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Purpose: To determine the relative expression of metallothionein isoforms and their differential induction by oxidative stress in cultured RPE cells and to localize the isoforms in the human chorioretinal complex., Methods: Total RNA was isolated from cultured human retinal pigment epithelial cells using TRI-Reagent. An "anchor-oligo-dT primer" was used for the synthesis of cDNA, reverse transcribed using avian reverse transcriptase and subsequently subjected to PCR analysis using oligonucleotides specific for metallothionein (MT) I, MT II, and MT III. The selected transcripts were then used to assess the expression of the above elements in fixed tissue sections by in situ hybridization. Cultured RPE cells were allowed to phagocytose bovine photoreceptor outer segments (ROS) or were treated with H(2)O(2) for 6 hours and then analyzed by RT-PCR or in situ hybridization to ascertain the effect of oxidative stress on metallothionein mRNA isoform expression., Results: Relative density analysis of amplified products demonstrate the presence of MT I, MT II and MT III in RPE cells, with an apparent relative expression MT II > MT I > MT III [corrected]. Expression of MT I and MT II mRNA was increased by both phagocytosis and hydrogen peroxide, however MT III was not induced by either stress. In situ hybridization corroborated the findings of the RT-PCR analysis and showed that MTs were mainly localized in the RPE and the photoreceptor layer of the retina., Conclusions: The localization of MT and the response of MT to oxidative stress are consistent with a role for MT as an antioxidant in the RPE and retina. Studies are ongoing to determine the specific mechanisms of action of these antioxidants in RPE cells.

Objective: Hyaluronic acid (HA) has a key role in the structure and organization of the extracellular matrix. We sought to identify the distribution of HA in human eye tissue with regard to age using a biotinylated HA-binding protein., Methods: Fetal and adult (from donors ranging from 28 to 94 years of age) eye tissues were fixed less than 24 hours post mortem and embedded in JB-4 medium (Polysciences, Warrington, Pa). Sections of 2-microns thickness were used. Control sections were pretreated either with Streptomyces hyaluronidase or HA-binding protein inactivated by HA. The binding of the protein to HA was detected with avidinbiotin alkaline phosphatase and developed by incubation with naphthol as-mx phosphate and Texas Red Salt (Pierce, Rockford, Ill)., Results: Specific staining for HA was observed in fetal eyes in the choroid, Bruch's membrane, sclera, retinal pigment epithelium, and developing retina from the vitreoretinal interface to the inner plexiform layer. Specific staining decreased with age in the choroid, retinal pigment epithelium, and Bruch's membrane. Hyaluronic acid-specific staining was undetectable in tissues from donors over 50 years of age., Conclusions: The localization of HA in the chorioretinal complex and its disappearance after the fifth decade of life may play a role in aging and age-related retinal disorders.

We studied zinc uptake by nonhuman primate retinal pigment epithelium (RPE) and choroid, using 65Zn as a probe. With intravenously administered 65ZnCl2, virtually all detectable tracer was lost from the plasma after 20 hours but the pigment epithelium-choroid showed prominent uptake and retention. Plasma concentrations of oral 65ZnO remained high 20 hours after feeding. Uptake and retention of orally administered 65Zn as 65ZnO from the bloodstream by the RPE/choroid was avid in both young and old animals. Excretion in urine and feces was minimal. All pigmented ocular tissues took up and retained 65Zn. A survey of total zinc content of human and nonhuman primate ocular tissues showed that the pigmented tissues had consistently higher concentrations of zinc. Our results demonstrate for the first time direct uptake and retention of zinc from the blood by primate RPE and other ocular tissues.

Bruch's membrane may provide a selective filtration barrier for nutrients coming from the choriocapillaris to the outer retina. Because proteoglycans have been shown to have structural and filtration properties in other tissues, we have been investigating the deposition of newly synthesized proteoglycans into Bruch's membrane and how these may be affected by aging and pathology. Proteoglycans deposited in human Bruch's membrane were metabolically labelled with 35SO4 and 3H-glucosamine using a whole-eye organ culture system. Labeled proteoglycans were extracted from dissected Bruch's membranes with 4 M guanidine and isolated by ion-exchange column chromatography on DEAE cellulose using a linear salt gradient. These molecules were subsequently chromatographed on Sepharose CL-4B and glycosaminoglycan content characterized by enzymatic and chemical degradation. The elution profiles for proteoglycans remains relatively unchanged with age, although in eyes from donors over age 70 there is a small change in size distribution toward higher molecular weights. However, the proportions of newly synthesized glycosaminoglycans remain unchanged with age, being approximately 75% chondroitin sulfate/dermatan sulfate and 25% heparan sulfate. Bruch's membrane proteoglycans from donors with different retinal pathologies, however, exhibited an increased proportion of heparan sulfate. Considering the structural and filtration properties of proteoglycans, such alterations could result in abnormal functioning of Bruch's membrane that could ultimately affect the maintenance of the outer retina.

We used specific antibodies to localize a variety of extracellular matrix components by indirect immunofluorescence reactions on cryostat sections of drusen-bearing human donor eye tissue. These included collagen types I through V, fibronectin, laminin, heparan sulfate-containing basement membrane proteoglycan as well as antibodies to human IgG and IgM. The composition of drusen with respect to these specific molecules varied greatly, even within the same region of an eye. Hard or nodular drusen were more likely to show a discrete organized layer of specific basement membrane molecules over their apical surfaces than were diffuse type drusen. Diffuse but not nodular drusen generally contained fibronectin. Immunohistochemical reactivity of Bruch's membrane showed age-related accumulation of type I collagen and localized changes associated with some drusen.

Proteoglycans, extracellular matrix molecules that have been shown to have filtration properties in some tissues, make up a significant proportion of the structural macromolecules of Bruch's membrane. Bruch's membrane may provide a selective filtration barrier between the choriocapillaris and the pigmented epithelium (PE) and outer retina. In this paper, we compare the proteoglycans extracted from metabolically-labeled normal and retinitis pigmentosa (RP) Bruch's membranes. Isolated RP Bruch's membrane proteoglycans were larger than those from normal donor eyes when chromatographed on a column of Sepharose CL-4B. In addition to the increased size, there was also a dramatic increase in the proportion of heparan sulfate proteoglycan being synthesized in RP. Considering the structural and filtration properties of proteoglycans, alterations such as these could affect the functioning of Bruch's membrane and, possibly, the PE and the outer retina.

The proteoglycan components of Bruch's membrane have not been well characterized to date. In this study, the glycosaminoglycans present in Bruch's membrane were identified and found to be heparan sulfate with small amounts of chondroitin and/or dermatan sulfate and hyaluronic acid. The biosynthesis of glycosaminoglycans and their incorporation into proteoglycans was investigated using an eye organ culture in which the cornea, iris, and sclera had been removed. The newly synthesized proteoglycan(s) extracted with 4 M guanidine hydrochloride bound to a DEAE-cellulose column and eluted with approximately 0.44 M NaCl. The proteoglycan(s) had a molecular weight ranging from 100,000 to 150,000 daltons. After papain treatment, the glycosaminoglycan side chains had a molecular weight of approximately 44,000 daltons. The newly synthesized proteoglycan(s) contained 65% chondroitin and/or dermatan sulfate and 35% heparan sulfate. This organ culture system should be useful in studying disease states of Bruch's membrane.

Aging changes known to occur in Bruch's membrane may be associated with drusen formation and retinal pigment epithelial mottling, which often have a peripheral as well as a macular distribution. Few details of peripheral Bruch's membrane aging changes have been reported. We conducted a histochemical investigation of Bruch's membrane in 31 postmortem donor eyes and an ultrastructural morphometric investigation of these regional changes in a subgroup of 22 eyes. The age of our donors ranged from 12 days to 80 years. When we reacted 5 micron paraffin embedded sections of chorio-retinal complex fixed in 2% glutaraldehyde-1% paraformaldehyde, we observed increased PAS positivity, and staining with Weigert's elastin and alcian blue at pH 2.5 in Bruch's membranes from donors over 46 years. In this older group, the macula showed increased histochemical reactivity for glycoconjugates, glycosaminoglycans, collagen and elastin as compared with the equator and periphery. Thickening of Bruch's membrane was first detected in the periphery in tissue from donors aged 10 to 45. Thickening of macular Bruch's membrane was first detected in tissue from donors older than 45. The major proportion of thickening, which appears to be due to the deposition of fibrillar and amorphous material, occurred earlier in the inner than the outer collagenous zone. The equatorial region was relatively spared. Our findings confirm and extend the observations of the timing, regional predeliction, and extent of Bruch's membrane aging changes.