Congenital microcoria deletion in mouse links Sox21 dysregulation to disease and suggests a role for TGFB2 in glaucoma and myopia.

Congenital microcoria (MCOR) is a rare hereditary developmental defect of the iris dilator muscle frequently associated with high axial myopia and high intraocular pressure (IOP) glaucoma. The condition is caused by submicroscopic rearrangements of chromosome 13q32.1. However, the mechanisms underlying the failure of iris development and the origin of associated features remain elusive. Here, we present a 3D architecture model of the 13q32.1 region, demonstrating that MCOR-related deletions consistently disrupt the boundary between two topologically associating domains (TADs). Deleting the critical MCOR-causing region in mice reveals ectopic Sox21 expression precisely aligning with Dct, each located in one of the two neighbor TADs. This observation is consistent with the TADs' boundary alteration and adoption of Dct regulatory elements by the Sox21 promoter. Additionally, we identify Tgfb2 as a target gene of SOX21 and show TGFΒ2 accumulation in the aqueous humor of an MCOR-affected subject. Accumulation of TGFB2 is recognized for its role in glaucoma and potential impact on axial myopia. Our results highlight the importance of SOX21-TGFB2 signaling in iris development and control of eye growth and IOP. Insights from MCOR studies may provide therapeutic avenues for this condition but also for glaucoma and high myopia conditions, affecting millions of people.

High throughput functional profiling of genes at intraocular pressure loci reveals distinct networks for glaucoma.

INTRODUCTION: Primary open angle glaucoma (POAG) is a leading cause of blindness globally. Characterized by progressive retinal ganglion cell degeneration, the precise pathogenesis remains unknown. Genome-wide association studies (GWAS) have uncovered many genetic variants associated with elevated intraocular pressure (IOP), one of the key risk factors for POAG. We aimed to identify genetic and morphological variation that can be attributed to trabecular meshwork cell (TMC) dysfunction and raised IOP in POAG. METHODS: 62 genes across 55 loci were knocked-out in a primary human TMC line. Each knockout group, including five non-targeting control groups, underwent single-cell RNA-sequencing (scRNA-seq) for differentially-expressed gene (DEG) analysis. Multiplexed fluorescence coupled with CellProfiler image analysis allowed for single-cell morphological profiling. RESULTS: Many gene knockouts invoked DEGs relating to matrix metalloproteinases and interferon-induced proteins. We have prioritized genes at four loci of interest to identify gene knockouts that may contribute to the pathogenesis of POAG, including ANGPTL2, LMX1B, CAV1, and KREMEN1. Three genetic networks of gene knockouts with similar transcriptomic profiles were identified, suggesting a synergistic function in trabecular meshwork cell physiology. TEK knockout caused significant upregulation of nuclear granularity on morphological analysis, while knockout of TRIOBP, TMCO1 and PLEKHA7 increased granularity and intensity of actin and the cell-membrane. CONCLUSION: High-throughput analysis of cellular structure and function through multiplex fluorescent single-cell analysis and scRNA-seq assays enabled the direct study of genetic perturbations at the single-cell resolution. This work provides a framework for investigating the role of genes in the pathogenesis of glaucoma and heterogenous diseases with a strong genetic basis.

Spaceflight-Induced Intracranial Hypertension and Visual Impairment: Pathophysiology and Countermeasures.

Visual impairment intracranial pressure (VIIP) syndrome is considered an unexplained major risk for future long-duration spaceflight. NASA recently redefined this syndrome as Spaceflight-Associated Neuro-ocular Syndrome (SANS). Evidence thus reviewed supports that chronic, mildly elevated intracranial pressure (ICP) in space (as opposed to more variable ICP with posture and activity on Earth) is largely accounted for by loss of hydrostatic pressures and altered hemodynamics in the intracranial circulation and the cerebrospinal fluid system. In space, an elevated pressure gradient across the lamina cribrosa, caused by a chronic but mildly elevated ICP, likely elicits adaptations of multiple structures and fluid systems in the eye which manifest themselves as the VIIP syndrome. A chronic mismatch between ICP and intraocular pressure (IOP) in space may acclimate the optic nerve head, lamina cribrosa, and optic nerve subarachnoid space to a condition that is maladaptive to Earth, all contributing to the pathogenesis of space VIIP syndrome. Relevant findings help to evaluate whether artificial gravity is an appropriate countermeasure to prevent this seemingly adverse effect of long-duration spaceflight.

Rare protective variants and glaucoma-relevant cell stressors modulate Angiopoietin-like 7 expression.

Rare missense and nonsense variants in the Angiopoietin-like 7 (ANGPTL7) gene confer protection from primary open-angle glaucoma (POAG), though the functional mechanism remains uncharacterized. Interestingly, a larger variant effect size strongly correlates with in silico predictions of increased protein instability (r = -0.98), suggesting that protective variants lower ANGPTL7 protein levels. Here, we show that missense and nonsense variants cause aggregation of mutant ANGPTL7 protein in the endoplasmic reticulum (ER) and decreased levels of secreted protein in human trabecular meshwork (TM) cells; a lower secreted:intracellular protein ratio strongly correlates with variant effects on intraocular pressure (r = 0.81). Importantly, accumulation of mutant protein in the ER does not increase expression of ER stress proteins in TM cells (P > 0.05 for all variants tested). Cyclic mechanical stress, a glaucoma-relevant physiologic stressor, also significantly lowers ANGPTL7 expression in primary cultures of human Schlemm's canal (SC) cells (-2.4-fold-change, P = 0.01). Collectively, these data suggest that the protective effects of ANGPTL7 variants in POAG stem from lower levels of secreted protein, which may modulate responses to physiologic and pathologic ocular cell stressors. Downregulation of ANGPTL7 expression may therefore serve as a viable preventative and therapeutic strategy for this common, blinding disease.

Enhanced Optic Nerve Expansion and Altered Ultrastructure of Elastic Fibers Induced by Lysyl Oxidase Inhibition in a Mouse Model of Marfan Syndrome.

Two major constituents of exfoliation material, fibrillin-1 and lysyl oxidase-like 1 (encoded by FBN1 and LOXL1), are implicated in exfoliation glaucoma, yet their individual contributions to ocular phenotype are minor. To test the hypothesis that a combination of FBN1 mutation and LOXL1 deficiency exacerbates ocular phenotypes, the pan-lysyl oxidase inhibitor ß-aminopropionitrile (BAPN) was used to treat adult wild-type (WT) mice and mice heterozygous for a missense mutation in Fbn1 (Fbn1C1041G/+) for 8 weeks and their eyes were examined. Although intraocular pressure did not change and exfoliation material was not detected in the eyes, BAPN treatment worsened optic nerve and axon expansion in Fbn1C1041G/+ mice, an early sign of axonal damage in rodent models of glaucoma. Disruption of elastic fibers was detected only in Fbn1C1041G/+ mice, which increased with BAPN treatment, as shown by histologic and immunohistochemical staining of the optic nerve pia mater. Transmission electron microscopy showed that Fbn1C1041G/+ mice had fewer microfibrils, smaller elastin cores, and a lower density of elastic fibers compared with WT mice in control groups. BAPN treatment led to elastin core expansion in both WT and Fbn1C1041G/+ mice, but an increase in the density of elastic fiber was confined to Fbn1C1041G/+ mice. LOX inhibition had a stronger effect on optic nerve and elastic fiber parameters in the context of Fbn1 mutation, indicating the Marfan mouse model with LOX inhibition warrants further investigation for exfoliation glaucoma pathogenesis.

DDX58 variant triggers IFN-ß-induced autophagy in trabecular meshwork and influences intraocular pressure.

Singleton-Merten syndrome (SMS) is a rare immunogenetic disorder affecting multiple systems, characterized by dental dysplasia, aortic calcification, glaucoma, skeletal abnormalities, and psoriasis. Glaucoma, a key feature of both classical and atypical SMS, remains poorly understood in terms of its molecular mechanism caused by DDX58 mutation. This study presented a novel DDX58 variant (c.1649A>C [p.Asp550Ala]) in a family with childhood glaucoma. Functional analysis showed that DDX58 variant caused an increase in IFN-stimulated gene expression and high IFN-ß-based type-I IFN. As the trabecular meshwork (TM) is responsible for controlling intraocular pressure (IOP), we examine the effect of IFN-ß on TM cells. Our study is the first to demonstrate that IFN-ß significantly reduced TM cell viability and function by activating autophagy. In addition, anterior chamber injection of IFN-ß remarkably increased IOP level in mice, which can be attenuated by treatments with autophagy inhibitor chloroquine. To uncover the specific mechanism underlying IFN-ß-induced autophagy in TM cells, we performed microarray analysis in IFN-ß-treated and DDX58 p.Asp550Ala TM cells. It showed that RSAD2 is necessary for IFN-ß-induced autophagy. Knockdown of RSAD2 by siRNA significantly decreased autophagy flux induced by IFN-ß. Our findings suggest that DDX58 mutation leads to the overproduction of IFN-ß, which elevates IOP by modulating autophagy through RSAD2 in TM cells.

Glaucoma-inducing retinal ganglion cell degeneration alters diurnal rhythm of key molecular components of the central clock and locomotor activity in mice.

Glaucoma is a chronic optic neuropathy characterized by the progressive degeneration of retinal ganglion cells (RGC). These cells play a crucial role in transmitting visual and non-visual information to brain regions, including the suprachiasmatic nucleus (SCN), responsible for synchronizing biological rhythms. To understand how glaucoma affects circadian rhythm synchronization, we investigated potential changes in the molecular clock machinery in the SCN. We found that the progressive increase in intraocular pressure (IOP) negatively correlated with spontaneous locomotor activity (SLA). Transcriptome analysis revealed significant alterations in the SCN of glaucomatous mice, including downregulation of genes associated with circadian rhythms. In fact, we showed a loss of diurnal oscillation in the expression of vasoactive intestinal peptide (Vip), its receptor (Vipr2), and period 1 (Per1) in the SCN of glaucomatous mice. These findings were supported by the 7-h phase shift in the peak expression of arginine vasopressin (Avp) in the SCN of mice with glaucoma. Despite maintaining a 24-h period under both light/dark (LD) and constant dark (DD) conditions, glaucomatous mice exhibited altered SLA rhythms, characterized by decreased amplitude. Taken altogether, our findings provide evidence of how glaucoma affects the regulation of the central circadian clock and its consequence on the regulation of circadian rhythms.

Glaucoma-Protective Human Single-Nucleotide Polymorphism in the Angpt2 Locus Increased ANGPT2 Expression and Schlemm Canal Area in Mice-Brief Report.

BACKGROUND: The ANGPT (angiopoietin)-TEK (tyrosine kinase, endothelial) vascular signaling pathway plays a key role in the formation of Schlemm canal, and loss-of-function mutations in the TEK or ANGPT1 gene are associated with primary congenital glaucoma in children. In genome-wide association studies, an association was identified between protection from primary open-angle glaucoma and the single-nucleotide polymorphism rs76020419 (G>T), located within a predicted miR-145-binding site in the 3' untranslated region of ANGPT2. To date, the functional impact of this variant in the anterior chamber of the eye remains largely unexplored. METHODS: MT (mutant) mice harboring an orthologous rs76020419 minor allele (T) were generated using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9). Plasma and tissue samples, including eyes, were collected, and ANGPT2 expression was quantified using ELISA. Anterior segments from eyes were collected from WT (wild-type) and MT mice, and Schlemm canal area was quantified. RESULTS: In the MT group, higher ANGPT2 concentrations were observed in the plasma, lungs, kidneys, and eyes (P=0.0212, P<0.001, P=0.0815, and P=0.0215, respectively). Additionally, the Schlemm canal was larger in MT mice compared with WT mice (P=0.0430). CONCLUSIONS: The rs76020419 minor allele (T) is associated with increased levels of ANGPT2 and a larger Schlemm canal in mice. These findings suggest a potential protective mechanism in glaucoma.

RNA-seq transcriptomic profiling of TGF-ß2-exposed human trabecular meshwork explants: Advancing insights beyond conventional cell culture models.

Primary open-angle glaucoma (POAG), a leading cause of irreversible vision loss, is closely linked to increased intraocular pressure (IOP), with the trabecular meshwork (TM) playing a critical role in its regulation. The TM, located at the iridocorneal angle, acts as a sieve, filtering the aqueous humor from the eye into the collecting ducts, thus maintaining proper IOP levels. The transforming growth factor-beta 2 (TGF-ß2) signaling pathway has been implicated in the pathophysiology of primary open-angle glaucoma POAG particularly, in the dysfunction of the TM. This study utilizes human TM explants to closely mimic in vivo conditions, thereby minimizing transcriptional changes that could arise from cell culture enabling an exploration of the transcriptomic impacts of TGF-ß2. Through bulk RNA sequencing and immunohistological analysis, we identified distinct gene expression patterns and morphological changes induced by TGF-ß2 exposure (5 ng/ml for 48 h). Bulk RNA sequencing identified significant upregulation in genes linked to extracellular matrix (ECM) regulation and fibrotic signaling. Immunohistological analysis further elucidated the morphological alterations, including cytoskeletal rearrangements and ECM deposition, providing a visual confirmation of the transcriptomic data. Notably, the enrichment analysis unveils TGF-ß2's influence on both bone morphogenic protein (BMP) and Wnt signaling pathways, suggesting a complex interplay of molecular mechanisms contributing to TM dysfunction in glaucoma. This characterization of the transcriptomic modifications on an explant model of TM obtained under the effect of this profibrotic cytokine involved in glaucoma is crucial in order to develop and test new molecules that can block their signaling pathways.

NAD+ supplementation improves mitochondrial functions and normalizes glaucomatous trabecular meshwork features.

Glaucoma is characterized by pathological elevation of intraocular pressure (IOP) due to dysfunctional trabecular meshwork (TM), which is the primary cause of irreversible vision loss. There are currently no effective treatment strategies for glaucoma. Mitochondrial function plays a crucial role in regulating IOP within the TM. In this study, primary TM cells treated with dexamethasone were used to simulate glaucomatous changes, showing abnormal cellular cytoskeleton, increased expression of extracellular matrix, and disrupted mitochondrial fusion and fission dynamics. Furthermore, glaucomatous TM cell line GTM3 exhibited impaired mitochondrial membrane potential and phagocytic function, accompanied by decreased oxidative respiratory levels as compared to normal TM cells iHTM. Mechanistically, lower NAD + levels in GTM3, possibly associated with increased expression of key enzymes CD38 and PARP1 related to NAD + consumption, were observed. Supplementation of NAD + restored mitochondrial function and cellular viability in GTM3 cells. Therefore, we propose that the aberrant mitochondrial function in glaucomatous TM cells may be attributed to increased NAD + consumption dependent on CD38 and PARP1, and NAD + supplementation could effectively ameliorate mitochondrial function and improve TM function, providing a novel alternative approach for glaucoma treatment.

Sirt6 protects retinal ganglion cells and optic nerve from degeneration during aging and glaucoma.

Glaucoma is characterized by the progressive degeneration of retinal ganglion cells (RGCs) and their axons, and its risk increases with aging. Yet comprehensive insights into the complex mechanisms are largely unknown. Here, we found that anti-aging molecule Sirt6 was highly expressed in RGCs. Deleting Sirt6 globally or specifically in RGCs led to progressive RGC loss and optic nerve degeneration during aging, despite normal intraocular pressure (IOP), resembling a phenotype of normal-tension glaucoma. These detrimental effects were potentially mediated by accelerated RGC senescence through Caveolin-1 upregulation and by the induction of mitochondrial dysfunction. In mouse models of high-tension glaucoma, Sirt6 level was decreased after IOP elevation. Genetic overexpression of Sirt6 globally or specifically in RGCs significantly attenuated high tension-induced degeneration of RGCs and their axons, whereas partial or RGC-specific Sirt6 deletion accelerated RGC loss. Importantly, therapeutically targeting Sirt6 with pharmacological activator or AAV2-mediated gene delivery ameliorated high IOP-induced RGC degeneration. Together, our studies reveal a critical role of Sirt6 in preventing RGC and optic nerve degeneration during aging and glaucoma, setting the stage for further exploration of Sirt6 activation as a potential therapy for glaucoma.

Early Proteomic Characteristics and Changes in the Optic Nerve Head, Optic Nerve, and Retina in a Rat Model of Ocular Hypertension.

The pathogenesis of glaucoma is still unknown. There are few studies on the dynamic change of tissue-specific and time-specific molecular pathophysiology caused by ocular hypertension (OHT). This study aimed to identify the early proteomic alterations in the retina, optic nerve head (ONH), and optic nerve (ON). After establishing a rat model of OHT, we harvested the tissues from control and glaucomatous eyes and analyzed the changes in protein expression using a multiplexed quantitative proteomics approach (TMT-MS3). Our study identified 6403 proteins after 1-day OHT and 4399 proteins after 7-days OHT in the retina, 5493 proteins after 1-day OHT and 4544 proteins after 7-days OHT in ONH, and 5455 proteins after 1-day OHT and 3835 proteins after 7-days OHT in the ON. Of these, 560 and 489 differential proteins were identified on day 1 and 7 after OHT in the retina, 428 and 761 differential proteins were identified on day 1 and 7 after OHT in the ONH, and 257 and 205 differential proteins on days 1 and 7 after OHT in the ON. Computational analysis on day 1 and 7 of OHT revealed that alpha-2 macroglobulin was upregulated across two time points and three tissues stably. The differentially expressed proteins between day 1 and 7 after OHT in the retina, ONH, and ON were associated with glutathione metabolism, mitochondrial dysfunction/oxidative phosphorylation, oxidative stress, microtubule, and crystallin. And the most significant change in retina are crystallins. We validated this proteomic result with the Western blot of crystallin proteins and found that upregulated on day 1 but recovered on day 7 after OHT, which are promising as therapeutic targets. These findings provide insights into the time- and region-order mechanisms that are specifically affected in the retina, ONH, and ON in response to elevated IOP during the early stages.

Intracellular Zn2+ promotes extracellular matrix remodeling in dexamethasone-treated trabecular meshwork.

Given the widespread application of glucocorticoids in ophthalmology, the associated elevation of intraocular pressure (IOP) has long been a vexing concern for clinicians, yet the underlying mechanisms remain inconclusive. Much of the discussion focuses on the extracellular matrix (ECM) of trabecular meshwork (TM). It is widely agreed that glucocorticoids impact the expression of matrix metalloproteinases (MMPs), leading to ECM deposition. Since Zn2+ is vital for MMPs, we explored its role in ECM alterations induced by dexamethasone (DEX). Our study revealed that in human TM cells treated with DEX, the level of intracellular Zn2+ significantly decreased, accompanied by impaired extracellular Zn2+ uptake. This correlated with changes in several Zrt-, Irt-related proteins (ZIPs) and metallothionein. ZIP8 knockdown impaired extracellular Zn2+ uptake, but Zn2+ chelation did not affect ZIP8 expression. Resembling DEX's effects, chelation of Zn2+ decreased MMP2 expression, increased the deposition of ECM proteins, and induced structural disarray of ECM. Conversely, supplementation of exogenous Zn2+ in DEX-treated cells ameliorated these outcomes. Notably, dietary zinc supplementation in mice significantly reduced DEX-induced IOP elevation and collagen content in TM, thereby rescuing the visual function of the mice. These findings underscore zinc's pivotal role in ECM regulation, providing a novel perspective on the pathogenesis of glaucoma.NEW & NOTEWORTHY Our study explores zinc's pivotal role in mitigating extracellular matrix dysregulation in the trabecular meshwork and glucocorticoid-induced ocular hypertension. We found that in human trabecular meshwork cells treated with dexamethasone, intracellular Zn2+ significantly decreased, accompanied by impaired extracellular Zn2+ uptake. Zinc supplementation rescues visual function by modulating extracellular matrix proteins and lowering intraocular pressure, offering a direction for further exploration in glaucoma management.

TRPV4 and chloride channels mediate volume sensing in trabecular meshwork cells.

Aqueous humor drainage from the anterior eye determines intraocular pressure (IOP) under homeostatic and pathological conditions. Swelling of the trabecular meshwork (TM) alters its flow resistance but the mechanisms that sense and transduce osmotic gradients remain poorly understood. We investigated TM osmotransduction and its role in calcium and chloride homeostasis using molecular analyses, optical imaging, and electrophysiology. Anisosmotic conditions elicited proportional changes in TM cell volume, with swelling, but not shrinking, evoking elevations in intracellular calcium concentration [Ca2+]TM. Hypotonicity-evoked calcium signals were sensitive to HC067047, a selective blocker of TRPV4 channels, whereas the agonist GSK1016790A promoted swelling under isotonic conditions. TRPV4 inhibition partially suppressed hypotonicity-induced volume increases and reduced the magnitude of the swelling-induced membrane current, with a substantial fraction of the swelling-evoked current abrogated by Cl- channel antagonists 4,4'-diisothiocyanato-2,2'-stilbenedisulfonic acid (DIDS) and niflumic acid. The transcriptome of volume-sensing chloride channel candidates in primary human was dominated by ANO6 transcripts, with moderate expression of ANO3, ANO7, and ANO10 transcripts and low expression of LTTRC genes that encode constituents of the volume-activated anion channel. Imposition of 190 mosM but not 285 mosM hypotonic gradients increased conventional outflow in mouse eyes. TRPV4-mediated cation influx thus works with Cl- efflux to sense and respond to osmotic stress, potentially contributing to pathological swelling, calcium overload, and intracellular signaling that could exacerbate functional disturbances in inflammatory disease and glaucoma.NEW & NOTEWORTHY Intraocular pressure is dynamically regulated by the flow of aqueous humor through paracellular passages within the trabecular meshwork (TM). This study shows hypotonic gradients that expand the TM cell volume and reduce the outflow facility in mouse eyes. The swelling-induced current consists of TRPV4 and chloride components, with TRPV4 as a driver of swelling-induced calcium signaling. TRPV4 inhibition reduced swelling, suggesting a novel treatment for trabeculitis and glaucoma.

A digoxin derivative that potently reduces intraocular pressure: efficacy and mechanism of action in different animal models.

Glaucoma is a blinding disease. Reduction of intraocular pressure (IOP) is the mainstay of treatment, but current drugs show side effects or become progressively ineffective, highlighting the need for novel compounds. We have synthesized a family of perhydro-1,4-oxazepine derivatives of digoxin, the selective inhibitor of Na,K-ATPase. The cyclobutyl derivative (DcB) displays strong selectivity for the human α2 isoform and potently reduces IOP in rabbits. These observations appeared consistent with a hypothesis that in ciliary epithelium DcB inhibits the α2 isoform of Na,K-ATPase, which is expressed strongly in nonpigmented cells, reducing aqueous humor (AH) inflow. This paper extends assessment of efficacy and mechanism of action of DcB using an ocular hypertensive nonhuman primate model (OHT-NHP) (Macaca fascicularis). In OHT-NHP, DcB potently lowers IOP, in both acute (24 h) and extended (7-10 days) settings, accompanied by increased aqueous humor flow rate (AFR). By contrast, ocular normotensive animals (ONT-NHP) are poorly responsive to DcB, if at all. The mechanism of action of DcB has been analyzed using isolated porcine ciliary epithelium and perfused enucleated eyes to study AH inflow and AH outflow facility, respectively. 1) DcB significantly stimulates AH inflow although prior addition of 8-Br-cAMP, which raises AH inflow, precludes additional effects of DcB. 2) DcB significantly increases AH outflow facility via the trabecular meshwork (TM). Taken together, the data indicate that the original hypothesis on the mechanism of action must be revised. In the OHT-NHP, and presumably other species, DcB lowers IOP by increasing AH outflow facility rather than by decreasing AH inflow.NEW & NOTEWORTHY When applied topically, a cyclobutyl derivative of digoxin (DcB) potently reduces intraocular pressure in an ocular hypertensive nonhuman primate model (Macaca fascicularis), associated with increased aqueous humor (AH) flow rate (AFR). The mechanism of action of DcB involves increased AH outflow facility as detected in enucleated perfused porcine eyes and, in parallel, increased (AH) inflow as detected in isolated porcine ciliary epithelium. DcB might have potential as a drug for the treatment of open-angle human glaucoma.

Influence of Growth Differentiation Factor 15 on Intraocular Pressure in Mice.

Growth differentiation factor 15 (GDF15), a stress-sensitive cytokine, and a distant member of the transforming growth factor ß superfamily, has been shown to exhibit increased levels with aging, and in various age-related pathologies. Although GDF15 levels are elevated in the aqueous humor (AH) of glaucoma (optic nerve atrophy) patients, the possible role of this cytokine in the modulation of intraocular pressure (IOP) or AH outflow is unknown. The current study addresses this question using transgenic mice expressing human GDF15 and GDF15 null mice, and by perfusing enucleated mouse eyes with recombinant human GDF15 (rhGDF15). Treatment of primary cultures of human trabecular meshwork cells with a telomerase inhibitor, an endoplasmic reticulum stress-inducing agent, hydrogen peroxide, or an autophagy inhibitor resulted in significant elevation in GDF15 levels relative to the respective control cells. rhGDF15 stimulated modest but significant increases in the expression of genes encoding the extracellular matrix, cell adhesion proteins, and chemokine receptors (C-C chemokine receptor type 2) in human trabecular meshwork cells compared with controls, as deduced from the differential transcriptional profiles using RNA-sequencing analysis. There was a significant increase in IOP in transgenic mice expressing human GDF15, but not in GDF15 null mice, compared with the respective wild-type control mice. The AH outflow facility was decreased in enucleated wild-type mouse eyes perfused with rhGDF15. Light microcopy-based histologic examination of the conventional AH outflow pathway tissues did not reveal identifiable differences between the GDF15-targeted and control mice. Taken together, these results reveal the modest elevation of IOP in mice expressing human GDF15 possibly stemming from decreased AH outflow through the trabecular pathway.

Automated AI labeling of optic nerve head enables insights into cross-ancestry glaucoma risk and genetic discovery in >280,000 images from UKB and CLSA.

Cupping of the optic nerve head, a highly heritable trait, is a hallmark of glaucomatous optic neuropathy. Two key parameters are vertical cup-to-disc ratio (VCDR) and vertical disc diameter (VDD). However, manual assessment often suffers from poor accuracy and is time intensive. Here, we show convolutional neural network models can accurately estimate VCDR and VDD for 282,100 images from both UK Biobank and an independent study (Canadian Longitudinal Study on Aging), enabling cross-ancestry epidemiological studies and new genetic discovery for these optic nerve head parameters. Using the AI approach, we perform a systematic comparison of the distribution of VCDR and VDD and compare these with intraocular pressure and glaucoma diagnoses across various genetically determined ancestries, which provides an explanation for the high rates of normal tension glaucoma in East Asia. We then used the large number of AI gradings to conduct a more powerful genome-wide association study (GWAS) of optic nerve head parameters. Using the AI-based gradings increased estimates of heritability by ∼50% for VCDR and VDD. Our GWAS identified more than 200 loci associated with both VCDR and VDD (double the number of loci from previous studies) and uncovered dozens of biological pathways; many of the loci we discovered also confer risk for glaucoma.

Glaucoma: now and beyond.

The glaucomas are a group of conditions leading to irreversible sight loss and characterised by progressive loss of retinal ganglion cells. Although not always elevated, intraocular pressure is the only modifiable risk factor demonstrated by large clinical trials. It remains the leading cause of irreversible blindness, but timely treatment to lower intraocular pressure is effective at slowing the rate of vision loss from glaucoma. Methods for lowering intraocular pressure include laser treatments, topical medications, and surgery. Although modern surgical innovations aim to be less invasive, many have been introduced with little supporting evidence from randomised controlled trials. Many cases remain undiagnosed until the advanced stages of disease due to the limitations of screening and poor access to opportunistic case finding. Future research aims to generate evidence for intraocular pressure-independent neuroprotective treatments, personalised treatment through genetic risk profiling, and exploration of potential advanced cellular and gene therapies.

Pathological high intraocular pressure induces glial cell reactive proliferation contributing to neuroinflammation of the blood-retinal barrier via the NOX2/ET-1 axis-controlled ERK1/2 pathway.

BACKGROUND: NADPH oxidase (NOX), a primary source of endothelial reactive oxygen species (ROS), is considered a key event in disrupting the integrity of the blood-retinal barrier. Abnormalities in neurovascular-coupled immune signaling herald the loss of ganglion cells in glaucoma. Persistent microglia-driven inflammation and cellular innate immune system dysregulation often lead to deteriorating retinal degeneration. However, the crosstalk between NOX and the retinal immune environment remains unresolved. Here, we investigate the interaction between oxidative stress and neuroinflammation in glaucoma by genetic defects of NOX2 or its regulation via gp91ds-tat. METHODS: Ex vivo cultures of retinal explants from wildtype C57BL/6J and Nox2 -/- mice were subjected to normal and high hydrostatic pressure (Pressure 60 mmHg) for 24 h. In vivo, high intraocular pressure (H-IOP) was induced in C57BL/6J mice for two weeks. Both Pressure 60 mmHg retinas and H-IOP mice were treated with either gp91ds-tat (a NOX2-specific inhibitor). Proteomic analysis was performed on control, H-IOP, and treatment with gp91ds-tat retinas to identify differentially expressed proteins (DEPs). The study also evaluated various glaucoma phenotypes, including IOP, retinal ganglion cell (RGC) functionality, and optic nerve (ON) degeneration. The superoxide (O2-) levels assay, blood-retinal barrier degradation, gliosis, neuroinflammation, enzyme-linked immunosorbent assay (ELISA), western blotting, and quantitative PCR were performed in this study. RESULTS: We found that NOX2-specific deletion or activity inhibition effectively attenuated retinal oxidative stress, immune dysregulation, the internal blood-retinal barrier (iBRB) injury, neurovascular unit (NVU) dysfunction, RGC loss, and ON axonal degeneration following H-IOP. Mechanistically, we unveiled for the first time that NOX2-dependent ROS-driven pro-inflammatory signaling, where NOX2/ROS induces endothelium-derived endothelin-1 (ET-1) overexpression, which activates the ERK1/2 signaling pathway and mediates the shift of microglia activation to a pro-inflammatory M1 phenotype, thereby triggering a neuroinflammatory outburst. CONCLUSIONS: Collectively, we demonstrate for the first time that NOX2 deletion or gp91ds-tat inhibition attenuates iBRB injury and NVU dysfunction to rescue glaucomatous RGC loss and ON axon degeneration, which is associated with inhibition of the ET-1/ERK1/2-transduced shift of microglial cell activation toward a pro-inflammatory M1 phenotype, highlighting NOX2 as a potential target for novel neuroprotective therapies in glaucoma management.

An Ultrasensitive Ti3C2Tx MXene-based Soft Contact Lens for Continuous and Nondestructive Intraocular Pressure Monitoring.

Wearable soft contact lens sensors for continuous and nondestructive intraocular pressure (IOP) monitoring are highly desired as glaucoma and postoperative myopia patients grow, especially as the eyestrain crowd increases. Herein, a smart closed-loop system is presented that combines a Ti3C2Tx MXene-based soft contact lens (MX-CLS) sensor, wireless data transmission units, display, and warning components to realize continuous and nondestructive IOP monitoring/real-time display. The fabricated MX-CLS device exhibits an extremely high sensitivity of 7.483 mV mmHg-1, good linearity on silicone eyeballs, excellent stability under long-term pressure-release measurement, sufficient transparency with 67.8% transmittance under visible illumination, and superior biocompatibility with no discomfort when putting the MX-CLS sensor onto the Rabbit eyes. After integrating with the wireless module, users can realize real-time monitoring and warning of IOP via smartphones, the demonstrated MX-CLS device together with the IOP monitoring/display system opens up promising platforms for Ti3C2Tx materials as the base for multifunctional contact lens-based sensors and continuous and nondestructive IOP measurement system.