Schlemm's canal-selective Tie2/TEK knockdown induces sustained ocular hypertension in adult mice.

Deficient Angiopoietin-Tie2 signaling is linked to ocular hypertension in glaucoma. Receptor Tie2/TEK expression and signaling at Schlemm's canal (SC) is indispensable for canal integrity and homeostatic regulation of aqueous humor outflow (AHO) and intraocular pressure (IOP), as validated by conditional deletion of Tie2, its ligands (Angpt1, Angpt2 and Angpt3/4) or regulators (Tie1 and PTPRB/VE-PTP). However, these Tie2/TEK knockouts and conditional knockouts are global or endothelial, preventing separation of systemic and ocular vascular defects that impact retinal or renal integrity. To develop a more targeted model of ocular hypertension induced by selective knockdown of Tie2/TEK expressed in SC, we combined the use of viral vectors to target the canal, and two distinct gene-editing strategies to disrupt the Tie2 gene. Adeno-associated virus (AAV2) is known to transduce rodent SC when delivered into the anterior chamber by intracameral injection. First, delivery of Cre recombinase via AAV2.Cre into R26tdTomato/+ reporter mice confirmed preferential and stable transduction in SC endothelium. Next, to disrupt Tie2 expression in SC, we injected AAV2.Cre into homozygous floxed Tie2 (Tie2FL/FL) mice. This led to attenuated Tie2 protein expression along the SC inner wall, decreased SC area and reduced trabecular meshwork (TM) cellularity. Functionally, IOP was significantly and steadily elevated, whereas AHO facility was reduced. In contrast, hemizygous Tie2FL/+ mice responded to AAV2.Cre with inconsistent and low IOP elevation, corroborating the dose-dependency of ocular hypertension on Tie2 expression/activation. In a second model using CRISPR/SaCas9 genome editing, wild-type C57BL/6 J mice injected with AAV2.saCas9-sgTie2 showed similar selective SC transduction and comparable IOP elevation in course and magnitude to that induced by AAV2.Cre in Tie2FL/FL mice. Together, our findings, demonstrate that selective Tie2 knockdown in SC is a targeted strategy that reliably induces chronic ocular hypertension and reproduces glaucomatous damage to the conventional outflow pathway, providing novel models of SC-Tie2 signaling loss valuable for preclinical studies.

Whole-genome sequencing unravels novel genetic determinants and regulatory pathways associated with triamcinolone acetonide-induced ocular hypertension.

Glucocorticosteroids commonly used to treat certain ocular inflammatory conditions cause an unwarranted elevation in intraocular pressure (IOP) leading to steroid-induced ocular hypertension (OHT). This study aims to identify novel genetic variants in the Indian population associated with steroid responsiveness, specifically to that of intravitreal Triamcinolone acetonide (TA) injections, which leads to OHT in 27% of the TA-treated Indian subjects. Genetic determinants and pathways regulating TA-OHT progression were investigated by applying whole-genome sequencing (WGS) on DNA extracted from 53 blood samples that included TA responders and non-responders. Sequencing analysis yielded 45 intronic and 49 exonic variants to be associated with TA-OHT, which are known to play a vital role in eye, heart, brain, and bone deformities. Of these, the most significant genetic variant associated with TA-OHT was further considered for molecular dynamics (MD) simulation studies. Variants in the CRPPA, PLOD1, ARHGAP1, TIMELESS and TNFSF4 genes were found to be directly implicating TA-OHT. Furthermore, these genes were enriched in pathways associated with cardiomyopathy, focal adhesion, extracellular matrix, and actin cytoskeleton reorganization. MD simulation studies revealed that the top significant variant (rs141625803) in the CRPPA gene possesses a high pathogenic and structurally destabilizing effect. Thus, novel genetic variants that could be significantly associated with the TA-OHT progression were identified in this study. Validation of these targets in a larger cohort of patients along with their functional analysis would inform on the disease, thereby adding to the existing knowledge on the pathophysiology of TA-OHT.

Genotype and Phenotype Influence the Personal Response to Prostaglandin Analogues and Beta-Blockers in Spanish Glaucoma and Ocular Hypertension Patients.

Analysis of the genotype that predicts the phenotypic characteristics of a cohort of glaucoma and ocular hypertension patients, and the correlation with their personal pharmacological response to beta-blockers (BB) and prostaglandin analogues (PGA). Prospective study that included 139 eyes from 72 patients under BB and/or PGA treatment, and in some cases other types of ocular hypotensive treatments. Five single-nucleotide polymorphisms were genotyped by real-time PCR assays: prostaglandin-F2α receptor (rs3766355, rs3753380); cytochrome-P450 2D6 (rs16947, rs769258); and beta-2-adrenergic receptor (rs1042714). Other studied variables were mean deviation (MD) of visual field, previous ocular interventions, medical treatment, baseline (bIOP), and treated intraocular pressure (tIOP). From a total of 139 eyes, 71 (51.1%) were left eyes. The main diagnosis was primary open angle glaucoma (66.2%). A total of 57 (41%) eyes were under three or more medications (PGA + BB + other) and, additionally, 57 eyes (41%) had had some kind of glaucoma surgery. The mean bIOP and tIOP were 26.55 ± 8.19 and 21.01 ± 5.54 mmHg, respectively. Significant differences in tIOP were found between heterozygous (HT) (21.07 ± 0.607 mmHg) and homozygous (HM) (20.98 ± 0.639 mmHg) rs3766355 with respect to wildtype individuals (16 ± 1.08 mmHg) (p = 0.031). The MD values presented significant differences between wildtype rs3766355 (-2 ± 2.2 dB), HT (-3.87 ± 4 dB), and HM carriers (-9.37 ± 9.51 dB) (p = 0.009). Significant differences were also observed between the MD in wildtype rs3753380 (-6.1 ± 8.67 dB), HT (-9.02 ± 8.63 dB), and HM carriers (-9.51 ± 7.44 dB) (p = 0.017). Patients carrying the variant rs3766355 in HM or HT presented clinically-significantly higher tIOP than wildtype patients. Additionally, some differences in MD were found in rs3766355 and rs3753380 carriers, and the more alleles that were affected, the worse the MD value, meaning greater severity of the glaucoma. Poor response to treatment and more visual field damage may be associated with being a carrier of these mutated alleles.

A Novel Mouse Model of TGFß2-Induced Ocular Hypertension Using Lentiviral Gene Delivery.

Glaucoma is a multifactorial disease leading to irreversible blindness. Primary open-angle glaucoma (POAG) is the most common form and is associated with the elevation of intraocular pressure (IOP). Reduced aqueous humor (AH) outflow due to trabecular meshwork (TM) dysfunction is responsible for IOP elevation in POAG. Extracellular matrix (ECM) accumulation, actin cytoskeletal reorganization, and stiffening of the TM are associated with increased outflow resistance. Transforming growth factor (TGF) ß2, a profibrotic cytokine, is known to play an important role in the development of ocular hypertension (OHT) in POAG. An appropriate mouse model is critical in understanding the underlying molecular mechanism of TGFß2-induced OHT. To achieve this, TM can be targeted with recombinant viral vectors to express a gene of interest. Lentiviruses (LV) are known for their tropism towards TM with stable transgene expression and low immunogenicity. We, therefore, developed a novel mouse model of IOP elevation using LV gene transfer of active human TGFß2 in the TM. We developed an LV vector-encoding active hTGFß2C226,228S under the control of a cytomegalovirus (CMV) promoter. Adult C57BL/6J mice were injected intravitreally with LV expressing null or hTGFß2C226,228S. We observed a significant increase in IOP 3 weeks post-injection compared to control eyes with an average delta change of 3.3 mmHg. IOP stayed elevated up to 7 weeks post-injection, which correlated with a significant drop in the AH outflow facility (40.36%). Increased expression of active TGFß2 was observed in both AH and anterior segment samples of injected mice. The morphological assessment of the mouse TM region via hematoxylin and eosin (H&E) staining and direct ophthalmoscopy examination revealed no visible signs of inflammation or other ocular abnormalities in the injected eyes. Furthermore, transduction of primary human TM cells with LV_hTGFß2C226,228S exhibited alterations in actin cytoskeleton structures, including the formation of F-actin stress fibers and crossed-linked actin networks (CLANs), which are signature arrangements of actin cytoskeleton observed in the stiffer fibrotic-like TM. Our study demonstrated a mouse model of sustained IOP elevation via lentiviral gene delivery of active hTGFß2C226,228S that induces TM dysfunction and outflow resistance.

TRPV4-induced Müller cell gliosis and TNF-α elevation-mediated retinal ganglion cell apoptosis in glaucomatous rats via JAK2/STAT3/NF-κB pathway.

BACKGROUND: Glaucoma, the leading cause of irreversible blindness worldwide, is a type of retinal disease characterized by the selective death of retinal ganglion cells (RGCs). However, the pathogenesis of glaucoma has not been fully elucidated. Transient receptor potential vanilloid 4 (TRPV4) is a pressure-sensitive and calcium-permeable cation channel. TRPV4 is widely distributed in the retina and its sustained activation leads to RGC death; indicating that TRPV4 may be a possible target for glaucoma treatment. Here, we investigated the effects of TRPV4 on RGC apoptosis in a rat model of chronic ocular hypertension (COH), then examined the mechanism underlying these effects. METHODS: The COH model was established by injection of micro-magnetic beads into the anterior chamber of adult male rats. The expression levels of TRPV4, glial fibrillary acidic protein, and inflammatory factors were assessed by immunohistochemistry and immunoblotting. RGC apoptosis and visual dysfunction were evaluated by TUNEL assay and photopic negative response. Functional expression of TRPV4 was examined by electrophysiology and calcium imaging. Real-time polymerase chain reaction and immunoblotting were employed to investigate the molecular mechanism underlying the effects of TRPV4 on tumor necrosis factor-α (TNF-α) release. RESULTS: We found that TRPV4 played an essential role in glaucoma, such that high levels of TRPV4 expression were associated with elevated intraocular pressure. Furthermore, TRPV4 activation was involved in glaucoma-induced RGC apoptosis and RGC-related reductions in visual function. Mechanistic investigation demonstrated that TRPV4 activation led to enhanced Müller cell gliosis and TNF-α release via the JAK2/STAT3/NF-kB pathway, while TRPV4 inhibition could reverse these effects. Finally, TRPV4 activation could lead to elevated expression of TNF receptor 1 in RGCs, while inhibition of TNF-α could reduce TRPV4-mediated RGC apoptosis. CONCLUSIONS: TRPV4 activation induces Müller cell gliosis and TNF-α elevation via the JAK2/STAT3/NF-κB pathway, which may exacerbate RGC apoptosis in glaucoma; these results suggest that TRPV4 can serve as a therapeutic target in glaucoma treatment.

Effects of topical TGF-ß1, TGF-ß2, ATX, and LPA on IOP elevation and regulation of the conventional aqueous humor outflow pathway.

PURPOSE: The effects of aqueous mediators possibly increasing the outflow resistance, transforming growth factor-ß1 (TGF-ß1), TGF-ß2, autotaxin (ATX), and lysophosphatidic acid (LPA) on human trabecular meshwork (hTM) cells and monkey Schlemm's canal endothelial (SCE) cells were characterized and compared, and the effects of intracameral application of these mediators on intraocular (IOP) elevation were also examined. METHODS: Cells were treated with TGF-ß1, TGF-ß2, ATX, LPA, or vehicle, and mRNA and protein expression levels of α-SMA, COL1A1, fibronectin, ß-catenin, and ZO-1 were examined with real-time quantitative PCR (RT-qPCR) or immunofluorescence analyses or both. The permeability of cell monolayers was measured by determining the transendothelial electrical resistance (TEER) or with the fluorescein isothiocyanate (FITC)-dextran permeability assay. IOP was evaluated in rabbit eyes after intracameral administration of the mediators. RESULTS: All mediators induced upregulation of α-SMA, COL1A1, and fibronectin in hTM cells. The effect of TGF-ß2 on mRNA expression of fibrotic markers was statistically significantly greater than that of TGF-ß1. The effects of ATX and LPA indicated the time-dependent difference in the upregulation of α-SMA, COL1A1, and fibronectin. The TEER and FITC-dextran permeability of the SCE cells was evaluated after treatment with TGF-ß1 and TGF-ß2, but no statistically significant change was observed within 24 h. ATX and LPA also reduced permeability statistically significantly after 3 h and 0.5 h, respectively, and the effect of LPA was more rapid compared to that of ATX. Statistically significant IOP elevation was observed in rabbit eyes as early as 0.5-2.0 h after ATX and LPA treatment and at 24 h after treatment with TGF-ß2. CONCLUSIONS: TGF-ß2 and ATX and LPA regulate aqueous outflow by modulation of hTM cells and SCE cells, and differences in timing between the effects of each mediator were observed. ATX and LPA showed more rapid effects on IOP elevation than TGF-ß2. It was suggested that TGF-ß2 and ATX/LPA are involved in increases of IOP, but the timing and sustainability differ between mediators, and they may play specific roles in different glaucoma subtypes.

Astragaloside IV Attenuates Ocular Hypertension in a Mouse Model of TGFß2 Induced Primary Open Angle Glaucoma.

Elevated intraocular pressure (IOP) is a major risk factor in developing primary open angle glaucoma (POAG), which is the most common form of glaucoma. Transforming growth factor-beta 2 (TGFß2) is a pro-fibrotic cytokine that plays an important role in POAG pathogenesis. TGFß2 induced extracellular matrix (ECM) production, deposition and endoplasmic reticulum (ER) stress in the trabecular meshwork (TM) contribute to increased aqueous humor (AH) outflow resistance and IOP elevation. Drugs which alter the glaucomatous fibrotic changes and ER stress in the TM may be effective in reducing ocular hypertension. Astragaloside IV (AS.IV), a novel saponin isolated from the roots of Astragalus membranaceus, has demonstrated antifibrotic and ER stress lowering effects in various tissues during disease conditions. However, the effect of AS.IV on glaucomatous TM fibrosis, ER stress and ocular hypertension has not been studied. Primary human TM cells treated with AS.IV decreased TGFß2 induced ECM (FN, Col-I) deposition and ER stress (KDEL, ATF4 and CHOP). Moreover, AS.IV treatment reduced TGFß2 induced NF-κB activation and αSMA expression in TM cells. We found that AS.IV treatment significantly increased levels of matrix metalloproteases (MMP9 and MMP2) and MMP2 enzymatic activity, indicating that the antifibrotic effects of AS.IV are mediated via inhibition of NF-κB and activation of MMPs. AS.IV treatment also reduced ER stress in TM3 cells stably expressing mutant myocilin. Interestingly, the topical ocular AS.IV eye drops (1 mM) significantly decreased TGFß2 induced ocular hypertension in mice, and this was associated with a decrease in FN, Col-1 (ECM), KDEL (ER stress) and αSMA in mouse TM tissues. Taken together, the results suggest that AS.IV prevents TGFß2 induced ocular hypertension by modulating ECM deposition and ER stress in the TM.

Neuroprotective Effect of Statins in a Rat Model of Chronic Ocular Hypertension.

Glaucoma is an optic neuropathy in which the degeneration of retinal ganglion cells (RGCs) results in irreversible vison loss. Therefore, neuroprotection of RGCs from glaucomatous afflictions is crucial for glaucoma treatment. In this study, we aimed to investigate the beneficial effects of statins in the protection of RGCs using a rat model. Glaucomatous injury was induced in rats by chronic ocular hypertension (OHT) achieved after performing a circumlimbal suture. The rats were given either statins such as simvastatin and atorvastatin or a solvent weekly for 6 weeks. Retina sections underwent hematoxylin and eosin, Brn3a, or cleaved casepase-3 staining to evaluate RGC survival. In addition, modulation of glial activation was assessed. While the retinas without statin treatment exhibited increased RGC death due to chronic OHT, statins promoted the survival of RGCs and reduced apoptosis. Statins also suppressed chronic OHT-mediated glial activation in the retina. Our results demonstrate that statins exert neuroprotective effects in rat retinas exposed to chronic OHT, which may support the prospect of statins being a glaucoma treatment.

Endogenous ocular lipids as potential modulators of intraocular pressure.

Elevated intraocular pressure (IOP) is a risk factor in glaucoma, a group of irreversible blinding diseases. Endogenous lipids may be involved in regulation of IOP homeostasis. We present comparative fold analysis of phospholipids and sphingolipids of aqueous humour and trabecular meshwork from human control vs primary open-angle glaucoma and mouse control (normotensive) vs ocular hypertensive state. The fold analysis in control vs disease state was based on ratiometric mass spectrometric data for above classes of lipids. We standardized in vitro assays for rapid characterization of lipids undergoing significant diminishment in disease state. Evaluation of lipids using in vitro assays helped select a finite number of lipids that may potentially expand cellular interstitial space embedded in an artificial matrix or increase fluid flow across a layer of cells. These assays reduced a number of lipids for initial evaluation using a mouse model, DBA/2J with spontaneous IOP elevation. These lipids were then used in other mouse models for confirmation of IOP lowering potential of a few lipids that were found promising in previous assessments. Our results provide selected lipid molecules that can be pursued for further evaluation and studies that may provide insight into their function.

Boosting the Power of the Sequence Kernel Association Test by Properly Estimating Its Null Distribution.

The sequence kernel association test (SKAT) is probably the most popular statistical test used in rare-variant association studies. Its null distribution involves unknown parameters that need to be estimated. The current estimation method has a valid type I error rate, but the power is compromised given that all subjects are used for estimation. I have developed an estimation method that uses only control subjects. Named SKAT+, this method uses the same test statistic as SKAT but differs in the way the null distribution is estimated. Extensive simulation studies and applications to data from the Genetic Analysis Workshop 17 and the Ocular Hypertension Treatment Study demonstrated that SKAT+ has superior power over SKAT while maintaining control over the type I error rate. This method is applicable to extensions of SKAT in the literature.

Role of Histamine H3 Receptor Antagonists on Intraocular Pressure Reduction in Rabbit Models of Transient Ocular Hypertension and Glaucoma.

Intraocular pressure (IOP) has a tendency to fluctuate throughout the day, reaching its peak in the early morning in healthy subjects or glaucoma patients. Likewise, histamine tone also fluctuates over time, being lower at nighttime. Numerous studies have demonstrated a correlation between short-term IOP fluctuation and glaucoma progression; however, it has not yet been determined whether histamine plays a role in IOP fluctuations. The aim of this research was to establish the distribution of the histamine receptor proteins and respective mRNAs in the eye by western blot, immunohistochemistry and RT-PCR in New Zealand rabbits. Furthermore, we used a transient ocular hypertension (OHT) model induced by injection of 50 µL of 5% hypertonic saline into the vitreous and a stable OHT model (100 µL 0.1% carbomer in the anterior chamber) to address the potential IOP-lowering ability of H3 receptor (H3R) antagonists (ciproxifan, DL76 and GSK189254). IOPs were performed with a Tono-Pen at baseline and 60, 120 and 240 min post treatment after transient OHT induction and, every day for 12 days in the stable OHT model. All histamine receptor subtypes were localized in the rabbit retina and ciliary body/trabecular meshwork. All the treatments lowered IOP in a dose-dependent fashion between 0.3% and 1%. More specifically, the effects were maximal with ciproxifan at 60 min post-dose (IOP60 change = -18.84 ± 4.85 mmHg, at 1%), remained stable until 120 min (IOP120 change = -16.38 ± 3.8 mmHg, at 1%) and decayed thereafter to reach baseline values at 240 min. These effects were highly specific and dependent on histamine release as pre-treatment with imetit (H3R agonist, 1%) or pyrilamine (H1R antagonist, 1%) largely blocked ciproxifan-mediated effects. Color Doppler ultrasound examination was performed to evaluate changes in ophtalmic artery resistivity index (RI) before and after repeated dosing with DL 76, GSK189254, ciproxifan and timolol. Chronic treatments with H3R antagonists and timolol improved the vascular performance of ophthalmic arteries and reduced retinal ganglion cell death. Oxidative stress was also reduced and measured 8-Hydroxy-2'-deoxyguanosine (8OHdG) expression, and by dihidroethydium (DHE) staining. These results demonstrated that the histamine system participates in IOP regulation and that H3R antagonists could represent a future promising therapy for glaucoma. Further studies should be focused on the long-term IOP circadian fluctuations.

Increased intraocular pressure alters the cellular distribution of HuR protein in retinal ganglion cells - A possible sign of endogenous neuroprotection failure.

The RNA-binding protein, HuR, modulates mRNA processing and gene expression of several stress response proteins i.e. Hsp70 and p53 that have been postulated to be involved in the pathogenesis of glaucoma, a chronic optic neuropathy leading to irreversible blindness. We evaluated HuR protein expression in retinas and optic nerves of glaucomatous rats and human primary open angle glaucoma patients and its possible impact on stress response mechanisms. We found that the cytoplasmic content of HuR was reduced more extensively in glaucomatous retinas than in optic nerves and this was linked with a declined cytoplasmic Hsp70 level and p53 nuclear translocation. In the optic nerve, the p53 content was decreased as a feature of reactive gliosis. Based on our findings, we conclude that the alteration in the HuR content, observed both in rat glaucoma model and human glaucoma samples, affects post-transcriptionally the expression of genes crucial for maintaining cell homeostasis; therefore, we postulate that HuR may be involved in the pathogenesis of glaucoma.

Conditional asymptotic inference for the kernel association test.

MOTIVATION: The kernel association test (KAT) is popular in biological studies for its ability to combine weak effects potentially of opposite direction. Its P-value is typically assessed via its (unconditional) asymptotic distribution. However, such an asymptotic distribution is known only for continuous traits and for dichotomous traits. Furthermore, the derived P-values are known to be conservative when sample size is small, especially for the important case of dichotomous traits. One alternative is the permutation test, a widely accepted approximation to the exact finite sample conditional inference. But it is time-consuming to use in practice due to stringent significance criteria commonly seen in these analyses. RESULTS: Based on a previous theoretical result a conditional asymptotic distribution for the KAT is introduced. This distribution provides an alternative approximation to the exact distribution of the KAT. An explicit expression of this distribution is provided from which P-values can be easily computed. This method applies to any type of traits. The usefulness of this approach is demonstrated via extensive simulation studies using real genotype data and an analysis of genetic data from the Ocular Hypertension Treatment Study. Numerical results showed that the new method can control the type I error rate and is a bit conservative when compared to the permutation method. Nevertheless the proposed method may be used as a fast screening method. A time-consuming permutation procedure may be conducted at locations that show signals of association. AVAILABILITY AND IMPLEMENTATION: An implementation of the proposed method is provided in the R package iGasso. CONTACT: kai-wang@uiowa.edu.

New Insight of Common Regulatory Pathways in Human Trabecular Meshwork Cells in Response to Dexamethasone and Prednisolone Using an Integrated Quantitative Proteomics: SWATH and MRM-HR Mass Spectrometry.

The molecular pathophysiology of corticosteroid-induced ocular hypertension (CIH) is not well understood. To determine the biological mechanisms of CIH, this study investigated protein expression profiles of human trabecular meshwork (hTM) cells in response to dexamethasone and prednisolone treatment. Both discovery-based sequential windowed data independent acquisition of the total high-resolution mass spectra (SWATH-MS) and targeted based high resolution multiple reaction monitoring (MRM-HR) confirmation were applied using a hybrid quadrupole-time-of-flight mass spectrometer. A comprehensive list of 1759 proteins (1% FDR) was generated from the hTM. Quantitative proteomics revealed 20 differentially expressed proteins (p-value ≤ 0.05 and fold-change ≥ 1.5 or ≤ 0.67) commonly induced by prednisolone and dexamethasone, both at 300 nM. These included connective tissue growth factor (CTGF) and thrombospondin-1 (THBS1), two proteins previously implicated in ocular hypertension, glaucoma, and the transforming growth factor-ß pathway. Their gene expressions in response to corticosteroids were further confirmed using reverse-transcription polymerase chain reaction. Together with other novel proteins identified in the data sets, additional pathways implicated by these regulated proteins were the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt) signaling pathway, integrin cell surface interaction, extracellular matrix (ECM) proteoglycans, and ECM-receptor interaction. Our results indicated that an integrated platform of SWATH-MS and MRM-HR allows high throughput identification and confirmation of novel and known corticosteroid-regulated proteins in trabecular meshwork cells, demonstrating the power of this technique in extending the current understanding of the pathogenesis of CIH.

Myocilin Predictive Genetic Testing for Primary Open-Angle Glaucoma Leads to Early Identification of At-Risk Individuals.

PURPOSE: To assess the difference in severity of disease in primary open-angle glaucoma (POAG) patients with a Myocilin (MYOC) disease-causing variant who presented through normal clinical pathways (Clinical cases) versus those who were examined following genetic testing (Genetic cases). DESIGN: Retrospective clinical and molecular study. PARTICIPANTS: Seventy-three MYOC mutation carriers identified through the Australian and New Zealand Registry of Advanced Glaucoma. METHODS: Individuals were classified based on how they first presented to an ophthalmologist: Clinical cases were referred by their general practitioner or optometrist, and Genetic cases were referred following positive results from genetic testing for the previously identified familial MYOC variant (cascade genetic testing). All cases were then sub-classified into 4 groups (unaffected, glaucoma suspect, glaucoma, advanced glaucoma) according to the severity of disease at the time of their first examination by an ophthalmologist. MAIN OUTCOME MEASURES: Glaucoma clinical parameters and age at presentation. RESULTS: At their first examination, 83% of Genetic cases were unaffected and 17% were glaucoma suspect, whereas among Clinical cases 44% were glaucoma suspect, 28% had glaucoma, and 28% had advanced glaucoma. Genetic cases were significantly younger at presentation than Clinical cases (40.6±12.5 vs. 47.5±16.7 years; P = 0.018). The mean highest intraocular pressure (32.2±9.7 vs. 17.6±3.6 mmHg; P < 0.001), cup-to-disc ratio (0.65±0.27 vs. 0.48±0.13; P = 0.006), and mean deviation on visual field testing (-10.0±10.3 vs. -1.2±1.2; P < 0.001) were all significantly worse in Clinical cases compared with Genetic cases. Individuals with common MYOC p.Gln368Ter variant were further analyzed separately to account for the phenotypic variability of different disease-causing variants. All findings remained significant after adjusting for the common MYOC p.Gln368Ter variant. CONCLUSIONS: Our findings demonstrated that MYOC cascade genetic testing for POAG allows identification of at-risk individuals at an early stage or even before signs of glaucoma are present. To our knowledge, this is the first study to demonstrate the clinical utility of predictive genetic testing for MYOC glaucoma.

Evaluation of the Myocilin Mutation Gln368Stop Demonstrates Reduced Penetrance for Glaucoma in European Populations.

PURPOSE: Sequence variations in the myocilin (MYOC) gene account for approximately 2% to 4% of glaucoma cases. One particular MYOC mutation, Gln368Stop (dbSNP accession number: rs74315329), is the most common genetic mutation causing glaucoma by increasing intraocular pressure (IOP). The objective of this study was to evaluate the effect of this MYOC mutation on IOP using data from large-scale European population panels (directly sequenced and imputation based). DESIGN: Cross-sectional, cohort study. PARTICIPANTS: For this study, the penetrance of the variant rs74315329 was estimated in 2 population-based cohorts, the TwinsUK (N = 6092) and the Rotterdam Study (RS) (N =11 189). METHODS: Carriers of the risk allele for rs74315329 were identified using whole-genome sequencing and imputation data (based on 1000 Genomes Project and Haplotype Reference Consortium panels). The penetrance of this variant was evaluated using IOP measurements and data on visual field testing/a diagnosis of glaucoma (if available). MAIN OUTCOME MEASURES: The penetrance of the variant rs74315329 was estimated from the percentage of the carriers of the risk allele of the variant who had high IOP (ocular hypertension) or glaucoma. RESULTS: In our study, the observed penetrance of the variant rs74315329 in relation to increased IOP was 12.5% and 19.4% in the TwinsUK and the RS, respectively. Thus, our study suggests a much lower penetrance for rs74315329 for ocular hypertension (and thus glaucoma), in comparison with that reported previously. CONCLUSIONS: The significance of this finding is that higher numbers of healthy individuals in the population are expected to be carriers of this mutation, which in turn reduces the utility of identifying carriers of this mutation as a screening tool for glaucoma.

RhoA GTPase-induced ocular hypertension in a rodent model is associated with increased fibrogenic activity in the trabecular meshwork.

Ocular hypertension arising from increased resistance to aqueous humor (AH) outflow through the trabecular meshwork is a primary risk factor for open-angle glaucoma, a leading cause of blindness. Ongoing efforts have found little about the molecular and cellular bases of increased resistance to AH outflow through the trabecular meshwork in ocular hypertension patients. To test the hypothesis that dysregulated Rho GTPase signaling and a resulting fibrotic activity within the trabecular meshwork may result in ocular hypertension, we investigated the effects of expressing a constitutively active RhoA GTPase (RhoAV14) in the AH outflow pathway in Sprague-Dawley rats by using lentiviral vector-based gene delivery. Rats expressing RhoAV14 in the iridocorneal angle exhibited a significantly elevated intraocular pressure. Elevated intraocular pressure in the RhoAV14-expressing rats was associated with fibrotic trabecular meshwork and increased levels of F-actin, phosphorylated myosin light chain, α-smooth muscle actin, collagen-1A, and total collagen in the trabecular AH outflow pathway. Most of these changes were ameliorated by topical application of Rho kinase inhibitor. Human autopsy eyes from patients with glaucoma exhibited significant increases in levels of collagen-1A and total collagen in the trabecular AH outflow pathway. Collectively, these observations indicate that increased fibrogenic activity because of dysregulated RhoA GTPase activity in the trabecular AH outflow pathway increases intraocular pressure in a Rho kinase-dependent manner.

Glaucoma Risk Alleles in the Ocular Hypertension Treatment Study.

PURPOSE: Primary open-angle glaucoma (POAG) is a major cause of blindness and visual disability. Several genetic risk factors for POAG and optic nerve features have been identified. We measured the relative risk for glaucoma that these factors contribute to participants in the Ocular Hypertension Treatment Study (OHTS). DESIGN: Comparative series. PARTICIPANTS: One thousand fifty-seven of 1636 participants (65%) of the OHTS were enrolled in this genetics ancillary study. METHODS: Samples of DNA were available from 1057 OHTS participants. Of these, 209 developed POAG (cases) and 848 did not develop glaucoma (controls) between 1994 and 2009. The frequencies of 13 risk alleles previously associated with POAG or with optic disc features in other cohorts were compared between POAG cases and controls in the OHTS cohort using analyses of variance. The 2 largest subgroups, non-Hispanic whites (n = 752; 70.7%) and blacks (n = 249, 23.7%), also were analyzed separately. The probability of glaucoma developing over the course of the OHTS was compared between participants stratified for transmembrane and coiled-coil domains 1 (TMCO1) risk alleles using Kaplan-Meier and Cox proportional hazards analyses. MAIN OUTCOME MEASURES: Association of POAG with known genetic factors. RESULTS: No association was detected between the known POAG risk alleles when the OHTS cohort was examined as a whole. However, in the subgroup of non-Hispanic whites, allele frequencies at the TMCO1 locus were statistically different between cases and controls (P = 0.00028). By 13 years, non-Hispanic white participants with TMCO1 risk alleles had a 12% higher cumulative frequency of glaucoma developing than participants with no TMCO1 risk alleles. Moreover, the Cox proportional hazard analysis demonstrated that TMCO1 alleles increased relative risk comparable with that of some previously analyzed clinical measures (i.e., intraocular pressure). CONCLUSIONS: The size of the OHTS cohort and its composition of 2 large racial subgroups may limit its power to detect some glaucoma risk factors. However, TMCO1 genotype was found to increase the risk of glaucoma developing among non-Hispanic whites, the largest racial subgroup in the OHTS cohort, at a magnitude similar to clinical predictors of disease that long have been associated with glaucoma.

Profiling IOP-Responsive Genes in the Trabecular Meshwork and Optic Nerve Head in a Rat Model of Controlled Elevation of Intraocular Pressure.

Purpose: The rat controlled elevation of intraocular pressure (CEI) model allows study of in vivo responses to short-term exposure to defined intraocular pressures (IOP). In this study, we used NanoString technology to investigate in vivo IOP-related gene responses in the trabecular meshwork (TM) and optic nerve head (ONH) simultaneously from the same animals. Methods: Male and female rats (N = 35) were subjected to CEI for 8 hours at pressures simulating mean, daytime normotensive rat IOP (CEI-20), or 2.5× IOP (CEI-50). Naïve animals that received no anesthesia or surgical interventions served as controls. Immediately after CEI, TM and ONH tissues were dissected, RNA was isolated, and samples were analyzed with a NanoString panel containing 770 genes. Postprocessing, raw count data were uploaded to ROSALIND for differential gene expression analyses. Results: For the TM, 45 IOP-related genes were significant in the CEI-50 versus CEI-20 and CEI-50 versus naïve comparisons, with 15 genes common to both comparisons. Bioinformatics analysis identified Notch and transforming growth factor beta (TGFß) pathways to be the most up- and downregulated Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, respectively. For ONH, 22 significantly differentially regulated genes were identified in the CEI-50 versus naïve comparison. Pathway analysis identified defense response and immune response as two significantly upregulated biological process pathways. Conclusions: This study demonstrated the ability to assay short-term IOP-responsive genes in both TM and ONH tissues simultaneously. In the TM, downregulation of TGFß pathway genes suggests that TM responses may reduce TGFß-induced extracellular matrix synthesis. For ONH, the initial response to short-term elevated IOP may be protective.

CRISPR-Cas9-mediated deletion of carbonic anhydrase 2 in the ciliary body to treat glaucoma.

The carbonic anhydrase 2 (Car2) gene encodes the primary isoenzyme responsible for aqueous humor (AH) production and plays a major role in the regulation of intraocular pressure (IOP). The CRISPR-Cas9 system, based on the ShH10 adenovirus-associated virus, can efficiently disrupt the Car2 gene in the ciliary body. With a single intravitreal injection, Car2 knockout can significantly and sustainably reduce IOP in both normal mice and glaucoma models by inhibiting AH production. Furthermore, it effectively delays and even halts glaucomatous damage induced by prolonged high IOP in a chronic ocular hypertension model, surpassing the efficacy of clinically available carbonic anhydrase inhibitors such as brinzolamide. The clinical application of CRISPR-Cas9 based disruption of Car2 is an attractive therapeutic strategy that could bring additional benefits to patients with glaucoma.