ALKBH1-demethylated DNA N6-methyladenine modification triggers vascular calcification via osteogenic reprogramming in chronic kidney disease
Liu Ouyang, … , Jie Chen, Hui Huang
Liu Ouyang, … , Jie Chen, Hui Huang
Published May 18, 2021
Citation Information: J Clin Invest. 2021;131(14):e146985. https://doi.org/10.1172/JCI146985.
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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 2

ALKBH1-demethylated DNA 6mA modification is reduced in human vascular calcifying progression during CKD.

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ALKBH1-demethylated DNA 6mA modification is reduced in human vascular ca...
(A) Leukocyte DNA 6mA level in patients with CKD with (VC, n = 106) or without (non-VC, n = 67) aortic arch calcification. (B and C) Leukocyte DNA 6mA level in subgroups defined by calcification Agatston score (B, n = 67 for non-VC; n = 61 for mild; n = 45 for severe) and Volume score (C, n = 67 for non-VC; n = 53 for mild; n = 53 for severe). (D) Correlation between leukocyte DNA 6mA level and calcification Agatston score from patients with CKD with aortic arch calcification (n = 106). (E) Quantitative real-time PCR analysis of ALKBH1 and N6AMT1 mRNA expression in leukocytes from patients with CKD with (VC, n = 40) or without (non-VC, n = 38) aortic arch calcification. (F and G) Leukocyte ALKBH1 mRNA expression level in subgroups defined by calcification Agatston score (F, n = 38 for non-VC; n = 21 for mild; n = 19 for severe) and Volume score (G, n = 38 for non-VC; n = 13 for mild; n = 27 for severe). (H) Scatterdot plot of correlation between leukocyte ALKBH1 mRNA expression level and calcification Agatston score from patients with CKD with aortic arch calcification (n = 40). (I–K) Representative immunofluorescence pictures (I) and quantification (J) and Western blot analysis (K) of ALKBH1, N6AMT1, and 6mA in radial arteries from CKD (n = 4) and control (n = 4) groups. Scale bars: 50 μm. Statistical significance was assessed using 2-tailed t tests (A, E, J, and K), 1-way ANOVA followed by Bonferroni’s test (B, C, F, and G), and Pearson’s correlation coefficient analysis (D and H). All values are presented as mean ± SD. *P < 0.05.