IL-17A Enhances Retinal Neovascularization

Abstract

Retinal neovascularization occurs in proliferative diabetic retinopathy, neovascular glaucoma, and age-related macular degeneration. This type of retinal pathology normally occurs in the later stages of these ocular diseases and is a prevalent cause of vision loss. Previously, we determined that Interleukin (IL)-17A plays a pivotal role in the onset and progression of non-proliferative diabetic retinopathy in diabetic mice. Unfortunately, none of our diabetic murine models progress to proliferative diabetic retinopathy. Hence, the role of IL-17A in vascular angiogenesis, neovascularization, and the onset of proliferative diabetic retinopathy was unclear. In the current study, we determined that diabetes-mediated IL-17A enhances vascular endothelial growth factor (VEGF) production in the retina, Muller glia, and retinal endothelial cells. Further, we determined that IL-17A can initiate retinal endothelial cell proliferation and can enhance VEGF-dependent vascular angiogenesis. Finally, by utilizing the oxygen induced retinopathy model, we determined that IL-17A enhances retinal neovascularization. Collectively, the results of this study provide evidence that IL-17A plays a pivotal role in vascular proliferation in the retina. Hence, IL-17A could be a potentially novel therapeutic target for retinal neovascularization, which can cause blindness in multiple ocular diseases.

Keywords: IL-17A; neovascularization; retina.

Figure 1

Inhibition of Diabetes-Mediated IL-17A Decreases VEGF Production. (A) Schematic of the 6-week analysis of STZ-diabetic C57BL/6 mice (n = 9/group) receiving an anti-IL-17A intravitreal injection. (B) Quantification of VEGF in retinas of untreated (grey) and anti-IL-17A treated (black) non-diabetic (ND) and diabetic (DB) mice 6 weeks post-diabetes. (C) Schematic of the 2- and 6-month analyses of Lepr^db mice (n = 9/group) receiving weekly intraperitoneal injections of anti-IL-17A. (D) Levels of VEGF in the retinas of untreated (grey) and anti-IL-17A treated (black) non-diabetic (ND) and diabetic (DB) Lepr^db mice 2- and 6-months post-diabetes. (E) Schematic of the 2- and 8-month analyses of STZ-diabetic C57BL/6 and IL17A^-/- mice (n = 9/group). (F) Quantification of VEGF in the retinas of C57BL/6 (grey) or IL17A^-/- (black) non-diabetic (ND) and diabetic (DB) mice 2- and 8-months post-diabetes. Error bars represent the SEM; * p < 0.01. The p-value was first equated by two-way ANOVA analysis and then unpaired Student’s t-test with Tukey’s post-hoc analysis.

Figure 2

VEGF Production by Retina Cells Stimulated with IL-17A. ELISA quantification of VEGF in spent media of unstimulated (grey) or IL-17A (black) stimulated: (A) murine Muller glia (mMG), (B) human Muller glia (hMG), (C) murine retinal endothelial cells (mREC), and (D) human retinal endothelial cells (hREC). Error bars represent the SEM. The p-values were equated by two-way ANOVA and then unpaired Student’s t-test with Tukey’s post-hoc analysis.

Figure 3

IL-17A Initiates Retinal Endothelial Cell Proliferation. (A) Representative images of WimTube analytical overlay of unstimulated and IL-17A-stimulated human retinal endothelial cells. Quantification of the number of angiogenic tubes (B) and branches (C) in unstimulated (grey) or IL-17A-stimulated (black) human retinal endothelial cells. Error bars represent the SEM. The p-values were equated using two-way ANOVA and then unpaired Student’s t-test with Tukey’s post-hoc analysis.

Figure 4

IL-17A Enhances Retinal Endothelial Cell Angiogenesis. (A) Representative phase contrast images of unstimulated or stimulated human retinal endothelial cells. Quantification of the percentage of proliferation area (B), or BrdU quantification of proliferation (C) in unstimulated (white), IL-17A-stimulated (light grey), VEGF-stimulated (dark grey), or IL-17A+VEGF-stimulated (black) human retinal endothelial cells. Error bars represent the SEM. All p-values were first equated by two-way ANOVA and then unpaired Student’s t-test with Tukey’s post-hoc analysis.

Figure 5

Retinal Neovascularization is Decreased in IL17A^-/- Mice with Oxygen-Induced Retinopathy. (A) Schematic of the murine model of oxygen induced retinopathy and retinal neovascularization. (B) Quantification of neovascularization in C57BL/6 and IL17A^-/- mice (n = 5/group). (C) Representative images of neovascularization in retina whole mounts. Red arrows highlight neovascularization. ND = not detected. Error bars represent the SEM. The p-value was first equated by two-way ANOVA analysis and then unpaired Student’s t-test with Tukey’s post-hoc analysis.