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.

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.

Role of Epithelial-to-Mesenchymal Transition of Retinal Pigment Epithelial Cells in Glaucoma Cupping.

Optic nerve head (ONH) cupping is a clinical feature of glaucoma associated with extracellular matrix (ECM) remodelling and lamina cribrosa (LC) fibrosis. Peripapillary atrophy (PPA) occurs commonly in glaucoma, and is characterised by the loss of retinal pigment epithelium (RPE) adjacent to the ONH. Under pro-fibrotic conditions, epithelial cells throughout the body can differentiate into fibroblast-like cells through epithelial-to-mesenchymal transition (EMT) and contribute to ECM fibrosis. This is investigated here in the context of glaucoma and PPA. Human-donor ONH sections were assessed for the presence of the RPE cell-specific marker RPE65 using immunofluorescence. We examined the EMT response of ARPE-19 cells to the following glaucoma-related stimuli: cyclic mechanical stretch, mechanical stiffness, transforming growth factor beta (TGFß), and tumour necrosis factor alpha (TNFα). The gene expression was measured using the PCR of the epithelial tight junction marker zona occludens 1 (ZO-1) and the mesenchymal markers alpha smooth muscle actin (αSMA) and vimentin. A scratch assay was used to assess the ARPE-19 migration. Significant RPE-65 staining was demonstrated in the glaucomatous ONH. The cyclic stretching and substrate stiffness of the ARPE-19 cells caused a significant decrease in ZO-1 (p = 0.04), and an increase in αSMA (p = 0.04). The scratch assays demonstrated increased migration of ARPE19 in the presence of TNFα (p = 0.02). Furthermore, ARPE-19 cells undergo an EMT-like transition (gain of αSMA, loss of ZO-1 and increased migration) in response to glaucomatous stimuli. This suggests that during PPA, RPE cells have the potential to migrate into the ONH and differentiate into fibroblast-like cells, contributing to glaucomatous ONH cupping.

The annual conference of the Irish College of Ophthalmologists: examining over a decade of trends.

BACKGROUND: The annual conference of the Irish College of Ophthalmologists (ICO) is a key calendar event for ophthalmology research in Ireland. AIMS: We investigated whether there were identifiable trends across various domains for the last twelve ICO meetings. Our objectives were to assess subspeciality and training centre representation, as well as the characteristics of the first author to include gender and stage of training. METHODS: A retrospective analysis of paper and poster presentations from the ICO annual conference yearbooks was conducted. The representation of subspecialties, affiliated institutions, and gender distribution were noted for both categories. For paper presentations, the author's career stage, full-text publication rates, and impact factors were also determined. RESULTS: A total of 306 paper presentations and 306 poster presentations were analysed. The subspecialty of retina had the highest representation within both sections. The overall mean publication rate was 38% (range, 6-39%), with a mean journal impact factor of 2.02. No statistically significant differences in gender noted with regard to poster, paper, or publications (p < 0.9, p < 0.1, p < 0.7, respectively). CONCLUSIONS: This is the first review of all research contributions to the ICO conference. We found that there is a need to promote research in some underrepresented subspecialities and training centres. No significant gender bias was found. There is scope to improve the publication conversion rate; this would allow for greater dissemination of the research presented at the ICO meeting.

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.

Glaucoma - 'A Stiff Eye in a Stiff Body'.

Glaucoma is a progressive, age-related optic neuropathy, whereby the prevalence increases sharply over the age of 60 and is associated with increased systemic tissue stiffness. On a molecular basis, this is associated with increased deposition of collagen and loss of elastin structure, resulting in aberrant biomechanical compliance and reduced tissue elasticity. Increased tissue stiffness is a known driver of myofibroblast activation and persistence, especially in chronic cellular injuries via mechanotransduction pathways mediated by integrins and focal adhesion kinases. Evidence from histological and imaging studies plus force measurements of glaucomatous eyes show that several ocular tissues are stiffer than normal, healthy age-matched controls including the trabecular meshwork, Schlemm's canal, cornea, sclera and the lamina cribrosa. This is associated with increased extracellular matrix deposition and fibrosis. This review reports on the evidence to support the concept that glaucoma represents 'a stiff eye in a stiff body' and addresses potential mechanisms to attenuate this.

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 Intertwined Roles of Oxidative Stress and Endoplasmic Reticulum Stress in Glaucoma.

Glaucoma is the leading cause of irreversible blindness worldwide, and the burden of the disease continues to grow as the global population ages. Currently, the only treatment option is to lower intraocular pressure. A better understanding of glaucoma pathogenesis will help us to develop novel therapeutic options. Oxidative stress has been implicated in the pathogenesis of many diseases. Oxidative stress occurs when there is an imbalance in redox homeostasis, with reactive oxygen species producing processes overcoming anti-oxidant defensive processes. Oxidative stress works in a synergistic fashion with endoplasmic reticulum stress, to drive glaucomatous damage to trabecular meshwork, retinal ganglion cells and the optic nerve head. We discuss the oxidative stress and endoplasmic reticulum stress pathways and their connections including their key intermediary, calcium. We highlight therapeutic options aimed at disrupting these pathways and discuss their potential role in glaucoma treatment.

Corneal Hysteresis, Intraocular Pressure, and Progression of Glaucoma: Time for a "Hyst-Oric" Change in Clinical Practice?

It is known that as people age their tissues become less compliant and the ocular structures are no different. Corneal Hysteresis (CH) is a surrogate marker for ocular compliance. Low hysteresis values are associated with optic nerve damage and visual field loss, the structural and functional components of glaucomatous optic neuropathy. Presently, a range of parameters are measured to monitor and stratify glaucoma, including intraocular pressure (IOP), central corneal thickness (CCT), optical coherence tomography (OCT) scans of the retinal nerve fibre layer (RNFL) and the ganglion cell layer (GCL), and subjective measurement such as visual fields. The purpose of this review is to summarise the current evidence that CH values area risk factor for the development of glaucoma and are a marker for its progression. The authors will explain what precisely CH is, how it can be measured, and the influence that medication and surgery can have on its value. CH is likely to play an integral role in glaucoma care and could potentially be incorporated synergistically with IOP, CCT, and visual field testing to establish risk stratification modelling and progression algorithms in glaucoma management in the future.

Matrix Mechanotransduction via Yes-Associated Protein in Human Lamina Cribrosa Cells in Glaucoma.

Purpose: Extracellular matrix stiffening is characteristic of both aging and glaucoma, and acts as a promoter and perpetuator of pathological fibrotic remodeling. Here, we investigate the role of a mechanosensitive transcriptional coactivator, Yes-associated protein (YAP), a downstream effector of multiple signaling pathways, in lamina cribrosa (LC) cell activation to a profibrotic, glaucomatous state. Methods: LC cells isolated from glaucomatous human donor eyes (GLC; n = 3) were compared to LC cells from age-matched nonglaucomatous controls (NLC; n = 3) to determine differential YAP expression, protein levels, and proliferation rates. NLC cells were then cultured on soft (4 kPa), and stiff (100 kPa), collagen-1 coated polyacrylamide hydrogel substrates. Quantitative real-time RT-PCR, immunoblotting, and immunofluorescence microscopy were used to measure the expression, activity, and subcellular location of YAP and its downstream targets, respectively. Proliferation rates were examined in NLC and GLC cells by methyl thiazolyl tetrazolium salt assays, across a range of incrementally increased substrate stiffness. Endpoints were examined in the presence or absence of a YAP inhibitor, verteporfin (2 µM). Results: GLC cells show significantly (P < 0.05) increased YAP gene expression and total-YAP protein compared to NLC cells, with significantly increased proliferation. YAP regulation is mechanosensitive, because NLC cells cultured on pathomimetic, stiff substrates (100 kPa) show significantly upregulated YAP gene and protein expression, increased YAP phosphorylation at tyrosine 357, reduced YAP phosphorylation at serine 127, increased nuclear pooling, and increased transcriptional target, connective tissue growth factor. Accordingly, myofibroblastic markers, α-smooth muscle actin (α-SMA) and collagen type I, alpha 1 (Col1A1) are increased. Proliferation rates are elevated on 50 kPa substrates and tissue culture plastic. Verteporfin treatment significantly inhibits YAP-mediated cellular activation and proliferation despite a stiffened microenvironment. Conclusions: These data demonstrate how YAP plays a pivotal role in LC cells adopting a profibrotic and proliferative phenotype in response to the stiffened LC present in aging and glaucoma. YAP provides an attractive and novel therapeutic target, and its inhibition via verteporfin warrants further clinical investigation.

Audit of outcomes following attendance at the City West drive-through IOP glaucoma clinic during the COVID-19 pandemic.

BACKGROUND: Glaucoma is the leading cause of irreversible blindness globally. During the COVID-19 pandemic, an enforced reduction in capacity resulted in the deferral of routine outpatient appointments for glaucoma patients. AIM: This study analyses patient outcomes following the establishment of a drive-through intra-ocular pressure (IOP) clinic during the COVID-19 pandemic to alleviate increased pressure on the tertiary glaucoma services at Royal Victoria Eye and Ear Hospital (RVEEH) and Mater Misericordiae University Hospital (MMUH) between August 2020 and June 2021. METHODS: A 1-lane driveway system was established in a marquee on the grounds of City West hotel. IOPs were measured in patients' cars using a hand held iCare100 tonometer. Results were reviewed by a consultant ophthalmologist. At hospital follow-up clinic visits, IOP was measured using the Goldmann applanation tonometer (GAT). RESULTS: Three hundred one patients of a total of 672 who attended the drive-through clinic have subsequently attended a designated hospital follow-up appointment. In this cohort, the mean drive-through iCare IOP of 19.4 mmHg ± 6.0 was significantly higher (< 0.005) than the mean GAT IOP at the pre-drive through clinic visit (16.3 mmHg ± 3.7) and the post drive-through hospital follow-up visit (17.2 mmHg ± 4.1). Two hundred twenty-six (75%) patients did not need any treatment change, 53 (18%) required eye drop medication changes, 10 (3%) underwent a laser procedure, 4(1%) required surgical intervention, and 8 (3%) were discharged. When patient outcomes were analysed according to IOP grade assigned at the drive-through clinic, those with an iCare IOP < 21 were significantly less likely to require a treatment change. The cohort with iCare IOP ≥ 30 were significantly more likely to have a laser or surgical intervention. CONCLUSION: The implementation of a drive-through IOP clinic was a safe and effective way to monitor glaucoma patients during COVID-19, and identify those at high risk of poor IOP control or requiring a change in treatment.

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.

Metformin and Glaucoma-Review of Anti-Fibrotic Processes and Bioenergetics.

Glaucoma is the leading cause of irreversible blindness globally. With an aging population, disease incidence will rise with an enormous societal and economic burden. The treatment strategy revolves around targeting intraocular pressure, the principle modifiable risk factor, to slow progression of disease. However, there is a clear unmet clinical need to find a novel therapeutic approach that targets and halts the retinal ganglion cell (RGC) degeneration that occurs with fibrosis. RGCs are highly sensitive to metabolic fluctuations as a result of multiple stressors and thus their viability depends on healthy mitochondrial functioning. Metformin, known for its use in type 2 diabetes, has come to the forefront of medical research in multiple organ systems. Its use was recently associated with a 25% reduced risk of glaucoma in a large population study. Here, we discuss its application to glaucoma therapy, highlighting its effect on fibrotic signalling pathways, mitochondrial bioenergetics and NAD oxidation.

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.

Abnormal corneal properties in osteogenesis imperfecta and glaucoma: a case series.

OBJECTIVE: We aimed to carry out ocular examination and genetic studies in a family in which some members are affected with osteogenesis imperfecta (OI) and primary open-angle glaucoma (POAG). We compared the corneal properties of affected and unaffected members (ie, cases and controls). METHODS: Eight family members from two generations, both affected and unaffected, were examined. Corneal hysteresis (CH), intraocular pressure (IOP) measured with Goldmann applanation tonometer, central corneal thickness (CCT) and cornea-corrected IOP (IOPcc) were recorded. Blood samples were obtained from seven family members, both affected and unaffected, and tested for a panel of genes associated with OI. RESULTS: Family members affected with OI (n=6) had a heterozygous splice site mutation in intron 26 of the COL1A1 gene. The family members affected with OI had reduced CCT (476.5±24.6 µm) and CH (7.9 ±1.4 mmHg) compared with the unaffected controls (CCT, 575.8±10.8 µm; CH, 12.3±0.8 mmHg). Two of the six patients affected with OI had a glaucoma diagnosis and were on topical therapy and under regular clinical review. CONCLUSIONS: Patients affected with OI have a significant risk of developing POAG due to the effects of abnormal collagen on various ocular structures. Two of these effects which place them at risk are reduced CCT and CH. They should be screened and monitored for glaucoma from a young age, and the examination should include corneal biomechanical measurements and CCT to identify those most at risk. IOPcc may be a more accurate way to monitor IOP in the presence of abnormal corneal properties.

Macular Pigment Response to Lutein, Zeaxanthin, and Meso-zeaxanthin Supplementation in Open-Angle Glaucoma: A Randomized Controlled Trial.

Purpose: To evaluate macular pigment response to carotenoid supplementation in glaucomatous eyes. Design: Double-masked, randomized, placebo-controlled clinical trial, the European Nutrition in Glaucoma Management Study (ClinicalTrials.gov identifier, NCT04460365). Participants: Sixty-two participants (38 men, 24 women) with a diagnosis of open-angle glaucoma were enrolled. Forty-two were randomized to receive the active supplement, 20 participants were allocated to placebo. Methods: Macular pigment optical density (MPOD) was measured by autofluorescence using the Heidelberg Spectralis scanning laser ophthalmoscope. Macular pigment optical density volume within the central 6° of retinal eccentricity as well as MPOD at 0.23°, 0.51°, 0.74°, and 1.02° were recorded at baseline and at 6-month intervals over 18 months. Visual function was assessed using visual acuity, mesopic and photopic contrast sensitivity under glare conditions, photo stress recovery time, microperimetry, and Glaucoma Activities Limitation 9 questionnaire. Advanced glaucoma module scans of retinal nerve fiber layer thickness and ganglion cell complex thickness over the central 6° of retinal eccentricity also were completed at each study visit. Main Outcome Measures: Change in MPOD after supplementation with 10 mg lutein, 2 mg zeaxanthin, and 10 mg meso-zeaxanthin or placebo over 18 months. Results: A mixed-model repeated measures analysis of variance revealed a statistically significant increase in MPOD volume (significant time effect: F(3,111) = 89.31, mean square error (MSE) = 1656.9; P < 0.01). Post hoc t tests revealed a significant difference in MPOD volume at each study visit for the treatment group (P < 0.01 for all), but no change in the placebo group (P > 0.05 for all). A statistically significant increase in mesopic contrast sensitivity under glare conditions was noted at 18 months in the treatment group, but not placebo. No other structural or functional changes were observed. No serious adverse events were noted during the trial. Conclusions: Macular pigment can be augmented in glaucomatous eyes by supplementation with a formulation containing the carotenoids lutein, zeaxanthin, and meso-zeaxanthin. The greatest relative benefit was observed in those with the lowest baseline levels, but increases were noted across all participants and each retinal eccentricity. The potential benefits of MP augmentation for macular health in glaucoma merit further long-term evaluation.

siRNA targeting Schlemm's canal endothelial tight junctions enhances outflow facility and reduces IOP in a steroid-induced OHT rodent model.

Systemic or localized application of glucocorticoids (GCs) can lead to iatrogenic ocular hypertension, which is a leading cause of secondary open-angle glaucoma and visual impairment. Previous work has shown that dexamethasone increases zonula occludens-1 (ZO-1) protein expression in trabecular meshwork (TM) cells, and that an antisense oligonucleotide inhibitor of ZO-1 can abolish the dexamethasone-induced increase in trans-endothelial flow resistance in cultured Schlemm's canal (SC) endothelial and TM cells. We have previously shown that intracameral inoculation of small interfering RNA (siRNA) targeting SC endothelial cell tight junction components, ZO-1 and tricellulin, increases aqueous humor outflow facility ex vivo in normotensive mice by reversibly opening SC endothelial paracellular pores. In this study, we show that targeted siRNA downregulation of these SC endothelial tight junctions reduces intraocular pressure (IOP) in vivo, with a concomitant increase in conventional outflow facility in a well-characterized chronic steroid-induced mouse model of ocular hypertension, thus representing a potential focused clinical application for this therapy in a sight-threatening scenario.

Intra-Cellular Calcium Signaling Pathways (PKC, RAS/RAF/MAPK, PI3K) in Lamina Cribrosa Cells in Glaucoma.

The lamina cribrosa (LC) is a key site of fibrotic damage in glaucomatous optic neuropathy and the precise mechanisms of LC change remain unclear. Elevated Ca2+ is a major driver of fibrosis, and therefore intracellular Ca2+ signaling pathways are relevant glaucoma-related mechanisms that need to be studied. Protein kinase C (PKC), mitogen-activated MAPK kinases (p38 and p42/44-MAPK), and the PI3K/mTOR axis are key Ca2+ signal transducers in fibrosis and we therefore investigated their expression and activity in normal and glaucoma cultured LC cells. We show, using Western immune-blotting, that hyposmotic-induced cellular swelling activates PKCα, p42/p44, and p38 MAPKs, the activity is transient and biphasic as it peaks between 2 min and 10 min. The expression and activity of PKCα, p38 and p42/p44-MAPKs are significantly (p < 0.05) increased in glaucoma LC cells at basal level, and at different time-points after hyposmotic stretch. We also found elevated mRNA expression of mRNA expression of PI3K, IP3R, mTOR, and CaMKII in glaucoma LC cells. This study has identified abnormalities in multiple calcium signaling pathways (PKCα, MAPK, PI3K) in glaucoma LC cells, which might have significant functional and therapeutic implications in optic nerve head (ONH) fibrosis and cupping in 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.

Reduced Oxidative Phosphorylation and Increased Glycolysis in Human Glaucoma Lamina Cribrosa Cells.

Purpose: The lamina cribrosa (LC) is a key site of damage in glaucomatous optic neuropathy. We previously found that glaucoma LC cells have an increased profibrotic gene expression, with mitochondrial dysfunction in the form of decreased mitochondrial membrane potential. Altered cell bioenergetics have recently been reported in organ fibrosis and in cancer. In this study, we carried out a systematic mitochondrial bioenergetic assessment and measured markers of alternative sources of cellular energy in normal and glaucoma LC cells. Methods: LC cells from three glaucoma donors and three age-matched normal controls were assessed using VICTOR X4 Perkin Elmer (Waltham, MA) plate reader with different phosphorescent and luminescent probes. adenosine triphosphate levels, oxygen consumption rate, and extracellular acidification were measured and normalized to total protein content. RNA and protein expression levels of MCT1, MCT4, MTFHD2, and GLS2 were quantified using real-time RT-PCR and Western blotting. Results: Glaucoma LC cells contain significantly less adenosine triphosphate (P < .05) when supplied with either glucose or galactose. They also showed significantly diminished oxygen consumption in both basal and maximal respiration with more lactic acid contribution in ECA. Both mRNA and protein expression levels of MCT1, MCT4, MTHFD2, and GLS2 were significantly increased in glaucoma LC cells. Conclusions: We demonstrate evidence of metabolic reprogramming (The Warburg effect) in glaucoma LC cells. Expression of markers of glycolysis, glutamine, and one carbon metabolism are elevated in glaucoma cells at both the mRNA and protein levels. A better understanding of bioenergetics in glaucoma may help in the development of new therapeutics.