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 CD14ϩ monocyte-derived cells, which secrete cytokines, and CD14Ϫ -derived cells, which contain high levels of CD34 ϩ KDR ϩ cells. OECs were cultured almost exclusively from CD14 Ϫ cells, not CD14 ϩ cells, and were distinct from mature endothelial cells in terms of proliferation potential, KDR ϩ expression level, and telomerase activity. A portion of cells from CD14 Ϫ 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.

Background-The colony number of early endothelial progenitor cells (EPCs) has been used as a quantitative indicator of the number of EPCs in the blood or as a biological marker of cardiovascular diseases. In the present study, we found a subset of T cells that were localized at the center of the EPC colony and played a pivotal role in colony formation and differentiation of early EPCs. Methods and Results-We found that CD3ϩ CD31 ϩ CXCR4 ϩ T cells (referred to as angiogenic T cells in the present study) constituted the center of EPC colonies during cultures of human peripheral blood mononuclear cells. These angiogenic T cells were required for colony formation and differentiation of early EPCs. They secreted high levels of angiogenic cytokines such as vascular endothelial growth factor, interleukin-8, and matrix metalloproteinases. Angiogenic T cells showed superior angiogenic potential to the other subset of T cells in the experiments with regard to Matrigel tube formation, adhesion, transendothelial migration, and collagen invasion assay, mainly through the stromal cell-derived factor 1/CXCR-4 axis. Furthermore, angiogenic T cells enhanced endothelial cell proliferation and function. In vivo study showed that angiogenic T cells play an important role in the process of vessel formation. Clinical study showed that the level of angiogenic T cells in the peripheral blood was well correlated with EPC colony numbers and had inverse relationships with age and the number of risk factors for coronary artery disease. Conclusions-These findings suggest that angiogenic T cells could be a potential therapeutic target for ischemic cardiovascular diseases.

The concept of reprogramming of somatic cells has opened a new era in regenerative medicine. Transduction of defined factors has successfully achieved pluripotency. However, during the generation process of induced pluripotent stem (iPS) cells, genetic manipulation of certain factors may cause tumorigenicity, which limits further application. We report that that a single transfer of embryonic stem (ES) cell-derived proteins into primarily cultured adult mouse fibroblasts, rather than repeated transfer or prolonged exposure to materials, can achieve full reprogramming up to the pluripotent state without the forced expression of ectopic transgenes. During the process, gene expression and epigenetic status were converted from somatic to ES-equivalent status. We verified that protein-based reprogramming was neither by the contamination of protein do-

E-selectin plays critical roles in tethering leukocytes to endothelial cells (ECs).We studied the role of E-selectin in endothelial progenitor cell (EPC) homing and vasculogenesis. After ischemia, the expression of E-selectin on ECs peaked 6 to 12 hours and returned to baseline at 24 hours, whereas the level of soluble E-selectin (sE-selectin) in serum increased over 24 hours and remained high at day 7. Mouse bone marrow-derived EPCs expressed not only E-selectin but also its ligand. Homing of circulating EPCs to ischemic limb was significantly impaired in E-selectin knock-out mice, as well as wild-type mice pretreated with blocking antibody against E-selectin, which was rescued by local sE-selectin injection. Mechanism for this is that sE-selectin stimulated not only ECs to express ICAM-1, but also EPCs to secrete interleukin-8 (IL-8), leading to enhanced migration and incorporation to ECs capillary formation. In therapeutic aspect, local treatment with sE-selectin enhanced efficacy of EPC transplantation for vasculogenesis and salvage of ischemic limb. Conversely, when E-selectin was knocked down by E-selectin small interfering RNA, blood flow recovery after EPC transplantation was significantly impaired. But this impaired vasculogenesis was rescued by sE-selectin. In conclusion, these data demonstrate E-selectin is a pivotal molecule for EPCs' homing to ischemic limb and vasculogenesis. IntroductionE-selectin is an inducible cell-adhesion molecule on endothelial cells (ECs), which mediates the binding of the neutrophils and functions as a calcium-dependent lectin. [1][2][3] It consists of 5 components: an amino-terminal "C type" lectin domain critical for ligand interaction, an epidermal growth factor-like domain, 6 complement regulatory repeats, a single transmembrane domain, and a cytoplasmic carboxyl-terminal tail. 4,5 E-selectin mediates adhesive interactions of circulating leukocytes with the vascular endothelium during inflammatory conditions such as rheumatoid arthritis and atherosclerosis. 6,7 It also plays a role in the homing of hematopoietic stem cells, and its constitutive expression on ECs of hematopoietic tissue is essential in the initial step of the homing process. 8,9 In addition, Koch et al have reported that soluble E-selectin (sE-selectin), which is thought to be a cleavage form of membrane-bound E-selectin and therefore lacks the trans-membrane and cytoplasmic domains, induces angiogenesis in the rat cornea and stimulates chemotaxis and tube formation of human dermal microvascular ECs through Src-and phosphatidylinositol 3-kinase-mediated pathways. 10,11 Endothelial progenitor cells (EPCs) have the potential to proliferate and to differentiate into mature ECs. 12 Recent studies in animal and humans suggest the ability of EPCs to home to the areas with reduced oxygen supply and to induce vasculogenesis and angiogenesis. 13,14 Because the number of circulating EPCs may limit the ultimate magnitude of therapeutic angiogenesis, strategy of ex vivo expansion of EPCs harvested from the patient...