A prototype protocol for evaluating the real-world data set using a structured electronic health record in glaucoma.

PURPOSE: As the first step in monitoring and evaluating day-to-day glaucoma care, this study reports all real-world data recorded during the first full year after the implementation of a prototype for glaucoma-specific structured electronic healthcare record (EHR). METHODS: In 2019, 4618 patients visited Tays Medical Glaucoma Clinic at Tays Eye Centre, Tampere University Hospital, Finland, that serves a population of 0.53 M. Patient data were entered into a glaucoma-specific EHR by trained nurses to be checked by glaucoma specialists. Tays Eye Centre follows the Finnish Current Care Guideline for glaucoma in which glaucoma is defined using a '2 out of 3' rule, that is, ≥2 findings evaluated as glaucomatous in optic nerve head (ONH), retinal nerve fibre layer (RNFL) and visual field (VF). RESULTS: The clinical evaluations of ONH, RNFL and VF were recorded in 95%-100% of all eyes. ONH was evaluated as glaucomatous more often (44%) than RNFL (33%) and VF tests (30%). Progressive changes in any of the three tests were recorded in 35% of the '≥2/3 glaucoma group' compared to 2%-9% in the other groups. The mean IOP at visit was 15 mmHg. The mean target IOP was 17 mmHg, and it was recorded in 94% of eyes. CONCLUSION: The developed structured data presentation enables comparisons between different population-based real-world glaucoma data sets and glaucoma clinics. Compared to a data set from the UK, the proportion of glaucoma suspicion-related visits was smaller in Tays Eye Centre and test intervals were longer.

Ten-year real-world outcomes of antivascular endothelial growth factor therapy in neovascular age-related macular degeneration using pro re nata regimen.

BACKGROUND/AIMS: To analyse long-term outcomes of antivascular endothelial growth factor (anti-VEGF) therapy for the treatment of neovascular age-related macular degeneration (nAMD) using pro re nata (PRN) regimen in a single-centre clinical practice. METHODS: All patients receiving intravitreal injection (IVI) for nAMD between 1 January 2008 and 31 December 2020 were searched from electronic medical records. All 3844 treatment-naïve eyes of 3008 patients were included with a total of 50 146 IVIs (87% bevacizumab) administered. Main outcome measures were mean change in visual acuity (VA) from baseline, proportion of eyes within 15 letters of baseline, proportion of eyes with VA ≥20/40 Snellen and ≤20/200 Snellen, number of annual visits and number of annual IVIs. RESULTS: The mean baseline VA was 55 Early Treatment Diabetic Retinopathy Study (ETDRS) letters and the mean change in VA from baseline was +2, +2, ±0, -2, -2 and -4 ETDRS letters at year 1, 2, 3, 5, 7 and 10, respectively. Proportions of eyes within 15 letters of baseline were 88%, 87%, 82%, 80%, 76% and 72% at the end of years 1, 2, 3, 5, 7 and 10, respectively. The median number of annual IVI was 6 at years 1-7 and 5 at year 10. The median number of annual total visits was 10 at year 1, 9 at years 2-7 and 8 at year 10, respectively. CONCLUSIONS: VA was maintained short-term and long-term with anti-VEGF therapy using PRN treatment regimen.

Proteomics Analysis of R-Ras Deficiency in Oxygen Induced Retinopathy.

Small GTPase R-Ras regulates vascular permeability in angiogenesis. In the eye, abnormal angiogenesis and hyperpermeability are the leading causes of vision loss in several ischemic retinal diseases such as proliferative diabetic retinopathy (PDR), retinal vein occlusion (RVO), and retinopathy of prematurity (ROP). Oxygen-induced retinopathy (OIR) is the most widely used experimental model for these ischemic retinopathies. To shed more light on how the R-Ras regulates vascular permeability in pathological angiogenesis, we performed a comprehensive (>2900 proteins) characterization of OIR in R-Ras knockout (KO) and wild-type (WT) mice by sequential window acquisition of all theoretical mass spectra (SWATH-MS) proteomics. OIR and age-matched normoxic control retinas were collected at P13, P17, and P42 from R-Ras KO and WT mice and were subjected to SWATH-MS and data analysis. The most significant difference between the R-Ras KO and WT retinas was an accumulation of plasma proteins. The pathological vascular hyperpermeability during OIR in the R-Ras KO retina took place very early, P13. This led to simultaneous hypoxic cell injury/death (ferroptosis), glycolytic metabolism as well compensatory mechanisms to counter the pathological leakage from angiogenic blood vessels in the OIR retina of R-Ras deficient mice.

Glaucoma progression in patients receiving intravitreal anti-VEGF treatment for neovascular age-related macular degeneration.

PURPOSE: The purpose of this study was to investigate how often glaucoma and neovascular age-related macular degeneration (nAMD) occur in the same patient and to evaluate whether glaucoma progression is faster in eyes treated with intravitreal anti-VEGF medications for nAMD. METHODS: This single-centre retrospective real-world data (RWD) consists of medical records of 6314 glaucoma and 2166 nAMD patients treated in 2008-2017 in Tays Eye Centre, Finland. To study glaucoma progression, changes in visual fields (mean deviation [MD], dB/year), IOP (mmHg/year) and fundus photographs (progression, yes/no) were compared in glaucoma eyes with and without anti-VEGF treatment for nAMD and ≥1 year follow-up. RESULTS: During the 10-year period, 147 patients with glaucoma received intravitreal anti-VEGF treatment for nAMD corresponding to 2% of glaucoma and 7% of nAMD patients. The mean change in MD was -0.70 dB/year (SD 1.8) vs. -0.27 dB/year (SD 1.7) (p = 0.027) in glaucoma eyes with (n = 37) and without (n = 4304) anti-VEGF injections, respectively. In patients with bilateral glaucoma and unilateral nAMD treated with anti-VEGF injections (n = 20), MD declined at -0.62 dB/year (SD 1.9) vs 0.33 dB/year (SD 1.5) (p = 0.654), and glaucoma progression was detected in 14/20 vs 10/20 (p = 0.219) fundus photographs in eyes with anti-VEGF treatment compared with their untreated fellow eyes. CONCLUSION: nAMD and glaucoma were found co-existing in the same eye at rates that were similar to the age-corrected prevalence of the two diseases in the general population. Our results suggest that intravitreal anti-VEGF treatment for nAMD may accelerate glaucoma progression.

Immediate sequential bilateral cataract surgery: a 13-year real-life report of 56 700 cataract operations.

BACKGROUND/AIMS: To assess the frequency of immediate sequential bilateral cataract surgery (ISBCS) and endophthalmitis during 13-year period in Tays Eye Centre, Tampere University Hospital, Tampere, Finland. METHODS: All cataract surgeries performed between 1 January 2008 and 31 December 2020, and all endophthalmitis cases during the same period were searched from electronic patient records. Numbers and frequencies of ISBCS, and complications, including endophthalmitis and vitreous loss, were recorded and compared with unilateral operations. RESULTS: The study included 56 700 cataract surgeries in 34 797 patients of whom 39% (n=13 445) had ISBCS. The median age of the patients was 75 (IQR 68-80, range 0.08-99) years at the time of surgery. The proportion of ISBCS patients increased from 4.2% in 2008 to 46% in 2020. Vitreous loss occurred in 480 (0.9%) of cataract surgeries. There were no postoperative endophthalmitis after cataract surgery (n=0) during the 13-year period. CONCLUSION: The proportion of patients undergoing ISBCS increased from 4.2% in 2008 to 46% in 2020. No endophthalmitis were found to be associated with ISBCS.

A comprehensive model for measuring real-life cost-effectiveness in eyecare: automation in care and evaluation of system (aces-rwm™).

This paper describes a holistic, yet simple and comprehensible, ecosystem model to deal with multiple and complex challenges in eyecare. It aims at producing the best possible wellbeing and eyesight with the available resources. When targeting to improve the real-world cost-effectiveness, what gets done in everyday practice needs be measured routinely, efficiently and unselectively. Collection of all real-world data of all patients will enable evaluation and comparison of eyecare systems and departments between themselves nationally and internationally. The concept advocates a strategy to optimize real-life effectiveness, sustainability and outcomes of the service delivery in ophthalmology. The model consists of three components: (1) resource-governing principles (i.e., to deal with increasing demand and limited resources), (2) real-world monitoring (i.e., to collect structured real-world data utilizing automation and visualization of clinical parameters, health-related quality of life and costs), and (3) digital innovation strategy (i.e., to evaluate and benchmark real-world outcomes and cost-effectiveness). The core value and strength of the model lies in the consensus and collaboration of all Finnish university eye clinics to collect and evaluate the uniformly structured real-world outcomes data. In addition to ophthalmology, the approach is adaptable to any medical discipline to efficiently generate real-world insights and resilience in health systems.

Selective Targeting and Tissue Penetration to the Retina by a Systemically Administered Vascular Homing Peptide in Oxygen Induced Retinopathy (OIR).

Pathological angiogenesis is the hallmark of ischemic retinal diseases among them retinopathy of prematurity (ROP) and proliferative diabetic retinopathy (PDR). Oxygen-induced retinopathy (OIR) is a pure hypoxia-driven angiogenesis model and a widely used model for ischemic retinopathies. We explored whether the vascular homing peptide CAR (CARSKNKDC) which recognizes angiogenic blood vessels can be used to target the retina in OIR. We were able to demonstrate that the systemically administered CAR vascular homing peptide homed selectively to the preretinal neovessels in OIR. As a cell and tissue-penetrating peptide, CAR also penetrated into the retina. Hyperoxia used to induce OIR in the retina also causes bronchopulmonary dysplasia in the lungs. We showed that the CAR peptide is not targeted to the lungs in normal mice but is targeted to the lungs after hyperoxia-/hypoxia-treatment of the animals. The site-specific delivery of the CAR peptide to the pathologic retinal vasculature and the penetration of the retinal tissue may offer new opportunities for treating retinopathies more selectively and with less side effects.

Pathological Angiogenesis Requires Syndecan-4 for Efficient VEGFA-Induced VE-Cadherin Internalization.

[Figure: see text].

Oxygen-Induced Retinopathy Model for Ischemic Retinal Diseases in Rodents.

One of the commonly used models for ischemic retinopathies is the oxygen-induced retinopathy (OIR) model. Here we describe detailed protocols for the OIR model induction and its readouts in both mice and rats. Retinal neovascularization is induced in OIR by exposing rodent pups either to hyperoxia (mice) or alternating levels of hyperoxia and hypoxia (rats). The primary readouts of these models are the size of neovascular (NV) and avascular (AVA) areas in the retina. This preclinical in vivo model can be used to evaluate the efficacy of potential anti-angiogenic drugs or to address the role of specific genes in the retinal angiogenesis by using genetically manipulated animals. The model has some strain and vendor specific variation in the OIR induction which should be taken into consideration when designing the experiments.

Exploration of Oxygen-Induced Retinopathy Model to Discover New Therapeutic Drug Targets in Retinopathies.

Oxygen-induced retinopathy (OIR) is a pure hypoxia-driven angiogenesis model and the most widely used model for ischemic retinopathies, such as retinopathy of prematurity (ROP), proliferative diabetic retinopathy (PDR), and retinal vein occlusion (RVO). OIR model has been used to test new potential anti-angiogenic factors for human diseases. We have recently performed the most comprehensive characterization of OIR by a relatively novel mass spectrometry (MS) technique, sequential window acquisition of all theoretical fragment ion mass spectra (SWATH-MS) proteomics and used genetically modified mice strains to identify novel molecular drug targets in angiogenic retinal diseases. We have confirmed the relevance of the identified molecular targets to human diseases by determining their expression pattern in neovascular membranes obtained from PDR and RVO patients. Based on our results, crystallins were the most prominent proteins induced by early hypoxic environment during the OIR, while actomyosin complex and Filamin A-R-Ras axis, that regulates vascular permeability of the angiogenic blood vessels, stood out at the peak of angiogenesis. Our results have revealed potential new therapeutic targets to address hypoxia-induced pathological angiogenesis and the associated vascular permeability in number of retinal diseases.

Expression of VEGFA-regulating miRNAs and mortality in wet AMD.

MicroRNAs (miRNAs) regulate gene expression; many of them act in the retinal pigment epithelium (RPE), and RPE degeneration is known to be a critical factor in age-related macular degeneration (AMD). Repeated injections with anti-VEGFA (vascular endothelial growth factor A) are the only effective therapy in wet AMD. We investigated the correlation between the expression of 18 miRNAs involved in the regulation of the VEGFA gene in serum of 76 wet AMD patients and 70 controls. Efficacy of anti-VEGFA treatment was evaluated by counting the number of injections delivered up to 12 years. In addition, we compared the relative numbers of deaths in patient with AMD and control groups. We observed a decreased expression of miR-34-5p, miR-126-3p, miR-145-5p and miR-205-5p in wet AMD patients as compared with controls. These miRNAs are involved in the regulation of angiogenesis, cytoprotection and protein clearance. No miRNA was significantly correlated with the treatment outcome. Wet AMD patients had greater mortality than controls, and their survival was inversely associated with the number of anti-VEGFA injections per year. No association was observed between miRNA expression and mortality. Our study emphasizes the need to clarify the role of miRNA regulation in AMD pathogenesis.

Viability of Mouse Retinal Explant Cultures Assessed by Preservation of Functionality and Morphology.

Purpose: Retinal explant cultures provide simplified systems where the functions of the retina and the effects of ocular therapies can be studied in an isolated environment. The purpose of this study was to provide insight into long-term preservation of retinal tissue in culture conditions, enable a deeper understanding of the interdependence of retinal morphology and function, and ensure the reliability of the explant technique for prolonged experiments. Methods: Retinal explants from adult mice were cultured as organotypic culture at the air-medium interface for 14 days in vitro (DIV). Retinal functionality was assessed by multielectrode array technique and morphology by immunohistochemical methods at several time points during culture. Results: Retinal explants retained viability for 14 DIV, although with diminishing neuronal activity, progressing neuronal loss, and increasing reactive gliosis. We recorded spontaneous retinal ganglion cell (RGC) activity up to 14 DIV with temporally changing distribution of RGC firing rates. Light responsiveness was measurable from RGCs for 7 DIV and from photoreceptors for 2 DIV. Apoptotic cells were detected beginning at 3 DIV with their density peaking at 7 DIV. The number of RGCs gradually decreased by 70% during 14 DIV. The change was accompanied by the loss of RGC functionality, resulting in 84% loss of electrically active RGCs. Conclusions: Retinal explants provide a valuable tool for studies of retinal functions and development of ocular therapies. However, critical for long-term use, retinal functionality was lost before structural loss, emphasizing a need for both functional and morphologic readouts to determine the overall state of the cultured retina.

R-Ras regulates vascular permeability, but not overall healing in skin wounds.

Wounds close by keratinocytes migrating from the edge of the wound and re-epithelializing the epidermis. It has been proposed that the major stimuli for wound closure are blood-derived growth factors, chemokines and cytokines. The small GTPase R-Ras, a known integrin activator, also regulates vascular permeability during angiogenesis, and blood vessels lacking R-Ras leak plasma proteins constantly. We explored whether the access to blood-derived proteins influences skin wound healing in R-Ras knockout (KO) mice. In skin wounds, R-Ras expression was mostly restricted to the vasculature in the granulation tissue. Angiogenic blood vessels in the R-Ras KO mice were significantly more permeable than in wild-type (WT) controls. Although the distances between epidermal tongues, and the panniculus carnosus muscles, were significantly longer in R-Ras KO than WT controls before the granulation tissue formation took place, there were no differences in the wound closure or re-epithelialization rates or granulation tissue formation. These findings were also corroborated in a special splint excision wound model. Our study shows that although R-Ras does not influence the skin wound healing itself, the blood vessels lacking R-Ras are leaky and thus could facilitate the access of blood-derived proteins to the wound.

Functional Voltage-Gated Calcium Channels Are Present in Human Embryonic Stem Cell-Derived Retinal Pigment Epithelium.

Retinal pigment epithelium (RPE) performs important functions for the maintenance of photoreceptors and vision. Malfunctions within the RPE are implicated in several retinal diseases for which transplantations of stem cell-derived RPE are promising treatment options. Their success, however, is largely dependent on the functionality of the transplanted cells. This requires correct cellular physiology, which is highly influenced by the various ion channels of RPE, including voltage-gated Ca2+ (CaV ) channels. This study investigated the localization and functionality of CaV channels in human embryonic stem cell (hESC)-derived RPE. Whole-cell patch-clamp recordings from these cells revealed slowly inactivating L-type currents comparable to freshly isolated mouse RPE. Some hESC-RPE cells also carried fast transient T-type resembling currents. These findings were confirmed by immunostainings from both hESC- and mouse RPE that showed the presence of the L-type Ca2+ channels CaV 1.2 and CaV 1.3 as well as the T-type Ca2+ channels CaV 3.1 and CaV 3.2. The localization of the major subtype, CaV 1.3, changed during hESC-RPE maturation co-localizing with pericentrin to the base of the primary cilium before reaching more homogeneous membrane localization comparable to mouse RPE. Based on functional assessment, the L-type Ca2+ channels participated in the regulation of vascular endothelial growth factor secretion as well as in the phagocytosis of photoreceptor outer segments in hESC-RPE. Overall, this study demonstrates that a functional machinery of voltage-gated Ca2+ channels is present in mature hESC-RPE, which is promising for the success of transplantation therapies. Stem Cells Translational Medicine 2019;8:179&15.

SWATH-MS Proteomic Analysis of Oxygen-Induced Retinopathy Reveals Novel Potential Therapeutic Targets.

Purpose: Oxygen-induced retinopathy (OIR) is the most widely used model for ischemic retinopathies such as retinopathy of prematurity (ROP), proliferative diabetic retinopathy (PDR), and retinal vein occlusion (RVO). The purpose of this study was to perform the most comprehensive characterization of OIR by a recently developed technique, sequential window acquisition of all theoretical mass spectra (SWATH-MS) proteomics. Methods: Control and OIR retina samples collected from various time points were subjected to SWATH-MS and detailed data analysis. Immunohistochemistry from mouse retinas as well as neovascular membranes from human PDR and RVO patients were used for the detection of the localization of the proteins showing altered expression in the retina and to address their relevance to human ischemic retinopathies. Results: We report the most extensive proteomic profiling of OIR to date by quantifying almost 3000 unique proteins and their expression differences between control and OIR retinas. Crystallins were the most prominent proteins induced by hypoxia in the retina, while angiogenesis related proteins such as Filamin A and nonmuscle myosin IIA stand out at the peak of angiogenesis. Majority of the changes in protein expression return to normal at P42, but there is evidence to suggest that proteins involved in neurotransmission remain at reduced level. Conclusions: The results reveal new potential therapeutic targets to address hypoxia-induced pathological angiogenesis taking place in number of retinal diseases. The extensive proteomic profiling combined with pathway analysis also identifies novel molecular networks that could contribute to the pathogenesis of retinal diseases.

Outcome of anti-vascular endothelial growth factor therapy for neovascular age-related macular degeneration in real-life setting.

AIMS: To evaluate outcome of anti-vascular endothelial growth factor (VEGF) therapy for the treatment of neovascular age-related macular degeneration (nAMD) in the real-life setting and to compare incidence of ocular serious adverse events (SAE) after injections administered by nurses and physicians. METHODS: Retrospective, single-centre study. Medical records of patients receiving anti-VEGF treatment for nAMD between 2008 and 2013 with three-loading-dose regimen were evaluated. Outcome measures were baseline visual acuity (VA), change in VA, number of intravitreal injections, incidence of ocular SAE and patients' baseline characteristics affecting VA change. In addition, the number of injections per 1000 citizens living in the serving area and per individuals over 65 years old were estimated. RESULTS: 1349 eyes in 1117 patients received a total of 11 562 intravitreal anti-VEGF injections. Twenty-one per cent of patients received treatment for both eyes. The mean baseline Snellen VA was 0.32. The mean change of VA from baseline was +2, +2 and ±0 Early Treatment Diabetic Retinopathy Study letters and the mean numbers of injections were 5.7, 4.7 and 4.9 at years 1, 2 and 3, respectively. There was a negative correlation between baseline VA and change of VA. Incidence of endophthalmitis was 0.086%. No difference in the incidence of ocular SAE was identified between injections given by nurses or by physicians. The number of intravitreal injections per all citizens was 9 per 1000 inhabitants and 45 per 1000 inhabitants over 65 years. CONCLUSION: The VA was maintained at the baseline level (±0 letters) with the mean of 15.3 anti-VEGF injections in real-world clinical practice during 3-year follow-up.

Furin deficiency in myeloid cells leads to attenuated revascularization in a mouse-model of oxygen-induced retinopathy.

Ischemic retinopathy is a vision-threatening disease associated with chronic retinal inflammation and hypoxia leading to abnormal angiogenesis. Furin, a member of the proprotein convertase family of proteins, has been implicated in the regulation of angiogenesis due to its essential role in the activation of several angiogenic growth factors, including vascular endothelial growth factor-C (VEGF-C), VEGF-D and transforming growth factor - ß (TGF- ß). In the present study, we evaluated expression of furin in the retina and its role in retinal angiogenesis. As both inflammation and hypoxia contribute to angiogenesis, the role of furin was evaluated using myeloid-cell specific furin knockout (KO) mice (designated LysMCre-fur(fl/fl)) both in developmental retinal angiogenesis as well as in hypoxia-driven angiogenesis using the oxygen-induced retinopathy (OIR) model. In the retina, furin expression was detected in endothelial cells, macrophages and, to some extent, in neurons. The rate of angiogenesis was not different in LysMCre-fur(fl/fl) mice when compared to their wild-type littermates during development. In the OIR model, the revascularization of retina was significantly delayed in LysMCre-fur(fl/fl) mice compared to their wild-type littermates, while there was no compensatory increase in the preretinal neovascularization in LysMCre-fur(fl/fl) mice. These results demonstrate that furin expression in myeloid cells plays a significant role in hypoxia-induced angiogenesis in retina.

The Finnish national guideline for diagnosis, treatment and follow-up of patients with wet age-related macular degeneration.

Age-related macular degeneration (AMD) is the main cause of visual impairment in developed countries. Several improvements in the visualization of posterior segment of the eye together with the introduction of intravitreal anti-VEGF treatment have revolutionized the prognosis of the wet form of AMD (wAMD). Increasing incidence of wAMD together with the limited resources of society and of the healthcare system poses challenges for the provision and development of care. In context of these current aspects, we aimed to set evidence-based medical guidelines for diagnosis, treatment and follow-up of patients with wAMD.