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Purpose: Eye development and photoreceptor maintenance is dependent on the retinal pigment epithelium (RPE), a thin layer of cells that underlies the neural retina. Despite its importance, development of RPE has not been studied by a genomic approach. In this study, a microarray expression-profiling methodology was established for studying RPE development., Methods: The intact retina with RPE attached was dissected from developing embryos, and differentially expressed genes in RPE were inferred by comparing the dissected tissues with retinas without RPE, in microarray and statistical analyses., Results: Of the probesets used, 8810 were significantly expressed in RPE at 52 hours postfertilization (hpf), of which 1443 may have biologically meaningful expression levels. Further, 78 and 988 probesets were found to be significantly over- or underexpressed in RPE, respectively, compared with retina. Also, 79.2% (38/48) of the known overexpressed probesets were independently validated as RPE-related transcripts., Conclusions: The results strongly suggest that this methodology can obtain in vivo RPE-specific gene expression from the zebrafish embryos and identify novel RPE markers.

Purpose: Mutations in the membrane frizzled-related protein (MFRP) gene cause nanophthalmos in humans, and a splice site mutation causes recessive retinal degeneration in the rd6 mouse. In human and mouse genomes, the MFRP gene lies adjoining to the complement 1q tumor necrosis factor-related protein 5 (CTRP5/C1QTNF5) gene involved in causing retinal degeneration and abnormal lens zonules in human. The purpose of this study was to characterize the spatial and temporal expression of the mouse Mfrp gene, determine tissue and subcellular localization of MFRP protein, and study its interaction with CTRP5., Methods: Expression of the Mfrp gene in the mouse was studied by quantitative (q)RT-PCR. MFRP protein expression and distribution were studied by Western blot analysis, immunohistochemistry, and immunoelectron microscopy. Interaction with CTRP5 was studied by immunoprecipitation and immunoblot analysis, using mouse eye and human retinal pigmented epithelium (RPE) choroid extracts and by expressing full-length CTRP5 and MFRP in a heterologous system., Results: The Mfrp gene is specifically expressed in RPE and ciliary body (CB), and its expression starts during early stages of embryogenesis. In the albino mouse eye, MFRP is localized to the apical and basal membranes of RPE and ciliary epithelium (CE). In addition, MFRP and CTRP5 were found to colocalize in RPE, CE, and MDCK cells, a general model of polarized epithelia. These proteins interact with each other in ocular tissues and also in a heterologous system., Conclusions: MFRP is localized to the plasma membrane of CE and RPE, and colocalizes and interacts with CTRP5 indicating a functional relationship between these two proteins.

Purpose: To study the possibility of generating photoreceptors through programming RPE transdifferentiation by examining cell differentiation after transplantation into the developing chick eye., Methods: RPE was isolated, and the cells were dissociated, cultured, and guided to transdifferentiate by infection with retrovirus expressing neuroD (RCAS-neuroD), using RCAS-green fluorescence protein (GFP) as a control. The cells were then harvested and microinjected into the developing eyes of day 5 to day 7 chick embryos, and their development and integration were analyzed., Results: Cells from the control culture integrated into the host RPE. When grafted cells were present in large number, multilayered RPE-like tissues were formed, and the extra tissues consisted of grafted cells and host cells. None of the cells from the control culture expressed photoreceptor-specific genes. In contrast, most cells from RCAS-neuroD-infected culture remained depigmented. A large number of them expressed photoreceptor-specific genes, such as visinin and opsins. Antibodies against red opsin decorated the apical tips and the cell bodies of the grafted, transdifferentiating cells. In the subretinal space, visinin(+) cells aligned along the RPE or an RPE-like structure. When integrated into the host outer nuclear layer, grafted cells emanated elaborate, axonal arborization into the outer plexiform layer of the host retina., Conclusions: Cultured RPE cells retained their remarkable regenerative capabilities. Cells guided to transdifferentiate along the photoreceptor pathway by neuroD developed a highly ordered cellular structure and could integrate into the outer nuclear layer. These data suggest that, through genetic programming, RPE cells could be a potential source of photoreceptor cells.

Purpose: Previous studies have demonstrated a role for the retinal pigment epithelium (RPE) in the development and maintenance of the choroidal vasculature, suggesting that RPE serves a trophic role for the choroidal vessels. The goal of this study was to determine the expression pattern of vascular endothelial growth factor (VEGF) and its receptors and their activation status in embryonic and adult choroid, with the purpose of providing cues regarding the role of VEGF in development and stabilization of the choroidal vasculature., Methods: Transgenic VEGF-LacZ mice were used to examine VEGF expression in embryonic and adult eyes. Expression of VEGF isoforms and receptors in the RPE-choroid complex was assessed by RT-PCR and real-time PCR. VEGF receptor 2 expression was assessed by immunohistochemistry and its activation state was examined by immunoprecipitation followed by phosphotyrosine blot., Results: VEGF is expressed by RPE throughout the choroidal vascular development and in the adult. The major VEGF isoforms detected in adult RPE were VEGF120 and VEGF164, with almost no detectable VEGF188. RT-PCR analysis showed expression of VEGF receptors and coreceptors in the RPE-choroid complex. VEGFR2 was detected in the choriocapillaris underlying the RPE. Immunoprecipitation and phosphotyrosine blot of this receptor revealed that VEGFR2 is activated in adult mouse and bovine choroids., Conclusions: The observations suggest that VEGF signaling is involved, not only in choroidal vessel formation, but perhaps also in the maintenance of the choriocapillaris.

Purpose: Best macular dystrophy is caused by mutations in the VMD2 gene, which encodes the protein bestrophin. The purpose of this study was to determine the postnatal onset of expression of bestrophin mRNA and protein in the mouse retinal pigment epithelium (RPE)., Methods: Rabbit anti-mouse bestrophin polyclonal antisera designated Pab-003 was generated against a peptide derived from the C terminus of mouse bestrophin and characterized by Western blot and immunofluorescence staining of transfected cells. Expression of bestrophin mRNA during ocular development was studied with quantitative PCR. Bestrophin protein expression in the developing eye was observed by using immunohistochemistry. The onset of mouse phototransduction was determined by conventional electroretinography (ERG)., Results: Bestrophin mRNA was detected at embryonic day 15 in whole mouse eyes by RT-PCR. Real-time quantification of mouse bestrophin mRNA levels indicated that the highest levels of mRNA were present in the early postnatal period. In contrast, bestrophin in the RPE was first detected at postnatal day (P)10 by immunohistochemistry. Phototransduction, as determined by the presence of an ERG a-wave, was first observed at P10., Conclusions: The results of this study show that mouse bestrophin mRNA is present in the eye during embryogenesis and significantly precedes the onset of bestrophin protein expression at P10. The appearance of bestrophin in the basolateral plasma membrane of the RPE is coincident with the first detectable ERG a-wave. Because bestrophin is thought to play a role in generating the light peak, a late response of the ERG, these data support a temporal role for bestrophin in RPE responses to light. Furthermore, bestrophin protein appears to be a very late marker of RPE differentiation and to be subject to strong translational control.

Purpose: To determine the localization of retinoic acid receptors (RAR) alpha, beta, and gamma and retinoid X receptors (RXR) alpha, beta, and gamma in developing and adult mouse eyes at the level of single cells., Methods: Immunohistochemistry was performed on paraformaldehyde-lysine-periodate-fixed cryosections of mouse eyes, from embryonic day 10.5 to adulthood, with polyclonal antibodies directed against each receptor isoform. Histologic sections from null mutant mice for each receptor served as negative controls., Results: RARalpha was present ubiquitously in the prenatal eye and preferentially located in the posnatal retina and ciliary body. RARbeta was detected predominantly in the periocular mesenchyme and ciliary body. RARgamma was distributed in the periocular mesenchyme, choroid, sclera, cornea, conjunctiva, and lids. RXRalpha was found preferentially in the prenatal periocular mesenchyme and retina and in the postnatal ciliary body, cornea, and conjunctiva. RXRbeta was ubiquitous at all the stages. RXRgamma was detected mainly in subsets of prenatal retinal cells and in postnatal ganglion cells as well as a subset of photoreceptor cells that were characterized as cones in adults., Conclusions: RARalpha, beta, and gamma and RXRalpha and gamma exhibit specific and dynamic patterns of distribution in ocular tissues throughout the course of development. The abundance of RARbeta, RARgamma, and RXRalpha in the periocular mesenchyme suggests that this tissue represents an important site of retinoid actions during eye development and in adulthood.

Purpose: Tissues derived from immune-privileged sites sometimes possess special characteristics that promote their own survival when transplanted to a nonprivileged site. This study was undertaken to evaluate whether retinal pigment epithelium (RPE) behaves as an immune-privileged tissue when transplanted extraocularly., Methods: RPE grafts were prepared from eyes of neonatal C57BL/6 or C57BL/6 gld/gld (deficient in CD95 ligand expression) mice. These grafts (or conjunctival grafts as positive controls) were transplanted into the anterior chamber, the subretinal space, the subconjunctival space, and underneath the kidney capsule of histoincompatible BALB/c mice. Transplant survival was evaluated by histology at selected time points after engraftment. Recipients were tested for acquisition of C57BL/6-specific delayed-type hypersensitivity (DH) and for the ability to suppress DH., Results: Allogeneic neonatal RPE grafts from normal donors showed significantly enhanced survival at all graft sites compared with conjunctival grafts. However, allogeneic RPE cell grafts from gld/gld mice were rapidly rejected after transplantation beneath the kidney capsule. Allogeneic RPE grafts placed in extraocular sites induced systemic DH directed at donor alloantigens, whereas RPE allografts placed intraocularly induced suppression of systemic DH., Conclusions: Allogeneic neonatal RPE grafts, through constitutive expression of CD95 ligand, promote their own survival at heterotopic sites. Paradoxically, these grafts also display immunogenicity. Thus, neonatal RPE tissue owes its immune privilege to the capacity to prevent immune rejection rather than to inhibit sensitization.

Purpose: Microphthalmia, a bHLH-zip transcription factor associated with the onset and maintenance of pigmentation, identifies the retinal pigment epithelial (RPE) compartment during optic vesicle and optic cup development. To determine a role for microphthalmia (mi) during eye development, the effects of an mi loss of function mutation on RPE and neural retinal were investigated in the mi/mi mouse., Methods: A series of embryonic and postnatal mi/mi and wild-type eyes were sectioned and labeled with neural retina- and RPE cell type-specific antibodies. Photoreceptor loss was quantified by counting the number of photoreceptor nuclei spanning the outer nuclear layer throughout postnatal retinal development., Results: Early neural retinal differentiation is not affected in the mi/mi mouse. The mi/mi ventral retinal pigment epithelial layer begins to develop normally, but does not pigment or attain a differentiated cuboidal morphology. The dorsal region of mi/mi retinal pigment epithelium expands and forms an ectopic retina, which develops all major retinal cell types along a similar time course as the wild type. After birth, mi/mi photoreceptors begin to form rosettes, outer segments fail to elongate, and over an extended time period, the retina degenerates., Conclusions: Together these results suggest that early retinal development can proceed normally in the mi/mi mutant, but later retinal histogenesis is dependent on the presence of a differentiated retinal pigment epithelium. Most importantly, loss of mi function permits a change in cell fate from RPE to retina in the dorsal eye.

Purpose: To measure the total amount of rhodopsin in human eyes across the life span and to test the hypothesis that the rhodopsin content of infants' and the elderly's eyes is lower than at other ages., Methods: Rhodopsin was extracted from retinal and pigment epithelial fractions of 196 eyes of 102 donors, ages 27 weeks' gestation through 94 years, using quantitative procedures. To recover photopigment bleached by unavoidable light exposure, the fractions from 78 eyes were incubated with 9-cis retinal. The total photopigment (retinal plus pigment epithelial fractions) per eye was examined for significant changes with age, using the higher value from pairs of eyes., Results: The median rhodopsin content of the higher eye of adults is 6.45 nmoles (range, 3.33-10.84 nmoles) with 8 nmoles or more recovered from 28% of all adult eyes. The rhodopsin content of infants' eyes (< 12 months post-term) is significantly lower than that of older individuals and increases with age. After infancy, no change with age is found. For both infants and adults, 9-cis retinal significantly increases the amount of photopigment recovered without reducing the variance in the amount of photopigment recovered. The rhodopsin content is estimated to be 50% of the median adult amount early in infancy, approximately 5 weeks postterm (95% confidence interval, 0-10 weeks postterm)., Conclusions: A developmental increase in rhodopsin content occurs during infancy. Thereafter rhodopsin content remains constant. The amount of rhodopsin recovered from human eyes is quite variable. Bleaching alone cannot explain the variability.

Purpose: To evaluate the attachment of human fetal rctinal pigment epithelial (HFRPE) cells to a biodegradable polymer film with subsequent formation of spheroids in vitro., Methods: Ten biodegradable polymer films with different compositions were examined for their physical properties and ease of manipulation under a dissecting microscope. The film with the most suitable handling characteristics was chosen, and a purely isolated sheet of HFRPE cells was attached to it. The purity of the cells was assessed by their pigmentation and expression of cytokeratin. Proliferation was assessed by incorporation of 5-bromo-2'-deoxyuridine (BrdtJ). Cellular structure was analyzed under light and electron microscopes, and the functional capability of the cells was evaluated by rod outer segment (ROS) phagocytosis., Results: The polymer film with composition 50:50 poly (DL-lactide) (PLA)/poly (DL-lactide-co-glycolide) (PLG) with an inherent viscosity of 1.03 dl/g was found to be the most suitable for handling under the microscope. Sheets of HFRPE cells attached to the polymer films within 48 hours and began to form spheroids. All the isolated cells were pigmented and expressed cytokeratin. They possessed a cuboidal morphology, numerous apical microvilli, and no sign of dedifferentiation. HFRPE cells produced extracellular matrix (collagen filaments) on their basal side, filling the cavities of the polymer film. The cells subsequently proliferated, incorporated BrdU, migrated onto the culture plate to form monolayers, and phagocytized ROS., Conclusions: Biodegradable polymer films can be used as a scaffold for the adhesion of the HFRPE sheet and formation of spheroids. Spheroids represent a source of high density and well-differentiated HFRPE cells that are easy to transfer. Furthermore, the stricture of the membrane makes it suitable for additional applications.

Purpose: To compare cell proliferation in vitiligo and control mouse retinal pigment epithelium (RPE) perinatally., Methods: C57BL/6J-mivit/mivit mice and congenic +/+ controls were injected once with bromodeoxyuridine 1 hour before they were killed between embryonic day 18 and postnatal day 8. Wholemounts of Carnoy-fixed posterior eyecups, minus lens and neural retina, were stained immunohistochemically to detect DNA synthesis (bromodeoxyuridine incorporation) and mitotic cells (R3 antibody binding). Cells were counted in carefully controlled sampling sites, and total RPE area and face-view cell areas were calculated. Retinal pigment epithelial cell heights were measured on light and electron micrographs., Results: Total surface areas of the mutant and control RPE monolayer were similar (I.E., RPE wholemount area was normal), but cell number was approximately doubled in the mutant RPE. By postnatal day 6, mutant cells had approximately 70% the face-view area as controls, but their heights were increased approximately 80%, so that cell volumes were near normal despite the higher packing density. Regional differences in cell size in the control RPE were absent in the mutant specimens. The mutant RPE showed an increased bromodeoxyuridine labeling index, as well as an absolute increase in the number of cells engaged in DNA synthesis and in mitosis., Conclusions: Cell genesis in the vitiligo RPE is quantitatively abnormal perinatally, well before the neural retina has been recognized to display functional or morphologic defects. Cells are being generated at an abnormally high rate, so that twice the normal number of cells are packed into a RPE of normal total area.

Purpose: The effects of adrenergic agonists and antagonists on Na+, K+, Cl- cotransport in fetal human nonpigmented ciliary epithelial (NPE) cells were investigated., Methods: 86Rb+ as a marker for K+ was used to study ouabain-insensitive, bumetanide-sensitive 86Rb+ uptake in cultured NPE monolayers. Cyclic adenosine monophosphate (cAMP) formation in NPE cells was determined by radioimmunoassay., Results: 1 microM isoproterenol caused a 1.65-fold stimulation in Na+,K+,Cl cotransport measured as bumetanide-sensitive, ouabain-insensitive 86Rb+ uptake. The half-maximal concentration for this effect was 6.4 nM, with maximal stimulation at 100 nM isoproterenol. Epinephrine stimulated Na+, K+, Cl- cotransport similarly to isoproterenol, whereas norepinephrine stimulated at much higher concentrations (half-maximal effective concentration = 1.4 microM). Stimulation of Na+, K+, Cl- cotransport by 1 microM isoproterenol was inhibited completely by the beta 2-adrenergic antagonist ICI-118,551 at 100 nM, with a half-maximal inhibitory concentration of 5 nM. Neither atenolol, a beta 1-specific adrenergic antagonist, prazosin, an alpha 1-adrenergic antagonist, nor yohimbine, an alpha 2-specific antagonist, was as effective. These four antagonists inhibited isoproterenol-stimulated cAMP formation with potencies similar to those observed against stimulated Na+, K+, Cl- cotransport. The hypotensive adrenergic antagonist timolol, propranolol, and betaxolol also inhibited Na+, K+, Cl- cotransport stimulated by isoproterenol in the order timolol > propranolol > betaxolol. Na+, K+, Cl- cotransport could be maintained in a stimulated state for at least 2 hours in the presence of agonist, but activity returned to basal levels within 20 minutes of isoproterenol removal. Adrenergic stimulation of Na+, K+, Cl- cotransport was blocked 80% to 85% by 70 microM H-89, a protein kinase A inhibitor., Conclusions: These data suggest that beta 2-adrenergic receptor activation results in increased cAMP formation and sustained stimulation of Na+, K+, Cl- cotransport in fetal human NPE cells. Protein kinase A activation is required for maximal stimulation of Na+, K+, Cl- cotransport by adrenergic agonists.

Purpose: To describe the abnormal phenotype in retinal pigment epithelium (RPE) and neural retina of vitiligo mutant mice from embryonic stages to old age., Methods: Eyes of wild-type controls and congenic vitiligo mutants were examined by light and electron microscopy from embryonic day (E) 12 to 2 years of age. The amount and distribution of pigment in the RPE was studied in wholemounts., Results: Earliest phenotypic expression of mivit is seen in the RPE, which is abnormally multilayered dorsally at E12 to E13, and contains both hyperpigmented and hypopigmented patches. Postnatally, most RPE cells have abnormally short, compact, apical microvilli not containing melanosomes and not interdigitating with rod outer segments (ROS). Rod outer segments begin to degenerate relatively late, at approximately postnatal day (P) 30, and fragments accumulate in the subretinal space; photoreceptor nuclei decrease in number progressively from approximately P60 to P500. Retinal detachment, more prominent than in most other retinal degenerations, begins as ROS break up. Additional unusual events are the appearance of macrophage-like cells in the subretinal space by P21 to P60 and extensive shedding of photoreceptor nuclei across the external limiting membrane and into the subretinal space from approximately P180 to P500. Photoreceptor cell degeneration follows a radial gradient, more severe centrally, and is more advanced superiorly than inferiorly. By 2 years, almost all rod and cone cells are gone, and the residual neural retina is invaded by heavily pigmented cells., Conclusions: The initial ocular target of the mivit gene is the RPE, which is abnormal for many weeks before photoreceptor cells differentiate and become demonstrably affected. The authors hypothesize that the slowly progressive photoreceptor cell degeneration is secondary to abnormal function of the RPE. This mutation serves to refocus attention on critical influences of the RPE on function and maintenance of photoreceptor cells.

Purpose: The authors investigated the presence of mitochondrial DNA (mtDNA) mutations in aging human retina., Methods: A quantitative polymerase chain reaction technique for studying the common mtDNA 4977-deletion (delta mtDNA4977) in retinal pigment epithelium (RPE) and neural retinal (NR) was developed., Results: Although no deletion was detected in the fetus, every adult RPE and NR had this common deletion. The ratio of the deleted delta mtDNA4977 to the total mtDNA increased significantly in elderly persons 60 to 110 years of age and was greater in peripheral than in central RPE., Conclusions: These results suggest that at least one type of mutation accumulates in the mtDNA in the retina during aging, reflecting a general phenomenon of genomic instability that could influence its function.

Purpose: The Royal College of Surgeons (RCS) rat suffers from a well-characterized, early-onset, and relentless form of photoreceptor cell degeneration. It has been shown that allografts of retinal pigment epithelial cells from normal perinatal rats have rescue effects in this condition. In preparation for human application, the authors determined whether human fetal retinal pigment epithelium (RPE) grafts have a photoreceptor rescue effect in RCS dystrophic rat retinas., Methods: Sheets of RPE from human fetal eyes (10 to 16 weeks gestational age) were isolated according to the authors' recently described method. Fragments of the RPE sheets were transplanted to the subretinal space within the superior hemisphere. Transplants were performed within the superior equatorial region of five dystrophic RCS rats, one eye per animal. A similar volume of vehicle was injected into the subretinal space of five age-matched control rats, again one eye per rat. All rats were immunosuppressed with daily injections of cyclosporine. Using light microscopy, photoreceptor cell nuclear profiles of superior equatorial (SE) and inferior equatorial (IE) regions of transplanted and sham-injected control animals were counted., Results: Four weeks after transplantation, a dramatic rescue effect was observed. Microscopically, presumptive donor RPE cells were seen as single pigmented cells and as cell clusters in the subretinal space. An outer nuclear layer three to four profiles thick was present in the area of the RPE transplant but was nearly absent in the rest of the retina, as well as in the retinas of control rats. The number of photoreceptor nuclear profiles per 100 microns was 34.7 +/- 2.2 (mean +/- SEM) in the SE region of transplanted rats and 3.5 +/- 1.4 in the same region of sham-injected rats. There were 3.0 +/- 1.0 photoreceptor nuclear profiles in the IE region of transplanted rats and 3.5 +/- 1.2 in the IE region of sham-injected eyes. No evidence of graft rejection was seen., Conclusions: This study provides the first indication that transplanted human fetal RPE cells are able to rescue photoreceptor cells in a model of hereditary retinal degeneration.

Purpose: To analyze the cytoskeletal development in neuroectodermally derived epithelial and muscle cells of the human eye during the second and third trimesters of pregnancy., Methods: Nine formalin-fixed, paraffin-embedded fetal autopsy eyes from the 13th to 40th week of gestation were studied with eight monoclonal antibodies (mAbs) to intermediate filaments and alpha-smooth muscle actin (alpha SMA) by the avidin-biotinylated-peroxidase complex method., Results: The epithelium of the iris reacted with mAbs Vim 3B4 and V9 to vimentin in all eyes. Reactivity with mAb CAM 5.2 to cytokeratin (CK) 8 and mAb CY-90 and KS-B17.2 to CK 18 disappeared from the iris epithelium by the 28th gestational week. mAb 1A4 to alpha SMA labeled its anterior layer from the 28th week on. mAbs DE-U-10 and D33 to desmin labeled dilator fibers by the 37th week, and focal reactivity for CK 8 and 18 concurrently appeared. The developing iris sphincter reacted for alpha SMA in all eyes. It was labeled increasingly for desmin from the 18th week on, whereas initial reactivity for vimentin gradually disappeared after the 22nd week. mAbs to vimentin, CK 8, and CK 18 labeled the ciliary epithelium and the retinal pigment epithelium in all eyes, except for lack of CK 8 and 18 in the nonpigmented ciliary epithelium at the 13th week. The ciliary muscle reacted uniformly for vimentin and alpha SMA and from the 16th week on for desmin., Conclusions: The results highlight the individuality of cytoskeletal profiles in the neuroectodermal epithelial and muscle cells of the eye and clarify the formation of the peculiar cytoskeleton typical of the adult human eye. They also offer a framework for detecting cytoskeletal changes in developmental anomalies of the eye after the first trimester of pregnancy.

Purpose: To determine if conditioned culture medium of photocoagulated human retinal pigment epithelial (RPE) cells contains inhibitors for the proliferation of bovine aortic endothelial cells (BAEC) and bovine retinal endothelial cells (BREC) and to characterize the nature of these inhibitory factors., Methods: Retinal pigment epithelial cells grown to confluence were photocoagulated (0.1 second, 50 microns, 350 mW) in serum-free medium. After 48 hours, the conditioned medium (PC-CM) was removed, and the effects of non-acid-treated and transiently acid-treated samples were determined on [3H]-thymidine uptake by BAEC and BREC. PC-CM was also subjected to size exclusion high-performance liquid chromatography (HPLC). Fractions were analyzed for the effects on the growth of the bovine endothelial cells before and after transient acid treatment., Results: The addition of non-acid-treated PC-CM (32% vol/vol) inhibited BREC [3H]-thymidine uptake to 18.5% of the control value. With HPLC, the inhibitory activity was recovered mainly in a fraction whose apparent molecular size was 25 kd. After transient acid treatment of the fractions, there also appeared a 100-kd inhibitor. Inhibitory effects were neutralized by pretreatment of the fractions with antiserum against transforming growth factor (TGF)-beta 2., Conclusions: Photocoagulated RPE cells secrete inhibitors of BAEC and BREC proliferation. Molecular size and immunologic properties of these inhibitors correspond to those of TGF-beta 2.

Purpose: To investigate the expression of alpha B-crystallin in the developing rat retina by immunohistochemical techniques., Methods: Rat eyes were enucleated on embryonic day 18 and on postnatal days 1, 4, 9, 15, 19, and 90. The avidin-biotin-peroxidase technique was applied to show alpha B-crystallin immunostaining. After the cornea and lens were removed, the developing rat eyeballs were solubilized by 1% sodium dodecyl sulfate. Western blot analysis and enzyme-linked immunosorbent assay (ELISA) were performed to detect alpha B-crystallin in the extracts., Results: From postnatal day 4 to postnatal day 9, alpha B-crystallin was present in retinal pigment epithelium, but it disappeared after postnatal day 15. alpha A-crystallin immunoreactivity was negative in retina and retinal pigment epithelium throughout development. The results of Western blot analysis and ELISA coincided with those of the immunostaining., Conclusions: These results show that retinal pigment epithelium in mammalian rat retina demonstrates alpha B-crystallin expression in the early developmental stages but loses it after 15 days of age.

Purpose: To determine the stoichiometry of Na+,K+,Cl- cotransport in fetal human nonpigmented ciliary epithelial cells and the effect of forskolin, an adenylyl cyclase activator, on Na+,K+,Cl- cotransport., Methods: 86Rb+ as a marker for K+ was used to study ouabain-insensitive, bumetanide-sensitive 86Rb+ uptake in cultured human nonpigmented epithelial (NPE) monolayers., Results: The dependence of ouabain-insensitive, bumetanide-sensitive 86Rb+ uptake upon Na+, K+, and Cl- concentrations was determined. Maximal uptake was observed at about 12 mM, 20 mM, and 120 mM of these ions, respectively. Analysis by Hill plot suggested that the stoichiometry of Na+,K+,Cl- cotransport is 1:1:2, making this an electroneutral process. Na+,K+,Cl- cotransport was found to be stimulated approximately 1.5- to 2-fold after incubation of cells for 15 minutes with 1 microM forskolin. neither ouabain-sensitive 86Rb+ uptake nor bumetanide-insensitive, ouabain-insensitive uptake was affected. 8-Bromoadenosine cAMP and 8-chlorophenylthio cAMP at 1 mM stimulated Na+,K+,Cl- cotransport approximately 30% to 40%, whereas 1,9 dideoxyforskolin, a non-adenylyl cyclase-activating analogue of forskolin, had little effect. Stimulation of Na+,K+,Cl- cotransport by forskolin was blocked by prior exposure of cells to 10 microM H-89, a protein kinase A inhibitor. Stimulation by forskolin was also observed in the presence of either 1 mM DIDS, 30 microM NPPB, 3 mM DPC, or 5 mM BaCl2, although all four channel blockers inhibited Na+,K+,Cl- cotransport to various degrees., Conclusions: The data suggest that the human NPE Na+,K+,Cl- cotransporter transports Na+, K+, and Cl- in the ratio of 1:1:2. Activation of adenylyl cyclase stimulates Na+,K+,Cl(-)-cotransport via a mechanism involving protein kinase A. Reduction of Na+,K+,Cl- cotransport by chloride channel blockers raises the possibility that activities of some ion channels can influence the rate of ion influx via Na+,K+,Cl- cotransport.

Purpose: Inhibition of carbonic anhydrase (CA) by acetazolamide causes a decrease in the standing potential of the retinal pigment epithelium (RPE) and an increase in the rate of subretinal fluid absorption, and it may improve cystoid macular edema. These effects are thought to be mediated by the RPE. Given the solubility coefficient of acetazolamide, the drug is most likely to act by direct inhibition of membrane-bound CA (CA IV). To identify a substrate for acetazolamide in the RPE, the distribution of CA activity and the isoform of CA in the RPE membrane were investigated., Methods: Carbonic anhydrase activity was determined by Hansson's technique in fresh human eyes from donors of both sexes and different ages. The presence of the membrane-bound isoform CA IV was investigated immunohistochemically at the light and electron microscopic level, as well as by Western blotting in fresh RPE, and in adult and fetal RPE cultures., Results: Hansson's histochemical method demonstrated CA activity on the apical and basolateral cell membrane of the RPE. Using the gamma-globulin fraction of a polyclonal antibody against pure CA IV, immunocytochemistry showed labeling for CA IV on the apical RPE membrane or morphologically polarized human adult and fetal RPE cultures. Gel electrophoresis and Western blotting demonstrated a major immunoreactive band at 55 kDa in homogenates, which was consistently reduced to approximately 35 kDa by incorporation of 0.1% Triton X-100 detergent., Conclusions: These results suggest that the clinical effects of carbonic anhydrase inhibitors on RPE function may be mediated via membrane-bound carbonic anhydrase activity in RPE and that CA IV is responsible for activity on the apical surface.

Purpose: Myosin-I is a nonfilamentous motor protein associated with the actin cytoskeleton and cellular membranes in several cell types. The occurrence and subcellular distribution of myosin-I in mammalian and fish RPE were investigated to examine the possible role of myosin-I in retinal pigment epithelium (RPE) motility processes., Methods: Antibodies directed against myosin-I proteins from bovine adrenal medulla or chicken intestinal brush border were used to examine cultured fetal human RPE cells and freshly isolated bovine or green sunfish RPE by Western immunoblots and immunocytochemistry, using both conventional and confocal fluorescence microscopy., Results: The monoclonal antibody directed against bovine adrenal myosin-I identified a single strong immunoreactive band at 116 kD in Western blots of homogenates of cultured human RPE cells and 114 kD in bovine RPE sheets. An immunoreactive band of similar molecular weight was also observed in bovine and rabbit retina. Cell fractionation studies of bovine RPE cells revealed that myosin-I was present in all fractions that included cell membranes. The polyclonal antibody directed against chicken brush border myosin-I identified doublet immunoreactive bands at 115/110 kD in Western blots of homogenates of fish retina but identified a strong predominant immunoreactive band at 140 kD in fish RPE and brain homogenates; minor bands at 115/110 kD were identified in fish RPE homogenates. Immunocytochemistry of cultured human RPE cells using the bovine adrenal myosin-I antibody revealed a broad distribution of myosin-I that appeared to be most concentrated along the length of the lateral membranes; no colocalization was seen with actin-rich stress fibers., Conclusions: Proteins immunoreactive with myosin-I antibodies are present in both RPE and retina of mammals and green sunfish. In confluent cultures of human RPE cells, myosin-I is concentrated along the lateral cell membranes of the cuboidal cells.

Purpose: The retinal pigment epithelium (RPE) lines the interface between the neural retina and the choroid. Early in chicken development, the beta 1 family of integrins resides in the apical (facing the neural retina) and basolateral (facing the choroid) membranes of RPE. Later in development, integrins reside only in the basolateral membranes, which is more typical of simple transporting epithelia. The authors examined whether the distribution of integrins is regulated by the neural retina., Methods: Individual integrins were examined by studying the individual alpha-subunits that form heterodimers with the beta 1 subunit. The expression and distribution of these subunits were determined by immunoblotting and immunocytochemistry., Results: Subunits alpha 3 and alpha 6 exemplified contrasting behaviors. Early and late in development, alpha 3 was found only in the basal membranes. As was beta 1, the distribution of alpha 6 was nonpolarized early in development but was basal later in development. The effect of the immature neural retina was determined by reconstituting the RPE:neural retinal interface in explant culture. Absent the neural retina, alpha 6 and beta 1 were removed from the apical membrane. When present, the immature neural retina maintained both subunits in the apical membrane. The neural retina was effective only if the outer (primordial photoreceptor) surface of the retina apposed the RPE., Conclusions: These data suggest that matrix or intercellular interactions determine the distribution of individual integrins. Further, the changes in integrin distribution during development reflect the maturation of the primordial interphotoreceptor matrix or photoreceptor cell layer.

Purpose: To determine the temporal and spatial localization of the messenger RNAs (mRNAs) for the two collagen alpha 1(IX) isoforms during early development of the embryonic chicken eye., Methods: The reverse transcription-polymerase chain reaction method was used to amplify mRNAs for the two collagen alpha 1(IX) isoforms. mRNA was extracted from optic vesicles of chicken embryos at stages 14 to 19 and from microdissected ocular tissues of older embryonic eyes. After synthesis of complementary DNA, the polymerase chain reaction was performed for 20, 25, or 30 cycles. This ensured a reliable estimate of the relative abundance of the two mRNAs at different stages and in different ocular tissues. Data from the polymerase chain reaction were confirmed by Northern blot analysis., Results: mRNA for the shorter alpha 1(IX) chain was present in the optic vesicle as early as stage 14, whereas mRNA for the longer alpha 1(IX) chain was not detectable until after the optic vesicle and lens started to invaginate (stage 15; day 2.5). In later stages of development, mRNAs for both alpha 1(IX) chains were present predominantly in the presumptive ciliary epithelium. They were just detectable in the neural retina at stages 20 and 23 (days 3 to 3.5). By E6 no mRNA for the shorter alpha 1(IX) isoform was detected in the retina, although a trace of the longer alpha 1(IX) isoform was still present. In the lens, mRNA for neither isoform was detectable at any stage., Conclusions: The two isoforms of alpha 1(IX) collagen mRNA are expressed differentially in space and time during early development of the embryonic chicken eye. These molecules may serve as markers for the early specialization of ciliary epithelium as a distinct region of the optic cup.

Human retinal pigment epithelium (RPE) contains two genetically distinct forms of superoxide dismutase (SOD) enzymes that scavenge harmful superoxide anions. Biochemical and immunochemical techniques were used to compare levels of copper-zinc- and manganese-containing forms of SOD (CuZn-SOD and Mn-SOD) in human adult and fetal RPE cells. It was found that Mn-SOD activity was higher in adult than fetal RPE cells, both in vivo and in vitro. Immunolocalization of Mn-SOD in cultured RPE cells showed a greater reactivity in the mitochondria of the adult cells. Primary cultures of adult RPE contained cells with various patterns of mitochondria as shown by immunolabeling for Mn-SOD. Adult RPE cells were more resistant to the effects of a superoxide generator, paraquat, which appeared to disrupt mitochondrial integrity as judged by staining with rhodamine 123. These results suggest that high levels of Mn-SOD protect mitochondria from oxidative damage that probably occurs with aging in the RPE.

Transthyretin (TTR) is a 55-kilodalton tetrameric protein that plays an important role in the plasma transport of thyroxine and retinol. Plasma TTR is synthesized in the liver, but major sites of synthesis also have been described in the choroid plexus (CP) epithelium, the visceral yolk sac, and the eye. Recently, the retinal pigment epithelium (RPE) was identified as the specific site of TTR synthesis in the rat eye, and it was suggested that this established a functional homology between the RPE and the CP epithelium. In this study, the temporal pattern of TTR mRNA expression was investigated in the rat eye and brain during development (embryonic day 10 [e10]-postnatal day 7 [P7]) by in situ hybridization and quantitative densitometry. The TTR mRNA was present in abundance in the primordial CP before organogenesis (e10-12), but in the eye, TTR mRNA first was detected at considerably lower levels after organogenesis (e16) and only in a subset of RPE cells in the equatorial region. The relative abundance of TTR mRNA in RPE rose gradually until e21, but on the first day of life a surge was seen, followed by stabilization at adult levels by P7. These findings suggest that the requirement for TTR in CP and RPE, and possibly its function, may differ during development. The postnatal surge in RPE TTR message levels raises the possibility that transcription of the TTR gene in the newborn animal may be responsive to newly encountered environmental stimuli in the perinatal period, such as incident light.

In many ocular diseases, macrophages are found in the subretinal space and probably play an important role in maintaining the disease process. Several issues concerning these cells are still unclear, such as their route of entry or their relation to the retinal pigment epithelium. The authors have found that the human fetal eye contains macrophage-like cells in the peripheral subretinal space. Their localization, distribution, and ultrastructural features are evaluated in 33 eyes from 17 specimens (12 to 22 weeks gestational age) by light microscopy and scanning and transmission electron microscopy. Subretinal macrophage-like cells occurred predominantly in the region of the ciliary folds. They were observed within the peripheral neural retina, beneath the retinal and ciliary pigmented epithelia, under Bruch's membrane, and in the choroid. This distribution suggests that one of the main entry pathways is through the vascular bed of the ciliary body. There were approximately nine subretinal space macrophages per 0.1 mm2 distributed in a regular fashion on the retinal pigment epithelial surface close to the ciliary folds, however, the incidence decreased further posteriorly where they were very rare. Their shapes varied from large flat cells with several long processes to more spherical cell bodies with a few membrane ruffles. There was also evidence that some of these cells had recently phagocytosed cell debris, including retinal pigment epithelial premelanosomes. Morphologically, these cells closely resemble supraependymal and epiplexus cells, the macrophage populations found on the cerebral ventricles, an environment that corresponds anatomically to the subretinal space.(ABSTRACT TRUNCATED AT 250 WORDS)

Although both the neural retina and the retinal pigment epithelium (RPE) arise as neighboring portions of the embryonic optic cup, these two tissues follow very different developmental pathways. In order to obtain probes for the analysis of RPE development from its earliest divergence from the neural retina to late stages of differentiation, we have developed a panel of monoclonal antibodies which recognize antigens specific to the RPE. These probes have been applied to an immunohistochemical analysis of RPE development. The results indicate that the RPE is antigenically distinct from the neural retina even before the onset of overt differentiation. In addition, the RPE layer of the retina becomes further subdivided antigenically as its distinct anterior and posterior derivatives develop. These antibodies will be useful markers in the analysis of RPE development.

The purpose of this study was to determine the ultrastructural features of optic fissure closure. Serial coronal sections of fetal eyes from the eleventh to the thirteenth gestational day were examined by light and electron microscopy. Fusion was associated with inversion of the retinal pigment epithelium at the optic fissure and it occurred first between undifferentiated cells at the junction of the retina and retinal pigment epithelium. It later extended to involve the entire thickness of the pigment epithelium and neuroretina, with the inner aspect of the latter being the last area to fuse. There was some evidence that closure does not always start at the center of the fissure as generally described, but may sometimes start near the developing papilla. At an ultrastructural level, there was multifocal disintegration of the basement membrane associated with the formation of cytoplasmic processes at these sites. Simple appositional contacts between processes on either side of the fissure comprised the first stages of fusion. Later intermediate-type junctions were formed between adjacent outer retinal cells (presumptive photoreceptors) and junctional complexes were formed at the apices of pigment epithelial cells at the site of fusion. This suggests an increase in mechanical adherence between cells. While basement membrane disintegrated at the center of the fusion site, there was a continuous layer of basement membrane at the internal and external limits of fusion. Cell death together with two morphological types of phagocytic cells were a constant feature at the fissure margins before, during and after fusion. The possible origins and roles of these cells in the fusion process is discussed.

The rate of membrane addition during outer segment development and the onset of membrane loss through shedding was evaluated for Xenopus laevis embryos reared at 23 degrees C in constant light (LL), constant darkness (DD), and cyclic light (12 hr L/12 hr D; LD). Embryos were placed under these lighting conditions 1 day following fertilization at open neural plate stages and were sampled each day for 9 days. Rod and cone outer segments were present in each treatment group on day 3 of development. At the end of 10 days of development, rod outer segment volumes were 708 +/- 195, 380 +/- 40, and 297 +/- 33 micrometer3 (mean +/- S.D.) for LL, LD, and DD treatment groups, respectively, whereas mean cone outer segment volumes were 57 +/- 8, 48 +/- 8, and 35 +/- 9 micrometer3 for these same treatment groups. The loss of outer segment material through shedding was assessed by monitoring the phagosome content of the pigment epithelium. Phagosomes were present in the pigment epithelium in DD embryos as early as 6 days of development, were present at day 5 only after the onset of the light cycle in LD embryos, and were rarely observed in LL embryos. The rate of outer segment membrane elaboration in rods (determined with 3H-leucine autoradiography) was virtually identical in LL and LD (100 and 98 micrometer3/day) but was greatly reduced in DD (70 micrometer3/day). These findings indicate that the relatively rapid rate of rod outer segment volume accumulation in LL embryos is due to a reduction in membrane loss through shedding and not to a higher rate of membrane addition as compared to animals reared in LD. In contrast, the reduced rate of rod outer segment development in DD embryos is the result of both a slower rate of membrane elaboration accompanied by the early onset and enhanced rate of membrane loss through shedding in darkness.

Cultured chick pigment epithelial (PE) cells from stages 29 to 31 chick embryos grown in normal Eagle's minimum essential medium (MEM) exhibit marked colonial organization and differentiation. Individual cells appear epithelial and heavily pigmented. In contrast, cells grown in Ham's modified F-12 medium appear fibrocytic with colonial disorganization and little visible pigmentation. Biochemically, cells grown in F-12 medium lack receptors for both 3H-retinol and 3H-retinoic acid, although cells grown in MEM exhibit specific 3H-retinol binding. Pathways of glucose utilization differ significantly in cells grown in the two media, with considerably lower overall respiratory activity and lower pentose phosphate pathway activity seen with the F-12 medium. Thus different nutritional states can markedly affect PE cell characteristics in culture and possibly in vivo as well.

The development of fibrous connective tissue in Bruch's membrane within the choroid of chronologically staged chick embryos was observed by means of transmission electron microscopy. The appearance of these connective tissue elements follows an orderly developmental sequence. The first component of Bruch's membrane to appear is a continuous basal lamina at the basal surface of the presumptive retinal pigment epithelium (RPE) at 21/2 days. Microfibrils and associated amorphous material are present in the adjacent connective tissue space. A discontinuous inner collagenous layer is observed at the basal aspect of the RPE during the fourth day. A definitive elastic layer is present during the ninth day and becomes more apparent following subsequent stages of development. An outer collagenous layer begins development during the tenth and twelfth days. Collagenous fibrils average 37 nm in diameter and display axial periodicity measuring 46 nm between major periods. These measurements increase with age as do the number of collagenous fibrils. Only isolated patches of basal lamina are observed in association with the choriocapillary endothelium by the twentieth day.

Observations on (postnatal) persistent hyperplastic tunica vasculosa lentis/persistent hyperplastic primary vitreous (PHTVL/PHPV) in man and dog have been published previously. Up to the present, no evidence on the etiology of this entity was available. The hereditary occurrence of the disease in the Dobermann pinscher dog and the similarity of ocular development in mammals has provided a useful model in providing ontogenetic data. The present study deals with the early morphogenesis of PHTVL/PHPV, from day 25 to 44 post-coitum (D25-D44), in genetically affected dog fetuses. Normal beagle dog fetuses served as reference material, which has been described separately. At D30, the hyaloid system, including the tunica vasculosa lentis posterior, had developed further than in the reference fetuses. From that stage onward, a retrolental fibrovascular membrane developed. In some of the eyes of D37, posterior polar subcapsular cataracts and preretinal glial proliferations were observed. Capsular anomalies and distortions of the lens shape as seen in clinical PHTVL/PHPV were not observed, and are believed to be secondary entities. Extrapolation of some of the obtained data from dog to man is possible by the use of comparable gestational time scales. The anterior form of (PHTVL/PHPV) in man probably develops its main features in the period of approximately 43 to 66 days of pregnancy. Recently, anti-angiogenetic properties of normal vitreous have been described. This, and the fact that overdevelopment and subsequent incomplete regression of the hyaloid system plays a major role in the pathogenesis of PHTVL/PHPV, gives rise to the hypothesis that a changed amount or effectiveness of such (humoral) factors is an important factor in the etiology of this disease.