Exosomes from retinal astrocytes contain antiangiogenic components that inhibit laser-induced choroidal neovascularization

Abstract

Exosomes released from different types of host cells have different biological effects. We report that exosomes released from retinal astroglial cells (RACs) suppress retinal vessel leakage and inhibit choroidal neovascularization (CNV) in a laser-induced CNV model, whereas exosomes released from retinal pigmental epithelium do not. RAC exosomes inhibit the migration of macrophages and the tubule forming of mouse retinal microvascular endothelial cells. Further, we analyzed antiangiogenic components in RAC exosomes using an angiogenesis array kit and detected several endogenous inhibitors of angiogenesis exclusively present in RAC exosomes, such as endostatin. Moreover, blockade of matrix metalloproteinases in the cleavage of collagen XVIII to form endostatin using FN-439 reverses RAC exosome-mediated retinal vessel leakage. This study demonstrates that exosomes released from retinal tissue cells have different angiogenic effects, with exosomes from RACs containing antiangiogenic components that might protect the eye from angiogenesis and maintain its functional integrity. In addition, by identifying additional components and their functions of RAC exosomes, we might improve the antiangiogenic therapy for CNV in age-related macular degeneration and diabetic retinopathy.

Keywords: Age-related Macular Degeneration; Angiogenesis; Choroidal Neovascularization; Endothelial Cell; Exosomes; Inflammation; Macrophages; Retinal Astrocytes; Retinal Pigmental Epithelium; Vascular Endothelial Growth Factor (VEGF).

Figures

FIGURE 1.

Characterization of RAC-derived exosomes.A, representative electron micrograph of exosomes (magnification, ×72,000; Scale bar, 100 nm). Images were acquired with a Phillips CM10 electron microscope. B, dose-dependent analysis of exosomes positive for CD63 by ELISA. Purified RAC exosomes (EX) were examined in indicated dilutions from the original concentration of 1 μg/ml. The control is the exosome-free cell culture medium.

FIGURE 2.

Periocular injected exosomes reach the choroid-retina.A–D, time course of the appearance of exosomes in the eye as seen by fluorescent microscopy. Mice preinjected with FITC-dextran, which labels blood vessels green, were injected periocularly with PKH26-labeled RAC-derived exosomes (10 μg) and examined in vivo using fluorescence microscopy at 15 (A), 30 (B), 60 (C), and 120 (D) min after injection (magnification, ×10). The arrows indicate PKH26-labeled (red) exosomes. E and F, confocal microscope of two-dimensional (E) and three-dimensional (F) view of frozen sections of the retina and choroid prepared after sacrifice of mice 60 min after periocular injection of RAC-derived exosomes labeled with PKH26 (red) (magnification, ×40; the blue stain of nucleus is DAPI. E, GCL, layer of ganglion cells; INL, inner nuclear layer; ONL, outer nuclear layer; CHO, choroid.

FIGURE 3.

RAC-derived exosomes inhibit retinal vascular leakage and choroid new vessel formation in a laser-induced CNV model.A and B, periocular injection of PBS (control) or RAC-derived exosomes (2 μg) was performed every other day or every day (n = 6 mice/group) starting at the same day of laser injury (d1). On day 7, retinal vascular leakage was determined in vivo by fluorescence microscopy (A) and average area of CNV in all four laser spots of six mice measured in choroidal flat mounts (B). C, on day 14, average area of CNV was determined in the mice treated with PBS or daily with RAC-derived exosomes from day 1 to 7 or from day 7 to14. **, p < 0.01 compared with control group treated with PBS in one-way ANOVA. Error bars, S.E.

FIGURE 4.

Exosomes derived from RACs are the most effective in suppressing active CNV. Exosomes (2 μg) derived from RACs, RPE, fibroblasts, dendritic cells (DCs), or PBS (control) were injected periocularly daily into mice with laser-induced CNV, then, after 7 days, retinal vascular leakage was determined in vivo by fluorescence microscopy (A and B) and average area of CNV in all four laser spots of six mice measured in choroidal flat mounts (C). *, p < 0.05 compared with control group treated with PBS in one-way ANOVA. Error bars, S.E.

FIGURE 5.

RAC-derived exosomes reduced the number of F4/80+ macrophages in the area of the laser-induced choroidal injury and in an in vitro chemoattractant migration assay.A, mice injected daily with 2 μg of RAC-derived exosomes were examined on day 7 for infiltrated macrophages in the choroidal flat mount using Cy3-conjugated F4/80 antibody; the average of infiltrated F4/80-positive macrophages per laser spot was counted and calculated (n = 24 laser spots). B and C, peritoneal macrophages, 86.5% pure (B) at the top well of a chemotaxis chamber that migrated to the lower wells containing the macrophage chemoattractant CCL2 (10 nm), alone or together with 1 or 10 μg/ml of RAC-derived exosomes, were collected after 2 h incubation and counted by flow cytometry (C). *, p < 0.05; **, p < 0.01 compared with control group treated with PBS in one-way ANOVA. Error bars, S.E.

FIGURE 6.

RAC exosomes inhibited the migration and vascular tubule forming, but not proliferation of mRMVECs.A, migration of mRMVECs. A crossed lesion was made in the 6-well plates cultured with 95% confluence mRMVECs in the medium with increasing dose of RAC exosomes. After 24 h of migration, three randomly selected fields at the lesion border were acquired using a 10× phase objective on an inverted microscope, and the distance between the margin of the lesion and the most distant point on migrating cells was analyzed. B, proliferation of mRMVECs. mRMVECs were cultured with 50 μg/ml VEGF in the presence of increasing doses of RAC exosomes. After 24 h, 20 μm BrdU was added to each well and cultured for additional 12 h. The green BrdU-positive proliferated cells were counted at five randomly selected fields in each well under a 5× lens. C and D, tubule forming of mRMVECs. mRMVECs were cultured as in B but for 6 h. Representative examples of micrographs (C) and total branch length (D) are shown. Data are mean ± S.E. (error bars) of triplicate experiments. *, p < 0.05; **, p < 0.01 compared with control group treated without RAC exosomes in one-way ANOVA.

FIGURE 7.

Differences of angiogenic related protein expression between RAC exosomes and RPE exosomes. Exosome extracts of RPE and RACs were incubated with angiogenesis antibody array according to the manufacturer's instructions. A, the numbers are indicated as differences in the expression of proteins between RAC and RPE exosomes. These are 1, endostatin; 2, ET-1; 3, KC; 4, MIP-1α; 5, MMP-3; 6, MMP-9; 7, NOV; 8, PTX3; 9, PlGF-2; 10, proliferin; 11, PEDF; and 12, TIMP-1. B, graphs represent quantification of antibody array using ImageJ software (National Institutes of Health). C, the levels of endostatin and PEDF in exosomes of RAC and RPE were quantitated by ELISA.

FIGURE 8.

Endostatin in RAC exosomes is responsible for suppression of retinal vessel leakage.A, endostatin level in the exosomes of RACs treated with or without FN-439 and RPE was examined by Western blotting using anti-endostatin Ab. B–E, PBS or exosomes derived from RACs treated with or without FN-439 were periocularly injected daily into mice with laser-induced CNV. After 7 days, retinal vascular leakage was determined (B and C), representative spot of CNV was photographed (D), and the average area of CNV in all four laser spots of six mice was measured (E) in choroidal flat mounts. **, p < 0.01 compared with control group treated with PBS in one-way ANOVA. Error bars indicate S.E.

FIGURE 9.

Internalization of exosomes by macrophages and mRMVECs. PKH-26 (red)-labeled exosomes were incubated with either mRMVECs or macrophages on a slide chamber for 2 h at 37 °C. Thereafter, the cells were washed with PBS, fixed in ethanol, stained with DAPI, and observed under a fluorescent microscope (magnification, ×40).