Pratap Challa

Cellular senescence in the glaucomatous outflow pathway.

The mechanisms responsible for the progressive malfunction of the trabecular meshwork (TM)-Schlemms canal (SC) conventional outflow pathway tissue in primary open angle glaucoma (POAG) are still not fully understood. To determine whether POAG is characterized by an accumulation of senescent cells, similar to what has been described in other diseases, we have compared the levels of the senescence marker senescence-associated-beta-galactosidase (SA-beta-gal) in the outflow pathway cells of POAG and age-matched control donors. POAG donors demonstrated a statistically significant fourfold increase in the percentage of SA-beta-gal positive cells. These results suggest a potential role for cellular senescence in the pathophysiology of the outflow pathway.

High failure rate associated with 180 degrees selective laser trabeculoplasty.

Purpose:To determine the efficacy of selective laser trabeculoplasty (SLT) in a tertiary care referral center. Patients and Methods:In this retrospective study of selective laser trabeculoplasty performed by five physicians, 94 eyes from 94 patients were included. A majority (83/92, 90%) underwent 180° selective laser trabeculoplasty. Selective laser trabeculoplasty failure was defined in two ways: (1) IOP decrease <3 mm Hg (definition one), or (2) IOP decrease <20% (definition two), on two successive visits ≥4 weeks after SLT. Results:Overall failure rates were 68% (64/94) and 75% (70/94) (by definitions one and two, respectively). By survival/life-table analysis, mean time to failure was 6 months and 5.5 months, by definitions one and two, respectively. By the end of the study (14.5 months), the failure rates were 86% and 92% by definitions one and two, respectively. By each definition, in both univariable and multivariable analysis, only lower baseline IOP was a significant predictor of failure. Conclusions:Selective laser trabeculoplasty had an overall low success rate in our tertiary clinic population, with overall failure rates of 68% to 74% in those who underwent 180° selective laser trabeculoplasty.

Mechanisms of action and efficacy of argon laser trabeculoplasty and selective laser trabeculoplasty.

Purpose of review Since the 1980s, laser trabeculoplasty has served as an effective way to lower intraocular pressure in patients with primary or secondary open angle glaucomas, both as an initial therapy or in conjunction with hypotensive medications. This manuscript will describe the proposed mechanisms of action of argon laser trabeculoplasty and selective laser trabeculoplasty, as well as review current studies of the therapeutic effect of these interventions. Recent findings The exact mechanisms by which argon laser and selective laser trabeculoplasty lower intraocular pressure are not known. There are several theories, however, and we discuss the three most common ones: the mechanical theory, the cellular (biologic) theory, and the cell division theory. Since both lasers are applied to the same tissue and produce similar results, they most likely produce their effects in comparable ways. We also describe the results of several studies comparing these devices. Most show them to be equally effective at lowering intraocular pressure; however, there are a few circumstances when selective laser trabeculoplasty may be a better option than argon laser trabeculoplasty. Summary Argon laser and selective laser trabeculoplasty are safe and effective procedures for lowering intraocular pressure. The results of ongoing clinical trials will help further define their role in the management of patients with open angle glaucoma.

Resveratrol prevents the expression of glaucoma markers induced by chronic oxidative stress in trabecular meshwork cells

Elevated intraocular pressure (IOP) constitutes the best characterized risk for primary open-angle glaucoma (POAG). Elevated IOP is believed to result from an increase in aqueous humor outflow resistance at the level of the trabecular meshwork (TM)/Schlemms canal. Malfunction of the TM in POAG is associated with the expression of markers for inflammation, cellular senescence, oxidative damage, and decreased cellularity. Current POAG treatments rely on lowering IOP, but there is no therapeutic approach available to delay the loss of function of the TM in POAG patients. We evaluated the effects of chronic administration of the dietary supplement resveratrol on the expression of markers for inflammation, oxidative damage, and cellular senescence in primary TM cells subjected to chronic oxidative stress (40% O2). Resveratrol treatment effectively prevented increased production of intracellular reactive oxygen species (iROS) and inflammatory markers (IL1alpha, IL6, IL8, and ELAM-1), and reduced expression of the senescence markers sa-beta-gal, lipofuscin, and accumulation of carbonylated proteins. Furthermore, resveratrol exerted antiapoptotic effects that were not associated with a decrease in cell proliferation. These results suggest that resveratrol could potentially have a role in preventing the TM tissue abnormalities observed in POAG.

Induction of TGF-β1 in the trabecular meshwork under cyclic mechanical stress

The pathophysiological mechanisms involved in the failure of the trabecular meshwork (TM) to maintain normal levels of aqueous outflow in glaucoma are not yet understood. Aberrant activation of the transforming growth factor beta‐1 (TGF‐β1) pathway has been implicated in several degenerative diseases. We investigated the possibility that chronic cyclic mechanical stress that affects the TM might result in increased production of TGF‐β1. Primary cultures of TM cells subjected to cyclic mechanical stress (5% stretching, 1 cycle/sec) demonstrate a significant increase in total and biologically active secreted TGF‐β1 that was associated with activation of the TGF‐β1 promoter, measured using a recombinant adenovirus expressing the secreted reporter gene secreted alkaline phosphatase protein (SEAP) under the TGF‐β1 gene promoter (AdTGFβ1‐SEAP). Associated changes in the transcription of MMP‐2, TIMP‐2, and CTGF were assessed by semiquantitative PCR. Immunohistochemical analysis of TGF‐β1 in organ culture of human eyes revealed a generalized accumulation of this protein in the extracellular matrix (ECM) of the TM, while expression of the TGF‐β1 promoter, analyzed using the LacZ reporter gene, was localized in some specific cells within the outflow pathway. Induction of the TGF‐β1 promoter in organ culture was demonstrated using a novel model for cyclic mechanical stress in human perfused anterior segments infected with AdTGFβ1‐SEAP. Given the relevant physiological and pathophysiological roles of TGF‐β1, its induction after cyclic mechanical stress in the TM supports the hypothesis that this cytokine might play a significant role in the physiology of the TM, and contribute to the pathological changes of this tissue in certain forms of glaucoma.

Investigation of Known Genetic Risk Factors for Primary Open Angle Glaucoma in Two Populations of African Ancestry

PURPOSE Multiple genes have been associated with primary open angle glaucoma (POAG) in Caucasian populations. We now examine the association of these loci in populations of African ancestry, populations at particularly high risk for POAG. METHODS We genotyped DNA samples from two populations: African American (1150 cases and 999 controls) and those from Ghana, West Africa (483 cases and 593 controls). Our analysis included 57 single nucleotide polymorphisms (SNPs) in five loci previously associated with POAG at the genome-wide level, including CDKN2B-AS1, TMCO1, CAV1/CAV2, chromosome 8q22 intergenic region, and SIX1/SIX6. We evaluated association in the full datasets, as well as subgroups with normal pressure glaucoma (NPG, maximum IOP ≤21 mm Hg) and high pressure glaucoma (HPG, IOP >21 mm Hg). RESULTS In African Americans, we identified an association of rs10120688 in the CDNK2B-AS1 region with POAG (P = 0.0020). Several other SNPs were nominally associated, but did not survive correction for multiple testing. In the subgroup analyses, significant associations were identified for rs10965245 (P = 0.0005) in the CDKN2B-AS1 region with HPG and rs11849906 in the SIX1/SIX6 region with NPG (P = 0.006). No significant association was identified with any loci in the Ghanaian samples. CONCLUSIONS POAG genetic susceptibility alleles associated in Caucasians appear to play a greatly reduced role in populations of African ancestry. Thus, the major genetic components of POAG of African origin remain to be identified. This finding underscores the critical need to pursue large-scale genome-wide association studies in this understudied, yet disproportionately affected population.

Lack of association between LOXL1 variants and primary open-angle glaucoma in three different populations.

PURPOSE Significant association has recently been reported between pseudoexfoliation glaucoma (XFG) and two single-nucleotide polymorphisms (SNPs), rs3825942, and rs1048661, in the lysyl oxidase-like 1 gene (LOXL1). The purpose of this study was to investigate whether XFG-associated variants of LOXL1 play a significant role in primary open-angle glaucoma in the Caucasian, African-American, and Ghanaian (West-African) populations. METHODS POAG was defined as the presence of glaucomatous optic nerve damage, associated visual field loss, and elevated intraocular pressure (>22 mm Hg in both eyes). Thirteen tagging SNPs were genotyped by allelic discrimination assays in the Caucasian (279 cases and 227 controls), African-American (193 cases and 97 controls), and Ghanaian (170 cases and 138 controls) populations. Allele and genotype frequencies were compared between the cases and controls from each population. RESULTS None of the SNPs associated with XFG in LOXL1 were significantly associated with POAG in these populations. The risk allele frequencies for rs2165241 and rs3825942 were significantly lower in the African-American and Ghanaian populations, compared with Caucasian individuals. CONCLUSIONS There was no association between SNPs in the LOXL1 gene and POAG. This is the first analysis of the LOXL1 gene in African-American and West-African populations. LOXL1 gene variants do not appear to play a significant role in the pathogenesis of POAG in populations of either Caucasian or West-African ancestry.

Extracellular Release of ATP Mediated by Cyclic Mechanical Stress Leads to Mobilization of AA in Trabecular Meshwork Cells

PURPOSE To investigate the mechanisms that mediate the release of ATP induced by cyclic mechanical stress (CMS) and the role of extracellular ATP in the mobilization of arachidonic acid (AA) and prostaglandin secretion. METHODS Porcine trabecular meshwork (pTM) cells were subjected to CMS. Extracellular ATP was detected with a luciferin-luciferase assay in the presence or absence of transport inhibitors and a lipid raft disrupter. ATP vesicles were visualized with quinacrine. The release of AA (AA 1-14C) was measured with and without ATP, ATP inhibitors, and phospholipase-A and -C inhibitors. Prostaglandin E2 (PGE2) and viability were measured with ELISA and a lactate dehydrogenase assay, respectively. RESULTS CMS induced ATP release that was inhibited by the vesicle inhibitors N-ethylmaleimide (NEM) and monensin. Lipid raft disruption significantly increased the extracellular ATP induced by CMS. CMS induced AA release (1-4-fold increase) and its metabolic product PGE2 (3.9-fold increase). The AA mobilization induced by CMS could be mimicked by the addition of extracellular ATP and was partially inhibited by a P2 antagonist, by an ATP inhibitor, and by inhibitors of phospholipase-A2 and -C. Addition of PGE2 (10 microM) to the media exerted cytoprotective effects against long-term CMS. CONCLUSIONS Extracellular release of ATP induced by CMS in TM cells is mediated by exocytosis of ATP-enriched vesicles into lipid rafts. The resulting activation of purinergic receptors leads to mobilization of AA from the plasma membrane. The subsequent release of PGE could exert protective effects by preventing TM cell loss that may result from chronic exposure to CMS.

Genetics of Pseudoexfoliation Syndrome

Purpose of review Pseudoexfoliation syndrome (XFS) is a late-onset and complex disorder that is strongly associated with the development of glaucoma. The purpose of this review is to discuss the inheritance patterns and recent genetic advances in the study of this disorder. Recent findings XFS has a strong familial association and recently, the lysyl oxidase-like 1 gene has been strongly associated with this disorder. This gene is involved in the synthesis and maintenance of elastic fibers and therefore has a strong biological rationale for being involved in this disorder. However, the exact relationship between lysyl oxidase-like 1 polymorphisms and the development of XFS has not been elucidated. Also, the value of genetic testing for this disorder has not been validated. Summary XFS is an important risk factor for glaucoma and lysyl oxidase-like 1 polymorphisms are strongly associated with XFS. The mechanisms behind glaucoma development and the value of genetic testing are not clear and further study is needed.

Autotaxin-Lysophosphatidic Acid Axis Is a Novel Molecular Target for Lowering Intraocular Pressure

Primary open-angle glaucoma is the second leading cause of blindness in the United States and is commonly associated with elevated intraocular pressure (IOP) resulting from diminished aqueous humor (AH) drainage through the trabecular pathway. Developing effective therapies for increased IOP in glaucoma patients requires identification and characterization of molecular mechanisms that regulate IOP and AH outflow. This study describes the identification and role of autotaxin (ATX), a secretory protein and a major source for extracellular lysophosphatidic acid (LPA), in regulation of IOP in a rabbit model. Quantitative proteomics analysis identified ATX as an abundant protein in both human AH derived from non-glaucoma subjects and in AH from different animal species. The lysophospholipase D (LysoPLD) activity of ATX was found to be significantly elevated (by ∼1.8 fold; n = 20) in AH derived from human primary open angle glaucoma patients as compared to AH derived from age-matched cataract control patients. Immunoblotting analysis of conditioned media derived from primary cultures of human trabecular meshwork (HTM) cells has confirmed secretion of ATX and the ability of cyclic mechanical stretch of TM cells to increase the levels of secreted ATX. Topical application of a small molecular chemical inhibitor of ATX (S32826), which inhibited AH LysoPLD activity in vitro (by >90%), led to a dose-dependent and significant decrease of IOP in Dutch-Belted rabbits. Single intracameral injection of S32826 (∼2 µM) led to significant reduction of IOP in rabbits, with the ocular hypotensive response lasting for more than 48 hrs. Suppression of ATX expression in HTM cells using small-interfering RNA (siRNA) caused a decrease in actin stress fibers and myosin light chain phosphorylation. Collectively, these observations indicate that the ATX-LPA axis represents a potential therapeutic target for lowering IOP in glaucoma patients.