INCREASING CELL-DENSITY DOWN-REGULATES THE EXPRESSION OF ACIDIC FGF BY HUMAN RPE CELLS IN-VITRO

Previous studies have reported the expression of acidic fibroblast gro wth factor (aFGF) by rat, bovine, and human retinal pigment epithelium (RPE) in vivo. To critically examine the expression of aFGF by RPE ce lls, we studied the density dependence of steady-state levels of mRNA and protein expression in vitro. Northern blot analysis demonstrated 5 transcripts ranging from 4.5 kB to 1 kB. Steady-state levels of all t he transcripts decreased as a function of culture density. A polyclona l antibody was raised against recombinant human aFGF and affinity puri fied on aFGF coupled to AffiGel-10. The resulting antibody crossreacte d with bFGF but not FGF-5, but this crossreactivity could be eliminate d by absorption of the antibody on bFGF coupled to AffiGel-10. The fin al antibody preparation recognized only a single band at approximately 18.5 kD in lysates of RPE. Immunohistochemical staining with this ant ibody preparation demonstrated a marked dependence on cell density aft er 3 days in culture. Low culture density yielded cells staining moder ately for aFGF, while confluent cells exhibited little or no staining. The reduction of aFGF from RPE cells in culture in a density-dependen t fashion could also be demonstrated by Western blot analysis.