Cora Roehlecke

Multi-Level Communication of Human Retinal Pigment Epithelial Cells via Tunneling Nanotubes

Background Tunneling nanotubes (TNTs) may offer a very specific and effective way of intercellular communication. Here we investigated TNTs in the human retinal pigment epithelial (RPE) cell line ARPE-19. Morphology of TNTs was examined by immunostaining and scanning electron microscopy. To determine the function of TNTs between cells, we studied the TNT-dependent intercellular communication at different levels including electrical and calcium signalling, small molecular diffusion as well as mitochondrial re-localization. Further, intercellular organelles transfer was assayed by FACS analysis. Methodology and Principal Findings Microscopy showed that cultured ARPE-19 cells are frequently connected by TNTs, which are not attached to the substratum. The TNTs were straight connections between cells, had a typical diameter of 50 to 300 nm and a length of up to 120 µm. We observed de novo formation of TNTs by diverging from migrating cells after a short time of interaction. Scanning electron microscopy confirmed characteristic features of TNTs. Fluorescence microscopy revealed that TNTs between ARPE-19 cells contain F-actin but no microtubules. Depolymerisation of F-actin, induced by addition of latrunculin-B, led to disappearance of TNTs. Importantly, these TNTs could function as channels for the diffusion of small molecules such as Lucifer Yellow, but not for large molecules like Dextran Red. Further, organelle exchange between cells via TNTs was observed by microscopy. Using Ca2+ imaging we show the intercellular transmission of calcium signals through TNTs. Mechanical stimulation led to membrane depolarisation, which expand through TNT connections between ARPE-19 cells. We further demonstrate that TNTs can mediate electrical coupling between distant cells. Immunolabelling for Cx43 showed that this gap junction protein is interposed at one end of 44% of TNTs between ARPE-19 cells. Conclusions and Significance Our observations indicate that human RPE cell line ARPE-19 cells communicate by tunneling nanotubes and can support different types of intercellular traffic.

Stress reaction in outer segments of photoreceptors after blue light irradiation.

The retina is prone to oxidative stress from many factors which are also involved in the pathogenesis of degenerative diseases. In this study, we used the application of blue light as a physiological stress factor. The aim of this study was to identify the major source of intracellular ROS that mediates blue light-induced detrimental effects on cells which may lead to cytotoxicity. We hypothesized that outer segments are the major source of blue light induced ROS generation. In photoreceptors, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) enzymes and the recently found respiratory chain complexes may represent a major source for reactive oxygen species (ROS), beside mitochondria and chromophores. Therefore, we investigated this hypothesis and analysed the exact localization of the ROS source in photoreceptors in an organotypic culture system for mouse retinas. Whole eyeball cultures were irradiated with visible blue light (405 nm) with an output power of 1 mW/cm2. Blue light impingement lead to an increase of ROS production (detected by H2DCFDA in live retinal explants), which was particularly strong in the photoreceptor outer segments. Nox-2 and Nox-4 proteins are sources of ROS in blue light irradiated photoreceptors; the Nox inhibitor apocynin decreased ROS stimulated by blue light. Concomitantly, enzyme SOD-1, a member of the antioxidant defense system, indicator molecules of protein oxidation (CML) and lipid oxidation (MDA and 4-HNE) were also increased in the outer segments. Interestingly, outer segments showed a mitochondrial-like membrane potential which was demonstrated using two dyes (JC-1 and TMRE) normally exclusively associated with mitochondria. As in mitochondria, these dyes indicated a decrease of the membrane potential in hypoxic states or cell stress situations. The present study demonstrates that ROS generation and oxidative stress occurs directly in the outer segments of photoreceptors after blue light irradiation.

Blue light stress in retinal neuronal (R28) cells is dependent on wavelength range and irradiance

The aim of our study was to elucidate the role of wavelength and irradiance in blue light retinal damage. We investigated the impact of blue light emitted from light‐emitting diode (LED) modules with peaks at either 411 nm (half bandwidth 17 nm) or 470 nm (half bandwidth 25 nm) at defined irradiances of 0.6, 1.5 and 4.5 W/m2 for 411 nm and 4.5 W/m2 for 470 nm on retinal neuronal (R28) cells in vitro. We observed a reduction in metabolic activity and transmembrane potential of mitochondria when cells were irradiated at 411 nm at higher irradiances. Furthermore, production of mitochondrial superoxide radicals increased significantly when cells were irradiated with 411 nm light at 4.5 W/m2. In addition, such irradiation caused an activation of the antioxidative glutathion system. Using vital staining, flow cytometry and western blotting, we were able to show that apoptosis only took place when cells were exposed to 411 nm blue light at higher irradiances; necrosis was not observed. Enhanced caspase‐3 cleavage product levels confirmed that this effect was dependent on light irradiance. Significant alterations of the above‐mentioned parameters were not observed when cells were irradiated with 471 nm light despite a high irradiance of 4.5 W/m2, indicating that the cytotoxic effect of blue light is highly dependent on wavelength. The observed phenomena in R28 cells at 411 nm (4.5 W/m2) point to an apoptosis pathway elicited by direct mitochondrial damage and increased oxidative stress. Thus, light of 411 nm should act via impairment of mitochondrial function by compromising the metabolic situation of these retinal neuronal cells.

Effects of advanced glycation end products‐inductor glyoxal and hydrogen peroxide as oxidative stress factors on rat retinal organ cultures and neuroprotection by UK‐14,304

Retinal ganglion cell degeneration is supposed to be mediated by reactive oxygen species (ROS) and advanced glycation end products (AGEs). The alpha2‐adrenergic agonist, 5‐bromo‐N‐(4,5‐dihydro‐1H‐imidazol‐2‐yl)‐6‐quinoxalinamine (brimonidine; UK‐14,304), is said to exert a neuroprotective effect. To investigate these mechanisms in detail, we exposed rat whole mounts to glyoxal or H2O2 and treated them with either UK‐14,304 alone or additionally with the phosphatidylinositide 3 kinase (PI3) kinase inhibitor, 2‐(4‐Morpholinyl)‐8‐phenyl‐4H‐1‐benzopyran‐4‐one (Ly 294002). The accumulation of Nε‐[carboxymethyl] lysine (CML) was assessed immunohistochemically and changes in intracellular pH (pHi), mitochondrial transmembrane potential (MTMP) and ROS production in cell bodies of multipolar ganglion cell layer were studied by intravital fluorescence microscopy and confocal laser scanning microscopy. Ultrastructural changes in mitochondria of multipolar ganglion cell layer cell bodies were determined by transmission electron microscopy. We found that glyoxal and H2O2 increased accumulation of CML‐modified proteins and ROS production and decreased pHi and MTMP in cell bodies of multipolar ganglion cell layer. UK‐14,304 could prevent production of ROS, accumulation of CML‐modified proteins, ameliorate acidification, preserve MTMP and attenuate ultrastructural damages of ganglion cell mitochondria. Ly 294002 reversed the UK‐14,304‐mediated attenuation of CML and ROS production. We conclude that the protective effects of UK‐14,304 seem partly to be mediated by PI3 kinase‐dependent pathways.

Effects of Temperature and Water-Filtered Infrared-A Alone or in Combination on Healthy and Glyoxal-Stressed Fibroblast Cultures

Water-filtered infrared-A (wIRA) radiation has been described as supportive for tissue regeneration. We sought to investigate in detail the wIRA effect at different temperatures in 3T3 fibroblasts that were treated with glyoxal to induce formation of advanced glycation end products (AGEs). Nonirradiated and nonglyoxal-treated cells served as controls. Experiments were carried out over a range of 37°–45°C with exact temperature monitoring to distinguish between temperature and wIRA effects. Metabolic activity was assessed by resazurin assay. Mitochondrial membrane potential was assessed by JC-1 vital staining. Apoptotic changes were determined by vital staining with annexin V and YO-PRO-1 and determination of subG1 DNA content. Temperature had a dominant effect overriding effects exerted by wIRA or glyoxal treatment. The number of apoptotic cells was significantly higher at 45°C, while the percentage of healthy cells was significantly lower at 45°C. WIRA irradiation itself or in combination with glyoxal treatment exerted no damaging effects on the fibroblasts at physiological (37°–40°C) or higher (42°–45°C) temperatures compared to untreated controls. Temperatures of 45°C, which can occur during inappropriate application of infrared irradiation, damage cells even in the absence of wIRA or glyoxal application.

Effects of Narrow-band IR-A and of Water-Filtered Infrared A on Fibroblasts

Exposures of the skin with electromagnetic radiation of wavelengths between 670 nm and 1400 nm are often used as a general treatment to improve wound healing and reduce pain, for example, in chronic diabetic skin lesions. We investigated the effects of water‐filtered infrared A (wIRA) and of narrow‐band IR‐A provided by a light‐emitting diode LED (LED‐IR‐A) irradiation in vitro on 3T3 fibroblast cultures under defined conditions with and without glyoxal administration. Glyoxal triggers the formation of advanced glycation end products, thereby mimicking a diabetic metabolic state. Cell viability and apoptotic changes were determined by flow cytometry after vital staining with Annexin V, YO‐PRO‐1 and propidium iodide (PI), and by SubG1 assay. Mitochondrial function and oxidative stress were examined by vital staining for radical production, mitochondrial membrane potential (MMP) and the ratio of reduced‐to‐oxidized glutathione (GSH/GSSG). The metabolic state was monitored by a resazurin conversion assay. The numbers of apoptotic cells were reduced in cultures irradiated with wIRA or LED‐IR‐A. More mitochondria showed a well‐polarized MMP after wIRA irradiation in glyoxal damaged cells. LED‐IR‐A treatment specifically restored the GSH/GSSG ratio. The immediate positive effects of wIRA and LED‐IR‐A observed in living cells, particularly on mitochondria, reflect the therapeutic benefits of wIRA and LED‐IR‐A.

Stress responses of human retinal pigment epithelial cells to glyoxal

PurposeIntracellular formation of advanced glycation end products (AGEs) is a crucial pathological process in retinal diseases such as age-related macular degeneration (AMD) or diabetic retinopathy (DR). Glyoxal is a physiological metabolite produced during formation of AGEs and has also been shown to derive from photodegraded bisretinoid fluorophores in aging retinal pigment epithelial (RPE) cells.MethodsFlow cytometry was combined with either: 1) immunocytochemical staining to detect glyoxal induced formation of Nε-carboxymethyllysine (CML)-modifications of intracellular proteins (AGEs) and changes in the production of stress response proteins; or 2) vital staining to determine apoptosis rates (annexin V binding), formation of intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and changes in intracellular pH upon treatment of cells with glyoxal. The percentage of apoptotic cells was further quantified by flow cytometry after staining of fixed cells with propidium iodide to determine cells with a subdiploid (fragmented) DNA content. Apoptosis related activation of caspase 3 was determined by Western blotting. Glyoxal induced changes in VEGF-A165a mRNA expression and protein production were determined by real-time PCR and by flow cytometry after immunocytochemical staining.ResultsIncreasing glyoxal concentrations resulted in enhanced formation of AGEs, such as CML modifications of proteins. This was associated with elevated levels of intracellular reactive oxygen species, a depolarized MMP, and a decreased intracellular pH, resulting in an increased number of apoptotic cells. Apoptosis related caspase 3 activation increased in a dose dependent manner after glyoxal incubation. In consequence, the cells activated compensatory mechanisms and increased the levels of the anti-oxidative and stress-related proteins heme oxygenase-1, osteopontin, heat shock protein 27, copper/zinc superoxide dismutase, manganese superoxide dismutase, and cathepsin D. Furthermore, VEGF-A165a mRNA expression and VEGF-A protein production were significantly increased after incubation with glyoxal in ARPE-19 cells.ConclusionsThe glyoxal-induced oxidative stress and apoptosis in ARPE-19 cells may provide a suitable in vitro model for studying RPE cellular reactions to AGEs that occur in AMD or in DR.

Outer Segments of Retinal Photoreceptors - A Review in the Light of Novel Findings

Aims: Recent studies have demonstrated that all molecules of the respiratory chain are present in the photoreceptor outer segment (see Calzia et al., Biol Cell 2013). Furthermore, our group (Roehlecke et al., PLoS One 2013) could show that, after blue light stress of the retina, the outer segments are significant sources of reactive oxygen species (ROS) ‐ even more so than the mitochondria in the inner segment. These two new findings have also important implications for degenerative diseases of the retina. Methodology: In this respect we revisited the literature regarding the photoreceptor reactions after blue light and radical stress. Furthermore, we refer to the common features of mitochondria and outer segments. Results: In the light of the recent findings many unique features of the photoreceptors get understandable: they are characterized by excessive oxygen consumption - even higher than that of other neuronal cells. Photoreceptors possess, in addition to their mitochondria ‐ rich neuronal component an outer segment packed with stacks of membrane discs harbouring the photo pigments, respiratory enzymes and enzymes of the visual cycle. Therapeutic use of red and near infrared light is often explained by ameliorating the