Epithelial-mesenchymal Transition (EMT) and the Effect of Atorvastatin on it in ARPE-19 cells.

Proliferative vitreoretinopathy (PVR) develops after an unsuccessful or complicated recovery from rhegmatogenous retinal detachment (RRD) surgery. Intraocular scar formation with the contribution of epithelial-mesenchymal transition (EMT) in RPE cells is prominent in the pathology of PVR. In the present study, the EMT process was experimentally induced in human retinal pigment epithelium (RPE; ARPE-19) cells, and the effect of atorvastatin on the process was studied. The mRNA and protein levels of mesenchymal markers actin alpha 2 (ACTA2) / alpha-smooth muscle actin (α-SMA) and fibronectin (FN), and epithelial markers occludin (OCLN) and zonula occludens-1 (ZO-1) were measured using quantitative real-time PCR (qRT-PCR) and western blot methods, respectively. In addition, α-SMA and FN were visualized using immunofluorescence staining. Cells were photographed under a phase contrast light microscope. Changes in the functionality of cells following the EMT process were studied using the IncuCyte scratch wound cell migration assay and the collagen cell invasion assay with confocal microscopy. The induction of EMT in ARPE-19 cells increased the expression of mesenchymal markers ACTA2/α-SMA and fibronectin and reduced the expression of epithelial marker OCLN both at mRNA and protein levels. The mRNA levels of ZO-1 were lower after EMT, as well. Increased levels of α-SMA and FN were confirmed by immunofluorescence staining. Atorvastatin further increased the mRNA levels of mesenchymal markers ACTA2 and FN as well as the protein levels of α-SMA and reduced the mRNA levels of epithelial markers OCLN and ZO-1 under the EMT process. EMT promoted wound closure and cell invasion into the 3D collagen matrix when compared to untreated control cells. These data present cellular changes upon the induction of the EMT process in ARPE-19 cells and the propensity of atorvastatin to complement the effect. More studies are needed to confirm the exact influence of the EMT process and atorvastatin treatment on the PVR development after RRD surgery.

Anti-inflammatory potential of simvastatin and amfenac in ARPE-19 cells; insights in preventing re-detachment and proliferative vitreoretinopathy after rhegmatogenous retinal detachment surgery.

PURPOSE: Rhegmatogenous retinal detachment is a severe vision-threatening complication that can result into proliferative vitreoretinopathy (PVR) and re-detachment of the retina if recovery from surgery fails. Inflammation and changes in retinal pigment epithelial (RPE) cells are important contributors to the disease. Here, we studied the effects of simvastatin and amfenac on ARPE-19 cells under inflammatory conditions. METHODS: ARPE-19 cells were pre-treated with simvastatin and/or amfenac for 24 h after which interleukin (IL)-1α or IL-1ß was added for another 24 h. After treatments, lactate dehydrogenase release, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) processing, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activity, prostaglandin E2 (PGE2) level, and extracellular levels of IL-6, IL-8, monocytic chemoattractant protein (MCP-1), vascular endothelial growth factor (VEGF), and pigment epithelium-derived factor, as well as the production of reactive oxygen species (ROS) were determined. RESULTS: Pre-treatment of human ARPE-19 cells with simvastatin reduced the production of IL-6, IL-8, and MCP-1 cytokines, PGE2 levels, as well as NF-κB activity upon inflammation, whereas amfenac reduced IL-8 and MCP-1 release but increased ROS production. Together, simvastatin and amfenac reduced the release of IL-6, IL-8, and MCP-1 cytokines as well as NF-κB activity but increased the VEGF release upon inflammation in ARPE-19 cells. CONCLUSION: Our present study supports the anti-inflammatory capacity of simvastatin as pre-treatment against inflammation in human RPE cells, and the addition of amfenac complements the effect. The early modulation of local conditions in the retina can prevent inflammation induced PVR formation and subsequent retinal re-detachment.

Incidence of glaucoma filtration surgery from disease onset of open-angle glaucoma.

AIMS: To investigate the rate and risk factors of undergoing glaucoma filtration surgery (GFS) in patients with newly diagnosed open-angle glaucoma (OAG). METHODS: This is a population-based historic cohort study, consisting of 9420 patients older than 45 years diagnosed with OAG during 1997-2010. Follow-up spanned from 1997 to 2017. We obtained data for trabeculectomy (TRE), deep sclerectomy (DS), and glaucoma drainage implant (GDI) surgeries from national administrative healthcare registers by hospital billing data. We plotted the cumulative incidence of GFS and carried out a multivariate Poisson regression analysis adjusted for age, sex, hospital district, systemic comorbidities, and the number of IOP-lowering drugs. We reported incidence rate ratios (IRR) with 95% confidence intervals (CI) for GFS after the onset of OAG. RESULTS: The cumulative incidence of GFS at 5 years from OAG onset was 3.1% and at 10 years 5.4%. Age over 80 years at baseline was associated with lower GFS incidence (IRR 0.51, CI 0.31-0.84). The number of IOP-lowering drugs in the first 2 years of treatment correlated with the risk of GFS increasing from (IRR 3.23, CI 2.32-4.50) for two drugs, (IRR 7.44, CI 5.28-10.47) for three and to (IRR 14.95, CI 10.38-21.52) for four drugs. CONCLUSION: This study characterized the treatment path of OAG from diagnosis to surgical intervention refining the role of GFS among glaucoma therapies.

Development of diabetic macular oedema shows associations with systemic medication - An epidemiological study.

PURPOSE: To identify associations between systemic drugs and the incidence of diabetic macular oedema (DME). Of interest was to find beneficial and/or deleterious associations of used drugs. METHODS: A historic cohort design based on administrative data. Study population consisted of 150 353 individuals with diabetes. Endpoint event was the development of DME (ICD-10 H36.01), censoring events were death or study end December 2017. The follow-up started between 1997 and 2010. The systemic medication consisted of 95 substances. We constructed a nested case-control study design comparing 2630 cases with DME to 13 144 age- and sex-matched controls without DME. Results are reported as odds ratios (ORs) with 95% confidence intervals (CIs) based on conditional logistic regression models. RESULTS: Incidence rate for DME was 1.80 per 1000 person-years (95% CI 1.73-1.87). In all, we observed a lower incidence rate of DME in females (IRR 0.57; 95% CI 0.52-0.62) compared to males. Exposure to hormone replacement therapy estradiol (OR 0.42; 0.25-0.68), temazepam (0.23; 0.08-0.62) and allopurinol (0.61; 0.43-0.86) were associated with lower risk of DME, while use of insulin or insulin analogue (3.30; 2.99-3.64), sulfonylureas (1.21; 1.05-1.40), diuretic furosemide (1.90; 1.61-2.24), calcium channel blocker amlodipine (1.53; 1.34-1.75), ACE inhibitors ramipril (1.66; 1.46-1.89) and enalapril (1.38; 1.16-1.64) were associated with an increased risk of DME. CONCLUSIONS: Large-scale studies examining the incidence of DME are lacking. Our findings suggest that associations of systemic medications with the incidence of DME may shed light on the pathogenesis of complex DME, encouraging further studies.

Diabetes mellitus and risk of open-angle glaucoma-A population-based follow-up study.

PURPOSE: The aim of this study was to investigate the association of diabetes mellitus (DM) and risk of open-angle glaucoma (OAG). METHODS: This population-based historic cohort consisted of individuals at age ≥ 40 years with DM treatment initiated 2001-2010 and a reference population matched by age, gender and hospital district. Incidence of OAG was compared between individuals with DM and their matched non-diabetic reference pairs. New glaucoma cases were identified from medication reimbursement certificates and hospital billing records. Incidence rate ratios (IRR) were analysed with Poisson regression models adjusted for age, sex, hospital district, socioeconomic status, systemic medications and chronic diseases. We analysed the sensitivity of the results with adapted input variables and performed a competing events analysis. RESULTS: Of the 244 100 study subjects meeting inclusion criteria, 2721 (1.1%) developed OAG. Follow-up spanned from 2001 to 2017. DM was associated with a modestly reduced incidence of OAG when adjusted for confounding factors (IRR 0.92, CI 0.85-0.99). CONCLUSIONS: In our longitudinal population-based study, we found a modest decrease in the risk of OAG for individuals with DM.

Association between NSAID and Statin Therapy and the Incidence of Intravitreal Anti-vascular Endothelial Growth Factor Injections and Nd:YAG Laser Treatment after Cataract Surgery in Finland.

Purpose: To examine the association between the use of topical non-steroidal anti-inflammatory (NSAID) medication, systemic statin therapy, and the incidence rate of two of the most common postsurgical procedures in adult patients undergoing cataract surgery in Finland between January 1, 2010 and December 31, 2016. Methods: This retrospective, nationwide cohort study considered 176,052 cataract operations coded with the International Classification of Disease coding: early adult (H25.0), normal (H25.1), other senile (H25.8), pre-senile (H26.02), or other (related to trauma, other eye disease, or medication). Operations were linked to purchased and reimbursed medications using Anatomical Therapeutic Chemical codes. The incidence rate of intravitreal anti-vascular endothelial growth factor (VEGF) injections, and neodymium-doped yttrium aluminum (Nd:YAG) laser treatments of posterior capsular opacification were evaluated using the Poisson regression model. Results: In our registry cohort, patients with a prescription of topical NSAID (ketorolac) at the time of cataract surgery were less likely treated with intravitreal anti-VEGF injections after surgery (adjusted Poisson regression model IRR 0.3; 95% CI: 0.15-0.60, P = 0.0007), and also had reduced incidence of Nd:YAG laser (0.59, CI: 0.43-0.81, P = 0.0011) treatments. Unlike topical NSAID, the use of systemic statin therapy was not associated with these two most common surgical procedures (RR 1.04, 95% CI: 0.96-1.12, P = 0.33). Conclusion: The use of topical NSAIDs is associated with reduced rates of intravitreal anti-VEGF injections and Nd:YAG laser treatments after cataract surgery. More observational and experimental studies are warranted to confirm possible benefits of topical NSAID administration after cataract surgery.

Risk of glaucoma after vitreoretinal surgery - Findings from a population-based cohort study.

PURPOSE: To investigate the association between different types of vitrectomy and risk of different types of glaucoma and to determine the effect of systemic medication and diabetes status on this risk. METHODS: A population-based nested case-control study included individuals of age ≥ 18 years who had undergone single vitrectomy, vitrectomy with retinal procedure, or combined phaco-vitrectomy between 2001 and 2010. End of follow-up was 2017. Odds ratio (OR) for the development of glaucoma after different types of vitrectomy and 95% confidence interval (CI) were based on conditional logistic regression models. For every glaucoma case, five controls were matched by age, sex, start of follow-up year, and hospital district. RESULTS: The cohort (n = 37 687), of which 52.8% was female, consisted of 6552 individuals diagnosed with glaucoma and 31 135 controls matched by age, sex, and hospital district. Vitrectomy was performed on 103 eyes in the glaucoma group and 158 eyes in the control group. As regards the risk of any glaucoma, the risk was lowest in eyes that underwent combined phaco-vitrectomy (OR: 2.7, 95% CI: 1.8-4.1), followed by single vitrectomy (OR: 3.15, 95% CI: 2.1-4.8), and highest in eyes that underwent vitrectomy with retinal procedure (OR: 4.5, 95% CI: 2.7-7.4). Diabetes had no effect (OR: 0.96, 95% CI: 0.92-1.01), but 5-year systemic statin use slightly decreased glaucoma risk (OR: 0.86, 95% CI: 0.77-0.97). CONCLUSIONS: Vitreoretinal surgery was associated with an increased glaucoma risk; the risk being related to the complexity of vitrectomy. Long-term systemic statin therapy may decrease glaucoma risk, while diabetes had no association.

Statin use and the reoperation rates in glaucoma filtration surgery - population-based cohort study.

PURPOSE: To examine the association of systemic statin therapy and reoperation rate after glaucoma filtration surgery (GFS). METHODS: This is a population-based, historic cohort study of 2705 eyes undergoing GFS in Finland between July 2009 and December 2016. GFSs were identified from national administrative healthcare registers. Baseline sociodemographic and health characteristics were documented. Reoperation rates of GFS subgroups were analysed, with statin users compared to non-users. The outcomes were modelled using a Poisson regression model adjusted for age, sex, education, statin use, chronic comorbidities, and cataract surgery with incident rate ratios (IRR) as the main outcome measure. RESULTS: The cohort contained 2263 subjects with open-angle glaucoma (OAG), 823 men and 1440 women. Surgery was performed on 2705 eyes. First documented procedures: deep sclerectomy (DS) (n = 1601), trabeculectomy (TRE) (799) and glaucoma drainage device (GDD) implantation (305) respectively. In total, 438 secondary operations were performed during the 7.5-year (median 2.25 years) follow-up period. The reoperation rates were 19% after DS, 12% after TRE, and 13% after GDD. Of the surgical procedures, 32% were performed on eyes of patients receiving statin therapy. Statin users showed no difference in reoperation rates (IRR 1.06, CI 0.82-1.37). In subgroups, no difference was observed in the reoperation rates adjusted with statin use after filtration surgery (DS, TRE) (IRR 1.06, CI 0.8-1.40) or GDD implantation (0.57, CI 0.20-1.63). CONCLUSION: Systemic statin therapy among surgically treated OAG patients had no impact on secondary surgery rates following DS, TRE or GDD implantation.

Associations between systemic medications and development of wet age-related macular degeneration.

PURPOSE: To examine whether systemic medications are associated with the subsequent development of wet age-related macular degeneration (AMD). METHODS: A retrospective study of 259 562 individuals based on registry data, from January 1, 2001, to December 31, 2017. End-point event was the International Classification of Diseases (ICD)-10 diagnosis for wet AMD. Association between use of systemic medication covering 85 generic drugs categorized according to Anatomical Therapeutic Chemical (ATC) codes and the incidence of wet AMD was evaluated using multivariate Poisson regression model (adjusted for age, sex, diabetes, cancer and socioeconomic group) and nested case-control design. RESULTS: The mean length of follow-up was 9.84 years. The number of cases with wet AMD was 2947 and incidence rate was 1.15 per 1000 person-years. After adjustment, we observed an increased risk for the development of wet AMD for patients exposed to amlodipine (IRR 1.33, 95% CI 1.16-1.53), or felodipine (1.24, 95% CI 1.02-1.50). Similarly, an increased risk of wet AMD was associated with the use of bicalutamide (2.14, 95% CI 1.14-4.02), estradiol (1.20, 95% CI 1.03-1.40) and atorvastatin (1.22, 95% CI 1.05-1.43). Of note, digoxin (0.72, 95% CI 0.57-0.91), and ramipril (0.80, 95% CI 0.65-0.99) users had a lower incidence of wet AMD. CONCLUSIONS: Our findings suggest that the use of second-generation calcium channel blockers could be associated with an increased risk for wet AMD development. Of note, the incidence of wet AMD seemed to be lower in patients using ramipril and digoxin. More studies are needed to elucidate the associations further.

Proliferative diabetic retinopathy transcriptomes reveal angiogenesis, anti-angiogenic therapy escape mechanisms, fibrosis and lymphatic involvement.

Proliferative diabetic retinopathy (PDR) is a sight-threatening diabetic complication in urgent need of new therapies. In this study we identify potential molecular mechanisms and target candidates in the pathogenesis of PDR fibrovascular tissue formation. We performed mRNA sequencing of RNA isolated from eleven excised fibrovascular membranes of type 1 diabetic PDR patients and two non-diabetic patients with rhegmatogenous retinal detachment with proliferative vitreoretinopathy. We determined differentially expressed genes between these groups and performed pathway and gene ontology term enrichment analyses to identify potential underlying mechanisms, pathways, and regulators. Multiple pro-angiogenic processes, including VEGFA-dependent and -independent pathways, as well as processes related to lymphatic development, epithelial to mesenchymal transition (EMT), wound healing, inflammation, fibrosis, and extracellular matrix (ECM) composition, were overrepresented in PDR. Overrepresentation of different angiogenic processes may help to explain the transient nature of the benefits that many patients receive from current intravitreal anti-angiogenic therapies, highlighting the importance of combinatorial treatments. Enrichment of genes and pathways related to lymphatic development indicates that targeting lymphatic involvement in PDR progression could have therapeutic relevance. Together with overrepresentation of EMT and fibrosis as well as differential ECM composition, these findings demonstrate the complexity of PDR fibrovascular tissue formation and provide avenues for the development of novel treatments.

Early middle age cholesterol levels and the association with age-related macular degeneration.

PURPOSE: To examine whether serum cholesterol in early middle age is associated with age-related macular degeneration (AMD) later in life. METHODS: A group of Helsinki Businessmen Study (HBS) participants (n = 209) were recruited for the study. Total cholesterol (TC), triglyceride and body mass index (BMI) were measured at the HBS baseline visit in 1964-1973. Lipid subfractions, BMI, smoking status and statin use were recorded in 2011 and fundus photographs graded for AMD in 2005-2012. The subjects were genotyped for the main AMD risk single nucleotide polymorphisms (SNPs). RESULTS: TC measured at baseline 1964-1973 was significantly higher in subjects later developing intermediate or late AMD (6.67 mmol/l versus 6.20 mmol/l, p = 0.024) or with drusen size of ≥125 µm (6.68 mmol/l versus 6.21 mmol/l, p = 0.030) compared with the rest of the study population. TC, LDL and TG values at follow-up 2011 were lower in subjects with AMD compared to those without, whereas HDL levels showed no difference. In multivariate analysis, baseline TC associated with intermediate or late AMD (OR 1.59, p = 0.004) and drusen size ≥ 125 µm (OR 1.57, p = 0.006) when corrected for age, BMI, AMD risk SNPs and smoking. Lipid values measured 2011 had no associations after correction. CONCLUSIONS: High systemic total cholesterol in early middle age may have a role in the initial development of AMD, especially in patients later developing large drusen.

Molecular pathogenesis of rhegmatogenous retinal detachment.

Rhegmatogenous retinal detachment (RRD) is an ophthalmic emergency, which usually requires prompt surgery to prevent further detachment and restore sensory function. Although several individual factors have been suggested, a systems level understanding of molecular pathomechanisms underlying this severe eye disorder is lacking. To address this gap in knowledge we performed the molecular level systems pathology analysis of the vitreous from 127 patients with RRD using state-of-the art quantitative mass spectrometry to identify the individual key proteins, as well as the biochemical pathways contributing to the development of the disease. RRD patients have specific vitreous proteome profiles compared to other diseases such as macular hole, pucker, or proliferative diabetic retinopathy eyes. Our data indicate that various mechanisms, including glycolysis, photoreceptor death, and Wnt and MAPK signaling, are activated during or after the RRD to promote retinal cell survival. In addition, platelet-mediated wound healing processes, cell adhesion molecules reorganization and apoptotic processes were detected during RRD progression or proliferative vitreoretinopathy formation. These findings improve the understanding of RRD pathogenesis, identify novel targets for treatment of this ophthalmic disease, and possibly affect the prognosis of eyes treated or operated upon due to RRD.

Statins for the prevention of proliferative vitreoretinopathy: cellular responses in cultured cells and clinical statin concentrations in the vitreous.

Proliferative vitreoretinopathy (PVR) with rhegmatogenous retinal detachment (RRD) is a complex inflammatory ocular disease. Statins are widely used cholesterol-lowering drugs with putative anti-inflammatory properties. In this study, we have explored their efficacy in controlling post-surgical PVR formation. Simvastatin (SIM), atorvastatin (ATV), or rosuvastatin (RSV) were added to cultures of human retinal pigment epithelial cells (ARPE-19) prior to exposure with the bacterial lipopolysaccharide (LPS), and the production of pro-inflammatory cytokines (IL-6, IL-8, MCP-1) was examined using an enzyme-linked immunosorbent assay. In addition, the concentrations of simvastatin, atorvastatin, rosuvastatin, and their metabolites were measured from the vitreal samples of 20 patients undergoing vitrectomy (16 of them receiving oral statin therapy) using an ultra-performance liquid chromatography-tandem mass spectrometer technique. All statins alleviated LPS-induced inflammation at 5 µM concentration in the ARPE-19 cell cultures. Statin levels in the vitreous samples ranged from 6 to 316 pg/mL (ca. 0.1-7 M-10). Vitreal statin concentrations were similar to the typical steady-state unbound statin concentrations in plasma, indicating that only the unbound drug distributes from the blood circulation into the vitreous. Pharmacokinetic simulations of the intravitreal delivery of statins indicate that the measured clinical statin concentrations could be maintained with existing drug delivery technologies for months. Our results suggest that intravitreal statin therapy may have the potential in alleviating the risk of post-surgical PVR.

Intravitreal anti-VEGF therapy is not associated with a higher risk of all-cause mortality in patients with macular oedema caused by posterior segment vascular diseases.

PURPOSE: To examine whether real-world clinical patients with macular oedema (MO) receiving intravitreal antivascular endothelial growth factor (VEGF) therapy have a higher mortality compared with a matched reference population. METHODS: A population-based, retrospective cohort study of 26 386 patients from Finland, from January 1, 2001, to December 31, 2017. Index patients were identified through the Caring Epidemiology Project database, receiving at least one intravitreal anti-VEGF injection for wet age-related macular degeneration (AMD, n = 2243, 48.61%), diabetic MO (n = 744, 16.12%), MO due to retinal vascular occlusion (n = 589, 12.77%), or other MO (n = 1038, 22.5%). For each individual treated with intravitreal injection (n = 4614), five age- , sex- , calendar year- and hospital district- matched control individuals (n = 21 772) were chosen. Baseline data of chronic conditions were available. All-cause and cause-specific mortality was analysed using Cox´s proportional hazards model. RESULTS: In general, the anti-VEGF treated patients had a higher prevalence of systemic conditions, including diabetes (60.1% vs. 46.8%, p < 0.001), chronic hypertension (38.4% vs. 34.6%, p < 0.001), in hospital-treated ischaemic heart disease (23.1% vs. 21.5%, p = 0.014), and glaucoma (11.1% vs. 6.3%, p < 0.001) than controls. There was no difference in all-cause mortality between the anti-VEGF treated patients and matched controls (p = 0.62). In unadjusted Kaplan-Meier analysis of wet AMD subgroup, all-cause mortality was lower in anti-VEGF treated patients than matched controls (p = 0.015), but adjusted Cox´s proportional hazards model showed no difference in the risk of all-cause mortality (HR 0.85, 95% CI 0.66-1.09). CONCLUSIONS: Intravitreal anti-VEGF therapy was not associated with an increase in the risk of mortality in patients with MO compared with age- and sex-matched controls.