Vascular calcification (VC), a pathological condition marked by aberrant mineral deposition in the arterial wall, elevates cardiovascular risk by promoting arterial stiffness and diminishing vascular compliance (Ghosh et al., 2020; Sutton et al., 2023). Prevalent in aging and metabolic disorders such as diabetes and chronic kidney disease (CKD) (Strauss et al., 2019; Atta, 2022), this process mirrors osteogenesis, where high calcium and phosphate levels trigger a phenotypic switch in vascular smooth muscle cells (VSMCs) from a contractile to an osteogenic state (Freise and Querfeld, 2014; Van den Bergh et al., 2019; Chen et al., 2020). This transition is characterized by the downregulation of contractile markers, including alpha-smooth muscle actin (α-SMA) and smooth muscle protein 22-alpha (SM22), alongside the upregulation of osteogenic transcription factors such as bone morphogenetic protein 2 (BMP2) and Runt-related transcription factor 2 (Runx2) (Alesutan et al., 2016). VC is driven by multiple dysregulated signaling pathways involving oxidative stress, inflammation (often mediated by the master transcriptional regulator NF-κB), apoptosis, and mitochondrial dysfunction (Dai et al., 2013; Freise and Querfeld, 2014; Alesutan et al., 2016; Van den Bergh et al., 2019; Chen et al., 2020; Zhang et al., 2020). These disturbances compromise cellular energy metabolism and amplify oxidative stress, thereby aggravating vascular injury. Notably, mitophagy, the selective autophagy of damaged mitochondria, is crucial for vascular homeostasis. Yet, its specific role in driving or preventing vascular calcification is poorly defined (Dai et al., 2013; Zhang et al., 2020).

Despite advances in understanding the molecular mechanisms of VC, effective therapeutic interventions remain limited. Natural compounds with multi-target properties represent promising alternative candidates. Leveraging our established network pharmacology platform (Lu et al., 2023), we identified luteolin (LU), a flavonoid recognized for its anti-inflammatory, antioxidant, and cardioprotective properties (Kampa et al., 2022; Shi et al., 2024), as a potential inhibitor of VC. Although LU has been reported to ameliorate VC via activation of the Sirtuin 1/C-X-C chemokine receptor type 4 (SIRT1/CXCR4) pathway and enhancement of autophagy (Yu et al., 2024), its influence on mitochondrial homeostasis—specifically the regulation of mitophagy—has not been explored. This knowledge gap motivates the present investigation into the role of mitochondrial mechanisms, particularly mitophagy, in the protective effects of LU against VC.

Mitophagy, a selective autophagic process that degrades damaged mitochondria, is essential for mitochondrial quality control and cellular energy homeostasis. In VC, impaired mitophagy has been associated with the accumulation of dysfunctional mitochondria, resulting in elevated oxidative stress and inflammation—both critical drivers of calcification. Consequently, restoring mitophagy has emerged as a promising therapeutic strategy for mitigating VC(Uoselis et al., 2023; Zhu et al., 2023), underscoring the relevance of investigating compounds such as LU that may modulate this process. The translocase of the outer mitochondrial membrane 6 (TOM6), a core component of the TOM complex, is known to regulate mitophagy (Kato and Mihara, 2008; Wang et al., 2020, 2023) and has been implicated in cardiovascular diseases (Pitt and Buchanan, 2021); however, its role in VC is entirely unknown. Elucidating the function of TOM6 in VC may therefore reveal novel therapeutic targets.

Given the established role of NF-κB signaling in both inflammation and VC pathogenesis, we reasoned that it acts as a key upstream regulator in this process (Voelkl et al., 2018). Based on these premises, we hypothesize that LU attenuates VC by restoring mitophagy through an NF-κB-dependent suppression of TOM6, leading to subsequent activation of the PTEN-induced kinase 1 (PINK1)/Parkin pathway. By employing integrated transcriptomic and functional analyses, we not only identify TOM6 as a novel regulator of VC but also establish the therapeutic efficacy of LU mediated through this newly delineated pathway.