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 2

FTO causes retinal vascular endothelial dysfunction in diabetic mice.

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FTO causes retinal vascular endothelial dysfunction in diabetic mice. (A...
(A) A schematic diagram showing the generation of endothelial cell–specific (EC-specific) Fto-deficient (EC FtoΔ/Δ) mice. (B) Immunofluorescence of FTO protein (red), cell nuclei (DAPI, blue), and retinal microvascular ECs (CD31, green) in EC Ftofl/fl and EC FtoΔ/Δ mice (scale bar: 5 μm). (C) PCR genotyping verified Fto exon 3 deletion in primary retinal microvascular ECs from EC FtoΔ/Δ mice. (D) Depletion of FTO protein in the primary retinal microvascular ECs from EC FtoΔ/Δ mice (n = 3). (E) Dot blot showing increased m6A content in EC FtoΔ/Δ mice (n = 6, Student’s t test). MB, methylene blue staining. (F) Endothelial knockout of Fto alleviated diabetes-induced retinal endothelium vascular leakage as shown in flat-mounted retinas stained with Evans blue dye. A representative image with the quantification of the fluorescence signal is shown (n = 4, scale bar: 1 mm). (G) EC FtoΔ/Δ mice presented with fewer acellular retinal capillaries after the induction of diabetes, as indicated by retinal trypsin digestion. Red arrows indicate acellular capillaries. Acellular capillaries were quantified in 20 high-power fields and averaged (n = 4, scale bar: 50 μm). (H) Transwell assays showing that FTO enhances the migration ability of human retinal microvascular ECs (HRMECs) cultivated in high glucose. The number of migrated cells was quantified (n = 4, scale bar: 200 μm). (I) FTO increased tube formation of HRMECs treated with high glucose. The average number of tube formation for each field was assessed (n = 4, scale bar: 200 μm). NG, normal glucose (5.5 mM) with D-mannitol as osmotic control; HG, high glucose (30 mM). For F–I, significant differences were determined by 1-way ANOVA or Kruskal-Wallis’s test followed by Bonferroni’s post hoc comparison test. Data are shown as the mean ± SD. *P < 0.05.