In vivo chamber angiogenesis assay: an optimized Matrigel plug assay for fast assessment of anti-angiogenic activity.
Efficient in vitro and in vivo angiogenesis assays, to assess and compare anti-angiogenic activity are a prerequisite for the discovery and characterization of anti-angiogenic targets. Here we describe an optimized Matrigel plug assay based on subcutaneously implanted chambers and two fast and reproducible measuring techniques. Plexiglas ring/nylon net filter-chambers (0.2 ml) containing growth factor-reduced Matrigel and 300 ng basic fibroblast growth factor (bFGF) were subcutaneously implanted into the right flank of rats. Chamber angiogenesis was scored on day 5 and day 10 post-implantation by computer image analysis of the chamber, and by optical density reading at 415 nm of a PBS solution of the chamber content. bFGF significantly induced chamber angiogenesis and histological examination confirmed that numerous blood vessels were present in the bFGF-induced chambers. The anti-angiogenic control compound TNP-470 (10 mg/kg/d s.c.) completely inhibited the bFGF-induced angiogenesis. In contrast, the anti-inflammatory or immuno-suppressive compounds cyclosporin A (15 mg/kg/d p.o.), indomethacin (1 mg/kg/d p.o.), and prednisolone (5 mg/kg/d p.o.) showed no anti-angiogenic activity, indicating that the bFGF-induced angiogenesis was not driven by an inflammatory response or by a foreign body reaction. Finally, two candidate anti-angiogenic compounds were tested in the assay. Continuous low-dose therapy with cyclophosphamide (25 mg/kg/d p.o.) significantly inhibited bFGF-induced angiogenesis, whereas 1alpha,25-dihydroxyvitamin D3 (0.5 micro g/kg/d p.o.) showed no significant anti-angiogenic activity. In conclusion, this in vivo chamber angiogenesis assay is a useful new tool for drug evaluation as well as research into anti-angiogenesis.
Kragh
Michael
M
Department of Pharmacology, LEO Pharma A/S, DK-2750, Ballerup, Denmark. michael.kragh@leo-pharma.com
Hjarnaa
Pernille-Julia V
PJ
Bramm
Erik
E
Kristjansen
Paul E G
PE
Rygaard
Jørgen
J
Binderup
Lise
L
D020533:Angiogenesis Inhibitors / Q000494:pharmacology*; D000818:Animals; D000893:Anti-Inflammatory Agents / Q000494:pharmacology; D065095:Calcineurin Inhibitors; D002117:Calcitriol / Q000494:pharmacology; D003094:Collagen; D003510:Cyclohexanes; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology; D003520:Cyclophosphamide / Q000494:pharmacology; D016572:Cyclosporine / Q000494:pharmacology; D004338:Drug Combinations; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005260:Female; D016222:Fibroblast Growth Factor 2 / Q000037:antagonists & inhibitors / Q000494:pharmacology; D007213:Indomethacin / Q000494:pharmacology; D007797:Laminin; D018919:Neovascularization, Physiologic / Q000187:drug effects*; D000077609:O-(Chloroacetylcarbamoyl)fumagillol; D011239:Prednisolone / Q000494:pharmacology; D011509:Proteoglycans; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D012717:Sesquiterpenes / Q000494:pharmacology*; D012884:Skin Window Technique
D020533:Angiogenesis Inhibitors; D000893:Anti-Inflammatory Agents; D065095:Calcineurin Inhibitors; D003510:Cyclohexanes; D016861:Cyclooxygenase Inhibitors; D004338:Drug Combinations; D007797:Laminin; D011509:Proteoglycans; D012717:Sesquiterpenes; D016222:Fibroblast Growth Factor 2; C059485:matrigel; D016572:Cyclosporine; D003520:Cyclophosphamide; D003094:Collagen; D011239:Prednisolone; D002117:Calcitriol; D000077609:O-(Chloroacetylcarbamoyl)fumagillol; D007213:Indomethacin