In Vivo Multimodal Retinal Imaging Correlates With Granular-specific Changes in the Organelles of Retinal Pigment Epithelium of a Murine Model of Stargardt Disease

2021 ◽  
Author(s):  
Ratheesh K. Meleppat ◽  
Kaitryn E. Ronning ◽  
Sarah J. Karlen ◽  
Marie E. Burns ◽  
Edward N. Pugh ◽  
...  
Keyword(s):  
Retinal Pigment Epithelium ◽  
Ex Vivo ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Age Related Macular Degeneration ◽  
Stargardt Disease ◽  
Rpe Cells ◽  
Age Related ◽  
Lipofuscin Granules

Abstract Melanosomes, lipofuscin, and melanolipofuscin are the three principal types of granular pigmented organelles found in the retinal pigment epithelium (RPE) cells. Changes in the density of melanosomes and lipofuscin granules in RPE cells are considered hallmarks of various retinal diseases, including Stargardt disease and age-related macular degeneration (AMD). Herein, we report the potential of an in vivo multimodal imaging technique based on directional back-scattering and short-wavelength fundus autofluorescence (SW-FAF) to study the disease-related changes in the density of melanosomes and lipofuscin granules in RPE cells. Changes in the concentration of these granules in Abca4-/- mice (a model of Stargardt disease) relative to age-matched wild-type (WT) controls were investigated. Directional optical coherence tomography (dOCT) was used to assess the melanosome density in vivo, whereas the AF images and emission spectra acquired with a spectrometer-integrated scanning laser ophthalmoscope (SLO) were used to characterize the lipofuscin and melanolipofuscin granules in the same RPE region. Subcellular-resolution ex vivo imaging using confocal fluorescence microscopy and electron microscopy was performed on the same tissue region to visualize and quantify melanosomes, lipofuscin, and melanolipofuscin granules. Comparisons between in vivo and ex vivo results confirmed an increased concentration of lipofuscin granules and decreased number of melanosomes in the RPE of Abca4-/- mice and provided an explanation for the differences in fluorescence and directionality of RPE scattering observed in vivo in the two mouse strains.

scholarly journals In vivo multimodal retinal imaging of disease-related pigmentary changes in retinal pigment epithelium

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ratheesh K. Meleppat ◽  
Kaitryn E. Ronning ◽  
Sarah J. Karlen ◽  
Marie E. Burns ◽  
Edward N. Pugh ◽  
...  
Keyword(s):  
Retinal Pigment Epithelium ◽  
Ex Vivo ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Age Related Macular Degeneration ◽  
Stargardt Disease ◽  
Rpe Cells ◽  
Age Related ◽  
Lipofuscin Granules

AbstractMelanosomes, lipofuscin, and melanolipofuscin are the three principal types of pigmented granules found in retinal pigment epithelium (RPE) cells. Changes in the density of melanosomes and lipofuscin in RPE cells are considered hallmarks of various retinal diseases, including Stargardt disease and age-related macular degeneration (AMD). Herein, we report the potential of an in vivo multimodal imaging technique based on directional back-scattering and short-wavelength fundus autofluorescence (SW-FAF) to study disease-related changes in the density of melanosomes and lipofuscin granules in RPE cells. Changes in the concentration of these granules in Abca4−/− mice (a model of Stargardt disease) relative to age-matched wild-type (WT) controls were investigated. Directional optical coherence tomography (dOCT) was used to assess melanosome density in vivo, whereas the autofluorescence (AF) images and emission spectra acquired with a spectrometer-integrated scanning laser ophthalmoscope (SLO) were used to characterize lipofuscin and melanolipofuscin granules in the same RPE region. Subcellular-resolution ex vivo imaging using confocal fluorescence microscopy and electron microscopy was performed on the same tissue region to visualize and quantify melanosomes, lipofuscin, and melanolipofuscin granules. Comparisons between in vivo and ex vivo results confirmed an increased concentration of lipofuscin granules and decreased concentration of melanosomes in the RPE of Abca4−/− mice, and provided an explanation for the differences in fluorescence and directionality of RPE scattering observed in vivo between the two mouse strains.

scholarly journals Regeneration of the zebrafish retinal pigment epithelium after widespread genetic ablation

2018 ◽  
Author(s):  
Nicholas J. Hanovice ◽  
Lyndsay L. Leach ◽  
Kayleigh Slater ◽  
Ana E. Gabriel ◽  
Dwight Romanovicz ◽  
...  
Keyword(s):  
Retinal Pigment Epithelium ◽  
Wnt Signaling ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Age Related Macular Degeneration ◽  
Rpe Cells ◽  
Genetic Ablation ◽  
Age Related ◽  
First Time

ABSTRACTThe retinal pigment epithelium (RPE) is a specialized monolayer of pigmented cells within the eye that is critical for maintaining visual system function. Diseases affecting the RPE have dire consequences for vision, and the most prevalent of these is atrophic (dry) age-related macular degeneration (AMD), which is thought to result from RPE dysfunction and degeneration. An intriguing possibility for treating RPE degenerative diseases like atrophic AMD is the stimulation of endogenous RPE regeneration; however, very little is known about the mechanisms driving successful RPE regeneration in vivo. Here, we developed a zebrafish transgenic model (rpe65a:nfsB-GFP) that enabled ablation of large swathes of mature RPE. RPE ablation resulted in rapid RPE degeneration, as well as degeneration of Bruch’s membrane and underlying photoreceptors. Using this model, we demonstrate for the first time that larval and adult zebrafish are capable of regenerating a functional RPE monolayer after RPE ablation. Regenerated RPE cells first appear at the periphery of the RPE, and regeneration proceeds in a peripheral-to-central fashion. RPE ablation elicits a robust proliferative response in the remaining RPE. Subsequently, proliferative cells move into the injury site and differentiate into RPE. BrdU pulse-chase analyses demonstrate that the regenerated RPE is likely derived from remaining peripheral RPE cells. Pharmacological inhibition of Wnt signaling significantly reduces cell proliferation in the RPE and delays overall RPE recovery. These data demonstrate that the zebrafish RPE possesses a robust capacity for regeneration and highlight a potential mechanism through which endogenous RPE regenerate in vivo.SIGNIFICANCE STATEMENTDiseases resulting in RPE degeneration are among the leading causes of blindness worldwide, and no therapy exists that can replace RPE or restore lost vision. One intriguing possibility is the development of therapies focused on stimulating endogenous RPE regeneration. For this to be possible, we must first gain a deeper understanding of the mechanisms underlying RPE regeneration. Here, we ablate mature RPE in zebrafish and demonstrate that zebrafish regenerate RPE after widespread injury. Injury-adjacent RPE proliferate and regenerate RPE, suggesting that they are the source of regenerated tissue. Finally, we demonstrate that Wnt signaling is required for RPE regeneration. These findings establish an in vivo model through which the molecular and cellular underpinnings of RPE regeneration can be further characterized.

scholarly journals Suppressor of Cytokine Signaling 2 Regulates Retinal Pigment Epithelium Metabolism by Enhancing Autophagy

2021 ◽  
Vol 15 ◽  
Author(s):  
Xi-Yuan Liu ◽  
Rui Lu ◽  
Jing Chen ◽  
Jie Wang ◽  
Hong-Mei Qian ◽  
...  
Keyword(s):  
Retinal Pigment Epithelium ◽  
Metabolic Regulation ◽  
Glycogen Synthase ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Age Related Macular Degeneration ◽  
Cytokine Signaling ◽  
Suppressor Of Cytokine Signaling ◽  
Rpe Cells ◽  
Age Related

Retinal pigment epithelium (RPE) serves critical functions in maintaining retinal homeostasis. An important function of RPE is to degrade the photoreceptor outer segment fragments daily to maintain photoreceptor function and longevity throughout life. An impairment of RPE functions such as metabolic regulation leads to the development of age-related macular degeneration (AMD) and inherited retinal degenerative diseases. As substrate recognition subunit of a ubiquitin ligase complex, suppressor of cytokine signaling 2 (SOCS2) specifically binds to the substrates for ubiquitination and negatively regulates growth hormone signaling. Herein, we explore the role of SOCS2 in the metabolic regulation of autophagy in the RPE cells. SOCS2 knockout mice exhibited the irregular morphological deposits between the RPE and Bruch’s membrane. Both in vivo and in vitro experiments showed that RPE cells lacking SOCS2 displayed impaired autophagy, which could be recovered by re-expressing SOCS2. SOCS2 recognizes the ubiquitylated proteins and participates in the formation of autolysosome by binding with autophagy receptors and lysosome-associated membrane protein2 (LAMP-2), thereby regulating the phosphorylation of glycogen synthase kinase 3β (GSK3β) and mammalian target of rapamycin (mTOR) during the autophagy process. Our results imply that SOCS2 participates in ubiquitin-autophagy-lysosomal pathway and enhances autophagy by regulating GSK3β and mTOR. This study provides a potential therapeutic target for AMD.

scholarly journals Clinical-grade stem cell–derived retinal pigment epithelium patch rescues retinal degeneration in rodents and pigs

2019 ◽  
Vol 11 (475) ◽  
pp. eaat5580 ◽  
Author(s):  
Ruchi Sharma ◽  
Vladimir Khristov ◽  
Aaron Rising ◽  
Balendu Shekhar Jha ◽  
Roba Dejene ◽  
...  
Keyword(s):  
Stem Cell ◽  
Retinal Pigment Epithelium ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Age Related Macular Degeneration ◽  
Clinical Grade ◽  
Functional Validation ◽  
Age Related

Considerable progress has been made in testing stem cell–derived retinal pigment epithelium (RPE) as a potential therapy for age-related macular degeneration (AMD). However, the recent reports of oncogenic mutations in induced pluripotent stem cells (iPSCs) underlie the need for robust manufacturing and functional validation of clinical-grade iPSC-derived RPE before transplantation. Here, we developed oncogenic mutation-free clinical-grade iPSCs from three AMD patients and differentiated them into clinical-grade iPSC-RPE patches on biodegradable scaffolds. Functional validation of clinical-grade iPSC-RPE patches revealed specific features that distinguished transplantable from nontransplantable patches. Compared to RPE cells in suspension, our biodegradable scaffold approach improved integration and functionality of RPE patches in rats and in a porcine laser-induced RPE injury model that mimics AMD-like eye conditions. Our results suggest that the in vitro and in vivo preclinical functional validation of iPSC-RPE patches developed here might ultimately be useful for evaluation and optimization of autologous iPSC-based therapies.

scholarly journals Avoiding the Pitfalls of siRNA Delivery to the Retinal Pigment Epithelium with Physiologically Relevant Cell Models

Pharmaceutics ◽  
2020 ◽  
Vol 12 (7) ◽  
pp. 667
Author(s):  
Eva Ramsay ◽  
Manuela Raviña ◽  
Sanjay Sarkhel ◽  
Sarah Hehir ◽  
Neil R. Cameron ◽  
...  
Keyword(s):  
Retinal Pigment Epithelium ◽  
Cell Model ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Sirna Delivery ◽  
Target Cells ◽  
Cell Models ◽  
Rpe Cells ◽  
Age Related

Inflammation is involved in the pathogenesis of several age-related ocular diseases, such as macular degeneration (AMD), diabetic retinopathy, and glaucoma. The delivery of anti-inflammatory siRNA to the retinal pigment epithelium (RPE) may become a promising therapeutic option for the treatment of inflammation, if the efficient delivery of siRNA to target cells is accomplished. Unfortunately, so far, the siRNA delivery system selection performed in dividing RPE cells in vitro has been a poor predictor of the in vivo efficacy. Our study evaluates the silencing efficiency of polyplexes, lipoplexes, and lipidoid-siRNA complexes in dividing RPE cells as well as in physiologically relevant RPE cell models. We find that RPE cell differentiation alters their endocytic activity and causes a decrease in the uptake of siRNA complexes. In addition, we determine that melanosomal sequestration is another significant and previously unexplored barrier to gene silencing in pigmented cells. In summary, this study highlights the importance of choosing a physiologically relevant RPE cell model for the selection of siRNA delivery systems. Such cell models are expected to enable the identification of carriers with a high probability of success in vivo, and thus propel the development of siRNA therapeutics for ocular disease.

scholarly journals Development-related mitochondrial properties of retinal pigment epithelium cells derived from hEROs

2021 ◽  
Vol 14 (8) ◽  
pp. 1138-1150
Author(s):  
Hao-Jue Xu ◽  
◽  
Ting Zou ◽  
Zheng-Qin Yin ◽  
◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Retinal Pigment Epithelium ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Embryonic Stem ◽  
Age Related Macular Degeneration ◽  
Level Energy ◽  
Rpe Cells ◽  
Age Related ◽  
High Level

AIM: To explore the temporal mitochondrial characteristics of retinal pigment epithelium (RPE) cells obtained from human embryonic stem cells (hESC)-derived retinal organoids (hEROs-RPE), to verify the optimal period for using hEROs-RPE as donor cells from the aspect of mitochondria and to optimize RPE cell-based therapeutic strategies for age-related macular degeneration (AMD). METHODS: RPE cells were obtained from hEROs and from spontaneous differentiation (SD-RPE). The mitochondrial characteristics were analyzed every 20d from day 60 to 160. Mitochondrial quantity was measured by MitoTracker Green staining. Transmission electron microscopy (TEM) was adopted to assess the morphological features of the mitochondria, including their distribution, length, and cristae. Mitochondrial membrane potentials (MMPs) were determined by JC-1 staining and evaluated by flow cytometry, reactive oxygen species (ROS) levels were evaluated by flow cytometry, and adenosine triphosphate (ATP) levels were measured by a luminometer. Differences between two groups were analyzed by the independent-samples t-test, and comparisons among multiple groups were made using one-way ANOVA or Kruskal-Wallis H test when equal variance was not assumed. RESULTS: hEROs-RPE and SD-RPE cells from day 60 to 160 were successfully differentiated from hESCs and expressed RPE markers (Pax6, MITF, Bestrophin-1, RPE65, Cralbp). RPE features, including a cobblestone-like morphology with tight junctions (ZO-1), pigments and microvilli, were also observed in both hEROs-RPE and SD-RPE cells. The mitochondrial quantities of hEROs-RPE and SD-RPE cells both peaked at day 80. However, the cristae of hEROs-RPE mitochondria were less mature and abundant than those of SD-RPE mitochondria at day 80, with hEROs-RPE mitochondria becoming mature at day 100. Both hEROs-RPE and SD-RPE cells showed low ROS levels from day 100 to 140 and maintained a normal MMP during this period. However, hEROs-RPE mitochondria maintained a longer time to produce high levels of ATP (from day 120 to 140) than SD-RPE cells (only day 120). CONCLUSION: hEROs-RPE mitochondria develop more slowly and maintain a longer time to supply high-level energy than SD-RPE mitochondria. From the mitochondrial perspective, hEROs-RPE cells from day 100 to 140 are an optimal cell source for treating AMD.

scholarly journals Transcriptomic and proteomic retinal pigment epithelium signatures of age-related macular degeneration.

2021 ◽  
Author(s):  
Anne Senabouth ◽  
Maciej Daniszewski ◽  
Grace Lidgerwood ◽  
Helena Liang ◽  
Damian Hernandez ◽  
...  
Keyword(s):  
Retinal Pigment Epithelium ◽  
Macular Degeneration ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Disease Status ◽  
Geographic Atrophy ◽  
Age Related Macular Degeneration ◽  
Rpe Cells ◽  
Age Related ◽  
Mitochondrial Functions

Induced pluripotent stem cells generated from patients with geographic atrophy as well as healthy individuals were differentiated to retinal pigment epithelium (RPE) cells. By integrating transcriptional profiles of 127,659 RPE cells generated from 43 individuals with geographic atrophy and 36 controls with genotype data, we identified 439 expression Quantitative Trait (eQTL) loci in cis that were associated with disease status and specific to subpopulations of RPE cells. We identified loci linked to two genes with known associations with geographic atrophy - PILRB and PRPH2, in addition to 43 genes with significant genotype x disease interactions that are candidates for novel genetic associations for geographic atrophy. On a transcriptome-only level, we identified molecular pathways significantly upregulated in geographic atrophy-RPE including in extracellular cellular matrix reorganisation, neurodegeneration, and mitochondrial functions. We subsequently implemented a large-scale proteomics analysis, confirming modification in proteins associated with these pathways. We also identified six significant protein (p) QTL that regulate protein expression in the RPE cells and in geographic atrophy - two of which share variants with cis-eQTL. Transcriptome-wide association analysis identified genes at loci previously associated with age-related macular degeneration. Further analysis conditional on disease status, implicated statistically significant RPE-specific eQTL. This study uncovers important differences in RPE homeostasis associated with geographic atrophy.

scholarly journals Comparative study of oxidant properties of oxidized and unoxidized bisretinoids of lipofuscine granules in the retinal pigment epithelium of human eye

2019 ◽  
pp. 66-71
Author(s):  
М.А. Яковлева ◽  
Н.Л. Сакина ◽  
И.Б. Кольчугина ◽  
П.М. Арбуханова ◽  
С.А. Борзенок ◽  
...  
Keyword(s):  
Lipid Peroxidation ◽  
Retinal Pigment Epithelium ◽  
Comparative Study ◽  
Degradation Products ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Retinal Pigment Epithelial Cells ◽  
Age Related Macular Degeneration ◽  
Age Related ◽  
Lipofuscin Granules

Актуальность. Недавно нами было показано, что при возрастной макулярной дегенерации сетчатки наблюдается повышенное содержание продуктов фотоокисления и фотодеградации бисретиноидов по сравнению с нормой. Поэтому на сегодняшний день вопрос о фототоксичности этих продуктов становится актуальным для решения проблемы поиска путей лечения и профилактики патологии. Цель. Провести сравнительное исследование фотосенсибилизирующего действия N-ретинилиден-N-ретинилэтаноламина (А2Е) и продуктов его фотоокисления и фотодеградации на индуцированную видимым светом пероксидацию липидов фоторецепторных мембран. Материалы и методы. При помощи метода высокоэффективной жидкостной хроматографии были получены отдельные фракции неокисленных и окисленных бисретиноидов в хлороформном экстракте липофусциновых гранул из ретинального пигментного эпителия кадаверных глаз. Результаты. Проведено сравнительное исследование фототоксических свойств неокисленных и окисленных бисретиноидов липофусциновых гранул из клеток ретинального пигментного эпителия глаза человека на пероксидацию липидов наружных сегментов фоторецепторных клеток. Выводы. Окисленные бисретиноиды липофусциновых гранул менее фототоксичны по сравнению с их неокисленными формами. Background. Recently we have shown that age-related macular degeneration is associated with higher than normal levels of bisretinoid photo-oxidation and photo-degradation products. Therefore, the issue of their phototoxicity currently becomes relevant for finding ways to treat and prevent this pathology. Aim. To conduct a comparative study of the photosensitizing effect of N-retinylidene-N-retinylethanolamine (A2E) and its photooxidation and photodegradation products on light-induced lipid peroxidation in photoreceptor membranes. Materials and methods. Using high-performance liquid chromatography fractions of unoxidized and oxidized bisretinoids were isolated in the chloroform extract of lipofuscin granules from the retinal pigment epithelium of cadaver eyes. Results. The study compared phototoxic effects of unoxidized and oxidized bisretinoids of lipofuscin granules from human retinal pigment epithelial cells on lipid peroxidation in rod outer segments. Conclusions. Oxidized bisretinoids of lipofuscin granules are less phototoxic compared to their unoxidized forms.

scholarly journals mTOR activity is essential for retinal pigment epithelium regeneration in zebrafish

2021 ◽  
Author(s):  
Jeff Gross ◽  
Fangfang Lu ◽  
Lyndsay Leach
Keyword(s):  
Retinal Pigment Epithelium ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Mtor Pathway ◽  
Age Related Macular Degeneration ◽  
Model Systems ◽  
Rpe Cells ◽  
Genetic Ablation ◽  
Age Related ◽  
Mtor Activity

The retinal pigment epithelium (RPE) plays numerous critical roles in maintaining vision and this is underscored by the prevalence of degenerative blinding diseases like age-related macular degeneration (AMD), in which visual impairment is caused by progressive loss of RPE cells. In contrast to mammals, zebrafish possess the ability to intrinsically regenerate a functional RPE layer after severe injury. The molecular underpinnings of this regenerative process remain largely unknown yet hold tremendous potential for developing treatment strategies to stimulate endogenous regeneration in the human eye.  In this study, we demonstrate that the mTOR pathway is activated in RPE cells post-genetic ablation. Pharmacological and genetic inhibition of mTOR activity impaired RPE regeneration, while mTOR activation enhanced RPE recovery post-injury, demonstrating that mTOR activity is necessary and sufficient for RPE regeneration in zebrafish. RNA-seq of RPE isolated from mTOR-inhibited larvae identified a number of genes and pathways dependent on mTOR activity at early and late stages of regeneration; amongst these were components of the immune system, which is emerging as a key regulator of regenerative responses across various tissue and model systems.  Our results identify crosstalk between macrophages/microglia and the RPE, wherein mTOR activity in the RPE is required for recruitment of macrophages/microglia to the injury site. In turn, these macrophages/microglia reinforce mTOR activity in regenerating RPE cells. Interestingly, the function of macrophages/microglia in maintaining mTOR activity in the RPE appeared to be inflammation-independent. Taken together, these data identify mTOR activity as a key regulator of RPE regeneration and link the mTOR pathway to immune responses in facilitating RPE regeneration.

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