Effect of angiogenic and antiangiogenic compounds on the outgrowth of capillary structures from fetal mouse bone explants

Fetal mouse metatarsals are well-known models to study cartilage differenti ation and osteoclastic resorption. We show here the outgrowth of PECAM-1 po sitive tubelike structures from the bone rudiments. This feature can be use d to study angiogenesis in vitro. The area of outgrowth significantly incre ased with culture time, as shown by computerized image analysis of PECAM-1 positive tubelike structures. Treatment with recombinant human vascular end othelial growth factor (rhVEGF-A) stimulated the formation of tubelike stru ctures. Treatment of explants with the angiogenesis inhibitor endostatin, t he chemokine IP-10, and the thalidomide derivative phatolyl glutamic acid ( PG-acid) resulted in an inhibition of the formation of PECAM-1 positive tub elike structures of 48.8% (+/- 4%), 50.2% (+/-12%), and 80.8% (+/-3%), resp ectively. Outgrowth of tubelike structures was partly dependent on endogeno us VEGF-A because treatment with anti-mVEGF-A and truncated VEGF receptor 1 (soluble fms-like tyrosine kinase 1, sFlt1) strongly inhibited the formati on of tubelike structures 74% (+/- 4%) and 38% (+/- 5%), respectively. Neit her onset of tube formation nor total area of tubelike structures were chan ged when metatarsals were cultured on a fibrin gel or collagen type I gel. Tube formation required activation of matrix metalloproteinases because tre atment of the bones with an inhibitor of matrix metalloproteinases complete ly inhibited migration and tube formation, whereas treatment with an inhibi tor of plasmin had no effect. In conclusion, we describe a new in vitro mod el to study angiogenesis that can be used to test the angiogenic or antiang iogenic potential of novel test compounds that also combines the multicellu larity of in vivo assays with the accessibility and flexibility of in vitro assays.