ALKBH1-demethylated DNA N6-methyladenine modification triggers vascular calcification via osteogenic reprogramming in chronic kidney disease
Liu Ouyang, Xiaoyan Su, Wenxin Li, Liangqiu Tang, Mengbi Zhang, Yongjun Zhu, Changming Xie, Puhua Zhang, Jie Chen, Hui Huang
Liu Ouyang, Xiaoyan Su, Wenxin Li, Liangqiu Tang, Mengbi Zhang, Yongjun Zhu, Changming Xie, Puhua Zhang, Jie Chen, Hui Huang
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Research Article Cardiology Vascular biology

ALKBH1-demethylated DNA N6-methyladenine modification triggers vascular calcification via osteogenic reprogramming in chronic kidney disease

Abstract

Vascular calcification (VC) predicts cardiovascular morbidity and mortality in chronic kidney disease (CKD). To date, the underlying mechanisms remain unclear. We detected leukocyte DNA N6-methyladenine (6mA) levels in patients with CKD with or without aortic arch calcification. We used arteries from CKD mice infected with vascular smooth muscle cell–targeted (VSMC-targeted) adeno-associated virus encoding alkB homolog 1 (Alkbh1) gene or Alkbh1 shRNA to evaluate features of calcification. We identified that leukocyte 6mA levels were significantly reduced as the severity of VC increased in patients with CKD. Decreased 6mA demethylation resulted from the upregulation of ALKBH1. Here, ALKBH1 overexpression aggravated whereas its depletion blunted VC progression and osteogenic reprogramming in vivo and in vitro. Mechanistically, ALKBH1-demethylated DNA 6mA modification could facilitate the binding of octamer-binding transcription factor 4 (Oct4) to bone morphogenetic protein 2 (BMP2) promoter and activate BMP2 transcription. This resulted in osteogenic reprogramming of VSMCs and subsequent VC progression. Either BMP2 or Oct4 depletion alleviated the procalcifying effects of ALKBH1. This suggests that targeting ALKBH1 might be a therapeutic method to reduce the burden of VC in CKD.

Authors

Liu Ouyang, Xiaoyan Su, Wenxin Li, Liangqiu Tang, Mengbi Zhang, Yongjun Zhu, Changming Xie, Puhua Zhang, Jie Chen, Hui Huang

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Figure 6

BMP2 mediates the procalcifying effects of ALKBH1.

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BMP2 mediates the procalcifying effects of ALKBH1. (A) Western blot anal...
(A) Western blot analysis of ALKBH1, BMP2, RUNX2, SOX9, and DLX5 expression in calcified mice primary VSMCs with AAV sh-Scr or AAV sh-ALKBH1 transfection (n = 4 per group). (B and C) Representative immunofluorescence images (B) and quantification (C) of α-SMA and BMP2 costained in aortas from indicated experimental cohorts (n = 6 per group). Scale bar: 50 μm. (D) Western blot analysis of osteogenic phenotype marker (RUNX2, OPN, OCN, and Collagen I) expression in mice primary VSMCs, which preinfected with AAV sh-Scr or AAV sh-BMP2 together with AAV-Vector or AAV-ALKBH1 and then incubated in calcifying medium for another 14 days. (E and F) Alizarin red staining (E) and ALP activity assay (F) performed in all of the groups for detecting calcification formation (n = 4–5 per group). (G) Quantification of calcium content in mice aortic ring cultured in calcifying medium with indicated transfection (n = 5 per group). Statistical significance was assessed using 1-way ANOVA followed by Dunnett’s test (A–C) or Bonferroni’s test (D–G). All values are presented as mean ± SD. *P < 0.05.