May 2004
Volume 45, Issue 5
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Retinal Cell Biology  |   May 2004
Localization of Collagen XVIII and the Endostatin Portion of Collagen XVIII in Aged Human Control Eyes and Eyes with Age-Related Macular Degeneration
Author Affiliations
  • Imran A. Bhutto
    From The Wilmer Ophthalmological Institute, Department of Ophthalmology, The Johns Hopkins Hospital, Baltimore, Maryland; and the
  • Sahng Y. Kim
    From The Wilmer Ophthalmological Institute, Department of Ophthalmology, The Johns Hopkins Hospital, Baltimore, Maryland; and the
  • D. Scott McLeod
    From The Wilmer Ophthalmological Institute, Department of Ophthalmology, The Johns Hopkins Hospital, Baltimore, Maryland; and the
  • Carol Merges
    From The Wilmer Ophthalmological Institute, Department of Ophthalmology, The Johns Hopkins Hospital, Baltimore, Maryland; and the
  • Naomi Fukai
    Department of Cell Biology, Harvard Medical School, Boston, Massachusetts.
  • Bjorn R. Olsen
    Department of Cell Biology, Harvard Medical School, Boston, Massachusetts.
  • Gerard A. Lutty
    From The Wilmer Ophthalmological Institute, Department of Ophthalmology, The Johns Hopkins Hospital, Baltimore, Maryland; and the
Investigative Ophthalmology & Visual Science May 2004, Vol.45, 1544-1552. doi:10.1167/iovs.03-0862
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      Imran A. Bhutto, Sahng Y. Kim, D. Scott McLeod, Carol Merges, Naomi Fukai, Bjorn R. Olsen, Gerard A. Lutty; Localization of Collagen XVIII and the Endostatin Portion of Collagen XVIII in Aged Human Control Eyes and Eyes with Age-Related Macular Degeneration. Invest. Ophthalmol. Vis. Sci. 2004;45(5):1544-1552. doi: 10.1167/iovs.03-0862.

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      © ARVO (1962-2015); The Authors (2016-present)

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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). 
Methods
Donor Eyes
Human donor eyes were obtained with the help of Janet Sunness and Carol Applegate at the Wilmer Ophthalmological Institute (Baltimore, MD), and the National Disease Research Interchange (NDRI; Philadelphia, PA). Eyes of the following donors were used in the study: nine subjects with AMD (age range, 74–105 years; mean age, 88.6 ± 8.3 years); six aged control donors (age range, 75–86 years; mean age, 80.5 ± 3.6 years) without evidence or history of chorioretinal disease. All donors were white. 1 includes the postmortem time and death to enucleation time, donor age, sex, cause of death, and the medical and ocular history. The protocol of the study adhered to the tenets of the Declaration of Helsinki regarding research involving human tissue. The diagnosis of AMD was made by reviewing systemic and ocular medical history on the eye bank transmittal sheet and the postmortem gross examination, using transmitted and reflected illumination with a dissecting microscope (Stemi; Carl Zeiss Meditec, Inc., Thornwood, NY). 
Tissue Preparation and Sectioning
After a deep incision was made 0.5 cm posterior to the limbus, the anterior segment of the eye was removed, and the eye cup was examined by stereomicroscopy (Stemi 2000; Carl Zeiss Meditec, Inc.). Gross images were obtained from a digital camera (Q-imaging; Vancouver, British Columbia, Canada) and imported directly into image-analysis software (Photoshop ver. 6.0; Adobe Systems Inc., San Jose, CA; running on a PowerMac G3; Apple Computer, Cupertino, CA). Eyes were fixed in 2% paraformaldehyde at room temperature for 1 hour, cryopreserved with increasing concentrations of sucrose, and serially sectioned as previously described. 44 Almost all sections used in this study were from the inferior macula. 
Immunohistochemistry
Streptavidin alkaline phosphatase (APase) immunohistochemistry was performed on cryopreserved tissue sections using a nitroblue tetrazolium (NBT) development system. In brief, 8-μm-thick cryosections were permeabilized with absolute methanol and blocked with 2% normal goat or rabbit serum in Tris-buffered saline (TBS; pH 7.4 with 1% BSA). Sections were also blocked with an avidin-biotin complex (ABC) blocking kit (Vector Laboratories, Inc., Burlingame, CA). After they were washed in TBS, the sections were incubated overnight at 4°C with one of the following primary antibodies: goat anti-human endostatin (1:4000; R&D Systems, Minneapolis, MN), mouse anti-human endostatin 45 (1:250), and rabbit anti-mouse coll XVIII 42 (1:2000). The coll XVIII antibody recognizes the common region of the amino terminus of coll XVIII. The endostatin antibodies recognize endostatin whether free (cleaved from coll XVIII) or part of the coll XVIII molecule. Therefore, when endostatin immunoreactivity is reported in this article, it represents endostatin that is free or part of coll XVIII. To assure that the endostatin immunoreactivity was bona fide, anti-endostatin antibody was preincubated with a 200-fold excess of human endostatin or human serum albumen overnight at 4°C before use. 14 Blood vessels were identified with mouse anti-human CD-34 (1:400; Signet Laboratory, Dedham, MA) antibody in adjacent sections. After they were washed in TBS, sections were incubated for 30 minutes at room temperature with the appropriate biotinylated secondary antibodies diluted 1:500 (Kirkegaard and Perry, Gaithersburg, MD). Finally, sections were incubated with streptavidin APase (1:500; Kirkegaard and Perry), and APase activity was developed with a 5-bromo-4-chloro-3-indoyl phosphate (BCIP)-NBT kit (Vector Laboratories, Inc.), yielding a blue reaction product. 
PAS–APase Staining Method
The PAS/APase staining method was used to identify viable choroidal capillaries (APase activity) and basement membranes and basal laminar deposits (BLDs; PAS staining). In brief, the sections were incubated for 15 minutes in APase medium 46 at 37°C followed by washing in several changes of distilled water. The sections were then placed into freshly prepared 0.5% periodic acid for 5 minutes, followed by a brief wash in distilled water. The sections were then treated in Schiff’s reagent for 10 minutes and developed in several changes of tap water until the water appeared clear. All reagents were purchased from Sigma-Aldrich (St. Louis, MO). 
Tissue Bleaching
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. 
Immunoreactivity Grading System
Three independent observers, using a grading system previously described, 47 48 scored the relative intensity of the immunoreactivity for each antibody in different structures blindly. The grades in the system were: 8, uniformly intense immunoreactivity; 7, patchy and intense; 6, uniform and moderate; 5, patchy and moderate; 4, uniform and weak; 3, patchy and weak; 2, uniform and very weak; 1, patchy and very weak; and 0, comparable to a nonimmune IgG control section. 
Statistical Analysis
Mean scores ± SD from the graders were calculated for each retinal and choroidal structure. Probabilities were determined by comparing mean scores from the aged control eyes with scores from eyes with AMD using the Students t-test and assuming unequal variance and two tails. P ≤ 0.05 was considered significant. 
Results
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
In retina, the immunostaining of coll XVIII and endostatin was most intense in the internal limiting membrane (ILM) and large retinal blood vessels 1 . There was no significant difference in immunoreactivity level between coll XVIII and endostatin antibodies in large retinal blood vessels. However, coll XVIII immunoreactivity was lower in retinal capillaries than in large retinal vessels. In contrast, endostatin was barely observed in retinal capillaries 1 . Immunostaining of endostatin and coll XVIII was patchy and very weak or absent in the inner neural retina and the interphotoreceptor matrix. 
Incubation of the anti-endostatin antibody with human endostatin before use on tissue eliminated binding of the antibody to tissue, whereas incubation of the coll XVIII antibody with human endostatin had no effect on coll XVIII antibody 2 . Use of control mouse IgG instead anti-endostatin antibody resulted in very little immunoreactivity. 
In the choroid, immunoreactivity was not uniform but rather heterogenous, and therefore the scores of immunoreactivity represent the grader’s overall impression of the immunoreactivity in a particular structure throughout the whole tissue section. Bruch’s membrane and choriocapillaris basement membrane had prominent coll XVIII and endostatin immunoreactivity 3 . At higher magnification 2 4 , it was apparent that the basal lamina of RPE cells was intensely labeled for both coll XVIII and endostatin. Large choroidal vessels, intercapillary septa, and the stroma had weaker labeling with both antibodies than choriocapillaris basement membranes. In choroidal arteries, the reaction product for coll XVIII and endostatin appeared more prominent on the abluminal side of the vessels adjacent to the smooth muscle cells (data not shown). A distinct but less intense endostatin and coll XVIII staining was also found in the abluminal surface of the choroidal veins and venules of variable sizes. No significant difference in immunoreactivity scores between arteries and veins was noted. In some choroidal tissue sections, leukocytes within choroidal vascular lumens were also weakly immunostained for coll XVIII and endostatin (data not shown). 
Expression of Endostatin and Collagen XVIII in AMD Retina and Choroid
Retinas of AMD eyes had a pattern of coll XVIII and endostatin staining similar to aged control eyes (data not shown). There was no significant difference in the retinal localization of both antibodies between control and AMD eyes. However, the immunoreactivity score of endostatin was significantly higher in ILM (P = 0.037) in AMD retinas than in aged control eyes. 
In the choroid, coll XVIII immunoreactivity was significantly lower in RPE basal lamina and Bruch’s membrane of AMD eyes compared with aged eyes (P = 0.02 and P = 0.0007 respectively; 5 ). There was no significant difference in immunoreactivity for coll XVIII in choriocapillaris and intercapillary septa in AMD compared with aged control eyes. Expression of endostatin was significantly reduced or nearly absent in the choroid-Bruch’s membrane-RPE complex of AMD eyes compared with aged control eyes 3 4 . The immunoreactivity scores for endostatin were significantly lower in the RPE basal lamina (P = 0.03), Bruch’s membrane (P < 0.001), choriocapillaris (P < 0.001), and intercapillary septa (P = 0.002) in AMD compared with aged control eyes 5 . Mean immunoreactivity scores for the choroidal structures of the control aged versus AMD eyes are shown in 5 . BLDs were positive for coll XVIII immunoreactivity 4 6 . Endostatin immunoreactivity was low in BLD. The identity of BLD was confirmed with PAS staining on serial sections 4
In advanced AMD cases (subjects 7, 10, 11, 12, 14) having disciform scars with small choroidal neovascularization (CNV) formations or geographic atrophy with subpigment epithelial neovascularization, there was intense coll XVIII immunoreactivity in scars and neovascularization, whereas the endostatin was nearly absent in the scar and CNV 6 7
Discussion
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. 
Table 1.
 
Characteristics of Human Donor Eyes
Table 1.
 
Characteristics of Human Donor Eyes
SubjectTime (hr)Age/SexPrimary Cause of DeathMedical HistoryOcular DiagnosisCryoprserved EyeOcular History
DETPMT
Aged
  13.01683/MCardiac respiratory arrestNormalOSIOL, OS
  22.52880/MCOPDNormalOSCataract, OU
  33.01582/MMetastatic Brain CANormalOSNone
  45.02686/FRespiratory failureNormalOSNone
  51.02677/MCOPDHTNNormalODUnknown
  62.53375/FHeart diseaseNormalODNone
AMD
  74.02093/FMulti system failureDM + HTNAMD (Disc. scar)ODMacular degeneration, OU
  84.03374/MProstate CAAMD (early)OSMacular degeneration; IOL, OU
  95.02981/FMyocardial infarctionHTNAMD (early)ODMacular Hole, OD
 104.5–5.011105/MCOPDAMD (Disc. scar)ODUnknown
 113.03694/MCardiac failureAMD (Disc. scar)OSMacular degeneration; IOL, OS
 127.03075/MAspiration pneumoniaAMD (GA)OSMacular degeneration, OS
 133.01283/MProstate CADM + HTNAMD (early)ODCataract + Maculopathy, OU
 143.5?95/MCardiomyopathyAMD (Disc. scar)OSLegally blind, OU
 152.03398/FOld ageAMD (early)ODIOL, OD
Figure 1.
 
Immunolocalization of endostatin and coll XVIII in aged control retina (case 3). Immunostaining of CD-34 was associated with the retinal blood vessels (A). Localization with rabbit anti-mouse coll XVIII in an adjacent section showed intense coll XVIII immunoreactivity in the ILM and in a large retinal blood vessel (B), with significantly less immunoreactivity in retinal capillaries. Both mouse anti-human and goat anti-human endostatin antibodies (C, D) showed a similar pattern of immunostaining, but the goat antibody reaction product was slightly more pronounced at the level of the ILM and large retinal blood vessels. The pattern and intensity of the immunoreaction product is similar between the coll XVIII and endostatin antibodies, but the endostatin immunoreactivity is less than coll XVIII immunoreactivity in retinal capillaries.
Figure 1.
 
Immunolocalization of endostatin and coll XVIII in aged control retina (case 3). Immunostaining of CD-34 was associated with the retinal blood vessels (A). Localization with rabbit anti-mouse coll XVIII in an adjacent section showed intense coll XVIII immunoreactivity in the ILM and in a large retinal blood vessel (B), with significantly less immunoreactivity in retinal capillaries. Both mouse anti-human and goat anti-human endostatin antibodies (C, D) showed a similar pattern of immunostaining, but the goat antibody reaction product was slightly more pronounced at the level of the ILM and large retinal blood vessels. The pattern and intensity of the immunoreaction product is similar between the coll XVIII and endostatin antibodies, but the endostatin immunoreactivity is less than coll XVIII immunoreactivity in retinal capillaries.
Figure 2.
 
Endostatin and coll XVIII localization in serial sections from an aged control eye (case 5). (A) A section of choroid immunostained with coll XVIII antibody preincubated overnight with BSA showed prominent labeling of Bruch’s membrane, choriocapillaris basement membrane, and intercapillary septa. This is considered a mock-blocked sample, because it was preincubated with a control protein. (B) Anti-coll XVIII that was preincubated overnight with recombinant human
 
endostatin. A pattern and intensity of immunoreaction product was observed similar to that in (A), and therefore endostatin did not block the binding of the coll XVIII antibody. (C) The choroid section was immunostained with goat anti-endostatin preincubated overnight with BSA, whereas choroidal immunoreactivity was eliminated by preincubating the antibody with the recombinant human endostatin overnight (D). There was no immunoreactivity when the section was incubated with nonimmune goat IgG at the same concentration as the endostatin antibody (E). A higher magnification of endostatin immunoreactivity in a mock-blocked section (C) clearly demonstrated immunoreactivity in the basal lamina of the RPE (F, arrow, RPE basal lamina; arrowhead, Bruch’s membrane).
Figure 2.
 
Endostatin and coll XVIII localization in serial sections from an aged control eye (case 5). (A) A section of choroid immunostained with coll XVIII antibody preincubated overnight with BSA showed prominent labeling of Bruch’s membrane, choriocapillaris basement membrane, and intercapillary septa. This is considered a mock-blocked sample, because it was preincubated with a control protein. (B) Anti-coll XVIII that was preincubated overnight with recombinant human
 
endostatin. A pattern and intensity of immunoreaction product was observed similar to that in (A), and therefore endostatin did not block the binding of the coll XVIII antibody. (C) The choroid section was immunostained with goat anti-endostatin preincubated overnight with BSA, whereas choroidal immunoreactivity was eliminated by preincubating the antibody with the recombinant human endostatin overnight (D). There was no immunoreactivity when the section was incubated with nonimmune goat IgG at the same concentration as the endostatin antibody (E). A higher magnification of endostatin immunoreactivity in a mock-blocked section (C) clearly demonstrated immunoreactivity in the basal lamina of the RPE (F, arrow, RPE basal lamina; arrowhead, Bruch’s membrane).
Figure 3.
 
Serial sections of choroid incubated with anti-coll XVIII (E, F) and anti-endostatin (G, H) from an aged control eye (case 2) and an AMD eye (case 15). Pigment in the sections was bleached from RPE and choroidal melanocytes. (A, B) Immunostaining of CD-34 was associated with choroidal vessels including the choriocapillaris. (C, D) The blue APase reaction product was present in the choriocapillaris and the pink PAS reaction product was present in Bruch’s membrane, vascular basement membranes and BLDs. In aged control choroid, immunostaining of coll XVIII (E) and endostatin (G) was prominent in Bruch’s membrane and the choriocapillaris basement membrane. In AMD choroid, expression of endostatin (H) was greatly reduced compared with the aged control (G), whereas coll XVIII immunoreactivity had a similar pattern and intensity in AMD and aged control choroid, but the reaction product appeared more diffuse (E, F).
Figure 3.
 
Serial sections of choroid incubated with anti-coll XVIII (E, F) and anti-endostatin (G, H) from an aged control eye (case 2) and an AMD eye (case 15). Pigment in the sections was bleached from RPE and choroidal melanocytes. (A, B) Immunostaining of CD-34 was associated with choroidal vessels including the choriocapillaris. (C, D) The blue APase reaction product was present in the choriocapillaris and the pink PAS reaction product was present in Bruch’s membrane, vascular basement membranes and BLDs. In aged control choroid, immunostaining of coll XVIII (E) and endostatin (G) was prominent in Bruch’s membrane and the choriocapillaris basement membrane. In AMD choroid, expression of endostatin (H) was greatly reduced compared with the aged control (G), whereas coll XVIII immunoreactivity had a similar pattern and intensity in AMD and aged control choroid, but the reaction product appeared more diffuse (E, F).
Figure 4.
 
Sections of choroid from an aged control eye (E, G; case 2) and an AMD eye (F, H; case 13) showing immunostaining of coll XVIII and endostatin antibodies at higher magnification. Pigment in sections was bleached from RPE and choroidal melanocytes (A, B, EH). Immunostaining of CD-34 was associated with choroidal vessels including the choriocapillaris (A, B). (C, D) PAS/APase reaction product and not bleached to demonstrate BLDs and basement membranes (pink) and viable, APase-positive, blood vessels (blue). In aged control, RPE basal lamina (arrow), Bruch’s membrane, and choroidal capillaries are prominently labeled with both antibodies (E, G). In AMD choroid (H), endostatin immunoreactivity was very weak compared with the aged control choroid (G), whereas coll XVIII immunoreactivity had a pattern and intensity in the AMD choroid (F) similar to that in the aged control (E). BLDs ( Image not available ) were labeled with coll XVIII antibody (F) and were prominently stained with PAS (D).
Figure 4.
 
Sections of choroid from an aged control eye (E, G; case 2) and an AMD eye (F, H; case 13) showing immunostaining of coll XVIII and endostatin antibodies at higher magnification. Pigment in sections was bleached from RPE and choroidal melanocytes (A, B, EH). Immunostaining of CD-34 was associated with choroidal vessels including the choriocapillaris (A, B). (C, D) PAS/APase reaction product and not bleached to demonstrate BLDs and basement membranes (pink) and viable, APase-positive, blood vessels (blue). In aged control, RPE basal lamina (arrow), Bruch’s membrane, and choroidal capillaries are prominently labeled with both antibodies (E, G). In AMD choroid (H), endostatin immunoreactivity was very weak compared with the aged control choroid (G), whereas coll XVIII immunoreactivity had a pattern and intensity in the AMD choroid (F) similar to that in the aged control (E). BLDs ( Image not available ) were labeled with coll XVIII antibody (F) and were prominently stained with PAS (D).
Figure 5.
 
Mean immunoreactivity scores ± SD for choroidal structures of aged control and AMD eyes. The immunoreactivity scores for both mouse anti-human endostatin and goat anti-human endostatin antibodies were significantly decreased in RPE basal lamina (P = 0.03), Bruch’s membrane (P < 0.001), choriocapillaris (P < 0.001), and intercapillary septa (P = 0.002) in AMD eyes compared with the aged control eyes. There was no significant difference in the immunoreactivity score for the coll XVIII in choriocapillaris and intercapillary septa between AMD choroids and those in aged control eyes, but there was a significant reduction in coll XVIII in RPE basal lamina (P = 0.02) and Bruch’s membrane (P = 0.0007) in AMD eyes.
Figure 5.
 
Mean immunoreactivity scores ± SD for choroidal structures of aged control and AMD eyes. The immunoreactivity scores for both mouse anti-human endostatin and goat anti-human endostatin antibodies were significantly decreased in RPE basal lamina (P = 0.03), Bruch’s membrane (P < 0.001), choriocapillaris (P < 0.001), and intercapillary septa (P = 0.002) in AMD eyes compared with the aged control eyes. There was no significant difference in the immunoreactivity score for the coll XVIII in choriocapillaris and intercapillary septa between AMD choroids and those in aged control eyes, but there was a significant reduction in coll XVIII in RPE basal lamina (P = 0.02) and Bruch’s membrane (P = 0.0007) in AMD eyes.
Figure 6.
 
AMD eye (case 12) with sub-RPE neovascularization showed intense coll XVIII immunoreactivity (B) associated with BLDs, whereas the endostatin (D) was negative in CNV and only weakly associated with BLD. CD-34 (A) and APase (C) labeling demonstrated the viable CNV (arrows).
Figure 6.
 
AMD eye (case 12) with sub-RPE neovascularization showed intense coll XVIII immunoreactivity (B) associated with BLDs, whereas the endostatin (D) was negative in CNV and only weakly associated with BLD. CD-34 (A) and APase (C) labeling demonstrated the viable CNV (arrows).
Figure 7.
 
AMD eye (case 14) with disciform scar with small CNV formation within the scar showed much more coll XVIII (B) immunoreactivity than did endostatin (D) in the scar and CNV. CD-34 (A) and APase (C) labeling demonstrated the viable CNV (arrows); (A, C, Image not available = scar).
Figure 7.
 
AMD eye (case 14) with disciform scar with small CNV formation within the scar showed much more coll XVIII (B) immunoreactivity than did endostatin (D) in the scar and CNV. CD-34 (A) and APase (C) labeling demonstrated the viable CNV (arrows); (A, C, Image not available = scar).
 
The authors are grateful to the eye donors and thank their relatives for their generosity. 
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