Matrigel model of in vivo angiogenesis

Growth factor-reduced Matrigel (Corning, New York, USA; 500 μl) supplemented with 200 ng ml⁻¹ VEGF-A 165 (VIB Protein Service Facility), 500 ng ml⁻¹ bFGF (Peprotech, London, UK) and 60 U ml⁻¹ heparin (Leo Pharma, Wilrijk, Belgium) was subcutaneously injected on the back of C57BL/6J mice (protocol adapted from ref. ³⁵). Pathological vasculature was allowed to develop during 6 days, followed by 5 consecutive days of treatment by intraperitoneal injection of control vehicle (DMSO, Merck Millipore, Overijse, Belgium), 3PO (ChemBridge Corporation, San Diego, USA; 25 mg kg⁻¹ per day) and/or the VEGFR2 tyrosine kinase inhibitor SU5416 (Sigma-Aldrich; 10 mg kg⁻¹ per day). These doses of 3PO and SU5416 were determined in pilot dose-response experiments. Three to five mice were used per group. Matrigel plugs were collected and stained for CD105 and NG2 to analyse vessel tortuosity and pericyte coverage.