PB1434 - Inhibition of thrombin receptor PAR-1 in endothelial colony forming cells modify their stemness and vasculogenic properties

Background: The importance of PAR-1 in blood vessel development has been demonstrated in knockout mice. By contrast, in adult mouse models of angiogenesis, PAR1 implication seems very moderate.

Aims: To explore the role of the thrombin receptor PAR-1 stemness effects on human cord blood endothelial colony forming cells (ECFCs).

Methods: ECFCs were expanded from human monocuclear cells from fresh cord blood.

Results: We first postulated that CD133 in ECFCs might be regulated by PAR-1. Indeed, PAR-1 activation with PAR-1 activating peptide induced a significant decrease in CD133 expression while inhibition of PAR-1 with siRNA induced an increase in CD133 at mRNA levels but also at the protein level with a significant increase in intracellular expression of CD133. To confirm relevance of relationship between CD133 and PAR-1, we explored association between levels of both PAR-1 and CD133 in fast and slow fibroblasts prone to reprogrammation. We observed an imbalance between PAR-1 and CD133 expression with a decreased expression of PAR-1 in fast reprogramming fibroblast expressing high CD133 expression. PAR-1 inhibition with specific siRNA induced ECFCs proliferation and Ki67 increase, while no modification of migration in a Boyden chamber assay or scratch test were observed. Moreover, PAR-1 inhibition did not modify ECFCs adhesion on smooth muscle cells or human arterial endothelial cells. In the model of mouse hind-limb ischemia, silencing of PAR-1 in ECFCs significantly increased post-ischemic revascularization induced by siCtrl-ECFCs; indeed, a significant increase in cutaneous blood flows (p = 0.01), capillary density (CD31) (p = 0.0022) and myofiber regeneration (p = 0.0001) was observed. Also, a significant decreased in necrosis (p = 0.0001) was observed in mice receiving siPAR-1-ECFCs compared to those treated with siCtrl-ECFCs.

Conclusion(s): In conclusion, our work describes for the first-time link between stemness, vasculogenesis and PAR-1 in human immature endothelial cells.