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 Association between a Decrease in On-Treatment Neutrophil-to-Eosinophil Ratio (NER) at Week 6 after Ipilimumab Plus Nivolumab Initiation and Improved Clinical Outcomes in Metastatic Renal Cell Carcinoma.

A lower baseline neutrophil-to-eosinophil ratio (NER) has been associated with improved responses to immune checkpoint inhibitors (ICI)-treated metastatic renal cell carcinoma (mRCC). This study investigated the decrease in NER at week 6 after ipilimumab/nivolumab (ipi/nivo) initiation and treatment responses in mRCC. A retrospective study of ipi/nivo-treated mRCC at two US academic cancer centers was conducted. A landmark analysis at week 6 was performed to assess the association between the change in NER and clinical responses (progression-free survival (PFS)/overall survival (OS)). Week 6 NER was modeled as a continuous variable, after log transformation (Ln NER), and a categorical variable by percent change. There were 150 mRCC patients included: 78% had clear cell histology, and 78% were IMDC intermediate/poor risk. In multivariable regression analysis, every decrease of 1 unit of Ln NER at week 6 was associated with improved PFS (adjusted hazard ratio (AHR): 0.78, p-value:0.005) and OS (AHR: 0.67, p-value: 0.002). When NER was modeled by percent change, decreased NER > 50% was associated with improved PFS (AHR: 0.55, p-value: 0.03) and OS (AHR: 0.37, p-value: 0.02). The decrease in week 6 NER was associated with improved PFS/OS in ipi/nivo-treated mRCC. Prospective studies are warranted to validate NER change as a biomarker to predict ICI responses.

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.

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.

The COVIRL002 Trial-Tocilizumab for management of severe, non-critical COVID-19 infection: A structured summary of a study protocol for a randomised controlled trial.

OBJECTIVES: Tocilizumab is a humanized monoclonal antibody which targets and inhibits interleukin-6 (IL-6) and has demonstrated efficacy in treating diseases associated with hyper-inflammation. Data are suggestive of tocilizumab as a potential treatment for patients with COVID-19 infection. The aim of this study is to determine the safety and efficacy of standard dose versus low dose tocilizumab in adults with severe, non-critical, PCR-confirmed COVID-19 infection with evidence of progressive decline in respiratory function and evolving systemic inflammation on time to intubation, non-invasive ventilation and/or all-cause mortality. TRIAL DESIGN: This trial is a phase 2, open label, two-stage, multicentre, randomised trial. PARTICIPANTS: Adult subjects with severe, non-critical, PCR-confirmed COVID-19 infection with evidence of progressive decline in respiratory function and evolving systemic inflammation requiring admission to hospital at St. Vincent's University Hospital and Mater Misericordiae University Hospital, Dublin, Ireland. Inclusion criteria Aged 18 years or older. Confirmed SARS-CoV2 infection (as defined by positive PCR). Evidence of hyper inflammatory state as evidenced by at least three of the following: Documented temperature >38°C in the past 48 hours, IL6 >40 pg/ml, or in its absence D-dimer >1.5 µgFEU /ml, Elevated CRP (>100mg/L) and/or a three-fold increase since presentation, Elevated ferritin X5 ULN, Elevated LDH (above the ULN), Elevated fibrinogen (above the ULN). Pulmonary infiltrates on chest imaging. Moderate to severe respiratory failure as defined by PaO2/FiO2≤300mmHg. INTERVENTION AND COMPARATOR: Intervention for participants in this trial is SOC plus Tocilizumab compared to SOC alone (comparator). For Stage 1, following randomisation, subjects will receive either (Arm 1) SOC alone or (Arm 2) SOC plus Tocilizumab (standard single dose - 8mg/kg, infused over 60 minutes. Once stage 1 has fully recruited, subsequent participants will be enrolled directly into Stage 2 and receive either (Arm 1) SOC plus Tocilizumab (standard single dose - 8mg/kg, infused over 60 minutes or (Arm 2) SOC plus Tocilizumab (standard single dose - 4mg/kg, infused over 60 minutes). MAIN OUTCOMES: The primary endpoint for this study is the time to a composite primary endpoint of progression to intubation and ventilation, non-invasive ventilation or death within 28 days post randomisation. RANDOMISATION: Eligible patients will be randomised (1:1) using a central register. Randomisation will be performed through an interactive, web-based electronic data capturing database. In stage 1, eligible participants will be randomised (1:1) to (Arm 1) SOC alone or to (Arm 2) SOC with single dose (8mg/kg, maximum 800mg) intravenous tocilizumab infused over 60 minutes. In stage 2, eligible participants will be randomised (1:1) to receive either (Arm 1) single, standard dose (8mg/kg, maximum 800mg) intravenous tocilizumab infused over 60 minutes or (Arm 2) reduced dose (4mg/kg, maximum 800mg) intravenous tocilizumab infused over 60 minutes. BLINDING: This study is open label. The study will not be blinded to investigators, subjects, or medical or nursing staff. The trial statistician will be blinded for data analysis and will be kept unaware of treatment group assignments. To facilitate this, the randomisation schedule will be drawn up by an independent statistician and objective criteria were defined for the primary outcome to minimize potential bias. NUMBERS TO BE RANDOMISED: In stage 1, 90 subjects will be randomised 1:1, 45 to SOC and 45 subjects to SOC plus Tocilizumab (8mg/kg, infused over 60 minutes). In stage 2, sample size calculation for the dose evaluation stage will use data generated from stage 1 using the same primary endpoint as in stage 1. TRIAL STATUS: The COVIRL002 trial (Protocol version 1.4, 13th May 2020) commenced in May 2020 at St. Vincent's University Hospital and Mater Misericordiae University Hospital, Dublin, Ireland. Recruitment is proceeding with the aim to achieve the target sample size on or before April 2021. TRIAL REGISTRATION: COVIRL002 was registered 25 June 2020 under EudraCT number: 2020-001767-86 and Protocol identification: UCDCRC/20/02. FULL PROTOCOL: The full protocol for COVIRL002 is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol. The study protocol has been reported in accordance with the Standard Protocol Items: Recommendations for Clinical Interventional Trials (SPIRIT) guidelines (Additional file 2).

Corneal Stiffness and Collagen Cross-Linking Proteins in Glaucoma: Potential for Novel Therapeutic Strategy.

Biomechanical properties of the cornea have recently emerged as clinically useful in risk assessment of diagnosing glaucoma and predicting disease progression. Corneal hysteresis (CH) is a dynamic tool, which measures viscoelasticity of the cornea. It represents the overall deformability of the cornea, and reduces significantly with age. Low CH has also been associated with optic nerve damage and progression of visual field loss in glaucoma. The extracellular matrix (ECM) constituents of the cornea, trabecular meshwork (TM), sclera, and lamina cribrosa (LC) are similar, as they are predominantly made of fibrillar collagen. This suggests that biomechanical changes in the cornea may also reflect optic nerve compliance in glaucomatous optic neuropathy, and in the known increase of TM tissue stiffness in glaucoma. Increased collagen cross-linking contributes to tissue stiffening throughout the body, which is observed in normal aging and occurs at an accelerated rate in systemic conditions such as fibrotic and cardiovascular diseases, cancer, and glaucoma. We reviewed 3 ECM cross-linking proteins that may have a potential role in the disease process of increased tissue stiffness in glaucoma, including lysyl oxidase (LOX)/lysyl oxidase-like 1 (LOXL1), tissue transglutaminase (TG2), and advanced glycation end products. We also report elevated messenger RNA (mRNA) levels of LOX and TG2 in glaucoma LC cells to support our proposed theory that increased levels of cross-linking proteins in glaucoma play a role in LC tissue stiffness. We highlight areas of research that are needed to better understand the role of cross-linking in glaucoma pathogenesis, leading potentially to a novel therapeutic strategy.

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.

Prevalence of Pseudoexfoliation Glaucoma Risk-associated Variants Within Lysyl Oxidase-like 1 in an Irish Population.

PRECIS: High-risk alleles of risk-associated single-nucleotide polymorphisms (SNPs) within the lysyl oxidase-like 1 (LOXL1) gene are associated with pseudoexfoliation in patients recruited from an Irish population. PURPOSE: SNPs within the LOXL1 gene have been identified as a major risk factor for pseudoexfoliation syndrome (PXF) and pseudoexfoliation glaucoma (PXFG), specifically SNPs within exon 1 and intron 1 regions of the gene. The common haplotype (G-G) of 2 SNPs within exon 1, rs1048661, and rs3825942, is the strongest associated risk factor for PXF in white populations, but is switched in some populations to act as protective or low risk. Herein, a study was undertaken to genotype an Irish population for PXF/PXFG risk-associated SNPs within LOXL1. MATERIALS AND METHODS: Patient cohorts of PXFG, PXF, and controls were recruited and genotyped for risk-associated SNPs within exon 1 (rs1048661 and rs3825942), along with 3 SNPs within intron 1 (rs1550437, rs6495085, and rs6495086) of LOXL1. RESULTS: The risk G alleles of rs1048661 and rs3825942 were most prevalent in PXFG patients, and a significant association was found between rs3825942 and pseudoexfoliation (P=0.04). Genotypes of several intron 1 SNPs were found to be present at higher frequencies within the pseudoexfoliation patient cohort (PXF/PXFG) compared with control patients, wherein rs6495085 showed statistical association (P=0.04). The G-G-G haplotype of rs1048661, rs3825942, and rs6495085 was the most prevalent in PXFG patients compared with control patients or patients with PXF alone. Patients with the G-G-G haplotype were more likely to need surgery, suggestive of a more severe form of disease. CONCLUSION: Collectively, these results represent the first study to assess the association of LOXL1 SNPs with PXFG in an Irish population.

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.

Problems with the WHO TB model.

WHO tuberculosis researchers recently published a mathematical model to predict TB incidence decline with fulfillment of Sustainable Development Goal (SDG) subtargets [1]. This model omitted the subtargets of land rights and basic services and of reduction in deaths from climatic disaster, likely highly influential factors, and retained only social insurance and reduction of extreme poverty as independent variables. The model predicted that fulfillment of these two subtargets will result in very large declines in TB incidence. This paper critiques the WHO model, reviews historic documents in TB social epidemiology, and examines dynamics of institutional effectiveness and efficiency in endemic disease control under conditions of systemic uncertainty associated with imbalances in population-level power relations, leading to exploding variance. These documents, and our own modeling exercise, indicate that the WHO model omits important determinations of TB incidence: war, civil conflict, and major upheaval such as rural and urban mass evictions; gross imbalance of power; accumulation of wealth into the hands of a tiny part of the global population; unequal female/male literacy and general low literacy level. Simple models should not be used for public policy, especially not-yet-validated models. The WHO model substitutes money for anti-TB medicines and leaves the underlying long-term causes of high TB incidence intact. Short-term reductions in TB incidence may be followed by increases as intervention effectiveness and efficiency ebb.

"The more you did, the more it made sense": Problem-based learning to improve early evidence-based practice in an undergraduate physiotherapy professional programme.

BACKGROUND AND PURPOSE: Evidence-based practice (EBP), which integrates clinical reasoning skills, research evidence, and patient preference, has become standard in curricula for health care professional programs. Students however perceive EBP as difficult and often irrelevant to clinical practice. METHODS: A problem-based learning (PBL) approach is trialled in an early stage module where EBP knowledge and skills are stated learning outcomes. Prior to this, the content-based approach to EBP teaching and learning received negative student feedback. The impact of delivering EBP through PBL is evaluated by comparing 5 standard Likert feedback scales and open-ended question responses relating to the EBP instruction in the module before and after a PBL approach was implemented. The impact of a PBL approach on EBP profiles is further examined under domains (relevance, sympathy, terminology, practice, and confidence) of the validated Evidence-based Practice Profile Questionnaire. RESULTS: All mean Likert scores relating to subject understanding, relevance of assessments, achievement of learning outcomes, teaching, and overall module satisfaction improved when the PBL approach was compared with the lecture-based format (p < 0.05). Student comment post-PBL continued to identify EBP as a difficult concept, but now comments on the teaching and assessment approach were mainly positive, addressing the collaborative nature of PBL, identifying EBP, communication and team-working skills acquired, praising the real life, practical application of EBP taken, and commenting on improvement in EBP self-efficacy. Within group change in the Evidence-based Practice Profile Questionnaire following a PBL approach identified significant improvement in EBP domains of terminology (mean change 3.38; p < 0.001); practice (mean change 16.5; p < 0.001), and confidence (mean change 10.1; p = 0.008). Conceptual links, based on constructivist underpinnings of PBL and EBP, are developed in the paper. CONCLUSIONS: Using mixed methods evaluation, PBL is effective at promoting early EBP. Students identified with the interactive, collaborative, and experiential nature of PBL to EBP instruction.

Response to Anti-PD-1 in Uveal Melanoma Without High-Volume Liver Metastasis.

Background: Uveal melanoma (UM) is an uncommon melanoma subtype with poor prognosis. Agents that have transformed the management of cutaneous melanoma have made minimal inroads in UM. Methods: We conducted a single-arm phase II study of pembrolizumab in patients with metastatic UM and performed bioinformatics analyses of publicly available datasets to characterize the activity of anti-PD-1 in this setting and to understand the mutational and immunologic profile of this disease. Results: A total of 5 patients received pembrolizumab in this study. Median overall survival was not reached, and median progression-free survival was 11.0 months. One patient experienced a complete response after one dose and 2 others experienced prolonged stable disease (20% response rate, 60% clinical benefit rate); 2 additional patients had rapidly progressing disease. Notably, the patients who benefited had either no liver metastases or small-volume disease, whereas patients with rapidly progressing disease had bulky liver involvement. We performed a bioinformatics analysis of The Cancer Genome Atlas for UM and confirmed a low mutation burden and low rates of T-cell inflammation. Note that the lack of T-cell inflammation strongly correlated with MYC pathway overexpression. Conclusions: Anti-PD-1-based therapy may cause clinical benefit in metastatic UM, seemingly more often in patients without bulky liver metastases. Lack of mutation burden and T-cell infiltration and MYC overexpression may be factors limiting therapeutic responses.ClinicalTrials.gov identifier: NCT02359851.

Increased Substrate Stiffness Elicits a Myofibroblastic Phenotype in Human Lamina Cribrosa Cells.

Purpose: Alteration in the extracellular matrix (ECM) of the optic nerve head (ONH) causes lamina cribrosa (LC) fibrosis and affects the mechanical integrity of the ONH. Increased ECM tissue stiffness drives myofibroblast activation leading to tissue fibrosis throughout the body. Here using primary human LC cells, we investigate the effect of substrate stiffness on profibrotic changes, which might be a key molecular mechanism driving ECM remodeling of the LC in primary open-angle glaucoma (POAG) glaucoma. Methods: Primary human LC cells from normal and age-matched POAG glaucoma donors were cultured on substrates with defined mechanical properties of 5 and 100 kPa to replicate the range of mechanical microenvironments that cells may experience in vivo. Cell morphology, spread area, actin stress fibers, vinculin-focal adhesion formation, and α-smooth muscle actin (α-SMA) signal were examined using immunofluorescence staining. The elastic modulus of cells was measured using atomic force microscopy (AFM). Results: Significantly greater cell spread area along with increased actin filament development, and vinculin-focal adhesion formation (number and size) were found in both normal and glaucoma LC cells cultured on stiff substrates. These changes were positively associated with elevated cell stiffness measured by AFM. Changes in spreading and cytoskeleton organization of glaucoma LC cells were significantly more pronounced than those in normal cells. The transformation to a myofibroblast-like cell phenotype was identified in both LC cells exposed to stiffer substrates, as indicated by an increased α-SMA signal and its colocalization with the actin stress fibers. Conclusions: These findings demonstrated that a stiffer cell microenvironment activates a myofibroblastic transformation in human LC cells, and therefore contributes to LC remodelling and fibrosis in glaucoma.

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.

Early Subretinal Allograft Rejection Is Characterized by Innate Immune Activity.

Successful subretinal transplantation is limited by considerable early graft loss despite pharmacological suppression of adaptive immunity. We postulated that early innate immune activity is a dominant factor in determining graft survival and chose a nonimmunosuppressed mouse model of retinal pigment epithelial (RPE) cell transplantation to explore this. Expression of almost all measured cytokines by DH01 RPE cells increased significantly following graft preparation, and the neutrophil chemoattractant KC/GRO/CINC was most significantly increased. Subretinal allografts of DH01 cells (C57BL/10 origin) into healthy, nonimmunosuppressed C57BL/6 murine eyes were harvested and fixed at 1, 3, 7, and 28 days postoperatively and subsequently cryosectioned and stained. Graft cells were detected using SV40 large T antigen (SV40T) immunolabeling and apoptosis/necrosis by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). Sections were also immunolabeled for macrophage (CD11b and F4/80), neutrophil (Gr1 Ly-6G), and T-lymphocyte (CD3-ɛ) infiltration. Images captured with an Olympus FV1000 confocal microscope were analyzed using the Imaris software. The proportion of the subretinal bolus comprising graft cells (SV40T+) was significantly (p < 0.001) reduced between postoperative day (POD) 3 (90 ± 4%) and POD 7 (20 ± 7%). CD11b+, F4/80+, and Gr1 Ly-6G+ cells increased significantly (p < 0.05) from POD 1 and predominated over SV40T+ cells by POD 7. Colabeling confocal microscopic analysis demonstrated graft engulfment by neutrophils and macrophages at POD 7, and reconstruction of z-stacked confocal images confirmed SV40T inside Gr1 Ly-6G+ cells. Expression of CD3-ɛ was low and did not differ significantly between time points. By POD 28, no graft cells were detectable and few inflammatory cells remained. These studies reveal, for the first time, a critical role for innate immune mechanisms early in subretinal graft rejection. The future success of subretinal transplantation will require more emphasis on techniques to limit innate immune-mediated graft loss, rather than focusing exclusively on suppression of the adaptive immune response.

Increased Global DNA Methylation and Decreased TGFß1 Promoter Methylation in Glaucomatous Lamina Cribrosa Cells.

BACKGROUND: Glaucoma is an optic neuropathy that affects 60 million people worldwide. There is an underlying fibrosis associated with the lamina cribrosa (LC) in glaucoma. DNA methylation is well established in regulating fibrosis and may be a therapeutic target for glaucoma. The purpose of this study was to compare global DNA methylation levels in primary human normal (NLC) and glaucomatous (GLC) cells, and to investigate DNA methylation in driving fibrosis through regulation of transforming growth factor ß1 (TGFß1). MATERIALS AND METHODS: LC cells were cultured from normal and glaucomatous human donors. Global methylation was assessed by ELISA. qPCR was conducted for DNA methyltransferases (DNMTs), methyl-CpG-binding protein 2 (MeCP2), TGFß 1 and 2, collagen 1α1 (COL1A1), and α-smooth muscle actin (αSMA). TGFß1 and DNMT1 were examined by immunofluorescence. Methylation of the TGFß1 promoter was determined by methylation-specific PCR (MSP). RESULTS: Global DNA methylation demonstrated an increase in GLC compared with NLC cells (P<0.05). The previously mentioned methylation and matrix genes were increased in GLC compared with NLC cells (P<0.05). Immunofluorescence showed increased TGFß1 and DNMT1 in GLC compared with NLC cells. MSP showed increased unmethylated DNA in the TGFß1 promoter of GLC compared with NLC cells. CONCLUSIONS: We found increased expression of fibrotic genes in GLC cells and demonstrated an increase in global DNA methylation and in associated enzymes in GLC cells. Furthermore, we showed decreased promoter methylation of TGFß1 in GLC cells. Determining a role for methylation in glaucoma and in regulating TGFß1 may provide a novel therapeutic approach.

Hypoxia-Induced Changes in DNA Methylation Alter RASAL1 and TGFß1 Expression in Human Trabecular Meshwork Cells.

PURPOSE: Fibrosis and a hypoxic environment are associated with the trabecular meshwork (TM) region in the blinding disease glaucoma. Hypoxia has been shown to alter DNA methylation, an epigenetic mechanism involved in regulating gene expression such as the pro-fibrotic transforming growth factor (TGF) ß1 and the anti-fibrotic Ras protein activator like 1 (RASAL1). The purpose of this study was to compare DNA methylation levels, and the expression of TGFß1 and RASAL1 in primary human normal (NTM) with glaucomatous (GTM) cells and in NTM cells under hypoxic conditions. METHODS: Global DNA methylation was assessed by ELISA in cultured age-matched NTM and GTM cells. qPCR was conducted for TGFß1, collagen 1α1 (COL1A1), and RASAL1 expression. Western immunoblotting was used to determine protein expression. For hypoxia experiments, NTM cells were cultured in a 1%O2, 5%CO2 and 37°C environment. NTM and GTM cells were treated with TGFß1 (10ng/ml) and the methylation inhibitor 5-azacytidine (5-aza) (0.5µM) respectively to determine their effects on DNA Methyltransferase 1 (DNMT1) and RASAL1 expression. RESULTS: We found increased DNA methylation, increased TGFß1 expression and decreased RASAL1 expression in GTM cells compared to NTM cells. Similar results were obtained in NTM cells under hypoxic conditions. TGFß1 treatment increased DNMT1 and COL1A1, and decreased RASAL1 expression in NTM cells. 5-aza treatment decreased DNMT1, TGFß1 and COL1A1 expression, and increased RASAL1 expression in GTM cells. CONCLUSIONS: TGFß1 and RASAL1 expression, global DNA methylation, and expression of associated methylation enzymes were altered between NTM and GTM cells. We found that hypoxia in NTM cells induced similar results to the GTM cells. Furthermore, DNA methylation, TGFß1 and RASAL1 appear to have an interacting relationship that may play a role in driving pro-fibrotic disease progression in the glaucomatous TM.

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.

The Function of Matricellular Proteins in the Lamina Cribrosa and Trabecular Meshwork in Glaucoma.

PURPOSE: To review the current literature regarding the role of matricellular proteins in glaucoma, specifically in the lamina cribrosa (LC) region of the optic nerve head (ONH) and the trabecular meshwork (TM). METHODS: A literature search was performed for published articles describing the expression and function of matricellular proteins such as thrombospondin (TSP), connective tissue growth factor (CTGF), secreted protein acidic and rich in cysteine (SPARC), and periostin in glaucoma. RESULTS: In glaucoma, there are characteristic extracellular matrix (ECM) changes associated with optic disc cupping in the ONH and subsequent visual field defects. Matricellular proteins are a family of nonstructural secreted glycoproteins, which enable cells to communicate with their surrounding ECM, including CTGF, also known as CCN2, TSPs, SPARC, periostin, osteonectin, and tenascin-C and -X, and other ECM proteins. Such proteins appear to play a role in fibrosis and increased ECM deposition. Importantly, most are widely expressed in tissues particularly in the TM and ONH, and deficiency of TSP1 and SPARC has been shown to lower intraocular pressure in mouse models of glaucoma through enhanced outflow facility. CONCLUSION: This article highlights the role of matricellular proteins in glaucoma pathology. The potential role of these proteins in glaucoma is emerging as some have an association with the pathophysiology of the TM and LC region and might therefore be potential targets for therapeutic intervention in glaucoma.