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Glucocorticoid-induced glaucoma is a secondary open-angle glaucoma. About 40% of the general population may develop elevated intraocular pressure on prolonged glucocorticoid treatment secondary to damages in the trabecular meshwork (TM), a tissue that regulates intraocular pressure. Therefore, identifying the key molecules responsible for glucocorticoid-induced ocular hypertension is crucial. In this study, Dickkopf-related protein 1 (Dkk1), a canonical Wnt signaling inhibitor, was found to be elevated in the aqueous humor and TM of glaucoma patients. At the signaling level, Dkk1 enhanced glucocorticoid receptor (GR) signaling, whereas Dkk1 knockdown or Wnt signaling activators decreased GR signaling in human TM cells as indicated by luciferase assays. Similarly, activation of the GR signaling inhibited Wnt signaling. At the protein level, glucocorticoid-induced extracellular matrix was inhibited by Wnt activation using Wnt activators or Dkk1 knockdown in primary human TM cells. In contrast, inhibition of canonical Wnt signaling by β-catenin knockdown increased glucocorticoid-induced extracellular matrix proteins. At the physiological level, adenovirus-mediated Wnt3a expression decreased glucocorticoid-induced ocular hypertension in mouse eyes. In summary, Wnt and GR signaling inhibit each other in the TM, and canonical Wnt signaling activators may prevent the adverse effect of glucocorticoids in the eye.

Glucocorticoids (GCs) have unique actions in their combined anti-inflammatory and immunosuppressive activities and are among the most commonly prescribed drugs, particularly for inflammatory condit...

Glaucoma is a group of optic neuropathies that leads to irreversible vision loss. The optic nerve head (ONH) is the site of initial optic nerve damage in glaucoma. ONH-derived lamina cribrosa (LC) cells synthesize extracellular matrix (ECM) proteins, however, these cells are adversely affected in glaucoma and cause detrimental changes to the ONH. LC cells respond to mechanical strain by increasing the profibrotic cytokine transforming growth factor-beta 2 (TGFβ2) and ECM proteins. Moreover, microRNAs (miRNAs or miR) regulate ECM gene expression in different fibrotic diseases, including glaucoma. A delicate homeostatic balance between profibrotic and anti-fibrotic miRNAs may contribute to the remodeling of ONH. This study aimed to determine whether modulation of miRNAs alters the expression of ECM in human LC cells. Primary human normal and glaucoma LC cells were grown to confluency and treated with or without TGFβ2 for 24 h. Differences in expression of miRNAs were analyzed using miRNA qPCR arrays. miRNA PCR arrays showed that the miR-29 family was significantly decreased in glaucomatous LC cell strains compared to age-matched controls. TGFβ2 treatment downregulated the expression of multiple miRNAs, including miR-29c-3p, compared to controls in LC cells. LC cells transfected with miR-29c-3p mimics or inhibitors modulated collagen expression.

Purpose In POAG, elevated IOP remains the major risk factor in irreversible vision loss. Increased TGFβ2 expression in POAG aqueous humor and in the trabecular meshwork (TM) amplifies extracellular matrix (ECM) deposition and reduces ECM turnover in the TM, leading to a decreased aqueous humor (AH) outflow facility and increased IOP. Inhibitor of DNA binding proteins (ID1 and ID3) inhibit TGFβ2-induced fibronectin and PAI-1 production in TM cells. We examined the effects of ID1 and ID3 gene expression on TGFβ2-induced ocular hypertension and decreased AH outflow facility in living mouse eyes. Methods IOP and AH outflow facility changes were determined using a mouse model of Ad5-hTGFβ2C226S/C288S-induced ocular hypertension. The physiological function of ID1 and ID3 genes were evaluated using Ad5 viral vectors to enhance or knockdown ID1/ID3 gene expression in the TM of BALB/cJ mice. IOP was measured in conscious mice using a Tonolab impact tonometer. AH outflow facilities were determined by constant flow infusion in live mice. Results Over-expressing ID1 and ID3 significantly blocked TGFβ2-induced ocular hypertension (P < 0.0001). Although AH outflow facility was significantly decreased in TGFβ2-transduced eyes (P < 0.04), normal outflow facility was preserved in eyes injected concurrently with ID1 or ID3 along with TGFβ2. Knockdown of ID1 or ID3 expression exacerbated TGFβ2-induced ocular hypertension. Conclusions Increased expression of ID1 and ID3 suppressed both TGFβ2-elevated IOP and decreased AH outflow facility. ID1 and/or ID3 proteins thus may show promise as future candidates as IOP-lowering targets in POAG.

Background Glaucoma is a leading neurodegenerative disease affecting over 70 million individuals worldwide. Early pathological events of axonal degeneration and retinopathy in response to elevated intraocular pressure (IOP) are limited and not well-defined due to the lack of appropriate animal models that faithfully replicate all the phenotypes of primary open angle glaucoma (POAG), the most common form of glaucoma. Glucocorticoid (GC)-induced ocular hypertension (OHT) and its associated iatrogenic open-angle glaucoma share many features with POAG. Here, we characterized a novel mouse model of GC-induced OHT for glaucomatous neurodegeneration and further explored early pathological events of axonal degeneration in response to elevated IOP. Methods C57BL/6 J mice were periocularly injected with either vehicle or the potent GC, dexamethasone 21-acetate (Dex) once a week for 10 weeks. Glaucoma phenotypes including IOP, outflow facility, structural and functional loss of retinal ganglion cells (RGCs), optic nerve (ON) degeneration, gliosis, and anterograde axonal transport deficits were examined at various stages of OHT. Results Prolonged treatment with Dex leads to glaucoma in mice similar to POAG patients including IOP elevation due to reduced outflow facility and dysfunction of trabecular meshwork, progressive ON degeneration and structural and functional loss of RGCs. Lowering of IOP rescued Dex-induced ON degeneration and RGC loss, suggesting that glaucomatous neurodegeneration is IOP dependent. Also, Dex-induced neurodegeneration was associated with activation of astrocytes, axonal transport deficits, ON demyelination, mitochondrial accumulation and immune cell infiltration in the optic nerve head (ONH) region. Our studies further show that ON degeneration precedes structural and functional loss of RGCs in Dex-treated mice. Axonal damage and transport deficits initiate at the ONH and progress toward the distal end of ON and target regions in the brain (i.e. superior colliculus). Most of anterograde transport was preserved during initial stages of axonal degeneration (30% loss) and complete transport deficits were only observed at the ONH during later stages of severe axonal degeneration (50% loss). Conclusions These findings indicate that ON degeneration and transport deficits at the ONH precede RGC structural and functional loss and provide a new potential therapeutic window for rescuing neuronal loss and restoring health of damaged axons in glaucoma.

The Hispanic population is underserved and underrepresented in health care. Epidemiological studies are cmcial for providing insight to identify disparities and unmet eye health needs in this vulnerable group. The purpose of our study is to examine the prevalence of ocular conditions in the elderly Hispanic population in North Texas and identify the frequency in which these conditions were undiagnosed. This study was ancillary to the Health and Aging Brain study among Latino Elders (HABLE). Seventy-three HABLE participants (aged > 50 years) underwent neuropsychological evaluation and an eye health screening at the University of North Texas Health Science Center study site. Descriptive analyses were performed for prevalence of ocular conditions, as well as a comparison of self-reported conditions and ocular Endings. Our results suggest the prevalence patterns for undetected ocular disease in the Hispanic population of North Texas are comparable with the epidemiological trends for this population group in other concentrated areas in the United States.

The lamina cribrosa is the initial site of glaucomatous injury. Pathological changes to the lamina cribrosa include posterior displacement of the lamina cribrosa, loss of trophic support, and remodeling of the extracellular matrix. Optic nerve head (ONH) astrocytes and lamina cribrosa cells synthesize extracellular matrix proteins to support and maintain the lamina cribrosa under physiological conditions. During glaucoma, these cells respond to mechanical strain and other stimuli, which leads to pathological remodeling of the ONH. Although ONH astrocytes and lamina cribrosa cells have been previously cultured, there is no well-accepted, straightforward technique to isolate both cell types from a single dissected human ONH. To better understand the pathophysiology of glaucoma, we obtained and cultured lamina cribrosa explants from human donor eyes. Initially, cells that grew out from the explant were ONH astrocytes and lamina cribrosa cells. Using a specialized medium, we isolated pure populations of lamina cribrosa cells and ONH astrocytes. ONH astrocytes expressed glial fibrillary acidic protein (GFAP). Lamina cribrosa cells expressed alpha-smooth muscle actin (α-SMA), but were negative for GFAP. This method of ONH cell isolation and cell-culture will provide a technique to better understand the molecular and cell-specific changes in glaucomatous damage to the ONH.

Optic nerve astrocytes play a major role in axonal degeneration and regeneration. Astrocyte lines are an important tool to elucidate the responsible cellular mechanisms. In this study, we established a conditionally immortalized mouse optic nerve astrocyte line. Astrocytes were cultured from explants derived from postnatal day 5–7 H-2k(b)-tsA58 transgenic mouse optic nerves. Cells were cultured in defined astrocyte culture medium under permissive (33°C) or non-permissive (38.5°C) temperature with or without interferon-v (IFN-ɤ). Astrocytes were characterized by immunocytochemistry staining using antibodies against glial fibrillary acidic protein (GFAP) and neural cell adhesion molecule (NCAM). Cell proliferation rates were determined by cell growth curves and percentage of Ki67 positive cells. Karyotyping was performed to validate the mouse origin of established cell line. Conditional immortalization was assessed by western blot-determined expression levels of SV40 large T antigen (TAg), p53, GFAP and NCAM in non-permissive culture conditions. In addition, phagocytic activity of immortalized cells was determined by flow cytometry-based pHrodo florescence analysis. After 5 days in culture, cells migrated out from optic nerve explants. Immunocytochemistry staining showed that migrating cells expressed astrocyte makers, GFAP and NCAM. In permissive conditions, astrocytes had increased expression levels of TAg and p53, exhibited a greater cell proliferation rate as well as a higher percentage of Ki67 positive cells (n=3, p

Due to their similarities in anatomy, physiology, and pharmacology to humans, mice are a valuable model system to study the generation and mechanisms modulating conventional outflow resistance and thus intraocular pressure. In addition, mouse models are critical for understanding the complex nature of conventional outflow homeostasis and dysfunction that results in ocular hypertension. In this review, we describe a set of minimum acceptable standards for developing, characterizing, and utilizing mouse models of open-angle ocular hypertension. We expect that this set of standard practices will increase scientific rigor when using mouse models and will better enable researchers to replicate and build upon previous findings.

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.

Purpose The trabecular meshwork (TM) has an important role in the regulation of aqueous humor outflow and IOP. Regulation of the extracellular matrix (ECM) by TGFβ2 has been studied extensively. Bone morphogenetic protein (BMP) and activin membrane-bound inhibitor (BAMBI) has been shown to inhibit or modulate TGFβ2 signaling. We investigate the role of TGFβ2 and BAMBI in the regulation of TM ECM and ocular hypertension. Methods Mouse TM (MTM) cells were isolated from B6;129S1-Bambitm1Jian/J flox mice, characterized for TGFβ2 and dexamethasone (DEX)-induced expression of fibronectin, collagen-1, collagen-4, laminin, α-smooth muscle actin, cross-linked actin networks (CLANs) formation, and DEX-induced myocilin (MYOC) expression. MTM cells were transduced with Ad5.GFP to identify transduction efficiency. MTM cells and mouse eyes were transduced with Ad5.Null, Ad5.Cre, Ad5.TGFβ2, or Ad5.TGFβ2 + Ad5.Cre to evaluate the effect on ECM production, IOP, and outflow facility. Results MTM cells express TM markers and respond to DEX and TGFβ2. Ad5.GFP at 100 MOI had the highest transduction efficiency. Bambi knockdown by Ad5.Cre and Ad5.TGFβ2 increased fibronectin, collagen-1, and collagen-4 in TM cells in culture and tissue. Ad5.Cre, Ad5.TGFβ2, and Ad5.TGFβ2 + Ad5.Cre each significantly induced ocular hypertension and lowered aqueous humor outflow facility in transduced eyes. Conclusions We show for the first time to our knowledge that knockdown of Bambi alters ECM expression in cultured cells and mouse TM, reduces outflow facility, and causes ocular hypertension. These data provide a novel insight into the development of glaucomatous TM damage and identify BAMBI as an important regulator of TM ECM and ocular hypertension.

With the combined purpose of facilitating useful vision over a lifetime, a number of ocular cells have evolved specialized features not found elsewhere in the body. The trabecular meshwork (TM) cell at the irido-corneal angle, which is a key regulator of intraocular pressure, is no exception. Examination of cells in culture isolated from the human TM has shown that they are unique in many ways, displaying characteristic features of several different cell types. Thus, these neural crest derived cells display expression patterns and behaviors typical of endothelia, fibroblasts, smooth muscle and macrophages, owing to the multiple roles and two distinct environments where they operate to maintain intraocular pressure homeostasis. In most individuals, TM cells function normally over a lifetime in the face of persistent stressors, including phagocytic, oxidative, mechanical and metabolic stresses. Study of TM cells isolated from ocular hypertensive eyes has shown a compromised ability to perform their daily duties. This review highlights the many responsibilities of the TM cell and its challenges, progress in our understanding of TM biology over the past 30 years, as well as discusses unanswered questions about TM dysfunction that results in IOP dysregulation and glaucoma.

Glucocorticoid (GC)-induced ocular hypertension (OHT) is a serious adverse effect of prolonged GC therapy that can lead to iatrogenic glaucoma and permanent vision loss. An appropriate mouse model can help us understand precise molecular mechanisms and etiology of GC-induced OHT. We therefore developed a novel, simple, and reproducible mouse model of GC-induced OHT. GC-induced myocilin expression in the trabecular meshwork (TM) has been suggested to play an important role in GC-induced OHT. We further determined whether myocilin contributes to GC-OHT. C57BL/6J mice received weekly periocular conjunctival fornix injections of a dexamethasone-21-acetate (DEX-Ac) formulation. Intraocular pressure (IOP) elevation was relatively rapid and significant, and correlated with reduced conventional outflow facility. Nighttime IOPs were higher in ocular hypertensive eyes compared to daytime IOPs. DEX-Ac treatment led to increased expression of fibronectin, collagen I, and α-smooth muscle actin in the TM in mouse eyes. No changes in body weight indicated no systemic toxicity associated with DEX-Ac treatment. Wild-type mice showed increased myocilin expression in the TM on DEX-Ac treatment. Both wild-type and Myoc −/− mice had equivalent and significantly elevated IOP with DEX-Ac treatment every week. In conclusion, our mouse model mimics many aspects of GC-induced OHT in humans, and we further demonstrate that myocilin does not play a major role in DEX-induced OHT in mice.

Additional file 1: Fig. S1. Dex-induced IOP elevation is higher during the night-time. C57BL/6 J mice were periocularly injected with Veh or Dex in both eyes, and IOPs were monitored weekly in the dark during the night time under isoflurane anesthetic conditions. Dex injections lead to sustained and significant IOP elevation and the observed mean difference of Dex-induced IOP is more pronounced at night compared to daytime IOP. Data are shown as mean ± SD (n = 8 in each group, 2-WAY ANOVA with multiple comparison, ***p = 0.0002, #p

Purpose Increased expression of TGF-β2 in primary open-angle glaucoma (POAG) aqueous humor (AH) and trabecular meshwork (TM) causes deposition of extracellular matrix (ECM) in the TM and elevated IOP. Bone morphogenetic proteins (BMPs) regulate TGF-β2-induced ECM production. The underlying mechanism for BMP4 inhibition of TGF-β2-induced fibrosis remains undetermined. Bone morphogenic protein 4 induces inhibitor of DNA binding proteins (ID1, ID3), which suppress transcription factor activities to regulate gene expression. Our study will determine whether ID1and ID3 proteins are downstream targets of BMP4, which attenuates TGF-β2 induction of ECM proteins in TM cells. Methods Primary human TM cells were treated with BMP4, and ID1 and ID3 mRNA, and protein expression was determined by quantitative PCR (Q-PCR) and Western immunoblotting. Intracellular ID1 and ID3 protein localization was studied by immunocytochemistry. Transformed human TM cells (GTM3 cells) were transfected with ID1 or ID3 expression vectors to determine their potential inhibitory effects on TGF-β2-induced fibronectin and plasminogen activator inhibitor-I (PAI-1) protein expression. Results Basal expression of ID1-3 was detected in primary human TM cells. Bone morphogenic protein 4 significantly induced early expression of ID1 and ID3 mRNA (P < 0.05) and protein in primary TM cells, and a BMP receptor inhibitor blocked this induction. Overexpression of ID1 and ID3 significantly inhibited TGF-β2-induced expression of fibronectin and PAI-1 in TM cells (P < 0.01). Conclusions Bone morphogenic protein 4 induced ID1 and ID3 expression suppresses TGF-β2 profibrotic activity in human TM cells. In the future, targeting specific regulators may control the TGF-β2 profibrotic effects on the TM, leading to disease modifying IOP lowering therapies.

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 β1 (TGFβ1).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), TGFβ 1 and 2, collagen 1α1 (COL1A1), and α-smooth muscle actin (αSMA). TGFβ1 and DNMT1 were examined by immunofluorescence. Methylation of the TGFβ1 promoter was determined by methylation-specific PCR (MSP).Global DNA methylation demonstrated an increase in GLC compared with NLC cells (P0.05). The previously mentioned methylation and matrix genes were increased in GLC compared with NLC cells (P0.05). Immunofluorescence showed increased TGFβ1 and DNMT1 in GLC compared with NLC cells. MSP showed increased unmethylated DNA in the TGFβ1 promoter of GLC compared with NLC cells.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 TGFβ1 in GLC cells. Determining a role for methylation in glaucoma and in regulating TGFβ1 may provide a novel therapeutic approach.

Graphical abstract, Oxidative stress-induced retinal pigment epithelial (RPE) cell damage is an important factor in the pathogenesis of age-related macular degeneration (AMD). Previous studies have shown that RTA 408, a synthetic triterpenoid compound, potently activates Nrf2. This study aimed to investigate the protective effects of RTA 408 in cultured RPE cells during oxidative stress and to determine the effects of RTA 408 on Nrf2 and its downstream target genes. Primary human RPE cells were pretreated with RTA 408 and then incubated in 200 μM H2O2 for 6 h. Cell viability was measured with the WST-8 assay. Apoptosis was quantitatively measured by annexin V/propidium iodide (PI) double staining and Hoechst 33342 fluorescent staining. Reduced (GSH) and oxidized glutathione (GSSG) were measured using colorimetric assays. Nrf2 activation and its downstream effects on phase II enzymes were examined by Western blot. Treatment of RPE cells with nanomolar ranges (10 and 100 nM) of RTA 408 markedly attenuated H2O2-induced viability loss and apoptosis. RTA 408 pretreatment significantly protected cells from oxidative stress-induced GSH loss, GSSG formation and decreased ROS production. RTA 408 activated Nrf2 and increased the expression of its downstream genes, such as HO-1, NQO1, SOD2, catalase, Grx1, and Trx1. Consequently, the enzyme activities of NQO1, Grx1, and Trx1 were fully protected by RTA 408 pretreatment under oxidative stress. Moreover, knockdown of Nrf2 by siRNA significantly reduced the cytoprotective effects of RTA 408. In conclusion, our data suggest that RTA 408 protect primary human RPE cells from oxidative stress-induced damage by activating Nrf2 and its downstream genes., Highlights • Oxidative stress contributes to the pathogenesis of AMD. • RTA408 in nanomolar concentrations increased survival and restored the redox state in H2O2-damaged RPE cells. • RTA408 stimulated Nrf2 and downstream antioxidant enzymes Grx1, NQO1, and HO-1. • Inhibition of Nrf2 abolished RTA408’s protective effects. • RTA408 may have the potential to treat AMD and other degenerative eye diseases.

Primary Open Angle Glaucoma (POAG) is an irreversible, vision-threatening disease that affects millions worldwide. The principal risk factor of POAG is increased intraocular pressure (IOP) due to pathological changes in the trabecular meshwork (TM). The TGFβ signaling pathway activator TGFβ2 and the Wnt signaling pathway inhibitor secreted frizzled-related protein 1 (sFRP1) are elevated in the POAG TM. In this study, we determined whether there is a crosstalk between the TGFβ/Smad pathway and the canonical Wnt pathway using luciferase reporter assays. Lentiviral luciferase reporter vectors for studying the TGFβ/Smad pathway or the canonical Wnt pathway were transduced into primary human non-glaucomatous TM (NTM) cells. Cells were treated with or without a combination of 5 μg/ml TGFβ2 and/or 100 ng/ml Wnt3a recombinant proteins, and luciferase levels were measured using a plate reader. We found that TGFβ2 inhibited Wnt3a-induced canonical Wnt pathway activation, while Wnt3a inhibited TGFβ2-induced TGFβ/Smad pathway activation (n = 6, p < 0.05) in 3 NTM cell strains. We also found that knocking down of Smad4 or β-catenin using siRNA in HTM5 cells transfected with similar luciferase reporter plasmids abolished the inhibitory effect of TGFβ2 and/or Wnt3a on the other pathway (n = 6). Our results suggest the existence of a cross-inhibition between the TGFβ/Smad and canonical Wnt pathways in the TM, and this cross-inhibition may be mediated by Smad4 and β-catenin.

Glaucoma patients have elevated aqueous humor and trabecular meshwork (TM) levels of transforming growth factor-beta2 (TGF-β2). TGF-β2 has been associated with increased extracellular matrix (ECM) deposition (i.e. fibronectin), which is attributed to the increased resistance of aqueous humor outflow through the TM. We have previously demonstrated that bone morphogenetic protein (BMP) 4 selectively counteracts the profibrotic effect of TGF-β2 with respect to ECM synthesis in the TM, and this action is reversed by the BMP antagonist gremlin. Thus, the BMP and TGF-β signaling pathways antagonize each other’s antifibrotic and profibrotic roles. The purpose of this study was to determine whether cultured human TM cells: (a) express other BMP antagonists including noggin, chordin, BMPER, BAMBI, Smurf1 and 2, and (b) whether expression of these proteins is regulated by exogenous TGF-β2 treatment. Primary human trabecular meshwork (TM) cells were grown to confluency and treated with TGF-β2 (5 ng/ml) for 24 or 48 h in serum-free medium. Untreated cell served as controls. qPCR and Western immunoblots (WB) determined that human TM cells expressed mRNAs and proteins for the BMP antagonist proteins: noggin, chordin, BMPER, BAMBI, and Smurf1/2. Exogenous TGF-β2 decreased chordin, BMPER, BAMBI, and Smurf1 mRNA and protein expression. In contrast, TGF-β2 increased secreted noggin and Smurf2 mRNA and protein levels. BMP antagonist members are expressed in the human TM. These molecules may be involved in the normal function of the TM as well as TM pathogenesis. Altered expression of BMP antagonist members may lead to functional changes in the human TM.

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 PKC&alpha, 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 PKC&alpha, p38 and p42/p44-MAPKs are significantly (p &lt, 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 (PKC&alpha, MAPK, PI3K) in glaucoma LC cells, which might have significant functional and therapeutic implications in optic nerve head (ONH) fibrosis and cupping in glaucoma.

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.

The lamina cribrosa (LC) in glaucoma is with augmented production of extracellular matrix proteins (ECM) and connective tissue fibrosis. Fundamental pathological mechanisms for this fibrosis comprise fibrotic growth factors and oxidative stress. Transient receptor potential canonical channels (TRPC) channels play a key role in ECM fibrosis. Here, we study TRPC expression in glaucomatous LC cells, and investigate the role of TRPC in oxidative stress induced-profibrotic ECM gene transcription and cell proliferation in normal LC cells. Age-matched human LC cells (normal, n = 3 donors; glaucoma, n = 3 donors) were used. Hydrogen peroxide (H2O2, 100 μM), was used to induce oxidative stress in LC cells in the presence or absence of the pan TRPC inhibitor SKF96365 (10 μM) or knockdown of TRPC1/6 with siRNA. After treatments, ECM gene transcription, LC cell viability and proliferation and the phosphorylation of the transcription factor NFATc3, were measured using real time RT-PCR, colorimetric cell counting with the methyl-thiazolyl tetrazolium salt (MTS) assay, and Western immunoblotting, respectively. Results showed that TRPC1/C6 transcript and protein expression levels were significantly (p

Glaucoma is one of the leading causes of vision impairment worldwide. In order to further understand the molecular pathobiology of this disease and to develop better therapies, clinically relevant animal models are necessary. In recent years, both the rat and mouse have become popular models in glaucoma research. Key reasons are: many important biological similarities shared among rodent eyes and the human eye; development of improved methods to induce glaucoma and to evaluate glaucomatous damage; availability of genetic tools in the mouse; as well as the relatively low cost of rodent studies. Commonly studied rat and mouse glaucoma models include intraocular pressure (IOP)-dependent and pressure-independent models. The pressure-dependent models address the most important risk factor of elevated IOP, whereas the pressure-independent models assess “normal tension” glaucoma and other “non-IOP” related factors associated with glaucomatous damage. The current article provides descriptions of these models, their characterizations, specific techniques to induce glaucoma, mechanisms of injury, advantages, and limitations.

Glaucoma is a leading cause of irreversible vision loss and is associated with fibrotic changes in two ocular tissues-the optic nerve head (ONH) and trabecular meshwork (TM). We investigated the differences in extracellular matrix components (ECM) including collagen, elastin, transforming growth factor beta-2, type-II receptor (TGFβRII) and Galectin3 (Gal3) in the glaucomatous human eyes to quantify fibrotic changes in ONH and TM. Glaucomatous and control human donor eyes were prepared for chemical and immunological staining to quantify ECM protein expression in the TM and ONH. Chemical staining included: Trichrome (collagen), Vernhoeff-Van Giesen (elastin) and Sirius Red (collagen). Immunohistochemistry was used to determine levels of Gal3 and TGFβ2RII. Quantitative analyses were performed using Image J software. Student's t-test was used to compare groups and Pearson's test was used to determine correlations P-values of 0.05 (or less) were considered statistically significant. Deposition of ECM proteins was elevated in glaucomatous tissues. There was increased collagen (P = 0.0469), Gal3 (P0.0001) and TGFβ2RII (P = 0.0005) in the TM of glaucomatous eyes. Likewise, collagen (P = 0.0517) and Galectin3 (P = 0.041) were increased in the ONH glaucomatous eyes. There was a correlation of TGFβRII with Gal3 in the TM (P0.0001) and optic nerve (P = 0.0003). The TM and ONH of glaucomatous eyes showed increased expression of ECM proteins supporting a fibrotic pathology. Galectin3 and TGFβ-2R II showed a positive correlation in TM and optic nerve supporting co-localization and suggesting their potential role in the glaucoma fibrotic process. Clin. Anat. 31:1031-1049, 2018. © 2018 The Authors. Clinical Anatomy published by Wiley Periodicals, Inc. on behalf of American Association of Clinical Anatomists.

Glucocorticoid (GC) therapy is widely used to treat a variety of inflammatory diseases and conditions. While unmatched in their anti-inflammatory and immunosuppressive activities, GC therapy is often associated with the significant ocular side effect of GC-induced ocular hypertension (OHT) and iatrogenic open-angle glaucoma. Investigators have generated GC-induced OHT and glaucoma in at least 8 different species besides man. These models mimic many features of this condition in man and provide morphologic and molecular insights into the pathogenesis of GC-OHT. In addition, there are many clinical, morphological, and molecular similarities between GC-induced glaucoma and primary open-angle glaucoma (POAG), making animals models of GC-induced OHT and glaucoma attractive models in which to study specific aspects of POAG.

Glucocorticoid (GC)-induced ocular hypertension (OHT) is a serious side-effect of prolonged GC therapy that can lead to glaucoma and permanent vision loss. GCs cause a plethora of changes in the trabecular meshwork (TM), an ocular tissue that regulates intraocular pressure (IOP). GCs act through the glucocorticoid receptor (GR), and the GR regulates transcription both through transactivation and transrepression. Many of the anti-inflammatory properties of GCs are mediated by GR transrepression, while GR transactivation largely accounts for GC metabolic effects and side effects of GC therapy. There is no evidence showing which of the two mechanisms play role in GC- OHT. Methods: GRdim transgenic mice (which have active transrepression and impaired transactivation) and wild type (WT) C57BL/6J mice received weekly periocular DEX-Ac injections. IOP, outflow facilities, and biochemical changes to the TM were determined. Results: GRdim mice did not develop GC-OHT DEX treatment, while WT mice had significantly increased IOP and decreased outflow facilities. Both TM tissue in eyes of DEX-treated GRdim mice and cultured TM cells isolated from GRdim mice had reduced or no change in the expression of fibronectin, myocilin, collagen type I, and α-SMA. GRdim MTM cells also had a significant reduction in DEX-induced cytoskeletal changes, which was clearly seen in WT MTM cells. Conclusion: We provide the first evidence for the role of GR transactivation in regulating GC-mediated gene expression in the TM and in the development of GC- OHT. This discovery suggests a novel therapeutic approach for treating ocular inflammation that will not cause the serious side effect of GC-OHT and glaucoma. Funding: NIH/NEI EY016242. Declaration of Interest: AFC is a consultant for Goodmans LLP, LayerBio, Kala Pharmacuticals and has received research support from Unity Biotechnology and Lung Therapeutics. However, none of these are related to the study presented. The other authors declare that they have no competing interests. Ethical Approval: All experimental protocols were approved by University of North Texas Health Science Center Institutional Animal Care and Use Committee regulations.

Prolonged glucocorticoid (GC) therapy can cause GC-induced ocular hypertension (OHT), which if left untreated progresses to iatrogenic glaucoma and permanent vision loss. The alternatively spliced isoform of glucocorticoid receptor GRβ acts as dominant negative regulator of GR activity, and it has been shown that overexpressing GRβ in trabecular meshwork (TM) cells inhibits GC-induced glaucomatous damage in TM cells. The purpose of this study was to use viral vectors to selectively overexpress the GRβ isoform in the TM of mouse eyes treated with GCs, to precisely dissect the role of GRβ in regulating steroid responsiveness. We show that overexpression of GRβ inhibits GC effects on MTM cells in vitro and GC-induced OHT in mouse eyes in vivo. Ad5 mediated GRβ overexpression reduced the GC induction of fibronectin, collagen 1, and myocilin in TM of mouse eyes both in vitro and in vivo. GRβ also reversed DEX-Ac induced IOP elevation, which correlated with increased conventional aqueous humor outflow facility. Thus, GRβ overexpression reduces effects caused by GCs and makes cells more resistant to GC treatment. In conclusion, our current work provides the first evidence of the in vivo physiological role of GRβ in regulating GC-OHT and GC-mediated gene expression in the TM.

Purpose Tissue transglutaminase (TGM2) is elevated in glaucomatous trabecular meshwork (TM) tissues. We investigated whether increased expression of TGM2 increases extracellular matrix crosslinking in the TM, thereby increasing aqueous humor outflow resistance and elevating intraocular pressure (IOP) in mouse eyes. Methods GTM3, primary human GTM 125-05, and cultured mouse TM cells were transduced with adenovirus serotype 5 expressing human transglutaminase 2 (Ad5.TGM2; multiplicity of infection [MOI]-75) and fixed for immunocytochemistry. To test the effect on IOP in living eyes, Ad5.TGM2 was injected intravitreally into one eye of BALB/cJ (n = 18) or C57BL/6J mice (n = 9). The uninjected contralateral eye and Ad5.GFP served as controls. Daytime conscious IOPs were measured twice per week. Aqueous outflow facility (C) was measured by constant flow infusion on completion of IOP measurements. Immunohistochemistry was performed on BALB/cJ mouse eyes to study TGM2 expression and activity. Results The treatment of cultured TM cells with Ad5.TGM2 increased immunostaining of N-e(γ-glutamyl) lysine crosslinks. Ad5.TGM2 injection significantly increased IOP in BALB/cJ (15.86 mm Hg [injected] vs. 10.70 mm Hg [control]) and in C57BL/6J mice (17.09 mm Hg [injected] vs. 12.01 mm Hg [control]). Mean aqueous outflow facility in the injected eyes of BALB/cJ (0.013 μL/min/mm Hg) and C57BL/6J mice (0.012 μL/min/mm Hg) was significantly lower than in the uninjected control eyes (BALB/cJ, 0.021 μL/min/mm Hg; C57BL/6J, 0.019 μL/min/mm Hg). The Ad5.TGM2 transduction of mouse eyes increased TGM2 expression in the TM region and increased N-e(γ-glutamyl) lysine crosslinks. Conclusions The increased expression of TGM2 in the TM increases N-e(γ-glutamyl) lysine crosslinking in the TM, increases aqueous outflow resistance, and elevates IOP in mice. TGM2 may be at least partially responsible for ocular hypertension in POAG.

Glaucoma is a vision threatening optic neuropathy that affects millions of people worldwide. In primary open angle, increased intraocular pressure (IOP) is the main risk factor for the development of this disease. Studies investigating the causes and mechanisms of increased IOP show fibrotic changes in the trabecular meshwork (TM) that are different from those of age-matched controls. Tissue transglutaminase (TGM2), an extracellular matrix (ECM) crosslinking enzyme, covalently crosslinks ECM proteins and causes excessive ECM protein deposition in the TM that could cause increased IOP. Previous literature reports increased expression of TGM2 in glaucomatous eyes compared to controls. We recently have shown that overexpression of TGM2 causes increased ECM crosslinking in the TM, increases IOP, and decreases aqueous humor (AH) outflow facility in mouse eyes. Therefore, we wanted to study the effect of TGM2 knockout (KO) on IOP in TGM2 floxed mice. Ad5.Cre transduction caused partial KO of TGM2, which decreased TGM2 expression in the TM region of mouse eyes. TGM2 KO significantly decreased IOP by itself and also in TGFβ2 induced ocular hypertensive mice. TGM2 KO also restores the outflow facility in TGFβ2 transduced eyes. Overall, TGM2 KO rescued the TGFβ2-induced ocular hypertensive phenotype. Thus, TGM2 may offer potential as a new therapeutic target for glaucoma.

Significance A mutation in myocilin is the most common known genetic cause of primary open-angle glaucoma (POAG). These mutations, which are dominant in nature, affect trabecular meshwork (TM) health and/or function and cause elevated intraocular pressure. Using in vitro human trabecular meshwork cells, an in vivo mouse model, and ex vivo human eyes, our study demonstrates the potential of clustered regularly interspaced short palindromic repeats (CRISPR)-mediated genome editing in human myocilin-associated POAG. By disrupting the mutant myocilin gene and its function using CRISPR-Cas9, we were able to reduce associated endoplasmic reticulum stress, lower intraocular pressure, and prevent further glaucomatous damage in mouse eyes. We also show the feasibility of using the CRISPR-Cas9 system in cultured human eyes.

Increased synthesis and deposition of extracellular matrix (ECM) proteins in the trabecular meshwork (TM) is associated with TM dysfunction and intraocular pressure (IOP) elevation in glaucoma. However, it is not understood how ECM accumulation leads to TM dysfunction and IOP elevation. Using a mouse model of glucocorticoid (GC)-induced glaucoma, primary human TM cells and human post-mortem TM tissues, we show that increased ECM accumulation leads to endoplasmic reticulum (ER) stress in the TM. The potent GC, dexamethasone (Dex) increased the secretory protein load of ECM proteins in the ER of TM cells, inducing ER stress. Reduction of fibronectin, a major regulator of ECM structure, prevented ER stress in Dex-treated TM cells. Overexpression of fibronectin via treatment with cellular fibronectin also induced chronic ER stress in primary human TM cells. Primary human TM cells grown on ECM derived from Dex-treated TM cells induced ER stress markers. TM cells were more prone to ER stress from ECM accumulation compared to other ocular cell types. Moreover, increased co-localization of ECM proteins with ER stress markers was observed in human post-mortem glaucomatous TM tissues. These data indicate that ER stress is associated with increased ECM accumulation in mouse and human glaucomatous TM tissues.

Mice are now routinely utilized in studies of aqueous humor outflow dynamics. In particular, conventional aqueous outflow facility (C) is routinely measured via perfusion of the aqueous chamber by a number of laboratories. However, in mouse eyes perfused ex-vivo, values for C are variable depending upon whether the perfusate is introduced into the posterior chamber (PC) versus the anterior chamber (AC). Perfusion via the AC leads to posterior bowing of the iris, and traction on the iris root/scleral spur, which may increase C. Perfusion via the PC does not yield this effect. But the equivalent situation in living mice has not been investigated. We sought to determine whether AC versus PC perfusion of the living mouse eye may lead to different values for C. All experiments were conducted in C57BL/6J mice (all ♀) between the ages of 20 and 30 weeks. Mice were divided into groups of 3–4 animals each. In all groups, both eyes were perfused. C was measured in groups 1 and 2 by constant flow infusion (from a 50 μL microsyringe) via needle placement in the AC, and in the PC, respectively. To investigate the effect of ciliary muscle (CM) tone on C, groups 3 and 4 were perfused live via the AC or PC with tropicamide (muscarinic receptor antagonist) added to the perfusate at a concentration of 100 μM. To investigate immediate effect of euthanasia, groups 5 and 6 were perfused 15–30 min after death via the AC or PC. To investigate the effect of CM tone on C immediately following euthanasia, groups 7 and 8 were perfused 15–30 min after death via the AC or PC with tropicamide added to the perfusate at a concentration of 100 μM. C in Groups 1 (AC perfusion) and 2 (PC perfusion) was computed to be 19.5 ± 0.8 versus 21.0 ± 2.1 nL/min/mmHg, respectively (mean ± SEM, p > 0.4, not significantly different). In live animals in which tropicamide was present in the perfusate, C in Group 3 (AC perfusion) was significantly greater than C in Group 4 (PC perfusion) (22.0 ± 4.0 versus 14.0 ± 2.0 nL/min/mmHg, respectively, p = 0.0021). In animals immediately following death, C in groups 5 (AC perfusion) and 6 (PC perfusion) was computed to be 21.2 ± 2.0 versus 22.8 ± 1.4 nL/min/mmHg, respectively (mean ± SEM, p = 0.1196, not significantly different). In dead animals in which tropicamide was present in the perfusate, C in group 7 (AC perfusion) was greater than C in group 8 (PC perfusion) (20.6 ± 1.4 versus 14.2 ± 2.6 nL/min/mmHg, respectively, p in situ in living mice or euthanized animals within 15–30 min post mortem is not significantly different when measured via AC perfusion or PC perfusion. In eyes of live or freshly euthanized mice, C is greater when measured via AC versus PC perfusion when tropicamide (a mydriatic and cycloplegic agent) is present in the perfusate.

Sigma-1 receptor (σ-1) activation and mitogen-activated protein kinases (MAPKs) have been shown to protect retinal ganglion cells (RGCs) from cell death. The purpose of this study was to determine if σ-1 receptor stimulation with pentazocine could promote neuroprotection under conditions of an ischemia-like insult (oxygen glucose deprivation (OGD)) through the phosphorylation of extracellular signal regulated kinase (pERK)1/2. Primary RGCs were isolated from P3-P7 Sprague-Dawley rats and purified by sequential immunopanning using Thy1.1 antibodies. RGCs were cultured for 7 days before subjecting the cells to an OGD insult (0.5% oxygen in glucose-free medium) for 6 h. During the OGD, RGCs were treated with pentazocine (σ-1 receptor agonist) with or without BD 1047 (σ-1 receptor antagonist). In other experiments, primary RGCs were treated with pentazocine in the presence or absence of an MEK1/2 inhibitor, PD098059. Cell survival/death was assessed by staining with the calcein-AM/ethidium homodimer reagent. Levels of pERK1/2, total ERK1/2, and beta tubulin expression were determined by immunoblotting and immunofluorescence staining. RGCs subjected to OGD for 6 h induced 50% cell death in primary RGCs (p 0.001) and inhibited pERK1/2 expression by 65% (p 0.001). Cell death was attenuated when RGCs were treated with pentazocine under OGD (p 0.001) and pERK1/2 expression was increased by 1.6 fold (p 0.05) compared to OGD treated RGCs without pentazocine treatment. The co-treatment of PD098059 (MEK1/2 inhibitor) with pentazocine significantly abolished the protective effects of pentazocine on the RGCs during this OGD insult. Activation of the σ-1 receptor is a neuroprotective target that can protect RGCs from an ischemia-like insult. These results also established a direct relationship between σ-1 receptor stimulation and the neuroprotective effects of the ERK1/2 pathway in purified RGCs subjected to OGD. These findings suggest that activation of the σ-1 receptor may be a therapeutic target for neuroprotection particularly relevant to ocular neurodegenerative diseases that effect RGCs.

Administration of glucocorticoids induces ocular hypertension in some patients. If untreated, these patients can develop a secondary glaucoma that resembles primary open-angle glaucoma (POAG). The underlying pathology of glucocorticoid-induced glaucoma is not fully understood, due in part to lack of an appropriate animal model. Here, we developed a murine model of glucocorticoid-induced glaucoma that exhibits glaucoma features that are observed in patients. Treatment of WT mice with topical ocular 0.1% dexamethasone led to elevation of intraocular pressure (IOP), functional and structural loss of retinal ganglion cells, and axonal degeneration, resembling glucocorticoid-induced glaucoma in human patients. Furthermore, dexamethasone-induced ocular hypertension was associated with chronic ER stress of the trabecular meshwork (TM). Similar to patients, withdrawal of dexamethasone treatment reduced elevated IOP and ER stress in this animal model. Dexamethasone induced the transcriptional factor CHOP, a marker for chronic ER stress, in the anterior segment tissues, and Chop deletion reduced ER stress in these tissues and prevented dexamethasone-induced ocular hypertension. Furthermore, reduction of ER stress in the TM with sodium 4-phenylbutyrate prevented dexamethasone-induced ocular hypertension in WT mice. Our data indicate that ER stress contributes to glucocorticoid-induced ocular hypertension and suggest that reducing ER stress has potential as a therapeutic strategy for treating glucocorticoid-induced glaucoma.

Glucocorticoid (GC)-induced ocular hypertension (OHT) is a serious side effect of GC therapy in susceptible individuals. This OHT is due to increased aqueous humor (AH) outflow resistance in the trabecular meshwork (TM) caused by GC-mediated changes in TM structure and function. GCs may also play a role in the development of primary open-angle glaucoma (POAG). Elevated cortisol levels in the AH or enhanced GC sensitivity may be one of the reasons for elevated intraocular pressure in POAG patients. The GC OHT responder population is at greater risk of developing POAG compared with non-responders. We recently have gained insight into the molecular mechanisms responsible for this differential GC responsiveness, which is attributed to differences in GC receptor isoform expression in the TM. This article summarizes current knowledge on alternative GC receptor splicing to generate GC receptor alpha (GRα) and GRβ and their roles in the regulation of GC responsiveness in normal and glaucoma TM.

Primary open-angle glaucoma (POAG) is the second leading cause of blindness worldwide. Elevated intraocular pressure (IOP) is a primary risk factor associated with POAG. Increased aqueous humor (AH) outflow resistance through the trabecular meshwork (TM) results in elevated IOP in POAG patients. Resistance to AH outflow is associated with increased accumulation of extracellular matrix (ECM) proteins in the TM. In addition, levels of transforming growth factor-beta2 (TGF-β2) are elevated in the AH and TM tissue of POAG patients. Elevated levels of TGF-β2 in other tissues have been associated with fibrosis and increased tissue stiffness. However, locally produced effectors that maintain homeostatic relationships must also be present. Bone morphogenetic proteins (BMPs) serve this purpose in the TM as they inhibit TGF-β2-induced ECM changes in TM cells. This review article first describes the TGF-β superfamily of growth factors including BMPs and their canonical and noncanonical signaling pathways. The article then addresses the role of TGF-β2 in the pathophysiology of POAG as related to the ECM and ECM crosslinking enzymes. This is followed by a discussion of potential homeostatic control mechanisms of TGF-β2 signaling in the TM including the inhibitory role of BMP-4 and BMP-7. We then describe the relationship of TGF-β2 and BMPs in TM fibrosis including the role of antagonists. Lastly, in future directions, we identify potential future studies that explore new and unique cellular interactions within the TM for potential therapeutic interventions.

Glaucoma is a neurodegenerative disease with elevated intraocular pressure as one of the major risk factors. Glaucoma leads to irreversible loss of vision and its progression involves optic nerve head cupping, axonal degeneration, retinal ganglion cell (RGC) loss, and visual field defects. Despite its high global prevalence, glaucoma still remains a major neurodegenerative disease. Introduction of mouse models of experimental glaucoma has become integral to glaucoma research due to well-studied genetics as well as ease of manipulations. Many established inherent and inducible mouse models of glaucoma are used to study the molecular and physiological progression of the disease. One such model of spontaneous mutation is the nee model, which is caused by mutation of the Sh3pxd2b gene. In both humans and mice, mutations disrupting function of the SH3PXD2B adaptor protein cause a developmental syndrome including secondary congenital glaucoma. The purpose of this study was to characterize the early onset nee glaucoma phenotype on the C57BL/6J background and to evaluate the pattern of RGC loss and axonal degeneration in specific RGC subtypes. We found that the B6.Sh3pxd2b(nee) mutant animals exhibit glaucoma phenotypes of elevated intraocular pressure, RGC loss and axonal degeneration. Moreover, the non-image forming RGCs survived longer than the On-Off direction selective RGCs (DSGC), and the axonal death in these RGCs was independent of their respective RGC subtype. In conclusion, through this study we characterized an experimental model of early onset glaucoma on a C57BL/6J background exhibiting key glaucoma phenotypes. In addition, we describe that RGC death has subtype-specific sensitivities and follows a specific pattern of cell death under glaucomatous conditions.

TGFβ2 induces extracellular matrix (ECM) remodeling and alters the cytoskeleton by both the canonical Smad and non-canonical signaling pathways. TGFβ2 regulates the expression of ECM proteins in trabecular meshwork (TM) cells, increases intraocular pressure (IOP) in an ex vivo perfusion organ culture model, and induces ocular hypertension in rodent eyes. A necessary step in the canonical Smad signaling pathway is phosphorylation of receptor protein Smad3 by the TGF-β receptor complex. The purpose of this study was to determine whether TGFβ2 signals in vivo through the canonical Smad signaling pathway in the TM using Smad3 knockout (KO) mice. Ad5.hTGFβ2(226/228) (2.5 × 10(7) pfu) was injected intravitreally into one eye of homozygous (WT), heterozygous (HET), and homozygous (KO) 129-Smad3(tm1Par)/J mice (n = 9-10 mice/group), with the uninjected contralateral eye serving as the control. IOP measurements were taken using a rebound tonometer. To test the effect of TGFβ2 signaling on the ECM, fibronectin expression was determined by immunohistochemistry and qPCR analysis. Transduction of the TM with viral vector Ad5.hTGFβ2(226/228) caused a statistically significant difference in IOP exposure between Smad3 genotypes: WT, 187.7 ± 23.9 mmHg*day (n = 9); HET, 95.6 ± 24.5 mmHg*day (n = 9); KO, 52.8 ± 25.2 mmHg*day (n = 10); (p 0.05 WT versus HET, p 0.01 WT versus KO). Immunohistochemistry and qPCR analysis showed that Ad5.hTGFβ2(226/228) increased fibronectin expression in the TM of WT mice (2.23 ± 0.24 fold) compared to Smad3 KO mice (0.99 ± 0.19 fold), p 0.05. These results demonstrate Smad3 is a necessary signaling protein for TGFβ2-induced ocular hypertension and fibronectin deposition in the TM.

Glucocorticoid (GC)-induced ocular hypertension (OHT) is a serious adverse effect of prolonged GC therapy that can lead to iatrogenic glaucoma and permanent vision loss. An appropriate mouse model can help us understand precise molecular mechanisms and etiology of GC-induced OHT. We therefore developed a novel, simple, and reproducible mouse model of GC-induced OHT. GC-induced myocilin expression in the trabecular meshwork (TM) has been suggested to play an important role in GC-induced OHT. We further determined whether myocilin contributes to GC-OHT. C57BL/6J mice received weekly periocular conjunctival fornix injections of a dexamethasone-21-acetate (DEX-Ac) formulation. Intraocular pressure (IOP) elevation was relatively rapid and significant, and correlated with reduced conventional outflow facility. Nighttime IOPs were higher in ocular hypertensive eyes compared to daytime IOPs. DEX-Ac treatment led to increased expression of fibronectin, collagen I, and α-smooth muscle actin in the TM in mouse eyes. No changes in body weight indicated no systemic toxicity associated with DEX-Ac treatment. Wild-type mice showed increased myocilin expression in the TM on DEX-Ac treatment. Both wild-type and Myoc−/− mice had equivalent and significantly elevated IOP with DEX-Ac treatment every week. In conclusion, our mouse model mimics many aspects of GC-induced OHT in humans, and we further demonstrate that myocilin does not play a major role in DEX-induced OHT in mice.

One of the major causes of decreased vision, irreversible vision loss and blindness worldwide is glaucoma. Increased intraocular pressure (IOP) is a major risk factor associated with glaucoma and its molecular mechanisms are not fully understood. The trabecular meshwork (TM) is the primary site of injury in glaucoma, and its dysfunction results in elevated IOP. The glaucomatous TM has increased extracellular matrix deposition as well as cytoskeletal rearrangements referred to as cross-linked actin networks (CLANs) that consist of dome like structures consisting of hubs and spokes. CLANs are thought to play a role in increased aqueous humor outflow resistance and increased IOP by creating stiffer TM cells and tissue. CLANs are inducible by glucocorticoids (GCs) and TGFβ2 in confluent TM cells and TM tissues. The signaling pathways of these induction agents give insight into the possible mechanisms of CLAN formation, but to date, the mechanism of CLANs regulation by these pathways has yet to be determined. Understanding the role CLANs play in IOP elevation and their mechanisms of induction and regulation may lead to novel treatment options to help prevent or intervene in glaucomatous damage to the trabecular meshwork.

Glaucoma is a major cause of blindness in China and the world. Currently, all therapeutic means in treating open-angle glaucoma are limited to control the progression of optic neuropathy by lowering intraocular pressure (IOP). Clinically available medicines lower IOP by either enhancing the uveoscleral pathway or inhibiting aqueous humor production. Since the primary cause of IOP elevation in POAG is elevated outflow resistance in the trabecular outflow pathway, current medicines are not able to correct the underlying pathogenesis and pathophysiology of the disease. In this review article, we discuss a series of new therapeutic targets and therapeutic approaches that are designed to directly modify the pathological changes related to the reduction in trabecular outflow in glaucoma patients. Some of these targets and approaches may produce a significant breakthrough in the treatment of this devastating disease. (Chin J Ophthalmol, 2016, 52: 471-475).

Oxidative injuries, such as those related to reactive oxygen species (ROS), have been implicated in various retinal and optic nerve disorders. Many ROS detection methods have been developed. Although widely utilized, many of these methods are useful only in post mortem tissues, or require relatively expensive equipment, or involve intraocular injection. In the present study, we demonstrated and characterized a chemiluminescent probe L-012 as a noninvasive, in vivo ROS detection agent in the mouse retina. Using optic nerve crush (ONC) and retinal ischemia/reperfusion (I/R) as mouse injury models, we show that L-012 produced intensive luminescent signals specifically in the injured eyes. Histological examination showed that L-012 administration was safe to the retina. Additionally, compounds that reduce tissue superoxide levels, apocynin and TEMPOL, decreased injury-induced L-012 chemiluminescence. The decrease in L-012 signals correlated with their protective effects against retinal I/R-induced morphological and functional changes in the retina. Together, these data demonstrate the feasibility of a fast, simple, reproducible, and non-invasive detection method to monitor in vivo ROS in the retina. Furthermore, the results also show that reduction of ROS is a potential therapeutic approach for protection from these retinal injuries.

Background The c-Jun N-terminal kinase (JNK) signaling pathway plays an important role in neuronal pathophysiology. Using JNK inhibitors, we examined involvement of the JNK pathway in cultured rat retinal ganglion cell (RGC) death and in mouse retinal ischemia/reperfusion (I/R) injury of the visual axis. The in vitro effects of JNK inhibitors were evaluated in cultured adult rat retinal cells enriched in RGCs. Retinal I/R was induced in C57BL/6J mice through elevation of intraocular pressure to 120 mmHg for 60 min followed by reperfusion. SP600125 was administered intraperitoneally once daily for 28 days. Phosphorylation of JNK and c-Jun in the retina was examined by immunoblotting and immunohistochemistry. The thickness of retinal layers and cell numbers in the ganglion cell layer (GCL) were examined using H&E stained retinal cross sections and spectral domain optical coherence tomography (SD-OCT). Retinal function was measured by scotopic flash electroretinography (ERG). Volumetric measurement of the superior colliculus (SC) as well as VGLUT2 and PSD95 expression were studied. Results JNK inhibitors SP600125 and TAT-JNK-III, dose-dependently and significantly (p