Bovine Retinal Pigment Epithelial Cells Research Articles

Biocompatibility of the vital dye Acid Violet-17 on retinal pigment epithelial cells.

PurposeTo examine the viability and differentiation of retinal pigment epithelial (RPE) cells after exposure to the vital dye Acid Violet-17 (AV-17).MethodsBovine RPE cells were incubated with AV-17 (0.0625–0.5 mg/mL) for 30 seconds or 5 minutes. Viability was determined by live/dead staining, cleaved CASP3 immunostainings, and MTT test. Actin cytoskeleton was visualized by Alexa 488-phalloidin. Immunocytochemistry was performed to determine the levels of ZO-1, CTNNB1, and KRT19.ResultsExposure to AV-17 at the concentrations of 0.25–0.5 mg/mL resulted in a dose-dependent decrease in viability, the loss of ZO-1 from tight junctions, translocation of CTNNB1 into the cytoplasm and nucleus, disarrangement of the actin cytoskeleton, and a slight increase in KRT19.ConclusionAV-17 at a concentration <0.125 mg/mL is likely to be well tolerated by the RPE cells, whereas the concentrations from 0.25 mg/mL onward can reduce viability and induce dedifferentiation particularly after long-term exposure.

Tadao Nakago, 1933-2016

Tadao Nakago died of bladder cancer on February 12, 2016, just 2 days before his 83rd birthday. He was born on February 14, 1933, in Sanda, north of the Rokko Mountains in Kobe, Japan. He graduated from Osaka University Dental School in 1957 and became a dentist. He chose orthodontics as his specialty and earned a PhD degree from Osaka University in 1965. His academic interest in the early phase of his career was the cephalometric assessment of craniofacial development in the Japanese population to qualify desirable tooth movement. He became a founding professor of orthodontics at Matsumoto Dental College in 1972. He moved to Okayama University in 1980 as a founding director of the dental school-affiliated hospital and served as president of the hospital until 1988. In 1982, he successfully moved the government of Japan to include, for the first time, orthodontic treatment for craniofacial anomalies, such as cleft palate, on the list for national health insurance reimbursement. He served as dean of the dental school from 1992 to 1994. He survived a bitter administrative storm at Okayama University in 1996 and restored its honor at the law court in 2002. He loved to educate young people and tried to disseminate practical thinking about the standards of orthodontic treatment and care. He translated Graber's Textbook of Orthodontics into Japanese, and it became a best-seller. He liked to organize laboratory studies to explain the mechanism of tooth movement. He designed a device to apply cyclic mechanical stretching to cultured cells in a Petri dish.1Matsuo T. Uchida H. Matsuo T. Bovine and porcine trabecular cells produce prostaglandin F2α in response to cyclic mechanical stretching.Jpn J Ophthalmol. 1996; 40: 289-296PubMed Google Scholar The cell-stretching machine was manufactured in 1983 at the workshop affiliated with the Faculty of Engineering of Okayama University. This unique original device preceded commercially available apparatuses and contributed to new knowledge in other fields of medicine.2Okada Y. Matsuo T. Ohtsuki H. Bovine trabecular cells produce TIMP-1 and MMP-2 in response to mechanical stretching.Jpn J Ophthalmol. 1998; 42: 90-94Crossref Scopus (44) Google Scholar, 3Sato Y. Matsuo T. Ohtsuki H. A novel gene (oculomedin) induced by mechanical stretching in human trabecular cells of the eye.Biochem Biophys Res Commun. 1999; 259: 349-351Crossref Scopus (30) Google Scholar, 4Yamaguchi K. Matsuo T. Shiraga F. Ohtsuki H. TIMP-1 production by bovine retinal pigment epithelial cells increases in response to cyclic mechanical stretch.Jpn J Ophthalmol. 2001; 45: 470-474Crossref Scopus (5) Google Scholar, 5Yamaoka A. Matsuo T. Shiraga F. Ohtsuki H. TIMP-1 production by human scleral fibroblast decreases in response to cyclic mechanical stretching.Ophthalmic Res. 2001; 33: 98-101Crossref Scopus (22) Google Scholar, 6Namba M. Matsuo T. Shiraga F. Ohtsuki H. Retinal cells produce TIMP-1 and TIMP-2 in response to cyclic mechanical stretching.Ophthalmic Res. 2001; 33: 163-169Crossref Scopus (11) Google Scholar His last work was focused on the threshold of hydraulic pressure to induce cellular response in periodontal ligament fibroblasts.7Nakago-Matsuo C. Matsuo T. Nakago T. Intracellular calcium response to hydraulic pressure in human periodontal ligament fibroblasts.Am J Orthod Dentofacial Orthop. 1996; 109: 244-248Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 8Nakago-Matsuo C. Matsuo T. Nakago T. Basal nitric oxide production is enhanced by hydraulic pressure in cultured human periodontal ligament fibroblasts.Am J Orthod Dentofacial Orthop. 2000; 117: 474-478Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar The threshold value was consistent with the force needed to move teeth in a clinical orthodontic setting. The work was continued by his daughter, an orthodontist, in collaboration with her husband, an ophthalmologist.9Matsuo T. Matsuo N. Intracellular calcium response to hydraulic pressure in human trabecular cells.Br J Ophthalmol. 1996; 80: 561-566Crossref Scopus (27) Google Scholar, 10Matsuo T. Basal nitric oxide production is enhanced by hydraulic pressure in cultured human trabecular cells.Br J Ophthalmol. 2000; 84: 631-635Crossref Scopus (15) Google Scholar Dr Nakago's funeral was held on his 83rd birthday, with family and friends present at the Buddhist temple where his wife's father had served as a priest.

KCNQ and KCNE potassium channel subunit expression in bovine retinal pigment epithelium

Human, monkey, and bovine retinal pigment epithelial (RPE) cells exhibit an M-type K+ current, which in many other cell types is mediated by channels composed of KCNQ α-subunits and KCNE auxiliary subunits. Recently, we demonstrated the expression of KCNQ1, KCNQ4, and KCNQ5 in the monkey RPE. Here, we investigated the expression of KCNQ and KCNE subunits in native bovine RPE. RT-PCR analysis revealed the expression of KCNQ1, KCNQ4, and KCNQ5 transcripts in the RPE, but, in Western blot analysis of RPE plasma membranes, only KCNQ5 was detected. Among the five members of the KCNE gene family, transcripts for KCNE1, KCNE2, KCNE3, and KCNE4 were detected in bovine RPE, but only KCNE1 and KCNE2 proteins were detected. Immunohistochemistry of frozen bovine retinal sections revealed KCNE1 expression near the apical and basal membranes of the RPE, in cone outer segments, in the outer nuclear layer, and throughout the inner retina. The localization of KCNE1 in the RPE basal membrane, where KCNQ5 was previously found to be present, suggests that this β-subunit may contribute to M-type K+ channels in this membrane.

Blue LED light exposure develops intracellular reactive oxygen species, lipid peroxidation, and subsequent cellular injuries in cultured bovine retinal pigment epithelial cells

The effects of blue light emitter diode (LED) light exposure on retinal pigment epithelial cells (RPE cells) were examined to detect cellular damage or change and to clarify its mechanisms. The RPE cells were cultured and exposed by blue (470 nm) LED at 4.8 mW/cm2. The cellular viability was determined by XTT assay and cellular injury was determined by the lactate dehydrogenase activity in medium. Intracellular reactive oxygen species (ROS) generation was determined by confocal laser microscope image analysis using dihydrorhodamine 123 and lipid peroxidation was determined by 4-hydroxy-2-nonenal protein-adducts immunofluorescent staining (HNE). At 24 h after 50 J/cm2 exposures, cellular viability was significantly decreased to 74% and cellular injury was significantly increased to 365% of control. Immediately after the light exposure, ROS generation was significantly increased to 154%, 177%, and 395% of control and HNE intensity was increased to 211%, 359%, and 746% of control by 1, 10, and 50 J/cm2, respectively. These results suggest, at least in part, that oxidative stress is an early step leading to cellular damage by blue LED exposure and cellular oxidative damage would be caused by the blue light exposure at even lower dose (1, 10 J/cm2).

The effect of OPTC-shRNA on the bovine retinal pigment epithelial cells and hyalocytes co-culture collagen gel contraction system

To investigate the OPTC-shRNA inhibiting effect on the opticin expression by the bovine hyalocytes and retina pigment epithelial (RPE) cells co-culture collagen gel contraction system. Experimental study. The OPTC-shRNA expression vector was designed and transfected into bovine RPE cells cultured in vitro. The relative expression and the inhibition rate of the opticin protein were measured by Western blot on days 3, 5 and 7. An in vitro cells co-culture bovine type I collagen gel contraction assay was constructed consisting of the hyalocytes and RPE cells. Six groups were established in this experiments:OPTC-shRNA plasmid transfected RPE cells and hyalocytes (group A), empty plasmid transfected RPE cells and hyalocytes (group B), non-transfected RPE cells and hyalocytes (group C), non-transfected RPE cells (group D), only hyalocytes (group E), and no cells (group F). The collagen gel contractile activities of these groups were compared by One-way ANOVA, SNK-q tests and regression analysis;and the influence of the hyalocytes density variance on the collagen gel contraction in groups A, B and C were also analyzed. The OPTC-shRNA expression vector with significant inhibition effect was constructed and transfected into bovine RPE cells successfully. The results of Western blot analysis showed that the inhibitory rates on the opticin expression on days 3, 5 and 7 were (83.91 ± 2.88), (84.71 ± 4.27) and (82.85 ± 2.72)%, respectively. Furthermore, the differences among days 3, 5 and 7 were insignificant (F = 1.15, P > 0.05). On day 3, the gel contraction rates for the sub-groups with various hyalocytes densities (2×10(7), 1×10(8) and 5×10(8)/L) in groups A, B and C were: group A: (23.52 ± 2.08), (56.00 ± 1.02), (61.62 ± 1.73)%; group B: (16.56 ± 2.01), (36.41 ± 1.33), (49.56 ± 1.75)%; group C: (15.75 ± 1.37), (37.45 ± 1.14), (48.45 ± 1.97)%. The gel contraction rates for groups D and E were (12.18 ± 0.95)% and (10.95 ± 0.93)%, respectively; no gel contraction was observed in group F. Pairwise comparisons of the gel contraction rates were performed by SNK-q test among groups A, B and C for various hyalocyte densities. In the 2×10(7)/L cell density group, the differences between groups A and B or C were significant (q = 11.38, 2.72, respectively, P both < 0.05), the differences between B and C were insignificant (q = 1.34, P > 0.05). In the 1×10(8)/L cell density group, the differences between groups A and B or C were significant (q = 8.83, 46.22, respectively, P both < 0.05), the differences between B and C were insignificant (q = 1.34, P > 0.05). In the 5×10(8)/L cell density group, the differences between groups A and B or C were significant (q = 48.83, 46.22, respectively, P both < 0.05), the differences between groups B and C were insignificant (q = 1.74, P > 0.05). Pairwise comparisons of the sub-groups with different hyalocyte densities in groups A, B and C (comparisons of 2×10(7)/L and 1×10(8)/L, 2×10(7)/L and 5×10(8)/L, 2×10(7)/L and 2×10(7)/L, respectively), the differences were all significant (group A:q = -55.97, -65.66, -9.69, respectively; group B: q = -34.53, -57.41, -22.88, respectively; group C: q = -41.94, -63.19, -21.25, P all < 0.05). Furthermore, the regression analysis was performed between the hyalocyte density and the collage gel contraction rates in each group. The results showed that there was a positive correlation between the gel contraction rates of the co-culture collagen gel contraction system and its hyalocyte density (groups A, B, C: r = 0.919, 0.981, 0.937, respectively, P all < 0.05). Pairwise comparison of groups D and E, D and F, E and F by SNK-q test revealed q = 54.87, 49.33, 5.54, respectively, P all < 0.05. Opticin is capable of regulating the contraction of bovine hyalocytes and RPE cells co-culture collagen gel.

UV-Absorption Spectra of Melanosomes Containing Varying 5,6-Dihydroxyindole and 5,6-Dihydroxyindole-2-Carboxylic Acid Content

Central to understanding the photochemical properties of melanosomes is a direct measurement of their absorption coefficients. Herein, the absorption spectra of intact melanosomes of varying molecular compositions and embryonic origins were measured and compared over the spectral range from 245 to 310 nm. The absorption spectra of melanosomes predominately comprised of the eumelanin pigment were found to differ significantly from their constituent precursor molecules, 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA). This difference was most notable in the UV-A region and indicates that the electronic structures of the monomeric building blocks, DHICA in particular, are significantly modified upon polymerization to the melanin pigment. Furthermore, in comparing embryonic differences, the absorption coefficients of melanosomes isolated from bovine retinal pigment epithelial (RPE) cells (originating from the primitive forebrain) were greater than those for bovine choroid or iris melanosomes (originating from the neural crest). This difference suggests that either the pigment is present in greater density in RPE melanosomes or that there is an underlying difference in molecular structure.

Evidence of alpha 7 nicotinic acetylcholine receptor expression in retinal pigment epithelial cells

Some evidence suggests that retinal pigment epithelium (RPE) can express nicotinic acetylcholine receptors (nAChRs) as described for other epithelial cells, where nAChRs have been involved in processes such as cell development, cell death, cell migration, and angiogenesis. This study is designed to determine the expression and activity of α7 nAChRs in RPE cells. Reverse transcriptase (RT)-PCR was performed to test the expression of nicotinic α7 subunit in bovine RPE cells. Protein expression was determined by Western blot and by immunocytochemistry. Expression of nicotinic α7 subunits was also analyzed in cryostat sections of albino rat retina. Changes in protein expression were tested under hypoxic conditions. Functional nAChRs were studied by examining the Ca2+ transients elicited by nicotine and acetylcholine stimulation in fura-2-loaded cells. Expression of endogenous modulators of nAChRs was analyzed by RT-PCR and Western blot in retina and RPE. Cultured bovine RPE cells expressed nicotinic receptors containing α7 subunit. RT-PCR amplified the expected specific α7 fragment. Western blotting showed expression at the protein level, with a specific band being found at 57 kDa in both cultured and freshly isolated RPE cells. Expression of nAChRs was confirmed for cultured cells by immunofluorescence. Immunohistochemistry confirmed α7 receptor expression in rat RPE retina. α7 receptor expression was down-regulated by long-term hypoxia. A small subpopulation of RPE cultured cells showed functional nAChRs, as evidenced by the selective response elicited by nicotine and acetylcholine stimulation. Expression of the endogenous nicotinic receptors' modulator lynx1 was confirmed in bovine retina and RPE, and expression of lynx1 and other endogenous nicotinic receptor modulators (SLURP1 and RGD1308195) were also confirmed in rat retina. These results suggest that nAChRs could have a significant role in RPE, which may not be related to the traditional role in nerve transmission but could more likely be related to the nonneuronal cholinergic system in the eye.

Gold nanoparticles downregulate VEGF-and IL-1β-induced cell proliferation through Src kinase in retinal pigment epithelial cells

Proliferative vitreo retinopathy (PVR) is one of the ocular complications, marked by the enhanced proliferation of various cells including retinal pigment epithelial cells (RPE). The aim of the present study is to analyze the effect of gold nanoparticles (Au-NP) on vascular endothelial growth factor (VEGF) and interleukin-1 beta (IL-1β)-induced cell spreading, migration and proliferation in RPE cells. Au-NP (300 nM) significantly blocked the VEGF-and IL-1β-induced cell spreading, migration and proliferation in bovine RPE cells (BRPEs). To elucidate the signaling mechanism of VEGF- and IL-1β-induced cell proliferation, BRPEs were treated with PP2, a Src inhibitor. Further, to clarify the possible involvement of the Src pathway on the inhibitory effect of Au-NPs, transient transfection assay was performed using dominant negative (DN) and constitutively active (CA) mutant plasmid of Src kinase. The results showed that VEGF and IL-1β exert their proliferative effects through the activation of Src kinase whereas CA Src rescued the inhibitory effect of Au-NP in presence or absence of VEGF and IL-1β in BRPEs. Further, an in vitro kinase assay was performed to identify the status of Src phosphorylation at Y419. We found that VEGF and IL-1β increased Src phosphorylation in BRPEs and Au-NP blocked the VEGF- and IL-1β-induced Src phosphorylation at Y419. Taken together, our result suggests that Au-NP could effectively inhibit the VEGF- and IL-1β-induced proliferation and migration by suppressing the Src kinase pathway in BRPEs and Au-NP might act as an effective therapeutic agent for the treatment of ocular diseases such as proliferative vitreo retinopathy.

Direct Measurement of the Ultraviolet Absorption Coefficient of Single Retinal Melanosomes

A novel approach to photoemission electron microscopy is used to enable the first direct measurement of the absorption coefficient from intact melanosomes isolated from bovine retinal pigment epithelial cells. The difference in absorption between newborn and adult melanosomes is in good agreement with that predicted from the relative amounts of the monomeric precursors present in the constituent melanin as determined by chemical degradation analyses. The results demonstrate that for melanosomes containing eumelanins, there is a direct relation between the absorption coefficient and the relative 5,6-dihydroxyindole: 5,6-dihydroxyindole-2-carboxylic acid (DHI:DHICA) content, with an increased UV absorption coefficient associated with increasing DHICA content.

Rpe65 Isomerase Associates with Membranes through an Electrostatic Interaction with Acidic Phospholipid Headgroups

Opsins are light-sensitive pigments in the vertebrate retina, comprising a G protein-coupled receptor and an 11-cis-retinaldehyde chromophore. Absorption of a photon by an opsin pigment induces isomerization of its chromophore to all-trans-retinaldehyde. After a brief period of activation, opsin releases all-trans-retinaldehyde and becomes insensitive to light. Restoration of light sensitivity to the apo-opsin involves the conversion of all-trans-retinaldehyde back to 11-cis-retinaldehyde via an enzyme pathway called the visual cycle. The critical isomerization step in this pathway is catalyzed by Rpe65. Rpe65 is strongly associated with membranes but contains no membrane-spanning segments. It was previously suggested that the affinity of Rpe65 for membranes is due to palmitoylation of one or more Cys residues. In this study, we re-examined this hypothesis. By two independent strategies involving mass spectrometry, we show that Rpe65 is not palmitoylated nor does it appear to undergo other post-translational modifications at significant stoichiometry. Instead, we show that Rpe65 binds the acidic phospholipids, phosphatidylserine, phosphatidylglycerol, and cardiolipin, but not phosphatidic acid. No binding of Rpe65 to basic phospholipids or neutral lipids was observed. The affinity of Rpe65 to acidic phospholipids was strongly pH-dependent, suggesting an electrostatic interaction of basic residues in Rpe65 with negatively charged phospholipid headgroups. Binding of Rpe65 to liposomes containing phosphatidylserine or phosphatidylglycerol, but not the basic or neutral phospholipids, allowed the enzyme to extract its insoluble substrate, all-trans-retinyl palmitate, from the lipid bilayer for synthesis of 11-cis-retinol. The interaction of Rpe65 with acidic phospholipids is therefore biologically relevant.

Regulation of Kir channels in bovine retinal pigment epithelial cells by phosphatidylinositol 4,5-bisphosphate

The inwardly rectifying K+ (Kir) current in mammalian retinal pigment epithelial (RPE) cells, which is largely mediated by Kir7.1 channels, is stable in cells dialyzed with MgATP but runs down when intracellular ATP is depleted. A potential mechanism for this rundown is a decrease in phosphatidylinositol 4,5-bisphosphate (PIP2) regeneration by ATP-dependent lipid kinases. Here, we used the whole cell voltage-clamp technique to investigate the membrane PIP2 dependence of Kir channels in isolated bovine RPE cells. When RPE cells were dialyzed with ATP-free solution containing PIP2 (25-50 microM), rundown persisted but was markedly reduced. Removal of Mg2+ from the pipette solution also slowed rundown, indicating that elevated intracellular Mg2+ concentration contributes to rundown. Cell dialysis with the PIP2 scavenger neomycin in MgATP solution diminished Kir current in a voltage-dependent manner, suggesting that it acted at least in part by blocking the Kir channel. Kir current in MgATP-loaded cells was partially inhibited by bath application of quercetin (100 microM), phenylarsine oxide (100 microM), or wortmannin (50 microM), inhibitors of phosphatidylinositol (PI) kinases, and was completely inhibited by cell dialysis with 2 mM adenosine, a PI4 kinase inhibitor. Both LY-294002 (100 microM), an inhibitor of PI3 kinases, and its inactive analog LY-303511 (100 microM) rapidly and reversibly inhibited Kir current, suggesting that these compounds act as direct channel blockers. We conclude that the activity of Kir channels in the RPE is critically dependent on the regeneration of membrane PIP2 by PI4 kinases and that this may explain the dependence of these channels on hydrolyzable ATP.

Regulated secretion of complement factor H by RPE and its role in RPE migration

Variants in the gene for complement factor H (CFH) have been implicated as a major risk factor for the development of age-related macular degeneration (AMD). Little is known, however, about the factors regulating local expression and secretion of CFH by retinal pigment epithelial cells (RPE). Cultured human early passage RPE cells, highly differentiated, polarized human RPE cultures, and bovine RPE explants were incubated in the presence or absence of recombinant human or bovine interferon-gamma (IFN-gamma; 25 ng/ml). CFH expression in cell lysates, and secretion into culture supernatants were examined by Western blot. CHF expression and localization was analyzed by confocal microscopy. Migration assay was performed in a modified Boyden chamber with early passage human RPE cells after stimulation with recombinant CFH protein (1-100 ng/ml). CFH was expressed in the cell lysates of RPE cells, and this expression was significantly upregulated by IFN-gamma. Immunoreactivity for CFH was detected in RPE cells of bovine explants and highly differentiated human RPE monolayers, and the level of immunoreactivity increased after IFN-gamma stimulation. Confocal microscopy revealed that CFH was predominantly localized in the apical cytoplasm of polarized human RPE. Western blot confirmed that IFN-gamma increased CFH secretion into RPE supernatants. Dose-dependent RPE cell chemotactic migration was induced by CFH. IFN-gamma promotes CFH expression in the apical compartment of RPE cells and increases secretion of CFH into RPE culture supernatants. Furthermore, CFH promotes chemotactic migration of RPE. This study suggests that interactions between CFH and IFN-gamma have the potential to play a role in the pathogenesis of AMD.

Selective Regulation of Heme Oxygenase-1 Expression and Function by Insulin through IRS1/Phosphoinositide 3-Kinase/Akt-2 Pathway

Heme oxygenase 1 (HO-1) is a representative mediator of antioxidants and cytoprotectants against various stress stimuli including oxidants in vascular cells. Intensive insulin treatment can delay the onset and progression of diabetic retinopathy and other vascularopathies, yet little is known about insulin regulation of anti-apoptotic and antioxidant molecules such as HO-1 in vascular cells. Intravitreous injection or in vitro addition of insulin increased HO-1 protein expression in rat retina and in cultured bovine retinal pericytes, retinal endothelial cells, and retinal pigment epithelial cells. In bovine retinal pericytes, insulin induced mRNA and protein expression of HO-1 in a time- and concentration-dependent manner. Using HO-1 promoter analysis, the luciferase reporter assay showed that induction of HO-1 expression by insulin is mediated by additional response elements in the ho-1 promoter gene, which was not responsive to antioxidants. Insulin-induced HO-1 mRNA expression through activation of PI3-kinase/Akt pathway without affecting ERK and p38 MAPK. Overexpression of an adenoviral vector of native IRS1, IRS2, and Akt dominant negative or small interfering RNA transfection of Akt1 and Akt2 targeted gene demonstrated that insulin regulated HO-1 expression via IRS1 and Akt2 pathway, selectively. Further, insulin treatment prevented H(2)O(2)-induced NF-kappaB and caspase-8 activation and apoptosis via the IRS1/PI3K/Akt2/HO-1 pathway in the pericytes. In conclusion, we suggest that the anti-apoptotic properties of insulin are mediated partly by increasing HO-1 expression at transcriptional level via IRS1/PI3K/Akt2 activation, a potential explanation for how insulin is retarding the progression of microvascular complications induced by diabetes.

Fibroblast Growth Factors Decrease Inducible Nitric Oxide Synthase mRNA Accumulation in Bovine Retinal Pigmented Epithelial Cells

Bovine retinal pigmented epithelial cells (RPE cells), after activation with interferon gamma (IFN gamma) and lipopolysaccharide (LPS), express an inducible nitric oxide (NO) synthase. This activity can be inhibited by the fibroblast growth factors (FGF), FGF-1 (acidic FGF) and FGF-2 (basic FGF). We have attempted to elucidate the molecular mechanisms involved in the FGF inhibition of NO synthase activity. Analysis by immunocytochemistry and Western blot using a polyclonal antibody against the inducible NO synthase of rat liver reveals that RPE cells co-stimulated with FGF-2 and LPS/IFN gamma accumulate lower levels of NO synthase than in the absence of FGF-2. Northern blot analysis by cross-species hybridization with a mouse macrophage NO synthase cDNA probe shows that a 4.4-kb mRNA accumulates when RPE cells are activated with LPS/IFN gamma. The level of inducible NO synthase mRNA in LPS/IFN gamma-activated RPE cells is markedly reduced by FGF-1 or FGF-2 treatment. Message stability studies revealed that the presence of FGF did not accelerate mRNA degradation, implying that FGF did not act on inducible NO synthase mRNA stability, but more probably on its expression. Furthermore an effect of FGF on IFN gamma receptors was excluded, since IFN gamma binding was not altered by FGF. Since NO acts as a cytostatic compound, FGF, by preventing NO synthase expression in RPE cells, may protect the retina from endotoxin and cytokine-mediated tissue damage.

Glypican 4, a Membrane Binding Protein for Bactericidal/Permeability-Increasing Protein Signaling Pathways in Retinal Pigment Epithelial Cells

Originally identified as a lipopolysaccharide binding protein with Gram-negative bactericidal activity in the leukocytes, bactericidal/permeability-increasing protein (BPI) has been shown to induce various effects in retinal cells in vivo and in vitro. The authors recently reported that BPI can induce ERK1/2 and Akt activity and that it increases DNA synthesis in the bovine retinal pigment epithelial (RPE) and pericyte cells. The authors have extended the characterization of BPI interaction with membrane proteins from bovine RPE. Crude membrane pools from RPE were isolated, solubilized, and bound to rBPI(21) affinity column. Bound proteins were separated by SDS-PAGE and stained with Coomassie blue, which showed an intense band at 36 kDa consistently displaced by rBPI(21). Tandem mass spectrometry of the 36-kDa band suggested that cell surface protein glypican 4 (GPC4) serves as a putative BPI-binding protein. Heparitinase, phosphatidylinositol-specific phospholipase C, and anti-GPC4 antibody suppressed BPI-induced ERK and Akt phosphorylation in bovine RPE. Moreover, heparitinase also inhibited BPI actions on VEGF and PDGF-B mRNA expression induced by H(2)O(2). These new findings suggest that GPC4 is a specific binding protein for BPI on RPE to mediate the activation of ERK1/2, Akt, and the mRNA expressions of PDGF-B and VEGF.

The Surface Oxidation Potential of Melanosomes Measured by Free Electron Laser-Photoelectron Emission Microscopy

A technique for measuring the photoionization spectrum and the photoelectron emission threshold of a microscopic structured material is presented. The theoretical underpinning of the experiment and the accuracy of the measurements are discussed. The technique is applied to titanium silicide nanostructures and melanosomes isolated from human hair, human and bovine retinal pigment epithelium cells, and the ink sac of Sepia officinalis. A common photothreshold of 4.5 +/- 0.2 eV is found for this set of melanosomes and is attributed to the photoionization of the eumelanin pigment. The relationship between the photoionization threshold and the electrochemical potential referenced to the normal hydrogen electrode is used to quantify the surface oxidation potential of the melanosome. The developed technique is used to examine the effect of iron chelation on the surface oxidation potential of Sepia melanosomes. The surface oxidation potential is insensitive to bound Fe(III) up to saturation, suggesting that the metal is bound to the interior of the granule. This result is discussed in relation to the age-dependent accumulation of iron in human melanosomes in both the eye and brain.

Functional Characteristics of Connective Tissue Growth Factor on Vitreoretinal Cells

Connective tissue growth factor (CTGF) level is elevated in eyes with proliferative vitreoretinal diseases, such as proliferative diabetic retinopathy and proliferative vitreoretinopathy (PVR), as we previously reported, but its functional characteristics on vitreoretinal cells are yet to be clarified. In this study, we demonstrated a growth-promoting effect of CTGF on cultured hyalocytes and bovine retinal pigment epithelial cells (BRPEs) with the induction of p44/p42 mitogen-activated protein kinase phosphorylation and [(3)H]thymidine incorporation. CTGF also stimulated the synthesis of fibronectin by hyalocytes and BRPEs without significant effect on collagen gel contraction by these cells. On the other hand, CTGF had no direct effects on the proliferation, migration, or in vitro tube formation by vascular endothelial cells. Nevertheless, CTGF promoted vascular endothelial growth factor (VEGF) gene expression by hyalocytes and BRPEs. Although the concentrations of both CTGF and VEGF in the human vitreous samples with proliferative vitreoretinal diseases were elevated, there was no significant correlation between these concentrations. These findings indicate that CTGF appears to be involved in the formation of proliferative membranes without direct regulation of their cicatricial contraction in the pathogenesis of proliferative vitreoretinal diseases. Whereas CTGF might have no direct effects or minimal effects, if any, on retinal neovascularization, it is possible that CTGF has indirect effects by modulating the expression of VEGF.

A Membrane Receptor for Retinol Binding Protein Mediates Cellular Uptake of Vitamin A

Vitamin A has diverse biological functions. It is transported in the blood as a complex with retinol binding protein (RBP), but the molecular mechanism by which vitamin A is absorbed by cells from the vitamin A-RBP complex is not clearly understood. We identified in bovine retinal pigment epithelium cells STRA6, a multitransmembrane domain protein, as a specific membrane receptor for RBP. STRA6 binds to RBP with high affinity and has robust vitamin A uptake activity from the vitamin A-RBP complex. It is widely expressed in embryonic development and in adult organ systems. The RBP receptor represents a major physiological mediator of cellular vitamin A uptake.

Bactericidal/permeability‐increasing protein's signaling pathways and its retinal trophic and anti‐angiogenic effects

Bactericidal/permeability-increasing protein (BPI) was originally identified as a lipopolysaccharide (LPS) binding protein with gram-negative bactericidal activity in the leukocytes. In this study, we characterized the previously unknown effects of BPI in the eye and the molecular mechanisms involved in its action. BPI mRNA was detected in bovine retina; retinal pigment epithelium; and primary cultures of bovine retinal pigment epithelial cells (RPE), pericytes (RPC), and endothelial cells (REC); while BPI protein was measured in human vitreous and plasma. BPI, but not control protein thaumatin, activated extracellular regulated kinase (ERK) and AKT, and increased DNA synthesis in RPE and RPC but not in REC. A human recombinant 21 kDa modified amino-terminal fragment of BPI (rBPI21) reduced H2O2-induced apoptosis in RPE and inhibited vascular endothelial growth factor (VEGF)-stimulated ERK phosphorylation in REC when preincubated with VEGF. Intraperitoneal (i.p.)-injected rBPI21 reduced ischemia-induced retinal neovascularization and diabetes-induced retinal permeability. Since BPI has unusual dual properties of promoting RPC and RPE growth while suppressing VEGF-induced REC growth and vascular permeability, the mechanistic understanding of BPI's action may provide novel therapeutic opportunities for diabetic retinopathy and age-related macular degeneration.

Degradation of extracellular ATP by the retinal pigment epithelium

Stimulation of ATP or adenosine receptors causes important physiological changes in retinal pigment epithelial (RPE) cells that may influence their relationship to the adjacent photoreceptors. While RPE cells have been shown to release ATP, the regulation of extracellular ATP levels and the production of dephosphorylated purines is not clear. This study examined the degradation of ATP by RPE cells and the physiological effects of the adenosine diphosphate (ADP) that result. ATP was readily broken down by both cultured human ARPE-19 cells and the apical membrane of fresh bovine RPE cells. The compounds ARL67156 and betagamma-mATP inhibited this degradation in both cell types. RT-PCR analysis of ARPE-19 cells found mRNA message for multiple extracellular degradative enzymes; ectonucleotide pyrophosphatase/phosphodiesterase eNPP1, eNPP2, and eNPP3; the ectoATPase ectonucleoside triphosphate diphosphohydrolase NTPDase2, NTPDase3, and some message for NTPDase1. Considerable levels of ADP bathed RPE cells, consistent with a role for NTPDase2. ADP and ATP increased levels of intracellular Ca(2+). Both responses were inhibited by thapsigargin and P2Y(1) receptor inhibitor MRS 2179. Message for both P2Y(1) and P2Y(12) receptors was detected in ARPE-19 cells. These results suggest that extracellular degradation of ATP in subretinal space can result in the production of ADP. This ADP can stimulate P2Y receptors and augment Ca(2+) signaling in the RPE.