Granulocyte Colony-Stimulating Factor–Mobilized Circulating c-Kit+/Flk-1+ Progenitor Cells Regenerate Endothelium and Inhibit Neointimal Hyperplasia After Vascular Injury

Abstract

BACKGROUND: Granulocyte colony-stimulating factor (G-CSF) treatment was shown to inhibit neointimal formation of balloon-injured vessels, whereas neither the identification of progenitor cells involved in G-CSF-mediated endothelial regeneration with a bone marrow (BM) transplant experiment nor the functional properties of regenerated endothelium have been studied. METHODS AND RESULTS: Recombinant human G-CSF (100 microg/kg per day) was injected daily for 14 days starting 3 days before balloon injury in the rat carotid artery. Neointimal formation of denuded vessels on day 14 was markedly attenuated by G-CSF (39% versus the control; P<0.05). Endothelial cell-specific immunostaining revealed an enhancement of re-endothelialization (1.8-fold increase versus the control; P<0.05) and inhibition of extravasation of Evans Blue dye (47%; P=0.02). The regenerated endothelium exhibited acetylcholine-mediated vasodilatation in NO-dependent manner. G-CSF increased the circulating c-Kit+/Flk-1+ cells (9.1-fold; P<0.02), which showed endothelial properties in vitro (acetylated low-density lipoprotein uptake and lectin binding) and incorporated into the regenerated endothelium in vivo. A BM replacement experiment with green fluorescent protein (GFP)-overexpressing cells showed that BM-derived GFP+/CD31+ endothelial cells occupied 39% of the total luminal length in the G-CSF-mediated neo-endothelium (2% in the control). CONCLUSIONS: The G-CSF-induced mobilization of BM-derived c-Kit+/Flk-1+ cells contributes to endothelial regeneration, and this cytokine therapy may be a feasible strategy for the promotion of re-endothelialization after angioplasty.