AAV-NDI1 Therapy Provides Significant Benefit to Murine and Cellular Models of Glaucoma.

Glaucoma, a leading cause of blindness, is a multifactorial condition that leads to progressive loss of retinal ganglion cells (RGCs) and vision. Therapeutic interventions based on reducing ocular hypertension are not always successful. Emerging features of glaucoma include mitochondrial dysfunction and oxidative stress. In the current study, NDI1-based gene therapy, which improves mitochondrial function and reduces reactive oxygen species, was delivered intraocularly via an adeno-associated viral vector (AAV). This AAV-NDI1 therapy protected RGCs from cell death in treated (1552.4 ± 994.0 RGCs/mm2) versus control eyes (1184.4 ± 978.4 RGCs/mm2, p < 0.05) in aged DBA/2J mice, a murine model of glaucoma. The photonegative responses (PhNRs) of RGCs were also improved in treated (6.4 ± 3.3 µV) versus control eyes (5.0 ± 3.1 µV, p < 0.05) in these mice. AAV-NDI1 also provided benefits in glaucomatous human lamina cribrosa (LC) cells by significantly increasing basal and maximal oxygen consumption rates and ATP production in these cells. Similarly, NDI1 therapy significantly protected H2O2-insulted primary porcine LC cells from oxidative stress. This study highlights the potential utility of NDI1 therapies and the benefits of improving mitochondrial function in the treatment of glaucoma.

Calcium-Signalling in Human Glaucoma Lamina Cribrosa Myofibroblasts.

Glaucoma is one of the most common causes of treatable visual impairment in the developed world, affecting approximately 64 million people worldwide, some of whom will be bilaterally blind from irreversible optic nerve damage. The optic nerve head is a key site of damage in glaucoma where there is fibrosis of the connective tissue in the lamina cribrosa (LC) extracellular matrix. As a ubiquitous second messenger, calcium (Ca2+) can interact with various cellular proteins to regulate multiple physiological processes and contribute to a wide range of diseases, including cancer, fibrosis, and glaucoma. Our research has shown evidence of oxidative stress, mitochondrial dysfunction, an elevated expression of Ca2+ entry channels, Ca2+-dependent pumps and exchangers, and an abnormal rise in cytosolic Ca2+ in human glaucomatous LC fibroblast cells. We have evidence that this increase is dependent on Ca2+ entry channels located in the plasma membrane, and its release is from internal stores in the endoplasmic reticulum (ER), as well as from the mitochondria. Here, we summarize some of the molecular Ca2+-dependent mechanisms related to this abnormal Ca2+-signalling in human glaucoma LC cells, with a view toward identifying potential therapeutic targets for ongoing optic neuropathy.

p53 and Myofibroblast Apoptosis in Organ Fibrosis.

Organ fibrosis represents a dysregulated, maladaptive wound repair response that results in progressive disruption of normal tissue architecture leading to detrimental deterioration in physiological function, and significant morbidity/mortality. Fibrosis is thought to contribute to nearly 50% of all deaths in the Western world with current treatment modalities effective in slowing disease progression but not effective in restoring organ function or reversing fibrotic changes. When physiological wound repair is complete, myofibroblasts are programmed to undergo cell death and self-clearance, however, in fibrosis there is a characteristic absence of myofibroblast apoptosis. It has been shown that in fibrosis, myofibroblasts adopt an apoptotic-resistant, highly proliferative phenotype leading to persistent myofibroblast activation and perpetuation of the fibrotic disease process. Recently, this pathological adaptation has been linked to dysregulated expression of tumour suppressor gene p53. In this review, we discuss p53 dysregulation and apoptotic failure in myofibroblasts and demonstrate its consistent link to fibrotic disease development in all types of organ fibrosis. An enhanced understanding of the role of p53 dysregulation and myofibroblast apoptosis may aid in future novel therapeutic and/or diagnostic strategies in organ fibrosis.

The Role of miR-29 Family in TGF-ß Driven Fibrosis in Glaucomatous Optic Neuropathy.

Primary open angle glaucoma (POAG), a chronic optic neuropathy, remains the leading cause of irreversible blindness worldwide. It is driven in part by the pro-fibrotic cytokine transforming growth factor beta (TGF-ß) and leads to extracellular matrix remodelling at the lamina cribrosa of the optic nerve head. Despite an array of medical and surgical treatments targeting the only known modifiable risk factor, raised intraocular pressure, many patients still progress and develop significant visual field loss and eventual blindness. The search for alternative treatment strategies targeting the underlying fibrotic transformation in the optic nerve head and trabecular meshwork in glaucoma is ongoing. MicroRNAs are small non-coding RNAs known to regulate post-transcriptional gene expression. Extensive research has been undertaken to uncover the complex role of miRNAs in gene expression and miRNA dysregulation in fibrotic disease. MiR-29 is a family of miRNAs which are strongly anti-fibrotic in their effects on the TGF-ß signalling pathway and the regulation of extracellular matrix production and deposition. In this review, we discuss the anti-fibrotic effects of miR-29 and the role of miR-29 in ocular pathology and in the development of glaucomatous optic neuropathy. A better understanding of the role of miR-29 in POAG may aid in developing diagnostic and therapeutic strategies in glaucoma.

The lysophosphatidic acid axis in fibrosis: Implications for glaucoma.

Glaucoma is a common progressive optic neuropathy that results in visual field defects and can lead to irreversible blindness. The pathophysiology of glaucoma involves dysregulated extracellular matrix remodelling in both the trabecular meshwork in the anterior chamber and in the lamina cribrosa of the optic nerve head. Fibrosis in these regions leads to raised intraocular pressure and retinal ganglion cell degeneration, respectively. Lysophosphatidic acid (LPA) is a bioactive lipid mediator which acts via six G-protein coupled receptors on the cell surface to activate intracellular pathways that promote cell proliferation, transcription and survival. LPA signalling has been implicated in both normal wound healing and pathological fibrosis. LPA enhances fibroblast proliferation, migration and contraction, and induces expression of pro-fibrotic mediators such as connective tissue growth factor. The LPA axis plays a major role in diseases such as idiopathic pulmonary fibrosis, where it has been identified as an important pharmacological target. In glaucoma, LPA is present in high levels in the aqueous humour, and its signalling has been found to increase resistance to aqueous humour outflow through altered trabecular meshwork cellular contraction and extracellular matrix deposition. LPA signalling may, therefore, also represent an attractive target for treatment of glaucoma. In this review we wish to describe the role of LPA and its related proteins in tissue fibrosis and glaucoma.

Fibrotic Changes to Schlemm's Canal Endothelial Cells in Glaucoma.

Previous studies have shown that glaucomatous Schlemm's canal endothelial cells (gSCECs) are stiffer and associated with reduced porosity and increased extracellular matrix (ECM) material compared to SCECs from healthy individuals. We hypothesised that Schlemm's canal (SC) cell stiffening was a function of fibrotic changes occurring at the inner wall of SC in glaucoma. This study was performed in primary cell cultures isolated from the SC lumen of human donor eyes. RNA and protein quantification of both fibrotic and endothelial cell markers was carried out on both healthy and gSCECs. Functional assays to assess cell density, size, migration, proliferation, and mitochondrial function of these cells were also carried out. Indeed, we found that gSCECs deviate from typical endothelial cell characteristics and exhibit a more fibrotic phenotype. For example, gSCECs expressed significantly higher protein levels of the fibrotic markers α-SMA, collagen I-α1, and fibronectin, as well as significantly increased protein expression of TGFß-2, the main driver of fibrosis, compared to healthy SCECs. Interestingly, we observed a significant increase in protein expression of endothelial marker VE-cadherin in gSCECs, compared to healthy SCECs. gSCECs also appeared to be significantly larger, and surprisingly proliferate and migrate at a significantly higher rate, as well as showing significantly reduced mitochondrial activity, compared to healthy SCECs.

The role of lamina cribrosa tissue stiffness and fibrosis as fundamental biomechanical drivers of pathological glaucoma cupping.

The primary biomechanical driver of pathological glaucomatous cupping remains unknown. Finite element modeling indicates that stress and strain play key roles. In this article, primarily a review, we utilize known biomechanical data and currently unpublished results from our lab to propose a three-stage, tissue stiffness-based model to explain glaucomatous cupping occurring at variable levels of translaminar pressure (TLP). In stage 1, a short-term increase in TLP gradient induces a transient increase in lamina cribrosa (LC) strain. Beyond a critical level of strain, the tissue stiffness rises steeply provoking cellular responses via integrin-mediated mechanotransduction. This early mechanoprotective cellular contraction reduces strain, which reduces tissue stiffness by return of the posteriorly deflected LC to baseline. In stage 2 a prolonged period of TLP increase elicits extracellular matrix (ECM) production leading to fibrosis, increasing baseline tissue stiffness and strain and diminishing the contractile ability/ability to return to the baseline LC position. This is supported by our three-dimensional collagen contraction assays, which show significantly reduced capacity to contract in glaucoma compared with normal LC cells. Second, 15% cyclic strain in LC cells over 24 h elicits a typical increase in ECM profibrotic genes in normal LC cells but a highly blunted response in glaucoma LC cells. Stage 3 is characterized by persistent fibrosis causing further stiffening and inducing a feed-forward ECM production cycle. Repeated cycles of increased strain and stiffness with profibrotic ECM deposition prevent optic nerve head (ONH) recoil from the new deflected position. This incremental maladaptive modeling leads to pathological ONH cupping.

Lysyl Oxidase Like 1: Biological roles and regulation.

Lysyl Oxidase Like 1 (LOXL1) is a gene that encodes for the LOXL1 enzyme. This enzyme is required for elastin biogenesis and collagen cross-linking, polymerising tropoelastin monomers into elastin polymers. Its main role is in elastin homeostasis and matrix remodelling during injury, fibrosis and cancer development. Because of its vast range of biological functions, abnormalities in LOXL1 underlie many disease processes. Decreased LOXL1 expression is observed in disorders of elastin such as Cutis Laxa and increased expression is reported in fibrotic disease such as Idiopathic Pulmonary Fibrosis. LOXL1 is also downregulated in the lamina cribrosa in pseudoexfoliation glaucoma and genetic variants in the LOXL1 gene have been linked with an increased risk of developing pseudoexfoliation glaucoma and pseudoexfoliation syndrome. However the two major risk alleles are reversed in certain ethnic groups and are present in a large proportion of the normal population, implying complex genetic and environmental regulation is involved in disease pathogenesis. It also appears that the non-coding variants in intron 1 of LOXL1 may be involved in the regulation of LOXL1 expression. Gene alteration may occur via a number of epigenetic and post translational mechanisms such as DNA methylation, long non-coding RNAs and microRNAs. These may represent future therapeutic targets for disease. Environmental factors such as hypoxia, oxidative stress and ultraviolet radiation exposure alter LOXL1 expression, and it is likely a combination of these genetic and environmental factors that influence disease development and progression. In this review, we discuss LOXL1 properties, biological roles and regulation in detail with a focus on pseudoexfoliation syndrome and glaucoma.

Differential Lysyl oxidase like 1 expression in pseudoexfoliation glaucoma is orchestrated via DNA methylation.

Pseudoexfoliation syndrome (PXF) is the most common cause of secondary open angle glaucoma worldwide. Single nucleotide polymorphisms (SNPs) in the gene Lysyl oxidase like 1 (LOXL1) are strongly associated with the development of pseudoexfoliation glaucoma (PXFG). However, these SNPs are also present in 50-80% of the general population, suggestive of other factors being involved in the pathogenesis of PXFG. In this study, we aimed to investigate the influence of epigenetic regulation, specifically DNA methylation, on LOXL1 expression in PXFG using human tenons fibroblasts (HTFs), aqueous humour and serum samples from donors with and without PXFG. LOXL1 expression in HTFs was measured by qPCR and Western Blotting and LOXL1 concentration in aqueous humour was determined by ELISA. Global DNA methylation levels were quantified using an ELISA for 5-methylcytosine. MeDIP assays assessed the methylation status of the LOXL1 promoter region. Expression of methylation-associated enzymes (DNMT1, DNMT3a and MeCP2) were determined by qPCR and inhibited by 0.3 µM 5-azacytidine (5-aza). Results showed that LOXL1 expression was significantly decreased in PXFG HTFs compared with Control HTFs at gene (Fold change 0.37 ± 0.05, P < 0.01) level and showed a decrease, when measured at the protein level (Fold change 0.65 ± 0.42, P = 0.22), however this was not found to be significant. LOXL1 concentration was increased in the aqueous of PXFG patients compared with Controls (2.76 ± 0.78 vs. 1.79 ± 0.33 ng/ml, P < 0.01). Increased global methylation (56.07% ± 4.87% vs. 32.39% ± 4.29%, P < 0.01) was observed in PXFG HTFs compared with Control HTFs, as was expression of methylation-associated enzymes (DNMT1 1.58 ± 0.30, P < 0.05, DNMT3a 1.89 ± 0.24, P < 0.05, MeCP2 1.63 ± 0.30, P < 0.01). Methylation-associated enzymes were also increased when measured at protein level (DNMT1 5.70 ± 2.64, P = 0.04, DNMT3a 1.79 ± 1.55, P = 0.42, MeCP2 1.64 ± 1.33, P = 0.45). LOXL1 promoter methylation was increased in patients with PXFG compared to Control patients in both blood (3.98 ± 2.24, 2.10 ± 1.29, P < 0.05) and HTF cells (37.31 ± 22.0, 8.66 ± 10.40, P < 0.01). Treatment of PXFG HTFs with in 5-azacytidine increased LOXL1 expression when compared with untreated PXFG HTFs (Fold change 2.26 ± 0.67, P < 0.05). These data demonstrate that LOXL1 expression is altered in PXFG via DNA methylation and that reversal of these epigenetic changes may represent future potential therapeutic targets in the management of PXFG.

Unravelling the transcriptional responses of TGF-ß: Smad3 and EZH2 constitute a regulatory switch that controls neuroretinal epithelial cell fate specification.

Cell differentiation is directed by extracellular cues and intrinsic epigenetic modifications, which control chromatin organization and transcriptional activation. Central to this process is PRC2, which modulates the di- and trimethylation of lysine 27 on histone 3; however, little is known concerning the direction of PRC2 to specific loci. Here, we have investigated the physical interactome of EZH2, the enzymatic core of PRC2, during retinoic acid-mediated differentiation of neuroepithelial, pluripotent NT2 cells and the dedifferentiation of neuroretinal epithelial ARPE19 cells in response to TGF-ß. We identified Smad3 as an EZH2 interactor in both contexts. Co-occupation of the CDH1 promoter by Smad3 and EZH2 and the cooperative, functional nature of the interaction were established. We propose that the interaction between Smad3 and EZH2 targets the core polycomb assembly to defined regions of the genome to regulate transcriptional repression and forms a molecular switch that controls promoter access through epigenetic mechanisms leading to gene silencing.-Andrews, D., Oliviero, G., De Chiara, L., Watson, A., Rochford, E., Wynne, K., Kennedy, C., Clerkin, S., Doyle, B., Godson, C., Connell, P., O'Brien, C., Cagney, G., Crean, J. Unravelling the transcriptional responses of TGF-ß: Smad3 and EZH2 constitute a regulatory switch that controls neuroretinal epithelial cell fate specification.

Therapeutic potential of AAV-mediated MMP-3 secretion from corneal endothelium in treating glaucoma.

Intraocular pressure (IOP) is maintained as a result of the balance between production of aqueous humour (AH) by the ciliary processes and hydrodynamic resistance to its outflow through the conventional outflow pathway comprising the trabecular meshwork (TM) and Schlemm's canal (SC). Elevated IOP, which can be caused by increased resistance to AH outflow, is a major risk factor for open-angle glaucoma. Matrix metalloproteinases (MMPs) contribute to conventional aqueous outflow homeostasis in their capacity to remodel extracellular matrices, which has a direct impact on aqueous outflow resistance and IOP. We observed decreased MMP-3 activity in human glaucomatous AH compared to age-matched normotensive control AH. Treatment with glaucomatous AH resulted in significantly increased transendothelial resistance of SC endothelial and TM cell monolayers and reduced monolayer permeability when compared to control AH, or supplemented treatment with exogenous MMP-3.Intracameral inoculation of AAV-2/9 containing a CMV-driven MMP-3 gene (AAV-MMP-3) into wild type mice resulted in efficient transduction of corneal endothelium and an increase in aqueous concentration and activity of MMP-3. Most importantly, AAV-mediated expression of MMP-3 increased outflow facility and decreased IOP, and controlled expression using an inducible promoter activated by topical administration of doxycycline achieved the same effect. Ultrastructural analysis of MMP-3 treated matrices by transmission electron microscopy revealed remodelling and degradation of core extracellular matrix components. These results indicate that periodic induction, via use of an eye drop, of AAV-mediated secretion of MMP-3 into AH could have therapeutic potential for those cases of glaucoma that are sub-optimally responsive to conventional pressure-reducing medications.

Glaucoma referral refinement in Ireland: managing the sensitivity-specificity paradox in optometric practice.

PURPOSE: Glaucoma referral refinement (GRR) has proven a successful demand management strategy for glaucoma suspect cases in the United Kingdom (UK). A GRR clinic was established in Dublin, Ireland to investigate the clinical viability of this pathway outside the UK's National Health Service (NHS) structures, and away from the influence of National Institute for Clinical Excellence (NICE) guidance. METHODS: Glaucoma suspect patients were recruited into the scheme following referral from community optometrists in the greater Dublin area. The GRR exam protocol was designed in consultation with the participating ophthalmology department. The refinement scheme optometrist, trained through apprenticeship style experience at a hospital outpatient clinic, made a tentative management decision after carrying out the GRR exam. The final management decision was made in a 'virtual clinic' by a glaucoma specialist consultant ophthalmologist. RESULTS: Two hundred and twenty-five glaucoma suspect patients were seen in the scheme. After their first GRR visit, 28% were discharged back to their own optometrist, 42% were monitored in the GRR clinic, and 30% were referred to ophthalmology. After this monitoring cohort were further assessed, a total of 38% of the patients seen within the scheme required referral to ophthalmology. Sixteen percent of the total participant group (n = 225) were lost to follow up. Cohen's κ was used to determine the level of agreement between the scheme optometrist and ophthalmologist. There was substantial agreement, with κ = 0.63 for the first visit management decisions (n = 225). Agreement increased for subsequent monitoring visits with κ = 0.85 for second visits (n = 65), and κ = 0.69 for all management decisions within the scheme (n = 301). We received management outcomes for 44 of the 86 patients referred to ophthalmology. Of these 44, 57% received medical treatment for glaucoma, 34% were monitored without treatment, 2% were discharged, and 7% had comorbidities that were assessed and managed. CONCLUSION: Of the patients seen within the scheme, 62% did not require referral onward to ophthalmology, thus releasing the significant majority of hospital clinic slots that would previously have been required to examine such patients. The high level of inter-professional decision agreement likely reflects the benefits of pre-scheme apprenticeship style training and ongoing hospital clinic participation by the scheme optometrist. The rate of loss to follow up compares favourably with ophthalmology led, hospital based, glaucoma clinics. Nevertheless, the losses indicate that patient education remains a key priority for future planning.

Corneal hysteresis in patients with glaucoma-like optic discs, ocular hypertension and glaucoma.

BACKGROUND: To compare corneal hysteresis (CH) measurements between patients with glaucoma, ocular hypertension (OHT) and glaucoma-like optic discs (GLD)- defined as a cup to disc ratio greater than or equal to 0.6 with normal intraocular pressure (IOP) and visual fields. The secondary aim was to investigate whether corneal resistance factor (CRF) and central corneal thickness (CCT) differ between patient groups. METHODS: In this cross sectional study a total of 123 patients (one eye each) were recruited from a glaucoma outpatient department to undergo ocular response analyser (ORA) testing and ultrasound pachymetry as well as clinical examination. A One-way Analysis of Covariance (ANCOVA) was conducted to evaluate the mean difference in CH between the three diagnostic groups (glaucoma, OHT and GLD) correcting for potential confounding factors, IOP and age. Analysis was repeated for CRF and CCT. RESULTS: There was a significant difference in mean CH across the three diagnosis groups; F(2, 115) = 96.95; p < 0.001. Mean CH significantly higher for GLD compared to glaucoma (mean difference 1.83, p < 0.001), and significantly higher for OHT compared to glaucoma (mean difference 2.35, p < 0.001). Mean CH was slightly lower in patients with GLD than those with OHT but this difference was not statistically significant. A similar pattern was seen when the analysis was repeated for CRF and CCT. CONCLUSIONS: Higher CH in GLD and OHT compared to glaucoma suggests increased viscoelasticity of ocular tissues may have a protective role against glaucoma.

Life under pressure: The role of ocular cribriform cells in preventing glaucoma.

Primary open-angle glaucoma is a multifactorial blinding disease often impacting the two pressure-sensitive regions of the eye: the conventional outflow pathway and the optic nerve head (ONH). The connective tissues that span these two openings in the globe are the trabecular meshwork of the conventional outflow pathway and the lamina cribrosa of the ONH. Resident cribiform cells of these two regions are responsible for actively remodeling and maintaining their connective tissues. In glaucoma, aberrant maintenance of the juxtacanalicular tissues (JCT) of the conventional outflow pathway results in ocular hypertension and pathological remodeling of the lamina cribrosa results in ONH cupping, damaging retinal ganglion cell axons. Interestingly, cells cultured from the lamina cribrosa and the JCT of the trabecular meshwork have similarities regarding gene expression, protein production, plus cellular responses to growth factors and mechanical stimuli. This review compares and contrasts the current knowledge of these two cell types, whose health is critical for protecting the eye from glaucomatous changes. In response to pressure gradients across their respective cribiform tissues, the goal is to better understand and differentiate healthy from pathological behavior of these two cell types.

The role of lamina cribrosa cells in optic nerve head fibrosis in glaucoma.

Glaucoma is a chronic progressive optic neuropathy. There are extracellular matrix (ECM) changes associated with optic disc cupping in the optic nerve head (ONH) and subsequent visual field defects. The primary risk factor for onset and progression of glaucoma is raised intraocular pressure (IOP). Elevated IOP causes deformation at the ONH specifically at the lamina cribrosa (LC) region where there is also deposition of ECM causing the LC to initially undergo thickening and posterior migration with eventual shearing and collapse of the LC plates leading to a thin fibrotic connective tissue structure/scar. Cells that populate the LC region of the ONH are those cells that are positive for GFAP (the astrocytes) and those negative for GFAP (the LC cells). The LC cell plays an integral role in ECM remodelling producing ECM when exposed to high level mechanical stretch, TGF- ß1 and a hypoxic environment.

Whole-mitochondrial genome sequencing in primary open-angle glaucoma using massively parallel sequencing identifies novel and known pathogenic variants.

PURPOSE: The aim of this study was to determine whether mutations in mitochondrial DNA play a role in high-pressure primary open-angle glaucoma (OMIM 137760) by analyzing new data from massively parallel sequencing of mitochondrial DNA. METHODS: Glaucoma patients with high-tension primary open-angle glaucoma and ethnically matched and age-matched control subjects without glaucoma were recruited. The entire human mitochondrial genome was amplified in two overlapping fragments by long-range polymerase chain reaction and used as a template for massively parallel sequencing on an Ion Torrent Personal Genome Machine. All variants were confirmed by conventional Sanger sequencing. RESULTS: Whole-mitochondrial genome sequencing was performed in 32 patients with primary open-angle glaucoma from India (n = 16) and Ireland (n = 16). In 16 of the 32 patients with primary open-angle glaucoma (50% of cases), there were 22 mitochondrial DNA mutations consisting of 7 novel mutations and 8 previously reported disease-associated sequence variants. Eight of 22 (36.4%) of the mitochondrial DNA mutations were in complex I mitochondrial genes. CONCLUSION: Massively parallel sequencing using the Ion Torrent Personal Genome Machine with confirmation by Sanger sequencing detected a pathogenic mitochondrial DNA mutation in 50% of the primary open-angle glaucoma cohort. Our findings support the emerging concept that mitochondrial dysfunction results in the development of glaucoma and, more specifically, that complex I defects play a significant role in primary open-angle glaucoma pathogenesis.

Lower Macular Pigment Optical Density in Foveal-Involved Glaucoma.

PURPOSE: To evaluate the relationship between macular pigment optical density (MPOD) and structural parameters of the macula and optic nerve head in glaucomatous eyes. DESIGN: A cross-sectional analysis of the baseline data collected during the Macular Pigment and Glaucoma Trial (ISRCTN registry number: 56985060). PARTICIPANTS: Eighty-eight subjects (48 male, 40 female) with a diagnosis of open-angle glaucoma and a median age of 67 years (interquartile range, 13; range, 36-84 years) were enrolled in this trial. METHODS: The MPOD at 0.25°, 0.5°, and 1° retinal eccentricity was measured using a customized heterochromatic flicker photometry technique. Glaucoma-related structural parameters were captured using RTVue Fourier-domain optical coherence tomography (FD-OCT). Statistical significance was set at P < 0.01, and P values ranging from 0.01 to 0.05 were considered borderline significant. MAIN OUTCOME MEASURES: The MPOD and its relationship to the macula and optic nerve head topography in glaucomatous eyes. RESULTS: The MPOD peaked centrally at 0.25° of retinal eccentricity (mean ± standard deviation, 0.23±0.14) and decreased at more peripheral eccentricities. For the overall group, borderline significant correlations were observed between MPOD and a range of topographic measures, including inferior peripapillary retinal nerve fiber layer (RNFL) thickness, inferior ganglion cell complex (GCC) thickness, foveal inner retinal thickness, cup-to-disc area ratio, and optic disc rim area. Glaucomatous eyes with GCC loss involving the foveal zone on FD-OCT imaging (n = 52) had lower MPOD at 0.25°, 0.5°, and 1° of retinal eccentricity compared with those without foveal GCC involvement (P < 0.001, for all). Those with foveal GCC loss also had greater glaucoma severity, and this was evident by lower GCC and RNFL thickness, greater cup-to-disc area ratio, and lower optic disc rim area (P < 0.001 for all). CONCLUSIONS: Our observations indicate that MPOD is lower in glaucomatous eyes with foveal GCC involvement relative to those without foveal involvement. A longitudinal evaluation of MPOD and structural change among patients with glaucoma is required to elucidate the nature of any causal relationship that might exist between MPOD and foveal damage in glaucoma.

Lipofuscin accumulation and autophagy in glaucomatous human lamina cribrosa cells.

BACKGROUND: Disease associated alterations in the phenotype of lamina cribrosa (LC) cells are implicated in changes occurring at the optic nerve head (ONH) in glaucoma. Lipofuscin, the formation of which is driven by reactive oxygen species (ROS), is an intralysosomal, non-degradable, auto-fluorescent macromolecule which accumulates with age and can affect autophagy - the lysosomal degradation of a cell's constituents. We aimed to compare the content of lipofuscin-like material and markers of autophagy in LC cells from normal and glaucoma donor eyes. METHODS: The number and size of peri-nuclear lysosomes were examined by transmission electron microscopy (TEM). Cellular auto-fluorescence was quantified by flow cytometry. Cathepsin K mRNA levels were assessed by PCR. Autophagy protein 5 (Atg5) mRNA and protein levels were analysed by PCR and Western blot. Protein levels of subunits of the microtubule associated proteins (MAP) 1A and 1B, light chain 3 (LC3) I and II were analysed by Western blot. Immunohistochemical staining of LC3-II in ONH sections from normal and glaucomatous donor eyes was performed. RESULTS: A significant increase in the number of peri-nuclear lysosomes [4.1 × 10,000 per high power field (h.p.f.) ± 1.9 vs. 2.0 × 10,000 per h.p.f. ± 1.3, p = 0.002, n = 3] and whole cell auto-fluorescence (83.62 ± 45.1 v 41.01 ± 3.9, p = 0.02, n = 3) was found in glaucomatous LC cells relative to normal LC cells. Glaucomatous LC cells possessed significantly higher levels of Cathepsin K mRNA and Atg5 mRNA and protein. Enhanced levels of LC3-II were found in both LC cells and optic nerve head sections from glaucoma donors. CONCLUSIONS: Increased lipofuscin formation is characteristic of LC cells from donors with glaucoma. This finding confirms the importance of oxidative stress in glaucoma pathogenesis. Intracellular lipofuscin accumulation may have important effects on autophagy the modification of which could form the basis for future novel glaucoma treatments.

The Role of αvß3 Integrin in Lamina Cribrosa Cell Mechanotransduction in Glaucoma.

Purpose: Glaucoma, one of the leading causes of irreversible blindness, is a common progressive optic neuropathy characterised by visual field defects and structural changes to the optic nerve head (ONH). There is extracellular matrix (ECM) accumulation and fibrosis of the lamina cribrosa (LC) in the ONH, and consequently increased tissue stiffness of the LC connective tissue. Integrins are cell surface proteins that provide the key molecular link connecting cells to the ECM and serve as bidirectional sensors transmitting signals between cells and their environment to promote cell adhesion, proliferation, and remodelling of the ECM. Here, we investigated the expression of αVß3 integrin in glaucoma LC cell, and its effect on stiffness-induced ECM gene transcription and cellular proliferation rate in normal (NLC) and glaucoma (GLC) LC cells, by down-regulating αVß3 integrin expression using cilengitide (a known potent αVß3 and αVß5 inhibitor) and ß3 integrin siRNA knockdown. Methods: GLC cells were compared to age-matched controls NLC to determine differential expression levels of αVß3 integrin, ECM genes (Col1A1, α-SMA, fibronectin, vitronectin), and proliferation rates. The effects of αVß3 integrin blockade (with cilengitide) and silencing (with a pool of four predesigned αVß3 integrin siRNAs) on ECM gene expression and proliferation rates were evaluated using both reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blotting in the human NLC cells cultured on soft (4 kPa) and stiff (100 kPa) substrate and in GLC cells grown on standard plastic plates. Results: αVß3 integrin gene and protein expression were enhanced (p < 0.05) in GLC cells as compared to NLC. Both cilengitide and siRNA significantly reduced αVß3 expression in GLC. When NLC were grown in the stiff substrate, cilengitide and siRNA also significantly reduced the increased expression in αVß3, ECM components, and proliferation rate. Conclusions: Here, we provide evidence of cilengitide- and siRNA-mediated silencing of αVß3 integrin expression, and inhibition of ECM synthesis in LC cells. Therefore, αVß3 integrin may be a promising target for the development of novel anti-fibrotic therapies for treating the LC cupping of the ONH in glaucoma.

Ability to Process Multisensory Information Is Impaired in Open Angle Glaucoma.

PRCIS: Patients with glaucoma demonstrated deficiencies in their ability to process multisensory information when compared with controls, with those deficiencies being related to glaucoma severity. Impaired multisensory integration (MSI) may affect the quality of life in individuals with glaucoma and may contribute to the increased prevalence of falls and driving safety concerns. Therapeutic possibilities to influence cognition in glaucoma should be explored. PURPOSE: Glaucoma is a neurodegenerative disease of the optic nerve that has also been linked to cognitive health decline. This study explored MSI as a function of glaucoma status and severity. METHODS: MSI was assessed in 37 participants with open angle glaucoma relative to 18 age-matched healthy controls. The sound-induced flash illusion was used to assess MSI efficiency. Participants were presented with various combinations of simultaneous visual and/or auditory stimuli and were required to indicate the number of visual stimuli observed for each of the 96 total presentations. Central retinal sensitivity was assessed as an indicator of glaucoma severity (MAIA; CenterVue). RESULTS: Participants with glaucoma performed with equivalent capacity to healthy controls on unisensory trials ( F1,53 =2.222, P =0.142). Both groups performed equivalently on congruent multisensory trials involving equal numbers of auditory and visual stimuli F1,53 =1.032, P =0.314). For incongruent presentations, that is, 2 beeps and 1 flash stimulus, individuals with glaucoma demonstrated a greater influence of the incongruent beeps when judging the number of flashes, indicating less efficient MSI relative to age-matched controls ( F1,53 =11.45, P <0.002). In addition, MSI performance was positively correlated with retinal sensitivity ( F3,49 =4.042, P <0.025), adjusted R ²=0.15). CONCLUSIONS: Individuals with open angle glaucoma exhibited MSI deficiencies that relate to disease severity. The type of deficiencies observed were similar to those observed among older individuals with cognitive impairment and balance issues. Impaired MSI may, therefore, be relevant to the increased prevalence of falls observed among individuals with glaucoma, a concept that merits further investigation.