Dual Anti-Inflammatory and Anti-Angiogenic Action of miR-15a in Diabetic Retinopathy

Abstract

Activation of pro-inflammatory and pro-angiogenic pathways in the retina and the bone marrow contributes to pathogenesis of diabetic retinopathy. We identified miR-15a as key regulator of both pro-inflammatory and pro-angiogenic pathways through direct binding and inhibition of the central enzyme in the sphingolipid metabolism, ASM, and the pro-angiogenic growth factor, VEGF-A. miR-15a was downregulated in diabetic retina and bone marrow cells. Over-expression of miR-15a downregulated, and inhibition of miR-15a upregulated ASM and VEGF-A expression in retinal cells. In addition to retinal effects, migration and retinal vascular repair function was impaired in miR-15a inhibitor-treated circulating angiogenic cells (CAC). Diabetic mice overexpressing miR-15a under Tie-2 promoter had normalized retinal permeability compared to wild type littermates. Importantly, miR-15a overexpression led to modulation toward nondiabetic levels, rather than complete inhibition of ASM and VEGF-A providing therapeutic effect without detrimental consequences of ASM and VEGF-A deficiencies.

Keywords: Diabetic retinopathy; Dyslipidemias; Sphingolipids; Vascular system injuries; microRNA.

Fig. 1

Identification and verification of miRNAs differentially expressed in the HRECs isolated from control and diabetic donors (A) Human RT² miRNA PCR arrays were used to profile the differential expression of total miRNAs in HRECs isolated from control (n = 6) and diabetic donors (n = 6). MiRNAs upregulated or downregulated by > 2.5-fold relative to control are shown. MiRNAs marked with red or green were top 15 miRNAs upregulated or downregulated by diabetes, respectively. (B) Relative expressions of miR-15a and selected miRNAs were determined by qPCR to validate the array result. Data are mean ± SEM (n = 6). *** P < 0.001; ** P < 0.01; * P < 0.05; not significant at P > 0.05.

Fig. 2

MiR-15a negatively regulates the expression of ASM by direct targeting 3′UTR of ASM mRNA (A) Expression profiles of miR-15a and ASM were determined by real-time PCR in HRECs from control donors (n = 6) and HRPE cells (n = 3). (B) ASM expression was examined in HRECs from diabetic donors (n = 6) compared with control donors (n = 6). (C) Expression profiles of miR-15a and ASM were detected by real-time PCR in HRPE treated with 25 mmol/l glucose for 24 h compared with 5 mmol/l glucose (n = 3). (D) HRPE cells were co-transfected with wild type human ASM 3′UTR or mutant ASM 3′UTR (200 ng) luciferase reporters and miR-15a mimic in increasing concentrations (30, 50 and 100 nM). Mutant human ASM 3′UTR and control mimic served as negative controls. Luciferase activity was measured 48 h post-transfection. (E) Real-time PCR analysis of ASM mRNA level after miR-15a mimic or inhibitor delivery in HRECs. (F) Real-time PCR analysis of ASM mRNA level after miR-15a mimic or inhibitor delivery in HRPE cells under normal and high glucose conditions for 24 h (n = 3). (G) Western blot analysis of ASM protein expression after miR-15a mimic or inhibitor delivery in HRPE cells under normal and high glucose conditions for 48 h (n = 3). α-tubulin serves as a loading control. Representative blots are from three independent experiments. Quantification of band intensity is relative to control. Data are mean ± SEM (n = 3). *** P < 0.001; ** P < 0.01; * P < 0.05; not significant at P > 0.05.

Fig. 3

MiR-15a regulates ceramide production in retina and retinal cells The immunoreactivity of ceramide was examined in HRPE cells under normal and high glucose conditions for 24 h (n = 3) after overexpression or inhibition of miR-15a by (A) miR mimics and (B) inhibitors respectively. Representative images show the immunoreactivity of ceramide. Signal detection and image acquisition were performed by fluorescence microscopy using Photometrics CoolSNAP HQ2 camera. The fluorescence intensity (red) of ceramide in the HRPE cells was normalized to DAPI (blue) nuclei staining and quantified in triplicates using the MetaMorph imaging software. (C) Orbitrap high resolution/accurate mass spectra were collected in positive ionization mode from monophasic lipid extracts of WT, ASM −/−, and Tie2-miR-15a TG mouse retina (n = 5 per group). Data are mean ± SEM. *** P < 0.001; ** P < 0.01; * P < 0.05; not significant at P > 0.05.

Fig. 4

MiR-15a negatively regulates the expression of VEGF-A in HRPE and HRECs Expression levels of VEGF-A were determined by real-time PCR in (A)HRECs from diabetic donors (n = 6) compared with control donors (n = 6) and (B) HRPE cells treated with 25 mmol/l glucose for 24 h compared with 5 mmol/l glucose (n = 3). RT-PCR analysis of VEGF-A mRNA levels after miR-15a mimic or inhibitor delivery in (C) HRECs and (D) HRPE cells under normal and high glucose conditions for 24 h (n = 3). (E) ELISA analysis of VEGF-A protein expression after miR-15a mimic or inhibitor delivery in HRPE cells under normal and high glucose conditions for 48 h (n = 3). Data are mean ± SEM (n = 3). *** P < 0.001; ** P < 0.01; * P < 0.05; not significant at P > 0.05.

Fig. 5

Upregulation of miR-15a in bone marrow improves CAC release, migration and homing to retinal vasculature (A) The expression levels of miR-15a were determined in mouse bone marrow (n = 6 per group), blood (n = 6 per group) and in human CD34⁺ cells isolated from diabetic patients (n = 8 per group) with DR. (B, C) CACs (green) isolated from control or STZ-induced diabetic gfp⁺ mice (duration of diabetes 8 weeks, n = 10 per group) were treated with control mimic, miR-15a mimic, control inhibitor or miR-15a inhibitor. Wild-type mice undergoing I/R model received intravitreal injections of CACs treated with mimic or inhibitor for miR-15a and the negative controls (scrambled) (n = 6 for each treatment). The retinal vasculature was stained with anti-collagen IV antibody (red). Confocal images of retina isolated from I/R injured mice treated with control mimic, miR-15a mimic, control inhibitor or miR-15a inhibitor and the quantification of co-localization of gfp⁺ cells associated with retinal vasculature (yellow) were shown in B. CACs migration towards either 100 nM SDF-1, PBS (negative control), or 10% FBS (positive control) were shown in C. RFU, relative fluorescence units. Data are mean ± SEM (n = 6). *** P < 0.001; ** P < 0.01; * P < 0.05; not significant at P > 0.05. (D, E) The in vitro tube formation assay was performed in HRECs after overexpression or inhibition of miR-15a by miR mimics and inhibitors respectively using Matrigel Matrix 96-well plate. (D) Photomicrographs of representative assays for control mimics, 15a mimics, control inhibitor and 15a inhibitor. The images of tubes were taken in ×10 magnifications and at least 5 different fields were randomly selected to collect images from each well. (E) Quantitative data for tube formation expressed as number of closed network of vessel-like tubes as percentage relative to control. Data is mean ± SEM (n = 3).

Fig. 6

MiR-15a overexpression in Tie-2 miR-15a TG mice prevents diabetes-induced increase in retinal vascular permeability. (A) Expression profiling of miR-15a, ASM and VEGF-A in retinas from control and STZ-induced diabetic rats (duration of diabetes for 8 weeks, n = 10 per group). (B) In situ hybridization was used to determine the location of miR-15a in the retinas of control and STZ-induced diabetic mice. n = 4 per group. (C) Expression profiling of miR-15a, ASM, VEGF, IL6, IL1β and TNFα in retinas isolated from littermate control and Tie2-miR-15a TG mice. n = 8 per group. (D) Permeability of retinas isolated from littermate control, STZ-induced diabetic littermate control and Tie2-miR-15a TG mice (duration of diabetes H 4 weeks, n = 6 per group) was examined. Leakage of FITC-albumin from blood vessels was measured. Data are mean ± SEM. *** P < 0.001; ** P < 0.01; * P < 0.05; not significant at P > 0.05.