EVects of single, short term exposures of human retinal pigment epithelial cells to thiotepa or 5-fluorouracil: implications for the treatment of proliferative vitreoretinopathy

Aim—To investigate the eVects of single, short term (5 or 30 minutes) exposures to thiotepa or 5-fluorouracil (5-FU) on collagen lattice contraction and retinal pigment epithelial (RPE) cell proliferation. Methods—For collagen contraction studies, RPE cells seeded into free floating type I collagen lattices were exposed to single 5 or 30 minute treatments with thiotepa (0.06–4 mg/ml), or 5-FU (0.25–25 mg/ml), or phosphate buVered saline alone as a control. For proliferation studies, RPE cell monolayers were similarly exposed to these agents. The degree of contraction, eVects on cell number, and viability were determined up to 14 days after treatment. Results—Contraction of collagen lattices containing RPE cells and proliferation of RPE cells were significantly inhibited (p<0.05) by thiotepa and 5-FU at concentrations above 0.06 mg/ml and 0.25 mg/ml respectively (for both 5 and 30 minute treatments), compared with controls. Cell death did not occur except for exposure of the RPE cells in collagen lattices to the highest concentration of thiotepa (4 mg/ ml). Conclusion—It was concluded that single 5 or 30 minute exposures to thiotepa or 5-FU significantly inhibited collagen contraction and the proliferation of RPE cells. These findings suggest that short, single, non-toxic exposures to thiotepa or 5-FU which can be reproduced clinically may be useful in the modulation of proliferative vitreoretinopathy. (Br J Ophthalmol 1998;82:554–560) Proliferative vitreoretinopathy (PVR) is the major cause of failure of retinal detachment surgery. Its pathophysiology involves the proliferation of a variety of cells and the production of extracellular matrix with the formation of epiretinal membranes and their subsequent contraction. There is evidence to suggest that retinal pigment epithelial (RPE) cells play an important part in this process. Recent research has focused on the use of pharmacological agents to prevent the occurrence of PVR. Many drugs have been suggested and used for their specific actions on the various stages of PVR formation but none is used in clinical practice because of the potential for toxicity and the insuYcient evidence for the safety of these drugs in human eyes. Thiotepa (N, N',N" triethylenethiophosphoramide) is a cytostatic, alkylating agent which has been used in the treatment of many tumours. It is non-vesicant and can be given by all parenteral routes as well as directly into the tumour mass. In ophthalmology, it is used both topically as eyedrops in the post-surgical prevention of recurrence of pterygium, and intravitreally for the treatment of retinoblastoma where data on its eVect on the retina have been favourable. 5-Fluorouracil (5-FU) is an antiproliferative and an antimetabolite which, after intracellular conversion, acts by interfering with DNA and RNA synthesis. It is increasingly being used as single, short term intraoperative applications to reduce conjunctival scarring after glaucoma surgery. Blumenkranz et al 10 have studied the use of intravitreal 5-FU in an in vivo experimental model of PVR with encouraging results. Although in their study the drug was left inside the eye, Khaw et al 11 have shown that short exposures to the agent also have long term eVects on scleral and subconjunctival fibroblast proliferation both in vitro and in vivo. If eVective, the benefits of such single, short term treatments in inhibiting the cellular behaviour which results in PVR, are clear: these drugs can be given as single intraoperative doses and washed away after the treatment period, thus controlling the therapeutic exposure more precisely. Therefore, we have investigated the effects of single 5 and 30 minute exposures to thiotepa or 5-FU on the ability of human RPE cells to contract collagen lattices and to proliferate. Materials and methods CULTURE OF HUMAN RPE CELLS RPE cells were isolated from human eyes up to 48 hours post mortem using the modified procedure outlined by Edwards. The cells were cultured in Ham’s F10 culture medium containing 20% (vol/vol) fetal bovine serum, 100 units/ml of penicillin streptomycin (Gibco, Scotland), 3 mg/ml glucose, and 0.12% (wt/vol) sodium bicarbonate. Cells between the fourth and fifth passage were used for experimentation. COLLAGEN MATRIX CONTRACTION STUDY A collagen solution (5 mg/ml) was prepared using collagen type I (Sigma, Dorset) dissolved in 0.1% (vol/vol) glacial acetic acid in sterile Br J Ophthalmol 1998;82:554–560 554 Wound Healing Research Unit, Department of Pathology, Institute of Ophthalmology and Moorfields Eye Hospital, London EC1V 9EL