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Effects of Cytokine Activation and Oxidative Stress on the Function of the Human Embryonic Stem Cell–Derived Retinal Pigment Epithelial Cells
摘要： PURPOSE. In several retinal complications, such as age-dependent macular degeneration (AMD), oxidative stress is increased and cytokine level is elevated. These are shown to alter the activation and expression of matrix metalloproteinase (MMP) both in human primary and immortalized retinal pigment epithelial (RPE) cells. However, the effects on human embryonic stem cell (hESC)–derived RPE cells remain to be elucidated. METHODS. The mature hESC-RPE cells were exposed to inflammatory cytokines (IFN-γ or TNF-α) for 24 hours or oxidative stress (H2O2) for 1 hour. Effects on barrier properties were analyzed with transepithelial electrical resistance (TEER), the expression of MMP-1, MMP-2, MMP-3, MMP-9, collagen I, and collagen IV genes with quantitative RT-PCR, and the expression of MMP-1 and MMP-3 proteins with Western blot or ELISA, respectively. Also, activation and secretion of MMP-2 and -9 proteins were analyzed with zymography. RESULTS. In normal state, mature hESC-RPE cells expressed MMP-1, -2, -3, and -9 genes in low levels, respectively. Tumor necrosis factor-α increased MMP-1 and -2 gene expression, and H2O2 increased MMP-3 and -9 gene expression. Zymography revealed IFN-γ– and TNF-α–induced secretion of MMP-2 and high-molecular-weight species of MMP (HMW MMP), but H2O2 decreased their secretion. Furthermore, TNF-α and H2O2 significantly decreased barrier properties. CONCLUSIONS. Here, cytokines induced the MMP-1 and -2 gene and protein expression. Also, H2O2 induced MMP-3 and -9 gene expression, but not their protein secretion. These data propose that under oxidative stress and cytokine stimuli, mature hESC-RPE cells resemble their native counterpart in the human eye in regard to MMP secretion and expression and could be used to model retinal disorders involving alterations in MMP activity such as AMD, diabetic retinopathy, or proliferative vitreoretinopathy in vitro.
关键词： matrix metalloproteinase,MMP,stem cells,retinal pigment epithelium
更新于2025-11-21 11:08:12
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Isolation and Culture of Primary Mouse Retinal Pigment Epithelial (RPE) Cells with Rho-Kinase and TGFβR-1/ALK5 Inhibitor
摘要： Primary RPE cells could be a reliable model for representing in vivo status of RPE compared with cell lines. We present a protocol for in vitro isolation and culture of primary RPE cells from C57BL mice. We used C57BL mice ages 7 days to 4 months. The RPE layer was separated from the neural retina layer by digestion with 2% Dispase for 45 min and scraped off from the choroid after 25-min incubation in 37°C. Collected RPE sheets were gently pipetted up into smaller sheets. RPE sheets were transferred into well plates and cultured in vitro for 2 weeks. To inhibit epithelial-mesenchymal transition (EMT) of RPE cells, we used Y27632 and Repsox to treat cultured primary RPE cells. RPE cells isolated from C57BL mice maintained pigmented and hexagonal morphology in culture. However, long-term in vitro culture lead to the periphery cells of a RPE sheet becoming mesenchymal-like cells. In contrast to the control group, Y27632 and Repsox, which are inhibitors of Rho-kinase or TGFbR-1/ALK5, promoted primary RPE cells to maintain epithelial-like morphology and eventually become confluent. RPE cells isolated from C57BL mice could be a powerful cell model to study the biological function of RPE. Especially, C57BL mice with different defective genetic background resulting in ocular diseases, would expand the genome type of RPE cells. The method presented here could be an efficient and applicable technique to obtain large numbers of primary RPE cells that maintain some characteristics of in vivo RPE.
关键词： Primary Cell Culture,Mice,Retinal Pigment Epithelium,Inbred C57BL
更新于2025-09-23 15:23:52
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Aberrant Activity of TAK1 is Associated with Retinal Pathology
摘要： Transforming growth factor-β-activated kinase-1 (TAK1) is a mitogen activated protein kinase kinase kinase that is involved in diverse biological roles across species. Functioning downstream of TGF-β, TAK1 mediates the activation of the c-Jun N-terminal kinase (JNK) signaling pathway, serves as the target of pro-inflammatory cytokines, such as TNF-α, mediates NF-κβ activation, and plays a role in Wnt signaling in mesenchymal stem cells. Still, the expression of TAK1 in the retina has not been defined. In our study, pathological and immunohistochemical assessments indicate a link between retinal pathology and TAK1 phosphorylation. We observed similar TAK1 expression both in non-obvious and obvious retinal pathologies. However, the phosphorylated form of TAK1 in the segments of retina with obvious pathology was hardly detected compared to its expression in the segments with non-obvious pathology. This finding indicates, for the first time, a possible involvement of TAK1 in human retinal pathologies. Better understanding the expression pattern of TAK1 may serve as a new therapeutic avenue for retinal pathologies.
关键词： Phosphorylation,Retinal pigment epithelium (RPE),Transforming growth factor-β-activated kinase 1 (TAK1)
更新于2025-09-23 15:23:52
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RefMoB, a Reflectivity Feature Model-Based Automated Method for Measuring Four Outer Retinal Hyperreflective Bands in Optical Coherence Tomography
摘要： PURPOSE. To validate a model-driven method (RefMoB) of automatically describing the four outer retinal hyperreflective bands revealed by spectral-domain optical coherence tomography (SDOCT), for comparison with histology of normal macula; to report thickness and position of bands, particularly band 2 (ellipsoid zone [EZ], commonly called IS/OS). METHODS. Foveal and superior perifoveal scans of seven SDOCT volumes of five individuals aged 28 to 69 years with healthy maculas were used (seven eyes for validation, five eyes for measurement). RefMoB determines band thickness and position by a multistage procedure that models reflectivities as a summation of Gaussians. Band thickness and positions were compared with those obtained by manual evaluators for the same scans, and compared with an independent published histological dataset. RESULTS. Agreement among manual evaluators was moderate. Relative to manual evaluation, RefMoB reported reduced thickness and vertical shifts in band positions in a band-specific manner for both simulated and empirical data. In foveal and perifoveal scans, band 1 was thick relative to the anatomical external limiting membrane, band 2 aligned with the outer one-third of the anatomical IS ellipsoid, and band 3 (IZ, interdigitation of retinal pigment epithelium and photoreceptors) was cleanly delineated. CONCLUSIONS. RefMoB is suitable for automatic description of the location and thickness of the four outer retinal hyperreflective bands. Initial results suggest that band 2 aligns with the outer ellipsoid, thus supporting its recent designation as EZ. Automated and objective delineation of band 3 will help investigations of structural biomarkers of dark-adaptation changes in aging.
关键词： age-related macular degeneration,retina,ellipsoid,segmentation,optical coherence tomography,photoreceptors,interdigitation,reflectivity,retinal pigment epithelium
更新于2025-09-23 15:23:52
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Isorhamnetin prevents H2O2?induced oxidative stress in human retinal pigment epithelial cells
摘要： Isorhamnetin, a 3-O-methylated metabolite of quercetin, exhibits antioxidant effects. However, to the best of our knowledge, no study to date has focused on the effects of isorhamnetin on retinal pigment epithelium (RPE) cells, and its underlying molecular mechanisms. Therefore, the present study aimed to examine the potential protective effect of isorhamnetin against oxidative stress in human RPE cells. The results demonstrated that pretreatment of RPE cells with isorhamnetin significantly protected cell viability against oxidative stress. In addition, isorhamnetin pretreatment inhibited hydrogen peroxide (H2O2)-induced reactive oxygen species (ROS) production and caspase-3 activation in RPE cells. Furthermore, isorhamnetin pretreatment significantly increased the phosphorylation of phosphoinositide 3-kinase (PI3K) and AKT serine/threonine kinase 1 (Akt) in RPE cells exposed to H2O2, compared with cells treated with H2O2 alone. Taken together, the present results demonstrated that isorhamnetin protected human RPE cells from oxidative stress-induced cell death, and this effect was associated with activation of the PI3K/Akt signaling pathway. Thus, isorhamnetin may be considered as a potential antioxidant useful for the prevention of age-related macular degeneration.
关键词： age-related macular degeneration,oxidative stress,isorhamnetin,retinal pigment epithelium cells
更新于2025-09-23 15:23:52
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Expression of ABCA4 in the retinal pigment epithelium and its implications for Stargardt macular degeneration
摘要： Recessive Stargardt disease (STGD1) is an inherited blinding disorder caused by mutations in the Abca4 gene. ABCA4 is a flippase in photoreceptor outer segments (OS) that translocates retinaldehyde conjugated to phosphatidylethanolamine across OS disc membranes. Loss of ABCA4 in Abca4?/? mice and STGD1 patients causes buildup of lipofuscin in the retinal pigment epithelium (RPE) and degeneration of photoreceptors, leading to blindness. No effective treatment currently exists for STGD1. Here we show by several approaches that ABCA4 is additionally expressed in RPE cells. (i) By in situ hybridization analysis and by RNA-sequencing analysis, we show the Abca4 mRNA is expressed in human and mouse RPE cells. (ii) By quantitative immunoblotting, we show that the level of ABCA4 protein in homogenates of wild-type mouse RPE is about 1% of the level in neural retina homogenates. (iii) ABCA4 immunofluorescence is present in RPE cells of wild-type and Mertk?/? but not Abca4?/? mouse retina sections, where it colocalizes with endolysosomal proteins. To elucidate the role of ABCA4 in RPE cells, we generated a line of genetically modified mice that express ABCA4 in RPE cells but not in photoreceptors. Mice from this line on the Abca4?/? background showed partial rescue of photoreceptor degeneration and decreased lipofuscin accumulation compared with nontransgenic Abca4?/? mice. We propose that ABCA4 functions to recycle retinaldehyde released during proteolysis of rhodopsin in RPE endolysosomes following daily phagocytosis of distal photoreceptor OS. ABCA4 deficiency in the RPE may play a role in the pathogenesis of STGD1.
关键词： Stargardt disease,retinal pigment epithelium,lipofuscin,ABCA4,bisretinoid
更新于2025-09-23 15:21:21
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Blood–Retinal Barrier Compromise and Endogenous <i>Staphylococcus aureus</i> Endophthalmitis
摘要： PURPOSE. To test the hypothesis that blood–retinal barrier compromise is associated with the development of endogenous Staphylococcus aureus endophthalmitis. METHODS. To compromise the blood–retinal barrier in vivo, streptozotocin-induced diabetes was induced in C57BL/6J mice for 1, 3, or 5 months. Diabetic and age-matched nondiabetic mice were intravenously injected with 108 colony-forming units (cfu) of S. aureus, a common cause of endogenous endophthalmitis in diabetics. After 4 days post infection, electroretinography, histology, and bacterial counts were performed. Staphylococcus aureus–induced alterations in in vitro retinal pigment epithelial (RPE) cell barrier structure and function were assessed by anti–ZO-1 immunohistochemistry, FITC-dextran conjugate diffusion, and bacterial transmigration assays. RESULTS. We observed one bilateral infection in a control, nondiabetic animal (mean ? 1.54 3 103 6 1.78 3 102 cfu/eye, 7% incidence). Among the 1-month diabetic mice, we observed culture-con?rmed unilateral infections in two animals (mean ? 5.54 3 102 6 7.09 3 102 cfu/ eye, 12% incidence). Among the 3-month diabetic mice, infections were observed in 11 animals, three with bilateral infections (mean ? 2.67 3 102 6 2.49 3 102 cfu/eye, 58% incidence). Among the 5-month diabetic mice, we observed infections in ?ve animals (mean ? 7.88 3 102 6 1.08 3 103 cfu/eye, 33% incidence). In vitro, S. aureus infection reduced ZO-1 immunostaining and disrupted the barrier function of cultured RPE cells, resulting in diffusion of ?uorophore-conjugated dextrans and transmigration of live bacteria across a permeabilized RPE barrier. CONCLUSIONS. Taken together, these results indicated that S. aureus is capable of inducing blood–retinal barrier permeability and causing endogenous bacterial endophthalmitis in normal and diabetic animals.
关键词： Staphylococcus aureus,endogenous endophthalmitis,blood–retinal barrier,bacteria,diabetes,retinal pigment epithelium
更新于2025-09-23 15:21:01
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Bisretinoids mediate light sensitivity resulting in photoreceptor cell degeneration in mice lacking the receptor tyrosine kinase Mer
摘要： The receptor tyrosine kinase Mer is expressed by retinal pigment epithelial (RPE) cells and participates in photoreceptor outer-segment phagocytosis, a process enabling membrane renewal. Mutations in the gene encoding MERTK cause blinding retinitis pigmentosa in humans. Targeted Mertk disruption in mice causes defective RPE-mediated phagocytosis of the outer segments, leading to deposition of autofluorescent debris at the RPE–photoreceptor cell interface, followed by photoreceptor cell degeneration. Here, we show that retinaldehyde adducts (bisretinoid fluorophores) that form in photoreceptor outer segments occupy the unphagocytosed outer segment debris that accumulates in Mertk-/- mice. Bisretinoids measured by HPLC were elevated in Mertk-/- mice compared with wild-type animals. Bisretinoids were also more abundant in albino Mertk-/- mice expressing leucine at position 450 of the isomerase RPE65 (Rpe65-Leu450) rather than the variant methionine (Rpe65-450Met) that yields lower bisretinoid levels. In Royal College of Surgeons rats having dysfunctional Mertk, bisretinoids were higher than in wild-type rats. Intensities of in vivo fundus autofluorescence were higher in Mertk-/- mice than in wild-type mice and peaked earlier in albino Mertk-/-/Rpe65-Leu450 mice than in albino Mertk-/-/Rpe65-450Met mice. Of note, the rate of photoreceptor cell degeneration was more rapid in albino Mertk-/- mice exposed to higher levels of intraocular light (albino versus pigmented mice) and in mice carrying Rpe65-Leu450 than in Rpe65-450Met mice, revealing a link between bisretinoid accumulation and light-mediated acceleration of photoreceptor cell degeneration. In conclusion, the light sensitivity of photoreceptor cell degeneration arising from Mertk deficiency is consistent with the known phototoxicity of bisretinoids.
关键词： Mertk-/-,Mertk,retinal pigment epithelium,lipofuscin,phagocytosis,retina,photodegradation,bisretinoid
更新于2025-09-23 15:21:01
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Simultaneous Investigation of Vascular and Retinal Pigment Epithelial Pathologies of Exudative Macular Diseases by Multifunctional Optical Coherence Tomography
摘要： To investigate exudative macular disease, multifunctional optical coherence tomography (MF-OCT) using a 1-lm probe band was developed. The clinical utility of MF-OCT was examined in a descriptive case series. METHODS. Ten eyes of nine subjects with exudative macular disease, including one eye with age-related macular degeneration (AMD), one eye with idiopathic neovascular maculopathy, and eight eyes with polypoidal choroidal vasculopathy (PCV), were investigated. Areas of 6 3 6 mm2 around the pathologic region were scanned with 512 3 1024 depth scans in 6.6 seconds. Structural OCT, Doppler optical coherence angiography (OCA), and cumulative phase retardation images were obtained with a single measurement. Each MF-OCT image visualized the structure, vasculature, and birefringence. Degree of polarization uniformity values were also obtained for selective visualization of the retinal pigment epithelium (RPE). The MF-OCT images were compared with conventional ophthalmic images. RESULTS. Abnormal vasculatures were observed with Doppler OCA in all eyes, which presented high similarity to indocyanine green angiography in the midphase. The RPE and exudation in the pathologic regions were discriminated in one eye with AMD and ?ve of eight eyes with PCV. Cumulative phase retardation visualized ?brosis scars in two of the PCV cases. CONCLUSIONS. Multifunctional OCT revealed depth-resolved abnormal vasculatures, the integrity of the RPE and choroid, discrimination of the RPE and exudation, and existence of ?brosis scars in exudative macular diseases. Interpretation of MF-OCT examination is well matched with conventional ophthalmic examination. These results suggest that MF-OCT can be used as a noninvasive ophthalmic examination tool prior to conventional examinations in clinical routines.
关键词： polarization-sensitive OCT,OCT angiography,optical coherence tomography,retinal pigment epithelium,exudative macular disease
更新于2025-09-23 15:21:01
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A simple, static preservation system for shipping retinal pigment epithelium cell sheets
摘要： The ability to move cells and tissues from bench to bedside is an essential aspect of regenerative medicine. In this study, we propose a simple, static shipping system to deliver tissue-engineered cell sheets. Notably, this system is electronic-device-free and simplified to minimize the number of packing and opening steps involved. Shipping conditions were optimized, and application and verification of the system were performed using human iPS-cells-derived or fetal retinal pigment epithelium (RPE) cell sheets. The temperature of the compartments within the insulated container was stable at various conditions, and filling up the cell vessel with medium effectively prevented turbulence-induced mechanical damage to the RPE cell sheets. Furthermore, no abnormal changes were observed in RPE morphology, transepithelial electrical resistance, or mRNA expression after transit by train and car. Taken together, our simple shipping system has the potential to minimize the costs and human error associated with bench to bedside tissue transfer. This specially designed regenerative tissue shipping system, validated for use in this field, can be used without any special training. This study provides a procedure for easily sharing engineered tissues with the goal of promoting collaboration between laboratories and hospitals and enhancing patient care.
关键词： shipping,induced pluripotent stem cells,product packaging,retinal pigment epithelium,human,regenerative medicine,temperature
更新于2025-09-19 17:15:36