Journal: Frontiers in Cardiovascular Medicine

Article Title: Endothelial-derived microvesicles promote pro-migratory cross-talk with smooth muscle cells by a mechanism requiring tissue factor and PAR2 activation

doi: 10.3389/fcvm.2024.1365008

Figure Lengend Snippet: Examination of the pro-migratory mechanism of TF-MV using blocking antibodies. MV were prepared from HCAEC (5 × 10 5 ) transfected to express TF Wt -tGFP and pre-incubated with mouse anti-human-TF antibodies, 10H10 (20 µg/ml) or HTF1 (20 µg/ml), or an inhibitory polyclonal rabbit anti-human fVIIa antibody (10 µg/ml). Additionally, HCASMC were incubated with an inhibitory mouse anti-human PAR2 antibody, SAM11 (20 µg/ml). HCASMC (3 × 10 4 ) were stimulated with MV at 37°C for 18 h. The cells were then fixed, washed and the cells on the upper side of the chamber were scraped off. ( A ) The cells were stained with crystal violet and photographed and ( B ) the numbers of migrating cells were determined by eluting the crystal violet and measuring the absorptions at 595 nm. Presented data include the calculated mean values ± the calculated standard error of the mean, from 5 experiments each examined in duplicate.

Article Snippet: In some experiments, the isolated MV were pre-incubated for 1 h, with a mouse anti-human-TF antibody, 10H10 (20 µg/ml; BD Bioscience, Wokingham, UK) capable of blocking TF signaling, a mouse anti-human-TF antibody, HTF-1 (20 µg/ml; eBioscience/Thermo Scientific, Warrington, UK) to block TF-fVIIa protease/procoagulant activity, an inhibitory polyclonal rabbit anti-human fVIIa antibody (10 µg/ml; Abcam, Cambridge, UK) or the respective control isotype IgG antibodies (20 µg/ml; New England Biolabs, Hitchin, UK).

Techniques: Blocking Assay, Transfection, Incubation, Staining