Donor Eyes Research Articles

Tissue-Specific Immune Transcriptional Signatures in the Bordering Tissues of the Mouse Retina and Brain.

Bordering the central nervous system (CNS) parenchyma are the choroid (underlying the retina) and the leptomeninges (the inner layers of the meninges enveloping the brain). Although near the neural parenchyma, the choroid and leptomeninges are external to the immune privileged environment of the retina and brain and thus are distinct immune compartments. This study aimed to characterize the transcriptomic signatures of immune cells within the choroid and leptomeninges bordering the healthy adult mouse CNS. Eyes and brains were obtained from 7-week-old C57Bl/6J mice. Choroid and leptomeninges were processed for isolation of CD45+ immune cells and single cell RNA-sequencing. Additionally, single cell RNA-sequencing was performed on immune cells isolated from choroid obtained from human donor eye tissue. Immunostaining and confocal microscopy of wholemount tissue were used to validate selected immune cell populations in situ. A total of 3606 cells were sequenced from mouse tissues, including 2125 CD45+ cells from choroid and 1481 CD45+ cells from leptomeninges. Clustering and differential gene expression analysis revealed heterogeneous subtypes of monocytes/macrophages, dendritic cells, T cells, and B cells. Whereas some clusters were common to both choroid and leptomeninges, others exhibited tissue-specific gene expression profiles and potential functional specializations. Analysis of 6501 CD45+ cells sequenced from human choroid identified similar immune cell populations to mouse choroid. This study provides a detailed characterization of the molecular signatures of immune cells within the vascular connective tissues bordering the healthy retina and brain, and their potential roles in immune protection.

Evaluation of Cross-Linked Actin Networks (CLANs) in Human Trabecular Meshwork Cells and Tissues.

Elevated intraocular pressure (IOP) is a major risk factor for the development and progression of glaucoma, the leading cause of irreversible vision loss and blindness. An overall increase in resistance to aqueous humor outflow causes sustained elevation in IOP. Glaucomatous insults in the aqueous humor outflow pathway, including the trabecular meshwork (TM), precede such chronic physiological changes in IOP. These insults include ultrastructural changes with excessive extracellular matrix deposition and actin cytoskeletal reorganization that leads to pathological stiffening of the ocular tissues. One of the most common cytoskeletal changes associated with TM tissue stiffness in glaucoma is the increased prevalence of cross-linked actin networks (CLANs) in cells of the trabecular meshwork (TM) and lamina cribrosa (LC). In glaucomatous cells, rearrangement of linear actin stress fibers leads to formation of polygonal arrays within the cytoplasm, resembling a geodesic dome-like structure, that we identified as CLANs. In addition to increased amounts of CLANs in POAG TM cells and tissues, we also discovered that glucocorticoid (GC) and TGFβ2 signaling pathways associated with the development of ocular hypertension (OHT) and glaucoma also induced CLANs in the TM. Despite a clear association, we are yet to completely understand the mechanisms involved in CLAN formation and their direct relevance to disease pathology. In this chapter, we will describe methods to identify and characterize CLANs using fluorescent microscopy in primary TM cell cultures, ex vivo perfusion cultured human anterior segments, and in situ in human donor eyes.

Human Anterior Segment Perfusion Organ Culture.

The human anterior segment perfusion organ culture is an ex vivo model system for studying the human conventional outflow pathway with reference to pressure regulation. In this model, anterior segments dissected from human donor eyes can be fixed to a modified petri dish and perfused with media along with various study agents at the physiological flow rate of 2.5μL/min. The model mimics the one-way flow of aqueous humor in human eyes and can be used to evaluate the effects of various drugs on eye pressure in real time. Using this model, cells and tissues of the anterior segment can be maintained for up to 28days, enabling histological and molecular evaluations.

Autologous Contralateral and Ipsilateral Rotational Penetrating Keratoplasty - A Case Series and Mini-Review.

Corneal stromal opacities can severely impact visual acuity if they are located in the visual axis. Homologous penetrating keratoplasty (HPK) is usually the preferred surgical option in such clinical circumstances. However, autologous penetrating keratoplasty (APK) could be an immunologically safer alternative. The purpose of this study was to report the surgical technique, indications, and (dis)advantages of ipsilateral rotational and contralateral APK, as well as pre- and postoperative clinical findings of four patients who underwent contralateral APK. In ipsilateral rotational APK, eccentric trephination places the central corneal opacity at the excision edge, whereafter the corneal button is rotated to clear the visual axis. Contralateral APK is suitable for more specific clinical situations with corneal opacity in a functionally much better eye. Clear cornea of the (almost) blind eye suffering from a noncorneal pathology is transplanted to the potentially better seeing eye suffering from a corneal pathology, followed by HPK in the (almost) blind donor eye. After 18 months, potentially better-seeing eyes improved from logMAR 1.3 to 0.6 in visual acuity, with most HPK-treated weaker eyes matching preoperative levels. Considering ipsilateral rotational and contralateral APK before moving on straight towards HPK is crucial in select cases. Autologous contralateral keratoplasty should be preferred for patients with (1) corneal scars in a potentially better seeing eye and (2) a clear cornea in an (almost) blind eye, especially in cases of high risk for graft rejection.

Innovative surgical treatment approaches for endothelial dysfunction : Descemet stripping only (DSO) and endothelial cell injection

Currently, due to arising number of patients Germany and many other countries experience alarge deficit of donor eyes for posterior lamellar keratoplasty procedures in the treatment of corneal endothelial diseases. To address this unmet need there is an ongoing investigation of treatment modalities which do not rely on donor tissue or enable clinicians to treat more patient eyes per donor eye. The authors introduce apromising approach for both treatment principles. First, the technique of Descemet stripping only (DSO) is detailed, in which acentral part of the Descemet's membrane including the endothelium is surgically removed without replacement with donor tissue. This then allows endothelial cells from the periphery of the cornea to migrate into the central area and can reduce corneal opacification and swelling. As arepresentative technique of the second group, the authors introduce endothelial cell injection, in which human corneal endothelial cells are cultivated in vitro and then, after removal of the diseased endothelium, injected into the anterior chamber of the recipient's eye to form anew and healthy endothelium. This is supported by injection of Rho kinase inhibitors and aface-down positioning of the patient after surgery. It is postulated that endothelial cell injection could possibly enable clinicians to treat up to 300 patient eyes with the tissue generated from 1 donor eye. Whether and how these novel approaches will become established in Europe remains to be seen.

Midterm results after allogeneic simple limbal epithelial transplantation from deceased-donor eyes in patients with persistent corneal epithelial defects due to limbal stem cell deficiency.

This study aims to characterize the clinical outcomes after allogeneic simple limbal epithelial transplantation (alloSLET) utilizing tissue from cadaveric donor eyes to address persistent corneal epithelial defects caused by limbal stem cell deficiency. We conducted a retrospective analysis of medical records from 20 patients, encompassing 24 eyes, who underwent alloSLET at least 2 years prior. The primary endpoint was the achievement of complete epithelialization of the corneal surface by corneal epithelium. Secondary endpoints included corrected distance visual acuity (CDVA) and postoperative adverse events. The median postoperative follow-up period was 36 months (range, 24-74 months). At 1, 3 and 6 months post-surgery, 96% of eyes demonstrated epithelialized corneal surfaces, which declined to 71% at 12 months, to 54% at 24 and 36 months after surgery, and to 50% thereafter. There were no significant differences in graft survival between alloSLET performed alone versus in combination with penetrating keratoplasty. However, instances of graft failure were associated with postoperative elevated intraocular pressure (IOP) and a history of multiple amniotic membrane and corneal graft transplants. AlloSLET emerges as a viable mid-term intervention for limbal stem cell deficiency-associated non-healing corneal epithelial defects in the absence of autologous limbal tissue. Our findings underscore the increased risk of graft failure in patients with elevated IOP and a background of multiple previous amniotic membrane and corneal graft procedures.

3D printed gelatin methacryloyl hydrogels for perfusion culture of human trabecular meshwork cells and glaucoma studies.

Glaucoma, a progressive eye disease leading to irreversible blindness, currently affects over 70 million people globally. Elevated intraocular pressure (IOP) is implicated in its development. IOP is carefully regulated by the trabecular meshwork (TM). However, studying TM behavior has been limited to traditional tissue culture studies or costly ex vivo cultures of animal and donor eyes. Developing novel functional TM models could enhance cell/tissue behavior understanding and aid therapeutic development for glaucoma. In this study, we 3D printed a simplified and reproducible model of the human TM (hTM) and studied hTM cell behavior under static and dynamic cultures. Gelatin Methacryloyl bioinks proved suitable for printing with viable and proliferative hTM cells expressing crucial marker genes in response to glucocorticoid induction. This, to our knowledge, is the first functional 3D printed hTM model and aims to facilitate TM research. Moreover, this easily reproducible model could also be applicable in the study of numerous other cell types throughout the body.

Evaluation of Sociomedical Factors on Corneal Donor Recovery Using Machine Learning

ABSTRACT Purpose To evaluate co-morbid sociomedical conditions affecting corneal donor endothelial cell density and transplant suitability. Method(s) Corneal donor transplant information was collected from the CorneaGen eye bank between June 1, 2012 and June 30, 2016. A natural language processing algorithm was applied to generate co-morbid sociomedical conditions for each donor. Variables of importance were identified using four machine learning models (random forest, Glmnet, Earth, nnet), for the outcomes of transplant suitability and endothelial cell density. SHAP (SHapley Additive exPlanations) values were generated, with beeswarm and box plots to visualize the contribution of each feature to the models. Results With a total of 23,522 unique donors, natural language processing generated 30,573 indices, which were reduced to 41 most common co-morbid sociomedical conditions. For transplant suitability, hypertension ranked the top overall variable of importance in two models. Hypertension, chronic obstructive pulmonary disease, history of smoking, and alcohol use appeared consistently in the top variables of importance. By SHAP feature importance, hypertension (0.042), alcohol use (0.017), ventilation of donor (0.011), and history of smoking (0.010) contributed the most to the transplant suitability model. For endothelial cell density, hypertension was the sociomedical condition of highest importance in three models. SHAP scores were highest among the sociomedical conditions of hypertension (0.037), alcohol use (0.013), myocardial infarction (0.012), and history of smoking (0.011). Conclusion In a large cohort of corneal donor eyes, hypertension was identified as the most common contributor to machine learning models examining sociomedical conditions for corneal donor transplant suitability and endothelial cell density.

βA3/A1-crystallin is an epigenetic regulator of histone deacetylase 3 (HDAC3) in the retinal pigmented epithelial (RPE) cells.

The retinal pigmented epithelial (RPE) cells maintain retinal homeostasis, and alterations in their function contribute to non-exudative age-related macular degeneration (AMD) 1,2 . Here, we explore the intricate relationship between RPE cells, epigenetic modifications, and the development of AMD. Importantly, the study reveals a substantial decrease in histone deacetylase 3 (HDAC3) activity and elevated histone acetylation in the RPE of human AMD donor eyes. To investigate epigenetic mechanisms in AMD development, we used a mouse model with RPE-specific Cryba1 knockout 3-5 , revealing that the loss of βA3/A1-crystallin selectively reduces HDAC3 activity, resulting in increased histone acetylation. βA3/A1-crystallin activates HDAC3 by facilitating its interaction with the casein kinase II (CK2) and phosphorylating HDAC3, as well as by regulating intracellular InsP6 (phytic acid) levels, required for activating HDAC3. These findings highlight a novel function of βA3/A1-crystallin as an epigenetic regulator of HDAC3 in the RPE cells and provide insights into potential therapeutic strategies in non-exudative AMD.

GSK3β Inhibitors Inhibit TGFβ Signaling in the Human Trabecular Meshwork.

Primary open-angle glaucoma (POAG) is a leading cause of blindness, and its primary risk factor is elevated intraocular pressure (IOP) due to pathologic changes in the trabecular meshwork (TM). We previously showed that there is a cross-inhibition between TGFβ and Wnt signaling pathways in the TM. In this study, we determined if activation of the Wnt signaling pathway using small-molecule Wnt activators can inhibit TGFβ2-induced TM changes and ocular hypertension (OHT). Primary human TM (pHTM) cells and transduced SBE-GTM3 cells were treated with or without Wnt and/or TGFβ signaling activators and used for luciferase assays; for the extraction of whole-cell lysate, conditioned medium, cytosolic proteins, and nuclear proteins for Western immunoblotting (WB); or for immunofluorescent staining. Human donor eyes were perfusion cultured to study the effect of Wnt activators on IOP. We found that the small-molecule Wnt activators (GSK3β inhibitors) (BIO, SB216763, and CHIR99021) activated canonical Wnt signaling in pHTM cells without toxicity at tested concentrations. This activation inhibited TGFβ signaling as well as TGFβ2-induced extracellular matrix deposition and formation of cross-linked actin networks in pHTM cells or SBE-GTM3 cells. We also observed nuclear translocation of both Smad4 and β-catenin in pHTM cells, which suggested that the cross-inhibition between the TGFβ and Wnt signaling pathways may occur in the nucleus. Using our ex vivo model, we found that CHIR99021 inhibited TGFβ2-induced OHT in perfusion-cultured human eyes. Our results showed that small-molecule Wnt activators have the potential for treating TGFβ signaling-induced OHT in patients with POAG.

Anterior Chamber Air Bubble Dynamics With Decreases in Atmospheric Pressure.

To evaluate the effect of decreasing barometric pressure on intracameral bubble size and intraocular pressure (IOP) in eyes with varying air fills in the anterior chamber. A total of 36 human donor eyes received 30%, 50%, or 90% anterior chamber air fill. The eyes were subjected to decreases in atmospheric pressure down to 750 hPa, equal to 2400 m in altitude, and were repeatedly imaged using anterior segment OCT while IOP was measured continuously. Eyes with 30% air fill initially showed moderate increases in IOP yet rising to an average of 30.83 mm Hg at 850 hPa (mimicking 1400 m altitude) and 42.08 mm Hg at 750 hPa. Eyes with larger air bubbles showed more acute increases in IOP with increases to an average of 47.25 mm Hg in eyes with 50% air fill at 850 hPa and 63.33 mm Hg at 750 hPa. In eyes with 90% air fill in the anterior chamber, IOP readings with an average of 113.42 mm Hg were observed already at 850 hPa, at which point additional pressure reduction was not performed. While severe increases in IOP were observed with decreased atmospheric pressure in eyes with large air bubbles in the anterior chamber, small and moderately sized bubbles seem to allow for travel over modest changes in altitude.

Neuritin 1 Drives Therapeutic Preservation of Retinal Ganglion Cells in an Ex Vivo Human Glaucoma Model.

Purpose: Glaucoma is a leading cause of irreversible blindness. Glaucomatous intraocular pressure (IOP) triggers deleterious effects, including gliosis, optic nerve (ON) axonal retraction, neurotrophic factor deprivation, inflammation, and other pathological events, leading to retinal ganglion cell (RGC) loss. Trophic factor impairment enhances RGC apoptosis susceptibility. Neuritin 1 (NRN1), a neurotrophic protein downstream of various neurotrophins, exhibited RGC protection and regeneration in axotomy models. We evaluated human recombinant NRN1's impact on human RGCs cultured in pressurized conditions within the ex vivo translaminar autonomous system to simulate glaucoma pathogenesis. Methods: Human glaucomatous and non-glaucomatous donor eyes were obtained from eye banks according to the Declaration of Helsinki. Initially, we evaluated NRN1and RGC marker expression in glaucoma and non-glaucomatous retina to determine the NRN1 level and its association with RGC loss. Further, we evaluated NRN1's therapeutic potential by treating pressurized human eyes at normal and high IOP for seven days. Retina, ON, and conditioned medium were analyzed for RGC survival (THY1, RBPMS), gliosis (GFAP), apoptosis (CASP3, CASP7), and extracellular matrix deposition (COLIV, FN) by qRT-PCR and western blotting. Paraphenylenediamine staining assessed ON axonal degeneration, whereas ex vivo electroretinogram assessed retinal activity. Results: Glaucomatous retinas exhibited significant reductions in both NRN1 (*p = 0.007, n = 5) and RGC marker expression (*p = 0.04, n = 5). NRN1 treatment reduced gliosis, extracellular matrix deposition, ON degeneration, and increased retinal activity in pressure-perfused eyes. Conclusions: Our study confirms that NRN1 enhances human RGC survival and improves retinal function in degenerative conditions, substantiating it as a promising candidate for rescuing human RGCs from degeneration.

Whole-eye transplantation: Current challenges and future perspectives.

Whole-eye transplantation emerges as a frontier in ophthalmology, promising a transformative approach to irreversible blindness. Despite advancements, formidable challenges persist. Preservation of donor eye viability post-enucleation necessitates meticulous surgical techniques to optimize retinal integrity and ganglion cell survival. Overcoming the inhibitory milieu of the central nervous system for successful optic nerve regeneration remains elusive, prompting the exploration of neurotrophic support and immunomodulatory interventions. Immunological tolerance, paramount for graft acceptance, confronts the distinctive immunogenicity of ocular tissues, driving research into targeted immunosuppression strategies. Ethical and legal considerations underscore the necessity for stringent standards and ethical frameworks. Interdisciplinary collaboration and ongoing research endeavors are imperative to navigate these complexities. Biomaterials, stem cell therapies, and precision immunomodulation represent promising avenues in this pursuit. Ultimately, the aim of this review is to critically assess the current landscape of whole-eye transplantation, elucidating the challenges and advancements while delineating future directions for research and clinical practice. Through concerted efforts, whole-eye transplantation stands to revolutionize ophthalmic care, offering hope for restored vision and enhanced quality of life for those afflicted with blindness.

PGC-1α repression dysregulates lipid metabolism and induces lipid droplet accumulation in the retinal pigment epithelium

Drusen, the yellow deposits under the retina, are composed of lipids and proteins, and represent a hallmark of age-related macular degeneration (AMD). Lipid droplets are also reported in the retinal pigment epithelium (RPE) from AMD donor eyes. However, the mechanisms underlying these disease phenotypes remain elusive. Previously, we showed that Pgc-1α repression, combined with a high-fat diet (HFD), induce drastic AMD-like phenotypes in mice. We also reported increased PGC-1α acetylation and subsequent deactivation in the RPE derived from AMD donor eyes. Here, through a series of in vivo and in vitro experiments, we sought to investigate the molecular mechanisms by which PGC-1α repression could influence RPE and retinal function. We show that PGC-1α plays an important role in RPE and retinal lipid metabolism and function. In mice, repression of Pgc-1α alone induced RPE and retinal degeneration and drusen-like deposits. In vitro inhibition of PGC1A by CRISPR-Cas9 gene editing in human RPE (ARPE19- PGC1A KO) affected the expression of genes responsible for lipid metabolism, fatty acid β-oxidation (FAO), fatty acid transport, low-density lipoprotein (LDL) uptake, cholesterol esterification, cholesterol biosynthesis, and cholesterol efflux. Moreover, inhibition of PGC1A in RPE cells caused lipid droplet accumulation and lipid peroxidation. ARPE19-PGC1A KO cells also showed reduced mitochondrial biosynthesis, impaired mitochondrial dynamics and activity, reduced antioxidant enzymes, decreased mitochondrial membrane potential, loss of cardiolipin, and increased susceptibility to oxidative stress. Our data demonstrate the crucial role of PGC-1α in regulating lipid metabolism. They provide new insights into the mechanisms involved in lipid and drusen accumulation in the RPE and retina during aging and AMD, which may pave the way for developing novel therapeutic strategies targeting PGC-1α.

Comparative 3D genome analysis between neural retina and retinal pigment epithelium reveals differential cis-regulatory interactions at retinal disease loci

BackgroundVision depends on the interplay between photoreceptor cells of the neural retina and the underlying retinal pigment epithelium (RPE). Most genes involved in inherited retinal diseases display specific spatiotemporal expression within these interconnected retinal components through the local recruitment of cis-regulatory elements (CREs) in 3D nuclear space.ResultsTo understand the role of differential chromatin architecture in establishing tissue-specific expression at inherited retinal disease loci, we mapped genome-wide chromatin interactions using in situ Hi-C and H3K4me3 HiChIP on neural retina and RPE/choroid from human adult donor eyes. We observed chromatin looping between active promoters and 32,425 and 8060 candidate CREs in the neural retina and RPE/choroid, respectively. A comparative 3D genome analysis between these two retinal tissues revealed that 56% of 290 known inherited retinal disease genes were marked by differential chromatin interactions. One of these was ABCA4, which is implicated in the most common autosomal recessive inherited retinal disease. We zoomed in on retina- and RPE-specific cis-regulatory interactions at the ABCA4 locus using high-resolution UMI-4C. Integration with bulk and single-cell epigenomic datasets and in vivo enhancer assays in zebrafish revealed tissue-specific CREs interacting with ABCA4.ConclusionsThrough comparative 3D genome mapping, based on genome-wide, promoter-centric, and locus-specific assays of human neural retina and RPE, we have shown that gene regulation at key inherited retinal disease loci is likely mediated by tissue-specific chromatin interactions. These findings do not only provide insight into tissue-specific regulatory landscapes at retinal disease loci, but also delineate the search space for non-coding genomic variation underlying unsolved inherited retinal diseases.Graphical

Isolation of Primary Porcine Retinal Pigment Epithelial Cells for In Vitro Modeling.

The retinal pigment epithelium (RPE) is a crucial monolayer in the outer retina responsible for supporting photoreceptors. RPE degeneration commonly occurs in diseases marked by progressive vision loss, such as age-related macular degeneration (AMD). Research on AMD often relies on human donor eyes or induced pluripotent stem cells (iPSCs) to represent the RPE. However, these RPE sources require extended differentiation periods and substantial expertise for culturing. Additionally, some research institutions, particularly those in rural areas, lack easy access to donor eyes. While a commercially available immortalized RPE cell line (ARPE-19) exists, it lacks essential in vivo RPE features and is not widely accepted in many ophthalmology research publications. There is a pressing need to obtain representative primary RPE cells from a more readily available and cost-effective source. This protocol elucidates the isolation and subculture of primary RPE cells obtained post-mortem from porcine eyes, which can be sourced locally from commercial or academic suppliers. This protocol necessitates common materials typically found in tissue culture labs. The result is a primary, differentiated, and cost-effective alternative to iPSCs, human donor eyes, and ARPE-19 cells.

Spatial targeted delivery of riboflavin with a controlled corneal iontophoresis delivery system in theranostic-guided UV-A light photo-therapy.

Seven human donor eye globes underwent corneal cross-linking using theranostic UV-A device with accessory corneal iontophoresis system for patterned delivery of a 0.22% riboflavin solution. Theranostic-guided UV-A light illumination assessed riboflavin distribution and treated corneas at 10 mW/cm2 for 9 min with a 5.0-mm beam size. Corneal topography maps were taken at baseline and 2-h post-treatment. Analysis utilized corneal topography elevation data, with results showing controlled riboflavin delivery led to a consistent gradient, with 40% higher levels centrally (248 ± 79 μg/cm3) than peripherally (180 ± 72 μg/cm3 at ±2.5 mm from the center). Theranostic-guided UV-A light irradiation resulted in significant changes in corneal topography, with a decrease in best-fit sphere value (-0.7 ± 0.2 D; p < 0.001) and consistent downward shift in corneal elevation map (-11.7 ± 3.7 μm). The coefficient of variation was 2.5%, indicating high procedure performance in achieving significant and reliable corneal flattening.

Levels of the HtrA1 Protein in Serum and Vitreous Humor Are Independent of Genetic Risk for Age-Related Macular Degeneration at the 10q26 Locus.

The purpose of this study was to determine if levels of the HtrA1 protein in serum or vitreous humor are influenced by genetic risk for age-related macular degeneration (AMD) at the 10q26 locus, age, sex, AMD status, and/or AMD disease severity, and, therefore, to determine the contribution of systemic and ocular HtrA1 to the AMD disease process. A custom-made sandwich ELISA assay (SCTM ELISA) for detection of the HtrA1 protein was designed and compared with three commercial assays (R&D Systems, MyBiosource 1 and MyBiosource 2) using 65 serum samples. Concentrations of HtrA1 were thereafter determined in serum and vitreous samples collected from 248 individuals and 145 human donor eyes, respectively. The SCTM ELISA demonstrated high specificity, good recovery, and parallelism within its linear detection range and performed comparably to the R&D Systems assay. In contrast, we were unable to demonstrate the specificity of the two assays from MyBioSource using either recombinant or native HtrA1. Analyses of concentrations obtained using the validated SCTM assay revealed that genetic risk at the 10q26 locus, age, sex, or AMD status are not significantly associated with altered levels of the HtrA1 protein in serum or in vitreous humor (P > 0.05). HtrA1 levels in serum and vitreous do not reflect the risk for AMD associated with the 10q26 locus or disease status. Localized alteration in HTRA1 expression in the retinal pigment epithelium, rather than systemic changes in HtrA1, is the most likely driver of elevated risk for developing AMD among individuals with risk variants at the 10q26 locus.

ANGPTL7 and Its Role in IOP and Glaucoma.

Loss-of-function variants in the ANGPTL7 gene are associated with protection from glaucoma and reduced intraocular pressure (IOP). We investigated the role of ANGPTL7 in IOP homeostasis and its potential as a target for glaucoma therapeutics. IOP, outflow facility, and outflow tissue morphology of Angptl7 knockout (KO) mice were assessed with and without dexamethasone (Dex). ANGPTL7 was quantified in conditioned media from human trabecular meshwork cells in response to Dex, in effluent from perfused human donor eyes, and in aqueous humor from human patients treated with steroids. Antibodies to ANGPTL7 were generated and tested in three-dimensional (3D) culture of outflow cells and perfused human donor eyes. Rabbits were injected intravitreally with a neutralizing antibody targeting ANGPTL7, and IOP was measured. IOP was significantly elevated, but outflow facility and outflow tissue morphology were not different between Angptl7 KO mice and littermates. When challenged with Dex, IOP increased in wild-type but not Angptl7 KO mice. In human samples, increased ANGPTL7 was seen in the aqueous humor of patients treated with steroids, regardless of glaucoma status. Using 3D culture, recombinant ANGPTL7 decreased, and ANGPTL7-blocking antibodies increased hydraulic conductivity. Significantly, outflow facility increased in human eyes treated ex vivo with ANGPTL7-blocking antibodies, and IOP decreased for 21 days in rabbits after a single injection of blocking antibodies. Using multiple models, we have demonstrated that excess ANGPTL7 increases outflow resistance and IOP and that neutralizing ANGPTL7 has beneficial effects in both naïve and steroid-induced hypertensive eyes, thus motivating the development of ANGPTL7-targeting therapeutics for the treatment of glaucoma.

Granzyme B degrades extracellular matrix and promotes inflammation and choroidal neovascularization

Age-related macular degeneration (AMD) is a common retinal neurodegenerative disease among the elderly. Neovascular AMD (nAMD), a leading cause of AMD-related blindness, involves choroidal neovascularization (CNV), which can be suppressed by anti-angiogenic treatments. However, current CNV treatments do not work in all nAMD patients. Here we investigate a novel target for AMD. Granzyme B (GzmB) is a serine protease that promotes aging, chronic inflammation and vascular permeability through the degradation of the extracellular matrix (ECM) and tight junctions. Extracellular GzmB is increased in retina pigment epithelium (RPE) and mast cells in the choroid of the healthy aging outer retina. It is further increased in donor eyes exhibiting features of nAMD and CNV. Here, we show in RPE-choroidal explant cultures that exogenous GzmB degrades the RPE-choroid ECM, promotes retinal/choroidal inflammation and angiogenesis while diminishing anti-angiogenic factor, thrombospondin-1 (TSP-1). The pharmacological inhibition of either GzmB or mast-cell degranulation significantly reduces choroidal angiogenesis. In line with our in vitro data, GzmB-deficiency reduces the extent of laser-induced CNV lesions and the age-related deterioration of electroretinogram (ERG) responses in mice. These findings suggest that targeting GzmB, a serine protease with no known endogenous inhibitors, may be a potential novel therapeutic approach to suppress CNV in nAMD.