Figure 3

Mesenchymal stem cells induce sprouting of endothelial cells by VEGF expression but do not differentiate into endothelial cells. (A) Vascular endothelial growth factor protein in supernatant of MSC cultured under hypoxic (black bars) or normoxic (white bars) conditions in the presence or absence of Avastin (25 μg ml⁻¹) was analysed by the ELISA assay. (B) RNA expression of VEGF was analysed by RT-PCR in MSC. The expression of α-SMA served as control for equal conditions. (C) A volume of 100 or 200 μl supernatant from MSC (as indicated), which have been cultured in medium containing 2% FCS for 48 h or recombinant VEGF (50 ng ml⁻¹), was transferred to spheroids consisting of 1 × 10³ HUVEC growing in wells of a 24-well plate 48 spheroids per well. The length of sprouts was analysed by cell^B 2.3 software and median lengths are given. Representative spheroids have been visualised by phase-contrast microscopy using an Olympus CKX41 microscope, × 10 magnification, and a colorview camera soft imaging system. (D) Mesenchymal stem cells were seeded on chamber slights covered with fibronectin and cultured in endothelial cell medium containing VEGF (50 ng ml⁻¹) for 2 weeks. Immunohistochemistry was performed using specific antibodies for the detection of the specific endothelial cell markers von-Willebrand factor (α-vWF) and CD31 (α-CD31). Human umbilical vein endothelial cells were used as positive control. A Leica DMRB microscope and × 1000 magnification were used.