Recombinant Human Clusterin Seals Damage to the Ocular Surface Barrier in a Mouse Model of Ophthalmic Preservative-Induced Epitheliopathy.

There is a significant unmet need for therapeutics to treat ocular surface barrier damage, also called epitheliopathy, due to dry eye and related diseases. We recently reported that the natural tear glycoprotein CLU (clusterin), a molecular chaperone and matrix metalloproteinase inhibitor, seals and heals epitheliopathy in mice subjected to desiccating stress in a model of aqueous-deficient/evaporative dry eye. Here we investigated CLU sealing using a second model with features of ophthalmic preservative-induced dry eye. The ocular surface was stressed by topical application of the ophthalmic preservative benzalkonium chloride (BAC). Then eyes were treated with CLU and sealing was evaluated immediately by quantification of clinical dye uptake. A commercial recombinant form of human CLU (rhCLU), as well as an rhCLU form produced in our laboratory, designed to be compatible with U.S. Food and Drug Administration guidelines on current Good Manufacturing Practices (cGMP), were as effective as natural plasma-derived human CLU (pCLU) in sealing the damaged ocular surface barrier. In contrast, two other proteins found in tears: TIMP1 and LCN1 (tear lipocalin), exhibited no sealing activity. The efficacy and selectivity of rhCLU for sealing of the damaged ocular surface epithelial barrier suggests that it could be of therapeutic value in treating BAC-induced epitheliopathy and related diseases.

GPR158 in the Visual System: Homeostatic Role in Regulation of Intraocular Pressure.

Purpose: GPR158 is a newly characterized family C G-protein-coupled receptor, previously identified in functional screens linked with biological stress, including one for susceptibility to ocular hypertension/glaucoma induced by glucocorticoid stress hormones. In this study, we investigated GPR158 function in the visual system. Methods: Gene expression and protein immunolocalization analyses were performed in mouse and human brain and eye to identify tissues where GPR158 might function. Gene expression was perturbed in mice, and in cultures of human trabecular meshwork cells of the aqueous outflow pathway, to investigate function and mechanism. Results:GPR158 is highly expressed in the brain, and in this study, we show prominent expression specifically in the visual center of the cerebral cortex. Expression was also observed in the eye, including photoreceptors, ganglion cells, and trabecular meshwork. Protein was also localized to the outer plexiform layer of the neural retina. Gpr158 deficiency in knockout (KO) mice conferred short-term protection against the intraocular pressure increase that occurred with aging, but this was reversed over time. Most strikingly, the pressure lowering effect of the acute stress hormone, epinephrine, was negated in KO mice. In contrast, no disruption of the electroretinogram was observed. Gene overexpression in cell cultures enhanced cAMP production in response to epinephrine, suggesting a mechanism for intraocular pressure regulation. Overexpression also increased survival of cells subjected to oxidative stress linked to ocular hypertension, associated with TP53 pathway activation. Conclusions: These findings implicate GPR158 as a homeostatic regulator of intraocular pressure and suggest GPR158 could be a pharmacological target for managing ocular hypertension.

Dynasore protects the ocular surface against damaging oxidative stress.

Water soluble "vital" dyes are commonly used clinically to evaluate health of the ocular surface; however, staining mechanisms remain poorly understood. Recent evidence suggests that sublethal damage stimulates vital dye uptake by individual living cells. Since cell damage can also stimulate reparative plasma membrane remodeling, we hypothesized that dye uptake occurs via endocytic vesicles. In support of this idea, we show here that application of oxidative stress to relatively undifferentiated monolayer cultures of human corneal epithelial cells stimulates both dye uptake and endocytosis, and that dye uptake is blocked by co-treatment with three different endocytosis inhibitors. Stress application to stratified and differentiated corneal epithelial cell cultures, which are a better model of the ocular surface, also stimulated dye uptake; however, endocytosis was not stimulated, and two of the endocytosis inhibitors did not block dye uptake. The exception was Dynasore and its more potent analogue Dyngo-4a, both small molecules developed to target dynamin family GTPases, but also having off-target effects on the plasma membrane. Significantly, while Dynasore blocked stress-stimulated dye uptake at the ocular surface of ex vivo mouse eyes when treatment was performed at the same time as eyes were stressed, it had no effect when used after stress was applied and the ocular surface was already damaged. Thus, Dynasore could not be working by inhibiting endocytosis. Employing cytotoxicity and western blotting assays, we went on to demonstrate an alternative mechanism. We show that Dynasore is remarkably protective of cells and their surface glycocalyx, preventing damage due to stress, and thus precluding dye entry. These unexpected and novel findings provide greater insight into the mechanisms of vital dye uptake and point the direction for future study. Significantly, they also suggest that Dynasore and its analogues might be used therapeutically to protect the ocular surface and to treat ocular surface disease.

Clusterin from human clinical tear samples: Positive correlation between tear concentration and Schirmer strip test results.

PURPOSE: To investigate the relationship between tear concentration of the homeostatic protein clusterin (CLU) and dry eye signs and symptoms, and to characterize tear CLU protein. METHODS: Two independent studies were conducted, one in Tucson (44 subjects), the other in Los Angeles (52 subjects). A cohort study design was employed to enroll patients without regard to dry eye diagnosis. Dry eye signs and symptoms were assessed using clinical tests. Tear samples were collected by Schirmer strip, and also by micropipette at slit lamp when possible. CLU from both sample types was quantified by immunoassay. The relationship between CLU concentration and clinical test scores was determined by Pearson's correlation coefficient (for individual eyes) and multiple linear regression analysis (including both eyes). CLU was also evaluated biochemically by western blotting. RESULTS: In the Tucson cohort, a positive correlation was observed between tear CLU concentration and results of the Schirmer strip test, a measure of tear flow (p = 0.021 includes both eyes). This result was corroborated in the Los Angeles cohort (p = 0.013). The mean tear CLU concentration was 31 ±â€¯14 µg/mL (n = 18 subjects, 33 eyes; range = 7-48 µg/mL). CLU from clinical tear samples appeared biochemically similar to CLU from a non-clinical tear sample and from blood plasma. CONCLUSIONS: Results support the hypothesis that an optimal concentration of tear CLU is important for ocular surface health, and that this drops below the effective threshold in dry eye. Tear CLU measurement might identify patients that could benefit from supplementation. Information about concentration will aid development of therapeutic dosage parameters.

PARP-1/PAR Activity in Cultured Human Lens Epithelial Cells Exposed to Two Levels of UVB Light.

This study investigated poly(ADP-ribose) polymerase-1 (PARP-1) activation in cultured human lens epithelial cells exposed to two levels of UVB light (312 nm peak wavelength), 0.014 and 0.14 J cm-2 ("low" and "high" dose, respectively). At the low dose, PARP-1 and poly(ADP-ribose) (PAR) polymers acted to repair DNA strand breaks rapidly with no subsequent major effects on either cell morphology or viability. However, following the high UVB dose, there was a dramatic second phase of PARP-1 activation, 90 min later, which included a sudden reappearance of DNA strand breaks, bursts of reactive oxygen species (ROS) formation within both the mitochondria and nucleus, a translocation of PAR from the nucleus to the mitochondria and an ultimate 70% loss of cell viability occurring after 24 h. The results provide evidence for an important role for PARP-1 in protecting the human lens epithelium against low levels of UVB light, and possibly participating in the triggering of cell death following exposure to toxic levels of radiation.

Down-Regulation of RNA 3'-Terminal Phosphate Cyclase Attenuates Toll-Like Receptor 3-Mediated Axonal Loss in the Retina and Optic Nerve.

PURPOSE: To investigate the role of RNA 3'-terminal phosphate cyclase (Rtca) in Toll-like receptor 3 (TLR3)-mediated loss of retinal ganglion cells (RGCs) and their axons. METHODS: Polyinosinic-polycytidylic acid (Poly[I:C]) or PBS was injected into the vitreous humor of C57BL/6J and Tlr3 knockout mice. C57BL/6J mouse eyes were treated with Rtca silencing RNA or control RNA, with or without PBS or Poly(I:C). At 24, 48, and 72 hours after treatments, RGC loss was determined with the brain-specific homeobox/POU domain protein 3a antibody, and axonal loss was assessed by using the neuronal class III beta-tubulin (Tuj1) antibody. Axonal loss in the optic nerves was determined by anterograde-labeling of Cholera Toxin B. Western blot assays were performed to determine TLR3, Rtca, c-jun N-terminal kinase 3 (JNK3), and phospho-JNK3 (pJNK3) levels, and immunohistochemistry assays were performed to determine the cells that synthesize Rtca. RESULTS: Poly(I:C) significantly up-regulated the protein levels of TLR3, Rtca, JNK3, and pJNK3 in the retina. Rtca levels were increased in RGCs, and an increase in Rtca levels promoted significant loss of RGCs and their axons. In Tlr3 knockout mouse retinas, Poly(I:C) failed to elevate Rtca, JNK3, and pJNK3 protein levels and did not promote significant axonal loss. Also, Rtca silencing RNA down-regulated Rtca, JNK3, and pJNK3 in C57BL/6J mouse retinas, and down-regulation of Rtca attenuated Poly(I:C)-mediated loss of RGCs and their axons. CONCLUSIONS: The results presented in this study show that the activation of TLR3 promotes the loss of RGCs and their axons by elevating Rtca levels in the retina. Also, the results presented in this study show that Rtca regulates JNK3 expression in the retina.

Tissue and urokinase plasminogen activators instigate the degeneration of retinal ganglion cells in a mouse model of glaucoma.

Elevated intraocular pressure (IOP) promotes the degeneration of retinal ganglion cells (RGCs) during the progression of Primary Open-Angle Glaucoma (POAG). However, the molecular mechanisms underpinning IOP-mediated degeneration of RGCs remain unclear. Therefore, by employing a mouse model of POAG, this study examined whether elevated IOP promotes the degeneration of RGCs by up-regulating tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA) in the retina. IOP was elevated in mouse eyes by injecting fluorescent-microbeads into the anterior chamber. Once a week, for eight weeks, IOP in mouse eyes was measured by using Tono-Pen XL. At various time periods after injecting microbeads, proteolytic activity of tPA and uPA in retinal protein extracts was determined by fibrinogen/plasminogen zymography assays. Localization of tPA and uPA, and their receptor LRP-1 (low-density receptor-related protein-1) in the retina was determined by immunohistochemistry. RGCs' degeneration was assessed by immunostaining with antibodies against Brn3a. Injection of microbeads into the anterior chamber led to a progressive elevation in IOP, increased the proteolytic activity of tPA and uPA in the retina, activated plasminogen into plasmin, and promoted a significant degeneration of RGCs. Elevated IOP up-regulated tPA and LRP-1 in RGCs, and uPA in astrocytes. At four weeks after injecting microbeads, RAP (receptor associated protein; 0.5 and 1.0 µM) or tPA-Stop (1.0 and 4.0 µM) was injected into the vitreous humor. Treatment of IOP-elevated eyes with RAP led to a significant decrease in proteolytic activity of both tPA and uPA, and a significant decrease in IOP-mediated degeneration of RGCs. Also, treatment of IOP-elevated eyes with tPA-Stop decreased the proteolytic activity of both tPA and uPA, and, in turn, significantly attenuated IOP-mediated degeneration of RGCs. Results presented in this study provide evidence that elevated IOP promotes the degeneration of RGCs by up-regulating the levels of proteolytically active tPA and uPA.

Activation of TLR3 promotes the degeneration of retinal ganglion cells by upregulating the protein levels of JNK3.

PURPOSE: To investigate whether activation of Toll-like receptor 3 (TLR3) promotes the degeneration of retinal ganglion cells (RGCs) by upregulating the protein levels of c-jun N-terminal kinase 3 (JNK3). METHODS: Toll-like receptor 3-specific activator, Poly(I:C) (polyinosinic-polycytidylic acid), or PBS was injected into the vitreous humor of Thy1-YFP mice. At 24, 48, and 72 hours after treatments, degeneration of RGCs was assessed by using antibodies against brain-specific homeobox/POU domain protein 3a (Brn3a). A TLR3-specific inhibitor was injected into the vitreous humor with or without Poly(I:C). Western blot assays were performed to determine relative levels of TLR3, JNK3, pJNK3, and sterile alpha and HEAT/Armadillo motif-containing 1 (SARM1) proteins in retinal protein extracts, and immunohistochemistry assays were performed to determine their cellular localization in the retina. Mouse eyes were treated with Poly(I:C) or PBS along with MitoTracker Red, and colocalization of MitoTracker Red and JNK3 in the retinas was determined by using antibodies against JNK3. RESULTS: Poly(I:C) activated TLR3 and upregulated its downstream target protein JNK3 but not SARM1 in the retina. Poly(I:C) activated TLR3 and upregulated JNK3 specifically in RGCs and promoted a significant degeneration of RGCs over a 72-hour time period. Toll-like receptor 3 upregulated the levels of JNK3 protein in the cytoplasm of RGCs, but not in the mitochondria. Toll-like receptor 3-specific inhibitor downregulated Poly(I:C)-mediated upregulation of JNK3 protein, and, in turn, significantly attenuated TLR3-induced degeneration of RGCs. CONCLUSIONS: Results presented in this study show that the activation of TLR3 alone promotes the degeneration of RGCs by upregulating the protein levels of JNK3.

Excitotoxicity upregulates SARM1 protein expression and promotes Wallerian-like degeneration of retinal ganglion cells and their axons.

PURPOSE: This study investigated the role of sterile alpha/Armadillo/Toll-Interleukin receptor homology domain 1 protein (SARM1) in Wallerian-like degeneration of retinal ganglion cells (RGCs) and their axons after inducing excitotoxicity. METHODS: To induce excitotoxicity, kainic acid (KA) was injected into the vitreous humor of B6.Cg-Tg(Thy1-YFP)HJrs/J mice. Control mice received PBS. At 24, 48, and 72 hours after injection, degeneration of RGCs and their axons in the retina was determined by fundus imaging, and axonal degeneration in the optic nerves was determined by fluorescence microscopy. SARM1 protein levels were determined by Western blot analysis and SARM1 tissue localization was determined by immunohistochemistry. Causal role of SARM1 in KA-mediated degeneration of RGCs and their axons was determined by treating the eyes with KA along with Sarm1 silencer siRNA. RESULTS: Fundus imaging and microscopic analysis indicated that KA promoted Wallerian-like degeneration of RGCs and axons in KA-treated eyes, but not in PBS-treated eyes. Quantitative analysis indicated a significant increase in degeneration of RGCs and their axons in KA-treated injected eyes, but not in PBS-treated eyes. Compared with low levels of SARM1 protein in retinal protein extracts, retinal cross sections, and optic nerve from PBS-treated eyes, SARM1 protein levels were increased in KA-treated eyes. Finally, treatment of eyes with KA along with a Sarm1 silencer siRNA attenuated KA-mediated degeneration of RGCs and their axons significantly. CONCLUSIONS: Results presented in this study, for the first time, show that KA-mediated upregulation of SARM1 protein promotes Wallerian-like degeneration of RGCs and their axons.

Curcumin attenuates staurosporine-mediated death of retinal ganglion cells.

PURPOSE: Staurosporine (SS) causes retinal ganglion cell (RGC) death in vivo, but the underlying mechanisms have been unclear. Since previous studies on RGC-5 cells indicated that SS induces cell death by elevating proteases, this study was undertaken to investigate whether SS induces RGC loss by elevating proteases in the retina, and curcumin prevents SS-mediated death of RGCs. METHODS: Transformed mouse retinal ganglion-like cells (RGC-5) were treated with 2.0 µM SS and various doses of curcumin. Two optimal doses of SS (12.5 and 100 nM) and curcumin (2.5 and 10 µM) were injected into the vitreous of C57BL/6 mice. Matrix metalloproteinase (MMP)-9, tissue plasminogen activator (tPA), and urokinase plasminogen activator (uPA) activities were assessed by zymography assays. Viability of RGC-5 cells was assessed by MTT assays. RGC and amacrine cell loss in vivo was assessed by immunostaining with Brn3a and ChAT antibodies, respectively. Frozen retinal cross sections were immunostained for nuclear factor-κB (NF-κB). RESULTS: Staurosporine induced uPA and tPA levels in RGC-5 cells, and MMP-9, uPA, and tPA levels in the retinas and promoted the death of RGC-5 cells in vitro and RGCs and amacrine cells in vivo. In contrast, curcumin attenuated RGC and amacrine cell loss, despite elevated levels of proteases. An NF-κB inhibitory peptide reversed curcumin-mediated protective effect on RGC-5 cells, but did not inhibit protease levels. Curcumin did not inhibit protease levels in vivo, but attenuated RGC and amacrine cell loss by restoring NF-κB expression. CONCLUSIONS: The results show that curcumin attenuates RGC and amacrine cell death despite elevated levels of proteases and raises the possibility that it may be used as a plausible adjuvant therapeutic agent to prevent the loss of these cells in retinal degenerative conditions.

A decrease in phosphorylation of cAMP-response element-binding protein (CREBP) promotes retinal degeneration.

Excitotoxicity, induced either by N-Methyl-d-aspartate (NMDA) or kainic acid (KA), promotes irreversible loss of retinal ganglion cells (RGCs). Although the intracellular signaling mechanisms underlying excitotoxic cell death are still unclear, recent studies on the retina indicate that NMDA promotes RGC death by increasing phosphorylation of cyclic AMP (cAMP) response element (CRE)-binding protein (CREBP), while studies on the central nervous system indicate that KA promotes neuronal cell death by decreasing phosphorylation of CREBP, suggesting that CREBP can elicit dual responses depending on the excitotoxic-agent. Interestingly, the role of CREBP in KA-mediated death of RGCs has not been investigated. Therefore, by using an animal model of excitotoxicity, the aim of this study was to investigate whether excitotoxicity induces RGC death by decreasing Ser(133)-CREBP in the retina. Death of RGCs was induced in CD-1 mice by an intravitreal injection of 20 nmoles of kainic acid (KA). Decrease in CREBP levels was determined by immunohistochemistry, western blot analysis, and electrophoretic mobility gel shift assays (EMSAs). Immunohistochemical analysis indicated that CREBP was constitutively expressed in the nuclei of cells both in the ganglion cell layer (GCL) and in the inner nuclear layer (INL) of CD-1 mice. At 6 h after KA injection, nuclear localization of Ser(133)-CREBP was decreased in the GCL. At 24 h after KA injection, Ser(133)-CREBP was decreased further in GCL and the INL, and a decrease in Ser(133)-CREBP correlated with apoptotic death of RGCs and amacrine cells. Western blot analysis indicated that KA decreased Ser(133)-CREBP levels in retinal protein extracts. EMSA assays indicated that KA also reduced the binding of Ser(133)-CREBP to CRE consensus oligonucleotides. In contrast, intravitreal injection of CNQX, a non-NMDA glutamate receptor antagonist, restored the KA-induced decrease in Ser(133)-CREBP both in the GCL and INL, and inhibited loss of RGCs and amacrine cells. These results, for the first time, suggest that KA promotes retinal degeneration by reducing phosphorylation of Ser(133)-CREBP in the retina.

Inhibition of reactive gliosis attenuates excitotoxicity-mediated death of retinal ganglion cells.

Reactive gliosis is a hallmark of many retinal neurodegenerative conditions, including glaucoma. Although a majority of studies to date have concentrated on reactive gliosis in the optic nerve head, very few studies have been initiated to investigate the role of reactive gliosis in the retina. We have previously shown that reactive glial cells synthesize elevated levels of proteases, and these proteases, in turn, promote the death of retinal ganglion cells (RGCs). In this investigation, we have used two glial toxins to inhibit reactive gliosis and have evaluated their effect on protease-mediated death of RGCs. Kainic acid was injected into the vitreous humor of C57BL/6 mice to induce reactive gliosis and death of RGCs. C57BL/6 mice were also treated with glial toxins, alpha-aminoadipic acid (AAA) or Neurostatin, along with KA. Reactive gliosis was assessed by immunostaining of retinal cross sections and retinal flat-mounts with glial fibrillary acidic protein (GFAP) and vimentin antibodies. Apoptotic cell death was assessed by TUNEL assays. Loss of RGCs was determined by immunostaining of flat-mounted retinas with Brn3a antibodies. Proteolytic activities of matrix metalloproteinase-9 (MMP-9), tissue plasminogen activator (tPA), and urokinase plasminogen activator (uPA) were assessed by zymography assays. GFAP-immunoreactivity indicated that KA induced reactive gliosis in both retinal astrocytes and in Muller cells. AAA alone or in combination with KA decreased GFAP and vimentin-immunoreactivity in Mϋller cells, but not in astrocytes. In addition AAA failed to decrease KA-mediated protease levels and apoptotic death of RGCs. In contrast, Neurostatin either alone or in combination with KA, decreased reactive gliosis in both astrocytes and Mϋller cells. Furthermore, Neurostatin decreased protease levels and prevented apoptotic death of RGCs. Our findings, for the first time, indicate that inhibition of reactive gliosis decreases protease levels in the retina, prevents apoptotic death of retinal neurons, and provides substantial neuroprotection.

Posterior vitreous detachment with microplasmin alters the retinal penetration of intravitreal bevacizumab (Avastin) in rabbit eyes.

PURPOSE: Intravitreal bevacizumab (BV) (Avastin, Genentech Inc., South San Francisco, CA) is frequently used for the treatment of age-related macular degeneration. Previous studies have demonstrated full-thickness retinal penetration. Intravitreal recombinant microplasmin (MP) has been shown to successfully induce a posterior vitreous detachment (PVD) and vitreous liquefaction in animals. It has been suggested that a PVD may alter the retinal penetration of molecules in the vitreous cavity. The aim of this study was to compare BV retinal penetration in rabbit eyes with and without an MP-induced PVD. METHODS: Twelve adult rabbits were injected with 0.1 mL (0.4 mg) of MP into the vitreous cavity of 1 eye. One week later, the rabbits were injected with 0.05 mL (1.25 mg) of BV into both eyes. Both eyes of 3 rabbits were harvested at 6 hours, 12 hours, 24 hours, and 72 hours after the BV injection. Frozen retinal cross sections were prepared, and BV retinal penetration was evaluated with immunohistochemistry using a fluorescence-labeled antibody against BV. Two eyes from one rabbit were not injected with either agent and used as controls to compare the background autofluorescence. Peripapillary retinal sections were recorded with a digital camera, and intraretinal BV fluorescence-labeled antibody was measured by qualitative photographic interpretation. Two additional rabbits received an intravitreal injection of 0.1 mL of MP in 1 eye. One week later, both eyes from each rabbit were enucleated, and frozen retinal sections were prepared and analyzed with light microscopy to evaluate histologic damage. RESULTS: Full-thickness BV retinal penetration was observed throughout the retina in both eyes of each rabbit. All the MP-injected eyes exhibited increased antibody labeling in retinas evaluated at 6 hours, 12 hours, and 24 hours after BV injection when compared with the contralateral non-MP-injected eyes. By 3 days after BV injection, all eyes demonstrated decreased antibody labeling compared with earlier periods. At 3 days, 1 rabbit showed increased antibody labeling in the retina of the non-MP-injected eye compared with the contralateral MP-injected eye, and 2 rabbits exhibited similar antibody labeling in both eyes. When compared with control eyes, light microscopy demonstrated normal retinal histologic findings in eyes injected only with MP. CONCLUSION: Increased BV retinal penetration is observed initially in eyes with an MP-induced PVD, and the mechanism is likely multifactorial. By 3 days, retinal penetration is similar in eyes with and without a PVD. Although it is difficult to directly extrapolate to humans, our study suggests that a PVD may alter the retinal penetration of BV.

Mechanisms regulating plasminogen activators in transformed retinal ganglion cells.

Irreversible loss of retinal ganglion cells (RGCs) is a major clinical issue in glaucoma, but the mechanisms that lead to RGC death are currently unclear. We have previously reported that elevated levels of tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA) cause the death of RGCs in vivo and transformed retinal ganglion cells (RGC-5) in vitro. Yet, it is unclear how secreted proteases such as tPA and uPA directly cause RGCs' death. In this study, by employing RGC-5 cells, we report that tPA and uPA elicit their direct effect through the low-density lipoprotein-related receptor-1 (LRP-1). We also show that blockade of protease-LRP-1 interaction leads to a complete reduction in autocrine synthesis of tPA and uPA, and prevents protease-mediated death of RGC-5 cells. RGC-5 cells were cultured in serum-free medium and treated with 2.0 microM Staurosporine to induce their differentiation. Neurite outgrowth was observed by a phase contrast microscope and quantified by NeuroJ imaging software. Proteolytic activities of tPA and uPA were determined by zymography assays. Cell viability was determined by MTT assays. Compared to untreated RGC-5 cells, cells treated with Staurosporine differentiated, synthesized and secreted elevated levels of tPA and uPA, and underwent cell death. In contrast, when RGC-5 cells were treated with Staurosporine along with the receptor associated protein (RAP), proteolytic activities of both tPA and uPA were significantly reduced. Under these conditions, a significant number of RGC-5 cells survived and showed increased neurite outgrowth. These results indicate that LRP-1 regulates autocrine synthesis of tPA and uPA in RGC-5 cells and suggest that the use of RAP to antagonize the effect of proteases may be a way to prevent RGC death in glaucoma.

Inhibition of plasminogen activators attenuates the death of differentiated retinal ganglion cells and stabilizes their neurite network in vitro.

PURPOSE: Although previous studies have indicated that elevated levels of the tissue plasminogen activator (tPA) and the urokinase plasminogen activator (uPA) associate with the death of retinal ganglion cells (RGCs), it was unclear whether these proteases directly cause cell death. With the use of a transformed and undifferentiated retinal ganglion cell line, RGC-5, which does not express tPA, and by treating this cell line with staurosporine, which induces not only the differentiation of RGC-5 cells but also the expression of uPA and tPA in other neuronal cells, the authors sought to determine whether these proteases regulate the differentiation of RGC-5 cells and whether elevated levels of these proteases directly cause the death of RGC-5 cells. METHODS: Transformed RGC-5 cells were cultured in serum-free medium and were treated with 0.5 muM to 2.0 muM staurosporine to induce their differentiation. Neurite outgrowth was assessed by phase-contrast microscopy and calcein AM staining and quantified with imaging software. Proteolytic activities of tPA and uPA were determined by zymography assays. Cell viability was determined by LIVE/DEAD viability assay kit. RESULTS: Compared with untreated RGC-5 cells, cells treated with staurosporine differentiated as early as 1 to 6 hours. However, proteolytic activities of neither tPA nor uPA were observed within this time frame. Differentiated RGC-5 cells expressed detectable levels of uPA proteolytic activity starting at 24 hours and tPA proteolytic activity only at 48 hours. RGC-5 cells synthesized and secreted uPA and tPA into the conditioned medium, depending on staurosporine concentration and treatment time. At lower concentrations of staurosporine, differentiated RGC-5 cells had longer neurites and expressed lower levels of tPA and uPA. At higher concentrations of staurosporine, differentiated RGC-5 cells expressed higher levels of tPA and uPA, had smaller neurites, and most of them died. In contrast, when RGC-5 cells were treated with staurosporine along with inhibitors specific to tPA and uPA, proteolytic activities of both PAs were significantly reduced. Under these conditions, a significant number of RGC-5 cells survived, showed increased neurite outgrowth, and established their neurite network in vitro. CONCLUSIONS: Results presented in this study indicate that RGC-5 cells do not require tPA and tPA for their differentiation. In fact, differentiated RGC-5 cells synthesize elevated levels of tPA and uPA, and elevated levels of these proteases acting in an autocrine-fashion in turn lead to the death of RGC-5 cells.

The emerging role of proteases in retinal ganglion cell death.

Retinal ganglion cell (RGC) death is an important issue in Primary Open Angle-Glaucoma (POAG) in terms of both vision loss and health care costs. Yet, the pathophysiology underlying RGC death in glaucoma is unclear. A growing body of evidence indicates that proteases that modulate the extracellular matrix (ECM) milieu in the retina, either directly or indirectly, play an important role in dictating the fate of RGCs. Recent evidence indicates that proteases, in addition to ECM-remodeling, have broader functional roles in glutamate receptor processing and predisposing RGCs to secondary damage. This review is focused on discussing the role of two groups of proteases, the matrix metalloproteinases (MMPs) and the plasminogen activators (PAs), in RGC death. In a long-run, a better understanding of the mechanisms involved in the regulation of proteases may lead to the development of adjunctive treatment options to attenuate RGC death and improve vision loss in glaucoma.

Matrix metalloproteinase gelatinase B (MMP-9) is associated with leaking glaucoma filtering blebs.

The goal of glaucoma filtering surgery is to create a low resistance pathway for aqueous outflow. The result is a blister or 'bleb' on the conjunctiva, from which fluid drains into the vasculature. Filtering surgery results may be compromised if blebs develop leaks, a problem that surfaces more frequently when antimetabolites are used to control the wound healing response. We investigated the role of tissue remodelling enzymes of the Matrix metalloproteinase (MMP) family in the development of bleb leaks. Our design was a case series. We enrolled glaucoma patients with leaking blebs, glaucoma patients with overhanging blebs and normal eyes. Leaking bleb tissues (n=11) and bleb leak fluid were collected from patients undergoing bleb revision surgery. Overhanging bleb tissues (from non-leaking blebs, n=3), normal conjunctiva (n=8), and aqueous humour (n=4) were collected for comparison. Samples were analysed for MMP content and proteinase activity by the methods of zymography, western blotting, immunohistochemistry, and in situ zymography. Our main outcome measures were presence and activity of MMP in sample. Zymography revealed the presence of a high molecular weight caseinase and a 92-kDa gelatinase of a size appropriate for the proenzyme form of gelatinase B (gelB; MMP-9), in extracts from leaking bleb tissue, but not in bleb leak fluid or aqueous humour samples. In contrast, a 65-kDa gelatinase of a size appropriate for gelatinase A (MMP-2) proenzyme was observed in all samples. All proteinases disappeared when 10mm EDTA was added to the development buffer, consistent with their identity as MMPs. Western blotting and immunohistochemical analyses confirmed the identity of the 92kDa proteinase as gelB, and further revealed its absence from extracts of overhanging bleb tissue and normal conjunctiva. In situ zymography demonstrated strong gelatinolytic activity in leaking bleb tissue, but not overhanging bleb tissue or normal conjunctiva. MMP-g may be involved in the mechanism of formation of bleb leaks. Precise description of the cascade of events leading to bleb leakage may allow the design of therapeutic interventions to prevent, stabilize or reverse bleb leakage.

Plasminogen activators promote excitotoxicity-induced retinal damage.

Increased levels of extracellular l-glutamate have been suggested to play a role in retinal damage in a number of blinding diseases such as glaucoma and diabetic retinopathy. Although glutamate can cause retinal damage in part by hyperstimulating its receptors ("excitotoxicity"), the downstream events that lead to retinal damage are poorly understood. In this study, we injected kainic acid (KA), a glutamate receptor agonist that specifically hyperstimulates non-NMDA-type receptors, into the vitreous humor of CD-1 mice and have investigated the role of plasminogen activators (PAs) [tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA)] in excitotoxicity-induced retinal damage. Injection of KA into the vitreous humor led to an up-regulation in tPA and an induction in uPA activity in the retina and this was associated with activation of zymogen plasminogen to active plasmin. Immunocytochemical analysis indicated that retinal ganglion cells (RGCs), constitutively express tPA and release it into the extracellular space upon KA injection. Immunocytochemical analysis also indicated an increase in uPA in the nerve fiber layer after KA injection that was absent in the control retinas. These events were associated with apoptotic death of cells initially in the ganglion cell layer and subsequently in the inner and outer nuclear layer, associated with loss of RGCs and amacrine cells. These phenomena were inhibited when recombinant plasminogen activator inhibitor (rPAI-1) or tPA-STOP were injected into the vitreous humor with KA, whereas a plasmin inhibitor, alpha-2-antiplasmin, failed to attenuate KA-induced retinal damage. Taken together, these results suggest that inhibition of plasminogen activators might attenuate retinal damage in blinding retinal diseases in which hyperstimulation of glutamate receptors is implicated as a causative factor to retinal damage.

Intravitreous injection of a membrane depolarization agent causes retinal degeneration via matrix metalloproteinase-9.

PURPOSE: Membrane depolarization and subsequent synaptic release of l-glutamate have been implicated in ischemic retinal damage. However, the mechanisms that lead to ischemia-induced retinal damage are poorly understood. In this study, KCl, a classic membrane depolarizing agent, was injected into the vitreous humor, and the role of matrix metalloproteinase (MMP)-9 in KCl-induced retinal damage was investigated. METHODS: Normal adult CD-1 mice were treated with KCl by intravitreal injection. MMP activity in retinal protein extracts was determined by gelatin zymography. Tissue localization of MMP-9 in the retina was determined by immunohistochemistry. MMP-9, MMP-2, tissue inhibitor of MMP (TIMP)-1, TIMP-2, Bax, and BCl-2 proteins in retinal extracts were determined by Western blot analysis. Apoptotic cell death in the retina was determined by TUNEL assays. Retinal damage was assessed by immunolocalization studies with antibodies against neurofilament-light (NF-L) and calretinin. RESULTS: Depolarizing concentrations of KCl induced a dose- and time-related upregulation in MMP-9 activity and protein in the retina. KCl-mediated MMP-9 upregulation was associated with an increase in proapoptotic protein Bax and apoptotic death of cells in the ganglion cell (GCL) and inner nuclear layer (INL), and subsequent loss of NF-L-positive ganglion cells and calretinin-positive amacrine cells. Intravitreal injection of KCl along with an N-methyl-d-aspartate (NMDA)-type glutamate receptor antagonist, MK-801, and a non-NMDA-type glutamate receptor antagonist, NBQX, resulted in a reduction in KCl-mediated MMP-9 upregulation in the retina. Furthermore, a synthetic MMP inhibitor inhibited KCl-mediated MMP-9 upregulation, which led to a significant attenuation of KCl-induced retinal damage. CONCLUSIONS: These results suggest that upregulation of MMP-9, in part, plays a causative role in KCl-induced retinal damage.