RPE Cells Research Articles

A novel lipophenol quercetin derivative to prevent macular degeneration: Intravenous and oral formulations for preclinical pharmacological evaluation

Drugs with properties against oxidative and carbonyl stresses are potential candidates to prevent dry age-related macular degeneration (Dry-AMD) and inherited Stargardt disease (STGD1). Previous studies have demonstrated the capacity of a new lipophenol drug: 3-O-DHA-7-O-isopropyl-quercetin (Q-IP-DHA) to protect ARPE19 and primary rat RPE cells respectively from A2E toxicity and under oxidative and carbonyl stress conditions. In this study, first, a new methodology has been developed to access gram scale of Q-IP-DHA. After classification of the lipophenol as BCS Class IV according to physico-chemical and biopharmaceutical properties, an intravenous formulation with micelles (M) and an oral formulation using lipid nanocapsules (LNC) were developed. M were formed with Kolliphor® HS 15 and saline solution 0.9 % (mean size of 16 nm, drug loading of 95 %). The oral formulation was optimized and successfully allowed the formation of LNC (25 nm, 96 %). The evaluation of the therapeutic potency of Q-IP-DHA was performed after IV administration of micelles loaded with Q-IP-DHA (M−Q−IP−DHA) at 30 mg/kg and after oral administration of LNC loaded with Q-IP-DHA (LNC-Q-IP-DHA) at 100 mg/kg in mice. Results demonstrated photoreceptor protection after induction of retinal degeneration by acute light stress making Q-IP-DHA a promising preventive candidate against dry-AMD and STGD1.

Elovanoid Precursor Inhibits Lipid Peroxidation in Retinal Cells: A Comprehensive Untargeted Lipidomic Approach

Age-related macular degeneration (AMD) accumulates amyloid-beta peptide as in senile plaques of Alzheimer's. Also, as in other neurodegenerative diseases, enhanced reactive oxygen species (ROS) and an overall uncompensated oxidative stress (UOS) environment evolve. Oxidized phospholipids (OxPLs) cause detrimental perturbations to photoreceptor cells (PRC) and to the retinal pigment epithelial cell (RPEC), a nondividing cell derived from the neuroectoderm, essential for PRC integrity. Elovanoids (ELVs) are lipid mediators that support PRC. They are oxygenated derivatives of very long-chain polyunsaturated fatty acids (VLC-PUFAs), specifically C32:6 and C34:6. PUFA-containing phospholipids in the cell membrane are susceptible to non-enzymatic (e.g., free radical and reactive oxygen species) and enzymatic oxidation of the unsaturated components, altering their structure and function. Studies have implicated oxidized phospholipids as relevant to the etiology of aging and neurodegeneration, including age-related macular degeneration and Alzheimer's. Due to high metabolic demands, RPE cells are under constant oxidative stress.

HAGLR, stabilized by m6A modification, triggers PTEN-Akt signaling cascade-mediated RPE cell pyroptosis via sponging miR-106b-5p.

Consistent hyperglycaemia on retinal microvascular tissues is recognized as a vital inducer of diabetic retinopathy (DR) pathogenesis. In view of the essential functionality of long noncoding RNAs (lncRNAs) in multiple human diseases, we aim to figure out the exact role and underlying mechanisms of lncRNA HOXD Cluster Antisense RNA 1 (HAGLR) in DR pathogenesis. Serum specimens from patients with proliferative DR and healthy volunteers were collected for measuring HAGLR levels. Human primary retinal pigment epithelium (HRPE) cells kept in high glucose (HG) condition were applied to simulating hyperglycaemia of DR pathology in vitro. Cell proliferation, apoptosis, either pyroptosis was assess using Cell Counting Kit-8 TUNEL, flow cytometry, and enzyme-linked immunoassay assays. Bioinformatics analysis was subjected to examine the interaction between HAGLR and N6-methyladenosine (m6A)-bind protein IGF2BP2, as determined using RNA immunoprecipitation and RNA pull-down. Luciferase reporter assay was performed to assess the HAGLR-miR-106b-5p-PTEN axis. Levels of pyroptosis-associated biomarkers were detected using western blotting. Aberrantly overexpressed HAGLR was uncovered in the serum samples of DR patients and HG-induced HRPE cells, of which knockdown attenuated HG-induced cytotoxic impacts on cell apoptosis and pyroptosis. Whereas, reinforced HAGLR further aggravated these effects. IGF2BP2 positively regulated HAGLR in a m6A-dependent manner. HAGLR served as a sponge for miR-106b-5p to upregulate PTEN, thereby activating Akt signaling cascade. Rescue assays demonstrated that PTEN overexpression abolished the inhibition of silenced HAGLR on pyroptosis in HRPE cells. HAGLR, epigenetically modified by IGF2BP2 in an m6A-dependent manner, functioned as a sponge for miR-106b-5p, thereby activating PTEN/Akt signaling cascade to accelerate DR pathology.

PEDF Prevents Mitochondrial Function Decay and ER Stress Induced by Rotenone in Aging RPE Cells.

Neurodegenerative diseases, including age-related macular degeneration (AMD), may be linked to mitochondrial dysfunction and endoplasmic reticulum (ER) stress. We examined whether Pigment epithelium-derived factor (PEDF) could prevent changes in the structure and function of these organelles by accelerating by rotenone (ROT), a mitochondrial inhibitor, in human retinal pigment epithelium (RPE) cells of chronological age. RPE cells from 9-20, 50-55, 60-70, and >70-year-old donors were isolated, grown as primary cultures, harvested, and treated with ROT and PEDF for electron microscope (EM), western blot analysis, and polymerase chain reaction (PCR). Reactive oxygen species (ROS) and cytoplasmic calcium [Ca2+]c and mitochondrial calcium [Ca2+]m levels were measured by flow cytometry using 2',7'-dichlorodihydrofluorescin diacetate (H2-DCF-DA), fluo-3/AM, and Rhod-2/AM, and ATP levels were measured using a luciferin/luciferase-based assay. Mitochondrial membrane potential (ΔΨm) was detected using 5,5',6,6'-tetrachloro1,1',3,3'-tetraethylbenzimid azolocarbocyanine iodide (JC-1), and susceptibility of the cells to ROT toxicity and PEDF-protective effect was determined by propidium iodide (PI) staining and lactate dehydrogenase (LDH) assay. The expression of ER stress-related genes was detected using real-time (RT)-PCR. We observed decay in the mitochondria of aged RPE cells, including matrix abnormalities, elongation, loss of cristae, and disruption of membrane integrity after ROT treatment. We also observed lower [Ca2+]c, higher ROS and [Ca2+]m levels, decreased ΔΨm after ROT treatment, and greater susceptibility to ROT toxicity in aged RPE cells. PEDF can protect the cristae and integrity of the mitochondrial membrane, increase ATP levels and ΔΨm, and lower ROS, [Ca2+]c, and [Ca2+]m in aged RPE cells induced by ROT. In addition, there was an increase in RDH expression in RPE cells with increasing age after PEDF treatment. Similarly, PEDF decreased the expression of ROT-induced ER stress-related genes. Our study provides evidence that PEDF can reduce bioenergetic deficiencies, mitochondrial decay, and ER stress in aging RPE, a condition that may trigger the onset of retinal diseases such as AMD.

Human Neural Progenitors Expressing GDNF Enhance Retinal Protection in a Rodent Model of Retinal Degeneration.

Stem cell therapy for retinal degenerative diseases has been extensively tested in preclinical and clinical studies. However, preclinical studies performed in animal models at the early stage of disease do not optimally translate to patients that present to the clinic at a later stage of disease. As the retina degenerates, inflammation and oxidative stress increase and trophic factor support declines. Testing stem cell therapies in animal models at a clinically relevant stage is critical for translation to the clinic. Human neural progenitor cells (hNPC) and hNPC engineered to stably express GDNF (hNPCGDNF) were subretinally injected into the Royal College of Surgeon (RCS) rats, a well-established model for retinal degeneration, at early and later stages of the disease. hNPCGDNF treatment at the early stage of retinal degeneration provided enhanced visual function compared to hNPC alone. Treatment with both cell types resulted in preserved retinal morphology compared to controls. hNPCGDNF treatment led to significantly broader photoreceptor protection than hNPC treatment at both early and later times of intervention. The phagocytic role of hNPC appears to support RPE cell functions and the secreted GDNF offers neuroprotection and enables the extended survival of photoreceptor cells in transplanted animal eyes. Donor cells in the RCS rat retina survived with only limited proliferation, and hNPCGDNF produced GDNF in vivo. Cell treatment led to significant changes in various pathways related to cell survival, antioxidative stress, phagocytosis, and autophagy. A combined stem cell and trophic factor therapy holds great promise for treating retinal degenerative diseases including retinitis pigmentosa and age-related macular degeneration.

MiR-21 Participates in Anti-VEGF-Induced Epithelial Mesenchymal Transformation in RPE Cells.

To explore the role and possible mechanism of miR-21 in anti-VEGF drug-induced epithelial-mesenchymal transformation (EMT) in human retinal pigment epithelium (ARPE-19) cells, and to seek more therapeutic targets to improve prognosis vision. ARPE-19 cells were exposed to clinical dosage of bevacizumab and miR-21 expression was measured by real-time polymerase chain reaction (RT-PCR) assay. MiR-21 mimic and inhibitor were transfected into bevacizumab-induced ARPE-19, the expression of α-smooth muscle actin (α-SMA), E-cadherin, and SNAI1 were detected by cell immunofluorescence and Western blotting. Clinical dosage of bevacizumab caused EMT and enhanced miR-21 expression in ARPE-19 cells (P<0.05). The inhibition of miR-21 attenuated the EMT effect of bevacizumab, while overexpression of miR-21 promoted this activity (P<0.05). The SNAI1 was up-regulated by bevacizumab and promotion was partially suppressed by the miR-21 inhibitor and aggravated by the miR-21 mimic (P<0.05). MiR-21 promotes bevacizumab-induced EMT in ARPE cells which is significantly positively correlated with SNAI1. MiR-21 might be a potential miRNA-based therapeutic target for reducing bevacizumab-induced subretinal fibrosis.

Paraoxonase 2 Deficiency Causes Mitochondrial Dysfunction in Retinal Pigment Epithelial Cells and Retinal Degeneration in Mice.

Although AMD is a complex disease, oxidative stress is a crucial contributor to its development, especially in view of the higher oxygen demand of the retina. Paraoxonase 2 (PON2) is a ubiquitously and constitutively expressed antioxidant protein that is found intracellularly associated with mitochondrial membranes and modulates mitochondrial ROS production and function. The contribution of PON2 to AMD has not been studied to date. In this study, we examined the role of PON2 in AMD utilizing both in vitro and in vivo models of AMD with emphasis on mitochondrial function. Mitochondrial localization and regulation of PON2 following oxidative stress were determined in human primary cultured retinal pigment epithelium (hRPE) cells. PON2 was knocked down in RPE cells using siRNA and mitochondrial bioenergetics were measured. To investigate the function of PON2 in the retina, WT and PON2-deficient mice were administered NaIO3 (20 mg/kg) intravenously; fundus imaging, optical coherence tomography (OCT), electroretinography (ERG) were conducted; and retinal thickness and cell death were measured and quantified. In hRPE, mitochondrial localization of PON2 increased markedly with stress. Moreover, a time-dependent regulation of PON2 was observed following oxidative stress, with an initial significant increase in expression followed by a significant decrease. Mitochondrial bioenergetic parameters (basal respiration, ATP production, spare respiratory capacity, and maximal respiration) showed a significant decrease with oxidative stress, which was further exacerbated in the absence of PON2. NaIO3 treatment caused significant retinal degeneration, retinal thinning, and reduced rod and cone function in PON2-deficient mice when compared to WT mice. The apoptotic cells and active caspase 3 significantly increased in PON2-deficient mice treated with NaIO3, when compared to WT mice. Our investigation demonstrates that deficiency of PON2 results in RPE mitochondrial dysfunction and a decline in retinal function. These findings imply that PON2 may have a beneficial role in retinal pathophysiology and is worthy of further investigation.

Dasatinib Plus Quercetin Alleviates Choroid Neovascularization by Reducing the Cellular Senescence Burden in the RPE-Choroid.

Wet AMD (wAMD) is associated with cellular senescence. However, senescent cell-targeted therapies for wAMD have rarely been comprehensively studied. This study aimed to explore the therapeutic effects of senolytic agents on choroidal neovascularization (CNV). RNA sequencing datasets were obtained from the Gene Expression Omnibus database and used to explore the association between senescence and wAMD. We explored the effects of senescent adult RPE cell line-19 cells on the proliferation, migration, invasion, and tube formation of human umbilical vein endothelial cells. A laser-induced CNV animal model was used to study wAMD. We studied a senescent cell elimination therapy for CNV progression using two types of senolytics and a transgenic method. Cells in the retinal pigment epithelium-choroid of the CNV model were enriched in senescence, inflammation, and angiogenesis gene sets. AP20187 was used to specifically eliminate senescent cells and proven to alleviate CNV progression in INK-ATTAC transgenic mice. Senescent adult RPE cell line-1 cells produced elevated levels of senescence-associated secretory phenotypes, including VEGFs; they also demonstrated increased proliferation, migration, invasion, and tube formation in human umbilical vein endothelial cells. The number of senescent cells increased in the laser-induced CNV rat model, and intravitreal injections of dasatinib with quercetin reduced the expression of p16 in CNV and alleviated neovascularization. Senescent RPE cells can accelerate pathological neovascularization; thus, senescent cell-targeting therapy has great clinical potential for wAMD.

Histology of Type 3 Macular Neovascularization and Microvascular Anomalies in Treated Age-Related Macular Degeneration: A Case Study.

To investigate intraretinal neovascularization and microvascular anomalies by correlating invivo multimodal imaging with corresponding exvivo histology in a single patient. A case study comprising clinical imaging from a community-based practice, and histologic analysis at a university-based research laboratory (clinicopathologic correlation). A White woman in her 90s treated with numerous intravitreal anti-VEGF injections for bilateral type 3 macular neovascularization (MNV) secondary to age-related macular degeneration (AMD). Clinical imaging comprised serial infrared reflectance, eye-tracked spectral-domain OCT, OCT angiography, and fluorescein angiography. Eye tracking, applied to the 2 preserved donor eyes, enabled the correlation of clinical imaging signatures with high-resolution histology and transmission electron microscopy. Histologic/ultrastructural descriptions and diameters of vessels seen in clinical imaging. Six vascular lesions were histologically confirmed (type 3 MNV, n= 3; deep retinal age-related microvascular anomalies [DRAMAs], n= 3). Pyramidal (n= 2) or tangled (n= 1) morphologies of type 3 MNV originated at the deep capillary plexus (DCP) and extended posteriorly to approach without penetrating persistent basal laminar deposit. They did not enter the subretinal pigment epithelium (RPE)-basal laminar space or cross the Bruch membrane. Choroidal contributions were not found. The neovascular complexes included pericytes and nonfenestrated endothelial cells, within a collagenous sheath covered by dysmorphic RPE cells. Deep retinal age-related microvascular anomaly lesions extended posteriorly from the DCP into the Henle fiber and the outer nuclear layers without evidence of atrophy, exudation, or anti-VEGF responsiveness. Two DRAMAs lacked collagenous sheaths. External and internal diameters of type 3 MNV and DRAMA vessels were larger than comparison vessels in the index eyes and in aged normal and intermediate AMD eyes. Type 3 MNV vessels reflect specializations of source capillaries and persist during anti-VEGF therapy. The collagenous sheath of type 3 MNV lesions may provide structural stabilization. If so, vascular characteristics may be useful in disease monitoring in addition to fluid and flow signal detection. Further investigation with longitudinal imaging before exudation onset will help determine if DRAMAs are part of the type 3 MNV progression sequence. Proprietary or commercial disclosure may be found after the references.

Parapapillary drusen of the retinal pigment epithelium.

To describe the occurrence, morphology and associations of parapapillary drusen of the retinal pigment epithelium (RPE-drusen). Using light microscopy, we histomorphometrically examined enucleated human eyes. The study included 83 eyes (axial length: 25.9 ± 3.2 mm; range: 20.0-35.0 mm). Eyes with parapapillary RPE-drusen (n = 29 (35%) eyes) as compared to those without drusen had a significantly shorter axial length (24.0 ± 1.8 mm vs 27.0 ± 3.3 mm; p < 0.001), higher prevalence (27/29 vs 12/54; p < 0.001) and longer width (213 ± 125 μm vs 96 ± 282 μm; p < 0.0001) of parapapillary alpha zone, and thicker BM in parapapillary beta zone (8.4 ± 2.7 μm vs 3.9 ± 2.0 μm; p < 0.001) and alpha zone (6.6 ± 3.9 μm vs 4.4 ± 1.5 μm; p = 0.02). Prevalence of parapapillary RPE-drusen was 27 (69%) out of 39 eyes with alpha zone. Beneath the RPE-drusen and in total alpha zone, choriocapillaris was open, while it was closed in the central part of parapapillary beta zone. BM thickness was thicker (p = 0.001) in alpha zone than beta zone, where it was thicker (p < 0.001) than in the region outside of alpha/beta zone. BM thickness outside of alpha/beta zone was not correlated with prevalence of parapapillary RPE-drusen (p = 0.47) or axial length (p = 0.31). RPE cell density was higher in alpha zone than in the region adjacent to alpha zone (22.7 ± 7.3 cells/240 μm vs 18.3 ± 4.1 cells/240 μm; p < 0.001). In the parapapillary RPE-drusen, RPE cells were connected with a PAS-positive basal membrane. Parapapillary RPE-drusen as fibrous pseudo-metaplasia of the RPE were associated with shorter axial length, higher prevalence and larger size of alpha zone, and thicker BM in alpha zone and beta zone. The RPE-drusen may be helpful to differentiate glaucomatous parapapillary beta zone from myopic beta zone.

A Cost-Effectiveness Analysis of Pegcetacoplan for the Treatment of Geographic Atrophy

To evaluate the cost-effectiveness of the treatment of geography atrophy (GA) with intravitreal pegcetacoplan and to identify utility measurement surrogates. cost analysis based on data from a published study SUBJECTS: none; based on data from published sham control compared to 2 treatment groups in the index study METHODS: Costs were based on 2022 Medicare reimbursement data. Specific outcomes were extrapolated from the DERBY and OAKS trials. Assumptions were made for the lifetime analysis based on a theoretical logistic growth model of the atrophy. cost, cost-utility, cost per QALY, cost per area of GA (in US$) RESULTS: The cost to treat GA in every month (EM) and every other month (EOM) treatment groups over the 2 years as reported was $70,000 and $34,600, respectively. The cost per area of delaying GA for 2 years in all patients was $87,300/mm2 (EM) and $49,200/mm2 (EOM), and in initially extrafoveal patients, $53,900/mm2 (EM) and $32,100/mm2 (EOM). The cost per day of delaying GA for 2 years was $295 (EM) and $170 (EOM); the marginal cost (EM vs EOM) per RPE cell saved was $30. The modeled lifetime costs were $350,000 (EM) and $172,000 (EOM), or $309,000/mm2 (EM) and $180,000 (EOM) /mm2 . The modeled time to 95% atrophy at 13 years was delayed by 2.5 years (EM) and 2.1 years (EOM). The costs/QALY gained based on modeled visual loss with 95% atrophy were $706,000 (EM) and $397,000 (EOM). Treatment of GA with intravitreal pegcetacoplan EOM was more cost effective than EM. Treatment of extrafoveal lesions yielded greater utility compared to the treatment of the entire group. As atrophy progression approaches an upper limit, the marginal cost/benefit ratios increase.

Impaired Lysosome Reformation in Chloroquine-Treated Retinal Pigment Epithelial Cells.

To model the in vivo effects of chloroquine on the retinal pigment epithelium in experimentally tractable cell culture systems and determine the effects of mild chloroquine treatment on lysosome function and turnover. Effects of low-dose chloroquine treatment on lysosomal function and accessibility to newly endocytosed cargo were investigated in primary and embryonic stem cell-derived RPE cells and ARPE19 cells using fluorescence and electron microscopy of fluorescent and gold-labeled probes. Lysosomal protein expression and accumulation were measured by quantitative PCR and Western blotting. Initial chloroquine-induced lysosome neutralization was followed by partial recovery, lysosomal expansion, and accumulation of undegraded endocytic, phagocytic, and autophagic cargo and inhibition of cathepsin D processing. Accumulation of enlarged lysosomes was accompanied by a gradual loss of accessibility of these structures to the endocytic pathway, implying impaired lysosome reformation. Chloroquine-induced accumulation of pro-cathepsin D, as well as the lysosomal membrane protein, LAMP1, was reproduced by treatment with protease inhibitors and preceded changes in lysosomal gene expression. Low-dose chloroquine treatment inhibits lysosome reformation, causing a gradual depletion of lysosomes able to interact with cargo-carrying vacuoles and degrade their content. The resulting accumulation of newly synthesized pro-cathepsin D and LAMP1 reflects inhibition of normal turnover of lysosomal constituents and possibly lysosomes themselves. A better understanding of the mechanisms underlying lysosome reformation may reveal new targets for the treatment of chloroquine-induced retinopathy.

LncRNA NORAD defects deteriorate the formation of age-related macular degeneration.

Long noncoding RNAs (lncRNAs) play important roles in the development of age-related macular degeneration (AMD). However, the effect of long non-coding RNA activated by DNA damage (NORAD) on AMD remains unknown. This study aimed to investigate the effect of NORAD on RPE cell senescence and degeneration. Irradiated adult retinal pigment epithelial cell line-19 (ARPE-19) and sodium iodate-treated mice were used as in vitro and in vivo AMD models. Results showed that irradiation-induced AMD characteristics of ARPE-19 and NORAD-knockdown aggravated cell cycle arrest in the G2/M phase, cell apoptosis and cell senescence along with the increased expression of phosphorylated P53 (p-P53) and P21. AMD factors C3, ICAM-1, APP, APOE, and VEGF-A were also increased by NORAD-knockdown. Moreover, NORAD-knockdown increased irradiation-induced reduction of mitochondrial homeostasis factors, (i.e., TFAM and POLG) and mitochondrial respiratory chain complex genes (i.e., ND1 and ND5) along with mitochondrial reactive oxygen species (ROS). We also identified a strong interaction of NORAD and PGC-1α and sirtuin 1 (SIRT1) in ARPE-19; that is, NORAD knockdown increases the acetylation of PGC-1α. In NORAD knockout mice, NORAD-knockout accelerated the sodium iodate-reduced retinal thickness reduction, function impairment and loss of retinal pigment in the fundus. Therefore, NORAD-knockdown accelerates retinal cell senescence, apoptosis, and AMD markers via PGC-1α acetylation, mitochondrial ROS, and the p-P53-P21signaling pathway, in which NORAD-mediated effect on PGC-1α acetylation might occur through the direct interaction with PGC-1α and SIRT1.

The role of mitochondrial genes on nuclear gene expression in neovascular age related macular degeneration: analysis of nuclear VEGF gene expression after ranibizumab treatment in cytoplasmic hybrid retinal pigment epithelial cell lines correlated with clinical evolution

PurposeThe present study tests the hypothesis that mitochondrial genes have retrograde signaling capacity that influences the expression of nuclear genes related to angiogenesis pathways. Cytoplasmic hybrid (cybrid) in vitro cell lines with patient specific mitochondria inserted into an immortalized retinal pigment epithelial cell line (ARPE-19) were used to test this hypothesis. This type of analysis can provide important information to identify the optimal regimen of anti-VEGF treatment, personalizing age-related macular degeneration (AMD) therapies.MethodsMitochondria deficient ARPE-19 cells (Rho0) were fused with AMD donor’s platelets to create individual cybrid cell lines containing mitochondria from patients with phenotypic AMD disease and nuclear DNA from the immortalized RPE cell line. The cybrids were treated with Ranibizumab (Lucentis, Genentech, San Francisco, CA), at 4 different concentrations for 24 h, and subsequently the levels of reactive oxygen species (ROS), gene expression for VEGF-A, hypoxia-inducible factor 1-alpha (HIF1-a) and manganese superoxide dismutase (SOD2) were measured. The clinical evolution of the two AMD-donors were correlated with the molecular findings found in their ‘personalized’ cybrids.ResultsCybrids from Patient-01 showed down-regulation of gene expression of VEGF-A and HIF-1a at both 1X and 4X Ranibizumab concentrations. Patient-01 AMD cybrid cultures had an increase in the ROS levels at 1X (P = 0.0317), no changes at 2X (P = 0.8350) and a decrease at 4X (P = 0.0015) and 10X (P = 0.0011) of Ranibizumab. Clinically, Patient-01 responded to anti-VEGF therapy but eventually developed geographic atrophy. Patient-02 cybrids demonstrated up-regulation of gene expression of VEGF-A and HIF-1a at Ranibizumab 1X and 4X concentrations. There was decreased ROS levels with Ranibizumab 1X (P = 0.1606), 2X (P = 0.0388), 4X (P = 0.0010) and 10X (P = < 0.0001). Clinically, Patient-02 presented with a neovascular lesion associated with a prominent production of intraretinal fluid in clinical follow-up requiring regular and repeated intravitreal injections of Ranibizumab with recurrent subretinal fluid.ConclusionsOur cybrid model has the potential to help personalize the treatment regimen with anti-VEGF drugs in patients with neovascular AMD. Further investigation is needed to better understand the role that the mitochondria play in the cellular response to anti-VEGF drugs. Future studies that focus on this model have the potential to help personalize anti-VEGF treatment.

Semi-automated quantification of vitreal hyperreflective foci in SD-OCT and their relevance in patients with peripheral retinal breaks

BackgroundRetinal breaks (RB) are emergencies that require treatment to prevent progression of rhegmatogenous retinal detachment. Vitreal hyperreflective foci (VHF) representing migration of RPE cell clusters or interphotoreceptor matrix from the RB are potential biomarkers. The aim of this study is to investigate VHF in RB-patients using SD-OCT.MethodsThe retrospective cross-sectional study included RB patients from our Department of Ophthalmology, HSK Wiesbaden who underwent macular SD-OCT (SPECTRALIS®, Heidelberg Engineering, Germany) on both eyes. VHF, defined and quantified as foci that differ markedly in size and reflectivity from the background speckle pattern, were assessed for presence and frequency. The RB-affected eyes were the study group (G1), the partner eyes the control group (G2).Results160 consecutive patients with RB were included. Age was 60 ± 10.2 years (52% female). 89.4% of G1 and 87.5% of G2 were phakic (p = 0.73). 94.4% (n = 151) were symptomatic. Symptom duration was 8.0 ± 10.1 days in G1, 94.4% (n = 151) showed VHF versus 5.6% (p < 0.0001) in G2, of which 75% (n = 6) showed asymptomatic lattice degenerations. Detectable VHF showed a strong association of OR = 320 (95% CI, 110–788, p < 0.0001)) with respect to symptomatic RB. Sensitivity and specificity were 94.7% and 94.7%, respectively.ConclusionsMost eyes with symptomatic RB show vitreal VHF in SD-OCT. Detected VHF are strongly associated with RB, and our semi-automated greyscale reflectivity analysis indicates that VHF likely originate from photoreceptor complexes torn out of the RB area that migrate into the vitreous cavity. The presence of VHF may indicate RB and should lead to a thorough fundus examination in both symptomatic and asymptomatic cases.

Aberrant Akt2 signaling in the RPE may contribute to retinal fibrosis process in diabetic retinopathy

Diabetic Retinopathy (DR) is a complication of diabetes that causes blindness in adults. Retinal fibrosis is closely associated with developing proliferative diabetic retinopathy (PDR). Clinical studies have shown that fibrotic membranes exhibit uncontrolled growth in PDR and contribute to retinal detachment from RPE cells, ultimately leading to vision loss. While anti-VEGF agents and invasive laser treatments are the primary treatments for PDR, retinal fibrosis has received minimal attention as a potential target for therapeutic intervention. Therefore, to investigate the potential role of Akt2 in the diabetes-induced retinal fibrosis process, we generated RPE-specific Akt2 conditional knockout (cKO) mice and induced diabetes in these mice and Akt2fl/fl control mice by intraperitoneal injection of streptozotocin. After an 8-month duration of diabetes (10 months of age), the mice were euthanized and expression of tight junction proteins, epithelial–mesenchymal transition (EMT), and fibrosis markers were examined in the RPE. Diabetes induction in the floxed control mice decreased levels of the RPE tight junction protein ZO-1 and adherens junction proteins occludin and E-cadherin; these decreases were rescued in Akt2 cKO diabetic mice. Loss of Akt2 also inhibited diabetes-induced elevation of RNA and protein levels of the EMT markers Snail/Slug and Twist1 in the RPE as compared to Akt2fl/fl diabetic mice. We also found that in Akt2 cKO mice diabetes-induced increase of fibrosis markers, including collagen IV, Connective tissue growth factor (CTGF), fibronectin, and alpha-SMA was attenuated. Furthermore, we observed that high glucose-induced alterations in EMT and fibrosis markers in wild-type (WT) RPE explants were rescued in the presence of PI3K and ERK inhibitors, indicating diabetes-induced retinal fibrosis may be mediated via the PI3K/Akt2/ERK signaling, which could provide a novel target for DR therapy.

Retinal pigment epithelial cells can be cultured on fluocinolone acetonide treated nanofibrous scaffold

Engineered tissue currently lacks requisite capacity to sustain cell viability and functionality. Here we demonstrate that human RPE cell lines (ARPE-19) can be cultured on ultrathin suspended electrospun nanofibre scaffolds (ENS) composed of hydrophobic polymer polyacrylonitrile (PAN) and a water-soluble aliphatic diamine, without (untreated) or with (treated) fluocinolone acetonide (FA). Cells survived and retained their characteristic morphology for up to 150 days with FA-treated ENS and manifested a morphological epithelial phenotype with expression of biomarkers critical for maintaining retinal physiological characteristics. This novel technique for producing culture substrates provides suitable hydrophilicity and a protective environment for prolonged RPE culture and has immense potential for subretinal transplantation. The findings indicate that FA-treated ENS is an excellent matrix for retaining the differentiated and epithelial phenotype.

Antioxidative Role of Heterophagy, Autophagy, and Mitophagy in the Retina and Their Association with the Age-Related Macular Degeneration (AMD) Etiopathogenesis.

Age-related macular degeneration (AMD), an oxidative stress-linked neurodegenerative disease, leads to irreversible damage of the central retina and severe visual impairment. Advanced age and the long-standing influence of oxidative stress and oxidative cellular damage play crucial roles in AMD etiopathogenesis. Many authors emphasize the role of heterophagy, autophagy, and mitophagy in maintaining homeostasis in the retina. Relevantly modifying the activity of both macroautophagy and mitophagy pathways represents one of the new therapeutic strategies in AMD. Our review provides an overview of the antioxidative roles of heterophagy, autophagy, and mitophagy and presents associations between dysregulations of these molecular mechanisms and AMD etiopathogenesis. The authors performed an extensive analysis of the literature, employing PubMed and Google Scholar, complying with the 2013-2023 period, and using the following keywords: age-related macular degeneration, RPE cells, reactive oxygen species, oxidative stress, heterophagy, autophagy, and mitophagy. Heterophagy, autophagy, and mitophagy play antioxidative roles in the retina; however, they become sluggish and dysregulated with age and contribute to AMD development and progression. In the retina, antioxidative roles also play in RPE cells, NFE2L2 and PGC-1α proteins, NFE2L2/PGC-1α/ARE signaling cascade, Nrf2 factor, p62/SQSTM1/Keap1-Nrf2/ARE pathway, circulating miRNAs, and Yttrium oxide nanoparticles performed experimentally in animal studies.

The alleviation effects of n-butylidenephthalide on apoptosis, senescence, and tight junction impairment of retinal pigment epithelium by activating Nrf-2/HO-1 signaling pathway in early diabetic retinopathy

AimsDiabetic retinopathy (DR) is a common complication of diabetes that causes visual impairment and blindness in adults. This study aimed to explore the protective effects of n-Butylidenephthalide (BP) on hyperglycemia-treated RPE in vitro and in vivo. Main methodsC57BL/6 mice were injected with STZ by intraperitoneal to induce early DR and orally administrated with 2 mg/kg BP every day for twelve weeks. Body weight and blood glucose were measured once a week. The level of retina damage was determined by TUNEL assay and H&E staining. The outer blood-retinal barrier integrity and RPE65 expression of retina were evaluated by immunofluorescence. In in vitro study, ARPE-19 cells were long-term cultured with high glucose and BP for 8 days and studied for cell survival, tight junction integrity, RPE65 expression, angiogenic factors, mitochondria membrane potential (MMP), and ROS by MTT assay, Western blot, β-galactosidase staining, immunofluorescence, JC-1, or DCFH-DA. Key findingsThe results indicate that BP suppressed the hyperglycemic effect and maintained retina anatomy normalization, as well as protected RPE cell survival, tight junction integrity, and RPE65 expression in vitro and in vivo. In vitro results showed BP stimulated high glucose-treated ARPE-19 cell proliferation and suppressed senescence via ERK pathway. Numerous ROS production and MMP imbalance were prevented by BP through Nrf-2/HO-1 pathway. BP inhibited high glucose-induced RPE neovascularization by VEGF dysregulation. SignificanceBP significantly protected tight junction integrity and RPE cellular physiology through ERK/Nrf-2/HO-1 pathway to prevent DR progression. Thus, BP has great potential to be developed therapeutic agents or adjuvants for DR.

Myopic Versus Glaucomatous Parapapillary Beta Zone in Myopic Eyes Versus Eyes With Secondary Angle-Closure Glaucoma.

To search for histologic differences in the beta zone between myopic eyes versus eyes with secondary angle-closure glaucoma. The histomorphometric study consisted of human eyes enucleated due to uveal melanomas or secondary angle-closure glaucoma. The study included 100 eyes (age: 62.1 ± 15.1 years; axial length: 25.6 ± 3.1 mm; range: 20.0-35.0 mm). In non-highly myopic glaucomatous eyes compared with non-highly myopic nonglaucomatous eyes, the parapapillary alpha zone was longer (223 ± 168 µm vs. 125 ± 128 µm; P = 0.03), beta zone prevalence (15/20 vs. 6/41; P < 0.001) and length (277 ± 245 µm vs. 44 ± 150 µm; P = 0.001) were higher, and RPE cell density in the alpha zone and alpha zone border was lower (all P < 0.05). In highly myopic nonglaucomatous eyes compared with non-highly myopic glaucomatous eyes, parapapillary RPE drusen prevalence (2/19 vs. 10/10; P = 0.01) and alpha zone prevalence (2/19 vs. 16/20; P < 0.001) and length (23 ± 68 µm vs. 223 ± 168 µm; P < 0.001) were lower. In non-highly myopic glaucomatous eyes, Bruch's membrane (BM) thickness decreased (P < 0.001) from the beta zone (6.0 ± 3.1 µm) to the alpha zone (5.1 ± 4.3 µm) and peripheral to it (3.0 ± 0.9 µm). In highly myopic nonglaucomatous eyes, BM thickness did not differ (P > 0.10) between all three regions. In the total study population, RPE cell density in the alpha zone (24.5 ± 9.3 cells/240 µm) was higher than at the alpha zone border (19.2 ± 4.8 cells/240 µm; P < 0.001) or peripheral to it (19.0 ± 3.6 cells/240 µm; P < 0.001). The glaucomatous beta zone in eyes with chronic angle-closure glaucoma (with the alpha zone, parapapillary RPE drusen, thickened BM, and higher RPE cell count in the adjacent alpha zone) differs histologically from the myopic beta zone (characterized by the absence of the alpha zone and parapapillary RPE drusen, unremarkable BM thickness, and unremarkable parapapillary RPE). The differences suggest different etiologies of the glaucomatous versus myopic beta zone.