1. ¹Department of Cardiac Surgery, University of Rostock, Rostock, Germany
2. ²Institute for Experimental Surgery, University of Rostock, Rostock, Germany
3. ³Department of General Surgery, University of Rostock, Rostock, Germany

Correspondence: Dr A Kaminski, MD, Klinik für Herzchirurgie, Universität Rostock, Schillingallee 35, 18057 Rostock, Germany. E-mail: alexander.kaminski@med.uni-rostock.de

Received 26 February 2007; Revised 6 September 2007; Accepted 10 September 2007; Published online 26 November 2007.

Abstract

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)-induced adhesion of c-kit⁺ cells to the vascular endothelium. SDF-1/tumor necrosis factor-alpha (TNF-)-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/TNF- 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 enhanced c-kit⁺-cell migration. In vivo, SDF-1 alone triggered endothelial rolling—not firm adherence—of c-kit⁺ cells in WT mice. While TNF- alone had little effect on adhesion of c-kit⁺ cells, it induced maximum endothelial EL adherence. However, after combined treatment with SDF-1+TNF-, 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-1 mediates firm adhesion c-kit⁺ cells only in the presence of TNF- 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.