Retinoid metabolism in cultured human retinal pigment epithelium.

Uptake, esterification and release of all-trans-retinol in primary cultures of human retinal epithelium were studied. Cultured cells were supplemented with 3H-labelled 11,12-all-trans-retinol, using fatty-acid-free albumin as the carrier. This led to incorporation of retinal and the formation of all-trans- and 11-cis-retinyl palmitate. The metabolism of the all-trans ester was monitored in a medium containing various concentrations of foetal-bovine serum (FBS). In 20% (v/v) FBS, the ester was hydrolysed, and all-trans-retinol was released into the culture medium. In the absence of FBS, little ester was hydrolysed and no retinol was found in the medium. Dialysed or heat-inactivated FBS or fatty-acid-free albumin was as effective as FBS in provoking ester hydrolysis and retinol release. The concentration-dependency of this effect on FBS was matched by the corresponding concentrations of albumin alone. A linear relationship was also found between interphotoreceptor retinoid-binding protein and retinoid release. Haemoglobin, which does not bind retinoids, is ineffective in this capacity. It is concluded that lipid-binding substances, mainly albumin, in FBS act as acceptors for retinol and drain the cultured cells of this molecule. The release of the retinol is coupled to the hydrolysis of retinyl esters in the cell, so that there is little or no net hydrolysis of ester if there is no acceptor for retinol in the culture medium. This effect explains why cultured human retinal epithelial cells are depleted of their stores of retinoids when maintained in medium supplemented with FBS.     

Decreased VEGF-A and sustained PEDF expression in a human retinal pigment epithelium cell line cultured under hypothermia

Background

Previous reports have described a decrease in retinal temperature and clinical improvement of wet age-related macular degeneration (AMD) after vitrectomy. We hypothesized that the retinal temperature decrease after vitrectomy plays a part in the suppression of wet AMD development. To test this hypothesis, we evaluated the temperature dependence of the expression of vascular endothelial growth factor-A (VEGF-A) and in vitro angiogenesis in retinal pigment epithelium (RPE).

Results

We cultured ARPE-19 cells at 37, 35, 33 and 31°C and measured the expression of VEGF-A, VEGF-A splicing variants, and pigment epithelium–derived factor (PEDF). We performed an in vitro tube formation assay. The dehydrogenase activity was also evaluated at each temperature. Expression of VEGF-A significantly decreased with decreased temperature while PEDF expression did not. VEGF165 expression and in vitro angiogenesis also were temperature dependent. The dehydrogenase activity significantly decreased as the culture temperature decreased.

Conclusions

RPE cultured under hypothermia that decreased cellular metabolism also had decreased VEGF-A and sustained PEDF expression, creating an anti-angiogenic environment. This mechanism may be associated with a beneficial effect after vitrectomy in patients with wet AMD.     

Puerarin decreases apoptosis of retinal pigment epithelial cells in diabetic rats by reducing peroxynitrite level and iNOS expression

The purpose of this study was to investigate the protective effect of puerarin on retina pigment epithelial (RPE) cells of diabetic rats against apoptosis. One hundred and eight Sprague-Dawley (SD) rats were randomly divided into 3 groups: control group, streptozotocin (STZ) group and puerarin group. STZ and puerarin groups received 3 d of STZ injection (45 mg/kg per day, i.p.). Additionally, puerarin groups were treated with puerarin (140 mg/kg, i.p.) from the 4th day to the end of experiment. The rats from different groups were sacrificed on 20, 40 and 60 d after STZ injection for harvesting RPE cells. Western blot analysis, DNA laddering, RT-PCR and immunohistochemistry were used for determining the expression of nitrotyrosine (NT, the foot print of peroxynitrite), cell apoptosis, iNOS mRNA and Fas/Fas ligand (FasL) signal transduction in RPE cells, respectively. The results showed that control group maintained low apoptosis level and little NT, iNOS mRNA, Fas/FasL protein expressions, as well as normal blood glucose and body weight during 60 d of the experiment. Compared with control group, STZ group showed obvious apoptosis and higher NT, iNOS mRNA, Fas/FasL protein expressions from 20 d after STZ injection. Puerarin relieved apoptosis of RPE cells and decreased NT, iNOS mRNA, Fas/FasL protein expressions in puerarin group 20 or 40 d after STZ injection, compared with STZ group. These results suggest puerarin can decrease RPE cells apoptosis in diabetic rats by reducing peroxynitrite level and iNOS expression, thus being a potential therapeutic agent in controlling of diabetic retinopathy.     

Retinoid-binding proteins and retinol esterification in cultured retinal pigment epithelium cells

The esterification of all-trans retinol and the occurrence of cytosolic retinoid-binding proteins was investigated in cultured bovine retinal pigment epithelium (RPE) cells. ³H-labeled all-trans retinyl ester (mainly palmitate) was formed at an initial rate of 0.1 nmol·mg protein⁻¹·min⁻¹ when ³H-labeled all-trans retinol was incubated with the 100,000 g pellet obtained from a homogenate of freshly-harvested cells. No esterification could be detected under the same conditions after 14 days in culture in defined medium (DM) or in medium containing 20% fetal bovine serum (CM). No enhancement or restoration of esterifying capacity was observed when the assay mixture was supplemented with palmitoyl CoA. As determined by specific, saturable binding of ³H-labeled all-trans retinol and ³H-labeled 11-cis retinal to proteins with mol. wts 16,000 and 33,000 dalton on calibrated Bio-Sil TSK 250 size-exclusion columns, the cytosol of freshly-harvested RPE cells contained cellular retinol-binding protein (CRBP) and cellular retinal-binding protein (CRAlBP). By comparison with the quantity of ³H-labeled all-trans retinol bound under identical conditions to pure dog liver CRBP, it was estimated that fresh RPE cells contained 102 ± 3 ng CRBP·μg cytosol protein⁻¹. In cultured and subcultured cells, CRBP was present at much lower levels (down to one-tenth of the initial amounts) and CRAlBP could not be detected. Since binding of ³H-labeled all-trans retinoic acid to a protein with molecular weight of 17,000 dalton was not observed in the cytosols of fresh or cultured cells, it was concluded that cellular retinoic acid binding protein (CRABP) was either present at very low levels or absent altogether. An unidentified peak of specific ³H-labeled all-trans-retinoic acid binding at mol. wt 61,000 dalton was prominent in subcultured cells. These results show that in RPE cells in culture the expression of differentiated phenotype with respect to retinoid utilization undergoes significant modification. It is postulated that changes in the composition of the extracellular matrix (e.g. absence of interstitial retinol-binding protein, IRBP) may be involved.     

Potassium currents in cultured cells of the rat retinal pigment epithelium

Whole-cell currents were investigated in cultured rat retinal pigment epithelial (RPE) cells. Two voltage-dependent conductances were discriminated. First, at potentials more positive than −30 mV, a time-dependent outward current was activated. Inhibition by Ba²⁺ (10 mM) and 4-aminopyridine (10 mM) indicated that this current was carried by potassium ions. This current showed no inactivation during 5 sec depolarizations. Second, an inward current, sensitive to Ba²⁺ (10 mM) and 4-aminopyridine (10 mM), was activated at potentials more negative than — 70 mV. Under extra- and intracellular potassium-free conditions, both currents disappeared. In summary, cultured rat RPE cells expressed one potassium conductance similar to the delayed rectifier and one similar to the inward rectifier. The delayed rectifier expressed characteristics comparable with those known in mammalian species and different from those in non-mammalian species.     

Regulation of the expression of lysyl oxidase mRNA in cultured rabbit retinal pigment epithelium cells.

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.     

Platelet-derived growth factor gene expression in cultured human retinal pigment epithelial cells.

Gene expression of platelet-derived growth factor (PDGF) and its receptors in cultured human retinal pigment epithelial (RPE) cells was studied by using semiquantitative polymerase chain reaction. The RPE cells were found to express PDGF A- and B-chain genes as well as alpha- and beta-receptor genes with dominant expression of B-chain and beta-receptor isoforms. Phorbol myristate acetate (PMA) and thrombin increased the expression of PDGF B-chain gene to 19.8 +/- 1.75 and 15.9 +/- 1.84 fold (n = 3) of the control without affecting beta-receptor gene expression. PDGF produced by the RPE cells may play an important role in the pathogenesis of some ocular proliferative diseases.     

High glucose induces mitochondrial dysfunction and apoptosis in human retinal pigment epithelium cells via promoting SOCS1 and Fas/FasL signaling

Diabetic retinopathy (DR) is one of the most serious complications of diabetes mellitus (DM), however, the contribution of high glucose (HG) or hyperglycemia to DR is far from fully understanding. In the present study, we examined the expression of Fas/FasL signaling and suppressors of cytokine signaling (SOCS)1 and 3 in HG-induced human retinal pigment epithelium cells (ARPE-19 cells). And then we investigated the regulatory role of both Fas and SOCS1 in HG-induced mitochondrial dysfunction and apoptosis. Results demonstrated that HG with more than 40 mM induced mitochondrial dysfunction via reducing mitochondrial membrane potential (MMP) and via inhibiting the Bcl-2 level, which is the upstream signaling of mitochondria in ARPE-19 cells. HG also upreuglated the Fas signaling and SOCS levels probably via promoting JAK/STAT signaling in ARPE-19 cells. Moreover, the exogenous Fas or entogenous overexpressed SOCS1 accentuated the HG-induced mitochondrial dysfunction and apoptosis, whereas the knockdown of either Fas or SOCS1 reduced the HG-induced mitochondria dysfunction and apoptosis. Thus, the present study confirmed that both Fas/FasL signaling and SOCS1 promoted the HG-induced mitochondrial dysfunction and apoptosis. These results implies the key regulatory role of Fas signaling and SOCS in DR.     

Precursor cystatin C in cultured retinal pigment epithelium cells: evidence for processing through the secretory pathway

Evidence was recently reported that the cysteine proteinase inhibitor, cystatin C, is highly expressed by cultured human retinal pigment epithelial (RPE) cells. As a step towards understanding possible functions of this protein associated with the RPE, the localization, targetting and trafficking of cystatin C were investigated. Constructs encoding an enhanced variant of green fluorescent protein (EGFP) fused to precursor cystatin C and to mature cystatin C were made and transfected into cultured human RPE cells. Expression of fusion proteins was monitored in vivo by fluorescence confocal microscopy. In cells transfected with precursor cystatin C-EGFP, fluorescence was initially targetted to the perinuclear zone, co-localizing with the Golgi apparatus. Transfected cells were observed at intervals over a period of up to 3 weeks, during which time fluorescent vesicles developed peripherally and basally while fluorescence continued to be detected in the Golgi region. Immunochemical analysis of cell lysates confirmed the expression of a fusion protein recognized by antibodies to both cystatin C and EGFP. Cells transfected with the construct lacking the leader peptide of precursor cystatin C presented a diffuse and weak fluorescence. Together, these results imply a leader sequence-dependent processing of cystatin C through the secretory pathway of RPE cells. This was confirmed by the detection, by Western blotting, of the chimaeric protein alongside endogenous cystatin C in the medium of transfected RPE cells.     

Morphogenesis and nature of the pigment granules in the adult human retinal pigment epithelium

Summary The pigment epithelial cells of the retina are a layer of highly specialized melanocytes. Beginning in the early embryonic period they produce melanin throughout the entire life. The Golgi apparatus plays a key role in the biosynthesis of melanin. The following steps can be distinguished morphologically: (a) Golgi-vesicles, (b) intermediate vesicles, (c) melanosomes, (d) melanin granules. Structures with a ringlike appearance that are described as lipofuscin granules in the literature prove to be altered intermediate vesicles and melanosomes.This investigation was carried out in part at the Francis I. Proctor Foundation for Research in Ophthalmology, San Francisco, California, U.S.A., and supported by United States Public Health Service Program Project Grant EY 00310, and Deutsche Forschungsgemeinschaft, Training Grant Nr. Sp 102/1.     

The glucose transport in retinal pigment epithelium is via passive facilitated diffusion

The glucose transport across the bovine retinal pigment epithelium (RPE) was studied in a modified Ussing chamber. Unidirectional fluxes were recorded with radioactive tracers L-[14C]-glucose (LG) and 3-O-methyl-D-[3H]-glucose (MDG). There was no significant difference between the unidirectional MDG fluxes (retina to choroid, and choroid to retina directions) with or without ouabain. The effects of two glucose transporter inhibitors, phloretin and cytochalasin B, on the glucose fluxes from choroid to retina cells were also investigated. The MDG flux was found to be inhibited by 45.5% by phloretin (10(-4) M) and 87.4% by cytochalasin B (10(-4) M). These inhibitory characteristics resembled the facilitated diffusion mode of glucose transport. The glucose transporter protein in the plasma membrane of RPE was located by means of photolabeling [3H]-cytochalasin B. The labeled plasma membrane enriched fraction was analysed by SDS-PAGE. The glucose transporter of bovine RPE was found to have a molecular weight range of 46-53 kDa. The molecular weight range of this transporter protein agreed with those of facilitated glucose transporters in other tissues indicating a molecular similarity between them. The results indicated that the glucose transport across the RPE is via passive facilitated diffusion.     

Regulation of taurine transporter expression by NO in cultured human retinal pigment epithelial cells

Taurine is activelytransported at the retinal pigment epithelial (RPE) apical membrane inan Na⁺- and Cl-dependent manner. Diabetes mayalter the function of the taurine transporter. Because nitric oxide(NO) is a molecule implicated in the pathogenesis of diabetes, we askedwhether NO would alter the activity of the taurine transporter incultured ARPE-19 cells. The activity of the transporter was stimulatedin the presence of the NO donor 3-morpholinosydnonimine. Thestimulatory effects of 3-morpholinosydnonimine were not observed duringthe initial 16-h treatment; however, stimulation of taurine uptake waselevated dramatically above control values with 20- and 24-htreatments. Kinetic analysis revealed that the stimulation wasassociated with an increase in the maximal velocity of the transporterwith no significant change in the substrate affinity. The NO-induced increase in taurine uptake was inhibited by actinomycin D and cycloheximide. RT-PCR analysis and nuclear run-on assays provided evidence for upregulation of the transporter gene. This study providesthe first evidence of an increase in taurine transporter geneexpression in human RPE cells cultured under conditions of elevatedlevels of NO.

     

Precursor cystatin C in cultured retinal pigment epithelium cells: evidence for processing through the secretory pathway.

Evidence was recently reported that the cysteine proteinase inhibitor, cystatin C, is highly expressed by cultured human retinal pigment epithelial (RPE) cells. As a step towards understanding possible functions of this protein associated with the RPE, the localization, targetting and trafficking of cystatin C were investigated. Constructs encoding an enhanced variant of green fluorescent protein (EGFP) fused to precursor cystatin C and to mature cystatin C were made and transfected into cultured human RPE cells. Expression of fusion proteins was monitored in vivo by fluorescence confocal microscopy. In cells transfected with precursor cystatin C-EGFP, fluorescence was initially targetted to the perinuclear zone, co-localizing with the Golgi apparatus. Transfected cells were observed at intervals over a period of up to 3 weeks, during which time fluorescent vesicles developed peripherally and basally while fluorescence continued to be detected in the Golgi region. Immunochemical analysis of cell lysates confirmed the expression of a fusion protein recognized by antibodies to both cystatin C and EGFP. Cells transfected with the construct lacking the leader peptide of precursor cystatin C presented a diffuse and weak fluorescence. Together, these results imply a leader sequence-dependent processing of cystatin C through the secretory pathway of RPE cells. This was confirmed by the detection, by Western blotting, of the chimaeric protein alongside endogenous cystatin C in the medium of transfected RPE cells.     

mTOR activity is essential for retinal pigment epithelium regeneration in zebrafish

The retinal pigment epithelium (RPE) plays numerous critical roles in maintaining vision and this is underscored by the prevalence of degenerative blinding diseases like age-related macular degeneration (AMD), in which visual impairment is caused by progressive loss of RPE cells. In contrast to mammals, zebrafish possess the ability to intrinsically regenerate a functional RPE layer after severe injury. The molecular underpinnings of this regenerative process remain largely unknown yet hold tremendous potential for developing treatment strategies to stimulate endogenous regeneration in the human eye. In this study, we demonstrate that the mTOR pathway is activated in RPE cells post-genetic ablation. Pharmacological and genetic inhibition of mTOR activity impaired RPE regeneration, while mTOR activation enhanced RPE recovery post-injury, demonstrating that mTOR activity is essential for RPE regeneration in zebrafish. RNA-seq of RPE isolated from mTOR-inhibited larvae identified a number of genes and pathways dependent on mTOR activity at early and late stages of regeneration; amongst these were components of the immune system, which is emerging as a key regulator of regenerative responses across various tissue and model systems. Our results identify crosstalk between macrophages/microglia and the RPE, wherein mTOR activity is required for recruitment of macrophages/microglia to the RPE injury site. Macrophages/microglia then reinforce mTOR activity in regenerating RPE cells. Interestingly, the function of macrophages/microglia in maintaining mTOR activity in the RPE appeared to be inflammation-independent. Taken together, these data identify mTOR activity as a key regulator of RPE regeneration and link the mTOR pathway to immune responses in facilitating RPE regeneration.