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Mechanisms of Glioma-Associated Neovascularization

2012; Elsevier BV; Volume: 181; Issue: 4 Linguagem: Inglês

10.1016/j.ajpath.2012.06.030

1525-2191

Matthew E. Hardee, David Zagzag,

Cancer, Hypoxia, and Metabolism

Glioblastomas (GBMs), the most common primary brain tumor in adults, are characterized by resistance to chemotherapy and radiotherapy. One of the defining characteristics of GBM is an abundant and aberrant vasculature. The processes of vascular co-option, angiogenesis, and vasculogenesis in gliomas have been extensively described. Recently, however, it has become clear that these three processes are not the only mechanisms by which neovascularization occurs in gliomas. Furthermore, it seems that these processes interact extensively, with potential overlap among them. At least five mechanisms by which gliomas achieve neovascularization have been described: vascular co-option, angiogenesis, vasculogenesis, vascular mimicry, and (the most recently described) glioblastoma-endothelial cell transdifferentiation. We review these mechanisms in glioma neovascularization, with a particular emphasis on the roles of hypoxia and glioma stem cells in each process. Although some of these processes are well established, others have been identified only recently and will need to be further investigated for complete validation. We also review strategies to target glioma neovascularization and the development of resistance to these therapeutic strategies. Finally, we describe how these complex processes interlink and overlap. A thorough understanding of the contributing molecular processes that control the five modalities reviewed here should help resolve the treatment resistance that characterizes GBMs. Glioblastomas (GBMs), the most common primary brain tumor in adults, are characterized by resistance to chemotherapy and radiotherapy. One of the defining characteristics of GBM is an abundant and aberrant vasculature. The processes of vascular co-option, angiogenesis, and vasculogenesis in gliomas have been extensively described. Recently, however, it has become clear that these three processes are not the only mechanisms by which neovascularization occurs in gliomas. Furthermore, it seems that these processes interact extensively, with potential overlap among them. At least five mechanisms by which gliomas achieve neovascularization have been described: vascular co-option, angiogenesis, vasculogenesis, vascular mimicry, and (the most recently described) glioblastoma-endothelial cell transdifferentiation. We review these mechanisms in glioma neovascularization, with a particular emphasis on the roles of hypoxia and glioma stem cells in each process. Although some of these processes are well established, others have been identified only recently and will need to be further investigated for complete validation. We also review strategies to target glioma neovascularization and the development of resistance to these therapeutic strategies. Finally, we describe how these complex processes interlink and overlap. A thorough understanding of the contributing molecular processes that control the five modalities reviewed here should help resolve the treatment resistance that characterizes GBMs. Glioblastomas (GBMs) are the most common and aggressive primary brain tumors in adults, with a median survival of only 14 months despite the best available treatments. GBMs are characterized by their resistance to radiotherapy and chemotherapy, as well as their abundant and aberrant vasculature. Neovascularization has long been implicated as a salient feature of glioma progression. In fact, high-grade gliomas are among the most vascular of all solid tumors, and vascular proliferation is a pathological hallmark of GBMs.1Brem S. Cotran R. Folkman J. Tumor angiogenesis: a quantitative method for histologic grading.J Natl Cancer Inst. 1972; 48: 347-356PubMed Google Scholar Tumor progression and resistance to both radiotherapy and chemotherapy lead to unfavorable clinical outcomes in glioma patients and are associated with the hypoxic tumor microenvironment known to exist within GBMs. Also contributing to resistance to traditional therapeutics are glioma stem cells (GSCs), which contain tumor-initiating functions and are thought to be responsible for replenishing and sustaining the glioma mass and promoting resistance to traditional cancer therapies. An increasing body of experimental evidence suggests that hypoxia and the hypoxia-inducible factors (HIFs) play a critical role in maintaining the stem-like fraction in gliomas by creating a microenvironment that provides the essential cellular interactions and environmental signals needed to prevent GSC differentiation and to support their survival and self-renewal.2Heddleston J.M. Li Z. Lathia J.D. Bao S. Hjelmeland A.B. Rich J.N. Hypoxia inducible factors in cancer stem cells.Br J Cancer. 2010; 102: 789-795Crossref PubMed Scopus (341) Google Scholar Although hypoxia is a well-known driver of neovascularization, there is also evidence demonstrating that non-hypoxia-driven mechanisms exist, including p53 and hypoxia-independent vascular endothelial growth factor (VEGF)-mediated pathways.Glioma cells are able to sense and adapt to hypoxic environments. HIF-1 is a heterodimeric nuclear transcription factor3Semenza G.L. Defining the role of hypoxia-inducible factor 1 in cancer biology and therapeutics.Oncogene. 2010; 29: 625-634Crossref PubMed Scopus (1307) Google Scholar that consists of two subunits, HIF-1α and HIF-1β. The HIF-1α subunit determines HIF-1 activity in response to changes in local O2 levels. Under normoxic conditions, the α subunit is rapidly degraded; under hypoxic conditions, however, this subunit remains intact and binds to the constitutively expressed β subunit to form HIF-1 in the cell nucleus, where it induces expression of many genes under the regulation of hypoxia response elements. This process triggers the up-regulation of multiple proangiogenic factors, the most studied and prominent of which is VEGF. The resulting migration and proliferation of endothelial cells are key events in the angiogenic cascade.Most studies have focused on the HIF-1α subunit, and less is known about the role of HIF-2α in tumor progression. Several studies have shown that HIF-2 may be involved in maintenance of GSCs. Both HIF-1α and HIF-2α are necessary for GSC maintenance. Furthermore, overexpression of HIF-2α promotes a cancer stem-cell like phenotype in preclinical models of GBM.4Heddleston J.M. Li Z. McLendon R.E. Hjelmeland A.B. Rich J.N. The hypoxic microenvironment maintains glioblastoma stem cells and promotes reprogramming towards a cancer stem cell phenotype.Cell Cycle. 2009; 8: 3274-3284Crossref PubMed Scopus (577) Google ScholarOriginally described simply as capillary sprouting from pre-existing host tissue capillaries (ie, angiogenesis), the process by which solid growing tumors generate an increasing blood supply to meet their ever-increasing nutrient and oxygen demand is now recognized as a highly complex spectrum of events. At least five distinct mechanisms of neovascularization in GBMs have been identified: i) vascular co-option, ii) angiogenesis, iii) vasculogenesis, iv) vascular mimicry, and v) glioblastoma-endothelial cell transdifferentiation. These mechanisms are not independent of one another, but rather are interlinked and are controlled, at least in part, by similar processes. Here, we review the evidence for and potential molecular mechanisms of each of these processes and discuss the experimental data for the roles of hypoxia and stem cells in each of the five mechanisms. In addition, we review the rationale for the targeting of neovascularization in gliomas (eg, antiangiogenic therapeutic strategies) and discuss the potential molecular mechanisms that could explain escape from antiangiogenic therapy. Finally, we describe how these complex mechanisms of vascularization might be interlinked. Although we focus our review on neovascularization in gliomas, there is evidence that many of these processes occur in a broad range of malignancies.Our objective here is to provide a thorough and comprehensive review of the mechanisms of glioma-associated neovascularization. We discuss processes that have now become widely accepted by the scientific community, such as vascular co-option, angiogenesis, and vasculogenesis. We also describe vascular mimicry and transdifferentiation, processes that are only beginning to be explored. Because they have been identified only rather recently, the vascular mimicry and transdifferentiation processes will require confirmation and validation before they can be widely accepted. Furthermore, the contribution of these two mechanisms to the process of neovascularization on a whole-tumor scale may vary considerably among tumor types and for now remains largely unknown.Vascular Co-OptionTemporally, vascular co-option is the first mechanism by which gliomas achieve their vasculature (Figure 1). This process involves organization of tumor cells into cuffs around normal microvessels. Holash et al5Holash J. Maisonpierre P.C. Compton D. Boland P. Alexander C.R. Zagzag D. Yancopoulos G.D. Wiegand S.J. Vessel cooption, regression, and growth in tumors mediated by angiopoietins and VEGF.Science. 1999; 284: 1994-1998Crossref PubMed Scopus (1889) Google Scholar were the first to definitively demonstrate vessel co-option, using a rat C6 glioma model. Early tumors were well vascularized through vessel co-option, and it was not until approximately 4 weeks after implantation that, after vascular regression, a robust angiogenic response was seen at the viable tumor periphery. In the interim, the majority of tumor vasculature was co-opted from normal brain vasculature. Co-opted vessels have been shown to express angiopoietin-2 (ANG-2).5Holash J. Maisonpierre P.C. Compton D. Boland P. Alexander C.R. Zagzag D. Yancopoulos G.D. Wiegand S.J. Vessel cooption, regression, and growth in tumors mediated by angiopoietins and VEGF.Science. 1999; 284: 1994-1998Crossref PubMed Scopus (1889) Google Scholar, 6Zagzag D. Amirnovin R. Greco M.A. Yee H. Holash J. Wiegand S.J. Zabski S. Yancopoulos G.D. Grumet M. Vascular apoptosis and involution in gliomas precede neovascularization: a novel concept for glioma growth and angiogenesis.Lab Invest. 2000; 80: 837-849Crossref PubMed Scopus (231) Google ScholarThe angiopoietins constitute a family of factors that bind competitively on TIE-2. ANG-2 functions mainly as an antagonist of ANG-1, but both pro- and antiangiogenic functions have been described for both ANG-1 and ANG-2. Although the situation is more complex, it is thought that ANG-1 acts predominantly in pericyte recruitment and maintenance of vessel integrity and that up-regulation of ANG-2 expression leads to vessel destabilization. At this stage, and in the presence of VEGF, angiogenic vessel sprouting occurs. In the absence of VEGF, however, ANG-2 promotes endothelial cell apoptosis and vessel regression.5Holash J. Maisonpierre P.C. Compton D. Boland P. Alexander C.R. Zagzag D. Yancopoulos G.D. Wiegand S.J. Vessel cooption, regression, and growth in tumors mediated by angiopoietins and VEGF.Science. 1999; 284: 1994-1998Crossref PubMed Scopus (1889) Google Scholar, 7Reiss Y. Machein M.R. Plate K.H. The role of angiopoietins during angiogenesis in gliomas.Brain Pathol. 2005; 15: 311-317Crossref PubMed Scopus (91) Google ScholarA temporal study of experimental glioma vascularization identified vascular co-option as an initial step of a cascade of events in implanted murine GL261 gliomas.6Zagzag D. Amirnovin R. Greco M.A. Yee H. Holash J. Wiegand S.J. Zabski S. Yancopoulos G.D. Grumet M. Vascular apoptosis and involution in gliomas precede neovascularization: a novel concept for glioma growth and angiogenesis.Lab Invest. 2000; 80: 837-849Crossref PubMed Scopus (231) Google Scholar As early as 1 week after glioma cell implantation, vascular co-option was observed, with endothelial cell apoptosis appearing by week 3, resulting in vascular regression and regions of necrosis followed by angiogenesis. Involution of co-opted vessels resulted in tumor hypoxia, up-regulation of proangiogenic factors, and a shift toward an angiogenic phenotype. Rong et al8Rong Y. Durden D.L. Van Meir E.G. Brat D.J. 'Pseudopalisading' necrosis in glioblastoma: a familiar morphologic feature that links vascular pathology, hypoxia, and angiogenesis.J Neuropathol Exp Neurol. 2006; 65: 529-539Crossref PubMed Scopus (386) Google Scholar reviewed pseudopalisading necrosis in GBM and described a similar sequence of events in human GBM.Using in vivo multiphoton laser scanning microscopy and the same GL261 murine glioma cell line, Winkler et al9Winkler F. Kienast Y. Fuhrmann M. Von Baumgarten L. Burgold S. Mitteregger G. Kretzschmar H. Herms J. Imaging glioma cell invasion in vivo reveals mechanisms of dissemination and peritumoral angiogenesis.Glia. 2009; 57: 1306-1315Crossref PubMed Scopus (150) Google Scholar described the invading potential of glioma cells when in close contact with brain microvessels. At the invasive border of the main tumor, vascularization occurred via co-option of pre-existing brain vessels, rather than by angiogenesis.Possible molecular links between hypoxia and vascular co-option include the up-regulation of ANG-2 by hypoxia through HIF-1–dependent mechanisms and the presence of a HIF-1 binding hypoxia response element location identified in the first intron of the ANG-2 gene (ANGPT2).10Simon M.P. Tournaire R. Pouyssegur J. The angiopoietin-2 gene of endothelial cells is up-regulated in hypoxia by a HIF binding site located in its first intron and by the central factors GATA-2 and Ets-1.J Cell Physiol. 2008; 217: 809-818Crossref PubMed Scopus (159) Google Scholar In addition, it has been shown that conditioned medium collected from neoplastic cells exposed to hypoxia promotes vascular co-option.11Das B. Yeger H. Tsuchida R. Torkin R. Gee M.F. Thorner P.S. Shibuya M. Malkin D. Baruchel S. A hypoxia-driven vascular endothelial growth factor/Flt1 autocrine loop interacts with hypoxia-inducible factor-1alpha through mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 pathway in neuroblastoma.Cancer Res. 2005; 65: 7267-7275Crossref PubMed Scopus (113) Google ScholarMontana and Sontheimer12Montana V. Sontheimer H. Bradykinin promotes the chemotactic invasion of primary brain tumors.J Neurosci. 2011; 31: 4858-4867Crossref PubMed Scopus (127) Google Scholar recently described a potential role for bradykinin in chemotaxis during vascular co-option in primary brain tumors. In glioma biopsy specimens, they demonstrated increased expression of bradykinin receptors in regions of tumor, with the highest levels in perivascular regions. Using in vitro assays, they also demonstrated increased glioma cell motility and migration/invasion in both Transwell and brain slice invasion assays in response to bradykinin, which was mediated by bradykinin-induced Ca2+ oscillations.AngiogenesisVascular co-option is followed by the development of new vessels from pre-existing ones (Figure 2), a process known as angiogenesis. This mechanism is integral to both physiological and pathological processes. Angiogenesis was described in GBM as early as 1976, when Brem13Brem S. The role of vascular proliferation in the growth of brain tumors.Clin Neurosurg. 1976; 23: 440-453PubMed Google Scholar observed intense neovascularization in rabbit corneas transplanted with GBMs, suggesting an in vivo production of a "vasoformative substance."Figure 2Angiogenesis. A: Angiogenesis follows vascular co-option during tumor vasculature development and is defined as the development of new vessels from pre-existing ones. Hypoxic pseudopalisading glioma cells around necrosis (inset) release proangiogenic factors. This results in the shift of the angiogenic balance toward a proangiogenic phenotype, inducing sprouting from pre-existing vessels. Hypoxia-independent mechanisms driving angiogenesis have also been described. B: Photomicrographs of a sectioned human GBM specimen stained for tenascin-C shows sprouting angiogenesis. Note the gradient from relatively poorly vascularized regions with little angiogenesis (upper left in each panel) to highly vascularized regions with hyperplastic vessels around necrosis (N) (lower right in each panel). Original magnification: ×20 (top); ×100 (bottom).View Large Image Figure ViewerDownload Hi-res image Download (PPT)A detailed and comprehensive description of the molecular and cellular mechanisms and biology of angiogenesis is beyond the scope of this review. Briefly, glioma-associated sprouting angiogenesis begins with an angiopoietin-mediated breakdown of existing vessels. After vascular co-option, persistent up-regulation of ANG-2 and TIE-2 in endothelial and tumor cells promotes disruption of endothelial and perivascular cell junctions, resulting in vessel disruption.5Holash J. Maisonpierre P.C. Compton D. Boland P. Alexander C.R. Zagzag D. Yancopoulos G.D. Wiegand S.J. Vessel cooption, regression, and growth in tumors mediated by angiopoietins and VEGF.Science. 1999; 284: 1994-1998Crossref PubMed Scopus (1889) Google Scholar, 7Reiss Y. Machein M.R. Plate K.H. The role of angiopoietins during angiogenesis in gliomas.Brain Pathol. 2005; 15: 311-317Crossref PubMed Scopus (91) Google Scholar, 14Zadeh G. Koushan K. Pillo L. Shannon P. Guha A. Role of Ang1 and its interaction with VEGF-A in astrocytomas.J Neuropathol Exp Neurol. 2004; 63: 978-989PubMed Google Scholar A key early event is the proteolysis of the basement membrane and extracellular matrix due to the activity of matrix metalloproteinases (MMPs). In the presence of ANG-2, VEGF promotes migration and proliferation of endothelial cells and stimulates sprouting of new blood vessels. Acquisition of the tip and stalk phenotypes among endothelial cells exposed to proangiogenic stimuli involves the delta-like 4 (DLL-4)/Notch pathway.15Hellström M. Phng L.K. Hofmann J.J. Wallgard E. Coultas L. Lindblom P. Alva J. Nilsson A.K. Karlsson L. Gaiano N. Yoon K. Rossant J. Iruela-Arispe M.L. Kalén M. Gerhardt H. Betsholtz C. Dll4 signalling through Notch1 regulates formation of tip cells during angiogenesis.Nature. 2007; 445: 776-780Crossref PubMed Scopus (1282) Google Scholar Recently, ephrin-B2 has been shown to regulate VEGF-induced endothelial tip cell guidance during angiogenesis, similar to its role in axonal guidance.16Sawamiphak S. Seidel S. Essmann C.L. Wilkinson G.A. Pitulescu M.E. Acker T. Acker-Palmer A. Ephrin-B2 regulates VEGFR2 function in developmental and tumour angiogenesis.Nature. 2010; 465: 487-491Crossref PubMed Scopus (406) Google ScholarThe final stages of angiogenesis involve capillary morphogenesis, mediated largely by integrins α3β1 and αvβ3, as well as by CD44.17Wang D. Anderson J.C. Gladson C.L. The role of the extracellular matrix in angiogenesis in malignant glioma tumors.Brain Pathol. 2005; 15: 318-326Crossref PubMed Scopus (41) Google Scholar Activated endothelial cells secrete platelet-derived growth factor (PDGF), which recruits pericytes to the newly formed vessel,18Lindahl P. Johansson B.R. Levéen P. Betsholtz C. Pericyte loss and microaneurysm formation in PDGF-B-deficient mice.Science. 1997; 277: 242-245Crossref PubMed Scopus (1716) Google Scholar aided by the ANG/TIE pathway. Negative feedback by endogenous antiangiogenic factors, as well as accumulation of extracellular matrix, may modulate the process of vascular modeling.19Kalluri R. Basement membranes: structure, assembly and role in tumour angiogenesis.Nat Rev Cancer. 2003; 3: 422-433Crossref PubMed Scopus (1312) Google Scholar A role for DLL-4/Notch in differentiation of endothelial cells during the final stages of angiogenesis has also been described,20Ridgway J. Zhang G. Wu Y. Stawicki S. Liang W.C. Chanthery Y. Kowalski J. Watts R.J. Callahan C. Kasman I. Singh M. Chien M. Tan C. Hongo J.A. de Sauvage F. Plowman G. Yan M. Inhibition of Dll4 signalling inhibits tumour growth by deregulating angiogenesis.Nature. 2006; 444: 1083-1087Crossref PubMed Scopus (816) Google Scholar, 21Noguera-Troise I. Daly C. Papadopoulos N.J. Coetzee S. Boland P. Gale N.W. Lin H.C. Yancopoulos G.D. Thurston G. Blockade of Dll4 inhibits tumour growth by promoting non-productive angiogenesis.Nature. 2006; 444: 1032-1037Crossref PubMed Scopus (859) Google Scholar and inhibition of that pathway has been proposed as a therapeutic target. Although intussusception, which does not require the steps described above, has been observed as a mechanism of angiogenesis in other tumor types, it has not been observed in glioma angiogenesis.The end result of the neoplastic angiogenic process is a characteristically abnormal vascular network, with dilated and tortuous vessels and abnormal branching and arteriovenous shunts, which can also lead to abnormal perfusion. GBMs in particular have immature vasculature, with excessive leakiness, that can contribute to the breakdown of the blood-brain barrier. In addition to physical disruption of existing vessels by lifting and displacement of astrocytic foot processes by glioma cells,6Zagzag D. Amirnovin R. Greco M.A. Yee H. Holash J. Wiegand S.J. Zabski S. Yancopoulos G.D. Grumet M. Vascular apoptosis and involution in gliomas precede neovascularization: a novel concept for glioma growth and angiogenesis.Lab Invest. 2000; 80: 837-849Crossref PubMed Scopus (231) Google Scholar induction of leakiness by VEGF and vesiculovacuolar organelles22Nagy J.A. Dvorak A.M. Dvorak H.F. VEGF-A and the induction of pathological angiogenesis.Annu Rev Pathol. 2007; 2: 251-275Crossref PubMed Scopus (302) Google Scholar contribute to an abnormal blood-brain barrier in the setting of glioma. The permeability of newly formed vascular channels is increased, compared with that of mature capillaries. Normal capillaries of the brain maintain the integrity of the blood-brain barrier, but the blood vessels of experimental and human brain tumors are structurally altered and have increased capillary permeability, in part due to lack of a basal lamina (resulting from persistent angiogenic stimuli leading to incomplete maturation).Several key pathways have been identified in the process of glioma-associated angiogenesis, including erythropoietin and its receptor, DLL4 and its receptor Notch, macrophage migration inhibitory factor (MIF), neuropilin-2 (NRP2), placental growth factor (PlGF), and basic fibroblast growth factor (bFGF), among others. The most studied and best characterized factor is VEGF, which is discussed in more detail below.Hypoxia-Induced Glioma AngiogenesisHypoxia has long been known as a major stimulator of angiogenesis in GBMs.3Semenza G.L. Defining the role of hypoxia-inducible factor 1 in cancer biology and therapeutics.Oncogene. 2010; 29: 625-634Crossref PubMed Scopus (1307) Google Scholar, 23Zagzag D. Zhong H. Scalzitti J.M. Laughner E. Simons J.W. Semenza G.L. Expression of hypoxia-inducible factor 1alpha in brain tumors: association with angiogenesis, invasion, and progression.Cancer. 2000; 88: 2606-2618Crossref PubMed Scopus (553) Google Scholar The extensive list of angiogenic factors (many of which are up-regulated by hypoxia) and the various mechanisms of angiogenesis in gliomas have been described in detail previously and have been reviewed by Fischer et al.24Fischer I. Gagner J.P. Law M. Newcomb E.W. Zagzag D. Angiogenesis in gliomas: biology and molecular pathophysiology.Brain Pathol. 2005; 15: 297-310Crossref PubMed Scopus (284) Google Scholar In particular, VEGF, which is up-regulated by hypoxia, stimulates vascularization during embryogenesis and in neoplastic tissues. The VEGF family consists of five members: VEGF-A (referred to here simply as VEGF), VEGF-B, VEGF-C, VEGF-D, and placental growth factor (PlGF). VEGF exerts its effects on the vascular endothelium through binding to several high-affinity receptors, including VEGFR-1 (also known as FLT-1) and VEGFR-2 (also known as FLK-1 and KDR). The expression of VEGF and VEGFR correlates with the grade of diffuse astrocytomas, is crucial for glioma growth, and displays a temporal and spatial correlation with the angiogenesis seen in human gliomas.25Lamszus K. Ulbricht U. Matschke J. Brockmann M.A. Fillbrandt R. Westphal M. Levels of soluble vascular endothelial growth factor (VEGF) receptor 1 in astrocytic tumors and its relation to malignancy, vascularity, and VEGF-A.Clin Cancer Res. 2003; 9: 1399-1405PubMed Google Scholar Hypoxia induces HIF-1α expression in GBMs and is the main molecular basis for the activation of VEGF gene transcription, leading to angiogenesis. The expression level of HIF-1α and VEGF in both human and murine gliomas is intense around areas of necrosis in pseudopalisading tumor cells,23Zagzag D. Zhong H. Scalzitti J.M. Laughner E. Simons J.W. Semenza G.L. Expression of hypoxia-inducible factor 1alpha in brain tumors: association with angiogenesis, invasion, and progression.Cancer. 2000; 88: 2606-2618Crossref PubMed Scopus (553) Google Scholar, 26Plate K.H. Breier G. Weich H.A. Risau W. Vascular endothelial growth factor is a potential tumour angiogenesis factor in human gliomas in vivo.Nature. 1992; 359: 845-848Crossref PubMed Scopus (2107) Google Scholar suggesting that this pattern of HIF-1α and VEGF expression is modulated by tumor oxygenation.Abundant experimental evidence suggests that the C-X-C chemokine receptor type 4/stromal-derived factor-1α (CXCR4/SDF-1α) pathway is also a crucial component of neovascularization in gliomas.27Zagzag D. Lukyanov Y. Lan L. Ali M.A. Esencay M. Mendez O. Yee H. Voura E.B. Newcomb E.W. Hypoxia-inducible factor 1 and VEGF upregulate CXCR4 in glioblastoma: implications for angiogenesis and glioma cell invasion.Lab Invest. 2006; 86: 1221-1232Crossref PubMed Scopus (308) Google Scholar CXCR4 is normally expressed at low levels in resting endothelial cells, but is increased in response to VEGF stimulation.27Zagzag D. Lukyanov Y. Lan L. Ali M.A. Esencay M. Mendez O. Yee H. Voura E.B. Newcomb E.W. Hypoxia-inducible factor 1 and VEGF upregulate CXCR4 in glioblastoma: implications for angiogenesis and glioma cell invasion.Lab Invest. 2006; 86: 1221-1232Crossref PubMed Scopus (308) Google Scholar SDF-1α is a ligand of the chemokine receptor CXCR4. Like VEGF, CXCR4 and SDF-1α are up-regulated by hypoxia, as are several other molecules that play a critical role in the angiogenic cascade (eg, MMPs). Knockout mouse experiments demonstrated that CXCR4 and SDF-1α are required for normal embryonic development of the nervous system; importantly, CXCR4 is required for vascularization of the gastrointestinal tract. The angiogenic effects of SDF-1α have been shown both in vitro and in vivo.28Strieter R.M. Belperio J.A. Phillips R.J. Keane M.P. CXC chemokines in angiogenesis of cancer.Semin Cancer Biol. 2004; 14: 195-200Crossref PubMed Scopus (182) Google Scholar SDF-1α acts as a chemoattractant for endothelial cells and induces endothelial cell proliferation in vitro, and promotes capillary sprouting and branching in vivo. SDF-1α expression by gliomas and vascular endothelial cells has been correlated with survival of endothelial cells,29Salmaggi A. Gelati M. Pollo B. Frigerio S. Eoli M. Silvani A. Broggi G. Ciusani E. Croci D. Boiardi A. De Rossi M. CXCL12 in malignant glial tumors: a possible role in angiogenesis and cross-talk between endothelial and tumoral cells.J Neurooncol. 2004; 67: 305-317Crossref PubMed Scopus (65) Google Scholar whereas CXCR4 expression has been shown to promote high levels of VEGF production by human astrocytic glioma cells.30Yang S.X. Chen J.H. Jiang X.F. Wang Q.L. Chen Z.Q. Zhao W. Feng Y.H. Xin R. Shi J.Q. Bian X.W. Activation of chemokine receptor CXCR4 in malignant glioma cells promotes the production of vascular endothelial growth factor.Biochem Biophys Res Commun. 2005; 335: 523-528Crossref PubMed Scopus (55) Google ScholarHypoxia-Independent Glioma AngiogenesisAlthough hypoxia has been shown to play a critical role in glioma angiogenesis, some experimental evidence also indicates the existence of hypoxia-independent mechanisms. Vascular proliferation can occur near the leading/invading edge of GBM, often remote from the central necrotic and hypoxic core of the tumor.For example, using fresh-frozen human tissue, Jubb et al31Jubb A.M. Pham T.Q. Hanby A.M. Frantz G.D. Peale F.V. Wu T.D. Koeppen H.W. Hillan K.J. Expression of vascular endothelial growth factor, hypoxia inducible factor 1alpha, and carbonic anhydrase IX in human tumours.J Clin Pathol. 2004; 57: 504-512Crossref PubMed Scopus (118) Google Scholar showed in several tumor types that VEGF-A was up-regulated in the absence of markers of hypoxia. Similarly, Arany et al32Arany Z. Foo S.Y. Ma Y. Ruas J.L. Bommi-Reddy A. Girnun G. Cooper M. Laznik D. Chinsomboon J. Rangwala S.M. Baek K.H. Rosenzweig A. Spiegelman B.M. HIF-independent regulation of VEGF and angiogenesis by the transcriptional coactivator PGC-1alpha.Nature. 2008; 451: 1008-1012Crossref PubMed Scopus (816) Google Scholar demonstrated induction of VEGF by peroxisome-proliferator-activated receptor-γ coactivator-1α (PGC-1-α) independently of HIF-1 through the estrogen-related receptor α (ERR-α). Hypoxia-independent mechanisms of HIF-1 stabilization have also been described. For example, several genetic mutations (including mutated genes encoding PDGFR, EGFR, p53, RB1, VHL, and PTEN) have been shown to result in HIF-1α stabilization3Semenza G.L. Defining the role of hypoxia-inducible factor 1 in cancer biology and therapeutics.Oncogene. 2010; 29: 625-634Crossref PubMed Scopus (1307) Google Scholar, 23Zagzag D. Zhong H. Scalzitti J.M. Laughner E. Simons J.W. Semenza G.L. Expression of hypoxia-inducible factor 1alpha in brain tumors: association with angiogenesis, invasion, and progression.Cancer. 2000; 88: 2606-2618Crossref PubMed Scopus (553)