Background:Cells of the retinal pigment epithelium(RPE)accumulate different kinds of granules(lipofuscin,melanolipofuscin,melanosomes)within their cell bodies,with lipofuscin and melanolipofuscin being autofluorescent...Background:Cells of the retinal pigment epithelium(RPE)accumulate different kinds of granules(lipofuscin,melanolipofuscin,melanosomes)within their cell bodies,with lipofuscin and melanolipofuscin being autofluorescent after blue light excitation.High amounts of lipofuscin granules within the RPE have been associated with the development of RPE cell death and age-related macular degeneration(AMD);however,this has not been confirmed in histology so far.Here,based on our previous dataset of RPE granule characteristics,we report the characteristics of RPE cells from human donor eyes that show either high or low numbers of intracellular granules or high or low autofluorescence(AF)intensities.Methods:RPE flatmounts of fifteen human donors were examined using high-resolution structured illumination microscopy(HR-SIM)and laser scanning microscopy(LSM).Autofluorescent granules were analyzed regarding AF phenotype and absolute number of granules.In addition,total AF intensity per cell and granule density(number of granules per cell area)were determined.For the final analysis,RPE cells with total granule number below 5th or above the 95th percentile,or a total AF intensity±1.5 standard deviations above or below the mean were included,and compared to the average RPE cell at the same location.Data are presented as mean±standard deviation.Results:Within 420 RPE cells examined,42 cells were further analyzed due to extremes regarding total granule numbers.In addition,20 RPE cells had AF 1.5 standard deviations below,28 RPE cells above the mean local AF intensity.Melanolipofuscin granules predominate in RPE cells with low granule content and low AF intensity.RPE cells with high granule content have nearly twice(1.8 times)as many granules as an average RPE cell.Conclusions:In normal eyes,outliers regarding autofluorescent granule load and AF intensity signals are rare among RPE cells,suggesting that granule deposition and subsequent AF follows intrinsic control mechanisms at a cellular level.The AF of a cell is related to the composition of intracellular granule types.Ongoing studies using AMD donor eyes will examine possible disease related changes in granule distribution and further put lipofuscin´s role in aging and AMD further into perspective.展开更多
Purpose:To investigate the mechanism and sequence of formation of ring-shaped mitochondria in retinal pigment epithelial ce3lls of a chick model of gyrate atro-phy.Methods:Electron microscopic analysis of the ultrastr...Purpose:To investigate the mechanism and sequence of formation of ring-shaped mitochondria in retinal pigment epithelial ce3lls of a chick model of gyrate atro-phy.Methods:Electron microscopic analysis of the ultrastructure of retinal pigment epithelial(RPE)mitochondria was carried out in chicks injected intravitreally with formoguanamine regularly(every4days)over the first 2weeksor4weeks post-hatching.Formoguanamine is a triazine drug which induces hyperor-nithinemic symptoms in the chick eye similar to those seen in human gyrate atro-phy.Results:A large population of irregularly shaped mitochondria was observed in the RPE of both peripheral and central retina.They showed extensive morpholog-ical changes.At 2wk,the mitochondria appeared enlarged and abnormal in shape with vacuolisation,partial loss of their double membrane and reduced mitochon-drial cristae.By 4wk,the mitochondria had assumed a rounder,almost circular profile,many with central holes,so-called ring mitochondria.Conclusion:The appearance of ring-shaped mitochondria has been previously as-cribed to the section of cupshaped three-dimensional structures.We present evi-dence that ring-shaped mitochondrial profiles arise through at least two different mechanisms of membrane breakdown and intraorganelle vacuoolisation.The nature of the three dimensional structures of these abnormal mit.展开更多
Background:Lesion to the retinal pigment epithelium(RPE)is a crucial event in age-related macular degeneration(AMD)development.Although the pathogenesis of this complex disease is poorly understood,sunlight exposure a...Background:Lesion to the retinal pigment epithelium(RPE)is a crucial event in age-related macular degeneration(AMD)development.Although the pathogenesis of this complex disease is poorly understood,sunlight exposure and smoking are major environmental risk factors associated with AMD.High-energy visible blue light(HEV;400-500 nm)is the most energetic and potentially harmful solar wavelengths reaching adults retina.On the other hand,RPE cells can be exposed to a large range of pollutants from cigarette smoke,with polycyclic aromatic hydrocarbons(PAH)being among the most toxic.Some PAH from cigarette smoke can absorb HEV light.This led us hypothesize that in RPE cells,the combination of PAH and HEV could synergize to exacerbate the stress caused by either factor alone.We thus investigate the combined effect of PAH and HEV light in RPE cells.Methods:Confluent RPE immortalized cells(ARPE19)were exposed to nanomolar concentrations of benzo[a]pyrene(BaP)or indeno[1,2,3-cd]pyrene(IcdP).While IcdP efficiently absorbs HEV wavelengths,BaP,the most studied PAH,does not significantly absorb HEV light and was used as a control.BaP or IcdP contaminated ARPE19 were then irradiated with increasing sub-lethal doses of HEV light(150-500 J/cm2)using a setup that mimics the light spectrum normally reaching the retina.Cytotoxicity,apoptosis and reactive oxygen species(ROS)generation were assessed in each condition.Results:In presence of low concentrations of IcdP,sub-lethal amounts of HEV light trigger,in a dose-dependent way,up to 70%of apoptotic cell death.Co-exposure to IcdP and HEV also leads to a synergistic ROS generation in ARPE19 cells,thus inducing oxidative stress.None of these effects were observed with BaP.Efficient inhibition of ROS production by specific antioxidants only decreases death by 20%in cells simultaneously exposed to both IcdP and HEV light.Conclusions:Low concentrations of IcdP synergize with HEV light to induce phototoxicity in ARPE19 cells.An increased oxidative stress results from the interaction between both agents and partially explains the enhanced HEV phototoxicity in IcdP contaminated ARPE19 cells.This suggests that another major mechanism is involved in the synergetic toxicity.For smokers,this synergy between HEV and PAH may accelerate RPE cells loss and contribute to their greater risk of developing AMD.展开更多
Diabetic retinopathy(DR)is a common cause of blindness all over the world.Bone marrow mesenchymal stem cells(BMSCs)have been considered as a promising strategy for retinal regeneration in the treatment of DR.However,t...Diabetic retinopathy(DR)is a common cause of blindness all over the world.Bone marrow mesenchymal stem cells(BMSCs)have been considered as a promising strategy for retinal regeneration in the treatment of DR.However,the poor viability and low levels of BMSCs engraftment limit the therapeutic potential of BMSCs.The present study aimed to examine the direct induction of BMSCs differentiation into the cell types related to retinal regeneration by using soluble cytokine ciliary neurotrophic factor(CNTF).We observed remarkably increased expression of cellular retinaldehyde-binding protein(CRALBP)and retinoid isomerohydrolase(RPE65)in BMSCs treated with CNTF in vitro,indicating the directional differentiation of BMSCs into the retinal pigment epithelium(RPE)cells,which are crucial for retinal healing.In vivo,the diabetic rat model was established by use of streptozotocin(STZ),and animals treated with BMSCs+CNTF exhibited better viability and higher delivery efficiency of the transplanted cells than those treated with BMSCs injection alone.Similar to the in-vitro result,treatment with BMSCs and CNTF combined led to the differentiation of BMSCs into beneficial cells(RPE cells),and accelerated retinal healing characterized by the activation of rod photoreceptor cells and phagocytosis function of RPE cells.In conclusion,CNTF contributes to the differentiation of BMSCs into RPE cells,which may help overcome the current stem cell therapy limitations in the field of retinal regeneration.展开更多
Age-related macular degeneration（AMD） is the leading cause of vision loss in the elderly throughout the world. Treatment of AMD utilizing retinal pigment epithelium（RPE） transplantation represents a promising ther...Age-related macular degeneration（AMD） is the leading cause of vision loss in the elderly throughout the world. Treatment of AMD utilizing retinal pigment epithelium（RPE） transplantation represents a promising therapy. However, simplex RPE transplantation can only replace the diseased RPE cells, but has no abilities to stop the development of AMD. It has been indicated that oxidization triggers the development of AMD by inducing the dysfunction and degeneration of RPE cells, which results in the upregulation of local monocyte chemotactic protein-1（MCP-1） expression. MCP-1 induces macrophage recruiment which triggers local inflammation. As a result, the expression of vascular endothelial growth factor（VEGF） is upregulated by MCP-1mediated inflammation and results in the formation of choroidal neovascularization（CNV）. We accordingly propose a targeted therapy of AMD by subretinal transplanting the compound of RPE cell, MCP-1 antibody, and VEGF antibody and using a magnetic system to guide RPE cell compounds conjugated with superparamagnetic iron oxide nanoparticles（SPIONs）. Furthermore, SPION-labelled RPE cells can be tracked and detected in vivo by non-invasive magnetic resonance imaging（MRI）. This novel RPE cell transplantation methodology seems very promising to provide a new therapeutic approach for the treatment of AMD.展开更多
Background:Age-related macular degeneration(AMD)is the second Canadian cause in visual deficiency.AMD is characterized by the death of photoreceptors and retinal pigmented epithelium(RPE)in the macular region of the r...Background:Age-related macular degeneration(AMD)is the second Canadian cause in visual deficiency.AMD is characterized by the death of photoreceptors and retinal pigmented epithelium(RPE)in the macular region of the retina,leading to the loss of central vision.Epidemiologic studies suggest an association between lifetime sun exposure and the probability to develop AMD even though mechanisms are unknown.Sunlight is made of about 30%of high-energy visible(HEV)light(blue light),the most energetic wavelength reaching the retina.These wavelengths can be absorbed by lipofuscin,an age pigment accumulating in RPE cells.Lipofuscin principal component is N-retinylidene-N-retinylethanolamine(A2E).Many research teams showed that absorption of HEV light by A2E in RPE cells at non-physiological doses produces free radicals and leads to cell death.Our earlier work shows that when A2E-loaded RPE cells are irradiated with HEV light at physiological doses,the same light does not lead to oxidative stress as measured by telomere and mitochondrial integrity.Our hypothesis is that HEV light,at physiological doses,modify or convert A2E in derived produces,inhibiting its photo-oxidant effect.Methods:In vitro,we irradiated A2E with HEV light with or without antioxidants and with varying irradiation regimen to observe the UV-Visible spectrum of A2E.In cellulo,we loaded ARPE-19 cells with A2E and irradiated cells at physiological levels for 4 consecutive days.We then observed A2E fluorescence using a fluorescence microscope with nucleus counterstaining with DRAQ5.Results:HEV light leads to the disappearance of A2E characteristic UV-Visible spectrum and the apparition of a new product suggesting that HEV light modifies A2E.Nor oxidation and irradiation regimen seem to have an impact in A2E’s conversion by HEV light.We observed and progressive diminution of A2E fluorescence in cellulo during physiological irradiations.Conclusions:The loss of A2E photo-oxidation capacities by HEV light seems to be caused by its conversion by HEV light.We suggest that HEV light,at physiological doses,may be protective rather than photo-toxic.The next step would be to identify A2E’derived produces and their cell toxicity.展开更多
文摘Background:Cells of the retinal pigment epithelium(RPE)accumulate different kinds of granules(lipofuscin,melanolipofuscin,melanosomes)within their cell bodies,with lipofuscin and melanolipofuscin being autofluorescent after blue light excitation.High amounts of lipofuscin granules within the RPE have been associated with the development of RPE cell death and age-related macular degeneration(AMD);however,this has not been confirmed in histology so far.Here,based on our previous dataset of RPE granule characteristics,we report the characteristics of RPE cells from human donor eyes that show either high or low numbers of intracellular granules or high or low autofluorescence(AF)intensities.Methods:RPE flatmounts of fifteen human donors were examined using high-resolution structured illumination microscopy(HR-SIM)and laser scanning microscopy(LSM).Autofluorescent granules were analyzed regarding AF phenotype and absolute number of granules.In addition,total AF intensity per cell and granule density(number of granules per cell area)were determined.For the final analysis,RPE cells with total granule number below 5th or above the 95th percentile,or a total AF intensity±1.5 standard deviations above or below the mean were included,and compared to the average RPE cell at the same location.Data are presented as mean±standard deviation.Results:Within 420 RPE cells examined,42 cells were further analyzed due to extremes regarding total granule numbers.In addition,20 RPE cells had AF 1.5 standard deviations below,28 RPE cells above the mean local AF intensity.Melanolipofuscin granules predominate in RPE cells with low granule content and low AF intensity.RPE cells with high granule content have nearly twice(1.8 times)as many granules as an average RPE cell.Conclusions:In normal eyes,outliers regarding autofluorescent granule load and AF intensity signals are rare among RPE cells,suggesting that granule deposition and subsequent AF follows intrinsic control mechanisms at a cellular level.The AF of a cell is related to the composition of intracellular granule types.Ongoing studies using AMD donor eyes will examine possible disease related changes in granule distribution and further put lipofuscin´s role in aging and AMD further into perspective.
基金a grant from the Australian Research Council (to DPC and SGC) a grant from the Australian Retinitis Pigmentosa Association.
文摘Purpose:To investigate the mechanism and sequence of formation of ring-shaped mitochondria in retinal pigment epithelial ce3lls of a chick model of gyrate atro-phy.Methods:Electron microscopic analysis of the ultrastructure of retinal pigment epithelial(RPE)mitochondria was carried out in chicks injected intravitreally with formoguanamine regularly(every4days)over the first 2weeksor4weeks post-hatching.Formoguanamine is a triazine drug which induces hyperor-nithinemic symptoms in the chick eye similar to those seen in human gyrate atro-phy.Results:A large population of irregularly shaped mitochondria was observed in the RPE of both peripheral and central retina.They showed extensive morpholog-ical changes.At 2wk,the mitochondria appeared enlarged and abnormal in shape with vacuolisation,partial loss of their double membrane and reduced mitochon-drial cristae.By 4wk,the mitochondria had assumed a rounder,almost circular profile,many with central holes,so-called ring mitochondria.Conclusion:The appearance of ring-shaped mitochondria has been previously as-cribed to the section of cupshaped three-dimensional structures.We present evi-dence that ring-shaped mitochondrial profiles arise through at least two different mechanisms of membrane breakdown and intraorganelle vacuoolisation.The nature of the three dimensional structures of these abnormal mit.
文摘Background:Lesion to the retinal pigment epithelium(RPE)is a crucial event in age-related macular degeneration(AMD)development.Although the pathogenesis of this complex disease is poorly understood,sunlight exposure and smoking are major environmental risk factors associated with AMD.High-energy visible blue light(HEV;400-500 nm)is the most energetic and potentially harmful solar wavelengths reaching adults retina.On the other hand,RPE cells can be exposed to a large range of pollutants from cigarette smoke,with polycyclic aromatic hydrocarbons(PAH)being among the most toxic.Some PAH from cigarette smoke can absorb HEV light.This led us hypothesize that in RPE cells,the combination of PAH and HEV could synergize to exacerbate the stress caused by either factor alone.We thus investigate the combined effect of PAH and HEV light in RPE cells.Methods:Confluent RPE immortalized cells(ARPE19)were exposed to nanomolar concentrations of benzo[a]pyrene(BaP)or indeno[1,2,3-cd]pyrene(IcdP).While IcdP efficiently absorbs HEV wavelengths,BaP,the most studied PAH,does not significantly absorb HEV light and was used as a control.BaP or IcdP contaminated ARPE19 were then irradiated with increasing sub-lethal doses of HEV light(150-500 J/cm2)using a setup that mimics the light spectrum normally reaching the retina.Cytotoxicity,apoptosis and reactive oxygen species(ROS)generation were assessed in each condition.Results:In presence of low concentrations of IcdP,sub-lethal amounts of HEV light trigger,in a dose-dependent way,up to 70%of apoptotic cell death.Co-exposure to IcdP and HEV also leads to a synergistic ROS generation in ARPE19 cells,thus inducing oxidative stress.None of these effects were observed with BaP.Efficient inhibition of ROS production by specific antioxidants only decreases death by 20%in cells simultaneously exposed to both IcdP and HEV light.Conclusions:Low concentrations of IcdP synergize with HEV light to induce phototoxicity in ARPE19 cells.An increased oxidative stress results from the interaction between both agents and partially explains the enhanced HEV phototoxicity in IcdP contaminated ARPE19 cells.This suggests that another major mechanism is involved in the synergetic toxicity.For smokers,this synergy between HEV and PAH may accelerate RPE cells loss and contribute to their greater risk of developing AMD.
文摘Diabetic retinopathy(DR)is a common cause of blindness all over the world.Bone marrow mesenchymal stem cells(BMSCs)have been considered as a promising strategy for retinal regeneration in the treatment of DR.However,the poor viability and low levels of BMSCs engraftment limit the therapeutic potential of BMSCs.The present study aimed to examine the direct induction of BMSCs differentiation into the cell types related to retinal regeneration by using soluble cytokine ciliary neurotrophic factor(CNTF).We observed remarkably increased expression of cellular retinaldehyde-binding protein(CRALBP)and retinoid isomerohydrolase(RPE65)in BMSCs treated with CNTF in vitro,indicating the directional differentiation of BMSCs into the retinal pigment epithelium(RPE)cells,which are crucial for retinal healing.In vivo,the diabetic rat model was established by use of streptozotocin(STZ),and animals treated with BMSCs+CNTF exhibited better viability and higher delivery efficiency of the transplanted cells than those treated with BMSCs injection alone.Similar to the in-vitro result,treatment with BMSCs and CNTF combined led to the differentiation of BMSCs into beneficial cells(RPE cells),and accelerated retinal healing characterized by the activation of rod photoreceptor cells and phagocytosis function of RPE cells.In conclusion,CNTF contributes to the differentiation of BMSCs into RPE cells,which may help overcome the current stem cell therapy limitations in the field of retinal regeneration.
基金Supported by the National Natural Science Foundation of China(No.81100670)the Scientific Research Foundation for the Returned Overseas Chinese Scholars,State Education Ministry of China
文摘Age-related macular degeneration（AMD） is the leading cause of vision loss in the elderly throughout the world. Treatment of AMD utilizing retinal pigment epithelium（RPE） transplantation represents a promising therapy. However, simplex RPE transplantation can only replace the diseased RPE cells, but has no abilities to stop the development of AMD. It has been indicated that oxidization triggers the development of AMD by inducing the dysfunction and degeneration of RPE cells, which results in the upregulation of local monocyte chemotactic protein-1（MCP-1） expression. MCP-1 induces macrophage recruiment which triggers local inflammation. As a result, the expression of vascular endothelial growth factor（VEGF） is upregulated by MCP-1mediated inflammation and results in the formation of choroidal neovascularization（CNV）. We accordingly propose a targeted therapy of AMD by subretinal transplanting the compound of RPE cell, MCP-1 antibody, and VEGF antibody and using a magnetic system to guide RPE cell compounds conjugated with superparamagnetic iron oxide nanoparticles（SPIONs）. Furthermore, SPION-labelled RPE cells can be tracked and detected in vivo by non-invasive magnetic resonance imaging（MRI）. This novel RPE cell transplantation methodology seems very promising to provide a new therapeutic approach for the treatment of AMD.
文摘Background:Age-related macular degeneration(AMD)is the second Canadian cause in visual deficiency.AMD is characterized by the death of photoreceptors and retinal pigmented epithelium(RPE)in the macular region of the retina,leading to the loss of central vision.Epidemiologic studies suggest an association between lifetime sun exposure and the probability to develop AMD even though mechanisms are unknown.Sunlight is made of about 30%of high-energy visible(HEV)light(blue light),the most energetic wavelength reaching the retina.These wavelengths can be absorbed by lipofuscin,an age pigment accumulating in RPE cells.Lipofuscin principal component is N-retinylidene-N-retinylethanolamine(A2E).Many research teams showed that absorption of HEV light by A2E in RPE cells at non-physiological doses produces free radicals and leads to cell death.Our earlier work shows that when A2E-loaded RPE cells are irradiated with HEV light at physiological doses,the same light does not lead to oxidative stress as measured by telomere and mitochondrial integrity.Our hypothesis is that HEV light,at physiological doses,modify or convert A2E in derived produces,inhibiting its photo-oxidant effect.Methods:In vitro,we irradiated A2E with HEV light with or without antioxidants and with varying irradiation regimen to observe the UV-Visible spectrum of A2E.In cellulo,we loaded ARPE-19 cells with A2E and irradiated cells at physiological levels for 4 consecutive days.We then observed A2E fluorescence using a fluorescence microscope with nucleus counterstaining with DRAQ5.Results:HEV light leads to the disappearance of A2E characteristic UV-Visible spectrum and the apparition of a new product suggesting that HEV light modifies A2E.Nor oxidation and irradiation regimen seem to have an impact in A2E’s conversion by HEV light.We observed and progressive diminution of A2E fluorescence in cellulo during physiological irradiations.Conclusions:The loss of A2E photo-oxidation capacities by HEV light seems to be caused by its conversion by HEV light.We suggest that HEV light,at physiological doses,may be protective rather than photo-toxic.The next step would be to identify A2E’derived produces and their cell toxicity.
