Cytoprotective Role of Mitogen-activated Protein Kinase Phosphatase-1 in Light-damaged Human Retinal Pigment Epithelial Cells

The role of the mitogen-activated protein (MAP) kinase phosphatases (MKPs) in light-damaged cells is unclear. Therefore we investigated the involvement of MKP-1 in the regulation of apoptosis and cell survival mediated by MAP kinase pathways in light-damaged human retinal pigment epithelial cells (ARPE-19). Light dose-dependent changes in the expression of MKP-1 and in the phosphorylation status of the MAP kinases, c-Jun-N-terminal kinase (JNK) and p38 were demonstrated. Low light doses up to 2 J cm⁻² led to an upregulation of MKP-1 which resulted in the prevention of cell death by inactivating JNK kinase. However, higher light doses (≥3 J cm⁻²) significantly reduced MKP-1 protein expression and subsequently led to an increased JNK kinase activity followed by a significant increase in cell death. JNK kinase inactivation by the JNK inhibitor SP600125 significantly reduced light-induced cell death, suggesting that the cytoprotective properties of MKP-1 are mediated mainly by the JNK MAP kinase pathway. Physiological concentrations of ascorbic acid or taurine were seen to prevent apoptosis and cell death in light-damaged ARPE-19 cells by reducing oxidative stress within cells, thus maintaining MKP-1 at high levels, leading to an inactivation of the JNK kinase pathway which resulted in an increased cell viability.     

Effects of Light‐emitting Diode Radiations on Human Retinal Pigment Epithelial Cells In Vitro

Human visual system is exposed to high levels of natural and artificial lights of different spectra and intensities along lifetime. Light‐emitting diodes (LEDs) are the basic lighting components in screens of PCs, phones and TV sets; hence it is so important to know the implications of LED radiations on the human visual system. The aim of this study was to investigate the effect of LEDs radiations on human retinal pigment epithelial cells (HRPEpiC). They were exposed to three light–darkness (12 h/12 h) cycles, using blue‐468 nm, green‐525 nm, red‐616 nm and white light. Cellular viability of HRPEpiC was evaluated by labeling all nuclei with DAPI; Production of reactive oxygen species (ROS) was determined by H2DCFDA staining; mitochondrial membrane potential was quantified by TMRM staining; DNA damage was determined by H2AX histone activation, and apoptosis was evaluated by caspases‐3,‐7 activation. It is shown that LED radiations decrease 75–99% cellular viability, and increase 66–89% cellular apoptosis. They also increase ROS production and DNA damage. Fluorescence intensity of apoptosis was 3.7% in nonirradiated cells and 88.8%, 86.1%, 83.9% and 65.5% in cells exposed to white, blue, green or red light, respectively. This study indicates three light–darkness (12 h/12 h) cycles of exposure to LED lighting affect in vitro HRPEpiC.     

Effect of Esculetin on Tert-Butyl Hydroperoxide-Induced Oxidative Injury in Retinal Pigment Epithelial Cells In Vitro

Esculetin is a coumarin-derived compound with antioxidant and anti-inflammatory properties. The current study aims to evaluate the therapeutic implications of esculetin on retinal dysfunction and uncover the underlying mechanisms. Tert-butyl hydroperoxide (t-BHP) at a concentration of 300 μM was used to induce oxidative stress in human retinal pigment epithelial cell line (ARPE-19) cells. Esculetin at concentrations below 250 μM did not cause cytotoxicity to ARPE-19 cells. Cell viability analysis confirmed that t-BHP induced oxidative injury of ARPE-19 cells. However, ARPE-19 cells were protected from t-BHP-induced oxidative injury by esculetin in a concentration-dependent manner. As a result of the TUNEL assay to confirm apoptosis, esculetin treatment reduced the number of TUNEL-positive cells. Esculetin down-regulated the expression levels of Bax, Caspase-3, and PARP and up-regulated the expression level of Bcl2. Collectively, this study demonstrates that esculetin exerts potent antioxidant properties in ARPE-19 cells, inhibiting t-BHP-induced apoptosis under the regulation of apoptotic factors.     

Effects of Light Exposure and Use of Intraocular Lens on Retinal Pigment Epithelial Cells In Vitro

To investigate the effect of a blue light-filtering intraocular lens (IOL) and a UV-absorbing IOL on light-induced damage to retinal pigment epithelial (RPE) cells laden with the lipofuscin fluorophore N -retinylidene- N -retinylethanolamine (A2E), A2E-laden RPE cells were exposed to white light which was filtered by either a blue light-filtering IOL or a UV-absorbing IOL. After 30 min of illumination the cell viability and the level of reactive oxygen species (ROS), free glutathione (GSH), vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF) were determined. In the absence of an IOL, the white light exposure decreased cell viability to 37.2% of the nonirradiated control. The UV-absorbing IOL tended to reduce light-induced cell death; however, the decrease was not significant. The blue light-filtering IOL significantly attenuated light-induced cell damage, increasing cell viability to 79.5% of the nonirradiated control. The presence of the blue light-filtering IOL significantly increased GSH and PEDF levels, and decreased ROS and VEGF levels. This study suggests that a blue light-filtering IOL may be more protective against A2E-induced light damage and inhibit more light-induced ROS and VEGF production than a conventional UV-absorbing IOL.     

Lipofuscin and A2E Accumulate with Age in the Retinal Pigment Epithelium of Nrl−/− Mice†

Lipofuscin is a fluorescent material with significant phototoxic potential that accumulates with age in the retinal pigment epithelium (RPE) of the eye. It is thought to be a factor in retinal degeneration diseases. The most extensively characterized lipofuscin component, N‐retinylidene‐N‐retinylethanolamine (A2E), has been proposed to be a byproduct of reactions involving the visual pigment chromophore. To examine the impact of the visual pigment and photoreceptor cell type on lipofuscin accumulation, we analyzed the RPE from Nrl^?/? mice of various ages for lipofuscin fluorescence and A2E levels. The photoreceptor cells of the Nrl^?/? retina contain only cone‐like pigments, and produce cone‐like responses to photostimulation. The cone‐like nature of these cells was confirmed by the presence of RPE65. Lipofuscin was measured with fluorescence imaging, whereas A2E was quantified by UV/VIS absorbance spectroscopy coupled to HPLC. The identity of A2E was corroborated with tandem mass spectrometry. Lipofuscin and A2E accumulated with age, albeit to lower levels compared with wild type mice. The emission spectra of RPE lipofuscin granules from Nrl^?/? mice were similar to those from wild type mice, with λ_maxca 610 nm. These results demonstrate that cone visual pigments can contribute to the production of lipofuscin and A2E.     

Fabrication and Evaluation of Gellan Gum/Hyaluronic Acid Hydrogel for Retinal Tissue Engineering Biomaterial and the Influence of Substrate Stress Relaxation on Retinal Pigment Epithelial Cells

Cell therapies for age-related macular degeneration (AMD) treatment have been developed by integrating hydrogel-based biomaterials. Until now, cell activity has been observed only in terms of the modulus of the hydrogel. In addition, cell behavior has only been observed in the 2D environment of the hydrogel and the 3D matrix. As time-dependent stress relaxation is considered a significant mechanical cue for the control of cellular activities, it is important to optimize hydrogels for retinal tissue engineering (TE) by applying this viewpoint. Herein, a gellan Gum (GG)/Hyaluronic acid (HA) hydrogel was fabricated using a facile physical crosslinking method. The physicochemical and mechanical properties were controlled by forming a different composition of GG and HA. The characterization was performed by conducting a mass swelling study, a sol fraction study, a weight loss test, a viscosity test, an injection force study, a compression test, and a stress relaxation analysis. The biological activity of the cells encapsulated in 3D constructs was evaluated by conducting a morphological study, a proliferation test, a live/dead analysis, histology, immunofluorescence staining, and a gene expression study to determine the most appropriate material for retinal TE biomaterial. Hydrogels with moderate amounts of HA showed improved physicochemical and mechanical properties suitable for injection into the retina. Moreover, the time-dependent stress relaxation property of the GG/HA hydrogel was enhanced when the appropriate amount of HA was loaded. In addition, the cellular compatibility of the GG/HA hydrogel in in vitro experiments was significantly improved in the fast-relaxing hydrogel. Overall, these results demonstrate the remarkable potential of GG/HA hydrogel as an injectable hydrogel for retinal TE and the importance of the stress relaxation property when designing retinal TE hydrogels. Therefore, we believe that GG/HA hydrogel is a prospective candidate for retinal TE biomaterial.     

Concentration Dependence of Vitamin C in Combinations with Vitamin E and Zeaxanthin on Light‐Induced Toxicity to Retinal Pigment Epithelial Cells

The purpose of this study was to determine the effects of increasing concentration of ascorbate alone and in combinations with α‐tocopherol and zeaxanthin on phototoxicity to the retinal pigment epithelium. ARPE‐19 cells were exposed to rose bengal and visible light in the presence and absence of antioxidants. Toxicity was quantified by an assay of cell‐reductive activity. A 20 min exposure to visible light and photosensitizer decreased cell viability to ca 42%. Lipophilic antioxidants increased viabilities to ca 70%, 61% and 75% for α‐tocopherol, zeaxanthin and their combination, respectively. Cell viabilities were ca 70%, 56% and 5% after exposures in the presence of 0.35, 0.7 and 1.4 mm ascorbate, respectively. A 45 min exposure increased cell death to ca 74% and >95% in the absence and presence of ascorbate, respectively. In the presence of ascorbate, zeaxanthin did not significantly affect phototoxicity. α‐Tocopherol and its combination with zeaxanthin enhanced protective effects of ascorbate, but did not prevent from ascorbate‐mediated deleterious effects. In conclusion, there is a narrow range of concentrations and exposure times where ascorbate exerts photoprotective effects, exceeding which leads to ascorbate‐mediated increase in photocytotoxicity. Vitamin E and its combination with zeaxanthin can enhance protective effects of ascorbate, but do not ameliorate its deleterious effects.     

Characterization of Retinal Pigment Epithelial Melanin and Degraded Synthetic Melanin Using Mass Spectrometry and In Vitro Biochemical Diagnostics

With increasing age, there is an observable loss of melanin in retinal pigment epithelial (RPE) cells. It is possible that degradation of the pigment contributes to the pathogenesis of retinal disease, as the cellular antioxidant material is depleted. Functionally, intact melanin maintains protective qualities, while oxidative degradation of melanin promotes reactive oxygen species (ROS) generation and formation of metabolic byproducts, such as melanolipofuscin. Understanding the structural and functional changes to RPE melanin with increasing age may contribute to a better understanding of disease progression and risk factors for conditions such as age‐related macular degeneration (AMD). In this study, human donor RPE melanin is characterized using MALDI mass spectrometry to follow melanin degradation trends. In vitro models using ARPE‐19 cells are used to assess photo‐reactivity in repigmented cells. Significant protection against intracellular ROS produced by blue light is observed in calf melanin‐pigmented cells versus unpigmented and black latex bead controls (P < 0.0001). UV‐B exposure to aged human melanin‐pigmented cells results in a significant increase in nitric oxide production versus control cells (P < 0.001). Peroxide‐treated synthetic melanin is characterized to elucidate degradation products that may contribute to RPE cell damage.     

Identification of the Large-Conductance Ca2+-Regulated Potassium Channel in Mitochondria of Human Bronchial Epithelial Cells

Mitochondria play a key role in energy metabolism within the cell. Potassium channels such as ATP-sensitive, voltage-gated or large-conductance Ca²⁺-regulated channels have been described in the inner mitochondrial membrane. Several hypotheses have been proposed to describe the important roles of mitochondrial potassium channels in cell survival and death pathways. In the current study, we identified two populations of mitochondrial large-conductance Ca²⁺-regulated potassium (mitoBK_Ca) channels in human bronchial epithelial (HBE) cells. The biophysical properties of the channels were characterized using the patch-clamp technique. We observed the activity of the channel with a mean conductance close to 285 pS in symmetric 150/150 mM KCl solution. Channel activity was increased upon application of the potassium channel opener NS11021 in the micromolar concentration range. The channel activity was completely inhibited by 1 µM paxilline and 300 nM iberiotoxin, selective inhibitors of the BK_Ca channels. Based on calcium and iberiotoxin modulation, we suggest that the C-terminus of the protein is localized to the mitochondrial matrix. Additionally, using RT-PCR, we confirmed the presence of α pore-forming (Slo1) and auxiliary β3-β4 subunits of BK_Ca channel in HBE cells. Western blot analysis of cellular fractions confirmed the mitochondrial localization of α pore-forming and predominately β3 subunits. Additionally, the regulation of oxygen consumption and membrane potential of human bronchial epithelial mitochondria in the presence of the potassium channel opener NS11021 and inhibitor paxilline were also studied. In summary, for the first time, the electrophysiological and functional properties of the mitoBK_Ca channel in a bronchial epithelial cell line were described.     

Metabolism of Pyrene and Chrysene in Epithelial Human Bronchial and Hamster Lung Cells

Abstract

The metabolism of pyrene and chrysene in epithelial human bronchial and in hamster lung cells has been studied and found to be very similar in both systems, although it differs from that observed in rat lung microsomes. Metabolite profiles have been analyzed by means of capillary GC and by GC/MS.     

UVB Irradiation Enhances TiO2 Nanoparticle‐induced Disruption of Calcium Homeostasis in Human Lens Epithelial Cells

Currently, titanium dioxide nanoparticles (TiO₂ NPs) have been widely used in various applications including cosmetics, food additives and biomedicine. However, there are few reports available using TiO₂ NPs to treat ocular diseases. Posterior capsular opacification (PCO) is the most frequent complication after cataract surgery, which is induced by the proliferation and migration of lens epithelial cells. Thus, inhibiting the proliferation of lens epithelial cells will efficiently reduce the occurrence of PCO. In this study, we investigated the effects of TiO₂ NPs on HLE B‐3 cells with or without ultraviolet B (UVB) irradiation in vitro. We found that TiO₂ NPs can inhibit HLE B‐3 cell growth, cause the elevation of intracellular [Ca²⁺], produce excessive reactive oxygen species (ROS), further reduce Ca²⁺‐ATPase activity and decrease the expression of plasma membrane calcium ATPase 1 (PMCA1), finally disrupt the intracellular calcium homeostasis and induce cell damage. Importantly, UVB irradiation can apparently enhance these effects on HLE B‐3 cells in the presence of TiO₂ NPs. Taken together, the generation of excessive ROS and the disruption of intracellular calcium homeostasis may be both involved in TiO₂ nanoparticle‐induced HLE B‐3 cell damage under UVB irradiation.     

Phototoxicity of Indocyanine Green on Human Retinal Pigment Epithelium in Vitro and its Reduction by Lutein¶

Previous work has shown that indocyanine green (ICG)‐assisted peeling of the internal limiting membrane during vitreoretinal surgery may damage the retinal pigment epithelium (RPE). The present study tested the direct toxic effects and phototoxic effects of ICG on cultured human RPE. RPE cells were exposed to ICG (0.5%, 5 min) with or without lutein (20 μM), followed by light irradiation at different doses of light energy (1.0,3.0 and 10.0 J/cm²). After 48 h, cells were collected and stained with trypan blue to obtain the number of viable and nonviable cells in different groups. Cultures exposed to ICG without light irradiation showed a significant decrease of viable cells (‐13.3%) and an increase of nonviable cells (x2.5‐fold) compared with cultures not exposed to either ICG or light, indicating the presence of direct toxic effects of ICG. In cultures exposed to ICG plus light irradiation (10.0 J/cm²), viable cells decreased significantly (‐45.0%) and nonviable cells increased significantly (x4.4‐fold) compared with cultures exposed to ICG alone. The damage to the RPE cells depended on the dose of light (1.0–10.0 J/cm²), indicating that ICG has a phototoxic effect as well as a toxic one. Lutein, an endogenous ocular antioxidant, had a protective effect on cultures exposed to ICG and light, cells treated with leutin showed an increase of viable cells (+74.6%) and decrease of nonviable cells (‐74.4%) compared with cultures without leutin but not on cultures exposed to ICG alone. Thus, it seems that photoactivated ICG kills cells through a photoxidative mechanism. Our study suggests that preoperative oral administration of lutein may protect against the phototoxic‐induced damage of ICG on the RPE cells.     

A Multi-action PtIV Conjugate with Oleate and Cinnamate Ligands Targets Human Epithelial Growth Factor Receptor HER2 in Aggressive Breast Cancer Cells

HER2-positive breast cancer is an aggressive subtype that typically responds poorly to standard chemotherapy. To design an anticancer drug selective for HER2-expressing breast cancer, a Pt^IV prodrug with axial oleate and cinnamate ligands was synthesized. We demonstrate its superior antiproliferative activity in monolayer and 3D spheroid models; the antiproliferative efficiency increases gradually with increasing expression of HER2. The results also suggest that the released Pt^II compound inhibits the proliferation of cancer cells by a DNA-damage-mediated mechanism. Simultaneously, the released oleic and cinnamic acid can effectively inhibit HER2 expression. To our knowledge, this is the first platinum-based complex inhibiting HER2 expression that does not contain protein or peptide. Moreover, this Pt^IV prodrug is capable of overcoming the resistance of cancer stem cells (CSCs), inducing death in both CSCs and differentiated cancer cells. Thus, the results substantiate our design strategy and demonstrate the potential of this approach for the development of new, therapeutically relevant compounds.     

Photochemical studies of A2-E.

Lipofuscin is thought to be involved in age-related macular degeneration as is one of its proposed components, an amphiphillic pyridinium-based bis-retinoid with a quaternary nitrogen atom, known as A2-E. We report the triplet state spectra obtained from photosensitisation using anthracene and 1-nitronaphthalene in benzene and methanol. The triplet state of A2-E has lambda(max) at 550 nm and a lifetime of approximately 30 micros, it is efficiently quenched by molecular oxygen with a second-order quenching rate constant of approximately 1 x 10(9) dm(3) mol(-1) s(-1). There is no significant triplet state formation from direct laser excitation of A2-E and hence its quantum yield of triplet state formation must be <0.01.     

Synthetic Studies of Et-743, Preparation of A Racemic Amino Alcohol Precursor

A mild two-step method was used to cleave the lactam ring of the tricyclic intermediate 1. A key racemic amino alcohol precursor 5 for the construction of Et-743 skeleton was synthesized from 1 through four steps in total yield of 47%.     

Crystal Structure,DFT Calculation and Photophysical Studies of a Schiff Base

A Schiff base DSH(DSH =(4-dipropylanilino-styryl) hydrazine) has been synthesized and fully characterized by ~1H NMR, ~(13)C NMR, ESI-MS, elemental analysis and single-crystal X-ray diffraction analysis. It crystallizes in triclinic, space group P1, with a = 8.447(5), b = 10.543(5), c = 14.540(5) ?, α = 78.600(5), β = 78.222(5), γ = 78.247(5)°, V = 1225(1) ?~3, Z = 2, D_c = 1.103 g/m~3, F(000) = 444, Μr = 406.60, μ = 0.066 mm~(-1), the final R = 0.0414 and wR = 0.1065 for 8691 observed reflections with I 2(I). The structural analysis revealed that the molecule possesses good planar structure. The absorption spectra and one-photon fluorescence in different solvents were experimentally studied. The result indicates that the compound exhibits intense fluorescent emission bands in protic solvents, such as ethanol and methanol. Quantum chemical calculations based on time-dependent density functional theory(TD-DFT) at the level of TD-DFT/B3LYP/6-31G were performed to investigate the UV absorption spectrum, and influence of solvents on the fluorescence spectra is analyzed based on the optimized structure at the level of B3LYP/6-31G(d).     

Epoxyfocardin and Its Putative Biogenetic Precursor,Focardin, Bioactive,New-Skeleton Diterpenoids of the Marine Ciliate Euplotes focardii from Antractica

From the ciliate Euplotes focardii, collected from Ross Sea coastal waters, Antarctica, a new-skeleton diterpenoid, epoxyfocardin, was isolated as a 85:15 mixture of hemiacetals 8a and 8b . The absolute configuration of 8a/8b was determined from Mosher's esters 11a/11b and 12a/12b . Focardin 9a/9b , most likely a biogenetic precursor of 8a/8b , was also isolated as a minor component. Focardin, and particularly epoxyfocardin, proved to be toxic towards representatives of ciliate communities from Antarctic, temperate, tropical, and equatorial environments, constituting the first example of ecologically relevant metabolites from ciliate species that inhabit polar ecosystems.     

RPE65 and the Accumulation of Retinyl Esters in Mouse Retinal Pigment Epithelium

The RPE65 protein of the retinal pigment epithelium (RPE) enables the conversion of retinyl esters to the visual pigment chromophore 11‐cis retinal. Fresh 11‐cis retinal is generated from retinyl esters following photoisomerization of the visual pigment chromophore to all‐trans during light detection. Large amounts of esters accumulate in Rpe65^?/? mice, indicating their continuous formation when 11‐cis retinal generation is blocked. We hypothesized that absence of light, by limiting the conversion of esters to 11‐cis retinal, would also result in the build‐up of retinyl esters in the RPE of wild‐type mice. We used HPLC to quantify ester levels in organic extracts of the RPE from wild‐type and Rpe65^?/? mice. Retinyl ester levels in Sv/129 wild‐type mice that were dark adapted for various intervals over a 4‐week period were similar to those in mice raised in cyclic light. In C57BL/6 mice however, which contain less Rpe65 protein, dark adaptation was accompanied by an increase in ester levels compared to cyclic light controls. Retinyl ester levels were much higher in Rpe65^?/? mice compared to wild type and kept increasing with age. The results suggest that the RPE65 role in retinyl ester homeostasis extends beyond enabling the formation of 11‐cis retinal.