FTO fuels diabetes-induced vascular endothelial dysfunction associated with inflammation by erasing m6A methylation of TNIP1
Chuandi Zhou, Xinping She, Chufeng Gu, Yanan Hu, Mingming Ma, Qinghua Qiu, Tao Sun, Xun Xu, Haibing Chen, Zhi Zheng
Chuandi Zhou, Xinping She, Chufeng Gu, Yanan Hu, Mingming Ma, Qinghua Qiu, Tao Sun, Xun Xu, Haibing Chen, Zhi Zheng
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Research Article Inflammation Vascular biology

FTO fuels diabetes-induced vascular endothelial dysfunction associated with inflammation by erasing m6A methylation of TNIP1

Abstract

Endothelial dysfunction is a critical and initiating factor of the vascular complications of diabetes. Inflammation plays an important role in endothelial dysfunction regulated by epigenetic modifications. N6-methyladenosine (m6A) is one of the most prevalent epigenetic modifications in eukaryotic cells. In this research, we identified an m6A demethylase, fat mass and obesity-associated protein (FTO), as an essential epitranscriptomic regulator in diabetes-induced vascular endothelial dysfunction. We showed that enhanced FTO reduced the global level of m6A in hyperglycemia. FTO knockdown in endothelial cells (ECs) resulted in less inflammation and compromised ability of migration and tube formation. Compared with EC Ftofl/fl diabetic mice, EC-specific Fto-deficient (EC FtoΔ/Δ) diabetic mice displayed less retinal vascular leakage and acellular capillary formation. Furthermore, methylated RNA immunoprecipitation sequencing (MeRIP-Seq) combined with RNA-Seq indicated that Tnip1 served as a downstream target of FTO. Luciferase activity assays and RNA pull-down demonstrated that FTO repressed TNIP1 mRNA expression by erasing its m6A methylation. In addition, TNIP1 depletion activated NF-κB and other inflammatory factors, which aggravated retinal vascular leakage and acellular capillary formation, while sustained expression of Tnip1 by intravitreal injection of adeno-associated virus alleviated endothelial impairments. These findings suggest that the FTO-TNIP1-NF-κB network provides potential targets to treat diabetic vascular complications.

Authors

Chuandi Zhou, Xinping She, Chufeng Gu, Yanan Hu, Mingming Ma, Qinghua Qiu, Tao Sun, Xun Xu, Haibing Chen, Zhi Zheng

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Figure 1

Diabetes induces decreased m6A modification and increased FTO expression in human and mice.

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Diabetes induces decreased m6A modification and increased FTO expression...
(A) Dot blot showing reduced m6A content in the retinal fibrovascular membranes of patients with diabetic retinopathy (DR) (control group, n = 30; DR group, type 1 diabetes [left 2 columns], n = 10; type 2 diabetes [right 4 columns], n = 20; Student’s t test). MB, methylene blue staining. (B) A heatmap of RNA expression showing an overview of m6A-related genes in diabetic retinas. Fto was elevated stressed by diabetes (n = 3, Mann-Whitney U test). (C and D) qRT-PCR revealed higher levels of FTO in retinal fibrovascular membranes of patients with retinopathy due to type 1 (C, n = 10) or type 2 (D, n = 10) diabetes (Mann-Whitney U test). (E and F) Western blotting showing elevated expression of FTO in retinal fibrovascular membranes of patients with retinopathy due to type 1 (E, n = 6) or type 2 (F, n = 6) diabetes (Student’s t test). (G) Evans blue dye displayed that silencing Fto alleviated diabetes-induced retinal endothelium vascular leakage and enhanced Fto aggravated endothelium vascular leakage. A representative image with the quantification of the fluorescence signal is shown (n = 4, scale bar: 1 mm). (H) Retinal trypsin digestion assays indicate that silencing Fto presented with fewer acellular retinal capillaries after the induction of diabetes, and overexpressed Fto increased the number of acellular retinal capillaries. Red arrows indicate acellular capillaries. Acellular capillaries are quantified in 20 high-power fields and averaged (n = 4, scale bar: 50 μm). For G and H, significant differences were assessed by Kruskal-Wallis’s test followed by Bonferroni’s post hoc comparison test. Data are shown as the mean ± SD. *P < 0.05.