Matrix metalloproteinase inhibition modulates fibroblast-mediated matrix contraction and collagen production in vitro.
OBJECTIVE
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 microM) 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.
Daniels
Julie T
JT
Wound Healing Research Unit, Department of Pathology and Glaucoma, Institute of Ophthalmology and Moorfields Eye Hospital, London, United Kingdom. j.daniels@ucl.ac.uk
Cambrey
Alison D
AD
Occleston
Nicholas L
NL
Garrett
Qian
Q
Tarnuzzer
Roy W
RW
Schultz
Gregory S
GS
Khaw
Peng T
PT
D002455:Cell Division; D002478:Cells, Cultured; D024042:Collagen Type I / Q000096:biosynthesis*; D004797:Enzyme-Linked Immunosorbent Assay; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D006877:Hydroxamic Acids; D007211:Indoles / Q000494:pharmacology; D061965:Matrix Metalloproteinase Inhibitors; D020782:Matrix Metalloproteinases / Q000235:genetics / Q000378:metabolism; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013876:Thiophenes / Q000494:pharmacology
D024042:Collagen Type I; D006877:Hydroxamic Acids; D007211:Indoles; D061965:Matrix Metalloproteinase Inhibitors; D011480:Protease Inhibitors; D012333:RNA, Messenger; D013876:Thiophenes; D010649:Phenylalanine; C080985:batimastat; D020782:Matrix Metalloproteinases; C119203:ilomastat