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(Investigative Ophthalmology and Visual Science. 2001;42:1963-1968.)
© 2001 by The Association for Research in Vision and Ophthalmology, Inc.

Active Matrilysin (MMP-7) in Human Pterygia: Potential Role in Angiogenesis

Nick Di Girolamo1, Minas T. Coroneo2 and Denis Wakefield1

1 From the Inflammation Research Unit, School of Pathology, University of New South Wales; and the 2 Department of Ophthalmology, Prince of Wales Hospital, Sydney, New South Wales, Australia.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. Pterygia are invasive, proliferative fibrovascular growths, with the matrix metalloproteinase (MMP) family of enzymes strongly implicated in the pathogenesis of these lesions. The purpose of this study was to determine the cellular distribution and activation status of matrilysin (MMP-7) in pterygia.

METHODS. Resected pterygia (n = 8) and normal conjunctiva (n = 8) were sectioned and analyzed immunohistochemically with two different epitope-specific anti-MMP-7 monoclonal antibodies (Abs) which differentiate pro- and active MMP-7. The specificity of each Ab was confirmed by Western blot analysis of p-aminophenylmercuric acetate (APMA)–activated and latent recombinant MMP-7. Pterygia (n = 4) and autologous normal conjunctiva (n = 4) were placed in organ culture to determine the activation status of secreted MMP-7.

RESULTS. Precursor and active forms of MMP-7 were detected in epithelial cells from both pterygia and normal conjunctiva. Intense immunoreactivity for pro- and active MMP-7 was also observed in the pterygium vasculature, but was essentially absent from conjunctival vessels. Pro-MMP-7 was also identified in the epithelial basement membrane and associated with matrix components in pterygia. The 141-7B2 Ab reacted with the 30-kDa latent MMP-7, and the IM47L Ab precipitated a 19-kDa active enzyme, thus confirming the differential specificity of each Ab. Pro- and active MMP-7 were increased 1.4- and 2.7-fold, respectively, in the supernatants from organ-cultured pterygia compared with conjunctiva.

CONCLUSIONS. This study is the first to specifically localize an active MMP species in pterygia and strengthens the hypothesis that these enzymes are involved in the pathogenesis of this disease. The data also suggest that MMP-7 may play a significant role in the angiogenesis that characterizes this lesion. Future studies will be directed at determining whether targeting MMP activity may be useful for treatment of pterygia.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Pterygia are inflammatory, invasive, and highly vascularized growths, thought to arise from activated and proliferating limbal epithelial stem cells.1 Despite the lack of knowledge regarding the pathogenesis of this disease, epidemiological studies have implicated UV light as a causative factor.2 3 Other popular theories include modifications in apoptosis pathways,4 5 the presence of active angiogenic factors,6 7 or the involvement of matrix metalloproteinases (MMPs).8 9 10 11 12 It is possible that elements of each of these theories are important in the pathogenesis of this disease.

MMPs are a family of zinc-binding endopeptidases, present as secreted, membrane-bound, or intracellularly stored proteins.13 They are considered to play a crucial role in physiological processes, such as embryo development and wound healing, and have been implicated in the pathogenesis of cancer,14 rheumatoid arthritis,15 diseases of the cornea,16 scleritis,17 and uveitis.18 MMPs are regulated transcriptionally, at the level of proenzyme activation and inhibition of enzymatic activity by naturally occurring tissue inhibitors (TIMPs).14 19 Activation of latent MMPs generally takes place in the extracellular space and involves the action of other proteases.20 Matrilysin (MMP-7) is the smallest member of the MMPs and is capable of denaturing a broad spectrum of matrix proteins, including fibronectin, vitronectin, elastin, collagen IV, aggrecan, and proteoglycans.21 In addition, this proteinase can amplify an inflammatory response through its ability to promote the cell-surface processing of cytokines such as TNF-{alpha}.22

Previous in vitro and in vivo studies have focused on the regulation of MMPs by cytokines and growth factors in cultured pterygium cells.8 10 It is conceivable that these and other cytokines regulate the inflammatory and angiogenic component in pterygia and may initiate tissue remodeling and cellular invasion that characterize this lesion. These data also suggest that MMPs may play a significant role in the pathogenesis of this disease.

Although currently there are several reports in the literature on the localization of MMPs in pterygia,8 9 11 these studies do not discriminate between the active and the latent enzymes. One biochemical technique, in situ zymography, discriminates the activation status of a particular MMP within a tissue specimen and identifies the secretory cell.23 Because of the significant overlap in substrate specificity between the MMPs, it can be difficult to identify enzymatic activity between, for example, the two different species of gelatinase enzymes. In the present study, we used an alternative histologic approach involving two antibodies (Abs) raised against different epitopes of MMP-7. One specifically detects the active enzyme and the other identifies the precursor.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Surgical Tissue Specimens
Resected pterygium (n = 8) and normal conjunctival tissue (n = 8) were obtained from Prince of Wales Hospital, Sydney, Australia. Autologous normal tissue was surgically removed from the superior bulbar conjunctiva (n = 5) and from patients undergoing cataract extraction (n = 3). Tissue was immediately fixed in formalin and paraffin embedded. Pterygium tissue was derived from six men and two women (mean age, 47.6 years). Conjunctival specimens were obtained from seven men and one woman (mean age, 44.6 years). In addition, fresh pterygium (n = 4) and autologous conjunctiva (n = 4) were surgically removed and immediately placed in organ culture, as previously described.9 Informed consent was obtained from each subject. All research protocols were approved by the University of New South Wales ethics committee and were performed in accordance with the tenets of the World Medical Association’s Declaration of Helsinki.

Immunohistochemical Analysis
Pterygium and normal human conjunctival tissue was sectioned (2–4 µm thick) and processed for immunohistochemistry, as previously described.8 9 In brief, tissue sections were deparaffinized, hydrated, equilibrated in 0.05 M Tris-buffered saline (TBS; pH 7.6), and blocked with 20% goat serum in 2% BSA-TBS for 30 minutes at room temperature, before applying either anti-human MMP-7 (clone 141-7B2; ICN Biomedicals, Sydney, Australia) that recognizes the 30-kDa latent enzyme or an anti-human MMP-7 (Ab 2, clone IM47L; Oncogene Research Products, Cambridge, MA) that reacts with the 19-kDa active MMP-7 species. All primary Abs were applied at the same final concentration (10 ng/ml), and then incubated overnight at 4°C in 2% BSA-TBS. Sections were extensively washed in TBS before the addition of a biotinylated goat anti-mouse secondary Ab (Vector Laboratories, Burlingame, CA). Sections were rinsed and incubated with horseradish peroxidase-conjugated streptavidin (Dako Inc., Carpinteria, CA) and the immunoreactivity revealed by adding 3-amino-9-ethyl-carbazole (Sigma, St. Louis, MO). Control reactions included incubating tissue sections with preabsorbed anti-pro-MMP-7 Ab, omitting the primary Ab, or replacing the primary Ab with a mouse isotype IgG1 negative control Ab (Dako). Sections were counterstained with hematoxylin, and immunoreactivity was assessed semiquantitatively using the grading system described in Table 1 . Comparisons of staining intensity between pterygium and conjunctival tissue using either MMP-7 Ab was possible because diseased and control tissue were analyzed in the same experimental run.


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  		Table 1. Semiquantitative Immunohistochemical Analysis

 
Western Blot Analysis
Western immunoblot analysis was performed as previously described.8 9 To determine the specificity of the two MMP-7 Abs (see Immunohistochemical Analysis), recombinant human (rh) pro-MMP-7 (Calbiochem-Novabiochem, Sydney, Australia) was used as the antigen. Active MMP-7 was generated by incubating rh-pro-MMP-7 (0.25 and 0.5 µg) with 10 mM p-aminophenylmercuric acetate (APMA, Sigma) for 90 minutes at 37°C. Both latent and APMA-activated MMP-7 were electrophoretically separated on 4% stacking and 12% resolving polyacrylamide gels under nondenaturing conditions and transferred to nitrocellulose membranes (Gelman, Ann Arbor, MI). Blots containing both active and latent MMP-7 were probed separately with the two anti-MMP-7 Abs (1 ng/ml, final concentration) and then placed in chemiluminescent reagent (NEN Life Science Products, Boston, MA) and exposed to film (BioMax; Kodak, Sydney, Australia). A prestained, low-molecular-weight protein ladder (Bio-Rad, Sydney, Australia) was run in adjacent lanes.

To further determine the specificity of the MMP-7 Abs, an alternative source for this enzyme was assayed. The supernatants derived from organ-cultured pterygia and normal conjunctiva were concentrated by using 10-kDa cutoff spin filters (Millipore, Bedford, MA), were standardized for total protein content (Pierce, Rockford, IL), and were analyzed by immunoblot to determine whether the Abs react with native MMP-7. Immunoreactive bands were subjected to semiquantitative analysis by computer (Gel Doc 2000 and Quantity One programs; Bio-Rad).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of Active and Latent MMP-7 in Pterygia
Immunohistochemical analysis was performed to determine the expression and activation status of MMP-7 in pterygia and normal conjunctiva. Pro-MMP-7 was constitutively expressed by the normal conjunctival epithelium, with minimal or no immunoreactivity observed in the vasculature (Fig. 1A , arrows). These observations are in accord with those recorded by other investigators who have localized this protease in abundance in the epithelium of other tissues.21 24 25 26 Despite localization of the active form of MMP-7 in the conjunctival epithelium (Fig. 1B) , the staining intensity for this enzyme species was considerably less than in the pterygium epithelium (Figs. 1D 1F) . Active MMP-7 was rarely detected in the conjunctival vascular endothelium (Fig. 1B , arrows), nor was it present in connective tissue fibroblasts. Sections of pterygium tissue demonstrated similar staining for pro-MMP-7 in the epithelium (Figs. 1C 1E) , with the occasional infiltrating inflammatory cell also illuminated (Fig. 1C , arrowhead). A sequential tissue section incubated with the IM47L Ab revealed intense immunoreactivity for active MMP-7 in the same large leukocyte (Fig. 1D , arrowhead), as well as in the more basal pterygium epithelium (Figs. 1D 1F) . Whereas the vasculature in the normal conjunctiva was minimally reactive or nonreactive to either of the two MMP-7 Abs (Figs. 1A 1B , arrows), abundant reactivity for latent MMP-7 was observed in both large and small pterygium blood vessels (Fig. 1G) and was similar to the staining for the active MMP-7 species in serial tissue sections (Fig. 1H) . Tissue sections incubated with a mouse isotype control IgG1 Ab (Figs. 1A 1C , insets), with rhMMP-7 preabsorbed Ab (Fig. 1E , inset) or in the absence of a primary Ab (Fig. 1G , inset) displayed no signal. The immunohistochemical staining from all diseased and control tissue specimens was subjected to semiquantitative analysis and the results summarized (Table 1) .



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  		Figure 1. Expression of active and latent MMP-7 in pterygia and normal conjunctiva. Normal conjunctiva (A, B) and pterygium tissue (C–H) were serially sectioned and analyzed immunohistochemically for the expression of latent (A, C, E, G) or active (B, D, F, H) MMP-7. Red stain: positive immunoreactivity; blue hematoxylin stain: cell nuclei. Sections incubated with a mouse isotype control Ab (A, C, insets), with preabsorbed Ab (E, inset) or with no primary Ab (G, inset) demonstrated no staining. (A, B, arrows) MMP-7–negative blood vessels; (C, D, arrowheads) the same large inflammatory cell. Sections in (A), (B), (G), and (H) were derived from patient 5; in (C) and (D) from patient 8; and in (E) and (F) from patient 1. These results are summarized in Table 1 . Original magnification, x500.

 
Localization of Latent MMP-7 to Pterygium Matrix Components
Latent MMP-7 was often detected along the pterygium epithelial basement membrane (Fig. 2A , arrows), associated with the collagenous matrix (Fig. 2B) , and specifically localized to the vascular endothelial basement membrane (Fig. 2C) . Active MMP-7 was observed in the pterygium vasculature (Fig. 1H) but was rarely detected in the connective tissue matrix (micrographs not shown).



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  		Figure 2. Matrix-associated latent MMP-7 in pterygia. Pterygium tissue derived from patient 3 was sectioned and analyzed immunohistochemically, as described in Figure 1 . Latent MMP-7 was identified on several matrix components, including the pterygium epithelial basement membrane (A, arrows), pterygium collagenous and elastotic matrix components (B), and the pterygium vascular endothelial basement membranes (C). Similar results were obtained from other pterygium specimens. Original magnification, x500.

 
Specificity of the MMP-7 Antibodies
Immunoblots were performed to confirm the differential binding ability of each Ab. APMA-activated and latent rhMMP-7 was analyzed in parallel by Western blot analysis (Fig. 3) . Blots reacted with the 141-7B2 Ab precipitated an intense 30-kDa protein corresponding to pro-MMP-7 (Fig. 3A , lanes 3, 4). Incubation with APMA resulted in significantly less immunoreactivity of the 30-kDa species (Fig. 3A , lanes 1, 2) because of the incomplete conversion of the proenzyme to the active form. The absence of an immunoreactive band at approximately 19 kDa suggests that this Ab does not cross-react with active MMP-7. Blots probed with the IM47L Ab precipitated an intense immunoreactive band in the APMA-treated pro-MMP-7 (Fig. 3B , lanes 1, 2), migrating to approximately 19 kDa and corresponding to the active MMP-7 species. This Ab also precipitated a 19-kDa active product from the untreated rh-pro-MMP-7 stock (Fig. 3B , lanes 3, 4), suggesting that some rh-pro-MMP-7 had been partially activated either in the purification procedure during the assay or at the time of freeze thawing the enzyme. The absence of an immunoreactive band at 30 kDa confirms that the IM47L Ab did not cross-react with pro-MMP-7.



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  		Figure 3. Specificity of the anti-MMP-7 antibodies. APMA-activated (lanes 1, 2) and nonactivated (lanes 3, 4) rh-pro-MMP-7 were electrophoretically separated on SDS-PAGE gels, transferred to nitrocellulose membranes, and probed with an anti-MMP-7 Ab (141-7B2) which recognizes the latent enzyme (A) or with an anti-MMP-7 Ab (IM47L) which distinguishes the active proteinase (B). Lanes 1 and 3 contained 250 ng; lanes 2 and 4 contained 500 ng rh-MMP-7. The two epitope-specific Abs are not cross-reactive. No immunoreactive bands were revealed when blots were incubated with normal preimmune mouse serum (data not shown). These data are representative of three separate experiments.

 
Secretion of Latent and Active MMP-7 from Pterygium Tissue
After standardizing the total protein content in each sample, pro-MMP-7 was 1.4 times more abundant in the supernatants derived from the four organ-cultured pterygium specimens (Fig. 4A , lanes 2, 4, 6, 8) compared with the corresponding normal conjunctiva (Fig. 4A , lanes 1, 3, 5, 7). Similarly, active MMP-7 was increased by approximately 2.7-fold in the pterygium supernatants (Fig. 4B , lanes 2, 4, 6, 8) compared with normal conjunctiva (Fig. 4B , lanes 1, 3, 5, 7). In addition, a significant difference in the amount of active MMP-7 was observed between pterygium specimens (Fig. 4B , lanes 2, 4, 6, 8). This may reflect differences related to stage of pterygium development, inflammation, or abundance of natural MMP-7 activators.



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  		Figure 4. Detection of active and latent MMP-7 from organ-cultured pterygia. Fresh pterygia were obtained from surgery and immediately placed in organ culture for 48 hours. Supernatants from four pterygium specimens (lanes 2, 4, 6, 8) and from their respective autologous normal conjunctiva (lanes 1, 3, 5, 7) were analyzed by Western blot analysis for latent (A) and active (B) MMP-7, by using the Abs described in Figure 3 .

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
To the best of our knowledge, this is the first report in which active MMP has been specifically localized in pterygia. Although our previous investigations illustrated the secretion of active gelatinase A (MMP-2), gelatinase B (MMP-9), and collagenase-1 (MMP-1) from ex vivo cultured pterygium tissue by gelatin-substrate zymography, no localization studies were performed to identify the cells producing these proteolytically active enzymes.9 The use of Abs that distinguish between active and proenzyme species is a significant advance in MMP biology. Such Abs have been used in few studies.27 28 At present, only one other assay (in situ zymography) has been used to make this distinction,23 29 30 but this assay allows the detection of limited MMPs, because of the similarity in substrate degradation between these enzymes. An alternative approach has been to raise Abs that detect specific MMP-mediated cleavage products of substrates such as collagen.31 However, although this technique is a reasonable measure of MMP activity, it does not identify the cellular source of the enzyme.

Recently, we documented the differential expression of MMPs at the advancing pterygium edge, where basal pterygium epithelial cells stained intensely for several MMPs.9 This apparent overexpression by the basal epithelial cells coincided with the loss of the Bowman layer.9 Active MMPs could be involved not only in the destruction of this collagenous natural barrier, but also in the extensive tissue invasion that characterizes pterygia. The present study has identified the abundant expression of active MMP-7 in both the pterygium epithelium and, of particular interest, in the pterygium vascular endothelium.

MMP-7 is the smallest member of the MMP family of enzymes without a C-terminal hemopexin-like domain. MMP-7 has broad substrate specificity and is included in the stromelysin subgroup because of its ability to denature fibronectin, laminin, elastin and proteoglycans.21 Unlike most MMPs, MMP-7 is constitutively expressed by epithelial cells.21 24 25 26 Investigators have reported the intense expression of this protease at the leading edge and in basal epithelial cells in a rat corneal model of wound healing.32 Similarly, in the present study we identified a row of intensely stained MMP-7–positive basal pterygium epithelial cells (Fig. 1D) . These data suggest that MMP-7 may be critical in the migratory and proliferative phase of wound healing and pterygium invasion. This hypothesis has been strengthened by studies that show that colon carcinoma cells transfected with MMP-7 DNA demonstrate increased invasiveness, whereas introduction of antisense RNA into MMP-7–producing cells significantly decreases their invasive potential.33 A similar study by Hasegawa et al.,34 demonstrates the suppression of metastatic tumor nodules after the administration of MMP-7–specific antisense oligonucleotide in a nude mouse model.

MMP-7 has been localized to the Bruch membrane and basement membrane–like structures around retinal pigment epithelial cells in tissue from patients with age-related macular degeneration.35 These data corroborate those presented in the present study, in which MMP-7 was localized in the epithelium, vascular endothelium, and basement membranes (Figs. 1 2) . Similarly, Nagashima et al.36 have localized MMP-7 mRNA and protein in vascular endothelial cells adjacent to MMP-7–positive tumors.

Active MMPs can function as tissue-destroying proteinases in pathologic states. However, other roles in inflammation should not be underestimated, because they have been shown to generate soluble cytokines. Recently, Haro et al.22 documented the MMP-7–dependent release of TNF-{alpha}, which in turn induced the expression of stromelysin-1 (MMP-3) in murine macrophages. The same investigators reported the requirement of MMP-7 for proteoglycan degradation. This is perhaps not surprising, because heparin sulfate proteoglycans act as extracellular docking molecules for MMP-7.37 Similarly, we observed the extensive matrix-associated binding of pro-MMP-7 in pterygia (Fig. 2) . It is notable that no active MMP-7 was localized to matrix components, perhaps because of its decreased affinity for substrates in the activate configuration.37 This could be a strategic mechanism that provides a reservoir of latent enzyme ready for proteolytic attack. It is of interest that MMP-7 has also been immunohistochemically identified in a bandlike pattern below basal keratinocytes in solar elastosis and in basal keratinocytes of UVB-irradiated human skin.38 The investigators in that study also noted a significant increase in staining intensity for MMP-7 in photoprovoked skin compared with control tissue. These results are relevant to those presented in the present study, because UV exposure is thought to be an important component in the pathogenesis of pterygia.2 3 Furthermore, a similar pattern of MMP-7 expression was noted in both basal pterygium epithelial cells (Fig. 1D) and in basement membranes (Figs. 2A 2C) .

This study has strengthened our hypothesis that MMPs play an important role in the pathogenesis of pterygia. Although previous studies have localized these enzymes in pterygium specimens,8 9 11 the Abs used in the present study have allowed us to discriminate between the expression of latent compared with active proteinase in diseased and control tissue. Knowledge of the activation status of a particular MMP may be relevant in pathologic conditions, including human pterygia, because treatment regimens could be tailored to inhibit specific proteinases.


   Footnotes
 
Supported by the National Health and Medical Research Council of Australia and the Ophthalmic Research Institute of Australia.

Submitted for publication January 8, 2001; revised April 16, 2001; accepted April 26, 2001.

Commercial relationships policy: N.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Nick Di Girolamo, Inflammation Research Unit, School of Pathology, The University of New South Wales, Sydney, 2052, Australia. n.digirolamo{at}unsw.edu.au


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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