Inhibitory effects of arresten on bFGF-induced proliferation, migration, and matrix metalloproteinase-2 activation in mouse retinal endothelial cells.
OBJECTIVE
The potential role of arresten (alpha1(IV)NC1) as an endogenous angiogenesis inhibitor in the prevention of bFGF mediated retinal angiogenesis and regulation of matrix metalloproteinase-2 activation has not been explored.


METHODS
Mouse retinal endothelial cells (MREC) were cultured on type IV collagen and treated with basic fibroblast growth factor (bFGF) alone or in the presence of arresten at concentrations ranging from 1 to 10 microg/ml. The proliferation of MRECs were evaluated using 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay, and bFGF stimulated endothelial cell migration was assessed using Boyden chamber. Expression of matrix metalloproteinase-2 (MMP-2) was assessed by reverse transcription polymerase chain reaction (RT-PCR) analysis using RNA isolated from MRECs. Secretion and activation of MMP-2 in arresten-treated conditioned MREC growth medium was determined by gelatin zymography and Western blotting.


RESULTS
Different doses of bFGF induced MREC proliferation was significantly inhibited upon arresten treatment (P < 0.005). The bFGF-induced migration was significantly inhibited by arresten at 1 and 10 microg/ml concentrations (P < 0.01). The bFGF stimulated expression of MMP-2 mRNA and secretion of MMP-2 in MREC was not affected and interestingly activation of MMP-2 was suppressed by arresten in a dose and time dependent manner.


CONCLUSIONS
Inhibitory effects of arresten on proliferation, migration and MMP-2 activation but not on expression and secretion of MMP-2 in MREC; this early work with arresten supports potential therapeutic action in retinal neovascularization dependent disorders.
Boosani
Chandra Shekar
CS
Department of Genetics, Boys Town National Research Hospital, Omaha, Nebraska 68131, USA.
Nalabothula
Narasimharao
N
Sheibani
Nader
N
Sudhakar
Akulapalli
A
D000818:Animals; D019393:Arrestin / Q000494:pharmacology*; D015153:Blotting, Western; D002465:Cell Movement / Q000187:drug effects*; D049109:Cell Proliferation / Q000187:drug effects*; D002478:Cells, Cultured; D003124:Colorimetry; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D004789:Enzyme Activation; D016222:Fibroblast Growth Factor 2 / Q000037:antagonists & inhibitors* / Q000494:pharmacology; D020778:Matrix Metalloproteinase 2 / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology; D012171:Retinal Vessels / Q000166:cytology*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D019393:Arrestin; D012333:RNA, Messenger; D011994:Recombinant Proteins; D016222:Fibroblast Growth Factor 2; D020778:Matrix Metalloproteinase 2