Localization of Collagen XVIII and the Endostatin Portion of Collagen XVIII in Aged Human Control Eyes and Eyes with Age-Related Macular Degeneration

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 ϭ 0.037) in AMD retina and significantly lower in the choriocapillaris, Bruch's membrane, and RPE basal lam-ina of AMD choroids (P Ͻ 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.

Developmental Basis of Nanophthalmos:MFRPIs Required for both Prenatal Ocular Growth and Postnatal Emmetropization

“…Sensitive detection of MFRP protein was achieved by alkaline phosphatasecoupled immunocytochemistry, followed by a bleach of overlying melanin pigment (17). We first examined RPE of of a 67 year-old eye donor in which the retina had detached before sectioning.…”

“…This was followed with biotinylated rabbit anti-goat, streptavidin alkaline phosphatase, and then was detected by a 30 minute BCIP-NBT reaction. Treatment with peracetic acid was then used to fully uncover the BCIP reaction product by bleaching RPE melanin (17).…”

The embryonic human choriocapillaris develops by hemo‐vasculogenesis

“…Streptavidin alkaline phosphatase (APase) immunohistochemistry was performed on eight-micron-thick sections of cryopreserved tissue using a nitroblue tetrazolium (NBT) system recently developed by Bhutto et al (2004). The sections were incubated overnight at 4°C with one of the following primary antibodies: mouse anti-CD34 (1:800; Signet Laboratory); mouse anti-CD31 (1:1,000; Dako); mouse anti-CD39 (1:400; Chemicon); mouse anti-CD45 (1:800, Chemicon); mouse anti-Ki67 (1:1,000; Zymed); and mouse anti-VEGFR-2 (1:600; Imclone Systems).…”

“…Melanin in RPE was bleached by a technique recently developed by Bhutto et al (2004) after streptavidin APase immunohistochemistry. When bleaching was complete, coverslips were mounted with Kaiser's glycerogel mounting medium without counterstaining.…”

The expression of advanced glycation endproduct receptors in rpe cells associated with basal deposits in human maculas

“…Streptavidin alkaline phosphatase (APase) immunohistochemistry was performed on cryopreserved tissue sections using a nitroblue tetrazolium (NBT) development system, as reported previously (Bhutto et al, 2004). 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).…”