Cultured trabecular meshwork (TM) cells are a valuable model system to study the cellular mechanisms involved in the regulation of conventional outflow resistance and thus intraocular pressure; and their dysfunction resulting in ocular hypertension. In this review, we describe the standard procedures used for the isolation of TM cells from several animal species including humans, and the methods used to validate their identity. Having a set of standard practices for TM cells will increase the scientific rigor when used as a model, and enable other researchers to replicate and build upon previous findings.

Exfoliation syndrome (XFS) is the commonest known risk factor for secondary glaucoma and a significant cause of blindness worldwide. Variants in two genes, LOXL1 and CACNA1A have been previously associated with XFS. To further elucidate the genetic basis of XFS, we collected a global sample of XFS cases to refine the association at LOXL1, which previously showed inconsistent results between populations, and to identify new variants associated with XFS. We identified a rare, protective allele at LOXL1 (p.407Phe, OR = 25, P =2.9 × 10−14) through deep resequencing of XFS cases and controls from 9 countries. This variant results in increased cellular adhesion strength compared to the wild-type (p.407Tyr) allele. A genome-wide association study (GWAS) of XFS cases and controls from 24 countries followed by replication in 18 countries identified seven genome-wide significant loci (P < 5 × 10−8). Index variants at the new loci map to chromosomes 13q12 (POMP), 11q23.3 (TMEM136), 6p21 (AGPAT1), 3p24 (RBMS3) and 5q23 (near SEMA6A). These findings provide biological insights into the pathology of XFS, and highlight a potential role for naturally occurring rare LOXL1 variants in disease biology.

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.

The role of Ca 2؉ in stimulus-response coupling in nonexcitable cells is still not well understood. The Ca 2؉ responses of individual cells are extremely diverse, often displaying marked oscillations, and almost nothing is known about the specific features of these Ca 2؉ signals that are important for the functional response of a cell. Using the RBL-2H3 mucosal mast cell as a model, we have studied the temporal relationship between changes in intracellular Ca 2؉ and serotonin secretion at the single-cell level using simultaneous indo-1 photometry and constant potential amperometry. Secretion in response to antigen never occurs until intracellular Ca 2؉ is elevated, nor is it seen during the first few oscillations in Ca 2؉ . Exocytotic events tend to be clustered around the peaks of oscillations, but excellent secretion is also seen in cells with sustained elevations in Ca 2؉ . Ca 2؉ release from stores in the absence of influx fails to elicit secretion. If refilling and continued release of Ca 2؉ from stores is prevented with thapsigargin, Ca 2؉ influx can still trigger secretion, suggesting that store-associated microdomains of Ca 2؉ are not required for exocytosis. Our findings demonstrate the importance of an amplitude-encoded Ca 2؉ signal and Ca 2؉ influx for stimulus-secretion coupling in these nonexcitable cells.Although it is generally agreed that an increase in Ca 2ϩ is both necessary and sufficient for the initiation of secretion in most excitable cells, the role of Ca 2ϩ in nonexcitable cells is less clear (1). The individual Ca 2ϩ responses of nonexcitable cells are often extremely heterogeneous, and many models have been proposed for the generation of these complex and often oscillatory patterns (2). Both amplitude-encoded and frequency-encoded Ca 2ϩ signals have been proposed (3, 4), and the availability of both mechanisms would allow multiple signaling pathways to be activated by Ca 2ϩ in a single cell (5). Hepatocytes, with their repetitive oscillations of constant amplitude but variable frequency are prime candidates for frequencymodulated Ca 2ϩ signaling (6), and it has now been shown that mitochondrial NAD(P)H production in these cells is indeed regulated by the frequency of Ca 2ϩ oscillations (7,8). In contrast, it seems clear that the secretory response of single rat salivary acinar cells is tightly coupled to the amplitude of the Ca 2ϩ response (9), as is ciliary beating in tracheal epithelial cells (10). Furthermore, it has recently been shown that differential activation of transcription factors in B lymphocytes is achieved via non-oscillatory Ca 2ϩ signals of different amplitudes and durations (11).In most cases, however, it has not been easy to determine the specific features of the Ca 2ϩ signal that are important for a physiological response. This is because sensitive methods for detecting function at the single-cell level and with high temporal resolution are not readily available. Furthermore, it now seems clear that additional signals, such as the activation of protein kinase C (12) ...

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.

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 ¼ 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.

Both exfoliation glaucoma (XFG) and primary open-angle glaucoma (POAG) have been linked to decreased conventional outflow of aqueous humor (AH). To better understand the molecular changes in the AH content under such conditions, we analyzed the miRNA profiles of AH samples from patients with POAG and XFG compared to non-glaucoma controls. Individual AH samples (n = 76) were collected from POAG and XFG patients and age-matched controls during surgical procedure. After RNA extraction, the miRNA profiles were individually determined in 12 POAG, 12 XFG and 11 control samples. We identified 205, 295 and 195 miRNAs in the POAG, XFG and control samples, respectively. Our differential expression analysis identified three miRNAs (miR-125b-5p, miR-302d-3p and miR-451a) significantly different between POAG and controls, five miRNAs (miR-122-5p, miR-3144-3p, miR-320a, miR-320e and miR-630) between XFG and controls and one miRNA (miR-302d-3p) between POAG and XFG. While none of these miRNAs have been previously linked to glaucoma, miR-122-5p may target three glaucoma-associated genes: OPTN, TMCO1 and TGF-ß1. Pathway analysis revealed that these miRNAs are involved in potential glaucoma pathways, including focal adhesion, tight junctions, and TGF-ß signaling. Comparison of the miRNA profile in AH to unrelated human serum (n = 12) exposed potential relationships between these two fluids, although they were not significantly correlated. In summary, we have successfully profiled the miRNA expression without amplification in individual human AH samples and identified several POAG or XFG-associated miRNAs. These miRNAs may play a role in pathways previously implicated in glaucoma and act as biomarkers for disease pathogenesis.

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 (TGFb) family of cytokines. Elevation of systemic TGFb and chronic activation of TGFb 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 microfibrilassociated genes.