PB1081 - Endothelial-derived microvesicles promote pro-migratory cross-talk with smooth muscle cells by a mechanism requiring tissue factor and PAR2 activation

Background: Microvesicles (MV) released by endothelial cells (EC) following injury or inflammation contain tissue factor (TF) and mediate communication with the underlying smooth muscle cells (SMC). Ser253-phosphorylated TF co-localizes with filamin A at the leading edge of migrating SMC and may be involved in the promotion of cell migration. TF-containing microvesicles (MV) act as messengers between different cells and can mediate repair. However, aberrant release of these MV can also give rise to vascular deformities.

Aims: In this study, the influence of endothelial-derived TF-MV, on human coronary artery SMC (HCASMC) migration was examined.

Methods: MV were isolated from human coronary artery EC (HCAEC) transfected to express Wild type or cytoplasmic domain-deleted TF, or control MV and the influence on HCASMC migration across examined using Transwells (0.8 µm pore). The MV were also pre-incubated with antibodies against TF (HTF1) and fVIIa (polyclonal) to block protease activity, or antibody to inhibit TF signalling (10H10). Alternatively, PAR2 activation was blocked on SMC by incubation with SAM11 antibody. In other experiments, HCASMC were transfected with peptides corresponding to the cytoplasmic domain of TF prior to stimulation with TF-fVIIa.

Results: TF-MV accelerated HCASMC migration, but was lower with cytoplasmic domain-deleted TF. Blocking TF-factor VIIa (TF-fVIIa) procoagulant/protease activity, or inhibiting PAR2 signalling on HCASMC, abolished the accelerated migration but 10H10 antibody had no effect. Incubation of HCASMC with fVIIa alone increased HCASMC migration, but was significantly enhanced on supplementation with recombinant TF. Neither recombinant TF alone, factor Xa alone, nor PAR2-acivating peptide (SLIGKV) influenced cell migration. Finally, cell migration was suppressed by the transfection of peptides that were phosphorylated at position of Ser253.

Conclusion(s): In conclusion, Ser253-phosphorylated TF and fVIIa released as MV-cargo by EC, act in conjunction with PAR2 on SMC to promote migration and may be crucial for normal arterial homeostasis as well as, during development of vascular abnormalities.