Expression of fibroblast growth factor-5 by bovine choroidal endothelial cells in vitro.
OBJECTIVE
To demonstrate the expression of fibroblast growth factor-5 (FGF-5) by bovine choroidal microvascular endothelial (BCME) cells and to investigate its possible role as an autocrine mitogen in these cells.


METHODS
Expression of FGF-5 by BCME cells was studied by a combination of Northern and Western blot analyses. Total RNA was isolated from BCME cultures at passages 5 through 8 and analyzed by Northern blot analysis for the presence of FGF-5 transcripts, using a 1-kb human complemetary DNA. Slot-blot analysis was performed to determine possible cross-reactivity between this probe and acidic and basic FGFs of human and bovine species. A previously characterized antibody directed against the aminoterminus of the human FGF-5 sequence was used in Western blot analyses to identify immunoreactive proteins released by BCME cells into the medium. Finally, the mitogenic activity of human recombinant FGF-5 on a variety of cell types was evaluated, using a cellular proliferation assay.


RESULTS
Northern blot analysis provided evidence for the expression of two major FGF-5 transcripts at 4 kb and 3 kb and two minor transcripts at 2.2 kb and 1.7 kb. A single immunoreactive protein with a molecular weight of 34 kDa was identified by Western blot analysis of conditioned media. In cellular proliferation assays, human recombinant FGF-5 was not mitogenic in BCME cells but exhibited an approximate ED50 of 1.8 to 3.7 nM in BALB/c3T3 fibroblasts. This ED50 was within the range reported by the manufacturer, using a thymidine incorporation assay and a similar embryonic fibroblast cell line. Fibroblast growth factor-5 also stimulated proliferation of human retinal pigment epithelial cells.


CONCLUSIONS
Bovine choroidal microvascular endothelial cells exhibit expression in vitro of FGF-5 at the messenger RNA and protein levels. Perivascular and endothelial cell staining for FGF-5 seen previously in choroidal neovascular membranes may therefore arise from expression by choroidal endothelial cells. Because nonglycosylated recombinant FGF-5 does not appear to be a mitogen in BCME cells in vitro, it is reasonable to question its role as an autocrine mitogen in vivo. Fibroblast growth factor-5 may instead be serving paracrine roles in the stimulation of fibroblasts and retinal pigment epithelial cells during the formation of choroidal neovascular membranes. Studies with fully glycosylated recombinant FGF-5 will be required, however, to assess the biologic activity of this member of the FGF gene family.
Keithahn
M A
MA
Department of Ophthalmology, University of California, Davis, Sacramento 95816, USA.
Aotaki-Keen
A E
AE
Schneeberger
S A
SA
Hjelmeland
L M
LM
Morse
L S
LS
D016475:3T3 Cells / Q000187:drug effects; D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002417:Cattle; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D002829:Choroid / Q000098:blood supply*; D015342:DNA Probes; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D051521:Fibroblast Growth Factor 5; D005346:Fibroblast Growth Factors / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D051379:Mice; D008807:Mice, Inbred BALB C; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000187:drug effects; D012333:RNA, Messenger / Q000096:biosynthesis
D015342:DNA Probes; C496450:Fgf5 protein, mouse; D012333:RNA, Messenger; D051521:Fibroblast Growth Factor 5; D005346:Fibroblast Growth Factors