Bradykinin Receptors Regulate Proliferation, Migration and Tube Formation of Endothelial Progenitor Cells

Bradykinin is a potent activator of endothelial cells and is known to induce vasodilation, vascular permeability, and nitric oxide production. Bradykinin signaling is initiated by binding to G protein-coupled bradykinin receptors 1 and 2 (B1R and B2R). In this study we investigated whether these two receptors are involved in regulation of endothelial progenitor cell (EPC) biology. First we isolated CD34⁺ cells from wild-type (WT) and double B1R and B2R knockout (B1RB2R^-/-) mice, and compared their capacities of proliferation, migration, as well as tube formation in a 3D cell culture system. Bone marrow-derived B1RB2R^-/- EPCs displayed a reduction in proliferation and migration through collagen-coated transwell, compared with EPCs from WT mice. Moreover, implanted B1RB2R^-/- EPCs into immunocompromised NOD-SCID mice showed significantly less vasculogenesis in matrigel plug. Human EPCs were isolated from peripheral blood of healthy donors using CD34⁺ selection. In a concentration-dependent manner, bardykinin stimulated proliferation, migration and tube formation of human EPCs. In contrast to human EPCs, human umbilicial vein endothelial cells (HUVECs) did not display a significant increase in proliferation, migration, and tube formation upon bradykinin stimulation, suggesting that bradykinin selectively enhances the function of EPCs, but not mature endothelial cells. Pre-incubation with a B2R selective antagonist (Icatibant) reduced these functions of human EPCs. When human EPCs were stimulated with [des-Arg⁹]-BK, a selective B1R agonist, their proliferation and tube formation remained unchanged. Moreover, preincubation of human EPCs with [des-Arg¹⁰]-Icatibant, a B1R selective antagonist, did not affect their proliferation, migration and tube formation. As detected by Western blot and real time RT-PCR, B2R was constitutively expressed in EPCs, while the expression of B1R was only induced when stimulated by inflammatory stimuli, such as TNFα and IL-1β. Take together, bradykinin regulates proliferation, migration and tube formation of EPCs through constitutively expressed B2R, whereas B1R is probably involved in inflammatory settings.