Hypoxia promotes the phenotypic change of aldehyde dehydrogenase activity of breast cancer stem cells.

Stable breast cancer cell (BCC) lines are valuable tools for the identification of breast cancer stem cell (BCSC) phenotypes that develop in response to several stimuli as well as for studying the basic mechanisms associated with the initiation and maintenance of BCSCs. However, the characteristics of individual, BCC-derived BCSCs varies and these cells show distinct phenotypes depending on the different BCSC markers used for their isolation. Aldehyde dehydrogenase (ALDH) activity is just such a recognized biomarker of BCSCs with a CD44+ /CD24- phenotype. We isolated BCSCs with high ALDH activity (CD44+ /CD24- /Aldefluorpos ) from a primary culture of human breast cancer tissue and observed that the cells had stem cell properties compared to BCSCs with no ALDH activity (CD44+ /CD24- /Aldefluorneg ). Moreover, we found Aldefluorpos BCSCs had a greater hypoxic response and subsequent induction of HIF-1α expression compared to the Aldefluorneg BCSCs. We also found that knocking down HIF-1α, but not HIF-2α, in Aldefluorpos BCSCs led to a significant reduction of the stem cell properties through a decrease in the mRNA levels of genes associated with the epithelial-mesenchymal transition. Indeed, HIF-1α overexpression in Aldefluorneg BCSCs led to Slug and Snail mRNA increase and the associated repression of E-cadherin and increase in Vimentin. Of note, prolonged hypoxic stimulation promoted the phenotypic changes of Aldefluorneg BCSCs including ALDH activity, tumorigenesis and metastasis, suggesting that hypoxia in the tumor environment may influence BCSC fate and breast cancer clinical outcomes.

In vivo retinal and choroidal hypoxia imaging using a novel activatable hypoxia-selective near-infrared fluorescent probe.

PURPOSE: Retinal hypoxia plays a crucial role in ocular neovascular diseases, such as diabetic retinopathy, retinopathy of prematurity, and retinal vascular occlusion. Fluorescein angiography is useful for identifying the hypoxia extent by detecting non-perfusion areas or neovascularization, but its ability to detect early stages of hypoxia is limited. Recently, in vivo fluorescent probes for detecting hypoxia have been developed; however, these have not been extensively applied in ophthalmology. We evaluated whether a novel donor-excited photo-induced electron transfer (d-PeT) system based on an activatable hypoxia-selective near-infrared fluorescent (NIRF) probe (GPU-327) responds to both mild and severe hypoxia in various ocular ischemic diseases animal models. METHODS: The ocular fundus examination offers unique opportunities for direct observation of the retina through the transparent cornea and lens. After injection of GPU-327 in various ocular hypoxic diseases of mouse and rabbit models, NIRF imaging in the ocular fundus can be performed noninvasively and easily by using commercially available fundus cameras. To investigate the safety of GPU-327, electroretinograms were also recorded after GPU-327 and PBS injection. RESULT: Fluorescence of GPU-327 increased under mild hypoxic conditions in vitro. GPU-327 also yielded excellent signal-to-noise ratio without washing out in vivo experiments. By using near-infrared region, GPU-327 enables imaging of deeper ischemia, such as choroidal circulation. Additionally, from an electroretinogram, GPU-327 did not cause neurotoxicity. CONCLUSIONS: GPU-327 identified hypoxic area both in vivo and in vitro.

Microvesicles derived from Alde-Low EPCs support the wound healing capacity of AT-MSCs.

Mesenchymal stem cells (MSCs) are defined as multipotent cells that can give rise to various kinds of differentiated mesenchymal cells, and are thus considered to be useful for clinical therapy. However, the big hurdles of MSC therapy are the inability of MSCs to reach the appropriate tissues or sites with high efficiency and engraftment after transplantation. In this study, we investigated how adipose tissue-derived MSCs (AT-MSCs) improve their homing ability after intravenous injection. We previously found that human endothelial progenitor cells with low aldehyde dehydrogenase activity (Alde-Low EPCs) are suitable for the treatment of ischemic tissues. In addition, we demonstrated that microvesicles (MVs) derived from Alde-Low EPCs possessed the ability to improve the homing ability of non-functional Alde-High EPCs, resulting in wound healing. We initially transfected MVs derived from Alde-Low EPCs (EMVs) to human AT-MSCs, which were originally unable to cure ischemic tissues by intravenous transplantation. Remarkably, AT-MSC transfected EMVs dramatically repaired the ischemic skin flap compared with AT-MSC derived-MV (MMVs) transfected AT-MSCs or control AT-MSCs. We then found that the expression of CXCR4, an important chemokine receptor for cell migration, was highly elevated in EMV-transfected AT-MSCs. Moreover, AT-MSCs transfected with EMVs, but not control AT-MSCs, migrated to wound sites after intravenous injection. Consequently, CD45(+) inflammatory cells were successfully recruited at the wound sites after the injection of EMV-transfected AT-MSCs. These results demonstrate that EMVs are a useful source to improve the homing ability and wound healing ability of MSCs at the wound sites.

A Chemokine Receptor, CXCR4, Which Is Regulated by Hypoxia-Inducible Factor 2α, Is Crucial for Functional Endothelial Progenitor Cells Migration to Ischemic Tissue and Wound Repair.

Endothelial progenitor cells (EPCs) have the ability to form new blood vessels and protect ischemic tissues from damage. We previously reported that EPCs with low activity of aldehyde dehydrogenase (Alde-Low EPCs) possess the greater ability to treat ischemic tissues compared with Alde-High EPCs. The expression level of the hypoxia-inducible factors (HIFs), HIF-1α and HIF-2α, was found to be greater in Alde-Low EPCs than in Alde-High EPCs. However, the precise role of the HIF factors in the regulation of EPC activity remains obscure. In this study, we demonstrate a critical role of HIF-2α and its target gene CXCR4 for controlling the migratory activity of EPC to ischemic tissue. We found that coculture of Alde-High EPCs with microvesicles derived from Alde-Low EPCs improved their ability to repair an ischemic skin flap, and the expression of CXCR4 and its ligand SDF1 was significantly increased following the coculture. In Alde-Low EPCs, the expression of CXCR4 was suppressed by short hairpin RNA (shRNA)-mediated HIF-2α, but not HIF-1α downregulation. Chromatin immunoprecipitation assays showed that HIF-2α, but not HIF-1α, binds to the promoter region of CXCR4 gene. The CXCR4 shRNA treatment in Alde-Low EPCs almost completely abrogated their migratory activity to ischemic tissues, whereas the reduction of vascular endothelial growth factor (VEGF) showed much less effect. The CXCR4 overexpression in Alde-High EPCs resulted in a partial, but significant improvement in their repairing ability in an ischemic skin flap. Collectively, these findings indicate that the CXCR4/SDF-1 axis, which is specifically regulated by HIF-2α, plays a crucial role in the regulation of EPC migration to ischemic tissues.

Impaired expression of HIF-2α induces compensatory expression of HIF-1α for the recovery from anemia.

Erythropoiesis is strongly influenced by the interactions between stromal cells and erythroid progenitors, as well as by a key regulatory factor, erythropoietin (EPO). We previously generated mice with a knockdown mutation of Hif-2α (referred to as kd/kd) and found that these kd/kd mice exhibited normocytic anemia, even though the EPO expression was not severely affected. However, the VCAM-1 expression in spleen endothelial cells (EC), which is regulated by HIF-2α, was impaired, resulting in defective erythroid maturation. A deficiency of HIF-2α clearly led to pancytopenia. However, the critical level of HIF-2α required for erythropoiesis has not yet been elucidated. In this study, we generated HIF-2α knockdown/knockout heterozygous mice (kd/null). Strikingly, anemia was observed in the kd/null mice, but the red blood cell indices were significantly improved compared to those of kd/kd mice. In the spleens of kd/null mice, higher HIF-1α activity and expansion of the red pulp area were observed compared to those of kd/kd mice. Importantly, EC isolated from kd/null spleens showed high expression of VEGF receptors, FLK-1 and FLT-1, which are regulated by HIF-1α instead of HIF-2α under hypoxic conditions. We also found higher expression of phosphorylated ERK and higher proliferative activity in the EC isolated from kd/null mice compared to those from kd/kd mice. While the HIF-2α expression was diminished, HIF-1α bound to the HRE region in the promoters of genes that are normally regulated by HIF-2α. These results suggest that there is a compensatory pathway involving HIF-1α that regulates the expression of some HIF-2α target genes.

Hypoxia-inducible factor-3α promotes angiogenic activity of pulmonary endothelial cells by repressing the expression of the VE-cadherin gene.

The variants of the hypoxia-inducible factor-3α gene HIF-3α and NEPAS are known to repress the transcriptional activities driven by HIF-1α and HIF-2α. Although NEPAS has been shown to play an important role in vascular remodeling during lung development, little is known about the roles of HIF-3α in adult lung function. Here, we examined pulmonary endothelial cells (ECs) isolated from wild-type (WT) and HIF-3α functional knockout (KO) mice. The expression levels of angiogenic factors (Flk1, Ang2 and Tie2) were significantly greater in the HIF-3α KO ECs than those in the WT ECs irrespective of oxygen tension. However, the HIF-3α KO ECs showed impaired proliferative and angiogenic activities. The impaired EC function was likely due to the excess vascular endothelial (VE)-cadherin, an inhibitor of Flk1/PI3 kinase/Akt signaling, as treatment of the cells to a neutralizing antibody partly restored the phenotype of the HIF-3α KO ECs. Importantly, we found that the mRNA levels of HIF-2α and Ets-1 were significantly increased by HIF-3α ablation. Given that both factors are known to activate the VE-cadherin gene, the transcriptional repression of these factors by HIF-3α might be important for silencing the irrelevant expression of the VE-cadherin gene. Collectively, these data show novel and unique roles of HIF-3α for angiogenic gene regulation in pulmonary ECs.

The role of CCL5 in the ability of adipose tissue-derived mesenchymal stem cells to support repair of ischemic regions.

Mesenchymal stem cells (MSC) are multipotent and possess high proliferative activity, and thus are thought to be a reliable cell source for cell therapies. Here, we isolated MSC from adult tissues--bone marrow (BM-MSC), dental tissue (DT-MSC), and adipose tissue (AT-MSC)--to compare how autotransplantation of these MSC effectively supports the repair of bone fracture and ischemic tissue. An analysis by in vitro differentiation assays showed no significant difference among these MSC. The degree of calcification at the joint region of bone fracture was higher in mice transplanted with AT-MSC than in mice transplanted with BM-MSC or DT-MSC. To compare the abilities of MSC, characterize how those MSC affect the repair of ischemic tissue, vascular occlusion was performed by ligation of the femoral artery and vein. Of note, the blood flow in the ischemic region rapidly increased in mice injected with AT-MSC, as contrasted with mice injected with BM- or DT-MSC. The number of CD45- and F4/80-positive cells at the femoral region was higher in AT-MSC recipients than in recipients of BM-MSC or DT-MSC. We evaluated the mRNA expression of angiogenic and migration factors in MSC and found the expression of CCL5 mRNA was higher in AT-MSC than in BM-MSC or DT-MSC. Transplantation of AT-MSC with impaired expression of CCL5 clearly showed a significant delay in the recovery of blood flow compared with the control. These findings have fundamental implications for the modulation of AT-MSC in the repair of vasculature and bone fracture.

Establishment of erythroleukemic GAK14 cells and characterization of GATA1 N-terminal domain.

GATA1 is a transcription factor essential for erythropoiesis and megakaryopoiesis. It has been found that Gata1 gene knockdown heterozygous female (Gata1(G1.05/+)) mice spontaneously develop erythroblastic leukemias. In this study, we have generated a novel Gata1 knockdown erythroblastic cell line, designated GAK14, from the leukemia cells in the Gata1(G1.05/+) mice. Although GAK14 cells maintain immature phenotype on OP9 stromal cells in the presence of erythropoietin and stem cell factor, the cells produce Gr-1-, Mac1-, B220-, CD3e- or CD49b-positive hematopoietic cells when co-cultured with DAS104-8 feeder cells. However, GAK14 cells did not produce erythroid and megakaryocytic lineages, perhaps due to the absence of GATA1. Indeed, GAK14 cells became capable of differentiating into mature erythroid cells when complemented with full-length GATA1 and co-cultured with fetal liver-derived FLS5 stromal cells. This differentiation potential was impaired when GATA1 lacking the N-terminal domain was complemented. The N-terminal domain is known to contribute to the pathogenesis of transient abnormal myelopoiesis and acute megakaryoblastic leukemia related to Down syndrome. These results thus showed that GAK14 cells will serve as a powerful tool for dissecting domain function of GATA1 and that the GATA1 N-terminal domain is essential for the erythroid differentiation of GAK14 cells.

Functional endothelial progenitor cells selectively recruit neurovascular protective monocyte-derived F4/80(+) /Ly6c(+) macrophages in a mouse model of retinal degeneration.

Retinitis pigmentosa is a group of inherited eye disorders that result in profound vision loss with characteristic retinal neuronal degeneration and vasculature attenuation. In a mouse model of retinitis pigmentosa, endothelial progenitor cells (EPC) from bone marrow rescued the vasculature and photoreceptors. However, the mechanisms and cell types underlying these protective effects were uncertain. We divided EPC, which contribute to angiogenesis, into two subpopulations based on their aldehyde dehydrogenase (ALDH) activity and observed that EPC with low ALDH activity (Alde-Low) had greater neuroprotection and vasoprotection capabilities after injection into the eyes of an rd1 mouse model of retinitis pigmentosa compared with EPC with high ALDH activity (Alde-High). Of note, Alde-Low EPC selectively recruited F4/80(+) /Ly6c(+) monocyte-derived macrophages from bone marrow into retina through CCL2 secretion. In addition, the mRNA levels of CCR2, the neurotrophic factors TGF-ß1 and IGF-1, and the anti-inflammatory mediator interleukin-10 were higher in migrated F4/80(+) /Ly6c(+) monocyte-derived macrophages as compared with F4/80(+) /Ly6c(-) resident retinal microglial cells. These results suggest a novel therapeutic approach using EPC to recruit neuroprotective macrophages that delay the progression of neural degenerative disease.

Umbilical cord blood-derived mesenchymal stem cells inhibit, but adipose tissue-derived mesenchymal stem cells promote, glioblastoma multiforme proliferation.

Mesenchymal stem cells (MSCs) possess self-renewal and multipotential differentiation abilities, and they are thought to be one of the most reliable stem cell sources for a variety of cell therapies. Recently, cell therapy using MSCs has been studied as a novel therapeutic approach for cancers that show refractory progress and poor prognosis. MSCs from different tissues have different properties. However, the effect of different MSC properties on their application in anticancer therapies has not been thoroughly investigated. In this study, to characterize the anticancer therapeutic application of MSCs from different sources, we established two different kinds of human MSCs: umbilical cord blood-derived MSCs (UCB-MSCs) and adipose-tissue-derived MSCs (AT-MSCs). We used these MSCs in a coculture assay with primary glioblastoma multiforme (GBM) cells to analyze how MSCs from different sources can inhibit GBM growth. We found that UCB-MSCs inhibited GBM growth and caused apoptosis, but AT-MSCs promoted GBM growth. Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick-end labeling assay clearly demonstrated that UCB-MSCs promoted apoptosis of GBM via tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). TRAIL was expressed more highly by UCB-MSCs than by AT-MSCs. Higher mRNA expression levels of angiogenic factors (vascular endothelial growth factor, angiopoietin 1, platelet-derived growth factor, and insulin-like growth factor) and stromal-derived factor-1 (SDF-1/CXCL12) were observed in AT-MSCs, and highly vascularized tumors were developed when AT-MSCs and GBM were cotransplanted. Importantly, CXCL12 inhibited TRAIL activation of the apoptotic pathway in GBM, suggesting that AT-MSCs may support GBM development in vivo by at least two distinct mechanisms-promoting angiogenesis and inhibiting apoptosis. The opposite effects of AT-MSCs and UCB-MSCs on GBM clearly demonstrate that differences must be considered when choosing a stem cell source for safety in clinical application.

A subpopulation of endothelial progenitor cells with low aldehyde dehydrogenase activity attenuates acute ischemic brain injury in rats.

Previous studies have examined the therapeutic effect of endothelial progenitor cells (EPCs) during the chronic phase of cerebral infarction in rats; however, few studies have investigated the effects of EPCs during the acute phase of infarction. In this study, we evaluated the therapeutic effect of EPCs with low aldehyde dehydrogenase activity (Alde-Low EPCs) in rats with acute cerebral infarction, and our results provide insight that may help to identify a therapeutic mechanism of EPCs for acute cerebral infarction. The administration of Alde-Low EPCs into rats with acute cerebral infarction results in the accumulation and migration of the Alde-Low EPCs into the infarct area and the subsequent decrease of infarct volume. Moreover, we found that the stromal cell-derived factor-1 (SDF-1) and CXC chemokine receptor 4 (CXCR4) signaling pathway may regulate the accumulation of Alde-Low EPCs. The transplantation of Alde-Low EPCs may represent a potential treatment strategy for acute cerebral infarction.

Parkinson's disease induced pluripotent stem cells with triplication of the α-synuclein locus.

A major barrier to research on Parkinson's disease is inaccessibility of diseased tissue for study. One solution is to derive induced pluripotent stem cells from patients and differentiate them into neurons affected by disease. Triplication of SNCA, encoding α-synuclein, causes a fully penetrant, aggressive form of Parkinson's disease with dementia. α-Synuclein dysfunction is the critical pathogenic event in Parkinson's disease, multiple system atrophy and dementia with Lewy bodies. Here we produce multiple induced pluripotent stem cell lines from an SNCA triplication patient and an unaffected first-degree relative. When these cells are differentiated into midbrain dopaminergic neurons, those from the patient produce double the amount of α-synuclein protein as neurons from the unaffected relative, precisely recapitulating the cause of Parkinson's disease in these individuals. This model represents a new experimental system to identify compounds that reduce levels of α-synuclein, and to investigate the mechanistic basis of neurodegeneration caused by α-synuclein dysfunction.

Identification of human placenta-derived mesenchymal stem cells involved in re-endothelialization.

Human placenta is an attractive source of mesenchymal stem cells (MSC) for regenerative medicine. The cell surface markers expressed on MSC have been proposed as useful tools for the isolation of MSC from other cell populations. However, the correlation between the expression of MSC markers and the ability to support tissue regeneration in vivo has not been well examined. Here, we established several MSC lines from human placenta and examined the expression of their cell surface markers and their ability to differentiate toward mesenchymal cell lineages. We found that the expression of CD349/frizzled-9, a receptor for Wnt ligands, was positive in placenta-derived MSC. So, we isolated CD349-negative and -positive fractions from an MSC line and examined how successfully cell engraftment repaired fractured bone and recovered blood flow in ischemic regions using mouse models. CD349-negative and -positive cells displayed a similar expression pattern of cell surface markers and facilitated the repair of fractured bone in transplantation experiments in mice. Interestingly, CD349-negative, but not CD349-positive cells, showed significant effects on recovering blood flow following vascular occlusion. We found that induction of PDGFß and bFGF mRNAs by hypoxia was greater in CD349-negative cells than in CD349-positive cells while the expression of VEGF was not significantly different in CD349-negative and CD349-positive cells. These findings suggest the possibility that CD349 could be utilized as a specialized marker for MSC isolation for re-endothelialization.

Hypoxia responsive mesenchymal stem cells derived from human umbilical cord blood are effective for bone repair.

Mesenchymal stem cells (MSCs) are highly useful in a variety of cell therapies owing to their multipotential differentiation capability. MSCs derived from umbilical cord blood are generally isolated by their plastic adherence without using specific cell surface markers and examined for their osteogenic, adipogenic, and chondrogenic differentiation properties retrospectively. Here, we report 2 subpopulations of MSCs, separated based on aldehyde dehydrogenase (ALDH) activity. MSCs with a high ALDH activity (Alde-High) proliferated more than those with a low ALDH activity (Alde-Low). Alde-High MSCs had a greater ability to differentiate than Alde-Low MSCs in in vitro culture. Transplantation of Alde-High MSCs into fractured mouse femurs enabled early repair of tissues and rapid bone substitution. Alde-High MSCs were also more responsive to hypoxia than Alde-Low MSCs, with the upregulation of Flt-1, CXCR4, and Angiopoietin-2. Thus, MSCs with a high ALDH activity might serve as an effective therapeutic tool for healing fractures within a short period of time.

Hypoxia-inducible transcription factor-2alpha in endothelial cells regulates tumor neovascularization through activation of ephrin A1.

The hypoxia-inducible transcription factors (HIF)-1alpha and -2alpha mediate responses to hypoxia, such as tumor neovascularization. To determine the function of HIF-2alpha in vascular endothelial cells (ECs), we examined vascular formation in HIF-2alpha knockdown (kd/kd) mice transplanted with tumors. We observed that both the tumor size and the number of large vessels growing within transplanted melanomas were significantly reduced in kd/kd recipients compared with wild-type (WT) mice. In contrast, we observed a similar extent of vascular formation within fibrosarcomas transplanted from either kd/kd or WT mice into WT recipients. Thus, HIF-2alpha expression in host animal ECs, but not in the tumor cells, is crucial for tumor neovascularization. HIF-2alpha may function through ephrin A1 as the expression of ephrin A1 and related genes was markedly reduced in kd/kd ECs, and HIF-2alpha specifically bound a hypoxia-response element sequence in the ephrin A1 promoter. Treatment of WT ECs with an ephrin A1 inhibitor (ephrin A1-Fc) also impaired neovascularization. We conclude that in ECs, HIF-2alpha plays an essential role in vascular remodeling during tumor vascularization through activation of at least ephrin A1.

Reduced BMP4 abundance in Gata2 hypomorphic mutant mice result in uropathies resembling human CAKUT.

Constitutive loss of transcription factor GATA-2 leads to embryonic lethality from primitive erythropoietic failure. We serendipitously discovered an essential contribution of GATA-2 to urogenital development when the hematopoietic deficiency of Gata2 null mutant animals was complemented by a Gata2 yeast artificial chromosome (YAC) transgene; these mice died from a perinatal lethal urogenital abnormality. Here, we report the generation and analysis of Gata2 hypomorphic mutant (Gata2(fGN)/(/fGN)) mice, which suffered from hydronephrosis and megaureter, as do the YAC-rescued Gata2 null mutants. Gata2(fGN)/(/fGN) mutants exhibit anteriorly displaced ureteric budding from the Wolffian duct as well as reduced BMP4 expression in the intermediate mesoderm derivatives in a manner that is temporally coincident with ureteric bud emergence. In Bmp4 mutant heterozygotes, rostral displacement of the initial bud site on the Wolffian duct results in abnormal urogenital development. We show here that Bmp4 mRNA is reduced approximately twofold in Gata2(fGN)/(/fGN) mice (as in Bmp4 null heterozygotes), and that GATA-2 trans-activates a Bmp4 first intron element-directed reporter plasmid in co-transfection assays. These experiments taken together implicate GATA-2 as a direct regulator of Bmp4 transcription. The pathophysiology described in Gata2 hypomorphic mutant animals resembles human congenital anomalies of the kidney and urinary tract.

Abnormal heart development and lung remodeling in mice lacking the hypoxia-inducible factor-related basic helix-loop-helix PAS protein NEPAS.

Hypoxia-inducible factors (HIFs) are crucial for oxygen homeostasis during both embryonic development and postnatal life. Here we show that a novel HIF family basic helix-loop-helix (bHLH) PAS (Per-Arnt-Sim) protein, which is expressed predominantly during embryonic and neonatal stages and thereby designated NEPAS (neonatal and embryonic PAS), acts as a negative regulator of HIF-mediated gene expression. NEPAS mRNA is derived from the HIF-3alpha gene by alternative splicing, replacing the first exon of HIF-3alpha with that of inhibitory PAS. NEPAS can dimerize with Arnt and exhibits only low levels of transcriptional activity, similar to that of HIF-3alpha. NEPAS suppressed reporter gene expression driven by HIF-1alpha and HIF-2alpha. By generating mice with a targeted disruption of the NEPAS/HIF-3alpha locus, we found that homozygous mutant mice (NEPAS/HIF-3alpha(-)(/)(-)) were viable but displayed enlargement of the right ventricle and impaired lung remodeling. The expression of endothelin 1 and platelet-derived growth factor beta was increased in the lung endothelial cells of NEPAS/HIF-3alpha-null mice. These results demonstrate a novel regulatory mechanism in which the activities of HIF-1alpha and HIF-2alpha are negatively regulated by NEPAS in endothelial cells, which is pertinent to lung and heart development during the embryonic and neonatal stages.

Role of hypoxia-inducible factor-2alpha in endothelial development and hematopoiesis.

Endothelial cells (EC) are important components for vessel formation and hematopoiesis. The proliferation and differentiation of EC are performed under the close influence of hypoxia-inducible factors (HIFs), which are master transcription factors that regulate vasculogenesis and angiogenesis in response to hypoxic stimuli. During early development of embryos, EC are directly involved in hematopoiesis and are known to act as stromal cells, which generate a variety of regulatory factors, including cytokines and growth factors, and maintain adhesive interactions with the hematopoietic cells essential for their survival and function in the microenvironment. Mouse gene-targeting technology provides us with the information that HIFs are crucial for the development of not only EC but also hematopoietic cells. Although we have determined some particular roles of HIF in association with neovascularization and hematopoiesis in the experiments using gene knockout mice, many crucial roles of HIFs in these processes still remain to be elucidated. Because of the complexity of vasculo/angiogenesis and hematopoiesis in vivo, it is very difficult to analyze distinct involvement of each of the HIFs in the regulation of vessel formation and development of hematopoietic cells. In this chapter, we review the role of HIFs in neovascularization and hematopoiesis with special attention to the usefulness of gene knockdown mice of HIF-2alpha (alpha) to analyze the respective roles of HIFs in these complex processes.

Identification of functional endothelial progenitor cells suitable for the treatment of ischemic tissue using human umbilical cord blood.

Umbilical cord blood (UCB) has been used as a potential source of various kinds of stem cells, including hematopoietic stem cells, mesenchymal stem cells, and endothelial progenitor cells (EPCs), for a variety of cell therapies. Recently, EPCs were introduced for restoring vascularization in ischemic tissues. An appropriate procedure for isolating EPCs from UCB is a key issue for improving therapeutic efficacy and eliminating the unexpected expansion of nonessential cells. Here we report a novel method for isolating EPCs from UCB by a combination of negative immunoselection and cell culture techniques. In addition, we divided EPCs into 2 subpopulations according to the aldehyde dehydrogenase (ALDH) activity. We found that EPCs with low ALDH activity (Alde-Low) possess a greater ability to proliferate and migrate compared to those with high ALDH activity (Alde-High). Moreover, hypoxia-inducible factor proteins are up-regulated and VEGF, CXCR4, and GLUT-1 mRNAs are increased in Alde-Low EPCs under hypoxic conditions, while the response was not significant in Alde-High EPCs. In fact, the introduction of Alde-Low EPCs significantly reduced tissue damage in ischemia in a mouse flap model. Thus, the introduction of Alde-Low EPCs may be a potential strategy for inducing rapid neovascularization and subsequent regeneration of ischemic tissues.

Transgene insertion in proximity to the c-myb gene disrupts erythroid-megakaryocytic lineage bifurcation.

The nuclear proto-oncogene c-myb plays crucial roles in the growth, survival, and differentiation of hematopoietic cells. We established three lines of erythropoietin receptor-transgenic mice and found that one of them exhibited anemia, thrombocythemia, and splenomegaly. These abnormalities were independent of the function of the transgenic erythropoietin receptor and were observed exclusively in mice harboring the transgene homozygously, suggesting transgenic disruption of a certain gene. The transgene was inserted 77 kb upstream of the c-myb gene, and c-Myb expression was markedly decreased in megakaryocyte/erythrocyte lineage-restricted progenitors (MEPs) of the homozygous mutant mice. In the bone marrows and spleens of the mutant mice, numbers of megakaryocytes were increased and numbers of erythroid progenitors were decreased. These abnormalities were reproducible in vitro in a coculture assay of MEPs with OP9 cells but eliminated by the retroviral expression of c-Myb in MEPs. The erythroid/megakaryocytic abnormalities were reconstituted in mice in vivo by transplantation of mutant mouse bone marrow cells. These results demonstrate that the transgene insertion into the c-myb gene far upstream regulatory region affects the gene expression at the stage of MEPs, leading to an imbalance between erythroid and megakaryocytic cells, and suggest that c-Myb is an essential regulator of the erythroid-megakaryocytic lineage bifurcation.