Daniels Julie T, Cambrey Alison D, Occleston Nicholas L, Garrett Qian, Tarnuzzer Roy W, Schultz Gregory S, Khaw Peng T
Wound Healing Research Unit, Department of Pathology and Glaucoma, Institute of Ophthalmology and Moorfields Eye Hospital, London, United Kingdom.
Invest Ophthalmol Vis Sci. 2003 Mar;44(3):1104-10. doi: 10.1167/iovs.02-0412.
To investigate the effect of matrix metalloproteinase (MMP) inhibition on fibroblast-mediated matrix contraction and production.
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).
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.
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.
研究基质金属蛋白酶（MMP）抑制对成纤维细胞介导的基质收缩和产生的影响。
用人眼Tenon囊成纤维细胞制备游离漂浮的成纤维细胞填充的I型胶原晶格。拍摄晶格区域并进行数字分析以指示晶格收缩程度。定量竞争性逆转录 - 聚合酶链反应（QCRT-PCR）和酶联免疫吸附测定（ELISA）分别用于定量成纤维细胞在晶格收缩过程中MMP-1、-2和-3的mRNA和蛋白质。明胶酶谱法显示释放到收缩晶格条件培养基中的MMPs活性。将广谱MMP抑制剂伊洛马司他、CellTech（英国斯劳）和BB-94的浓度添加到收缩的成纤维细胞填充的胶原晶格中。通过ELISA在收缩晶格的条件培养基中测量I型胶原分泌的C末端前肽。使用试剂水溶性四氮唑-1（WST-1）测量在伊洛马司他浓度存在下的成纤维细胞增殖。
在I型胶原晶格收缩过程中，Tenon囊成纤维细胞表达MMP-1、-2和-3的mRNA和蛋白质。酶谱法显示有四种明胶溶解物质释放到收缩晶格的条件培养基中（57、72、91和100 kDa）。在酶谱显影缓冲液中加入MMP抑制剂可降低检测条带的蛋白水解活性。MMP抑制（1 - 100 microM）显著降低成纤维细胞介导的胶原晶格收缩（P < 0.05），并且发现这种作用是可逆的。伊洛马司他还以剂量依赖性方式显著抑制胶原蛋白的产生（P < 0.05）。在伊洛马司他存在下未发现对成纤维细胞增殖有影响。
在Tenon囊成纤维细胞介导的胶原晶格收缩过程中产生MMPs。广谱MMP抑制可显著降低基质收缩和产生，且无细胞毒性。由于MMP抑制可显著降低与收缩性瘢痕形成相关的成纤维细胞功能，未来MMP抑制剂可能有临床应用价值。
