Vet Clin Small Anim 34 (2004) 611–622 Matrix metalloproteinase inhibition in corneal ulceration Dennis E. Brooks, DVM, PhD*, Franck J. Ollivier, DVM, PhD Department Clinical Sciences, College of Veterinary Medicine, University of Florida, 2015 SW 16th Avenue, Gainesville, FL 32608, USA The cornea and precorneal tear film combine to function as a strong refractive lens. To produce such an optically powerful structure, the corneal microanatomy consists of an epithelium and thin epithelial basement membrane, a thick relatively acellular stroma, Descemet’s membrane, and a monolayered endothelium. The corneal epithelium is 10 to 15 cell layers thick. The basement membrane of the epithelium is composed of type IV, VI, and VII collagen as well as laminin, hyaluronans, and fibronectin [1]. The stroma comprises most of the corneal thickness and consists of multiple contiguous layers of thin and uniformly positioned collagen microfibrils surrounded by an extracellular matrix (ECM) of collagen types I, III, V, VI, and XII; stromal glycosaminoglycans (keratan sulfates, dermatan sulfates, and chondroitin sulfates); and glycoproteins [1]. A limited number of stromal keratocyte/fibroblasts are present. Descemet’s membrane is the acellular basement membrane of the endothelium. This ever-thickening basement membrane contains collagen types I, III, IV, V, VI, and VIII; laminin; fibronectin; and heparan sulfates [1]. The corneal endothelium produces Descemet’s membrane and contains an energy-dependent pump to maintain corneal deturgescence. The precorneal tear film is a lipid-bilayered and aqueous mucin– dominated gel that aids lubrication, transfers oxygen to the cornea, and smoothes out small surface irregularities in the anterior corneal epithelium to maintain a uniform optical surface. The tear film also removes exfoliating corneal epithelial cells and provides a means for inflammatory cells to reach the central cornea. Inflammatory conditions of the cornea or conjunctiva tend to produce a shift toward a more acid tear film [2]. Tear film proteins * Corresponding author. E-mail address: brooksd@mail.vetmed.ufl.edu (D.E. Brooks). 0195-5616/04/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.cvsm.2003.12.005 612 D.E. Brooks, F.J. Ollivier / Vet Clin Small Anim 34 (2004) 611–622 serve to control infectious agents and lower the surface tension, enabling the tear film to cover the conjunctival and corneal surfaces better. Proteins in the tears are species specific and include albumin; the immune globulins IgA, IgG, IgD, and IgE; ceruloplasmin and transferrin; histamine; plasminogen activator; complement; interferon; prostaglandins; the antimicrobial proteins lysozyme, b-lysin, and lactoferrin; and the tear film proteinases [2]. Damage to the cells of the corneal epithelium and stromal ECM results in what is referred to as an abrasion or ulcer. Most superficial uncomplicated corneal ulcers in animals heal without incident, because corneal wound healing is remarkably well regulated thanks to a natural harmonious balance between mechanisms of corneal repair and degradation. Infected corneal ulcers or ulcers with extensive stromal involvement may, however, progress to corneal perforation in as little as 24 hours. This rapidly progressive corneal deterioration is described clinically as keratomalacia, or corneal ‘‘melting,’’ with the primary mediators of this corneal stromal degeneration being microbial-, leukocyte-, tear film–, and corneal cell–derived enzymes called proteinases [3–5]. Function of tear film proteinases Tear film and corneal proteinases provide a mechanism for the surveillance, detection, repair, and removal of damaged corneal epithelial cells, altered corneal stromal collagen, and abnormal components of the ECM caused by normal wear and tear of the cornea [6–8].The maintenance and repair of the corneal ECM involve a tightly coordinated balance of collagen and ECM synthesis, degradation, and remodeling. Proteinases exist in inactive latent forms, become activated during inflammation, and can be produced by corneal and inflammatory cells [9–11]. They are involved in leukocyte chemotaxis, pathogen destruction after corneal infection, corneal epithelial cell migration in corneal ulceration, and corneal angiogenesis [8,9,12–17]. Proteinases are divided into the following categories: matrix metalloproteinases (MMPs), serine proteases, aspartic proteinases, and cysteine proteinases [18]. The MMPs and serine proteinases seem to predominate in the disease processes and wound healing of the cornea of the horse and dog [5,19,20]. Theses proteinases normally exist in balance with proteinase inhibitors or antiproteinases. In the normal cornea and some cases of ulcerative keratitis, antiproteinases serve to prevent excessive tissue degeneration [7,8]. In corneal ulcers, however, the combination of overexpression of certain destructive proteinases and reduction in antiprotease activity can lead to rapid degradation of collagen and other components of the corneal ECM [5,12,21,22]. Normal controls of routine degradative activity seem to be lost, leading to pathologic destruction of the ulcerated cornea (Fig. 1). Corneal ulceration can thus be considered a disorder of proteinase homeostasis [3,5]. D.E. Brooks, F.J. Ollivier / Vet Clin Small Anim 34 (2004) 611–622 613 Fig. 1. This melting ulcer in a dog is associated with elevated metalloproteinase levels in the tear film. Matrix metalloproteinases The MMPs and serine proteinases play an important role in the normal and diseased corneal metabolism of human beings and animals [5,9,21,22]. The MMPs are a multigene family of tightly regulated zinc-dependent enzymes classified according to their substrate specificity [9]. They include gelatinases, collagenases, stromelysins, and membrane-type MMPs (Table 1). All MMPs degrade at least one component of the ECM, share genetic homology with that of the collagenase enzyme, are secreted in a latent form (Fig. 2), possess zinc at the active catalytic site, and are inhibited by naturally occurring tissue inhibitors of metalloproteinases (TIMPs) [9]. MMPs are present at relatively low levels in normal corneal tissues, because little is constitutively expressed [9]. Their expression is upregulated or activated in response to cytokines and growth factors [9]. MMP activity is highly regulated by control of transcription and proenzyme (Pro-MMP) activation and by inhibition of the active enzyme by TIMPs [9]. Pro-MMP can be activated by nonproteolytic compounds, serine proteinases (eg, plasmin), or another MMP (eg, MMP-14) on the cell surface, or it can be activated intracellularly [9]. Two gelatinases, 72-kd gelatinase A (MMP-2) and 92-kd gelatinase B (MMP-9), demonstrate activity against unwound collagen degradation products (gelatin) as well as against collagen types IV, V, and VII [5,12,17,23,24]. Stromelysins degrade the type IV collagen of basement membranes and ECM components [9]. The membrane type MMPs 614 D.E. Brooks, F.J. Ollivier / Vet Clin Small Anim 34 (2004) 611–622 Table 1 Matrix metalloproteinase types and substrate categories MMP number Enzyme name Preferred substrate Collagenase MMP-1 Interstitial collagenase MMP-8 MMP-13 Neutrophil collagenase Collagenase-3 Collagens I, II, III, VII, X; pro-MMP-2; pro-MMP-9 Collagens I, II, III Collagens I, II, III, IV; gelatin; fibronectin Gelatinases MMP-2 Gelatinase A MMP-9 Gelatinase B Stromelysins MMP-3 Stromelysin-1 MMP-10 MMP-11 Membrane type MMPs MMP-14 MMP-16 MMP-17 MMP-24 MMP-25 Others MMP-7 MMP-26 MMP-12 Stromelysin-2 Stromelysin-3 MT1-MMP Gelatin; collagens IV, V, VII, XI; fibronectin; elastin; pro-MMP-9 Gelatin; collagens IV, V; elastin; fibronectin Pro-MMPs-1, -7, -8, -9, -13; laminin; fibronectin Similar to stromelysin-1 a1-proteinase inhibitor MT3-MMP MT4-MMP MT5-MMP MT6-MMP Pro-MMPs-2, -13; collagens; fibronectin Pro-MMP-2 Pro-MMP-2 Pro-MMP-2 ProgelatinaseA Matrilysin Matrilysin-2 Macrophage metalloelastase Collagen IV, elastin, fibronectin Gelatinase, collagen IV, fibronectin Elastin Abbreviations: MMP, matrix metalloproteinase; Pro-MMP, proenzyme matrix metalloproteinase. (MT-MMPs) are anchored to cell membranes by an amino acid tail and play a role in angiogenesis [9]. MMP-2 exists in an inactive form in the normal noninjured cornea, and MMP-9 is undetectable [11]. The synthesis of MMP-9 is induced during corneal injury [9–11]. MMP-9 is involved in the early stages of corneal epithelial wound healing, and MMP-2 is important in the remodeling of the corneal ECM in the later stages of corneal wound healing [8]. MMP-2 is secreted by corneal epithelial cells, stromal keratocytes/ fibroblasts, and polymorphonuclear (PMN) leukocytes [7,11,25] and performs a custodial or housekeeping function in normal cornea by degrading collagen fibers that occasionally become damaged [8,11–13,26]. MMP-9 is produced by corneal epithelial and stromal cells, destroys the adhesive structure of the epithelial cell basement membrane before overt stromal ulceration, and delays the re-epithelialization of the ulcerated cornea [7–9,11]. D.E. Brooks, F.J. Ollivier / Vet Clin Small Anim 34 (2004) 611–622 615 Fig. 2. Gelatin zymogram of tears from an ulcerated eye. This image of a gelatin zymogram gel shows three zymograms on the right as well as the molecular weight markers on the left hand of the gel. The red line on the right lane (lane 1) shows the seven detected bands. The proteinases could be identified according to their location on the gel based on their molecular weight. Pro, latent form; Act, active form. TIMPs are important in maintaining a balance between ECM deposition and breakdown [9,17]. They exist in four isoforms (TIMP-1, TIMP-2, TIMP-3, and TIMP-4) and can bind to pro-MMPs and MMPs. Each TIMP can inhibit most MMPs and is produced by the same cells as those that make the MMPs [9,10]. TIMP-1 has high affinity for MMP-9, and TIMP-2 has high affinity for MMP-2 [8]. The serine proteinases are another class of enzyme that can affect the cornea. Neutrophil elastase (NE) is the most abundant serine proteinase in human, canine, and equine tears and is synthesized by PMN leukocytes and macrophages [5,27]. It degrades native type III and IV collagen as well as the corneal ECM compounds laminin and fibronectin [24,28]. The clinical significance of NE and the serine proteinases remains to be determined, 616 D.E. Brooks, F.J. Ollivier / Vet Clin Small Anim 34 (2004) 611–622 because significant inherent antiproteinase activity against NE is present in horses despite high tear film levels of NE [5,18]. Matrix metalloproteinases and corneal ulcers The response to corneal injury is mediated by leukocytes, fibroblasts, and vascular endothelial cells and includes spatiotemporal phases of inflammation, angiogenesis, re-epithelialization, granulation tissue formation, and ECM deposition [9]. Many of these responses are caused by or modified by MMPs and other proteinases [4,11,22]. The corneal epithelial basement membrane has been shown to degenerate as a result of overexpression of MMPs just before the onset of stromal ulceration [7]. Stromal ulceration cannot occur until after the epithelial basement membrane disappears and is the controlling step leading to corneal stromal ulceration [7]. In severely damaged corneas, proteinase activities are highly enhanced because of activation of MMPs, elevated plasmin activity, and secreted proteinases from inflammatory cells [5,9,16,19,22,29,30]. Tumor necrosis factor-a (TNFa) and transforming growth factor-b (TGFb) stimulate MMP production [31]. If infection is present, the proteinases secreted by infectious organisms are also responsible for the severe corneal damage that is associated with the disease [3,5,10,16,32]. The bacteria Pseudomonas produce two types of MMPs, elastase and alkaline proteases, which play an important role in the aggressive ulcerative keratitis associated with this microbe [16]. In alkali-induced corneal ulcerations of human beings and rabbits, proteinases have long been recognized as mediators of progressive stromal tissue destruction [17,33,34]. Initially, the primary source of these proteinases was believed to be the cornea itself [7,11,12], but a study evaluating the role of PMN leukocytes in corneal collagen degradation found that PMN leukocytes, through the release of proteinases, play an important role in the rapid keratomalacia of collagen and ECM after alkali burns in rabbits [34]. MMPs that contribute to corneal ulceration in the early stages of infection could be of bacterial or corneal cell origin [3,5,35]. Bacterial and fungal pathogens produce MMPs and also induce corneal epithelial cells, corneal stromal fibroblasts, and PMN leukocytes in the tear film to upregulate cytokines (interleukin [IL]-1, IL-6, and IL-8) that induce MMP production and leukocyte infiltration and elicit inflammatory, angiogenic, and destructive processes such that there is an explosion of proteinase activity [10,30,36]. In the later stages, as PMN leukocytes and monocytes invade and accumulate, leukocyte-derived proteinases predominate as the main factor in corneal tissue destruction [17,35]. MMPs are important mediators of enzymatic activity during corneal repair and collagen remodeling [4,11,21,22]. MMP-9 is released in large amounts into the microenvironment of injured corneal tissue from recruited leukocytes and leaking plasma in response to tissue factors and chemoattractants [9]. MMP-2 and MMP-9 are overexpressed at the healing edge of D.E. Brooks, F.J. Ollivier / Vet Clin Small Anim 34 (2004) 611–622 617 re-epithelializing corneal ulcers in dogs and horses [19,20]. MMP-9 is expressed in migrating basal epithelial cells after tissue wounding [8]. Some evidence indicates that TIMP levels are low in nonhealing chronic ulcers [9,10]. MMPs facilitate keratocyte migration and proliferation as well as myofibroblast transformation in the stromal ECM at the corneal injury site to complete corneal ECM synthesis, deposition, contraction, and remodeling [9]. Remodeled stroma is opaque because of disorganization of the newly synthesized ECM. MMPs derived from fibroblasts cause corneal stromal remodeling and increased clarity [9]. Overexpression of stromal MMPs by IL-1, TNFa, and TGFb may lead to stromal scarring and loss of corneal transparency [9,10,30]. Tear film MMP-2, MMP-9, and NE are dramatically elevated in the eyes of horses with corneal ulcers [5,19]. Tear film MMP levels parallel the severity of the corneal disease. These levels diminish when treatment is initiated and as the ulcer heals [19]. A study in dogs found that precorneal tear film serine proteinase levels were significantly higher in dogs with indolent ulcers versus normal controls [27]. MMP-2 and MMP-9 are increased in the corneal epithelium of dogs with refractory superficial ulcers [20]. Matrix metalloproteinases and corneal neovascularization Vascular endothelial cells from limbal venules are stimulated by vascular endothelial growth factor (VEGF) and other angiogenic factors to produce active MMP-2 and MMP-9 that digest the surrounding endothelial cell basement membrane and corneal tissue ECM [9]. Stromal tissue ECM breakdown allows the endothelial cells to migrate and penetrate toward the corneal angiogenic stimulus, where they proliferate and form new capillaries. Neutralizing antibodies to VEGF inhibits this activity [37,38]. Therapeutic use of matrix metalloproteinase inhibitors MMP inhibitors are recommended for treatment of ulcerative keratitis and progressive keratomalacia to reduce the progression of stromal ulceration, speed epithelial healing, and minimize corneal scarring [5,16,31,33, 39–45]. Specific antiproteinases for ophthalmic use include N-acetylcysteine (NAC), disodium ethylene diamine tetraacetate (EDTA), tetracycline antibiotics, and autogenous serum. Serum contains a2-macroglobulin, which has activity against MMPs and serine proteinases, and a1-antitrypsin, which inhibits serine proteinases [5,18,46]. NAC, disodium EDTA, and tetracyclines are metal-chelating agents and seem to inhibit MMPs specifically [5,18,47–54]. EDTA, doxycycline, and NAC inhibit MMPs by chelation of the zinc and calcium that MMPs require as a cofactor and stabilizing ion, respectively [13,18,22,42,53,55]. By chelating the calcium ion, EDTA interferes with the stability of MMPs and thus decreases the stimulation for the 618 D.E. Brooks, F.J. Ollivier / Vet Clin Small Anim 34 (2004) 611–622 migration of PMN leukocytes to the corneal ulcer site. EDTA also interferes with the attachment of the MMPs to the PMN cell membrane, leaving the PMN cell membrane in a resting inactivated state [53]. It seems to be well tolerated by the canine and equine eye when used topically at 0.05% to 0.2% concentrations for corneal ulceration [44] and exhibits a high rate (99.4%) of in vitro anti-MMP activity [19]. Tetracycline type drugs exhibit MMP inhibition activity independent of their antimicrobial properties [49]. The proposed mechanism of action of these antimicrobial agents is that tetracyclines bind to the zinc and calcium cations necessary for MMP activation and thus cause reduced MMP activity [49,54]. Doxycycline, in particular, inhibits the synthesis of MMPs in endothelial cells [54] and reduces the breakdown of cornea mediated by excessive collagenolytic activity [49,54]. It inhibits TGFb-induced MMP-9 production and activity in epithelial cell culture [56]. Doxycycline promotes healing of persistent ulcers and epithelial defects in human beings [47,48,57] and inhibits alkali-induced corneal ulceration in rabbits [48,50]. Its topical and systemic use is recommended in corneal ulceration, because 0.025% to 0.1% doxycycline exhibits a high rate (96.3%) of in vitro anti-MMP activity [19]. NAC is an MMP inhibitor commonly used in human as well as veterinary ophthalmology [3,22,45,51,52]. Topical application of 10% NAC every 1 to 4 hours has been recommended in the dog [45] and the horse [44,45]. No adverse effects of 10% and 20% NAC were found in studies on the rate of re-epithelialization of superficial epithelial ulcers in rabbits [52], although the negative effect of NAC on the stability of the tear film restricts its use to adjunctive therapy. NAC at 10% proved to be effective (98.8%) in vitro in inhibiting tear film MMPs [19]. Autogenous serum contains a number of antiproteinases. a2-Macroglobulin is a nonspecific proteinase inhibitor produced in the liver that reduces the activity of proteinases from all major proteinase classes [18,22,42,58]. It is a tetrameric molecule composed of two pairs of identical disulfide-linked subunits. Binding of the proteinase to the bait region of each subunit leads to a change in the conformation of the a2-macroglobulin molecule and the entrapment of the proteinase within the inhibitor [18,58]. This mechanism results in tenacious binding of two proteinase molecules per one a2macroglobulin molecule and causes this circulating agent to be one of the strongest known inhibitors of MMPs [18,22,42]. This multifunctional inhibitor is present at high levels in blood, comprising 8% to 10% of the total serum proteins [18,59]. For this reason, the topical application of autogenous serum (one or two drops every 1 to 2 hours) is highly recommended for the treatment of corneal ulcerations in human beings [22,42, 60–62] and animals [22,42,44,45]. Blood drawn into sterile containers containing no anticoagulants rapidly clots and yields serum that can be separated by centrifugation [45]. The serum can be used at room temperature or refrigerated until needed, and its inhibitory effect remains high even after several days of storage [44]. It is important to replace it with freshly collected D.E. Brooks, F.J. Ollivier / Vet Clin Small Anim 34 (2004) 611–622 619 serum at least every 8 days, because it may provide a medium for bacterial growth if it becomes contaminated and may show some decline in inhibitory activity [44]. The fibronectin in serum may reduce the discomfort in corneal ulcers and is known as the ‘‘feel good factor’’ [61,62,63]. Serum also contains platelet-derived growth factors that could aid corneal ulcer healing. High levels (90%) of in vitro inhibition of MMP activity by serum have been noted [19]. Efforts have been made to design more powerful synthetic inhibitors of proteinases [40]. Ilomostat, a hydroxamic acid–containing modified dipeptide also called Galardin, seems to be a promising and powerful MMP inhibitor in the treatment of rapid corneal stromal degradation [40]. It is effective against Pseudomonas elastase, MMP-1, and MMP-9. Because the structure of MMPs is highly conserved between animal species, it is reasonable to expect that the inhibitory effects of this synthetic MMP inhibitor could be reproduced in other domestic animals. Ilomostat (0.1%) exhibits a high rate (98.9%) of in vitro anti-MMP activity [19]. Determination of the relative efficacies of proteinase inhibitors indicates that EDTA is about 100 times more effective on a molar basis than NAC. The a2-macroglobulin on a molar basis is superior as an inhibitor to the metal-binding agents [42]. Summary The primary objective of current treatment strategies for infectious keratitis is to sterilize the ulcer as rapidly as possible with topically administered antibiotics. Ulcerative processes can proceed in some cases, despite the absence of microbes, as a result of remaining corneal and tear film MMPs. 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