Retinal angiogenesis is mediated by an interaction between the angiotensin type 2 receptor, VEGF, and angiopoietin

Abstract

There is evidence that angiotensin II, vascular endothelial growth factor (VEGF), angiopoietins, and their cognate receptors participate in retinal angiogenesis. We investigated whether angiotensin type 2-receptor blockade (AT2-RB) reduces retinal angiogenesis and alters the expression of VEGF/VEGF-R2 and angiopoietin-Tie2. Retinopathy of prematurity (ROP) was induced in Sprague Dawley (SD) rats by exposure to 80% oxygen from postnatal (P) days 0 to 11, followed by 7 days in room air. ROP shams were in room air from P0-18. A group of ROP rats received the AT2-RB, PD123319, by mini-osmotic pump (5 mg/kg/day) from P11-18 (angiogenesis period). Evaluation of the retinal status of the AT2 receptor indicated that this receptor, as assessed by real-time PCR, immunohistochemistry, and in vitro autoradiography, was present in the retina, was more abundant than the AT1 receptor in the neonatal retina, and was increased in the ROP model. AT2-RB reduced retinal angiogenesis. VEGF and VEGF-R2 mRNA were increased in ROP and localized to blood vessels, ganglion cells, and the inner nuclear layer, and were decreased by PD123319. Angiopoietin2 and Tie2, but not angiopoietin1 mRNA were increased with ROP, and angiopoietin2 was reduced with PD123319. This study has identified a potential retinoprotective role for AT2-RB possibly mediated via interactions with VEGF- and angiopoietin-dependent pathways.

Figure 1.

Autoradiographic analysis of AT1 and AT2 receptor binding sites in the retina of neonatal and adult Sprague Dawley rats. Values are mean ± SEM. N = 6 rats per group. dpm/mm², represents the disintegration per minute of I¹²⁵ per millimeter² of tissue. *, P < 0.05 compared to AT1 receptor binding at a particular time point; ^†, P < 0.05 compared to AT1 receptor binding at postnatal day 1; ^#, P < 0.05 compared to AT2 receptor binding at postnatal days 1, 14, and 21.

Figure 2.

A–C: Three-μm paraffin sections of inner retina from Sprague Dawley rats following retinopathy of prematurity (ROP) and AT2 receptor blockade. Stain, hematoxylin and eosin. ILM, inner limiting membrane; GCL, ganglion cell layer; IPL, inner plexiform layer. Magnification, ×400. Bar, 30 μm. A: ROP sham. B: ROP. C: ROP + PD123319. Arrows denote blood vessels in the inner retina. D: Quantitation of blood vessel profiles (BVPs) per high-powered field of inner retina (BVPs/stereological test grid). Values are mean ± SEM. N = 6 to 8 rats per group. *, P < 0.001 compared to sham; ^#, P < 0.01 compared to ROP.

Figure 3.

Real-time PCR showing gene expression for VEGF, VEGF-R2, Ang1, Ang2, and Tie2 in retina from Sprague Dawley rats following retinopathy of prematurity (ROP) and AT2 receptor blockade. Values are mean ± SEM. N = 6 to 8 rats per group. *, P < 0.01 compared to sham and ROP + PD123319; ^#, P < 0.05 compared to sham and ROP+PD123319; ^†, P < 0.05 compared to sham.

Figure 4.

Dark field sections showing VEGF (A–C) and VEGF-R2 (a–c) gene expression in retina from 18-day-old Sprague Dawley rats following retinopathy of prematurity (ROP) and AT2 receptor blockade. Stain, hematoxylin and eosin. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium. Magnification, ×180. Bar, 55 μm. Weak hybridization signal is observed in the GCL, blood vessels, IPL, and INL of sham animals (A and a). Hybridization signal for both VEGF and VEGFR-2 is increased in the GCL, blood vessels, IPL, and INL with ROP (B and b) and reduced in ROP rats with the AT2-RB, PD123319 (C and c). In all sections, specific hybridization signal is not observed in the ONL. Arrow denotes hybridization signal on blood vessels. Double arrows denote blood vessels with minimal or no hybridization signal.

Figure 5.

Optical density of VEGF (A) and VEGF-R2 (B) mRNA in Sprague Dawley rats following retinopathy of prematurity (ROP) and AT2 receptor blockade. Values are mean ± SEM. N = 6 to 8 rats per group. *, P < 0.05 compared to all groups, ^#, P < 0.05 compared to sham.

Figure 6.

Real-time PCR for renin and AT1 and AT2 receptors in retina from 18-day-old Sprague Dawley rats following retinopathy of prematurity (ROP) and AT2 receptor blockade. Values are mean ± SEM. N = 6 to 8 rats per group. *, P < 0.05 compared to sham.

Figure 7.

Immunolabeling for AT1 and AT2 receptors in the retina of Sprague Dawley rats following retinopathy of prematurity and treated with AT2 receptor blockade. Counterstain, hematoxylin. ILM, inner limiting membrane; IPL, inner plexiform layer; INL, inner nuclear layer. Magnification, ×320. Bar, 25 μm. A: AT1 receptor. B: AT2 receptor. C: Negative control. Arrows denote labeling on blood vessels. Asterisks denotes labeling in the cytoplasm of cells in the INL.