An Adam15 amplification loop promotes vascular endothelial growth factor-induced ocular neovascularization

Abstract

Proteins with a disintegrin and a metalloproteinase domain (ADAMs) are a family of membrane-bound proteinases that bind integrins through their disintegrin domain. In this study, we have found modest expression of ADAM15 in pericytes in normal retina and strong up-regulation of ADAM15 in retinal vascular endothelial cells in ischemic retina. Increased expression of vascular endothelial growth factor (VEGF) in the retina in the absence of ischemia also increased ADAM15 levels, and knockdown of Vegf mRNA in ischemic retina reduced Adam15 mRNA. Mice deficient in ADAM15 showed a significant reduction in ischemia-induced retinal neovascularization, choroidal neovascularization at rupture sites in Bruch's membrane, and VEGF-induced subretinal neovascularization. ADAM15-deficient mice also showed reduced levels of VEGF(164), VEGF receptor 1, and VEGF receptor 2 in ischemic retina. These data suggest that ADAM15 and VEGF participate in an amplification loop; VEGF increases expression of ADAM15, which in turn increases expression of VEGF and its receptors. Perturbation of the loop by elimination of ADAM15 suppresses ocular neovascularization in 3 different model systems, and thus ADAM15 provides a new therapeutic target for diseases complicated by neovascularization.

Figure 1.

Immunofluorescent staining for Adam15, PECAM-1, and PDGFR-β in normal and ischemic retina. A) A nonischemic retina from a P17 mouse reared in room air shows small areas of staining for Adam15 visualized with a Cy3-labeled secondary antibody (red) throughout the inner retina (arrows). B) Visualization of PECAM-1 staining in the same section shown in A with an FITC-labeled secondary antibody shows superficial, intermediated, and deep capillaries. C) Merge of A and B shows close association of Adam15 and PECAM-1 staining but not exact colocalization. D–F) An ocular section from a P17 mouse with oxygen-induced ischemic retinopathy shows areas of increased staining throughout the inner retina that colocalized with PECAM-1-stained vascular proliferation within and on the surface of the retina. G–I) Ocular sections from P21 transgenic mice expressing VEGF in photoreceptors (rho/VEGF mice) show several small areas of Adam15 staining that colocalize with PECAM-1. Adam15 was localized in retinal vessels, choroidal vessels (arrows), and subretinal neovascularization (arrowhead). J–L) Staining of a section from a P17 mouse with ischemic retinopathy without primary anti-Adam15 antibody (J) shows elimination of the staining seen in D, whereas PECAM-1 staining of the same section shows extensive vascular proliferation (K). Merge of Adam15 and PDGFR-β staining of a P17 nonischemic retina (L) shows colocalization (yellow, arrows), indicating that the ADAM15 expression is in pericytes.

Figure 2.

Adam15 mRNA is increased in the retina by ischemia or VEGF. At P15, total RNA was isolated from the retinas of mice reared in room air (P15 control, n=7), mice with ischemic retinopathy (P15 ischemic, n=8), rho/VEGF mice (n=5), and P15 littermate controls of the rho/VEGF mice (n=7). Total RNA was also isolated from double-transgenic mice with doxycycline (Dox) -inducible expression of VEGF in the retina (Tet/opsin/VEGF) that were given 2 mg/ml of Dox for 4 days in their drinking water (Dox+, n=6) or unsupplemented water (Dox−, n=6). Real-time RT-PCR was performed to measure the level of Adam15 and cyclophilin A mRNA. Cyclophilin A mRNA is generally modulated very little and served as an endogenous reference for normalization. Bars represent mean ± se of Adam15 transcripts per 10⁵ copies of cyclophilin A transcripts. The level of Adam15 mRNA was significantly higher in ischemic retina than nonischemic retina (A). The level of Adam15 mRNA was not significantly different in the retinas of rho/VEGF transgenics compared with littermate controls (B); however, compared with untreated Tet/opsin/VEGF double transgenics, those treated with Dox had significantly higher levels of Adam15 mRNA (C).

Figure 3.

Ischemia-induced retinal neovascularization is reduced in mice deficient in Adam15. Adam15^−/−, Adam15^+/−, or Adam15^+/+ mice were placed in 75% oxygen at P7 and returned to room air at P12. At P17, mice were given an intravitreous injection of anti-PECAM-1 antibody. After 12 h, retinas were dissected, washed, and incubated with secondary antibody (goat anti-rat IgG conjugated with FITC) at room temperature for 45 min, and retinal flat mounts were examined by fluorescence microscopy. Retinas from Adam15^−/− mice (A) appeared to have less neovascularization than retinas from Adam15^+/− (B) or Adam15^+/+ mice (C). Measurement of the area of retinal neovascularization by image analysis confirmed that there was a significant reduction in Adam15^−/− retinas (n=12) compared with Adam15^+/− (n=12) or Adam15^+/+ (n=12) retinas. Bars represent means ± se; statistical comparisons were made by ANOVA with Dunnett’s correction for multiple comparisons (D).

Figure 4.

Choroidal neovascularization at Bruch’s membrane rupture sites is reduced in mice deficient in Adam15. Adult Adam15^−/−, Adam15^+/−, or Adam15^+/+ mice had rupture of Bruch’s membrane in 3 locations in each eye. After 2 wk, the mice were perfused with fluorescein-labeled dextran, and choroidal flat mounts were examined by fluorescence microscopy. The area of choroidal neovascularization appeared smaller at rupture sites in Adam15^−/− mice (A) compared with those in Adam15^+/− (B) or Adam15^+/+ mice (C). Image analysis confirmed that the area of choroidal neovascularization was significantly smaller (n=54 for each; ANOVA with Dunnett’s correction) in Adam15^−/− mice compared with Adam15^+/− or Adam15^+/+ mice (D).

Figure 5.

Mice deficient in Adam15 have reduced VEGF-induced subretinal neovascularization. Adam15^−/− mice that also carried the rho/VEGF transgene appeared to have less subretinal neovascularization (A) than did rho/VEGF mice that were not deficient in Adam15 (B). Image analysis confirmed that rho/VEGF-Adam15^−/− mice (n=14) had fewer subretinal neovascular sprouts per eye (C) and smaller total area of subretinal neovascularization per eye (D) than did rho/VEGF mice that also expressed Adam15 (n=14).

Figure 6.

Mice deficient in Adam15 show reduced levels of mRNA and protein for VEGF₁₆₄, VEGFR1, and VEGFR2. Adam15^−/− (n=7) and Adam15^+/+ (n=7) mice were placed in 75% oxygen at P7 and returned to room air at P12. At P17, total retinal RNA was isolated from one eye, and retinal homogenates were prepared from the other eye. Real-time RT-PCR was done with primers specific for Vegf₁₆₄, Vegfr1, Vegfr2, and cyclophilin A, and retinal homogenates were used in ELISAs for VEGF₁₆₄, VEGFR1, and VEGFR2. Bars represent means ± se. There were significant reductions in the mRNA ratios Vegf₁₆₄:cyclophilin A (A) and Vegfr2:cyclophilin A (E), but not Vegfr1:cyclophilin A (C), in ischemic retinas from Adam15^−/− mice compared with Adam15^+/+ mice. There were significant reductions in VEGF₁₆₄ (B), VEGFR1 (D), and VEGFR2 (F) proteins.

Figure 7.

Reduction of Adam15 mRNA in ischemic retina by knockdown of Vegf₁₆₄ mRNA. At P12, C57BL/6 mice (n=8) with ischemic retinopathy were given an intravitreous injection of Vegf₁₆₄ siRNA in one eye and Gfp siRNA in the other eye and returned to room air. At P15, total retinal RNA was isolated, and real-time RT-PCR was done with primers specific for Vegf₁₆₄, Adam15, and cyclophilin A. Bars represent means ± se of Vegf₁₆₄ and Adam15 transcripts per 10⁵ copies of cyclophilin A transcripts. The levels of Vegf₁₆₄ (A) and Adam15 (B) mRNA were significantly decreased in the retinas with intravitreous injection of Vegf siRNA compared with those with injection of Gfp siRNA.