human aortic VSMCs (HASMCs). Knockdown of AK4 suppressed the proliferation and synthetic phenotype of HASMCs, while AK4 overexpression accelerated it. Mechanistically, AK4 interacted with protein kinase AMP-activated catalytic subunit alpha (AMPKα) and promoted the phosphorylation of AMPKα at Thr172, which reduced mitochondrial oxidative damage and improved mitochondrial function. Furthermore, activation of AMPKα by metformin or AICAR (acadesine) reversed the inhibitory effects of AK4 deficiency on HASMC phenotypic switching. Moreover, overexpression of wild-type AMPKα counteracted the effects of AK4 knockdown, whereas mutational inactivation of AMPKα (AMPKα^T172A) was not effective in reversing the effect on HASMCs.

Conclusions: Our findings suggest that AK4 is a novel regulator of AMPKα activity and positively regulates VSMC dedifferentiation, proliferation, and migration. Targeted inhibition of AK4 may be a potential approach for the treatment of neointima formation and restenosis.