ABSTRACT
The edible mushroom lectin from Agaricus bisporus (ABL) has antiproliferative effects on a range of cell types. This investigation was undertaken to test whether it might have inhibitory activity on Tenon's capsule fibroblasts in in vitro models of wound healing and therefore have a use in the modification of scar formation after glaucoma surgery.Human ocular fibroblasts in monolayers and in three-dimensional collagen lattices were exposed to ABL (0-100 microg ml(-1)). Proliferation was studied by the MTS assay and by counting haematoxylin-stained cells; contraction was measured as a change in the diameter of three-dimensional collagen lattices. Toxicity was investigated using a fluorescent viability assay. FITC-labelled lectin was used to study cell binding and internalization of ABL.ABL caused a dose-dependent inhibition of proliferation and lattice contraction without significant toxicity. Proliferation was inhibited by 5-40% in the dose range 20-100 microg ml(-1) Significant inhibition of lattice contraction was achieved with 40 microg ml(-1) ABL, and at 100 microg ml(-1) contraction was completely prevented. FITC-ABL binds to the cell surface and accumulates around the nuclear envelope when internalized. These experiments have shown that ABL possesses key features required of an agent that might control scarring processes and suggest that ABL might be especially useful where subtle modification of healing is needed. Further evaluation is warranted.
Subject(s)
Collagen/drug effects , Conjunctiva/drug effects , Fibroblasts/drug effects , Lectins/pharmacology , Wound Healing/drug effects , Cell Death/drug effects , Cell Division/drug effects , Cells, Cultured , Conjunctiva/cytology , Dose-Response Relationship, Drug , Fibroblasts/cytology , Humans , Lectins/pharmacokinetics , TrabeculectomyABSTRACT
Epidermal growth factor (EGF) has been implicated in the control of embryonic development, but although the receptor is expressed from an early stage, there is little evidence of embryonic expression of EGF. In order to investigate the role of maternally derived EGF during organogenesis, rat embryos were explanted at d 9.5 and cultured in serum depleted of low molecular weight molecules (retenate) which was then supplemented with EGF. Serum depleted of low molecular weight molecules by prolonged filtration loses its capacity to support normal embryonic development, possibly due to the loss of growth promoting factors. The addition of EGF to retenate significantly improved embryonic development with a maximal effect at 8 ng/ml. The addition of an analogue of EGF, long EGF, to retenate also caused a significant increase in development, although at higher concentrations a decrease in its effect was observed, possibly due to down regulation of the EGF receptor. Therefore, embryos may be able to utilise maternally derived EGF during organogenesis. To test the effects of inhibiting the EGF receptor during organogenesis, d 9.5 embryos were cultured in the presence of tyrphostin 47, a specific EGF receptor inhibitor. Tyrphostin 47 caused a significant dose-dependent decrease in the development of embryos which was also observed when tyrphostin 47 was injected into the vitelline circulation at d 11.5 to bypass the effects of the yolk sac. These findings suggest that the EGF receptor is essential for normal organogenesis and may play a role in the control of proliferation and differentiation. Although EGF is not expressed in the rat embryo at this stage, maternally derived EGF may be the ligand for the embryonic EGF receptor.
