FIG. 6.

S1P1 receptor signaling is required for human and ovine endothelial cell sprouting responses in 3-D collagen matrices. In all experiments, endothelial invasion was stimulated with 1 μM S1P, 40 ng/ml VEGF, and 40 ng/ml FGF2. A) FTY720, an S1P receptor antagonist, blocked HUVEC invasion in a dose-dependent manner. B) HUVEC invasion was conducted with increasing doses of the S1PR1 and S1PR3 antagonist VPC23019. RT-PCR reactions using primer sets designed for human (C) and ovine (D) S1PR1–S1PR5 sequences using HUVEC and oUAEC cDNA as a template, respectively. Also pictured are GAPDH and beta actin (ACTB) positive controls. Primer sequences are available upon request. E) oUAEC invasion cultures were established in the presence of S1P in combination, with doses of FTY720 and VPC23019 indicated. DMSO and bovine serum albumin served as vehicle controls for FTY720 and VPC23019, respectively. Dose-responses were conducted four times, and duplicate experiments were performed on endothelial cell lines isolated from separate individuals.