CULTURED OCULAR CELLS AND EXTRACELLULAR MATRICES - ROLE OF GROWTH-FACTORS, RETINOIC ACID AND CELL POLARITY

Culture of various types of cells on gelled, reconstituted extracellul ar matrices results in decreased cellular proliferation. In the presen t study, we evaluated several possible mechanisms for this inhibition, as applied to cultured bovine retinal microvascular endothelial cells (EC) or to retinal pigment epithelial (RPE) cells: whether the inhibi tion might be related to (a) inactivation of fibroblast growth factor (FGF) by binding of the molecules present in the medium to a matrix co mponent; (b) release of an inhibitor by the matrix in culture; or (c) inhibitory properties of the matrix macromolecules themselves. Our res ults suggest that mechanism (c) is most likely. The reasons are, first , that culture of EC or RPE cells on several different extracellular m atrix substrates in the presence of logarithmically increasing concent rations of acidic or basic fibroblast growth factors (aFGF or bFGF) le ads to a vertical shift of the plots of cell number after 4 days in cu lture vs. log growth factor concentration for both types of cells. The same result obtains when cells are cultured with logarithmically incr easing concentrations of all-trans retinoic acid, which inhibits EC bu t not RPE cell proliferation in a dose-dependent fashion. This is cons istent with mechanism (b) or (c), but not (a), for which one would exp ect a horizontal shift. Second, washing the matrices prior to the plat ing of cells with 1M NaCl, which elutes aFGF and partially elutes bFGF molecules from basement membranes, does not alter the growth of cells plated after the wash. This suggests also that growth factor binding to the matrix is not a likely mechanism for the observed inhibition. I ncubation of matrices with culture medium prior to plating cells does not usually alter the ability of the medium thus ''conditioned'' to su pport cell growth, arguing against the possibility that the matrices r elease a soluble activator or inhibitor of such growth. However, in so me experiments performed with lots of Matrigel(R) (a commercially avai lable basement membrane extract from a murine tumor) obtained prior to mid-1991, media ''conditioned'' by incubation with this matrix did sh ow enhanced ability to facilitate EC and RPE cell proliferation. Final ly, if RPE cells or EC are plated on various substrates, allowed to at tach for 24 hr., and then the same or other substrates are poured over the cells, the effect on proliferation of the matrices plated on the apical surfaces of the cells is often less than that of matrices plate d adjacent to their basal surfaces. Although in most cases these diffe rences are not statistically significant, there is an apparent trend. The overall results of this experiment suggest an asymmetrical cell-su bstrate interaction, mediated through receptors located predominantly on the basal surfaces of the cells.