Influence of pigment content, intracellular calcium and cyclic AMP on the ability of human retinal pigment epithelial cells to contract collagen gels.
OBJECTIVE
The aim of the study was to determine to what extent collagen gel contraction could be reduced by calcium and calmodulin antagonists and agents that elevate cyclic AMP in order to develop a pharmacological approach to prevent/arrest RPE contraction of epiretinal membranes in proliferative vitreoretinopathy. We also explored a possible role of pigment in collagen gel contraction.


METHODS
We measured RPE mediated contraction of 3D collagen gels in the presence 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 contraction mediated by RPE cells re-pigmented with melanin with that mediated by unpigmented RPE. The effect of IBMX on RPE proliferation was assessed using 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 calmodulin antagonists reduce RPE mediated collagen gel contraction. Cyclic AMP elevation was more effective than a reduction in calcium in reducing contraction. There were no significant advantages in combining both approaches. The presence of melanin had no effect on gel contraction. Calcium antagonists and 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 arborized cell processes. Immunostaining for actin and cytokeratin revealed changes in cytoskeletal organisation in response to IBMX in that cells contained less actin than untreated cells and concentrated cytokeratins more centrally.


CONCLUSIONS
We have identified two possible pharmacological approaches which may provide a new direction for preventing or slowing down the development of PVR.
Smith-Thomas
L C
LC
Clinical Sciences Center, Section of Medicine, Northern General Hospital, Division of Clinical Sciences, Sheffield, United Kingdom.
Richardson
P S
PS
Rennie
I G
IG
Palmer
I
I
Boulton
M
M
Sheridan
C
C
MacNeil
S
S
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D048429:Cell Size; D002470:Cell Survival / Q000187:drug effects; D003094:Collagen / Q000648:ultrastructure; D000242:Cyclic AMP / Q000378:metabolism*; D003598:Cytoskeletal Proteins / Q000378:metabolism; D005782:Gels; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism*; D008543:Melanins / Q000502:physiology; D008855:Microscopy, Electron, Scanning; D010587:Phagocytosis / Q000502:physiology; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D012168:Retinal Pigments / Q000378:metabolism*; D014018:Tissue Distribution
D002121:Calcium Channel Blockers; D003598:Cytoskeletal Proteins; D005782:Gels; D008543:Melanins; D012168:Retinal Pigments; D003094:Collagen; D000242:Cyclic AMP; D002118:Calcium; D015056:1-Methyl-3-isobutylxanthine