Preparation of fluorescent basic fibroblast growth factor: localization in living retinal microvascular endothelial...

A biologically active fluorescent derivative of recombinant human basic fibroblast growth factor (bFGF) was prepared by immobilization on heparin-Sepharose 4B (HS) and derivatization with the fluorophore, Texas Red (TR). TR-bFGF was separated from free dye and carrier protein by elution from HS using 1.5 M NaCl. TR-bFGF contained an average of two dye molecules bound per bFGF, retained its mitogenic activity and was visible using a fluorescence microscope equipped with silicon intensified target camera (SIT). TR-bFGF stimulated the growth of bovine aortic endothelial cells (BAEC), microvessel endothelial cells (MVEC) and BHK-21 cells grown in culture. BAEC, MVEC and BHK-21 cells treated with 20 ng ml-1 (1 nM) TR-bFGF for 72 hr were stimulated over serum controls by 87, 26 and 6%, respectively. TR-bFGF stimulated EC growth was inhibited in a dose-dependent fashion when cells were coincubated with microM chloroquine. When EC were treated with TR-bFGF at 4 degrees C and then monitored at 37 degrees C, bright, focal, cytoplasmic spots were observed, which accumulated as punctate, perinuclear fluorescence. EC internalization of TR-bFGF was inhibited 80% by the addition of 100-fold molar excess unlabeled bFGF or by maintaining cultures at 4 degrees C. TR-bFGF colocalized with an EC lysosomal marker, but TR-bFGF was not detected in the nucleus. Results of these localization studies suggest that TR-bFGF stimulates EC proliferation without entering the nucleus.