Rachel W. Kuchtey

Multiplex Cytokine Analysis Reveals Elevated Concentration of Interleukin-8 in Glaucomatous Aqueous Humor

PURPOSE To test the hypothesis that immune activation occurs in glaucoma by comparing concentrations of multiple cytokines in aqueous humor (AH) from patients with primary open angle glaucoma (POAG) and from cataract patients without glaucoma as controls. METHODS Cytokine concentrations in AH obtained during surgery were measured using microparticle-based immunoassays. Localized expression of IL-8 protein was investigated by immunohistochemistry of human eyes. RESULTS Eight cytokines (IL-1β, IL-2, IL-4, IL-5, IL-10, IL-12, IFN-γ, and TNF-α) were below the limits of detection, and two cytokines (IL-18 and IL-15) were detected at low levels or in only a few patients. Although IL-6 was detected in 26 of 30 control patients (median, 2.7 pg/mL) and in 23 of 29 POAG patients (median, 1.6 pg/mL), the difference was not statistically significant. IL-8 was detected in 28 of 30 control patients (median, 1.8 pg/mL) and in all 29 POAG patients (median, 4.9 pg/mL). The higher IL-8 concentration in the AH of POAG patients was statistically significant (P < 0.001). In pairs of eyes from patients with asymmetric glaucomatous optic nerve damage, IL-8 concentration was higher in the AH of the more severely affected eye (P < 0.05). Patients with severe visual field defects had higher IL-8 concentrations in the AH than did patients with mild visual field defects. IL-8 protein expression was found in human retina and optic nerve. CONCLUSIONS Concentration of the inflammatory cytokine IL-8 is significantly elevated in the AH of POAG patients, supporting the hypothesis that immune activation occurs in glaucoma.

Mapping of the Disease Locus and Identification of ADAMTS10 As a Candidate Gene in a Canine Model of Primary Open Angle Glaucoma

Primary open angle glaucoma (POAG) is a leading cause of blindness worldwide, with elevated intraocular pressure as an important risk factor. Increased resistance to outflow of aqueous humor through the trabecular meshwork causes elevated intraocular pressure, but the specific mechanisms are unknown. In this study, we used genome-wide SNP arrays to map the disease gene in a colony of Beagle dogs with inherited POAG to within a single 4 Mb locus on canine chromosome 20. The Beagle POAG locus is syntenic to a previously mapped human quantitative trait locus for intraocular pressure on human chromosome 19. Sequence capture and next-generation sequencing of the entire canine POAG locus revealed a total of 2,692 SNPs segregating with disease. Of the disease-segregating SNPs, 54 were within exons, 8 of which result in amino acid substitutions. The strongest candidate variant causes a glycine to arginine substitution in a highly conserved region of the metalloproteinase ADAMTS10. Western blotting revealed ADAMTS10 protein is preferentially expressed in the trabecular meshwork, supporting an effect of the variant specific to aqueous humor outflow. The Gly661Arg variant in ADAMTS10 found in the POAG Beagles suggests that altered processing of extracellular matrix and/or defects in microfibril structure or function may be involved in raising intraocular pressure, offering specific biochemical targets for future research and treatment strategies.

Angiopoietin-like 7 secretion is induced by glaucoma stimuli and its concentration is elevated in glaucomatous aqueous humor.

PURPOSE To investigate the possibility that Angiopoietin-like 7 (ANGPTL7) protein is involved in the pathogenesis of glaucoma. METHODS Primary human trabecular meshwork (TM) cells and corneoscleral explants were stimulated with either dexamethasone (DEX) or transforming growth factor beta (TGFbeta), and ANGPTL7 protein secreted into culture medium was determined by Western blot analysis. The effect of stable overexpression of ANGPTL7 in transfected immortalized TM cell lines on collagen expression was investigated by immunocytochemistry. Localization of ANGPTL7 protein in human eyes was determined by immunohistochemistry. The concentration of ANGPTL7 protein in aqueous humor (AH) from patients with glaucoma and control patients was compared by Western blot analysis. The beagle model of primary open-angle glaucoma (POAG) was used to correlate ANGPTL7 protein levels in canine AH with disease progression. RESULTS TGFbeta and DEX stimulated secretion of ANGPTL7 protein by TM cells and corneoscleral explants. Overexpression of ANGPTL7 by immortalized TM cell lines increased expression of type I collagen. Expression of ANGPTL7 protein was located in the corneal stroma, near the limbus, and throughout the sclera, with lower expression in the TM. In the lamina cribrosa, ANGPTL7 expression was associated with the cribriform plates. The concentration of ANGPTL7 protein was elevated in AH from patients with glaucoma and increased as disease progressed in POAG beagle dogs. CONCLUSIONS Induction of ANGPTL7 secretion by glaucoma stimuli and increased concentration of ANGPTL7 in glaucomatous AH suggest that ANGPTL7 is overexpressed in glaucoma. Since overexpression of ANGPTL7 increases collagen expression, a potential disease mechanism, ANGPTL7 could have a pathogenic role in glaucoma, and may serve as a potential therapeutic target.

The Microfibril Hypothesis of Glaucoma: Implications for Treatment of Elevated Intraocular Pressure

Microfibrils are macromolecular aggregates located in the extracellular matrix of both elastic and nonelastic tissues that have essential functions in formation of elastic fibers and control of signaling through the transforming growth factor beta (TGFβ) family of cytokines. Elevation of systemic TGFβ and chronic activation of TGFβ signal transduction are associated with diseases caused by mutations in microfibril-associated genes, including FBN1. A role for microfibrils in glaucoma is suggested by identification of risk alleles in LOXL1 for exfoliation glaucoma and mutations in LTBP2 for primary congenital glaucoma, both of which are microfibril-associated genes. Recent identification of a mutation in another microfibril-associated gene, ADAMTS10, in a dog model of primary open-angle glaucoma led us to form the microfibril hypothesis of glaucoma, which in general states that defective microfibrils may be an underlying cause of glaucoma. Microfibril defects could contribute to glaucoma through alterations in biomechanical properties of tissue and/or through effects on signaling through TGFβ, which is well established to be elevated in the aqueous humor of glaucoma patients. Recent work has shown that diseases caused by microfibril defects are associated with increased concentrations of TGFβ protein and chronic activation of TGFβ-mediated signal transduction. In analogy with other microfibril-related diseases, defective microfibrils could provide a mechanism for the elevation of TGFβ2 in glaucomatous aqueous humor. If glaucoma shares mechanisms with other diseases caused by defective microfibrils, such as Marfan syndrome, therapeutic interventions to inhibit chronic activation of TGFβ signaling used in those diseases may be applied to glaucoma.

Screening ADAMTS10 in dog populations supports Gly661Arg as the glaucoma-causing variant in beagles.

PURPOSE Previously, we mapped the disease locus in the beagle model of autosomal recessive primary open angle glaucoma (POAG) to a 4-Mb interval on chromosome 20, and identified a Gly661Arg variant in ADAMTS10 as the candidate disease-causing variant. The purpose of this study was to test the hypothesis that the Gly661Arg variant of ADAMTS10 causes glaucoma by genotyping dogs of various breeds affected and unaffected by primary glaucoma. METHODS Dogs of various breeds, affected or unaffected with primary glaucoma, were genotyped for the Gly661Arg variant of ADAMTS10, as well as 7 other nonsynonymous single nucleotide polymorphisms (SNPs) in other genes in the beagle POAG locus that segregate with disease. Alternate allele frequencies were calculated with 95% confidence intervals and comparisons made to expected allele frequency relative to disease prevalence or between cases and controls. RESULTS For the nonsynonymous SNPs other than the ADAMTS10 variant, control dogs were identified that were homozygous for the alternative alleles, ruling out those variants as causative. None of the nonsynonymous SNPs were found associated with primary glaucoma in American cocker spaniels. The Gly661Arg variant of ADAMTS10 was the only variant with minor allele frequency consistent with the prevalence of primary glaucoma in the general beagle population. The only dog found homozygous for the Gly661Arg variant of ADAMTS10 was an affected beagle, unrelated to the POAG colony. CONCLUSIONS These findings support the Gly661Arg mutation of ADAMTS10 as the likely cause of POAG in beagles.

Prospective Retinal and Optic Nerve Vitrectomy Evaluation (PROVE) Study: Twelve-Month Findings

PURPOSE To report 1-year outcomes of the Prospective Retinal and Optic Nerve Vitrectomy Evaluation study. DESIGN Prospective, controlled, observational study. PARTICIPANTS Eighty eyes of 40 participants undergoing pars plana vitrectomy for epiretinal membrane (ERM), macular hole (MH), or vitreous opacities. METHODS Enrolled participants underwent baseline evaluation of the study (surgical) and fellow (control) eyes by a masked fellowship-trained glaucoma specialist; evaluation included intraocular pressure (IOP; Goldmann applanation and Tono-Pen), central corneal thickness, gonioscopy, and cup-to-disc ratio measurement. Baseline testing included bilateral color fundus and optic disc photography, fundus autofluorescence, automated perimetry, and optical coherence tomography (OCT) of the macula and optic nerve. Evaluations were repeated at 3 months and 1 year after surgery. MAIN OUTCOME MEASURES The primary outcome measure was changes in peripapillary retinal nerve fiber layer (pRNFL) thickness. Secondary outcomes included changes in macular thickness and IOP. RESULTS Thirty-eight of 40 patients completed 1 year of follow-up. Mean visual acuity (VA) improved in study eyes from baseline (P = 0.003) but remained worse than fellow eyes (P<0.001). Study eyes had thinner inferior pRNFL thickness (114±16.8 μm) compared with fellow eyes (123±14.7 μm; P = 0.004). Mean IOP difference between study eyes and fellow eyes increased from baseline to 1 year. At 1 year, MH study eyes had higher mean IOP (16.0±3.7 mmHg) compared with fellow eyes (14.8±3.4 mmHg; P = 0.08). Mean IOP for pseudophakic study eyes increased from 14.5±3.2 mmHg at baseline to 16.0±2.8 mmHg at 1 year (P = 0.04). Central subfield thickness (CST) and cube volume decreased in study eyes at 1 year but remained greater than that of fellow eyes (P<0.05). Reduction in CST from baseline correlated with degree of VA improvement (P<0.05). Mean deviation (MD) improved in ERM study eyes at 1 year when compared with baseline (-2.2 vs. -4.0; P = 0.02) but remained worse than fellow eyes (-1.2; P = 0.002). CONCLUSIONS One year after vitrectomy, VA, CST, and MD improved in study eyes but not to the level of fellow eyes. Inferior pRNFL thickness decreased in study eyes. Reduction in CST from baseline correlated with degree of VA improvement. Pseudophakic study eyes demonstrated increased IOP when compared with baseline.

Metabolome-Wide Association Study of Primary Open Angle Glaucoma.

PURPOSE To determine if primary open-angle glaucoma (POAG) patients can be differentiated from controls based on metabolic characteristics. METHODS We used ultra-high resolution mass spectrometry with C18 liquid chromatography for metabolomic analysis on frozen plasma samples from 72 POAG patients and 72 controls. Metabolome-wide Spearman correlation was performed to select differentially expressed metabolites (DEM) correlated with POAG. We corrected P values for multiple testing using Benjamini and Hochberg false discovery rate (FDR). Hierarchical cluster analysis (HCA) was used to depict the relationship between participants and DEM. Differentially expressed metabolites were matched to the METLIN metabolomics database; both DEM and metabolites significantly correlating with DEM were analyzed using MetaboAnalyst to identify metabolic pathways altered in POAG. RESULTS Of the 2440 m/z (mass/charge) features recovered after filtering, 41 differed between POAG cases and controls at FDR = 0.05. Hierarchical cluster analysis revealed these DEM to associate into eight clusters; three of these clusters contained the majority of the DEM and included palmitoylcarnitine, hydroxyergocalciferol, and high-resolution METLIN matches to sphingolipids, other vitamin D-related metabolites, and terpenes. MetaboAnalyst also indicated likely alteration in steroid biosynthesis pathways. CONCLUSIONS Global ultrahigh resolution metabolomics emphasized the importance of altered lipid metabolism in POAG. The results suggest specific metabolic processes, such as those involving palmitoylcarnitine, sphingolipids, vitamin D-related compounds, and steroid precursors, may contribute to POAG status and merit more detailed study with targeted methods.

Marfan syndrome caused by a novel FBN1 mutation with associated pigmentary glaucoma

Mutations in fibrillin‐1 (FBN1) cause a wide spectrum of disorders, including Marfan syndrome, which have in common defects in fibrillin‐1 microfibrils. Ectopia lentis and myopia are frequently observed ocular manifestations of Marfan syndrome. Glaucoma is also associated with Marfan syndrome, though the form of glaucoma has not been well‐characterized. In this report, ocular examination of a patient diagnosed with Marfan syndrome based on family history and aortic dilatation was performed, including measurement of facility of aqueous humor outflow by tonography. The patient did not have ectopia lentis at the age of 42 years. Based on optic nerve appearance, reduced outflow facility, elevated IOP with open angles and clear signs of pigment dispersion, the patient was diagnosed with pigmentary glaucoma. The patient was heterozygous for a novel truncating mutation in FBN1, p.Leu72Ter. Histology of normal human eyes revealed abundant expression of elastic fibers and fibrillin‐1 in aqueous humor outflow structures. This is the first report of a patient with Marfan syndrome that is caused by a confirmed FBN1 mutation with associated pigmentary glaucoma. In addition to identifying a novel mutation of FBN1 and broadening the spectrum of associated ocular phenotypes in Marfan syndrome, our findings suggest that pigmentary glaucoma may involve defects in fibrillin‐1 microfibrils.

Elevated Transforming Growth Factor β1 in Plasma of Primary Open-Angle Glaucoma Patients

PURPOSE To test the hypothesis that primary open-angle glaucoma (POAG) patients have a systemic elevation of transforming growth factor β1 (TGFβ1). METHODS Plasma was prepared from blood samples drawn from patients of the Vanderbilt Eye Institute during clinic visits. Concentrations of total TGFβ1 and thrombospondin-1 (TSP1) in plasma were determined by ELISA. Statistical significance of differences between POAG and control samples was evaluated by Mann-Whitney test. Regression analysis was used to evaluate correlations between plasma TGFβ1 and patient age and between plasma TGFβ1 and TSP1. RESULTS Plasma samples were obtained from 148 POAG patients and 150 controls. Concentration of total TGFβ1 in the plasma of POAG patients (median = 3.25 ng/mL) was significantly higher (P < 0.0001) than in controls (median = 2.46 ng/mL). Plasma TGFβ1 was not correlated with age of patient (P = 0.17). Thrombospondin-1 concentration was also significantly higher (P < 0.0001) in POAG patients (median = 0.774 μg/mL) as compared to controls (median = 0.567 μg/mL). Plasma total TGFβ1 and TSP1 concentrations were linearly correlated (P < 0.0001). CONCLUSIONS Plasma samples from POAG patients display elevated total TGFβ1 compared to controls, consistent with elevated systemic TGFβ1 in POAG patients.

A Common Variant in MIR182 Is Associated With Primary Open-Angle Glaucoma in the NEIGHBORHOOD ConsortiumAssociation of miR-182 and POAG in NEIGHBORHOOD

Purpose Noncoding microRNAs (miRNAs) have been implicated in the pathogenesis of glaucoma. We aimed to identify common variants in miRNA coding genes (MIR) associated with primary open-angle glaucoma (POAG). Methods Using the NEIGHBORHOOD data set (3853 cases/33,480 controls with European ancestry), we first assessed the relation between 85 variants in 76 MIR genes and overall POAG. Subtype-specific analyses were performed in high-tension glaucoma (HTG) and normal-tension glaucoma subsets. Second, we examined the expression of miR-182, which was associated with POAG, in postmortem human ocular tissues (ciliary body, cornea, retina, and trabecular meshwork [TM]), using miRNA sequencing (miRNA-Seq) and droplet digital PCR (ddPCR). Third, miR-182 expression was also examined in human aqueous humor (AH) by using miRNA-Seq. Fourth, exosomes secreted from primary human TM cells were examined for miR-182 expression by using miRNA-Seq. Fifth, using ddPCR we compared miR-182 expression in AH between five HTG cases and five controls. Results Only rs76481776 in MIR182 gene was associated with POAG after adjustment for multiple comparisons (odds ratio [OR] = 1.23, 95% confidence interval [CI]: 1.11–1.42, P = 0.0002). Subtype analysis indicated that the association was primarily in the HTG subset (OR = 1.26, 95% CI: 1.08–1.47, P = 0.004). The risk allele T has been associated with elevated miR-182 expression in vitro. Data from ddPCR and miRNA-Seq confirmed miR-182 expression in all examined ocular tissues and TM-derived exosomes. Interestingly, miR-182 expression in AH was 2-fold higher in HTG patients than nonglaucoma controls (P = 0.03) without controlling for medication treatment. Conclusions Our integrative study is the first to associate rs76481776 with POAG via elevated miR-182 expression.