Lipofuscin is a cytologic hallmark of aging in metabolically active postmitotic cells including n... more Lipofuscin is a cytologic hallmark of aging in metabolically active postmitotic cells including neurons, cardiac muscle cells, and the retinal pigment epithelium (RPE). High levels of lipofuscin are involved in the pathogenesis of age-related macular degeneration (AMD), the main cause of blindness in the elderly population in the western world. Degradation and exocytosis of lipofuscin by RPE cells have not been observed in vivo until now, and no drug is known to eliminate the intracellular amount of lipofuscin. Here, we show that in monkeys treated with a small molecule belonging to the tetrahydropyridoethers class (n = 36 of 48 monkeys), RPE cells significantly release lipofuscin. In 4 eyes, macrophages were detected which had taken up lipofuscin. They were located between the Bruch's membrane and the RPE, and in the choroid. The quantification of pigment granules was performed by transmission electron microscopy. Our findings open the way to develop therapeutic strategies to r...
Graefe's Archive for Clinical and Experimental Ophthalmology, 2014
This study reports the clinicopathologic findings of leaky sites in pathological vessels after su... more This study reports the clinicopathologic findings of leaky sites in pathological vessels after submacular removal of choroidal neovascular membranes (CNV). As the site that causes fluid exudation from neovascular vessels is unknown, specific attention was focused on the formation of fenestrations, cellular junctions, and morphologic alteration which can cause endothelial leakage. Choroidal neovascular membranes of 15 patients who underwent submacular surgery for CNV were investigated. Five patients received bevacizumab treatment before surgery, and another five received photodynamic therapy before surgery. The remaining five did not receive any other treatment before surgery. All membranes were embedded for transmission electron microscopy. CNVs were analyzed for pathological cell-to-cell connections, fenestrations, or other pathological conditions which can cause leakage of plasma. The morphology of the newly formed blood channels was very variable, and in principle was not different in treated and untreated patients. The sources of leakage in neovascular choroidal vessels were caused by insufficient endothelial cell connections and by capillaries with microvillar projections into the vessel lumen which blocked cellular perfusion but still allowed the flow of plasma. Fenestrations were only infrequently observed. A newly discovered type of pathological capillary, called a labyrinth capillary, is very likely responsible for the permanent leakage of fluid. Due to the small vessel lumen, thrombocytes cannot enter these capillaries to close the leakages. Fenestrations did not appear to play a significant role in vascular leakiness.
Superoxide dismutase 1 (SOD1) is a Cu/Zn metalloenzyme that aggregates in amyotrophic lateral scl... more Superoxide dismutase 1 (SOD1) is a Cu/Zn metalloenzyme that aggregates in amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder. Correct metal insertion during SOD1 biosynthesis is critical to prevent misfolding; however Zn(2+) can bind to the copper-site leading to an aberrantly metallated protein. These effects of Zn(2+) misligation on SOD1 aggregation remain to be explored, even though Zn(2+) levels are upregulated in ALS motor neurons. Here we use complementary biophysical methods to investigate Zn(2+) binding and its effects on the aggregation of three immature metal-free SOD1 conformers that represent biogenesis intermediates: dimeric, monomeric and reduced monomeric SOD1. Using isothermal titration calorimetry we determined that Zn(2+) binds to all conformers both at the zinc- as well as to the copper-site; however Zn(2+) binding mechanisms to the zinc-site have distinct characteristics across immature conformers. We show that this 'zinc overload' of immature SOD1 promotes intermolecular interactions, as evidenced by dynamic light scattering and ThT fluorescence kinetic studies. Analysis of aged zinc-induced aggregates by energy-dispersive X-ray and electron energy-loss spectroscopy shows that aggregates integrate some Zn(2+). In addition, electron diffraction analysis identifies nano-scaled crystalline materials and amyloid fibril-like reflections. Transmission electron microscopy reveals that Zn(2+) diverts the SOD1 aggregation pathway from fibrils to amorphous aggregate, and electrophoretic analysis evidences an increase in insoluble materials. Overall, we provide evidence that aberrant zinc coordination to immature conformers broadens the population of SOD1 misfolded species at early aggregation stages and provide evidence for a high structural polymorphism and heterogeneity of SOD1 aggregates.
This work presents a combined light and electron microscopical approach to investigate the initia... more This work presents a combined light and electron microscopical approach to investigate the initial breakdown of the retinal pigment epithelium (RPE) and choriocapillaris (CC) in age-related macular degeneration (AMD). Perimacular sections of 12 dry and wet AMD eyes (82 ± 15 years) and 7 age-matched controls (75 ± 10 years) without retinal pathology were investigated. Disease progression was classified into 5 stages of retinal degeneration to investigate the concurrent CC breakdown. Special emphasis was laid on transitions where intact CC-RPE-retina complexes went over into highly atrophied areas. AMD sections showed elevated loss of photoreceptors, RPE and CC (p < 0.01), and thickened Bruch's membrane with increased basal laminar and linear deposits compared with controls. Up to 27% of the CC was lost in controls although RPE and retina were still intact. This primary loss of CC further increased with AMD (up to 100%). The data implicate that CC breakdown already occurs during normal aging and precedes degeneration of the RPE and retina with AMD, defining AMD as a vascular disease. Particular attention should be given to the investigation of early AMD stages and transitional stages to the late stage that reveal a possible sequence of degenerative steps with aging and AMD.
Melanosomes in retinal tissues of a human, monkey and rat were analyzed by EDX in the TEM. Sample... more Melanosomes in retinal tissues of a human, monkey and rat were analyzed by EDX in the TEM. Samples were prepared by ultramicrotomy at different thicknesses. The material was mounted on Al grids and samples were analyzed in a Zeiss 912 TEM equipped with an Omega filter and EDX detector with ultrathin window. Melanosomes consist of C and O as main components, mole fractions are about 90 and 3-10 at.%, respectively, and small mole fraction ratios, between 2 and 0.1 at.%, of Na, Mg, K, Si, P, S, Cl, Ca. All elements were measured quantitatively by standardless EDX with high precision. Mole fractions of transition metals Fe, Cu and Zn were also measured. For Fe a mole fraction ratio of less than 0.1at.% was found and gives the melanin its paramagnetic properties. Its mole fraction is however close to or below the minimum detectable mass fraction of the used equipment. Only in the human eye and only in the retinal pigment epitelium (rpe) the mole fractions of Zn (0.1 at.% or 5000 microg/g) and Cu were clearly beyond the minimum detectable mass fraction. In the rat and monkey eye the mole fraction of Zn was at or below the minimum detectable mass fraction and could not be measured quantitatively. The obtained results yielded the chemical composition of the melanosomes in the choroidal tissue and the retinal pigment epitelium (rpe) of the three different species. The results of the chemical analysis are discussed by mole fraction correlation diagrams. Similarities and differences between the different species are outlined. Correlation behavior was found to hold over species, e.g. the Ca-O correlation. It indicates that Ca is bound to oxygen rich sites in the melanin. These are the first quantitative analyses of melanosomes by EDX reported so far. The quantitative chemical analysis should open a deeper understanding of the metabolic processes in the eye that are of central importance for the understanding of a large number of eye-related diseases. The chemical analysis also allows a correlation with structural changes observed at the various regions of the eye.
Long Evans rats were treated by a low-Zinc-Diet (ZD) and the ultrastructure and elemental composi... more Long Evans rats were treated by a low-Zinc-Diet (ZD) and the ultrastructure and elemental composition of melanosomes of the RPE and choroidal melanocytes and RPE lipofuscin granules were investigated using Analytical Electron Microscopy (AEM). In controls the Zn mole fraction of melanosomes was 0.05 at% (RPE) and 0.06 at% (choroid), respectively. For ZD-rats the Zn mole fraction of these granules was almost unchanged in the choroid but decreased and was below the detection limit (0.02 at%) in the RPE. ZD-rats produced also giant melanosomes (diameter 1-3 μm) in the choroid and greatly increased amounts of RPE lipofuscin. The giant melanosomes contained about six times as much Cu as control melanosomes. Lipofuscin granules were identified by AEM and could be clearly distinguished from melanosomes by their chemical composition. Thus, changes of the ultrastructure and transition metal storage of melanosomes due to a ZD can be directly traced using AEM.
Journal of Photochemistry and Photobiology B: Biology, 2008
To investigate the effects of zinc supplementation on human amelanotic (ARPE-19) and native pigme... more To investigate the effects of zinc supplementation on human amelanotic (ARPE-19) and native pigmented retinal pigment epithelial cells (hRPE) under normal light conditions and after ultraviolet A light exposure. hRPE cells, containing both melanin and lipofuscin granules, were prepared from human donor eyes of 60-70 year old patients. Cells of the amelanotic ARPE-19 cell line and pigmented hRPE cells were treated with zinc chloride and subjected to oxidative stress by UV-A irradiation. Intracellular H(2)O(2) formation was measured using a fluorescence oxidation assay. Additionally, apoptosis and viability assays were performed. Control cells were treated identically except for irradiation and zinc supplementation. Under normal light conditions, zinc treated hRPE cells produced less H(2)O(2) than unsupplemented hRPE cells. Viability and apoptosis events did not change. After UV-A irradiation, ARPE and hRPE cells were greatly impaired in all tests performed compared to the non-irradiated controls. No differences were found after zinc supplementation. hRPE cells showed a higher apoptosis and mortality rate than non-pigmented cells when stressed by UV-A light. ARPE cells never showed any zinc related effects. In contrast, without irradiation, zinc supplementation reduced H(2)O(2) production in pigmented hRPE cells slightly. We did not find any zinc effect in irradiated hRPE cells. After UV light exposure, pigmented cells showed a higher apoptosis and mortality than cells lacking any pigmentation. We conclude that cells with pigmentation consisting of melanin and lipofuscin granules have more prooxidative than antioxidative capacity when stressed by UV light exposure compared to cells lacking any pigmentation.
Graefe's Archive for Clinical and Experimental Ophthalmology, 2007
Tyrosinase (EC 1.14.18.1) is the key enzyme of melanin pigment formation and it is unclear whethe... more Tyrosinase (EC 1.14.18.1) is the key enzyme of melanin pigment formation and it is unclear whether it is synthesized in human postnatal retinal pigment epithelium (RPE). In this study, we investigated if phagocytosis of rod outer segments (ROS) can increase tyrosinase expression in vitro. Primary cultures of human RPE cells were fed with isolated ROS from cattle and with latex particles. After phagocytosis, RPE cells were tested for tyrosinase presence and activity with several independent methods: (1) immunocytochemistry with anti-tyrosinase antibodies and (2) ultrastructural as well as light microscopic DOPA histochemistry; (3) mRNA was isolated from human RPE before incubation with ROS and 5, 20 and 40 h after feeding with ROS. The amount of tyrosinase mRNA was determined quantitatively by real-time reverse transcription polymerase chain reaction (RT-PCR), and the tyrosinase activity was investigated by measuring tyrosine hydroxylase activity using [(3)H]tyrosine. Tyrosinase was found in fed RPE cells using these methods, but was absent without feeding. Furthermore, we showed co-localization of rhodopsin and tyrosinase in the fed RPE cells. Contrary to tyrosinase activity, the mRNA for tyrosinase was clearly present in the cultured RPE cells which had not been exposed to ROS, decreased significantly from 5 h after exposure to ROS and returned to its original non-fed level 40 h after ROS feeding. Our study does not present new evidence that de novo melanogenesis takes place in the adult differentiated RPE. However, in contrast to the classic hypothesis, which states that tyrosinase is only detected in embryos, we provide evidence with several independent methods that the expression of tyrosinase and its enzymatic activity are induced in cultured human adult RPE by phagocytosis of ROS.
Energy-filtered analytical transmission electron microscopy was used to image the ultrastructure ... more Energy-filtered analytical transmission electron microscopy was used to image the ultrastructure and determine quantitatively the chemical composition of pigment granules of the choroid and retinal pigment epithelium of two healthy human donors, aged 68 and 85 years. The electron microscopy preparation procedure did not affect the autofluorescence of melanolipofuscin and lipofuscin granules, since staining was omitted during sample preparation. Oval melanosomes, melanolipofuscin and lipofuscin granules were observed, having sizes of about 1.5 μm×0.5 μm, and were analyzed using energy-dispersive X-ray microanalysis and electron energy loss spectroscopy. Up to now, these pigments could only be identified by scattering contrast in bright field images, with melanosomes having dark contrast and lipofuscin being much brighter. High-precision energy-dispersive X-ray microanalysis of pigment granules (>15,000 integrated counts in the oxygen K(α) peak) yielded minimum detectable mole fractions of about 0.02 at% for copper and zinc. For the first time, quantitative analytical electron microscopy yielded the chemical composition of the different pigments without prior isolation from the tissue. This is important to better understand physical and chemical properties of the pigments and their metabolism and turnover. The composition of melanosomes and lipofuscin can clearly be distinguished by the applied methods. Melanosomes were the pigments with largest oxygen (about 5 at%) and nitrogen (about 10 at%) mole fractions. The S/N ratio determination demonstrated a high pheomelanin content of the melanosomes. Lipofuscin had a significantly smaller oxygen mole fraction (about 4 at%) and nitrogen was found to be only slightly above the limit of detection (0.4 at%). For comparison, the cytoplasm contained oxygen and nitrogen mole fractions of 3 at% and 0.8 at%. Bright field images showed melanolipofuscin granules having a core-shell structure with a dark inner and a bright outer fraction. The dark fraction had a chemical composition close to the melanosomes and the composition of the bright fraction could be distinguished from that of lipofuscin due to a significantly increased nitrogen mole fraction in the melanolipofuscin granule. For all pigments observed the oxygen mole fraction yielded a positive correlation with the calcium mole fraction as previously established for melanosomes. Only lipofuscin contained measurable phosphorus mole fractions, which also correlated positively with oxygen. In lipofuscin, mole fractions of nitrogen were significantly smaller than in melanosomes and only indicated a small fraction of proteins. In contrast, the phosphorus mole fraction was significantly larger indicating the presence of significant amounts of phospholipids. Copper and zinc mole fractions were larger than 0.1at% in the melanosomes, but were below the detection limit in the lipofuscin granules. Compared to melanosomes of monkeys and rats analyzed beforehand, human retinal pigment epithelium melanosomes contained the highest amount of zinc, which even exceeded the calcium mole fraction. Trace elements like zinc are of great importance for metabolism and anti-oxidative mechanisms and also play a role in the progression of age related macular degeneration. They can now be investigated by quantitative analytical electron microscopy.
Premelanosomes are presumed to be essential for melanogenesis in melanocytes and pre-natal retina... more Premelanosomes are presumed to be essential for melanogenesis in melanocytes and pre-natal retinal pigment epithelium (RPE) cells. We analysed melanin synthesis in adenoviral-transduced tyrosinase-gene-expressing amelanotic RPE (ARPE) 19 cells to determine whether premelanosome formation is needed for post-natal melanogenesis. The synthesis of melanogenic proteins and melanin granules was investigated by immunocytochemistry and light and electron microscopy. The occurrence of tyrosinase was analysed ultrastructurally by dihydroxyphenylalanine histochemistry. The viability of transduced cell cultures was examined via MTT assay. We found active tyrosinase in small granule-like vesicles throughout the cytoplasm and in the endoplasmic reticulum and nuclear membrane. Tyrosinase was also associated with multi-vesicular and multi-lamellar organelles. Typical premelanosomes, structural protein PMEL17, tyrosinase-related protein 1 and classic melanosomal stages I-IV were not detected. Instead, melanogenesis took place inside multi-vesicular and multi-lamellar bodies of unknown origin. Viability was not affected up to 10 days after transduction. We thus demonstrate a pathway of melanin formation lacking typical hallmarks of melanogenesis.
Since there is evidence that the Fc domain of antivascular endothelial growth factor drugs may ca... more Since there is evidence that the Fc domain of antivascular endothelial growth factor drugs may cause unexpected consequences in retinal and choroidal vessels, the effects of intravitreal ranibizumab and aflibercept on monkey eyes were investigated. Four cynomolgus monkeys were intravitreally injected with 0.5 mg of ranibizumab and another four with 2 mg of aflibercept. Two untreated monkeys served as controls. Funduscopy, fluorescein angiography (FA), spectral-domain-optical coherence tomography (SD-OCT) and measurement of intraocular pressure (IOP) were performed. The eyes were inspected by light, fluorescence and electron microscopy. The diameter of the choriocapillaris (CC) was measured by morphometry, and the areas of the CC with free haemoglobin, CC fenestrations and endothelial thickness were quantified. Analysis showed ranibizumab permeated the retina via intercellular clefts, whereas aflibercept was taken up by ganglion cells, cells of the inner and outer retinal layers and the retinal pigment epithelium (RPE). Stasis and haemolysis in the choriocapillaris and choroidal vessels were more frequent after aflibercept treatment, which caused hypertrophy and death of individual RPE cells. The area of the CC was significantly reduced after both drugs compared with controls, but the reduction of the CC endothelium thickness, number of fenestrations and the areas with haemolysis were more pronounced after aflibercept. Ranibizumab permeated the retina through intercellular spaces, whereas aflibercept was taken up by neuronal and RPE cells. Aflibercept induced protein complex formation and more haemolysis in the choriocapillaris, leading to individual RPE cell death. The clinical significance and relation of these findings to the Fc domain or to other characteristics of aflibercept remain to be investigated.
By investigating the effects of intravitreal bevacizumab on retinal vessels of monkeys, we found ... more By investigating the effects of intravitreal bevacizumab on retinal vessels of monkeys, we found that bevacizumab accumulated locally at high concentration within individual blood vessels. It formed electron-dense fibrous deposits between endothelial cells and erythrocytes or granulocytes inducing retinal vein thrombosis. To better characterise the observed deposits, we investigated in vitro whether these deposits result from a complex between bevacizumab, vascular endothelial growth factor (VEGF)-A(165) and heparin. Cynomolgus monkeys were intravitreally injected with 1.25 mg bevacizumab. The eyes were enucleated between 1 and 14 days after injection and investigated by electron microscopy and immunohistochemistry. Human umbilical vein endothelial cells (HUVEC) were incubated with bevacizumab, VEGF-A(165) and heparin at different concentrations. Treatments with ranibizumab served as control. Bevacizumab and ranibizumab were detected immunohistochemically using Cy-3 or immunogold labelled antibodies. Treated animals showed bevacizumab locally at high concentration within retinal blood vessels. Electron-dense deposits inside retinal vessels and between erythrocytes were detected in three out of four treated monkeys. In vitro, many globular aggregates heavily stained with anti-human IgG were only observed with equimolar amounts (240 nM) of bevacizumab and VEGF-A(165) and 0.2 U/ml heparin and not after ranibizumab treatment. The immunogold labelling specifically localised ultrastructurally the complexes formed between bevacizumab, VEGF-A(165) and heparin at the surfaces of HUVEC cells. Heparin promotes bevacizumab immune complex deposition on to endothelial cells. Our in vitro results could explain the presence of deposits observed on endothelial veins in monkey eyes intravitreally injected with bevacizumab.
Lipofuscin is a cytologic hallmark of aging in metabolically active postmitotic cells including n... more Lipofuscin is a cytologic hallmark of aging in metabolically active postmitotic cells including neurons, cardiac muscle cells, and the retinal pigment epithelium (RPE). High levels of lipofuscin are involved in the pathogenesis of age-related macular degeneration (AMD), the main cause of blindness in the elderly population in the western world. Degradation and exocytosis of lipofuscin by RPE cells have not been observed in vivo until now, and no drug is known to eliminate the intracellular amount of lipofuscin. Here, we show that in monkeys treated with a small molecule belonging to the tetrahydropyridoethers class (n = 36 of 48 monkeys), RPE cells significantly release lipofuscin. In 4 eyes, macrophages were detected which had taken up lipofuscin. They were located between the Bruch's membrane and the RPE, and in the choroid. The quantification of pigment granules was performed by transmission electron microscopy. Our findings open the way to develop therapeutic strategies to r...
Graefe's Archive for Clinical and Experimental Ophthalmology, 2014
This study reports the clinicopathologic findings of leaky sites in pathological vessels after su... more This study reports the clinicopathologic findings of leaky sites in pathological vessels after submacular removal of choroidal neovascular membranes (CNV). As the site that causes fluid exudation from neovascular vessels is unknown, specific attention was focused on the formation of fenestrations, cellular junctions, and morphologic alteration which can cause endothelial leakage. Choroidal neovascular membranes of 15 patients who underwent submacular surgery for CNV were investigated. Five patients received bevacizumab treatment before surgery, and another five received photodynamic therapy before surgery. The remaining five did not receive any other treatment before surgery. All membranes were embedded for transmission electron microscopy. CNVs were analyzed for pathological cell-to-cell connections, fenestrations, or other pathological conditions which can cause leakage of plasma. The morphology of the newly formed blood channels was very variable, and in principle was not different in treated and untreated patients. The sources of leakage in neovascular choroidal vessels were caused by insufficient endothelial cell connections and by capillaries with microvillar projections into the vessel lumen which blocked cellular perfusion but still allowed the flow of plasma. Fenestrations were only infrequently observed. A newly discovered type of pathological capillary, called a labyrinth capillary, is very likely responsible for the permanent leakage of fluid. Due to the small vessel lumen, thrombocytes cannot enter these capillaries to close the leakages. Fenestrations did not appear to play a significant role in vascular leakiness.
Superoxide dismutase 1 (SOD1) is a Cu/Zn metalloenzyme that aggregates in amyotrophic lateral scl... more Superoxide dismutase 1 (SOD1) is a Cu/Zn metalloenzyme that aggregates in amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder. Correct metal insertion during SOD1 biosynthesis is critical to prevent misfolding; however Zn(2+) can bind to the copper-site leading to an aberrantly metallated protein. These effects of Zn(2+) misligation on SOD1 aggregation remain to be explored, even though Zn(2+) levels are upregulated in ALS motor neurons. Here we use complementary biophysical methods to investigate Zn(2+) binding and its effects on the aggregation of three immature metal-free SOD1 conformers that represent biogenesis intermediates: dimeric, monomeric and reduced monomeric SOD1. Using isothermal titration calorimetry we determined that Zn(2+) binds to all conformers both at the zinc- as well as to the copper-site; however Zn(2+) binding mechanisms to the zinc-site have distinct characteristics across immature conformers. We show that this 'zinc overload' of immature SOD1 promotes intermolecular interactions, as evidenced by dynamic light scattering and ThT fluorescence kinetic studies. Analysis of aged zinc-induced aggregates by energy-dispersive X-ray and electron energy-loss spectroscopy shows that aggregates integrate some Zn(2+). In addition, electron diffraction analysis identifies nano-scaled crystalline materials and amyloid fibril-like reflections. Transmission electron microscopy reveals that Zn(2+) diverts the SOD1 aggregation pathway from fibrils to amorphous aggregate, and electrophoretic analysis evidences an increase in insoluble materials. Overall, we provide evidence that aberrant zinc coordination to immature conformers broadens the population of SOD1 misfolded species at early aggregation stages and provide evidence for a high structural polymorphism and heterogeneity of SOD1 aggregates.
This work presents a combined light and electron microscopical approach to investigate the initia... more This work presents a combined light and electron microscopical approach to investigate the initial breakdown of the retinal pigment epithelium (RPE) and choriocapillaris (CC) in age-related macular degeneration (AMD). Perimacular sections of 12 dry and wet AMD eyes (82 ± 15 years) and 7 age-matched controls (75 ± 10 years) without retinal pathology were investigated. Disease progression was classified into 5 stages of retinal degeneration to investigate the concurrent CC breakdown. Special emphasis was laid on transitions where intact CC-RPE-retina complexes went over into highly atrophied areas. AMD sections showed elevated loss of photoreceptors, RPE and CC (p < 0.01), and thickened Bruch's membrane with increased basal laminar and linear deposits compared with controls. Up to 27% of the CC was lost in controls although RPE and retina were still intact. This primary loss of CC further increased with AMD (up to 100%). The data implicate that CC breakdown already occurs during normal aging and precedes degeneration of the RPE and retina with AMD, defining AMD as a vascular disease. Particular attention should be given to the investigation of early AMD stages and transitional stages to the late stage that reveal a possible sequence of degenerative steps with aging and AMD.
Melanosomes in retinal tissues of a human, monkey and rat were analyzed by EDX in the TEM. Sample... more Melanosomes in retinal tissues of a human, monkey and rat were analyzed by EDX in the TEM. Samples were prepared by ultramicrotomy at different thicknesses. The material was mounted on Al grids and samples were analyzed in a Zeiss 912 TEM equipped with an Omega filter and EDX detector with ultrathin window. Melanosomes consist of C and O as main components, mole fractions are about 90 and 3-10 at.%, respectively, and small mole fraction ratios, between 2 and 0.1 at.%, of Na, Mg, K, Si, P, S, Cl, Ca. All elements were measured quantitatively by standardless EDX with high precision. Mole fractions of transition metals Fe, Cu and Zn were also measured. For Fe a mole fraction ratio of less than 0.1at.% was found and gives the melanin its paramagnetic properties. Its mole fraction is however close to or below the minimum detectable mass fraction of the used equipment. Only in the human eye and only in the retinal pigment epitelium (rpe) the mole fractions of Zn (0.1 at.% or 5000 microg/g) and Cu were clearly beyond the minimum detectable mass fraction. In the rat and monkey eye the mole fraction of Zn was at or below the minimum detectable mass fraction and could not be measured quantitatively. The obtained results yielded the chemical composition of the melanosomes in the choroidal tissue and the retinal pigment epitelium (rpe) of the three different species. The results of the chemical analysis are discussed by mole fraction correlation diagrams. Similarities and differences between the different species are outlined. Correlation behavior was found to hold over species, e.g. the Ca-O correlation. It indicates that Ca is bound to oxygen rich sites in the melanin. These are the first quantitative analyses of melanosomes by EDX reported so far. The quantitative chemical analysis should open a deeper understanding of the metabolic processes in the eye that are of central importance for the understanding of a large number of eye-related diseases. The chemical analysis also allows a correlation with structural changes observed at the various regions of the eye.
Long Evans rats were treated by a low-Zinc-Diet (ZD) and the ultrastructure and elemental composi... more Long Evans rats were treated by a low-Zinc-Diet (ZD) and the ultrastructure and elemental composition of melanosomes of the RPE and choroidal melanocytes and RPE lipofuscin granules were investigated using Analytical Electron Microscopy (AEM). In controls the Zn mole fraction of melanosomes was 0.05 at% (RPE) and 0.06 at% (choroid), respectively. For ZD-rats the Zn mole fraction of these granules was almost unchanged in the choroid but decreased and was below the detection limit (0.02 at%) in the RPE. ZD-rats produced also giant melanosomes (diameter 1-3 μm) in the choroid and greatly increased amounts of RPE lipofuscin. The giant melanosomes contained about six times as much Cu as control melanosomes. Lipofuscin granules were identified by AEM and could be clearly distinguished from melanosomes by their chemical composition. Thus, changes of the ultrastructure and transition metal storage of melanosomes due to a ZD can be directly traced using AEM.
Journal of Photochemistry and Photobiology B: Biology, 2008
To investigate the effects of zinc supplementation on human amelanotic (ARPE-19) and native pigme... more To investigate the effects of zinc supplementation on human amelanotic (ARPE-19) and native pigmented retinal pigment epithelial cells (hRPE) under normal light conditions and after ultraviolet A light exposure. hRPE cells, containing both melanin and lipofuscin granules, were prepared from human donor eyes of 60-70 year old patients. Cells of the amelanotic ARPE-19 cell line and pigmented hRPE cells were treated with zinc chloride and subjected to oxidative stress by UV-A irradiation. Intracellular H(2)O(2) formation was measured using a fluorescence oxidation assay. Additionally, apoptosis and viability assays were performed. Control cells were treated identically except for irradiation and zinc supplementation. Under normal light conditions, zinc treated hRPE cells produced less H(2)O(2) than unsupplemented hRPE cells. Viability and apoptosis events did not change. After UV-A irradiation, ARPE and hRPE cells were greatly impaired in all tests performed compared to the non-irradiated controls. No differences were found after zinc supplementation. hRPE cells showed a higher apoptosis and mortality rate than non-pigmented cells when stressed by UV-A light. ARPE cells never showed any zinc related effects. In contrast, without irradiation, zinc supplementation reduced H(2)O(2) production in pigmented hRPE cells slightly. We did not find any zinc effect in irradiated hRPE cells. After UV light exposure, pigmented cells showed a higher apoptosis and mortality than cells lacking any pigmentation. We conclude that cells with pigmentation consisting of melanin and lipofuscin granules have more prooxidative than antioxidative capacity when stressed by UV light exposure compared to cells lacking any pigmentation.
Graefe's Archive for Clinical and Experimental Ophthalmology, 2007
Tyrosinase (EC 1.14.18.1) is the key enzyme of melanin pigment formation and it is unclear whethe... more Tyrosinase (EC 1.14.18.1) is the key enzyme of melanin pigment formation and it is unclear whether it is synthesized in human postnatal retinal pigment epithelium (RPE). In this study, we investigated if phagocytosis of rod outer segments (ROS) can increase tyrosinase expression in vitro. Primary cultures of human RPE cells were fed with isolated ROS from cattle and with latex particles. After phagocytosis, RPE cells were tested for tyrosinase presence and activity with several independent methods: (1) immunocytochemistry with anti-tyrosinase antibodies and (2) ultrastructural as well as light microscopic DOPA histochemistry; (3) mRNA was isolated from human RPE before incubation with ROS and 5, 20 and 40 h after feeding with ROS. The amount of tyrosinase mRNA was determined quantitatively by real-time reverse transcription polymerase chain reaction (RT-PCR), and the tyrosinase activity was investigated by measuring tyrosine hydroxylase activity using [(3)H]tyrosine. Tyrosinase was found in fed RPE cells using these methods, but was absent without feeding. Furthermore, we showed co-localization of rhodopsin and tyrosinase in the fed RPE cells. Contrary to tyrosinase activity, the mRNA for tyrosinase was clearly present in the cultured RPE cells which had not been exposed to ROS, decreased significantly from 5 h after exposure to ROS and returned to its original non-fed level 40 h after ROS feeding. Our study does not present new evidence that de novo melanogenesis takes place in the adult differentiated RPE. However, in contrast to the classic hypothesis, which states that tyrosinase is only detected in embryos, we provide evidence with several independent methods that the expression of tyrosinase and its enzymatic activity are induced in cultured human adult RPE by phagocytosis of ROS.
Energy-filtered analytical transmission electron microscopy was used to image the ultrastructure ... more Energy-filtered analytical transmission electron microscopy was used to image the ultrastructure and determine quantitatively the chemical composition of pigment granules of the choroid and retinal pigment epithelium of two healthy human donors, aged 68 and 85 years. The electron microscopy preparation procedure did not affect the autofluorescence of melanolipofuscin and lipofuscin granules, since staining was omitted during sample preparation. Oval melanosomes, melanolipofuscin and lipofuscin granules were observed, having sizes of about 1.5 μm×0.5 μm, and were analyzed using energy-dispersive X-ray microanalysis and electron energy loss spectroscopy. Up to now, these pigments could only be identified by scattering contrast in bright field images, with melanosomes having dark contrast and lipofuscin being much brighter. High-precision energy-dispersive X-ray microanalysis of pigment granules (>15,000 integrated counts in the oxygen K(α) peak) yielded minimum detectable mole fractions of about 0.02 at% for copper and zinc. For the first time, quantitative analytical electron microscopy yielded the chemical composition of the different pigments without prior isolation from the tissue. This is important to better understand physical and chemical properties of the pigments and their metabolism and turnover. The composition of melanosomes and lipofuscin can clearly be distinguished by the applied methods. Melanosomes were the pigments with largest oxygen (about 5 at%) and nitrogen (about 10 at%) mole fractions. The S/N ratio determination demonstrated a high pheomelanin content of the melanosomes. Lipofuscin had a significantly smaller oxygen mole fraction (about 4 at%) and nitrogen was found to be only slightly above the limit of detection (0.4 at%). For comparison, the cytoplasm contained oxygen and nitrogen mole fractions of 3 at% and 0.8 at%. Bright field images showed melanolipofuscin granules having a core-shell structure with a dark inner and a bright outer fraction. The dark fraction had a chemical composition close to the melanosomes and the composition of the bright fraction could be distinguished from that of lipofuscin due to a significantly increased nitrogen mole fraction in the melanolipofuscin granule. For all pigments observed the oxygen mole fraction yielded a positive correlation with the calcium mole fraction as previously established for melanosomes. Only lipofuscin contained measurable phosphorus mole fractions, which also correlated positively with oxygen. In lipofuscin, mole fractions of nitrogen were significantly smaller than in melanosomes and only indicated a small fraction of proteins. In contrast, the phosphorus mole fraction was significantly larger indicating the presence of significant amounts of phospholipids. Copper and zinc mole fractions were larger than 0.1at% in the melanosomes, but were below the detection limit in the lipofuscin granules. Compared to melanosomes of monkeys and rats analyzed beforehand, human retinal pigment epithelium melanosomes contained the highest amount of zinc, which even exceeded the calcium mole fraction. Trace elements like zinc are of great importance for metabolism and anti-oxidative mechanisms and also play a role in the progression of age related macular degeneration. They can now be investigated by quantitative analytical electron microscopy.
Premelanosomes are presumed to be essential for melanogenesis in melanocytes and pre-natal retina... more Premelanosomes are presumed to be essential for melanogenesis in melanocytes and pre-natal retinal pigment epithelium (RPE) cells. We analysed melanin synthesis in adenoviral-transduced tyrosinase-gene-expressing amelanotic RPE (ARPE) 19 cells to determine whether premelanosome formation is needed for post-natal melanogenesis. The synthesis of melanogenic proteins and melanin granules was investigated by immunocytochemistry and light and electron microscopy. The occurrence of tyrosinase was analysed ultrastructurally by dihydroxyphenylalanine histochemistry. The viability of transduced cell cultures was examined via MTT assay. We found active tyrosinase in small granule-like vesicles throughout the cytoplasm and in the endoplasmic reticulum and nuclear membrane. Tyrosinase was also associated with multi-vesicular and multi-lamellar organelles. Typical premelanosomes, structural protein PMEL17, tyrosinase-related protein 1 and classic melanosomal stages I-IV were not detected. Instead, melanogenesis took place inside multi-vesicular and multi-lamellar bodies of unknown origin. Viability was not affected up to 10 days after transduction. We thus demonstrate a pathway of melanin formation lacking typical hallmarks of melanogenesis.
Since there is evidence that the Fc domain of antivascular endothelial growth factor drugs may ca... more Since there is evidence that the Fc domain of antivascular endothelial growth factor drugs may cause unexpected consequences in retinal and choroidal vessels, the effects of intravitreal ranibizumab and aflibercept on monkey eyes were investigated. Four cynomolgus monkeys were intravitreally injected with 0.5 mg of ranibizumab and another four with 2 mg of aflibercept. Two untreated monkeys served as controls. Funduscopy, fluorescein angiography (FA), spectral-domain-optical coherence tomography (SD-OCT) and measurement of intraocular pressure (IOP) were performed. The eyes were inspected by light, fluorescence and electron microscopy. The diameter of the choriocapillaris (CC) was measured by morphometry, and the areas of the CC with free haemoglobin, CC fenestrations and endothelial thickness were quantified. Analysis showed ranibizumab permeated the retina via intercellular clefts, whereas aflibercept was taken up by ganglion cells, cells of the inner and outer retinal layers and the retinal pigment epithelium (RPE). Stasis and haemolysis in the choriocapillaris and choroidal vessels were more frequent after aflibercept treatment, which caused hypertrophy and death of individual RPE cells. The area of the CC was significantly reduced after both drugs compared with controls, but the reduction of the CC endothelium thickness, number of fenestrations and the areas with haemolysis were more pronounced after aflibercept. Ranibizumab permeated the retina through intercellular spaces, whereas aflibercept was taken up by neuronal and RPE cells. Aflibercept induced protein complex formation and more haemolysis in the choriocapillaris, leading to individual RPE cell death. The clinical significance and relation of these findings to the Fc domain or to other characteristics of aflibercept remain to be investigated.
By investigating the effects of intravitreal bevacizumab on retinal vessels of monkeys, we found ... more By investigating the effects of intravitreal bevacizumab on retinal vessels of monkeys, we found that bevacizumab accumulated locally at high concentration within individual blood vessels. It formed electron-dense fibrous deposits between endothelial cells and erythrocytes or granulocytes inducing retinal vein thrombosis. To better characterise the observed deposits, we investigated in vitro whether these deposits result from a complex between bevacizumab, vascular endothelial growth factor (VEGF)-A(165) and heparin. Cynomolgus monkeys were intravitreally injected with 1.25 mg bevacizumab. The eyes were enucleated between 1 and 14 days after injection and investigated by electron microscopy and immunohistochemistry. Human umbilical vein endothelial cells (HUVEC) were incubated with bevacizumab, VEGF-A(165) and heparin at different concentrations. Treatments with ranibizumab served as control. Bevacizumab and ranibizumab were detected immunohistochemically using Cy-3 or immunogold labelled antibodies. Treated animals showed bevacizumab locally at high concentration within retinal blood vessels. Electron-dense deposits inside retinal vessels and between erythrocytes were detected in three out of four treated monkeys. In vitro, many globular aggregates heavily stained with anti-human IgG were only observed with equimolar amounts (240 nM) of bevacizumab and VEGF-A(165) and 0.2 U/ml heparin and not after ranibizumab treatment. The immunogold labelling specifically localised ultrastructurally the complexes formed between bevacizumab, VEGF-A(165) and heparin at the surfaces of HUVEC cells. Heparin promotes bevacizumab immune complex deposition on to endothelial cells. Our in vitro results could explain the presence of deposits observed on endothelial veins in monkey eyes intravitreally injected with bevacizumab.
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