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. Author manuscript; available in PMC: 2013 May 1.
Published in final edited form as: Arch Ophthalmol. 2012 May 1;130(5):615–620. doi: 10.1001/archopthalmol.2011.1892

Lack of Thrombospondin-1 and Exacerbation of Choroidal Neovascularization

Shoujian Wang 1, Christine M Sorenson 2, Nader Sheibani 1,3,*
PMCID: PMC3404202  NIHMSID: NIHMS371771  PMID: 22232368

Abstract

Purpose

To assess the impact of thrombospondin-1 (TSP1) deficiency on choroidal neovascularization (CNV) and determine whether administration of a TSP1 antiangiogenic mimetic peptide attenuates CNV.

Methods

The impact of TSP1-deficiency on laser induced CNV was assessed using wild type (TSP1+/+) and TSP1-deficient (TSP1−/−) mice. Three laser burns were placed in each eye of TSP1+/+ and TSP1−/− mice to induce CNV. Intravitreal injection of the TSP1 mimetic peptide was performed on the day 1 and day 7 post laser in the mice. For quantitative measurements of neovascularization, ICAM-2 staining was performed at 14 days post-laser of the choroidal-sclera flatmounts. The recruitment of macrophages to the sites of damage was investigated by immunohistochemistry. The CNV area was measured by ICAM-2 staining and use of image J software.

Results

TSP1−/− mice exhibited significantly larger areas of neovascularization on choroidal flatmounts compared to TSP1+/+ mice. This was consistent with enhanced recruitment of macrophages in TSP1−/− mice compared to TSP1+/+ mice 3 days post-laser. The development of CNV was significantly attenuated in mice receiving the TSP1-antiangiogenic mimetic peptide compared to those receiving vehicle alone.

Conclusions and Clinical Relevance

TSP1 deficiency contributes to enhanced choroidal neovascularization. This is consistent with the anti-inflammatory and antiangiogenic activity of TSP1. TSP1 antiangiogenic peptide was effective in attenuation of CNV. Therefore, intravitreal injection of TSP1 antiangiogenic mimetic peptides may provide alternative treatment for CNV.

Introduction

Age-related macular degeneration (AMD) affects millions of individuals worldwide, with approximately 90% of severe vision loss attributed to choroidal neovascularization (CNV) (1, 2). The global prevalence of CNV is expected to double in the next decade due to the aging population. AMD is characterized by a progressive degeneration of the macula, usually bilateral, leading to a central scotoma and severe decrease in vision. The visual deficit results initially from retinal degeneration called geographic atrophy (dry or nonexudative AMD) often complicated by the secondary effects of CNV (wet or exudative AMD). An early sign of AMD is the appearance of drusen, which are extracellular deposits that accumulate below the retinal pigment epithelium (RPE) and are known to be a risk factor for developing CNV(2).

Angiogenesis, the formation of new capillaries from preexisting capillaries, is associated with the pathogenesis of exudative AMD (3, 4). Therefore, inhibition of angiogenesis has become a viable strategy to inhibit CNV. Targeting of the proangiogenic factor VEGF-A has been validated in patients with CNV (5, 6). However, significant improvement in vision is only observed in 30% of patients treated with VEGF-A antagonist, with 20% of treated patients still progressing to legal blindness. Furthermore, safety concerns about the conditional blockade of VEGF-A, which is constitutively expressed in the normal human retina (7, 8) and glomeruli (9, 10) are emerging. Thus, additional treatment strategies on the basis of more specific targeting of CNV are desirable.

It is now well accepted that endogenous inhibitors of angiogenesis play a significant role in regulation of angiogenesis and their decreased production may contribute to pathological neovascularization. Thrombospondin-1 (TSP1), a member of TSP gene family, was the first endogenous inhibitor of angiogenesis identified whose expression was decreased during malignant transformation (11). We have shown that TSP1 is an important modulator of retinal vascular homeostasis and its increased production results in attenuation of retinal vascular development and neovascularization (12, 13). We have also shown that TSP1 is present at very high levels in vitreous samples from various species and its level decreased during diabetes, perhaps contributing to the pathogenesis of diabetic retinopathy (14, 15). However, TSP1 expression changes and its contribution to the pathogenesis of AMD need further investigation.

TSP1 is synthesized and secreted by retinal pigment epithelial (RPE) cells and its expression is up-regulated by vitamin A (16, 17). Recent studies also suggest an important role for TSP1 in choroidal vascular homeostasis and pathogenesis of CNV (18). Immunohistochemical analysis of sections prepared from human eyes indicated that TSP1 is present in the macula region of the Bruch’s membrane, the choroidal capillaries, and the larger choroidal vessels. In addition, a significant decrease in the level of TSP1 was detected in Bruch’s membrane and choroidal capillaries from AMD eyes (19). We, therefore, hypothesized that TSP1 deficiency will exacerbate the pathogenesis of CNV and it may be an important target for inhibition of CNV. Here we investigated the impact of TSP1-deficiency on laser-induced CNV using TSP1−/− mice. We also determined the potential therapeutic role of TSP1 in treatment of CNV using a TSP1 antiangiogenic mimetic peptide.

Materials and Methods

Animals

All animal studies were conducted in accordance with an animal protocol reviewed and approved by University of Wisconsin-Madison Animal Care and Use Committee and in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Six- week-old female wild type (TSP1+/+) or TSP1-deficient (TSP1−/−) C57BL/6j mice (10 per group) were used in these studies and housed on a 12-hour light–dark cycle, with food and water provided ad libitum.

Laser induced choroidal neovascularization (CNV) and its quantification

Various animal models have been instrumental in advancing our understanding of the pathogenesis of AMD and the development and testing of effective treatments (20). An early and important model was the laser induced CNV model, developed by Ryan et al. in 1979 (21), and it remains one of the commonly used models for wet AMD research. The CNV was induced in mice by laser photocoagulation-induced rupture of Bruch's membrane on Day 0 as previously described (22, 23). Mice were anesthetized with ketamine hydrochloride (100 mg/kg) and xylosine, and the pupils were dilated using a drop of 1% tropicamide. Laser photocoagulation (75 µm spot size, 0.1 s duration, 120 mW) was performed in the 9, 12, and 3 o'clock positions of the posterior pole of each eye with the slit lamp delivery system of an OcuLight GL diode laser (Iridex, Mountain View, CA) and a handheld cover slip as a contact lens to view the retina. After 14 days, the eyes were removed and fixed in 4% paraformaldehyde at 4°C for 2 h. Following three washes in PBS, the eyes were sectioned at the equator, and the anterior half, the vitreous, and the retina was removed. The remaining eye tissue was incubated in blocking buffer (20% fetal calf serum, 20% normal goat serum in PBS) for 1 hours at room temperature, followed by incubation with anti-ICAM-2 (BD Pharmagen 1:500 in PBS containing 20% fetal calf serum, 20% normal goat serum) overnight at 4 °C. The remaining eye tissue was then washed three times with PBS and incubated with the appropriate secondary antibody. The retinal pigment epithelium–choroid–sclera complex was dissected through five to six relaxing radial incisions and flatmounted on a slide with VectaMount™ AQ (Vector Laboratories, Burlingame, CA). The samples were viewed by fluorescence microscopy and images were captured in digital format using a Zeiss microscope (Zeiss, Chester, VA). Image J software (National Institute of Mental Health, Bethesda, MD; http://rsb.info.nih.gov/ij/) was used to measure the total area (in µm2) of CNV associated with each burn.

Immunohistochemistry for detection of macrophages

Eyes were enucleated 3 d after laser injury, when maximum number of macrophages is observed (24), fixed in 4% paraformaldehyde (PFA) for 2 h, and RPE-choroid-sclera complexes were dissected. Flatmounts were treated with blocking solution (20% fetal calf serum, 20% normal goat serum in PBS) for 1 h at room temperature, followed by incubation with anti-F4/80 (BD Pharmagen; 1:500 in PBS containing 20% fetal calf serum, 20% normal goat serum) overnight at 4°C. The remaining eye tissue was then washed three times with PBS and incubated with the appropriate secondary antibody. The tissue was viewed by fluorescence microscopy and images were captured in digital format using a Zeiss microscope (Zeiss, Chester, VA). For quantitative analysis of macrophage recruitment, the area of fluorescence and average pixel intensities (mean gray value) were determined using Photoshop software. The integrated density was calculated by multiplying the area of florescence by the mean gray value.

TSP1 antiangiogenic mimetic peptide treatment

The antiangiogenic activity of TSP1 is mapped to peptides from type 1 repeats and procollagen homology domain (25). An overlapping peptide that expands these regions has shown good efficacy for inhibition of angiogenesis in various tumor models (26) and was the basis for development of ABT510, and most recently a newer generation (27), ABT898. The amino acid sequence of ABT-898 is N-acetyl-glycine-valine-D-alloisoleucine-serine-glutamine-isoleucine-argenine-prolin-ethylamid and was synthesized at UW-Biotechnology peptide synthesis core facility (Madison, WI). The purity and sequence of the peptide were confirmed using standard methods. The peptide was dissolved in 5%-dextran solution and used for intravitreal injections.

TSP1+/+ mice and TSP1−/− mice were injected intravitreally with TSP1 antiangiogenic mimetic peptide at day 0, right after laser rupture of Bruch’s membrane (2 µl of 100 µg/mL). This dose was found to be most effective. Intravitreous injections were performed with a pump microinjection apparatus (Harvard Apparatus, Holliston, MA) and pulled glass micropipettes. Each micropipette was calibrated to deliver 2 µL of vehicle containing the peptide on depression of a foot switch. The mice were anesthetized and the pupils were dilated. Under a dissecting microscope, the sharpened tip of the micropipette was passed through the sclera, just behind the limbus into the vitreous cavity, and the foot switch was depressed. The injections were repeated 7 days after Bruch’s membrane rupture to replenish the peptide concentration in the vitreous cavity. After 14 days the samples were prepared for quantification of area of neovascularization as described above.

Statistical analysis

Statistical differences between groups were evaluated with the student's unpaired t-test (two-tailed). Mean ± standard deviation is shown. P ≤ 0.05 is considered significant.

Results

TSP1-deficiency results in increased areas of CNV

TSP1 is a potent inhibitor of angiogenesis and can directly act on endothelial cells inhibiting their proliferation and migration and promoting their apoptosis. The impact of TSP1 on choroidal endothelial cells, and more specifically on choroidal neovascularization remains elusive. To determine whether lack of TSP1 expression would impact the degree of neovascularization upon laser-induced CNV, six-week-old C57BL/6 TSP1+/+ and TSP1−/− female mice underwent laser-mediated photocoagulation and CNV neovascularization. The areas of neovascularization were assessed as described in Methods. Images obtained with rat anti–ICAM-2 staining of the choroidal flatmounts showed the typical morphology of blood vessels in the CNV lesions two weeks after laser photocoagulation. The TSP1−/− mice showed significantly larger neovascular choroidal outgrowths (Figs. 1A, B) compared to TSP1+/+ mice. The average CNV area per eye was significantly increased in TSP1−/− mice (23750±4200 µm2, n=15 eyes) compared with TSP1+/+ mice, approximately 8-fold (3235±910 µm2, n=15 eyes) (Fig. 1C; *P< 0.05). Thus, enhanced CNV is observed in the absence of TSP1.

Fig. 1.

Fig. 1

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Increased CNV lesion size in TSP1−/− mice following laser photocoagulation. Immunohistochemical visualization of the laser scars on RPE/choroidal flatmounts two weeks after photocoagulation of TSP1+/+ (A) and TSP1−/− (B) were visualized with ICAM-2 staining. Quantification of the data is shown in (C). A significant increase in the area of CNV was observed in TSP1−/− mice compared to TSP1+/+ mice (*P<0.05; n=15 eyes). Bar=50 µm.

Enhanced infiltration of macrophages in mice lacking TSP1 during CNV

Recent studies have established an important role for inflammatory processes, particularly recruitment of macrophages, in pathogenesis of CNV. Macrophages participate in all stages of CNV and modulate the severity of CNV. TSP1 is an important modulator of ocular immune privilege and in its absence enhanced inflammation has been reported (28). We next analyzed the degree of macrophage infiltration in TSP1+/+ and TSP1−/− mice subjected to laser induced CNV by staining choroidal flat mounts with the antibody to macrophage-specific antigen F4/80. We observed a significant increase in density of F4/80 positive macrophages in TSP1−/− mice following laser treatment compared to TSP1+/+ mice (Fig. 2).

Fig. 2.

Fig. 2

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Enhanced infiltration of macrophages in TSP1−/− mice after laser injury. Representative immunohitochemical images of lesions in TSP1+/+ (A) and TSP1−/− mice (B), following F4/80 staining is shown. The density of infiltrating macrophages 3 days after laser application was markedly increased in TSP1−/− mice compared with TSP1+/+ mice (C; *P < 0.05; n=15 eyes). Bar=50 µm.

Attenuation of CNV by administration of the TSP1-antiangiogenic mimetic peptide

Our results in Figures 1 and 2 coupled with studies demonstrating decreased levels of TSP1 in choroidal membranes prepared from eyes with CNV (18, 19) suggest that TSP1 may suppress CNV. We next determined whether administration of TSP1 mimetic antiangiogenic peptide can inhibit CNV and/or reduce area of neovascularization in TSP1−/− mice. Peptides derived from TSP1 type 1 repeats and pro-collagen homology domain, including the peptide used here, can signal through CD36 (TSP1 receptor) inhibiting angiogenesis (29, 30). Recent studies have demonstrated that this may also require interaction of TSP1 with CD47 (receptor for C-terminal domain of TSP1 (31). TSP1-antiangiogenic mimetic peptide was administered intravitreally to TSP1−/− mice subjected to the laser-induced CNV. We observed a significant decrease in mean area of CNV in TSP1−/− mice which received the TSP1 mimetic peptide compared with vehicle control (TSP1−/− with peptide: 2254±1040 µm2 vs. TSP1−/− with vehicle: 11340±3315 µm2, n=15) (Figs. 3A, B). The mean CNV area in eyes treated with the TSP1 peptide was decreased by approximately 80% compared to that seen in eyes receiving vehicle alone (Fig. 3C; *P<0.05). Administration of TSP1 peptide in TSP1+/+ mice also significantly inhibited CNV lesion formation (TSP1+/+ with peptide: 1840±379 µm2 vs. TSP1+/+ with vehicle: 3235±910 µm2, n=15) (Fig. 4; *P<0.05). Thus, the TSP1 antiangiogenic mimetic peptide may provide an alternative treatment for CNV.

Fig. 3.

Fig. 3

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Attenuation of CNV following intravitreal administration of the TSP1 antiangiogenic mimetic peptide in TSP1−/− mice exposed to laser-induced photocoagulation. Representative choroidal flatmounts after staining with ICAM-2, two weeks after laser photocoagulation, is shown. TSP1−/− mice were subjected to laser photocoagulation and received vehicle (A) or TSP1 peptide (B) on day 1 and 7 post laser by intravitreal injection (2 µl of 100 µg/ml in 5% dextran). A significant decrease in area of CNV was observed in mice that received TSP1 peptide compared with those that received vehicle (*P<0.05; n=15 eyes). Bar=50 µm.

Fig. 4.

Fig. 4

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Attenuation of CNV following intravitreal administration of the TSP1 antiangiogenic mimetic peptide in TSP1+/+ mice exposed to laser-induced photocoagulation. Representative choroidal flatmounts after staining with ICAM-2, two weeks after laser photocoagulation, is shown. TSP1+/+ mice were subjected to laser photocoagulation and received vehicle (A) or TSP1 peptide (B) on day 1 and 7 post laser by intravitreal injection (2 µl of 100 µg/ml in 5% dextran). A significant decrease in area of CNV was observed in mice that received TSP1 peptide compared with those that received vehicle (*P<0.05; n=15 eyes). Bar=50 µm.

Discussion

The studies presented here demonstrate that lack of TSP1 results in enhanced CNV, and this was associated with a significant increase in number of macrophages recruited to sites of laser lesions. These observations are consistent with the important role of inflammation processes in pathogenesis of CNV and the ocular anti-inflammatory role previously demonstrated for TSP1. In addition, we showed that administration of TSP1-antiangiogenic mimetic peptide inhibited CNV in both TSP1+/+ and TSP1−/− mice. Thus, modulation of TSP1 expression or its antiangiogenic mimetic peptides may provide a novel approach for the treatment of CNV associated with AMD.

Histological studies have demonstrated the immediate arrival of macrophages at laser rupture sites within 1 hour of laser application, and macrophages accumulate in areas of disruption of Bruch’s membrane (24, 32–34). Laser-induced CNV is an appropriate model for investigating the relationship between inflammation and angiogenesis. Macrophages secret IL-1β, among other inflammatory cytokines, in response to tissue injury and inflammation, and promote angiogenesis (33, 34).

There has been considerable discussion of the role of inflammation in promoting ocular angiogenesis, particularly in neovascular AMD. Inflammation is critically involved in the formation of CNV lesions, and contributes to the pathogenesis of AMD. Inflammatory cells are found in surgically excised CNV lesions from AMD patients and in autopsied eyes with CNV. In particular, macrophages have been implicated in the pathogenesis of AMD due to their spatiotemporal distribution in the proximity of the CNV lesions in experimental models and humans, making significant contribution to the pathogenesis of CNV (24, 32).

The majority of the macrophages found in the proximity of the laser-induced CNV lesions is derived from newly recruited peripheral blood monocytes and are not resident macrophages (34, 35). Because macrophages play such a critical role in CNV formation, prevention of monocyte recruitment and infiltration into ocular tissues may ameliorate the development of CNV, a function that could be exploited therapeutically (32, 36–38). Here we showed that lack of TSP1 exaggerates CNV and this was associated with increased recruitment of macrophages into the sites of lesions. These results are consistent with previous reports of impaired TSP1 expression in Bruch’s membrane and choroidal vessels of eyes with AMD (18, 39) and identification of TSP1 as a genetic loci that controls the size of CNV (40).

The anti-inflammatory nature of the intraocular environment is critical to the immune privilege of the eye and pathogenesis of CNV. Investigation into the role of inflammation in neovascular eye disease often overlook the principles of immune privilege in the eye. The ocular environment is generally not pro-inflammatory and it prohibits inflammation at the expense of certain immune effector mechanisms. It has been suggested that the loss of immune privilege as the eye ages may contribute to the increases in neovascular disease (41, 42). These concepts challenge the idea that neovascular disease is simply an inflammatory process, and support the idea that these diseases may result from the loss or dysfunction of important components of the cellular immune system (36).

Inflammatory mechanisms and immune activation have been implicated in the pathogenesis of CNV. Retinal laser burns disrupt the immune privilege in the eye resulting in inflammation (43, 44). In addition, TSP1 plays a vital role in the modulation of immune privilege such that in its absence the retinal microenvironment is more proinflammatory and supports an activated state of microglia with poor recovery from injury (43). This is consistent with the increased recruitment of macrophages and enhanced severity of neovascularization observed here in TSP1−/− mice.

The anti-inflammatory activity of TSP1 in the eye is mainly attributed to the enhanced levels of active TGF-β in the presence of TSP1 (45, 46). A major physiological function of TSP1 is activation of latent TGF-β with important function during normal developmental process (47). The TSP1 antiangiogenic peptide used here lacks the TSP1 sequence that is responsible for activation of TGF-β. Thus, the angioinhibitory activity observed here may be independent of the TSP1 effects on activation of TGF-β and its anti-inflammatory effects on CNV. Thus, intact TSP1 molecule may impact CNV through both its anti-inflammatory and antiangiogenic activities. The potential synergistic and/or additive effects of these TSP1 activities on CNV associated with AMD are subject of current investigation in our laboratory.

In summary, TSP1 deficient mice exhibited significantly enhanced choroidal neovascular membrane formation associated with increased inflammation and recruitment of macrophages. Together these results suggest that modulation of TSP1 plays an important role in CNV associated with AMD. Furthermore, TSP1 mimetic peptides could be used as novel therapeutics to inhibit CNV perhaps by modulating both the inflammatory and angiogenic state of choroidal vessels.

Acknowledgments

This work was supported by grants EY16995, EY18179, EY21357 (NS), and P30-EY16665, from the National Institutes of Health and an unrestricted departmental award from Research to Prevent Blindness. NS is a recipient of a Research Award from American Diabetes Association, 1-10-BS-160 and Retina Research Foundation. CMS is supported by a grant from American Heart Association, 0950057G.

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