Metformin Alleviates Inflammation and Induces Mitophagy in Human Retinal Pigment Epithelium Cells Suffering from Mitochondrial Damage.

Mitochondrial malfunction, excessive production of reactive oxygen species (ROS), deficient autophagy/mitophagy, and chronic inflammation are hallmarks of age-related macular degeneration (AMD). Metformin has been shown to activate mitophagy, alleviate inflammation, and lower the odds of developing AMD. Here, we explored the ability of metformin to activate mitophagy and alleviate inflammation in retinal pigment epithelium (RPE) cells. Human ARPE-19 cells were pre-treated with metformin for 1 h prior to exposure to antimycin A (10 µM), which induced mitochondrial damage. Cell viability, ROS production, and inflammatory cytokine production were measured, while autophagy/mitophagy proteins were studied using Western blotting and immunocytochemistry. Metformin pre-treatment reduced the levels of proinflammatory cytokines IL-6 and IL-8 to 42% and 65% compared to ARPE-19 cells exposed to antimycin A alone. Metformin reduced the accumulation of the autophagy substrate SQSTM1/p62 (43.9%) and the levels of LC3 I and II (51.6% and 48.6%, respectively) after antimycin A exposure. Metformin also increased the colocalization of LC3 with TOM20 1.5-fold, suggesting active mitophagy. Antimycin A exposure increased the production of mitochondrial ROS (226%), which was reduced by the metformin pre-treatment (84.5%). Collectively, metformin showed anti-inflammatory and antioxidative potential with mitophagy induction in human RPE cells suffering from mitochondrial damage.

VEGF and ELAVL1/HuR protein levels are increased in dry and wet AMD patients. A new tile in the pathophysiologic mechanisms underlying RPE degeneration?

Age-related macular degeneration (AMD) is a common retinal pathology characterized by degeneration of macula's retinal pigment epithelium (RPE) and photoreceptors, visual impairment, or loss. Compared to wet AMD, dry AMD is more common, but lacks cures; therefore, identification of new potential therapeutic targets and treatments is urgent. Increased oxidative stress and declining antioxidant, detoxifying systems contribute to the pathophysiologic mechanisms underlying AMD. The present work shows that the Embryonic Lethal Abnormal Vision-Like 1/Human antigen R (ELAVL1/HuR) and the Vascular Endothelial Growth Factor (VEGF) protein levels are higher in the RPE of both dry and wet AMD patients compared to healthy subjects. Moreover, increased HuR protein levels are detected in the retina, and especially in the RPE layer, of a dry AMD model, the nuclear factor erythroid 2-related factor 2 (Nrf2) / peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) double knock-out mouse. The crosstalk among Nrf2, HuR and VEGF has been also studied in ARPE-19 cells in basal and stressful conditions related to the AMD context (i.e., oxidative stress, autophagy impairment, Nrf2 deficit), offering new evidence of the mutual influence between Nrf2 and HuR, of the dependence of VEGF expression and secretion by these two factors, and of the increased susceptibility of cells to stressful conditions in Nrf2- or HuR-impaired contexts. Overall, this study shows evidence of the interplay among Nrf2, HuR and VEGF, essential factors for RPE homeostasis, and represents an additional piece in the understanding of the complex pathophysiologic mechanisms underlying AMD.

Autophagy in drusen biogenesis secondary to age-related macular degeneration.

Age-related macular degeneration (AMD) is an emerging cause of blindness in aged people worldwide. One of the key signs of AMD is the degeneration of the retinal pigment epithelium (RPE), which is indispensable for the maintenance of the adjacent photoreceptors. Because of impaired energy metabolism resulting from constant light exposure, hypoxia, and oxidative stress, accumulation of drusen in AMD-affected eyes is observed. Drusen contain damaged cellular proteins, lipoprotein particles, lipids and carbohydrates and they are related to impaired protein clearance, inflammation, and extracellular matrix modification. When autophagy, a major cellular proteostasis pathway, is impaired, the accumulations of intracellular lipofuscin and extracellular drusen are detected. As these aggregates grow over time, they finally cause the disorganisation and destruction of the RPE and photoreceptors leading to visual loss. In this review, the role of autophagy in drusen biogenesis is discussed since impairment in removing cellular waste in RPE cells plays a key role in AMD progression. In the future, means which improve intracellular clearance might be of use in AMD therapy to slow the progression of drusen formation.

The AKT2/SIRT5/TFEB pathway as a potential therapeutic target in non-neovascular AMD.

Non-neovascular or dry age-related macular degeneration (AMD) is a multi-factorial disease with degeneration of the aging retinal-pigmented epithelium (RPE). Lysosomes play a crucial role in RPE health via phagocytosis and autophagy, which are regulated by transcription factor EB/E3 (TFEB/E3). Here, we find that increased AKT2 inhibits PGC-1α to downregulate SIRT5, which we identify as an AKT2 binding partner. Crosstalk between SIRT5 and AKT2 facilitates TFEB-dependent lysosomal function in the RPE. AKT2/SIRT5/TFEB pathway inhibition in the RPE induced lysosome/autophagy signaling abnormalities, disrupted mitochondrial function and induced release of debris contributing to drusen. Accordingly, AKT2 overexpression in the RPE caused a dry AMD-like phenotype in aging Akt2 KI mice, as evident from decline in retinal function. Importantly, we show that induced pluripotent stem cell-derived RPE encoding the major risk variant associated with AMD (complement factor H; CFH Y402H) express increased AKT2, impairing TFEB/TFE3-dependent lysosomal function. Collectively, these findings suggest that targeting the AKT2/SIRT5/TFEB pathway may be an effective therapy to delay the progression of dry AMD.

Ventriculoperitoneal shunt patients and glaucoma: a cohort analysis of the NPH registry.

BACKGROUND: Idiopathic Normal Pressure Hydrocephalus (iNPH) is a chronic condition affecting the elderly. It is characterized by a triad of symptoms and radiological findings. Glaucoma is the leading cause of irreversible blindness worldwide. Earlier studies have proposed that the rate of glaucoma is higher in iNPH patients, and of a possible link between ventriculoperitoneal shunt (VP) treatment and the development of glaucoma. OBJECTIVES: This study aimed to determine the prevalence of glaucoma among iNPH patients and assess the impact of VPs on glaucoma prevalence. METHODS: A cohort study was conducted at Kuopio University Hospital (KUH), including 262 patients with a ventriculoperitoneal shunt. Clinical data were obtained from the Kuopio NPH Registry and medical records. Patients were grouped by iNPH status: iNPH (+) - probable/possible iNPH (n = 192), and iNPH (-) - other causes of hydrocephalus (congenital, secondary, obstructive) (n = 70). We conducted statistical analysis using the Independent Samples T-test, Fisher's exact test, and Pearson Chi-Square. We compared demographics, glaucoma prevalence, brain biopsies positive for Amyloid-ß (Aß) and hyperphosphorylated tau (HPτ) as well as comorbidities for hypertension and diabetes medication. Age stratification assessed glaucoma prevalence in the full cohort. RESULTS: Both iNPH (+) and iNPH (-) groups had comparable demographic and comorbidity profiles. The prevalence of glaucoma in the iNPH (+) group was 11.5% (n = 22) and 11.4% (n = 8) in the iNPH (-) group without a statistically significant difference (p = 1.000). Brain biopsies positive for Amyloid-ß (Aß) and hyperphosphorylated tau (HPτ) were similar. CONCLUSIONS: Neither shunted iNPH patients nor those with a comorbid condition other than iNPH showed a markedly higher prevalence of glaucoma. Instead, both groups exhibited age-related increases in glaucoma prevalence, similar to the trends observed in population-based studies. Our data does not suggest a correlation between VP shunts and an elevated rate of glaucoma.

Exploring the Association Between Visual Field Testing and CERAD Neuropsychological Battery in Idiopathic Normal Pressure Hydrocephalus Patients.

Background: Association between visual field test indices and The Consortium to Establish a Registry for Alzheimer's Disease Neuropsychological Battery (CERAD-NB) is unknown. Idiopathic normal pressure hydrocephalus (iNPH) patients provide a unique set of patient data for analysis. Objective: To assess the reliability of visual field testing using the CERAD-NB in patients with iNPH and to investigate the association between visual field test results and cognitive function. Methods: 62 probable iNPH patients were subjected to comprehensive ophthalmological examination, ophthalmological optical coherence tomography imaging studies, visual field testing, and CERAD-NB. Based on visual field indices, the patients were divided into two groups: unreliable (n = 19) and reliable (n = 43). Independent T-test analysis was performed to examine the relationship between visual field test results and cognitive function. Pearson Chi-square test was used for non-continuous variables. Results: The unreliable group performed worse in CERAD-NB subtests compared to the reliable group. Statistically significant differences were observed in nine out of ten subtests, with only Clock Drawing showing no statistical significance. Pairwise comparison of the groups showed no statistical significance between amyloid-ß (Aß) biopsy, hyperphosphorylated tau biopsy, apolipoprotein E allele or the ophthalmological status of the patient. But there was a statistically significant difference in cerebrospinal fluid Aß42 and age between the groups. Conclusions: Patients with unreliable visual field tests performed worse on CERAD-NB subtests. CERAD-NB subtests do not provide a specific cut-off value to refrain patients from visual field testing. Should patients with unreliable visual field tests be screened for cognitive impairment?

Oxidative Stress, Persistent Inflammation and Blood Coagulation Alterations in Serum Proteome of Patients with Neovascular Age-Related Macular Degeneration.

Neovascular age-related macular degeneration (AMD) is a major cause of irreversible blindness in elderly populations in developed countries. AMD's etiopathology is multifactorial, with strong environmental and genetic components, but the exact molecular pathomechanisms underlying the disease are still unknown. In this study, we analyzed blood serum collected from 74 neovascular AMD patients and 58 healthy controls to identify proteins that may serve as potential biomarkers and expand our knowledge about the etiopathogenesis of the disease. The study revealed 17 differentially expressed proteins-11 up-regulated and 6 down-regulated-in neovascular AMD, which are involved in the biological processes previously linked with the disease-oxidative stress and persistent inflammation, impaired cellular transport, lipid metabolism and blood coagulation. In conclusion, the differences in the expressions of the proteins identified in this study may contribute to our understanding of the mechanisms underlying AMD and possibly serve in future as promising biomarkers.

Rare genetic variation in VE-PTP is associated with central serous chorioretinopathy, venous dysfunction and glaucoma.

Central serous chorioretinopathy (CSC) is a fluid maculopathy whose etiology is not well understood. Abnormal choroidal veins in CSC patients have been shown to have similarities with varicose veins. To identify potential mechanisms, we analyzed genotype data from 1,477 CSC patients and 455,449 controls in FinnGen. We identified an association for a low-frequency (AF=0.5%) missense variant (rs113791087) in the gene encoding vascular endothelial protein tyrosine phosphatase (VE-PTP) (OR=2.85, P=4.5×10-9). This was confirmed in a meta-analysis of 2,452 CSC patients and 865,767 controls from 4 studies (OR=3.06, P=7.4×10-15). Rs113791087 was associated with a 56% higher prevalence of retinal abnormalities (35.3% vs 22.6%, P=8.0×10-4) in 708 UK Biobank participants and, surprisingly, with varicose veins (OR=1.31, P=2.3×10-11) and glaucoma (OR=0.82, P=6.9×10-9). Predicted loss-of-function variants in VEPTP, though rare in number, were associated with CSC in All of Us (OR=17.10, P=0.018). These findings highlight the significance of VE-PTP in diverse ocular and systemic vascular diseases.

Long-term cumulative incidence of clinically diagnosed retinopathy in the Finnish Diabetes Prevention Study.

CONTEXT: Lifestyle intervention reduces the incidence of type 2 diabetes (T2D) in people with impaired glucose tolerance. OBJECTIVE: To find out whether participation in the earlier lifestyle intervention had an effect on the occurrence of clinically diagnosed retinopathy during a median of 22 years of follow-up time. METHODS: The study included 505 individuals from the Finnish Diabetes Prevention Study (DPS) (mean 55, range 40-64 years at the onset of the study) with impaired glucose tolerance who were originally randomized into the intervention (weight loss, healthy diet and physical activity (N=257) and usual care control groups (N=248). The median follow-up was 22 years. Clinical retinopathy diagnoses were obtained from the Finnish national hospital Care Register for Health. Data on glycemic parameters, serum lipids and blood pressure were available from both the intervention (median 4 years) and post-intervention period (until the year 7). RESULTS: No significant difference was found in the cumulative incidence of clinically diagnosed diabetic retinopathy between the original intervention (N=23, 8.9%) and control groups (N=19, 7.7%) during the extended follow-up (Odds ratio: 1.15, 95% confidence interval: 0.61-2.21). A higher cumulative HbA1c was significantly associated with a higher risk of retinopathy (Hazard ratio 1.4; 1.02-1.88, 95% posterior interval, adjusted for group, age and sex). Furthermore, the incidence of retinopathy diagnosis was numerically more common among individuals who had developed diabetes during the follow-up (33/349) compared with those who had not (9/156), however, the comparison was not statistically significant (Odds ratio: 1.86, 95% confidence interval: 0.89-4.28, adjusted for group, age and sex). CONCLUSION: A higher cumulative HbA1c was significantly associated with a higher risk of retinopathy. No evidence was found for a beneficial effect of a 4-year lifestyle intervention on the long-term occurrence of clinically diagnosed retinopathy during a median of 22-year follow-up.

PGC-1α regulates the interplay between oxidative stress, senescence and autophagy in the ageing retina important in age-related macular degeneration.

We previously showed that mice with knockout in the peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PPARGC1A) gene encoding the PGC-1α protein, and nuclear factor erythroid 2 like 2 (NFE2L2) gene, exhibited some features of the age-related macular degeneration (AMD) phenotype. To further explore the mechanism behind the involvement of PGC-1α in AMD pathogenesis we used young (3-month) and old (12-month) mice with knockout in the PPARGC1A gene and age-matched wild-type (WT) animals. An immunohistochemical analysis showed age-dependent different expression of markers of oxidative stress defence, senescence and autophagy in the retinal pigment epithelium of KO animals as compared with their WT counterparts. Multivariate inference testing showed that senescence and autophagy proteins had the greatest impact on the discrimination between KO and WT 3-month animals, but proteins of antioxidant defence also contributed to that discrimination. A bioinformatic analysis showed that PGC-1α might coordinate the interplay between genes encoding proteins involved in antioxidant defence, senescence and autophagy in the ageing retina. These data support importance of PGC-1α in AMD pathogenesis and confirm the utility of mice with PGC-1α knockout as an animal model to study AMD pathogenesis.

Tissue fibroblasts are versatile immune regulators: An evaluation of their impact on the aging process.

Fibroblasts are abundant stromal cells which not only control the integrity of extracellular matrix (ECM) but also act as immune regulators. It is known that the structural cells within tissues can establish an organ-specific immunity expressing many immune-related genes and closely interact with immune cells. In fact, fibroblasts can modify their immune properties to display both pro-inflammatory and immunosuppressive activities in a context-dependent manner. After acute insults, fibroblasts promote tissue inflammation although they concurrently recruit immunosuppressive cells to enhance the resolution of inflammation. In chronic pathological states, tissue fibroblasts, especially senescent fibroblasts, can display many pro-inflammatory and immunosuppressive properties and stimulate the activities of different immunosuppressive cells. In return, immunosuppressive cells, such as M2 macrophages and myeloid-derived suppressor cells (MDSC), evoke an excessive conversion of fibroblasts into myofibroblasts, thus aggravating the severity of tissue fibrosis. Single-cell transcriptome studies on fibroblasts isolated from aged tissues have confirmed that tissue fibroblasts express many genes coding for cytokines, chemokines, and complement factors, whereas they lose some fibrogenic properties. The versatile immune properties of fibroblasts and their close cooperation with immune cells indicate that tissue fibroblasts have a crucial role in the aging process and age-related diseases.

Analysis of prognostic and predictive factors in neovascular age-related macular degeneration Kuopio cohort.

PURPOSE: The aim of the study was to explore factors affecting the progression of neovascular age-related macular degeneration (nAMD) and identify predictive factors that can estimate the duration of intravitreal treatments. METHODS: This retrospective real-world study included 421 nAMD patients treated at the Kuopio University Hospital during years 2007-2021. The collected data included background demographics, treatment history, visual acuity and retinal biomarker analysis. Impact of baseline factors on age at diagnosis, treatment duration, received treatment intensity and visual acuity gains were analysed. RESULTS: Heavy smoking and high body mass index (BMI) were associated with an earlier onset, while the use of anticoagulation and anti-aggregation medication were associated with a later onset of nAMD. A low number of injections during the first year of treatment and the presence of intraretinal fluid (IRF) at baseline were associated with shorter treatment duration. Interestingly, when IRF only patients were compared to subretinal fluid (SRF) only patients, IRF patients showed higher occurrences of subretinal drusenoid deposits (43.5% vs. 15%, p = 0.04). In addition, when all patients with IRF were compared to SRF only patients, more hyperreflective foci (HRF) and complete RPE and outer retinal atrophy (cRORA; 20.7% vs. 5%, p = 0.02) were observed in patients with IRF. CONCLUSIONS: Our results reveal that heavy smoking and high BMI are accelerating factors for earlier emergence of nAMD, while the presence of IRF results in a fast-progressing disease. More intriguingly, the link between IRF and appearance of subretinal drusenoid deposits, HRF, and increased retinal atrophy was observed.

The incidence and prevalence of diabetic macular edema and proliferative diabetic retinopathy, their progression to visual impairment and patterns in their intravitreal treatment in the Finnish population.

PURPOSE: The worldwide prevalence of diabetes mellitus (DM) continues to increase. As DM is linked to various ophthalmological comorbidities, it is crucial to understand the incidence and the treatment patterns of these complications to minimise the treatment burden for the patient and the healthcare system. This study aims to evaluate the incidence and prevalence of diabetic macular oedema (DME) and proliferative diabetic retinopathy (PDR) and to analyse intravitreal (IVT) treatment patterns and responses in the Finnish population with diabetes. METHODS: A nationwide data register containing details of over 20-year-old individuals with diabetes was used in the analyses. RESULTS: The incidence and prevalence of DME and PDR among the Finnish population with diabetes either declined or remained stable during 2007-2017 (Incidence rate: DME -40.8%, PDR -65.3%; prevalence rate: DME +4.7%, PDR -11.2%). During the same period, number of persons suffering from diabetes increased by +58.3%. The total number of IVT injections increased by 261.7%; the number of patients receiving IVT treatments increased by 133.6% from 2011 to 2017, reflecting changes in patient numbers in the ophthalmology departments. Furthermore, irrespective of the rising number of patients with diabetes, the numbers with visual impairment declined by 75.8% among DME and by 75.7% among PDR patients in 2007-2017. CONCLUSIONS: Regardless of the considerable increase in the workload of ophthalmology departments, the healthcare system has been able to reduce both the age and sex standardised incidence of DME and PDR among the diabetic population suffering from a visual impairment associated with this disease.

A New Generation of Gene Therapies as the Future of Wet AMD Treatment.

Age-related macular degeneration (AMD) is an eye disease and the most common cause of vision loss in the Western World. In its advanced stage, AMD occurs in two clinically distinguished forms, dry and wet, but only wet AMD is treatable. However, the treatment based on repeated injections with vascular endothelial growth factor A (VEGFA) antagonists may at best stop the disease progression and prevent or delay vision loss but without an improvement of visual dysfunction. Moreover, it is a serious mental and financial burden for patients and may be linked with some complications. The recent first success of intravitreal gene therapy with ADVM-022, which transformed retinal cells to continuous production of aflibercept, a VEGF antagonist, after a single injection, has opened a revolutionary perspective in wet AMD treatment. Promising results obtained so far in other ongoing clinical trials support this perspective. In this narrative/hypothesis review, we present basic information on wet AMD pathogenesis and treatment, the concept of gene therapy in retinal diseases, update evidence on completed and ongoing clinical trials with gene therapy for wet AMD, and perspectives on the progress to the clinic of "one and done" therapy for wet AMD to replace a lifetime of injections. Gene editing targeting the VEGFA gene is also presented as another gene therapy strategy to improve wet AMD management.

Acta Ophthalmologica 100 years-Overview of selected articles during Acta Ophthalmologica history.

We selected and discussed 10 articles in Acta Ophthalmologica since 1923 that changed clinical ophthalmology and treatment protocols, or provided novel findings and perspectives. We are aware that the selection of articles may be debatable and we invite readers to suggest other significant Acta articles. For historians, the article archive of Acta Ophthalmologica is located in Copenhagen.

Extracellular vesicles in degenerative retinal diseases: A new therapeutic paradigm.

Nanoscale extracellular vesicles (EVs), consisting of exomers, exosomes and microvesicles/ectosomes, have been extensively investigated in the last 20 years, although their biological role is still something of a mystery. EVs are involved in the transfer of lipids, nucleic acids and proteins from donor to recipient cells or distant organs as well as regulating cell-cell communication and signaling. Thus, EVs are important in intercellular communication and this is not limited to sister cells, but may also mediate the crosstalk between different cell types even over long distances. EVs play crucial functions in both cellular homeostasis and the pathogenesis of diseases, and since their contents reflect the status of the donor cell, they represent an additional valuable source of information for characterizing complex biological processes. Recent advances in isolation and analytical methods have led to substantial improvements in both characterizing and engineering EVs, leading to their use either as novel biomarkers for disease diagnosis/prognosis or even as novel therapies. Due to their capacity to carry biomolecules, various EV-based therapeutic applications have been devised for several pathological conditions, including eye diseases. In the eye, EVs have been detected in the retina, aqueous humor, vitreous body and also in tears. Experiences with other forms of intraocular drug applications have opened new ways to use EVs in the treatment of retinal diseases. We here provide a comprehensive summary of the main in vitro, in vivo, and ex vivo literature-based studies on EVs' role in ocular physiological and pathological conditions. We have focused on age-related macular degeneration, diabetic retinopathy, glaucoma, which are common eye diseases leading to permanent blindness, if not treated properly. In addition, the putative use of EVs in retinitis pigmentosa and other retinopathies is discussed. Finally, we have reviewed the potential of EVs as therapeutic tools and/or biomarkers in the above-mentioned retinal disorders. Evidence emerging from experimental disease models and human material strongly suggests future diagnostic and/or therapeutic exploitation of these biological agents in various ocular disorders with a good possibility to improve the patient's quality of life.

Association of APOE Haplotypes With Common Age-Related Ocular Diseases in 412,171 Individuals.

Purpose: Apolipoprotein E4 (APOE4), a known risk factor for Alzheimer's disease, has controversially been associated with reduced risk of primary open-angle glaucoma (POAG) and age-related macular degeneration (AMD). Here, we sought to systematically quantify the associations of APOE haplotypes with age-related ocular diseases and to assess their scope and age-dependency. Methods: We included genetic and registry data from 412,171 Finnish individuals in the FinnGen study. Disease endpoints were defined using nationwide registries. APOE genotypes were directly genotyped using Illumina and Affymetrix arrays or imputed using a custom Finnish reference panel. We evaluated the disease associations of APOE genotypes containing ε2 (without ε4) and ε4 (without ε2) compared with the ε3ε3 genotype using logistic regressions stratified by age. Results: APOE ε4 enriched haplotypes were inversely associated with overall glaucoma (odds ratio [OR] = 0.95, 95% confidence interval [CI] = 0.92-0.99, P = 0.0047), and its subtypes POAG (OR = 0.95, P = 0.027), normal-tension glaucoma (OR = 0.87, P = 0.0058), and suspected glaucoma (OR = 0.95, P = 0.014). Individuals with the ε4 allele also had lower odds for AMD (OR = 0.80, 95% CI = 0.76-0.84, P < 0.001), seen both in dry and neovascular subgroups. A slight negative association was also detected in senile cataract, but this was not reproducible in age-group analyses. Conclusions: Our results support prior evidence of the inverse association of APOE ε4 with glaucoma, but the association was weaker than for AMD. We could not show an association with exfoliation glaucoma, supporting the hypothesis that APOE may be involved in regulating retinal ganglion cell degeneration rather than intraocular pressure.