Copper, Lysyl Oxidase Activity, and Collagen in Aqueous Humour of Primary Glaucoma: An Association with Clinical Parameters.

INTRODUCTION: To measure copper (Cu), lysyl oxidase (LOX) activity, and collagen levels in aqueous humour (AH) of primary glaucoma patients and correlate with clinical parameters. METHODS: 120 patients with 40 each of primary open angle glaucoma (POAG), primary angle closure glaucoma (PACG), and cataract controls were recruited in this case-control study. AH samples were collected during the trabeculectomy and cataract surgeries. Cu levels were measured using an atomic absorption spectrophotometer. LOX unit activity was determined by Amplex Red assay and collagen concentration by Sirius red assay. RESULTS: Significantly higher levels of Cu expressed as median (IQR) µmol/L were observed in POAG (p = 0.008) and PACG (p = 0.005) compared to controls. The LOX activity was increased in POAG and PACG (p = 0.04) compared to controls represented as median (IQR) µmol/min. The collagen levels given as median (IQR) mg/ml showed an insignificant increase in POAG and PACG compared to controls (p = 0.78). The LOX unit activity was correlated with visual field index (VFI), which showed a significant increase with the progression of the diseases (p < 0.05), whereas Cu levels were negatively correlated with LOX activity in AH. Cu and LOX activity showed weak correlation with YAG peripheral iridotomy (YAGPI), duration of anti-glaucoma medications, and highest preoperative intraocular pressure. CONCLUSION: Elevated Cu and LOX activity was observed in both POAG and PACG groups compared to controls. LOX activity showed notable increase with VFI as the severity of the disease. Although Cu levels are increased in glaucoma, it's insufficient to significantly increase the activity of LOX.

Anti-glaucoma medications lowered decorin and altered profibrotic proteins in human tenon's fibroblasts.

Long term exposure to anti-glaucoma medications (AGMs) leads to an increase in extracellular matrix (ECM) accumulation in primary glaucoma patients. This study aims to evaluate the effect of topical AGMs in primary human tenon's fibroblasts (HTFs) and analyze the expression of profibrotic and anti-fibrotic proteins. Primary HTFs were cultured from patients undergoing cataract (control) and trabeculectomy. The different types of AGMs in single/multiple combinations (BB, PG, AA, CAI, CH, combinations of 3- PG + AA + CAI, 4A- BB + PG + AA + CAI, 4B- BB + PG + CAI + CH and 5- BB + PG + AA + CAI + CH) on chronic exposure were tested for cell viability using MTT assay and morphological alterations. Profibrotic proteins mainly SPARC, LOXL2, COL1A1 and anti-fibrotic DCN were analyzed in treated HTFs using q-PCR and ELISA. Sirius red staining and collagen gel contraction (CGC) assay were performed to assess collagen synthesis and the contractility of HTFs, respectively. Except for AA and CH, the other AGMs at a higher concentration were found to decrease the cell viability of HTFs. The morphology of HTFs were altered on exposure to BB, CH and AA; Profibrotic proteins i.e., SPARC, LOXL2 and COL1A1 were significantly increased (p < 0.05) on exposure to a combination of AGMs with TGF-ß1, whereas the anti-fibrotic DCN expression was significantly lowered (p < 0.05) in single/multiple AGM exposure. Sirius red staining showed increased collagen synthesis with combinations of AGMs with TGF-ß1. Meanwhile, HTFs showed increased collagen gel contraction with TGF-ß1, CAI and CH. This study reveals that altered profibrotic proteins, with significantly lowered DCN on chronic exposure of AGMs in HTFs.

A common variant near TGFBR3 is associated with primary open angle glaucoma.

Primary open angle glaucoma (POAG), a major cause of blindness worldwide, is a complex disease with a significant genetic contribution. We performed Exome Array (Illumina) analysis on 3504 POAG cases and 9746 controls with replication of the most significant findings in 9173 POAG cases and 26 780 controls across 18 collections of Asian, African and European descent. Apart from confirming strong evidence of association at CDKN2B-AS1 (rs2157719 [G], odds ratio [OR] = 0.71, P = 2.81 × 10(-33)), we observed one SNP showing significant association to POAG (CDC7-TGFBR3 rs1192415, ORG-allele = 1.13, Pmeta = 1.60 × 10(-8)). This particular SNP has previously been shown to be strongly associated with optic disc area and vertical cup-to-disc ratio, which are regarded as glaucoma-related quantitative traits. Our study now extends this by directly implicating it in POAG disease pathogenesis.

ABCC5, a gene that influences the anterior chamber depth, is associated with primary angle closure glaucoma.

Anterior chamber depth (ACD) is a key anatomical risk factor for primary angle closure glaucoma (PACG). We conducted a genome-wide association study (GWAS) on ACD to discover novel genes for PACG on a total of 5,308 population-based individuals of Asian descent. Genome-wide significant association was observed at a sequence variant within ABCC5 (rs1401999; per-allele effect size =  -0.045 mm, P = 8.17 × 10(-9)). This locus was associated with an increase in risk of PACG in a separate case-control study of 4,276 PACG cases and 18,801 controls (per-allele OR = 1.13 [95% CI: 1.06-1.22], P = 0.00046). The association was strengthened when a sub-group of controls with open angles were included in the analysis (per-allele OR = 1.30, P = 7.45 × 10(-9); 3,458 cases vs. 3,831 controls). Our findings suggest that the increase in PACG risk could in part be mediated by genetic sequence variants influencing anterior chamber dimensions.

Understanding the treatment paradigm and the sequencing of antiglaucoma fixed-dose combinations in a tertiary center in South India.

PURPOSE: To analyze the medical management of primary open-angle glaucoma (POAG) and ocular hypertension (OHT) and the placement of fixed-drug combination drugs (FDC) in the treatment paradigm at a tertiary eye care center in South India. METHODS: Retrospective study (January 2011-December 2015) of newly diagnosed POAG and OHT patients (≥18 years) with ≥5 years follow-up. Primary outcome included percentage use of different antiglaucoma drugs (at initiation) and FDCs at the first, second, and third progression (sequencing). Secondary outcomes: Percentage discontinuation for different FDCs, efficacy parameters (decrease in intraocular pressure (IOP)/visual field), adverse events, and the median number of antiglaucoma medications (AGMs) after 5 years. RESULTS: Three hundred and seventy eyes (198 patients) were analyzed; 84% of them had POAG. Prostaglandin analogs (PGAs) were the most common (66.2%). FDCs were started in 48 eyes (12.9%), with brimonidine + timolol combination being the most common. FDC usage at subsequent modifications was 10.1%, 24.04%, and 30.0%. Beta-blockers and PGAs were the most frequently prescribed AGMs in our practice pattern, with ß-blockers being the most consistent one. CONCLUSION: This study is a fairly large study with a minimum of a 5-year follow-up of patients with POAG and OHT and gives insights into the treatment patterns, use of FDCs, and the need for multiple medications over time.

Change in iris parameters with physiological mydriasis.

PURPOSE: A pilot study to analyze the association of change in iris parameters with physiological mydriasis in angle closure eyes compared with normal control eyes among south Indian participants. METHODS: Sixteen eyes diagnosed with primary angle closure and 14 age, gender-matched open-angle control eyes were selected. All participants underwent anterior segment imaging using anterior segment optical coherence tomography in dim and bright illumination. The iris area (IA), iris volume (IV), and pupillary diameter were calculated using customized image processing software. RESULTS: The IA and IV were found to be lesser in angle closure compared with normal controls. Average IA (dim and bright) was found to be 3.4 and 3.7 mm(2) in normals and 3.03 and 3.35 mm(2) in angle closure, and average IV (dim and bright) was found to be 24.28 and 26.09 mm(2) in normals and 21.38 and 22.6 mm(2) in angle closure. The IA and IV decreased significantly with pupillary dilation in normals and in angle closures. However, decrease in the IA (slope = 0.52 and 0.42; p = 0.012) and IV (slope = 1.97 and 1.41; p = 0.014) with pupillary dilation was significantly higher in normals compared with angle closures, respectively. CONCLUSIONS: IA and IV were found to decrease as pupil dilates in normal and angle closure eyes among South Indians. Loss in the IV was found to be lower in angle closure eyes.

Elastin modulation and modification by homocysteine: a key factor in the pathogenesis of Pseudoexfoliation syndrome?

BACKGROUND: Pseudoexfoliation syndrome (PXF) is an idiopathic, elastogenesis-associated systemic disease characterised by amyloid-like material aggregates in the eye. Elevated plasma and aqueous humour (aqH) homocysteine (Hcy) is reportedly associated with PXF. This study is aimed to probe Hcy-mediated alterations in elastin expression. METHODOLOGY: Lens level of Hcy (total Hcy (tHcy)), mRNA expression of Eln, CBS and MTR in lens capsule, protein expression of elastin in aqH were estimated by enzyme immunoassay, quantitative PCR and western blot, respectively in PXF, PXF with glaucoma (PXF-G) cases, in comparison with cataract-alone disease controls. Human lens epithelial cells (hLECs) were exposed to Hcy and homocysteine thiolactone (HCTL) to evaluate elastin expression in vitro. Furthermore, elastin recombinant protein was incubated with Hcy and HCTL to assess secondary and tertiary structural modifications based on circular dichroism spectroscopy, spectrophotometric and SEM studies. RESULTS: The lens tHcy was significantly high in PXF (p=0.02) and PXF-G (p=0.009). Eln expression was elevated in PXF and PXF-G (p=0.0007). Elastin level in aqH was elevated in PXF (p=0.01) and PXF-G (p=0.002). Hcy (200 µM) and HCTL (1 µM) promoted elastin expression at mRNA level by 36-fold (p=0.02) and 10-fold (p=0.05), respectively, and at protein level by nearly two-fold in cultured hLECs. Secondary structure changes in elastin protein caused by Hcy were evident from 34.11% drop in α-helix and 6.17% gain in ß-sheet. Fluorescence, spectral assays and SEM analyses showed aggregation and amyloid formation of elastin with homocysteinylation. CONCLUSION: The study reveals that lens accumulation of Hcy associated with hyperhomocysteinaemia is characteristic of PXF that augments elastin expression. Hcy causes structural changes promoting elastin aggregation, thereby contributing to defective elastin in PXF and PXF-G.

Lowered Decorin With Aberrant Extracellular Matrix Remodeling in Aqueous Humor and Tenon's Tissue From Primary Glaucoma Patients.

Purpose: To evaluate the inflammatory cytokine, growth factors, extracellular matrix (ECM) remodeling genes, profibrotic and antifibrotic molecules in patients undergoing glaucoma filtration surgery (GFS). Additionally, the effect of preoperative antiglaucoma medications (AGMs) and postoperative bleb status were related to these parameters. Methods: Tenon's tissue and aqueous humour (AH) were collected from 207 patients undergoing GFS with primary open-angle glaucoma (POAG) (n = 77), primary angle-closure glaucoma (PACG) (n = 62), and cataract controls (n = 68). Monocyte chemoattractant protein-1 (MCP-1), connective tissue growth factor (CTGF), transforming growth factor ß1/2 (TGF-ß1/2), lysyl oxidase (LOX), lysyl oxidase L2 (LOXL2), elastin (ELN), collagen type 1 α 1 (COL1A1), secreted protein acidic and rich in cysteine (SPARC), α-smooth muscle actin (α-SMA), and decorin (DCN) were determined in tenon's tissue by real-time PCR and in AH using ELISA. Results: A significant increase was observed in the transcripts of MCP-1, TGF-ß2, and SPARC in POAG and PACG (P < 0.05); CTGF, TGF-ß1, LOX, LOXL2, ELN, COL1A1, and α-SMA in PACG (P < 0.05) compared with control. DCN transcript was significantly decreased in POAG and PACG (P < 0.05) compared with control. The protein levels of CTGF, TGF-ß1/ß2, ELN, SPARC, and LOXL2 was significantly elevated in POAG and PACG (P < 0.05); DCN was decreased (P < 0.05) compared with control. These parameters showed significant association with duration of preoperative AGMs and postoperative bleb status. Conclusions: This study demonstrates increased expression of growth factors and ECM molecules, both at protein and transcript levels in GFS patients. A decreased DCN in AH seems striking, and if restored might have a therapeutic role in minimizing postoperative scarring to improve GFS outcome.

Detection of Proteins Associated with Extracellular Matrix Regulation in the Aqueous Humour of Patients with Primary Glaucoma.

Purpose: The protein composition of aqueous humour (AH) has held significant relevance and remains to be the prime sample in the discovery of biomarkers in glaucoma. The purpose of this study is to analyze the AH protein concentrations in primary open angle glaucoma (POAG) and primary angle closure glaucoma (PACG) and further examine the proteome changes compared to cataract control. Methods: AH was collected from 90 POAG, 72 PACG, 78 cataracts (controls) in this study. The total protein was quantified using Bradford's assay. Samples were subjected to trypsin digestion followed by liquid chromatography-mass spectrometry (LC-MS) for proteomic studies (n = 3 per group). The extracellular matrix has a major influence on the AH outflow, and the regulator proteins osteopontin (OPN), cathepsin D, and cystatin C detected by mass spectrometry are validated in AH samples by Western blot and turbidimetric immunoassay. Results: We observed a significant increase in protein levels of POAG (p = .0009); interestingly, a similar increase in PACG compared to cataract (p < .0001) and POAG (p = .02). Proteomics analysis identified 184, 190, and 299 proteins in control, POAG and PACG. OPN was increased in POAG (p = .0319) and PACG (p = .0103) compared to control. The precursor form of cathepsin D was increased in POAG and decreased in PACG, though not significant compared to control. Cystatin C was also increased in both POAG (p = .0310) and PACG (p = .0125) compared to control. Conclusion: In this study, we report for the first time that PACG cohort had higher total protein compared to controls. A qualitative comparison of proteomes revealed increased numbers of proteins identified in PACG. We assume that elevated levels of OPN and cystatin C in POAG and PACG along with altered cathepsin levels may contribute to ECM aberration in glaucoma.

Ahmed glaucoma valve in eyes with preexisting episcleral encircling element.

BACKGROUND: To describe the use of Ahmed glaucoma valve (AGV) in the management of intractable glaucoma in eyes with a preexisting episcleral encircling element. MATERIALS AND METHODS: This is a retrospective, consecutive, noncomparative study. The study included 12 eyes of 12 patients with a preexisting episcleral encircling element that underwent implantation of silicone AGV to treat intractable glaucoma during January 2009 to September 2010. RESULTS: The mean patient age was 25.6 (standard deviation 17.1) years. Five (41.6%) patients were monocular. The indications for AGV were varied. The mean duration between placement of episcleral encircling element and implantation of AGV was 30.5 (33.8) months. The mean follow-up was 37.4 (22.9) weeks. Preoperatively, the mean intraocular pressure (IOP) was 31.4 (7.9) mmHg and the mean antiglaucoma medications were 2.8. At the final postoperative follow-up, the mean IOP was 12.5 (3.5) mmHg and the mean number of antiglaucoma medications was 0.8 (P < 0.001). The complications observed over the follow-up period did include corneal graft failure in three eyes, tube erosion in two eyes and rhegmatogenous retinal detachment in one eye. CONCLUSION: AGV is an effective option in the management of intractable glaucoma in eyes with a preexisting episcleral encircling element keeping in mind the possibility of significant postoperative complications.

Genome-wide association analyses identify three new susceptibility loci for primary angle closure glaucoma.

Primary angle closure glaucoma (PACG) is a major cause of blindness worldwide. We conducted a genome-wide association study including 1,854 PACG cases and 9,608 controls across 5 sample collections in Asia. Replication experiments were conducted in 1,917 PACG cases and 8,943 controls collected from a further 6 sample collections. We report significant associations at three new loci: rs11024102 in PLEKHA7 (per-allele odds ratio (OR)=1.22; P=5.33×10(-12)), rs3753841 in COL11A1 (per-allele OR=1.20; P=9.22×10(-10)) and rs1015213 located between PCMTD1 and ST18 on chromosome 8q (per-allele OR=1.50; P=3.29×10(-9)). Our findings, accumulated across these independent worldwide collections, suggest possible mechanisms explaining the pathogenesis of PACG.

Outcomes of bleb excision with free autologous conjunctival patch grafting for bleb leak and hypotony after glaucoma filtering surgery.

PURPOSE: To determine the outcomes of bleb excision with free autologous conjunctival patch grafting for bleb leak and hypotony after glaucoma filtering surgery. PATIENTS AND METHODS: Retrospective, consecutive, noncomparative case series. Outcome measures were closure of bleb leak, intraocular pressure (IOP), and best corrected visual acuity after patch grafting and complications from intervention. Complete success was defined as resolution of the bleb leak or hypotony, with IOP between 6 and 18 mm Hg. RESULTS: Fifty-eight eyes (57 patients) were included: 51 with bleb leaks and 7 with hypotonous maculopathy without a bleb leak. Eight eyes required scleral flap resuturing and 2 required scleral patch grafts in addition to free conjunctival patch grafting. The mean postoperative follow-up period was 112.65 ± 128.74 weeks (median 80.0 wk). At 6 weeks and final follow-up, the IOP increased from baseline of 4.41 ± 4.61 mm Hg (median 4.00 mm Hg) to 11.98 ± 6.25 mm Hg (median 11.50 mm Hg) (P<0.001) and 12.67 ± 4.83 mm Hg (median 12.00) (P<0.001), respectively. Visual acuity increased from baseline of 0.87 ± 0.95 logMAR (median 0.60) to 0.65 ± 0.80 logMAR (median 0.48) (P=0.001) and 0.76 ± 0.93 logMAR (median 0.48) (P=0.35) at 6 weeks and last follow-up, respectively. The complete and qualified success rates at the final follow-up were 75.8% and 79.3%, respectively. Failure events occurred in 12 (20.6%) eyes, including 2 eyes with hypotony, 5 with raised IOP, and 3 with postoperative bleb leaks. CONCLUSIONS: Free conjunctival patch grafting is a successful procedure for bleb repair and hypotony providing moderate IOP control with minimal postoperative complications in majority of patients.