Tae-Youn Kim

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.

Identification of a novel role of T cells in postnatal vasculogenesis : Characterization of endothelial progenitor cell colonies

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 CD3+CD31+CXCR4+ 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.

Integrin-Linked Kinase, a Hypoxia-Responsive Molecule, Controls Postnatal Vasculogenesis by Recruitment of Endothelial Progenitor Cells to Ischemic Tissue

Background— Recruitment and adhesion of endothelial progenitor cells (EPCs) to hypoxic endothelial cells (ECs) is essential for vasculogenesis in ischemic tissue; little is known, however, about the key signals or intracellular signaling pathways involved in orchestrating the expression of adhesion molecules by ECs in response to hypoxia and how this is related to the recruitment of EPCs to the ischemic tissue. Here, we report that endogenous integrin-linked kinase (ILK) is a novel molecule that responds to hypoxia in ECs that regulates the expression of stromal cell–derived factor-1 (SDF-1) and intercellular adhesion molecule-1 (ICAM-1) through nuclear factor-&kgr;B and hypoxia-inducible factor-1α and induces recruitment of EPCs to ischemic areas. Methods and Results— Under hypoxia, both the endogenous amount and kinase activity of ILK were time-dependently upregulated in ECs, which was associated with increased ICAM-1 and SDF-1. This upregulation of ILK was mediated by stabilization of ILK by heat shock protein 90. ILK overexpression in normoxic ECs resulted in ICAM-1 and SDF-1 upregulation through dual control by nuclear factor-&kgr;B and hypoxia-inducible factor-1α. Blockade of ILK in hypoxic ECs significantly abrogated the expression of both molecules, which led to decreased EPC incorporation into ECs. A hindlimb ischemia model showed that ILK blockade significantly reduced EPC homing to ischemic limb and consequently led to poor neovascularization. Overexpression of ILK in the Matrigel plug significantly improved neovascularization in vivo, whereas the blockade of ILK resulted in the opposite effect. Conclusions— Endogenous ILK is a novel and physiological upstream responder of numerous intracellular molecules involved in hypoxic stress in ECs and may control the recruitment of EPCs to ischemic tissue.

Intercellular Adhesion Molecule-1 Is Upregulated in Ischemic Muscle, Which Mediates Trafficking of Endothelial Progenitor Cells

Background—Trafficking of transplanted endothelial progenitor cells (EPCs) to an ischemic organ is a critical step in neovascularization. This study was performed to elucidate the molecular mechanism of EPC trafficking in terms of adhesion molecules. Methods and Results—Using murine hindlimb ischemia model, we examined expressions of E-selectin, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and platelet-endothelial cell adhesion molecule-1 (PECAM-1) in ischemic muscle by immunofluorescence. ICAM-1 was overexpressed in ischemic muscle compared with nonischemic muscle, whereas expressions of E-selectin, VCAM-1, and PECAM-1 did not show that much difference. ICAM-1 was also upregulated by hypoxia in murine endothelial cells (ECs) as assessed by immunoblot and flow cytometry. EPCs were attached to ECs specifically through ICAM-1/β-2 integrin interaction in vitro. When EPCs were labeled with fluorescent dye or radioisotope (Tc-99m-HMPAO) and systemically administrated in vivo, EPCs preferentially homed to ischemic muscle. By blocking ICAM-1, EPCs entrapment to ischemic limb in vivo was significantly reduced and neovascularization induced by EPC transplantation was attenuated. Conclusions—ICAM-1 is upregulated by ischemia, and this is closely associated with EPCs entrapment to ischemic limb. Our findings suggest that ICAM-1 expression might be important in regulating the process of neovascularization through its ability to recruit EPCs.

Akt is a key modulator of endothelial progenitor cell trafficking in ischemic muscle.

Trafficking of transplanted endothelial progenitor cells (EPCs) to ischemic tissue is enhanced by stromal‐derived factor 1 (SDF‐1) and vascular endothelial growth factor (VEGF). However, it has not been studied how these cytokines modulate the local milieu to entrap EPCs. This study was performed to elucidate a molecular pathway of trafficking EPCs through Akt and to test its application as an adjuvant modality to increase EPC homing. In a mouse hind limb ischemia model, systemically administered 1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethylindocarbocyanine‐labeled mouse EPCs showed three stages of homing to ischemic limb: adhesion to endothelial cells (ECs), incorporation to capillary, and transendothelial migration into extravascular space. As an underlying mechanism to control adhesion of EPCs to ECs, we found that Akt was activated in ECs of ischemic muscle by ischemia‐induced VEGF and SDF‐1. In vitro and in vivo experiments using adenoviral vector for constitutively active or dominant‐negative Akt genes showed that activated Akt enhanced intercellular adhesion molecule 1 (ICAM‐1) expression on ECs. Akt activation in ECs also enhanced EPC incorporation to ECs and transendothelial migration in vitro experiments. Activated Akt was sufficient for induction of EPC homing even in normal hind limb, where VEGF or SDF‐1 was not increased. Finally, local Akt gene transfer to ischemic limb significantly enhanced homing of systemically administered EPCs, new vessel formation, blood flow recovery, and tissue healing. Akt plays a key role in EPC homing to ischemic limb by controlling ICAM‐1 and transendothelial migration. Modulation of Akt in the target tissue may be an adjunctive measure to enhance homing of systemically administered stem cells, suggesting a possibility of cell‐and‐gene hybrid therapy.

Regulation of Endothelial Cell and Endothelial Progenitor Cell Survival and Vasculogenesis by Integrin-Linked Kinase

Objective—New vessel formation is a dynamic process of attachment, detachment, and reattachment of endothelial cells (ECs) and endothelial progenitor cells (EPCs) with each other and with the extracellular matrix (ECM). Integrin-linked kinase (ILK) plays a pivotal role in ECM-mediated signaling. Therefore, we investigated the role of ILK in ECs and EPCs during neovascularization. Methods and Results—In human umbilical cord vein ECs and EPCs, endogenous ILK expression, along with subsequent cell survival signals phospho-Akt and phospho–glycogen synthase kinase 3&bgr;, was reduced after anchorage or nutrient deprivation. Even brief anchorage deprivation resulted in retarded capillary tube formation by ECs. Adenoviral ILK gene transfer in ECs and EPCs reversed the decrease in cell survival signals after anchorage or nutrient deprivation, leading to enhanced survival, reduced apoptosis, and significantly accelerated the functional recovery after reattachment. And ILK overexpressing EPCs significantly improved blood flow recovery and prevented limb loss in nude mice hindlimb ischemia model. Furthermore, the efficacy of systemic delivery was equivalent to local injection of ILK-EPCs. Conclusions—ILK overexpression protects ECs and EPCs from anchorage- or nutrient-deprived stress and enhances neovascularization, suggesting that ILK is an optimal target gene for genetically modified cell-based therapy. Neovascularization is a dynamic process of detachment and reattachment of ECs and EPCs. Endogenous ILK expression was decreased in various stress conditions, and the gene transfer of ILK protected ECs and EPCs from temporary anchorage or nutrient deprivation. Furthermore, ILK gene transfer in EPCs significantly enhanced neovascularization in vivo.

β-Catenin Overexpression Augments Angiogenesis and Skeletal Muscle Regeneration Through Dual Mechanism of Vascular Endothelial Growth Factor–Mediated Endothelial Cell Proliferation and Progenitor Cell Mobilization

Objective—&bgr;-Catenin plays a critical role in directing cell fate during embryogenesis, and uncontrollable activation leads to cancers, suggesting its importance in cell survival and proliferation. However, little is known regarding its role in endothelial cell (EC) and skeletal muscle proliferation and progenitor cell mobilization. Methods and Results—&bgr;-Catenin enhanced ECs proliferation, protected ECs from apoptosis, and increased the capillary forming capabilities, which was completely blocked by inhibition of its nuclear translocation. In addition, the increased proliferation by &bgr;-catenin was associated with increased expression of cyclin E2. In skeletal myocytes, &bgr;-catenin overexpression increased proliferation with cyclin D1 expression, decreased apoptosis, and induced hypertrophy. Furthermore, &bgr;-catenin induced the expression of vascular endothelial growth factor (VEGF) in skeletal myocytes, resulting in EC proliferation. In a mouse hindlimb ischemia model, &bgr;-catenin significantly increased recovery of blood perfusion, capillary density along with enhanced VEGF expression, and the number of proliferating ECs and myocytes. Local delivery of &bgr;-catenin also promoted angiogenic progenitor cell mobilization and increased the number of satellite cells. Conclusions—&bgr;-Catenin may be an important modulator of angiogenesis and myocyte regeneration not only by directly enhancing proliferation and survival of ECs and skeletal myocytes but also by inducing VEGF expression and promoting angiogenic progenitor cell mobilization and muscle progenitor cell activation.

New Mechanism of Rosiglitazone to Reduce Neointimal Hyperplasia: Activation of Glycogen Synthase Kinase-3β Followed by Inhibition of MMP-9

Objective—Mechanism of neointimal hyperplasia after vascular injury includes activation of signaling pathways and matrix metalloproteinases (MMPs) that are involved in cell proliferation and migration. Rosiglitazone, a synthetic peroxisome proliferator-activated receptor-&ggr; (PPAR-&ggr;) agonist, was reported to inhibit neointimal hyperplasia in diabetic animals and humans. But the underlying mechanism has not been clarified. In this study, we examined how rosiglitazone inhibited neointimal hyperplasia. Methods and Results—The proliferation and survival of cultured rat VSMCs were reduced by rosiglitazone, which was mediated by inhibition of ERK and activation GSK-3&bgr;, without change of Akt. The antiproliferative effect of rosiglitazone was reversed by GSK-3&bgr; inactivation. The migration of VSMCs was also suppressed by rosiglitazone that inhibited the expression and activity MMP-9 through GSK-3&bgr; activation. Thus migration of MMP-9(−/−) VSMCs from MMP-9 knockout mice was not affected by rosiglitazone. The underlying mechanism of MMP-9 suppression by rosiglitazone was that it inhibited NF-&kgr;B DNA binding activity, which was also dependent on GSK-3&bgr;. In rat carotid artery, balloon injury significantly inactivated GSK-3&bgr; with induction of MMP-9, which was effectively prevented by rosiglitazone. Thus, rosiglitazone significantly decreased the ratio of intima to media by reducing proliferation and inducing apoptosis of VSMCs at neointima, which was reversed by inactivation of GSK-3&bgr; with adenoviral transfer of catalytically-inactive GSK-KM gene. Conclusions—Rosiglitazone activates GSK-3&bgr;, which inhibits not only proliferation of VSMCs but also migration of VSMCs through blocking NF-&kgr;B–dependent MMP-9 activation.

Priming With Angiopoietin-1 Augments the Vasculogenic Potential of the Peripheral Blood Stem Cells Mobilized With Granulocyte Colony-Stimulating Factor Through a Novel Tie2/Ets-1 Pathway

Background— The low engraftment rate of stem/progenitor cells infused via the intracoronary route to the ischemic myocardium is one of the most important factors limiting the efficacy of cell therapy. We investigated the concept of priming peripheral blood stem cells enriched by granulocyte colony-stimulating factor mobilization and apheresis (mobPBSCs) with angiopoietin-1 (Ang1), to enhance the engraftment into the ischemic tissue and neovasculogenic potential. Methods and Results— The expression of Tie2, the Ang1 receptor, was significantly higher in mobPBSCs than naïve peripheral blood mononuclear cells (19.2±3.0% versus 1.2±0.8% versus 1.2±0.2%; P<0.001 for mobPBSCs from acute myocardial infarction (AMI) patients with granulocyte colony-stimulating factor treatment for 3 days versus peripheral blood mononuclear cells from AMI patients versus peripheral blood mononuclear cells from stable angina patients). After 4 hours of cartilage oligomeric matrix protein (COMP)-Ang1 stimulation, mobPBSCs committed to the endothelial lineage with the induction of CD31 and VE-cadherin expression, mediated by Tie2/Ets-1 pathway. Priming of mobPBSCs with COMP-Ang1 induced the expression of &agr;4&bgr;1 and &agr;5&bgr;1 integrins, which are also Ets-1 downstream molecules, leading to enhanced adhesion to endothelial cells or fibronectin. In a rabbit ear ischemia/reperfusion model, priming of mobPBSCs with COMP-Ang1 improved first-pass engraftment to the distal vascular bed after intraarterial delivery. In a murine ischemic hind-limb model, intravascular delivery of primed mobPBSCs enhanced both engraftment and neovascularization. Conclusions— The short-term priming with COMP-Ang1 may be a feasible and promising option to activate mobPBSCs by enhancing differentiation and adhesiveness and to improve the efficacy of cell therapy for ischemic diseases.

Magnetic Bionanoparticle Enhances Homing of Endothelial Progenitor Cells in Mouse Hindlimb Ischemia

Background and Objectives Poor homing efficiency is one of the major limitations of current stem cell therapy. Magnetic bionanoparticles (MPs) obtained from Magnetospirillum sp. AMB-1 have a lipid bilayer membrane and ferromagnetic properties. We evaluated a novel priming strategy using MPs to enhance the homing of transplanted progenitor cells to target tissue. Materials and Methods Effects of MP on proliferation, viability, and migration of late human endothelial progenitor cells (EPCs) were examined in vitro. Additionally, effects of MP on gene and protein expression related to survival and adhesion were evaluated. Homing and angiogenic efficiency of MP transferred late EPCs was evaluated in nude mouse hindlimb ischemia model. Results Below threshold concentration, MP transfer did not influence proliferation or survival of late EPCs, but enhanced migration and trans-endothelial migration of late EPCs toward magnet. Below threshold concentration, MP transfer did not influence gene and protein expression related to survival. In the mouse hindlimb ischemia model, late EPCs treated with high dose MP (5 ug/mL) showed enhanced homing of injected late EPCs in the ischemic limb by magnet, compared to low dose MP (1 ug/mL) treated late EPCs. In addition, high dose MP transferred EPC showed significantly better improvement of perfusion in ischemic limb compared to untreated EPC. Conclusion MP transfer with magnet application can be a promising novel strategy to enhance homing efficacy and outcomes of current stem cell therapy.