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PURPOSE. The aim of the study was to determineto what extent collagen gel contraction could be reduced by calcium and calmodulinantagonists and agents that elevate cyclic AMP in order to develop a pharmacologicalapproach to prevent/arrest RPE contraction of epiretinal membranes in proliferativevitreoretinopathy. We also explored a possible role of pigment in collagengel contraction. METHOD. We measured RPE mediated contraction of 3D collagen gels in thepresence and absence of the calcium and calmodulin antagonists TMB8, Verapamiland Tamoxifen and the cAMP elevating agents IBMX and Forskolin. The effectof pigment on collagen gel contraction was assessed by comparing gel contractionmediated by RPE cells re-pigmented with melanin with that mediated by unpigmentedRPE. The effect of IBMX on RPE proliferation was assessed using a BrdU ELISAand the effects of IBMX on RPE cytoskeleton and cell shape were assessed usingActin and Cytokeratin immunocytochemistry. RESULTS. We report that both cAMP elevating agents and calcium and calmodulinantagonists reduce RPE mediated collagen gel contraction. Cyclic AMP elevationwas more effective than a reduction in calcium in reducing contraction. Therewere no significant advantages in combining both approaches. The presenceof melanin had no effect on gel contraction. Calcium antagonists and particularlyagents which elevate cAMP caused RPE cells in collagen gels to extend fewerand shorter processes. cAMP elevation in particular caused RPE cells to becomemore rounded and develop arborised cell processes. Immunostaining for actinand cytokeratin revealed changes in cytoskeletal organisation in responseto IBMX in that cells contained less actin than untreated cells and concentratedcytokeratins more centrally. CONCLUSION. We have identified two possible pharmacological approacheswhich may provide a new direction for preventing or slowing down the developmentof PVR. [ABSTRACT FROM AUTHOR]

PURPOSE. The aim of this study is to explore therole of intra-cellular calcium in the mechanism of co-regulation of retinalpigment epithelial cells (RPE) by vitreous fluid and platelet mitogens, inorder to evaluate the use of calcium modulating drugs in preventing RPE cellproliferation and contraction of fibrocellular membranes. METHODS. Monolayers of human RPE cells were loaded with Fura-2-AM and examinedin a fluorimeter for changes in intra-cellular free calcium in response toplatelet mitogens (PDG-FAB or TGFβ1) and vitreous fluid (containing vitreoussubstrate proteins), both alone or in combination. The effect of the calciumantagonists TMB8 and verapamil and the calmodulin antagonists J8 and tamoxifenwere then examined on RPE cell proliferation and pigmentation, both in thepresence and absence of vitreous substrate and platelet mitogens. RESULTS. We report that co-exposure of RPE cells to platelet mitogens andvitreous fluid produces an increase in intracellular free calcium of greaterduration than that with either PDG-FAB, TGFβ1 or vitreous fluid alone.Calcium and calmodulin antagonists significantly reduce RPE cell proliferationin both the presence and absence of vitreous substrate and platelet mitogens.Calcium antagonists also stimulate the accumulation of autofluorescent granuleswithin RPE cells. CONCLUSIONS. Calcium signalling plays a role in the co-regulation of RPEcells by vitreous substrate and platelet mitogens. Drugs that lower intracellularcalcium or inhibit calmodulin may offer an additional approach to preventingthe hyperproliferation of RPE cells in PVR. [ABSTRACT FROM AUTHOR]