miR-15a/16 inhibits TGF-beta3/VEGF signaling and increases retinal endothelial cell barrier proteins

Abstract

Hyperglycemia is a significant risk factor for diabetic retinopathy and induces multiple biochemical changes, including inflammation and endothelial dysfunction in the retina. Alterations in microRNA expression have been implicated in the pathological responses of diabetic retinopathy and the manipulation of microRNA may provide powerful strategy for therapeutics. Among the predicted targets of miR-15a and -16 are TGF-beta3, SMAD2/3, and VEGF, all of which are known to play a role in vascular endothelial functions. The purpose of this study was to investigate the hypothesis that miR-15a/16 inhibits TGF-beta3/VEGF signaling to maintain retinal endothelial cell barrier protein levels. Human primary retinal endothelial cells (REC) were maintained in normal (5mM) glucose or transferred to high glucose medium (25mM) for 3days. REC were transfected with miRNA mimics (hsa-miR-15a-5p and -16-5p). Retinal lysates from miR-15a-transgenic mice were also analyzed. We demonstrated that overexpression of miR-15a/16 resulted in decreased TGF-beta3 signaling and VEGF levels in cultured REC grown in high glucose conditions. In addition, the levels of tight junction proteins, zonula occludens-1 (ZO-1) and occludin, were elevated in REC following overexpression of miR-15a and -16. Overexpression of miR-15a and -16 played a role in reducing cellular permeability through inhibition of VEGF signaling in REC cultured under high glucose conditions. Using miR-15a-transgenic mice, we demonstrated the regulatory role of miR-15a on TGF-beta3 signaling and tight junction proteins in vivo. Our outcomes suggest that miR-15a/16 maintain the retinal endothelial cell barrier by reducing TGFbeta3/VEGF signaling and increasing levels of key tight junction proteins.

Keywords: Retinal endothelial permeability; SMAD2/3; TGF-beta3; VEGF; miR-15a/16.

Fig. 1

Effects of miR-15a/16 on TGF-beta3 and SMAD2/3 levels in REC. REC were cultured in normal glucose (5 mM, NG) or high glucose (25 mM, HG). Transfection with miR-15a, -16, and Neg. mimics were performed under HG conditions. HG increased levels of TGF-beta3 (A) and SMAD2/3 phosphorylation (B) as compared to NG. REC transfected with miR-15a and/or -16 mimics (labeled as miR-15a, miR-16, and miR-15a/16) had reduced levels of TGF-beta3 (A) and SMAD2/3 phosphorylation (B), compared to that of control groups (HG and Neg.). Neg. control indicates REC transfected with a negative miR-mimic. ^#p < 0.05 versus NG, ^*p < 0.05 versus HG, ^$p < 0.05 versus Neg. control, N = 4; Data are mean ± S.E.M.

Fig. 2

Changes of VEGF levels in high glucose conditions. Western blot results for VEGF on REC. Transfection with miR-15a, -16, and Neg. mimics were performed under HG conditions. VEGF levels, which were elevated in control HG condition, but were reduced when REC was transfected with miR-15a and/or -16 mimics (labeled as miR-15a, miR-16, and miR-15a/16) as compared to that of control groups (HG and Neg.). ^#p < 0.05 versus NG, ^*p < 0.05 versus HG, ^$p < 0.05 versus Neg. control, N = 3; Data are mean ± S.E.M.

Fig. 3

Elevated levels of ZO-1 and occludin in REC. Western blot results for tight junction proteins on REC. Transfection with miR-15a, -16, and Neg. mimics were performed under HG conditions. HG decreased the levels of ZO-1 (A) and occludin (B) as compared to NG. REC transfected with miR-15a and/or -16 mimics (labeled as miR-15a, miR-16, and miR-15a/16) had increased levels of ZO-1 (A) and occludin (B), compared to that of control groups (HG and Neg.). ^#p < 0.05 versus NG, ^*p < 0.05 versus HG, ^$p < 0.05 versus Neg. control, N = 4; Data are mean ± S.E.M.

Fig. 4

Transendothelial permeability assay using REC. Treatments with miR-mimics and/or VEGF were done in HG conditions. The rate of diffusive flux (Po) was calculated from the absorbance measured at 30, 60, 90, 120, 150, 180, 210, and 240 min after adding RITC-dextran onto REC monolayer. REC transfected with miR-15a and -16 showed reduced levels of Po in high glucose conditions, compared to HG only and VEGF-treated groups. ^*p < 0.05 versus HG, ^$p < 0.05 versus Neg., ^#p < 0.05 versus NG, N = 3; Data are mean ± S.E.M.

Fig. 5

Changes of TGF-beta3 signaling, ZO-1, and occludin levels in the retina of miR-15a overexpressing TG mice. Retinal lysates were examined by Western blot. The retinas of miR-15a-TG mice showed decreased levels of TGF- beta3 (A) and SMAD2/3 phosphoryation (B), with elevated levels of ZO-1 (C) and occludin (D) compared to control wild type (WT) mice. ^*p < 0.05 versus control mice, N = 3; Data are mean ± S.E.M.