Search Results (27)

This study aimed to assess outer retinal layer (ORL), retinal pigment epithelium (RPE), choroid (Ch) and choriocapillaris (CC) modifications in adolescents with long-lasting (>10 years) type 1 diabetes (T1D) without (noDR) or with diabetic retinopathy (DR). ORL and RPE thickness were measured at optical coherence tomography (OCT) macular scans. Vascular parameters of Ch and CC were quantified after elaboration of macular OCT-angiography (OCTA) images. Insulin dose and auxological and metabolic parameters were correlated with OCT and OCTA findings in patients. ORL thickness was higher in DR eyes than in noDR and healthy controls (HC), and RPE thickness was higher in noDR and DR eyes than in HC, with statistical significance for some sectors in noDR versus HC. No OCTA parameters of CC and Ch differed among groups, and no significant correlation was observed with auxological and metabolic parameters. In conclusion, ORL and RPE were both increased in adolescents with long-lasting T1D. Such changes were not associated with insulin dose and glycemia control, nor to any choroid or choriocapillaris flow change clinically detectable at OCTA, and they could be potential imaging biomarkers of disease progression. Full article

Pathogenic variants in the RPE65 gene cause the only known form of inherited retinal degenerations (IRDs) that are prone to gene therapy. The current study is aimed at the evaluation of the prevalence of RPE65-associated retinopathy in the Russian Federation, the characterization of known variants in the RPE65 gene, and the establishment of the specificities of the mutation spectrum in Russian patients. Methods: The analysis was carried out on blood samples obtained from 1053 non-related IRDs patients. The analysis, which consisted of 211 genes, was carried out based on the method of massive parallel sequencing (MPS) for all probands. Variant validation, as well as biallelic status verification, were carried out using direct automated Sanger sequencing. The number of copies of RPE65 exons 1–14 was analyzed with quantitative MLPA using an MRC-Holland SALSA MLPA probemix. Results: Out of 1053 non-related patients, a molecular genetic diagnosis of IRDs has been confirmed in 474 cases, including 25 (5.3%) patients with RPE65-associated retinopathy. We detected 26 variants in the RPE65 gene, nine of which have not been previously described in the literature. The most common mutations in the Russian population were c.304G>T/p.(Glu102*), c.370C>T/p.(Arg124*), and c.272G>A/p.(Arg91Gln), which comprised 41.8% of all affected chromosomes. Conclusions: The current study shows that pathogenic variants in the RPE65 gene contribute significantly to the pathogenesis of IRDs and comprise 5.3% of all patients with a confirmed molecular genetic diagnosis. This study allowed for the formation of a cohort for target therapy of the disorder; such therapy has already been carried out for some patients. Full article

Dysfunction of the retinal pigment epithelium (RPE) is associated with several diseases characterized by retinal degeneration, such as diabetic retinopathy (DR). However, it has recently been proposed that outer retinal neurons also participate in the damage triggering. Therefore, we have evaluated the possible crosstalk between RPE and photoreceptors in priming and maintaining oxidative damage of the RPE. For this purpose, we used ARPE-19 cells as a model of human RPE, grown in normal (NG, 5.6 mM) or high glucose (HG, 25 mM) and unoxidized (UOx) or oxidized (Ox) mammalian retinal rod outer segments (OSs). ARPE-19 cells were efficient at phagocytizing rod OSs in both NG and HG settings. However, in HG, ARPE-19 cells treated with Ox-rod OSs accumulated MDA and lipofuscins and displayed altered LC3, GRP78, and caspase 8 expression compared to untreated and UOx-rod-OS-treated cells. Data suggest that early oxidative damage may originate from the photoreceptors and subsequently extend to the RPE, providing a new perspective to the idea that retinal degeneration depends solely on a redox alteration of the RPE. Full article

The method of quantitative fundus autofluorescence (qAF) can be used to assess the levels of bisretinoids in retinal pigment epithelium (RPE) cells so as to aid the interpretation and management of a variety of retinal conditions. In this review, we focused on seven retinal diseases to highlight the possible pathways to increased fundus autofluorescence. ABCA4- and RDH12-associated diseases benefit from known mechanisms whereby gene malfunctioning leads to elevated bisretinoid levels in RPE cells. On the other hand, peripherin2/RDS-associated disease (PRPH2/RDS), retinitis pigmentosa (RP), central serous chorioretinopathy (CSC), acute zonal occult outer retinopathy (AZOOR), and ceramide kinase like (CERKL)-associated retinal degeneration all express abnormally high fundus autofluorescence levels without a demonstrated pathophysiological pathway for bisretinoid elevation. We suggest that, while a known link from gene mutation to increased production of bisretinoids (as in ABCA4- and RDH12-associated diseases) causes primary elevation in fundus autofluorescence, a secondary autofluorescence elevation also exists, where an impairment and degeneration of photoreceptor cells by various causes leads to an increase in bisretinoid levels in RPE cells. Full article

Our purpose is to describe blue-light fundus autofluorescence (BAF) features of inflammatory diseases of the outer retina characterised by photoreceptor damage. BAF from patients diagnosed with secondary and primary inflammatory photoreceptor damage were retrospectively analyzed and compared to other imaging modalities including fluorescein angiography (FA), indocyanine green angiography (ICGA), and spectral domain optical coherence tomography (SD-OCT). Multiple evanescent white dot syndrome (MEWDS), idiopathic multifocal choroiditis (MFC), acute posterior multifocal placoid pigment epitheliopathy (APMPPE), serpiginous choroiditis (SC), and acute syphilitic posterior placoid chorioretinitis (ASPPC), all cases corresponding to secondary photoreceptor diseases caused by inflammatory choriocapillaris nonperfusion, were included and compared to primary photoreceptor disease entities, including acute zonal occult outer retinopathy (AZOOR) and cancer-associated retinopathy (CAR). Both groups showed increased BAFs of variable intensity. In severe cases of APMPPE and ASPPC, BAF also showed hypoautofluorescent areas. In group 1 (secondary diseases) BAF hyperautofluorescent areas were associated with colocalized ICGA hypofluorescent areas, indicating choriocapillaris nonperfusion; whereas in group 2 (primary diseases), no ICGA signs were detected. The associated colocalized areas of hypofluorescence on ICGA in the first group, which were absent in the second group, were crucial to allow the differentiation between primary (photoreceptoritis) and secondary (choriocapillaritis) photoreceptor diseases. BAF patterns in inflammatory diseases of the outer retina can give relevant information on the photoreceptor and RPE involvement, with ICGA being crucial to detect concurring choriocapillaris damage and differentiating the two pathologies. Full article

Diabetic retinopathy (DR) is the leading cause of vision loss and a major complication of diabetes. Hyperglycemia-induced accumulation of reactive oxygen species (ROS) is an important risk factor for DR. β-asarone, a major component of volatile oil extracted from Acori graminei Rhizoma, exerts antioxidant effects; however, its efficacy in DR remains unknown. In this study, we investigated whether β-asarone inhibits high-glucose (HG)-induced oxidative damage in human retinal pigment epithelial (RPE) ARPE-19 cells. We found that β-asarone significantly alleviated cytotoxicity, apoptosis, and DNA damage in HG-treated ARPE-19 cells via scavenging of ROS generation. β-Asarone also significantly attenuated the excessive accumulation of lactate dehydrogenase and mitochondrial ROS by increasing the manganese superoxide dismutase and glutathione activities. HG conditions markedly increased the release of interleukin (IL)-1β and IL-18 and upregulated their protein expression and activation of the nuclear factor-kappa B (NF-κB) signaling pathway, whereas β-asarone reversed these effects. Moreover, expression levels of the NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome multiprotein complex molecules, including thioredoxin-interacting protein, NLRP3, apoptosis-associated speck-like protein containing a caspase-recruitment domain, and cysteinyl aspartate-specific proteinase-1, were increased in ARPE-19 cells under HG conditions. However, their expression levels remained similar to those in the control group in the presence of β-asarone. Therefore, β-asarone protects RPE cells from HG-induced injury by blocking ROS generation and NF-κB/NLRP3 inflammasome activation, indicating its potential as a therapeutic agent for DR treatment. Full article

Mutations in the RCBTB1 gene cause inherited retinal disease; however, the pathogenic mechanisms associated with RCBTB1 deficiency remain poorly understood. Here, we investigated the effect of RCBTB1 deficiency on mitochondria and oxidative stress responses in induced pluripotent stem cell (iPSC)-derived retinal pigment epithelial (RPE) cells from control subjects and a patient with RCBTB1-associated retinopathy. Oxidative stress was induced with tert-butyl hydroperoxide (tBHP). RPE cells were characterized by immunostaining, transmission electron microscopy (TEM), CellROX assay, MitoTracker assay, quantitative PCR and immunoprecipitation assay. Patient-derived RPE cells displayed abnormal mitochondrial ultrastructure and reduced MitoTracker fluorescence compared with controls. Patient RPE cells displayed increased levels of reactive oxygen species (ROS) and were more sensitive to tBHP-induced ROS generation than control RPE. Control RPE upregulated RCBTB1 and NFE2L2 expression in response to tBHP treatment; however, this response was highly attenuated in patient RPE. RCBTB1 was co-immunoprecipitated from control RPE protein lysates by antibodies for either UBE2E3 or CUL3. Together, these results demonstrate that RCBTB1 deficiency in patient-derived RPE cells is associated with mitochondrial damage, increased oxidative stress and an attenuated oxidative stress response. Full article

Diabetic retinopathy is a form of diabetic microangiopathy, and vascular hyperpermeability in the macula leads to retinal thickening and concomitant reduction of visual acuity in diabetic macular edema (DME). In this review, we discuss multimodal fundus imaging, comparing the pathogenesis and interventions. Clinicians diagnose DME using two major criteria, clinically significant macular edema by fundus examination and center-involving diabetic macular edema using optical coherence tomography (OCT), to determine the appropriate treatment. In addition to fundus photography, fluorescein angiography (FA) is a classical modality to evaluate morphological and functional changes in retinal capillaries, e.g., microaneurysms, capillary nonperfusion, and fluorescein leakage. Recently, optical coherence tomography angiography (OCTA) has allowed us to evaluate the three-dimensional structure of the retinal vasculature and newly demonstrated that lamellar capillary nonperfusion in the deep layer is associated with retinal edema. The clinical application of OCT has accelerated our understanding of various neuronal damages in DME. Retinal thickness measured by OCT enables us to quantitatively assess therapeutic effects. Sectional OCT images depict the deformation of neural tissues, e.g., cystoid macular edema, serous retinal detachment, and sponge-like retinal swelling. The disorganization of retinal inner layers (DRIL) and foveal photoreceptor damage, biomarkers of neurodegeneration, are associated with visual impairment. Fundus autofluorescence derives from the retinal pigment epithelium (RPE) and its qualitative and quantitative changes suggest that the RPE damage contributes to the neuronal changes in DME. These clinical findings on multimodal imaging help to elucidate the pathology in the neurovascular units and lead to the next generation of clinical and translational research in DME. Full article

Epithelial-mesenchymal transition (EMT), which is well known for its role in embryonic development, malignant transformation, and tumor progression, has also been implicated in a variety of retinal diseases, including proliferative vitreoretinopathy (PVR), age-related macular degeneration (AMD), and diabetic retinopathy. EMT of the retinal pigment epithelium (RPE), although important in the pathogenesis of these retinal conditions, is not well understood at the molecular level. We and others have shown that a variety of molecules, including the co-treatment of human stem cell-derived RPE monolayer cultures with transforming growth factor beta (TGF–β) and the inflammatory cytokine tumor necrosis factor alpha (TNF–α), can induce RPE–EMT; however, small molecule inhibitors of RPE–EMT have been less well studied. Here, we demonstrate that BAY651942, a small molecule inhibitor of nuclear factor kapa-B kinase subunit beta (IKKβ) that selectively targets NF-κB signaling, can modulate TGF–β/TNF–α-induced RPE–EMT. Next, we performed RNA-seq studies on BAY651942 treated hRPE monolayers to dissect altered biological pathways and signaling events. Further, we validated the effect of IKKβ inhibition on RPE–EMT-associated factors using a second IKKβ inhibitor, BMS345541, with RPE monolayers derived from an independent stem cell line. Our data highlights the fact that pharmacological inhibition of RPE–EMT restores RPE identity and may provide a promising approach for treating retinal diseases that involve RPE dedifferentiation and EMT. Full article

Mitochondrial open reading frame of the 12S rRNA type-c (MOTS-c) is the most unearthed peptide encoded by mitochondrial DNA (mtDNA). It is an important regulator of the nuclear genome during times of stress because it promotes an adaptive stress response to maintain cellular homeostasis. Identifying MOTS-c specific binding partners may aid in deciphering the complex web of mitochondrial and nuclear-encoded signals. Mitochondrial damage and dysfunction have been linked to aging and the accelerated cell death associated with many types of retinal degenerations. Furthermore, research on MOTS-c ability to revive oxidatively stressed RPE cells has revealed a significant protective role for the molecule. Evidence suggests that senescent cells play a role in the development of age-related retinal disorders. This review examines the links between MOTS-c, mitochondria, and age-related diseases of the retina. Moreover, the untapped potential of MOTS-c as a treatment for glaucoma, diabetic retinopathy, and age-related macular degeneration is reviewed. Full article

Epithelial-mesenchymal transition (EMT) is a dedifferentiation program in which polarized, differentiated epithelial cells lose their cell-cell adhesions and transform into matrix-producing mesenchymal cells. EMT of retinal pigment epithelial (RPE) cells plays a crucial role in many retinal diseases, including age-related macular degeneration, proliferative vitreoretinopathy, and diabetic retinopathy. This dynamic process requires complex metabolic reprogramming to accommodate the demands of this dramatic cellular transformation. Both transforming growth factor-beta 2 (TGFβ2) and tumor necrosis factor-alpha (TNFα) have the capacity to induce EMT in RPE cells; however, little is known about their impact on the RPE metabolome. Untargeted metabolomics using high-resolution mass spectrometry was performed to reveal the metabolomic signatures of cellular and secreted metabolites of primary human fetal RPE cells treated with either TGFβ2 or TNFα for 5 days. A total of 638 metabolites were detected in both samples; 188 were annotated as primary metabolites. Metabolomics profiling showed distinct metabolomic signatures associated with TGFβ2 and TNFα treatment. Enrichment pathway network analysis revealed alterations in the pentose phosphate pathway, galactose metabolism, nucleotide and pyrimidine metabolism, purine metabolism, and arginine and proline metabolism in TNFα-treated cells compared to untreated control cells, whereas TGFβ2 treatment induced perturbations in fatty acid biosynthesis metabolism, the linoleic acid pathway, and the Notch signaling pathway. These results provide a broad metabolic understanding of the bioenergetic rewiring processes governing TGFβ2- and TNFα-dependent induction of EMT. Elucidating the contributions of TGFβ2 and TNFα and their mechanistic differences in promoting EMT of RPE will enable the identification of novel biomarkers for diagnosis, management, and tailored drug development for retinal fibrotic diseases. Full article

Aim of the study: To report optical coherence tomography angiography (OCTA) findings in patients affected by CRB1-associated retinal dystrophies. Method: Patients affected by a genetically confirmed CRB1-associated retinal dystrophy were prospectively enrolled in an observational study, along with age- and sex-matched healthy volunteers as control subjects. All study and control subjects received a complete ophthalmic examination and multimodal retinal imaging, including OCTA. Result: A total of 12 eyes from 6 patients were included in the study. The mean BCVA of patients was 0.42 ± 0.25 logMAR. Two patients showed large central atrophy, with corresponding definite hypo-autofluorescence on fundus autofluorescence (FAF). Another four patients disclosed different degrees of RPE mottling, with uneven FAF. On OCTA, the macular deep capillary plexus and choriocapillaris had a lower vessel density in eyes affected by CRB1-associated retinopathy when compared to healthy controls. On the other hand, vessel density at the peripapillary radial capillary plexus, superficial capillary plexus, and deep capillary plexus was significantly altered with respect to control eyes. Statistical analyses disclosed a negative correlation between the deep capillary plexus and both LogMAR best corrected visual acuity and central retinal thickness. Conclusion: Our study reveals that CRB1-associated retinal dystrophies are characterized by vascular alterations both in the macular and peripapillary region, as assessed by OCTA. Full article

Relationships between retinal disease, diet, and the gut microbiome have started to emerge. In particular, high-fat diets (HFDs) are associated with the prevalence and progression of several retinal diseases, including age-related macular degeneration (AMD) and diabetic retinopathy (DR). These effects are thought to be partly mediated by the gut microbiome, which modulates interactions between diet and host homeostasis. Nevertheless, the effects of HFDs on the retina and adjacent retinal pigment epithelium (RPE) and choroid at the transcriptional level, independent of gut microbiota, are not well-understood. In this study, we performed the high-throughput RNA-sequencing of germ-free (GF) mice to explore the transcriptional changes induced by HFD in the RPE/choroid. After filtering and cleaning the data, 649 differentially expressed genes (DEGs) were identified, with 616 genes transcriptionally upregulated and 33 genes downregulated by HFD compared to a normal diet (ND). Enrichment analysis for gene ontology (GO) using the DEGs was performed to analyze over-represented biological processes in the RPE/choroid of GF-HFD mice relative to GF-ND mice. GO analysis revealed the upregulation of processes related to angiogenesis, immune response, and the inflammatory response. Additionally, molecular functions that were altered involved extracellular matrix (ECM) binding, ECM structural constituents, and heparin binding. This study demonstrates novel data showing that HFDs can alter RPE/choroid tissue transcription in the absence of the gut microbiome. Full article

Emerging evidence shows that peroxisome proliferator-activated receptor delta (PPARδ) plays a pivotal role in cellular aging. However, its function in retinal disease processes such as hyperglycemia-associated diabetic retinopathy is unclear. Here, we demonstrate that PPARδ inhibits premature senescence of retinal pigment epithelial (RPE) cells induced by high glucose (HG) through SIRT1 upregulation. A specific ligand GW501516-activation of PPARδ suppressed premature senescence and production of reactive oxygen species induced by HG in ARPE-19 cells, a spontaneously arising human RPE cell line. These effects were accompanied by the regulation of the premature senescence-associated genes p53, p21, and SMP-30. Furthermore, GW501516-activated PPARδ almost completely abolished the effects of HG treatment on the formation of phosphorylated H2A histone family member X (γ-H2A.X) foci, a molecular marker of aging. These inhibitory effects of GW501516 were significantly reversed in ARPE-19 cells stably expressing small hairpin RNA targeting PPARδ. Notably, GW501516 significantly increased the mRNA and protein levels of SIRT1, indicating that GW501516-activated PPARδ exerted its beneficial effects through SIRT1. In addition, GW501516 restored HG-suppressed SIRT1 expression, corroborating the role of SIRT1 in the anti-senescence function of PPARδ. The effects of PPARδ on HG-induced premature senescence and the expression of the senescence-associated genes p53, p21, and SMP-30 were mimicked by the SIRT1 activator resveratrol, but blocked by the SIRT1 inhibitor sirtinol. Collectively, these results indicate that GW501516-activated PPARδ inhibits HG-triggered premature senescence of RPE cells by modulating SIRT1 signaling. Full article

Retinitis pigmentosa 11 (RP11) is caused by dominant mutations in PRPF31, however a significant proportion of mutation carriers do not develop retinopathy. Here, we investigated the relationship between CNOT3 polymorphism, MSR1 repeat copy number and disease penetrance in RP11 patients and non-penetrant carriers (NPCs). We further characterized PRPF31 and CNOT3 expression in fibroblasts from eight RP11 patients and one NPC from a family carrying the c.1205C>T variant. Retinal organoids (ROs) and retinal pigment epithelium (RPE) were differentiated from induced pluripotent stem cells derived from RP11 patients, an NPC and a control subject. All RP11 patients were homozygous for the 3-copy MSR1 repeat in the PRPF31 promoter, while 3/5 NPCs carried a 4-copy MSR1 repeat. The CNOT3 rs4806718 genotype did not correlate with disease penetrance. PRFP31 expression declined with age in adult cadaveric retina. PRPF31 and CNOT3 expression was reduced in RP11 fibroblasts, RO and RPE compared with controls. Both RP11 and NPC RPE displayed shortened primary cilia compared with controls, however a subpopulation of cells with normal cilia lengths was present in NPC RPE monolayers. Our results indicate that RP11 non-penetrance is associated with the inheritance of a 4-copy MSR1 repeat, but not with CNOT3 polymorphisms. Full article