Search Results (460)

 Galectin-3 is a multifunctional protein that is associated with diseases of the chorioretinal interface, in which the retinal pigment epithelium (RPE) plays a central role in disease development and progression. Since galectin-3 can function extracellularly as well as intracellularly via different mechanisms, we developed an immortalized human RPE cell line (ARPE-19) with a knockdown for galectin-3 expression (ARPE-19/LGALS3^+/−) using a sgRNA/Cas9 all-in-one expression vector. By Western blot analysis, a reduced galectin-3 expression of approximately 48 to 60% in heterozygous ARPE-19/LGALS3^+/− cells was observed when compared to native controls. Furthermore, ARPE-19/LGALS3^+/− cells displayed a flattened, elongated phenotype with decreased E-cadherin as well as enhanced N-cadherin and α-smooth muscle actin mRNA expression, indicating an epithelial–mesenchymal transition of the cells. Compared to wildtype controls, ARPE-19/LGALS3^+/− cells had significantly reduced metabolic activity to 86% and a substantially decreased proliferation to 73%. Furthermore, an enhanced cell adhesion and a diminished migration of immortalized galectin-3 knockdown RPE cells was observed compared to native ARPE-19 cells. Finally, by Western blot analysis, reduced pAKT, pERK1/2, and β-catenin signaling were detected in ARPE-19/LGALS3^+/− cells when compared to wildtype controls. In summary, in RPE cells, endogenous galectin-3 appears to be essential for maintaining the epithelial phenotype as well as cell biological functions such as metabolism, proliferation, or migration, effects that might be mediated via a decreased activity of the AKT, ERK1/2, and β-catenin signaling pathways.  Full article

Background: Choroidal nevi are common, benign tumors. These tumors rarely cause adverse retinal sequalae, but when they do, they can lead to disruption of the outer retina and vision loss. In this paper, we used high-resolution retinal imaging modalities, optical coherence tomography (OCT) and adaptive optics scanning laser ophthalmoscopy (AOSLO), to longitudinally monitor retinal sequelae of a submacular choroidal nevus. Methods: A 31-year-old female with a high-risk choroidal nevus resulting in subretinal fluid (SRF) and a 30-year-old control subject were longitudinally imaged with AOSLO and OCT in this study over 18 and 22 months. Regions of interest (ROI) including the macular region (where SRF was present) and the site of laser photocoagulation were imaged repeatedly over time. The depth of SRF in a discrete ROI was quantified with OCT and AOSLO images were assessed for visualization of photoreceptors and retinal pigmented epithelium (RPE). Cell-like structures that infiltrated the site of laser photocoagulation were measured and their count was assessed over time. In the control subject, images were assessed for RPE visualization and the presence and stability of cell-like structures. Results: We demonstrate that AOSLO can be used to assess cellular-level changes at small ROIs in the retina over time. We show the response of the retina to SRF and laser photocoagulation. We demonstrate that the RPE can be visualized when SRF is present, which does not appear to depend on the height of retinal elevation. We also demonstrate that cell-like structures, presumably immune cells, are present within and adjacent to areas of SRF on both OCT and AOSLO, and that similar cell-like structures infiltrate areas of retinal laser photocoagulation. Conclusions: Our study demonstrates that dynamic, cellular-level retinal responses to SRF and laser photocoagulation can be monitored over time with AOSLO in living humans. Many retinal conditions exhibit similar retinal findings and laser photocoagulation is also indicated in numerous retinal conditions. AOSLO imaging may provide future opportunities to better understand the clinical implications of such responses in vivo. Full article

The retinal pigment epithelium (RPE), a monolayer of pigmented cells, is critical for visual function through its interaction with the neural retina. In healthy eyes, RPE cells exhibit a uniform hexagonal arrangement, but under stress or disease, such as age-related macular degeneration (AMD), dysmorphic traits like cell enlargement and apparent multinucleation emerge. Multinucleation has been hypothesized to result from cellular fusion, a compensatory mechanism to maintain cell-to-cell contact and barrier function, as well as conserve resources in unhealthy tissue. However, traditional two-dimensional (2D) imaging using apical border markers alone may misrepresent multinucleation due to the lack of lateral markers. We present high-resolution confocal images enabling three-dimensional (3D) visualization of apical (ZO-1) and lateral (α-catenin) markers alongside nuclei. In two RPE damage models, we find that seemingly multinucleated cells are often single cells with displaced neighboring nuclei and lateral membranes. This emphasizes the need for 3D analyses to avoid misidentifying multinucleation and underlying fusion mechanisms. Lastly, images from the NaIO₃ oxidative damage model reveal variability in RPE damage, with elongated, dysmorphic cells showing increased ZsGreen reporter protein expression driven by EMT-linked CAG promoter activity, while more regular RPE cells displayed somewhat reduced green signal more typical of epithelial phenotypes. Full article

The extracellular matrix (ECM) is a collagen-based scaffold that provides structural support and regulates nutrient transport and cell signaling. ECM homeostasis depends on a dynamic balance between synthesis and degradation, the latter being primarily mediated by matrix metalloproteinases (MMPs). These enzymes are secreted as pro-forms and require activation to degrade ECM components. Their activity is modulated by tissue inhibitors of metalloproteinases (TIMPs). Aging disrupts this balance, leading to the accumulation of oxidized, cross-linked, and denatured matrix proteins, thereby impairing ECM function. Bruch’s membrane, a penta-laminated ECM structure in the eye, plays a critical role in supporting photoreceptor and retinal pigment epithelium (RPE) health. Its age-related thickening and decreased permeability are associated with impaired nutrient delivery and waste removal, contributing to the pathogenesis of age-related macular degeneration (AMD). In AMD, MMP dysfunction is characterized by the reduced activation and sequestration of MMPs, which further limits matrix turnover. This narrative review explores the structural and functional changes in Bruch’s membrane with aging, the role of MMPs in ECM degradation, and the relevance of these processes to AMD pathophysiology, highlighting emerging regulatory mechanisms and potential therapeutic targets. Full article

Chronic kidney disease (CKD)-associated anemia is a global health concern and is linked to vascular and ocular complications. Hypoxia-inducible factor (HIF) stabilizers, or HIF prolyl hydroxylase inhibitors (PHIs), are promising candidates for the treatment of CKD-associated anemia. Since hypoxia and angiogenesis are involved in eye diseases, this study examined the effects of HIF-PHIs on metabolism and gene expression in retinal pigment epithelium (RPE) cells. Results revealed that PHIs differentially induced angiogenic (VEGFA, ANG) and glycolytic (PDK1, GLUT1) gene expression, with Roxadustat causing the strongest transcriptional changes. However, Roxadustat-induced angiogenic signals did not promote endothelial tube formation. Moreover, it did not induce oxidative stress, inflammation, or significant antioxidant gene responses in ARPE-19 cells. Roxadustat also reduced the inflammatory cytokine response to tumor necrosis factor-α, including IL-6, IL-8, and MCP-1, and did not exacerbate VEGF expression under high-glucose conditions. Overall, Roxadustat triggered complex gene expression changes without promoting inflammation or oxidative stress in RPE cells. Despite these findings, ophthalmologic monitoring is advised during PHI treatment in CKD patients receiving HIF-PHIs. Full article

Saffron, branded as Repron^®, is effective in slowing the progression of several neurodegenerative diseases. Its production, however, requires specific cultivation techniques and procedures that, together with low yields, make it expensive. To address this challenge, hydroponic cultivation has been adopted. Previous studies have shown that hydroponically cultivated saffron and Repron^® share comparable chemical compositions and neuroprotective effects under oxidative stress conditions. In this study, we evaluated the protective properties of extracts derived from Crocus sativus L. waste, compared with those of saffron derived from stigmas. Human retinal pigment epithelium (ARPE-19) cells were pre-treated with extracts of various plant waste fractions before being subjected to three stress conditions: H₂O₂-induced oxidative stress (500 μM, 3 h), lipopolysaccharide (LPS; 0.25 mg/mL, 24 h), and hyperglycemia (25 mM glucose, 96 h). Saffron Repron^® served as a positive control. The results revealed that the extract derived from C. sativus waste had superior protective effects against oxidative stress and inflammation by preserving the state of the mitochondria and tight junctions (ZO-1); conversely, the tepal extract alone was more effective under hyperglycemic conditions by also modulating acrolein levels. These results suggest that different plant fractions contain bioactive compounds with specific protective actions, which together lead to increased cell survival. Full article

Age-related macular degeneration (AMD) is the main cause of blindness in Western nations. AMD models addressing specific pathological pathways are desired. Through this study, a best-practice protocol for polarized porcine single-eye retinal pigment epithelium (RPE) preparation for AMD-relevant models of RPE barrier and polarity is established. Single-eye porcine primary RPE cells (from one eye for one well) were prepared in 12-well plates including Transwell inserts. Different coatings (laminin (Lam), Poly-ᴅ-Lysine (PDL), fibronectin (Fn) and collagens) and varying serum contents (1%, 5% and 10%) were investigated to determine optimal culture parameters for this model. Success rates of cultures, cell number (trypan-blue exclusion assay), morphology/morphometry (light and fluorescence microscopy), protein secretion/expression (ELISA, Western blot), gene expression (qPCR), transepithelial electric resistance (TEER) and polar location of bestrophin 1 (BEST1) by cryosectioning (IHC-Fr) were assessed. Cells seeded on Lam exhibited the highest level of epithelial cells and confluence properties. Fn resulted in the highest cell number growth. Lam and Fn exhibited the highest culture success rates. TEER values and vascular endothelial growth factor secretion were highest when Lam was used. For the first time, polar (Transwell) porcine single-eye RPE morphometry parameters were determined. RPE on Lam showed bigger cells with a higher variety of cell shapes. CIV displayed the lowest claudin 19 expression. The highest basolateral expression of BEST1 was achieved with Lam coating. The higher the serum, the better the cell number increase and confluence success. A reduction in serum on Lam showed positive results for RPE morphology, while morphometry remained stable. A five percent serum on Lam showed the highest culture success rate and best barrier properties. RPE65 expression was reduced by using 10% serum. Altogether, the most suitable coating of Transwell inserts was Lam, and a reduction in serum to 5% is recommended, as well as a cultivation time of 28 days. A protocol for the use of polar porcine single-eye cultures with validated parameters was established and is provided herein. Full article

The retinal pigment epithelium (RPE) is a cellular source of retinal regeneration in lower vertebrates and a cellular source of retinal diseases in mammals, including humans. Both processes are based on a genetic program for the conversion of RPE cells into cells of other phenotypes: neural in the first case and mesenchymal in the second. RPE reprogramming in the neural direction is realized in tailed amphibians and bird embryos in vivo, but in higher vertebrates and humans, this process is realized in vitro. Epigenetic regulation determines the phenotypic plasticity of RPE cells, i.e., their choice of the cell differentiation pathway in animals of different classes. It has been suggested that the implementation of the genetic program for RPE reprogramming into different types of retinal neurons in adult amphibians and birds at the early stages of embryogenesis is conditioned by the specificity of the epigenetic landscape. The retinal RPE-dependent pathologies in mammals are characterized by different epigenetic signatures, and have a shared characteristic: specifically, a deficient epigenetic landscape (dysregulations in DNA methylation and histone modifications). Knowledge of the patterns and features of the epigenetic regulation of RPE cell behavior will allow us to obtain RPE cells that are in demand in medicine, from direct reprogramming with the possibility of epigenetically maintaining the cellular identities to the creation of neuro-regenerative technologies for the replacement therapy of RPE-dependent retinal pathologies in humans. Full article

Background: Adaptive optics transscleral flood illumination (AO-TFI) enables in vivo, non-invasive, high-resolution imaging of retinal pigment epithelium (RPE) and photoreceptor (PR) cells, paving the way for a new potential characterization of retinal diseases. This study aimed to analyze RPE and PR cells in a case of acute zonal occult outer retinopathy (AZOOR) using AO-TFI. Methods: A patient affected by AZOOR underwent a comprehensive eye examination, perimetry, electroretinography (ERG), autofluorescence, and optical coherence tomography (OCT) during the acute phase (T0). After three years (T1), OCT angiography (OCTA) and AO-TFI were also performed. Voronoi analysis was utilized to quantify RPE and PR cells. Results: At T0, OCT revealed interruptions in the ellipsoid zone (EZ) of the right eye, while the structure of the left eye appeared normal. Perimetry and ERG were abnormal in both eyes. At T1, OCT indicated recovery of the EZ in the right eye, while thinning of the ONL persisted. Perimetry and mfERG values remained below normative limits. OCTA exhibited globally reduced vessel density in the inner retina of the right eye. AO-TFI demonstrated reduced PR density in affected areas despite preserved EZ, while RPE cell density appeared unaffected. Conclusion: AO-TFI enabled a detailed visualization and quantification of macular RPE and PR cells, providing valuable insights into the pathophysiology of AZOOR. Full article

Age-related macular degeneration (AMD) is a common cause of blindness worldwide, and it is projected to affect several million individuals by 2040. The human retinal pigment epithelium (hRPE) degenerates in dry AMD, prompting the need to develop stem cell therapies to replace the lost tissue by autologous transplantation and restore the visual function. Nevertheless, the molecular factors behind the hRPE cell fate determination have not been elucidated. Here we identify all molecular determinants of the hRPE cell fate identity by comprehensive and unbiased screening of predicted pioneer factors in the human genome: such TFs mediate coordinated transitions in chromatin accessibility and transcriptional outcome along three major stages of the hRPE genesis. Furthermore, we compile a complete census of all transcription factor-specific binding sites by footprinting analysis of the human epigenome along the RPE developmental trajectory. Gene regulatory networks were found to be involved in cellular responses to glucose and hypoxia, RPE nitrosative stress, type II epithelial-to-mesenchymal transition (EMT), and type III tumorigenic EMT, providing routes for therapeutic intervention on pleiotropic targets dysregulated in AMD, diabetic retinopathy, and cancer progression. Genome editing technologies may leverage this repository to devise functional screenings of regulatory elements and pharmacogenomic therapies in complex diseases, paving the way for strategies in precision medicine. Full article

Current evidence indicates that most types of autophagy represent a pivot in promoting retinal integrity. In healthy conditions, autophagy acts on multiple pathways, which are fundamental for the biochemistry and the fine structure of the retina. Autophagy is essential in granting visual processes. On the other hand, autophagy dysfunction characterizes several retinal disorders. This is mostly evident in age-related macular degeneration (AMD), which represents the most common degenerative disease leading to blindness. The involvement of autophagy in AMD is documented in vitro and in vivo experiments, and it is strongly suggested by clinical findings in humans. The present manuscript provides an overview of the specific types of autophagy, which prevail in the retina and their alterations in retinal degeneration with an emphasis on AMD. The dysfunction of specific autophagy steps was analyzed in relation to hallmarks of AMD pathology and symptoms. An extended session of the manuscript analyzes the connection between altered autophagy and cell pathology within retinal pigment epithelium, as well as the site and structure of extracellular aggregates named drusen. The significance of the drusen in relation to visual function is discussed in the light of the role of autophagy in regulating key steps of phototransduction. Full article

The retinal pigment epithelium (RPE) plays a crucial role in maintaining retinal homeostasis, and dysregulation of the transforming growth factor-beta (TGF-β) signaling pathways contributes to retinal fibrosis and inflammatory diseases, including proliferative vitreoretinopathy (PVR). Tacrolimus (FK506), an immunosuppressant, has shown potential antifibrotic properties, but its effects on TGF-β-related genes and microRNAs (miRNAs) in RPE cells remain unclear. Human RPE (H-RPE) cells were treated with lipopolysaccharide (LPS) to induce inflammation and subsequently exposed to tacrolimus. Gene and miRNA expression profiling related to TGF-β signaling pathways were conducted using microarrays, followed by Quantitative Reverse-Transcription Polymerase Chain Reaction (RT-qPCR) validation. Protein levels were assessed via enzyme-linked immunosorbent assay (ELISA), and interactions were analyzed using STRING database network analysis. Tacrolimus modulated key components of the TGF-β pathway, upregulating TGF-β2, TGF-β3, SMAD2, and SMAD4 while downregulating TGF-βR1 and SMAD7. JAK/STAT and MAPK pathways were also affected, indicating broad regulatory effects. miRNA profiling identified hsa-miR-200a-3p, hsa-miR-589-3p, hsa-miR-21, and hsa-miR-27a-5p as key regulators. STRING analysis confirmed strong functional interactions within the TGF-β network. In conclusion, tacrolimus modulates both canonical (upregulation of SMAD2/4 and downregulation of SMAD7) and non-canonical (JAK/STAT and MAPK) TGF-β signaling pathways in LPS-stimulated RPE cells. These changes collectively suggest a dual anti-inflammatory and anti-fibrotic effect. The increased TGF-β2 and decreased SMAD7 levels, alongside altered miRNA expression (e.g., downregulation of miR-200a-3p), indicate that tacrolimus may inhibit key profibrotic mechanisms underlying PVR. These findings support the potential therapeutic repurposing of tacrolimus in PVR and warrant further in vivo validation. Full article

Age-related macular degeneration (AMD), the leading cause of irreversible blindness worldwide, represents a complex neurodegenerative disorder whose pathogenesis remains elusive. At the core of AMD pathophysiology lies the retinal pigment epithelium (RPE), whose epithelial–mesenchymal transition (EMT) has emerged as a critical pathological mechanism driving disease progression. This transformative process, characterized by RPE cell dedifferentiation and subsequent extracellular matrix remodeling, is orchestrated through a sophisticated network of molecular interactions and cellular signaling cascades. Our review provides a comprehensive analysis of the molecular landscape underlying RPE EMT in AMD, with particular emphasis on seven interconnected pathological axes: (i) oxidative stress and mitochondrial dysfunction, (ii) hypoxia-inducible factor signaling, (iii) autophagic flux dysregulation, (iv) chronic inflammatory responses, (v) complement system overactivation, (vi) epigenetic regulation through microRNA networks, and (vii) key developmental signaling pathway reactivation. Furthermore, we evaluate emerging therapeutic strategies targeting EMT modulation, providing a comprehensive perspective on potential interventions to halt AMD progression. By integrating current mechanistic insights with therapeutic prospects, this review aims to bridge the gap between fundamental research and clinical translation in AMD management. Full article

Lipid metabolism and oxidative stress are major contributors to ocular diseases, including drusen formation and photoreceptor damage. Aster-B, encoded by GRAMD1B, mediates the non-vesicular transport of cholesterol and carotenoids and is highly expressed in the human eye, though its specific ocular functions remain unknown. We investigated Aster-B’s role in ARPE-19 cells, a model of the retinal pigment epithelium (RPE), using CRISPR/dCas9 to generate an Aster-B-expressing cell line. Aster-B expression significantly improved cell survival under oxidative stress induced by hydrogen peroxide (H₂O₂) and was associated with the activation of the p53 and TGFβ signaling pathways, indicating a role in modulating stress responses. To confirm its lipid transport activity, we treated cholesterol-depleted cells with carotenoids and tracked their localization. In Aster-B-expressing cells, carotenoids accumulated in mitochondria, while in control cells, they remained in other cellular compartments. Under oxidative stress, mitochondrial carotenoid levels declined in Aster-B-expressing cells but not in control cells. Interestingly, carotenoids enhanced survival in control cells exposed to H₂O₂ but had a detrimental effect in Aster-B-expressing cells, suggesting that carotenoid function is context and location dependent. These findings highlight Aster-B’s role in coordinating lipid transport and stress responses in the RPE, with implications for oxidative stress-related eye diseases. Full article

Background: Hyperreflective retinal foci (HRF) are small, discrete, hyperreflective elements observed in the retina using optical coherence tomography (OCT). They appear in many retinal diseases and have been linked to disease progression, treatment response, and prognosis. However, their definition and clinical use vary widely, not just between different diseases, but also within a single disorder. Methods: This perspective is based on a review of peer-reviewed studies examining HRF across different retinal diseases. The studies included analyzed HRF morphology, distribution, and clinical relevance using OCT. Particular attention was given to histopathological correlations, disease-specific patterns, and advancements in automated quantification methods. Results: HRF distribution and features vary with disease type and even within the same disease. A variety of descriptions have been proposed with different characteristics in terms of dimensions, reflectivity, location, and association with back shadowing. Automated OCT analysis has enhanced HRF detection, enabling quantitative analysis that may expand their use in clinical practice. However, differences in software and methods can lead to inconsistent results between studies. HRF have been linked to microglial cells and may be defined as neuro-inflammatory cells (Inflammatory, I-HRF), migrating retinal pigment epithelium cells (Pigmentary, P-HRF), blood vessels (Vascular, V-HRF), and deposits of proteinaceous or lipid elements leaking from vessels (Exudative, E-HRF). Conclusions: HRF are emerging as valuable imaging biomarkers in retinal diseases. Four main types have been identified, with different morphological features, pathophysiological origin, and, therefore, different implications in the management of retinal diseases. Advances in imaging and computational analysis are promising for their incorporation into personalized treatment strategies. Full article