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Sequence comparisons and functional analysis of the 5' upstream regions of tyrosinase genes have revealed the importance of cis-regulatory elements acting to control the spatiotemporal expression of tyrosinase in the melanocytes and retinal pigmented epithelium of developing embryos. To date there are no reports addressing the control of tyrosinase gene transcription in zebrafish, a vertebrate model organism of increasing importance. To exploit the tyrosinase gene as a marker in zebrafish we set out to clone its promoter and analyse its regulation during embryogenesis. Amplification of a zebrafish tyrosinase complementary DNA fragment by reverse transcriptase polymerase chain reaction allowed us to isolate and sequence a 1041 nt genomic DNA fragment that includes a transcription initiation site and 73 nt of the open reading frame. Bioinformatic analysis of this genomic sequence revealed five E-box motifs, including one CATGTG type E-box present in a putative initiation region. These are conserved positive regulatory elements in vertebrate tyrosinase promoters. We show that a region of 814 nt upstream from the translation start site of the zebrafish tyrosinase gene can drive expression in retinal pigmented epithelium in transiently transgenic zebrafish embryos but that its activity is not restricted to melanin-producing cells. This region is unable to drive transcription in human melanoma cell lines. Ectopic expression from this zebrafish tyrosinase promoter fragment is probably due to the absence of positive and negative cis-regulatory elements, such as a tyrosinase distal element, which is known to function as a pigment cell-specific enhancer.

The CYP39A1 oxysterol 7alpha-hydroxylase preferentially catalyzes the 7alpha-hydroxylation of 24-hydroxycholesterol and has been suggested to play a role in the alternative bile acid synthesis pathway in the liver. The presence of CYP39A1 oxysterol 7alpha-hydroxylase has been reported only in the liver. To investigate the physiologic characteristics of the ciliary processes in bovine ocular tissues, we raised a mAb, 42C, against nonpigmented epithelial (NPE) cells, which have tight junctions that act as a blood-aqueous barrier and are involved in producing aqueous humor and maintaining ocular homeostasis. Immunohistochemical analysis showed that 42C antibody reacted intensely with an antigen in the NPE cells of the ciliary processes but not with other ocular tissues. The SDS-PAGE profile of immunoaffinity-purified antigens from bovine ciliary processes showed a predominant protein of molecular mass of 44.0 kDa. The amino acid sequence of this antigenic protein was identical to human CYP39A1 oxysterol 7alpha-hydroxylase. Immunoreactivity with 42C antibody was found only in hepatocytes and ocular tissues. These data suggest a new physiologic function for the CYP39A1 oxysterol 7alpha-hydroxylase in addition to the production of bile acids and provide new insight into the physiologic role of the ciliary NPE cells concerning the metabolism of sterols in the eye.

Purpose: Retinal pigment epithelial (RPE) cell death is an important step in the pathogenesis of ocular diseases. JNK1 and P38 kinase, two stress-activated kinases, play key roles relaying stress signals leading to cell death through cyclin D1 and c-Myc. Recently, stress-activated kinases have been shown to regulate cell proliferation. In the current study, the involvement of the JNK1 and P38 kinase signaling pathways in RPE cell proliferation and death was investigated., Methods: RPE cell proliferation was stimulated with 10% fetal calf serum (FCS). Activation of the JNK1 and P38 kinase cascades and their potential targets was detected by Western blot analysis. Pharmacologic inhibitors and activators, and antisense oligodeoxynucleotides (ODN) directed against the stress kinases were used to analyze the signaling involved in RPE cell death., Results: P38 and JNK1 and their respective upstream activating kinases, MKK3/6 and -4, were all transiently activated in FCS-stimulated RPE cell cultures. Ras controlled only the activation of JNK1, whereas Rho transmitted the activation of both JNK1 and P38, suggesting parallel signaling pathways and cross talk between the two kinases. Pharmacologic inhibition of JNK1 did not affect cell proliferation in FCS-stimulated cells. Inactivation of P38 kinase and antisense ODN-induced downregulation of P38 kinase also had no affect on cell proliferation. Long-term, high-level activation of JNK1 and P38 kinase occurred during serum depletion-induced RPE cell death. Overactivation of JNK1 and P38 kinase was also observed during pharmacologically induced cell death, suggesting that this process is common to RPE cell-death-signaling pathways induced by various stress stimuli. Cell death mediated by the overactivation of JNK1 and P38 kinase was cyclin D1- and c-Myc-independent., Conclusions: The inhibition of JNK1 or P38 kinase had no effect on FCS-stimulated proliferation of RPE cells, whereas the overactivation of these two enzymes was involved in RPE cell death in FCS-depleted cultures. Parallel upstream signaling pathways and cross talk between the two kinases suggest that the regulation of signaling in RPE cell death is complex.

Purpose: Estrogen status influences the incidence and severity of many diseases in women. Because women with early menopause appear at risk for worse ARMD, estrogen deficiency may also contribute to the onset or severity of ARMD in women. It has been observed that aged male C57BL/6 mice fed a high-fat diet and briefly exposed to blue-green light exhibit development of significant sub-RPE deposits and mild Bruch's membrane (BrM) thickening. This model was used in an attempt to delineate the role of gender and estrogen status in this model., Methods: C57BL/6 male and female mice of 9 or 16 months were fed a high-fat diet for 4.5 months. Several groups of 9-month-old female mice underwent estrogen depletion by ovariectomy, with or without supplementation with exogenous 17beta-estradiol. After 4 weeks of a high-fat diet, the eyes were exposed to seven 5-second doses of nonphototoxic levels of blue-green light over 2 weeks. Three and a half months after cessation of blue light treatment, transmission electron microscopy was performed to assess severity of deposits, BrM changes, and choriocapillaris endothelial morphology. In some mice, gelatin zymography and Western blot analyses were performed on protein extracts of freshly isolated RPE to determine the effect of estrogen on matrix metalloproteinase (MMP)-2 activity in the RPE., Results: Both male and female 16-month-old mice showed qualitatively similar basal laminar deposit morphology, but the severity of thickness, continuity, and content was significantly greater in female mice. Aged female mice also demonstrated a trend toward more severe endothelial changes and increased BrM thickening compared with age-matched male mice. Ovariectomized middle-aged mice showed more severe deposits than sham-surgery control animals. However, ovariectomized mice that received high-dose estrogen supplementation also showed significant deposits, although they had thinner BrMs than did the estrogen-deficient mice. Loss of RPE MMP-2 activity correlated with deposit severity, with estrogen-deficient mice expressing less MMP-2 than ovary-intact control mice., Conclusions: Female gender in aged mice and estrogen deficiency in middle-aged mice appears to increase the severity of sub-RPE deposit formation. Estrogen deficiency may increase susceptibility to formation of sub-RPE deposits by dysregulating turnover of BrM, contributing to collagenous thickening and endothelial changes. Estrogen supplementation at the dosages used in this study does not appear to protect against formation of sub-RPE deposits.

Purpose: To investigate the effects on telomerase activity, human telomerase reverse transcriptase(hTERT) gene and TEP1mRNA of retinal pigment epithelial(RPE) cells were treated by TGF-beta 1 of different concentration., Methods: The cultured human RPE cells were treated with TGF-beta 1 at different concentration (0 ng/ml, 0.01 ng/ml, 0.1 ng/ml, 1 ng/ml, 10 ng/ml) for 24 h, then telomerase activity was detected by telomerase repeat amplification protocol (TRAP). The expression of hTERT mRNA and TEP-1mRNA were detected by reverse transcription polymerase chain reaction (RT-PCR)., Results: TRAP and RT-PCR showed when the concentration of TGF-beta 1 was gradually increased, telomerase activity and the expression of hTERTmRNA were gradually reduced, TEP1mRNA showed no apparent differential expression., Conclusion: TGF-beta 1 can down-regulate telomerase activity and the expression of hTERT mRNA, but no effection on TEP-1mRNA, hTERTmRNA expression was in accordance with telomerase activity in hRPE cells, hTERT gene plays a crucial role in the expression of telomerase activity, while TEP1 plays a much smaller role.

Regeneration of 11-cis retinal from all-trans retinol in the retinal pigment epithelium (RPE) is a critical step in the visual cycle. The enzyme(s) involved in this isomerization process has not been identified and both all-trans retinol and all-trans retinyl esters have been proposed as the substrate. This study is to determine the substrate of the isomerase enzyme or enzymatic complex. Incubation of bovine RPE microsomes with all-trans [(3)H]-retinol generated both retinyl esters and 11-cis retinol. Inhibition of lecithin retinol acyltransferase (LRAT) with 10-N-acetamidodecyl chloromethyl ketone (AcDCMK) or cellular retinol-binding protein I (CRBP) diminished the generation of both retinyl esters and 11-cis retinol from all-trans retinol. The 11-cis retinol production correlated with the retinyl ester levels, but not with the all-trans retinol levels in the reaction mixture. When retinyl esters were allowed to form prior to the addition of the LRAT inhibitors, a significant amount of isomerization product was generated. Incubation of all-trans [(3)H]-retinyl palmitate with RPE microsomes generated 11-cis retinol without any detectable production of all-trans retinol. The RPE65 knockout (Rpe65(-/-)) mouse eyecup lacks the isomerase activity, but LRAT activity remains the same as that in the wild-type (WT) mice. Retinyl esters in WT mice plateau at 8 weeks-of-age, but Rpe65(-/-) mice continue to accumulate retinyl esters with age (e.g., at 36 weeks, the levels are 20x that of WT). Our data indicate that the retinyl esters are the substrate of the isomerization reaction.

Otx2 is a member of homeodomain-containing transcription factors and is essential for eye morphogenesis in mice. Here we show the expression of OTX2, the human counterpart of Otx2, in cell lines of retinal pigment epithelium (RPE) and in Y79 retinoblastoma cells that exhibit the property of presumptive RPE. These RPE cells express DOPAchrome tautomerase (DCT) that is an enzyme involved in melanin biosynthesis. DCT may contribute to the homeostasis of RPE by detoxifying DOPA-derived metabolites. OTX2 binds to the DCT gene promoter in vivo, as judged by chromatin immunoprecipitation assays. Furthermore, repression of endogenous OTX2 expression in Y79 cells by an anti-sense OTX2 oligonucleotide resulted in the decrease of DCT protein contents. Transient expression assays revealed that OTX2 activated the DCT gene promoter through the OTX-2-binding site in an RPE-specific manner. Therefore, OTX2 may regulate RPE-specific target genes, such as DCT, thereby maintaining the homeostasis of RPE.

Nitric oxide (NO) has been implicated in many physiological and pathological conditions in the eyes. The induction of inducible NO synthase (iNOS) and NO production have been noted in immunostimulated retinal pigment epithelial (RPE) cells. Cellular NO production depends on the availability of arginine, a substrate for NOS. Arginine can be regenerated from citrulline, another product of the NOS reaction, by argininosuccinate synthetase and argininosuccinate lyase, forming the citrulline-NO cycle. When rat RPE-J cells were treated with interferon-gamma (IFNgamma), tumor necrosis factor-alpha (TNFalpha) and lipopolysaccharide (LPS), and expression of the citrulline-NO cycle enzymes and related enzymes was analyzed, iNOS and argininosuccinate synthetase were highly induced at both mRNA and protein levels. On the other hand, argininosuccinate lyase was not induced. Among other related enzymes and transporters, mRNA for cationic amino acid transporter (CAT)-1 was weakly induced, whereas those for CAT-2, arginase I and II, ornithine aminotransferase and ornithine decarboxylase remained little changed. NO was produced by cells after stimulation with TNFalpha, IFNgamma and LPS. The induction of iNOS mRNA and the production of NO by these immunostimulated cells was further enhanced by cAMP. NO was produced from citrulline as well as from arginine. Our findings indicate that in activated RPE-J cells citrulline-arginine recycling is important for NO production.

Purpose: To investigate the localization and the activity of membrane dipeptidase (MDP) in the bovine eye., Methods: A monoclonal antibody (mAb), 49C mAb, raised against bovine ciliary process was used to examine the localization of MDP. Conversion of leukotriene (LT)D4 to LTE4 was evaluated by enzyme-linked immunosorbent assay for LTE4. Hydrolytic activity (beta-lactamase activity) was evaluated with a fluorometric assay. To clarify the contribution of MDP to conversion of LTD4 and beta-lactamase activity, we separated MDP from other enzymes by 49C mAb-conjugated gel., Results: The antigenic molecule of 49C mAb was shown to be MDP by amino acid sequencing. MDP was immunohistochemically detected in the ciliary pigmented and nonpigmented epithelial cells. Conversion of LTD4 to LTE4 in the ciliary process was much greater than that of the neural retina (NR). beta-Lactamase activity in the ciliary process was apparent, but that in the NR or the retinal pigment epithelium was negligible. Approximately 100% of beta-lactamase activity in the ciliary process was catalyzed by the 49C mAb-bound fraction. Conversion of LTD4 was catalyzed by the 49C mAb-bound fraction (55% of total activity) and by the unbound fraction (45% of total activity)., Conclusions: This study produced the first evidence of the presence of MDP in ciliary epithelial cells. The ciliary epithelium converts LTD4 to LTE4 and shows beta-lactamase activity. Conversion of LTD4 is catalyzed by at least two enzymes, and a major part of the conversion is induced by MDP.

The retinal pigment epithelium (RPE) shows cell heterogeneity in morphology and enzymatic activity. Routine isolation procedures for RPE cells may reduce enzymatic activity and prevent the quantification of regional enzymatic differences in vivo. We developed a new technique for the isolation of RPE cells based on adhesion of the cells to agarose. The morphology of the isolated cells resembled that of RPE cells in vivo. The cells were viable in the dye exclusion test and showed a histochemical staining pattern as RPE cells in vivo. With this technique, quantitative regional differences in the enzymatic activities were detected.

Purpose: Beta-carotene 15,15' monooxygenase (beta-CM) catalyzes the central cleavage of beta-carotene to all-trans-retinal, the first step in vitamin A synthesis. This study was conducted to determine the expression of beta-CM in the mammalian retina and RPE, to assess its relevance in carotenoid-retinoid metabolism in the retina and RPE., Methods: RT-PCR was used to detect expression of beta-CM mRNA in the retina and RPE-choroid of the mouse, cow, human, and monkey and in RPE cells and other cell lines. Immunofluorescence microscopy was used to localize beta-CM in mouse and monkey retina with an anti-peptide antibody specific for beta-CM., Results: By RT-PCR, beta-CM mRNA was detected at a low level in mouse and monkey retina and in the RPE-choroid of the monkey but not of the mouse. Conversely, beta-CM mRNA was expressed at a low level in both human and bovine RPE-choroid, but not in the retina of either. RPE primary cultured cells of the monkey also showed beta-CM mRNA expression, although the three human lines did not. In addition, of nine other cell lines tested, only COS-7 was positive for beta-CM. Immunofluorescence microscopy showed weak immunoreactivity in the inner retina in both the mouse and monkey. beta-CM immunoreactivity was not detectable in RPE of the mouse. Use of a long-wavelength exciting and emitting secondary probe to mitigate lipofuscin autofluorescence, facilitated the detection of a low level of beta-CM immunoreactivity in monkey RPE., Conclusions: Beta-CM mRNA and protein are expressed at low levels in the mammalian retina and RPE-choroid. Given the low and variable expression of beta-CM in the retina and RPE, it can be concluded that beta-CM is not necessary for a conserved retina or RPE-specific function, but may be necessary for a species-specific function.

Purpose: Proliferative vitreoretinopathy (PVR) is characterized by the development of epi- and subretinal fibrocellular membranes containing modified retinal pigment epithelial (RPE) cells among others. In the present study, the role of transglutaminases in accumulation of extracellular matrix (ECM) proteins in these membranes was investigated. Transglutaminases are enzymes capable of cross-linking ECM proteins to proteolysis-resistant complexes., Methods: PVR membranes were incubated with dansyl-cadaverine to demonstrate active transglutaminase. Localization of tissue transglutaminase (tTgase), its reaction product epsilon-(gamma-glutamyl)-lysine, and fibronectin was investigated immunohistochemically. Colocalization was studied with a confocal laser scanning microscope. PVR membranes were also analyzed by RT-PCR for the presence of tTgase mRNA. In vitro, RPE cells were treated with transforming growth factor-beta2 (TGF-beta2), basic fibroblast growth factor, interleukin-6, and interleukin-1beta. Their effect was studied using immunohistochemistry and Northern and Western blot analyses., Results: Transglutaminase activity and expression of tTgase were present in all PVR membranes. Staining was most prominent at the rim of the membranes. The enzyme was colocalized with epsilon-(gamma-glutamyl)-lysine and fibronectin. No staining differences were found between epi- and subretinal membranes. Although native RPE cells contained only a basal level of tTgase mRNA, the expression and activity of tTgase was increased under culture conditions and further stimulated by TGF-beta2 treatment., Conclusions: The findings demonstrate that in PVR membranes tTgase is present and functionally active. The amount and activity of this enzyme appears to be related to the differentiation state of the RPE cells and their stimulation by TGF-beta2, a growth factor known to be increased in the vitreous of PVR. Intervention at this pathway may open a new approach for PVR prevention and therapy.

Prohormone convertases PC1 and PC2 are enzymes involved in the intracellular processing of pro-neurotensin/neuromedin N (pro-NT/NN) through the regulated secretory pathway. In this study, we present evidence of the differential gene expression of pro-NT/NN, pro-PC1 and pro-PC2 in two cell lines established from the neuroendocrine ocular ciliary epithelium. Dexamethasone and forskolin were found to synergistically up-regulate NT/NN mRNA expression in both cell types. The pigmented cells released NT, and this release was enhanced by agents that induced its biosynthesis. In contrast, nonpigmented cells exhibited a significantly reduced neurotensin secretion in response to inducers, leading to an accumulation of the peptide. PC1 and PC2 mRNA expression was induced in a cell-specific manner by the same agents that enhanced pro-NT/NN biosynthesis. These results demonstrate cell-specific processing of pro-NT/NN by the ciliary epithelium., (Copyright 2002 Elsevier Science Ireland Ltd.)

Purpose: In the photic visual cycle, retinal G protein-coupled receptor (RGR) isomerizes all-trans retinal to 11-cis retinal in the retinal pigment epithelium (RPE) after illumination. It is unclear, however, how all-trans retinal, the substrate for RGR, is generated in the RPE, because no all-trans retinol dehydrogenase (atRDH) has been identified in the RPE. This study was conducted to identify the atRDH that generates all-trans retinal in the RPE., Methods: The full-length cDNA encoding a novel atRDH, RDH10, was cloned by PCR based on an expressed sequence tag (EST). Cellular localization was determined at the mRNA level by Northern blot analysis, RT-PCR, and in situ hybridization and at the protein level by immunohistochemistry with an antibody specific to RDH10. The activity was measured by an RDH activity assay with recombinant RDH10 expressed in COS cells., Results: The full-length RDH10 was cloned from the human, cow, and mouse. These cDNAs encode a protein of 341 amino acids and have significant sequence homology with other short-chain dehydrogenases/reductases (SDRs). The human RDH10 shares 100% and 98.6% amino acid sequence identity with the bovine and mouse proteins, respectively, suggesting a highly conserved sequence during evolution. RDH10 is predominantly expressed in the microsomal fraction of the RPE. Human RDH10 expressed in COS cells oxidized all-trans retinol to all-trans retinal. RDH10 displayed substrate specificity for all-trans retinol and preferred nicotinamide adenine dinucleotide phosphate (NADP) as the cofactor., Conclusions: RDH10 is a novel retinol oxidase expressed in the RPE. This enzyme can generate all-trans retinal from all-trans retinol and may play an important role in the photic visual cycle.

Background: Mucopolysaccharidosis VI (MPS VI), due to recessively inherited 4-sulfatase (4S) deficiency, results in lysosomal storage of dermatan sulfate in numerous tissues. Retinal involvement is limited to the retinal pigment epithelium (RPE). This study aimed to determine whether recombinant adeno-associated virus (AAV)-mediated delivery of 4S would reverse the RPE pathology seen in MPS VI cats., Methods: AAV.f4S, containing the feline 4S cDNA, was delivered unilaterally to eyes of affected cats by subretinal or intravitreal injection. Contralateral eyes received AAV with the green fluorescent protein (GFP) reporter gene as control. At 2-11 months post-injection, the cats were sacrificed and the treatment effects were evaluated histologically., Results: By ophthalmoscopy and histological analyses, GFP was evident as early as 4 weeks and persisted through the latest time point (11 months). Untreated and AAV.GFP-treated diseased retinas contained massively hypertrophied RPE cells secondary to accumulation of dilated lysosomal inclusions containing dermatan sulfate. MPS VI eyes treated subretinally with AAV.f4S had minimal RPE cell inclusions and, consequently, were not hypertrophied., Conclusions: AAV-mediated subretinal delivery of f4S provided correction of the disease phenotype in RPE cells of feline MPS VI, supporting the utility of AAV as a vector for the treatment of RPE-specific as well as lysosomal storage diseases., (Copyright 2002 John Wiley & Sons, Ltd.)

Purpose: Retinal pigment epithelial (RPE) cell proliferation plays a key role in the pathogenesis of ocular diseases involving the posterior segment. Mitogen-activated protein kinases (MAPKs) are involved in the control of cell proliferation. This study was conducted to investigate the involvement of the extracellular signal-regulated kinase (ERK) pathway, the major MAPK pathway implicated in cell growth during the induction of RPE cell proliferation., Methods: RPE cell proliferation was stimulated with 10% fetal calf serum (FCS). Activation of the Ras/Raf/MAP kinase-ERK kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway was detected by Western blot analysis and immunochemistry with specific anti-phosphosignaling protein antibodies. Pharmacologic and antisense (AS) oligonucleotide (ODN) strategies were used to analyze the ERK signaling involved in serum-induced cell proliferation., Results: FCS (10%) induced more vigorous RPE cell proliferation than did FGF2, VEGF, platelet-derived growth factor (PDGF), or epidermal growth factor (EGF), alone or in combination. Pharmacologic inhibition of Ras and Raf-1 reduced cell proliferation by 67% to 100% and by 62% to 79%, respectively, demonstrating that activation of the Ras/Raf-1 pathway was essential for FCS-induced RPE cell proliferation. MEK1/2, ERK2, and P90 ribosomal S6 kinase (P90(RSK)), the kinases downstream from ERK2, were strongly activated during cell proliferation. Pharmacologic inhibition of MEK1/2 abolished activation of ERK2, but reduced cell proliferation by only 32%, showing that MEK/ERK participates in the signaling involved in RPE cell growth. Both inhibition of ERK2 activation, which reduced cyclin D1 production, and inhibition of cyclin D1 by AS ODN decreased cell proliferation, suggesting that RPE cell proliferation is mediated by cyclin D1 through ERK2., Conclusions: The requirement for Ras and the regulatory role of ERK2 in cyclin D1 production and in cell proliferation suggest that the Ras/Raf/MEK/ERK pathway plays a key role in the control of RPE cell proliferation. These data may have important implications for the development of more selective methods for the inhibition of retinal proliferation.

During vertebrate eye development, the optic vesicle originating from the neuroectoderm is partitioned into a domain that will give rise to the neural retina (NR) and another that will give rise to the retinal pigmented epithelium (RPE). Previous studies have shown that ectopic expression of FGFs in the RPE induces RPE-to-NR transdifferentiation. Similarly, a naturally occurring mutation of the transcription factor Mitf in mouse resulted in the formation of a second neural retina in place of the dorsal RPE, but the putative signaling pathway linking FGF to Mitf regulation is presently unknown. In cultures of neural crest-derived melanocytes, the MAPK pathway was recently shown to target the Mitf transcription factor for ubiquitin-dependent proteolysis, resulting in a rapid degradation and downregulation. In the present study, we show that ectopic expression of a constitutively activated allele of MEK-1, the immediate upstream activator of the MAPK ERK, in chicken embryonic retina in ovo, induces transdifferentiation of the RPE into a neural-like epithelium that is correlated with a downregulation of Mitf expression in the presumptive RPE.

Conditional gene targeting using the Cre/loxP system enables specific deletion of a gene in a tissue of interest. For application of Cre-mediated recombination in pigment cells, Cre expression has to be targeted to pigment cells in transgenic mice. So far, no pigment cell-specific Cre transgenic line has been reported and we present and discuss our first results on use of Cre recombinase in pigment cells. A construct was generated where Cre recombinase is controlled by the promoter of the mouse dopachrome tautomerase (Dct) gene. The construct was functionally tested in vitro and introduced into mice. Following breeding to two reporter mouse strains, we detected Cre recombinase activity in telencephalon, melanoblasts, and retinal pigment epithelium (RPE). Our data demonstrate the feasibility of pigment cell-specific Cre/loxP-mediated recombination.

To study the relationship among tyrosinase activity, melanin production, and the routing of retinal ganglion cell (RGC) axons at the optic chiasm, we analysed mice with varying doses of the tyrosinase gene. These include the dark-eyed albino (Tyrc44H), a radiation-induced hypomorphic allele of tyrosinase; and transgenic mice carrying 1 or 2 alleles of a tyrosinase minigene on both wild-type (Tyr+) and albino (Tyrc) backgrounds. Melanization of the retinal pigment epithelium (RPE) occurred gradually even at <2% wild-type tyrosinase activity and was sensitive to tyrosinase activity up to <35% of wild-type levels, beyond which melanin synthesis appeared to be saturated. Overexpression of tyrosinase led to tyrosinase activity above wild type level, but did not increase melanin production. Although a loss of melanin because of a mutation in tyrosinase is associated with a decrease in the number of uncrossed fibers, elevating tyrosinase levels does not appear to cause an increase in the size of the uncrossed retinal projection. Our results suggest that replacing less than 35% of wild-type tyrosinase activity is sufficient to restore normal pigmentation of the RPE, and potentially, to allay visual defects.

Lysyl oxidase, an extracellular amine oxidase, controls the maturation of collagen and elastin. We examined the regulation of lysyl oxidase mRNA in cultured rabbit retinal pigment epithelium (RPE) cells in relation to the changes in subretinal fluid transport and phenotype of RPE cells. The level of the mRNA in cells grown on microporous membranes was markedly increased by application of hyperosmotic mannitol solution on the apical side (191% of control), implying that RPE cells express more lysyl oxidase in the condition which may cause the accumulation of subretinal fluid. Platelet-derived growth factor increased the mRNA level in subconfluent cells in culture (137% of control) and basic fibroblast growth factor decreased it (79% of control). In addition, exposure of cells to retinoic acid alone or in combination with dibutyryl cAMP for 22 days markedly decreased the level of lysyl oxidase mRNA (52 or 35% of control) while increasing the level of mRNA of N-acetylglucosaminidase (NAG), a marker enzyme for lysosomes (162 or 142% of control). Moreover, the level of lysyl oxidase mRNA in cells grown on microporous membranes was lower than that in cells grown on plastic dishes, while the level of NAG mRNA in the former cells was higher than that in the latter. Taken together, the expression of lysyl oxidase seemed to increase during proliferation of RPE cells and decrease toward differentiation. beta-Aminopropionitrile, an inhibitor of lysyl oxidase, significantly inhibited the contraction of collagen gels by fetal calf serum, suggesting that lysyl oxidase may be involved in pathogenesis caused by RPE cells.

Transformation with viral oncogene extends the lifespan of normal cells beyond replicative senescence called M1, but most of them eventually succumb to second crisis called M2 when telomeres become critically short. To acquire an infinite growth capacity, these cells have to overcome M2 crisis, which is known to follow telomerase activation. We have investigated if telomerase expression is required for virus-transformed pre-M2 cells to avert M2 crisis. Human retinal pigment epithelial (RPE) cells were transformed with simian virus 40 large T antigen and a VR3 clone in pre-M2 stage was obtained. Then, VR3 cells were transfected with a telomerase-containing vector and two cell lines that expressed telomerase temporarily or continuously were cloned and designated as ST1 and ST2, respectively. Normal RPE cells went into senescence after 36 population doublings. Although the lifespan was extended in the VR3 clone about 20 times more, it eventually underwent second crisis. The telomere length of VR3 decreased compared to that of normal RPE cells and the decrease continued during subculture. However, the ST1 and ST2 clones that expressed both T antigen and telomerase could avert this crisis. The initial telomere length of ST1 and ST2 was longer than that of normal cells. The ST1 underwent growth arrest again as telomerase expression faded out and elongated telomere was shortened, but the ST2 that maintained telomerase activity and telomere length proliferated continuously. In conclusion, telomerase activation is definitely required to overcome M2 crisis and acquire an infinite lifespan in human somatic epithelial cells and this mechanism is independent from M1 crisis escape in cell immortalization.

Membrane-bound lecithin retinol acyltransferase (LRAT), an essential enzyme in vitamin A processing, catalyzes the formation of retinyl esters from vitamin A and lecithin. Cloned and expressed LRAT has a molecular mass of 25.3 kDa. The enzyme is not homologous to known enzymes and is, therefore, of substantial interest mechanistically. Along these lines, the functional protomeric state of LRAT is of importance. Gel electrophoretic studies on LRAT in the presence of SDS and disulfide reducing agents show the expected 25 kDa monomer. However, gel electrophoresis in the absence of a reducing agent and/or strong denaturing conditions reveals substantial dimer formation. LRAT monomers can be efficiently and irreversibly cross-linked by thiol reactive bismaleimides in retinal pigment epithelial (RPE) membranes generating LRAT homodimers. Cross-linked LRAT homodimers are fully active catalytically. The experiments suggest that LRAT monomers interact in membranes and form functional homodimers through protein-protein interactions and disulfide bond formation.

Purpose: To investigate conditions promoting the differentiation of cultured human retinal pigment epithelial (RPE) cells and assess the differentiation potential of telomerase-immortalized RPE cells., Methods: Serially passaged RPE 340 (parental) cells have limited replicative ability and senesce after 50 to 60 population doublings (PD)s. RPE 340 cells transfected with the catalytic component of human telomerase (hTERT) have an extended lifespan. RPE 340 and hTERT-transfected RPE (hTERT-RPE) cells were maintained at confluence without serum for up to 12 weeks. Morphologic, immunocytochemical, flow cytometric, and spectrophotometric analyses were performed to examine the extent of RPE differentiation., Results: Parental RPE 340 and hTERT-RPE cells underwent growth arrest and differentiated in the absence of serum. In early-passage parental (PD11) and hTERT-RPE (PD115 and PD300) cells, serum deprivation for 4 weeks or more induced terminal differentiation as characterized by mature, growth-arrested, confluent sheets of polygonal and melanized cells that demonstrated diminished 5'-bromo-2'-deoxyuridine (BrdU) uptake and positive reactivity with antibodies to cellular retinaldehyde-binding protein (CRALBP), cytokeratin, and vimentin. Reintroduction of serum at 4 or 8 weeks allowed the cells to reenter the cell cycle and demelanize. Midpassage (PD 25) parental cells, however, were irreversibly arrested at G(1) after 8 weeks of serum deprivation., Conclusions: Cultured parental and hTERT-RPE cells express RPE-associated proteins and become stably melanized when density is arrested in the absence of serum. Moreover, hTERT-immortalized RPE cells retain the capacity to undergo terminal differentiation in vitro, even after long-term culture.

Purpose: Generate site-specific somatic mutations selectively in the retinal pigment epithelium (RPE) in mice., Methods: A transgenic mouse line expressing the Cre recombinase under the control of the tyrosinase-related protein (TRP)-1 promoter was generated. The presence of Cre was determined by in situ hybridization, and Cre-mediated excision of DNA was analyzed by PCR and alkaline phosphatase (AP) histochemistry in reporter mice carrying a loxP-flanked (floxed) retinoid X receptor alpha (RXRa) gene and in Z/AP mice, respectively., Results: Cre was expressed in the RPE from embryonic day 10.5 to postnatal day 12, resulting in efficient floxed excision of DNA in the RPE from embryonic day 10.5 to adulthood in TRP1-Cre mice. Expressed Cre and excision of DNA were also detected in the ciliary margin of the retina and in some cells in the neural retina, but not in the embryonic periocular mesenchyme or in the choroid., Conclusions: The TRP1-Cre mouse line, which induces efficient Cre-mediated excision of DNA selectively in the RPE, provides a new, powerful tool to study gene functions in the RPE in vivo.

Vascular endothelial growth factor (VEGF) is among the cytokines which have been implicated in the pathogenesis of choroidal neovascularization secondary to age-related macular degeneration (ARMD). There is, however, evidence that intercellular signaling molecules, such as nitric oxide (NO), are involved in this process. NO is synthesized via the inducible isoform of NO synthase (iNOS), which is expressed after induction by cytokines. In the current study, we investigated whether VEGF and iNOS are coexpressed in choroidal neovascular membranes (n = 7) from patients with ARMD. Immunohistochemistry was performed on cryosections with anti-iNOS and anti-VEGF. Moderate to intense immunostaining for iNOS and VEGF was observed in retinal pigment epithelial cells, macrophages, and in spatial relation to vessel walls. As scored by light microscopy, we found a significant correlation between immunoreactivity for VEGF and iNOS (p < 0.0341) in vascular endothelial cells. Our study supports a significant role for iNOS in the pathogenesis of neovascularization and membrane growth in ARMD. Moreover, our findings suggest a possible relationship between NO and VEGF in the regulation of pathologic angiogenesis in this disease., (Copyright 2002 S. Karger AG, Basel)

Purpose: Migration of retinal pigment epithelial (RPE) cells can be triggered by disruption of the RPE monolayer or injury to the neural retina. Migrating cells may re-establish a confluent monolayer, or they may invade the neural retina and disrupt visual function. The purpose of this study was to examine the role of endogenous polyamines in mechanisms of RPE migration., Methods: Endogenous polyamine levels were determined in an immortalized RPE cell line, D407, using HPLC. Activities of the two rate-limiting enzymes for polyamine synthesis, ornithine decarboxylase (ODC), and S-adenosylmethionine decarboxylase (SAMdc), were measured by liberation of ((14)CO(2))(.) Migration was assessed in confluent cultures by determining the number of cells migrating into a mechanically denuded area. All measurements were obtained both in control cultures and in cultures treated with synthesis inhibitors that deplete endogenous polyamines. Subcellular localization of endogenous polyamines was determined using a polyamine antibody., Results: The polyamines, spermidine and spermine, as well as their precursor, putrescine, were normal constituents of RPE cells. The two rate-limiting synthetic enzymes were also present, and their activities were stimulated dramatically by addition of serum to the culture medium. Cell migration was similarly stimulated by serum exposure. When endogenous polyamines were depleted, migration was blocked. When polyamines were replenished through uptake, migration was restored. Polyamine immunoreactivity was limited to membrane patches in quiescent cells. In actively migrating and dividing cells, immunoreactivity was enhanced throughout the cytoplasm., Conclusions: Polyamines are essential for RPE migration. Pharmacologic manipulation of the polyamine pathway could provide a therapeutic strategy for regulating anomalous migration.

Purpose: To evaluate the expression pattern of matrix metalloproteinases (MMPs) from retinal pigment epithelial (RPE) cells in culture, and to determine their ability to alter the transport properties of human Bruch's membrane., Methods: Human RPE cells from either primary cultures or a cell line were maintained under culture conditions. At different time intervals after subculturing of cells the presence of MMPs in the bathing medium was determined by zymography. Cellular MMP activity was determined in a similar series of experiments where serum was omitted from the culture medium. Cultured cells were introduced onto Bruch's membrane, mounted in a modified Ussing chamber, to assess entry of MMPs into the membrane. Fluid flow across Bruch's membrane was determined by hydraulic conductivity for different ages of donor tissue, before and after 24 hours' incubation with active MMPs from the RPE-conditioned medium or after incubation with purified activated MMPs. Latent (inactive) MMPs from medium containing serum were used in control experiments., Results: Cultured RPE cells expressed both MMP-2 and -9, with active MMP-2 becoming detectable from 4 days after subculture through to confluence and activated MMP-9 becoming abundant up to 24 hours after subculture. Both active MMPs significantly increased hydraulic conductivity of Bruch's membrane, with the increase after MMP-9 incubation being far greater than that for MMP-2. Both enzymes showed a trend in hydraulic conductivity change with age such that, MMP-2 produced a relatively constant change, whereas MMP-9 showed a greater increase in older eyes., Conclusions: Activation of both MMP-2 and -9 by cultured RPE cells appeared to show a temporal relationship with the growth cycle of the cells. The activated enzymes increased fluid flow of Bruch's membrane, and the marked effect observed with MMP-9 in older eyes suggests a mechanism that may allow debris removal.

Purpose: To examine the nitric oxide (NO) system in the choroid of normal rats and rats with streptozotocin (STZ)-induced diabetes., Methods: We assayed NO synthase (NOS) activity by monitoring the conversion of L-[(14)C] arginine to L-[(14 )C] citrulline, identified the NOS isoforms by immunoblotting, and examined the effects of STZ-induced diabetes on NOS in the rat choroid., Results: Calcium-independent NOS activity was insignificant in the choroid of normal and diabetic rats. Choroidal calcium-dependent NOS activity was high and comparable to that in the cerebellum. Neuronal (n) NOS protein in the choroid was found in both the membrane and cytosolic fractions and showed similar subcellular distribution as NOS activity, while endothelial (e) NOS protein in the choroid was present almost solely in the membrane fraction. Total NOS activity (nNOS + eNOS) and protein levels of nNOS and eNOS in the choroid were significantly reduced 6 weeks after the induction of diabetes., Conclusions: The high NOS activity in the choroid measured in vitro appears to come mostly from nNOS. Because choroidal nNOS exists in the parasympathetic perivascular nerve fibers, the decrease in choroidal nNOS in diabetic eyes suggests that the choroid undergoes a diabetes-induced neuronal disorder. Thus, diabetic choroidopathy encompasses diabetic neuropathy and microangiopathy.

Aims: To determine if mild hyperoxia induces senescence in retinal pigment epithelium (RPE) cells in vitro., Methods: RPE340 cells and WI38 cells were grown in 20% oxygen and 40% oxygen until proliferative exhaustion. A combined senescence associated beta galactosidase (SABG) and 5-bromo-2'-deoxyuridine (BrdU) double labelling technique was performed at different times and labelled cells were counted., Results: Cells grown in 40% oxygen stopped proliferating at an earlier population doubling level than when grown in 20% oxygen. An increase in SABG positive cells and decrease of BrdU positive cells in 40% oxygen developed at an earlier time than when grown in 20% oxygen., Conclusion: Mild hyperoxia induces premature senescence in a specific RPE cell line.

The regulation of stearoyl-CoA desaturase (SCD), a rate-limiting enzyme in the synthesis of unsaturated fatty acids, is physiologically important because the ratio of saturated to unsaturated fatty acids is thought to control cellular functions by modulating the structural integrity and fluidity of cell membranes. Transforming growth factor-beta (TGF-beta), a multifunctional cytokine, increased SCD mRNA expression in cultured human retinal pigment epithelial cells. This response was elicited by all three TGF-beta isoforms, beta1, beta2, and beta3. However, SCD mRNA expression was not increased either by other members of the TGF-beta family or by other growth factors or cytokines. TGF-beta also increased SCD mRNA expression in several other cell lines tested. The increase in SCD mRNA expression was preceded by a marked increase in Smad2 phosphorylation in TGF-beta-treated human retinal pigment epithelial cells. TGF-beta did not induce SCD mRNA expression in a Smad4-deficient cell line. However, Smad4 overexpression restored the TGF-beta effect in this cell line. Moreover, TGF-beta-induced SCD mRNA expression was effectively blocked by the overexpression of Smad7, an inhibitory Smad. Thus, a TGF-beta signal transduction pathway involving Smad proteins appears to regulate the cellular expression of the SCD gene, and this regulation may play an important role in lipid metabolism.

Purpose: To show that fish oil-derived omega-3 polyunsaturated fatty acids, delivered to the retinal pigment epithelium (RPE) by circulating low-density lipoproteins (LDL), enhance already considerable RPE lysosomal acid lipase activity, providing for more efficient hydrolysis of intralysosomal RPE lipids, an effect that may help prevent development of age-related macular degeneration (ARMD)., Methods: Colorimetric biochemical and histochemical techniques were used to demonstrate RPE acid lipase in situ, in vitro, and after challenge with phagocytic stimuli. Receptor-mediated RPE uptake of fluorescently labeled native, aceto-acetylated, and oxidized LDL was studied in vitro and in vivo. LDL effects on RPE lysosomal enzymes were assessed. Lysosomal enzyme activity was compared in RPE cells from monkeys fed diets rich in fish oil to those from control animals and in cultured RPE cells exposed to sera from these monkeys., Results: RPE acid lipase activity was substantial and comparable to that of mononuclear phagocytes. Acid lipase activity increased significantly following phagocytic challenge with photoreceptor outer segment (POS) membranes. Receptor-mediated RPE uptake of labeled lipoproteins was determined in vitro. Distinctive uptake of labeled lipoproteins occurred in RPE cells and mononuclear phagocytes in vivo. Native LDL enhanced RPE lysosomal enzyme activity. RPE lysosomal enzymes increased significantly in RPE cells from monkeys fed fish oil-rich diets and in cultured RPE cells exposed to their sera., Conclusions: RPE cells contain substantial acid lipase for efficient metabolism of lipids imbibed by POS phagocytosis and LDL uptake. Diets rich in fish oil-derived omega-3 fatty acids, by enhancing acid lipase, may reduce RPE lipofuscin accumulation, RPE oxidative damage, and the development of ARMD.

To investigate the possible role of matrix metalloproteinase-7 in choroidal neovascularization associated with age-related macular degeneration, immunoelectron microscopy using ultrathin frozen sections and conventional transmission electron microscopy were performed in subfoveal fibrovascular membranes from patients with age-related macular degeneration. immunoelectron microscopy revealed that matrix metalloproteinase-7 was expressed within basal laminar deposits and amorphous materials around the retinal pigment epithelial cells. The results support a role for matrix metalloproteinase-7 in the development of choroidal neovascularization in age-related macular degeneration.

Drusen are abnormal extracellular matrix deposits characteristic of age-related macular degeneration (AMD), a leading cause of blindness in the aging human population. The mechanisms underlying drusen formation are not well characterized. The purpose of this study was to examine the expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in drusen, and in the surrounding cells and tissue. To assess the extent of MMP and TIMP expression by retinal pigment epithelial (RPE) cells, cDNA arrays were screened with probes generated from cultured human RPE cells. The distribution of MMP-1, -2 and -3 and TIMP-1, -2, -3 and -4 was determined using immunohistochemistry in human RPE choroid from donor eyes with and without a clinical history of AMD. Gelatinase activity was assessed in unfixed frozen sections using in situ zymography. In cultured RPE cells, expression of 10 MMP and all four known TIMP mRNAs was detected. MMP immunoreactivity was widespread in the RPE choroid, but was absent from the interior of drusen. TIMP-3, but not other TIMPs, was detected in the drusen interior. Likewise, metal ion dependent gelatinase activity could be detected in RPE choroid, but not in drusen. These results show that, while metalloproteinase activity is widespread throughout the RPE choroid, drusen are cold spots for proteolysis. The data lead to the speculation that high TIMP-3 concentrations within drusen could inhibit MMPs and as a result slow the proteolytic degradation of these deposits., (Copyright 2002 Elsevier Science Ltd.)

This work aims to investigate the effect of compromised lysosomal enzyme activity on the accumulation of photoreceptor-derived debris in the retinal pigment epithelial (RPE) cells and to examine if this accelerated debris accumulation can induce retinal abnormalities similar to those observed in aged individuals. A mutated, enzymatically inactive form of cathepsin D (CatD), generated by site-directed mutagenesis was used to produce stable cell lines and transgenic mice. There was a strong increase in enzymatically inactive CatD protein production in the mutated CatD DNA transfected D407 cells (D407MCD). The presence of the inactive CatD has been linked to an impairment in bovine rod outer segment (BROS) digestion and was confirmed by a statistically significant increase of undigested residual BROS in the medium of D407MCD when compared to the control vector-transfected D407 cells (t-test, P < or = 0.016, P < or = 0.003) or untransfected D407 cells (t-test, P < or = 0.008, P < or = 0.003). The impairment was also confirmed in vivo by demonstration of BROS-derived debris accumulation in the RPE cell layer of transgenic mice. These results demonstrated that the mutated and inactive CatD form could lead to impairment of photoreceptor outer segments (POS) proteolysis. It is proposed that this initial impairment of POS proteolysis may result in the accumulation of CatD-opsin-like complexes in the pigment epithelium, which further compromises RPE cell functions and thus causes the changes observed in aging humans.

Mammalian cells are equipped with elaborate systems for protection against the toxicity of reactive oxygen and nitrogen species and electrophiles that are constant dangers to the integrity of their DNA. Phase 2 enzymes (e.g., glutathione transferases, NAD(P)H:quinone reductase) and glutathione synthesis are widely recognized as playing major protective roles against electrophilic carcinogens, but their antioxidant functions have attracted far less attention. The cytotoxicities of four oxidative stressors (menadione, tert-butyl hydroperoxide, 4-hydroxynonenal, and peroxynitrite) for human adult retinal pigment epithelial cells (ARPE-19) were quantified by measuring the concentration dependence of cell death and were expressed as the median effect dose (D(m)) for each oxidant. After treatment of ARPE-19 cells for 24 h with 0-5 microM concentrations of sulforaphane (the powerful Phase 2 enzyme inducer isolated from broccoli), the toxicities of the oxidants were markedly reduced as shown by 1.5- to 3-fold increases in D(m) values. The magnitude of protection was a function of the nature of the oxidants and the concentrations of both the oxidants and sulforaphane. Protection was prolonged and persisted for several days after removal of sulforaphane before returning to control levels. The sulforaphane-dependent increases in specific activities of cytosolic quinone reductase and the glutathione levels were highly significantly correlated with the degree of protection as measured by D(m) values. Antioxidant protection was also demonstrated for human HaCaT keratinocytes and L1210 murine leukemia cells. It is therefore highly likely that the multifaceted and prolonged antioxidant protection provided by sulforaphane is a general phenomenon that is mediated through induction of the Phase 2 enzyme response.

We measured the activities of total Na+, K+-ATPase (Na, K-ATPase), its alpha1 and alpha2/alpha3 isoforms and the angiotensin-converting enzyme (ACE) in the microvascular and neural compartments of the retina, and/or retinal pigment epithelium (RPE) of streptozotocin (STZ)-diabetic rats. The effect of captopril, an ACE inhibitor on Na, K-ATPase activities was also determined and correlated to morphological changes. Insulin-dependent diabetes mellitus was induced by a single intraperitoneal injection of STZ (60 mg/kg) in male Long-Evans rats. ACE activity was inhibited by captopril (10 mg/kg given in the drinking water) for 1 month. Na, K-ATPase activity was measured spectrophotometrically or by a radioassay (32P-labeled ATP). The activity of ACE was determined by a radioassay using tritiated benzoyl-gly-gly-gly as substrate. Both the alpha1 and alpha2/alpha3 isoforms of Na, K-ATPase were present in the microvascular and neural compartments of retinas, whereas only one isoform, the alpha2/alpha3, was found in the RPE. In 2-month diabetic rats, the activity of the alpha2/alpha3 isoform was reduced in both the microvascular and neural compartments of retinas, while the activity of the alpha1 isoform was reduced only in the neural isolates. ACE activity was significantly decreased in the retinal neural compartment and unaltered in the microvascular compartment from 2-month diabetic rats. In 5-month diabetic rats, Na, K-ATPase activity was moderately but not significantly reduced in RPE preparations. Ultrastructural studies revealed a significant deepening of basal infoldings in the RPE and a noticeable increase in the size of the extracellular space between the basal infoldings of 5-month diabetic animals. Captopril stimulated Na, K-ATPase activity in the neural retina, but not in the RPE. Diabetes-induced morphological changes in the RPE were not improved by captopril. An enlargement of intercellular space between the RPE cells was a frequent finding in the treated group. In conclusion, captopril stimulated Na, K-ATPase activity in the neural retina of diabetic rats. This stimulation seems to be beneficial to the neural retina. ACE inhibition, however, did not improve RPE morphological changes. Although the clinical significance of increased intercellular spacing between RPE cells in treated animals is not clearly established, we speculate that it might contribute to an increased alteration of their barrier function. Additional studies are necessary to assess both the desirable and adverse effects of captopril and other ACE inhibitors in the retinas of diabetic patients.

Background: The aim of this study is to see the effect of interferon beta (IFN-beta) on cell proliferation and the protein kinase C (PKC) signaling pathway., Methods: Proliferation of cultured human retinal pigment epithelium (RPE) cells, with various concentrations of IFN-beta, and with or without 3% fetal calf serum (FCS), was assessed by cell counting. Effects of short (3 h) or prolonged (48 h) exposure of RPE cells to natural human IFN-beta were assessed by (3)H-thymidine uptake. Cytosolic and membranous PKC activity over time in cells treated with IFN-beta and calphostin C was also measured., Results: IFN-beta inhibited the increased proliferation by FCS in the prolonged-exposure assay. The PKC inhibitor calphostin C also showed an inhibitory effect on RPE cell growth and (3)H-thymidine uptake in the chronic exposure with FCS. Short treatment with IFN-beta had no inhibitory or stimulatory effect on (3)H-thymidine uptake. Cytosolic and membranous PKC activity was strongly upregulated after short IFN-beta exposure but returned to original levels after 1 h. PKC activity was downregulated both in the cytosol and membrane after 24 or 48 h., Conclusion: IFN-beta inhibited RPE proliferation in vitro and the effect is mediated by upregulation of the PKC pathway., (Copyright 2001 S. Karger AG, Basel)

Purpose: To develop an effective therapy for treating glycosaminoglycan (GAG) storage in mucopolysaccharidosis VII (MPS VII) retinal pigment epithelium (RPE) in vitro using adenoviral vector mediated human beta-glucuronidase cDNA (Ad-GUSB) transfer., Methods: Ad-GUSB was used to infect RPE at confluency. The transduction condition was optimized varying time of infection and number of infectious particles. The beta-glucuronidase (GUSB) activity was measured in transduced cells and media using a fluorogenic substrate. The GAG profiles were examined by metabolically labeling RPE with (35)Na(2)SO(4)., Results: Transduced RPE, irrespective of species or disease status, expressed a high level of beta-glucuronidase. The expressed enzyme restored normal levels of GAGs in the RPE cells of homozygous affected MPS VII dogs by metabolizing stored GAGs. The over-expressed enzyme (>10 000 nmoles/hr/mg) failed to restore normal level of GAGs. A high level of GUSB expression was maintained in vitro at least nine weeks., Conclusions: Adenoviral vector could mediate transfer of GUSB in MPS VII affected RPE and RPE of various species, and the expression was observed to be stable in vitro. However, controlled expression of GUSB was essential for the metabolism of stored GAGs to achieve normal levels.

We studied the composition of molecular forms of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in normal and streptozotocin-induced diabetic rat retinal pigment epithelium (RPE). Tissues were sequentially extracted with saline (S(1)) and saline-detergent buffers (S(2)). About a 50% decrease in AChE molecular forms was observed in the diabetic RPE compared to the controls. Approximately 70% of the BChE activity in normal RPE was brought into solution and evenly distributed in S(1) and S(2). Analysis of the fractions from RPE revealed the presence of G(A)(1), G(A)(4) and a small proportion of G(H)(4) BChE forms in S(1); whereas G(A)(4) and G(A)(1) molecules predominate in S(2). A 40% decrease in the activity of G(A)(4) in S(2) was observed in the diabetic RPE. Our results show that diabetes caused a remarkable decrease in the activity of cholinesterases molecular forms in the RPE. This might be related to the alterations observed in diabetic retinopathy.

Purpose: To evaluate potential toxic effects of indocyanine green dye on cultured human retinal pigment epithelial cells., Methods: Controlled laboratory experiment. Cultured human retinal pigment epithelial cells were exposed to balanced saline solution, balanced saline solution with endoillumination, indocyanine green or indocyanine green with endoillumination. Cells were evaluated by light microscopy, electron microscopy, and a mitochondrial dehydrogenase assay., Results: Retinal pigment epithelial cells exposed to indocyanine green showed no histologic or ultrastructural changes. Those exposed to indocyanine green alone or indocyanine green plus light demonstrated a significant decrease in mitochondrial enzyme activity (P = 0.0002 and 0.005, respectively)., Conclusion: Brief exposure of cultured human retinal pigment epithelial cells to indocyanine green results in decreased mitochondrial enzyme activity but does not appear to influence cellular morphology or ultrastructure.

Purpose: Cyclooxygenases (COX) orchestrate a variety of homeostatic processes and participate in various pathophysiological conditions. The retinal pigment epithelium (RPE) cell performs a variety of regulatory functions within the retina. The conditions under which COX-1 and COX-2 are expressed and upregulated in human RPE (HRPE) cells were determined., Methods: COX gene expression was examined using RT-PCR analysis of untreated HRPE cultures or cultures exposed to bacterial lipopolysaccharide or various cytokines. COX proteins were detected by immunohistochemistry and Western blot analysis. Prostaglandin (PG) production was analyzed by EIA., Results: Examination of untreated RPE cells revealed the presence of COX-2 mRNA and the absence of COX-1 mRNA. Moreover, cytokine stimulation more readily enhanced COX-2 gene expression than COX-1 gene expression. IL-1 beta, the most potent inducer of COX-2, also resulted in detection of COX-2 protein by immunocytochemical staining and Western blot analysis. There was a direct relationship between both the appearance and amount of COX-2 mRNA and protein synthesis and the degree of PG synthesis by RPE cells. Furthermore, COX inhibitors significantly decreased PG production. Pretreatment of RPE cells with a NF-kappa B inhibitor, PDTC, resulted in dose-dependent decrease in IL-1 beta-induced COX-2 gene expression and PG production., Conclusions: COX-2 was the predominant isoform of cyclooxygenase in untreated HRPE cells. When HRPE cells were treated with proinflammatory cytokines, COX-2 gene expression and synthesis of PGs were enhanced. NF-kappa B mediated the induction of COX-2 gene expression in HRPE cells. These studies indicate that RPE cells may participate in normal and pathologic retinal conditions through the induction of COX-2.

In the vertebrate retina, the final step of visual chromophore production is the oxidation of 11-cis-retinol to 11-cis-retinal. This reaction is catalyzed by 11-cis-retinol dehydrogenases (11-cis-RDHs), prior to the chromophore rejoining with the visual pigment apo-proteins. The RDH5 gene encodes a dehydrogenase that is responsible for the majority of RDH activity. In humans, mutations in this gene are associated with fundus albipunctatus, a disease expressed by delayed dark adaptation of both cones and rods. In this report, an animal model for this disease, 11-cis-rdh-/- mice, was used to investigate the flow of retinoids after a bleach, and microsomal membranes from the retinal pigment epithelium of these mice were employed to characterize remaining enzymatic activities oxidizing 11-cis-retinol. Lack of 11-cis-RDH leads to an accumulation of cis-retinoids, particularly 13-cis-isomers. The analysis of 11-cis-rdh-/- mice showed that the RDH(s) responsible for the production of 11-cis-retinal displays NADP-dependent specificity toward 9-cis- and 11-cis-retinal but not 13-cis-retinal. The lack of 13-cis-RDH activity could be a reason why 13-cis-isomers accumulate in the retinal pigment epithelium of 11-cis-rdh-/- mice. Furthermore, our results provide detailed characterization of a mouse model for the human disease fundus albipunctatus and emphasize the importance of 11-cis-RDH in keeping the balance between different components of the retinoid cycle.

The human CMV (HCMV) is a persistent virus that may cause severe inflammatory responses especially in immunocompromised hosts. In different cell types, HCMV infection leads to the activation of the pleiotropic transcription factor, NF-kappaB, which triggers virus replication but also propagates cell-mediated inflammatory mechanisms that largely depend on PG synthesis. We investigated the interactions of HCMV and the NF-kappaB-dependent PG synthesis pathway in cultures of retinal pigment epithelial (RPE) cells that are known to be infected in HCMV retinitis patients. Unlike in other cell types, HCMV increased neither NF-kappaB activity nor p65 and p105/50 mRNA levels in RPE cells. Both TNF-alpha and phorbol ester 12,0-tetradecanoylphorbol 13-acetate (TPA) enhanced NF-kappaB activity but only TPA increased HCMV replication. Cyclooxygenase-2 expression and PGE2 release was increased by TPA and TNF-alpha but not by HCMV infection. Stimulatory activity of TPA on HCMV replication was suppressed by protein kinase C inhibitors and inhibitors of p42/44 and p38 mitogen-activated protein kinases but not by NF-kappaB inhibitors. In conclusion, HCMV circumvents the NF-kappaB route in favor of the protein kinase C-dependent mitogen-activated protein kinase pathway in RPE cells. This virus/host cell interaction might be a mechanism that promotes HCMV persistence in immune-privileged organs such as the eye.

Stearoyl-CoA desaturase (SCD) is a regulatory enzyme involved in the synthesis of the monounsaturated fatty acids palmitoleate and oleate. The regulation of SCD is of physiological importance because the ratio of saturated fatty acids to unsaturated fatty acids is thought to modulate membrane fluidity. Differential display analysis of retinal pigment epithelial (ARPE-19) cells identified SCD as a gene regulated by retinoic acid. Two SCD transcripts of 3.9 and 5.2 kilobases in size were found to be expressed in these cells by Northern blot analysis. All-trans-retinoic acid (all-trans-RA) increased SCD mRNA expression in a dose- and time-dependent manner; an approximately 7-fold increase was observed with 1 microm all-trans-RA at 48 h. SCD mRNA expression was also increased by 9-cis-retinoic acid (9-cis-RA) as well as 4-(E-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl)benzoic acid (TTNPB), a retinoic acid receptor (RAR)-specific agonist. AGN194301, a RAR alpha-specific antagonist, suppressed the SCD expression induced by all-trans-RA, TTNPB, and 9-cis-RA. These results indicate the involvement of RAR alpha in the induction of SCD expression by retinoic acid. However, AGN194204, a RXR (retinoid X receptor) pan agonist, also increased SCD mRNA expression. This increase was not blocked by AGN194301, suggesting that an RAR-independent mechanism may also be involved. Thus, SCD expression in retinal pigment epithelial cells is regulated by retinoic acid, and the regulation appears to be mediated through RAR and RXR.

Purpose: The present study investigates whether beta-adrenoreceptor agents such as isoproterenol and propranolol can regulate Na(+)-K(+)-ATPase activity in cultured human non-pigmented ciliary epithelial cells., Methods: Human non-pigmented ciliary epithelial cells (ODM2) were grown to confluence. The active ion transport mediated by the Na(+)-K(+)-ATPase was evaluated by measuring ouabain-sensitive rubidium (Rb+) uptake. In a first set of experiments, cells were exposed to the beta-adrenoreceptor agonist isoproterenol (0.01-10 microM). In a second set of experiments, cells were exposed to isoproterenol (1 microM) in the presence of different concentrations of the beta-adrenoreceptor antagonist propranolol (0.01, 0.1, 1 microM)., Results: In a concentration-dependent manner, isoproterenol induced an increase in Na(+)-K(+)-ATPase activity. The maximal Na(+)-K(+)-ATPase activity was observed at a concentration of 1 microM of isoproterenol (283 +/- 58%, P < 0.001). The increase in Na(+)-K(+)-ATPase activity evoked by isoproterenol (1 microM) was inhibited. In a concentration dependent manner, by propranolol (maximum: 659 +/- 39 vs. 141 +/- 42 pM/mg protein/min, P < 0.01)., Conclusion: The beta-adrenoreceptor agents isoproterenol and propranolol are apparently able to modulate Na(+)-K(+)-ATPase activity in cultured human non-pigmented ciliary epithelial cells.

Retinoids play a critical role in vision, as well as in development and cellular differentiation. beta,beta-Carotene-15,15'-dioxygenase (Bcdo), the enzyme that catalyzes the oxidative cleavage of beta,beta-carotene into two retinal molecules, plays an important role in retinoid synthesis. We report here the first cloning of a mammalian Bcdo. Human BCDO encodes a protein of 547 amino acid residues that demonstrates 68% identity with chicken Bcdo. It is expressed highly in the retinal pigment epithelium (RPE) and also in kidney, intestine, liver, brain, stomach, and testis. The gene spans approximately 20 kb, is composed of 11 exons and 10 introns, and maps to chromosome 16q21-q23. A mouse orthologue was also identified, and its predicted amino acid sequence is 83% identical with human BCDO. Biochemical analysis of baculovirus expressed human BCDO demonstrates the predicted beta,beta-carotene-15,15'-dioxygenase activity. The expression pattern of BCDO suggests that it may provide a local supplement to the retinoids available to photoreceptors, as well as a supplement to the retinoid pools utilized elsewhere in the body. In addition, the finding that many of the enzymes involved in retinoid metabolism are mutated in retinal degenerations suggests that BCDO may also be a candidate gene for retinal degenerative disease., (Copyright 2001 Academic Press.)

Endothelin-1 (ET-1), a potent vasoconstrictor, lowers intraocular pressure in mammals, either by enhancing the outflow of aqueous humor (AH) via the trabecular meshwork and Schlemm's canal or by reducing AH formation at the ciliary epithelium. Aqueous humor production occurs by passive diffusion of water coupled with active transport of ions, mainly involving Na(+):K(+):2Cl(-) cotransporter and Na(+)/K(+)-ATPase pump from serosal to aqueous side. Presently, we have evaluated the effects of ET-1 on Na(+):K(+):2Cl(-) cotransport and Na(+)/K(+)-ATPase activity in HNPE cells using (86)Rb(+) uptake. ET-1 (100 pM-100 nM) decreased mean (86)Rb(+) uptake by 15% during a 15-min uptake period. ET-1's effect was not prevented by BQ610, an ET(A) receptor antagonist, but was blocked by BQ788, an ET(B) receptor antagonist. ET-1's effect was mimicked by sarafotoxin, an ET(B) agonist. ET-1-induced reduction in (86)Rb(+) uptake was additive with bumetanide, a selective inhibitor of Na(+):K(+):2Cl(-) cotransporter but not with ouabain, a selective inhibitor of the Na(+)/K(+)-ATPase. ET-1 did not affect iberiotoxin-sensitive maxi K(+) channels. This suggests that ET-1-induced reduction in (86)Rb(+) uptake is mediated through the inhibition of the Na(+)/K(+)-ATPase via an ET(B)-like receptor. These findings are consistent with an ET-1 effect on active ion transport activity in HNPE cells that could explain the reduction in aqueous humor production and the lowering of intraocular pressure.