Localization of insulin-like growth factor-1 binding sites in the embryonic chicken eye.

Insulin-like growth factor-1 (IGF-1) binding sites were localized in the embryonic chicken lens, retina, and retinal pigmented epithelium (RPE) with the use of autoradiography. Each of the ocular tissues exhibited specific binding of radiolabeled ligand. Labeling occurred over the entire surface of the lens from 6-day-old embryos, including the epithelium, basal ends of the fiber cells, and the annular pad. There was relatively little labeling over the lens nucleus. A similar pattern was seen in lenses from 19-day-old embryos. In isolated retinas from embryos of this age, the inner and outer plexiform layers were most heavily labeled. In the 19-day-old RPE, only the apical surface of the cells was heavily labeled. Electron microscopic studies revealed an apical layer of membranous material that may represent outer photoreceptor segments that remained attached to the RPE during dissection. It was uncertain, therefore, whether the IGF-1 was binding to sites on the RPE or to these membrane fragments.

Identification and localization of talin in chick retinal pigment epithelial cells.

Retinal pigmented epithelial cells are adherent at their basal surface to Bruch's membrane and at their apical surface to the neural retina. We examined the expression and distribution of two proteins that are found in regions of cell-matrix interaction, talin and integrin. Talin is a 235-kDa cytoplasmic protein that has been localized to regions of cell-substrate adhesion. It binds to both integrin, a transmembrane glycoprotein complex, and to vinculin, a cytoskeletal protein. In the present study, we produced a polyclonal antibody to chicken gizzard talin. Using this antibody we showed by western blot analysis that talin is expressed by RPE cells and is found in the triton-soluble fraction. Talin was shown to co-localize with integrin and vinculin in the basal region of chick RPE cells isolated from 18-day-old chick embryos. Neither talin nor integrin was found in the apical processes or in the zonula adherens. Antibodies to vinculin showed staining both in the apical and basal regions of the RPE cells. The localization of integrin, talin and vinculin along the basal membrane suggests that this complex is important in the attachment of the RPE cells to the basement membrane. The distribution of integrin and talin was examined in primary cultures of RPE cells grown on permeable filters. In these cells, a polarized distribution of integrin and talin was not observed. This may suggest that the neural retina may be important for maintaining the differentiated state of the RPE cells.

Immunohistochemical localization of cathepsin D in ocular tissues.

Cathepsin D has been believed to play an important role in the catabolism of protein in various tissues. In retinal pigment epithelium, cathepsin D degrades rod outer segments and rhodopsin into glycopeptides. To our knowledge, no reports have described the immunohistochemical localization of cathepsin D in whole ocular tissues. We investigated the reaction of bovine, rat, and human eyes with a polyclonal antibody to cathepsin D from bovine spleen. Cathepsin D immunoreactivity was observed in the cytoplasm of the following cells: epithelium and endothelium of the cornea; keratocytes; pigmented and nonpigmented epithelium of the ciliary body; epithelium and cortex of the lens; epithelium and sphincter and dilator muscles of the iris; Müller cells; ganglion cells and pigment epithelium of the retina; and endothelium of various vessels. Positively stained ocular tissues were believed to have a high activity of protein catabolism. Since cathepsin D was closely associated with phagosomes in retinal pigment epithelium, we concluded that cathepsin D probably contributes to the physiologic degradation of rod outer segments.

Identification of an adhesion-associated protein of the retinal pigment epithelium.

The normal function of the retinal pigment epithelium (RPE) is dependent on the maintenance of tight adhesions between cells. In order to identify cell surface molecules which may be important for maintaining the integrity of the RPE, we have undertaken a combined functional, biochemical, and immunohistochemical analysis of cell surface proteins of the RPE. These studies have led to the identification of a 100-kD cell surface protein whose presence correlates with the maintenance of calcium-dependent adhesions between RPE cells. In intact RPE tissue the protein is concentrated at the junctions between RPE cells. The properties of the protein suggest that it may be a member of the cadherin family of calcium-dependent cell adhesion proteins.

Neural cell adhesion molecule (NCAM) in adult vertebrate retinas: tissue localization and evidence against its role in retina-pigment epithelium adhesion.

The presence of neural cell adhesion molecule (NCAM) was examined in the neural retina, interphotoreceptor matrix (IPM), and retinal pigment epithelium (RPE) of adult bovine and frog eyes. Using polyclonal antibodies raised against adult isoforms of NCAM. Western blot analyses revealed the presence of NCAM in the neural retina, but not in the IPM or RPE of these species. As a control, Western blot analysis was used to demonstrate the presence of interphotoreceptor retinoid-binding protein (IRBP) in the IPM preparations. NCAM immunoreactivity was detected by light microscopic immunocytochemistry primarily in the plexiform layers and nerve fibre layer of the frog retina. Minor immunoreactivity was also detected in the inner and outer nuclear layers, but there was no detectable NCAM immunoreactivity in the IPM, outer segments, or RPE. These results indicate that NCAM is not a likely participant in the process of retina-RPE adhesion in the adult eye.

Identification and localization of a beta-1 receptor from the integrin family in mammalian retinal pigment epithelial cells.

The distribution of an adhesion receptor from the integrin family was mapped in cultured mammalian retinal pigment epithelial cells (RPE), and in primate RPE cells in vivo, using antibodies to the human fibronectin receptor (FnR) in conjunction with indirect immunofluorescence and immunoelectron microscopy. Protein homogenates from human RPE or MG-63 cells were separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions. On Western blots of proteins from both cell types, anti-FnR and a monoclonal antibody to the beta 1 subunit of human FnR each recognized a single band with a molecular mass of approximately 115 kDa. After 1-6 weeks in culture human, monkey and feline RPE cells gradually acquired a morphology and cytoskeletal arrangement typical of a mature cuboidal epithelium. The distribution of anti-FnR labeling changed dramatically in accordance with the cells' phenotype. In preconfluent cells, labeling consisted of streaks and flecks of fluorescence at the termini of stress fibers and at putative sites of cell substratum attachment. As the cells became confluent and acquired an epithelioid morphology, the bulk of anti-FnR labeling shifted to the peripheral cytoplasm and appeared as a cross-hatched meshwork. In fully differentiated RPE cells anti-FnR labeling consisted of a dense punctate pattern on or close to the apical cell surface that coincided with the distribution of f-actin as shown by phalloidin staining, and to the distribution of apical microvilli as identified by scanning electron microscopy. In addition, a compacted rim of fluorescence appeared at the cells' lateral margins that was also virtually identical to the phalloidin staining pattern. No basal surface labeling was apparent at this stage. At the ultrastructural level, FnR was localized to the apical surface of nonpermeabilized RPE cells and, in particular, to the plasma membrane of apical microvilli in vitro and in vivo using an indirect, pre-embedding method. The results strongly suggest that: (1) a membrane receptor/s containing the integrin beta 1 subunit is normally present on the plasma membrane of apical microvilli and on the lateral cell surfaces of cultured mammalian RPE cells; and (2) this receptor also is present on the plasmalemma of RPE apical microvilli in vivo.

Distributions of elements in the human retinal pigment epithelium.

Distributions of elements above the atomic number of sodium were mapped in the retinal pigment epithelia of eight human eyes. X-ray energy spectra and maps were collected from cryofixed, freeze-dried, and epoxy-embedded tissues using energy-dispersive x-ray microanalysis. All eyes had high concentrations of phosphorus in the nuclei of retinal pigment epithelial cells. Melanosomes were rich in sulfur, zinc, calcium, and iron. Lipofuscin and cytoplasm contained only phosphorus and sulfur in detectable amounts. Drusen, when present, contained phosphorus and calcium. Six eyes had a prominent aluminum peak recorded from melanosomes, nuclei, and Bruch's membrane. In one pair of 90-year-old eyes, small, electron-dense deposits surrounded many melanosomes and contained mercury and selenium. Retinal pigment epithelial melanosomes may bind and accumulate metals and other potentially toxic ions over time, preventing them from reaching the neural retina.

Interphotoreceptor retinoid-binding protein (IRBP) in the postnatal developing rds mutant mouse retina: EM immunocytochemical localization.

The distribution of IRBP was examined in postnatal developing retinas of rds (020/A) mutant mice and Balb/c controls by EM immunocytochemistry. Light labeling for IRBP was detected in mutant and control retinas by postnatal day 9 (P9) largely in the interphotoreceptor matrix (IPM). At P14, some photoreceptors in the rds retina showed a higher density of label in the Golgi for IRBP than neighboring cells and those of controls processed simultaneously. This high density of label for IRBP was observed also in the Golgi of a small population of photoreceptor cells at P18, P19 and P21 in rds retinas. These cells were found to represent approximately 3-5% of the photoreceptor population. The density of label for IRBP at the apical RPE region was obviously low in the rds retinas by P18, P19 and P21. However, this same region in controls of the same ages was densely labeled for IRBP. The low density of labeling at the apical RPE region in the rds retinas may indicate a change in the rate of synthesis, secretion, distribution and/or degradation in the IPM. The high density of intracellular labeling in a small population of cells may be indicative of impaired secretion, an increase in IRBP synthesis or the initiation of photoreceptor deterioration. Whether the differences observed in the distribution of IRBP in the rds mutant are primary or secondary effects of the genetic lesion remains undetermined.

Localization of gamma glutamyl transpeptidase in bovine retina.

Gamma-glutamyl transpeptidase (GGTP) is a membrane bound enzyme which has an important role in regulation of glutathione and glutamate in the retina. We have used histochemical and colorimetric enzyme assays to localize GGTP in the bovine retina and choroid. Our results demonstrate that (i) GGTP is present in retinal microvessels but not choroidal microvessels. (ii) Retinal microvascular endothelium loses the ability to express GGTP in cultured cells. (iii) GGTP is present in Muller cells. (iv) Isolated and purified rod outer segments contain high levels of GGTP. (v) Retinal pigment epithelial cells (RPE) in vivo and in culture contain GGTP. The findings of this study lend support to the concept that GGTP may be a biochemical marker for cellular systems which are part of specialized diffusion barriers.

Bovine retinal pigment epithelium promotes proliferation of choroidal endothelium in vitro.

The invasion of Bruch's membrane and subsequently the subpigment epithelium and subretinal spaces by proliferating choroidal capillaries is a prominent feature of age-related macular degeneration. The ability to isolate and propagate pure cultures of bovine choroidal microvascular endothelium, choroidal pericytes, turbinate fibroblasts, and bovine retinal pigment epithelium allowed us to identify a growth-promoting activity produced by the retinal pigment epithelium that stimulated the proliferation of these cells in vitro. This mitogenic activity is present in both retinal pigment epithelium-conditioned medium and retinal pigment epithelium extract and enhances the mitogenic activity of retinal-derived extract. The activity is resistant to extremes of temperature and pH but partially sensitive to trypsin inactivation.

Immunogold localization of extracellular matrix molecules in Bruch's membrane of the rat.

The distribution of extracellular matrix molecules in Bruch's membrane of the rat was studied by a post-embedding immunogold labeling method. These molecules included laminin, types I, III and IV collagen, fibronectin and heparan sulfate proteoglycan. Laminin and type IV collagen were localized in the basal lamina of the pigment epithelium and choriocapillaris; types I and III collagen, to the inner and outer collagenous zones; fibronectin, to the elastic layer; heparan sulfate proteoglycan was found diffusely throughout the entire Bruch's membrane. These results not only agree with previous biochemical and immunofluorescence studies of these molecules in Bruch's membrane of other species but also provide a more precise localization of these molecules in the various layers of Bruch's membrane.

Analysis of proteins in developing rd (retinal degeneration) chick retina using two-dimensional gel electrophoresis.

The rd (retinal degeneration) chick possesses an autosomal recessive mutation which results in behavioral and electrophysiological blindness at hatch. Using the technique of two-dimensional gel electrophoresis, we have identified two groups of proteins whose expression in the retina/pigment epithelium/choroid (RET/PE/CH) of +/+, +/rd and rd/rd chicks is related to genotype. The two proteins within Group 1 had an apparent mass (Mr) of 63 kDa and isoelectric points (pI) of 6.48 and 6.55. The four proteins in Group 2 had an apparent Mr of 98 kDa and pI values ranging from 6.08 to 6.25. Quantities of the Group 1 proteins in RET/PE/CH of each of the three types of animals were found to be related to genotype; the amounts of each did not change with development. The expression of the Group 2 proteins in PET/PE/CH was found to change during retinal development. Proteins P98-6.08 (Mr-pI) and P98-6.13 were found in all rd/rd and +/rd RET/PE/CH and in most +/+ embryonic tissues. Proteins P98-6.19 and P98-6.25, which were present in the majority of +/+ embryonic and in all +/+ E21 (hatch RET/PE/CH, did not appear in +/rd tissues until hatch. P98-6.19 and P98-6.25 were never observed in rd/rd RET/PE/CH.

Transferrin receptors on the surfaces of retinal pigment epithelial cells are associated with the cytoskeleton.

Retinal pigment epithelial cells, derived from human donor eyes, have been grown in culture as monolayers on membrane filters or plastic surfaces and shown to possess transferrin receptors with a monomeric molecular mass of 93,000. These receptors internalize 125I-labelled transferrin and recycle it to the surrounding medium in a similar manner to other cell types. Scatchard analyses show that there are about 100,000 high-affinity receptors on the surface of each cell and most of these receptors are associated with the cytoskeleton. In total cell extracts, there are additional low-affinity binding sites that do not appear to be strongly associated with the cytoskeleton. The apparent interaction of transferrin receptors with the cytoskeleton was confirmed in two ways: first, using 200 kV electron microscopy for stereo analyses, skeleton-associated transferrin receptors were detected by a monoclonal anti-receptor antibody and a colloidal gold-conjugated second antibody after Triton X-100 extraction of pigment epithelial cells grown directly on laminin-coated gold grids; and, second, when cell surface receptors were labelled with radioiodinated transferrin and then incubated for various periods of time, the labelled transferrin was observed to move from a Triton X-100-insoluble fraction (a putative cytoskeletal compartment) to a Triton-soluble compartment that was not associated with the cytoskeleton. Using either horseradish peroxidase or colloidal gold-labelled transferrin, it has been shown that basolateral and apical surface-located receptors participate in receptor-mediated endocytosis via clathrin-coated pits, endosomes and tubular structures. Initially, transferrin internalized from the apical surface is observed in small endosomes that often appear to be embedded in an apical layer of microfilaments. From these peripheral regions of the cells, the labelled receptors move to larger endosomes and multivesicular bodies deeper in the cytoplasm. These structures have no apparent association with cytoskeletal elements.

Activation of Mg-ATPase of skeletal-muscle myosin by bovine retinal pigment epithelial actin.

The morphology and function of actin in cultured bovine retinal pigment epithelial (RPE) cells were studied. Filamentous actin was identified with a fluorescent mushroom toxin, nitrobenzoxadiazole (NBD)-phallacidin, specific for actin. Dark-field microscopy of cultured RPE cells revealed numerous pigment granules; fluorescent microscopy identified scattered lipofuscin granules. One-dimensional SDS polyacrylamide gel electrophoresis of urea-soluble proteins extracted from RPE cells showed a 46,000-dalton protein band which comigrated with authentic muscle actin. Densitometric scanning showed that this protein band comprised 7.6% of the total urea-soluble proteins. An actin-activated skeletal-muscle myosin Mg-ATPase assay, using skeletal-muscle heavy meromyosin as enzyme and [gamma-32P]-ATP as substrate, demonstrated functional actin in RPE cell extracts after DEAE-cellulose anion exchange chromatography. The actin-containing protein fractions were eluted at ionic strengths between 0.19 and 0.36 M KCl. The activation of myosin ATPase by actin in RPE cells provides a molecular basis for the phagocytic activity which is important in maintaining the integrity of retinal photoreceptor cells.

Attempts of tissue typing from human donor eyes.

A method for HLA-ABC typing using pigmented retinal epithelial and uveal cells of the donor eye is described and the possible implications for corneal grafting are mentioned.

Retinol-binding protein is synthesized in the mammalian eye.

As the chromophoric component of the visual pigment, retinol plays an essential role in vision. In the plasma, retinol is transported by retinol-binding protein (RBP) in complex with transthyretin (TTR, prealbumin). In previous work we demonstrated intraocular synthesis of TTR. To determine whether RBP is also synthesized in the eye, we performed Northern and Western blot analysis of rat eye, and detected both RBP mRNA and immunoreactive RBP. Regional Northern analysis of bovine eye localized RBP mRNA to ciliary body/iris and retina/RPE. Preliminary immunohistochemical studies revealed a widespread but heterogeneous distribution of RBP in rat eye. We postulate that ocular RBP and TTR are involved in the intraocular translocation of retinol.

Cytoskeletons of retinal pigment epithelial cells: interspecies differences of expression patterns indicate independence of cell function from the specific complement of cytoskeletal proteins.

In vertebrate tissue development a given cell differentiation pathway is usually associated with a pattern of expression of a specific set of cytoskeletal proteins, including different intermediate filament (IF) and junctional proteins, which is identical in diverse species. The retinal pigment epithelium (RPE) is a layer of polar cells that have very similar morphological features and practically identical functions in different vertebrate species. However, in biochemical and immunolocalization studies of the cytoskeletal proteins of these cells we have noted remarkable interspecies differences. While chicken RPE cells contain only IFs of the vimentin type and do not possess desmosomes and desmosomal proteins RPE cells of diverse amphibian (Rana ridibunda, Xenopus laevis) and mammalian (rat, guinea pig, rabbit, cow, human) species express cytokeratins 8 and 18 either as their sole IF proteins, or together with vimentin IFs as in guinea pig and a certain subpopulation of bovine RPE cells. Plakoglobin, a plaque protein common to desmosomes and the zonula adhaerens exists in RPE cells of all species, whereas desmoplakin and desmoglein have been identified only in RPE desmosomes of frogs and cows, including bovine RPE cell cultures in which cytokeratins have disappeared and vimentin IFs are the only IFs present. These challenging findings show that neither cytokeratin IFs nor desmosomes are necessary for the establishment and function of a polar epithelial cell layer and that the same basic cellular architecture can be achieved by different programs of expression of cytoskeletal proteins. The differences in the composition of the RPE cytoskeleton further indicate that, at least in this tissue, a specific program of expression of IF and desmosomal proteins is not related to the functions of the RPE cell, which are very similar in the various species.

The cytoskeletal elements of human retinal pigment epithelium: in vitro and in vivo.

The cytoskeletal elements of normal (in situ) and cultured human retinal pigment epithelium (RPE) were studied by a variety of immunocytochemical techniques. Primary antibodies to vimentin and cytokeratins were used. Positive immunoreactivity for vimentin was obtained with in situ and cultured material. The pattern of reactivity obtained with antisera and monoclonals to cytokeratins was more complex. Cytokeratin immunoreactivity could be demonstrated in situ and in cultured cells. The pattern of cytokeratin expression was similar to that of simple or glandular epithelia. A monoclonal antibody that specifically recognizes cytokeratin 18 identified a population of cultured RPE cells that had particularly well-defined filamentous networks within their cytoplasm. Freshly isolated RPE was cytokeratin 18 negative by immunofluorescence, but upon culture cytokeratin 18 positive cells were identifiable. Cytokeratin 18 positive cells were identified in all RPE cultures (other than early primaries), regardless of passage number, age or sex of the donor. In post-confluent cultures cytokeratin 18 cells were identified growing over cytokeratin 18 negative cells, suggesting an association of cytokeratin 18 immunoreactivity with cell proliferation. Immunofluorescence studies of retinal scar tissue from two individuals revealed the presence of numerous cytokeratin 18 positive cells. These findings indicate that RPE cells can be identified by their cytokeratin immunoreactivity and that the overt expression of cytokeratin 18 may be associated with proliferation of human RPE both in vitro and in vivo.

Fluorophores of the human retinal pigment epithelium: separation and spectral characterization.

Ten fluorescent fractions originating from the chloroform extracts of retinal pigment epithelial (RPE) cells of human donor eyes (ages 52-98 yr) have been separated and characterized by UV-vis absorbance and corrected fluorescence spectroscopy. The semipurified fluorophores fall into four categories based upon their spectral properties: green-emitting fluorophores, a golden yellow-emitting fluorophore, yellow-green-emitting fluorophores and orange-red-emitting fluorophores. All share common absorbance peaks around 280- and 330 nm, and the orange-red-emitting fluorophores also exhibit a strong absorbance peak at 420 nm. No significant visible-emitting fluorophores were detected in the methanol-water phase of these extracts. While these fluorophores are abundant in extracts from adult-derived RPE, most of the fluorophores occur in much lower amounts in RPE extracts from human donors under 10 yr of age. Eyes from child human donors also have much less RPE lipofuscin than those from adult donors, suggesting that most of the fluorophores are lipofuscin derived. This interpretation is supported by the previous finding that all of the fluorophores from whole RPE are also present in extracts of purified lipofuscin granules. Characterization of the chromatographic and spectral properties of the chloroform-soluble fluorescent components from the human RPE provides an important tool for determining the mechanism of RPE lipofuscin fluorophore formation. The absorbance properties defined here are of significance to investigations into the photobiology of the RPE and to those using laser therapy in treatment of age-related retinal diseases.