Search Results (470)

The relevance of well-structured mitochondria in sustaining the integrity of the retinal pigment epithelium (RPE) is increasingly evident. Conversely, altered mitochondria are a culprit of age-related macular degeneration (AMD), which is influenced by the activity of mechanistic target of rapamycin (mTOR). In the present manuscript, the mitochondrial status of RPE cells was investigated by light and electron microscopy following the administration of various doses of compounds, which modulate mTOR. The study combines MitoTracker dyes and mitochondrial immunohistochemistry with in situ mitochondrial morphometry. Various doses of 3-methyladenine (3-MA), curcumin, and rapamycin were administered alone or in combination. The activity of autophagy and mTOR was quantified following each treatment. Administration of 3-MA led to activation of mTOR, which was associated with severe cell death, altered membrane permeability, and altered ZO-1 expression. In this condition, mitochondrial mass was reduced, despite a dramatic increase in damaged mitochondria being reported. The decrease in healthy mitochondria was concomitant with alterations in key mitochondria-related antigens such as Tomm20, Pink1, and Parkin. Specific mitochondrial alterations were quantified through in situ ultrastructural morphometry. Both curcumin and rapamycin counteract mTOR activation and rescue mitochondrial status, while preventing RPE cell loss and misplacement of decreased ZO-1 expression. Mitigation of mTOR may protect mitochondria in retinal degeneration. Full article

Age-related macular degeneration (AMD) is a leading cause of irreversible vision loss in the elderly, characterized by progressive degeneration of the retinal pigment epithelium (RPE) and photoreceptors in the macula. Current treatment options primarily focus on slowing disease progression in neovascular AMD, while effective therapies for dry AMD remain limited. Optogenetics, a revolutionary technique utilizing light-sensitive proteins (opsins) to control the activity of genetically targeted cells, has emerged as a promising therapeutic strategy for restoring vision in retinal degenerative diseases. In retinal disease models, adeno-associated viruses (AAVs) serve as delivery vectors via intravitreal or subretinal injections. This review explores the principles of optogenetics, its application in preclinical AMD models, and the potential for clinical translation of this approach. We discuss the various optogenetic tools, delivery methods, and the challenges and future directions in harnessing this technology to combat AMD-related vision loss. Full article

Oxidative stress destabilizes microRNA homeostasis in the retinal pigment epithelium (RPE), driving apoptosis and the epithelial-to-mesenchymal transition, which contribute to age-related macular degeneration. We investigated whether Quantum Molecular Resonance (QMR^®) electrostimulation, alone or combined with Patient Blood-Derived (PBD) secretoma, can reprogram the RPE miRNome and mitigate stress-induced damage. Human ARPE-19 cells were exposed to tert-butyl-hydroperoxide and treated with QMR^®, PBD secretome, or their combination. The deep sequencing of small RNAs at 24 h and 72 h, followed by differential expression and pathway enrichment analyses, delineated treatment-driven miRNA signatures. Oxidative stress deregulated > 50 miRNAs, enriching pro-apoptotic, fibrotic, and inflammatory pathways. QMR^® restored roughly 40% of these miRNAs and upregulated additional cytoprotective species such as miR-590-3p, a known regulator of the NF-κB and NLRP3 pathways according to validated target databases. While these observations suggest the potential involvement of inflammatory and stress-related cascades, functional assays will be required to directly confirm such effects. Secretome treatment preferentially increased anti-inflammatory miR-146a-5p and regenerative miR-204-5p while suppressing pro-fibrotic let-7f-5p. Combined QMR^® + secretome triggered the broadest miRNA response, normalizing over two-thirds of stress-altered miRNAs. These changes are predicted to influence antioxidant, anti-apoptotic, and anti-fibrotic pathways, although they did not translate into additional short-term cytoprotection compared with QMR^® alone. These data indicate that QMR^® and PBD secretome modulate complementary miRNA programs that converge on stress response networks. This broader molecular reprogramming may reflect regulatory complementarity, but functional validation is needed to determine whether it provides benefits beyond those observed with QMR^® alone. These findings offer molecular insights into potential non-invasive, cell-free strategies for retinal degeneration, although in vivo validation will be required before any clinical translation to Age-Related Macular Degeneration (AMD) therapy. Full article

Retinal Pigment Epithelium (RPE), a component of the blood–retinal barrier, plays a pivotal role in maintaining retinal homeostasis and visual function. Dysfunction of the RPE is an early event that triggers photoreceptor death, in Age-related Macular Degeneration (AMD), a multifactorial disorder primarily caused by an imbalance between endogenous antioxidant defenses and reactive oxygen species production. Our in vitro study investigated the hormetic effects of curcumin in human RPE cells (ARPE-19), focusing on its capability to modulate two enzymes related to the onset of AMD: Sirtuin 1 (SIRT1), a NAD+-dependent deacetylase enzyme involved in cellular metabolism, aging, and stress response, and caspase-3, a crucial enzyme in programmed cell death. Curcumin exhibited classic hormetic doseresponses, with low concentrations (5–10 μM) providing cytoprotection while at high doses (≥20 μM) inducing toxicity. Under moderate oxidative stress, acetylated p53 was significantly reduced, indicating SIRT1 activation; curcumin 10 μM restored basal SIRT1 activity, while 5 µM did not. Both concentrations significantly decreased cleaved caspase-3 levels, demonstrating the anti-apoptotic effects of curcumin. Our results reveal curcumin’s hormetic mechanisms of RPE protection and emphasize the critical importance of dose optimization within the hormetic window for AMD therapeutic development. Full article

Pyroptosis is a programmed cell death pathway that initiates and sustains inflammation to protect the host against invading pathogens or stress. Activation of caspase-1-mediated canonical pyroptosis takes place via formation of multi-protein cytoplasmic immune signaling complexes known as inflammasomes. Because we have shown previously that the canonical pyroptosis pathway plays a significant role in the pathogenesis of experimental murine cytomegalovirus (MCMV) retinal necrosis in mice with retrovirus-induced immunosuppression (MAIDS), we performed additional studies to determine whether this pathogenic involvement extends to inflammasomes as initiators of the canonical pyroptosis pathway. Initial studies demonstrated significant transcription of three different pyroptosis-associated inflammasomes, NLRP3, NLRP1b, and AIM2, within the ocular compartments of MCMV-infected eyes of MAIDS mice. Subsequent histopathologic findings revealed MCMV-infected eyes of groups of NLRP3^−/− MAIDS mice, NLRP1b^−/− MAIDS mice, or AIM2^−/− MAIDS mice each exhibited a similar atypical retinal pathology characterized by loss of photoreceptors and proliferation and/or loss of retinal pigmented epithelium but with relative sparing of the neurosensory retina, an outcome different from typical full-thickness retinal necrosis of MCMV-infected eyes of wildtype MAIDS mice. We conclude that multiple inflammasomes are individually stimulated within MCMV-infected eyes of MAIDS mice and each independently contributes to MAIDS-related MCMV full-thickness retinal necrosis pathogenesis. Full article

Purpose: We previously reported that the systemic administration of preprogrammed mouse hematopoietic bone marrow-derived progenitor cells (HSPCs) improved visual function and restored a functional retinal pigment epithelial (RPE) layer. Here, we investigated the potential impact of donor vs. host age on systemic cellular therapy in a murine model of retinal degeneration. Methods: HSPCs from young (8 weeks) and old (15 months) mice were programmed ex vivo with a lentiviral vector expressing the RPE65 gene (LV-RPE65) and systemically administering into young or old SOD2 KD mice. Visual loss and pathological changes were evaluated by electroretinogram (ERG), optical coherence tomography (OCT), histology, and immunohistochemistry. Results: Old donor HSPCs administered to old manganese superoxide dismutase (SOD2) knockdown (KD) recipient mice offered the least benefit. This was exemplified by the reduced recruitment and incorporation of LV-RPE65 HSPC into the RPE layer, as well as decreased improvement in visual function, retinal thinning, and limited reduction in oxidative damage and microglial activation. LV-RPE65 HSPC from young mice incorporated into the RPE layer of old SOD2 KD mice, though to a lesser extent than young cells administered to young hosts, offered some level of protection. By contrast, LV-RPE65 HSPCs from old mice, located to the subretinal space of young host mice, reduced visual loss, although some retinal pathology was observed. Conclusions: The administration of LV-RPE65 HSPC from old donors to old SOD2 KD mice offered the least improvement. Translational Relevance: Our findings highlight how both donor and recipient age impact the success of HSPC-based retinal therapy and using cells from aged donors for AMD treatment may have some limitations. Full article

The retinal pigment epithelium (RPE) is strongly involved in the pathogenesis of several retinal diseases, such as age-related macular degeneration (AMD). RPE models addressing specific pathological pathways are of high importance for understanding cellular pathomechanisms and pre-clinical screening of potential new therapeutics. The goal of this study is to establish standard operation protocols for single-eye porcine RPE preparation for AMD-relevant models of oxidative stress (RPE-Ox) and inflammation (RPE-Inf). Porcine primary RPE were prepared from one eye and seeded into one well of 12-well plates or, for polar differentiation, in transwell inserts. Different coatings (Poly-ᴅ-Lysine and laminin) and serum content of media (10%, 5%, and 1%) were tested to determine optimal culture parameters. For RPE-Ox, cells were treated with NaIO₃, CoCl₂, or erastin; cell viability (thiazolyl blue tetrazolium bromide, MTT), and gene expression (RT-qPCR) were determined. For RPE-Inf, cells were treated with lipopolysaccharide (LPS), polyinosinic/polycytidylic acid (Poly I:C), or tumor necrosis factor alpha (TNF-α); cell viability (MTT), cytokine secretion (ELISA), and gene expression (RT-qPCR) were determined. For transwell plates in RPE-Inf, cell viability (MTT), polar cytokine secretion (ELISA), gene expression (RT-qPCR), and transepithelial electrical resistance (TEER) for barrier assessment were conducted. For RPE-Ox, effective LD₅₀ could be achieved by using 24 h stimulation with 25 µm erastin, seven days after preparation in 5% serum cultures, without coating. For gene expression assessment, the use of Poly-ᴅ-Lysine is recommended. For RPE-Inf, three days of LPS stimulation (1 µg/mL) showed effective cytokine activation with 5% serum on uncoated 12-well plates. Transwell plates are not recommended for cytokine secretion assessment. It can be used for cell barrier assays in which LPS also showed effective cell barrier decrease and gene expression assays. Two specific best practice protocols for the use of porcine single-eye cultures in AMD research concerning oxidative stress and inflammation with optimized parameters were established and are provided. Full article

Background: Age-related macular degeneration (AMD) is a major cause of irreversible vision loss in the developed world, and the approved products for geographic atrophy (GA), a late-stage form of dry AMD, have shown limited efficacy and require frequent administration. Therefore, longer-lasting therapies with improved efficacy would be a welcome addition to AMD treatment. One potential therapeutic is ONL1204, a small peptide inhibitor of the Fas receptor that has prevented cell death and inflammation in retinal disease models. This study characterizes the pharmacokinetics (PK) and durability of protection conferred by ONL1204. Methods: Ocular pharmacokinetic profiles were generated over 3 months in rabbit and minipig following a single intravitreal (IVT) injection of ONL1204 at multiple doses. Ocular pharmacodynamics were evaluated in two models: a rabbit model using a single IVT injection of ONL1204 with a delayed sodium iodate challenge coupled with fluorescein angiography to quantify RPE loss, and a chronic mouse model that reflects key features of dry AMD disease pathology to assess the efficacy of repeat IVT administrations of ONL1204. Results: ONL1204 had prolonged residence in the ocular tissues of rabbit and minipig, with a vitreous humor half-life of over 100 days. ONL1204 demonstrated significant protection of the retinal pigment epithelium (RPE) in the rabbit sodium iodate model. In the chronic mouse model, two administrations of ONL1204 preserved RPE morphology, reduced caspase-8 activity, and decreased inflammation. Conclusions: These data represent key characteristics of ONL1204, highlighting its clinical potential as a therapeutic for chronic retinal diseases, including GA. Full article

Replacement of the retinal pigment epithelium (RPE) is emerging as a promising approach to treat degenerative retinal diseases, including age-related macular degeneration and Stargardt disease, in which RPE function cannot otherwise be restored. Despite the limitations of existing treatments, advances in cell sourcing and surgical methods have enabled initial human trials of RPE transplantation, with early results indicating potential efficacy. This review comprehensively examines the evolution of RPE transplantation in recent decades, highlighting the advantages and limitations of different cell sources and delivery methods. Current clinical trial data are analyzed with a particular focus on immune rejection risks, surgical complications, and long-term safety. Despite encouraging safety profiles, achieving consistent and sustained visual improvement remains a challenge, as vision outcomes might be influenced by factors such as disease stage at intervention, transplantation site, number of cells transplanted, and duration of follow-up. Key challenges, such as cell or graft survival and integration with the host retina, are discussed in depth, as overcoming these obstacles is essential for achieving stable and effective RPE replacement. Future research directions, including innovations in biomaterials, molecular modification strategies, and personalized approaches, hold promise for enhancing the efficacy and durability of RPE transplantation for retinal disease. Full article

The retina is highly sensitive to oxygen and blood supply, and hypoxia plays a key role in retinal diseases such as diabetic retinopathy (DR) and age-related macular degeneration (AMD). Müller glial cells, which are essential for retinal homeostasis, respond to injury and hypoxia with reactive gliosis, characterized by the upregulation of the glial fibrillary acidic protein (GFAP) and vimentin, cellular hypertrophy, and extracellular matrix changes, which can impair retinal function and repair. The retinal pigment epithelium (RPE) supports photoreceptors, forms part of the blood–retinal barrier, and protects against oxidative stress; its dysfunction contributes to retinal degenerative diseases such as AMD, retinitis pigmentosa (RP), and Stargardt disease (SD). Extracellular vesicles (EVs) play a crucial role in intercellular communication, protein homeostasis, and immune modulation, and have emerged as promising diagnostic and therapeutic tools. Understanding the role of extracellular vesicles’ (EVs’) signaling machinery of glial cells and the retinal pigment epithelium (RPE) is critical for developing effective treatments for retinal degeneration. In this study, we investigated the bidirectional EV-mediated crosstalk between RPE and Müller cells under hypoxic conditions and its impact on cellular metabolism and retinal cell integrity. Our findings demonstrate that RPE-derived extracellular vesicles (RPE EVs) induce time-dependent metabolic reprogramming in Müller cells. Short-term exposure (24 h) promotes pathways supporting neurotransmitter cycling, calcium and mineral absorption, and glutamate metabolism, while prolonged exposure (72 h) shifts Müller cell metabolism toward enhanced mitochondrial function and ATP production. Conversely, Müller cell-derived EVs under hypoxia influenced RPE metabolic pathways, enhancing fatty acid metabolism, intracellular vesicular trafficking, and the biosynthesis of mitochondrial co-factors such as ubiquinone. Proteomic analysis revealed significant modulation of key regulatory proteins. In Müller cells, hypoxic RPE-EV exposure led to reduced expression of Dyskerin Pseudouridine Synthase 1 (DKc1), Eukaryotic Translation Termination Factor 1 (ETF1), and Protein Ser/Thr phosphatases (PPP2R1B), suggesting alterations in RNA processing, translational fidelity, and signaling. RPE cells exposed to hypoxic Müller cell EVs exhibited elevated Ribosome-binding protein 1 (RRBP1), RAC1/2, and Guanine Nucleotide-Binding Protein G(i) Subunit Alpha-1 (GNAI1), supporting enhanced endoplasmic reticulum (ER) function and cytoskeletal remodeling. Functional assays also revealed the compromised barrier integrity of the outer blood–retinal barrier (oBRB) under hypoxic co-culture conditions. These results underscore the adaptive but time-sensitive nature of retinal cell communication via EVs in response to hypoxia. Targeting this crosstalk may offer novel therapeutic strategies to preserve retinal structure and function in ischemic retinopathies. Full article

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