Expression of MatrixMetalloproteinases and Tissue

Inhibitors of Metalloproteinases inHuman Optic Nerve Head Astrocytes

OLGA A. AGAPOVA,1 CYNTHIA S. RICARD,1 MERCEDES SALVADOR-SILVA,1AND M. ROSARIO HERNANDEZ1,2*

1Department of Ophthalmology and Visual Sciences, Washington University School of Medicine,St. Louis, Missouri

2Department of Anatomy and Neurobiology, Washington University School of Medicine,St. Louis, Missouri

KEY WORDS MMPs; TIMPs; glaucoma; optic nerve head; lamina cribrosa; axons;extracellular matrix; glia

ABSTRACT Glaucomatous optic neuropathy is a common blinding disease charac-terized by remodeling of the extracellular matrix (ECM) and loss of retinal ganglion cell(RGC) axons at the level of the optic nerve head (ONH). Astrocytes, the major cell typein ONH, may participate in this process by production of matrix metalloproteinases(MMPs) and their inhibitors (TIMPs). In normal and glaucomatous ONH, we detectedMMP and TIMP expression by immunohistochemistry. Cultured astrocytes were used tocharacterize expression of MMPs and TIMPs by zymography, Western blot, and RNaseprotection assay. MMP production was stimulated with phorbol 12-myristate 13-acetate(PMA). Astrocytes expressed MMP1, MT1-MMP, MMP2, TIMP1, and TIMP2 in normaland glaucomatous ONH. MMP2, TIMP1, and TIMP2 localized to RGCs and their axons.Increased MMP1 and MT1-MMP expression was demonstrated in glaucoma. Culturedastrocytes constitutively expressed MMP2, MT1-MMP, TIMP1, and TIMP2, whereasMMP3, MMP7, MMP9, and MMP12 were not detectable in tissues or in culturedastrocytes. Our findings demonstrate the presence of specific MMPs and TIMPs in theONH that may participate in the homeostasis and remodeling of the ECM in glaucoma.Expression of the same MMPs and TIMPs in cultured ONH astrocytes will allow furtherstudies on the mechanisms regulating these enzymes. GLIA 33:205–216, 2001.© 2001 Wiley-Liss, Inc.

INTRODUCTION

Matrix metalloproteinases (MMPs) or matrixins area family of Zn–dependent metalloproteinases, whichhave the specialized function degrading the extracellu-lar matrix (ECM) components such as collagens, pro-teoglycans, elastin, laminin, fibronectin, and other gly-coproteins in normal and pathological conditions(Woessner, 1991). A multigene family encodes for se-creted and membrane-associated MMPs. MMPs aresynthesized in preproenzyme form and most of themare secreted from cells as proenzymes and activated inthe extracellular compartment. Currently, more then

20 MMPs are described, which are involved in normaland pathological processes connected with remodelingand destruction of the ECM (Nagase and Woessner,1999). Some MMPs are expressed constantly at certainlevel and in most cell types, while others are inducibleand tissue-specific (Fini et al., 1998). The expression

Grant sponsor: NIH; Grant number: EY-06416 and EY-02687.

*Correspondence to: M. Rosario Hernandez, Department of Ophthalmologyand Visual Sciences, Washington University School of Medicine, 660 S. EuclidAvenue, Box 8096, St. Louis, MO 63110. E-mail: hernandez@vision.wustl.edu

Received 28 September 2000; Accepted 20 November 2000

GLIA 33:205–216 (2001)

© 2001 Wiley-Liss, Inc.

and activity of MMPs is tightly regulated (Borden andHeller, 1997; Nagase, 1997). Specific proteins known asthe tissue inhibitors of metalloproteinases (TIMPs) arethe physiological regulators of these enzymes. TheTIMP family comprises four members (TIMP1-4) withhigh gene and protein homology (Douglas et al., 1997).

The optic nerve head (ONH) is thought to be the siteof retinal ganglion cell (RGC) damage in primary openangle glaucoma (POAG), a common blinding neurode-generative disease. POAG is characterized clinically bycupping or excavation of the optic disk and in manypatients by elevated intraocular pressure (IOP) (Quig-ley et al., 1983). At the microscopic level, disk cuppingin POAG is due to loss of the RGC axons and extensiveremodeling of the ECM at the level of the lamina cri-brosa of the ONH (Hernandez and Pena, 1997).

In humans and nonhuman primates, the lamina cri-brosa consists of several fibroelastic connective tissuelamellae, the cribriform plates. They are arranged inregister, leaving channels to allow the exit of the non-myelinated axons of the RGCs. Astrocytes, orientedhorizontally across the nerve head perpendicular to theaxons, line the cribriform plates (Anderson, 1969). Col-lagen fibers, elastic fibers, basement membranes, andproteoglycans form the ECM of the cribriform plates.Basement membranes separate the astrocytes from theunderlying ECM (Hernandez and Pena, 1997). In ad-dition, during development of the retina and opticnerve, the lamina cribrosa is thought to constitute abarrier to prevent migration of myelinating cells andmyelination of the retina (Perry and Lund, 1990).

In POAG, there is extensive remodeling of the ECMof the lamina cribrosa with increased expression ofECM, cell adhesion molecules, and neurotoxic media-tors by reactive astrocytes (Hernandez, 2000). Astro-cytes detach from their basement membranes and mi-grate into the nerve bundles (Varela and Hernandez,1997). There are marked changes in elastic fibers at thelevel of the lamina cribrosa, which lead to elastoticdegeneration of the ECM (Pena et al., 1998). In addi-tion, there is a decrease in the density of collagen fibersin the tissue (Quigley et al., 1991; Hernandez, 1992).However, in POAG, despite all these changes, there isno neovascularization, no breakdown of the blood-nerve barrier, no obvious sign of inflammation, and noformation of a typical glial scar. This pathophysiologysuggests a very specific and tightly regulated activity ofECM-degrading enzymes and their inhibitors.

The important role of MMPs and TIMPs preservingthe homeostasis of the ECM in most tissues led us toidentify and localize the major MMPs and TIMPs in thenormal and glaucomatous ONH and in cultured type1B astrocytes. In this study, we used immunohisto-chemistry to screen human normal and glaucomatoustissues for expression and distribution of severalMMPs and TIMPs. In addition, we characterized theexpression, synthesis, and enzymatic activity of MMPsand TIMPs in cultured human ONH astrocytes (type1B) using casein and gelatin zymography, Westernblots, and RNase protection assays.

MATERIALS AND METHODSTissue Preparation

Human eyes from nine donors, ages 54–85 (70 6 12years, mean 6 SD), with no history of eye disease,diabetes, or neurodegenerative disease, were obtainedfrom eye banks throughout the United States throughNational Disease Research Interchange (NDRI) andthe Mid-America Transplant Services (Saint Louis,MO). Eyes from 14 donors, ages 47–89 (71 6 14 years),with well-documented POAG, were obtained throughthe Glaucoma Research Foundation and NDRI. Theeyes with glaucoma had extensive histories, whichwere evaluated by an ophthalmologist to ascertainPOAG and degree of damage. In addition, cross-sec-tions of the myelinated nerves stained with p-phenyl-enediamine were routinely checked to confirm axonalloss (Pena et al., 1998). The eyes with POAG wereseparated into three groups according to the clinicalhistories into mild, moderate, and advanced POAG.Due to the nature of this study, there was no history ofsepsis or infections in any of the donor used. The causeof death for all donors was myocardial infarction orcardiopulmonary failure.

The eyes were enucleated shortly after death (2–4 h)and fixed in 10% neutral buffered formalin and trans-ported to the laboratory on ice within 24 h after death.The optic nerves were dissected, washed several timesin phosphate buffered saline (PBS) containing 20 mMof glycine, and paraffin-embedded according to stan-dard protocol. Four eyes with POAG and two normalage-matched controls were embedded in O.C.T. Com-pound (Miles, Elkhart, IN) and flash-frozen in methylbutane cooled in liquid nitrogen.

Immunochistochemistry

Six mm sagittal sections were used for MMP1,MMP2, MMP3, MMP7, MMP9, MMP12, MT1-MMP,and TIMP1 immunodetection. For immunoperoxidasedetection, sections after blocking endogenous peroxi-dase activity were incubated with primary antibodies(Table 1) and then with appropriate biotinylated sec-ondary antibodies, avidin–biotinylated peroxidase,and DAB (39,39-diaminobenzidine) substrate fromVestastain ABC Kit (Vector Laboratories, Burlingame,CA); nuclei were counterstained with hematoxylin. ForTIMP1 detection, antigen retrieval in 1 mM EDTA, pH8.0, at 100°C 20 min was performed. For TIMP2 andTIMP1 detection, we used 15 mm sagittal frozen sec-tions followed by immunofluorescence staining. Fordouble immunofluorescence staining, we used mousemonoclonal antibody against human glial fibrillaryacid protein (GFAP) and rabbit polyclonal antibodiesagainst MMP1, MT1-MMP, TIMP1, and TIMP2 (Sig-ma, St. Louis, MO) and Alexa 488 and Alexa 568–labeled secondary antibodies (Molecular Probes, Eu-gene, OR). For negative control, the primary antibodywas replaced for nonimmune serum. To control for

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cross-reactivity in double immunofluorescence, sec-tions were incubated with primary antibody followedby the wrong species secondary antibody. Serial sec-tions of normal and glaucomatous eyes were stainedsimultaneously to control for variations in immuno-staining. Formalin-fixed, paraffin-embedded humanplacenta or human breast carcinoma sections, knownto contain most of the MMPs and TIMPs, served as apositive control for immunostaining.

Visualization and Photography

Slides were examined by microscopy (BHS, Olympus,Tokyo, Japan) and images were recorded using a digi-tal camera (Spot, Diagnostic Instruments, SterlingHeights, MI) and stored as computer files in Photoshop(Adobe, version 5.0). Double immunofluorescent im-ages were obtained by a Zeiss LSM 410 laser-scanningconfocal microscope (Carl Zeiss, Thornwood, NY). Im-ages were stored as TIF files and subsequently ana-lyzed by Adobe.

Cell Culture

Three pairs of human eyes from donors without his-tory of eye disease (ages 42–46) were used to obtainprimary cultures of type 1B astrocytes from the ex-planted human optic nerve heads (Kobayashi et al.,1997). Type 1B astrocytes (passages 3–5) were platedat 2 3 105 cells per 100 mm dish and grown to conflu-ence in DMEM/F12 with 10% FBS at 37°C and 5% CO2.Cells were preincubated (20 h) in DMEM/F12 withInsulin-Transferrin-Selenite (ITS; Sigma) and treatedin fresh media with PMA (5 3 1027 M) 6 or 24 h.Control cells were preincubated and grown in freshDMEM/F12 with ITS 6 or 24 h. Conditioned mediawere collected in test tubes and stored at 280°C. ForWestern blot and casein zymography, media were con-centrated 20 times with the Centricon 10 CentrifugalFilter Devices (Amicon, Millipore, Bedford, MA)

Protein Extraction

Cells were washed twice in cold 1 3 PBS and incu-bate in 500 ml of ice-cold RIPA buffer (50 mM Tris HCl,pH 7.5, 150 mM NaCl, 1 mM EGTA, 1% IGEPAL CA-630, 0.5% deoxycholate, Roche protease inhibitors,Roche Molecular Biochemicals, Indianapolis, IN). After15-min incubation, cells were scraped with disposablecell lifters and centrifuged for 15 min at 4°C and 14,000RPM. The supernatant was recovered and protein con-centration in cell lysates determined by Bio-Rad (Her-cules, CA) Protein Assay Kit (Bradford method). Sam-ples were stored at 280°C.

Zymography

Proteins (20 ml of conditioned media for gelatin and20 ml 20 3 media for casein zymography) were sepa-rated by SDS–polyacrylamide gel electrophoresis(SDS-PAGE) on 8% separating gels with 0.1% gelatin,or with 0.2% casein, without reduction. After electro-phoresis, gels were washed in 2.5% Triton X-100 twotimes on the shaker for 15 min at room temperature toremove the SDS. Gels were incubated overnight at37°C in incubation buffer (50 mM Tris pH 8.2, 5 mMCaCl2, 0.5 mM ZnCl2), stained with 0.25% Coomassieblue, and destained in 5% acetic acid and 10% metha-nol in H2O. Areas of proteolytic activity were detectedas transparent bands. Some samples were treated 1 hat 37°C with 10 mM APMA (p-aminophenylmercuricacetate–MMP activator) in 50 mM NaOH, or with 50mM NaOH as negative control of activation. In a fewexperiments, 10 mM EDTA was added to incubationbuffer to inhibit MMP activity. Gelatinase zymographystandard (Chemicon, Temecula, CA) and MMP control1 (Sigma) were used as positive controls for gelatin andcasein zymography.

Western Blot

Samples (10 mg of cell lysates and 20 ml 20 3 condi-tioned media) were run on 4%–15% gradient SDS poly-

TABLE 1. Antibodies for immunohistochemistry and western blots

Antigen Antibodies Source and catalog number Immunohistochemistry Western blot

TIMP1 Mouse mono Neomarkers, MS-608 1:50TIMP1 Rabbit poly Sigma, T8187 1:100 1:1,000TIMP2 Rabbit poly Sigma, T8062 1:100 1:1,000TIMP3 Rabbit poly Sigma, T7812 1:1,000MMP1 Rabbit poly Sigma, M4177 1:200 1:1,000MMP2 Rabbit poly Chemicon, AB809 1:1,000MMP2 Mouse mono Neomarkers, MS-567 1:100MMP3 Mouse mono Neomarkers, MS-810 1:25MMP3 Rabbit poly Chemicon, AB810 1:1,000MMP7 Rabbit poly Gift from Dr. Parks 1:1,000MMP9 Mouse mono Chemicon, MAB3300 1:200 1:1,000MMP12 Rabbit poly Gift from Dr. Shapiro 1:350MT1-MMP Rabbit poly Sigma, M3927 1:400 1:1,000GFAP Mouse mono Sigma, G3893 1:400

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acrylamide gel under reduction conditions and trans-ferred to Bio-Rad nitrocellulose membrane. Membranewas blocked for 1 h in blocking solution (PBS, 0.05%Tween-20, 5% Amersham blocking agent, AmershamPharmacia Biotech, Piscataway, NJ) and incubated 1 hwith primary antibody (Table 1) diluted in PBS with0.05% Tween-20 and 2.5% blocking agent. After washingwith PBS/0.05% Tween-20, membrane was incubatedwith goat anti-rabbit or goat anti-mouse horseradishperoxidase-labeled secondary antibody (Amersham)diluted in blocking solution. After additional washes,binding of the peroxidase-labeled antibody was visual-ized by using ECL Western blotting detection system(Amersham).

RNA Isolation

Total cytoplasmic RNA was isolated as previouslydescribed (Favaloro et al., 1980). Cells were rinse twicein saline and then scraped into saline. Cells were re-covered by centrifugation and resuspended in TSM (10mM Tris, pH 7.4, 150 mM NaCl, 2 mM MgCl2) 1 0.5%IGEPAL CA-630. After incubation on ice (2–3 min),cells debris were pelleted. An equal volume of TSE1S(10 mM Tris, pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.2%SDS) was mixed with supernatant. The samples wereextracted twice with 50% phenol 1 50% chloroform/isoamyl alcohol (24:1) and twice with chloroform/isoamyl alcohol (24:1). The supernatant was adjustedto 0.1 M NaCl, 2 volumes of ethanol were added andprecipitated overnight at 220°C. RNA was quantifiedby measuring absorbence at 260 nm and purity wasassessed by calculating the absorbence ratio 260/280nm.

Plasmid Constructs for Riboprobes

Probe cDNA fragments for MMP2, MMP3, MMP7,and MMP9 were cut from cDNA (a gift from Dr. W.C.Parks) with appropriate restriction enzymes andcloned into vectors for in vitro transcription. Differentvectors were used for cloning of different cDNA frag-ments due to restriction enzyme site compatibility.MMP3 and MMP7 cDNA fragments were cloned intopBluescript KS (Stratagene, La Jolla, CA), MMP9 intopBluescript SK (Stratagene), and MMP2 intopGEM3Zf(1) (Promega, Madison, WI). Probe cDNAfragments for MT1-MMP, MMP1, TIMP1, and TIMP2were synthesized by RT-PCR of total RNA from cul-tured ONH astrocytes treated with PMA. MMP1, MT1-MMP, and TIMP1 PCR fragments were cloned intopGEM(R)-T Easy (Promega), cut with EcoRI, andcloned into pBluescript KS. TIMP2 PCR fragment wascloned directly into pBluescript KS into SmaI site. Se-quencing proved fragment orientation and sequence.The specificity of these sequences was confirmed bycomputer-assisted alignment with the reported cDNAsequences for MMPs and TIMPs (see accession num-

bers in Table 2). One mg of each linearized plasmid wasused for making antisense transcripts (riboprobes).The linearized Ambion pTRI RNA18S plasmid (Austin,TX) was used to produce 18SrRNA antisense probethat serve as internal control for the amount of RNA ineach sample. Positions of the specific target sequencesused to generate riboprobes for MMP and TIMP mRNAdetection and also probe and protected fragment sizesare detailed in Table 2.

RNase Protection Assay

Radioactively labeled antisense transcripts (ribo-probes) were produced utilizing MEGAscript (for low-specific-activity 18S RNA probe, ; 5 3 104 cpm/mg) orMAXIscript (for high-specific-activity MMPs andTIMPs probes, ; 109 cpm/mg) in vitro transcriptionKits (Ambion). a-[32P] UTP (ICN, Costa Mesa, CA) andappropriate RNA polymerase (T3 or T7) were used forin vitro transcription. RNase protection assay was per-formed using RPA III Kit from Ambion. The riboprobes(5 3 104 cpm) were annealed to 5 mg total RNA 5 minat 85°C and then at 54°C overnight in Hybridization IIIBuffer. RNase digestion was performed with RNaseA/RNaseT1 diluted 1:50 in RNase Digestion III Buffer at37°C 30 min. After RNase inactivation and RNA du-plexes precipitation, samples were dissolved in GelLoading Buffer II, denatured at 90°C for 5 min, and runon 8% polyacrylamide sequencing gel. Probe sets (2 3103 cpm each) and transcribed RNA Century MarkerTemplates (Ambion; 3 3 103 cpm) were run on theseparate lanes as size standard.

RESULTSMMP and TIMP Expression in Normal and

Glaucomatous Optic Nerve Head

The distribution of MMPs and TIMPs in normal andglaucomatous human optic nerve heads were analyzedusing immunoperoxidase staining for MMP1, MMP2,MMP3, MMP7, MMP9, MMP12, MT-MMP1, andTIMP1. Immunofluorescence staining was used forTIMP1 and TIMP2 detection. Double immunofluores-

TABLE 2. Positions of the specific target sequences used to generateriboprobes for MMPs and TIMPs mRNA detection and probe and

protected fragment sizes

SequenceAccessionnumber

Probe size(base)

Protectedfragment

size (base)

MMP1 1053-1342 NM_002421 388 290MMP7 403-679 NM_002423 320 277MMP2 1502-1752 J03210 308 251TIMPI 302-491 S68252 316 190MMP3 1585-1757 NM_002422 227 173TIMP2 571-739 S48568 255 169MMP9 1982-2123 NM_004994 206 142MT1-MMP 1364-1486 NM_004995 185 12318SrRNA 128 80

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cence staining for MMPs or TIMPs and the astrocytemarker, GFAP, was performed to determine whetherthe cells expressing MMPs and TIMPs were astrocytes.Three distinct histological regions of the optic nervehead, the prelaminar region, lamina cribrosa, and post-laminar region, were analyzed.

MMP1

In the normal adult ONH, very low levels of MMP1immunoreactivity was observed in association with as-trocytes in the glial columns in the prelaminar region(Fig. 1A) and in the cribriform plates in the laminacribrosa (Fig. 1B). There were few labeled astrocytes inthe postlaminar myelinated optic nerve (Fig. 1C). Inthe glaucomatous ONH, immunoreactivity for MMP1was markedly stronger than in normals in the laminarand postlaminar regions (Fig. 1). Immunostaining forMMP1 was associated with astrocytes and ECM in thecribriform plates. In glaucoma, MMP1 was also local-ized to the reactive astrocytes and axons in nerve bun-dles in the laminar and postlaminar regions (Fig. 1Eand F). MMP1 immunoreactivity was detected in smallvessels throughout the ONH. Double immunofluores-cent staining for GFAP and MMP1 showed localization

of MMP1 to astrocytes in the cribriform plates and inthe nerve bundles. MMP1 also stained the ECM in thecribriform plates and pial septa (Fig. 2A and B).

MT1-MMP

In the normal adult ONH granular immunostainingfor MT1-MMP was associated with blood vessels (Fig.3A and C) and with a few astrocytes lining the cribri-form plates in the lamina cribrosa (Fig. 3B). No MT1-MMP immunoreactivity was detected in axons andECM. In the glaucomatous ONH, high level of MT1-MMP immunoreactivity was observed in the laminacribrosa and the postlaminar optic nerve (Fig. 3E andF). Double immunofluorescence staining for GFAP andMT1-MMP demonstrated that MT1-MMP was associ-ated with astrocytes in the optic nerve head (Fig. 2Cand D).

MMP2

MMP2 was expressed in the ONH and retina. Therewas no difference in the level of expression betweennormal and glaucomatous ONH (Fig. 4). Retinal gan-glion cell bodies and their axons in the nerve fiber layerwere MMP2-positive (Fig. 4A and E). In the ONH,MMP2 was associated with the astrocytes in the glialcolumns and axons in the prelaminar region (Fig. 4Band F), astrocytes in the cribriform plates, and axons inthe lamina cribrosa (Fig. 4C and G). MMP2 immuno-reactivity in the nonmyelinated axons in the prelami-

Fig. 1. MMP1 immunoperoxidase staining in normal (A–C) andglaucomatous (D–F) ONH. A and D, prelaminar region; B and E,lamina cribrosa; C and F, postlaminar region. In glaucomatous ONH(D–F), significantly higher level of MMP1 immunoreactivity than incontrol (A–C) was detected in laminar (E) and postlaminar (F) regionsin association with astrocytes (arrows) and extracellular (arrowheads)in glial columns, cribriform plates, pial septa, and nerve bundles. GC,glial columns; CP, cribriform plates; PS, pial septa; NB, nerve bun-dles; V, blood vessels. Scale bar, 20 mm.

Fig. 2. Colocalization of MMP1 and MT1-MMP with GFAP in theglaucomatous lamina cribrosa. Double immunofluorescence stainingfor GFAP (green) and MMP1 (red) (A and B) and for GFAP (green)and MT1-MMP (red; C and D). Arrows point to the cells that showdouble staining. Arrowheads point to the extracellular localization ofMMP1. NB, nerve bundles; CP, cribriform plates; PS, pial septa. Scalebar, 25 mm.

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nar and laminar regions was stronger than in the post-laminar myelinated nerve (Fig. 4D and H).

MMP3, MMP7, MMP9, MMP12

In normal and glaucomatous adult ONH, no detect-able immunoreactivity for MMP3 was observed in as-sociation with astrocytes, ECM, or nerve bundles. How-ever, MMP3-positive staining was detected in the bloodvessels throughout the region. Immunoreactivity forMMP3 was associated with the perivascular cells (datanot shown). No immunoreactivity for MMP7, MMP9,and MMP12 localized in normal and glaucomatousadult ONH (data not shown).

TIMP1 and TIMP2

In the normal and glaucomatous ONH, immuno-staining for TIMP1 was associated with astrocytes andaxons in the prelaminar region (Fig. 5A and D) and inthe lamina cribrosa (Fig. 5B and E). In the postlaminarnerve, TIMP1 was associated with the axons and withastrocytes lining the pial septa (Fig. 5C and F). Double

immunofluorescence staining for TIMP2 and GFAPdemonstrated TIMP2 localization in the ONH similarto TIMP1 (Fig. 6). There were no apparent differencesin the level of staining for TIMP1 and TIMP2 in nor-mals and glaucomatous samples using either fluores-cence or peroxidase detection (Figs. 5 and 6). Clearpositive immunoreactivity for both TIMP1 and TIMP2was localized to the soma of RGCs and their axons innerve fiber layer (Fig. 7). Astrocytes in the nerve fiberlayer did not express TIMP1 or TIMP2 (Fig. 7).

MMP and TIMP Expression in Cultured HumanType 1B Astrocytes

Zymography

Gelatin and casein zymography were performed fordetection of the proteolytic activity in conditioned me-

Fig. 3. MT1-MMP immunoperoxidase staining in normal (A–C)and glaucomatous (D–F) ONH. A and D, prelaminar region; B and E,lamina cribrosa; C and F, postlaminar region. Positive immunoreac-tivity to MT1-MMP was localized to blood vessels (A, C, and D).MT1-MMP immunoreactivity is remarkably higher in glaucomatousOHN in lamina cribrosa (E) and postlaminar region (F) compare tonormal (B and C). Arrows point to immunoreactive astrocytes. V,blood vessels; GC, glial columns; NB, nerve bundles; CP, cribriformplates; PS, pial septa. Scale bar, 20 mm.

Fig. 4. MMP2 immunoperoxidase staining in normal (A–D) andglaucomatous (E–H) ONH and retina. A and E, retina; B and F,prelaminar region; C and G, lamina cribrosa; D and H, postlaminarregion. MMP2 immunoreactivity localized in RGCs (arrows) and theiraxons in nerve fiber layer in retina (A and E), in glial columns,cibriform plates, pial septa, and nerve bundles in prelaminar (B andF), laminar (C and G), and postlaminar regions (D and H). NFL, nervefiber layer; GC, glial columns; CP, cribriform plates; PS, pial septa;NB, nerve bundles; V, blood vessels. Scale bar, 20 mm.

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dia from cultured ONH astrocytes (Fig. 8). Gelatino-lytic activity at 68 kDa (Fig. 8A, lane 1) and lightcaseinolytic activity at 50 kDa (Fig. 8B, lane 1) weredetected in 24 h conditioned media from nontreatedcells. Media from cells treated 24 h with PMA demon-strated the same level activity at 68 kDa and a newactivity at 92 kDa by gelatin zymography (Fig. 8A, lane2). Caseinolytic activity at 50 kDa was apparently in-creased after PMA treatment and an additional case-inolytic activity at 78 kDa was detected (Fig. 8B, lane 2).

To determine whether proteolytic activities detectedby zymography were indeed MMPs, conditioned mediafrom astrocytes were treated with p-aminophenylmer-curic acetate (APMA) for MMP activation or withEDTA to inhibit MMP activity. After treatment withAPMA, the gelatinolytic and caseinolytic bands at 92,68, and 50 kDa partly converted to species of lowermolecular weight at 86, 64, and 47 kDa, respectively(Fig. 8A and B, lanes 3). However, the band at 78 kDadid not change after APMA treatment (Fig. 8B, lane 3),indicating that this proteolytic activity does not corre-spond to MMPs. EDTA inhibited protease activity at92, 68, and 50 kDa (data not shown). According theirmobility, these MMPs are MMP9 (gelatinase B, 92kDa), MMP2 (gelatinase A, 68 kDa), and MMP1 (inter-stitial collagenase, 50 kDa).

Western blots

In order to confirm our zymography results, Westernblot detection of MMPs and TIMPs in conditioned me-dia and cell lysates was carried out.

MMP2 was detected in media and cell lysates fromcontrol and PMA-treated cells. No additional inductionof MMP2 expression was observed after PMA treat-ment (data not shown). MMP9 was not detectable inconditioned media and cell lysates in control samples.However, after 24 h of PMA treatment, a light bandabout 92 kDa appeared in media and cell lysates (datanot shown). Low expression of MMP1 was found inconditioned media from control astrocytes (Fig. 9, lanes1 and 2), but a clear induction of MMP1 both in celllysates and media was observed after PMA treatment(Fig. 9, lanes 3 and 4). MMP3 was not detectable byWestern blot of cell proteins or media from controlastrocytes or after PMA treatment (data not shown).MT1-MMP was detected as a 65 kDa band in cell ly-sates from control astrocytes (Fig. 9, lane 5). A slightupregulation of MT1-MMP was observed after 24 hPMA treatment (Fig. 9, lane 7).

TIMP1 and TIMP2 antibodies recognized bands at 30and 24 kDa in conditioned media (Fig. 9). TIMP2 ex-pression did not change after PMA treatment. A slightincrease of TIMP1 in the media was detected afterPMA treatment for 24 h compared with untreated con-trols (Fig. 9, lanes 2 and 4). In control cells, TIMP1 wasidentified as a band at approximately 35 kDa, but afterPMA treatment, an additional band 30 kDa was ap-peared in cell lysates (Fig. 9, lanes 5–7).

MMP and TIMP m-RNA Expression

MMP and TIMP m-RNA expression in cultured type1B astrocytes was characterized using RNAse protec-tion assay. Five mg of total RNA from ONH astrocyteswas hybridized with antisense riboprobes for MMP1,MMP2, MMP3, MMP7, MMP9, MT1-MMP, TIMP1,TIMP2, and 18SrRNA (internal control; Table 2). Pro-tected fragments of expected size for MMP2, MT1-MMP, TIMP1, TIMP2, and 18SrRNA were visible incells grown in control conditions (Fig. 10, lanes 1, 4, 5).No detectable bands for MMP1, MMP3, MMP7, andMMP9 mRNA were visible in control cells. There wereno differences in band intensity and detection between6- and 24-h controls (data not shown).

When total RNA from cells treated with PMA wasused for hybridization, we detected the MMP1 mRNAafter 6 and 24 h of treatment (Fig. 10, lanes 2 and 3).The amount of MT1-MMP mRNA was clearly increasedafter 6- and 24-h PMA treatment (Fig. 10, lanes 6 and7). TIMP1, MMP2, and TIMP2 mRNA were detected atthe same level as in control, untreated cells (Fig. 10,lanes 1–3, 5–7). MMP3, MMP7, and MMP9 mRNAwere not detectable after 6- or 24-h PMA treatment, asin control (Fig. 10).

Fig. 5. TIMP1 immunoperoxidase staining in normal (A–C) andglaucomatous (D–F) ONH. A and D, prelaminar region; B and E,lamina cribrosa; C and F, postlaminar region. TIMP1 immunoreac-tivity in normal and glaucomatous ONH is localized in glial columns,cribriform plates, pial septa, and nerve bundles in prelaminar (A andD), laminar (B and E), and postlaminar (C and F) regions. GC, glialcolumns; CP, cribriform plates; PS, pial septa; NB, nerve bundles; V,blood vessels. Scale bar, 20 mm.

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DISCUSSION

This study reports the immunohistochemical local-ization of MMPs and TIMPs in normal and glaucoma-tous ONH. MMP and TIMP expression was furthercharacterized at the protein and mRNA level in type1B astrocytes cultured from human ONH. The resultsindicate that, as in the central nervous system (CNS),a restricted group of MMPs is expressed in normal andglaucomatous ONH and in cultured type 1B astrocytes.MMPs were expressed at low level in normal ONH. Theexpression of some MMPs was increased in glaucoma.

However, TIMP expression was detected at high levelin both normal and glaucomatous tissues. Interest-ingly, TIMP1 and TIMP2 localized to the RGCs andtheir axons in the retina and ONH, suggesting thatinhibition of MMP activity plays an important role inmaintaining the intactness of these tissues in normaland pathological conditions.

The main role of MMP1 or interstitial collagenase isdegradation of fibrillar collagen type I, II, and III (Im-per and Van Wart, 1998). The presence of increasedamounts of MMP1 in reactive astrocytes and in theECM of the glaucomatous lamina cribrosa is in agree-

Fig. 6. Double immunofluorescence staining for GFAP (green) andTIMP2 (red) in lamina cribrosa in normal (A) and glaucomatous (B)ONH. TIMP2 immunostaining of nerve bundles and colocalization

(yellow) of GFAP and TIMP2 in astrocytes (arrows) in cribriformplates and nerve bundles. CP, cribriform plates; NB, nerve bundles.Scale bar, 25 mm.

Fig. 7. Double immunofluores-cence staining for TIMP1 (red) andGFAP (green; A), and TIMP2 (red)and GFAP (green; B) of retina inglaucomatous eye. TIMP1 andTIMP2 staining of RGC bodies (ar-rows) and their axons in nerve fiberlayer and GFAP-positive astrocytes(green) in nerve fiber layer (A andB). NFL, nerve fiber layer; VS, vit-real surface. Scale bar, 25 mm.

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ment with previous ultrastructural qualitative andquantitative studies that demonstrated loss of collagenfibers in the ECM of the cribriform plates (Quigley etal., 1991; Hernandez, 1992). Our study indicates thattype 1B astrocytes are most likely to be the source ofMMP1 in glaucoma. As was shown recently, culturedhuman brain astrocytes produced increased amounts ofMMP1 after stimulation with IL-1b (Vos et al., 2000).The authors also reported that MMP1 was cytotoxic toorganotypical spinal cord and neuronal cultures and

hypothesized that MMP1 exerted its effects throughthe destruction of ECM or by activation of membrane-bound receptors/cytokines as well as soluble cytokines(Vos et al., 2000).

MT1-MMP is an integral membrane MMP that canparticipate in ECM degradation by activation ofproMMP2 on the cell surface (Strongin et al., 1995).Recently, it was shown that MT1-MMP might cleavefibrillar collagens, proteoglycans, and various ECMglycoproteins in vitro (Ohuchi et al., 1997). The associ-ation between MT1-MMP and invasion of astrocytictumors in the CNS has been studied in detail (Belien etal., 1999; Nakada et al., 1999). MT1-MMP enables in-vasive migration of glioma cells not only throughproMMP2 activation, but also by direct degradation ofproteins that inhibit cell migration in CNS (Belien etal., 1999). MT1-MMP is expressed constitutively at lowlevel in normal ONH tissues and in type 1B astrocytesin culture. Increased expression of this enzyme was

Fig. 8. Gelatinolytic (A) and caseinolytic (B) activity of conditionedmedia (CM) from cultured ONH astrocytes. Lane 1, untreated cells;lanes 2–4, CM from cells PMA-treated for 24 h; lane 3, CM incubatedwith 10 mM APMA (p-aminophenylmercuric acetate) in 50 mM NaOHfor 1 h at 37°C for MMP activation; lane 4, CM incubated in 50 mMNaOH for 1 h at 37°C as a negative control of MMP activation.Prestained Molecular Weight Standard (Bio-Rad) of low range wasrun in each gel for estimating the molecular weights of sample pro-teins. 92 kDa, proMMP9; 86 kDa, active MMP9; 68 kDa, proMMP2; 64kDa, active MMP2; 78 kDa, protease, but not MMP; 50 kDa,proMMP1; 47 kDa, active MMP1.

Fig. 9. Western blots of ONH astrocyte conditioned media (CM) andcell lysates (CL) with antibodies to TIMP2, TIMP1, MMP1, and MT1-MMP. Lanes 1–4, CM; lanes 5–6, CL. Lanes 1 and 2: 6- and 24-h CMfrom control cells. Lanes 3 and 4: 6- and 24-h CM from PMA-treatedcells. Lane 5, untreated cells. Lanes 6 and 7: 6- and 24-h PMA-treatedcells. Prestained Molecular Weight Standard (Bio-Rad) of broad rangewas run in each gel for estimating the molecular weights of sampleproteins. Seven Western blots are shown and 65 kDa, 52 kDa, 30 kDa,and 24 kDa indicate MT1-MMP, MMP1, TIMP1, and TIMP2 molecu-lar weights, respectively.

Fig. 10. RNase protection assay of total RNA from control cells andcells treated with PMA for 6 or 24 h with riboprobes for MMPs andTIMPs. M, RNA Century Size Markers a set of five in vitro transcriptsof 100, 200, 300, 400, and 500 bases (Ambion). Lanes 1, 4, 5: hybrid-ization with 5 mg of total RNA from 24-h control cells. Lanes 2 and 6and lanes 3 and 7, hybridization with 5 mg of total RNA from 6- and24-h PMA-treated cells, respectively. Lanes 1–3, hybridization withriboprobes for MMP1, MMP3, MMP7, MMP9, TIMP1, and 18SrRNA;lane 4, hybridization with riboprobes for MMP1, MMP2, MMP3,MMP7, MMP9, MT1-MMP, TIMP1, TIMP2, and 18SrRNA; lanes 5–7,hybridization with riboprobes for MMP2, MT1-MMP, TIMP2, and18SrRNA. Protected fragments for MMP1 (290 bases), MMP2 (251,235 bases), TIMP1 (190 bases), TIMP2 (169 bases), MT1-MMP (140,123 bases), and 18SrRNA (80 bases) are indicated. No protectedfragments for MMP3 (173 bases), MMP7 (277 bases), and MMP9 (142bases) are visible.

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evident in the glaucomatous ONH and was associatedwith reactive astrocytes. MT1-MMP may be a compo-nent of the transition of quiescent astrocytes to thereactive phenotype in glaucoma, which requires achange in cell shape, detachment from the basementmembrane, and migration through the ECM. MT1-MMP activities are likely to be involved in this transi-tion by degrading cell surface adhesion molecules and,through disruption of the extracellular domains, byaltering the cytoskeleton (Werb, 1997). Expression ofMT1-MMP and proMMP2 may be required to localizeactivity of MMP2 to the cell surface to accomplish thistransition (Chen and Wang, 1999).

MMP2 or gelatinase A degrades gelatin, type IV col-lagen, and elastin (Matrisian, 1992; Yu et al., 1998).The ubiquitous distribution, lack of significant tran-scriptional regulation, and specific cellular mechanismfor the control of MMP2 activation make this enzymeunique among matrixins (Yu et al., 1998). As in othertissues, MMP2 is expressed constantly in brain. Lowlevel of MMP2 mRNA expression was demonstrated inthe mouse brain (Pagenstecher et al., 1998), culturedrat brain astrocytes (Wells et al., 1996), and in humanbrain neurons and astrocytes (Del Bigio and Jacque,1995; Anthony et al., 1997). MMP2 expression in brainneurons and astrocytes did not change after experi-mental cerebral focal ischemia in rats or in humanbrains with multiple sclerosis or stroke (Del Bigio andJacque, 1995; Anthony et al., 1997; Romanic et al.,1998). MMP2 was detected in astrocytes and axons inONH tissues and in RGC in retina at the same level innormal and glaucomatous eyes. The similar levels ofMMP2 in normal and glaucomatous ONH may implythat MMP2 in ONH, as in other tissues, plays impor-tant role in tissue homeostasis in normal and patho-logical conditions.

A previous report described the localization ofMMP1, MMP2, and MMP3 in human eyes with glau-coma (Yan et al., 2000). Our results confirm the pres-ence of MMP2 and MMP1 in human normal and glau-comatous tissues. In our study, however, the presenceof MMP3 or stromelysin 1 was not detectable in neuraltissues. MMP3 immunoreactivity was limited to perivas-cular cells in the ONH, perhaps smooth muscle cells, ormicroglia (Neufeld, 1999). In addition, MMP3 activitiesand mRNA were also undetectable in cultured ONHastrocytes with or without PMA stimulation.

MMP9 or gelatinase B has substrate specificity sim-ilar to MMP2 (Matrisian, 1992) and was first identifiedas a product of neutrophils and macrophages (Hibbs, etal., 1987). In the CNS, MMP9 has been detected inendothelial cells and in infiltrating neutrophils andmacrophages after experimental rat brain injury(Rosenberg et al., 1998) and in human brain in multi-ple sclerosis and stroke (Anthony et al., 1997). MMP9expression in the CNS correlates with the opening ofthe blood-brain barrier and inflammation (Mun-Bryceand Rosenberg, 1998; Pagenstecher et al., 1998). Dur-ing CNS inflammation, MMP9 and MMP12 were ex-pressed within the inflammatory lesions, most likely by

infiltrating leukocytes or activated microglia (Pagen-stecher et al., 1998). MMP7, MMP9, and MMP12 werenot detectable in normal and glaucomatous ONH or incultured type 1B astrocytes. The absence of these en-zymes suggests the lack of inflammatory cells and neo-vascularization in glaucomatous ONH.

TIMPs are the major endogenous regulators of MMPactivities in tissues. TIMP1 inhibits active MMP1,MMP3, and MMP9 and also binds to proMMP9,whereas TIMP2 preferentially binds to MMP2. TIMPsare multifunctional proteins that also can act asgrowth factors and inhibitors of angiogenesis (Gomezet al., 1997). In adult rat brain, TIMP1 and TIMP2mRNA but not TIMP3 or TIMP4 were markedly in-creased after stab injury. In situ hybridization demon-strated increased expression of TIMP1 mRNA in reac-tive astrocytes and TIMP2 mRNA in microglia andneurons under these experimental conditions (Jawor-ski, 2000). TIMP1 neuronal/axonal localization was re-ported in comparable levels in both the control andischemic cortical region of rat brain by immunohisto-chemistry (Romanic et al., 1998). In our study, TIMP1and TIMP2 were expressed at high levels in the ONHand in cultured astrocytes. In ONH, TIMP1 and TIMP2are present in both astrocytes and the nonmyelinatedaxons of the RGCs. Furthermore, their localization tothe RGC soma suggests that both TIMPs are synthe-sized in the RGC bodies and transported to the axons.TIMP1 and TIMP2 expressed by RGCs can protectnerve axons in the ONH from structural damage, dueto MMP proteolysis, in normal and pathological condi-tions. In ONH astrocytes, TIMP2 may be important foractivation of membrane-bound proMMP2. Culturedtype 1B astrocytes expressed only proMMP2 and activeMMP2 was detected in media only after APMA activa-tion in vitro. Considerably high level of TIMP2 expres-sion by type 1B astrocytes may explain the lack ofactive MMP2 in culture.

Growth factors and cytokines regulate MMP andTIMP expression, whereas MMPs can release growthfactors from the storage sites in the ECM and activatethe growth factor/cytokines or their receptors(Gottschall and Deb, 1996; Alexander et al., 1998; Qinet al., 1998; Rooprai et al., 2000; Yu and Stamenkovic,2000). TGFb2 is a multifunctional cytokine that ispresent at high level in the glaucomatous optic nervehead (Pena et al., 1999). Latent TGFb activation can bemediated by proteolysis of ECM-binding proteins byMMPs (Yu and Stamenkovic, 2000). Perhaps MMP2 inONH is required to release and activate TGFb2, whichin turn may increase ECM synthesis and inhibit MMPsand thus regulate tissue homeostasis. TNFa has alsobeen detected in the glaucomatous ONH (Yan et al.,2000; Yuan and Neufeld, 2000). TNFa is a potent cy-tokine that may play a neurodestructive role in glau-coma. In a rat model of experimental brain damage,TNFa induced marked upregulation of MMPs thatcause neuronal damage, which can be reduced by treat-ment with MMP inhibitors (Leib et al., 2000). Thus, theregulation and interplay of MMPs and TIMPs with

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growth factors and cytokines in the glaucomatous ONHrepresent important areas for future research.

In conclusion, the data described here represent theinitial characterization of the role of MMPs and TIMPsin the pathophysiology of glaucomatous optic neuropa-thy. It is well known that elevated IOP is a major riskfactor in glaucoma. IOP-related stress has been shownto be considerable at the tissue level (Bellezza et al.,2000). Induction of MT1-MMP in response to stretchhas been reported using cultured cardiac fibroblasts(Tyagi et al., 1998). Our laboratory demonstrated thathydrostatic pressure applied in vitro and that elevatedIOP in a monkey model of glaucoma induced increasedexpression and deposition of elastin by astrocytes inthe optic nerve head (Hernandez et al., 2000; Pena etal., 2000). Perhaps elevated IOP in glaucoma can in-duce increased expression of MT-MMP1 leading to de-tachment of astrocytes from the underlying basementmembranes and migration through the ECM. Subse-quently, MMP1 permits migration of astrocytesthroughout the ECM of the lamina cribrosa into thenerve bundles, where MMP1, if not counterbalanced byTIMP1, will continue to degrade the scant ECM aroundthe axons and interfere with axon survival. Our datasuggest that feedback regulation of MMP activity orexpression in reactive astrocyte occurs by interactionsbetween MMPs, TIMPs, growth factors/cytokines, andECM substrates with astrocyte membrane or intracel-lular components. Our results are consistent with thepathophysiology of POAG, a chronic neurodegenerativedisease that spans years, in which focal areas of dam-age to the optic nerve axons expand slowly, leading toRGC lost and blindness.

ACKNOWLEDGMENTS

The authors thank Mrs. Belinda McMahan and Ms.Ping Yang for excellent technical assistance. The Na-tional Disease Research Interchange (NDRI) and theGlaucoma Foundation (San Francisco, CA) have pro-vided the human eyes used in this study. The authorsalso thank Dr. William C. Parks, Washington Univer-sity School of Medicine, for helpful discussion of ourexperimental results and for the gift of the MMP7antibodies and human MMPs cDNA; Dr. Steven D.Shapiro, Washington University School of Medicine,for the gift of the MMP12 antibodies; and Dr. Ted S.Acott, Oregon Health Sciences University, for the giftof the MT1-MMP and TIMP2 PCR primers. Supportedby an unrestricted grant from the National Research toPrevent Blindness (RPB) to the Department of Oph-thalmology and Visual Sciences, Washington Univer-sity School of Medicine.

REFERENCES

Alexander JP, Samples JR, Acott TS. 1998. Growth factor and cyto-kine modulation of trabecular meshwork matrix metalloproteinaseand TIMP expression. Curr Eye Res 17:276–285.

Anderson DA. 1969. Ultrastructure of human and monkey laminacribrosa and optic nerve head. Arch Ophthalmology 82:800–814.

Anthony DC, Ferguson B, Matyzak MK, Miller KM, Esiri MM, PerryVH. 1997. Differential matrix metalloproteinase expression in casesof multiple sclerosis and stroke. Neuropathol Appl Neurobiol 23:406–415.

Belien AT, Paganetti PA, Schwab ME. 1999. Membrane-type 1 matrixmetalloprotease (MT1-MMP) enables invasive migration of gliomacells in central nervous system white matter. J Cell Biol 144:373–384.

Bellezza AJ, Hart RT, Burgoyne CF. 2000. The optic nerve head as abiomechanical structure: initial finite element modeling. InvestOphthalmol Vis Sci 41:2991–3000.

Borden P, Heller RA. 1997. Transcriptional control of matrix metal-loproteinases and the tissue inhibitors of matrix metalloprotein-ases. Crit Rev Eukaryot Gene Expr 7:159–178.

Chen WT, Wang JY. 1999. Specialized surface protrusions of invasivecells, invadopodia and lamellipodia, have differential MT1-MMP,MMP-2, and TIMP-2 localization. Ann NY Acad Sci 878:361–371.

Del Bigio MR, Jacque CM. 1995. Localization of proteinase expressionin the developing rabbit brain. Brain Res Dev Brain Res 86:345–347.

Douglas DA, Shi YE, Sang QA. 1997. Computational sequence anal-ysis of the tissue inhibitor of metalloproteinase family. J ProteinChem 16:237–255.

Favaloro J, Treisman R, Kamen R. 1980. Transcription maps of poly-oma virus-specific RNA: analysis by two-dimensional nuclease S1gel mapping. Methods Enzymol 65:718–749.

Fini ME, Cook JR, Mohan R, Brinckerhoff CE. 1998. Regulation ofmatrix metalloproteinase gene expression. In: Parks WC, MechamRP, editors. Matrix metalloproteinases. New York: Academic Press.p 299–356.

Gomez DE, Alonso DF, Yoshiji H, Thorgeirsson UP. 1997. Tissueinhibitors of metalloproteinases: structure, regulation and biologi-cal functions. Eur J Cell Biol 74:111–122.

Gottschall PE, Deb S. 1996. Regulation of matrix metalloproteinaseexpressions in astrocytes, microglia and neurons. Neuroimmuno-modulation 3:69–75.

Hernandez MR. 1992. Ultrastructural immunocytochemical analysisof elastin in the human lamina cribrosa: changes in elastic fibers inprimary open-angle glaucoma. Invest Ophthalmol Vis Sci 33:2891–2903.

Hernandez MR, Pena JD. 1997. The optic nerve head in glaucomatousoptic neuropathy. Arch Ophthalmol 115:389–395.

Hernandez MR. 2000. The optic nerve head in glaucoma: role ofastrocytes in tissue remodeling. Prog Retin Eye Res 19:297–321.

Hernandez MR, Pena JD, Selvidge JA, Salvador-Silva M, Yang P.2000. Hydrostatic pressure stimulates synthesis of elastin incultired optic nerve head astrocytes. Glia 32:122–136.

Hibbs MS, Hoidal JR, Kang AH. 1987. Expression of a metallopro-teinase that degrades native type V collagen and denatured colla-gens by cultured human alveolar macrophages. J Clin Invest 80:1644–1650.

Imper V, Van Wart HE. 1998. Substrate specificity and mechanismsof substrate recognition of the matrix metalloproteinases. In: ParksWC, Mecham RP, editors. Matrix metalloproteinases. New York:Academic Press. p 219–242.

Jaworski DM. 2000. Differential regulation of tissue inhibitor of met-alloproteinase mRNA expression in response to intracranial injury.Glia 30:199–208.

Kobayashi S, Vidal I, Pena JD, Hernandez MR. 1997. Expression ofneural cell adhesion molecule (NCAM) characterizes a subpopulationof type 1 astrocytes in human optic nerve head. Glia 20:262–273.

Leib SL, Leppert D, Clements J, Tauber MG. 2000. Matrix metallo-proteinases contribute to brain damage in experimental pneumo-coccal meningitis. Infect Immun 68:615–620.

Matrisian LM. 1992. The matrix-degrading metalloproteinases.Bioessays 14:455–463.

Mun-Bryce S, Rosenberg GA. 1998. Gelatinase B modulates selectiveopening of the blood-brain barrier during inflamation. Am J Physiol274:R1203–R1211.

Nagase H. 1997. Activation mechanisms of matrix metalloprotein-ases. Biol Chem 378:151–160.

Nagase H, Woessner JF Jr. 1999. Matrix metalloproteinases. J BiolChem 274:21491–21494.

Nakada M, Nakamura H, Ikeda E, Fujimoto N, Yamashita J, Sato H,Seiki M, Okada Y. 1999. Expression and tissue localization of mem-brane-type 1, 2, and 3 matrix metalloproteinases in human astro-cytic tumors. Am J Pathol 154:417–428.

Neufeld AH. 1999. Microglia in the optic nerve head and the region ofparapapillary chorioretinal atrophy in glaucoma. Arch Ophthalmol117:1050–1056.

215MATRIX METALLOPROTEINASES

Ohuchi E, Imai K, Fujii Y, Sato H, Seiki M, Okada Y. 1997. Membranetype 1 matrix metalloproteinase digests interstitial collagens andother extracellular matrix macromolecules. J Biol Chem 272:2446–2451.

Pagenstecher A, Stalder AK, Kincaid CL, Shapiro SD, Campbell IL.1998. Differential expression of matrix metalloproteinase and tis-sue inhibitor of matrix metalloproteinase genes in the mouse cen-tral nervous system in normal and inflammatory states. Am JPathol 152:729–741.

Pena JD, Netland PA, Vidal I, Dorr DA, Rasky A, Hernandez MR.1998. Elastosis of the lamina cribrosa in glaucomatous optic neu-ropathy. Exp Eye Res 67:517–524.

Pena JD, Taylor AW, Ricard CS, Vidal I, Hernandez MR. 1999.Transforming growth factor beta isoforms in human optic nerveheads. Br J Ophthalmol 83:209–218.

Pena JD, Kaufman PL, Kiland JA, Gabelt BT, Hernandez MR. 2000.Elastin expression in the lamina cribrosa of monkeys with experi-mental glaucoma and with optic nerve transection. Invest Ophthal-mol Vis Sci 41:S949.

Perry VH, Lund RD. 1990. Evidence that the lamina cribrosa preventsintraretinal myelination of retinal ganglion cell axons. J Neurocytol19:265–272.

Qin H, Moellinger JD, Wells A, Windsor LJ, Sun Y, Benveniste EN.1998. Transcriptional suppression of matrix metalloproteinase-2gene expression in human astroglioma cells by TNF-alpha andIFN-gamma. J Immunol 161:6664–6673.

Quigley HA, Hohman RM, Addicks EM, Massof RW, Green WR. 1983.Morphologic changes in the lamina cribrosa correlated with neuralloss in open-angle glaucoma. Am J Ophthalmol 95:673–691.

Quigley HA, Dorman-Pease ME, Brown AE. 1991. Quantitative studyof collagen and elastin of the optic nerve head and sclera in humanand experimental monkey glaucoma. Curr Eye Res 10:877–888.

Romanic AM, White RF, Arleth AJ, Ohlstein EH, Barone FC. 1998.Matrix metalloproteinase expression increases after cerebral focalischemia in rats: inhibition of matrix metalloproteinase-9 reducesinfarct size. Stroke 29:1020–1030.

Rooprai HK, Rucklidge GJ, Panou C, Pilkington GJ. 2000. The effectsof exogenous growth factors on matrix metalloproteinase secretionby human brain tumour cells. Br J Cancer 82:52–55.

Rosenberg GA, Estrada EY, Dencoff JE. 1998. Matrix metalloprotein-ases and TIMPs are associated with blood-brain barrier openingafter reperfusion in rat brain. Stroke 29:2189–2195.

Strongin AY, Collier I, Bannikov G, Marmer BL, Grant GA, GoldbergGI. 1995. Mechanism of cell surface activation of 72-kDa type IVcollagenase. Isolation of the activated form of the membrane met-alloprotease. J Biol Chem 270:5331–5338.

Tyagi SC, Lewis K, Pikes D, Marcello A, Mujumdar VS, Smiley LM,Moore CK. 1998. Stretch-induced membrane type matrix metallo-proteinase and tissue plasminogen activator in cardiac fibroblastcells. J Cell Physiol 176:374–382.

Varela HJ, Hernandez MR. 1997. Astrocyte responses in human opticnerve head with primary open-angle glaucoma. J Glaucoma 6:303–313.

Vos CM, Sjulson L, Nath A, McArthur JC, Pardo CA, Rothstein J,Conant K. 2000. Cytotoxicity by matrix metalloprotease-1 in orga-notypic spinal cord and dissociated neuronal cultures. Exp Neurol163:324–330.

Wells GM, Catlin G, Cossins JA, Mangan M, Ward GA, Miller KM,Clements JM. 1996. Quantitation of matrix metalloproteinases incultured rat astrocytes using the polymerase chain reaction with amulti-competitor cDNA standard. Glia 18:332–340.

Werb Z. 1997. ECM and cell surface proteolysis: regulating cellularecology. Cell 91:439–442.

Woessner JF Jr. 1991. Matrix metalloproteinases and their inhibitorsin connective tissue remodeling. FASEB J 5:2145–2154.

Yan X, Tezel G, Wax MB, Edward DP. 2000. Matrix metalloprotein-ases and tumor necrosis factor alpha in glaucomatous optic nervehead. Arch Ophthalmol 118:666–673.

Yu AE, Murphy AN, Stetler-Stevenson WG. 1998. 72-kDa gelatinase(gelatinase A): structure, activation,regulation, and substrate spec-ificity. In: Parks WC, Mecham RP, editors. Matrix metalloprotein-ases. New York: Academic Press. p 85–113.

Yu Q, Stamenkovic I. 2000. Cell surface-localized matrix metallopro-teinase-9 proteolytically activates TGF-beta and promotes tumorinvasion and angiogenesis. Genes Dev 14:163–176.

Yuan L, Neufeld AH. 2000. Tumor necrosis factor-alpha: a potentiallyneurodestructive cytokine produced by glia in the human glauco-matous optic nerve head. Glia 32:42–50.

216 AGAPOVA ET AL.