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 4

ALKBH1 is essential for the regulation of vascular calcification.

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ALKBH1 is essential for the regulation of vascular calcification. (A) We...
(A) Western blot analysis identifying the ALKBH1 deficiency in arteries (n = 6 per group). Mice were injected via tail vein with AAV carrying scrambled shRNA (sh-Scr) or Alkbh1 shRNA (sh-ALKBH1) at 4 weeks after adenine diet and then fed for another 4 weeks. (B–D) Von Kossa staining (B and C) and calcium content quantification of aortic arch (D) were performed in different experimental groups for detecting mineralization (n = 10–12 per group). Scale bar: 100 μm. (E) Photomicrographs of Alizarin red staining in mice primary VSMCs pretransfected with indicated treatment and exposed in osteogenic medium for another 14 days (n = 6 per group). (F and G) Bar graphs representative of calcium content (F) and ALP activity (G) in mice primary VSMCs from all of the experimental cohorts (n = 6 per group). (H) ALKBH1 overexpression in arteries confirmed by Western blot (n = 6 per group). Mice were injected with AAV-Vector or AAV-ALKBH1 at 4 weeks after the adenine diet and then fed for another 4 weeks. (I–K) Percentage of positive von Kossa staining (I and J) and calcium content (K) quantified in the aortic arch from the different cohorts (n = 10–12 per group). Scale bar: 100 μm. (L) Representative images of Alizarin red staining in mice primary VSMCs after indicated transfection and osteogenic medium exposure for another 14 days (n = 6 per group). (M and N) Scatter dot plots representative of calcium content (M) and ALP activity (N) in mice primary VSMCs from all of the experimental cohorts (n = 6 per group). Statistical significance was assessed using 2-tailed t tests (A and H) and 1-way ANOVA followed by Dunnett’s test (C–G, and J–N). All values are presented as mean ± SD. *P < 0.05.