Abstract 629: EphB4 Activation Increases Pro-Angiogenic Potential of Endothelial Progenitor Cells. Role of P-Selectin Glycoprotein Ligand-1
Therapeutic neovascularization of ischemic tissues has been achieved through infusion of endothelial progenitor cells (EPCs). However, only a minor percentage of injected cells are able to home and incorporate into the neo-capillaries. Eph receptors and their ephrin ligands are key regulators of endothelial cell proliferation, migration, adhesion and repulsion during vascular development. We hypothesized that activation of Eph receptor may enhance EPCs pro-angiogenic potential. We previously demonstrated that EPCs-derived from human umbilical cord blood expressed EphB4 receptor tyrosine kinase and its ligand ephrinB2. Unilateral hindlimb ischemia was surgically induced in athymic nude mice, and ex vivo expanded human EPCs activated by ephrin-B2-Fc (to activate EphB4), EphB4-Fc (to activate ephrinB2) or CD6-Fc (control) fusion proteins were injected intravenously, 5 hours after the onset of ischemia. Administration of ephrinB2-Fc-treated EPCs improved angiographic score, capillary density and blood flow perfusion compared to untreated- and EphB4-Fc-treated EPCs (+49%, p<0.01; +73.7%, p<0.01; +50.2%, p<0.05, n=8, respectively versus EPCs-treated mice). EPCs express several Eph receptors such as, EphB1, EphB2 and EphB4 that can bind ephrinB2-Fc. To assess the involvement of EphB4 in the ephrin-B2-Fc related effects, we used siRNA targeting EphB4. Infusion of EPCs transfected with EphB4 siRNA and pre-treated by ephrinB2-Fc abrogated ephrinB2-Fc-induced activation of EPCs pro-angiogenic potential. In vitro studies indicated that EphB4 stimulation by ephrinB2-Fc increased EPCs adhesion on IL-1β-activated HUVECs (+32%, p<0.05, n=3, versus control EPCs), and upregulated P-selectin glycoprotein ligand -1 (PSGL-1) expression. In addition, PSGL-1 mediated adhesion of ephrinB2-Fc-treated EPCs on recombinant CD62-E and -P coated onto tissue culture wells. Interestingly, CD62-E and CD62-P were upregulated in ischemic muscles suggesting that PSGL-1/CD62-E and CD62-P and interaction may mediate the homing of ephrin-B2-Fc-treated EPCs towards ischemic tissues. Our results demonstrate, for the first time, that ephrin-B2-Fc treatment could improve efficacy of EPCs-based therapy in severe peripheral vascular disease.