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The role of adrenomedullin in angiogenesis
Peptides 26 (2005) 1670–1675
Review
The role of adrenomedullin in angiogenesis Domenico Ribatti a,∗ , Beatrice Nico a , Raffaella Spinazzi b , Angelo Vacca c , Gastone G. Nussdorfer b b
a Department of Human Anatomy and Histology, Piazza Giulio Cesare, 11, Policlinico, I-70124 Bari, Italy Department of Human Anatomy and Physiology, Section of Anatomy, University of Padua, Medical School, Padua, Italy c Department of Internal Medicine and Clinical Oncology, University of Bari Medical School, Bari, Italy
Received 28 December 2004; received in revised form 12 February 2005; accepted 15 February 2005 Available online 17 March 2005
Abstract Adrenomedullin (AM) is a 52 amino acid peptide originally isolated from human pheochromocytoma. It was initially demonstrated to have profound effects in vascular cell biology, since AM protects endothelial cells from apoptosis, promotes angiogenesis and affects vascular tone and permeability. This review article summarizes the literature data concerning the relationship between AM and angiogenesis and describes the relationship between vascular endothelial growth factor, hypoxia and AM and tumor angiogenesis. Finally, the role of AM as a potential target of antiangiogenic therapy is discussed. © 2005 Elsevier Inc. All rights reserved. Keywords: Adrenomedullin; Angiogenesis; HIF; Tumor progression; VEGF
Contents 1. 2. 3. 4. 5. 6.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . AM and the development of the vascular system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . AM and tumor angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . AM and VEGF expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . AM, hypoxia and tumor angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . AM as a potential target of antiangiogenic therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction Adrenomedullin (AM) is a potent hypotensive peptide originally isolated from extracts of human pheochromocytomas [36]. It is produced by post-translational proteolytic cleavage of a prohormone called prepro(pp)AM. A 20amino acid sequence in the NH2-terminus of ppAM exerts a ∗
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0196-9781/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2005.02.017
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transient hypotensive effect and has been named pAM Nterminal 20 peptide (PAMP) [37]. AM belongs to a large regulatory peptide family includes calcitonin gene-related peptide (CGRP), amylin and calcitonin, and acts via selective receptors derived from the calcitonin receptor-like receptor (CRLR). CRLR acts as either a CGRP or an AM receptor depending on its interaction with the members of a family of single transmembrane domain proteins referred to as receptor-activity-modifying proteins (RAMPs). RAMP1 generates CGRP receptors from CRLR, whereas
D. Ribatti et al. / Peptides 26 (2005) 1670–1675 Table 1 Pleiotropic effects of adrenomedullin on vascular smooth muscle cells and endothelial cells Regulation of vascular permeability Regulation of blood coagulation Regulation of fibrinolysis Modulation of cell shape Modulation of cell metabolism Modulation of cell growth Modulation of cell movement Modulation of cell fate Angiogenesis Vascular remodeling
RAMP2 and RAMP3 produce selective AM receptors, provisionally named AM1 and AM2 receptors respectively [23,24,64]. AM and its receptors are expressed in several tissues and organs, including the heart and blood vessels, kidneys, lungs, gastrointestinal tract, spleen and thymus, endocrine glands and brain. This extensive distribution suggests that AM is involved in the regulation of several body functions [13,25,40]. Its biological actions concern fluid and electrolyte homeostasis, and the cardiovascular system (Table 1). AM enhances Na+ excretion either directly by modulating kidney tubular function or indirectly by inhibiting agonist-stimulated adrenal aldosterone secretion [59,70,79]. The bulk of evidence indicates that AM “protects” the cardiovascular system [5,56,81]. Under physiological conditions, AM dilates blood vessels, including the coronaries, and increases cardiac output [10,35,73,80]. Its overexpression inhibits experimental cuff-induced arterial intimal proliferation [2,31,84] and hypoxia-induced pulmonary vascular damage [43]. Plasma AM is increased in patients with congestive heart failure and myocardial infarction [30,38,53] and AM has a therapeutic use in the treatment of heart failure [47,65], while chronic pressure overload upregulates AM mRNA expression concomitantly with the development of left ventricular hypertrophy [3,15,26,41,46,66,85]. AM1 and AM2 receptor expression, too, is enhanced in the left ventricular hypertrophy induced by malignant hypertension [48,54,77,82], and AM infusion attenuates the transition from left ventricular hypertrophy to heart failure in hypertensive rats [55] and reduces infarct size in the rat myocardial ischemia/reperfusion injury [63]. AM knockout mice have been used to show that endogenous AM provides strong protection against stressinduced cardiac hypertrophy [57]. Due to the well-known deleterious (profibrotic) effect of aldosterone excess on the heart [11,74–76], AM may indirectly benefit the cardiovascular system through its aldosterone antisecretagogue action (see above). AM also plays an important modulatory role in organ growth and differentiation during embryogenesis [19], and promotes the growth of several normal and neoplastic tissues [4,9,87]. Moreover, evidence is accumulating that AM possesses a clearcut angiogenic effect, though only one brief review of this topic has been published so far [51].
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2. AM and the development of the vascular system The cardiovascular system is the first functional organ system to develop in the vertebrate embryo. A widely accepted view is that blood vessels are the result of vasculogenesis followed by angiogenesis [69]. Vasculogenesis is the formation of capillaries from endothelial cells (EC) differentiating in situ from groups of mesodermal cells. The primitive heart and primitive vascular plexus are formed in this way [68]. The term angiogenesis, applied to the formation of capillaries from pre-existing vessels, i.e. capillaries and post-capillary venules [69], is based on endothelial sprouting or intussusceptive (non-sprouting) microvascular growth [6]. The angiogenic cascade is a multistep process that includes sequential basement membrane degradation, EC migration and invasion of the surrounding extracellular matrix (ECM) and EC proliferation and capillary lumen formation, while investment of the vessel wall with pericytes and subsequent inhibition of endothelial proliferation, basement membrane reconstitution, and junctional complex formation stabilize the newly formed microvasculature. A genetically determined absence of AM may be one cause of non-immune hydrops fetalis and hemorrhage, as a result of cardiovascular abnormalities and disturbance of lymphangiogenesis and angiogenesis [7,28,72]. Shindo et al. [72] generated a strain of AM knockout mice and demonstrated that homozygotes (AM −/−) die in utero at approximately embryonic day (E) 13.5 to 14. The most apparent abnormality in AM −/− embryos at this stage was severe hemorrhage under the skin and in the lung and liver. This was not detectable at E 12.5 to E 13, although histologic examination showed poor vascularization of the yolk sac, very thin umbilical cord and few fully formed, angiographically leaky placental vessels. AM exerts its angiogenic activity on EC through activation of Akt, MAPK, CRLR/RAMP2-CRLR/RAMP3 receptors and focal adhesion kinase [14,34,45], and may have an anti-inflammatory role in controlling vascular endothelial growth factor (VEGF)-induced adhesion molecule gene expression and adhesiveness towards leukocytes in EC [33]. AM augments collateral development in response to acute ischemia [1,58], and AM gene transfer induces therapeutic angiogenesis in a rabbit model of chronic hind limb ischemia [78].
3. AM and tumor angiogenesis Angiogenesis and the production of angiogenic factors are fundamental for tumor growth, invasion and metastasis [16]. Tumor angiogenesis is linked to a switch in the equilibrium between positive and negative regulators [21], and mainly triggered by the release by neoplastic cells of ECspecific growth factors that stimulate growth of the host’s blood vessels. The switch depends on increased production of one or more of the positive regulators of angiogenesis, such
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as VEGF, fibroblast growth factor-2 (FGF-2), interleukin-8 (IL-8), placental growth factor (PlGF), transforming growth factor-beta (TGF-), platelet derived endothelial growth factor (PDEGF), pleiotrophins and others exported from tumor cells, mobilized from the ECM, or released from host cells recruited to the tumor. It clearly involves something more than simple upregulation of angiogenic activity and has thus been regarded as the result of a net balance of positive and negative regulators. Detection of high levels of AM expression in various types of cancer cell suggests that it is involved in tumor growth [17,29,39,42,44,60]. Its presence of is associated with a more aggressive tumor phenotype in some cancer cell lines [22]. Expression of AM has been demonstrated in small and non-small lung carcinomas, astrocytomas and glioblastomas, pheochromocytomas, aldosteronomas, non-functioning adrenal adenomas, cortisol-secreting adenomas and adrenal carcinomas, and ectopic ACTH-secreting and human prostate adenocarcinomas [4]. In addition, AM expression and AM receptors have been detected in several tumor cell lines derived from lung large- and small-cell, squamous and bronchioalveolar carcinomas, breast carcinomas, colon adenocarcinomas, glioblastomas, neuroblastomas, ovarian adenocarcinomas, prostate carcinomas, adrenocortical carcinomas, chondrosarcomas, monocytic leukemia and skin melanomas [4]. Martinez et al. [42] stably transfected the human breast cancer cell lines T47D and MCF7, both of which express low basal levels of AM, with an expression construct that contained the coding region of human AM gene or with empty expression vector. Cells that overexpressed AM displayed a more pleiotropic morphology and increased angiogenic potential both in vitro and in vivo compared with those transfected with the empty vector. AM-overexpressing tumors are characterized by increased vascularity [61,62], and an increased expression of AM mRNA in ovarian tumors has been statistically associated with a poor prognosis [22]. AM produced from tumor cells is thought to promote angiogenesis in vivo in the chick embryo chorioallantoic membrane (CAM) assay [67,86].
4. AM and VEGF expression VEGF is a major regulator of tumor-associated angiogenesis and promotes tumor growth, invasion and metastasis [12]. Its amount (as measured by immunohistochemistry, in situ hybridization, quantitative immunoassay, Western blotting or reverse-transcriptase polymerase chain reaction) expressed by cancer cells correlates with tumor size, metastasis and poor prognosis in many types of solid and hematological tumors [12]. Hague et al. [20] showed that AM and VEGF are the most widely expressed angiogenic factors in uterine leiomyomas, and that these tumors have a higher vascular density and endothelial proliferative index than the normal myometrium
and endometrium. A striking finding of this study was that expression of AM, but not that of VEGF, correlated with the vascular density of the leiomyomas. High VEGF and AM expression in leiomyomas may provide a target for treatment with specific anti-VEGF and anti-AM antibodies. Iimuro et al. [27] demonstrated that AM administration upregulates the expression of VEGF in both in vitro and in vivo models. They used laser doppler perfusion imaging to show that AM stimulates recovery of blood flow to the affected limb in the mouse hind-limb ischemia model, partly by promoting local expression of VEGF. Immunostaining for CD31 revealed that this enhanced flow reflected increased capillary density. By enhancing tumor angiogenesis, AM also promoted the growth of subcutaneously transplanted sarcoma 180 tumor cells. Heterozygotic AM knockout mice (AM +/−) showed significantly less blood flow recovery with less collateral capillary development and VEGF expression. In EC and fibroblast cocultures, AM enhanced VEGF-induced capillary formation, whereas in EC cultures it enhanced VEGF-induced Akt activation. However, blocking antibodies to VEGF can not significantly inhibit AM-induced capillary tube formation by human umbilical vein endothelial cells (HUVEC) [14], indicating that AM does not function indirectly through up-regulation of VEGF. These findings suggest that the angiogenic activity of AM may translate into enhanced vascularization in vivo and identify AM and its receptors acting as potential targets for antiangiogenic therapy.
5. AM, hypoxia and tumor angiogenesis Tumors are thought to become hypoxic when their growth rate exceeds that of neovascularization, or when their fragile, poorly organized vasculature partially collapses under interstitial pressure. Many tumors contain a hypoxic microenvironment, a condition that is associated with poor prognosis and resistance to treatment. There is a complex interrelationship between tumor hypoxia and tumor angiogenesis. Hypoxia is an important stimulus of angiogenesis. Low oxygen levels activate type alphahypoxia-inducible transcription factors (HIF-1␣, -2␣, -3␣) that bind as heterodimers with HIF-1␣ onto hypoxia-response elements in the promoter region of various genes encoding angiogenesis factors [83]. Tumors are often hypoxic and, by driving angiogenesis, HIFs are generally believed to promote tumor growth [71]. CRLR is also up-regulated under hypoxic conditions in microvascular endothelial cells, although expression of RAMPs is not activated by hypoxia in microvascular endothelial cells [52]. Besides VEGF, endothelin-1 and -2, angiogenin and AM are also induced by hypoxia. The wide range of angiogenic pathways regulated by hypoxia suggests that drugs targeting the HIF pathways should be considered as antitumor agents, whereas drugs activating HIF would be proangiogenic. AM produced from tumor cells is thought to inhibit their hypoxic death by acting as an antiapoptotic factor [60]. AM is
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upregulated by hypoxia [8,49] and its expression is controlled by HIF-1␣ [18,50]. Oehler et al. [60] studied the role of AM in endometrial carcinoma cells under hypoxia. They found that AM confers resistance to hypoxic cell death in an autocrine/paracrine manner. This antiapoptotic action appears to be mediated by upregulation of the bcl.2 oncoprotein. 6. AM as a potential target of antiangiogenic therapy Antiangiogenesis was proposed as a cancer therapy over 20 years ago and many compounds display antiangiogenic activity. Inhibitors are direct or indirect. A direct inhibitor, such as endostatin, targets genetically stable EC recruited to a tumor and can bring about their apoptosis. An indirect inhibitor generally inhibits the products of a tumor cell or its receptor. Antiangiogenic therapy is applicable to a wide variety of solid and hematologic tumors. Because of the low mutagenic potential of EC, tumors do not develop resistance to the effects of many of these compounds. Inhibitors block several steps in the angiogenic cascade, including proliferation and attachment of EC to the ECM proteins, migration and invasion via the ECM required for capillary sprouting, morphogenesis, differentiation and stabilization [32]. We have looked to see whether vinblastine (VBL), as demonstrated in other experimental conditions, is angiostatic in the angiogenic response induced by AM in two assays, namely Matrigel tube formation in vitro and angiogenesis in the CAM assay in vivo [67]. When tested on Matrigel, AM caused a morphogenetic effect. EC spread and aligned with each other to form branching anastomosing tubes with multicentric junctions that gave rise to a meshwork of capillary-like tubes. When AM was administered in the presence of VBL, these tubes were interrupted and most cells were spherical, either isolated or aggregated in small clumps. In the CAM assay, AM induced a strong angiogenic response that was counteracted by VBL. Overall, these observations implicate AM as a promoter of tumor growth and a possible target for anticancer strategies, such as the use of VBL at very low, non-toxic doses. Iimuro et al. [27] demonstrated that heterozygotic AM knockout mice (AM +/−) treated with AM 22-52, a competitive inhibitor of AM, showed reduced capillary development, and growth of sarcoma 180 tumors was inhibited. Moreover, administration of VEGF or AM rescued blood flow recovery and capillary formation in AM +/− and AM 22-52 treated mice. Acknowledgements Supported in part by Associazione Italiana per la Ricerca sul Cancro (AIRC), Milan, Fondazione Italiana per la Lotta al Neuroblastoma, Genoa, Ministero per la Salute – Regione Puglia (grant BS2) and Ministero dell’Istruzione, dell’Universit`a e della Ricerca (Consorzio Carso Grant No. 72/2), Rome, Italy.
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Review
The role of adrenomedullin in angiogenesis Domenico Ribatti a,∗ , Beatrice Nico a , Raffaella Spinazzi b , Angelo Vacca c , Gastone G. Nussdorfer b b
a Department of Human Anatomy and Histology, Piazza Giulio Cesare, 11, Policlinico, I-70124 Bari, Italy Department of Human Anatomy and Physiology, Section of Anatomy, University of Padua, Medical School, Padua, Italy c Department of Internal Medicine and Clinical Oncology, University of Bari Medical School, Bari, Italy
Received 28 December 2004; received in revised form 12 February 2005; accepted 15 February 2005 Available online 17 March 2005
Abstract Adrenomedullin (AM) is a 52 amino acid peptide originally isolated from human pheochromocytoma. It was initially demonstrated to have profound effects in vascular cell biology, since AM protects endothelial cells from apoptosis, promotes angiogenesis and affects vascular tone and permeability. This review article summarizes the literature data concerning the relationship between AM and angiogenesis and describes the relationship between vascular endothelial growth factor, hypoxia and AM and tumor angiogenesis. Finally, the role of AM as a potential target of antiangiogenic therapy is discussed. © 2005 Elsevier Inc. All rights reserved. Keywords: Adrenomedullin; Angiogenesis; HIF; Tumor progression; VEGF
Contents 1. 2. 3. 4. 5. 6.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . AM and the development of the vascular system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . AM and tumor angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . AM and VEGF expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . AM, hypoxia and tumor angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . AM as a potential target of antiangiogenic therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction Adrenomedullin (AM) is a potent hypotensive peptide originally isolated from extracts of human pheochromocytomas [36]. It is produced by post-translational proteolytic cleavage of a prohormone called prepro(pp)AM. A 20amino acid sequence in the NH2-terminus of ppAM exerts a ∗
Corresponding author. Tel.: +39 080 5478240; fax: +39 080 5478310. E-mail address: [email protected] (D. Ribatti).
0196-9781/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2005.02.017
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transient hypotensive effect and has been named pAM Nterminal 20 peptide (PAMP) [37]. AM belongs to a large regulatory peptide family includes calcitonin gene-related peptide (CGRP), amylin and calcitonin, and acts via selective receptors derived from the calcitonin receptor-like receptor (CRLR). CRLR acts as either a CGRP or an AM receptor depending on its interaction with the members of a family of single transmembrane domain proteins referred to as receptor-activity-modifying proteins (RAMPs). RAMP1 generates CGRP receptors from CRLR, whereas
D. Ribatti et al. / Peptides 26 (2005) 1670–1675 Table 1 Pleiotropic effects of adrenomedullin on vascular smooth muscle cells and endothelial cells Regulation of vascular permeability Regulation of blood coagulation Regulation of fibrinolysis Modulation of cell shape Modulation of cell metabolism Modulation of cell growth Modulation of cell movement Modulation of cell fate Angiogenesis Vascular remodeling
RAMP2 and RAMP3 produce selective AM receptors, provisionally named AM1 and AM2 receptors respectively [23,24,64]. AM and its receptors are expressed in several tissues and organs, including the heart and blood vessels, kidneys, lungs, gastrointestinal tract, spleen and thymus, endocrine glands and brain. This extensive distribution suggests that AM is involved in the regulation of several body functions [13,25,40]. Its biological actions concern fluid and electrolyte homeostasis, and the cardiovascular system (Table 1). AM enhances Na+ excretion either directly by modulating kidney tubular function or indirectly by inhibiting agonist-stimulated adrenal aldosterone secretion [59,70,79]. The bulk of evidence indicates that AM “protects” the cardiovascular system [5,56,81]. Under physiological conditions, AM dilates blood vessels, including the coronaries, and increases cardiac output [10,35,73,80]. Its overexpression inhibits experimental cuff-induced arterial intimal proliferation [2,31,84] and hypoxia-induced pulmonary vascular damage [43]. Plasma AM is increased in patients with congestive heart failure and myocardial infarction [30,38,53] and AM has a therapeutic use in the treatment of heart failure [47,65], while chronic pressure overload upregulates AM mRNA expression concomitantly with the development of left ventricular hypertrophy [3,15,26,41,46,66,85]. AM1 and AM2 receptor expression, too, is enhanced in the left ventricular hypertrophy induced by malignant hypertension [48,54,77,82], and AM infusion attenuates the transition from left ventricular hypertrophy to heart failure in hypertensive rats [55] and reduces infarct size in the rat myocardial ischemia/reperfusion injury [63]. AM knockout mice have been used to show that endogenous AM provides strong protection against stressinduced cardiac hypertrophy [57]. Due to the well-known deleterious (profibrotic) effect of aldosterone excess on the heart [11,74–76], AM may indirectly benefit the cardiovascular system through its aldosterone antisecretagogue action (see above). AM also plays an important modulatory role in organ growth and differentiation during embryogenesis [19], and promotes the growth of several normal and neoplastic tissues [4,9,87]. Moreover, evidence is accumulating that AM possesses a clearcut angiogenic effect, though only one brief review of this topic has been published so far [51].
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2. AM and the development of the vascular system The cardiovascular system is the first functional organ system to develop in the vertebrate embryo. A widely accepted view is that blood vessels are the result of vasculogenesis followed by angiogenesis [69]. Vasculogenesis is the formation of capillaries from endothelial cells (EC) differentiating in situ from groups of mesodermal cells. The primitive heart and primitive vascular plexus are formed in this way [68]. The term angiogenesis, applied to the formation of capillaries from pre-existing vessels, i.e. capillaries and post-capillary venules [69], is based on endothelial sprouting or intussusceptive (non-sprouting) microvascular growth [6]. The angiogenic cascade is a multistep process that includes sequential basement membrane degradation, EC migration and invasion of the surrounding extracellular matrix (ECM) and EC proliferation and capillary lumen formation, while investment of the vessel wall with pericytes and subsequent inhibition of endothelial proliferation, basement membrane reconstitution, and junctional complex formation stabilize the newly formed microvasculature. A genetically determined absence of AM may be one cause of non-immune hydrops fetalis and hemorrhage, as a result of cardiovascular abnormalities and disturbance of lymphangiogenesis and angiogenesis [7,28,72]. Shindo et al. [72] generated a strain of AM knockout mice and demonstrated that homozygotes (AM −/−) die in utero at approximately embryonic day (E) 13.5 to 14. The most apparent abnormality in AM −/− embryos at this stage was severe hemorrhage under the skin and in the lung and liver. This was not detectable at E 12.5 to E 13, although histologic examination showed poor vascularization of the yolk sac, very thin umbilical cord and few fully formed, angiographically leaky placental vessels. AM exerts its angiogenic activity on EC through activation of Akt, MAPK, CRLR/RAMP2-CRLR/RAMP3 receptors and focal adhesion kinase [14,34,45], and may have an anti-inflammatory role in controlling vascular endothelial growth factor (VEGF)-induced adhesion molecule gene expression and adhesiveness towards leukocytes in EC [33]. AM augments collateral development in response to acute ischemia [1,58], and AM gene transfer induces therapeutic angiogenesis in a rabbit model of chronic hind limb ischemia [78].
3. AM and tumor angiogenesis Angiogenesis and the production of angiogenic factors are fundamental for tumor growth, invasion and metastasis [16]. Tumor angiogenesis is linked to a switch in the equilibrium between positive and negative regulators [21], and mainly triggered by the release by neoplastic cells of ECspecific growth factors that stimulate growth of the host’s blood vessels. The switch depends on increased production of one or more of the positive regulators of angiogenesis, such
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as VEGF, fibroblast growth factor-2 (FGF-2), interleukin-8 (IL-8), placental growth factor (PlGF), transforming growth factor-beta (TGF-), platelet derived endothelial growth factor (PDEGF), pleiotrophins and others exported from tumor cells, mobilized from the ECM, or released from host cells recruited to the tumor. It clearly involves something more than simple upregulation of angiogenic activity and has thus been regarded as the result of a net balance of positive and negative regulators. Detection of high levels of AM expression in various types of cancer cell suggests that it is involved in tumor growth [17,29,39,42,44,60]. Its presence of is associated with a more aggressive tumor phenotype in some cancer cell lines [22]. Expression of AM has been demonstrated in small and non-small lung carcinomas, astrocytomas and glioblastomas, pheochromocytomas, aldosteronomas, non-functioning adrenal adenomas, cortisol-secreting adenomas and adrenal carcinomas, and ectopic ACTH-secreting and human prostate adenocarcinomas [4]. In addition, AM expression and AM receptors have been detected in several tumor cell lines derived from lung large- and small-cell, squamous and bronchioalveolar carcinomas, breast carcinomas, colon adenocarcinomas, glioblastomas, neuroblastomas, ovarian adenocarcinomas, prostate carcinomas, adrenocortical carcinomas, chondrosarcomas, monocytic leukemia and skin melanomas [4]. Martinez et al. [42] stably transfected the human breast cancer cell lines T47D and MCF7, both of which express low basal levels of AM, with an expression construct that contained the coding region of human AM gene or with empty expression vector. Cells that overexpressed AM displayed a more pleiotropic morphology and increased angiogenic potential both in vitro and in vivo compared with those transfected with the empty vector. AM-overexpressing tumors are characterized by increased vascularity [61,62], and an increased expression of AM mRNA in ovarian tumors has been statistically associated with a poor prognosis [22]. AM produced from tumor cells is thought to promote angiogenesis in vivo in the chick embryo chorioallantoic membrane (CAM) assay [67,86].
4. AM and VEGF expression VEGF is a major regulator of tumor-associated angiogenesis and promotes tumor growth, invasion and metastasis [12]. Its amount (as measured by immunohistochemistry, in situ hybridization, quantitative immunoassay, Western blotting or reverse-transcriptase polymerase chain reaction) expressed by cancer cells correlates with tumor size, metastasis and poor prognosis in many types of solid and hematological tumors [12]. Hague et al. [20] showed that AM and VEGF are the most widely expressed angiogenic factors in uterine leiomyomas, and that these tumors have a higher vascular density and endothelial proliferative index than the normal myometrium
and endometrium. A striking finding of this study was that expression of AM, but not that of VEGF, correlated with the vascular density of the leiomyomas. High VEGF and AM expression in leiomyomas may provide a target for treatment with specific anti-VEGF and anti-AM antibodies. Iimuro et al. [27] demonstrated that AM administration upregulates the expression of VEGF in both in vitro and in vivo models. They used laser doppler perfusion imaging to show that AM stimulates recovery of blood flow to the affected limb in the mouse hind-limb ischemia model, partly by promoting local expression of VEGF. Immunostaining for CD31 revealed that this enhanced flow reflected increased capillary density. By enhancing tumor angiogenesis, AM also promoted the growth of subcutaneously transplanted sarcoma 180 tumor cells. Heterozygotic AM knockout mice (AM +/−) showed significantly less blood flow recovery with less collateral capillary development and VEGF expression. In EC and fibroblast cocultures, AM enhanced VEGF-induced capillary formation, whereas in EC cultures it enhanced VEGF-induced Akt activation. However, blocking antibodies to VEGF can not significantly inhibit AM-induced capillary tube formation by human umbilical vein endothelial cells (HUVEC) [14], indicating that AM does not function indirectly through up-regulation of VEGF. These findings suggest that the angiogenic activity of AM may translate into enhanced vascularization in vivo and identify AM and its receptors acting as potential targets for antiangiogenic therapy.
5. AM, hypoxia and tumor angiogenesis Tumors are thought to become hypoxic when their growth rate exceeds that of neovascularization, or when their fragile, poorly organized vasculature partially collapses under interstitial pressure. Many tumors contain a hypoxic microenvironment, a condition that is associated with poor prognosis and resistance to treatment. There is a complex interrelationship between tumor hypoxia and tumor angiogenesis. Hypoxia is an important stimulus of angiogenesis. Low oxygen levels activate type alphahypoxia-inducible transcription factors (HIF-1␣, -2␣, -3␣) that bind as heterodimers with HIF-1␣ onto hypoxia-response elements in the promoter region of various genes encoding angiogenesis factors [83]. Tumors are often hypoxic and, by driving angiogenesis, HIFs are generally believed to promote tumor growth [71]. CRLR is also up-regulated under hypoxic conditions in microvascular endothelial cells, although expression of RAMPs is not activated by hypoxia in microvascular endothelial cells [52]. Besides VEGF, endothelin-1 and -2, angiogenin and AM are also induced by hypoxia. The wide range of angiogenic pathways regulated by hypoxia suggests that drugs targeting the HIF pathways should be considered as antitumor agents, whereas drugs activating HIF would be proangiogenic. AM produced from tumor cells is thought to inhibit their hypoxic death by acting as an antiapoptotic factor [60]. AM is
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upregulated by hypoxia [8,49] and its expression is controlled by HIF-1␣ [18,50]. Oehler et al. [60] studied the role of AM in endometrial carcinoma cells under hypoxia. They found that AM confers resistance to hypoxic cell death in an autocrine/paracrine manner. This antiapoptotic action appears to be mediated by upregulation of the bcl.2 oncoprotein. 6. AM as a potential target of antiangiogenic therapy Antiangiogenesis was proposed as a cancer therapy over 20 years ago and many compounds display antiangiogenic activity. Inhibitors are direct or indirect. A direct inhibitor, such as endostatin, targets genetically stable EC recruited to a tumor and can bring about their apoptosis. An indirect inhibitor generally inhibits the products of a tumor cell or its receptor. Antiangiogenic therapy is applicable to a wide variety of solid and hematologic tumors. Because of the low mutagenic potential of EC, tumors do not develop resistance to the effects of many of these compounds. Inhibitors block several steps in the angiogenic cascade, including proliferation and attachment of EC to the ECM proteins, migration and invasion via the ECM required for capillary sprouting, morphogenesis, differentiation and stabilization [32]. We have looked to see whether vinblastine (VBL), as demonstrated in other experimental conditions, is angiostatic in the angiogenic response induced by AM in two assays, namely Matrigel tube formation in vitro and angiogenesis in the CAM assay in vivo [67]. When tested on Matrigel, AM caused a morphogenetic effect. EC spread and aligned with each other to form branching anastomosing tubes with multicentric junctions that gave rise to a meshwork of capillary-like tubes. When AM was administered in the presence of VBL, these tubes were interrupted and most cells were spherical, either isolated or aggregated in small clumps. In the CAM assay, AM induced a strong angiogenic response that was counteracted by VBL. Overall, these observations implicate AM as a promoter of tumor growth and a possible target for anticancer strategies, such as the use of VBL at very low, non-toxic doses. Iimuro et al. [27] demonstrated that heterozygotic AM knockout mice (AM +/−) treated with AM 22-52, a competitive inhibitor of AM, showed reduced capillary development, and growth of sarcoma 180 tumors was inhibited. Moreover, administration of VEGF or AM rescued blood flow recovery and capillary formation in AM +/− and AM 22-52 treated mice. Acknowledgements Supported in part by Associazione Italiana per la Ricerca sul Cancro (AIRC), Milan, Fondazione Italiana per la Lotta al Neuroblastoma, Genoa, Ministero per la Salute – Regione Puglia (grant BS2) and Ministero dell’Istruzione, dell’Universit`a e della Ricerca (Consorzio Carso Grant No. 72/2), Rome, Italy.
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