Influence of pigment content, intracellular calcium and cyclic AMP on the ability of human retinal pigment epithelial cells to contract collagen gels

Purpose. The aim of the study was to determine to what extent collagen gel contraction could be reduced by calcium and calmodulin antagonists and agen ts that elevate cyclic AMP in order to develop a pharmacological approach t o prevent/ arrest RPE contraction of epiretinal membranes in proliferative vitreoretinopathy. We also explored a possible role of pigment in collagen gel contraction. Method. We measured RPE mediated contraction of 3D collagen gels in the pre sence and absence of the calcium and calmodulin antagonists TMB8, Verapamil and Tamoxifen and the cAMP elevating agents IBMX and Forskolin. The effect of pigment on collagen gel contraction was assessed by comparing gel contr action mediated by RPE cells re-pigmented with melanin with that mediated b y unpigmented RPE. The effect of IBMX on RPE proliferation was assessed usi ng a BrdU ELISA and the effects of IBMX on RPE cytoskeleton and cell shape were assessed using Actin and Cytokeratin immunocytochemistry. Results. We report that both cAMP elevating agents and calcium and calmodul in antagonists reduce RPE mediated collagen gel contraction. Cyclic AMP ele vation was more effective than a reduction in calcium in reducing contracti on. There were no significant advantages in combining both approaches. The presence of melanin had no effect on gel contraction. Calcium antagonists a nd particularly agents which elevate cAMP caused RPE cells in collagen gels to extend fewer and shorter processes. cAMP elevation in particular caused RPE cells to become more rounded and develop arborised cell processes. Imm unostaining for actin and cytokeratin revealed changes in cytoskeletal orga nisation in response to IBMX in that cells contained less actin than untrea ted cells and concentrated cytokeratins more centrally. Conclusion. We have identified two possible pharmacological approaches whic h may provide a new direction for preventing or slowing down the developmen t of PVR.