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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.

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

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.

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) 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.

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.

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.

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.

Elevated intraocular pressure (IOP) is a causative risk factor for the development and progression of glaucoma. Glaucomatous mutations in myocilin (MYOC) damage the trabecular meshwork and elevate IOP in humans and in mice. Animal models of glaucoma are important to discover and better understand molecular pathogenic pathways and to test new glaucoma therapeutics. Although a number of different animal models of glaucoma have been developed and characterized, there are no true models of human primary open angle glaucoma (POAG). The overall goal of this work is to develop the first inducible mouse model of POAG using a human POAG relevant transgene (i.e. mutant MYOC) expression in mouse eyes to elevate IOP and cause pressure-induced damage to the optic nerve. Four mouse strains (A/J, BALB/cJ, C57BL/6J, and C3H/HeJ) were used in this study. Ad5.MYOC.Y437H (5 × 10(7) pfu) was injected intravitreally into one eye, with the uninjected contralateral eye serving as the control eye. Conscious IOP measurements were taken using a TonoLab rebound tonometer. Optic nerve damage was determined by scoring PPD stained optic nerve cross sections. Retinal ganglion cell and superior colliculus damage was assessed by Nissl stain cell counts. Intravitreal administration of viral vector Ad5.MYOC.Y437H caused a prolonged, reproducible, and statistically significant IOP elevation in BALB/cJ, A/J, and C57BL/6J mice. IOPs increased to approximately 25 mm Hg for 8 weeks (p < 0.0001). In contrast, the C3H/HeJ mouse strain was resistant to Ad5.MYOC.Y437H induced IOP elevation for the 8-week time period. IOPs were stable (12-15 mm Hg) in the uninjected control eyes. We also determined whether there were any strain differences in pressure-induced optic nerve damage. Even though IOP was similarly elevated in three of the strains tested (BALB/cJ, C57BL/6J, and A/J) only the A/J strain had considerable and significant optic nerve damage at the end of 8 weeks with optic nerve damage score of 2.64 ± 0.19 (n = 18, p < 0.001) in the injected eye. There was no statistical difference in retinal ganglion cell death or superior colliculus damage at the 8-week time point in any of the strains tested. These results demonstrate strain dependent responses to Ad5.MYOC.Y437H-induced ocular hypertension and pressure-induced optic nerve damage.

Glaucoma is a neurodegenerative disease characterized by the apoptotic death of retinal ganglion cells (RGCs). The primary insult to RGCs in glaucoma is thought to occur to their axons as they exit the eye in the optic nerve head. However, pathological signaling pathways that exert central roles in triggering RGC death following axonal injury remain unidentified. It is likely that the first changes to occur following axonal injury are signal relay events that transduce the injury signal from the axon to the cell body. Here we focus on the c-Jun N-terminal kinase (JNK1–3) family, a signaling pathway implicated in axonal injury signaling and neurodegenerative apoptosis, and likely to function as a central node in axonal injury-induced RGC death. We show that JNK signaling is activated immediately after axonal injury in RGC axons at the site of injury. Following its early activation, sustained JNK signaling is observed in axonally-injured RGCs in the form of JUN phosphorylation and upregulation. Using mice lacking specific Jnk isoforms, we show that Jnk2 and Jnk3 are the isoforms activated in injured axons. Combined deficiency of Jnk2 and Jnk3 provides robust long-term protection against axonal injury-induced RGC death and prevents downregulation of the RGC marker, BRN3B, and phosphorylation of JUN. Finally, using Jun deficient mice, we show that JUN-dependent pathways are important for axonal injury-induced RGC death. Together these data demonstrate that JNK signaling is the major early pathway triggering RGC death after axonal injury and may directly link axon injury to transcriptional activity that controls RGC death.

Glucocorticoid treatment can lead to the development of glaucomatous ocular hypertension and a secondary open-angle glaucoma due to increased aqueous humor outflow resistance that is associated with morphological and biochemical changes in the trabecular meshwork (TM). The cellular responses of glucocorticoids are achieved by binding to the glucocorticoid receptor alpha (GRalpha), a ligand-activated transcription factor. An alternatively spliced variant, glucocorticoid receptor beta (GRbeta), has dominant negative activity on GRalpha and has been implicated in a variety of steroid-resistant diseases. We previously showed that GRbeta can block dexamethasone (DEX) responsiveness in TM cells. TM cells are actively phagocytic and function in the removal of debris, pigment and other materials from the aqueous outflow drainage pathway. A decrease in phagocytic activity has been proposed in the pathogenesis of glaucoma and glucocorticoid-induced glaucoma. In this study, we investigated the effect of DEX and GRbeta on phagocytosis in normal and glaucomatous TM cells. Human transformed normal NTM-5 and primary normal NTM174-00 cells, which express relatively high amounts of GRbeta, and transformed glaucomatous GTM-3 and primary glaucomatous GTM520-05 cells, which have lower GRbeta expression, were treated with 100 nM DEX or vehicle control for 24h. NTM cells also were transfected with a control or GRbeta expression plasmid to examine the effect of GRbeta on phagocytic activity. The cells were incubated with Alexa 488 conjugated Staphylococcus aureus bioparticles opsonized with rabbit IgG for 1h, followed by fixation and incubation with Alexa 633 conjugated goat anti-rabbit IgG to distinguish ingested from extracellular bioparticles. DAPI nuclear staining was used to quantify cell numbers. Cells and bioparticles were visualized by confocal microscopy. We found that NTM-5 cells ingested more bioparticles than GTM-3 cells. DEX treatment significantly decreased the phagocytosis of bioparticles in NTM-5 and GTM-3 cells, while GTM-3 cells were more responsive to DEX, compared to NTM-5 cells. In primary cell culture, NTM174-00 also engulfed more bioparticles than GTM520-05 cells. DEX treatment significantly decreased the phagocytic activity in GTM520-05, but not in NTM174-00 cells. Transient transfection of pCMX-hGRbeta plasmid increased the expression of GRbeta and consequently maintained the phagocytotic activity of NTM-5 cells in the presence of DEX. Our data demonstrated that the expression level of GRbeta in TM cells can regulate DEX-induced suppression of phagocytotic activity. The lower expression of GRbeta in glaucomatous TM cells may contribute to the altered phagocytic function of TM cells, and may lead to the increased aqueous humor outflow resistance mediated by glucocorticoids.

Anecortave acetate is a unique ocular angiostatic cortisene that has broad-based anti-angiogenic activity in 14 different preclinical models of neovascularization, across multiple species and inducers of neovascularization. Anecortave acetate is being tested clinically for inhibition of choroidal neovascularization associated with age-related macular degeneration.

Glucocorticoid effects on the human trabecular meshwork can be used as a model system in which to study glaucomatous damage to the trabecular meshwork. One of the most important risk factors for glaucoma is an elevated intraocular pressure. The administration of glucocorticoids also can cause elevated intraocular pressure in some individuals. In addition, there is suggestive evidence linking glucocorticoids with the development of glaucoma. Glucocorticoids cause multiple effects on the human trabecular meshwork including changes in extracellular matrix metabolism, organisation of the cytoskeleton, and changes in gene expression and cell function. New discoveries on the molecular mechanisms of glucocorticoid receptor action provide new opportunities to study the possible role of this receptor in the development of glaucoma. For example, alternate spliced forms of the glucocorticoid receptor, glucocorticoid receptor response element halve-sites, numerous modulatory factors, and direct effects of nuclear transcription factors have been recently described. Other recent information has shown that the new glaucoma gene (GLC1A/myocilin) is induced in the human trabecular meshwork by glucocorticoids. Although the exact function of myocilin is currently unknown, it offers the opportunity to dissect the molecular pathways regulating aqueous humor outflow. Future challenges include determining (1) which glucocorticoid effects in the human trabecular meshwork are responsible for elevated intraocular pressure; and (2) the significance of these findings to the development of glaucoma.

Glucocorticoid treatment in vivo can produce a glaucoma similar in many ways to POAG. Treatment of trabecular meshwork cells in culture with dexamethasone allows the study of biochemical aspects of this disease process. The effects of dexamethasone on the expression of integrins and laminin in both normal and glaucomatous cultured human trabecular meshwork cells were evaluated. Human trabecular meshwork cell lines were cultured for 18 days in the presence or absence of 10(-7) m dexamethasone. Radioimmunoprecipitation was used to determine the relative expression of five alphaintegrin subunits. Laminin expression was evaluated with Western blots. Laminin was increased in all cell lines following dexamethasone treatment. alpha2, alpha5 and alphaV integrin chains showed consistent dexamethasone-induced changes in expression, while alpha3 and alpha4 subunits did not. There were no differences in the expression patterns for any of these integrin subunits between normal and glaucomatous cell lines. Increased laminin deposition as seen in this study with dexamethasone treatment may be partially responsible for the decreased outflow facility seen in both steroid-induced glaucoma and in POAG.

The prevailing view regarding the mechanism of steroid cataract formation holds that glucocorticoids are covalently bound to lens proteins resulting in destabilization of the protein structure allowing further modification (i.e. oxidation) leading to cataract. Alternative hypotheses (e.g. that cataracts result from glucocorticoid receptor mediated effects) have been difficult to test since protein binding does in fact occur for many cataractogenic steroids. A glucocorticoid lacking the typical glucocorticoid hydroxy group at C21 (fluorometholone, FML), other steroids which can bind to proteins but lack glucocorticoid activity, and a glucocorticoid antagonist (RU486) have been utilized to discriminate between these two hypotheses. Purified bovine beta-crystallin incubated with three different 3H-steroids, dexamethasone (Dex), aldosterone or progesterone demonstrated that the C-21 hydroxyl group is not essential for steroid binding. Progesterone (with no C-21 OH) bound to the greatest extent. Pretreatment of the protein with aspirin to acetylate the free protein amino groups blocked this binding, demonstrating the probability of a Schiff base mechanism. Lens culture studies with the same three radiolabeled steroids demonstrated much the same result. Rat lenses cultured for 48 hr-11 days, demonstrated that loss of GSH is an early and significant effect of several glucocorticoids (Dex, prednisolone and FML) but is not seen with other non-glucocorticoid steroids. However, none of the steroids tested consistently produced lenticular opacification (i.e. cataracts) in this in vitro system, nor did they alter rubidium transport. We suggest that a mechanism other than covalent binding of steroids to lens proteins is responsible for glucocorticoid induced cataracts because: (1) non-glucocorticoids were demonstrated to bind lens proteins as well or better than the glucocorticoid Dex and (2) only glucocorticoids, and not other steroids, lowered lens reduced glutathione content which has been demonstrated to be associated with other forms of cataract.

Previous studies using cultured bovine trabecular meshwork cells demonstrated at least two different cell types which were distinguishable morphologically and electrophysiologically. The purpose of the present study was to evaluate the electrical membrane properties of cultured human trabecular meshwork cells. Seven different human trabecular meshwork cell lines were grown from four different donors. One trabecular meshwork cell line was transformed by microinjection with SV40 DNA. The electrochemical properties of these TM cells were determined by micropuncture with glass microelectrodes. The mean membrane voltage at resting conditions differed between the cell lines (- 33·3 to - 58·7 mV). Application of 10-mmol l -1 Ba 2+ induced repetitive voltage spikes in all cell lines. The voltage transients similar to action potentials were inhibited by nifedipine, but insensitive to tetrodotoxin. Acetylcholine evoked depolarizations in three cell lines which were blocked by atropine. In one cell line isoproterenol caused sustained depolarizations sensitive to metipranolol. All three of the cell lines tested depolarized upon application of the vasoactive peptide endothelin-1. All untransformed cell lines showed voltage spikes typical for smooth muscle cells and functional receptors for endothelin-1 and cholinergic agonists. One out of three cell lines tested possessed β-adrenergic receptors influencing the membrane voltage.

There is considerable interest in the discovery of compounds which inhibit angiogenesis dependent (neovascular) diseases. The chick embryo, due to the rapid development of an extensive vascular capillary network in the chorioallantoic membrane (CAM), has been used extensively as a model for studying angiogenesis. Angiostatic steroids are a new class of compounds which inhibit the growth of new capillaries in the chick CAM and in other models of neovascularization. Despite the potential therapeutic importance of these compounds, little is known about the ability of the CAM to metabolize these steroids. We have evaluated the ability of the chick CAM to metabolize cortisol which is both an angiostatic steroid as well as a glucocorticoid. When CAM homogenate was incubated with [3H]cortisol and NADPH at 37 degrees C and pH 7.4, and the reaction products analyzed by reverse phase HPLC, [3H]cortisol was converted exclusively to 20 beta-dihydrocortisol (4-pregnen-11 beta,17 alpha,20 beta,21-tetrol-3-one). The cortisol metabolite, 20 beta-dihydrocortisol, has very little glucocorticoid activity, but shows significant angiostatic activity in the CAM comparable to cortisol. The apparent Km determined for cortisol metabolism was 12 microM and the observed Vmax was 1.4 mumol cortisol/mg protein/min. The majority of the 20 beta-reductase activity was found in the soluble (242,000 g) fraction of CAM homogenate. 20 beta-Reductase activity in chick embryo CAM has not been previously reported.

1. StructureLysyl Oxidase (LOX, also called protein-lysine 6-oxidase)(Genbank accession numbers: Nucleotide NM_002317.5, ProteinNP_002308.2, EC 1.4.3.13) is one of the five LOX family members(LOX and LOX Like1-4). The human LOX gene is located onchromosome 5q23.2. LOX is synthesized as a 50 kDa pre-proteincontaining 3 domains; the N-terminal signal peptide sequence(aa1-21), the N-terminal pro-peptide domain (aa 22-162), and theC-terminal catalytic domain (aa 162-417). The signal peptide isremoved by cleavage at the Cys21-Ala22 bond and the pro-peptidedomain is N-glycosylated in the endoplasmic reticulum and Golgiapparatus yielding a pro-enzyme, which is then secreted fromcells as a catalytically inactive protein. The 32 kDa active enzyme(C-terminal domain) is released by proteolytic cleavage of thepro-peptide at Gly162eAsp163 by procollagen C-proteinase (bonemorphogenetic protein 1; BMP-1) (Kagan and Li, 2003)(Fig. 1).2. FunctionLOX belongs to a family of amine oxidases. LOX was initiallyreported to be expressed andsecreted by fibrogenic cells but is nowknown to be expressed in several other cell types. LOX oxidizespeptidyl lysine and hydroxylysine residues in collagen and lysineresidues in elastin to produce peptidyl alpha-aminoadipic-delta-semialdehydes. These aldehyde modifications can spontaneouslycombine with vicinal peptidyl aldehydes or with epsilon-aminogroups of peptidyl lysine to form covalent cross-links that stabilizeand cause collagen and elastin fibers to be insoluble in the extra-cellular matrix (ECM). The significance of LOX was demonstrated inthe LOX knockout mouse, which could not survive at birth, due torupture of the aorta and diaphragm from incomplete elastin cross-linking. LOX is also essential for development of the distal andproximal airways, and the formation of alveoli in the lungs. LOX isalso critical for notochord formation and muscle development. LOXaffects differentiation of osteoblasts by forming cross-links in thesurrounding collagen matrix. These results underline the impor-tance of LOX in the morphogenesis and repair of connective tissuesof the cardiovascular, respiratory, skeletal, and other organ systems(Fongetal.,2009).LOXcanalsoinducecross-linkingbetweenlysinesin histones in the nucleus (Kagan and Li, 2003). LOX has also beenreported as a potent chemotactic agent for monocytes and vascularsmooth muscle cells.3. Disease involvementDuetotherangeofLOXbiologicalfunctions,abnormalitiesofLOXexpression underlie the development of a number of pathologicalprocesses related with an imbalance in ECM synthesis/degradation.LOX is expressed in several ocular tissues, and the followingdiseases of the eye have been linked with LOX:A. Keratoconus: Keratoconus is a corneal degeneration in whichthe extracellular matrix of the cornea loses its integrity, slowlychanging from the normal round shape to a cone shape. Onestudy reported that corneal LOX mRNA expression was signif-icantly lower in keratoconus patients when compared to theirage-matched controls. Another study showed that reducingcopper levels in tears results in keratoconus in an animalmodel. Copper is an essential cofactor for LOX activity.B. Diabetic retinopathy (DR): There are a number of reports oflower expression of LOX co-factors in DR retina samples andlower LOX cross-linking activity in vitreous samples of patientswith DR. However, theeffectofhigh glucose onLOXexpressionand enzymatic activity in cultured human retinal endothelialcells is less clear, with conflicting evidence suggesting eitherincreased or decreased LOX expression. Although discrepant,these studies indicate a potential involvement of LOX in theobserved DR phenotype.

Transglutaminase 2, also known as TGM2, tTG, TG2, TGC, and G-alpha-h (Accession numbers: Nucleotide NM_004613.2, Protein NP_004604.2; EC 2.3.2.13) has two isoforms due to alternative splicing: (a) has 13 exons encoding 687 amino acids (77kDa) and (b) has 10 exons encoding 548 amino acids (61.7kDA). The human transglutaminase 2 gene (TGM2) is located on chromosome 20q12. Transglutaminase 2 is secreted by an unknown non-conventional mechanism, since it lacks a signal sequence. Although potential glycosylation sites are present, no evidence of glycosylation has been reported. X-ray crystallography has revealed that TGM2 has four domains: an N-terminal β-sandwich domain (1-139), a transamidation core domain (140-454), and two C-terminal β-barrels (479-585 and 586-687, respectively) (Figure1). All four domains have particular roles. For example, the N-terminal domain interacts with fibronectin (Collighan RJ and Griffin M, 2009). The transamidation core domain is involved in GTP binding (Ser171 and Lys173), has an active triad site (Cys277, His335, and Asp358), and a calcium binding region. The Cterminal domain regulates transamidation activity, GTPase activity, as well as concealing the active site from contact with possible substrates. Upon calcium binding, the interaction between the active site and C-terminal domains is released, allowing transamidation enzyme activity.

R.R. Krishnamoorthy, P. Agarwal, G. Prasanna, K. Vopat, W. Lambert, H.J. Sheedlo, I.-H. Pang, D. Shade, R.J. Wordinger, T. Yorio, A.F. Clark, N. Agarwal Department of Pathology and Anatomy, UNT Health Science Center, 3500 Camp Bowie Blvd., Fort Worth, TX 76107, USA Texas Academy of Math and Science, UNT, Denton, TX, USA Department of Pharmacology, UNT Health Science Center, Fort Worth, TX, USA Alcon Research Ltd., Fort Worth, TX, USA

Purpose To describe the intraocular pressure (IOP)-lowering effects in eyes with open-angle glaucoma (OAG) after treatment with an anterior juxtascleral depot of anecortave acetate. Design Prospective, interventional case series. Methods Seven eyes of six subjects with OAG, with uncontrolled IOP while being administered one or more topical medications, received 24 mg anecortave acetate delivered by anterior juxtascleral depot. IOP was assessed at baseline and regularly after treatment for up to 24 months. Results Mean IOP before anecortave acetate treatment was 31.3 ± 11.3 mm Hg and dropped by 9.5 ± 4.5 mm Hg (32.7% ± 16.8%) within one week after treatment. This IOP reduction was sustained through six months (8.4 ± 5.4 mm Hg [29.6% ± 12.4%]) and 12 months (9.5 ± 5.7 mm Hg [34.0% ± 15.9%]) after a single anecortave acetate treatment. The injection process was well tolerated, and no eyes experienced any injection-related or drug-related serious adverse events. Conclusions Both the anterior juxtascleral depot of a drug and anecortave acetate may be promising candidates for IOP reduction in eyes with OAG. Additional studies are required to establish better their efficacy and safety, optimal dosing frequency, mechanism of action, and potential additivity to other IOP-lowering therapies.

We examined the synthesis of proteins in rat myocardium after starvation. Rates of total protein synthesis in myofibrillar and nonmyofibrillar fractions of myocardium of starved animals were reduced similarly (to 70-80% of the rates in hearts of fed animals, p less than 0.002), but rates of synthesis of some individual proteins were affected discoordinately. Radiolabeled proteins from atrial and ventricular explants, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealed that starvation for 2 days reduced the rate of cardiac actin synthesis to 26-38% of control levels, while the rate of myosin heavy chain synthesis in the same hearts was only moderately reduced (74-80% of control levels). This starvation-induced reduction in actin synthesis could be accounted for at least in part by disproportionately decreased levels of actin mRNA in starved hearts, as revealed by Northern blot hybridization and by in vitro translation analysis. The dramatic decrease in cardiac actin synthesis was rapidly reversible, and actin synthesis returned to normal after a single day of refeeding. The selective reduction of actin synthesis after starvation was specific for the heart: rates of myosin heavy chain and actin synthesis in skeletal muscles (soleus and extensor digitorum longus) were coordinately reduced in response to starvation. To our knowledge, this is the first example of such dramatic discoordinate regulation of myofibrillar protein synthesis in response to a physiological stimulus.