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All-trans retinoic acid stimulates the secretion of TGF-β2 via the phospholipase C but not the adenylyl cyclase signaling pathway in retinal pigment epithelium cells
摘要： Background: By investigating that (i) all-trans retinoic acid (ATRA) affects human retinal pigment epithelium (RPE) in expressing and secreting transforming growth factor (TGF)-β2 and (ii) U73122 (phospholipase C inhibitor) and SQ22536 (adenylyl cyclase inhibitor) regulate the ATRA-induced secretion of TGF-β2 in human RPE, we sought to interpret the signaling pathway of ATRA in promoting the development of myopia. Methods: The RPE cell line (D407) was treated with (i) ATRA (10 μM), (ii) U73122 (5–40 μM) and ATRA (10 μM), or (iii) SQ22536 (5–40 μM) and ATRA (10 μM). The control group was no-treated. After stimulated at 2, 4, 8, 16, 24, and 48 h, The expression and secretion of TGF-β2 was detected. Results: TGF-β2 in the cytoplasm was time-dependent increased by ATRA (p < 0.001). A time-dependent increase in the TGF-β2 protein of the supernatant was induced by ATRA (p < 0.001). U73122 (in the range of 5 to 40 μM) could suppress the secretion of TGF-β2 induced by ATRA (p < 0.001), and 40 μM U73122 could completely inhibit the up-regulated effect of 10 μM ATRA. However, SQ22536 (in the range of 5 to 40 μM) had no impact on the secretion of TGF-β2 induced by ATRA (p > 0.05). Conclusions: In RPE cells, ATRA stimulates the secretion of TGF-β2 via the phospholipase C signaling pathway but not the adenylyl cyclase signaling pathway. U73122 may inhibit the promotion of ATRA in the development of myopia.
关键词： Retinal pigment epithelium cells,All-trans retinoic acid,SQ22536,Transforming growth factor-β2,U73122
更新于2025-09-19 17:15:36
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Inhibitor of growth 4 affects hypoxia-induced migration and angiogenesis regulation in retinal pigment epithelial cells
摘要： Inhibitor of growth 4 (ING4), a potential tumor suppressor, is implicated in cell migration and angiogenesis. However, its effects on diabetic retinopathy (DR) have not been elucidated. In this study, we aimed to evaluate ING4 expression in normal and diabetic rats and clarify its effects on hypoxia‐induced dysfunction in human retinal pigment epithelial (ARPE‐19) cells. A Type 1 diabetic model was generated by injecting rats intraperitoneally with streptozotocin and then killed them 4, 8, or 12 weeks later. ING4 expression in retinal tissue was detected using western blot analysis, reverse transcription quantitative real‐time polymerase chain reaction (RT‐qPCR), and immunohistochemistry assays. After transfection with an ING4 overexpression lentiviral vector or small interfering RNA (siRNA), ARPE‐19 migration under hypoxia was tested using wound healing and transwell assays. The angiogenic effect of conditioned medium (CM) from ARPE‐19 cells was examined by assessing human retinal endothelial cell (HREC) capillary tube formation. Additionally, western blot analysis and RT‐qPCR were performed to investigate the signaling pathways in which ING4, specificity protein 1 (Sp1), matrix metalloproteinase 2 (MMP‐2), MMP‐9, and vascular endothelial growth factor A (VEGF‐A) were involved. Here, we found that ING4 expression was significantly reduced in the diabetic rats’ retinal tissue. Silencing ING4 aggravated hypoxia‐induced ARPE‐19 cell migration. CM collected from ING4 siRNA‐transfected ARPE‐19 cells under hypoxia promoted HREC angiogenesis. These effects were reversed by ING4 overexpression. Furthermore, ING4 suppressed MMP‐2, MMP‐9, and VEGF‐A expression in an Sp1‐dependent manner in hypoxia‐conditioned ARPE‐19 cells. Overall, our results provide valuable mechanistic insights into the protective effects of ING4 on hypoxia‐induced migration and angiogenesis regulation in ARPE‐19 cells. Restoring ING4 may be a novel strategy for treating DR.
关键词： migration,angiogenesis,diabetic retinopathy,retinal pigment epithelium cells,hypoxia,inhibitor of growth 4
更新于2025-09-19 17:15:36
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Fluorescence Lifetime Imaging Ophthalmoscopy of the Retinal Pigment Epithelium During Wound Healing After Laser Irradiation
摘要： Purpose: To investigate the change in fluorescence lifetime of retinal pigment epithelium (RPE) after laser irradiation by using an organ culture model. Methods: Porcine RPE-choroid-sclera explants were irradiated with selective retina treatment laser (wavelength: 527 nm, beam diameter: 200 lm, energy: 80–150 lJ). At 24 and 72 hours after irradiation, the mean fluorescence lifetime (sm) was measured with fluorescence lifetime imaging ophthalmoscopy (FLIO) (excitation wavelength: 473 nm, emission: short spectral channel: 498-560 nm, long spectral channel: 560–720 nm). For every laser spot, central damaged zone (zone 1: 120 3 120 lm), area including wound rim (280 3 280 lm except zone 1), and environmental zone (440 3 440 lm except zone 1 and 2) were analyzed. Peripheral zone at a distance from laser spots longer than 2000 lm was examined for comparison. Cell viability was evaluated with calcein-acetoxymethyl ester and morphology with fluorescence microscopy for filamentous-actin. Results: The RPE defect after selective retina treatment was mostly closed within 72 hours. FLIO clearly demarcated the irradiated region, with prolonged sm at the center of the defect decreasing with eccentricity. In short spectral channel, but not in long spectral channel, sm in the environmental zone after 72 hours was still significantly longer than in the peripheral zone. Conclusions: FLIO may clearly demarcate the RPE defect, demonstrate its closure, and, moreover, indicate the induced metabolic changes of surrounding cells during wound healing. Translational Relevance: This ex vivo study showed that FLIO may be used to evaluate the extent and quality of restoration of the damaged RPE and to detect its metabolic change in human fundus noninvasively.
关键词： selective retina therapy,retinal pigment epithelium,fluorescence lifetime,energy metabolism,wound healing
更新于2025-09-16 10:30:52
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Evaluation of Retinal Pigment Epithelium and Choroidal Neovascularization in Rats Using Laser-Scanning Optical-Resolution Photoacoustic Microscopy
摘要： Purpose: To demonstrate the value of the laser-scanning optical-resolution (LSOR)-photoacoustic (PA) microscopy (PAM) system and the conventional multimodal imaging techniques in the evaluation of laser-induced retinal injury and choroidal neovascularization (CNV) in rats. Methods: Different degrees of retinal injury were induced using laser photocoagulation. We compared the LSOR-PAM system with conventional imaging techniques in evaluating retinal injury with or without CNV. Six additional rats, treated with an anti-VEGF antibody or immunoglobulin G immediately after photocoagulation, were imaged 7 and 14 days after injection, and CNV lesion areas were compared. Results: In the retinal injury model, fundus autofluorescence showed well-defined hyperreflection, while the lesion displayed abundant PA signals demonstrating nonuniform melanin distribution in retinal pigment epithelium (RPE). RPE was detected with higher contrast in the PAM B-scan image than optical coherence tomography (OCT). Additionally, the CNV lesion was present with multiple PA signal intensi- ties which distinctly characterized the location and area of CNV as found in fundus fluorescein angiography. Further- more, the decreased PA signals extending from the CNV le- sion were similar to those of the vascular bud in ex vivo im- aging, which was invisible in other in vivo images. When treated with anti-VEGF agents, statistically significant differ- ences can be demonstrated by PAM similar to other mo- dalities. Conclusions: LSOR-PAM can detect the melanin distribution of RPE in laser-induced retinal injury and CNV in rats. PAM imaging provides a potential new tool to evalu- ate the vitality and functionality of RPE in vivo as well as to monitor the development and treatment of CNV.
关键词： Age-related macular degeneration,Retinal pigment epithelium,Multimodal imaging,Photoacoustic microscopy,Choroidal neovascularization
更新于2025-09-12 10:27:22
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UNRAVELED FRINGE-LIKE MARGINS AND BIPHASIC AUTOFLUORESCENCE OF UNILATERAL RETINAL PIGMENT EPITHELIUM DYSGENESIS
摘要： To illustrate the features of unilateral retinal pigment epithelium dysgenesis (URPED) in an African-American male patient. A 47-year-old asymptomatic African-American man was referred for an atypical subretinal pigmented mass in the left eye. On examination, visual acuity was 20/20 in both eyes. The right eye was unremarkable. The left eye revealed a darkly pigmented grey-black lesion at the level of the RPE with irregular, unraveled fringe-like margins, consistent with URPED. The lesion measured 5 mm in basal dimension and was located 400 mm from the foveola. The dark portion of the lesion was grey-black and demonstrated homogeneous hypoauto?uorescence, particularly at the site of grey-white peripheral fringe of ?brous metaplasia. By contrast, there was an additional, subtle lacey arrangement of normal-appearing RPE traversing over the entire lesion demonstrating isoauto?uorescence. On ?uorescein angiography, the lesion was generally hypo?uorescent, particularly in the dark portion of the lesion, but the peripheral fringe of ?brous metaplasia displayed angiographic hyper?uorescent staining, and the subtle lacey normal RPE showed iso?uorescence. Optical coherence tomography demonstrated RPE hyperplasia and shallow RPE detachment interspersed with normal-appearing RPE and thinning of outer retina and preservation of the foveola and choroid. In this case, URPED demonstrated biphasic auto?uorescence implying RPE dysfunction in the hypoauto?uorescent area and partial RPE function in the lacey isoauto?uorescent region.
关键词： optical coherence tomography,African-American,retinal pigment epithelium,auto?uorescence,race,dysgenesis
更新于2025-09-12 10:27:22
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Longitudinal Assessment of Progressive Retinal Pigment Epithelium Disruption in a 26 Year Old A MultiSpectral Imaging Case Study
摘要： Background: Progressive retinal pigment epithelium (RPE) disruption in the absence of drusen or visual disturbances in young people is rarely reported in the literature, except for cases of juvenile macular degenerative diseases, pattern dystrophies or white dot syndromes. In age-related macular degeneration (AMD), RPE disruption typically does not become manifest clinically before age 55. This case report presents a young, healthy 26 year old Caucasian male with asymptomatic progressive RPE disruption in the absence of drusen, as detected by multi-spectral imaging (MSI) technology. Methods: A 26 year old Caucasian male was followed for progressive changes in RPE atrophy and melanin clumping over three visits in a three year period. His dilated fundus examination, fundus photography and optical coherence tomography were within normal limits. Long wavelength MSI revealed progressive RPE changes in the form of RPE atrophy and melanin clumping. MSI fundus auto-fluorescence (FAF) showed corresponding hyperautofluorescence and hypoautofluorescence in the left. Findings: Macular RPE changes can result from phototoxic effects and vary by ethnicity. Functional biomarkers to determine the risk of future vision loss with AMD are frequently sought and include complement factor H and Age-Related Maculopathy Susceptibility 2. FAF, for example is highly indicative of RPE dysfunction and progression. Long wavelength MSI, 620 nm to 740 nm, enhances visualization of the RPE, more specifically atrophy and melanin clumping, which may be indicative of asymptomatic progressive early AMD or retinal dystrophies. Conclusion: This case shows a longitudinal example of progressive RPE disruption in a 26 year old male. This is only one example, but multiple cases of RPE disruption in young people exist. Using MSI to further investigate RPE disruption and progression in a young population may provide a potential biomarker for early AMD, photo toxicity or other retinal dystrophies and degenerations.
关键词： Retinal Pigment Epithelium,Fundus Autofluorescence,Age-related Macular Degeneration,Multi-Spectral Imaging,Progressive retinal pigment epithelium disruption
更新于2025-09-11 14:15:04
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Self-Formation of RPE Spheroids Facilitates Enrichment and Expansion of hiPSC-Derived RPE Generated on Retinal Organoid Induction Platform
摘要： PURPOSE. Retinal pigment epithelium (RPE) and neural retina could be generated concurrently through retinal organoid induction approaches using human induced pluripotent stem cells (hiPSCs), providing valuable sources for cell therapy of retinal degenerations. This study aims to enrich and expand hiPSC-RPE acquired with this platform and explore characteristics of serially passaged RPE cells. METHODS. RPE has been differentiated from hiPSCs with a published retinal organoid induction method. After detachment of neural retina on the 4th week, the remaining mixture was scraped from the dish and subjected to suspension culture for the formation of RPE spheroids. RPE sheets were isolated and digested for expansion. The cellular, molecular, and functional features of expanded RPE cells were evaluated by different assays. RESULTS. Under suspension culture, hiPSC-RPE spheroids with pigmentation self-formed were readily enriched by removing the non-retinal tissues. RPE sheets were further dissected and purified from the spheroids. The individualized RPE cells could be passaged every week for at least 5 times in serum medium, yielding large numbers of cells with high quality in a short period. In addition, when switched to a serum-free medium, the passaged RPE cells could mature in cellular, molecular, and physiological levels, including repigmentation, markers expression, and phagocytosis. CONCLUSIONS. We developed a simple and novel RPE spheroids formation approach to enrich and expand hiPSC-RPE cells generated along with retinal neurons on a universal retinal organoid induction platform. This achievement will reduce the cost and time in producing retinal cells for basic and translational researches, in particular for retinal cell therapy.
关键词： differentiation,human induced pluripotent stem cells,retinal pigment epithelium,retinal degenerations,organoids
更新于2025-09-11 14:15:04
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Nanosekundenlaserbehandlung bei Chorioretinopathia centralis serosa ohne RPE-Defekte: eine retrospektive Fallserie
摘要： Background Chorioretinopathia centralis serosa (CCS) is a uni- or bilateral disease of the macula which is characterized by detachment of the neuro-sensory retina. The spontaneous resolution rate is 68 % after four months and 84 % after six months. Purpose To investigate the efficacy of subthreshold nanopulse laser treatment for central serous chorioretinopathy (CCS) in the absence of any atrophy in the retinal pigment epithelium (RPE). Methods This retrospective study comprised 23 eyes of 23 patients without previous treatment. Patients were followed up to 12 months. Laser treatment was performed with the 2RT? nanolaser using a grid stimulation. Changes in corrected visual acuity (VA), microperimetry and subretinal fluid height in optical coherence tomographic images were measured. Saliences in autofluorescence images and angiographic images were observed. All results were documented 1, 3, 6 and 12 months after the first treatment. Patients did not receive any supplementary treatment. Results Two months after the first treatment, 74 % of the patients showed complete SRF resolution and 91 % of the patients within 6 months had complete resorption of the SRF. Central visual acuity and macula sensitivity significantly improved from 0.18 ± 0.16 logMAR to 0.09 ± 0.17 logMAR and 24.19 ± 3.96 dB to 27.59 ± 2.89 dB. The SRF decreased within one month significantly. No CNV was documented during the observation time. The baseline subretinal fluid height is a predictive factor of faster resolution. Conclusion The evaluation of our treatment results shows that the therapy is a safe and promising method. Patients with a CCS without existing RPE defects benefit from the treatment with the 2RT? nanolaser, which is associated with an improvement of the macula function.
关键词： Retina,central serous chorioretinopathy,Nanolaser,microperimetry,retinal pigment epithelium,Subthreshold?Laser,Chorioretinopathia centralis serosa,subthreshold nanopulse laser,subretinal fluid,Mikroperimetrie,subretinale Flüssigkeit
更新于2025-09-11 14:15:04
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Quantitative Fundus Autofluorescence and Optical Coherence Tomography in <i>PRPH2/RDS</i> - and <i>ABCA4</i> -Associated Disease Exhibiting Phenotypic Overlap
摘要： PURPOSE. To assess whether quantitative fundus autofluorescence (qAF), a measure of RPE lipofuscin, and spectral-domain optical coherence tomography (SD-OCT) can aid in the differentiation of patients with fundus features that could either be related to ABCA4 mutations or be part of the phenotypic spectrum of pattern dystrophies. METHODS. Autofluorescence images (308, 488-nm excitation) from 39 patients (67 eyes) were acquired with a confocal scanning laser ophthalmoscope equipped with an internal fluorescent reference and were quantified as previously described. In addition, horizontal SD-OCT images through the fovea were obtained. Patients were screened for ABCA4 and PRPH2/RDS mutations. RESULTS. ABCA4 mutations were identified in 19 patients (mean age, 37 ± 12 years) and PRPH2/RDS mutations in 8 patients (mean age, 48 ± 13 years); no known ABCA4 or PRPH2/RDS mutations were found in 12 patients (mean age, 48 ± 9 years). Differentiation of the groups using phenotypic SD-OCT and AF features (e.g., peripapillary sparing, foveal sparing) was not reliable. However, patients with ABCA4 mutations could be discriminated reasonably well from other patients when qAF values were corrected for age and race. In general, ABCA4 patients had higher qAF values than PRPH2/RDS patients, while most patients without mutations in PRPH2/RDS or ABCA4 had qAF levels within the normal range. CONCLUSIONS. The high qAF levels of ABCA4-positive patients are a hallmark of ABCA4-related disease. The reason for high qAF among many PRPH2/RDS-positive patients is not known; higher RPE lipofuscin accumulation may be a primary or secondary effect of the PRPH2/RDS mutation.
关键词： scanning laser ophthalmoscope,PRPH2/RDS,optical coherence tomography,quantitative fundus autofluorescence,retinal pigment epithelium,lipofuscin,recessive Stargardt disease,ABCA4,pattern dystrophy
更新于2025-09-11 14:15:04
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Light-Induced Thickening of Photoreceptor Outer Segment Layer Detected by Ultra-High Resolution OCT Imaging
摘要： PURPOSE. We examined if light induces changes in the retinal structure that can be observed using optical coherence tomography (OCT). METHODS. Normal C57BL/6J mice (age 3–6 months) adapted to either room light (15 minutes to ~5 hours, 50–500 lux) or darkness (overnight) were imaged using a Bioptigen UHR-OCT system. Confocal histologic images were obtained from mice killed under light- or dark-adapted conditions. RESULTS. The OCT image of eyes adapted to room light exhibited signi?cant increases (6.1 6 0.8 lm, n ? 13) in total retina thickness compared to the same eyes after overnight dark adaptation. These light-adapted retinal thickness changes occurred mainly in the outer retina, with the development of a hypore?ective band between the RPE and photoreceptor-tip layers. Histologic analysis revealed a light-evoked elongation between the outer limiting membrane and Bruch’s membrane from 45.8 6 1.7 lm in the dark (n ? 5) to 52.1 6 3.7 lm (n ? 5) in the light. Light-adapted retinas showed an increase of actin staining in RPE apical microvilli at the same location as the hypore?ective band observed in OCT images. Elongation of the outer retina could be detected even with brief light exposures, increasing 2.1 6 0.3 lm after 15 minutes (n ? 9), and 4.1 6 1.0 lm after 2 hours (n ? 6). Conversely, dark-adaptation caused outer retinal shortening of 1.4 6 0.4 lm (n ? 7) and 3.0 6 0.5 lm (n ? 8) after 15 minutes and 2 hours, respectively. CONCLUSIONS. Light-adaption induces an increase in the thickness of the outer retina and the appearance of a hypore?ective band in the OCT image. This is consistent with previous reports of light-induced ?uid accumulation in the subretinal space.
关键词： optical coherence tomography,subretinal space,retinal pigment epithelium,light/dark adaptation,outer segments
更新于2025-09-10 09:29:36