Lens epithelial cells cultured on plastic are protected from Fas mediated apoptosis by conditioned media from lens epithelial cells cultured on collagen IV

Abstract

Abstract: : Purpose: Our aim is to covalently modify intraocular lens implants (IOLs) with death receptor ligands to induce apoptosis in contacting lens epithelial cells (LECs). The clinical goals are prevention of posterior capsular opacification (PCO) and preservation of normal capsule tissue compliance for accommodating IOLs. We have previously demonstrated that primary LECs on lens capsule are resistant to Fas stimulated apoptosis; whereas HLE–B3 LECs are sensitive to Fas ligation when cultured on tissue culture plastic but become Fas resistant when cultured on lens capsule or collagen IV. Here we determine whether Fas ligand modified surfaces can induce apoptosis in LECs using conditions under which they are Fas sensitive. We also initiate exploration of the mechanism of Fas resistance for primary LECs adherent to capsule. Methods: Prototype surfaces were constructed by coupling Fas ligand (FasL) via heterobifunctional polyethylene glycol (PEG) chains to hydroxyethyl methacrylate (HEMA). HLE–B3 cells were cultured on 1.0µm pore transparent tissue culture inserts (Falcon) and either treated with soluble anti–Fas IgM (Upstate), control IgM (500ng/ml), test (PEG–FasL) or control (PEG) surfaces gently lowered onto the apical cell surface. After 48 hours culture the number of living cells remaining was estimated by Hoechst staining and cell counting. The effect of conditioned media on LEC Fas sensitivity was determined by incubating HLE–B3 cells cultured on either TC plastic or collagen IV with either anti–Fas or control IgM in the presence of conditioned medium from HLE–B3 cells cultured on either TC plastic or collagen IV. %apoptotic cell counts were determined at 48 hours by Hoechst staining. Results: FasL–coated surfaces induced a 50% reduction in LEC numbers compared to PEG modified control surfaces after 48 hours of exposure to the surface. HLE–B3 cells cultured on TC plastic which are normally Fas sensitive (55% apoptotic) became protected from Fas–dependent apoptosis in the presence of conditioned medium obtained from HLE–B3 cells cultured on collagen IV (26% apoptotic). This protection was almost as great as that provided by direct culture on collagen IV (22% apoptotic). Conclusions: IOL materials covalently modified with FasL kill some but not all contacting LECs after 48 hours in experimental conditions. Blocking one or more soluble survival factors may significantly enhance the potential clinical utility of these novel biomaterial surfaces.

Keywords: cell death/apoptosis • cataract • wound healing