Abstract Body: Introduction: Vascular calcification (VC) is a major contributor to cardiovascular morbidity and mortality and is driven in part by osteogenic reprogramming of vascular smooth muscle cells (VSMCs). Although microbiota-derived metabolites influence systemic physiology, their roles in vascular mineralization are not well defined. Cyclic dinucleotides (CDNs), metabolites, produced by bacteria and host cells activate innate immune signaling, yet their impact on vascular remodeling remains unclear.
Hypothesis: CDNs modulate VSMC osteogenic differentiation and VC through STING-dependent signaling.
Methods: Primary murine VSMCs were cultured under osteogenic conditions with increasing concentrations of c-di-AMP, c-di-GMP, or cGAMP to assess effects on mineralization. Mineral deposition was quantified by Von Kossa and Alizarin Red staining. Findings were validated using an ex vivo mouse aortic ring calcification model. To define STING dependence, VSMCs from wild-type and STING-deficient mice were compared. Activation of the STING–TBK1–IRF3 pathway and induction of IFNβ were assessed by immunoblotting and/or ELISA. The effects of recombinant IFNβ or IFNγ on calcification were tested. STAT1 activation and Runx2 nuclear localization were evaluated in calcifying VSMCs. All experiments included three biological replicates.
Results: CDNs dose-dependently inhibited VSMC mineralization and reduced calcium deposition in vitro and in aortic rings ex vivo. This effect required STING, as CDN-mediated inhibition was lost in STING-deficient VSMCs. Mechanistically, CDNs activated TBK1 and IRF3 and markedly increased IFNβ production. Recombinant IFNβ dose-dependently suppressed VC, whereas IFNγ had no effect, supporting a selective role for Type I interferon signaling. CDNs and IFNβ increased STAT1 activation and reduced nuclear localization of the osteogenic transcription factor Runx2, consistent with suppression of osteogenic reprogramming.
Conclusions: Bacterial and host-derived CDNs inhibit VC through a STING–IFNβ–STAT1–Runx2 axis. These findings identify CDN-triggered innate immune signaling as a previously unrecognized protective mechanism in vascular calcification and suggest that CDN-based immunomodulators could be explored as therapeutic candidates for VC-associated cardiovascular disease.