Matrix Metalloproteinases and their Inhibitors in Aqueous Humor

Exp. Eye Res. (1996) 62, 481–490

Matrix Metalloproteinases and their Inhibitors in Aqueous Humor S H E R L E E N H. H U A N Ga, b, A N T H O N Y P. A D A M I Sa, b*, D M I T R I G. W I E D E R S C H A INa, D A V I D T. S H I M Aa, c, Y U E N S H I NGa, d    M A R S H A A. M O S E Sa, d a

Laboratory for Surgical Research, Department of Surgery, Children’s Hospital, b Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, c Program in Cell and Developmental Biology and d Department of Surgery, Harvard Medical School, 300 Longwood Avenue, Boston, MA 02115, U.S.A. (Received Columbia 2 August 1995 and accepted in revised form 13 November 1995) Matrix metalloproteinase activity is the rate-limiting step in extracellular matrix degradation. One mechanism by which metalloproteinases are regulated is through the activity of their endogenous inhibitors, the tissue inhibitors of metalloproteinases. Since metalloproteinase activity is a key component of the angiogenic process and many anterior segment structures are largely avascular, we became interested in examining aqueous humor for the presence of metalloproteinases and their endogenous inhibitors. Using zymography, we have identified the presence of several metalloproteinases in normal aqueous humor. Treatment with 4-aminophenylmercuric acetate, an organomercurial which activates latent metalloproteinases, revealed that all metalloproteinases were in their active state. By Western blot analysis, normal aqueous humor was also found to contain at least two tissue inhibitors of metalloproteinases. Subsequent partial purification by two successive chromatographic steps revealed the presence of inhibitory activity against collagenase, endothelial cell DNA synthesis, and angiogenesis on the chick chorioallantoic membrane. The presence of metalloproteinases and their inhibitors in normal aqueous humor, a fluid which bathes avascular ocular structures, suggests that future studies should examine whether an imbalance in this protease}inhibitor family may contribute to the anterior chamber extracellular matrix alterations associated with diseases such as ocular neovascularization and glaucoma. # 1996 Academic Press Limited Key words : tissue inhibitors of matrix metalloproteinases ; extracellular matrix ; collagenase inhibitor ; DNA synthesis inhibitor ; angiogenesis.

1. Introduction Degradation and remodeling of the extracellular matrix (ECM) are essential, tightly-regulated components of many physiologic processes, including embryonic development, wound healing, and angiogenesis. While many enzymes can degrade ECM components, the family of matrix metalloproteinases (MMPs), a group of zinc-dependent enzymes, are considered to be physiologically-relevant, endogenous mediators of ECM degradation in vivo (Woessner, 1994 ; Birkedal-Hansen, 1993a). The MMPs have been classified into at least three broad categories based on substrate specificity—the collagenases, the gelatinases (Type IV collagenases), and the stromelysins (rev. Woessner, 1994). Specific examples include interstitial collagenase (MMP-1), 72kDa gelatinase (MMP-2), stromelysin (MMP-3), matrilysin (MMP-7), neutrophil collagenase (MMP-8), 92kDa gelatinase (MMP-9) (Matrisian, 1992) and the newly described membrane-type matrix metalloproteinase (MT-MMP) (Sato et al., 1994). As a family of enzymes, the MMPs are controlled at a minimum of three levels, including gene expression, proteolytic activation of the secreted zymogen, and suppression by endogenous inhibitors. The MMPs are * For correspondence at : Massachusetts Eye and Ear Infirmary, 243 Charles Street, Boston, MA 02114, U.S.A.

0014–4835}96}050481­09 $18.00}0

inhibited by a specific class of inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), which bind tightly and exclusively to the MMPs, as well as the ubiquitous serum protein α2-macroglobulin. TIMP-1 was the first endogenous MMP inhibitor to be identified (Stricklin and Welgus, 1983). It is a 28kDa glycoprotein detected in many tissues, including bone, kidney and the conditioned media of fibroblasts (Nomura et al., 1989). TIMP-2 was purified from human melanoma cells and endothelial cells (StetlerStevenson, Krutzsch and Liotta, 1989 ; Boone et al., 1990). It is a 20-kDa, non-glycosylated protein whose cDNA shows 38 % sequence identity to TIMP-1 (Boone et al., 1990). A cartilage-derived metalloproteinase inhibitor (CDI) has been purified from bovine cartilage (Moses, Sudhalter and Langer, 1990) and scapular chondrocytes (Moses, Sudhalter and Langer, 1992) as a 35-kDa glycosylated protein. Recently, the newest member of the TIMP family, TIMP-3, was purified (Pavloff et al., 1992) and identified in brain, lung, kidney (Leco et al., 1994), placental (Apte, Mattei and Olsen, 1994) and uterine tissues (Uria et al., 1994). To maintain the integrity of the ECM, MMP activity must be tightly controlled and regulated. Disturbances in the proteolytic balance between MMPs and their inhibitors have been implicated in the pathogenesis of several human diseases, including arthritis (Dean et al., 1989), periodontal disease (Birkedal-Hansen, 1993b), pulmonary fibrosis (Pardo et al., 1992), # 1996 Academic Press Limited

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hereditary macular dystrophy (Weber et al., 1994), and tumor invasion and metastasis (Liotta, Steeg and Stetler-Stevenson, 1991 ; Lokeshwar et al., 1993 ; Polette et al., 1993). Similarly, aberrations in this enzyme system might be involved in the extracellular matrix alterations associated with glaucoma (Tripathi, 1972 ; Samuelson, Gum and Gelatt, 1989 ; Lu$ tjenDrecoll et al., 1989). Since MMP activity is the ratelimiting step in the degradation of ECM components, and ECM degradation is an important step in angiogenesis, it has been hypothesized that inhibition of MMP activity may inhibit the process of angiogenesis (Moses and Langer, 1991). With the demonstration that matrix metalloproteinase inhibitors prevent angiogenesis in vitro and in vivo (Moses, Sudhalter and Langer, 1990 ; Johnson et al., 1994 ; Murphy, Unsworth and Stetler-Stevenson, 1993 ; Mignatti et al., 1989) and the finding that aqueous humor contains several species of MMPs (Ando et al., 1993), we became interested in examining the aqueous humor, a fluid that bathes avascular ocular structures, for the presence and activity of MMPs and their endogenous inhibitors. 2. Materials and Methods Aqueous Humor Collection and Preparation Four hundred adult bovine eyes obtained from a local abattoir (E. L. Blood and Son, West Groton, MA, U.S.A.) were harvested within 5 min of death and stored at 4°C for 3–6 hr. Taking care to avoid injury to the cornea and lens, aqueous humor was collected from the anterior chamber using a 23-gauge needle, yielding an average of 0±5 ml per eye. The aqueous humor was diluted 1 : 1 with 20 m Tris (pH 8) with leupeptin, aprotinin, and phenylmethylsulfonyl fluoride (added to final concentrations of 1 µg ml−", 1 µg ml−" and 2 m, respectively), and stirred at 4°C for 30 min. The solution was centrifuged at 12 000 g for 30 min and the supernatant collected and stored at 4°C until further study. The protein concentration of these aqueous humor samples was approximately 2 mg ml−" as determined by both Lowry (Lowry et al., 1951) and Bradford (Bio-Rad, Melville, NY, U.S.A.) protein determination assays.

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Heparin–Sepharose Chromatography Fractions from the Bio-Rex cation exchange column enriched in collagenase inhibitor activity were applied to a heparin-Sepharose column (1¬8 cm) pre-equilibrated with 50 m NaCl, 10 m Tris, pH 7. After rinsing with the equilibration buffer, the column was eluted with 330 ml of a gradient from 0±05 to 2  NaCl in 10 m Tris, pH 7 at a flow rate of 1 ml min−". Eight-milliliter fractions were collected and separately assayed for inhibition of collagenase and capillary endothelial cell DNA synthesis. Substrate Gel Electrophoresis (Zymography) To evaluate the presence of gelatinolytic activity, samples of aqueous humor were electrophoresed on an SDS–polyacrylamide gel copolymerized with gelatin as described by Herron et al. (1986). Type I gelatin was added to the standard Laemmli acrylamide polymerization mixture to a final concentration of 1 mg ml−". The aqueous humor preparation was affinity-purified with a heparin-Sepharose slurry preequilibrated with 75 m NaCl, eluted from the beads with 400 m NaCl, mixed with substrate gel buffer, and loaded into wells of a 4 % acrylamide stacking gel. Following electrophoresis under non-reducing conditions, the gel was soaked in 2±5 % Triton X-100 for 30 min at room temperature and then rinsed and incubated overnight at 37°C in substrate buffer (50 m Tris–HCl buffer, pH 8±0, 5 m CaCl , 0±02 % # NaN ). After incubation, the gel was then stained for $ 15–30 min with 0±5 % Coomassie blue R-250 in acetic acid–isopropyl alcohol–water (1 : 3 : 6), destained in water, and photographed. Proteolytic activity appears as a clear band of lysis against the dark background of stained gelatin. To verify that the gelatinase activities were specifically metalloproteinase activities, samples were exposed to 1,10 phenanthroline, a metal chelator and specific inhibitor of matrix metalloproteinases. Treatment with the organomercurial, 4-aminophenylmercuric acetate (APMA), which activates MMPs, distinguished latent from active forms of metalloproteinases.

Cation Exchange Chromatography

Radiometric Enzyme Assay for Collagenase and Collagenase Inhibitor

The aqueous preparation was diluted 1 : 1 with 20 m Tris, pH 7±6, and applied to a Bio-Rex 70 cation exchange column (2±5 cm¬48 cm) pre-equilibrated with 10 m Tris, pH 7±6, 50 m NaCl, and 0±02 % NaN . After rinsing with the equilibration buffer, the $ column was eluted with 1280 ml of a gradient from 50 to 750 m NaCl in 10 m Tris, pH 7±6 at a flow rate of 1 ml min−". Fractions of 5±3 ml were collected and separately assayed for inhibition of collagenase and capillary endothelial cell DNA synthesis.

In order to determine whether aqueous humor contained metalloproteinase inhibitory activity, unfractionated aqueous humor samples were screened in the radiometric enzyme assay for collagenase inhibition. Since metalloproteinase inhibitors have been shown to have an affinity for heparin and since heparin–sepharose affinity chromatography has previously been used to purify TIMPs (Murphy, Cawston and Reynolds, 1981 ; Morales et al., 1983 ; Bunning et al., 1984 ; Moses and Shing, 1994), aqueous humor

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extract was first incubated with heparin–sepharose in a slurry (sample}resin ¯ 100 : 1) overnight at 4°C in order to enrich the samples for inhibitory activity. The aqueous humor extract was then batch-eluted from the resin using 400 m NaCl, and enzyme (interstitial collagenase) and inhibitor activities were determined by a method (Murray et al., 1986) modified from that previously described by Johnson-Wint (Johnson-Wint, 1980). Specifically, aliquots of each fraction were dialysed against collagenase assay buffer (50 m Tris–HCl, pH 7±6, 0±2  NaCl, 1 m CaCl , and 0±02 % # NaN ). To assay for collagenase inhibitory activity, $ samples of 100 µl were diluted with 100 µl of bovine corneal collagenase and added to microtiter wells containing "%C-radiolabeled Type I collagen. After incubation at 37°C for 2±5 hr, the supernatant, containing soluble radiolabeled collagen, was transferred to scintillation vials and analysed using a Beckman model LS 3801 scintillation counter. In order to partially purify collagenase inhibitory activity from aqueous humor, it was empirically determined that a heparin–sepharose chromatography step was more efficient following a Biorex-70 cation exchange chromatography step. Thus, the radiometric enzyme assay was also performed on aqueous humor fractions from both chromatography columns. Western Blot Analysis The aqueous humor preparation was affinitypurified with a heparin–sepharose slurry pre-equilibrated with 75 m NaCl, eluted from the beads with 400 m NaCl, dialysed against three changes of distilled water, and concentrated by lyophilization. After reconstitution in Laemmli sample buffer (Laemmli, 1970), samples were electrophoresed in a 15 % SDS–polyacrylamide gel electrophoresis using a Mini-Protean II (Bio-Rad, Melville, NY, U.S.A.) apparatus in the presence of β-mercaptoethanol and electrophoretically transferred to a nitrocellulose membrane. The nitrocellulose was then incubated with a polyclonal rabbit IgG raised against the highlyconserved amino-terminus of TIMPs which crossreacts with TIMP-1, TIMP-2 (Braunhut and Moses, 1994), and CDI (Moses and Shing, 1994). Immunoreactive bands were visualized by incubation with a donkey anti-rabbit IgG horseradish peroxidase conjugate and substrate system (ECL system, Amersham, Arlington Heights, IL, U.S.A.). Western blot analysis was also performed on aqueous humor fractions from the heparin–sepharose column which were enriched in collagenase inhibitor activity. Endothelial Cell DNA Synthesis Assay The effect of Bio-Rex and heparin–sepharose chromatography fractions on endothelial cell DNA synthesis was assayed. Bovine capillary endothelial (BCE)

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cells, the kind gift of Dr Judah Folkman and Catherine Butterfield, were previously isolated from bovine adrenal glands and grown on gelatin-coated dishes as described by Folkman and Haudenschild (1980). DNA synthesis was assessed by incorporation of [$H]thymidine into the capillary endothelial cells as previously described (Shing, 1991). Inhibition of DNA synthesis was determined as percentage decrease in [$H]-thymidine incorporation from bFGF-stimulated controls. Chick Chorioallantoic Membrane Assay (CAM ) Fractions from the heparin–sepharose column enriched in collagenase inhibitor activity were pooled, dialysed against three changes of distilled water, and concentrated by lyophilization. Samples were mixed with methylcellulose, allowed to air dry, and placed on the surface of 6-day-old chicken chorioallantoic membranes (CAM) as previously described by Taylor and Folkman (1982). CAMs were graded 48 hr later and photographed with a Zeiss stereoscope. Samples were performed in duplicate. 3. Results Substrate Gel Electrophoresis Substrate gel electrophoresis revealed several major bands of gelatinolytic activity consistent with the molecular weights of known MMPs (Fig. 1). Specifically, at least two bands of gelatinolytic activity

97.4 66.2

42.7

31

21.5

14.4 1

2

F. 1. Metalloproteinase activities in aqueous humor. Representative zymogram of unfractionated aqueous humor samples demonstrates several gelatinolytic species, appearing as clear bands of lysis in a polyacrylamide gel impregnated with gelatin and stained with Coomassie R-250 (lane 1). Treatment of samples with 4-aminophenylmercuric acid (APMA), an organomercurial which activates latent MMPs (lane 2). Molecular weights (non-prestained low molecular weight standards, Bio-Rad, Melville, NY, U.S.A.) are indicated to the left.

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80

40

0.4

20

0.2

0

20

30

40 50 60 Fraction number

70

80

40

0

20

A280 (× 10–3)

60

60

NaCl (M)

Collagenase (% inhibition) DNA synthesis

484

0

0.8

60

0.6

40

0.4

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20

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4

8

12 16 Fraction number

20

24

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A280 (× 10–3)

80

NaCl (M)

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F. 2. Fractionation of aqueous humor by cation exchange chromatography. Pooled bovine aqueous humor was purified on a Bio-Rex 70 cation exchange column. Fractions of 5±3 ml were collected and analysed for absorbance at 280 nm and conductivity at 200 mS cm−" to determine protein and salt concentrations, respectively. Fractions were assayed for the ability to inhibit collagenase and BCE cell DNA synthesis.

F. 3. Fractionation of partially-purified aqueous humor by heparin–sepharose affinity chromatography. Fractions from the cation exchange column enriched in collagenase inhibitory activity were pooled and applied to a heparin–sepharose column. Fractions of 8 ml were collected and analysed for absorbance at 280 nm and conductivity at 200 mS cm−" to determine protein and salt concentrations, respectively. Fractions were assayed for the ability to inhibit collagenase and BCE cell DNA synthesis.

migrated at molecular weights corresponding to species of the MMP-9 family of metalloenzymes. Additionally, two bands of gelatinolytic activity migrated at molecular weights corresponding to species of the MMP-2 family of enzymes. Several other gelatinolytic bands were identified at molecular weights below 50 kDa. These may represent members of the MMP-1 and -7 families of enzymes. A gelatinolytic zone was also observed at C 100 kDa. Treatment with 1,10 phenanthroline eliminated all bands of gelatinolytic activity previously detected, demonstrating that all gelatinase activity identified represents MMP activity (data not shown). After APMA treatment, no change was observed in the pattern of lysis, demonstrating that all metalloproteinases detected were in their active forms (Fig. 1).

from the heparin–sepharose resin using 0±4  NaCl. Subsequent analysis of aqueous humor fractions eluted from the cation exchange column revealed a peak of 80 % collagenase inhibitory activity at 0±15  [NaCl] (Fig. 2). These same fractions revealed two peaks of inhibition of BCE DNA synthesis, with 57 % inhibition at 0±18  NaCl and 48 % inhibition at 0±2  NaCl (Fig. 2). These peaks of inhibitory activity were subsequently pooled and further purified by heparin– sepharose affinity chromatography. Chromatographed samples were then tested for collagenase inhibition and zones enriched in collagenase activity were tested for inhibition of BCE DNA synthesis. Analysis of these fractions revealed overlapping peaks of collagenase inhibition (90 %) and endothelial cell DNA synthesis inhibition (52 %) (Fig. 3).

Collagenase and Growth Factor Inhibition

Western Blot Analysis

Collagenase inhibitory activity was detected in the aqueous humor samples which were batch-eluted

Western blot analysis identified three immunoreactive bands in unfractionated aqueous humor [Fig.

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(B)

(A) Mr (kDa)

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106

107 76

80 52 49.5

32.5

36.8

27.5

TIMP-1 27.2 TIMP-2

18.5

19 1

2

3

F. 4. Representative Western blot of aqueous humor using a polyclonal IgG raised against the highly-conserved aminoterminus of TIMPs. (A) Unfractionated aqueous humor samples (lane 3). Lane 1 : recombinant TIMP-1 standard (glycosylated). Lane 2 : unglycosylated TIMP-2 standard. (B) Pooled aqueous humor fractions enriched in collagenase inhibitory activity (lane 2). Lane 1 : Protein standards from cartilage extracts containing deglycosylated CDI (Moses, Sudhalter and Langer, 1990) and TIMP-2 (Murray et al., 1986) controls. Lane 3 : recombinant TIMP-1 standard (glycosylated). Molecular weights (prestained low molecular weight standards, Bio-Rad, Melvile, NY, U.S.A.) are indicated at left for both blots.

4(A)]. One immunoreactive species comigrated at MW 21-kDa with the TIMP-2 standard. Another species co-migrated at MW 28-kDa with the glycosylated TIMP-1 standard. A third, currently unidentified, immunoreactive band appeared at 25-kDa. Analysis of fractions from the heparin-sepharose column enriched in collagenase inhibitory activity [Fig. 4(B)] revealed two immunoreactive bands comigrating with the TIMP-2 and TIMP-1 standards at 21- and 28-kDa, respectively. Chick Chorioallantoic Membrane Assay Aqueous humor fractions from the heparin– sepharose column enriched in collagenase inhibitory activity were dialysed, concentrated, and mixed in methylcellulose discs which were then placed on the surface of growing chick chorioallantoic membranes (CAMs) for 48 hr. A zone of angiogenesis inhibition, characterized by pruning of vessels and capillary loss, was observed [Fig. 5(B)]. 4. Discussion Deregulated angiogenesis is involved in many ocular diseases, including diabetic retinopathy, neovascular glaucoma, retinopathy of prematurity and macular degeneration. The aqueous humor is a clear fluid which bathes the avascular cornea, lens, and trabecular meshwork. Since the maintenance of this avascularity is crucial to vision as well as to ocular physiology, we became interested in examining the

aqueous humor for modulators of the angiogenic process. An important control point for angiogenesis is extracellular matrix turnover. Using zymography we have established the presence of several gelatinolytic species in normal bovine aqueous humor. The addition of a specific inhibitor of MMPs (1,10 phenanthroline) suppressed all bands of gelatinolysis, demonstrating that the enzymes identified were specifically MMPs. The molecular weights and bioactivity of these gelatinases are consistent with their identities as members of the MMP-2 and MMP-9 families. Other bands of gelatinolytic activity were detected between molecular weights of 28- and 40-kDa, and likely represent members of the MMP-1 and -7 families. The presence, among other enzymes, of MMP-2 and -9 in aqueous humor from patients undergoing elective cataract surgery has been reported (Ando et al., 1993). In this study, we extend these prior observations by showing that all aqueous humor MMPs detected in our assays are in their active forms, an important finding because these metalloenzymes are secreted as latent proenzymes which must be extracellularly activated before becoming proteolytic. The discovery of active MMPs in the aqueous humor, a fluid which surrounds and nourishes tissues with minimal steady-state changes in the extracellular matrix, was paradoxical and thus led us to examine the aqueous for the presence of the endogenous MMP inhibitors, the TIMPs. After detecting two immunoreactive bands comigrating with TIMP-1 and TIMP-2 standards in our aqueous humor samples, we sought

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F. 5. Inhibition of angiogenesis by aqueous humor in the chick chorioallantoic membrane assay (CAM). Aqueous humor samples enriched in collagenase inhibitory activity were mixed in methylcellulose discs which were then placed on the surface of growing chick chorioallantoic membranes (CAMs) for 48 hr. CAMs were then photographed at 10¬ magnification. Arrows indicate zone of angiogenesis inhibition. (A) Normal CAM with empty methylcellulose disc. (B) CAM with aqueous humorimpregnated disc. (C) CAM with heparin hydrocortisone-impregnated disc used as a positive control (Crum, Szabo and Folkman, 1985).

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to further characterize the inhibitory activity of this fluid since imbalances in the MMP}TIMP family have been associated with a number of angiogenic disease processes, including hereditary macular dystrophy (Weber et al., 1994) and tumor invasion and metastasis (Liotta, Steeg and Stetler-Stevenson, 1991 ; Lokeshwar et al., 1993 ; Polette et al., 1993). Two necessary steps in the process of angiogenesis are basement membrane collagenolysis and capillary endothelial cell proliferation. Using two successive chromatographic steps, we partially purified fractions of aqueous humor which inhibited endothelial cell growth, collagenase, and angiogenesis in vivo and which copurified with TIMP-1 and TIMP-2. To our knowledge this is the first demonstration of TIMPs in aqueous humor. Our findings are consistent with what is currently known about the TIMPs. TIMP-1 has been shown to inhibit the process of basement membrane invasion by BCE cells, a crucial step in the angiogenic process (Mignatti et al., 1989). A cartilage-derived TIMP-1like protein has been shown to inhibit endothelial cell proliferation and migration in vitro and angiogenesis in vivo (Moses, Sudhalter and Langer, 1990). TIMP-2 was subsequently found to inhibit bFGF-induced endothelial cell proliferation and DNA synthesis (Murphy, Unsworth and Stetler-Stevenson, 1993). Interestingly, in the same study TIMP-1 did not inhibit endothelial cell proliferation or DNA synthesis. Most recently, TIMP-1 was shown to inhibit bFGF-stimulated angiogenesis in the rat corneal micropocket (Johnson et al., 1994). Aqueous humor from normal rabbits was shown to inhibit both endothelial cell proliferation in vitro and angiogenesis on the CAM in vivo, whereas aqueous from diabetic rabbits did not (Okamoto, Likawa and Toyota, 1990). While Okamoto and colleagues did not attempt to further identify the source of this inhibitory activity in rabbit aqueous humor, we suggest that the TIMPs identified in our samples of bovine aqueous humor are likely candidates. Given that TIMP-1 does not inhibit endothelial cell DNA synthesis (Murphy, Unsworth and StetlerStevenson, 1993), TIMP-2 is one candidate potentially responsible for the endothelial cell DNA synthesis inhibition described herein. Silver stain analysis revealed that the immunoreactive bands identified by our anti-TIMP antibodies were two protein bands among several (data not shown). As such, definitive conclusions that the TIMPs are solely responsible for the inhibitory activity detected against collagenase, DNA synthesis, and angiogenesis in normal aqueous humor must await neutralizing studies. Of interest is the observation that TGF-β has also been detected in aqueous humor (Grainstein et al., 1990 ; Eisenstein and GrantBertacchini, 1991 ; Jampel et al., 1990) and its presence correlated with the ability to inhibit endothelial cell growth (Eisenstein and Grant-Bertacchini, 1991). However, the majority of TGF-β in

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aqueous humor is in its latent, inactive form (Granstein et al., 1990 ; Eisenstein and Grant-Bertacchini, 1991) and even after heat or acid treatment was used to activate TGF-β, TGF-β neutralizing antibodies abrogated only 39 % of the inhibitory activity detected. In the present study, the presence of TGF-β alone probably cannot account for our findings on the CAM, since TGF-β actually stimulates angiogenesis in several in vivo assays (Roberts et al., 1985 ; Fiegel and Knighton, 1988) including the CAM (Yang and Moses, 1990). We therefore suggest that in aqueous humor, the TIMPs may play a role in the inhibition of endothelial cell growth. Although our aqueous humor samples were obtained within hours of death, the possibility remains that the MMPs and TIMPs detected were artifacts from the breakdown of other anterior chamber tissues after death. However, MMPs and α2-macroglobulin have been previously detected in aqueous humor samples from living cataract patients (Ando et al., 1993). Moreover, the MMPs and TIMPs are efficiently secreted proteins such that significant release secondary to cell death would be unlikely. In conclusion, we have isolated extracts of normal aqueous humor which inhibit collagenase, capillary endothelial cell growth, as well as angiogenesis in vivo, and which copurify with TIMP-1 and TIMP-2. The simultaneous presence of MMPs and these inhibitory activities in normal aqueous humor raises the question of whether in this fluid, extracellular matrix turnover and neovascularization may be inhibited in part by the activity of TIMP family members. Since an imbalance in this protease system may contribute to the ECM alterations that characterize ocular neovascularization and glaucoma, future studies are needed to examine the presence and relative levels of MMPs and TIMPs in normal and diseased eyes. Acknowledgements The authors gratefully acknowledge Cecilia Fernandez and Geri Jackson for excellent technical assistance, Yihai Cao for helpful discussions, and Tony Maciag and Lori DeSantis for photography.

References Ando, H., Twinning, S. S., Yue, B. Y. J. T., Zhou, X., Fini, M. E., Kaiya, T., Higginbotham, E. J. and Sugar, J. (1993). MMPs and proteinase inhibitors in the human aqueous humor. Invest. Ophthalmol. Vis. Sci. 34, 3541–48. Apte, S. S., Mattei, M. G. and Olsen, B. R. (1994). Cloning of the cDNA encoding human tissue inhibitor of metalloproteinases-3 (TIMP-3) and mapping of the TIMP-3 gene to chromosome 22. Genomics 19, 86–90. Birkedal-Hansen, H. (1993a). Role of matrix metalloproteinases in human periodontal diseases. J. Periodontol 64 (suppl.), 474–80. Birkedal-Hansen, H. (1993b). Role of MMPs in human periodontal disease. J. Periodontol. 64, 474–84. Boone, T. C., Johnson, M. J., DeClerck, Y. A. and Langley, K. E. (1990). cDNA cloning and expression of a metallo-

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