The role of matrix metallproteinases in pathogenesis of proliferative vitreoretinopathy
Cellular migration and membrane contraction are two vital steps in pathogenesis of proliferative vitreoretinopathy (PVR). Several factors, related to cell activity, are involved in induction of cell migration and cell-induced membrane contraction. However, the roles of some of these factors in PVR pathophysiology are still to be clarified. The studies described in this thesis were undertaken to investigate the factors affecting cellular migration, mainly HRPE cells, which may modify cellular migration during proliferative vitreoretinopathy. The mechanisms of cell-induced collagen gel contraction, which resembles membrane contraction in PVR, was studied. The role of matrix metallproteinases (MMPs) in induction or inhibition of cell migration and cell-mediated gel contraction is one major goal; this study tried to clarify. In chapter III, HRPE cells were cultured from human male eyes, aged 43 years, and passed several times to create a cell line. To study the effect of different ECMs (fibronectin, laminin and different subtypes of collagen) on cell migration, I developed a cell migration assay "agarose droplet". This assay was established by incorporating HRPE cells into small amount of agarose and the solution was left to set as a gel. The drops were observed, photographed and scanned for 48 hours. The distance migrated by the cells, on plastic or substrate coated plastic, was estimated using both microscopic scale and computer analysis program (Coral Xara) by converting the pixels into metric unites. The results showed that fibronectin at a dose of 10 ,g/ml significantly enhanced cell migration in comparison with cells migrated on plastic. On the other hand, laminin at a dose of 10 g/ml markedly inhibited cell migration and this inhibition was statistically significant. These results suggest that HRPE cells require fibronecin but not laminin to migrate out of agarose droplet. This alteration in cell reaction to different ECM substrates may represent a similar phenomenon of cell-matrix interactions, which stimulate cell migration and survival in PVR environment. Cell migration assay was used again in Chapter IV to determine the role of matrix metalloproteinases (MMPs) 1, 2 9 in directing the behaviour of HRPE cells during migration from agarose droplets. Expression of MMP-1,2 9 by HRPE cells was determined by immunocytochemistry using monoclonal antibodies. In addition, expression of MMPs in cultured HRPE cells was confirmed by western blotting in the absence and in the presence of MMP synthetic inhibitor: Batimastat (BB-94). Finally, cell migration from the agarose was studied in presence of Batimastat. The results showed that MMP-2 9 were expressed by primary HRPE cell cultures and the strong expression of the duplex bands of the same enzymes were shown by western blotting technique. Expression of the MMP-2 9 bands was inhibited completely after treatment of the HRPE cells with BB-94 (500nM and 5 M). BB-94, also inhibited cellular migration from agarose droplets in a dose dependent manner. These results suggest that expression of MMP-2 9 by HRPE cells during their migration from the agarose might resemble the expression of the same enzymes during migration of the cells in the PVR and the inhibition of these enzymes by BB-94 might prevent cellular migration, a critical step in PVR pathogenesis. Therefore, we concluded that migration of HRPE cells from their resident basement membrane into periretinal surfaces requires an interplay between certain ECMs and enzymes as, MMPs to permit cellular invasion. There was an intention to use this assay to study the effect of MMPs on the cellular migration in the presence of these ECMs but because of the tiny size of the drop, it was very difficult to retrieve any materials from the drops. In chapter V, I used cell-mediated collagen gel contraction model to determine the effect of HRPE cells, seeded on the surface of 3-D collagen gels, on the integrity of the gel and to observe the behaviour of human retinal pigment epithelial (HRPE) cells during this time. A comparison between the effect of the HRPE cells on the collagen gel integrity and other cell types as human skin fibroblasts (HSF) and human monocytic cell line (THP-1) has been performed after standardisation of the HRPE cell-mediated collagen gel contraction assay. Also, the effects of coating the collagen gel with different ECM proteins on the cell-mediated collagen gel contraction were studied. The extent of cellular invasion and the degree of gel contraction were quantified for up to three days using phase contrast microscopy and digital image analysis. These experiments showed that HRPE cells mostly proliferated as sheets of cells on the surface of the collagen gel and only minimally invaded the gel, some cells were seen to invade the gel as single cells and invaded the gel to a depth of several tens of microns.