Investigation of Inhibitors of MMP-2 Activity in a Scleral Fibroblast Cell Line

Abstract

Abstract: : Purpose: Matrix metalloproteinase-2 (MMP-2) is a degradative enzyme involved in regulation of the extracellular matrix in many tissues. Increases in MMP-2 activity are associated with diseases that involve matrix degradation and remodelling. In the eye, increases in MMP-2 activity have been implicated in proliferative vitreoretinopathy and corneal ulceration. In vitro and in vivo studies have shown that MMP-2 inhibition can decrease its activity and associated pathological consequences. An increase in MMP-2 activity and expression occurs during the development of myopia in the tree shrew, thus an in vitro model was established to investigate MMP-2 inhibition in the sclera. Methods: Primary scleral fibroblast cell lines were established by explant culture of tree shrew tissue and cell phenotype assessed by morphology and PCR over several passages. The mRNA sequence of tree shrew MMP-2 was obtained using PCR amplification and sequencing and used in the design of selective antisense inhibitors of MMP-2 expression. The effectiveness of these inhibitors and two commercially available MMP inhibitors was evaluated in the cell line using both gelatin zymography and PCR. Results: Scleral fibroblast cells were grown for 9 passages in culture and expressed MMP-2, TIMP-1, TIMP-2, collagen I, III, V; and α-smooth muscle actin mRNA for at least six passages after primary culture. Sequencing of MMP-2 mRNA gave 2587 nucleic acids that predicted a protein of 660 amino acids. These mRNA and protein sequence were closely related to MMP-2 of other species, particularly human (97% identity). The activity of MMP-2 from scleral fibroblasts was inhibited in a dose-dependent manner by MMP inhibitors, known to be effective in other species. Assessment of antisense oligonucleotides, targeted to MMP-2, revealed a decrease in MMP-2 activity after treatment with at least 2 oligonucleotides. Conclusion: The development of a tree shrew scleral fibroblast cell line, which expresses mRNA species found in vivo, enables the evaluation of the effect of MMP-2 regulation on tree shrew MMP-2 protein. This study demonstrates that tree shrew MMP-2 activity can be regulated with synthetic MMP-2 inhibitors and the expression of MMP-2 inhibited by antisense oligonucleotides. The ability to selectively regulate MMP-2 in vivo could prevent the degradation and remodelling of the extracellular matrix in diseases such as myopia, that involve increases in MMP-2 activity.

Keywords: myopia • sclera • gene/expression