In the course of conducting a series of studies whose goal was to discover novel endogenous angiogenesis inhibitors, we have purified matrilin-1 (MATN-1) and have demonstrated, for the first time, that it inhibits neovascularization both in vitro and in vivo. Proteins were extracted from cartilage using a 2 m NaCl, 0.01 m HEPES buffer at 4 °C, followed by concentration of the extract. The concentrate was fractionated by size exclusion chromatography, and fractions were then screened for their ability to inhibit capillary endothelial cell (EC) proliferation in vitro. Fractions containing EC inhibitory activity were pooled and further purified by cation exchange chromatography. The resulting fractions from this step were then screened to isolate the antiangiogenic activity in vitro. This activity was identified by tandem mass spectrometry as being MATN-1. Human MATN-1 was cloned and expressed in Pichia pastoris and purified to homogeneity. Purified recombinant MATN-1, along with purified native protein, was shown to inhibit angiogenesis in vivo using the chick chorioallantoic membrane assay by the inhibition of capillary EC proliferation and migration. Finally, using a MATN-1-deficient mouse, we showed that angiogenesis during fracture healing was significantly higher in MATN-1(-/-) mice compared with the wild type mice as demonstrated by in vivo imaging and by elevated expression of angiogenesis markers including PECAM1, VEGFR, and VE-cadherin.
