DOI: 10.1161/atvbaha.126.324532 ISSN: 1079-5642
M
6
A Demethylase FTO Targets Pcsk9 to Inhibit Vascular Calcification in Chronic Kidney Disease
Yuchen Xie, Huan Zhao, Lan Lan, Rumin Liu, Minhui Zheng, Guoli Zhong, Liang Yuan, Kun Li, Vicky E. Macrae, Shengping He, Yao Hao, Liangxi Yuan, Guojun Chen, Dongxing Zhu, Hongjiao Yu
BACKGROUND:
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.
METHODS:
High phosphate (Pi) was used to induce calcification in vascular smooth muscle cells (VSMCs) and aortic rings, and high-dose vitamin D
3
(vD
3
) 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.
RESULTS:
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 vD
3
-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 vD
3
. 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.
CONCLUSIONS:
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.