IOVS
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

(Investigative Ophthalmology and Visual Science. 2003;44:1104-1110.)
© 2003 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.02-0412
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Daniels, J. T.
Right arrow Articles by Khaw, P. T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Daniels, J. T.
Right arrow Articles by Khaw, P. T.

Matrix Metalloproteinase Inhibition Modulates Fibroblast-Mediated Matrix Contraction and Collagen Production In Vitro

Julie T. Daniels,1 Alison D. Cambrey,1 Nicholas L. Occleston,1 Qian Garrett,1 Roy W. Tarnuzzer,2 Gregory S. Schultz,2 and Peng T. Khaw1,3

1From the Wound Healing Research Unit, Department of Pathology and Glaucoma, Institute of Ophthalmology, London, United Kingdom; the 2Institute for Wound Research, University of Florida, Gainesville, Florida; and the 3Glaucoma Unit, Moorfields Eye Hospital, London, United Kingdom.


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
PURPOSE. To investigate the effect of matrix metalloproteinase (MMP) inhibition on fibroblast-mediated matrix contraction and production.

METHODS. Free-floating fibroblast–populated type I collagen lattices were prepared with human Tenon’s capsule fibroblasts. Lattice areas were photographed and digitally analyzed to indicate the degree of lattice contraction. Quantitative competitive reverse transcription–polymerase chain reaction (QCRT-PCR) and enzyme-linked immunosorbent assay (ELISA) were used to quantify mRNA and protein respectively for MMP-1, -2, and -3 by fibroblasts during lattice contraction. Gelatin zymography demonstrated activity of MMPs released into the conditioned medium of contracting lattices. Concentrations of the broad-spectrum MMP inhibitors ilomastat, CellTech (Slough, UK), and BB-94 were added to the contracting fibroblast-populated collagen lattices. Secreted C-terminal propeptide of type I collagen was measured in conditioned medium of contracting lattices by ELISA. Fibroblast proliferation in the presence of concentrations of ilomastat was measured by using the reagent water-soluble tetrazolium-1 (WST-1).

RESULTS. During contraction of type I collagen lattices, Tenon’s capsule fibroblasts expressed MMP-1, -2, and -3 mRNA and protein. Zymography demonstrated the release of four gelatinolytic species into the conditioned medium of contracting lattices (57, 72, 91, and 100 kDa). Inclusion of MMP inhibitors in the zymogram-developing buffer reduced the proteolytic activity of the detected bands. MMP inhibition (1–100 µM) significantly reduced fibroblast-mediated collagen lattice contraction (P < 0.05), and this effect was found to be reversible. Ilomastat also significantly inhibited production of collagen in a dose-dependent manner (P < 0.05). No effect on fibroblast proliferation was found in the presence of ilomastat.

CONCLUSIONS. MMPs are produced during Tenon’s capsule fibroblast-mediated collagen lattice contraction. Broad-spectrum MMP inhibition significantly reduced matrix contraction and production without cell toxicity. Future clinical use of MMP inhibitors may be possible, because MMP inhibition significantly reduces fibroblast functions associated with contractile scarring.

The movement of cells through extracellular matrix (ECM) and contraction of collagen-containing tissues are fundamental to the biology of morphogenesis,1 development,2 wound healing, and scarring throughout the body.3 4 5 Fibroblasts are key players in tissue repair. After injury, quiescent fibroblasts in the surrounding matrix are activated to proliferate and migrate into the wound site to deposit and remodel new matrix.6 In some individuals, this process proceeds to contractile scarring.

Scarring is involved in either the pathogenesis or treatment failure of most visually disabling and blinding conditions worldwide. Successful filtration surgery for the treatment of glaucoma depends directly on an individual’s wound healing response. Maintenance of intraocular pressure in the low teens can prevent long-term progression of glaucoma.7 However, the development of scar tissue over the drainage site can lead to an increase in intraocular pressure and surgical failure.

Use of antimetabolites such as single intraoperative applications of mitomycin-C or subconjunctival injections of 5-fluorouracil has reduced the degree of postoperative scarring.8 9 The initial rationale of these treatments is to reduce the healing response, primarily by suppressing fibroblast proliferation.10 11 12 Clinically, however, surgery still fails in some high-risk patients, even after antiproliferative treatment,8 in part due to residual activity of the growth-arrested cells13 and their potential interaction with surrounding untreated fibroblasts.14 For this reason, new targets for antiscarring therapy, such as neutralization of scar-promoting transforming growth factor-ß15 are still sought. To find alternative targets for antiscarring therapy, identification of factors that facilitate fibroblast activity during wound healing is necessary.

The matrix metalloproteinases (MMPs) are a family of enzymes capable of cleaving components of the ECM. They are regulated at the level of transcription, activation, and inhibition of proteolytic activity.2 16 Collagenases, gelatinases, stromelysins, and membrane-type MMPs are produced by a variety of cell types in disease and after injury, including neutrophils, macrophages, endothelial cells, keratinocytes, and fibroblasts.17 18 19 20 21 22 The requirement of MMP activity for the penetration and movement of several cell types through the ECM has been identified.23 24 25 26 27 28 29 30 31 32 These reports suggest that movement of cells through ECM and subsequent matrix contraction (processes involved in scarring) may involve MMPs. Hence, MMP inhibitors may provide future antiscarring therapies for glaucoma filtration surgery.

The profound antiscarring effect of inhibition of MMP in an experimental model of glaucoma filtration surgery has been described elsewhere.33 Application of the broad-spectrum MMP inhibitor ilomastat in the rabbit resulted in reduced matrix (scar) deposition and cellularity at the filtration surgery site, compared with the control. Fibroblast-populated collagen lattices have long been used to study the fibroblast-mediated contraction phase of wound healing in vitro. Free-floating fibroblast-populated type I collagen lattices originally described by Bell et al.34 are believed to represent contraction resulting from fibroblast migration through matrix.3 Previous studies have demonstrated that broad-spectrum inhibition of MMP can reduce matrix contraction mediated by dermal fibroblasts31 and retinal pigmented epithelial cells.32 These reports did not include an assessment of the production of collagen matrix, a crucial element of the scarring response. Using Tenon’s capsule fibroblast-populated collagen lattices, we investigated the effect of MMP inhibition on fibroblast-mediated matrix contraction and collagen production to determine how ilomastat may act as an antiscarring agent in vivo after glaucoma filtration surgery.


   Methods
Top
 Abstract
 Methods
Results
 Discussion
 References
 
Cell Culture
Human Tenon’s capsule fibroblasts were isolated and cultured as previously described.10 The cells were maintained in fibroblast culture medium (FCM) composed of Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (vol/vol) newborn calf serum, 2 mM L-glutamine, 100 IU/mL penicillin, 100 µg/mL streptomycin, 50 mg/mL gentamicin, and 0.25 mg/mL amphotericin B (all from Gibco Life Technologies, Paisley, Scotland, UK) at 37°C with 5% (vol/vol) CO2 in air. Cultures were used between passages 3 and 7 for the experiments. The tenets of the Declaration of Helsinki were followed, and institutional human experimentation committee approval was granted.

Collagen Contraction
To assess matrix contraction free-floating collagen lattice models were used.3 Three-dimensional fibroblast-populated type I collagen (Sigma Chemical Co., Poole, Dorset, UK) lattices were prepared with 500,000 cells/mL of lattice as previously described.35 The lattices were fed with FCM and detached before addition of test substances. Reduction in lattice area due to contraction was determined as previously described at intervals up to 7 days.36

MMP mRNA Expression
Total RNA was isolated from cells within contracting collagen lattices (3 x 106) by the method of Chomczynski and Sacchi.37 Levels of mRNA for MMP-1, -2, -3, and -9 were determined by quantitative competitive reverse transcription-polymerase chain reaction (QCRT-PCR).38 39 This technique allows the calculation of message copy number/103 cells in a sample.

Production of MMP Protein
MMP levels were measured in conditioned medium collected from contracting fibroblast-populated collagen lattices by enzyme-linked immunosorbent assay (ELISA). MMP-1 (pro and active forms) was quantified with a double sandwich ELISA kit (Amersham, Buckinghamshire, UK). MMP-2 (pro form) and MMP-3 (pro form) were quantified with a specific one-step immunoassay system (Fuji Chemicals Ltd., Toyama, Japan).

MMP Activity
Samples of conditioned medium collected from contracting lattices (fed with FCM containing gelatinase-free serum)40 were stored at -70°C, lyophilized, and reconstituted in phosphate-buffered saline (PBS) before use. The lattices were homogenized.41 Both medium and homogenate samples were analyzed by gelatin zymography using a commercial system and buffers (Novex Mini Cell; InVitrogen, Groningen, The Netherlands) in accordance with the manufacturer’s instructions. Samples activated with amino phenyl mercuric acid (APMA) were included to detect characteristic MMP molecular weight shifts after activation.42 Conditioned medium collected from fibroblast-populated lattices at day 7 was also subjected to zymography. However, before the development stage, the zymograms were cut into individual lanes and then developed in the presence of MMP inhibitor concentrations to determine the effects of the inhibitors on MMP activity.

Inhibition of MMP
The effect of several broad-spectrum, hydroxamic-acid–derived MMP inhibitors on lattice contraction were investigated: ilomastat,43 BB-94,44 and an MMP inhibitor, BMS-275291, referred to as CellTech (CellTech, Slough, UK). These compounds are capable of inhibiting the MMPs detected in this study—that is MMP-1, -2, and -3.43 45 All inhibitors were prepared as stock solutions in dimethyl sulfoxide (DMSO) and used at concentrations between 0.1 nM and 100 µM. Hydroxamic acid (100 µM) in 0.1% (vol/vol) DMSO served as a control. The MMP inhibitors were added individually to the culture medium of the lattices at the start of the experiment.

Reversibility of Lattice Contraction by Inhibition of MMP
The effects of the addition of ilomastat to contracting lattices (containing 100,000 cells/lattice) and removal of ilomastat from lattices previously inhibited from contracting were investigated over 7 days. In the addition experiments, ilomastat (10 µM) was added to lattices at 1 day after seeding. In the removal experiments, lattices that had been exposed to ilomastat (10 µM) for 2 days after seeding were washed in PBS and cultured in FCM containing hydroxamic acid (10 µM).

Collagen Production
Matrix synthesis was determined over 7 days in the presence or absence of ilomastat (100 µM). Secreted C-terminal propeptide of collagen type I (CICP) was measured in culture supernatant 24 hours after feeding, using an ELISA (Quidel Corp., Oxford, UK).46 CICP levels therefore provide an index for formation of fibroblast matrix in vitro. Data were corrected for the presence of background CICP in fresh culture medium.

Proliferation
Fibroblast proliferation, in the presence of concentrations of ilomastat, was monitored with a kit incorporating the reagent water-soluble tetrazolium (WST-1; Roche Molecular Diagnostics and Biochemicals, Lewes, UK) according to the manufacturer’s instructions. Briefly, fibroblasts were seeded into 96-well tissue culture plates, at a density of 2.5 x 103 per well in FCM, and cultured for 4 hours. FCM was removed and the cells were washed three times with PBS before addition of FCM containing concentrations of ilomastat (0–100 µm). Four plates per assay were seeded for harvesting at 2 hours and 1, 3, and 7 days. At each time interval, 10 µL WST-1 was added to each well, and the plate was incubated at 37°C for 2 hours. The absorbance, related to the number of viable cells converting the reagent to colored formazan crystals, was read at 450 nm. On day 3 the cells for harvesting on day 7 were refed.

Statistical Analysis
All experiments were repeated at least three times. The cell culture experiments were analyzed by one-way analysis of variance (ANOVA) incorporating the Bonferroni correction on computer (SPSS for Windows; SPSS Inc., Chicago, IL). P < 0.05 was considered to be statistically significant. Standard error of the mean was plotted on the graphs.


   Results
Top
 Abstract
 Methods
 Results
Discussion
 References
 
MMP Production during Fibroblast-Mediated Collagen Lattice Contraction
Using QCRT-PCR, fibroblasts within contracting lattices were found to express mRNA for MMP-1, -2, and -3 (mRNA for MMP-9 was not detected). The expression levels peaked at day 1 and was then reduced by day 7 when contraction of the lattices had virtually ceased (Table 1) . Protein production for each MMP, quantified by ELISA, peaked at day 7 (Table 1) . Zymography demonstrated that fibroblasts seeded within the collagen lattices released four gelatinolytic species into the medium (57, 72, 91, and 100 kDa; Fig. 1A ) during contraction. Similar profiles were also found for homogenized lattices (data not shown). Because MMP-9 mRNA was not detected using the very sensitive QCRT-PCR method, MMP-9 protein was not measured by ELISA. However, the very faint high-molecular-mass bands of 91 and 100 kDa detected by zymography were similar in molecular mass to pro-MMP-9 (92 kDa). Therefore, it is possible that these bands represent the 92-kDa pro-MMP-9 enzyme. Because the levels were very low and did change throughout contraction, they were not studied further. APMA-induced activation of MMPs in the conditioned medium (Fig. 1B) resulted in a reduction of the 100-kDa species and partial reduction of the 72-kDa (corresponding to the molecular mass of MMP-2) species to a 65-kDa band (active MMP-2) and increased levels of the 57-kDa species (corresponding to the molecular masses of MMP-1 and -3). The reduction of the 100 and partial reduction of the 72-kDa bands is demonstrated by the disappearance of a band at 100 kDa (compared with Fig. 1A ) and a reduction in size of the 72-kDa band (Fig. 1B compared with Fig. 1A ). Adding increasing concentrations of ilomastat, BB-94, and CellTech (Fig. 1C) to the developing buffer of zymograms increasingly inhibited the activity of the 72- and 57-kDa MMP bands produced during fibroblast-mediated collagen lattice contraction. On the zymograms, the white bands represent MMP activity. The dark bands that are seen in some of the lanes are the MMP proteins, detected by the Coomassie blue stain, that have no gelatinolytic activity because of the presence of the MMP inhibitors.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Quantification of Fibroblast MMP during Collagen Contraction

 


View larger version (40K):
[in this window]
[in a new window]
  		FIGURE 1. Analysis and inhibition of gelatinolytic activity produced during ocular fibroblast-mediated collagen contraction by gelatin zymography. (A) MMP activity produced during collagen contraction, by ocular fibroblasts seeded within collagen lattices, in samples of conditioned medium. Lane 1: day 1; lane 2: day 3; and lane 3: day 7 after seeding. Gelatinolytic activity increased during the 7-day contraction assay. Four bands of gelatinolytic activity were produced (molecular masses to the right). (B) Effects of APMA on MMP profiles produced by ocular fibroblasts within collagen lattices, by gelatin zymography. Lane 1: day 1; lane 2: day 3; and lane 3: day 7 after seeding. Activities at 100-, 91-, and 72-kDa were reduced after incubation with APMA, suggesting these activities were proenzymes. The 57-kDa activity was not affected by treatment with APMA, indicating active-form enzyme(s). (C) Effects of MMP inhibitors on MMP activity produced during collagen contraction, by gelatin zymography. Photographs show that exposure to increasing concentrations of MMP inhibitors (ilomastat, BB-94, and CellTech) reduced the MMP (72 and 57 kDa) activity present in samples of day-7 conditioned medium of contracting lattices in a concentration-dependent manner.

 
Effect Of MMP Inhibitors on Fibroblast-Mediated Collagen Contraction
Exposure of fibroblast-populated collagen lattices to concentrations of ilomastat (Fig. 2A) , BB-94 (Fig. 2B) , or CellTech (Fig. 2C) significantly inhibited contraction in a concentration-dependent manner by day 7, compared with the control (P < 0.05). The results for ilomastat were of the most interest to us, because this inhibitor was found to reduce fibroblast-collagen matrix contraction over the widest concentration range and also was very effective for reducing scarring in an animal model of glaucoma filtration surgery.33 Ilomastat is one of the most potent inhibitors of collagenase (MMP-1 which is required for cleavage for the type I collagen triple helix) with an inhibition constant (Ki) of 0.4 nM.43



View larger version (18K):
[in this window]
[in a new window]
  		FIGURE 2. Effects of MMP inhibitors on ocular fibroblast-mediated collagen contraction. Fibroblast-mediated collagen lattice contraction was analyzed in the presence of the MMP inhibitors ilomastat (A), BB-94 (B), or CellTech (C). The concentrations were () 0, ({blacksquare}) 0.1 nM, ({blacktriangleup}) 1 nM, ({blacktriangledown}) 10 nM, ({diamondsuit}) 100 nM, () 1 µM, (•) 10 µM, and ({blacksquare}) 100 µM. Exposure to MMP inhibitors significantly inhibited contraction compared with the control (P < 0.05). Error bars, SE.

 
Effect of Removal and Addition of MMP Inhibitor on Fibroblast-Mediated Collagen Contraction
The anticontractile effects of ilomastat (10 µM) were further investigated in the fibroblast-populated collagen lattices. Removal of ilomastat from inhibited lattices resulted in their continued contraction (Fig. 3 , top). Addition of ilomastat to contracting lattices significantly inhibited (P < 0.05) contraction compared with the control (Fig. 3 , bottom).



View larger version (13K):
[in this window]
[in a new window]
  		FIGURE 3. Effect of removal and addition (bottom) of ilomastat on ocular fibroblast-mediated collagen contraction. Top: in the removal experiment, fibroblast-populated collagen lattices were continuously fed with (•) 10 µm ilomastat ({blacktriangleup}) or 10 µm hydroxamic acid (control) or ({blacksquare}) had the ilomastat removed after 2 days of culture (arrow). The inhibitory effects of ilomastat on fibroblast-mediated collagen contraction were reversible. Bottom: In the addition experiment, fibroblast-populated lattices were allowed to contract for 1 day (arrow) before addition of (•) 10 µm ilomastat, ({blacksquare}) 10 µm hydroxamic acid (control), or ({blacktriangleup}) fibroblast culture medium only. The results showed that the inhibitory effects of ilomastat on fibroblast-mediated collagen contraction can also take effect subsequent to initiation of the contraction process. Error bars, SE.

 
Collagen Production
Secreted CICP was detected by ELISA in the culture supernatant of contracting fibroblast-populated collagen lattices (Fig. 4) . Fibroblasts actively secreted CICP during lattice contraction up to 3 days. By day 7, when fibroblast-mediated lattice contraction had virtually ceased, CICP secretion was also reduced. The MMP inhibitor ilomastat (100 µm) significantly reduced CICP secretion at each time point (P < 0.05).



View larger version (15K):
[in this window]
[in a new window]
  		FIGURE 4. Effect of ilomastat on type I collagen production. Secreted CICP was detected by ELISA in the culture supernatant of contracting fibroblast-populated collagen lattices. Fibroblasts actively secreted CICP during lattice contraction up to 3 days. At day 7, secretion of CICP was reduced. The MMP inhibitor ilomastat (100 µM) significantly reduced secreted CICP at each time point (*P < 0.05, compared with the control).

 
Fibroblast Proliferation
Fibroblasts were found to proliferate in the presence of concentrations of ilomastat (0–100 µm) as detected by the WST-1 assay (Fig. 5) . No significant difference was found between the rates of proliferation in each concentration of ilomastat compared with the control. The assay measures the ability of cells to convert the WST-1 reagent to formazan and hence requires the cells to be viable. The data indicate that any influence that ilomastat has on fibroblast function is unlikely to be due to toxicity of the MMP inhibitor. To confirm fibroblast viability, cells were cultured in the presence of concentrations of ilomastat as described earlier. Viability was confirmed (data not shown) by trypan blue dye exclusion47 and with a lactate dehydrogenase activity kit (Roche Diagnostics and Biochemicals).



View larger version (19K):
[in this window]
[in a new window]
  		FIGURE 5. Effect of ilomastat on fibroblast proliferation. Fibroblast proliferation in the presence of ({blacktriangleup}) 0, ({blacksquare}) 1 nM, ({blacktriangleup}) 10 nM, () 100 nM, () 1 µm, (•) 10 µm, and () 100 µm ilomastat was measured with the reagent WST-1. No significant difference was found between the rates of proliferation. Error bars, SEM.

 

   Discussion
Top
 Abstract
 Methods
 Results
 Discussion
References
 
Members of the MMP family are upregulated during a number of biological processes, including tissue morphogenesis, embryonic development, and wound healing. In our study of scarring after glaucoma filtration surgery in the rabbit, reduced cellularity was observed in the group treated with the MMP inhibitor ilomastat, suggesting that inhibition of MMP may impair the ability of fibroblasts to migrate into the wound site.33 The free-floating fibroblast-populated collagen in vitro lattice model is thought to represent matrix contraction exerted by tractional forces generated by cells as they migrate through matrix in vivo.48 49 50

Data obtained in an in vitro model of fibroblast-mediated matrix contraction demonstrated involvement of members of the MMP family in this process. Both mRNA and protein production were markedly upregulated during fibroblast-mediated collagen lattice contraction. The main substrate for MMP-1 is type I collagen; hence, identification of MMP-1 in the conditioned medium of the contracting lattices suggests utilization of this enzyme during matrix contraction. MMP-2, also known as gelatinase-A, because of its gelatinolytic activity, was also detected in the conditioned media. When MMP-1 makes a single site-specific cleavage through the triple helix of type I collagen, the resultant fragments become thermally unstable at body temperature making them susceptible to further degradation by gelatinases such as MMP-2.51 Detection of MMP-2 in the in vitro contraction model may indicate a role for this enzyme in further processing and remodeling of the collagen lattice by fibroblasts during contraction. Indeed, much less MMP-1 was detected than MMP-2, perhaps preventing all the collagen from being susceptible to further proteolysis and possible dissolution, yet allowing remodeling to occur.

The presence of MMP-3, the major substrates of which are the proteoglycans, may be explained by the presence of small amounts of proteoglycan material in the commercially prepared collagen used. It was noted that between days 1 and 7 the amount of MMP mRNA detected decreased, yet protein production increased. The MMPs are regulated on a number of levels, including transcription, activation, and proteolytic inhibition.2 By day 7, fibroblast-mediated matrix contraction had virtually ceased, and further transcription of MMP mRNA no longer appeared to be needed. The MMP proteins detected at day 7 were likely to have been translated from mRNA messages previously transcribed by the cells during active contraction before day 7. The ELISA kits used to measure production of MMP detected both the pro and active forms of MMP-1 but only the pro forms of MMPs-2 and -3, therefore the presence of high levels of MMP protein at day 7 did not necessarily reflect activation and utilization of the total amount of enzyme protein produced. Gelatin zymography detects activity of both pro and active forms of MMPs with gelatinolytic substrate specificity. The normally nonactive enzyme pro forms are detected, because sodium dodecyl sulfate within the zymogram gels unfolds the pro-MMP protein exposing the active catalytic site of the enzyme. Activation of MMPs in the conditioned medium by APMA showed characteristic molecular weight shifts from pro to active enzyme forms.42 This experiment also demonstrated that much of the MMP proteins released into the conditioned medium of contracting lattices remained in their latent forms. The metalloproteinase activity of the proteins released was confirmed by the addition of broad-spectrum MMP inhibitors into the zymogram-developing buffer, which inhibited gelatinolytic activity in a concentration-dependent manner.

In agreement with our previous findings and those of other investigators,31 52 the broad-spectrum MMP inhibitors significantly reduced fibroblast-populated collagen lattice contraction in a dose-dependent manner. Ilomastat, which is one of the most potent inhibitors of MMP-1 with a Ki of 0.4 nM,43 was found to be more effective than BB-94 or CellTech in reducing contraction over the widest concentration range and therefore received more attention throughout the study.

This effect did not appear to be due to cytotoxicity, because the cells were able to proliferate normally and remained viable in the presence of all concentrations of the MMP inhibitors used. Indeed, the effect on fibroblast-mediated matrix contraction was found to be reversible, in that removal of MMP inhibitor allowed previously inhibited contraction to proceed. The addition of MMP inhibitor reduced contraction of fibroblast-populated collagen lattices. Other studies have indicated the clinical relevance and importance of MMPs in fibroblast and endothelial cell-mediated collagen matrix contraction.53 54 Because matrix contraction plays such a significant role in contractile scarring,55 56 it is possible that inhibition of MMP may reduce scarring in vivo, as demonstrated in our other study, by reducing fibroblast-mediated matrix contraction. The in vivo filtration surgery model showed decreased cellularity at the wound site in the MMP inhibitor-treated group compared with the control by day 30, perhaps suggesting that the inhibitor had impaired the ability of cells to migrate into the wound site. The in vitro data corroborate this hypothesis, in that contraction of free-floating lattices is thought to be mediated by tractional forces generated on the matrix by migrating fibroblasts.48 49 57 If indeed inhibition of MMP reduces fibroblast migration, it could serve to reduce scarring at a number of levels, by reducing contraction brought about by cell locomotion and by reducing the number of fibroblasts arriving at the wound site, hence reducing the number of fibroblasts available to differentiate into myofibroblasts that are capable of contracting matrix in situ without locomotion.58

Our filtration surgery model showed less matrix deposition in the MMP inhibitor-treated groups compared with the control.33 This may have been due partly to decreased cellularity at the wound site and hence less matrix production. However, the in vitro data showed that application of the MMP inhibitor ilomastat to contracting fibroblast-populated collagen lattices also reduced synthesis of new collagen by the fibroblasts. Previously, it was thought that synthetic MMP inhibitors exerted their effects by simply neutralizing the activity of MMPs to which they bound. Our data show for the first time the ability of a synthetic MMP inhibitor to alter matrix production by fibroblasts. This is a surprising and very important finding in the context of antiscarring therapy, because excess matrix deposition and contraction significantly contribute to the formation of contractile scar tissue.55 56 Procollagen consists of mature collagen with extension peptides, or propeptides, that are cleaved by specific proteases from the collagen molecule before incorporation into a growing collagen fibril.59 The release of these peptides into the conditioned medium of the collagen lattices provides a stoichiometric representation of the production of collagen detectable by ELISA. It is possible that the MMP inhibitor prevented cleavage of the collagen propeptides; hence, there were less to detect in the conditioned medium. Nevertheless, the synthetic MMP inhibitor appeared to reduce collagen deposition in vivo and in vitro by an as yet unknown mechanism. This is currently under investigation in our laboratory.

Contractile scarring plays a role in the pathogenesis and treatment failure of many visually disabling or blinding conditions including glaucoma (conjunctival scarring after surgery), cataracts (capsular contraction), trachoma (entropion and corneal scarring), burns, proliferative vitreoretinopathy, and even age-related macular degeneration. The movement of cells through ECM and the subsequent contraction of collagen-containing tissues are crucial components of the scarring response. As such, our findings that inhibition of MMP can reduce deposition and contraction in the ECM may have important implications for the development of new antiscarring therapies not only for the eye but other parts of the body.


   Acknowledgements
 
The authors would like to thank Glycomed Inc. (San Diego, CA) for providing ilomastat, and British Technology (Oxford, UK) for providing BB-94.


   Footnotes
 
Supported in part by the Royal National Institute for the Blind, the Mr. and Mrs. Magnus Bequest of Fight for Sight (JTD), The Eranda Foundation (JTD), The Hayman Trust (JTD), Guide Dogs for the Blind Association (NLO, ADC, PTK), International Glaucoma Association (NLO), National Eye Institute Grant EY05587 (GSS), and The Wellcome Trust (QG). The views expressed in this publication are those of the authors and not necessarily those of the grant-awarding bodies.

Submitted for publication April 24, 2002; revised August 21, 2002; accepted September 5, 2002.

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: Julie T. Daniels, Wound Healing Research Unit, Department of Pathology and Glaucoma, Institute of Ophthalmology and Moorfields Eye Hospital, 11-43 Bath Street, London EC1V 9EL, UK; j.daniels{at}ucl.ac.uk.


   References
Top
 Abstract
 Methods
 Results
 Discussion
 References
 

  1. Werb, Z, Chin, JR. (1998) Extracellular matrix remodelling during morphogenesis Ann NY Acad Sci 857,110-118[Abstract/Free Full Text]
  2. Sternlicht, MD, Werb, Z. (2001) How matrix metalloproteinases regulate cell behaviour Annu Rev Cell Dev Biol 17,463-516[CrossRef][Medline][Order article via Infotrieve]
  3. Grinnell, F. (1994) Fibroblasts, myofibroblasts, and wound contraction J Cell Biol 124,401-404[Free Full Text]
  4. Pilcher, BK, Wang, M, Qin, XJ, et al (1999) Role of matrix metalloproteinases and their inhibition in cutaneous wound healing and allergic contact hypersensitivity Ann NY Acad Sci 878,12-24[Abstract/Free Full Text]
  5. Ravanti, L, Kahari, VM. (2000) Matrix metalloproteinases in wound repair (review) Int J Mol Med 4,391-407
  6. Clark, RAF, Henson, PM. (1988) The Molecular and Cellular Biology of Wound Repair Plenum Press New York.
  7. . AGIS Investigators (2000) The relationship between control of intraocular pressure and visual field deterioration Am J Ophthalmol 130,429-440[CrossRef][Medline][Order article via Infotrieve]
  8. Skuta, GL, Beeson, CC, Higginbotham, EJ. (1992) Intraoperative mitomycin versus postoperative 5-fluorouracil in high-risk filtering surgery Ophthalmology 99,438-444[Medline][Order article via Infotrieve]
  9. . Fluorouracil Filtering Surgery Group (1996) Five-year follow-up of the fluorouracil filtering surgery study Am J Pathol 121,349-366[Abstract]
  10. Khaw, PT, Ward, S, Porter, A, et al (1992) The long-term effects of 5-fluorouracil and sodium butyrate on human Tenon’s fibroblasts Invest Ophthalmol Vis Sci 33,2043-2052[Abstract/Free Full Text]
  11. Khaw, PT, Sherwood, MB, MacKay, SL, et al (1992) Five-minute treatments with fluorouracil, floxuridine, and mitomycin have long-term effects on human Tenon’s capsule fibroblasts Arch Ophthalmol 110,1150-1154[Abstract]
  12. Khaw, PT, Doyle, JW, Sherwood, MB, et al (1993) Prolonged localized tissue effects from 5-minute exposures to fluorouracil and mitomycin C Arch Ophthalmol 111,263-267[Abstract]
  13. Occleston, NL, Daniels, JT, Tarnuzzer, RW, et al (1997) Single exposures to antiproliferatives: long-term effects on ocular fibroblast wound-healing behaviour Invest Ophthalmol Vis Sci 38,1998-2007[Abstract/Free Full Text]
  14. Daniels, JT, Occleston, NL, Crowston, JG, et al (1999) Effects of antimetabolite induced cellular growth arrest on fibroblast-fibroblast interactions Exp Eye Res 69,117-127[CrossRef][Medline][Order article via Infotrieve]
  15. Cordeiro, MF, Gay, JA, Khaw, PT. (1999) Human anti-transforming growth factor-beta2 antibody: a new glaucoma anti-scarring agent Invest Ophthalmol Vis Sci 40,2225-2234[Abstract/Free Full Text]
  16. Nagase, H, Woessner, F. (1999) Matrix metalloproteinases J Biol Chem 274,21491-21494[Free Full Text]
  17. Rudolph-Owen, LA, Matrisian, LM. (1998) Matrix metalloproteinases in remodelling of the normal and neoplastic mammary gland J Mammary Gland Biol Neoplasia 2,177-189
  18. Fini, ME, Cook, JR, Mohan, JJ. (1998) Proteolytic mechanisms in corneal ulceration and repair Arch Dermatol Res 290,S12-S23
  19. Luttan, A, Dewerchin, M, Collen, D, et al (2000) The role of proteinases in angiogenesis, heart development, restenosis, atherosclerosis, myocardial ischemia, and stroke: insights from genetic studies Curr Atheroscler Rep 5,407-416
  20. Kahari, VM, Saarialho-Kere, U. (1997) Matrix metalloproteinases in skin Exp Dermatol 5,199-213
  21. Vaalamo, M, Karjalainen-Lindsberg, ML, Puolakkainen, P, et al (1998) Distinct expression profiles of stromelysin-2 (MMP-10), collagenase-3 (MMP-13), macrophage metalloelastase (MMP-12), and tissue inhibitor of metalloproteinase-3 (TIMP-3) in intestinal ulcerations Am J Pathol 152,1005-1014[Abstract]
  22. Suomela, S, Kariniemi, AL, Snellman, E, et al (2001) Metalloelastase (MMP-12) and 92-kDa gelatinase (MMP-9) as well as their inhibitors, TIMP-1 and -3, are expressed in psoriatic lesions Exp Dermatol 10,175-183[CrossRef][Medline][Order article via Infotrieve]
  23. Bendeck, MP, Zempo, N, Clowes, AW, et al (1994) Smooth muscle cell migration and matrix metalloproteinase expression after arterial injury in the rat Circ Res 75,539-545[Abstract/Free Full Text]
  24. Pauly, RR, Passaniti, A, Bilato, C, et al (1994) Migration of cultured vascular smooth muscle cells through a basement membrane barrier requires type IV collagenase activity and is inhibited by cellular differentiation Circ Res 75,41-54[Abstract/Free Full Text]
  25. Leppert, D, Hauser, SL, Kishiyama, JL, et al (1995) Stimulation of matrix metalloproteinase-dependent migration of T cells by eicosanoids FASEB J 9,1473-1481[Abstract]
  26. Leppert, D, Waubant, E, Galardy, R, et al (1995) T cell gelatinases mediate basement membrane transmigration in vitro J Immunol 154,4379-4389[Abstract]
  27. Romanic, AM, Madri, JA. (1994) The induction of 72-kD gelatinase in T cells upon adhesion to endothelial cells is VCAM-1 dependent J Cell Biol 125,1165-1178[Abstract/Free Full Text]
  28. Nordstrom, LA, Lochner, J, Yeung, W, et al (1995) The metalloproteinase stromelysin-1 (transin) mediates PC12 cell growth cone invasiveness through basal laminae Mol Cell Neurosci 6,56-68[CrossRef][Medline][Order article via Infotrieve]
  29. Nakagawa, T, Kubota, T, Kabuto, M, et al (1995) Captopril inhibits glioma cell invasion in vitro: involvement of matrix metalloproteinases Anticancer Res 15,1985-1989[Medline][Order article via Infotrieve]
  30. Terada, T, Okada, Y, Nakanuma, Y. (1995) Expression of matrix proteinases during human intrahepatic bile duct development: a possible role in biliary cell migration Am J Pathol 147,1207-1213[Abstract]
  31. Scott, KA, Wood, EJ, Karran, EH. (1998) A matrix metalloproteinase inhibitor which prevents fibroblast-mediated collagen lattice contraction FEBS Lett 441,137-140[CrossRef][Medline][Order article via Infotrieve]
  32. Sheridan, CM, Occleston, NL, Hiscott, P, et al (2001) Matrix metalloproteinases: a role in the contraction of vitreo-retinal scar tissue Am J Pathol 159,1555-1566[Abstract/Free Full Text]
  33. Wong, TTL, Mead, AL, Khaw, PT. (2003) Matrix metalloproteinase inhibition modulates postoperative scarring after experimental glaucoma filtration surgery Invest Ophthalmol Vis Sci 44,1097-1103[Abstract/Free Full Text]
  34. Bell, E, Ivarsson, B, Merrill, C. (1979) Production of a tissue-like structure by contraction of collagen lattices by human fibroblasts of different proliferative potential in vitro Proc Natl Acad Sci USA 76,1274-1278[Abstract/Free Full Text]
  35. Mazure, A, Grierson, I. (1992) In vitro studies of the contractility of cell types involved in proliferative vitreoretinopathy Invest Ophthalmol Vis Sci 33,3407-3416[Abstract/Free Full Text]
  36. Occleston, NL, Alexander, RA, Mazure, A, et al (1994) Effects of single exposures to antiproliferative agents on ocular fibroblast-mediated collagen contraction Invest Ophthalmol Vis Sci 35,3681-3690[Abstract/Free Full Text]
  37. Chomczynski, P, Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction Anal Biochem 162,156-159[Medline][Order article via Infotrieve]
  38. Tarnuzzer, RW, Macauley, SP, Farmerie, WG, et al (1996) Competitive RNA templates for detection and quantitation of growth factors, cytokines, extracellular matrix components and matrix metalloproteinases by RT-PCR Biotechniques 20,670-674[Medline][Order article via Infotrieve]
  39. Ashcroft, G, Herrick, S, Tarnuzzer, R, et al (1997) Human ageing impairs injury-induced in vivo expression of tissue inhibitor of matrix metalloproteinases (TIMP)-1 and -2 proteins and mRNA J Pathol 183,169-176[CrossRef][Medline][Order article via Infotrieve]
  40. Azzam, HS, Thompson, EW. (1992) Collagen-induced activation of the M(r) 72, 000 type IV collagenase in normal and malignant human fibroblastoid cells Cancer Res 52,4540-4544[Abstract/Free Full Text]
  41. Goldberg, MA, Gaut, CC, Bunn, HF. (1991) Erythropoietin mRNA levels are governed by both the rate of gene transcription and posttranscriptional events Blood 77,271-277[Abstract/Free Full Text]
  42. Nagase, H, Suzuki, K, Morodomi, T, et al (1992) Activation mechanisms of the precursors of matrix metalloproteinases 1, 2 and 3 Matrix Suppl 1,237-244[Medline][Order article via Infotrieve]
  43. Grobelny, D, Poncz, L, Galardy, RE. (1992) Inhibition of human skin fibroblast collagenase, thermolysin, and Pseudomonas aeruginosa elastase by peptide hydroxamic acids Biochemistry 31,7152-7154[CrossRef][Medline][Order article via Infotrieve]
  44. Davies B, Brown PD, East, N, et al (1993) A synthetic matrix metalloproteinase inhibitor decreases tumor burden and prolongs survival of mice bearing human ovarian carcinoma xenografts [published correction appears in Cancer Res. 1993;53:3652] Cancer Res 53,2087-2091[Abstract/Free Full Text]
  45. Fini, ME, Cui, TY, Mouldovan, A, et al (1991) An inhibitor of the matrix metalloproteinases synthesized by rabbit corneal epithelium Invest Ophthalmol Vis Sci 32,2997-3001[Abstract/Free Full Text]
  46. Siggelkow, H, Niedhart, C, Kurre, W, et al (1998) In vitro differentiation potential of a new human osteosarcoma cell line (HOS 58) Differentiation 63,81-91[CrossRef][Medline][Order article via Infotrieve]
  47. Hunt, SV. (1987) Kavovs, GGB eds. The Lymphocytes: a Practical Approach ,1-35 IRL Press Oxford, UK.
  48. Harris, AK, Stopak, D, Wild, P. (1981) Fibroblast traction as a mechanism for collagen morphogenesis Nature 290,249-251[CrossRef][Medline][Order article via Infotrieve]
  49. Ehrlich, HP, Rajaratnam, JB. (1990) Cell locomotion forces versus cell contraction forces for collagen lattice contraction: an in vitro model of wound contraction Tissue Cell 22,407-417[CrossRef][Medline][Order article via Infotrieve]
  50. Eastwood, M, McGrouther, DA, Brown, RA. (1994) A culture force monitor for measurement of contraction forces generated in human dermal fibroblast cultures: evidence for cell-matrix mechanical signalling Biochim Biophys Acta 1201,186-192[Medline][Order article via Infotrieve]
  51. Jeffrey, JJ. (1998) Parks, WC Mecham, RP. eds. Matrix Metalloproteinases ,15-42 Academic Press, Inc. San Diego, CA.
  52. Stephens, P, Davies, KJ, Occleston, N, et al (2001) Skin and oral fibroblasts exhibit phenotypic differences in extracellular matrix reorganisation and matrix metalloproteinase activity Br J Dermatol 144,229-237[CrossRef][Medline][Order article via Infotrieve]
  53. Davis, GE, Pintar Allen, KA, Salazar, R, et al (2001) Matrix metalloproteinase-1 and -9 activation by plasmin regulates a novel endothelial cell-mediated mechanism of collagen gel contraction and capillary tube regression in three-dimensional collagen matrices J Cell Sci 114,917-930[Abstract]
  54. Rittie, L, Berton, A, Monboisse, JC, et al (1999) Decreased contraction of glycated collagen lattices coincides with impaired matrix metalloproteinase production Biochem. Biophys Res Commun 264,488-492[CrossRef][Medline][Order article via Infotrieve]
  55. Daniels, JT, Occleston, NL, Crowston, JG, et al (1998) Understanding and controlling the scarring response: the contribution of histology and microscopy Microsc Res Tech 42,317-333[CrossRef][Medline][Order article via Infotrieve]
  56. Cordeiro, MF, Chang, L, Lim, KS, et al (2000) Modulating conjunctival wound healing Eye 14,536-547
  57. Eastwood, M, Porter, R, Khan, U, et al (1996) Quantitative analysis of collagen gel contractile forces generated by dermal fibroblasts and the relationship to cell morphology J Cell Physiol 166,33-42[CrossRef][Medline][Order article via Infotrieve]
  58. Jester, JV, Petroll, WM, Barry, PA, et al (1995) Temporal, 3-dimensional, cellular anatomy of corneal wound tissue J Anat 186,301-311
  59. Olsen, BR. (1991) Hay, ED eds. Cell Biology of Extracellular Matrix ,177-220 Plenum Press New York.



This article has been cited by other articles:


Home page
 IOVSHome page
W.-C. Wu, C.-C. Lai, H. S.-L. Chen, M.-H. Sun, L.-M. Lee, C.-P. Shih, H. W.-H. Lee, and W.-C. Hsu
Efficacy and Safety of Biodegradable Collagen-Glycosaminoglycan Polymer as a Material for Scleral Buckling
Invest. Ophthalmol. Vis. Sci., June 1, 2008; 49(6): 2673 - 2678.
[Abstract] [Full Text] [PDF]

Home page
 IOVSHome page
N. Awasthi, S. T. Wang-Su, and B. J. Wagner
Downregulation of MMP-2 and -9 by Proteasome Inhibition: A Possible Mechanism to Decrease LEC Migration and Prevent Posterior Capsular Opacification
Invest. Ophthalmol. Vis. Sci., May 1, 2008; 49(5): 1998 - 2003.
[Abstract] [Full Text] [PDF]

Home page
 J. Dent. Res.Home page
S.T.W. McKeown, J.J. Barnes, P.L. Hyland, F.T. Lundy, M.J. Fray, and C.R. Irwin
Matrix Metalloproteinase-3 Differences in Oral and Skin Fibroblasts
J. Dent. Res., May 1, 2007; 86(5): 457 - 462.
[Abstract] [Full Text] [PDF]

Home page
 Am J Sports MedHome page
S. P. Arnoczky, M. Lavagnino, M. Egerbacher, O. Caballero, and K. Gardner
Matrix Metalloproteinase Inhibitors Prevent a Decrease in the Mechanical Properties of Stress-Deprived Tendons: An In Vitro Experimental Study
Am. J. Sports Med., May 1, 2007; 35(5): 763 - 769.
[Abstract] [Full Text] [PDF]

Home page
 IOVSHome page
S. G. Priglinger, C. S. Alge, D. Kook, M. Thiel, R. Schumann, K. Eibl, A. Yu, A. S. Neubauer, A. Kampik, and U. Welge-Lussen
Potential role of tissue transglutaminase in glaucoma filtering surgery.
Invest. Ophthalmol. Vis. Sci., September 1, 2006; 47(9): 3835 - 3845.
[Abstract] [Full Text] [PDF]

Home page
 IOVSHome page
T. T. L. Wong, A. L. Mead, and P. T. Khaw
Prolonged Antiscarring Effects of Ilomastat and MMC after Experimental Glaucoma Filtration Surgery
Invest. Ophthalmol. Vis. Sci., June 1, 2005; 46(6): 2018 - 2022.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Pathol.Home page
G. A. Limb, K. Matter, G. Murphy, A. D. Cambrey, P. N. Bishop, G. E. Morris, and P. T. Khaw
Matrix Metalloproteinase-1 Associates with Intracellular Organelles and Confers Resistance to Lamin A/C Degradation during Apoptosis
Am. J. Pathol., May 1, 2005; 166(5): 1555 - 1563.
[Abstract] [Full Text] [PDF]

Home page
 IOVSHome page
D. W. Esson, M. P. Popp, L. Liu, G. S. Schultz, and M. B. Sherwood
Microarray Analysis of the Failure of Filtering Blebs in a Rat Model of Glaucoma Filtering Surgery
Invest. Ophthalmol. Vis. Sci., December 1, 2004; 45(12): 4450 - 4462.
[Abstract] [Full Text] [PDF]

Home page
 Br. J. Ophthalmol.Home page
T T L Wong, J T Daniels, J G Crowston, and P T Khaw
MMP inhibition prevents human lens epithelial cell migration and contraction of the lens capsule
Br. J. Ophthalmol., July 1, 2004; 88(7): 868 - 872.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Pathol.Home page
J. T. Daniels, G. S. Schultz, T. D. Blalock, Q. Garrett, G. R. Grotendorst, N. M. Dean, and P. T. Khaw
Mediation of Transforming Growth Factor-{beta}1-Stimulated Matrix Contraction by Fibroblasts: A Role for Connective Tissue Growth Factor in Contractile Scarring
Am. J. Pathol., November 1, 2003; 163(5): 2043 - 2052.
[Abstract] [Full Text] [PDF]

Home page
 IOVSHome page
T. T. L. Wong, A. L. Mead, and P. T. Khaw
Matrix Metalloproteinase Inhibition Modulates Postoperative Scarring after Experimental Glaucoma Filtration Surgery
Invest. Ophthalmol. Vis. Sci., March 1, 2003; 44(3): 1097 - 1103.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Daniels, J. T.
Right arrow Articles by Khaw, P. T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow