Fibrinogen stimulates in vitro angiogenesis by choroidal endothelial cells via autocrine VEGF.
BACKGROUND
The purpose of this study is to investigate the effect of fibrinogen on angiogenesis in vitro formed by cultured bovine choroidal endothelial cells (BCECs) and the involvement of vascular endothelial growth factor (VEGF) in this mechanism.


METHODS
For in vitro tube formation assay, BCECs were seeded on collagen gel containing fibrinogen (0-1.5 mg/ml). After 3 days of cultivation, the total length of the tubular structure was measured using Macscope Analyzer. Total RNA and conditioned media were collected after fibrinogen treatment and subjected to Northern and Western blot analyses, respectively. Transcription factor HIF-1alpha was also analyzed by Western blot analysis using cytosolic and nuclear fraction of BCECs. Involvement of VEGF in fibrinogen-dependent in vitro tube formation was evaluated using anti-VEGF neutralizing antibody or VEGF receptor 2-selective inhibitor (SU5416).


RESULTS
Formation of the tubular structure was enhanced 20 to approximately 50 times in fibrinogen-containing gel in a concentration-dependent manner. The treatment of BCECs with fibrinogen resulted in a significant increase in VEGF gene and protein expression. Accumulation of HIF-1alpha protein in the nuclear fraction was also detected after the treatment with fibrinogen. Finally, fibrinogen-induced tube formation was significantly inhibited in the presence of anti-VEGF-neutralizing antibody (52.0% inhibition at the concentration of 1 microg/ml, P<0.05) or SU5416 (54.8% inhibition at the concentration of 3 microM, P<0.05).


CONCLUSIONS
Extravasated fibrinogen might play an important role in the development of choroidal neovascularization associated with age-related macular degeneration, at least in part, through the function of VEGF in an autocrine manner. Transcription factor HIF-1 appears to be involved in fibrinogen-induced VEGF expression.
Shiose
Satomi
S
Department of Ophthalmology of the Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi Higashi-ku, 812-8582 Fukuoka, Japan. asshy@med.kyushu-u.ac.jp
Hata
Yasuaki
Y
Noda
Yoshihiro
Y
Sassa
Yukio
Y
Takeda
Atsunobu
A
Yoshikawa
Hiroshi
H
Fujisawa
Kimihiko
K
Kubota
Toshiaki
T
Ishibashi
Tatsuro
T
D020533:Angiogenesis Inhibitors / Q000494:pharmacology; D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002417:Cattle; D002478:Cells, Cultured; D002829:Choroid / Q000098:blood supply*; D020256:Choroidal Neovascularization / Q000209:etiology* / Q000378:metabolism; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000378:metabolism; D005340:Fibrinogen / Q000494:pharmacology*; D015870:Gene Expression; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007211:Indoles / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D011758:Pyrroles / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism*
D020533:Angiogenesis Inhibitors; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007211:Indoles; D011758:Pyrroles; D012333:RNA, Messenger; D014157:Transcription Factors; D042461:Vascular Endothelial Growth Factor A; C116890:Semaxinib; D005340:Fibrinogen; D011505:Protein-Tyrosine Kinases