Preparation of fluorescent basic fibroblast growth factor: localization in living retinal microvascular endothelial cells.
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.
Healy
A M
AM
Program in Cell, Molecular and Developmental Biology, Tuft University Health Science Schools, Boston, MA 02111.
Herman
I M
IM
D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D004727:Endothelium / Q000378:metabolism; D004730:Endothelium, Vascular / Q000187:drug effects; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism*; D006801:Humans; D008247:Lysosomes / Q000378:metabolism; D008856:Microscopy, Fluorescence; D012171:Retinal Vessels / Q000254:growth & development / Q000378:metabolism*; D014966:Xanthenes / Q000378:metabolism
D014966:Xanthenes; D016222:Fibroblast Growth Factor 2; C034657:Texas red