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. 2008 Aug;22(8):2775–2783. doi: 10.1096/fj.07-099283

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

Bing Xie *,†,1, Jikui Shen †,1, Aling Dong †, Mara Swaim †, Sean F Hackett †, Lorenza Wyder ‡, Susanne Worpenberg ‡, Samuel Barbieri ‡, Peter A Campochiaro †,2
PMCID: PMC2493454  PMID: 18381816

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 VEGF164, 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.—Xie, B., Shen, J., Dong, A., Swaim, M., Hackett, S. F., Wyder, L., Worpenberg, S., Barbieri, S., Campochiaro, P. A. An Adam15 amplification loop promotes VEGF-induced ocular neovascularization.

Keywords: age-related macular degeneration, angiogenesis, chemokines, diabetic retinopathy, inflammation, stromal-derived factor-1, vascular endothelial growth factor

Disintegrins are protein components of snake venom that contain an RGD sequence that binds platelet integrin αIIbβ3 and interferes with platelet aggregation (1). Proteins with a disintegrin and a metalloproteinase domain (ADAMs) are a family of membrane-bound proteinases that bind integrins through their disintegrin domain. The family is large and diverse, and various members have been implicated in cell adhesion, cell fusion, proteolysis, signal transduction, and ectodomain shedding (2). ADAM15 was independently isolated from a human mammary epithelial cell line and from human smooth muscle and vascular endothelial cells using degenerate PCR primer approaches (3, 4). Human ADAM15 has an RGD sequence in its integrin-binding domain and binds αvβ3 and α5β1 (5, 6). One report suggests that the mouse ortholog of ADAM15 does not contain an RGD sequence and does not bind αvβ3 or α5β1 (7). ADAM15 is up-regulated in atherosclerotic plaques and in new vessels within rheumatoid synovium, suggesting that it might play a role in neovascular disease processes (4, 8). Despite expression of ADAM15 in developing vasculature, brain, and bone, Adam15−/− mice are viable and appear normal, indicating that ADAM15 is not required for vascular development (9). However, Adam15−/− mice show reduced ischemia-induced retinal neovascularization and tumor growth after implantation of melanoma cells, suggesting a role in pathological neovascularization (9). In this study, we explored the role of ADAM15 in 3 different types of ocular neovascularization.

MATERIALS AND METHODS

Mice

All mice were treated in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research and the guidelines of the Animal Care and Use Committee at the Johns Hopkins University Medical School. Mice used in the study included pathogen-free C57BL/6 mice (Charles River, Wilmington, MA, USA), Adam15−/− mice (Deltagen, San Mateo, CA, USA), rhodopsin promoter/vascular endothelial growth factor (rho/VEGF) transgenic mice (10), and mice with doxycycline-inducible expression of VEGF in the retina (11), all in a C57BL/6 background. The Adam15−/− mice were generated at Deltagen (San Mateo, CA, USA) by homologous recombination. An Adam15 allele lacking the codons that code for amino acids A348–L351 of the mouse Adam15 gene was introduced into embryonic stem cells derived from the 129/OlaHsd mouse substrain. After Adam15−/− mice were obtained, they were backcrossed into a C57BL/6 background.

Immunoblots for ADAM15

Adult Adam15−/− and wild-type mice were euthanized and lungs were dissected and homogenized in radioimmunoprecipitation assay buffer (50 mM Tris/HCl, pH 7.2; 120 mM NaCl; 1mM EDTA; 6 mM EGTA; 1% Nonidet P-40; and 20 mM NaF) supplemented with 1 mM Na-Vanadate and Complete Mini Protease inhibitor cocktail (1 tablet/50 ml buffer; Roche, Basel, Switzerland). Protein concentrations were determined with the Pierce BCA kit (Pierce, Rockford, IL, USA), and 500 μg of protein were immunoprecipitated by incubation with 2 μg of a polyconal goat antibody directed against the ectodomain of mouse ADAM15 (AF945; R&D Systems, Minneapolis, MN, USA), electrophoresed in 8% sodium dodecyl sulfate (SDS) gel, and transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). The membrane was blocked with PBS + 0.05% Tween20 and 3% BSA and incubated with a monoclonal antibody directed against mouse ADAM15 (MAB945; 1:2000; R&D Systems). After washing, the membrane was incubated with goat anti-rat-horseradish peroxidase (Amersham Biosciences; Amersham, UK) and developed on film with ECL™ (Amersham Biosciences).

Immunohistochemical staining for ADAM15

C57BL/6 mice with or without oxygen-induced ischemic retinopathy (12) and rho/VEGF mice (10) were euthanized at postnatal day 17 (P17), and eyes were rapidly removed and frozen in optimum cutting temperature embedding compound (Miles Diagnostics, Elkhart, IN, USA). Frozen sections (10 μm) were thawed, air-dried, and fixed in prechilled acetone. Sections were respectively incubated in 10% normal donkey serum followed by overnight incubation at 4°C in polyclonal goat anti-mouse Adam15 polycolonal antibody (R&D Systems) rat anti-mouse platelet endothelial cell adhesion molecule-1 (PECAM-1, Pharmingen, San Jose, CA, USA) or monoclonal rabbit anti-mouse platelet-derived growth factor receptor β (PDGFR-β, Abcam Inc., Cambridge, MA, USA). Sections were then respectively incubated in Cy3-conjugated donkey anti-goat immunoglobulin G (IgG; 1:800, Jackson Immuno Research Laboratories Inc., West Grove, PA, USA), fluorescein isothiocynate (FITC) -conjugated donkey anti-rat IgG (1:500, Biosource International, Camarillo, CA, USA), or FITC-conjugated donkey anti-rabbit IgG (1:500, Jackson ImmunoResearch Laboratories Inc.) for 45 min at room temperature. The sections were thoroughly washed with phosphate-buffered saline containing 0.25% Triton X-100 (PBST) between all incubations, and primary and secondary antibodies were diluted in PBS containing 1% donkey serum. Sections were examined under a Nikon microscope and captured as digital files with a Nikon Digital Still Camera DXM1200 (Nikon Instruments Inc., Melville, NY, USA).

Measurement of mRNA levels in the retina by real-time reverse transcriptase-polymerase chain reaction (RT-PCR)

Adam15−/− or Adam15+/+ mice with or without ischemic retinopathy and rho/VEGF transgenic mice were euthanized at P17. Six-week-old rtTA-rhodopsin promoter/TRE-VEGF (Tet/opsin/VEGF) double-transgenic mice with doxycycline-inducible expression of VEGF in the retina (11) were treated with 2 mg/ml of doxycycline in their drinking water or left untreated and, after 4 days, euthanized. Eyes were removed, retinas were dissected, and total retinal RNA was isolated using RNeasy kits (Quigen Inc., Chatsworth, CA, USA). RNA concentration was measured by spectrophotometry (Gen SpecIII; Hitachi, Tokyo, Japan), and after treatment with DNase I (Ambion Inc., Austin, TX, USA), 1 μg of RNA was incubated with reverse transcriptase (SuperScript II, Life Technologies, Gaithersburg, MD, USA) and 5 μM oligo-d(T) primer. Samples of cDNA were aliquoted and stored at −80°C.

Real-time polymerase chain reaction was performed using a Light Cycler rapid thermal cycler system (Roche Applied Bioscience) according to the manufacturer’s instructions. Primers specific for Adam15 (forward: 5′-AAA ACT GCT GCT ACC GAG GA-3′ and reverse: 5′-GGA TCC GAG AAA TGA CAG GA-3′), Vegf164 (forward: 5′-CAG GCT GCT GTA ACG ATG AA-3′ and reverse: 5′-AAT GCT TTC TCC GCT CAG AA-3′), Vegf receptor 1 (Vegfr1; forward: 5′-GAG CTA AAA ATC TTG ACC CAC ATT G-3′ and reverse: 5′-CAG TAT TCA ACA ATC ACC ATC AGA G-3′), Vegfr2 (forward: 5′-CAC CTG CCA GGC CTG CAA-3′ and reverse: 5′-GCT TGG TGC AGG CGC CTA-3′), and cyclophilin A (forward: 5′-CAG ACG CCA CTG TCG CTT T-3′ and reverse: 5′-TGT CTT TGG AAC TTT GTC TGC AA-3′) were used. Cyclophilin A was used as a control for normalization. Standard curves generated with purified cDNA were used to calculate copy number according to the Roche absolute quantification technique manual. Values are expressed as copies of mRNA of interest per 105 copies of cyclophilin A mRNA.

Silencing VEGF with small interfering RNAs (siRNAs) targeting Vegf mRNA

Four pairs of 21 base pair RNA duplexes with a TT overhang at the 3′ end that specifically target Vegf mRNA were purchased (VEGF Smartpool, Dharmacon, Chicago, IL, USA). For control, siRNA directed against green fluorescent protein (Gfp) siRNA was used. At P12, mice with ischemic retinopathy were given an intravitreous injection of Vegf siRNA in one eye and Gfp siRNA in the other eye. At P15, retinas were dissected; total RNA was isolated; and Vegf164, Adam15, and cyclophilin mRNA were measured by quantitative real-time RT-PCR as described above.

Measurement of VEGF164, VEGFR1, and VEGFR2 protein levels in the retinas of Adam15 −/− and Adam15+/+ mice

Adam15−/− and Adam15+/+ mice with ischemic retinopathy were euthanized at P15. The retinas were removed and placed in 300 μl lysis buffer (50 μl 1 M Tris-HCl, pH 7.4; 50 μl 10% SDS; 5 μl 100 nM phenylmethaneculfonyl; and 5 ml sterile, deionized water). Retinas were homogenized by pipetting, sonicated at 4°C for 5 s, and centrifuged at 13,000 g for 5 min at 4°C. The pellet was discarded and the supernatant was transferred to a fresh tube. The protein concentration of the supernatants was measured using a Bio-Rad Protein Assay Kit (Bio-Rad, Hercules, CA, USA). ELISAs were performed using the Quantikine VEGF164, VEGFR1, VEGFR2 assay kits (R&D Systems) using the manufacturer’s instructions. Serial dilutions of recombinant VEGF164, VEGFR1, VEGFR2 were assayed to generate standard curves with limits of detection of 3, 9.8, and 28 pg/ml, respectively.

Mouse model of choroidal neovascularization

Choroidal neovascularization was induced by laser photocoagulation-induced rupture of Bruch’s membrane as described previously (13). Briefly, 5- to 6-wk-old female Adam15+/+, Adam15−/−, and Adam15+/− mice were anesthetized with ketamine hydrochloride (100 mg/kg body weight), and pupils were dilated with 1% tropicamide. Burns (75 μm spot size, 0.1 s duration, 120 mW) were performed in the 9, 12, and 3 o’clock positions of the posterior pole of the retina with the slit lamp delivery system of an OcuLight GL diode laser (Iridex, Mountain View, CA, USA) using a handheld coverslip as a contact lens to view the retina. Production of a bubble at the time of laser, which indicates rupture of Bruch’s membrane, is an important factor in obtaining choroidal neovascularization; therefore, only burns in which a bubble was produced were included in the study. After 14 days, the mice were perfused with 1 ml of PBS containing 50 mg/ml of fluorescein-labeled dextran (2×106 average molecular weight; Sigma-Aldrich, St. Louis, MO, USA), and choroidal flat mounts were examined by fluorescence microscopy. Images were captured with a Nikon Digital Still Camera DXM1200. Image analysis software (Image-Pro Plus; Media Cybernetics, Silver Spring, MD, USA) was used to measure the total area of choroidal neovascularization at each rupture site, with the investigator masked with respect to treatment group.

Model of ischemia-induced retinal neovascularization

Litters containing Adam15−/−, Adam15+/−, and Adam15+/+ mice were exposed to 75% oxygen between P7 and P12, and then returned to room air. At P17, the retinal neovascularization on the surface of the retina was selectively stained by in vivo immunostaining for PECAM-1 as described previously (14). Briefly, the mice were given an intraocular injection of 1 μl of anti-PECAM-1 antibody under a dissecting microscope with a Harvard Pump Microinjection System (Harvard Apparatus, Holliston, MA, USA) and pulled glass micropipettes (15) and euthanized 12 h after injection. Eyes were fixed in formalin for 5 h. Retinas were dissected, washed, and incubated with secondary antibody (goat anti-rat IgG conjugated with FITC, 1:500) at room temperature for 45 min and then flat mounted. An observer masked with respect to treatment group measured the area of neovascularization per retina by image analysis.

Model of subretinal neovascularization in rho/VEGF transgenic mice

Rho/VEGF mice (transgenic mice in which the rhodopsin promoter drives expression of VEGF in photoreceptors) were crossed with Adam15−/− mice, and Adam15+/− offspring that carried a rho/VEGF transgene were crossed with Adam15−/− mice. At P21, Adam15−/− and Adam15+/− mice that carried a rho/VEGF transgene were anesthetized and perfused with fluorescein-labeled dextran. Retinal flat mounts were examined by fluorescence microscopy at ×200, which provides a narrow depth of field so that when focusing on neovascularization on the outer edge of the retina, the remainder of the retinal vessels are out of focus, allowing easy delineation of the neovascularization. The outer edge of the retina, which corresponds to the subretinal space in vivo, is easily identified, ensuring standardization of the focal plane from slide to slide. Image-Pro Plus software was used to measure the number of lesions per retina and the total area of neovascularization per retina.

RESULTS

Increased staining for ADAM15 in endothelial cells of retinal vessels and new vessels in ischemic retina

Eyes of normal P17 mice showed small areas of staining for ADAM15 throughout the inner retina (Fig. 1A, arrows). The staining showed close association with PECAM-1 staining but not exact colocalization (Fig. 1B, C). Eyes from P17 mice with oxygen-induced ischemic retinopathy showed many areas of increased staining throughout the inner retina that colocalized with PECAM-1 on the surface of the retina, indicating that it occurred in vascular endothelial cells, including those in new vessels (Fig. 1D–F). This staining was eliminated by omitting the primary antibody in the staining procedure (Fig. 1J), despite prominent areas of vascular proliferation on the same section indicated by PECAM-1 staining (Fig. 1K). The close association of ADAM15 and PECAM-1 staining in nonischemic retina caused us to hypothesize that it occurred in pericytes. To test this hypothesis, we utilized an antibody directed against PDGFR-β, a marker for pericytes (16, 17). In nonischemic retina, ADAM15 colocalized with PDGFR-β (Fig. 1L), indicating that under normal conditions ADAM15 is expressed in pericytes, and in the presence of ischemia it is strongly induced in endothelial cells.

Figure 1.

Figure 1.

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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.

Increased staining for ADAM15 in endothelial cells of retinal vessels, choroidal vessels, and new vessels beneath the retina in mice with overexpression of VEGF

Rho/VEGF mice develop subretinal neovascularization by P21 (10). Eyes from P21 rho/VEGF mice showed several small areas of ADAM15 staining that colocalized with PECAM-1 (Fig. 1G–I). ADAM15 was localized in retinal vessels, choroidal vessels (arrows), and subretinal neovascularization (arrowhead). This result indicates that VEGF is at least partially responsible for the up-regulation of ADAM15 because overexpression of VEGF induces expression of ADAM15 in endothelial cells in nonischemic retina and choroid.

Adam15 mRNA is increased in the retina by ischemia or VEGF

The level of Adam15 mRNA in the retinas of P15 mice with ischemic retinopathy was significantly higher than that in the retinas of P15 mice that had been maintained in room air (Fig. 2A). Compared with littermate controls, rho/VEGF transgenic mice did not show a statistically significant increase in Adam15 mRNA (Fig. 2B). However, Tet/opsin/VEGF double-transgenic mice with doxycycline-induced expression of VEGF in the retina, which is at least 5-fold higher than the level of VEGF in rho/VEGF mice (11), showed a large increase in Adam15 mRNA in the retina (Fig. 2C).

Figure 2.

Figure 2.

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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).

Mice deficient in ADAM15 show reduced ischemia-induced retinal neovascularization

Immunoblots of lung extracts from wild-type and Adam15−/− mice showed high levels of ADAM15 in lungs from wild-type mice that were absent in Adam15−/− mice (data not shown). The Adam15−/− mice appeared healthy, were fertile, and did not display any evident pathological phenotypes similar to other published Adam15−/− mice (9).

Oxygen-induced ischemic retinopathy (12) is a model that is relevant to retinopathy of prematurity, diabetic retinopathy, and other diseases in which areas of nonperfused retina release VEGF and other factors that stimulate growth of new vessels on the surface of the retina. Adam15−/− mice with oxygen-induced ischemic retinopathy appeared to have less retinal neovascularization (Fig. 3A) than that seen in Adam15+/− (Fig. 3B) or Adam15+/+ mice with ischemic retinopathy (Fig. 3C). The retinal neovascularization is visualized by in vivo immunostaining for PECAM-1, which selectively stains endothelial cells of new vessels without staining the normal vessels in the retina (14). Whereas the structural detail of the neovascularization is not discernable with this technique, the selective staining greatly facilitates and objectifies measurement of the amount of neovascularization by image analysis, which confirms that there was a significant reduction in the amount of retinal neovascularization in Adam15−/− mice compared with Adam15+/− or Adam15+/+ mice (Fig. 3D).

Figure 3.

Figure 3.

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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).

Choroidal neovascularization at Bruch’s membrane rupture sites is reduced in mice deficient in ADAM15

Choroidal neovascularization occurs in diseases in which there are defects or abnormalities in Bruch’s membrane and/or the retinal pigmented epithelium. Rupture of Bruch’s membrane with laser photocoagulation in mice provides a model that is useful for investigating the role of particular gene products in the development of choroidal neovascularization (13, 18). The area of choroidal neovascularization at Bruch’s membrane rupture sites appeared smaller in Adam15−/− mice (Fig. 4A) compared with that seen in Adam15+/− (Fig. 4B) or Adam15+/+ mice (Fig. 4C). Image analysis confirmed that the area of choroidal neovascularization was significantly smaller in mice deficient in ADAM15 (Fig. 4D).

Figure 4.

Figure 4.

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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).

Mice deficient in ADAM15 have reduced VEGF-induced subretinal neovascularization

Rho/VEGF transgenic mice develop neovascularization that originates from the deep capillary bed of the retina and grows into the subretinal space (10, 19). The same type of neovascularization occurs in patients with age-related macular degeneration and is referred to as retinal angiomatous proliferation (20). Adam15−/− mice that also expressed the rho/VEGF transgene (Fig. 5A) appeared to have less subretinal neovascularization than did littermates that expressed the rho/VEGF transgene but were not deficient in ADAM15 (Fig. 5B). Image analysis confirmed that rho/VEGF mice that were deficient in ADAM15 had fewer tufts of subretinal neovascularization per eye (Fig. 5C) and smaller total area of subretinal neovascularization per eye (Fig. 5D) than did rho/VEGF mice that did not lack ADAM15.

Figure 5.

Figure 5.

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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).

Mice deficient in ADAM15 show reduced levels of mRNA and protein for VEGF164, VEGFR1, and VEGFR2 in ischemic retina

At P17, Adam15−/− and Adam15+/+ mice with ischemic retinopathy had measurement of Vegf164, Vegfr1, Vegfr2, and cyclophilin A mRNA in one retina and measurement of VEGF164, VEGFR1, and VEGFR2 protein by ELISA in the retina from the other eye. There were significant reductions in the mRNA ratios Vegf164:cyclophilin A (Fig. 6A) and Vegfr2:cyclophilin A (Fig. 6E), but not Vegfr1:cyclophilin A (Fig. 6C), in ischemic retinas from Adam15−/− mice compared with Adam15+/+ mice. There were also significant reductions in VEGF164 (Fig. 6B), VEGFR1 (Fig. 6D), and VEGFR2 protein (Fig. 6F).

Figure 6.

Figure 6.

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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.

Knockdown of Vegf mRNA results in reduction of Adam15 mRNA in ischemic retina

At P12, mice with ischemic retinopathy were given an intravitreous injection of a mixture of siRNAs that specifically target Vegf mRNA in one eye and siRNAs targeting Gfp mRNA in the other eye. At P15, the mice were euthanized, and the levels of Vegf, Adam15, and cyclophilin mRNA in the retina were measured by real-time quantitative RT-PCR. The level of Vegf mRNA was significantly reduced in retinas from eyes injected with Vegf siRNA compared with those from eyes injected with Gfp siRNA (Fig. 7A), and the level of Adam15 mRNA was significantly decreased in the retinas with reduced Vegf mRNA (Fig. 7B). This result further supports the link between VEGF and ADAM15 levels in the retina.

Figure 7.

Figure 7.

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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.

DISCUSSION

In this study, we have shown that ADAM15 is increased in ischemic retina, and at least part of the increase is the result of stimulation by VEGF, because ADAM15 levels are also elevated in the retinas of transgenic mice with overexpression of VEGF in the retina. ADAM15 is also up-regulated in rheumatoid synovium by VEGF (8). In normal retinas, there is some staining for ADAM15 in perivascular cells and minimal staining in endothelial cells, but in ischemic retina there is strong staining in endothelial cells. Interestingly, Adam15 mRNA was not detected in quiescent endothelial cells from normal aorta but was present in substantial amounts in activated endothelial cells (4). We have confirmed that mice deficient in ADAM15 have reduced ischemia-induced retinal neovascularization (9), indicating that the up-regulation of ADAM15 is an important part of the proangiogenic cascade. Furthermore, ADAM15 also contributes to subretinal and choroidal neovascularization because those types of neovascularization are also reduced in mice deficient in ADAM15. It appears that ADAM15 is part of an amplification loop because its expression is increased by VEGF and it, in turn, increases expression of VEGF, VEGFR1, and VEGFR2.

The exact mechanism by which the ADAM15 amplification loop functions is uncertain. Whereas hypoxia and VEGF stimulate the up-regulation of ADAM15 in endothelial cells in vivo, VEGF does not stimulate increased expression of Adam15 in cultured endothelial cells (9), suggesting that VEGF-induced up-regulation of ADAM15 in vivo is indirect. Once ADAM15 is increased in endothelial cells, it must activate intracellular signaling that mediates up-regulation of VEGF and its receptors. The intracellular domain of ADAM15 contains SH3 ligand-binding domains that functionally interact with Src family protein tyrosine kinases (21). Src and its family members are involved in up-regulation of VEGF (22, 23); therefore, activation of Src family members by ADAM15 may increase expression of VEGF. Membrane-type 1 matrix metalloproteinase is another transmembrane protein with proteinase activity that increases transcription of VEGF through Src tyrosine kinases (24). Src activates hypoxia-inducible factor-1 and STAT3, which form a transcriptional complex that activates VEGF and other hypoxia-regulated genes, including VEGF receptor 1 (25, 26). VEGF receptor 2 expression is increased by VEGF through Src (27); therefore, Src is involved in up-regulation of VEGF, VEGFR1, and VEGFR2 and may mediate the effects of ADAM15.

ADAM15 has other activities that could contribute to its proangiogenic effects in the retina. It is a metalloproteinase and also binds integrins. A synthetic inhibitor, GL 129471, which blocks the proteolytic activity of ADAMs as well as other matrix metalloproteinases, inhibited endothelial tube formation in vitro, which is considered an in vitro assay of angiogenesis (28). In contrast, tissue inhibitor of metalloproteinases (TIMP)-2, which inhibits other matrix metalloproteinases but not ADAMs, failed to block endothelial tube formation, suggesting that the metalloproteinase activity of ADAM15 is important in this in vitro model of angiogenesis. It appears that the proteolytic activity of ADAM15 may be important for regression as well as formation of endothelial tubules because siRNA directed against Adam15 mRNA or pericyte-derived TIMP-3, which blocks the proteolytic activity of ADAM15, prevents regression and stabilizes the tubules (29). The integrin-binding activity of the disintegrin domain may also be important. Human ADAM15 binds α5β1 and αvβ3, two integrins that are minimally expressed in quiescent endothelial cells of ocular vessels and strongly induced in endothelial cells participating in angiogenesis (30, 31). A soluble ADAM15 disintegrin domain blocked endothelial cell migration, proliferation, and tube formation in vitro and suppressed tumor growth and angiogenesis in vivo (32). This result suggests that blockade of integrin binding by ADAM15 reduces its proangiogenic activity (3, 5, 6). It appears likely that ADAM15 promotes ocular neovascularization by multiple mechanisms.

Monthly intraocular injections of an antibody fragment that binds VEGF is the first treatment to cause visual improvement in a significant number of patients with choroidal neovascularization caused by age-related macular degeneration (33). VEGF antagonists also appear to provide benefit in patients with choroidal neovascularization resulting from causes other than age-related macular degeneration (34) and in patients with retinal neovascularization resulting from diabetic retinopathy or diabetic macular edema (35). Blockade of the ADAM15 amplification loop could potentially enhance the effectiveness of VEGF antagonists and thereby reduce the required frequency of intraocular injections. Further work is needed to explore this approach.

Acknowledgments

This work was supported by Novartis (grant EY012609), the National Eye Institute (core grant P30EY1765), a Senior Scientist Award from Research to Prevent Blindness, and Dr. and Mrs. William Lake. P.A.C. is the George S. and Dolores Dore Eccles Professor of Ophthalmology.

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