Characterization of Endothelial Progenitor Cell: Past, Present, and Future.

Endothelial progenitor cells (EPCs) are currently being studied as candidate cell sources for revascularization strategies. Despite these promising results, widespread clinical acceptance of EPCs for clinical therapies remains hampered by several challenges. The challenges and issues surrounding the use of EPCs and the current paradigm being developed to improve the harvest efficiency and functionality of EPCs for application in regenerative medicine are discussed. It has been observed that controversies have emerged regarding the isolation techniques and classification and origin of EPCs. This manuscript attempts to highlight the concept of EPCs in a sequential manner, from the initial discovery to the present (origin, sources of EPCs, isolation, and identification techniques). Human and murine EPC marker diversity is also discussed. Additionally, this manuscript is aimed at summarizing our current and future prospects regarding the crosstalk of EPCs with the biology of hematopoietic cells and culture techniques in the context of regeneration-associated cells (RACs).

Overview of Basic Mechanisms of Notch Signaling in Development and Disease.

Notch signaling is an evolutionarily conserved pathway associated with the development and differentiation of all metazoans. It is needed for proper germ layer formation and segmentation of the embryo and controls the timing and duration of differentiation events in a dynamic manner. Perturbations of Notch signaling result in blockades of developmental cascades, developmental anomalies, and cancers. An in-depth understanding of Notch signaling is thus required to comprehend the basis of development and cancer, and can be further exploited to understand and direct the outcomes of targeted cellular differentiation into desired cell types and complex tissues from pluripotent or adult stem and progenitor cells. In this chapter, we briefly summarize the molecular, evolutionary, and developmental basis of Notch signaling. We will focus on understanding the basics of Notch signaling and its signaling control mechanisms, its developmental outcomes and perturbations leading to developmental defects, as well as have a brief look at mutations of the Notch signaling pathway causing human hereditary disorders or cancers.

The Hedgehog Signaling Pathway in Ischemic Tissues.

Hedgehog (Hh) proteins are prototypical morphogens known to regulate epithelial/mesenchymal interactions during embryonic development. In addition to its pivotal role in embryogenesis, the Hh signaling pathway may be recapitulated in post-natal life in a number of physiological and pathological conditions, including ischemia. This review highlights the involvement of Hh signaling in ischemic tissue regeneration and angiogenesis, with particular attention to the heart, the brain, and the skeletal muscle. Updated information on the potential role of the Hh pathway as a therapeutic target in the ischemic condition is also presented.

Sonic Hedgehog Signaling Pathway in Endothelial Progenitor Cell Biology for Vascular Medicine.

The Hedgehog (HH) signaling pathway plays an important role in embryonic and postnatal vascular development and in maintaining the homeostasis of organs. Under physiological conditions, Sonic Hedgehog (SHH), a secreted protein belonging to the HH family, regulates endothelial cell growth, promotes cell migration and stimulates the formation of new blood vessels. The present review highlights recent advances made in the field of SHH signaling in endothelial progenitor cells (EPCs). The canonical and non-canonical SHH signaling pathways in EPCs and endothelial cells (ECs) related to homeostasis, SHH signal transmission by extracellular vesicles (EVs) or exosomes containing single-strand non-coding miRNAs and impaired SHH signaling in cardiovascular diseases are discussed. As a promising therapeutic tool, the possibility of using the SHH signaling pathway for the activation of EPCs in patients suffering from cardiovascular diseases is further explored.

The sulfated polysaccharide fucoidan rescues senescence of endothelial colony-forming cells for ischemic repair.

The efficacy of cell therapy using endothelial colony-forming cells (ECFCs) in the treatment of ischemia is limited by the replicative senescence of isolated ECFCs in vitro. Such senescence must therefore be overcome in order for such cell therapies to be clinically applicable. This study aimed to investigate the potential of sulfated polysaccharide fucoidan to rescue ECFCs from cellular senescence and to improve in vivo vascular repair by ECFCs. Fucoidan-preconditioning of senescent ECFCs was shown by flow cytometry to restore the expression of functional ECFC surface markers (CD34, c-Kit, VEGFR2, and CXCR4) and stimulate the in vitro tube formation capacity of ECFCs. Fucoidan also promoted the expression of cell cycle-associated proteins (cyclin E, Cdk2, cyclin D1, and Cdk4) in senescent ECFCs, significantly reversed cellular senescence, and increased the proliferation of ECFCs via the FAK, Akt, and ERK signaling pathways. Fucoidan was found to enhance the survival, proliferation, incorporation, and endothelial differentiation of senescent ECFCs transplanted in ischemic tissues in a murine hind limb ischemia model. Moreover, ECFC-induced functional recovery and limb salvage were markedly improved by fucoidan pretreatment of ECFCs. To our knowledge, the findings of our study are the first to demonstrate that fucoidan enhances the neovasculogenic potential of ECFCs by rescuing them from replicative cellular senescence. Pretreatment of ECFCs with fucoidan may thus provide a novel strategy for the application of senescent stem cells to therapeutic neovascularization.

Selective Interference Targeting of Lnk in Umbilical Cord-Derived Late Endothelial Progenitor Cells Improves Vascular Repair, Following Hind Limb Ischemic Injury, via Regulation of JAK2/STAT3 Signaling.

The Lnk adaptor protein is a strong negative regulator that affects self-renewal of hematopoietic stem cells and vascular repair in injured tissues. However, the signaling mechanisms through which these proteins influence the vascular regeneration function of endothelial progenitor cells (EPCs) remain unknown. In this study, we investigated the effect of Lnk-targeted small interfering RNA (si-lnk) on the clonogenic proliferative potential and vascular regenerative function of EPCs and the activation of the JAK/STAT3 signaling pathway. Treatment with stem cell factor (SCF) increased the clonogenic proliferation of si-lnk EPCs. Importantly, activation of the JAK2/STAT3 pathway was enhanced in SCF-sensitized si-lnk EPCs. In a hind limb model of ischemia, transplantation of si-lnk EPCs increased the blood flow ratio, capillary density, proliferation, and survival of transplanted cells, and the secretion of pivotal angiogenic cytokines at ischemic sites. These results provide strong evidence that si-lnk regulates the clonogenic proliferative potential of EPCs through the activation of the JAK2/STAT3 signaling pathway, thereby accelerating angiogenesis and promoting repair in injured hind limb ischemia. Stem Cells 2014;33:1490-1500.

Tauroursodeoxycholic acid, a bile acid, promotes blood vessel repair by recruiting vasculogenic progenitor cells.

Although serum bile acid concentrations are approximately 10 µM in healthy subjects, the crosstalk between the biliary system and vascular repair has never been investigated. In this study, tauroursodeoxycholic acid (TUDCA) induced dissociation of CD34(+) hematopoietic stem cells (HSCs) from stromal cells by reducing adhesion molecule expression. TUDCA increased CD34(+) /Sca1(+) progenitors in mice peripheral blood (PB), and CD34(+) , CD31(+) , and c-kit(+) progenitors in human PB. In addition, TUDCA increased differentiation of CD34(+) HSCs into EPC lineage cells via Akt activation. EPC invasion was increased by TUDCA, which was mediated by fibroblast activating protein via Akt activation. Interestingly, TUDCA induced integration of EPCs into human aortic endothelial cells (HAECs) by increasing adhesion molecule expression. In the mouse hind limb ischemia model, TUDCA promoted blood perfusion by enhancing angiogenesis through recruitment of Flk-1(+) /CD34(+) and Sca-1(+) /c-kit(+) progenitors into damaged tissue. In GFP(+) bone marrow-transplanted hind limb ischemia, TUDCA induced recruitment of GFP(+) /c-kit(+) progenitors to the ischemic area, resulting in an increased blood perfusion ratio. Histological analysis suggested that GFP(+) progenitors mobilized from bone marrow, integrated into blood vessels, and differentiated into VEGFR(+) cells. In addition, TUDCA decreased cellular senescence by reducing levels of p53, p21, and reactive oxygen species and increased nitric oxide. Transplantation of TUDCA-primed senescent EPCs in hind limb ischemia significantly improved blood vessel regeneration, as compared with senescent EPCs. Our results suggested that TUDCA promoted neovascularization by enhancing the mobilization of stem/progenitor cells from bone marrow, their differentiation into EPCs, and their integration with preexisting endothelial cells.

CD34 hybrid cells promote endothelial colony-forming cell bioactivity and therapeutic potential for ischemic diseases.

OBJECTIVE: Although endothelial progenitor cells (EPCs) have been reported to promote neovessel formation during vascular injury, the function of supporting cells of EPCs and their interaction with EPCs during EPC isolation remain unclear. APPROACH AND RESULTS: We investigated the functional properties of 2 types of EPCs, also known as endothelial colony-forming cells (ECFCs), CD34(-)/CD34(+) cell-derived ECFCs (hybrid-dECFCs) and CD34(+) cell-derived ECFCs (stem-dECFCs), isolated using different methods, to elucidate the role of CD34(-) cell populations as cell-supporting niches. Using EPC colony-forming and insert coculture assays, we found that CD34(-) accessory cells dynamically modulate hematopoietic stem cell-derived endothelial cell progenitor commitment via angiogenic cytokines secreted by CD34(-)/CD11b(+) macrophages. On the basis of these findings, we isolated 2 types of ECFCs and investigated their bioactivities. We found that stem-dECFCs showed remarkably retarded cell growth, enhanced senescence, and decreased characteristics of ECFCs, whereas hybrid-dECFCs showed greater proliferative properties but delayed senescence. In a murine hind-limb ischemia model, hybrid-dECFCs showed significantly enhanced blood perfusion, capillary density, transplanted cell survival and proliferation, and angiogenic cytokine secretion compared with stem-dECFCs. In particular, the migratory capacity of hybrid-dECFCs was significantly enhanced, in part mediated via an augmented phosphorylation cascade of focal adhesion kinase and Src, resulting in a highly increased incorporation capacity of hybrid-dECFCs compared with stem-dECFCs. CD34(-) accessory cells of hybrid-dECFCs might be niche-supporting cells that facilitate cell survival, increase the secretion of angiogenic cytokines, and increase incorporation. CONCLUSIONS: This study provided important insight into blood vessel formation and repair in ischemic diseases for ECFC-based cell therapy.

Human cardiac stem cells with reduced notch signaling show enhanced therapeutic potential in a rat acute infarction model.

BACKGROUND: Because human cardiac stem cells (CSC) have regeneration potential in damaged cardiac tissue, there is increasing interest in using them in cell-based therapies for cardiac failure. However, culture conditions, by which CSCs are expanded while maintaining their therapeutic potential, have not been optimized. We hypothesized that the plating cell-density would affect proliferation activity, differentiation and therapeutic potential of CSCs through the Notch signaling pathway. METHODS AND RESULTS: Human CSCs were plated at 4 different densities. The population doubling time, C-KIT positivity, and dexamethasone-induced multidifferentiation potential were examined in vitro. The therapeutic potential of CSCs was assessed by transplanting them into a rat acute myocardial infarction (AMI) model. The low plating density (340cells/cm(2)) maintained the multidifferentiation potential with greater proliferation activity and C-KIT positivity in vitro. On the other hand, the high plating density (5,500cells/cm(2)) induced autonomous differentiation into endothelial cells by activating Notch signaling in vitro. CSCs cultured at low or high density with Notch signal inhibitor showed significantly greater therapeutic potential in vivo compared with those cultured at high density. CONCLUSIONS: CSCs cultured with reduced Notch signaling showed better cardiomyogenic differentiation and therapeutic potentials in a rat AMI model. Thus, reducing Notch signaling is important when culturing CSCs for clinical applications.

Phase II clinical trial of CD34+ cell therapy to explore endpoint selection and timing in patients with critical limb ischemia.

BACKGROUND: A prior phase I/IIa clinical trial provided evidence for safety, feasibility and potential efficacy of i.m. injection of granulocyte colony-stimulating factor (G-CSF)-mobilized CD34+ cells in patients with critical limb ischemia (CLI). METHODS AND RESULTS: A phase II trial of CD34+ cell therapy was conducted in patients with CLI to explore endpoint selection and timing. No-option CLI patients (n=11) underwent i.m. transplantation of G-CSF-mobilized CD34+ cells isolated by magnetic sorting. Ischemic rest pain scales improved from week 2 vs. baseline (P<0.05). Skin perfusion pressure (P=0.0175), transcutaneous partial oxygen pressure (P=0.0446) and pain-free walking distance (P=0.0056) improved from week 2, total walking distance from week 8 (P=0.0182) and toe brachial pressure index from week 12 (P=0.0174) vs. baseline. These parameters peaked at week 36 or 52. Rutherford's category improved from week 24 vs. baseline (P=0.0065). CLI-free ratio serially increased and peaked (85.7%) at week 36. Serial change in Rutherford's category correlated with that in Rest Pain Scale (P=0.0374), but not with that in any physiological parameters. CONCLUSIONS: Ischemic rest pain scales and physiological parameters improved relatively early after cell therapy, then plateaued later accompanied by recovery from the CLI state. Rutherford's category and CLI-free ratio at week 36 or later may be suitable endpoints in cell therapy clinical trials for CLI. Functional parameters should be evaluated independently of such clinical endpoints for ischemia severity. ( CLINICAL TRIAL REGISTRATION: URL: https://dbcentre3.jmacct.med.or.jp/jmactr/Default.aspx. Unique identifier: JMA-IIA00022)

CD34(+) cell therapy is safe and effective in slowing the decline of hepatic reserve function in patients with decompensated liver cirrhosis.

BACKGROUND AND AIM: Preclinical studies in rodent models of chronic liver fibrosis have shown that transplantation of peripheral blood (PB) CD34(+) cells leads to hepatic regeneration and a reduction of liver fibrosis by suppressing hepatic stellate cell activity and increasing matrix metalloproteinase activity. The aim of this study was to examine the safety and clinical efficacy of intrahepatic transplantation of autologous granulocyte colony-stimulating factor (G-CSF)-mobilized PB-CD34(+) cells in patients with decompensated liver cirrhosis. METHODS: PB-CD34(+) cells were isolated from G-CSF-mobilized apheresis products. Ten patients were treated with G-CSF-mobilized PB-CD34(+) cells (treatment group) and seven patients were treated with standard medical therapy. For mobilization, patients in the treatment group received subcutaneous injections of 10 µg G-CSF/kg/day for 5 days. The cells were then injected at three different doses (5 × 10(5) , 1 × 10(6) and 2 × 10(6) cells/kg) through the hepatic artery. Thereafter, all patients were followed up for 24 months. RESULTS: G-CSF treatment and leukapheresis were well tolerated, and no serious adverse events were observed. Patients in the treatment group had a significant but transient splenomegaly. After 24 weeks, serum albumin was significantly increased in patients who had received middle or high doses of CD34(+) cells compared with baseline. Doppler ultrasound showed a significant increase in hepatic blood flow velocity and blood flow volume after CD34(+) cell therapy. The hepatic vein pressure gradient decreased in two patients who received high-dose CD34(+) cells at week 16. CONCLUSIONS: CD34(+) cell therapy is feasible, safe and effective in slowing the decline of hepatic reserve function.

A small interfering RNA targeting Lnk accelerates bone fracture healing with early neovascularization.

Lnk, an intracellular adapter protein, is expressed in hematopoietic cell lineages, which has recently been proved as an essential inhibitory signaling molecule for stem cell self-renewal in the stem cell factor-c-Kit signaling pathway with enhanced hematopoietic and osteogenic reconstitution in Lnk-deficient mice. Moreover, the therapeutic potential of hematopoietic stem/endothelial progenitor cells (EPCs) for fracture healing has been demonstrated with mechanistic insight into vasculogenesis/angiogenesis and osteogenesis enhancement in the fracture sites. We report here, Lnk siRNA-transfected endothelial commitment of c-kit+/Sca-1+/lineage- subpopulations of bone marrow cells have high EPC colony-forming capacity exhibiting endothelial markers, VE-Cad, VEGF and Ang-1. Lnk siRNA-transfected osteoblasts also show highly osteoblastic capacity. In vivo, locally transfected Lnk siRNA could successfully downregulate the expression of Lnk at the fracture site up to 1 week, and radiological and histological examination showed extremely accelerated fracture healing in Lnk siRNA-transfected mice. Moreover, Lnk siRNA-transfected mice exhibited sufficient therapeutic outcomes with intrinstic enhancement of angiogenesis and osteogenesis, specifically, the mice demonstrated better blood flow recovery in the sites of fracture. In our series of experiments, we clarified that a negatively regulated Lnk system contributed to a favorable circumstance for fracture healing by enhancing vasculogenesis/angiogenesis and osteogenesis. These findings suggest that downregulation of Lnk system may have the clinical potential for faster fracture healing, which contributes to the reduction of delayed unions or non-unions.

Methodological development of a clonogenic assay to determine endothelial progenitor cell potential.

The precise and conceptual insight of circulating endothelial progenitor cell (EPC) kinetics is hampered by the absence of an assay system capable of evaluating the EPC differentiation cascade. An assay system for EPC colony formation was developed to delineate circulating EPC differentiation. EPC colony-forming assay using semisolid medium and single or bulk CD133(+) cells from umbilical cord blood exhibited the formation of two types of attaching cell colonies made of small or large cells featuring endothelial lineage potential and properties, termed small EPC colony-forming units and large EPC colony-forming units, respectively. In vitro and in vivo assays of each EPC colony-forming unit cell revealed a differentiation hierarchy from small EPC to large EPC colonies, indicating a primitive EPC stage with highly proliferative activity and a definitive EPC stage with vasculogenic properties, respectively. Experimental comparison with a conventional EPC culture assay system disclosed EPC colony-forming unit cells differentiate into noncolony-forming early EPC. The fate analysis of single CD133(+) cells into the endothelial and hematopoietic lineage was achieved by combining this assay system with a hematopoietic progenitor assay and demonstrated the development of colony-forming EPC and hematopoietic progenitor cells from a single hematopoietic stem cell. EPC colony-forming assay permits the determination of circulating EPC kinetics from single or bulk cells, based on the evaluation of hierarchical EPC colony formation. This assay further enables a proper exploration of possible links between the origin of EPC and hematopoietic stem cells, representing a novel and powerful tool to investigate the molecular signaling pathways involved in EPC biology.

Effect of ionizing radiation induced damage of endothelial progenitor cells in vascular regeneration.

OBJECTIVE: A number of studies have revealed that stress signaling and subsequent stress responses in stem/progenitor cells are responsible for attenuated regeneration or degenerative disease. Because ionizing radiation (IR), which sensitizes diverse types of stem cells, reportedly induces cardio-circulatory diseases, we hypothesized that IR-induced vascular abnormalities are associated with defects in endothelial progenitor cells (EPCs) that are responsible for vascular homeostasis. METHODS AND RESULTS: We used an irradiated mouse model to mimic the IR effect on vasculogenesis. Mouse EPCs isolated from irradiated mice and human EPCs exposed to IR were used for functional analysis and gene expression study. Under IR exposure, EPCs were depleted, and their function for vasculogenesis in vitro and in vivo was significantly reduced. In such IR-mediated stress responses, upregulating p21Cip1 and downregulating vascular endothelial growth factor (VEGF) were mediated by p53 transcriptional activity. CONCLUSIONS: The results of the present study suggest that suppression of p53 would be clinically applicable to (1) minimize the functional defects in EPCs in order to prevent the onset of vascular diseases caused by radiation therapy or radiation exposure and also to (2) provide novel insight into the mechanisms of IR-induced vascular damage and a possible strategy to minimize vascular damage by IR.

p53-induced inhibition of Hif-1 causes cardiac dysfunction during pressure overload.

Cardiac hypertrophy occurs as an adaptive response to increased workload to maintain cardiac function. However, prolonged cardiac hypertrophy causes heart failure, and its mechanisms are largely unknown. Here we show that cardiac angiogenesis is crucially involved in the adaptive mechanism of cardiac hypertrophy and that p53 accumulation is essential for the transition from cardiac hypertrophy to heart failure. Pressure overload initially promoted vascular growth in the heart by hypoxia-inducible factor-1 (Hif-1)-dependent induction of angiogenic factors, and inhibition of angiogenesis prevented the development of cardiac hypertrophy and induced systolic dysfunction. Sustained pressure overload induced an accumulation of p53 that inhibited Hif-1 activity and thereby impaired cardiac angiogenesis and systolic function. Conversely, promoting cardiac angiogenesis by introducing angiogenic factors or by inhibiting p53 accumulation developed hypertrophy further and restored cardiac dysfunction under chronic pressure overload. These results indicate that the anti-angiogenic property of p53 may have a crucial function in the transition from cardiac hypertrophy to heart failure.

Impact of cardiac stem cell sheet transplantation on myocardial infarction.

PURPOSE: Myocardial infarction (MI) remains a major cause of mortality because of the limited regenerative capacity of the myocardium. Transplantation of somatic tissue-derived cells into the heart has been shown to enhance the endogenous healing process, but the magnitude of its therapeutic effects is dependent upon the cell-source or cell-delivery method. We investigated the therapeutic effects of C-Kit positive cardiac cell (CSC) cell-sheet transplantation therapy in a rat model of MI. METHODS AND RESULTS: CSCs of human origin were sorted and cultured to generate scaffold-free CSC cell-sheets. One-layered or 3-layered cell-sheets were transplanted into nude rats 1 h after left coronary artery ligation. We observed a significant increase in the left ventricular ejection fraction and a significant decrease in left ventricular systolic dimension at 2 and 4 weeks in the 3-layer group, but not in the 1-layer or sham groups. Consistently, there was less accumulation of interstitial fibrosis in the 3-layer group than in the 1-layer or sham groups. Moreover, capillary density was significantly greater in the 3-layer group than in the 1-layer or sham groups. CONCLUSIONS: The 3-layered cell-sheet improved cardiac function associated with angiogenic and anti-fibrotic effects. Thus, CSC is a promising cell-source to use with the cell-sheet method for the treatment of cardiac failure, as long as a sufficient number of cells are delivered.

Therapeutic application of regeneration-associated cells: a novel source of regenerative medicine.

Chronic diseases with comorbidities or associated risk factors may impair the function of regenerative cells and the regenerative microenvironment. Following this consideration, the vasculogenic conditioning culture (VCC) method was developed to boost the regenerative microenvironment to achieve regeneration-associated cells (RACs), which contain vasculogenic endothelial progenitor cells (EPCs) and anti-inflammatory/anti-immunity cells. Preclinical and clinical studies demonstrate that RAC transplantation is a safe and convenient cell population for promoting ischemic tissue recovery based on its strong vasculogenicity and functionality. The outputs of the scientific reports reviewed in the present study shed light on the fact that RAC transplantation is efficient in curing various diseases. Here, we compactly highlight the universal features of RACs and the latest progress in their translation toward clinics.

CD34 positive cells as endothelial progenitor cells in biology and medicine.

CD34 is a cell surface antigen expressed in numerous stem/progenitor cells including hematopoietic stem cells (HSCs) and endothelial progenitor cells (EPCs), which are known to be rich sources of EPCs. Therefore, regenerative therapy using CD34+ cells has attracted interest for application in patients with various vascular, ischemic, and inflammatory diseases. CD34+ cells have recently been reported to improve therapeutic angiogenesis in a variety of diseases. Mechanistically, CD34+ cells are involved in both direct incorporation into the expanding vasculature and paracrine activity through angiogenesis, anti-inflammatory, immunomodulatory, and anti-apoptosis/fibrosis roles, which support the developing microvasculature. Preclinical, pilot, and clinical trials have well documented a track record of safety, practicality, and validity of CD34+ cell therapy in various diseases. However, the clinical application of CD34+ cell therapy has triggered scientific debates and controversies in last decade. This review covers all preexisting scientific literature and prepares an overview of the comprehensive biology of CD34+ cells as well as the preclinical/clinical details of CD34+ cell therapy for regenerative medicine.

Repetitive administration of cultured human CD34+ cells improve adenine-induced kidney injury in mice.

BACKGROUND: There is no established treatment to impede the progression or restore kidney function in human chronic kidney disease (CKD). AIM: To examine the efficacy of cultured human CD34+ cells with enhanced proliferating potential in kidney injury in mice. METHODS: Human umbilical cord blood (UCB)-derived CD34+ cells were incubated for one week in vasculogenic conditioning medium. Vasculogenic culture significantly increased the number of CD34+ cells and their ability to form endothelial progenitor cell colony-forming units. Adenine-induced tubulointerstitial injury of the kidney was induced in immunodeficient non-obese diabetic/severe combined immunodeficiency mice, and cultured human UCB-CD34+ cells were administered at a dose of 1 × 106/mouse on days 7, 14, and 21 after the start of adenine diet. RESULTS: Repetitive administration of cultured UCB-CD34+ cells significantly improved the time-course of kidney dysfunction in the cell therapy group compared with that in the control group. Both interstitial fibrosis and tubular damage were significantly reduced in the cell therapy group compared with those in the control group (P < 0.01). Microvasculature integrity was significantly preserved (P < 0.01) and macrophage infiltration into kidney tissue was dramatically decreased in the cell therapy group compared with those in the control group (P < 0.001). CONCLUSION: Early intervention using human cultured CD34+ cells significantly improved the progression of tubulointerstitial kidney injury. Repetitive administration of cultured human UCB-CD34+ cells significantly improved tubulointerstitial damage in adenine-induced kidney injury in mice via vasculoprotective and anti-inflammatory effects.

Fluid shear stress induces differentiation of circulating phenotype endothelial progenitor cells.

Endothelial progenitor cells (EPCs) are mobilized from bone marrow to peripheral blood, and contribute to angiogenesis in tissue. In the process, EPCs are exposed to shear stress generated by blood flow and tissue fluid flow. Our previous study showed that shear stress induces differentiation of mature EPCs in adhesive phenotype into mature endothelial cells and, moreover, arterial endothelial cells. In this study we investigated whether immature EPCs in a circulating phenotype differentiate into mature EPCs in response to shear stress. When floating-circulating phenotype EPCs derived from ex vivo expanded human cord blood were exposed to controlled levels of shear stress in a flow-loading device, the bioactivities of adhesion, migration, proliferation, antiapoptosis, tube formation, and differentiated type of EPC colony formation increased. The surface protein expression rate of the endothelial markers VEGF receptor 1 (VEGF-R1) and -2 (VEGF-R2), VE-cadherin, Tie2, VCAM1, integrin α(v)/ß(3), and E-selectin increased in shear-stressed EPCs. The VEGF-R1, VEGF-R2, VE-cadherin, and Tie2 protein increases were dependent on the magnitude of shear stress. The mRNA levels of VEGF-R1, VEGF-R2, VE-cadherin, Tie2, endothelial nitric oxide synthase, matrix metalloproteinase 9, and VEGF increased in shear-stressed EPCs. Inhibitor analysis showed that the phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signal transduction pathway is a potent activator of adhesion, proliferation, tube formation, and differentiation in response to shear stress. Western blot analysis revealed that shear stress activated the VEGF-R2 phosphorylation in a ligand-independent manner. These results indicate that shear stress increases differentiation, adhesion, migration, proliferation, antiapoptosis, and vasculogenesis of circulating phenotype EPCs by activation of VEGF-R2 and the PI3K/Akt/mTOR signal transduction pathway.