Mer silencing suppresses both ProS-induced SHP2 activation and inhibition of VEGF-A–induced EC BrdU incorporation and tube formation. (A) Changes in BrdU incorporation resulting from silencing of Tyro3, Axl, or Mer gene expression. ECs were seeded at a density of 6 × 103 cells/well in 96-well plates in EGM-2 for 24 hours. Cells were then transfected with either the specified siRNA used either at 5nM for Tyro3 and Axl or at 50nM for Mer or with vehicle (0.5 μL/well siPORT) or as a control with 50nM nontargeting siRNA. At 24 hours after transfection, cells were switched to EBM-2 containing the indicated factors at the specified concentrations. BrdU incorporation was measured by ELISA. Data obtained from 3 independent experiments (3 distinct independent cell culture 96-well plates) each in triplicate wells are expressed as percentages of the same ratio in siPORT-transfected ECs ± SD. ***P < .001. *P < .05. ns indicates not significant. (B) The in vitro morphogenesis assay. ECs (2 × 105 cells/well) were cultured for 24 hours in complete medium and then transfected with 5nM of either Mer or control siRNA. At 24 hours after transfection, cells were harvested and seeded at the density of 105 cells/well in EBM-2 containing 0.5% FCS and 20 ng/mL VEGF-A, in 24-well plates coated with Matrigel containing either human ProS (20 μg/mL) or vehicle. The cell 3-dimensional organization was photographed using a MVX10 microscope. Black scale bar on pictures represents 100 μm length. (C) Quantifications of total tube length of capillary-like structures by automatic counting using the AngioQuant software. Data obtained from 3 independent experiments each in duplicates are expressed as percentages of control ± SD. *P < .05. ns indicates not significant. (D) Mer receptor tyrosine phosphorylation after treatment with ProS (10 μg/mL) for the indicated time. GAPDH was used as loading control. Because the data were obtained from 3 independent experiments each in triplicate and only 1 representative result for each time point is depicted in the figure, vertical lines have been inserted to indicate repositioned gel lanes. The intensity of bands was quantified and is represented in panel E as a percentage of control of the ratio of phosphorylated Mer over GAPDH ± SD. **P < .01. *P < .05. ns indicates not significant. (F) Western blotting analysis of Mer receptor expression and changes in EC response to ProS, in term of SHP2 activation, after silencing of Mer. (G) The intensity of bands was quantified and is represented as a percentage of control of the ratio of phosphorylated SHP2 over SHP2; some error bars were too small to be visible. Data obtained from 4 independent experiments each in duplicate are expressed as percentages of control ± SD. *P < .05. ns indicates not significant. (H) The proposed mechanism through which ProS interferes with VEGF-A-induced VEGFR2 signaling. VEGF-A triggers VEGFR2, Akt, and Erk 1/2 activation, leading to EC migration proliferation and subsequently to vascular tube formation (in green). ProS activates its tyrosine kinase receptor Mer, leading to SHP-2 activation, which dephosphorylates VEGFR2 on the SHP2-sensitive site Tyr996, thereby inhibiting VEGFR2 recruitment of Akt and Erk 1/2 pathways and subsequently VEGFR2 induced EC migration and proliferation necessary for tube formation and angiogenesis (in red). A recent report45 has described that, similarly to ProS, its structural homolog Gas6 activates Mer tyrosine kinase receptor, leading to the inhibition of EC migration and angiogenesis (in blue). Tumor cells release soluble Mer, which acts as a decoy receptor for Gas6, thereby reducing the suppressive effects of Gas6 on endothelial cell recruitment.
