M⁶A Demethylase FTO Targets Pcsk9 to Inhibit Vascular Calcification in Chronic Kidney Disease

FTO (fat mass and obesity-associated) protein, a key N6-methyladenosine demethylase, plays a critical role in cardiovascular disease. However, its function in vascular calcification remains poorly understood.

High phosphate (Pi) was used to induce calcification in vascular smooth muscle cells (VSMCs) and aortic rings, and high-dose vitamin D₃ (vD₃) was used to induce vascular calcification in mice. FTO expression was modulated using adenovirus, adeno-associated virus, and siRNA. To identify FTO targets, we performed integrated MeRIP sequencing and RNA sequencing analyses.

FTO expression was significantly downregulated in calcified VSMCs, mouse arteries, and human arteries relative to their noncalcified counterparts. Silencing of Fto increased high Pi-induced osteogenic gene expression and calcification in VSMCs. Conversely, overexpression of Fto resulted in the opposite effect. These results were confirmed in high Pi-induced rat aortic ring calcification and a vD₃-induced mouse model of vascular calcification through treatment with the FTO inhibitor F,B-2,3. Intriguingly, the ALP (alkaline phosphatase) inhibitor TNAP-IN-1 or levamisole hydrochloride partially reduced FTO inhibition-induced increases in VSMC calcification, rat aortic ring calcification, and vascular calcification in mice. Adeno-associated virus-mediated VSMC-specific overexpression of Fto significantly attenuated vascular calcification in mice with overloaded vD₃. Integrated MeRIP sequencing and RNA sequencing identified that Pcsk9 is a potential target of FTO. Knockdown of FTO increased N6-methyladenosine methylation and mRNA stability of Pcsk9, and its secretion by VSMCs. In addition, knockdown of Fto enhanced the activation of NF-κB (nuclear factor-κB) signaling and increased the expression of inflammatory cytokines, including IL-6, Icam1, and Vcam1, which were attenuated by concomitant knockdown of Pcsk9. Finally, knockdown of Pcsk9 or p65 abolished silencing of the Fto-induced increase in osteogenic transition and calcification in VSMCs.

Our results suggest that FTO may inhibit vascular calcification at least in part through Pcsk9-mediated modulation of NF-κB signaling and altered ALP activity. FTO may therefore be an attractive target for the treatment of vascular calcification.