Endothelial NOS is required for SDF-1 alpha/CXCR4-mediated peripheral endothelial adhesion of c-kit(+) bone marrow stem cells

In the era of intravascular approaches for regenerative cell therapy, the underlying mechanisms of stem cell migration to non-marrow tissue have not been clarified. We hypothesized that next to a local inflammatory response implying adhesion molecule expression, endothelial nitric oxide synthase (eNOS)-dependent signaling is required for stromal-cell-derived factor-1 alpha (SDF-1 alpha)-induced adhesion of c-kit(+) cells to the vascular endothelium. SDF-1 alpha/tumor necrosis factor-alpha (TNF-alpha)-induced c-kit(+) cell shape change and migration capacity was studied in vitro using immunohistochemistry and Boyden chamber assays. In vivo interaction of c-kit(+) cells from bone marrow with the endothelium in response to SDF-1 alpha/TNF-alpha stimulation was visualized in the cremaster muscle microcirculation of wild-type (WT) and eNOS (-/-) mice using intravital fluorescence microscopy. In addition, NOS activity was inhibited with N-nitro-L-arginine-methylester-hydrochloride in WT mice. To reveal c-kit(+)-specific adhesion behavior, endogenous leukocytes (EL) and c-kit(+) cells from peripheral blood served as control. Moreover, intercellular adhesion molecule-1 (ICAM-1) and CXCR4 were blocked systemically to determine their role in inflammation-related c-kit(+)-cell adhesion. In vitro, SDF-1 alpha enhanced c-kit(+)-cell migration. In vivo, SDF-1 alpha alone triggered endothelial rolling-not firm adherence-of c-kit(+) cells in WT mice. While TNF-a alone had little effect on adhesion of c-kit(+) cells, it induced maximum endothelial EL adherence. However, after combined treatment with SDF-1a+TNF-alpha, endothelial adhesion of c-kit(+) cells increased independent of their origin, while EL adhesion was not further incremented. Systemic treatment with anti-ICAM-1 and anti-CXCR4-monoclonal antibody completely abolished endothelial c-kit(+)-cell adhesion. In N-nitro-L-arginine-methylester-hydrochloride-treated WT mice as well as in eNOS (-/-) mice, firm endothelial adhesion of c-kit(+) cells was entirely abrogated, while EL adhesion was significantly increased. The chemokine SDF-1a mediates firm adhesion c-kit(+) cells only in the presence of TNF-alpha stimulation via an ICAM-1- and CXCR4-dependent mechanism. The presence of eNOS appears to be a crucial and specific factor for firm c-kit(+)-cell adhesion to the vascular endothelium.