Regulation of Angiogenesis by Hypoxia and Hypoxia‐Inducible Factors

Regulation of Angiogenesis by Hypoxia and Hypoxia‐Inducible Factors

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Regulation of Angiogenesis by Hypoxia and Hypoxia‐Inducible Factors Michele M. Hickey and M. Celeste Simon Department of Cell and Molecular Biology Abramson Family Cancer Research Institute, and Howard Hughes Medical Institute, University of Pennsylvania Philadelphia, Pennsylvania 19104

I. Introduction II. Mechanisms of Angiogenesis III. The Hypoxia‐Inducible Factor Family A. Structure of HIF Proteins B. Regulation of HIF Activity C. HIF Target Genes IV. V. VI. VII.

The Role of HIF in Developmental Angiogenesis The Role of HIF in Adult Tissues HIF and Ischemic Injury HIF and Cancer A. Tumor Angiogenesis and Hypoxia B. HIF Expression in Tumors C. von Hippel‐Lindau Disease D. The Role of HIF1 and HIF2 in Tumor Angiogenesis E. Therapeutic Implications

VIII. Conclusions Acknowledgments References

Maintenance of oxygen homeostasis is critical for the survival of multicellular organs. As a result, both invertebrates and vertebrates have developed highly specialized mechanisms to sense changes in oxygen levels and to mount adequate cellular and systemic responses to these changes. Hypoxia, or low oxygen tension, occurs in physiological situations such as during embryonic development, as well as in pathological conditions such as ischemia, wound healing, and cancer. A primary eVector of the adaptive response to hypoxia in mammals is the hypoxia‐inducible factor (HIF) family of transcription regulators. These proteins activate the expression of a broad range of genes that mediate many of the responses to decreased oxygen concentration, including enhanced glucose uptake, increased red blood cell production, and the formation of new blood vessels via angiogenesis. This latter process is dynamic and results in the establishment of a mature Current Topics in Developmental Biology, Vol. 76 Copyright 2006, Elsevier Inc. All rights reserved.

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0070-2153/06 $35.00 DOI: 10.1016/S0070-2153(06)76007-0

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vascular system that is indispensable for proper delivery of oxygen and nutrients to all cells in both normal tissue and hypoxic regions. Angiogenesis is essential for normal development and neoplastic disease as tumors must develop mechanisms to stimulate vascularization to meet increasing metabolic demands. The link between hypoxia and the regulation of angiogenesis is an area of intense research and the molecular details of this connection are still being elaborated. This chapter will provide an overview of current knowledge and highlight new insights into the importance of HIF and hypoxia in angiogenesis in both physiological and pathophysiological conditions. ß 2006, Elsevier Inc.

I. Introduction In the absence of a functional blood supply, the growth of both multicellular organisms and tumors is limited by the ability of oxygen to diVuse to cells from blood vessels. Decreased oxygen levels, or hypoxia, can develop within rapidly proliferating tissues or as the result of occlusion of blood vessels. Hypoxia leads to insuYcient cellular energy production as oxygen is essential for oxidative phosphorylation. At the same time, however, excessively high levels of oxygen are detrimental and can result in the production of reactive oxygen species (ROS) that damage cellular organelles and DNA. Therefore, it is imperative that oxygen concentrations be tightly regulated. At the systemic level, oxygen tension is detected by highly sensitive tissues, such as the carotid body, to eVect a rapid physiological response to acute hypoxia that includes increased ventilation and cardiac output. More prolonged hypoxia is also sensed at the cellular level, leading to the activation of molecular pathways to cope with this stress. The key mediators of this response are members of the hypoxia‐inducible factor (HIF) family of proteins. The hypoxic response and the HIF pathway are conserved from Caenorhabitis elegans and Drosophila melanogaster to mice and humans, emphasizing its importance in the maintenance of oxygen homeostasis. These proteins function as transcriptional regulators that stimulate the expression of a multitude of genes important for adaptation to hypoxia, including those encoding glucose transporter‐1 (Glut‐1), which increases cellular glucose uptake, and glycolytic enzymes, which mediate enhanced glycolysis to maintain ATP production in the face of lower oxygen levels. In addition, HIF‐stimulated erythropoietin (Epo) expression improves the oxygen carrying capacity of the blood by enhancing the production of erythrocytes. Another mechanism by which cells can alleviate the increasing metabolic demands presented by hypoxia is via new blood vessel formation through vasculogenesis and angiogenesis. These processes are complex and occur in a stepwise fashion. Angiogenesis is regulated by a balance of positive‐ and

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negative‐acting growth factors and by physiological stresses such as alterations in oxygen levels. Hypoxia stimulates the expansion and remodeling of the existing vasculature to enhance blood flow to oxygen‐deprived tissues. This is accomplished primarily through the activation of HIF target genes involved in various steps of angiogenesis such as vascular endothelial growth factor (VEGF). This regulation of angiogenesis by hypoxia and HIFs is essential for proper embryonic development and recovery after ischemic injury. In addition, numerous lines of evidence suggest that tumors develop regions of hypoxia and that the HIF pathway is an important component of tumor growth and angiogenesis.

II. Mechanisms of Angiogenesis The vascular network mediates the delivery of oxygen and nutrients to all cells of an organism and is essential for normal development and survival. Figure 1 outlines the mechanisms by which the vasculature is formed and highlights several of the key molecules involved in regulating these processes. Mesodermal progenitor cells known as hemangioblasts represent bipotential precursor cells that give rise to both endothelial and hematopoietic cells (Sabin, 1920). A putative hemangioblast, the blast colony‐forming cell (BL‐CFC), has been identified in vitro in embryoid bodies derived from embryonic stem (ES) cells and has been shown to express the VEGF‐receptor‐2 (VEGF‐R2/Flk1) (Choi et al., 1998; Kennedy et al., 1997; Yamaguchi et al., 1993). Hemangioblasts

Figure 1 Overview of vascular development. During embryonic development, mesoderm‐ derived precursor cells known as hemangioblasts are stimulated by VEGF and FGF signaling to give rise to both hematopoietic and endothelial cells (ECs). A primary capillary network is formed by the process of vasculogenesis in which endothelial progenitors, or angioblasts, diVerentiate to form primitive blood vessels in the yolk sac and embryo. VEGF signaling through VEGF‐R2/Flk‐1 is essential for this process. Subsequently, new capillaries are formed by sprouting or splitting of existing vessels through angiogenesis. VEGF is also critical for this process and acts together with angiopoietins to stimulate the proliferation and migration of ECs. These vessels are stabilized through the recruitment of supporting cells to form the mature vasculature.

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have also been detected in vivo in the mouse embryo during gastrulation as mesodermal cells that express brachyury and VEGF‐R2 (Huber et al., 2004). Signaling through VEGF and members of the fibroblast growth factor (FGF) family is required for the diVerentiation of mesoderm into these precursor cells (Faloon et al., 2000; Kennedy et al., 1997). In addition, hypoxia has been shown to enhance the production of BL‐CFCs in a HIF‐dependent manner (Ramirez‐Bergeron et al., 2004). Endothelial cell (EC) precursors, or angioblasts, aggregate within the yolk sac and embryo and subsequently diVerentiate to give rise to a primary capillary plexus in the process of vasculogenesis (Conway et al., 2001). Additional capillaries can be generated by sprouting from and splitting of existing vessels through angiogenesis. Degradation of the extracellular matrix (ECM) by matrix metalloproteinases (MMPs) allows ECs to migrate in response to chemotactic growth factors. VEGF and the angiopoietins, together with FGF proteins and platelet‐derived growth factor (PDGF), mediate the migration and proliferation of ECs to newly formed vessels (Conway et al., 2001; Jain, 2003; Risau, 1997). Primitive blood vessels synthesize ECM proteins and recruit supporting cells, such as pericytes and smooth muscle cells, as they mature. PDGF, angiopoietin‐1 (Ang‐1), and transforming growth factor (TGF)‐ are important in the regulation of these processes (Jain, 2003). Angiogenesis is controlled by a balance between stimulatory and inhibitory factors and the so‐called ‘‘angiogenic switch’’ occurs when this balance shifts in favor of positive stimuli (Carmeliet, 2005; Hanahan and Folkman, 1996). Among these factors, VEGF is essential for most steps in vasculogenesis and angiogenesis, acting in a paracrine manner to stimulate diVerentiation of VEGF‐R2þ angioblasts, proliferation and survival of ECs, and sprouting of new blood vessels (Carmeliet, 2000; Ferrara and Gerber, 2001; Veikkola et al., 2000). The importance of VEGF signaling is supported by the finding that embryos lacking VEGF‐R2 do not form hematopoietic cells or ECs (Shalaby et al., 1995). In contrast, loss of VEGF‐R1 expression results in defective formation of mature blood vessels, demonstrating the role of this receptor later in angiogenesis and vessel maturation (Fong et al., 1995). Regulation of VEGF expression itself is also essential in order to sustain proper angiogenesis during development and to control angiogenesis in pathogenic situations. Deficiency in Vegf expression is early embryonic lethal due to severe defects in vascularization; importantly loss of a single Vegf allele is suYcient to disrupt angiogenesis and cause lethality (Carmeliet et al., 1996; Damert et al., 2002; Duan et al., 2003; Ferrara et al., 1996). Therefore, the dosage of VEGF expression in the embryo is absolutely critical, as overexpression of Vegf leads to cardiac defects and developmental lethality (Miquerol et al., 2000). As a result, there are multiple mechanisms by which VEGF is controlled by hypoxia, including transcriptional upregulation,

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mRNA stabilization, and the presence of an internal ribosomal entry site to allow for maintenance of translation under hypoxic conditions (Forsythe et al., 1996; Levy et al., 1998; Stein et al., 1998). Although mature vessels are mostly quiescent, angiogenesis also occurs in the adult, for example, during wound healing and in response to hypoxic stress such as myocardial ischemia, and VEGF signaling is critical in triggering this process. In addition, abnormal angiogenesis is a feature of a number of pathological conditions including diabetes, hypertension, and cancer (Carmeliet, 2003). Dysregulation of the signals governing angiogenesis and the maturation of vessels can result either in insuYcient vascularization or in excessive formation of vessels that are often abnormal, immature, or leaky (Carmeliet, 2003). It has been suggested that a population of bone marrow‐ derived endothelial progenitor cells (EPCs) exists in the adult and is recruited by VEGF for neovascularization (Asahara et al., 1997; Grunewald et al., 2006; Khakoo and Finkel, 2005; Lyden et al., 2001; Rafii and Lyden, 2003; Reyes et al., 2002; Takahashi et al., 1999; Urbich and Dimmeler, 2004). In addition, hematopoietic cells of the myeloid lineage that express the angiopoietin receptor Tie‐2 (Tie2‐expressing monocytes or TEMs) have been shown to originate from the bone marrow and circulate in the peripheral blood (De Palma et al., 2003, 2005; Grunewald et al., 2006). Elimination of these TEM cells resulted in a lack of angiogenesis and tumor growth, suggesting that these cells may play an important role in tumor vascularization (De Palma et al., 2003, 2005; Lyden et al., 2001). The potential importance of bone marrow‐derived myeloid cells in tumor angiogenesis will be further discussed in Section VII.

III. The Hypoxia‐Inducible Factor Family A. Structure of HIF Proteins HIF proteins are members of a larger, evolutionarily conserved group of proteins known as bHLH‐PAS (basic helix loop helix‐Per ARNT Sim) proteins (Crews, 1998). These proteins function as sensors of environmental stimuli and activate the expression of genes important for angiogenesis, as well as circadian rhythms, xenobiotic detoxification, and adaptation to hypoxia (Crews and Fan, 1999; Gu et al., 2000; Kewley et al., 2004). Each member of this family contains an N‐terminal bHLH domain that mediates binding to consensus DNA sequences in the promoters of target genes (Murre et al., 1989). HIF proteins heterodimerize via their HLH and PAS domains in the center of each protein to form functionally active transcription factors (Wang and Semenza, 1995; Wang et al., 1995). The C‐terminus consists of one or two transactivation domains (TADs) that bind transcription

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Figure 2 Generalized structure of human HIF proteins. HIF proteins bind DNA at target gene promoters via the basic helix‐loop‐helix (bHLH) domain at the N‐terminus. The HLH and PAS (Per‐ARNT‐Sim) domains mediate heterodimerization of and subunits to form an active transcription complex. HIF1 and HIF2 each contain two transactivation domains (TADs); the C‐TAD binds to the coactivator proteins p300 and CBP. Stability of the HIF proteins is regulated by hydroxylation of two proline residues located within the oxygen‐ dependent degradation domain (ODD) via the prolyl hydroxylase (PHD) enzymes. In addition, hydroxylation of an asparagine residue located within the C‐terminus by the factor inhibiting HIF‐1 (FIH‐1) enzyme prevents association of HIF with p300/CBP.

cofactors such as p300 and CREB binding protein (CBP) (Fig. 2) (Arany et al., 1996; Carrero et al., 2000; Ebert and Bunn, 1998; Ema et al., 1999; Jiang et al., 1997b; O’Rourke et al., 1999; Pugh et al., 1997). The HIF family is comprised of three subunits: HIF1 [also known as MOP1 (member of PAS1)], HIF2 [also known as EPAS1 (endothelial PAS domain protein 1), MOP2, HLF (HIF1 ‐like factor), and HRF (HIF‐related factor)], and HIF3 (also known as MOP3); and three subunits: HIF / ARNT (aryl hydrocarbon nuclear translocator), ARNT2, and ARNT3 (Ema et al., 1997; Flamme et al., 1997; Gu et al., 1998; Tian et al., 1997; Wang and Semenza, 1995; Wang et al., 1995). HIF proteins form heterodimers of and subunits; HIF1 /ARNT and HIF2 /ARNT complexes have been shown to be primarily responsible for the hypoxic induction of angiogenesis (Jiang et al., 1996; Wenger and Gassmann, 1997). These heterodimeric protein complexes activate transcription by binding to hypoxia response elements (HREs), the first of which was identified in the Epo gene and contains a core sequence of CTACGTGCT (Semenza and Wang, 1992; Semenza et al., 1991; Wang et al., 1995). HIF1 and HIF2 are highly homologous proteins that share approximately 48% overall identity with the highest degree of similarity in the amino‐terminus (Ema et al., 1997; Tian et al., 1997). In contrast, there is some divergence in the C‐terminal sequences, which may help to explain diVerences in the genes activated by each subunit (see later). Expression of the principal subunit, ARNT, is constitutive, whereas subunit levels are tightly regulated and tissue‐restricted, allowing for spatial and temporal control of HIF‐dependent gene activation. HIF1 has been shown to be expressed nearly ubiquitously in humans and mice, whereas HIF2 expression is more spatially restricted. For example, HIF2 is highly

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expressed in tissues such as ECs, from which it was first identified (Ema et al., 1997; Jain et al., 1998; Tian et al., 1997). HIF2 mRNA is also detected in highly vascularized organs such as the heart, placenta, and lung (Ema et al., 1997; Tian et al., 1997). HIF2 protein is expressed during development in the lung and neural crest derivatives as well as in hypoxic tissues in the adult including bone marrow macrophages, kidney epithelial cells, liver parenchyma, cardiac myocytes, and pancreatic parenchymal cells (Ema et al., 1997; Talks et al., 2000; Wiesener et al., 2003).

B. Regulation of HIF Activity Activation of the HIF pathway is regulated at many levels, including mRNA expression, protein stability, subcellular localization, and activity, to ensure timely and eYcient induction of the hypoxic response. Changes in cellular oxygen concentrations are the major stimuli governing the regulation of HIF activity, but several nonhypoxic factors can also stimulate HIF. For example, a number of growth factors, such as insulin, epidermal growth factor, and PDGF, as well as cytokines, like tumor necrosis factor‐ and interleukin‐ 1 , can increase expression of the HIF1 protein (Haddad and Land, 2001; Stiehl et al., 2002; Treins et al., 2002; Zelzer et al., 1998; Zhou et al., 2003). Importantly, however, the amplitude of HIF induction by such stimuli is much lower than hypoxic activation of HIF (Arsham et al., 2002). In addition, oncogenes such as HER2neu, H‐ras, and v‐Src enhance HIF1 activity (Chen et al., 2001; Jiang et al., 1997a; Laughner et al., 2001). These results emphasize the importance of HIF activation during normal growth and development, as well as during the pathological processes of inflammation and tumorigenesis. The principal mechanism of HIF regulation is its posttranslational modification and subsequent degradation under normoxia. Both HIF and HIF subunits are constitutively transcribed and translated; however, regulation of the stability of the HIF subunits controls HIF‐dependent gene expression. Under normoxic conditions the subunits have a very short half‐life and are undetectable, whereas these proteins are rapidly stabilized in response to hypoxic stimulation (Salceda and Caro, 1997; Wang et al., 1995). The subunits contain an oxygen‐dependent degradation domain (ODD) (Fig. 2) that contains two conserved proline residues (Pro402 and Pro564 in humans) as part of an LXXLAP consensus motif. The ODD is suYcient for destabilization and degradation of HIF under normoxia (Huang et al., 1998; Kaelin, 2002). These proline residues are hydroxylated by a family of iron‐dependent dioxygenases in oxygenated cells known as the prolyl hydroxylase domain proteins (PHD1, ‐2, and ‐3 in humans) (Bruick and McKnight, 2001; Epstein

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et al., 2001; Ivan et al., 2001; Jaakkola et al., 2001; Masson et al., 2001; Yu et al., 2001). The C. elegans homolog of the PHD enzymes was first identified as a gene (egl‐9) associated with an abnormal egg‐laying phenotype, and orthologs have subsequently been described in D. melanogaster, mice, rats, and humans (Bruick and McKnight, 2001; Epstein et al., 2001). The hydroxylation reaction adds one atom of oxygen to form hydroxyproline with the production of succinate and carbon dioxide as by‐products. PHD enzymes function similarly to collagen prolyl hydroxylases and require molecular oxygen as a substrate in addition to iron, ascorbate, and 2‐oxoglutarate as cofactors. Therefore, these enzymes are catalytically inactive in the absence of oxygen or in the presence of iron chelators and cobalt ions that abolish iron binding (Masson and RatcliVe, 2003). In addition to the PHD enzymes, other studies have demonstrated that mitochondria act as cellular oxygen sensors that activate HIF through the generation of ROS (Brunelle et al., 2005; Chandel et al., 1998, 2000; Guzy et al., 2005; Mansfield et al., 2005). Hydroxylation of HIF1 and HIF2 subunits at these key proline residues under normal oxygen tensions provides a recognition site for binding of the von Hippel‐Lindau protein (pVHL), which is the substrate‐recognition component of a complex consisting of elongins B and C, Cul2, and Rbx‐1 that mediates the polyubiquitination of HIF proteins (Fig. 3) (Cockman et al., 2000; Ivan et al., 2001; Iwai et al., 1999; Jaakkola et al., 2001; Kamura et al., 1999; Kibel et al., 1995; Lisztwan et al., 1999; Lonergan et al., 1998; Maxwell et al., 1999; Pause et al., 1997; Yu et al., 2001). Following binding of pVHL, HIF subunits are rapidly degraded via the 26S proteasome, thereby preventing transactivation of HIF target genes under normoxia (Kim and Kaelin, 2003). PHD enzymatic activity is inhibited in hypoxic cells and unhydroxylated HIF proteins escape recognition by pVHL. Stabilized HIF then translocates to the nucleus, where it binds to its dimerization partner ARNT to form an active transcription complex (Fig. 3) (Kallio et al., 1999; Tanimoto et al., 2000). pVHL is encoded by the VHL tumor suppressor gene, which is mutated in a hereditary cancer syndrome known as VHL disease (see Section VII for more detail) (Gnarra et al., 1997; Iliopoulos et al., 1995; Latif et al., 1993). Briefly, germline mutation of VHL results in predisposition to the development of a spectrum of tumors including clear cell renal cell carcinoma (RCC), cerebellar and retinal hemangiomas, and pheochromocytomas (Ivan and Kaelin, 2001). These tumors arise on loss or inactivation of the second allele of VHL in somatic tissues and are highly vascularized with elevated levels of VEGF and Epo, two important HIF target genes. Loss of pVHL activity mimics a hypoxic state and allows for constitutive HIF activity under normoxic conditions (Iliopoulos et al., 1996; Maxwell et al., 1999). A second level of HIF regulation occurs through the hydroxylation of asparagine 803 in the C‐terminal activation domain in the presence of

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Figure 3 Regulation of HIF activity. Under normoxic conditions, HIF subunits are polyubiquitinated at two proline residues within the oxygen‐dependent degradation domain (ODD) by a family of enzymes known as prolyl hydroxylases (PHDs). This promotes recognition by the pVHL E3 ubiquitin ligase complex and subsequent degradation of HIF via the proteasome. In addition, hydroxylation of a C‐terminal asparagine residue of HIF by factor inhibiting HIF‐1 (FIH‐1) prevents binding of cofactors required for HIF activity. However, hypoxia inhibits the activity of the PHD and FIH‐1 enzymes, allowing HIF proteins to escape recognition by pVHL, be stabilized, and translocate to the nucleus. There they dimerize with HIF /ARNT and bind hypoxia response elements (HREs) within the promoters of target genes. Together with the coactivator proteins p300 and CBP, the HIF complex activates the transcription of a panel of genes required for the response to hypoxia.

oxygen. This reaction is accomplished by a dioxygenase known as factor inhibiting HIF‐1 (FIH‐1) (Mahon et al., 2001). Hydroxylation of this asparagine residue inhibits HIF activity by preventing the recruitment of the coactivators p300 and CBP (Hewitson et al., 2002; Lando et al., 2002; McNeill et al., 2002). Since FIH‐1 requires oxygen for catalytic activity, the C‐terminus of HIF remains unmodified under hypoxia and can interact with its cofactors to productively activate transcription of its target genes.

C. HIF Target Genes The first HIF target gene identified was Epo, which stimulates red blood cell production and is markedly upregulated on hypoxic exposure (Semenza and Wang, 1992; Wang et al., 1995). It was soon realized, however, that HIF functions as a master regulator of the long‐term response and adaptation to

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hypoxia by activating the transcription of close to 200 genes (Semenza, 2003). These genes are involved in a broad range of cellular processes as shown in Table I. For example, HIF induces the expression of a number of metabolic genes that maintain glucose uptake and ATP production through glycolysis in the face of oxygen deprivation. These genes include, but are not limited to, Glut‐1, lactate dehydrogenase A (LDHA), phosphoglycerate kinase (PGK), and aldolase A (Hu et al., 2003, 2006; Semenza, 2003). HIF can also enhance the expression of genes that modulate cell proliferation, viability, and apoptosis, such as insulin‐like growth factor‐2 (IGF‐2), the cyclin dependent kinase inhibitor p21, and the proapoptotic gene Bnip3 (Bruick, 2000; Feldser et al., 1999; Goda et al., 2003; Hu et al., 2003, 2006; Sowter et al., 2001). Importantly, a large percentage of genes induced in the hypoxic response is involved in various steps of angiogenesis. One of the most important of such genes is VEGF, which is a primary regulator of the formation of new blood vessels (Forsythe et al., 1996). HIF also induces the expression of VEGF‐R1 (Flt‐1), VEGF‐R2 (Flk‐1), plasminogen activator inhibitor‐1 (PAI‐1), Ang‐1 and ‐2, the Tie‐2 receptor, and MMP‐2 and ‐9 (Ben‐Yosef et al., 2002; Currie et al., 2002; Elvert et al., 2003; Gerber et al., 1997; Kietzmann et al., 1999, 2003; Melillo et al., 1997; Tian et al., 1997). Furthermore, hypoxic stabilization of HIF results in the induction of genes that control vascular tone and blood flow, such as nitric oxide synthases (iNOS) and adrenomedullin (ADM), as well as inflammatory cytokines such as interleukin‐8 (IL‐8) (Cejudo‐Martin et al., 2002; Desbaillets et al., 1999; Garayoa et al., 2000; Melillo et al., 1995, 1997). The net result of the activation of these genes is the stimulation of increased vessel formation and remodeling to provide adequate oxygen delivery to hypoxic tissues. HIF‐ dependent expression of these genes is essential for developmental angiogenesis and the response to ischemic insults, and upregulation of proangiogenic HIF target genes has been demonstrated to occur in tumorigenesis. As stated earlier, HIF1 and HIF2 are the primary regulators of genes essential for the hypoxic response. In addition to the tissue‐specific expression patterns of HIF1 and HIF2 , there are diVerences in the target genes activated by each subunit. These two proteins share a number of targets, suggesting that there may be redundancy in the function of HIF subunits (Hu et al., 2003). However, there are likely diVerences in the genes activated by each protein depending on the cell type, even when both subunits are present. HIF1 has been shown to uniquely activate the glycolytic enzymes, including PGK and aldolase A, while Oct‐4, a transcription factor involved in maintaining pluripotentiality of ES cells, appears to be regulated by HIF2 but not HIF1 (Covello et al., 2005, 2006; Hu et al., 2003). The mechanisms governing target gene specificity are the subject of investigation.

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Table I Representative Examples of Genes that Are Regulated by HIF Signaling Under Hypoxia Partial List of Genes Transcriptionally Activated by HIF Cell proliferation and apoptosis Insulin‐like growth factor‐2 (IGF‐2) Transforming growth factor‐ (TGF‐ ) Transforming growth factor‐ (TGF‐ ) p21 Bnip3 Glucose metabolism Phosphoglycerate kinase 1 (PGK) Glucose transporter‐1 (Glut‐1) Glucose transporter‐3 (Glut‐3) Lactate dehydrogenase A (LDHA) Aldolase A Phosphofructokinase L (PFKL) pH regulation Carbonic anhydrase IX (CAIX) Erythropoiesis Erythropoietin (Epo) Iron metabolism Transferrin Transferrin receptor Extracellular matrix metabolism Matrix metalloproteinase‐2 (MMP‐2) Fibronectin Inflammation Interleukin‐8 (IL‐8) Chemokine receptor CXCR4 Stromal cell‐derived factor‐1 (SDF‐1 ) Transcription factors Oct‐4/Pou5F1 Angiogenesis and control of vascular tone Vascular endothelial growth factor (VEGF) VEGF‐receptor 1 (VEGF‐R1/Flt‐1) VEGF‐receptor 2 (VEGF‐R2/Flk‐1) Platelet‐derived growth factor‐ (PDGF‐ ) Angiopoietin‐2 (Ang‐2) Tie‐2 Endothelin‐1 (ET‐1) Plasminogen activator inhibitor‐1 (PAI‐1) Inducible nitric oxide synthase 2 (iNOS2) Adrenomedullin (ADM)

Although the primary role of HIF is the direct activation of target genes, it has been appreciated that there is substantial cross‐talk between the HIF pathway and other signaling pathways. This interaction between HIF and

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other cellular signal transduction molecules may serve to amplify and broaden the hypoxic response. For example, hypoxia has been shown to inhibit the diVerentiation of cell types including myogenic cells and neural stem cells through enhanced Notch signaling in a manner that is dependent on HIF1 function (Gustafsson et al., 2005). HIF also has an eVect on genes regulated by c‐Myc; HIF1 inhibits the ability of c‐Myc to repress p21, resulting in hypoxia‐induced cell cycle arrest (Goda et al., 2003; Koshiji et al., 2004; Mack et al., 2005). HIF1 interacts directly with and stabilizes the tumor suppressor p53, whereas p53 targets HIF1 for ubiquitination (An et al., 1998; Ravi et al., 2000). Furthermore, links between HIF and other transcription factors, including c‐Jun and NF‐B, have been suggested (Alfranca et al., 2002; Bracken et al., 2005; Gerald et al., 2004; Jung et al., 2003a,b; Laderoute et al., 2002; Walmsley et al., 2005; Zhou et al., 2003). It is likely that the eVect of hypoxia on gene expression and the role of HIF in regulating transcription will become increasingly complex as additional mechanisms of cross‐talk are elucidated.

IV. The Role of HIF in Developmental Angiogenesis Hypoxia is an important feature of embryonic development: as the embryo grows and develops, it quickly outstrips the oxygen and nutrients provided by diVusion alone (Maltepe and Simon, 1998). These naturally occurring hypoxic gradients trigger the expression of genes critical for the formation of a complex network of blood vessels to provide adequate oxygenation of the dividing tissues. A number of HIF target genes has been shown to be essential for proper vascular development, including Vegf and Epo (Carmeliet et al., 1996; Ferrara et al., 1996; Kieran et al., 1996; Lin et al., 1996; Wu et al., 1995, 1999). Mice lacking these genes die during embryonic development with severe vascular and hematopoietic defects. Furthermore, gene dosage of Vegf in the yolk sac and embryo is critical for proper angiogenesis and development (Damert et al., 2002; Duan et al., 2003; Miquerol et al., 2000). These results demonstrate the importance of the expression of HIF targets during development. In agreement with these findings and with the role of HIF as the central mediator of the hypoxic response, inactivation of HIF subunits in mouse models also results in embryonic lethality. Targeted deletion of the Arnt gene encoding HIF1 /ARNT leads to lethality between days 9.5 and 10.5 of embryonic development (E9.5–10.5). The absence of Arnt expression resulted in defective blood vessel formation in the yolk sac and ineYcient vessel remodeling and maturation in the embryo (Kozak et al., 1997; Maltepe et al., 1997). Loss of Arnt resulted in significantly reduced hematopoietic progenitor development in the yolk sac; this defect was cell extrinsic

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and could be rescued with exogenous VEGF (Adelman et al., 1999). Explants from Arnt/ embryos also displayed abnormal hematopoiesis as well as a failure to undergo vasculogenesis or angiogenesis (Ramirez‐Bergeron et al., 2006). Again, administration of recombinant VEGF rescued these vascular defects, confirming that VEGF secreted by hematopoietic cells is necessary for vessel development (Ramirez‐Bergeron et al., 2006). Furthermore, Arnt deficiency resulted in aberrant architecture of the placenta, the vascular tissue that provides nutrients to the growing embryo (Adelman et al., 2000). Aggregation of Arnt/ ES cells with tetraploid wild‐type embryos rescued the placental defect; however, these embryos still exhibited defects in yolk sac vascularization and malformations in the endocardial cushion, leading to impaired cardiac function (Adelman et al., 2000). These phenotypes strongly support the essential role of HIF signaling, through the activation of targets such as VEGF and Epo, in both extraembryonic and embryonic tissues for the establishment of the vasculature and the hematopoietic system. Another important molecule in the oxygen‐sensing pathway, VHL, also plays an important role in angiogenesis. Vhl / mice die between E10.5 and E12.5 due to defects in placental vascularization with an absence of embryonic blood vessels in the placental labyrinth, hemorrhaging, and necrosis (Gnarra et al., 1997). EC‐specific deletion of the Vhl gene is embryonic lethal at E12.5 with significant hemorrhaging in the head and heart regions, a disorganized and dilated yolk sac vasculature, and defective embryonic vasculogenesis in the placenta (Tang et al., 2006). Deletion of Hif1a expression in these mice did not rescue this phenotype, suggesting that the role of Vhl in embryonic angiogenesis is independent of HIF1 . pVHL has been shown to bind to fibronectin, an ECM protein that is necessary for proper vessel formation (Ohh et al., 1998; Stickle et al., 2004). Tang et al. (2006) showed that loss of Vhl expression in ECs resulted in reduced fibronectin deposition around vessels both in the yolk sac and embryo, leading to increased EC permeability and impaired EC migration. Therefore, pVHL regulation of fibronectin assembly, in addition to its role in negatively regulating the HIF pathway, is required for embryonic angiogenesis. Deficiency in Hif1a results in embryonic lethality during midgestation at around E10.5 similar to loss of Arnt (Iyer et al., 1998; Ryan et al., 1998). Hif1a/ ES cells expressed decreased levels of glycolytic enzymes and VEGF mRNA in response to hypoxia, confirming the role of HIF1 in regulating hypoxic gene expression. In addition, loss of Hif1a resulted in decreased ES cell proliferation. Hif1a/ embryos were developmentally delayed with fewer somites than wild‐type embryos, and displayed a disorganized yolk sac vasculature lacking branching. Mutant embryos developed severe defects in neuronal development, such as failure of the neural tube to close, and significant cardiovascular abnormalities, including defective

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ventricle formation (Compernolle et al., 2003; Iyer et al., 1998; Ryan et al., 1998). These phenotypes correlated with increased hypoxia in the neural ectoderm and enhanced apoptosis of cephalic mesenchymal cells compared to wild‐type embryos (Iyer et al., 1998; Ryan et al., 1998). Moreover, although vascularization of the embryo initiated properly in Hif1a‐deficient embryos, these vessels, especially those in the cephalic region, regressed by E9.5 and did not form a complete capillary network (Iyer et al., 1998; Ryan et al., 1998). In agreement with these results, neural cell‐specific deletion of Hif1a led to reduced expression of HIF targets, including VEGF, and decreased vessel density in the brain at E19, which was accompanied by enhanced apoptosis (Tomita et al., 2003). These findings demonstrate that HIF1 is necessary for cardiovascular development, neural crest cell migration, and embryonic angiogenesis. Four independent mouse models for Hif2a deficiency have been generated, each of which is embryonic lethal but with dramatically diVerent phenotypes (Compernolle et al., 2002; Peng et al., 2000; Scortegagna et al., 2003a; Tian et al., 1998). In the first of these models, Hif2a‐deficient embryos died by E16.5 due to bradycardia and cardiac failure. This was attributed to decreased production of catecholamines from the Hif2a‐expressing organ of Zuckerkandl, possibly due to reduced tyrosine hydroxylase expression (Tian et al., 1998). Despite the initial identification of the Hif2a gene in ECs, no vascular defects were observed in these embryos. Mutant embryos in the second model died between E9.5 and E13 with abnormal vessel remodeling postvasculogenesis and hemorrhaging in the yolk sac and embryo (Peng et al., 2000). The third model demonstrated 50% lethality by E13.5 as a result of cardiac failure, while the Hif2a/ mice that survived died later as neonates of respiratory distress syndrome (Compernolle et al., 2002). This phenotype was found to be the result of reduced VEGF levels and consequently decreased surfactant production by alveolar type 2 cells within the lung. This result suggests a role for HIF2 in the regulation of VEGF expression in and development of the embryonic lung, which expresses high levels of HIF2 (Ema et al., 1997). In contrast to these previous models, Scortegagna et al. (2003a) were able to obtain viable Hif2a‐deficient adult mice, although at a significantly reduced frequency, by intercrossing mice from two genetic backgrounds. These mice presented with multiple organ pathology, the phenotype of which is discussed in greater detail in Section V. The diVerences in these phenotypes are likely attributable to modifying genes in the various genetic backgrounds used in each model. Nevertheless, the impaired embryonic survival observed in the absence of HIF2 expression emphasizes that HIF1 and HIF2 have distinct and nonoverlapping, essential functions in developmental angiogenesis. Further evidence of the importance of HIF2 in development comes from recent work that identified Oct‐4/Pou5F1 as a HIF2 ‐unique target gene

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(Covello et al., 2006). Using a knock‐in approach, the Hif1a locus was replaced with Hif2a, resulting in expanded HIF2 expression and a corresponding increase in Oct‐4 levels. Enhanced HIF2 expression is embryonic lethal, suggesting that there is a dose‐dependent requirement for HIF2 ‐controlled Oct‐4 expression during hematopoietic stem cell diVerentiation and for the survival of primordial germ cells (Covello et al., 2006).

V. The Role of HIF in Adult Tissues In addition to its role in development, HIF is important for the physiological hypoxic response in the adult. Mice heterozygous for Hif1a have an impaired response to chronic hypoxic exposure, including delayed erythropoiesis and decreased pulmonary vascular remodeling (Yu et al., 1999). HIF1 is induced in keratinocytes during wound healing of the skin, overlapping to some extent with VEGF expression (Elson et al., 2000). Several groups have used conditional gene targeting of a floxed Hif1a allele to study the function of HIF1 in specific adult tissues. For example, deletion of Hif1a in the brain resulted in decreased numbers of cells in the cortex and defective spatial memory (Tomita et al., 2003). Hif1a has also been shown to be necessary for chondrocyte survival and for mammary gland diVerentiation and lactation, independent of its role in inducing VEGF expression and vessel formation (Schipani et al., 2001; Seagroves et al., 2003). Deletion of Hif1a in cardiac myocytes impaired contraction of cardiac muscle and decreased ATP levels in the heart. Hif1a deficiency in the heart also resulted in a reduction in VEGF expression and in the number of vessels in the left ventricle (Huang et al., 2004). Loss of Hif1a is also detrimental for the function of macrophages, as HIF1 is required for glycolysis and ATP production in these cells (Cramer et al., 2003). In the absence of Hif1a, the expression of genes, including PGK and VEGF, is significantly decreased and the migration and invasion of macrophages is impaired both in vitro and in physiologic assays. A number of the molecules released by macrophages are proangiogenic, such as VEGF, angiopoietins, IL‐8, MMP‐2, and MMP‐9, emphasizing the importance of these cells in stimulating the formation of new blood vessels in hypoxic tissues (Pollard, 2004). Hypoxia and HIF have been shown to regulate the expression of many of these factors, suggesting a role for the HIF pathway in macrophage‐induced angiogenesis (Semenza, 2003). The potential implications of HIF1 and HIF2 expression in macrophages in the context of tumor angiogenesis will be discussed more thoroughly in Section VII. Importantly, an EC‐specific deletion using Tie2‐Cre demonstrated a role for Hif1a in angiogenesis (Tang et al., 2004). Mice lacking Hif1a expression in ECs developed normally, suggesting that HIF2 may be important or

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may compensate for loss of HIF1 during formation of the embryonic vasculature. However, ECs lacking Hif1a displayed reduced proliferation and decreased tubular network formation under hypoxia, as well as an inability to eYciently form new capillaries in wound healing assays. Furthermore, mutant ECs exhibited impaired ECM penetration in matrigel and defective chemotactic migration, which was attributable to decreased VEGF expression (Tang et al., 2004). These results support the importance of HIF1 in the function of ECs in response to hypoxia and the potential role of HIF1 in neovascularization during wound healing and tumor formation (Elson et al., 2000). It remains to be determined whether these angiogenic phenotypes are secondary to the glycolytic defects that are observed in the absence of HIF1 . As described earlier, Hif2a deficiency was found to be embryonic lethal in several independent studies. However, by mating mice of two diVerent backgrounds (129 and C57), Scortegagna et al. (2003a) generated viable homozygous Hif2a‐null mice, although at less than the expected frequency. Mice lacking Hif2a were smaller in size and had shorter lifespans due to pathological phenotypes in multiple organs. For example, mutant mice developed cardiac hypertrophy, steatosis of the liver, and retinopathy. In addition, mitochondrial dysfunction was observed with increased oxidative stress, as measured by elevated levels of ROS. This finding suggests that HIF2 may regulate the expression of antioxidant enzymes that eliminate ROS. These mutant mice also had a hypocellular bone marrow with significantly reduced hematocrit levels and red blood cell numbers, likely due to a defect in the bone marrow stroma (Scortegagna et al., 2003b). In addition, Epo production was decreased in Hif2a/ kidneys, demonstrating that HIF2 is essential for the regulation of renal Epo expression and thus for global hematopoiesis (Scortegagna et al., 2005). Future studies using conditional, tissue‐specific Hif2 knockout models will likely reveal additional functions for this protein in the adult.

VI. HIF and Ischemic Injury Levels of HIF1 mRNA and protein increase following ischemic insults in a number of tissues, including the retina, heart, brain, and lung, in both mice and humans (Bergeron et al., 1999; Lee et al., 2000; Ozaki et al., 1999; Yu et al., 1998). This finding supports a role for the HIF pathway in pathophysiological angiogenesis. For example, HIF activity has been associated with ischemia in the retina that is associated with diabetic retinopathy and retinopathy of prematurity (ROP), in which retinal vessels become ischemic due to occlusion, resulting in neovascularization. This increased angiogenesis can lead to blindness if uncontrolled, and is dependent on

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enhanced VEGF expression, possibly mediated by HIF1 (Ferrara, 1999; Ozaki et al., 1999). In order to study HIF2 function in the retina, Morita et al. (2003) generated a hypomorphic Hif2a allele via the insertion of a floxed neomycin gene into exon 1. Mice homozygous for the interrupted Hif2a allele were viable and fertile without developmental abnormalities. Using an animal model of ROP in which mice were exposed to hyperoxia followed by a return to room air, these authors demonstrated that mice with reduced Hif2a expression did not undergo retinal angiogenesis and displayed a lower induction of Epo compared to wild‐type mice (Morita et al., 2003). This finding suggests that HIF2 is an important mediator of the vascular phenotype associated with ROP and therefore may represent a useful therapeutic target. In the case of myocardial tissue, blockage of coronary arteries inhibits blood flow to the heart, resulting in ischemia and induction of HIF1 expression (Lee et al., 2000). HIF1 expression correlates with enhanced VEGF production and neovascularization in animal models and in humans (Lee et al., 2000; Martin et al., 1998). Similarly, increased HIF1 mRNA is associated with expression of HIF target genes, such as glycolytic enzymes and VEGF, in brain tissue surrounding the site of ischemia (Bergeron et al., 1999; Marti et al., 2000). In patients with chronic obstructive lung disease, hypoxia results in vascular remodeling that reduces blood flow and causes heart failure. HIF1 appears to play a role in this process, as mice heterozygous for this gene display reduced pulmonary remodeling (Yu et al., 1999). HIF1 signaling has also been implicated in the pathogenesis of preeclampsia, in which placental trophoblasts fail to invade the maternal decidua, leading to defective vascular remodeling of the uterine arteries. Hypoxia in early pregnancy stimulates proliferation of placental trophoblasts through HIF1 ‐dependent activation of TGF‐ 3 expression (Caniggia et al., 1999, 2000). Downregulation of HIF activity is thought to be necessary for trophoblasts to become invasive and to allow for perfusion of the placenta through maternal blood vessels. Administration of angiogenic factors, including VEGF and FGF, and transplantation of bone marrow cells have been tested as potential treatments to enhance vascularization in ischemic disease (Rafii and Lyden, 2003; Simons, 2005; Tateishi‐Yuyama et al., 2002). The above‐mentioned findings suggest that increasing HIF1 levels may also be of therapeutic value in treating ischemic disorders through the induction of neovascularization. Evidence supporting this hypothesis comes from the finding that transgenic mice overexpressing active HIF1 in the skin have a marked increase in VEGF expression and vascularization; importantly, these vessels were functionally sound and leakage‐resistant (Elson et al., 2001). Treatment with DNA encoding the N‐terminus of HIF1 fused to the VP16 transactivation domain stimulated angiogenesis in both a rabbit hindlimb ischemia model

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and a model of myocardial infarction in the rat (Shyu et al., 2002; Vincent et al., 2000). In an alternative approach, a macrophage‐derived peptide, PR39, that inhibits HIF degradation by interacting with the proteasome has been shown to increase angiogenesis in ischemic mouse cardiac tissue (Li et al., 2000). In addition, overexpression of peptides containing the HIF prolyl hydroxylation sites impeded degradation of endogenous HIF1 , resulting in induction of HIF target genes and angiogenesis (Willam et al., 2002). Prolyl hydroxylase inhibitors may also prove to be useful in this regard (Ivan et al., 2002; Jaakkola et al., 2001). Activation of the HIF pathway may also be beneficial in ischemic preconditioning. Preconditioning by exposure to mild hypoxia has been suggested as a means to provide protection against subsequent challenge by stimulating angiogenesis or by preventing apoptosis, as is the case in the retina (Grimm et al., 2002, 2005). Exposure to lower oxygen levels or treatment with cobalt chloride induces HIF1 protein expression and oVers protection to the brain, heart, and retina in various animal models (Bergeron et al., 2000; Bolli et al., 1997; Gidday et al., 1994, 1999; Kietzmann et al., 2001; Ozaki et al., 1999; Schulz et al., 2001).

VII. HIF and Cancer A. Tumor Angiogenesis and Hypoxia The induction of angiogenesis is essential for tumor growth, survival, and progression to an invasive phenotype (Hanahan and Folkman, 1996). One of the critical factors responsible for mediating the activation of the so‐called angiogenic switch is the presence of hypoxia within solid tumors. As tumors proliferate beyond 1–2 mm3, oxygen and nutrients become limiting, resulting in the development of hypoxia. Tumor hypoxia correlates with aggressive disease and a poor prognosis in patients, and leads to resistance to radiation therapy, since this treatment requires oxygen free radicals to induce cell death (Hockel and Vaupel, 2001; Hockel et al., 1996, 1999). Cancer cells adapt to this hypoxic environment through the activation of a number of cellular pathways that stimulate glycolysis, genetic instability, invasion, and neovascularization. These processes provide the tumor with adequate energy and blood supply to allow for continued growth in the face of lower oxygen. As the master regulator of the hypoxic transcriptional response, HIF has been implicated as an important player in tumor growth and angiogenesis. HIF activates the expression of a broad range of genes that can contribute to tumorigenesis such as glucose transporters that support the enhanced rate of glycolysis in tumors, growth factors that stimulate proliferation, and metalloproteinases and chemokines that may influence metastasis and EC

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recruitment (Ben‐Yosef et al., 2002; Ceradini et al., 2004; Dang et al., 1997; Harris, 2002; Staller et al., 2003; Zagzag et al., 2005). Importantly, as mentioned earlier, HIF induces multiple genes involved in angiogenesis, most notably VEGF, which is essential for initiating and maintaining new vessel formation (Forsythe et al., 1996; Semenza, 2000).

B. HIF Expression in Tumors HIF1 is overexpressed in a variety of human cancers and correlates with highly vascular tumors (Brahimi‐Horn and Pouyssegur, 2005; Semenza, 2002). For example, HIF1 protein levels were increased in glioblastoma multiforme, prostate cancer, breast cancer, lung cancer, and pancreatic cancer (Bos et al., 2001; Giatromanolaki et al., 2001; Talks et al., 2000; Zagzag et al., 2000; Zhong et al., 1999). In addition, HIF1 is induced in a transgenic mouse model of epidermal carcinogenesis (Elson et al., 2000). HIF2 overexpression is also detected in these tumor types, with more pronounced expression than HIF1 in hepatocellular carcinoma (Bangoura et al., 2004; Talks et al., 2000). Furthermore, both subunits are expressed in RCC (see later), with progressively higher expression in more advanced tumors and a bias toward HIF2 expression (Mandriota et al., 2002; Turner et al., 2002; Wiesener et al., 2001). Immunostaining for both HIF subunits is predominantly nuclear and generally strongest in necrotic regions; there is often significant colocalization with increased levels of VEGF mRNA, especially in the case of HIF2 (Favier et al., 2001; Talks et al., 2000; Zhong et al., 1999). In several studies, HIF1 expression is associated with decreased survival and resistance to radiation therapy, suggesting that evaluation of HIF levels may have prognostic value (Aebersold et al., 2001; Brahimi‐Horn and Pouyssegur, 2005; Unruh et al., 2003). In addition to enhanced expression in tumor parenchymal cells, HIF2 is also upregulated in tumor‐associated macrophages (TAMs) within the stroma (Talks et al., 2000). TAMs are recruited to hypoxic, avascular regions of many diVerent tumors by growth factors and chemokines, and the degree of TAM infiltration correlates with tumor progression and poor prognosis (Leek and Harris, 2002; Leek et al., 2002; Pollard, 2004). For example, high expression of HIF2 in TAMs was shown to significantly correlate with high tumor vascularity and tumor grade in breast cancer and bladder cancer (Leek et al., 2002; Onita et al., 2002). Macrophages likely promote tumor growth and angiogenesis by secreting growth factors that stimulate tumor cell proliferation, MMPs that induce invasion and metastasis, and proangiogenic cytokines (Pollard, 2004). These angiogenic factors include basic fibroblast growth factor (bFGF), iNOS, angiopoietins, IL‐8, and VEGF, many of which are HIF target genes (Pollard, 2004). In particular, macrophage infiltration

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has been associated with increased VEGF expression, potentially through HIF activation, and tumor angiogenesis in several studies both in vitro and in vivo (Bingle et al., 2006; Leek et al., 1996, 2000; Lewis et al., 2000). VEGF in turn is a chemotactic factor for macrophages and monocytic endothelial precursor cells (TEMs), which may serve to further enhance vascularization within the tumor (Barleon et al., 1996; Grunewald et al., 2006; Leek et al., 2000). There are several mechanisms by which HIF is overexpressed in tumors, the primary of which is hypoxic activation. In addition, mutations in tumor suppressor genes, such as VHL (Section VII.C), p53, and PTEN, result in increased HIF1 and HIF2 (Maxwell et al., 1999; Ravi et al., 2000; Zundel et al., 2000). Conversely, activation of oncogenes, including v‐SRC, EGFR, and HER2neu, and subsequent signaling through the phosphatidylinositol‐3‐ kinase (PI‐3K) and mitogen‐activated protein (MAP) kinase pathways mediate HIF1 accumulation (Chen et al., 2001; Jiang et al., 1997a; Laughner et al., 2001). In each of these cases, activation of the HIF pathway promotes the production of angiogenic factors, such as VEGF, and therefore stimulates tumor angiogenesis (Harris, 2002; Semenza, 2000).

C. von Hippel‐Lindau Disease One of the principal lines of evidence supporting a role for HIF in tumor angiogenesis comes from analysis of the eVect of mutations in the VHL tumor suppressor gene. The hereditary cancer syndrome known as VHL disease is caused by germline mutations in VHL. Patients inherit an inactivating mutation in one allele of VHL and develop tumors on mutation or loss of the remaining allele in somatic tissue (Kaelin, 2002). The disease is characterized by a defined spectrum of highly vascular tumors that arise within specific tissues, including hemangioblastomas of the central nervous system and retina, pheochromocytoma aVecting the adrenal medulla, and RCC. In addition, VHL is also inactivated in a large percentage of sporadic hemangioblastomas and RCC through mutations, deletions, loss of heterozygosity, or methylation (Kaelin, 2002). Loss of pVHL function results in constitutive HIF activation throughout RCC tumors, even under normoxia (Wiesener et al., 2001). Enhanced expression of HIF target genes, including VEGF and TGF‐ , mediates proliferation, angiogenesis, and increased vascular permeability in these tumors (de Paulsen et al., 2001; Gunaratnam et al., 2003; Kaelin, 2002). Cell lines derived from RCC tumors display high levels of HIF activity (especially HIF2 ), and the introduction of wild‐type VHL into these cells restores proper regulation of the HIF pathway and suppresses tumor growth (Iliopoulos et al., 1995).

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Most of the tumor‐associated mutations in VHL dysregulate HIF activity, either by disrupting binding of pVHL to HIF subunits or by impairing the formation of the complete E3 ubiquitin ligase complex (CliVord et al., 2001; Duan et al., 1995; Stebbins et al., 1999). Hydroxylated HIF is bound by the ‐domain of pVHL through several conserved residues; mutations of these amino acids abrogate this interaction with HIF and are associated with tumor development. The ‐domain of pVHL is responsible for binding to elongins B and C and formation of an active E3 complex, and mutations in this domain are also found in VHL disease (Duan et al., 1995; Kamura et al., 2000; Ohh et al., 2000; Stebbins et al., 1999; Tanimoto et al., 2000). In addition, an ‐helix at the C‐terminus of pVHL is important, although not required, for stabilization of substrate binding and thus for complete ubiquitination of HIF (Lewis and Roberts, 2004). However, additional mutations are required for Vhl‐mediated tumorigenesis, since although loss of Vhl in these cells resulted in constitutive HIF stabilization, Vhl/ tumors displayed a growth disadvantage (Mack et al., 2003, 2005). In addition, HIF‐ independent functions of pVHL, such as fibronectin matrix assembly, likely play an important role in tumor development (CliVord et al., 2001; HoVman et al., 2001; Ohh et al., 1998; Stickle et al., 2004). Several groups have tried to model VHL disease in the mouse in order to gain a better understanding of the mechanisms associated with tissue‐specific tumor development. Although mice completely lacking Vhl die during embryogenesis, targeted inactivation of one allele of Vhl in the liver resulted in the formation of cavernous hemangiomas that resemble hemangioblastomas as well as polycythemia (Gnarra et al., 1997; Haase et al., 2001). HIF2 , but not HIF1 , expression was detectable in these tumors, and VEGF mRNA levels were significantly elevated (Haase et al., 2001). HIF signaling was required for the development of both hemangiomas and polycythemia in this model. However, inactivation of Hif1a alone did not suppress these phenotypes, further suggesting the importance of HIF2 in VHL disease (Rankin et al., 2005). Another mouse model has been generated in which kidney‐specific deletion of Vhl enhanced renal cyst development (Rankin et al., 2006). Furthermore, mice expressing various mutant forms of Vhl associated with VHL disease have been generated; analysis of this allelic series should help elucidate the relative contributions of HIF‐dependent and HIF‐independent functions of pVHL to tumorigenesis (Rathmell et al., 2004; Rathmell, W. K., Hickey, M. M., and Simon, M. C., unpublished observations). D. The Role of HIF1a and HIF2a in Tumor Angiogenesis Inactivation of VHL and subsequent HIF activation is detected in the early stages of RCC development and is associated with increased vascularization

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in early foci, suggesting that HIF promotes angiogenesis in these tumors (Mandriota et al., 2002). In agreement with this, loss of Vhl in murine ES cells and murine embryonic fibroblasts (MEFs) resulted in constitutive HIF activation and increased VEGF expression. As a result, Vhl/ teratocarcinomas and fibrosarcomas derived from these cells were more hemorrhagic and exhibited a higher microvessel density compared to tumors derived from wild‐type cells (Mack et al., 2003, 2005). A number of mouse xenograft studies have investigated the role of the HIF1 subunit in tumor growth and angiogenesis. These studies obtained conflicting results with regard to the role of HIF1 in tumor cell proliferation and apoptosis, probably due to diVerences in the cell types used. Some groups found that tumors lacking HIF1 grew more slowly (Maxwell et al., 1997; Ryan et al., 1998, 2000; Tang et al., 2004), while others reported that loss of HIF1 enhanced tumor growth and decreased apoptosis (Blouw et al., 2003; Carmeliet et al., 1998). However, in each case HIF1 inactivation resulted in impaired tumor angiogenesis. For example, hepatoma cells deficient for HIF1 /ARNT formed less vascular, slow‐growing tumors with reduced VEGF levels compared to tumors derived from wild‐type cells (Maxwell et al., 1997). Deletion of HIF1 in ES cells also led to decreased VEGF expression and defective vascularization of tumors in nude mice (Carmeliet et al., 1998; Ryan et al., 1998). Similarly, Lewis lung carcinoma cells gave rise to necrotic tumors with significantly reduced microvessel density when injected into mice lacking HIF1 in ECs (Tang et al., 2004). Therefore, HIF1 likely promotes tumor vascularization by inducing the expression of VEGF as well as other angiogenic factors such as the angiopoietins and VEGF receptors. Importantly, this eVect is dependent on the tumor microenvironment; subcutaneous tumors derived from Hif1a‐ deficient astrocytes were poorly vascularized with extensive necrosis, while brain tumors derived from these cells exhibited increased microvessel density compared to wild‐type tumors (Blouw et al., 2003). HIF2 is highly expressed in ECs and neural crest cells, from which pheochromocytoma originates, as well as a number of tumors and RCC cell lines, suggesting that HIF2 may be the more important subunit in the pathogenesis of VHL disease and tumorigenesis in a broader sense. Several genes involved in tumor growth and angiogenesis, including cyclin D1, Glut‐1, TGF‐ , and VEGF, were found to be specifically regulated by HIF2 , not HIF1 , in RCC cell lines (Raval et al., 2005). Expression of a constitutively stable form of HIF2 , in which one of the critical proline residues was mutated to alanine, in VHL‐rescued RCC cells resulted in the formation of larger tumors in nude mice as compared to controls (Kondo et al., 2002, 2003). In contrast, stabilization of HIF1 alone by similar means did not induce tumor formation (Maranchie et al., 2002). Furthermore, downregulation of HIF2 with short hairpin RNAs (shRNAs) blocked the ability of VHL‐deficient RCC cells to

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form tumors in nude mice (Kondo et al., 2003). These findings demonstrate that HIF2 signaling is necessary and suYcient to induce tumorigenesis in the case of RCC cells. Further experiments are necessary to address the role of HIF2 in other types of tumors. Additional studies have reported that HIF2 contributes more significantly to tumor angiogenesis. Teratocarcinomas generated from ES cells in which Hif1a was replaced with Hif2a were larger in size with increased vascularization. This phenotype correlated with enhanced expression of genes such as VEGF, cyclin D1, and TGF‐ (Covello et al., 2005). Similarly, Acker et al. (2005) found that overexpression of HIF2 in glioma tumors stimulated VEGF expression, while inhibition of HIF2 resulted in decreased VEGF levels and significantly less vascular teratocarcinomas. Therefore, both HIF1 and HIF2 regulate tumor angiogenesis in response to hypoxia, although their respective contributions in the context of diVerent cell types needs to be further explored.

E. Therapeutic Implications The use of antiangiogenic agents to treat cancer was first proposed in 1971 by Judah Folkman, sparking a period of intense research in this area (Folkman, 1971). Despite some initial setbacks, the results of clinical trials have given new life to this idea and suggest that the inhibition of angiogenesis may in fact be a promising therapeutic strategy. For example, a monoclonal antibody against VEGF known as bevacizumab or Avastin has been shown to increase overall survival in metastatic colorectal and non‐small‐cell lung cancer patients when administered together with standard chemotherapy (Hurwitz et al., 2004; Kim et al., 1993). In addition, small molecule inhibitors, such as Sorafenib, have been developed that target multiple receptor tyrosine kinases, including the VEGF‐Rs and PDGF‐R, in both ECs and tumor cells. Treatment with Sorafenib has been shown to be eVective in patients with advanced RCC, resulting in a significant increase in progression‐free survival (Carmeliet, 2005; Ferrara and Kerbel, 2005). Other approaches to targeting tumor angiogenesis are also being developed and are in the early phases of clinical trials, including a soluble, chimeric VEGF receptor known as the VEGF‐trap, and recombinant human endostatin, an endogenous inhibitor of angiogenesis (Herbst et al., 2002; Holash et al., 2002). The success of these studies has led to the suggestion that the inhibition of upstream regulators of angiogenesis may also be eVective in the treatment of cancer. Given the significant role of HIF activity in promoting tumor growth and angiogenesis, this pathway represents an exciting target for therapeutic intervention. Modulation of HIF signaling, perhaps in combination with

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more traditional chemotherapy and/or other more direct antiangiogenic treatments may provide eYcient inhibition of tumor vascularization and proliferation. However, it will be essential to take into account the varied and complicated eVects of HIF on gene expression, such as its role in regulating apoptosis, when designing novel cancer therapies. One possibility that has been proposed is the use of HIF1 antisense therapy, which blocked T‐cell lymphoma growth in combination with T‐cell‐specific activation (Sun et al., 2001). Antisense therapy against HIF also synergized with overexpression of VHL in gliomas to increase tumor cell apoptosis and decrease tumor angiogenesis (Sun et al., 2006). In addition, blockade of the interaction between HIF and its coactivators CBP/p300 by polypeptides corresponding to the HIF1 transactivation domain suppressed hypoxia‐inducible gene expression and attenuated tumor growth in a xenograft model (Kung et al., 2000). Tumor‐specific death may be achieved by using gene therapy systems in which HREs drive the expression of proapoptotic or antiproliferation genes (Dachs et al., 1997). Alternatively, hypoxic tumor cells can be specifically targeted by anaerobic bacteria expressing prodrugs that inhibit tumor growth (Agrawal et al., 2004; Bettegowda et al., 2003; Dang et al., 2001; Lemmon et al., 1997). Macrophages engineered to express adenoviral vectors may also be useful for targeting therapeutic genes and drugs to hypoxic tumors (GriYths et al., 2000). Inhibition of HIF could also enhance the response of tumors to radiation therapy. Irradiation has been shown to induce HIF1 through the production of ROS, resulting in the expression of VEGF and enhanced survival of ECs (Moeller et al., 2004). Inhibition of HIF1 with a small molecule, YC‐1, slowed tumor growth following irradiation (Moeller et al., 2004; Yeo et al., 2003). However, HIF can also sensitize tumors to radiation through the activation of p53‐dependent apoptosis, emphasizing the complex eVects of HIF on tumors. Therefore, radiation treatment followed by HIF blockade may prove to be an eVective cancer therapy (Moeller et al., 2005).

VIII. Conclusions The HIF pathway is an essential eVector of the cellular response to changes in oxygen concentration. Activation of HIF under hypoxia results in the induction of a broad program of gene expression that is necessary for adaptation to low oxygen. HIF signaling mediates the hypoxic response through cell autonomous mechanisms, for example, by regulating proliferation, apoptosis, and metabolism, as well as through cell nonautonomous eVects on angiogenesis. HIF‐stimulated expression of proangiogenic factors, such as VEGF, angiopoietins, VEGF receptors, MMPs, and IL‐8, is critical

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for the formation of new blood vessels in both physiological and pathophysiological settings. HIF signaling is required for proper embryonic development, particularly in the formation of the vasculature, and is important for the function of a variety of cell types in the adult. In addition, HIF expression and activity are increased under conditions of pathological hypoxia thereby leading to enhanced vascularization. Therefore, modulation of HIF represents a potential therapeutic target that may have benefits in the treatment of both ischemic disease and cancer. It is hoped that ongoing and future studies will help to further elucidate the mechanisms of HIF regulation and the importance of the hypoxic response in both normal physiology and disease.

Acknowledgments We thank members of the lab, especially Dr. Brian Keith, for helpful discussions and proofreading assistance. We apologize to those colleagues whose work could not be cited directly.

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