Molecular Vision 2003; 9:238-248 <http://www.molvis.org/molvis/v9/a35/> Received 04 March 2003 | Accepted 11 June 2003 | Published 12 June 2003

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Inhibition of plasminogen activation protects against ganglion cell loss in a mouse model of retinal damage

Xu Zhang, Aisha Chaudhry, Shravan K. Chintala
 
 

Eye Research Institute, Oakland University, Rochester, MI

Correspondence to: Shravan K. Chintala, PhD, Eye Research Institute, 409 Dodge Hall, Oakland University, Rochester, MI, 48309; Phone: (248) 370-2532; FAX: (248) 370-2006; email: Chintala@oakland.edu

Abstract

Purpose: The mechanisms that trigger ganglion cell death in ischemic retinal diseases are not clearly understood. Using a mouse optic nerve ligation model, the objective of this study was to test the hypothesis that extracellular matrix (ECM) modulating plasminogen activators (PAs) potentiate ganglion cell loss.

Methods: Optic nerve ligation was performed to initiate ganglion cell loss in the retina. Urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) activity in retinal extracts was determined by plasminogen/fibrinogen zymography. Immunostaining and western blot analysis was performed to detect uPA and tPA proteins. Plasmin activity was determined by casein gel-zymography. Plasminogen and plasmin proteins were detected and quantified by western blotting. Morphology was assessed using hematoxylin and eosin stained retinal cross sections, and programmed cell death was monitored by an apoptotic assay. Laminin degradation in retinal extracts was assessed by western blot analysis.

Results: Optic nerve ligation led to a transient increase in uPA and plasmin proteolytic activity in the retina. Urokinase inhibitor, amiloride, blocked uPA activity in retinal extracts. We found a correlation between the increased uPA activity, and conversion of zymogen plasminogen to active plasmin in retinal extracts with laminin degradation in the retina and apoptosis of ganglion cells. We found that by adding exogenous plasmin, in vitro, laminin present in control retinal extracts could be degraded in similar fashion. In addition, uPA or tPA failed to degrade laminin in control retinal extracts unless plasminogen was added, indicating that plasminogen activation is necessary for laminin degradation, in vitro. After intravitreal injection of plasmin inhibitor, α-2 antiplasmin, we found a significant protection against optic nerve ligation-induced ganglion cell loss.

Conclusions: Optic nerve ligation-induced plasmin(ogen) activation that precedes ganglion cell loss suggest that specific targeting of plasmin activity may have therapeutic potential in preventing ganglion cell loss in retinal diseases.

Introduction

Ganglion cells, located in the innermost cellular layer of the neural retina, are vulnerable to damage caused by occlusion of the vessels that supply blood and oxygen to the retina, retinal ischemia. While the mechanisms underlying ganglion cell death following retinal ischemia are not clearly understood, current opinion holds that retinal pathology following ischemia [1-6] is due to neurotrophic deprivation, high concentrations of glutamate [7,8], oxidative stress, and inflammation [3,4,9]. Ischemia, however, could play a role in damaging the retina in conjunction with some or all of the events listed above.

Cells can respond to damaging stimuli by activating tissue repair and remodeling mechanisms that are strictly regulated through interaction with their extracellular matrix. This feedback is mediated primarily through two enzyme systems, the plasminogen activator (PA)-plasminogen (PG) system and matrix metalloproteinase (MMP) system [10-12]. The PA-PG system modulates fibrin matrices but also converts inactive pro-MMPs to their active forms [13,14]. Plasminogen activators (PAs), uPA and tPA are serine proteases that convert a physiological zymogen plasminogen into active plasmin, a trypsin-like endopeptidase of broad substrate specificity [10]. In the central nervous system (CNS) tPA is associated with neuronal plasticity [15,16] and excitotoxic cascade [17], whereas uPA is associated with astrocyte proliferation and remodeling [18,19]. Plasminogen activators have also been associated with neural crest cell migration, growth cones of cultured neurons, and neuronal regeneration [20-22]. A number of recent studies have linked PAs with excitotoxicity-mediated neuronal apoptosis in the CNS [23,24]. Following excitotoxin-induced seizures [25] tPA expression is induced in the hippocampus and tPA or PG deficient mice are resistant to excitotoxin-induced neuronal degeneration [17,26]. Plasmin, produced from plasminogen activation cleaves extracellular matrix (ECM) components such as laminin and fibronectin [27]. Laminin loss, in response to excitotoxin injection into the hippocampus results from PA-PG proteolytic activity and contributes to neuronal cell death [17,28]. Additional studies suggest that tPA cleaves the NR1 subunit of the NMDA receptor, and contributes to neuronal death from enhanced NMDA-evoked Ca⁺² influx [29].

Although immunolocalization studies have reported the presence of plasminogen activators in normal retinal tissues [30,31], mounting evidence suggest that PAs also play a role in neovascular diseases of the retina and choroid [32-34]. However, pathological role of plasminogen activators in a progressive retinal disease model has not been reported until now. Therefore, using a mouse optic nerve ligation model to deplete ganglion cells, we tested whether optic nerve ligation-mediated plasminogen activation in the retina causes ECM degradation and subsequent ganglion cell death.

Methods

Optic nerve ligation

We performed all surgical manipulations on mice under general anesthesia according to the institutional protocol guidelines that are comparable to those published by the Institute for Laboratory Animal Research and the ARVO statement for the use of Animals in Ophthalmology and Vision Research. Normal adult CD-1 mice (6-8 weeks old; Charles River Breeding Labs, Wilmington, MA) were anesthetized by an intraperitonial injection of 1.25% avertin (2,2,2-tribromoethanol in tert-amyl alcohol; 0.017 ml/g body weight). Retinal ischemia-reperfusion injury was induced in mice [35] by a method that has been described previously for rats [36]. While observing under an operating microscope, lateral conjunctival peritomy was performed to disinsert the lateral rectus muscle, and the optic nerve of the right eye was then exposed by blunt dissection. A 6-0-nylon suture was placed around the optic nerve and tightened until blood flow in all the retinal vessels was stopped. Non-perfusion of the blood flow was confirmed using an operating microscope. After 30 min, re-perfusion was allowed by removing the suture. Sham-operated mice underwent similar surgery without tightening the suture.

Tissue extraction

Following 12 h, day 1, day 2, day 4, and day 6 after optic nerve ligation, animals were anesthetized with an overdose of avertin, and their eyes were enucleated. Enucleated eyes were cut in half at the equator and the lenses were removed. Retinas were carefully peeled off using forceps, and washed three times with phosphate buffered saline (pH 7.4). Two to three retinas each were placed in eppendorf tubes containing 40 μl of extraction buffer (1% (v/v) nonidet-P40, 20 mM Tris-HCl, 150 mM NaCl, 1 mM Na₃VO₄, pH 7.4) and the tissues were homogenized. Tissue homogenates were centrifuged at 10,000 rpm for 5 min at 4 °C and the supernatants were collected. The total protein concentration in supernatants was determined using the Bio-Rad protein assay (Bio-Rad laboratories, Hercules, CA).

Gel Zymography

Activities of uPA, tPA, and plasmin were determined by zymography according to methods described in published papers [24,37]. Retinal extracts containing equal amounts of protein (25 μg) were mixed with SDS gel-loading buffer [38], and then loaded without reduction or heating onto 10% SDS polyacrylamide gels containing fibrinogen/plasminogen (5.5 mg/ml and 50 μg/ml, respectively) as substrates for uPA and tPA or 0.2% beta-casein as substrate for plasmin. Following electrophoresis, the gels were washed with 2.5% (v/v) Triton-X 100 for 45 min to remove SDS. Finally the gels were placed in glycine buffer (0.1 M, pH 8.0) and incubated overnight at 37 °C to allow proteolysis of the substrates in the gels. The gels were stained with 0.1% (w/v) Coomassie Brilliant Blue-R250 (25% (v/v) methanol, 10% (v/v) acetic acid in water) and then destained (25% (v/v) methanol, 10% (v/v) acetic acid in water without Coomassie blue). Upon staining with Coomassie blue and destaining, the final gel had a uniform background except in regions to which uPA, tPA, and plasmin had migrated and cleaved their respective substrates. Samples containing purified murine tPA (American Diagnostica, CT), murine uPA or murine plasmin (Innovative Research, Southfield, MI) were co-electrophoresed for comparison. In addition, a reduced molecular weight size standard was included on all gels (Life Technologies, Gaithersburg, MD). In separate sets of experiments, uPA inhibitor amiloride (200 μM) was added during incubation period to differentiate between uPA and tPA.

Immunohistochemistry

Eyes enucleated after optic nerve ligation were embedded in OCT compound (Sakura Finetek USA, Torrance, CA). Traverse, 10 μm thick cryostat sections were cut, placed onto super-frost plus slides (Fisher Scientific, Pittsburgh, PA), and fixed with 4% (v/v) paraformaldehyde for 30 min at room temperature. Sections were subsequently processed for indirect immunofluorescent localization using antibodies against uPA (1: 100 dilution; Innovative Research, MI) or tPA (1:50 dilution, Innovative Research, MI). Specific binding of the primary antibody was visualized using appropriate biotin-labeled secondary antibodies and an ABC reagent kit (Vector Labs, Burlingame, CA). Staining was visualized using diaminobenzidine tetrahydrochloride (DAB) reagent according to the manufacturer's instructions (Vector Labs). Negative controls included omitting the primary antibody. Sections were then observed under a Nikon bright field microscope and digitized images were obtained using a SPOT digital camera.

Apoptosis assay

Traverse, 10 μm thick cryostat sections were cut and placed into superforst slides as described above. Sections were fixed with 4% (v/v) paraformaldehyde for 30 min at room temperature. Apoptotic cell death was detected by TdT-mediated dUTP nick-end labeling (TUNEL) assay, using a kit (In situ cell death detection kit with fluorescein; Roche Biochemicals, Mannheim, Germany) and the protocol provided by the manufacturer. Hoechst dye (0.1 mg/ml) counter staining was performed for 2 min to determine the nuclei of cells. Tissue sections were examined using a Nikon microscope equipped with epifluorescence, and digital images were obtained with a SPOT digital camera.

Western blot analysis

Aliquots containing equal amount of protein (25 μg) were mixed with gel loading buffer, and separated on 10% SDS-polyacrylamide gels. After electrophoresis, the proteins were transferred out of the gels onto PVDF membranes. The membranes were blocked with 10% non-fat dry milk in Tris-buffered saline containing 0.1% Tween-20 (TBS-T), and then probed with antibodies against mouse plasmin(ogen), 1:2000 dilution (Innovative Research), mouse uPA (1:1000 dilution, Innovative Research), mouse tPA (1:250 dilution, Innovative Research), and murine albumin (1:1000 dilution, Nordic Immunologics, Tilburg, Netherlands). The membranes were washed with TBS-T and incubated with appropriate peroxidase-conjugated secondary antibodies for 1 h at room temperature. Finally, the proteins on the membranes were detected using an ECL chemiluminescence kit (Amersham Pharmacia Biotech, Piscataway, NJ). Purified mouse plasminogen, plasmin (Innovative Research, MI), and mouse uPA (American Diagnostica, Greenwich, CT) were co-electrophoresed as positive standards. To determine quantitative amounts of plasminogen, we separated a range of mouse plasminogen standards (7.81, 15.625, 31.25, 62.5, and 125 ng, data not shown) by electrophoresis and the protein bands were detected by western blot analysis as described above. After optimizing to film exposure and scanning parameters, plasminogen standard bands were scanned by densitometry to obtain a linear densitometric curve (r²=0.975). Amount of plasminogen present in retinal extracts was then calculated by comparing to the standard curve.

In vitro analysis of laminin degradation

To determine the role of PA-mediated plasminogen activation in degrading endogenous laminin, retinal proteins from unligated control retinas were prepared as described above. Aliquots containing equal amount proteins were then mixed as appropriate to the experiment and incubated at 37 °C for 4 h in 20 mM Tris-HCl, pH 7.5, 150 mM NaCl. To determine the capacity of plasmin to initiate laminin degradation in retinal extracts, 100 ng of purified mouse plasmin (Innovative Research) was added to equal amount of retinal extracts (25 μg) in the presence or absence of plasmin inhibitor, α2-antiplasmin (1 μg). In separate sets of experiments, equal amount (25 μg) of control retinal extracts were incubated with of uPA (10 IU) or tPA (10 IU) in the presence or absence of 100 ng plasminogen. For analysis of laminin content after incubation, all extracts were loaded onto 4-20% gradient SDS-polyacrylamide gels and electrophoresed under reducing conditions. After electrophoresis the proteins were transferred to nylon membranes and probed with anti-laminin antibodies (Product number L9393; 1:1000 dilution; Sigma, St. Louis, MO) and binding of the primary antibody was detected using appropriate secondary antibody and an ECL western blot system as described above.

Intravitreal injections

Intravitreal injection of phosphate buffered saline, or α2-antiplasmin (Calbiochem, San Diego, CA) was performed as described previously [39]. Briefly, mice were anesthetized with avertin and a small incision was made with a 30-guage needle behind the limbus, through the conjunctiva and sclera. After dilation, a Hamilton syringe was passed through the incision to a 40°to 50°angle to the equator to inject the solutions. For control experiments, eyes were injected with 2 μl of phosphate buffered saline alone and for treatment groups, eyes were injected with 2 μl of α2-antiplasmin (4 μg/2 μl). Four days after optic nerve ligation, eyes were enucleated, embedded in JB-4 and processed for morphological evaluation as described below.

Morphometry

To determine the morphological changes in retinas after intravitreal injection of α-2 antiplasmin, eyes enucleated 4 days after optic nerve ligation were embedded in JB-4 plus (Polyscience Inc, Warrington, PA). Briefly, after removing cornea and lens, eyecups were fixed overnight with fixative containing 1.2% (v/v) paraformaldehyde and 0.8% (v/v) glutaraldehyde in PBS. Eyes cups were washed for 3-4 h in PBS, and dehydrated with increasing concentrations of ethanol (50-95%). After dehydration, eyecups were embedded in JB-4 and cut into 4 μm cross sections and then stained with hematoxylin and eosin (H&E). Retinal ganglion cell loss was quantified by counting the cells in the ganglion cell layer in a 10x field at a distance of 1-2 mm from the optic disc. Data from 8-10 sections from 3 different eyes were analyzed by a paired Student's t-test using Slidewrite software (version 6.0; Advanced Graphics Software Inc., Encinitas, CA).

Results

Optic nerve ligation causes uPA induction in the retina

To investigate whether optic nerve ligation-induced plasminogen activation plays a role in ganglion cell loss in the retina, we adapted a well established rat optic nerve ligation [36] model to mice as previously reported [35]. Majority of the studies performed using this model [2,40-43] have suggested that optic nerve ligation causes retinal damage primarily due to ischemia-reperfusion injury and this model has been accepted as a useful model for studying mechanisms underlying ganglion cell loss in the retina [6]. Total proteins extracted from control and optic nerve ligated retinas were subjected to zymography analysis to determine the proteolytic activity. Plasminogen/ fibrinogen zymography indicated low levels of a serine protease with an apparent molecular weight (65 kDa) appropriate for tPA in control retinal extracts (Figure 1A). The levels of this protease species were quite similar to those found in retinal extracts after optic nerve ligation. In contrast, optic nerve ligation-led to an increase in another serine protease with an apparent molecular weight (55 kDa) appropriate to be uPA. Levels of this protease increased as early as 12 h after ligation reached a peak 1-2 days and returned to basal levels by day 6 (Figure 1A). In a separate experiment extracts from control (12 h and day 1) and ligated (12 h and day 1) retinas, which showed constitutive levels of 65 kDa protease and induced levels of 55 kDa protease (Figure 1A) were subjected to plasminogen/fibrinogen zymography but the zymograms were allowed to develop in the presence of uPA inhibitor, amiloride (200 μM). Inclusion of amiloride inhibited appearance of the 55 kDa protease species, suggesting the identity of this inhibited protease as uPA (Figure 1B). In contrast, amiloride had no effect on the constitutive levels of the 65 kDa protease band, suggesting its identity as tPA (Figure 1B). In a separate experiment, known amounts of mouse tPA (0.125 ng) and uPA (0.125 ng) were subjected to plasminogen/fibrinogen zymography and the zymograms were allowed to develop in the presence of 200 μM amiloride. Inclusion of amiloride inhibited the appearance of uPA but not tPA indicating the specificity of amiloride (Figure 1C). Western blot analysis confirmed the identity of the induced 55 kDa protease as uPA (Figure 2A) and constitutive levels of the 65 kDa protease as tPA (Figure 2C). Compared to controls increased immunostaining of uPA was observed in the inner limiting membrane and in the inner plexiform layer (Figure 2B) after optic nerve ligation, consistent with increased trend of uPA observed by zymography and western blot analysis. In contrast, a diffusive pattern of tPA immunostaining was observed in the inner plexiform layer in both control and optic nerve ligated retinas, in addition to positive staining in the pigment epithelium (Figure 2D). Overall tPA levels seem to be similar in control and optic nerve ligated retinas, consistent with the low levels observed by both zymography and western blot analysis.

Plasmin(ogen) activation results in ganglion cell loss

The major role of plasminogen activators, uPA and tPA, is to convert a physiological zymogen plasminogen into active plasmin, which subsequently modulates the extracellular matrix proteins present in the retina. Therefore, using retinal extracts casein-gel zymography was performed to determine plasmin(ogen) activation, and western blot analysis was performed to confirm whether this was due to proteolytic conversion of physiological zymogen plasminogen into active plasmin. Casein zymography indicated undetectable level of plasmin activity in control retinal extracts (Figure 3A). In contrast, plasmin activity appeared as early as day 1, peaked around day 2 and returned to undetectable levels by day 6 in optic nerve ligated retinal extracts (Figure 3A,B). Note that although uPA activity was noticed as early as 12 h (Figure 1A), detectable levels of plasmin could not be observed until day 1 indicating that either plasminogen is not present in the retina or its activation is not taking place until day 1. Western blot analysis performed with an antibody against mouse plasminogen (that detects both a higher molecular weight plasminogen and a lower molecular weight active plasmin), indicated very low or undetectable levels of plasminogen in control retinal extracts (Figure 3C). Optic nerve ligation led to an increase in plasminogen protein levels in optic nerve ligated retinal extracts as early as 12 h, reached a peak around day 1 and decreased thereafter (Figure 3C). Increased appearance of plasminogen protein in optic nerve-ligated extracts was accompanied by its conversion to the lower molecular weight plasmin (Figure 3C), consistent with plasmin activity shown in Figure 3A. These results indicate that presence of plasminogen is critical for optic nerve ligation-induced plasmin generation. Therefore, a range of mouse plasminogen standards was separated by electrophoresis and quantitative levels of plasminogen were determined by western blot analysis (data not shown) as described in materials methods section. After optimizing to film exposure, standard plasminogen bands were scanned to obtain a liner densitometric curve (Figure 3D). Amount of plasminogen in retinal extracts shown in Figure 3C were then calculated by comparing to the standard curve. Quantitative levels of plasminogen in Figure 3E indicate a significant increase in plasminogen levels after optic nerve ligation.

Although the origin of plasminogen is currently not clear, blood retinal barrier (BRB) breakdown associated with ischemia [44,45] could result in increased plasminogen levels in the retina. Determination of extravascular albumin has been a widely accepted technique to demonstrate BRB breakdown. Therefore, albumin western blot analysis was performed on retinal extracts to determine whether increased plasminogen was associated with increased albumin (as an indication of BRB leak). Western blot analysis indicated (Figure 3F) increased levels of serum albumin in retinal extracts after optic nerve ligation, suggesting BRB leakage. It is interesting to note the appearance of increased albumin in retinal extracts (Figure 3F) coincident with the appearance of plasminogen (Figure 3C).

Tissue localization of plasmin activity determined by in situ casein zymography indicated its localization in the inner retina (data not shown). In addition, optic nerve ligation was associated with degradation of extracellular matrix component, laminin from the nerve fiber layer (data not shown) similar to our previous observations [35]. Interestingly laminin is a major ECM component of the nerve fiber layer and loss of laminin precedes neuronal cell death in the central nervous system (CNS). To determine whether optic nerve ligation-induced plasminogen activation leads to ganglion cell loss, retinal cryosections obtained 2 days after optic nerve ligation were stained with H&E. Morphological examination of H&E stained retinal cross sections from control and optic nerve ligated retinas indicated a decrease in inner retinal thickness due to progressive loss of the ganglion cells (Figure 4A), consistent with our previous report [35]. TdT-mediated dUTP nick-end labeling (TUNEL) assay confirmed that the cell death is due to apoptosis (Figure 4B). Few TUNEL positive cells were also observed in the inner nuclear layer due to secondary degeneration. Essentially no TUNEL positive cells were observed in control retinal sections.

Plasmin(ogen) activation contributes to laminin degradation in vitro

The above data suggest that once plasminogen is present in the retina (in part, due to BRB leak), both uPA and tPA could convert zymogen plasminogen to active plasmin, which in turn can degrade or modulate extracellular matrix components such as laminin (a known substrate for plasmin) in the nerve fiber layer. Therefore to provide a mechanistic role of plasmin in degrading endogenous laminin in vitro, control retinal extracts (that contain undegraded laminin) were mixed with 100 ng of purified mouse plasmin and incubated in the presence or absence of 1 μg α2-antiplasmin and laminin degradation was assessed by western blot analysis. The antibody employed for this experiment was raised against the α (440 kDa) and β/γ (220 kDa) chains of purified mouse laminin, but this antibody does not detect the α1 chain on western blots consistent with previous reports [46-48]. Western blot analysis detected a 200-220 kDa protein band appropriate to be the size of β/γ chains in control retinal extracts (Figure 5). Addition of plasmin degraded laminin in control retinal extracts, which could be blocked by a plasmin inhibitor, α2-antiplasmin. In contrast, uPA or tPA failed to degrade laminin in vitro, indicating that PAs themselves cannot degrade laminin. Therefore, exogenous plasminogen was added to the control retinal extracts in combination with uPA or tPA to determine whether addition of plasminogen, presumably due to generation of active plasmin, degrades laminin. Indeed, uPA and tPA in the presence of plasminogen caused laminin degradation suggesting that plasmin(ogen) activation plays a major role in this process (Figure 5).

α2-antiplasmin attenuates ganglion cell loss

The data presented above suggest that plasmin(ogen) activation might be a key event that causes degradation of extracellular matrix component laminin and, in part, promotes ganglion cell loss. As stated before, once plasminogen is extravasated into the retina after optic nerve ligation, it can be converted into active plasmin by induced levels of uPA. Inhibition of uPA alone might not be retinal protective because low levels of tPA can also convert plasminogen to plasmin. Therefore, we reasoned that inhibition of plasmin activity might be an effective strategy to determine neuroprotective effects after optic nerve ligation. α2-antiplasmin is a specific inhibitor of plasmin, which has been found to block neuordegeneration in the CNS [49]. To determine whether inhibition of plasmin activity leads to neuroprotection, we injected α2-antiplasmin in to the vitreous 10 min before optic nerve ligation. Morphometric analysis of retinal cross sections four days after optic nerve ligation indicated a significant reduction in mean number of ganglion cells in eyes that had undergone intravitreal injection of PBS alone (vehicle) or no injection at all (Figure 6A,B). In contrast, a significant (p<0.005) protection in ganglion cell loss was observed in eyes that have undergone α2-antiplasmin injection (Figure 6A,B), suggesting that inhibition of plasminogen activation, in part, might be neuroprotective.

Discussion

Although the retina is considered to be an extension of the brain, and considerable progress has been made in our understanding of the mechanisms underlying neuronal death during ischemia in the central nervous system, the mechanisms that underlie ganglion cell death during retinal ischemia are not well understood. Based on the data presented in this study, our working hypothesis (Figure 7) is that optic nerve ligation induces uPA levels in the retina and causes plasminogen leak, in part, due to BRB breakdown. Once plasminogen is extravasated, optic nerve ligation-induced uPA can convert plasminogen into active plasmin. This in turn can degrade extracellular matrix component laminin in the nerve fiber layer and promote ganglion cell loss. Although the exact mechanisms are not clear at this time, laminin is a major ECM component of the inner limiting membrane [50-53] and laminin loss has been shown to precede neuronal cell death in the CNS. Several reports have shown that retinal ganglion cells synthesize laminins in the inner limiting membrane [52,54] and enzymatic removal of ECM components of the inner limiting membrane has been shown to disrupt axon extension [55]. Laminins associated with neurons in the brain disappear from the site of excitotoxin injection, and their loss is temporally and spatially coincident with neuronal cell loss [49]. Therefore, it is possible that plamin(ogen)-mediated proteolytic modifications in the ECM may alter ganglion cell-laminin interactions and trigger a cascade of cell-detachment induced signaling events leading to "anoikis"-related ganglion cell death [56] or predispose ganglion cells to glutamate-induced excitotoxicity in the absence of ECM. In support of this idea, studies of the CNS have shown that tPA itself cannot induce neuronal cell death unless laminin-neuronal cell interactions are compromised by plasmin [49]. Laminin degradation in the mouse model described in this study could be direct, through plasmin activation, or it may be indirectly through activation of other MMPs [57]. Although it remains to be determined whether similar mechanisms exist in the retina, in a previous study we have shown that deficiency in MMP-9 prevents laminin degradation in the retina and prevents ganglion cell loss after optic nerve ligation [35].

Controlled extracellular matrix remodeling mediated by proteases constitutes a physiological phenomenon whereas excessive protease expression results in pathological remodeling. Such a role for plasminogen activators has been reported in tumor cell invasion [10,58] tumor angiogenesis [59] and neuronal degeneration [49]. The plasminogen activators uPA and tPA are traditionally linked to blood clot dissolution [60,61] by their conversion of the zymogen plasminogen to active plasmin, a protease that finally dissolves blood clots. Subsequently, tPA, often referred as a "clot busting agent", has been successfully used to treat myocardial infarction and stroke. However, accumulating evidence suggests that in addition to their beneficial role, plasminogen activators may also be involved in neuronal damage [28,29,62-64].

Traditionally, ischemic damage has been associated with neurotropohic deprivation, glutamate release, and calcium influx [62,65,66]. In support of this idea, injection of glutamate agonist into the posterior chamber of the eye induces specific death of ganglion cells [7,8]. This in turn leads to secondary effects such as an increase in cytokines, the expression of immediate early genes, and the generation of free radicals [67,68]. These complex events also cause an induction of proteases which could participate in the disruption of the extracellular matrix of basement membranes and surrounding tissues [69]. For instance, laminin loss has been observed in response to the injection of glutamate in to the CNS, and this loss was mediated by the plasminogen/plasmin activator system [26,28]. In agreement with these studies, the data presented in this study also support the role of plasmin(ogen) activation in laminin degradation.

Although the presence of uPA and tPA proteins has been demonstrated by immunlocalization studies [30,31,33,70], little is known about their physiological and pathological roles in the retina. The results presented in this study represent, to our knowledge, the first description of the role of PAs in ischemic damage of the retina in an in vivo mouse model of optic nerve ligation. The data presented strongly indicate an association of increased level of uPA and uPA-mediated plasmin(ogen) activation with ganglion cell loss. While it is possible that constitutive levels of tPA might also contribute to retinal damage, based on normal retinal morphology in control retinas, basal level of tPA activity does not seem to play a degenerative role in the normal retina. It is possible, however, that this low level of protease activity might be involved with normal ECM remodeling, or normal neuronal function [71].

Although the results presented suggest the pivotal role of plasminogen in retinal damage, the source of plasminogen in the retina after optic nerve ligation is also not clear at this time. Previous results suggest the contribution of endothelial and glial cells to plasminogen synthesis in the retina [72-74]. It is possible that plasminogen could be originated from vitreous contamination. The absence of plasminogen in control retinal extracts at any given time period after optic nerve ligation, however, indicates that this might not be the case. In addition, BRB breakdown associated with ischemia [44,45] could also reflect a nonselective increase in the permeability of retinal capillaries to high molecular weight proteins, including plasminogen, which is present mostly in the blood plasma. Our data suggest that increased levels of plasminogen in retinal extracts might be due to BRB breakdown. In support of this, we observed the presence of increased levels of serum albumin (Figure 3F), a protein that has been used to demonstrate BRB breakdown, consistently in retinal extracts after optic nerve ligation, coincident with the appearance of plasminogen. Once plasminogen is extravasated after BRB breakdown, uPA can degrade plasminogen to produce active plasmin, which could then act on ECM substrates such as laminins. In addition, tPA, which we observed in control retinal extracts, could also convert extravasated plasminogen to plasmin after optic nerve ligation and exacerbate retinal damage. This is one of the reasons for using α-2 antiplasmin to inhibit overall plasmin activity rather than uPA or tPA. Our results suggest that inhibition of plasmin activity might significantly protect ganglion cell loss after optic nerve ligation (Figure 6).

In summary, the data presented here, identifies for the first time the plasminogen activator-mediated pathological mechanisms responsible for retinal damage in a mouse optic nerve ligation model. These results demonstrate that the optic nerve ligation-induced plasminogen/plasmin system contributes to ganglion cell death in the retina, and inhibition of plasmin(ogen) activity in part, might represent a retinal protective strategy in ischemic blinding diseases of this tissue.

Acknowledgements

This work was supported by the National Eye Institute project grant, EY13643 (SKC). We thank Mrs. Mei Cheng for her technical assistance, and Drs. Seetaramayya Ari, Barry Winkler, and Kenneth Mitton for critical reading of the manuscript.

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