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. 2017 Sep 12;25(12):2689–2704. doi: 10.1016/j.ymthe.2017.09.009

Figure 1.

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.