Our morphological analysis of the cells and matrix during contraction strongly suggested that doxycycline could act through a modulation of matrix degradation and thus likely MMP release. MMPs have long been connected to scarring processes. We have previously shown that matrix remodeling by MMPs plays an important role in tissue contraction, both in in vitro,
8,23 ex vivo,
8 as well as in an ocular scarring model in the rabbit model of glaucoma filtration surgery.
27 In addition, our recent studies have suggested a role for MMPs in the development of the fibrotic phenotype in trachoma.
10,11 We thus used real-time PCR to evaluate MMP expression during matrix contraction. We chose to investigate levels of MMP1 as a well-known collagenase previously implicated in our standard collagen contraction assay
26 ; MMP2 as a standard gelatinase; and MMP7, MMP9, and MMP12 as these particular MMPs have been found enriched in trichiasis samples.
12 The C
T values from the RT-PCR study demonstrated that all of the above MMPs were present in both F10 and F11 at day 0. MMP1 and MMP2 were expressed at significant levels, while MMP7 and MMP9 were naturally low (
Table). All MMPs showed an increased expression during contraction in the control group, although to a different extent and kinetics. While MMP1, MMP2, and MMP12 showed a sustained increase throughout the contraction kinetics, MMP9 expression peaked at day 3 (
Fig. 3). Continuous treatment with 416 μM doxycycline did not significantly affect MMP2 expression (
Figs. 3C,
3D). However, MMP1 (
Figs. 3A,
3B), MMP9 (
Fig. 3E,
3F), and MMP12 (
Figs. 3G,
3H) all show a strong reduction in expression in the presence of the drug. We also observed a similar trend for MMP7 in F10 (
Table, normalized expression data not shown), but could not confirm this effect in F11 due to its lower expression of MMP7 and the technical limitation of RT-PCR. To confirm that the effect of doxycycline on MMP gene expression led to a reduction in protein expression and activity, we measured the total MMP activity released in the medium during contraction. As expected, the total MMP activity releasable from the medium increased significantly during contraction, particularly in F10, matching the gene expression profile (
Fig. 4). Treatment with doxycycline completely abrogated MMP activity, even in medium at day 0, suggesting that doxycycline affected both the MMP protein levels and the activity of the MMPs present in the medium.