An endothelial-cell-enriched primary culture system to study vascular endothelial growth factor (VEGF A) expression in a teleost, the Japanese eel (Anguilla japonica).
A partial gene for eel (Anguilla japonica) vascular endothelial growth factor (VEGF) has been cloned and an endothelial-cell-enriched primary culture derived from rete mirabile established to study regulation of the expression of the eel VEGF gene. Cells were cultured in M199 medium containing 0.1% fetal calf serum (FCS) and serum-free M199 medium for long-and short-term experiments, respectively. Cells were separately treated with cobalt ions (Co2+), basic fibroblast growth factor (bFGF), and estradiol (E2), which have been demonstrated to stimulate mammalian VEGF A expression, followed by quantification of the VEGF mRNA levels by real-time reverse transcription polymerase chain reaction. Our results show that: (1) the deduced eel VEGF protein encoded by the cloned gene is about 130 amino acids in length, and is closely related to a zebrafish (Danio rerio) VEGF A; (2) the endothelial-cell-enriched rete mirabile primary culture containing mainly (over 70%) the capillary endothelial cells; (3) the expression levels of the eel VEGF transcript were increased by Co2+, bFGF, and E2 treatments in a dose-and time-dependent manner. Our data demonstrate that an eel partial VEGF gene has been cloned and its regulation of expression in endothelial-cell-enriched rete mirabile cell culture is similar to that in higher vertebrates.
Huang
Yung-Sen
YS
Department of Life Science, National University of Kaohsiung, No. 700, Kaohsiung University Road, Nan Tzu Dist., 811 Kaohsiung, Taiwan. yshuang@nuk.edu.tw
Huang
Wen-Lian
WL
Lin
Wei-Fan
WF
Chen
Ming-Chyuan
MC
Jeng
Shan-Ru
SR
D000595:Amino Acid Sequence; D000811:Anguilla / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D003035:Cobalt / Q000494:pharmacology; D003470:Culture Media / Q000737:chemistry / Q000494:pharmacology; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D004958:Estradiol / Q000494:pharmacology; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D007150:Immunohistochemistry; D008077:Lipoproteins, LDL / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D010802:Phylogeny; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D017422:Sequence Analysis, DNA; D013997:Time Factors; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics* / Q000378:metabolism
D003470:Culture Media; D018076:DNA, Complementary; D008077:Lipoproteins, LDL; D012333:RNA, Messenger; D042461:Vascular Endothelial Growth Factor A; D016222:Fibroblast Growth Factor 2; D003035:Cobalt; D004958:Estradiol