Epigenetic Modification of MicroRNA-200b Contributes to Diabetic Vasculopathy

Abstract

Hyperglycemia (HG) induces genome-wide cytosine demethylation. Our previous work recognized miR-200b as a critical angiomiR, which must be transiently downregulated to initiate wound angiogenesis. Under HG, miR-200b downregulation is not responsive to injury. Here, we demonstrate that HG may drive vasculopathy by epigenetic modification of a miR promoter. In human microvascular endothelial cells (HMECs), HG also lowered DNA methyltransferases (DNMT-1 and DNMT-3A) and compromised endothelial function as manifested by diminished endothelial nitric oxide (eNOS), lowered LDL uptake, impaired Matrigel tube formation, lower NO production, and compromised VE-cadherin expression. Bisulfite-sequencing documented HG-induced miR-200b promoter hypomethylation in HMECs and diabetic wound-site endothelial cells. In HMECs, HG compromised endothelial function. Methyl donor S-adenosyl-L-methionine (SAM) corrected miR-200b promoter hypomethylaton and rescued endothelial function. In vivo, wound-site administration of SAM to diabetic mice improved wound perfusion by limiting the pathogenic rise of miR-200b. Quantitative stable isotope labeling by amino acids in cell culture (SILAC) proteomics and ingenuity pathway analysis identified HG-induced proteins and principal clusters in HMECs sensitive to the genetic inhibition of miR-200b. This work presents the first evidence of the miR-200b promoter methylation as a critical determinant of diabetic wound angiogenesis.

Keywords: DNA methylation; diabetic vasculopathy; miR-200b; wound.

Graphical abstract

Figure 1

Hyperglycemia-Induced MicroRNA-200b Elevation Causes Endothelial Cell Dysfunction (A) qRT-PCR analysis of miR-200b expression in HMECs exposed to high D-glucose (HG) (25 mM, 4 days); osmotic control (L-glucose, LG); or cultured in normal glucose (NG) conditions. n = 3, *p < 0.05, F = 12.04 (one-way ANOVA). (B) qRT-PCR analysis of miR-200b levels in HMECs challenged with methylglyoxal (MG, 500 μM, 4 days). n = 3, *p < 0.05 (Student’s t test). (C) qRT-PCR analysis of VEGF expression under NG/HG and co-treatment with miRIDIAN microRNA hsa-miR-200b-3p hairpin inhibitor (MI) or miRIDIAN microRNA hairpin inhibitor negative control (control inhibitor, CI) (100 nM, 48 hr). n = 3, *p < 0.001, F = 22.17 (one-way ANOVA). (D) Total nitrate/nitrite production in HMECs under NG/HG and co-treatment with control (CI) or miR-200b inhibitor (MI). n = 4, *p < 0.05, F = 9.03 (one-way ANOVA). (E and F) Immunocytochemical analysis of eNOS production (E) and its quantitation (F) in HMECs under NG/HG and co-treatment with control (CI) or miR-200b inhibitor (MI). Scale bar, 20 μm. n = 5, *p < 0.001, F = 27.22 (one-way ANOVA). (G and H) Matrigel tube formation (G) and tube length analysis (H) in HMECs in NG/HG and co-treatment with control (CI) or miR-200b inhibitor (MI). Scale bar, 100 μm. n = 4, *p < 0.05, F = 22.24 (one-way ANOVA). (I and J) Ac-LDL uptake assay in HMECs (I) and intensity quantitation (J) under NG/HG and co-treatment with control (CI) or miR-200b inhibitor (MI). Scale bar, 100 μm. n = 6, *p < 0.05, F = 5.86 (one-way ANOVA). (K and L) Immunofluorescence staining of VE-cadherin (green) (K) and intensity quantitation (L) in HMECs under NG/HG and co-treatment with control (CI) or miR-200b inhibitor (MI). Scale bar, 100 μm. n = 4, *p < 0.05, F = 12.94 (one-way ANOVA). Data are represented as the mean ± SD. HMEC, human microvascular endothelial cells; LDL, low density lipoprotein; AU, arbitrary unit.

Figure 2

Hyperglycemia-Induced Promoter Hypomethylation Elevates Endothelial MicroRNA-200b (A) Schematic diagram showing the regions (1, 2) of miR-200b promoter analyzed through bisulfite genomic sequencing of DNA. Methylation profile of the miR-200b promoter in HMECs cultured in normal glucose (NG), exposed to high glucose (HG) and HG supplemented with S-adenosylmethionine (SAM) (80 μM, 48 hr) conditions (methylated CpG, black; unmethylated CpG, white). Number of clones = 5. (B) Total number of methylated CpG sites obtained from bisulfite sequencing analysis. n = 5, *p < 0.05 (Student’s t test). (C) qRT-PCR analysis of miR-200b expression in HMECs treated with SAM under HG condition (80 μM, 48 hr). n = 3, *p < 0.05 (Student’s t test). (D) Total number of methylated CpG sites obtained from bisulfite sequencing analysis in HG condition with or without SAM administration. n = 5. (E) miR-200b promoter luciferase activity was measured by calculating the GLUC/SEAP ratio in NG- or HG-exposed HMECs with or without SAM. n = 4, *p < 0.05, F = 10.80 (one-way ANOVA). (F) Endothelial function analysis: matrigel tube length (upper panel), ac-LDL uptake (middle panel), and VE-cadherin expression (lower panel) after SAM treatment in HG condition. n = 5, *p < 0.05 (Student’s t test). (G) Immunofluorescence staining of vWF in SAM-treated HMECs in HG condition. n = 5, *p < 0.001 (Student’s t test). Data represented as the mean ± SD.

Figure 3

Promoter Hypomethylation Renders MicroRNA-200b Non-responsive to Injury in Diabetic Wounds (A) Schematic diagram showing experimental design of miR-200b promoter methylation analysis in human chronic wounds. (B) Representative figure shows the selection of CD31⁺ tissue elements (red) and their collection before and after the laser capture microdissection (LCM). Scale bar, 150 μm. (C and D) Methylation profile (C) and quantitation of methylated CpG islands in miR-200b promoter (D) in diabetic wounds compared to normoglycemic wounds. N = 5, *p < 0.001 (Student’s t test). (E) qRT-PCR analysis of miR-200b levels in LCM captured endothelial elements from diabetic and non-diabetic wounds. n = 3, *p < 0.05 (Student’s t test). (F) Schematic diagram showing the region of miR-200c promoter analyzed by bisulfite genomic sequencing. Methylation profile of the miR-200c gene promoter in LCM captured endothelial elements from diabetic and non-diabetic wounds with methylated CpGs shown in black. (G) Quantitation of methylated CpG islands in miR-200c promoter in diabetic wounds compared to normoglycemic wounds. n = 5, p = NS (Student’s t test). (H) qRT-PCR analysis of miR-200c expression in diabetic wounds compared to normoglycemic wounds. n = 4, p = NS (Student’s t test). (I) qRT-PCR analysis of miR-200b levels in HMECs after exposure to wound fluid (10%, 4 days). n = 3, *p < 0.05 (Student’s t test). (J and K) qRT-PCR analysis of VEGFR-2 (J) and vWF (K) in HMECs after exposure to wound fluid. n = 3, p = NS (Student’s t test). Data represented as the mean ± SD.

Figure 4

SAM Reversed Diabetes-Associated Impairment in Wound Vascularization (A) Schematic diagram showing intradermal delivery of S-adenosylmethionine (SAM) or vehicle to the dorsal skin of db/db mice. Two 8-mm diameter full thickness stent wounds were created on dorsal skin on day 0. (B) Representative figure shows the selection of CD31⁺ tissue elements (red), and their collection before and after the LCM from the wound edge tissues. Scale bar, 150 μm. (C) qRT-PCR analysis of miR-200b levels in endothelial elements of SAM-treated diabetic wounds compared to placebo. n = 3, *p < 0.05 (Student’s t test). (D) Perimed Laser speckle-assisted wound perfusion analysis of diabetic wounds administered with SAM or placebo. n = 5, *p < 0.05 (Student’s t test). (E and F) Immunohistochemical analysis of eNOS⁺/CD31⁺ co-expression (E) and its co-localization analysis (F) in SAM- or PBS-administered diabetic wounds. (G and H) Immunohistochemical analysis of vWF⁺/CD31⁺ co-expression (G) and its co-localization analysis (H) in SAM- or PBS-administered diabetic wounds. n = 5, *p < 0.05 (Student’s t test). Data represented as the mean ± SD.

Figure 5

Quantitative Proteomics Identify Novel Targets of MicroRNA-200b during Hyperglycemia (A) Schematic diagram showing SILAC quantitative proteomics workflow. (B) Total number of identified (up) and differentially expressed (down) unique proteins by SILAC analysis. (C) Respective mass spectra of peptides FEDEELQQILDDIQTK (m/z = 982.4762²⁺) and FEDEELQQILDDIQTK (13C(6)15N(2)) (m/z = 986.4826²⁺); the ratio between heavy isotopic-labeled peptide and light isotopic-labeled peptide represents the differential expression of the peptide (protein) in two different conditions. (D and E) Ingenuity pathway analysis (IPA) showing the inhibition of miR-200b during HG-exposed HMEC target proteins like core histones and heat shock protein (D) and in mitochondrial dysfunction and fatty acid metabolism pathways (E). (F) Oxygen consumption rate measurement in HMECs under NG/HG and co-treatment with control (CI) or miR-200b inhibitor (MI). n = 5, *p < 0.05, F = 9.30 (one-way ANOVA). (G) Normalized ATP production in HMECs under NG/HG and co-treatment with control (CI) or miR-200b inhibitor (MI). n = 3, *p < 0.001, F = 260.90 (one-way ANOVA). Data represented as the mean ± SD.