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Hypoxia-Inducible Factor and the Development of Stem Cells of the Cardiovascular System

^a Abramson Family Cancer Research Institute,
^b Howard Hughes Medical Institute,
^c Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania

Key Words. HIF-1 • Hemangioblast • Hypoxia • Hematopoietic development • Vascular development

M. Celeste Simon, Ph.D., Howard Hughes Medical Institute, Abramson Family Cancer Research Institute, University of Pennsylvania School of Medicine, BRBII/III, Room 456, 421 Curie Blvd., Philadelphia, Pennsylvania 19104, USA. Telephone: 215-746-5532; Fax: 215-746-5511; e-mail: celeste2{at}mail.med.upenn.edu

	Abstract

Top Abstract Introduction HIF-1: Genetic Regulation of... Hypoxia and Cardiovascular... Hemangioblast Stem Cells Hypoxia and Development Genetic Models of O2... Hypoxia and In Vitro... Summary Acknowledgements References

 
Decreased oxygen (O₂) levels activate hypoxia-inducible factor (HIF-1) to induce genes involved in glycolysis, glucose transport, erythropoiesis, and angiogenesis. Mutations in various HIF-1 subunits have contributed to our understanding of the role hypoxia plays during early embryonic development in general and the cardiovascular system in particular. We propose that HIF-1 is important for the generation, proliferation, maintenance, and differentiation of the early cardiovascular system. Understanding aberrations in these hypoxic responses is important since they contribute to serious human disease such as ischemia and tumorigenesis. In this review we will focus on the critical role of O₂ in regulating cardiovascular events during early embryonic development.

	INTRODUCTION

Top Abstract Introduction HIF-1: Genetic Regulation of... Hypoxia and Cardiovascular... Hemangioblast Stem Cells Hypoxia and Development Genetic Models of O2... Hypoxia and In Vitro... Summary Acknowledgements References

 
A hypoxic environment occurs in adult animals during solid tumor growth, tissue ischemia, and infection. During embryogenesis, the natural progression of organogenesis involves hypoxia or "low O₂ levels"; diffusion of oxygen (O₂) in the embryo is limited by its size shortly after gastrulation. In turn, molecular responses to O₂ gradients by hypoxia-inducible factor (HIF)-1 are responsible for the proper differentiation and maintenance of the cardiovascular system. Composed of a heart, vascular network, and blood supply, O₂ and nutrients are provided through the cardiovascular system to allow proper growth and development of the embryo. Thus, improper response to low O₂ tension can lead to lesions in multiple aspects of cardiovascular development.

	HIF-1: GENETIC REGULATION OF OXYGEN RESPONSES

Top Abstract Introduction HIF-1: Genetic Regulation of... Hypoxia and Cardiovascular... Hemangioblast Stem Cells Hypoxia and Development Genetic Models of O2... Hypoxia and In Vitro... Summary Acknowledgements References

 
The primary molecular mechanism of gene activation during hypoxia is through HIF-1. HIF-1 is activated during development, as well as during the normal homeostasis of most tissues, since organs naturally generate a gradient of O₂ [1, 2]. Many genes involved in cellular differentiation are directly or indirectly regulated by hypoxia. These include erythropoietin (EPO), transferrin, transferrin receptor, vascular endothelial growth factor (VEGF), Flk-1, Flt-1, platelet-derived growth factor-ß (PDGF-ß), basic fibroblast growth factor (bFGF), and others genes affecting glycolysis [3-9].

HIF-1 is a member of the basic helix-loop-helix (bHLH)-PAS family of transcription factors known to induce gene expression by binding to a ~50-bp hypoxia response element (HRE) containing a core 5'-ACGTG-3' sequence [1, 2, 10]. bHLH-PAS proteins heterodimerize to form transcription complexes that regulate O₂ homeostasis, circadian rhythms, neurogenesis, and toxin metabolism. Three bHLH-PAS proteins in vertebrates respond to hypoxia: HIF-1, EPAS (HIF-2), and HIF-3 [11-13]. These dimerize with ARNT (aryl hydrocarbon receptor nuclear translocator protein), ARNT-2, or ARNT-3 [14].

HIF-1 was initially characterized while investigating hypoxic induction of the EPO gene [15]. As shown in Figure 1, this subunit of HIF-1 is ubiquitinated and subsequently degraded in less than 5 minutes under normoxic conditions [16]. Although several candidate O₂-sensing molecules have emerged in the literature, the molecular basis of how cells sense O₂ levels is poorly characterized [17]. pVHL, the protein product of a tumor-suppressor gene responsible for von Hippel Lindau disease, is implicated in this O₂-sensing system by its association with HIF-1, targeting it for ubiquitin-mediated degradation [18-21]. HIF-1 is stabilized under low O₂ (<5% O₂) leading to the formation of a functional transcription factor complex with ARNT [22]. Both HIF1- and ARNT are widely expressed [23]. This complex is the master regulator of O₂ homeostasis and induces a network of genes involved in angiogenesis, erythropoiesis, and glucose metabolism.

View larger version (28K): [in this window] [in a new window]   Figure 1. Model of HIF-1 regulation by oxygen. In the presence of oxygen, HIF-1 is ubiquinated by pVHL and proteosomally degraded. Under hypoxic conditions, HIF-1 is translocated to the nucleus and dimerized to ARNT forming the HIF-1 transcriptional activator of genes containing HRE.

	HYPOXIA AND CARDIOVASCULAR DISEASE

Top Abstract Introduction HIF-1: Genetic Regulation of... Hypoxia and Cardiovascular... Hemangioblast Stem Cells Hypoxia and Development Genetic Models of O2... Hypoxia and In Vitro... Summary Acknowledgements References

Hypoxic conditions affect various pathologies [25]. First, tissue ischemia, a variation in O₂ tension caused by hypoxia/reoxygenation, can lead to endothelial cell changes. For example, long periods of ischemia result in endothelial changes, such as vascular leakage, resulting in varicose veins. In more severe situations, ischemia can lead to myocardial or cerebral infarction and retinal vessel occlusion [26]. Of interest, HIF-1 is stabilized prior to induction of VEGF expression during acute ischemia in the human heart [27, 28]. Second, pulmonary hypertension associated with chronic respiratory disorders results from persistent vasoconstriction and vascular remodeling. Third, hypoxic gradients created in enlarging solid tumors trigger expression of genes containing HREs such as those involved in angiogenesis. This allows subsequent delivery of O₂, nutrients, and further tumor growth [29-31]. Vascular remodeling is an important component to tumorigenesis; without proper blood supply, delivery of oxygen may occur by diffusion, but becomes inefficient in tumors greater than 1 mm in diameter [32]. Short-term hypoxia can also elevate platelet numbers, while prolonged exposure may cause some degree of thrombocytopenia in response to increased levels of EPO [33]. Another disorder involving inadequate responses to hypoxia is preeclampsia, a pathology of pregnancy thought to be caused by improper differentiation of placental trophoblast cells due to poorly controlled O₂ tension or improper HIF-mediated responses [34-37]. Thus, understanding the role of hypoxia in development is pertinent to human disease as well.

	HEMANGIOBLAST STEM CELLS

Top Abstract Introduction HIF-1: Genetic Regulation of... Hypoxia and Cardiovascular... Hemangioblast Stem Cells Hypoxia and Development Genetic Models of O2... Hypoxia and In Vitro... Summary Acknowledgements References

 
Hematopoiesis is closely linked to angiogenesis (endothelial cell development), both spatially and temporally during development. In fact, both hematopoietic and endothelial cells emerge from a common progenitor termed the "hemangioblast" [38]. The notion that endothelial and hematopoietic cells are derived from a common precursor is based on the observation that such lineages form simultaneously and in proximity to each other in order to establish the body's O₂ delivery system during organogenesis. The proximity of differentiating endothelial and hematopoietic cells may be crucial for establishment of the vascular system. In a Para-Aortic-Splanchnopleural explant assay, it was demonstrated that hematopoietic stem cells (HSC) are required for angiogenesis to occur [39].

Hemangioblasts are stem cells characterized by their ability to give rise to primitive and definitive hematopoietic and endothelial cells. These progeny cells also share expression of multiple genes including CD34 [40], CD31 [41], Flk-1 [42-46], Flt-1 [47], SCL [48], Tie-2 [49], and Gata-2 [50]. In the avian system, a putative hemangioblast has been reported to arise from the splanchnopleural mesoderm. Contact with associated endoderm promotes hematopoietic cell production, while the ectoderm promotes more of an angioblastic cell type [51]. Primitive endocardium has also been suggested to arise from this common progenitor in avian systems [52]. An in vitro murine embryonic stem (ES) cell differentiation assay identified "blast cell colonies" (BL-CFC) that generate both endothelial and hematopoietic precursors [53]. The signals and transcriptional controls that specify the differentiation events of endothelial and hematopoietic cells are not completely defined. Furthermore, isolated Flk-1⁺ cells derived from ES cells generate endothelial, hematopoietic and smooth muscle cells in vitro suggesting that hemangioblasts exhibit even greater plasticity than originally thought [54]. Therefore, any disregulation of this early progenitor will likely impact on numerous aspects of development.

	HYPOXIA AND DEVELOPMENT

Top Abstract Introduction HIF-1: Genetic Regulation of... Hypoxia and Cardiovascular... Hemangioblast Stem Cells Hypoxia and Development Genetic Models of O2... Hypoxia and In Vitro... Summary Acknowledgements References

 
In mice, Flk-1⁺ hemangioblasts are first detected at E7.0 [55]. They originate in a region where mesoderm is in contact with visceral endoderm and contribute to the extraembryonic vasculature of the yolk sac. These early progenitors are mesodermal cell aggregates that contain both endothelial and hematopoietic precursors. Such endothelial precursors go on to become the linings of yolk sac blood islands by the process of vasculogenesis, while hematopoietic precursors contribute to primitive blood cells that produce embryonic globins [56, 57]. Meanwhile, in the embryo proper, paraxial mesoderm precursor cells, (perhaps the putative hemangioblasts) proliferate, sprout, and interconnect to form a loose meshwork forming the primary vascular plexus [58]. This layer of endothelial/mesodermal cells is thin enough to allow for the proper diffusion of nutrients and O₂. However, as the embryo grows in size and becomes hypoxic, delivery of these factors requires a more extensive network [59, 60]. Vascular development proceeds via angiogenesis, which involves the remodeling of the initial vascular plexus into a more mature appearing vascular network followed by recruitment of supporting cells, i.e., smooth muscle cells and pericytes [61, 62].

The pumping mechanism of the heart allows for the circulation of blood, thus providing the nutrients and O₂ required for survival of all cells within the organism. Like the yolk sac, cardiac morphogenesis occurs in the epithelial/mesenchymal transitional areas of the epicardial mesothelium. Hypoxia is also an important event in the development of the placenta allowing for exchange of O₂ and nutrients [34, 36, 37, 62, 63]. Oxygen levels are critical in regulating the differentiation of trophoblast cells required for proper vascular networking, critical for the remodeling process interdigitating the connection between maternal and fetal vessels [63].

	GENETIC MODELS OF O2 REGULATION

Top Abstract Introduction HIF-1: Genetic Regulation of... Hypoxia and Cardiovascular... Hemangioblast Stem Cells Hypoxia and Development Genetic Models of O2... Hypoxia and In Vitro... Summary Acknowledgements References

 
Little is known about the events allowing commitment of hemangioblasts to either the hematopoietic or endothelial lineages. However, it is not surprising to find that mutagenesis of genes encoding various growth factors or their receptors contribute to defects in both lineages. These include VEGF and EPO, both of which are strongly activated by hypoxic events and direct HIF-1 target genes. Vegf^+/– and ^–/– mice die during early embryonic development [64, 65]. Vegf^+/– mice die at E11-12, exhibiting a rudimentary dorsal aorta, extensive vascular defects, and a reduced number of blood vessels and blood cells within yolk sac blood islands. Furthermore, yolk sac veins fail to fuse. Vegf^–/– mice exhibit more severe defects, including lethality at E8.5-9.5 and a complete absence of the dorsal aorta. Epo^–/– or EpoR^–/– mice die at E13.5 with decreased numbers of circulating erythrocytes and severe fetal liver hypocellularity [66-68]. EPO expression is an essential regulator of erythrocyte production in mammals, with a pivotal role in their proliferation, survival, and differentiation [69]. Although mutant mice are highly hypoxic due to lack of these O₂-providing cells, they suffer from ventricular hypoplasia independent of the hypoxic state of the embryo, caused by a decreased proliferative capacity of myocytes in the myocardium [70]. Vegf^–/– mice also suffer from a hypoplastic myocardium and malformed endothelial cushions.

Receptors for VEGF are also direct HIF-1 target genes, or indirectly regulated by HIF-1 due to VEGF induction of its receptors [5, 71-73]. Flk-1^–/– mice, lacking the earliest expressed VEGF receptor on both hematopoietic and angiogenic cells, die between E8.5-9.5 with defects in both lineages [74, 75]. These mice lack yolk sac blood islands and vessels in the embryo proper. A similar phenotype is observed in Scl^–/– mice; SCL is a helix-loop-helix transcription factor also expressed on both cell types [76, 77]. In contrast, Flt-1^–/– mice, deficient in a second VEGF receptor expressed in more differentiated endothelial cells, exhibit increased numbers of hemangioblast progenitors leading to vascular disorganization [78]. In summary, mutations of direct or indirect HIF-1 target genes affect multiple components of early cardiovascular development.

Mice with HIF mutations also develop extensive cardiovascular pathologies. Hif-1^–/–– and Arnt^–/– exhibit lethality by E10.5. Both have intact endothelial cell differentiation but aberrant vessel maturation. Hif-1^–/– mice are arrested at about E8.5 and die by E10.5 with neural tube defects, cephalic mesenchymal cellular death, dilated vasculature, and hyperplastic myocardium [3, 7, 79, 80]. The Arnt^–/– mice also exhibit yolk sac, placental, cardiac, and vascular defects [63, 81-84]. Further analysis has shown that Arnt^–/– yolk sac cells exhibit decreased numbers of hematopoietic progenitors in clonogenic assays [85]. A significant difference between Hif-1^–/– and Arnt^–/– mice, however, is expression levels of VEGF—deficient in Arnt^–/– but not in Hif-1^–/– [80, 84]. Analysis of VEGF expression in Arnt^–/– tissues performed by in situ hybridization demonstrated regional differences in VEGF mRNA levels, i.e., normal in the neural tube and gut, but decreased in yolk sac and branchial arches compared to expression in control embryos [84]. On the other hand, analysis of VEGF mRNA levels in Hif-1^–/– mice was performed by Northern blot on whole embryos making direct comparisons between the two mutants impossible [80]. Other possible explanations include localized differences in expression between ARNT and HIF-1 [23] or in the possible regulation of VEGF by ARNT independent of HIF-1. Nonetheless, both mutant strains are unable to establish a proper vessel network in response to hypoxia.

EPAS-1 (HIF-2), a second HIF, was originally thought to be exclusively expressed in endothelial cells, but is also expressed in the organ of Zuckerkandl and carotid body, the embryonic and postnatal sites of catecholamine release (responsible for the control of heart rates), respectively [12, 86]. Hif-2^–/– mice exhibit markedly distinct phenotypes, depending on the mouse strain used during mutagenesis [86, 87]: Hif-2^–/– mice die by E9.5-13.5 due to vascular disorganization in the yolk sac and embryo proper [87]; or die by E15.5 of heart failure due to deficiency in catecholamine production [12, 86]. In either case, an O₂-mediated defect causes developmental defects in such mutants.

pVHL is linked to oxygen-regulated ubiquitination of HIF. VHL^–/– mice die in utero at E10-12.5 due to placental defects: abnormal embryonic blood vessel formation in the placental labyrinth, failure of proper trophoblast differentiation, hemorrhaging, and necrosis [88]. Although it would follow that loss of VHL would lead to an increase of HIF-1 and thus an increase of VEGF levels, VEGF protein levels were greatly reduced in VHL^–/– placentas, perhaps secondary to improper differentiation. Further understanding of molecular and cellular mechanisms of HIF-1 regulation and regulatory events of cardiovascular development is required to complement the phenotypes of the precedent mouse genetic models of O₂ regulation.

	HYPOXIA AND IN VITRO ASSAYS

Top Abstract Introduction HIF-1: Genetic Regulation of... Hypoxia and Cardiovascular... Hemangioblast Stem Cells Hypoxia and Development Genetic Models of O2... Hypoxia and In Vitro... Summary Acknowledgements References

 
Much interest lies in defining mechanisms of maintenance, growth, and differentiation of HSC and their derivatives. Low levels of O₂ (1%) increase the number of CD34⁺, BFU-E, but decrease their size and level of hemoglobinization in vitro [89]. Thus, it appears that hypoxia induces erythropoiesis in two phases. First, low O₂ promotes immature BFU-E maintenance, and secondly, hypoxia inhibits terminal expansion of these clones. In embryoid bodies (EB), hypoxia increases EPO expression, and also modulates ß-globin production [90]. Other reports describe the expansion of not only BFU-E, but also colony-forming units-granulocyte, macrophage (CFU-GM) after hypoxic exposure of human bone marrow cells [91-93]. Oxygen tension also influences the differentiation, maturation and apoptosis of human megakaryocytes (CFU-Mk), which are involved in platelet generation [94]. Progenitor cells maintained under hypoxia are able to repopulate in a marrow-repopulating ability assay presumably due to elevated EPO production [95]. These compelling data provide evidence that low O₂ levels have the potential to optimize cell expansion and survival in culture.

Although such models contribute to a mechanistic understanding of signaling pathways governing developmental events, in vivo models are necessary to confirm O₂-mediated processes. Further in vivo studies of the role of HIF-1 have been limited by the inability of the mutant embryos to survive. Therefore, in vitro differentiation of yolk sac and ES cells allows for further analysis of the role of HIF-1 in hematopoietic development. The differentiation of ES cells into EB closely resembles early embryonic development [96]. These differentiating cell masses produce many embryonic cell lineages including neuronal, muscle, endothelial and hematopoietic. Thus, one can mimic early development by differentiating ES cells under "physiologic" hypoxic conditions.

Indeed, by manipulating the EB differentiation environment to resemble normal physiological O₂ levels (3%), 9 days of differentiation promotes the increased production of hematopoietic progenitors including CFU-E, CFU-GM, CFU-M, and CFU-granulocyte, erythroid, macrophage, megakaryocyte [85]. Arnt^–/– ES cells are defective in generating appropriate numbers of various hematopoietic lineage progenitors, a phenotype clearly recapitulated in the yolk sac of the Arnt^–/– mice. Thus, hypoxic responses are critical for the proliferation and/or survival of hematopoietic progenitor cells. Interestingly, progenitor cells in these assays are rescued by the addition of VEGF during EB differentiation, but not by EPO.

A similar in vitro clonogenic system has been developed which reveals the existence of temporal hemangioblast cells in early differentiating 2.5-3.5-day EB [53, 97]. These BL-CFU colonies, generated in methylcellulose containing VEGF and endothelial-conditioned media, consist of bilineage cells. The assay provides a model of the regulation of early progenitor cells in either the extraembryonic yolk sac or intraembryonic aorta-gonad-mesonephron regions where mesoderm differentiates into cardiovascular stem cells, including the putative hemangioblast. Cells from these blast colonies give rise to primitive and definitive hematopoietic cells and endothelial cells when replated in medium containing the appropriate growth factors. This assay allows the dissection of early cardiovascular events. For example, although Flk-1^–/– mice fail to develop any yolk sac blood islands or blood vessels, in vitro differentiation of Flk-1^–/– ES cells in this blast assay gave rise to BL-CFC. Although fewer in number, Flk-1^–/– BL-CFC maintain the potential to differentiate into both endothelial or hematopoietic cells [98]. These results suggest that hemangioblasts can arise independent of Flk-1 but further survival, differentiation, or migration is dependent on this VEGF signaling pathway.

We have found that hypoxia stimulates not only production of BL-CFC progenitor colonies, but also accelerates the kinetics of BL-CFC appearance. Along these lines, Arnt^–/– ES are defective in generating these BL-CFC (D. Ramirez Bergeron and M.C. Simon, unpublished data). Similar phenotypes were obtained from SCL^–/– and bFGF-receptor^–/– ES cells which, instead of BL-CFC, generate a dominant blast-transitional hemangioblast subpopulation [98, 99]. These colonies represent an earlier stage of development than the original BL-CFC and generate cells with predominantly mesodermal and endothelial markers. Thus, lack of HIF-1 affects early progression of hemangioblast cells that promote endothelial cell maturation and hematopoietic cell differentiation. This is consistent with the phenotype in the Arnt^–/– and Hif-1^–/– mice, in that although they form early endothelial cells, they are deficient in proper differentiation of the vascular system. These findings suggest that hypoxia-mediated generation and proliferation of progenitor cells is critical for early cardiovascular events.

	SUMMARY

Top Abstract Introduction HIF-1: Genetic Regulation of... Hypoxia and Cardiovascular... Hemangioblast Stem Cells Hypoxia and Development Genetic Models of O2... Hypoxia and In Vitro... Summary Acknowledgements References

 
Although it has been suggested that a key feature of HIF-1 mutant mice is their lack of a mature vasculature, lethality may actually stem from improper production or differentiation of a common progenitor of the cardiovascular system, the hemangioblast. Various factors respond to hypoxia, i.e., EPO, VEGF, bFGF, TGF-ß3, and PDGF, and collectively regulate the generation and differentiation of these stem cells. The use of in vitro BL-CFU assays allows a further analysis of the role of HIF-1 in early development. Future studies include determining the physiological and molecular mechanism(s) by which hypoxia influences these early events. For example, does HIF-1: A) influence the expression of early progenitor markers; B) stimulate production of growth factors that influence mesoderm differentiation into early stem cells, or C) mediate hemangioblast proliferation or differentiation during establishment of the cardiovascular system? A very important element to this puzzle is to identify hemangioblasts in the embryonic splachnopleural region since mesoderm from this region gives rise to endothelial, hematopoietic, and cardiac progenitors. An understanding of the biological role of hypoxia in the early events of progenitor cell generation, lineage commitment, and development is critical to assess O₂ delivery during organogenesis and disease.

	Acknowledgements

Top Abstract Introduction HIF-1: Genetic Regulation of... Hypoxia and Cardiovascular... Hemangioblast Stem Cells Hypoxia and Development Genetic Models of O2... Hypoxia and In Vitro... Summary Acknowledgements References

 
This research was supported in part by a grant HL63310 from the National Institutes of Health (M.C.S.). M.C.S. is an investigator of the Howard Hughes Medical Institute.

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Top Abstract Introduction HIF-1: Genetic Regulation of... Hypoxia and Cardiovascular... Hemangioblast Stem Cells Hypoxia and Development Genetic Models of O2... Hypoxia and In Vitro... Summary Acknowledgements References

 

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