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Multilineage embryonic hematopoiesis requires hypoxic ARNT activity
Abstract
Although most cells undergo growth arrest during hypoxia, endothelial cells and placental cytotrophoblasts proliferate in response to low O(2). We demonstrate that proliferation of embryonic multilineage hematopoietic progenitors is also regulated by a hypoxia-mediated signaling pathway. This pathway requires HIF-1 (HIF-1alpha/ARNT heterodimers) because Arnt(-/-) embryoid bodies fail to exhibit hypoxia-mediated progenitor proliferation. Furthermore, Arnt(-/-) embryos exhibit decreased numbers of yolk sac hematopoietic progenitors. This defect is cell extrinsic, is accompanied by a decrease in ARNT-dependent VEGF expression, and is rescued by exogenous VEGF. Therefore, "physiologic hypoxia" encountered by embryos is essential for the proliferation or survival of hematopoietic precursors during development.
... Oxygen acts as a potent regulator of vascular development. Endothelial cells (Phillips et al. 1995) and hematopoietic progenitors (Adelman et al. 1999) proliferate in response to low O 2 . Human placental cells also proliferate during hypoxic culture conditions, but high O 2 levels inhibit proliferation and promote differentiation (Genbacev et al. 1997;Caniggia et al. 2000). ...
... Arnt −/− animals die by E10.5, displaying deficiencies in yolk sac and/or placental vascularization (Kozak et al. 1997;Maltepe et al. 1997). Furthermore, Arnt −/− yolk sacs show decreased numbers of multilineage hematopoietic progenitors (Adelman et al. 1999). HIF-1 activity is therefore essential for the proliferation, survival, and/or differentiation of multiple embryonic tissues. ...
... We have shown previously that Arnt −/− embryos show defects in angiogenesis and hematopoiesis (Maltepe et al. 1997;Adelman et al. 1999). Spurred by recent studies describing the role of O 2 tension in regulating human placental cell differentiation in vitro (Genbacev et al. 1996(Genbacev et al. , 1997Caniggia et al. 2000), we characterized placental development in Arnt −/− embryos. ...
Placental development is profoundly influenced by oxygen (O 2 ) tension. Human cytotrophoblasts proliferate in vitro under low O 2 conditions but differentiate at higher O 2 levels, mimicking the developmental transition they undergo as they invade the placental bed to establish the maternal–fetal circulation in vivo. Hypoxia-inducible factor-1 (HIF-1), consisting of HIF-1α and ARNT subunits, activates many genes involved in the cellular and organismal response to O 2 deprivation. Analysis of Arnt −/− placentas reveals an aberrant cellular architecture due to altered cell fate determination of Arnt −/− trophoblasts. Specifically, Arnt −/− placentas show greatly reduced labyrinthine and spongiotrophoblast layers, and increased numbers of giant cells. We further show that hypoxia promotes the in vitro differentiation of trophoblast stem cells into spongiotrophoblasts as opposed to giant cells. Our results clearly establish that O 2 levels regulate cell fate determination in vivo and that HIF is essential for mammalian placentation. The unique placental phenotype of Arnt −/− animals also provides an important tool for studying the disease of preeclampsia. Interestingly, aggregation of Arnt −/− embryonic stem (ES) cells with tetraploid wild-type embryos rescues their placental defects; however, these embryos still die from yolk sac vascular and cardiac defects.
... erythropoiesis; renal ischemia-reperfusion injury; renal fibrosis; renal cell cancer OVERVIEW OF HIF SIGNALING TISSUE HYPOXIA, ASIDE FROM being a frequently encountered clinical problem as a result of pulmonary or cardiovascular conditions, is also an important microenvironmental factor that is critical for the regulation of normal embryonic development and stem cell maintenance (1,2,21,22,86,123,140). Over the last decade, major advances have been made in deciphering the molecular mechanisms that allow cells to respond and to adapt to low PO 2 . ...
... We and others shown that hypoxia increased the percentage of transitioned renal epithelial cells in a HIFdependent fashion in vitro and in vivo (48,62,78), supporting a role for HIF-1 in the dedifferentiation and transition of renal epithelial to mesenchymal fibroblast-like cells. The observa-tion that hypoxia, through HIF, influences the differentiation state of cells has also been made in other biological systems (1,2,21,22,86,123,140). Although the underlying molecular mechanisms may differ between cell types, an increase in Notch signaling as a result of direct biochemical interaction between HIF-1? and the Notch ICD may be one of the mechanisms by which cells are maintained in an undifferentiated state, as has been suggested by Gustaffsson et al. (39). ...
... 56,57 Deletion of ARNT, which prevents HIF-1a and HIF-2a function, reduces proliferation in hematopoietic progenitors. 58 Exogenous VEGF rescues the hematopoietic proliferative defects in Arnt À/À mice. 58 VEGF expression is also activated under conditions of hypoxia. ...
... 58 Exogenous VEGF rescues the hematopoietic proliferative defects in Arnt À/À mice. 58 VEGF expression is also activated under conditions of hypoxia. 59 The VEGF promoter contains canonical HIF response elements. ...
... Our previous study has demonstrated that DMAG can be separated from a traditional Chinese medicine Sanguisorba officinalis L.. 27 Sanguisorba officinalis L. is reported to possess hemostatic and anti-leukopenia activities, 28 21 Previous studies have demonstrated that HIF-1β is crucial for hematopoiesis. [33][34][35] However, its role in megakaryocyte differentiation and platelet formation is largely unknown. Here, we did not find any change in HIF-1a expression between the control and DMAG-treated groups. ...
Thrombocytopenia is a TPO-related life-threatening disorder with very limited treatment options. Typical thrombopoietic agents targeting TPO signaling encounter a huge challenge. Thus, it is urgent to discover a novel TPO-independent mechanism involving thrombopoiesis and its potential targeted medications. Here, we developed a drug screening model by the Multi-Grained Cascade Forest (gcForest) algorithm and identified that 3,8-Di-O-methylellagic acid 2-O-glucoside (DMAG) (10, 20 and 40 μM) promoted megakaryocyte differentiation in vitro. Subsequent investigations revealed that DMAG (40 μM) activated ERK1/2, HIF-1β and NF-E2. Inhibition of ERK1/2 blocked megakaryocyte differentiation and attenuated the upregulation of HIF-1β and NF-E2 induced by DMAG. Megakaryocyte differentiation induced by DMAG was inhibited via knockdown of NF-E2. In vivo studies showed that DMAG (5 mg/kg) accelerated platelet recovery and megakaryocyte differentiation in mice with thrombocytopenia. The platelet level of DMAG-treated group recovered to almost 72% and 96% of control group at day 10 and 14. The platelet counts in the DMAG-treated group exhibited almost 1.5 and 1.3 fold higher compared with the irradiation (IR) group at day 10 and 14. Moreover, DMAG (10, 25 and 50 μM) stimulated thrombopoiesis in zebrafish. DMAG (5 mg/kg) could also increase platelet levels in c-MPL knockout (c-MPL-/-) mice. In summary, we establish a drug screening model through gcForest and demonstrate DMAG promotes megakaryocyte differentiation via the ERK/HIF1/NF-E2 pathway, which is more importantly independent of the TPO/c-MPL classic pathway. The present study may provide new insights into drug discovery for thrombopoiesis, TPO-independent regulation of thrombopoiesis and a promising avenue for thrombocytopenia treatment.
... Erythropoietin is up-regulated by HIF (Semenza et al., 1991) increasing erythrocyte production. HIF has also been shown to be involved in multilineage haematopoiesis during embryonic development (Adelman et al., 1999). Results suggest possible SDF-1 involvement in the effects of HIF on haematopoiesis. ...
Inflammation is a key defence response to infection or injury. However, dysregulation of inflammation can lead to chronic inflammation and tissue damage, making resolution of critical importance. Inflammatory diseases are responsible for considerable morbidity and mortality in the developed world and few effective treatments are currently available. The neutrophil is a key leukocyte in the acute inflammatory response. However, failure of clearance of neutrophils can cause tissue damage owing to their highly destructive nature and may be implicated in the pathogenesis of inflammatory diseases. This dissertation focuses on the neutrophilic component of acute inflammation. In this project, I investigated whether stress signals delayed the clearance of neutrophils and the mechanisms underlying these effects. Using a zebrafish model of tailfin transection, I investigated the effects of pathogens, pathogen-associated molecular patterns (PAMPs) and inhibition of caspase enzymes and the inflammasome on resolution. Stabilisation of hypoxia inducible factor (HIF), the major hypoxic transcription factor, has previously been shown to delay resolution of neutrophilic inflammation. I examined the effects of HIF on resolution in this model and investigated the signalling pathways involved. The interaction between danger signals and HIF were explored to determine if danger signals act via HIF. A greater understanding of the regulation of inflammation resolution may uncover novel therapeutic targets for treating inflammatory diseases. 3
... HIF-1a is an important regulator of VEGFA expression in local hypoxia, such as tumor microenvironment (83) and on the development and survival of the hematopoietic system (84,85). HIF-1a also regulates VEGFA transcription, and mobilization of HSPC increases VEGF-A expression (86). ...
Although it is well known that hypoxia incites unleashed cellular inflammation, the mechanisms of exaggerated cellular inflammation in hypoxic conditions are not known. We observed augmented proliferation of hematopoietic stem and progenitor cells (HSPC), precursors of inflammatory leukocytes, in mice under hypoxia. Consistently, a transcriptomic analysis of human HSPC exposed to hypoxic conditions revealed elevated expression of genes involved in progenitor proliferation and differentiation. Additionally, bone marrow cells in mice expressed high amount of vascular endothelial growth factor (VEGF), and HSPC elevated VEGF receptor 1 (VEGFr1) and its target genes in hypoxic conditions. In line with this, VEGFr1 blockade in vivo and in vitro decreased HSPC proliferation and attenuated inflammation. In silico and ChIP experiments demonstrated that HIF-1α binds to the promoter region of VEGFR1. Correspondingly, HIF1a silencing decreased VEGFr1 expression in HSPC and diminished their proliferation. These results indicate that VEGF signaling in HSPC is an important mediator of their proliferation and differentiation in hypoxia-induced inflammation and represents a potential therapeutic target to prevent aberrant inflammation in hypoxia-associated diseases.
... For instance, studies examining Arnt conditional deletion within myeloid cells revealed dysregulated immune responses that enhanced or reduced inflammation in a tissue-specific fashion (6)(7)(8). Additionally, ARNT is required for long-term HIF-dependent hematopoietic stem cell homeostasis (9,10), and mice null for Arnt in fetal liver or bone marrow hematopoietic stem cells displayed abnormal numbers of B and T cells in the bone marrow, spleen, and thymus (11). Moreover, the presence of altered T cell subsets in mice with Arnt-deficient CD4 + T cells predicts a role for ARNT in T cell differentiation (12). ...
Significance
Nontoxic agonists and antagonists of the aryl hydrocarbon receptor (AhR) hold high therapeutic potential for treating autoimmune disease and cancer. However, AhR activation by different ligands can lead to opposing phenotypical outcomes in a cell- and tissue-specific manner. In this study, we demonstrate that proportional flux in the levels of aryl hydrocarbon receptor nuclear translocator (ARNT) isoforms 1 and 3 modulates AhR signaling in terms of amplitude and expression of distinct gene programs. These results delineate a molecular mechanism of ARNT isoform–mediated AhR regulation, simplify our understanding of a complex AhR signaling pathway, and provide feasibility for ARNT-targeted therapies that could be used in conjunction with nontoxic AhR ligands for the purpose of immunomodulation.
... HSC heterogeneity is influenced by oxygen gradients within the BM niche including influences on HSC self-renewal, differentiation, metabolism, division, and multi-lineage potential [42,43]. Early studies demonstrated that hypoxic signaling pathways regulate multilineage hematopoietic progenitors during embryogenesis as upon deletion of hypoxia response pathway protein arylhydrocarbon receptor nuclear translocator (ARNT; HIF-1β), hypoxia-mediated HSPC proliferation is ablated [44]. Further, more recent studies have demonstrated heterogeneity not only within HSC/HSPC populations, but also signaling factors (secreted and expressed on the cell surface) within the BM niche themselves [38,45]. ...
Purpose of Review
Hematopoietic stem and progenitor cells (HSC/HSPCs) originate in bone marrow (BM) niches and facilitate immune cell production throughout life. Critical signals from physical and soluble factors in the niche, including the native low oxygen (“hypoxic”) environment, regulate the phenotype, function, and cell fate decisions of HSCs. In this review, we highlight foundational knowledge, recent insights detailing the implications of varied oxygen levels in the BM niche and how manipulation of the oxygen environment in experimental conditions impact the data/interpretation of HSC/HSPC populations.
Recent Findings
Using novel techniques, characterization of oxygen tension in distinct BM niches has shaped foundational studies of HSC/HSPC localization and biology. Additional studies utilizing sorted, live HSCs that remain in their physiologic low oxygen environment, rather than exposing them to air and placing them back into low oxygen conditions, continue to advance our knowledge of endogenous HSC phenotype, heterogeneity, signaling, and function/fate decisions.
Summary
The low oxygen (O2) environment of the BM niche plays a vital role in normal and malignant hematopoiesis including HSC/HSPC metabolism, signaling/regulatory mechanisms, quiescence, stem cell fate/differentiation, engraftment/transplantation ability, and therapeutic response. Technological innovations have advanced our current understanding of HSC localization and regulation within the BM niche with respect to the low O2 environment. Initial studies identified unique oxygen gradients as well as loss of HSC after removal from low O2 (i.e., exposure to room air using whole BM). Recent studies being performed with sorted, live HSC/HSPC populations remaining in their native low O2 environment have provided novel insight, direct comparisons, and identification of unique HSC/HSPC subpopulations demonstrating alterations in signaling, heterogeneity, cell fidelity, and enhancement in the HSC/HSPC phenotype/function not identified in previous studies. Further studies of hematopoietic cell subpopulations in their native low O2 environment will shed additional light on endogenous HSC/HSPC biology leading to translational implications for transplantation and therapeutic intervention.
... Other key transcription regulators included HIF1A (and its cofactor ARNT), whose binding is facilitated by demethylation of the binding motif 123 ; HIF1A/ARNT are critical factors for HSC quiescence, through maintenance of the anaerobic glycolysisdependent metabolic activity in the bone marrow niche [124][125][126][127][128][129][130] . USF1/2 were also among the highlighted TFs, which have been shown to regulate chromatin architecture in erythroid differentiation and the betaglobin locus 131,132 . ...
Somatic mutations in cancer genes have been ubiquitously detected in clonal expansions across healthy human tissue, including in clonal hematopoiesis. However, mutated and wildtype cells are morphologically and phenotypically similar, limiting the ability to link genotypes with cellular phenotypes. To overcome this limitation, we leveraged multi-modality single-cell sequencing, capturing the mutation with transcriptomes and methylomes in stem and progenitors from individuals with DNMT3A R882 mutated clonal hematopoiesis. DNMT3A mutations resulted in myeloid over lymphoid bias, and in expansion of immature myeloid progenitors primed toward megakaryocytic-erythroid fate. We observed dysregulated expression of lineage and leukemia stem cell markers. DNMT3A R882 led to preferential hypomethylation of polycomb repressive complex 2 targets and a specific sequence motif. Notably, the hypomethylation motif is enriched in binding motifs of key hematopoietic transcription factors, serving as a potential mechanistic link between DNMT3A R882 mutations and aberrant transcriptional phenotypes. Thus, single-cell multi-omics pave the road to defining the downstream consequences of mutations that drive human clonal mosaicism.
... Under 'physiologic hypoxia' of the early embryo, the hypoxia response pathway was shown to control HSPC emergence (Adelman et al., 1999;Gerri et al., 2018;Harris et al., 2013;Imanirad et al., 2014;Lim et al., 2017) alongside promoting endothelial development that results in increased oxygen levels in the embryo (Michiels et al., 2000;Wong et al., 2017). In the absence of circulation these processes are perturbed, and oxygen levels cannot rise. ...
Hematopoietic stem cells (HSCs) emerge during development from the vascular wall of the main embryonic arteries. The onset of circulation triggers several processes that provide critical external factors for HSC generation. Nevertheless, it is not fully understood how and when the onset of circulation affects HSC emergence. Here we show that in Ncx1−/− mouse embryos devoid of circulation the HSC lineage develops until the phenotypic pro-HSC stage. However, these cells reside in an abnormal microenvironment, fail to activate the hematopoietic program downstream of Runx1, and are functionally impaired. Single-cell transcriptomics shows that during the endothelial-to-hematopoietic transition, Ncx1−/− cells fail to undergo a glycolysis to oxidative phosphorylation metabolic switch present in wild-type cells. Interestingly, experimental activation of glycolysis results in decreased intraembryonic hematopoiesis. Our results suggest that the onset of circulation triggers metabolic changes that allow HSC generation to proceed.
... They found that under this condition the maintenance and cloning efficiencies are enhanced. Adelman et al. [112] used Arnt −/− (null) mutants to demonstrate that embryonic multilineage hematopoietic progenitors are also regulated under hypoxia via a HIF complex. Embryoid bodies with Arnt KO mutants were unable to proliferate, and the numbers of hematopoietic progenitors in the yolk sac were significantly decreased. ...
Oxygen levels in the placental microenvironment throughout gestation are not constant, with severe hypoxic conditions present during the first trimester. This hypoxic phase overlaps with the most critical stages of placental development, i.e., blastocyst implantation, cytotrophoblast invasion, and spiral artery remodeling initiation. Dysregulation of any of these steps in early gestation can result in pregnancy loss and/or adverse pregnancy outcomes. Hypoxia has been shown to regulate not only the self-renewal, proliferation, and differentiation of trophoblast stem cells and progenitor cells, but also the recruitment, phenotype, and function of maternal immune cells. In this review, we will summarize how oxygen levels in early placental development determine the survival, fate, and function of several important cell types, e.g., trophoblast stem cells, extravillous trophoblasts, syncytiotrophoblasts, uterine natural killer cells, Hofbauer cells, and decidual macrophages. We will also discuss the cellular mechanisms used to cope with low oxygen tensions, such as the induction of hypoxia-inducible factor (HIF) or mammalian target of rapamycin (mTOR) signals, regulation of the metabolic pathway, and adaptation to autophagy. Understanding the beneficial roles of hypoxia in early placental development will provide insights into the root cause(s) of some pregnancy disorders, such as spontaneous abortion, preeclampsia, and intrauterine growth restriction.
... HIF family of proteins are also intricately connected to angiogenesis. On the one hand, they have been shown to be the master regulators of angiogenesis, while on the other hand, defective vasculature leads to hypoxia which in turn stabilizes HIF1a (61,62). Since TGC-specific GATA factor-loss resulted in a vasculature defect, ...
The placenta acts as a major organ for hematopoiesis. It is believed that placental hematopoietic stem and progenitor cells (HSPCs) migrate to the fetal liver to ensure optimum hematopoiesis in the developing embryo. The labyrinth vasculature in a mid-gestation mouse placenta provides a niche for the definitive hematopoietic stem cell (HSC) generation and expansion. It has been proposed that these processes are regulated by a host of paracrine factors secreted by trophoblast giant cells (TGCs) at the maternal-fetal interface. However, the molecular mechanism by which the TGCs regulate the hematoendothelial niche in a developing placenta is yet to be defined. Using a TGC-specific Gata2 and Gata3 double knockout mouse model, we show that the loss of GATA2 and GATA3 at the TGC layer leads to fetal growth retardation and embryonic death due to disruptions in the delicate hematopoietic-angiogenic balance in the developing placenta. Using single-cell RNA-Seq analyses, we also show that the loss of GATA factors in the TGCs results in the loss of HSC population within the placental labyrinth and is associated with defective placental angiogenesis. Interestingly, we also found that this TGC-specific GATA factor-loss leads to impaired differentiation and distribution of trophoblast progenitor cells. Our study helps to define the GATA-dependent non-autonomous signaling mechanisms of the primary parietal trophoblast giant cells by which it regulates the delicate hematopoietic-angiogenic balance in the developing placenta.
... tumeur, il a été montré que toutes les cellules n'ont pas le même apport en O2, en effet, certaines zones tumorales sont largement hypoxiques. Il est établi que l'hypoxie influence le comportement des cellules souche saines et cancéreuses. Par exemple, inhiber HIF-1± ou HIF-2± conduit à un défaut de production des cellules souche hématopoïétiques.(Adelman et al., 1999) De plus, comme évoqué avant, l'activation des facteurs HIF favorise l'expression des gènes impliqués dans les programmes spécifiques des CS tels qu'Oct4, Klf4 et Sox2 dans des cellules de cancer des poumons.(Iida et al., 2012) Il a été également montré que dans les gliomes, l'hypoxie via HIF1± augmente le nombre de cellules CD133+.(Soed ...
Les cellules souche cancéreuses contribuent au développement des sarcomes, mais le manque de marqueurs spécifiques empêche leur caractérisation et la possibilité de cibler ce type de cellules. Nous avons utilisé la séquence régulatrice de la calpaïne-6 dans des systèmes rapporteurs pour identifier les cellules exprimant la calpaïne-6. Ces cellules étaient des cellules initiatrices de tumeurs et se comportaient comme des cellules souche, au sommet de la hiérarchie cellulaire. L'expression de la calpaïne-6 dépend d’un programme génique de cellules souche qui implique Oct4, Nanog et Sox2 et est activée par l'hypoxie. L’inhibition de la calpaïne-6 a bloqué le développement tumoral et a induit la diminution du nombre de cellules souche cancéreuses dans les sarcomes osseux. L’expression de la calpaïne-6 était inversement corrélée à l'expression de marqueurs de sénescence mais était associé à un flux autophagique dynamique. L’inhibition de la calpaïne-6 a induit l'entrée des cellules en sénescence et a supprimé le flux autophagique. Nos résultats révèlent que le calpaïne-6 identifie les cellules souche des sarcomes et joue un rôle important dans le maintien des cellules souche cancéreuses en contrôlant les processus d’autophagie et de sénescence. La calpaïne-6 semble être une cible thérapeutique prometteuse pour éradiquer les cellules souche dans les sarcomes
... An oxygen gradient emerges in early development, which guides cellular differentiation and morphogenesis [141]. While the O 2 uptake of early embryonic cells relies on the simple diffusion of oxygen, hypoxia starts to be observed in different regions as the embryo expands [5,141]. The initial vascularization, vasculogenesis, starts with the differentiation of angioblasts (embryonic progenitors of ECs), which surround hemopoietic cells to form blood islands. ...
The substantial majority of cell types are known to be capable of sensing changes in O2 tension and in the extracellular matrix (ECM), resulting in various responses depending on the cell type and other factors in the microenvironment, such as cell-cell interactions. A growing body of evidence suggests that hypoxia greatly influences angiogenesis and vasculogenesis through the transcription of numerous genes, including vascular endothelial growth factor (VEGF), the major regulatory protein of these processes. At the same time, the spatial and temporal distribution of ECM components affects ECM properties and growth factor (GF) availability, which, in turn, regulates vascular development. This chapter will discuss how hypoxia and the ECM influence vascular morphogenesis. It seeks to provide a better understanding of vascular development by considering recent research and emerging technologies focused on controlling O2 tension and manipulating ECM properties. We will first focus on the influences of O2 tension and ECM composition on neovascularization. Then we present strategies for manipulating the microenvironment using both synthetic and naturally derived biomaterials. Control over O2 in three-dimensional (3D) microenvironments is thoroughly highlighted, along with the currently available O2 measurement techniques and mathematical models that are necessary to monitor O2 gradients in 3D microenvironments. Finally, we discuss the state-of-the-art technology in microfluidics and smart biomaterials to provide insight into future directions of these exciting research areas.
... We found that the enrichment pattern is cell-type-specific ( Supplementary Fig. 6b), suggesting CTCF hubs may be associated with distinct sets of transcription factors in different cell types. Notably, ZFX, ARNT, and MZF1 are only enriched in GM12878 and K562 cells, consistent with their roles in the development of hematopoietic system [50][51][52] . ...
The CCCTC-binding zinc-finger protein (CTCF)-mediated network of long-range chromatin interactions is important for genome organization and function. Although this network has been considered largely invariant, we find that it exhibits extensive cell-type-specific interactions that contribute to cell identity. Here, we present Lollipop, a machine-learning framework, which predicts CTCF-mediated long-range interactions using genomic and epigenomic features. Using ChIA-PET data as benchmark, we demonstrate that Lollipop accurately predicts CTCF-mediated chromatin interactions both within and across cell types, and outperforms other methods based only on CTCF motif orientation. Predictions are confirmed computationally and experimentally by Chromatin Conformation Capture (3C). Moreover, our approach identifies other determinants of CTCF-mediated chromatin wiring, such as gene expression within the loops. Our study contributes to a better understanding about the underlying principles of CTCF-mediated chromatin interactions and their impact on gene expression.
... Additionally, low levels of oxygen and ROS enhance the self-renewal capacity of normal stem cells. Deregulation of pathways that maintain levels of oxygen and ROS can impair stem-cell function [152][153][154][155][156][157]. Inflammatory cytokines produce high levels of ROS, and interestingly, cancer stem cells in acute myeloid leukemia (AML) have higher level of ROS compared to normal stem cells [158]. ...
Introduction The use of mutiparametric MRI (MpMRI) guided fusion biopsy is becoming an increasingly popular investigation in an aid to increase diagnostic yield in those suspected of having prostate cancer (PCa). Before adopting this technology, it is necessary to confirm the accuracy, so that PCa can be reliably diagnosed with characterisation. Materials and Methods This chapter analysed the evidences, which varied from well-designed randomised controlled trials to case series to detect the accuracy of MpMRI compared with biopsy/ histology. Results MpMRI incorporating T2 and diffusion weighted imaging only detects tumours in around 92% cases. When dynamic contrast enhancement is added, cancer diagnosis is significantly improved. Fusion biopsy increases the detection of high-risk PCa by 32% over conventional biopsy alone. Conclusion This review also revealed that fusion biopsy did not increase cancer detection rate but combined biopsy (Systematic and fusion) provide the highest detection rate for the diagnosis of PCa.
... HIF-1α, -1β, and HIF-2α are also involved in hematopoiesis. 20,21) The embryo encounters physiologic hypoxia in the uterine endometrium, and HIF is essential for the proliferation or survival of hematopoietic precursor cells during embryonic development. Furthermore, the levels of HIF-1α could modulate the mobilization of hematopoietic stem cells (HSCs) from or to the bone marrow. ...
Oxidative stress is required for the development of hypoxic injury and has been investigated as a key factor in the pathogenesis of preeclampsia. In preeclampsia, hypoxia at the implantation site can contribute to lesion formation, leading to poor placentation. Hypoxia-inducible factor-1α (HIF-1α) is a known transcription factor involved in placentation. Increased levels of HIF-1α can induce secretion of soluble vascular endothelial growth factor receptor-1 (sFlt-1), which is known to be a major factor influencing the pathogenesis of preeclampsia. Meanwhile, HIF-1α and HIF-1β mediate the maintenance and production of endothelial progenitor cells and bone marrowderived stem cells, which play important roles in placental vascular development. Related dysfunction can result in preeclampsia. It is also known that glycolytic enzyme phosphoglycerate mutase (PGAM) is deacetylated and activated by reactive oxygen species (ROS), the former of which may regulate cell proliferation through the effects of ROS without the participation of HIF-1α. In this review, we characterize the roles of HIF and PGAM in the pathogenesis of preeclampsia.
... Emerging evidence suggests that the metabolic state of HSCs regulates haematopoiesis, although the mechanisms that link metabolism to HSC function remain poorly understood 1,2 . Many of the metabolic studies on haematopoiesis have focused on the role of glycolysis and its major regulator HIF-1 in maintaining functional stem and progenitor cell populations 3,4 ; however, it is not known whether mitochondrial respiration is essential for HSC function. ...
Adult and fetal haematopoietic stem cells (HSCs) display a glycolytic phenotype, which is required for maintenance of stemness; however, whether mitochondrial respiration is required to maintain HSC function is not known. Here we report that loss of the mitochondrial complex III subunit Rieske iron-sulfur protein (RISP) in fetal mouse HSCs allows them to proliferate but impairs their differentiation, resulting in anaemia and prenatal death. RISP-null fetal HSCs displayed impaired respiration resulting in a decreased NAD(+)/NADH ratio. RISP-null fetal HSCs and progenitors exhibited an increase in both DNA and histone methylation associated with increases in 2-hydroxyglutarate (2HG), a metabolite known to inhibit DNA and histone demethylases. RISP inactivation in adult HSCs also impaired respiration resulting in loss of quiescence concomitant with severe pancytopenia and lethality. Thus, respiration is dispensable for adult or fetal HSC proliferation, but essential for fetal HSC differentiation and maintenance of adult HSC quiescence.
... In addition to regulating iron metabolism, hypoxia and the HIF pathway stimulate erythropoiesis through direct effects on the bone marrow via stimulation of EPOR expression, hemoglobin synthesis [42][43][44][45][46] and modulation of stem cell maintenance, lineage differentiation and maturation [47,48]. ...
The oxygen-sensitive hypoxia-inducible factor (HIF) pathway plays a central role in the control of erythropoiesis and iron metabolism. The discovery of prolyl hydroxylase domain (PHD) proteins as key regulators of HIF activity has led to the development of inhibitory compounds that are now in phase 3 clinical development for the treatment of renal anemia, a condition that is commonly found in patients with advanced chronic kidney disease. This review provides a concise overview of clinical effects associated with pharmacologic PHD inhibition and was written in memory of Professor Lorenz Poellinger.
... In addition to regulating iron metabolism, hypoxia and the PHD/HIF pathway stimulate erythropoiesis through direct effects on the bone marrow via stimulation of EPO receptor expression, hemoglobin synthesis, [63][64][65][66][67] and modulation of stem cell maintenance, lineage differentiation, and maturation. 68,69 ...
A classic response to systemic hypoxia is the increase in red blood cell production. This response is controlled by the prolyl hydroxylase domain/hypoxia-inducible factor (HIF) pathway, which regulates a broad spectrum of cellular functions. The discovery of this pathway as a key regulator of erythropoiesis has led to the development of small molecules that stimulate the production of endogenous erythropoietin and enhance iron metabolism. This review provides a concise overview of the cellular and molecular mechanisms that govern HIF-induced erythropoietic responses and provides an update on clinical experience with compounds that target HIF-prolyl hydroxylases for anemia therapy.
... The measurement of low partial pressures of oxygen in various stem cell niches raises question whether HIF-1a and iPSCs pathways were converged. It was described initially in Embryonic stem cells (ESCs) [106], hematopoietic stem cells (HSCs) [107], neural stem cells (NSCs) [108], and cancer stem cells (CSCs) [109], which now further expanded to include iPSCs [110]. Remarkably, Yamanaka first reprogrammed fibroblasts to iPSCs using only four transcription factors (Oct4, Sox2, c-Myc, and Klf4) [105] in the same year that Oct4 was shown to be a specific target gene of HIF-2a [111]. ...
Mesenchymal stem cells (MSCs) are non-hematopoietic multipotent stem cells with selfrenewal
properties and ability to differentiate into a variety of mesenchymal tissues.
This chapter overviews effects of hypoxia on MSCs, makes it promising therapy to
various diseases. Cultivation of MSCs under hypoxic condition results in variety of
outcome that is important to be noted in clinical use. In most studies, hypoxic condition
appears to increase proliferation, differentiation, and immune regulatory performance
of MSCs without affecting its characteristic. Those benefits are therefore utilized in
clinical application. However, there are also studies that report on negative effects of
hypoxia in MSCs such as chromosomal instability. Molecular mechanism of MSCs in
hypoxic condition is provided for better understanding, which is crucial for further
development with better outcome.
... HIF signaling, when stabilized by hypoxic conditions, upregulates several fetal genes to promote survival in low-oxygen conditions. Recently, HIFs have been shown to activate specific signaling pathways, such as Notch, and the expression of transcription factors, such as Oct4, that control stem cell self-renewal and multipotency [29][30][31][32][33]. Embryoid-like metamorphosis represents an adaptive mechanism to promote the survival of clusters of somatic cells under conditions that are not survivable for individual cells. ...
Emergent biological responses develop via unknown processes dependent on physical collision. In hypox-
ia, when the tissue architecture collapses but the geometric core is stable, actin cytoskeleton filament components
emerge, revealing a hidden internal order that identifies how each molecule is reassembled into the original mold,
using one common connection, i.e., a fractal self-similarity that guides the system from the beginning in reverse
metamorphosis, with spontaneous self-assembly of past forms that mimics an embryoid phenotype. We captured
this hidden collective filamentous assemblage in progress: Hypoxic deformed cells enter into intercellular colli-
sions, generate migratory ejected filaments, and produce self-assembly of triangular chiral hexagon complexes; this dynamic geometry guides the microenvironment scaffold in which this biological process is incubated, recapitulat-ing embryonic morphogenesis. In all injured tissues, especially in damaged skeletal (striated) muscle cells, visibly hypertrophic intercalated actin-myosin filaments are organized in zebra stripe pattern along the anterior-posterior axis in the interior of the cell, generating cephalic-caudal polarity segmentation, with a high selective level of immu-nopositivity for Actin, Alpha Skeletal Muscle antibody and for Neuron-Specific Enolase expression of ectodermal dif-ferentiation. The function of actin filaments in emergent responses to tissue injury is to reconstitute, reactivate and orchestrate cellular metamorphosis, involving the re-expression of fetal genes, providing evidence of the reverse flow of genetic information within a biological system. The resultant embryoid phenotype emerges as a microscopic fractal template copy of the organization of the whole body, likely allowing the modification and reprogramming of the phenotype of the tumor in which these structures develop, as well as establishing a reverse primordial micro-scopic mold to collectively re-form cellular building blocks to regenerate injured tissues. Tumorigenesis mimics a self-organizing process of early embryo development. All malignant tumors produce fetal proteins, we now know from which these proteins proceed. Embryoid-like metamorphosis phenomena would represent the anatomical and functional entity of the injury stem cell niche. The sufficiently fast identification, isolation, culture, and expansion of these self-organized structures or genetically derived products could, in our opinion, be used to develop new thera-peutic strategies against cancer and in regenerative medicine
... This term is used for the cell lines obtained after ex vivo propagation of the cell clones isolated from the internal mass of blastocyst (reviewed in [12]). Using both ES culture at 3% O 2 and genetic interventions (targeted disruption of the Arnt locus-coding for HIF1b) it was shown much earlier that the intact "HIF system," the main part of the "hypoxic cell response machinery," is necessary for multilineage embryonic hematopoiesis [13]. Further experiments with murine ES showed a great metabolic flexibility of these cells, but, although providing some advantage in terms of growth, the physiologically relevant O 2 concentration (dissolved O 2 = 36 mmHg; i.e., 4.7%) did not significantly decrease the amount of DNA damage nor enhance the primitiveness of these cells (as judged on the basis of morphology and molecular markers) [14]. ...
After the historic review concerning the ex vivo culture of living cells and their oxygenation, in this chapter the available data on embryonic and several adult stem/progenitor cells systems were analyzed. After taking into consideration (1) the heterogeneity of cell populations studied, (2) type of stimulation of culture, and (3) application of a “correction” of the viewpoint (i.e., considering the atmospheric O2 concentrations as not physiologic but the increased ones), it can be concluded that the O2 concentration to which the culture is exposed has a great impact on the self-renewal, commitment, and differentiation processes. If properly stimulated, the primitive stem cell subsets respond to the physiologically relevant O2 concentrations by maintaining their proliferative capacities, either setting the self-renewal/commitment balance toward self-renewal or by maintaining the quiescent state. Increase in O2 concentration from the lowest values (corresponding to the ones in the in vivo stem cells niche) toward the intermediary physiologically relevant values is premising for the commitment of stem cells. Stem cell maintenance, decline, or total exhaustion is a function of actual oxygenation level. The response of committed progenitors is rather different: the very low O2 concentrations induce the quiescence of these cells, the intermediate O2 concentrations maintain the proliferative progenitors with a decreased differentiation rate, and the higher ones enhance the differentiation process. Some committed progenitors whose differentiation specifically demands reduced oxygenation (i.e. endothelial) are stimulated to differentiate in low O2 concentrations. Altogether the ex vivo data suggest that O2 requirement and sensitivity to low oxygen condition depend on the developmental stage of stem cells and the way and level of commitment of progenitor cells.
... Although we cannot find these genes or gene blocks directly as a result of a lack of expression profile data or quantified research, we can resort to GO annotation and publications to find some cluesfor example, the pleiotropic gene TNFAIP3 that we defined in the third level has the GO terms 'negative regulation of B cell activation' and 'negative regulation of innate immune response,' but it has been proven to be a stimulus of cell growth, especially in cancer cells (Vereecke et al., 2009). In addition, the gene HIF1A (Supplementary Table S1) is annotated as 'negative regulation of growth,' but this gene actually improves the immune capacity by activating embryonic hematopoiesis (Adelman et al., 1999). There are some pleiotropic genes that have the same functional direction as wellfor example, the pleiotropic gene SIX1 (Supplementary Table S1) plays a positive role in many organs' development, including the thymus, which is very important for immune capacity (Klein and Nikolaidis, 2005), and the gene DAB2IP, which we defined in the third level with the GO term 'negative regulation of toll-like receptor 4 signaling pathway,' can also decrease the development speed of hepatocellular carcinoma (Zhang et al., 2012). ...
Both growth and immune capacity are important traits in animal breeding. The animal quantitative trait loci (QTL) database is a valuable resource and can be used for interpreting the genetic mechanisms that underlie growth and immune traits. However, QTL intervals often involve too many candidate genes to find the true causal genes. Therefore, the aim of this study was to provide an effective annotation pipeline that can make full use of the information of Gene Ontology terms annotation, linkage gene blocks and pathways to further identify pleiotropic genes and gene sets in the overlapping intervals of growth-related and immunity-related QTLs. In total, 55 non-redundant QTL overlapping intervals were identified, 1893 growth-related genes and 713 immunity-related genes were further classified into overlapping intervals and 405 pleiotropic genes shared by the two gene sets were determined. In addition, 19 pleiotropic gene linkage blocks and 67 pathways related to immunity and growth traits were discovered. A total of 343 growth-related genes and 144 immunity-related genes involved in pleiotropic pathways were also identified, respectively. We also sequenced and genotyped 284 individuals from Chinese Meishan pigs and European pigs and mapped the single nucleotide polymorphisms (SNPs) to the pleiotropic genes and gene sets that we identified. A total of 971 high-confidence SNPs were mapped to the pleiotropic genes and gene sets that we identified, and among them 743 SNPs were statistically significant in allele frequency between Meishan and European pigs. This study explores the relationship between growth and immunity traits from the view of QTL overlapping intervals and can be generalized to explore the relationships between other traits.
... In mice, the deletion of Hif1a 16 * and Hif1b 17 * results in embryonic lethality at E10.5, whereas the embryonic or perinatal lethality resulting from deletion of Epas1 (name of HIF-2 protein) depends on the murine microenvironment 18 * , 19 . HIF1α is necessary for the generation and expansion of HSPCs at early embryonic stages 20 *. ...
Introduction:
Hypoxia inducible factors (HIF-1α and HIF-2α) are the main mediators of hypoxic responses that operate in both normal and pathological conditions. Recent evidence indicates that HIF-1α and HIF-2α could have overlapping, unique and even sometimes opposing activities in both normal physiology and disease. Despite an increase in our understanding of the different pathways regulated by HIF-1α and HIF-2α, the role played by each factor in HSC maintenance and leukemogenesis is still controversial. Areas covered: This review summarizes our current understanding of HIF-1α and HIF-2α activities and discusses the implications and challenges of using HIF inhibitors therapeutically in blood malignancies. Expert opinion: As HIF inhibitors are currently under clinical evaluation in different cancers, including hematological malignancies, a more thorough understanding of the unique roles performed by HIF-1α and HIF-2α in human neoplasia is warranted.
... Hypoxia regulates also the number of cells in internal blastocyst mass and accelerates the development of hematopoiesis and hemangioblast precursors. 1,25,63 Mammalian adaptation to hypoxia at the cellular and systemic levels is primarily provided by transcriptional regulators, hypoxia-inducible factors (HIFs). HIFs are heterodimers consisting of a regulatory α-subunits (HIF-1α, HIF-2α, HIF-3α) and a constitutive β-subunit (HIFβ), which is also known as ARNT (aryl hydrocarbon nuclear translocator). ...
The purpose of this chapter is to describe the role of hypoxia in functioning of various stem cell types-embryonic, hematopoietic, mesenchymal and neural, paying special attention to the very limited data concerning intermittent hypoxia (IH) effects. All stem cells and their precursors exist in microenvironment named stem cell niches. The most crucial factor for their normal functioning is hypoxia, which contributes to maintain the stem cells in quiescent state with necessary rate of self-renewal. The level of hypoxia in cells and tissues are generally estimated by following methods: 1) direct oxygen pressure measured in tissues; 2) evaluating the distribution of hematopoietic cell subsets along an in vitro Hoechst 33342 (Ho) dye perfusion gradient; 3) immunofluorescent detection of hypoxic marker pimonidazole penetrated into cells; 4) detection of a lysed cell number under specific hypoxic cytotoxin terapazamine. Using these methods it has been established that hypoxia significantly affects stem cells proliferation and differentiation, either attenuating or enhancing these processes in certain circumstances. The key mechanism of cell adaptation to reduced oxygen pressure is the synthesis of hypoxia-inducible transcription factors. These factors control a great number of genes involved in cell metabolism regulation, hematopoiesis and angiogenesis associated with stem cells self-renewal. They also include the telomerase gene and key cytokine genes that indicate the direction of migration and intensity of repair processes. Few data testify that exogenous hypoxic impact could increase stem cells number and govern their differentiation. Very scant information about IH effects on stem cells reveals that IH (at certain duration and intensity) is a much more potent trigger of transcription activation than continuous hypoxia. Therefore, via mobilizing stem cells, IH could become a potential therapeutic approach for treatment of many pathological disorders.
... In this review, we have summarized recent advances in our understanding of the nature of the hypoxic BM and the effects of hypoxia signaling on HSCs, niches, and leukemia. During development, hypoxia signaling contributes to the primitive and definitive hematopoiesis [3,14,27]. The dependence of adult HSCs on hypoxia signaling might be a remnant of their embryonic origin. ...
Bone marrow, the site of hematopoiesis throughout adulthood, is a physiologically hypoxic organ. Thus, various biological oxygen sensors and their signaling cascades play a pivotal role in hematopoietic systems in the bone marrow under both physiologic and pathologic conditions. Hypoxia-inducible factors (HIFs) are hypoxic stress sensor proteins that are stabilized under homeostatic or stress-induced hypoxia. In the hypoxic bone marrow, HIFs play crucial roles in hematopoietic stem cells (HSCs) and in the cells of the HSC niche. The signals downstream of the HIFs maintain HSC quiescence, survival, and metabolic homeostasis through both cell-autonomous and non-cell-autonomous mechanisms. Leukemic stem cells (LSCs) hijack these delicate hypoxia-sensing mechanisms to sustain their self-renewal potential, promoting disease progression and drug resistance even under normoxic conditions. This review focuses on HIF-mediated oxygen-sensing mechanisms of adult HSCs and LSCs and their niche cells in the hypoxic bone marrow.
... AdCA5, an adenovirus encoding a constitutively active form of HIF-1α, blocks diabetes-induced vasculopathy, demonstrating that HIF-1α reduction contributes to diabetes-induced vasculopathy 39 . Mice that lack HIF-1 activity due to HIF-1α− or HIF-1β-null mutations develop extensive vascular defects, similar to those observed in diabetic yolk sac vasculopathy, including inadequate vessel formation and aberrant vascular remodeling 54,55 . HIF-1 deficiency also decreases cell survival, leading to abnormal vasculogenesis 56 . ...
Maternal diabetes is a significant risk factor for structural birth defects, including congenital heart defects and neural tube defects (NTDs). With the rising prevalence of type 2 diabetes and obesity in women of childbearing age, diabetes-induced birth defects have become an increasingly significant public health problem. Maternal diabetes in vivo and high glucose in vitro induce yolk sac injuries by damaging the morphology of cells and altering the dynamics of organelles. The yolk sac vascular system is the first system to develop during embryogenesis, therefore, it is the most sensitive to hyperglycemia. The consequences of yolk sac injuries include impairment of nutrient transportation due to vasculopathy. Although the functional relationship between yolk sac vasculopathy and structural birth defects has not yet been established, a recent study reveals that the quality of yolk sac vasculature is inversely related to embryonic malformation rates. Studies in animal models have uncovered key molecular intermediates of diabetic yolk sac vasculopathy, including hypoxia-inducible factor-1α (HIF-1α), apoptosis signal-regulating kinase 1 (ASK1) and its inhibitor thioredoxin-1 (Trx), c-Jun-N-terminal kinases (JNK), nitric oxide (NO) and nitric oxide synthase (NOS). Yolk sac vasculopathy is also associated with abnormalities in arachidonic acid and myo-inositol. Dietary supplementation with fatty acids that restore lipid levels in the yolk sac lead to reduction in diabetes-induced malformations. Although the role of the human yolk in embryogenesis is less extensive than in rodents, nevertheless, human embryonic vasculogenesis is negatively affected by maternal diabetes. Mechanistic studies have identified potential therapeutic targets for future intervention against yolk sac vasculopathy, birth defects, and other complications associated with diabetic pregnancies.
... Similarly, lack of HIF-1 leads to a defective vascularization of the yolk sack and/or the embryonic part of the placenta, and death of the embryo by the E10.5 day (99,100). In addition HIF-1 deficiency results in a decreased number of hematopoietic progenitors of the yolk sac (101). On the other hand, the results of global HIF-2 deletion remain model-dependent. ...
... Under in vitro conditions, HIF-1 promotes the arterial differentiation of endothelial progenitor cells (EPCs) over venous differentiation by regulating the expression of genes that inhibit the venous specification factor Coup-TFII (Hey2 and delta-like ligand 4 (DII4)) [67]. In contrast, HIF-1 enhances the differentiation of hemangioblasts from mesodermal progenitors [68]. Mouse model studies have demonstrated the participation of both HIF-1 dimer subunits in vasculogenesis [69,70]. ...
The cardiovascular system ensures the delivery of oxygen and nutrients to all cells, tissues, and organs. Under extended exposure to reduced oxygen levels, cells are able to survive through the transcriptional activation of a series of genes that participate in angiogenesis, glucose metabolism, and cell proliferation. The oxygen-sensitive transcriptional activator HIF-1 (hypoxia-inducible factor-1) is a key transcriptional mediator of the response to hypoxic conditions. The HIF-1 pathway was found to be a master regulator of angiogenesis. Whether the process is physiological or pathological, HIF-1 seems to participate in vasculature formation by synergistic correlations with other proangiogenic factors such as VEGF (vascular endothelial growth factor), PlGF (placental growth factor), or angiopoietins. Considering the important contributions of HIF-1 in angiogenesis and vasculogenesis, it should be considered a promising target for treating ischaemic diseases or cancer. In this review, we discuss the roles of HIF-1 in both physiological/pathophysiological angiogenesis and potential strategies for clinical therapy.
Proteins that contain basic helix-loop-helix (bHLH) and Per-Arnt-Sim motifs (PAS) function as transcription factors. bHLH–PAS proteins exhibit essential and diverse functions throughout the body, from cell specification and differentiation in embryonic development to the proper function of organs like the brain and liver in adulthood. bHLH–PAS proteins are divided into two classes, which form heterodimers to regulate transcription. Class I bHLH–PAS proteins are typically activated in response to specific stimuli, while class II proteins are expressed more ubiquitously. Here, we discuss the general structure and functions of bHLH–PAS proteins throughout the animal kingdom, including family members that do not fit neatly into the class I-class II organization. We review heterodimerization between class I and class II bHLH–PAS proteins, binding partner selectivity and functional redundancy. Finally, we discuss the evolution of bHLH–PAS proteins, and why a class I protein essential for cardiovascular development in vertebrates like chicken and fish is absent from mammals.
Oxygen is vital for the normal function of cells and is transported to all parts of the body through red blood cells in the vasculature. Abnormal oxygen concentrations can lead to many complications, and reestablishing oxygen balance is essential for cell biological functions. Mammalian cells have evolved to adapt to hypoxia and sense oxygen levels during hypoxia to maintain and coordinate different biological responses. The best mechanism for studying the hypoxia response involves hypoxia-inducible factors (HIFs), which control the expression of many hypoxia-inducible genes. Recent studies have found that other epigenetic modifier enzymes and metabolite signaling pathways can also sense dynamic changes in oxygen. The mammalian intrauterine oxygen concentration and the mechanisms controlling oxygen homeostasis affect placental development, structure and function. In this review, we focus on how mammalian cells sense oxygen levels and produce anoxic-dependent adaptive responses and explore the relationship between hypoxia and placental development during early pregnancy.
Under normoxia, hypoxia inducible factor (HIF) α subunits are hydroxylated by PHDs (prolyl hydroxylase domain proteins) and subsequently undergo polyubiquitylation and degradation. Normal embryogenesis occurs under hypoxia, which suppresses PHD activities and allows HIFα to stabilize and regulate development. In this Primer, we explain molecular mechanisms of the oxygen-sensing pathway, summarize HIF-regulated downstream events, discuss loss-of-function phenotypes primarily in mouse development, and highlight clinical relevance to angiogenesis and tissue repair.
The human hematopoietic differentiation in vitro of human pluripotent stem cells (hPSCs) has provided new tools to elucidate the mechanisms of related genetic abnormalities, such as congenital diseases and acquired hematopoietic malignancies, and to discover new treatments. The differentiation can also be applied to developing a stable source of blood products for transfusion with minimal risk of several blood-borne infections. We previously proposed a method for hematopoietic progenitor cell (HPC) differentiation, the "hPSC-sac method", in which hPSCs are cocultured with C3H10T1/2 mouse stromal cells and mixed with a single cytokine, VEGF. The hPSC-sac method can differentiate hPSCs to multiple blood lineages. Here we describe improvements in the method by adding bFGF, TGFβ inhibitor and heparin to the culture, which increases the yield of CD34+CD43+ HPCs 50-fold compared with the original protocol. This revised hPSC-sac method is expected to contribute to the development of disease models and regenerative medicine using hematopoietic lineage cells.
The present study in sheep model was to find out the interaction of apoptotic transcripts, that is, Bcl2, Bax, Casp3, PCNA and p53 and pluripotency related transcripts, that is, Sox2, Nanog and Oct4 in ovine embryos produced in vitro at different O2 concentrations (20% and 5% O2) to compare their developmental potential. Oxygen concentrations did not influence the maturation and cleavage rate but the percentage of morula and blastocysts was significantly more at 5% as compared to 20% O2. A significant upregulated expression of Bcl2 and PCNA genes and significantly downregulated expression of Casp3 and p53 were observed in the blastocysts at 5% than those at 20% O2. The expression of Bax was not influenced by the O2 concentration. Among the pluripotency related transcripts, the expression of Oct4 was significantly upregulated and the expression of Sox2 and Nanog was significantly downregulated in embryos at 5% than at 20% O2. The study concluded that the embryos produced in vitro at low O2 (5%) concentration regulate the expression of developmental genes related to apoptosis and pluripotency to improve the developmental potential of embryos as compared to high O2 (20%) concentration.
Chronic inflammation resulting from infections, altered metabolism, inflammatory diseases or other environmental factors can be a major contributor to the development of several types of cancer. In fact around 20% of all cancers are linked to some form of inflammation. Evidence gathered from genetic, epidemiological and molecular pathological studies suggest that inflammation plays a crucial role at various stages of prostatic carcinogenesis and tumor progression. These include initiation, promotion, malignant conversion, invasion, and metastasis. Detailed basic and clinical research in these areas, focused towards understanding the etiology of prostatic inflammation, as well as the exact roles that various signaling pathways play in promoting tumor growth, is critical for understanding this complex process. The information gained would be useful in developing novel therapeutic strategies such as molecular targeting of inflammatory mediators and immunotherapy-based approaches.
Lifelong generation of blood and immune cells depends on hematopoietic stem cells ( HSCs ). Their function is precisely regulated by complex molecular networks that integrate and respond to ever changing physiological demands of the body. Over the past several years, significant advances have been made in understanding the extrinsic regulation of HSCs during development and in homeostasis. Propelled by technical advances in the field, the cellular and molecular components of the microenvironment that support HSCs in vivo are emerging. In addition, the interaction of HSCs with their niches is appreciated as a critical contributor to the pathogenesis of a number of hematologic disorders. Here, we review these advances in detail and highlight the extrinsic regulation of HSCs in the context of development, homeostasis, and diseases. WIREs Dev Biol 2017, 6:e279. doi: 10.1002/wdev.279
This article is categorized under: Adult Stem Cells, Tissue Renewal, and Regeneration > Tissue Stem Cells and Niches
Adult Stem Cells, Tissue Renewal, and Regeneration > Regeneration
Adult Stem Cells, Tissue Renewal, and Regeneration > Stem Cells and Disease
One of the pivotal problems in experimental and clinical medicine is adaptation to hypoxia. Almost any pathologic process is more or less accompanied with the development of a particular type of hypoxia. The hypoxic stimulation exerts a powerful effect on the transport of blood gases resulting in the functional and then the structural rearrangements in the mechanisms supplying oxygen for an organism. On the whole, these changes sustain the energy metabolism [1, 10, 14, 124, 126, 147, 159, 235].
Hematopoietic stem cells (HSCs) reside at the apex of the hematopoietic hierarchy, giving rise to each of the blood lineages found throughout the lifetime of the organism. Since the genetic programs regulating HSC development are highly conserved between vertebrate species, experimental studies in zebrafish have not only complemented observations reported in mammals but have also yielded important discoveries that continue to influence our understanding of HSC biology and homeostasis. Here, we summarize findings that have established zebrafish as an important conserved model for the study of hematopoiesis, and describe methods that can be utilized for future investigations of zebrafish HSC biology.
Above all other elements, molecules, and compounds, O2 is the critical environmental substrate that must be delivered to all cells of metazoan organisms on a virtually continuous basis for survival. The evolution of multicellular animals in which oxygen could no longer be acquired by all cells via simple diffusion necessitated the co-evolution of physiological systems specialized for O2 delivery, which became progressively more complex as body size increased, most notably with the appearance of the vertebrates. The HIF-1 (hypoxia-inducible factor 1) family of bHLH-PAS transcription factors appears to have evolved as a specialized system for regulating oxygen homeostasis at the level of gene expression in metazoans. HIF-1 is present in simple invertebrates such as the roundworm Caenorhabditis elegans, which consists of ~103 cells and relies on simple diffusion for O2 transport, more complex invertebrates such as the fruit fly Drosophila melanogaster, in which O2 is distributed through the body via a set of specialized tracheal tubes, and in complex vertebrates such as Homo sapiens, which consist of > 1013 cells that are supplied with O2 via the combined functioning of highly complex and specialized circulatory and respiratory systems.
Once thought of exclusively as damaging molecules, reactive oxygen species (ROS) are becoming increasingly appreciated for the role they play in cellular signaling through redox biology. Notably, mitochondria are a major source of ROS within a cell (mROS). Mounting evidence now clearly shows that mROS are critical for intracellular redox signaling by which they contribute to a plethora of cellular processes such as proliferation. mROS are essential for physiological cell proliferation, particularly by the regulation of hypoxia inducible factors (HIFs) under hypoxia. mROS are also vital mediators of growth factor signaling cascades such as angiotensin II (Ang II) and T-cell receptor (TCR) signaling. Pathological proliferative diseases such as cancer utilize mROS to their advantage, aberrantly activating growth factor signaling cascades and perpetuating angiogenesis under hypoxia. This review discusses how mROS positively regulate mitogenic cellular signaling through redox biology, which is critical for both physiological and pathological proliferation.
Tight regulation of the stem cell features, quiescence, self-renewal, and multipotency, ensures tissue regeneration and longevity throughout the lifetime of the organism. Numerous microenviromental and intracellular factors are being constantly put in action in order to maintain tissue homeostasis in physiological and stress conditions. Genetic, biochemical, and three-dimensional culture analyses have identified a number of signal transduction pathways (Notch, Wnt, Hedgehog, etc.), which are provided by niche cells and regulate stem cell properties. Also, residing in the low O2 niche, the stem cells exhibit the “hypoxic” profile. Such a profile imposes hypoxia inducible factors (HIFs), transcription factors essential for the setting of cellular metabolism in low O2 availability, as the key players for stem cell maintenance and survival. Recent progress in the genetic approaches and high-resolution isolation of the phenotypically defined cellular subpopulations enabled establishment of molecular signatures associated with stem cell quiescence/self-renewal or differentiation states. Finally, fine-tuned molecular regulation of stem cells is accomplished by the epigenetic modifications that coupled cell nutrient and energetic status with gene expression.
Hypoxia-inducible factor (HIF)-deficient placentas exhibit a number of defects, including changes in cell fate adoption, lack of fetal angiogenesis, hypocellularity, and poor invasion into maternal tissue. HIF is a heterodimeric transcription factor consisting of alpha and beta aryl hydrocarbon receptor nuclear translocator or ARNT) subunits. We used undifferentiated trophoblast stem (TS) cells to characterize HIF-dependent adhesion, migration, and invasion. Arnt(-/-) and Hif alpha(-/-) TS cells exhibit reduced adhesion and migration toward vitronectin compared with wild-type cells. Furthermore, this defect is associated with decreased cell surface expression of integrin alpha v beta 3 and significantly decreased expression of this integrin in focal adhesions. Because of the importance of adhesion and migration in tumor progression (in addition to placental development), we examined the affect of culturing B16F0 melanoma cells in 1.5% oxygen (O-2). Culturing B16F0 melanoma cells at 1.5% O-2 resulted in increased alpha v beta 3 integrin surface expression and increased adhesion to and migration toward vitronectin. Together, these data suggest that HIF and 0, tension influence placental invasion and tumor migration by increasing cell surface expression of alpha v beta 3 integrin.
In vertebrate embryos, hematopoiesis takes place in two successive waves of hematopoietic precursors (HP). While the forerunners of the first wave, generated in the yolk sac (YS), present maintenance capacity and reduced differentiation, those from the latter have all the characteristics of hematopoietic stem cells (HSC). They are able to differentiate into all lineages of blood and help ensure long-term hematopoietic reconstitution of irradiated mice lethal.
The transcription factor GATA-3 is expressed in the embryo during the stages of determination and generation of HSCs. It's never expressed to equivalent levels in the YS. GATA-3 could play an important role in the generation of HSCs and the acquisition of various properties of hematopoietic precursors of the two sites.
We first developed an experimental approach to perform functional analysis of genes with different expression between the two sites generated hematopoietic precursors of the embryo.
The in situ electroporation technique is most effective to disrupt gene expression at the time the choice of precursors and retroviral transduction permit HP target at the time of their generation.
The analysis of ectopic expression of GATA-3 in the HP YS using retroviral infection show that GATA-3 is able to amplify and maintain immatures HP (article in progress). Tests carried out in parallel by performing the forced expression of GATA-5 (naturally absent from SV), or GATA-1 overexpression, we have concluded to the specificity of these effects. Dramatically, GATA-3 is also capable of amplifying a population responding to a phenotype of type mesodermal / pre-hematopoietic. In conclusion, these results show that GATA-3 could be involved in phenomena related to the maintenance of the quota of HSCs.
Placental vasculogenesis is essential for fetal growth and development, and is affected profoundly by oxygen tension (hypoxia). Hypoxia-inducible factor-1α (HIF-1α), which is stabilized at the protein level in response to hypoxia, is essential for vascular morphogenesis in the placenta. Many studies suggested that responses to hypoxia is influenced by O-GlcNAcylation. O-GlcNAcylation is regulated by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) that catalyze the addition and removal of O-GlcNAc respectively.
We generated OGA deficient mice and evaluated OGA(-/-) placentas. The analysis of OGA(-/-) placentas was focused on morphological change and placental vasculogenesis. HIF-1α protein stability or transcriptional activity under dysregulation of O-GlcNAcylation were evaluated by Western blot, RT-qPCR and luciferase reporter gene assays in MEFs or MS1 cell line.
Deletion of OGA results in defective placental vasculogenesis. OGA(-/-) placentas showed an abnormal placental shape and reduced vasculature in the labyrinth, which caused a developmental delay in the embryos. OGA deletion, which elevates O-GlcNAcylation and downregulates O-GlcNAc transferase (OGT), suppressed HIF-1α stabilization and the transcription of its target genes. In contrast, the overexpression of O-GlcNAc cycling enzymes enhanced the expression and transcriptional activity of HIF-1α.
These results suggest that OGA plays a critical role in placental vasculogenesis by modulating HIF-1α stabilization. Control of O-GlcNAcylation is essential for placental development.
Copyright © 2015. Published by Elsevier Ltd.
The origin and potentiality of the hematopoietic stem cell has been debated with two main concepts: the monophyletic hypothesis recognizing a common stem cell for all lympho-myeloid lineages; and the polyphyletic hypothesis recognizing a variety of distinct stem cells. The current consensus recognizes a pluripotential lympho-myeloid stem cell, and a hierarchy of progressively lineage-restricted progenitor cells with a major bifurcation at the level of the common lymphoid and common myeloid progenitor. In mammalian systems, hematopoiesis is clearly initiated in the YS, and subsequently in the aorta–gonad–mesonephros (AGM) region in association with the dorsal aorta and vitelline veins where the first stem cells arise with the capacity to engraft adult recipients. There has been extensive debate as to the role of the yolk sac (YS) in mammalian hematopoietic ontogeny with the site viewed as the initial source of stem cells that then migrated into the embryo, and, the other extreme that views the YS as a transient source of primitive erythroid progenitors, with definitive generation stem cells arising exclusively in the AGM region. There is increasing evidence that stem cells arise independently in both sites, and that both contribute to the subsequent colonization of the fetal liver. A number of studies have revived the concept of a YS origin of intraembryonic hematopoiesis in murine development. In murine ontogeny, primitive macrophage progenitors appear in the proximal region of the egg cylinder associated.
Inefficient vascular supply and the resultant reduction in tissue oxygen tension often lead to neovascularization in order to satisfy the needs of the tissue. Examples include the compensatory development of collateral blood vessels in ischaemic tissues that are otherwise quiescent for angiogenesis and angiogenesis associated with the healing of hypoxic wounds. But the presumptive hypoxia-induced angiogenic factors that mediate this feedback response have not been identified. Here we show that vascular endothelial growth factor (VEGF; also known as vascular permeability factor) probably functions as a hypoxia-inducible angiogenic factor. VEGF messenger RNA levels are dramatically increased within a few hours of exposing different cell cultures to hypoxia and return to background when normal oxygen supply is resumed. In situ analysis of tumour specimens undergoing neovascularization show that the production of VEGF is specifically induced in a subset of glioblastoma cells distinguished by their immediate proximity to necrotic foci (presumably hypoxic regions) and the clustering of capillaries alongside VEGF-producing cells.
We have developed a simple method for producing embryonic stem (ES) cell lines whereby both alleles have been inactivated
by homologous recombination and which requires a single targeting construct. Four different ES cell lines were created that
were heterozygous for genes encoding two guanine nucleotide-binding protein subunits, alpha i2 and alpha i3, T-cell receptor
alpha, and beta-cardiac myosin heavy chain. When these heterozygous cells were grown in high concentrations of G418, many
of the surviving cells were homozygous for the targeted allele and contained two copies of the G418 resistance gene. This
scheme provides an easy method for obtaining homozygous mutationally altered cells, i.e., double knockouts, and should be
generally applicable to other genes and to cell lines other than ES cells. This method should also enable the production of
cell lines in which more than one gene have had both alleles disrupted. These mutant cells should provide useful tools for
defining the role of particular genes in cell culture.
We report that embryonic stem cells efficiently undergo differentiation in vitro to mesoderm and hematopoietic cells and that this in vitro system recapitulates days 6.5 to 7.5 of mouse hematopoietic development. Embryonic stem cells differentiated as embryoid bodies (EBs) develop erythroid precursors by day 4 of differentiation, and by day 6, more than 85% of EBs contain such cells. A comparative reverse transcriptase-mediated polymerase chain reaction profile of marker genes for primitive endoderm (collagen alpha IV) and mesoderm (Brachyury) indicates that both cell types are present in the developing EBs as well in normal embryos prior to the onset of hematopoiesis. GATA-1, GATA-3, and vav are expressed in both the EBs and embryos just prior to and/or during the early onset of hematopoiesis, indicating that they could play a role in the early stages of hematopoietic development both in vivo and in vitro. The initial stages of hematopoietic development within the EBs occur in the absence of added growth factors and are not significantly influenced by the addition of a broad spectrum of factors, including interleukin-3 (IL-3), IL-1, IL-6, IL-11, erythropoietin, and Kit ligand. At days 10 and 14 of differentiation, EB hematopoiesis is significantly enhanced by the addition of both Kit ligand and IL-11 to the cultures. Kinetic analysis indicates that hematopoietic precursors develop within the EBs in an ordered pattern. Precursors of the primitive erythroid lineage appear first, approximately 24 h before precursors of the macrophage and definitive erythroid lineages. Bipotential neutrophil/macrophage and multilineage precursors appear next, and precursors of the mast cell lineage develop last. The kinetics of precursor development, as well as the growth factor responsiveness of these early cells, is similar to that found in the yolk sac and early fetal liver, indicating that the onset of hematopoiesis within the EBs parallels that found in the embryo.
Angiogenesis is required for a wide variety of physiological and pathological processes. The endothelial cell-specific mitogen vascular endothelial growth factor (VEGF) is a major mediator of pathological angiogenesis. Also, the expression of VEGF and its two receptors, Flt-1 and Flk-1/KDR, is related to the formation of blood vessels in mouse and rat embryos. Mice homozygous for mutations that inactivate either receptor die in utero between days 8.5 and 9.5. However, ligand(s) other than VEGF might activate such receptors. To assess the role of VEGF directly, we disrupted the VEGF gene in embryonic stem cells. Here we report the unexpected finding that loss of a single VEGF allele is lethal in the mouse embryo between days 11 and 12. Angiogenesis and blood-island formation were impaired, resulting in several developmental anomalies. Furthermore, VEGF-null embryonic stem cells exhibit a dramatically reduced ability to form tumours in nude mice.
The endothelial cell-specific vascular endothelial growth factor (VEGF) and its cellular receptors Flt-1 and Flk-1 have been implicated in the formation of the embryonic vasculature. This is suggested by their colocalized expression during embryogenesis and the impaired vessel formation in Flk-1 and Flt-1 deficient embryos. However, because Flt-1 also binds placental growth factor, a VEGF homologue, the precise role of VEGF was unknown. Here we report that formation of blood vessels was abnormal, but not abolished, in heterozygous VEGF-deficient (VEGF+/-) embryos, generated by aggregation of embryonic stem (ES) cells with tetraploid embryos (T-ES) and even more impaired in homozygous VEGF-deficient (VEGF-/-) T-ES embryos, resulting in death at mid-gestation. Similar phenotypes were observed in F1-VEGF+/- embryos, generated by germline transmission. We believe that this heterozygous lethal phenotype, which differs from the homozygous lethality in VEGF-receptor-deficient embryos, is unprecedented for a targeted autosomal gene inactivation, and is indicative of a tight dose-dependent regulation of embryonic vessel development by VEGF.
Blastocyst-derived pluripotent mouse embryonic stem cells can differentiate in vitro to form so-called embryoid bodies (EBs), which recapitulate several aspects of murine embryogenesis. We used this in vitro model to study oxygen supply and consumption as well as the response to reduced oxygenation during the earliest stages of development. EBs were found to grow equally well when cultured at 20% (normoxia) or 1% (hypoxia) oxygen during the first 5 days of differentiation. Microelectrode measurements of pericellular oxygen tension within 13- to 14-day-old EBs (diameter 510-890 micron) done at 20% oxygen revealed efficient oxygenation of the EBs' core region. Confocal laser scanning microscopy analysis of EBs incubated with fluorescent dyes that specifically stain living cells confirmed that the cells within an EB were viable. To determine the EBs' capability to sense low oxygen tension and to specifically respond to low ambient oxygen by modulating gene expression we quantified aldolase A and vascular endothelial growth factor (VEGF) mRNAs, since expression of these genes is upregulated by hypoxia in a variety of cells. Compared with the normoxic controls, we found increased aldolase A and VEGF mRNA levels after exposing 8- to 9-day-old EBs to 1% oxygen. We propose that EBs represent a powerful tool to study oxygen-regulated gene expression during the early steps of embryogenesis, where the preimplantation conceptus resides in a fluid environment with low oxygen tension until implantation and vascularization allow efficient oxygenation.
Expression of vascular endothelial growth factor (VEGF) is induced in cells exposed to hypoxia or ischemia. Neovascularization stimulated by VEGF occurs in several important clinical contexts, including myocardial ischemia, retinal disease, and tumor growth. Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric basic helix-loop-helix protein that activates transcription of the human erythropoietin gene in hypoxic cells. Here we demonstrate the involvement of HIF-1 in the activation of VEGF transcription. VEGF 5'-flanking sequences mediated transcriptional activation of reporter gene expression in hypoxic Hep3B cells. A 47-bp sequence located 985 to 939 bp 5' to the VEGF transcription initiation site mediated hypoxia-inducible reporter gene expression directed by a simian virus 40 promoter element that was otherwise minimally responsive to hypoxia. When reporters containing VEGF sequences, in the context of the native VEGF or heterologous simian virus 40 promoter, were cotransfected with expression vectors encoding HIF-1alpha and HIF-1beta (ARNT [aryl hydrocarbon receptor nuclear translocator]), reporter gene transcription was much greater in both hypoxic and nonhypoxic cells than in cells transfected with the reporter alone. A HIF-1 binding site was demonstrated in the 47-bp hypoxia response element, and a 3-bp substitution eliminated the ability of the element to bind HIF-1 and to activate transcription in response to hypoxia and/or recombinant HIF-1. Cotransfection of cells with an expression vector encoding a dominant negative form of HIF-1alpha inhibited the activation of reporter transcription in hypoxic cells in a dose-dependent manner. VEGF mRNA was not induced by hypoxia in mutant cells that do not express the HIF-1beta (ARNT) subunit. These findings implicate HIF-1 in the activation of VEGF transcription in hypoxic cells.
Homozygosity for a null mutation in the scl gene causes mid-gestational embryonic lethality in the mouse due to failure of development of primitive hematopoiesis. Whilst this observation established the role of the scl gene product in primitive hematopoiesis, the death of the scl null embryos precluded analysis of the role of scl in later hematopoietic development. To address this question, we created embryonic stem cell lines with a homozygous null mutation of the scl gene (scl-/-) and used these lines to derive chimeric mice. Analysis of the chimeric mice demonstrates that the scl-/- embryonic stem cells make a substantial contribution to all non-hematopoietic tissues but do not contribute to any hematopoietic lineage. These observations reveal a crucial role for the scl gene product in definitive hematopoiesis. In addition, in vitro differentiation assays with scl-/- embryonic stem cells showed that the scl gene product was also required for formation of hematopoietic cells in this system.
Hypoxia-inducible factor 1 (HIF-1) is a basic helix-loop-helix transcription factor which is expressed when mammalian cells are subjected to hypoxia and which activates transcription of genes encoding erythropoietin, vascular endothelial growth factor, and other proteins that are important for maintaining oxygen homeostasis. Previous studies have provided indirect evidence that HIF-1 also regulates transcription of genes encoding glycolytic enzymes. In this paper we characterize hypoxia response elements in the promoters of the ALDA, ENO1, and Ldha genes. We demonstrate that HIF-1 plays an essential role in activating transcription via these elements and show that although absolutely necessary, the presence of a HIF-1 binding site alone is not sufficient to mediate transcriptional responses to hypoxia. Analysis of hypoxia response elements in the ENO1 and Ldha gene promoters revealed that each contains two functionally-essential HIF-1 sites arranged as direct and inverted repeats, respectively. Our data establish that functional hypoxia-response elements consist of a pair of contiguous transcription factor binding sites at least one of which contains the core sequence 5'-RCGTG-3' and is recognized by HIF-1. These results provide further evidence that the coordinate transcriptional activation of genes encoding glycolytic enzymes which occurs in hypoxic cells is mediated by HIF-1.
The arylhydrocarbon-receptor nuclear translocator (ARNT) is a member of the basic-helix-loop-helix-PAS family of heterodimeric transcription factors which includes the arylhydrocarbon receptor (AHR), hypoxia-inducible factor-1alpha (HIF-1alpha) and the Drosophila single-minded protein (Sim). ARNT forms heterodimeric complexes with the arylhydrocarbon receptor, HIF-1alpha, Sim and the PAS protein Per. In response to environmental pollutants, AHR-ARNT heterodimers regulate genes involved in the metabolism of xenobiotics, whereas ARNT-HIF-1alpha heterodimers probably regulate those involved in the response to oxygen deprivation. By generating a targeted disruption of the Arnt locus in the mouse, we show here that Arnt-/- embryonic stem cells fail to activate genes that normally respond to low oxygen tension. Arnt-/- ES cells also failed to respond to a decrease in glucose concentration, indicating that ARNT is crucial in the response to hypoxia and to hypoglycaemia. Arnt-/- embryos were not viable past embryonic day 10.5 and showed defective angiogenesis of the yolk sac and branchial arches, stunted development and embryo wasting. The defect in blood vessel formation in Arnt-/- yolk sacs is similar to the angiogenic abnormalities reported for mice deficient in vascular endothelial growth factor or tissue factor. On the basis of these findings, we propose a model in which increasing tissue mass during organogenesis leads to the formation of hypoxic/nutrient-deprived cells, the subsequent activation of ARNT, and a concomitant increase in the expression of genes (including that encoding vascular endothelial growth factor) that promote vascularization of the developing yolk sac and solid tissues.
The hypoxia-inducible factor 1 transcriptional activator complex (HIF-1) is involved in the activation of the erythropoietin and several other hypoxia-responsive genes. The HIF-1 complex is composed of two protein subunits: HIF-1beta/ARNT (aryl hydrocarbon receptor nuclear translocator), which is constitutively expressed, and HIF-1alpha, which is not present in normal cells but induced under hypoxic conditions. The HIF-1alpha subunit is continuously synthesized and degraded under normoxic conditions, while it accumulates rapidly following exposure to low oxygen tensions. The involvement of the ubiquitin-proteasome system in the proteolytic destruction of HIF-1 in normoxia was studied by the use of specific inhibitors of the proteasome system. Lactacystin and MG-132 were found to protect the degradation of the HIF-1 complex in cells transferred from hypoxia to normoxia. The same inhibitors were able to induce HIF-1 complex formation when added to normoxic cells. Final confirmation of the involvement of the ubiquitin-proteasome system in the regulated degradation of HIF-1alpha was obtained by the use of ts20TGR cells, which contain a temperature-sensitive mutant of E1, the ubiquitin-activating enzyme. Exposure of ts20 cells, under normoxic conditions, to the non-permissive temperature induced a rapid and progressive accumulation of HIF-1. The effect of proteasome inhibitors on the normoxic induction of HIF-1 binding activity was mimicked by the thiol reducing agent N-(2-mercaptopropionyl)-glycine and by the oxygen radical scavenger 2-acetamidoacrylic acid. Furthermore, N-(2-mercaptopropionyl)-glycine induced gene expression as measured by the stimulation of a HIF-1-luciferase expression vector and by the induction of erythropoietin mRNA in normoxic Hep 3B cells. These last findings strongly suggest that the hypoxia induced changes in HIF-1alpha stability and subsequent gene activation are mediated by redox-induced changes.
Embryonic stem cell-derived embryoid bodies contain a unique precursor population which, in response to vascular endothelial growth factor, gives rise to blast colonies in semi-solid medium. Upon transfer to liquid culture with appropriate cytokines, these blast colonies generate both hematopoietic and adherent, stromal-type cells. Cells within the adherent population display characteristics of endothelial lineage including the expression of CD31, flk-1, flt-1, tie-2, the capacity to take up acetylated LDL and the presence of cytoplasmic Weibel-Palade bodies. Mixing studies demonstrated that the hematopoietic and endothelial precursors within the blast colonies develop from the same cell, the blast colony-forming cell. Kinetic analysis showed that the blast colony-forming cell represents a transient cell population that develops early and is lost quickly during embryoid body development. These findings provide strong evidence that the blast colony-forming cell represents the long-hypothesized hemangioblast, the common precursor of the hematopoietic and endothelial lineages.
The transcriptional response to lowered oxygen levels is mediated by the hypoxia-inducible transcription factor (HIF-1), a heterodimer consisting of the constitutively expressed aryl hydrocarbon receptor nuclear translocator (ARNT) and the hypoxic response factor HIF-1alpha. To study the role of the transcriptional hypoxic response in vivo we have targeted the murine HIF-1alpha gene. Loss of HIF-1alpha in embryonic stem (ES) cells dramatically retards solid tumor growth; this is correlated with a reduced capacity to release the angiogenic factor vascular endothelial growth factor (VEGF) during hypoxia. HIF-1alpha null mutant embryos exhibit clear morphological differences by embryonic day (E) 8.0, and by E8.5 there is a complete lack of cephalic vascularization, a reduction in the number of somites, abnormal neural fold formation and a greatly increased degree of hypoxia (measured by the nitroimidazole EF5). These data demonstrate the essential role of HIF-1alpha in controlling both embryonic and tumorigenic responses to variations in microenvironmental oxygenation.
The TEL (translocation-Ets-leukemia or ETV6) locus, which encodes an Ets family transcription factor, is frequently rearranged in human leukemias of myeloid or lymphoid origins. By gene targeting in mice, we previously showed that TEL-/- mice are embryonic lethal because of a yolk sac angiogenic defect. TEL also appears essential for the survival of selected neural and mesenchymal populations within the embryo proper. Here, we have generated mouse chimeras with TEL-/- ES cells to examine a possible requirement in adult hematopoiesis. Although not required for the intrinsic proliferation and/or differentiation of adult-type hematopoietic lineages in the yolk sac and fetal liver, TEL function is essential for the establishment of hematopoiesis of all lineages in the bone marrow. This defect is manifest within the first week of postnatal life. Our data pinpoint a critical role for TEL in the normal transition of hematopoietic activity from fetal liver to bone marrow. This might reflect an inability of TEL-/- hematopoietic stem cells or progenitors to migrate or home to the bone marrow or, more likely, the failure of these cells to respond appropriately and/or survive within the bone marrow microenvironment. These data establish TEL as the first transcription factor required specifically for hematopoiesis within the bone marrow, as opposed to other sites of hematopoietic activity during development.
We reported previously that vascular endothelial growth factor (VEGF) inhibits the apoptotic death of hematopoietic cells that is induced by exposure to ionizing radiation (O. Katoh et al., Cancer Res., 55: 5687-5692, 1995). In this study, we show that VEGF also inhibits apoptotic cell death that is induced by exposure to the chemotherapeutic drugs etoposide and doxorubicin. To elucidate the molecular mechanisms underlying this inhibitory effect of VEGF, we examined expression levels of BCL2 family proteins in CMK86, a human leukemia cell line, after treatment with VEGF. Northern blotting and immunoblotting analyses revealed that the expression level of MCL1, a member of the BCL2 family, was increased by VEGF. Moreover, to examine the effects of MCL1 on apoptotic cell death induced by exposure to etoposide, we generated a clonal U937 myeloid leukemia cell line transfected with vectors that promoted the constitutive expression of MCL1. MCL1 decreased the caspase 3 activity induced by exposure to etoposide and increased the viability of the transfected cells after etoposide exposure. Therefore, MCL1 may be involved in the inhibitory effect of VEGF on apoptotic cell death.
Mice deficient in the Flk-1 receptor tyrosine kinase are known to die in utero because of defective vascular and hematopoietic development. Here, we show that flk-1(-/-) embryonic stem cells are nevertheless able to differentiate into hematopoietic and endothelial cells in vitro, although they give rise to a greatly reduced number of blast colonies, a measure of hemangioblast potential. Furthermore, normal numbers of hematopoietic progenitors are found in 7.5-day postcoitum flk-1(-/-) embryos, even though 8. 5-day postcoitum flk-1(-/-) embryos are known to be deficient in such cells. Our results suggest that hematopoietic/endothelial progenitors arise independently of Flk-1, but that their subsequent migration and expansion require a Flk-1-mediated signal.
Genetic studies in mice have previously demonstrated an intrinsic requirement for the vascular endothelial growth factor (VEGF) receptor Flk-1 in the early development of both the hematopoietic and endothelial cell lineages. In this study, embryonic stem (ES) cells homozygous for a targeted null mutation in flk-1 (flk-1 (-/-)) were examined for their hematopoietic potential in vitro during embryoid body (EB) formation or when cultured on the stromal cell line OP9. Surprisingly, in EB cultures flk-1 (-/-) ES cells were able to differentiate into all myeloid-erythroid lineages, albeit at half the frequency of heterozygous lines. In contrast, although flk-1 (-/-) ES cells formed mesodermal-like colonies on OP9 monolayers, they failed to generate hematopoietic clusters even in the presence of exogenous cytokines. However, flk-1 (-/-) OP9 cultures did contain myeloid precursors, albeit at greatly reduced percentages. This defect was rescued by first allowing flk-1 (-/-) ES cells to differentiate into EBs and then passaging these cells onto OP9 stroma. Thus, the requirement for Flk-1 in early hematopoietic development can be abrogated by alterations in the microenvironment. This finding is consistent with a role for Flk-1 in regulating the migration of early mesodermally derived precursors into a microenvironment that is permissive for hematopoiesis.
Hypoxia results in adaptive changes in the transcription of a range of genes including erythropoietin. An important mediator is hypoxia-inducible factor-1 (HIF-1), a DNA binding complex shown to contain at least two basic helix-loop-helix PAS-domain (bHLH-PAS) proteins, HIF-1α and aryl hydrocarbon nuclear receptor translocator (ARNT). In response to hypoxia, HIF-1α is activated and accumulates rapidly in the cell. Endothelial PAS domain protein 1 (EPAS-1) is a recently identified bHLH-PAS protein with 48% identity to HIF-1α, raising the question of its role in responses to hypoxia. We developed specific antibodies and studied expression and regulation of EPAS-1 mRNA and protein across a range of human cell lines. EPAS-1 was widely expressed, and strongly induced by hypoxia at the level of protein but not mRNA. Comparison of the effect of a range of activating and inhibitory stimuli showed striking similarities in the EPAS-1 and HIF-1α responses. Although major differences were observed in the abundance of EPAS-1 and HIF-1α in different cell types, differences in the inducible response were subtle with EPAS-1 protein being slightly more evident in normoxic and mildly hypoxic cells. Functional studies in a mutant cell line (Ka13) expressing neither HIF-1α nor EPAS-1 confirmed that both proteins interact with hypoxically responsive targets, but suggest target specificity with greater EPAS-1 transactivation (relative to HIF-1α transactivation) of the VEGF promoter than the LDH-A promoter.
This review focuses on the molecular stratagems utilized by bacteria, yeast, and mammals in their adaptation to hypoxia. Among this broad range of organisms, changes in oxygen tension appear to be sensed by heme proteins, with subsequent transfer of electrons along a signal transduction pathway which may depend on reactive oxygen species. These heme-based sensors are generally two-domain proteins. Some are hemokinases, while others are flavohemoproteins [flavohemoglobins and NAD(P)H oxidases]. Hypoxia-dependent kinase activation of transcription factors in nitrogen-fixing bacteria bears a striking analogy to the phosphorylation of hypoxia inducible factor-1 (HIF-1) in mammalian cells. Moreover, redox chemistry appears to play a critical role both in the trans-activation of oxygen-responsive genes in unicellular organisms as well as in the activation of HIF-1. In yeast and bacteria, regulatory operons coordinate expression of genes responsible for adaptive responses to hypoxia and hyperoxia. Similarly, in mammals, combinatorial interactions of HIF-1 with other identified transcription factors are required for the hypoxic induction of physiologically important genes.
Hypoxia results in adaptive changes in the transcription of a range of genes including erythropoietin. An important mediator is hypoxia-inducible factor-1 (HIF-1), a DNA binding complex shown to contain at least two basic helix-loop-helix PAS-domain (bHLH-PAS) proteins, HIF-1 alpha and aryl hydrocarbon nuclear receptor translocator (ARNT), In response to hypoxia, HIF-1 alpha is activated and accumulates rapidly in the cell. Endothelial PAS domain protein 1 (EPAS-1) is a recently identified bHLH-PAS protein with 48% identity to HIF-1 alpha, raising the question of its role in responses to hypoxia. We developed specific antibodies and studied expression and regulation of EPAS-1 mRNA and protein across a range of human cell lines. EPAS-1 was widely expressed, and strongly induced by hypoxia at the level of protein but not mRNA. Comparison of the effect of a range of activating and inhibitory stimuli showed striking similarities in the EPAS-1 and HIF-1 alpha responses. Although major differences were observed in the abundance of EPAS-1 and HIF-1 alpha in different cell types, differences in the inducible response were subtle with EPAS-1 protein being slightly more evident in normoxic and mildly hypoxic cells. Functional studies in a mutant cell line (Ka13) expressing neither HIF-1 alpha nor EPAS-1 confirmed that both proteins interact with hypoxically responsive targets, but suggest target specificity with greater EPAS-1 transactivation (relative to HIF-1 alpha transactivation) of the VEGF promoter than the LDH-A promoter. (C) 1998 by The American Society of Hematology.
We have previously shown using gene targeting that PU.1 is essential for the development of lymphoid and myeloid lineages during fetal liver hematopolesis. We now show that PU.1 is required for the maturation of yolk sac-derived myeloid progenitors and for the differentiation of ES cells into macrophages. The role of PU.1 in regulating target genes, thought to be critical in the development of monocytes and granulocytes, has been analyzed. Early genes such as GM-CSFR, G-CSFR, and myeloperoxidase are expressed in PU.1−/− embryos and differentiated PU.1−/− ES cells. However, the expression of genes associated with terminal myeloid differentiation (CD11b, CD64, and M-CSFR) is eliminated in differentiated PU.1−/− ES cells. Development of macrophages is restored with the introduction of a PU.1 cDNA regulated by its own promoter. The PU.1−/− ES cells represent an important model for analyzing myeloid cell development.
Hypoxia is an essential developmental and physiological stimulus that plays a key role in the pathophysiology of cancer, heart attack, stroke, and other major causes of mortality. Hypoxia inducible factor 1 (HIF-1) is the only known mammalian transcription factor expressed uniquely in response to physiologically relevant levels of hypoxia. We now report that in Hif1 alpha(-/-) embryonic stem cells that did not express the O-2-regulated HIF-1 alpha subunit, levels of mRNAs encoding glucose transporters and glycolytic enzymes were reduced, and cellular proliferation was impaired. Vascular endothelial growth factor mRNA expression was also markedly decreased in hypoxic Hif1 alpha(-/-) embryonic stem cells and cystic embryoid bodies. Complete deficiency of HIF-1 alpha resulted in developmental arrest and lethality by E11 of Hif1 alpha(-/-) embryos that manifested neural tube defects, cardiovascular malformations, and marked cell death within the cephalic mesenchyme. In Hif1 alpha(+/+) embryos, HIF-1 alpha expression increased between E8.5 and E9.5, coincident with the onset of developmental defects and cell death in Hif1 alpha(-/-)embryos. These results demonstrate that HIF-1 alpha is a master regulator of cellular and developmental O-2 homeostasis.
Although hypoxia (lack of oxygen in body tissues) is perhaps the most physiological inducer of the wild-type p53 gene, the mechanism of this induction is unknown. Cells may detect low oxygen levels through a haem-containing sensor protein. The hypoxic state can be mimicked by using cobalt chloride and the iron chelator desferrioxamine: like hypoxia, cobalt chloride and desferrioxamine activate hypoxia-inducible factor 1alpha (HIF-1alpha), which stimulates the transcription of several genes that are associated with hypoxia. Here we show that these treatments induce accumulation of wild-type p53 through HIF-1alpha-dependent stabilization of p53 protein. Induction of p53 does not occur in either a mutant hepatoma cell line that is unable to induce HIF-1alpha or embryonic stem cells derived from mice lacking HIF-1beta. HIF-1alpha is found in p53 immunoprecipitates from MCF7 cells that express wild-type p53 and are either hypoxic or have been exposed to desferrioxamine. Similarly, anti-haemagglutinin immunoprecipitates from lysates of normoxic PC3M cells that had been co-transfected with haemagglutinin-tagged HIF-1alpha and wild-type p53 also contain p53. Transfection of normoxic MCF7 cells with HIF-1alpha stimulates a co-transfected p53-dependent reporter plasmid and increases the amount of endogenous p53. Our results suggest that hypoxic induction of transcriptionally active wild-type p53 is achieved as a result of the stabilization of p53 by its association with HIF-1alpha.
The effect of varying cellular oxygenation on L6 muscle cell 2-deoxy-D-glucose transport, glucose utilization, lactate production, and expression of GLUT1 and GLUT4 transport proteins was investigated. Incubation of L6 myotubes in 3% O2 (mimicking a state of hypoxia) elevated glucose uptake by 6.5-fold over 48 h relative to cells incubated in 21% O2 (normoxia). Incubation of L6 cells in hyperoxic conditions (50% O2) significantly depressed glucose uptake by 0.4-fold. These effects were fully reversible. Incubation in 3% O2 also caused lactate accumulation and enhanced glucose consumption from the medium. Hypoxia elevated 2-deoxy-D-glucose transport even when the concentration of glucose in the medium was kept constant, suggesting that glucose deprivation alone was not responsible for increased cellular glucose uptake. Incubation in 3% O2 also elevated 3-O-methylglucose uptake but not amino acid uptake. Cycloheximide prevented the hypoxia-induced increase in glucose uptake, indicating that de novo synthesis of glucose transport-related proteins was the major means by which cells increased glucose uptake. The content of GLUT1 glucose transporter was significantly elevated in total membranes of cells incubated in 3% O2 and depressed in membranes from cells incubated in hyperoxic conditions, whereas GLUT4 expression was not affected. These results indicate that hypoxia induces an adaptive response of increasing cellular glucose uptake through elevated expression of GLUT1 in an attempt to maintain supply of glucose for utilization by nonoxidative pathways.
Placental and endometrial partial pressures of oxygen (PO2) were measured using a polarographic oxygen electrode during the first trimester of pregnancy. Between 8-10 weeks' gestation, placental PO2 levels were significantly lower (P less than .001) than endometrial levels. A significant (P less than .001) increase was observed for placental PO2 values measured at 12-13 weeks compared with those obtained at 8-10 weeks. We suggest that the increase of placental PO2 at the end of the first trimester is related to the establishment of continuous maternal blood flow in the intervillous space.
Human erythropoietin gene expression in liver and kidney is inducible by anemia or hypoxia. DNase I-hypersensitive sites were identified 3' to the human erythropoietin gene in liver nuclei. A 256-base-pair region of 3' flanking sequence was shown by DNase I protection and electrophoretic mobility-shift assays to bind four or more different nuclear factors, at least two of which are induced by anemia in both liver and kidney, and the region functioned as a hypoxia-inducible enhancer in transient expression assays. These results provide insight into the molecular basis for the regulation of gene expression by a fundamental physiologic stimulus, hypoxia.
In general, cell cultures, including hemopoietic stem cells, are produced in an atmosphere of various CO2 concentrations in air, although most cells in vivo proliferate and differentiate at lower oxygen tensions. We therefore investigated the effect of reduced oxygen tension on the in vitro colony growth of committed and multipotential hemopoietic progenitor cells from human bone marrow. All hemopoietic progenitor cells (CFU-mix, BFU-E, CFU-E, and CFU-GM) investigated showed enhanced colony growth at lower oxygen tension. CFU-E showed the highest enhancement, followed in order by BFU-E, CFU-mix and CFU-GM. At reduced oxygen tension, the sensitivity of early and late erythroid progenitor cells to erythropoietin was significantly increased, and this can be one of the mechanisms for the enhanced colony growth of erythroid progenitors. In the colony growth of CFU-GM, plating efficiency was also enhanced by the predominant increment of neutrophilic colonies. The lowering of oxygen tension would presumably reduce oxygen toxicity and result in the increased colony growth of human bone marrow stem cells, although the precise mechanisms of oxygen toxicity at the level of hemopoietic stem cells have yet to be elucidated. However, this clonal culture system, using a low oxygen tension, can be a useful means for elucidating the regulatory mechanisms involved in the proliferation and differentiation of hemopoietic progenitor cells in physiological and pathological conditions.
The development of new vessels (angiogenesis) is essential to wound healing. The center of a wound space is hypoxic, a condition that has been shown to stimulate angiogenesis in animal models of coronary artery occlusion. Because the mechanisms involved in this complex process are difficult to study in situ, an in vitro model would provide a useful complement to in vivo studies. This laboratory has developed and characterized calf pulmonary microvessel endothelial cell (PMVEC) cultures and an in vitro model system of angiogenesis using collagen three-dimensional gels that permit migration of cells into vessel networks. This system was used to study the direct effect of normoxia (20% O2) or hypoxia (5% O2) on PMVEC ability to undergo angiogenesis in vitro. Major changes leading to formation of capillary-like networks occurred during the first 3 days of hypoxic exposure only and included restructuring of actin filament networks, focal changes in distribution of basic fibroblast growth factor, and orientation and migration of cell tracts into a collagen gel matrix to form vessel networks.
Erythropoietin (EPO) is the principal growth factor regulating the production of circulating erythrocytes. We introduced null mutations into both Epo and the EPO receptor (EpoR) gene. Both heterozygotes appeared normal. Homozygous animals exhibited reduced primitive erythropoiesis and died around embryonic day 13, owing to failure of definitive fetal liver erythropoiesis. Both types of mutations exhibited identical phenotypes, indicating that EPO and the EPOR are crucial for definitive erythropoiesis in vivo and that no other ligands or receptors can replace them. Committed erythroid BFU-E and CFU-E progenitors were present in both homozygous fetal livers. Thus, neither EPO nor the EPOR is required for erythroid lineage commitment or for the proliferation and differentiation of BFU-E to CFU-E progenitors. EPO and the EPOR are crucial in vivo for the proliferation and survival of CFU-E progenitors and their irreversible terminal differentiation.
Oxygen tension was measured using flexible polarographic microelectrodes within the oviductal and uterine lumen in rhesus monkeys (n = 9), golden hamsters (n = 21) and rabbits (n = 6), during the reproductive cycle (monkey), during oestrus and pseudopregnancy (hamsters, rabbits) and during pregnancy (hamsters). In general, oxygen tensions in each species were much less than half of atmospheric O2, ranging from high values of about 60 mm Hg (8.7% O2) in the rabbit oviduct, rabbit and hamster uterus, to as low as 11 mm Hg (1.5% O2) in the monkey uterus. Oxygen tensions did not vary significantly between left and right sides of the reproductive tracts (all species), nor between pregnant and pseudopregnant states nor between oviduct and uterus (hamsters). Differences owing to reproductive stage were found in the monkey oviduct, hamster oviduct and uterus, and rabbit uterus. Oxygen tensions were consistently very low (11-14 mm Hg) in the monkey uterus throughout the menstrual cycle. In hamsters and rabbits, intrauterine O2 decreased significantly at about the normal time of blastocyst formation and implantation, to 37 mm Hg (5.3% O2) and 24 mm Hg (3.5% O2), respectively. This study indicates that embryos develop in vivo under low oxygen concentrations, especially during the peri-implantation period. The data have implications for investigations of embryo metabolism and for improving embryo development in vitro.
Apoptosis is a genetically encoded programme of cell death that can be activated under physiological conditions and may be an important safeguard against tumour development. Regions of low oxygen (hypoxia) and necrosis are common features of solid tumours. Here we report that hypoxia induces apoptosis in oncogenically transformed cells and that further genetic alterations, such as loss of the p53 tumour-suppressor gene or overexpression of the apoptosis-inhibitor protein Bcl-2, substantially reduce hypoxia-induced cell death. Hypoxia also selects for cells with defects in apoptosis, because small numbers of transformed cells lacking p53 overtake similar cells expressing wild-type p53 when treated with hypoxia. Furthermore, highly apoptotic regions strongly correlate with hypoxic regions in transplanted tumours expressing wild-type p53, whereas little apoptosis occurs in hypoxic regions of p53-deficient tumours. We propose that hypoxia provides a physiological selective pressure in tumours for the expansion of variants that have lost their apoptotic potential, and in particular for cells acquiring p53 mutations.
Although the hormone erythropoietin (Epo) and its receptor (EpoR) are known to play important roles in the regulation of erythropoiesis, several questions remain concerning the developmental role of Epo/EpoR signaling. As the functions of Epo have been defined primarily through studies of definitive erythroid cells, its importance for primitive, embryonic erythropoiesis remains uncertain, as does the significance of EpoR expression in several nonerythroid cell types. To address these questions, mouse embryonic stem cells and embryos lacking a functional EpoR gene were produced by gene targeting. The effects of the mutation were examined in embryos developing in vivo, in chimeric adult mice produced with homozygous mutant embryonic stem cells, and in hemopoietic cells cultured in vitro. No defects were apparent in nonerythroid cell lineages in which the EpoR normally is expressed, including megakaryocytes and endothelial cells. In the mutant yolk sac, primitive erythrocytes were produced in normal numbers, they underwent terminal differentiation, and expressed near normal levels of embryonic globins, although they were reduced in size and their proliferation was severely retarded after E9.5. In contrast, in the fetal liver, definitive erythropoiesis beyond the late progenitor (CFU-E) stage was drastically inhibited by the EpoR mutation, and virtually no definitive erythrocytes were produced in vivo, leading to embryonic death by E13.5. Thus, our results suggest a fundamental difference in the molecular mechanisms stimulating primitive and definitive erythropoiesis. It was also observed that a few mutant definitive erythroid cells could terminally differentiate when cultured with additional cytokines, demonstrating that although Epo/EpoR signaling is important for definitive erythroid cell survival and proliferation, it is not an obligatory step in differentiation.
Hypoxia-inducible factor-1 (HIF-1), a DNA-binding complex implicated in the regulation of gene expression by oxygen, has been shown to consist of a heterodimer of two basic helix-loop-helix Per-AHR-ARNT-Sim (PAS) proteins, HIF-1alpha, and HIF-1beta. One partner, HIF-1beta, had been recognized previously as the aryl hydrocarbon receptor nuclear translocator (ARNT), an essential component of the xenobiotic response. In the present work, ARNT-deficient mutant cells, originally derived from the mouse hepatoma line Hepa1c1c7, have been used to analyze the role of ARNT/HIF-1beta in oxygen-regulated gene expression. Two stimuli were examined: hypoxia itself and desferrioxamine, an iron-chelating agent that also activates HIF-1. Induction of the DNA binding and transcriptional activity of HIF-1 was absent in the mutant cells, indicating an essential role for ARNT/HIF-1beta. Analysis of deleted ARNT/HIF-1beta genes indicated that the basic, helix-loop-helix, and PAS domains, but not the amino or carboxyl termini, were necessary for function in the response to hypoxia. Comparison of gene expression in wild type and mutant cells demonstrated the critical importance of ARNT/HIF-1beta in the hypoxic induction of a wide variety of genes. Nevertheless, for some genes a reduced response to hypoxia and desferrioxamine persisted in these mutant cells, clearly distinguishing ARNT/HIF-1beta-dependent and ARNT/HIF-1beta-independent mechanisms of gene activation by both these stimuli.
This review focuses on the molecular stratagems utilized by bacteria, yeast, and mammals in their adaptation to hypoxia. Among this broad range of organisms, changes in oxygen tension appear to be sensed by heme proteins, with subsequent transfer of electrons along a signal transduction pathway which may depend on reactive oxygen species. These heme-based sensors are generally two-domain proteins. Some are hemokinases, while others are flavohemoproteins [flavohemoglobins and NAD(P)H oxidases]. Hypoxia-dependent kinase activation of transcription factors in nitrogen-fixing bacteria bears a striking analogy to the phosphorylation of hypoxia inducible factor-1 (HIF-1) in mammalian cells. Moreover, redox chemistry appears to play a critical role both in the trans-activation of oxygen-responsive genes in unicellular organisms as well as in the activation of HIF-1. In yeast and bacteria, regulatory operons coordinate expression of genes responsible for adaptive responses to hypoxia and hyperoxia. Similarly, in mammals, combinatorial interactions of HIF-1 with other identified transcription factors are required for the hypoxic induction of physiologically important genes.
The adult hematopoietic system of mammals is a dynamic hierarchy of cells with the hematopoietic stem cell at its foundation. During embryonic development, the source and expansion potential of this cell remain unclear. Two sites of hematopoietic activity, the yolk sac and aorta-gonad-mesonephros (AGM) region, function in mouse ontogeny at the pre-liver stage of hematopoiesis. However, cellular interchange between these tissues obscures the embryonic site of hematopoietic stem cell generation. Here we present the results of a novel in vitro organ culture system demonstrating that, at day 10 in gestation, hematopoietic stem cells initiate autonomously and exclusively within the AGM region. Furthermore, we provide evidence for the in vitro expansion of hematopoietic stem cells within the AGM region. These results strongly suggest that the AGM region is the source of the definitive adult hematopoietic system, which subsequently colonizes the liver.
The generation of blood cells, haematopoiesis, in the mouse embryo begins with the development of primitive nucleated erythroid cells in the yolk sac followed by the appearance of precursors for multiple definitive haematopoietic lineages. The later developing lineages arise from multipotential stem cells, but the relationship of primitive erythroid cells to these other haematopoietic populations is unknown. Using an in vitro embryonic stem (ES) cell differentiation system, we show that primitive erythrocytes and other haematopoietic lineages arise from a common multipotential precursor that develops within embryoid bodies generated from differentiated ES cells. In response to vascular endothelial growth factor and c-kit ligand these precursors give rise to colonies containing immature cells (blasts) expressing marker genes characteristic of haematopoietic precursors. Many blast colonies also expressed betaH1 and beta major globins but not Brachyury, a mesodermal marker. Kinetic analysis demonstrated that the blast colony-forming cells represent a transient population, preceding the establishment of the primitive erythroid and other lineage-restricted precursors. This precursor population may represent the earliest stage of embryonic haematopoietic commitment.
Mouse embryos lacking the receptor tyrosine kinase, Flk1, die without mature endothelial and hematopoietic cells. To investigate the role of Flk1 during vasculogenesis and hematopoiesis, we examined the developmental potential of Flk1-/- embryonic stem cells in chimeras. We show that Flk1 is required cell autonomously for endothelial development. Furthermore, Flk1-/- cells do not contribute to primitive hematopoiesis in chimeric yolk sacs or definitive hematopoiesis in adult chimeras and chimeric fetal livers. We also demonstrate that cells lacking Flk1 are unable to reach the correct location to form blood islands, suggesting that Flk1 is involved in the movement of cells from the posterior primitive streak to the yolk sac and, possibly, to the intraembryonic sites of early hematopoiesis.
The hypoxia-inducible factor-1 (HIF-1) is a basic-helix-loop-helix (bHLH) heterodimeric transcription factor activated by reductions in oxygen concentration (hypoxia). Activated HIF-1 upregulates expression of genes involved in the adaptation of higher organisms to hypoxic conditions, caused by e.g. high altitude, anemia, wound healing or during development. Examples of these oxy-genes include erythropoietin, a hormone regulating erythropoiesis and hence the oxygen transport capacity, and vascular endothelial growth factor, a potent inducer of angiogenesis leading to increased blood capillary density. The HIF-1 heterodimer is composed of a HIF-1alpha and an ARNT subunit, both belonging to the explosively growing PAS subfamily of bHLH transcription factors. Closely related, but differentially expressed, factors have recently been cloned, at least one of which can also be activated by hypoxia. In this review, we present a survey of the bH LH-PAS family as well as of the genes regulated by HIF-1, and we summarize our current knowledge on the oxygen-dependent activation of this fascinating transcription factor.
Cytotrophoblasts, specialized placental cells, proliferate early in pregnancy and then differentiate into tumor-like cells that establish blood flow to the placenta by invading the uterus and its vasculature. In this study, cytotrophoblasts cultured under hypoxic conditions (2 percent oxygen), mimicking the environment near the uterine surface before 10 weeks of gestation, continued proliferating and differentiated poorly. When cultured in 20 percent oxygen, mimicking the environment near uterine arterioles, the cells stopped proliferating and differentiated normally. Thus, oxygen tension determines whether cytotrophoblasts proliferate or invade, thereby regulating placental growth and cellular architecture.
We used homologous recombination in embryonic stem cells to generate mice heterozygous for an aryl hydrocarbon nuclear translocator (ARNT) null mutation. These mice were intercrossed, but no live homozygous Arnt-/- knockout mice were produced among 64 newborns. Homozygotes die in utero between 9.5 and 10.5 days of gestation. Abnormalities included neural tube closure defects, forebrain hypoplasia, delayed rotation of the embryo, placental hemorrhaging, and visceral arch abnormalities. However, the primary cause of lethality appears to be failure of the embryonic component of the placenta to vascularize and form the labyrinthine spongiotrophoblast. This may be related to ARNT's known role in hypoxic induction of angiogenesis. We found no defects in yolk sac circulation.
Development of the mammalian cardiovascular system is a complex process guided by both genetic and environmental components. Significant advances in the genetics of vascular development have been accomplished most recently by the analysis of multiple "knockout" and transgenic mice which exhibit varying degrees of impaired vascularity. This review focuses on the potential of the environment of the developing embryo to affect its development. In particular we analyze the evidence implicating the ability of physiological parameters such as oxygen and glucose concentrations within and surrounding the embryo to affect the expression of genes critical for vascular development. We conclude that the vascularization of a developing mammalian embryo is a plastic process dependent on the dynamic interaction between fundamental genetic and physiological factors.
As a result of deprivation of oxygen (hypoxia) and nutrients, the growth and viability of cells is reduced. Hypoxia-inducible factor (HIF)-1alpha helps to restore oxygen homeostasis by inducing glycolysis, erythropoiesis and angiogenesis. Here we show that hypoxia and hypoglycaemia reduce proliferation and increase apoptosis in wild-type (HIF-1alpha+/+) embryonic stem (ES) cells, but not in ES cells with inactivated HIF-1alpha genes (HIF-1alpha-/-); however, a deficiency of HIF-1alpha does not affect apoptosis induced by cytokines. We find that hypoxia/hypoglycaemia-regulated genes involved in controlling the cell cycle are either HIF-1alpha-dependent (those encoding the proteins p53, p21, Bcl-2) or HIF-1alpha-independent (p27, GADD153), suggesting that there are at least two different adaptive responses to being deprived of oxygen and nutrients. Loss of HIF-1alpha reduces hypoxia-induced expression of vascular endothelial growth factor, prevents formation of large vessels in ES-derived tumours, and impairs vascular function, resulting in hypoxic microenvironments within the tumour mass. However, growth of HIF-1alpha tumours was not retarded but was accelerated, owing to decreased hypoxia-induced apoptosis and increased stress-induced proliferation. As hypoxic stress contributes to many (patho)biological disorders, this new role for HIF-1alpha in hypoxic control of cell growth and death may be of general pathophysiological importance.
Hypoxia results in adaptive changes in the transcription of a range of genes including erythropoietin. An important mediator is hypoxia-inducible factor-1 (HIF-1), a DNA binding complex shown to contain at least two basic helix-loop-helix PAS-domain (bHLH-PAS) proteins, HIF-1alpha and aryl hydrocarbon nuclear receptor translocator (ARNT). In response to hypoxia, HIF-1alpha is activated and accumulates rapidly in the cell. Endothelial PAS domain protein 1 (EPAS-1) is a recently identified bHLH-PAS protein with 48% identity to HIF-1alpha, raising the question of its role in responses to hypoxia. We developed specific antibodies and studied expression and regulation of EPAS-1 mRNA and protein across a range of human cell lines. EPAS-1 was widely expressed, and strongly induced by hypoxia at the level of protein but not mRNA. Comparison of the effect of a range of activating and inhibitory stimuli showed striking similarities in the EPAS-1 and HIF-1alpha responses. Although major differences were observed in the abundance of EPAS-1 and HIF-1alpha in different cell types, differences in the inducible response were subtle with EPAS-1 protein being slightly more evident in normoxic and mildly hypoxic cells. Functional studies in a mutant cell line (Ka13) expressing neither HIF-1alpha nor EPAS-1 confirmed that both proteins interact with hypoxically responsive targets, but suggest target specificity with greater EPAS-1 transactivation (relative to HIF-1alpha transactivation) of the VEGF promoter than the LDH-A promoter.
Hematopoietic stem cells (HSCs) are the rare cells from which all hematopoietic cells are derived. The absence of HSCs is not compatible with life because many essential cells, such as myeloid and erythroid cells, are short lived. The hematopoietic system is the first essential organ system that fails following cytotoxic treatments. It is the vulnerability of HSCs that prevents regeneration following treatment and thus long-term survival. Because HSCs have the capacity to regenerate a functional hematopoietic system, the manipulation of these cells in vitro holds many promises for gene-therapeutic and other applications; however, these are severely curtailed by current difficulties in maintaining and expanding HSCs in culture. This review focuses on recent approaches towards understanding how the HSC compartment is regulated in vivo and discusses how this knowledge might be applied to manipulating HSC numbers.
Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis
- D Schweiki
- A Itin
- D Soffer
- E Keshet
Schweiki, D., A. Itin, D. Soffer, and E. Keshet. 1992. Vascular endothelial
growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis. Nature 359: 843-845.