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.

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.

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.

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.

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.

Elevated maxi-K(+) ion channel current in glaucomatous lamina cribrosa cells.

The connective tissue plates of the lamina cribrosa (LC) region are continuously exposed to a mechanically dynamic environment. To study how the LC cells respond to these mechanical forces, we measured the mechano-sensitive calcium dependent maxi-K(+) ion channel current in the cell membrane of LC cells of glaucoma and normal subjects. Primary culture LC cells from 7 normal and 7 age matched glaucoma donors were studied. Perfusion of cells with hypotonic solution was used to stretch the cell membrane. Whole-cell patch-clamp technique was used to measure the basal (non stretched) and hypotonic stretch-induced changes in maxi-K(+) ion channel activity in normal and glaucoma LC cells. The role of membrane-type Ca(2+) entry channel inhibition (verapamil) and internal Ca(2+) store re-uptake blockade (2-APB) on maxi-K(+) activity was also examined. Basal and stretched-induced maxi-K(+) current were significantly elevated in the glaucoma LC cells compared to normal controls (p < 0.05). In normal LC cells hypotonic stretch elevated the mean maxi-K(+) current from 18.5 ± 5.7 pA/pF (at Vp = +100 mV) to 88.4 ± 12.4 pA/pF (P < 0.05), and from 39.5 ± 7.3 pA/pF to 133.1 ± 18.5 pA/pF in glaucoma LC cells (P < 0.02). Verapamil and 2-APB significantly reduced basal maxi-K(+) current in glaucoma LC cells (33.1 ± 8.2 pA/pF to 17.9 ± 5.6 pA/pF; and 32.2 ± 8.3 pA/pF to 17.3 ± 5.4 pA/pF, P < 0.05, respectively) but not in normal LC cells (P > 0.05). Following hypotonic stretch, verapamil and 2-APB significantly (P < 0.05) reduced the maxi-K(+) current in both normal and glaucoma LC cells. Baseline and hypotonic stretch induced Ca(2+)-dependent maxi-K(+) channel activity are elevated in LC cells of glaucoma patients, which may result from the abnormally high levels of intracellular calcium in glaucoma LC cells.

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.

bFGF-mediated redox activation of the PI3K/Akt pathway in retinal photoreceptor cells.

In many retinal diseases, it is the death of photoreceptors that leads to blindness. In previous in vitro and in vivo studies, basic fibroblast growth factor (bFGF) has been shown to increase retinal cell survival. More recently, reactive oxygen species (ROS) have also been shown to promote cell survival, contrary to the traditional view that they are solely destructive molecules. Due to this possible link, we hypothesised that bFGF could stimulate the production of ROS, which in turn stimulates the protein kinase B (Akt) survival pathway. Flow cytometry was used to measure the fluorescence of oxidised dihydrorhodamine, a ROS indicator, in the murine 661W photoreceptor cell line under several different conditions. Expression of cyclooxygenase (Cox) enzymes was evaluated by immunohistochemistry, and the response of photoreceptor cells to exogenous bFGF in the explanted mouse retina was studied by confocal microscopy. Exogenous addition of bFGF to 661W cells resulted in an increase in ROS production that lasted for 24 h. When this ROS production was inhibited, bFGF-induced phosphorylation of Akt was prevented. Through the use of inhibitors and small interfering RNA in the cell line, the source of this production was shown to be Cox and to involve the activation of phospholipases A(2) + C. This pathway may also occur in the mouse retina, as we showed that the retina expressed Cox1&2, and that photoreceptors in explanted retina respond to bFGF by increasing their ROS levels. These results demonstrate that exogenous bFGF can stimulate ROS production through the activation of Cox, and activate the Akt pathway.

Age-dependent rat retinal ganglion cell susceptibility to apoptotic stimuli: implications for glaucoma.

BACKGROUND: This paper seeks to investigate differences between the neonatal and adult retinal ganglion cell populations to apoptotic death stimuli. DESIGN AND SAMPLES: In vitro and ex vivo paradigms involving P6 and P60 Sprague-Dawley rat retinal explants and retinal ganglion cells were employed. METHODS: Postnatal day 6 (P6) and 60 (P60) Sprague-Dawley retinal ganglion cells and retinal explants were either serum starved or subjected to excitotoxicity using calcium ionophore A23187. MAIN OUTCOME MEASURES: Apoptosis was detected in both models using terminal dUTP nick end labelling. Expression of Apaf-1, active caspases-3 and 9 in P6 and P60 retinas, and in the ganglion cell layer was examined using Western blotting. RESULTS: In both the dissociated retinal ganglion cell and retinal explant models, P60 retinal ganglion cells were significantly less susceptible to excitoxicity and serum starvation than their P6 counterparts. Western blotting indicated that active caspase-3 and Apaf-1 are downregulated in the Sprague-Dawley rat retina at P60 compared with P6. CONCLUSIONS: We demonstrate that neonatal Sprague-Dawley retinal ganglion cells are more susceptible to glaucoma-related death stimuli than their adult counterparts in dissociated retinal ganglion cells and axotomized retinal explant models. It is apparent that these different retinal ganglion cell populations are inherently designed to react differently to death stimuli. Thus caution should be exercised when noting the high susceptibility of neonatal retinal ganglion cells to glaucomatous death stimuli.

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.

Differential global and extra-cellular matrix focused gene expression patterns between normal and glaucomatous human lamina cribrosa cells.

PURPOSE: Marked extracellular matrix (ECM) remodeling occurs in the human optic nerve head in primary open angle glaucoma (POAG). The glial fibrillary acid protein (GFAP) negative lamina cribrosa cell may play an important role in this remodeling process. We report the first study of global and ECM-focused gene transcription differentials between GFAP-negative lamina cribrosa (LC) cells from normal and POAG human donors. METHODS: GFAP-negative LC cell lines were generated from the optic nerve tissue of four normal (n=4) and four POAG (n=4) human donors. Using Affymetrix U133A arrays the transcriptional profile between the normal and diseased groups were compared. Bioinformatic analysis was performed using robust multichip average (RMA Express) and EASE/David. Real time TaqMan PCR and immunohistochemistry analyses were performed to validate the microarray data. RESULTS: 183 genes were upregulated by greater than 1.5 fold and 220 were down regulated by greater than 1.5 fold in the POAG LC cells versus normal controls. Upregulated genes in POAG LC cells included, transforming growth factor beta 1 (TGFbeta1), secreted acid protein cysteine rich (SPARC), periostin (POSTN), thrombospondin-1 (THBS1), cartilage linking protein-1 (CRTL-1), and collagen type I (COL1A1), collagen type V (COL5A1), and collagen type XI (COL11A1). Downregulated ECM genes in POAG included fibulin 1 (FBLN1), decorin (DCN), and collagen type XVIII (COL18A1). All TaqMan PCR validation assays were significant (*p<0.05) and consistent with the array data. Immunohistochemistry of one target (periostin) confirmed its differential expression at the protein level in POAG optic nerve head tissue compared with non-glaucomatous controls. Functional annotation and over-representation analysis identified ECM genes as a statistically over-represented class of genes in POAG LC cells compared with normal LC cells. CONCLUSIONS: This study reports for the first time that POAG LC cells in-vitro demonstrate upregulated ECM and pro-fibrotic gene expression compared with normal LC cells. This may be a pathological characteristic of this cell type in POAG in-vivo. We believe that the LC cell may be a pivotal regulator of optic nerve head ECM remodeling in POAG and an attractive target for molecular therapeutic strategies in the future.

Worldwide geographical distribution of ophthalmology publications.

International peer-reviewed publications form the basis of evidence-based medicine and are one of the main indicators of ophthalmology research activity. This paper examines the origins of such articles in relation to geographical location, population demographics and economic research profiles. Publications from five ophthalmology journals over 5 years were analysed using the Medline/Pubmed search engine. Country of origin was ascertained based on the address of the corresponding author. Worldwide ophthalmology research output was analysed in relation to population demographics and research expenditure. In total, 7,754 articles from 67 countries from 2002 to 2006 were analysed. The overall number of articles published increased by over 29% during this period. The United States (US) produced the greatest number of articles. Singapore produced the most publications per 10(6) population. There was a relationship between gross domestic product (GDP) greater than $20,000 and population-adjusted output. The US followed by the United Kingdom and Japan were the greatest gross contributors. Population-adjusted figures revealed that Singapore, Iceland and Australia were the most prolific nations. There was a relationship between GDP and the top-ranked population-adjusted countries. The top 10 most productive population-adjusted countries spend relatively more on research and development than the top 10 gross contributors.

Basic fibroblast growth factor-induced protection from light damage in the mouse retina in vivo.

Basic fibroblast growth factor (bFGF) has proven neuroprotective efficacy in the rodent retina against a diverse array of injurious stimuli. However, there is no consensus to date as to the molecular mechanisms underlying this neuroprotection. The study presented herein demonstrates increased expression of endogenous bFGF in the albino mouse retina in response to acute exposure to sublethal levels of light stress. The increased expression correlates with significant photoreceptor protection from light damage. The neuroprotection is likely to be mediated by bFGF as we demonstrate that a shorter exposure to bright light stress that does not up-regulate bFGF fails to protect photoreceptors from light damage. Furthermore, intravitreal bFGF injection into the retina of mice 3 h prior to light damage affords almost complete photoreceptor protection from light-induced degeneration. In addition, injected bFGF induces the activation of protein kinase B and extracellular signal-regulated kinase 1/2 signalling which correlate directly with the pathways we find to be activated in response to light stress and up-regulated bFGF. Moreover, we demonstrate that both bright light pre-conditioning and intravitreal bFGF injection result in dramatic increases in levels of inactive glycogen synthase kinase 3beta and cyclic AMP response element binding protein phosphorylation indicating a potential mechanism by which bFGF promotes survival of photoreceptors in vivo.

Aging and ocular tissue stiffness in glaucoma.

Glaucoma is a progressive and chronic neurodegenerative disorder characterized by damage to the inner layers of the retina and deformation of the optic nerve head. The degeneration of retinal ganglion cells and their axons results in an irreversible loss of vision and is correlated with increasing age. Extracellular matrix changes related to natural aging generate a stiffer extracellular environment throughout the body. Altered age-associated ocular tissue stiffening plays a major role in a significant number of ophthalmic pathologies. In glaucoma, both the trabecular meshwork and the optic nerve head undergo extensive extracellular matrix remodeling, characterized by fibrotic changes associated with cellular and molecular events (including myofibroblast activation) that drive further tissue fibrosis and stiffening. Here, we review the literature concerning the role of age-related ocular stiffening in the trabecular meshwork, lamina cribrosa, sclera, cornea, retina, and Bruch membrane/choroid and discuss their potential role in glaucoma progression. Because both trabecular meshwork and lamina cribrosa cells are mechanosensitive, we then describe molecular mechanisms underlying tissue stiffening and cell mechanotransduction and how these cellular activities can drive further fibrotic changes within ocular tissues. An improved understanding of the interplay between age-related tissue stiffening and biological responses in the trabecular meshwork and optic nerve head could potentially lead to novel therapeutic strategies for glaucoma treatment.

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.

A key role for calpains in retinal ganglion cell death.

PURPOSE: The purpose of this study was to examine the importance of calpains in retinal ganglion cell (RGC) apoptosis and the protection afforded by calpain inhibitors against cell death. METHODS: Two different models of RGC apoptosis were used, namely the RGC-5 cell line after either intracellular calcium influx or serum withdrawal and retinal explant culture involving optic nerve axotomy. Flow cytometry analysis with Annexin V/PI staining was used to identify RGC-5 cells undergoing apoptosis after treatment. TdT-mediated dUTP nick end labeling (TUNEL) was used to identify cells undergoing apoptosis in retinal explant sections under various conditions. Serial sectioning was used to isolate the cell population of the ganglion cell layer (GCL). Western blotting was used to demonstrate calpain cleavage and activity by detecting cleaved substrates. RESULTS: In the RGC-5 cell line, the authors reported the activation of mu-calpain and m-calpain after serum starvation and calcium ionophore treatment, with concurrent cleavage of known calpain substrates. They found that the inhibition of calpains leads to the protection of cells from apoptosis. In the second model, after a serial sectioning method to isolate the cells of the ganglion cell layer (GCL) on a retinal explant paradigm, protein analysis indicated the activation of calpains after axotomy, with concomitant cleavage of calpain substrates. The authors found that inhibition of calpains significantly protected cells in the GCL from cell death. CONCLUSIONS: These results suggest that calpains are crucial for apoptosis in RGCs after calcium influx, serum starvation, and optic nerve injury.

Altered endothelin-1 vasoreactivity in patients with untreated normal-pressure glaucoma.

PURPOSE: Vasospasm, resulting from a generalized dysfunction in the vascular endothelium, is implicated in the development of normal-pressure glaucoma (NPG). Impaired endothelium-derived nitric oxide activity and abnormalities of the endothelin system suggest systemic endothelial cell dysfunction in patients with NPG. Endothelin (ET)-1 vasoreactivity was assessed in the peripheral circulation of patients with NPG. METHODS: Forearm blood flow was measured using venous occlusion plethysmography in eight patients with untreated NPG and eight age- and sex-matched healthy volunteers during intra-arterial infusion of ET-1 (5 pmol/min) and, on a separate occasion, to BQ123, a selective endothelin-A receptor antagonist, (100 nmol/min). Blood pressure and heart rate were measured in the noninfused arm, and plasma ET-1 concentrations were measured using a radioimmunoassay. RESULTS: Forearm blood flow fell during infusion of ET-1 (P<0.001 for both) to a similar extent in both groups (P=0.7; patients versus control subjects). In contrast, BQ123 increased forearm blood flow in both groups (P<0.001 for both), although the vasodilatation was lower in patients than in control subjects (P<0.001; patients versus control subjects). There was no difference in basal plasma ET-1 concentrations between the two groups (P=0.81; patients versus control subjects). CONCLUSIONS: Despite normal responses to ET-1, patients with NPG have reduced vasodilatation in response to ETA-receptor antagonism. This could be due to attenuated ETA-receptor-mediated tone, increased ETB-receptor-mediated contraction or impaired ETB-receptor-mediated release of endothelial nitric oxide. These results are consistent with the authors' previous demonstration of systemic vascular dysfunction in patients with NPG.

Age-dependent susceptibility of the retinal ganglion cell layer to cell death.

PURPOSE: The purpose of this study was to determine the susceptibility of the retinal ganglion cell layer (GCL) to apoptosis after optic nerve transection and excitotoxic stimulus and to investigate the regulation of apoptosis in the GCL during development. The authors also sought to determine the role played by caspases in cell death and their expression during development. METHODS: TdT-mediated dUTP nick end labeling (TUNEL) was used to identify cells undergoing apoptosis during mouse retinal development from postnatal day (P)3 to P5 and in retinal explant sections under various conditions. The expression of active caspases was determined by immunohistochemistry (IHC) using an antibody that detects the cleaved large subunit. IHC was also used to detect the expression levels of procaspase-3, procaspase-9, and Apaf-1 in P6 and P60 whole eye sections. Retinal ganglion cells at ages P6 and P60 were purified by immunopanning, total RNA was extracted, and mRNA levels of the above proteins were determined by semiquantitative PCR. RESULTS: After optic nerve transection, a significant number of TUNEL-positive cells were seen 24 hours after lesion in P6 retinas. This death was caspase dependent, as shown by IHC and caspase inhibition with zVAD-fmk. In contrast, adult GCL was resistant to apoptosis under these conditions. Similarly, after excitotoxic stimulus, the GCL of the P6 retinas underwent apoptosis at 6 hours and was caspase dependent, whereas adult GCL was resistant. Developmental apoptosis in the GCL between P2 and P6 was shown to involve caspase-3 and caspase-9. Significant downregulation of Apaf-1 and caspase-3 was detected in the P60 GCL at both the mRNA and the protein levels. CONCLUSIONS: Adult GCL is more resistant to apoptosis than neonatal GCL after ON transection and excitotoxic stimulus. The expression of caspase-3 and Apaf-1 is significantly reduced in adult GCL. The authors suggest that age-dependent susceptibility to apoptosis may be caused by this reduced expression.