Synergistic Neovascularization by mixed transplantation of early endothelial progenitor cells and late outgrowth endothelial cells - The role of angiogenic cytokines and matrix metalloproteinases

Background - Two types of cells are cultured from the human peripheral blood, early endothelial progenitor cells (EPCs) and outgrowth endothelial cells (OECs), as previously reported. Here, we further characterize these cells, especially with respect to their different origins and functions both in vitro and in vivo. We also investigated whether the combination of these different cell types shows synergism during neovascularization. Methods and Results - Early EPCs were heterogeneously made up of both CD14(+) monocyte-derived cells, which secrete cytokines, and CD14(-)-derived cells, which contain high levels of CD34(+)KDR(+) cells. OECs were cultured almost exclusively from CD14(-) cells, not CD14(-) cells, and were distinct from mature endothelial cells in terms of proliferation potential, KDR+ expression level, and telomerase activity. A portion of cells from CD14(-) cells and early EPCs produced rapidly proliferating, capillary-forming cells in both the Matrigel plug and the ischemic hind limb similar to OECs. Early EPCs and OECs expressed receptors for vascular endothelial growth factor and interleukin-8, cytokines secreted by early EPCs. There was a differential increase in matrix metalloproteinases ( MMPs): MMP-9 in early EPCs and MMP-2 in OECs. In vitro, the angiogenic capability of the 2 cell types was augmented by mutual interaction through cytokines and MMPs. Injection of a mixture of the 2 cells resulted in superior neovascularization in vivo to any single-cell-type transplantation. Conclusions - Distinct origins of the different types of EPCs exist that have different functions in neovascularization. Mixed transplantation of these cells results in synergistic neovascularization through cytokines and MMPs.