Abstract
purpose. Endostatin, a C-terminal fragment of collagen XVIII (coll XVIII) formed by proteolysis, specifically inhibits endothelial cell migration and proliferation in vitro and potently inhibits angiogenesis and tumor growth in vivo. The purpose of this study was to examine the immunolocalization of endostatin and coll XVIII in the retina and choroid of human donor tissue sections from aged control donor eyes and to determine whether the localization or relative levels are changed in age-related macular degeneration (AMD).
methods. Ocular tissues were obtained from six aged control donors (age range, 75–86 years; mean age, 80.5 years) without evidence or history of chorioretinal disease and from nine donors with AMD (age range, 74–105 years; mean age, 88.6 years). Tissues were cryopreserved, and streptavidin alkaline phosphatase immunohistochemistry was performed with goat anti-human and mouse anti-human endostatin antibodies and rabbit anti-mouse coll XVIII. Blood vessels were identified with mouse anti-human CD-34 antibody in adjacent sections. Pigment in RPE and choroidal melanocytes was bleached. Three independent observers scored the immunohistochemical reaction product.
results. In aged control eyes, coll XVIII and endostatin (the endostatin portion of coll XVIII) immunoreactivity was observed in large retinal blood vessels and in capillaries in some individuals, but the internal limiting membrane (ILM) had the most intense retinal immunostaining. There was no significant difference in immunoreactivity to both antibodies in retinal blood vessels in aged control eyes. In the choroid, endostatin and coll XVIII were localized to blood vessels, Bruch’s membrane, and RPE basal lamina. AMD retina and choroid had a similar pattern and intensity of coll XVIII immunostaining, as observed in control eyes but reaction product was more diffuse in the choroid. Endostatin immunoreactivity was significantly higher in ILM (P = 0.037) in AMD retina and significantly lower in the choriocapillaris, Bruch’s membrane, and RPE basal lamina of AMD choroids (P < 0.05) and completely negative in some areas of AMD choroids.
conclusions. These data suggest that reduced levels of the endostatin portion of coll XVIII in Bruch’s membrane, RPE basal lamina, intercapillary septa, and choriocapillaris in eyes with AMD may be permissive for choroidal neovascularization.
Angiogenesis, the outgrowth of new capillaries from preexisting vessels, is essential for embryonic development, organ formation, tissue regeneration, and remodeling.
1 2 It also contributes to the development and progression of a variety of pathologic conditions, including tumor growth and metastasis, cardiovascular diseases, diabetic retinopathy, rheumatoid arthritis, psoriasis, and exudative AMD.
3 Angiogenesis is a complex multistep process that includes degradation of the extra cellular matrix, migration, proliferation and tubule formation by endothelial cells.
3 4 The complexity of the angiogenic processes suggests the existence of multiple controls of the system that can be switched on and off transiently.
A switch to the angiogenic phenotype in tissues is thought to depend on a local change in the balance between angiogenic stimulators and inhibitors.
5 A large number of cytokines have been shown to stimulate angiogenesis under experimental conditions. Among the most important mediators of angiogenesis, both in normal and pathologic conditions, are vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and interleukin (IL)-18.
6 7 8 Angiogenesis is also controlled by a large variety of endogenous inhibitors such as pigment epithelium-derived factor (PEDF), angiostatin, endostatin, and thrombospondin (TSP). They are predominantly extracellular proteins that quite frequently require proteolytic processing for their activation.
9
Ocular neovascularization, the pathologic growth of new blood vessels in the eye, is a leading cause of blindness worldwide. Neovascularization is responsible for many severe ocular disorders, such as corneal neovascularization, neovascular glaucoma, diabetic and sickle cell retinopathy, age-related macular degeneration, and retinopathy of prematurity.
10 11 Similar to the well-known angiogenesis in tumors, neovascularization in the eye is considered to result from an imbalance between stimulatory and inhibitory angiogenic factors.
12 13 The elevated expression of stimulatory factors and/or the downregulation of inhibitory factors has often been observed in pathologic conditions such as ocular inflammation and ischemia.
13 14 15 In contrast to these pathologic conditions, ocular tissues are maintained physiologically without the occurrence of neovascularization, and the vasculature in the eye is highly restricted, despite constitutive expression of many angiogenic molecules, such as bFGF,
16 insulin-like growth factor (IGF)-1,
17 and VEGF.
18 19 20 21 These findings in the eye suggest the physiological existence of angiogenesis inhibitors to counterbalance these stimulators. Potent inhibitory factors have been thought to exist in the retinal pigment epithelium (RPE),
22 the vitreous body,
23 24 25 26 27 and the lens.
28 Some have been purified from vitreous—for example, TGF-β
29 and PEDF.
30
One endogenous inhibitor of angiogenesis recently isolated is endostatin. Endostatin is the proteolytically cleaved carboxyl terminus globular domain of collagen XVIII (coll XVIII).
31 Coll XVIII is an integral proteoglycan in endothelial and epithelial basement membranes.
32 33 Endostatin blocks mitogen-activated protein kinase (MAPK) activation in endothelial cells.
31 It specifically inhibits endothelial cell proliferation and migration in vitro and potently inhibits angiogenesis in vivo.
34 Human serum and tissue forms of endostatin have been identified.
35 36 It has recently been shown that endostatin is a potent angiogenesis inhibitor that controls the growth of many different types of solid tissue tumors in animal models.
35 37 38 Endostatin is also currently being aggressively pursued as a candidate for cancer therapy in humans.
39 40
Coll XVIII is widely expressed in mice and humans.
33 37 41 In mouse, coll XVIII has been demonstrated prominently in all ocular basement membranes except Descemet’s membrane.
31 42 Also, coll XVIII knockout mice show delayed regression of the vasa hyaloidea propria (VHP) portion of the hyaloid vasculature.
42 This suggests that a lack of coll XVIII/endostatin results in vascular phenotypic changes in mouse. The production and liberation of endostatin in vivo is still poorly understood, but it has been found to be expressed in some differentiated tissues (e.g., kidney and liver) and freely circulating in serum.
34 36 43 These observations, combined with the emerging data on endostatin as a potential tumor angiogenesis inhibitor, prompted us to examine the localization of coll XVIII and endostatin in human retina and choroid and to determine whether the localization or relative levels were changed in donors with age-related macular degeneration (AMD).
For qualitative and quantitative assessment of immunohistochemistry at the level of choroid-Bruch’s membrane-RPE complex, the removal of melanin pigment was desirable. We developed a technique to bleach melanin from RPE and choroidal melanocytes that was compatible with our immunohistochemical procedure, so that the confounding presence of RPE and choroidal pigment could be reduced or eliminated. Sections were fixed in 4% paraformaldehyde overnight at 4°C immediately after streptavidin APase immunohistochemistry. Slides were washed in distilled water at room temperature, immersed in a 0.05% potassium permanganate solution (Aldrich Chemical Co., Milwaukee, WI) for 25 minutes, and then rinsed in distilled water for 5 minutes. Sections were covered with 35% peracetic acid (FMC Corp., Philadelphia, PA) in a humidified container for 15 minutes at room temperature followed by washing in distilled water for 10 minutes. Finally, coverslips were mounted with Kaiser’s glycerogel without counterstaining.
It appeared that the relative amount of reaction product did not change with the potassium permanganate bleaching protocol. Most of the pigment was bleached with the good retention of tissue morphology and little loss of reaction product, allowing us to evaluate precisely the immunoreactivity in the choroid-Bruch’s membrane-RPE complex. Furthermore, both mouse anti-human endostatin and goat anti-human endostatin antibodies showed similar staining patterns, localization, and immunoreactivity in the retina and choroid. Most of the photographs presented are from goat anti-human endostatin. The term endostatin is used to represent endostatin that has been cleaved from coll XVIII or still remains part of coll XVIII, since our endostatin antibodies cannot distinguish the difference.
Immunolocalization of Collagen XVIII and the Endostatin Portion of Coll XVIII in Aged Control Human Retina and Choroid
We investigated the expression pattern of coll XVIII and the presence of the endostatin portion of coll XVIII in the retina and choroid of aged control and AMD eyes. Coll XVIII is an integral proteoglycan in endothelial and epithelial basement membranes
32 33 and, as expected, was prominently localized in the ILM of the retina, the basement membranes of retinal and choroidal blood vessels, and Bruch’s membrane. Immunohistochemistry demonstrated that coll XVIII was similar in level in control aged and AMD eyes, but the endostatin portion of coll XVIII was significantly reduced. To our knowledge, this is the first demonstration of retinal and choroidal localization of endostatin and coll XVIII in human eyes.
The permanganate bleaching protocol did not affect the specificity of the antigen detection nor did it affect the sensitivity of immunostaining based on a comparison of bleached and nonbleached sections. This technique resulted in a method that may be useful for applications other than histochemical procedures, such as in situ hybridization. By fixing sections extensively after streptavidin APase immunohistochemistry, we bleached most of the pigment, with tissue morphology and reaction production retained. Because color-based APase protocols for immunohistochemistry can use chromogens that are resistant to bleaching, our protocol was directly applicable to this method.
The normal localization of coll XVIII and endostatin that we observed was predominantly in matrix (choroidal stroma and intercapillary septa) and basement membranes (vascular basement membranes as well as Bruch’s and internal limiting membrane). This is a logical location for a basement membrane component that prevents endothelial migration and subsequent neovascularization.
49 The human localization was comparable to that observed by Fukai et al.
42 in the mouse. One shortcoming of our study is that our endostatin antibodies do not distinguish between endostatin cleaved from coll XVIII and the endostatin portion of intact coll XVIII. The coll XVIII antibody recognizes the amino terminus and the endostatin antibodies recognize endostatin at the carboxyl terminus. The levels and location of coll XVIII and endostatin immunoreactivity appeared comparable in the control eyes, suggesting that the endostatin detected may not be a soluble form. It cannot be determined from the present study whether endostatin is actually produced in the eye, but expression of coll XVIII/endostatin in developing and postnatal ocular basement membranes has already been reported.
42 Finding the two molecules colocalized in control eyes suggests that this is true in humans. The importance of a reduction in endostatin immunoreactivity is that there is potentially less of this endogenous antiangiogenic agent in AMD choroids.
Some of the endostatin detected in this study may have been the soluble form
50 that had bound at these sites. For example, we observed higher endostatin in the internal limiting membrane of AMD eyes compared with aged eyes, but not increased coll XVIII at that site. Endostatin may bind, not only to blood vessel basement membranes, but also to a subset of epithelial basement membrane. For example, renal cortical tubular and Bowman’s capsule basement membranes of kidney strongly bind endostatin, whereas glomerular basement membrane largely fails to do so.
50 Breast ductal and acinar basement membranes bind endostatin strongly. Binding to epidermal basement membrane in skin is more variable. Some areas of the dermal-epidermal junction showing weak binding alternate with sections lacking any binding interaction.
50 We observed this variability in the level of endostatin and coll XVIII in choroidal structures but not in the retina. In the eye, coll XVIII/endostatin-deficient mice showed a rupture of the posterior iris pigment epithelial (IPE) cell layer with pigment dispersion suggesting the importance of coll XVIII/endostatin in stabilizing or adherence of ocular epithelial cells on their basement membrane.
51 The localization of coll XVIII and endostatin to RPE basal lamina suggests that they are important in RPE stabilization and function as well. This has recently been demonstrated in the aged coll XVIII knockout mice where there is massive accumulation of sub-RPE deposits that are very similar to BLDs in human AMD.
45 Overall, the endostatin antibody-binding pattern closely resembled the distribution reported for coll XVIII, endostatin’s parent molecule, in our study and the study of others.
33
The importance of endostatin as an antiangiogenic factor in the eye has been reinforced recently by several studies. In mice, coll XVIII has been demonstrated prominently in all ocular basement membranes except Descemet’s membrane.
42 In knockout mice without coll XVIII and therefore endostatin, there is a delayed regression of the hyaloid vessels in the vitreous along the ILM of the retina (VHP) after birth, when they have completely disappeared in wild-type mice.
42 Therefore, the absence of coll XVIII/endostatin results in vascular phenotypic changes in mice. Recently, Mori et al.
52 reported that intravenous injection of adenoviral vectors containing the endostatin gene significantly reduced laser-induced CNV in mice.
The localization of endostatin reported herein suggests that endostatin could potentially be an antagonist for the angiogenic factors present in the eyes of patients with AMD and proliferative retinopathies. To further clarify the clinical significance of endostatin, we must understand the regulation of its expression and cleavage from coll XVIII. Cathepsin L,
43 matrilysin (MMP-7),
53 and elastase
54 cleave endostatin from coll XVIII. Although cathepsin L and elastase have not been reported to be associated with the RPE-Bruch’s membrane-choriocapillaris complex, matrilysin has been demonstrated in choroidal neovascular membranes.
55 In addition, MMP-2 and −9 have been found to increase with age in this complex and are associated with areas of CNV in AMD,
55 56 57 and both of these MMPs are known to cleave endostatin from coll XVIII.
58 Therefore, during endothelial cell and RPE activation, increased production of proteolytic enzymes may result in the release of endostatin, which serves to control local angiogenesis, as proposed by Zatterstrom et al.
49 This could account for the low levels of endostatin in AMD choriocapillaris, whereas levels of coll XVIII were not significantly different from those in aged control eyes. Finally, our study suggests that the low levels of endostatin, whether free or part of coll XVIII, in AMD choroid may be permissive for formation of CNV.
Supported by National Eye Institute Grants EY01765 (to the Wilmer Ophthalmological Institute) and AR36820 (BRO), the Michael B. Panitch Fund, the Steinbach Foundation, and the Foundation Fighting Blindness.
Submitted for publication August 8, 2003; revised December 29, 2003, and February 3, 2004; accepted February 10, 2004.
Disclosure:
I.A. Bhutto, None;
S.Y. Kim, None;
D.S. McLeod, None;
C. Merges, None;
N. Fukai, None;
B.R. Olsen, None;
G.A. Lutty, None
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “
advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Corresponding author: Gerard A. Lutty, 170 Woods Research Building, Johns Hopkins Hospital, 600 North Wolfe Street, Baltimore, MD 21287-9115;
glutty@jhmi.edu.
Table 1. Characteristics of Human Donor Eyes
Table 1. Characteristics of Human Donor Eyes
| Subject | Time (hr) | | Age/Sex | Primary Cause of Death | Medical History | Ocular Diagnosis | Cryoprserved Eye | Ocular History |
|---|
| DET | PMT | | | | | | |
|---|
| Aged | | | | | | | | |
| 1 | 3.0 | 16 | 83/M | Cardiac respiratory arrest | | Normal | OS | IOL, OS |
| 2 | 2.5 | 28 | 80/M | COPD | | Normal | OS | Cataract, OU |
| 3 | 3.0 | 15 | 82/M | Metastatic Brain CA | | Normal | OS | None |
| 4 | 5.0 | 26 | 86/F | Respiratory failure | | Normal | OS | None |
| 5 | 1.0 | 26 | 77/M | COPD | HTN | Normal | OD | Unknown |
| 6 | 2.5 | 33 | 75/F | Heart disease | | Normal | OD | None |
| AMD | | | | | | | | |
| 7 | 4.0 | 20 | 93/F | Multi system failure | DM + HTN | AMD (Disc. scar) | OD | Macular degeneration, OU |
| 8 | 4.0 | 33 | 74/M | Prostate CA | | AMD (early) | OS | Macular degeneration; IOL, OU |
| 9 | 5.0 | 29 | 81/F | Myocardial infarction | HTN | AMD (early) | OD | Macular Hole, OD |
| 10 | 4.5–5.0 | 11 | 105/M | COPD | | AMD (Disc. scar) | OD | Unknown |
| 11 | 3.0 | 36 | 94/M | Cardiac failure | | AMD (Disc. scar) | OS | Macular degeneration; IOL, OS |
| 12 | 7.0 | 30 | 75/M | Aspiration pneumonia | | AMD (GA) | OS | Macular degeneration, OS |
| 13 | 3.0 | 12 | 83/M | Prostate CA | DM + HTN | AMD (early) | OD | Cataract + Maculopathy, OU |
| 14 | 3.5 | ? | 95/M | Cardiomyopathy | | AMD (Disc. scar) | OS | Legally blind, OU |
| 15 | 2.0 | 33 | 98/F | Old age | | AMD (early) | OD | IOL, OD |
The authors are grateful to the eye donors and thank their relatives for their generosity.
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