Angiopoietins in malignancy

EJSO 33 (2007) 7e15

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Angiopoietins in malignancy F. Bach, F.J. Uddin, D. Burke* Department of Academic Surgery, Room B40, Clarendon Wing, Leeds General Infirmary, Great George Street, Leeds, West Yorkshire LS1 3EX, UK Accepted 28 July 2006 Available online 7 September 2006

Abstract Background: Tumour growth is dependant upon the development of an adequate blood supply. This, in turn, is thought to depend upon a switch by the tumour, from a dormant to angiogenic state. Recent data suggest that this switch may occur when the balance of proand anti-angiogenic agents alters to promote angiogenesis. Angiopoietins may be involved in this balance. Methods: An electronic literature search was performed with respect to angiopoietins from 1996 to the present. Published data from in-vitro and in-vivo studies were critically analysed. A specific focus was made of studies relating to tumour growth and vasculature. Results: Since angiopoietin-1 was first described in 1996, three more angiopoietins have been described. All family members bind to the Tie-2 receptor. There is now a considerable accumulation of data that suggests they play a pivotal role in the development and stabilisation of tumour vasculature. angiopoietin-2 appears to be pro-angiogenic whilst angiopoietin-1 appears to be a stabilising factor. Conclusions: Recent trials of anti-angiogenic agents show promise in the treatment of solid human cancers. The angiopoietins are a new family of proteins that appear to be influential in the development of the tumour vasculature. Manipulation of the angiopoietin balance may provide a potential therapeutic target in human cancer. Ó 2006 Elsevier Ltd. All rights reserved. Keyword: Angiopoietins; Cancer; Tumour vascularity

Introduction Angiogenesis is the highly ordered formation of new blood vessels from pre-existing vessels.1,2 It is seen throughout growth, in wound healing and menses, and is important in cancer, where pro- and anti-angiogenic signals can be released by cancer cells, endothelial cells, stromal cells, blood and the extracellular matrix.3 These signals form the basis of the theoretical ‘angiogenic switch’ Abbreviations: Ang1, angiopoietin 1; Ang2, angiopoietin 2; Ang3, angiopoietin 3; Ang4, angiopoietin 4; EGF, epidermal growth factor; FGF, fibroblast growth factor; GS, Gleason score; HCC, hepatocellular cancer; MMP, matrix metalloproteinase; NSCLC, non-small cell lung carcinoma; OSCC, oral squamous cell carcinoma; PCR, polymerase chain reaction; PDGF, platelet derived growth factor; PECAM, platelet endothelial cells adhesion molecule; RT-PCR, reverse transcriptase polymerase chain reaction; SCC, squamous cell carcinoma; TGF-a, transforming growth factor-a; Tie1, Tie 1 receptor; Tie2, Tie 2 receptor; TNF-a, tumour necrosis factor-a; VEGF, vascular endothelial growth factor. * Corresponding author. Tel.: þ44 113 392 3465; fax: þ44 113 392 3635. E-mail address: [email protected] (D. Burke). 0748-7983/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.ejso.2006.07.015

suggested by Hanahan and Folkmann.4 This switch activates or inhibits angiogenesis depending on the relative levels of angiogenesis inducers or inhibitors. Recent studies suggest that a new family of angiogenic proteins, the angiopoietins, may be involved in this angiogenic switch. This review outlines the function of the major angiopoietins and focuses on their role in malignancy. Physiological angiogenesis Physiological angiogenesis (Fig. 1) is accomplished by angiopoietin 2 (Ang2) binding to the tie 2 receptor (Tie2), which, in the presence of vascular endothelial growth factor (VEGF), induces removal of pericytes from the endothelium. This causes the vessels to become dilated and leaky and therefore allows local extravasation of proteases and matrix components from the blood stream. Ang2 and proteases dissolve the basement membrane and the interstitial matrix leading to further destabilisation. The endothelial cells can now be acted upon by numerous angiogenic molecules

F. Bach et al. / EJSO 33 (2007) 7e15

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1 Destabilisation Ang2

2 Proliferation VEGF, FGF, EGF

3 Cell migration V 3 Integrin,

VEGF, FGF

Key Endothelial cell (EC) Pericyte

4 Vessel Formation FGF, PDGF, TNF- , ephrin 2A

Tube formation of a new blood vessel

Ang2 binds to Tie2 on EC to remove pericytes and destabilise vessels

5 Cell Migration PDGF, Ang1/Tie2

Mesenchymal cells proliferate and migrate to the ablumenal side of the vessel

Destabilisation allows stimulation by VEGF, FGF and EGF to occur which stimulates EC proliferation

6 Differentiation TGF-

TGF- causes cells to differentiate into mature pericytes

Integrin, VEGF and FGF stimulate cell migration and cell:cell interaction

V 3

7 Stabilisation VEGF, Ang1

Stabilisation occurs. VEGF and Ang1 are survival factors for EC and therefore for vessels

Figure 1. Physiological angiogenesis.

such as VEGF and platelet derived growth factor (PDGF) which cause them to proliferate, migrate, form cell:cell interactions and eventually form tubes through which blood can flow. Stabilisation of the new vessel is achieved by reformation of the basement membrane and the addition of pericytes and smooth muscle cells. VEGF and angiopoietin 1 (Ang1) are survival factors for endothelial cells,5 which ensure the survival of the new vessel.6

functions.9 Ang1 is mainly produced by endothelial cells and pericytes and is widely expressed in adult tissue5 in which it has a stabilising effect. Ang1 produces a paracrine stabilisation or survival signal by low-level constitutive expression in normal tissues.10 Signalling occurs through P13-K and this appears to be essential for Ang1 function.9

Angiopoietins and Tie2 receptors: their discovery and properties

Ang2 is mainly produced by endothelial cells and pericytes. It is expressed at the site of vascular remodelling and promotes vessel destabilisation. 6,11e13 It is seen alone at sites of frank vessel regression (e.g. atretic follicles) and is seen, in conjunction with VEGF, at sites of vessel sprouting and growth.6,13,14 Maisonpierre et al.6 cloned Ang2 and showed that it also binds to Tie2 but does not induce phosphorylation. They found that Ang1 activity on endothelial cells could be blocked by increasing the concentration of Ang2 to a molar excess of four to eight-fold that of Ang1, demonstrating that Ang2 is an antagonist of Ang1. Recent evidence suggests that Ang2 may be able to act as an agonist depending on cell type and experimental context.9

Four angiopoietins have been identified and form a family of secreted proteins that all bind to an endotheliumspecific receptor tyrosine kinase e the Tie2 (tyrosine kinase with immunoglobulin and epidermal growth factor homology domains 2) receptor. The best characterised are angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2), which are both w70 kDa secreted ligands for the receptor Tie2.5 They are 60% identical and both contain an NH2-terminal coiled-coil domain and a COOH-terminal fibrinogen-like domain.5e8

Angiopoietin 2

Angiopoietin 1 Angiopoietin 3 and angiopoietin 4 Davis et al.5 discovered Ang1 and determined that it binds specifically to Tie2 thus activating it by inducing phosphorylation. This interaction does not promote the growth of cultured endothelial cells which is the common response of an angiogenic substance5 however it does induce migration, tube formation, sprouting and survival.9 Ang1 also has anti-permeability and anti-inflammatory

Angiopoietins 3 and 4 (Ang3, Ang4) were described as ligands for the Tie-2 receptor in 1999 by homology cloning.15 Ang3 appears to be the mouse equivalent of the human protein Ang4. Ang4 has been shown to increase Tie2 phosphorylation and induce survival and migration unlike Ang3 which did not produce significant changes.15,16

F. Bach et al. / EJSO 33 (2007) 7e15

Ang4 has been demonstrated in both gastric17 and colorectal18 tumours, but its biological role is presently uncertain. Tie2 receptors The Tie receptor family consists of a novel class of tyrosine kinase receptors19,20 with two members, Tie1 and Tie2, that are closely related but contain unique extracellular domains The expression of these receptors is largely restricted to endothelial cells,5,8,21 however they are also expressed in haematopoietic progenitors and differentiating megakaryoblasts.22,23 Tie1 is an orphan receptor i.e. no ligand has been identified to date.6 Tie2 is seen both in areas of vessel formation and in areas of vessel quiescence, suggesting a dual action of angiogenesis and vascular stabilisation. Expression of the Tie2 receptor is stimulated by hypoxia and proinflammatory cytokines in human endothelial cells.24 Ang1 and Ang2 bind to Tie2 with the same affinity,21 but they have very different actions, due to differences in binding activity.13 Summary of Ang1, Ang2 and Tie2 Ang1, Ang2 and Tie2 are not involved in vasculogenesis as the primary vasculature is present when any of the three are knocked out. However they have an essential function in vessel remodelling, maturation and growth. The angiopoietins are known for their antagonistic roles on Tie2 in physiological vasculature7 where Ang1 stabilises the blood vessel by maintaining periendothelial cell coverage and Ang2 permits the removal of these cells from the vessel, which in the presence of VEGF facilitates the angiogenic response and in the absence of VEGF induces vessel regression. VEGF expression is not modulated by overexpression of Ang1 or Ang2.11 Table 1 summarises the levels of angiogenic factor needed for changes in the blood vessel to occur. Table 2 summarises the main functions of the angiopoietins. Is the balance of the angiopoietins important in cancer? The switch to an angiogenic phenotype occurs early to midstage in tumour development and it is thought that neovascularisation must occur before the rapid clonal expansion associated with the development of a macroscopic tumour.25 There is no doubt that VEGF is critical in tumour vasculature but it is not clear what role the angiopoietins have. VEGF increases microvascular permeability and Table 1 Response of vessels to angiogenic factors

Maturation Proliferation Regression

Ang1

Ang2

VEGF

High Low Low

Low High High

High High Low

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Table 2 Demonstrating the main functions of the angiopoietins Ang1 Agonist of Tie2. Promotes structural integrity of blood vessels Ang2 Antagonist of Tie2. Natural competitor of Ang1. Promotes vessel growth in presence of VEGF or vessel regression alone Ang3 Antagonist Ang4 Agonist

Expressed by many tissues in adults. Primary cellular source is non-endothelial cells Expressed at sites of vascular remodelling. Primary source is endothelial cells

Expressed in many tissues. Mouse orthologue of Ang4 Expressed at high levels in the lungs

promotes survival, migration and proliferation of endothelial cells through interaction with transmembrane tyrosine kinase receptors VEGFR-1 and VEGFR-2.26 The involvement of the angiopoietins in the angiogenic switch has been suggested by Tanaka et al.27,28 and others.29 The angiogenic switch4 is described as a balance, in which a higher relative level of angiogenic inducers leads to angiogenesis and a higher relative level of inhibitors leads to inhibition of angiogenesis. Tumour angiogenesis is thought to occur when Ang2 destabilises vessels by removal of pericytes. This allows the vessels, which were previously in a quiescent state, to be acted upon by other angiogenic factors, such as VEGF. Therefore in keeping with the notion of the angiogenic switch, it is logical to conclude that when the imbalance between the two angiopoietins in tumours results in a higher relative level of Ang2 compared with Ang1, angiogenesis and growth of the tumour are initiated. This may be created in the tumour by overexpression of Ang2, a reduction of Ang1 or a combination of both. The three areas of interest concerning the angiopoietins are: the effect of induced overexpression in the laboratory; the differential expression in human tumours in-vivo compared to normal tissue; and their potential as a prognostic marker in human malignancy. The effects of experimental overexpression of Ang1 and Ang2 in the laboratory Several authors have investigated the effects of experimental overexpression of each of the angiopoietins in a variety of tumours. Overexpression of Ang1 in tumours leads to a reduction in tumour size and weight Hawighorst et al. discovered that overexpression of Ang1 in human SCC resulted in more than 70% inhibition of tumour growth which was associated with enhanced Tie2 phosphorylation levels compared to low levels in control transfected tumours.11 The Ang1 overexpressing tumours exhibited an increased number of mature blood vessels surrounded by SMA-positive periendothelial cells, as compared with control and Ang2 overexpressing tumours. A three fold retardation of tumour growth was seen in a breast cancer cell line when Ang1 was overexpressed.30 Stoeltzing

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et al. investigated human colon cancer cells (HT29 & KM12L4) stably transfected with Ang1 construct or a vector, injected into the livers or peritoneal cavities of nude mice. Liver weights and tumour volume respectively along with tumour cell proliferation were significantly lower in the Ang1 transfected group than in the control group.31,32 Ang1 transfected tumours developed a significantly higher degree of pericyte coverage, which is known to stabilise vessels, and therefore inhibits tumour mediated angiogenesis.31 Conditioned medium from Ang1 transfected cells decreased vascular permeability, and vessel counts were significantly lower in the Ang1 transfected group than in the control group.31,32 Tian et al.33 found that overexpression of Ang1 in a breast cancer xenograft led to stabilisation of blood vessels at the tumour edge with inhibition of both vessel dilation and dissociation of smooth muscle cells from existing vessels. Additionally Ang1 encouraged mesenchymal cell infiltration into the tumour which increased the pericyte coverage of microvessels.33 The tumour growth in the xenograft was arrested by increased levels of Ang1 as the rate of tumour cell proliferation decreased. Overexpression of Ang1 in human colon cancer produced both significantly fewer PCNA positive cancer cells and significantly fewer ( p < 0.05) vessels.28

Overexpression of Ang2 leads to a more invasive tumour Colorectal tumours transfected with Ang2 grew significantly larger and heavier in nude mice compared to those transfected with the Ang1 construct or the vector alone.28 A significantly higher cellular proliferation rate was seen in the Ang2 transfected group compared to the vector ( p < 0.05). A similar result was found when Ang2-transfected gastric cancer cells were injected into nude mice.12 The resulting tumours developed into highly metastatic tumours with hypervascularity when compared to the tumours produced by cells transfected with the vector alone. Overexpression of Ang2 in intracranial xenografts was found to create a tumour that was highly invasive into adjacent brain parenchyma compared to isogenic control tumours. The areas of invasion in the intracranial xenograft showed increased angiogenesis.34 Yoshiji et al. studied the relationship of Ang2 combined with VEGF in murine HCC. In vivo, Ang2 overexpression alone did not increase tumour development however simultaneous expression of Ang2 and VEGF synergistically augmented HCC tumour growth and angiogenesis.35 This action was attenuated by treatment with neutralising monoclonal antibodies against VEGF receptors. The synergistic combination of VEGF and Ang2 resulted in tumour growth, due to a marked decrease in apoptosis whereas it did not significantly affect tumour cell proliferation. The combination of overexpression of Ang2 and VEGF significantly increased the activities of matrix metalloproteinase (MMP)-2 and MMP-9 indicating a mediating role and equally the

activities of MMP were virtually abolished by suppression of intra-tumoural VEGF.35 Inhibition of Ang2 leads to suppression of angiogenesis and tumour growth Oliner et al. generated antibodies and peptide-Fc fusion proteins that selectively neutralised the interaction between Ang2 and its receptor. Tumour bearing mice treated systemically with these agents resulted in tumour stasis followed by elimination of all measurable tumour in a subset of animals.36 VEGF-stimulated neovascularisation in a rat corneal model of angiogenesis was also prevented using the anti Ang2 therapy.36 In summary, overexpression of Ang1 leads to smaller tumours with less tumour cell proliferation. These tumours contain fewer vessels and the vessels that are present are stabilised by high pericyte coverage. The precise mechanism by which Ang1 stabilisation of blood vessels reduces tumour growth remains to be confirmed. It may result from decreased permeability of the blood vessels so that the extravascular matrix is less suited to tumour growth,11 or from the reduced number of vessels. Overexpression of Ang2 results in larger and heavier tumours with a higher rate of tumour cell proliferation. These tumours contain more vessels and can be highly metastatic. The change to a more vascular and aggressive tumour may therefore be due to the balance between Ang1 and Ang2. Synergistic combination of VEGF and Ang2 leads to tumour growth due to decrease in apoptosis. Inhibition of Ang2 leads to suppression of angiogenesis and tumour growth. Encouraging though these data are, a degree of caution must remain. Machein et al. introduced transfected glioma cells into the intracerebral tissue of rats and showed that increased Ang1 expression produced a significant increase of tumour growth in vivo by 50% compared to control tumours ( p ¼ 0.014) with more numerous highly branched mature vessels. Increased Ang2 expression, however, inhibited tumour growth by 40%, with these smaller tumours containing few vessels.37 This effect may be explained by a lack of VEGF in this particular tumour. As discussed later, VEGF may be play a critical role when Ang2 binds to its receptor. Shim et al.38 found that overexpression of Ang1 promoted growth of human cervical cancer in mice by promoting tumour angiogenesis and inhibiting tumour cell apoptosis. In their study, increased Ang1 resulted in tumour vessel plasticity with a large number of vessels lacking periendothelial cells. They suggest that there may be multifunctional roles played by the Ang1/Tie2 pathway in various aspects of angiogenesis and that the exact role of Ang1 remains elusive. Differential expression of the angiopoietins in human tumours compared to normal tissue It is thought that there is an alteration in the expression of the angiopoietins as tumours develop which leads to the angiogenic phenotype.

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Overexpression of Ang2 in tumours A number of studies have examined angiopoietin levels or expression in human malignancies. In hepatocellular carcinoma (HCC), frequently an extremely vascular tumour, both Tanaka et al.,39 measuring protein levels, and Mitsuhashi et al.,40 measuring mRNA levels, found a significant increase in Ang2 and no difference in Ang1 expression in tumour, when compared to adjacent normal liver tissue. When hypo- and hypervascular HCC tumours were compared, Ang2 expression was seen in a significantly greater number of hypervascular than hypovascular tumours ( p < 0.05).39 A high Ang2:Ang1 ratio in HCC was related to tumour portal vein invasion, tumour diameter and microvessel density.40 Zhao et al. found similar results with a significantly ( p < 0.01) higher expression level of both VEGF and Ang2 in hepatocellular carcinoma compared to noncancerous liver41 whereas there was no significant difference in Ang1 or Tie2. In renal tissues Currie et al.21 found there was no significant difference in the Ang1 expression when comparing tumour to normal tissue. However, Ang2 mRNA was significantly overexpressed in tumour compared to normal renal tissue ( p < 0.001). Immunohistochemistry subsequently showed that it was expressed strongly on tumour endothelium and weakly in tumour cells.21 Similarly in gastric tissue Etoh et al.12 found a significant increase in Ang2 expression in cancer cells when compared to normal gastric mucosa, with Ang2 expressed in both tumour endothelial and epithelial cells. Ang1 was not significantly increased in gastric cancer in this study. A further study of gastric cancer by Wang et al. again showed Ang2 expression, but also Ang1 expression, were significantly increased in gastric cancer tissue compared to normal controls.42 Ang2 was identified in normal epithelial cells if present in normal tissue. Conversely in tumours, variously differentiated cancerous tissues and some microvessels expressed Ang2.42 In both breast cancer,43 and angiosarcoma,44 Ang2 levels were significantly higher in tumour than in normal tissue. Ahmad et al.13 found that a significantly greater number of colon carcinoma specimens expressed Ang2 compared to Ang1 ( p < 0.05). Durkin et al. showed using microarray that Ang2 was upregulated in neuroendocrine tumours of the pancreas (8 times normal pancreas tissue) and in pancreatic adenocarcinoma (5 times normal). RT-PCR validated this finding showing strong Ang2 expression in 9/9 neuroendocrine tumours, 8/9 adenocarcinoma of the pancreas and weak expression in 2/9 normal pancreas.45 There was no difference with Ang1 and Tie2. Takanami found by RT-PCR in non-small cell lung cancer that Ang2 mRNA expression was significantly greater than that in normal lung ( p ¼ 0.0178).46 Immunohistochemical analysis demonstrated that Ang2 was expressed predominantly in cancer tissues and that normal tissue showed zero or very little expression of Ang2.45 Positive expression of Ang2 was seen mainly in the edges of tumour tissues and in cytoplasm of tumour cells and in endothelial

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cells.45 Hu et al.34 detected a high level of Ang2 in invasive areas of human glioma biopsies compared to central areas. Ang2 expression was prominent in areas surrounding necrosis and the periphery of glioblastoma and was found to be higher in areas of hypoxia. Reduction of Ang1 in tumours Hata et al.47 studied angiopoietin expression in ovarian tumours. A higher expression of Ang1 was found in normal ovary with corpus luteum compared to ovarian cancer. The gene expression of Ang2 was unchanged. Transcripts for Ang1 were found in corpus luteum cells, in endothelial cells within the corpus luteum, and in tumour cells and endothelial cells at the periphery of tumour invasion. Ang2 transcripts were found in the same pattern. In normal ovary with corpus luteum the Ang1/Ang2 gene expression ratio was significantly higher than in ovarian cancer.47 Ahmad et al.13 detected Ang1 in 78% of samples of normal colon mucosa studied, however it was seen in only 54% of colon carcinoma specimens. Ang2 overexpression and Ang1 reduction Wong et al.48 studied non-small cell lung carcinoma (NSCLC) and normal lung. Normal lung expressed a high level of Ang1 that was significantly lower in the carcinomas. An increased intensity of Ang2 expression was found in the tumour vessels compared to normal lung. A significantly higher average CD105-IMVD (a marker of proliferative endothelial cells) was found for Ang2 positive NSCLC compared to Ang2 negative tumours in the presence of high VEGF expression. When VEGF expression was low, the average CD105-IMVD was similar in both high and low Ang2 expression tumours.49 Using immunohistochemistry, Lind et al. showed that Ang2 was generally upregulated in prostate cancer epithelial cells compared with non-malignant epithelial cells.50 Ang2 immunoexpression correlated with immature vessel formation, but not with mature vessels, as was similarly seen in lung tumours.49 On the contrary, Ang1 was expressed more strongly in non-malignant prostate tissue than in tumours.50 This resulted in overall increase in Ang2:Ang1 expression in tumours. In oral squamous cell carcinoma (OSCC) Ang2 expression was significantly ( p < 0.05) higher in OSCC than in adjacent non-cancerous oral tissues and normal oral mucosa.51,52 Conversely the positive rate of Ang1 detected by immunohistochemistry was significantly ( p < 0.05) lower in OSCC than in adjacent non-cancerous oral tissues and normal oral mucosa.51 In an elegant experiment in OSCC, Ang1 overexpression inhibited the growth of the tumour, but when a mixture of Ang2 overexpressing cells to Ang1 overexpressing cells (1:1) was used, a greatly diminished inhibition of tumour growth was seen.11 These studies support the concept of an angiogenic switch where the switch is controlled by the balance of angiogenic proteins, in these cases, angiopoietins. A relatively

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high level of Ang1 creates a stable vasculature in normal tissue and a relatively high level of Ang2 is a destabilising influence in tumour vasculature leaving it exposed for further angiogenic factors to act on it. When the balance is in favour of the inducers, angiogenesis can occur. Studies on brain tumours however, appear to provide conflicting data. Stratmann et al.53 detected cell type-specific upregulation of both Ang1 mRNA and Ang2 mRNA in glioblastoma. Ang1 mRNA was highly expressed in high-grade gliomas but was absent or faint in normal brain tissue and in low-grade gliomas. Ang2 was expressed in glioblastoma but in lower levels than Ang1 and was absent or faint in low-grade gliomas or normal brain. In glioblastoma, Ang1 was expressed in almost all tumour cells with areas of more intense staining alternating with areas of less intense or very weak staining. Ang2 transcripts were not observed in glioblastoma cells but were found in small tumour vessels and capillaries with few periendothelial support cells (unstable vasculature) whereas larger vessels with many periendothelial support cells (stable vasculature) showed only weak staining or were completely negative.53 A similar result was found by Zagzag et al.54 in human astrocytomas where neither Ang1 nor Ang2 were detected in normal brain but Ang1 mRNA was found in tumour cells and Ang2 mRNA was found in endothelial cells of tumour vessels. Those vessels that showed dynamic changes in smooth muscle actin expression (which demonstrates active angiogenesis) expressed high levels of Ang2.54 It would appear therefore that the tissue localisation of the angiopoietins may be very important, but there are very few data regarding distribution of angiopoietins within tissues.

The prognostic value of the angiopoietins Given that tumour development and growth appears to depend upon the Ang1:Ang2 ratio, analysis of this ratio may help to indicate the clinical prognosis. A number of studies in various tumour types have examined this. In renal tumours, Currie et al. demonstrated a significantly higher expression of Ang1 mRNA in low grade (Fuhrman 1 and 2) than in high grade (Fuhrman 3 and 4) tumours in all renal tumour types ( p ¼ 0.02).21 A study in gastric carcinoma12 showed that the average level of Ang2 mRNA expression in groups showing increased vascular involvement and advanced stage disease was significantly ( p < 0.05) higher than in those with less vascular involvement and those showing a less advanced stage of disease. A significantly ( p < 0.05) increased survival time for patients with a low expression of Ang2 compared to those with high expression was observed.12 A later study in gastric cancer showed Ang2 expression correlated with higher TNM stage ( p < 0.01), lymph node metastasis ( p < 0.05) and distant metastasis ( p < 0.01).42 Expression of Tie1 in gastric cancer cells was shown to be associated with reduced survival of the patients and served as an

independent predictor of survival.55 Tie1, Tie2, Ang1 and Ang4 correlated significantly with the degree of histological differentiation and with the depth of tumour invasion. Expression of Tie2 and Ang2 correlated with the degree of venous invasion and the expression of Tie1, Tie2 and Ang1 correlated with the presence of lymphatic invasion. In OSCC,51 the positive rate of Ang2 detected by immunohistochemistry was significantly ( p < 0.01) higher in lymph node positive tumours and level of Ang2 expression was related to pathologic classification.52 Sfiligoi et al. demonstrated that level of expression of Ang2 in breast cancer correlated positively with lymph node invasion, shorter disease free time and overall survival.43 In both patients with HCC40 and those with NSCLC,56 a high relative overexpression of Ang2 was associated with a significantly worse prognosis. In the latter study, high VEGF together with high Ang2 expression resulted in an even lower five year post operative survival.56 In NSCLC the overall ( p < 0.0001) and pathological stage I survival ( p < 0.0201) rates for patients with high Ang2 mRNA expression was significantly worse when compared to those with low Ang2 mRNA expression.46 Multivariate analysis indicated that Ang2 gene expression was an independent prognostic indicator of overall survival.46 The average level of Ang2 mRNA expression, in groups showing positive lymph node metastasis ( p ¼ 0.0009) and advanced stage ( p ¼ 0.0002) was significantly higher than in other groups.46 Oka et al. studied human bladder cancer and found overall survival of patients with Ang2 þ ve tumours was significantly lower than those with Ang2  ve tumours ( p < 0.05). Ang2 þ ve expression was significantly correlated with histological grade ( p ¼ 0.026), histological stage ( p ¼ 0.009) and poor prognosis ( p < 0.05). There was no significant difference in survival according to Ang1 status in the patient. On multivariate analysis Ang2 þ ve expression was an independent negative predictor for survival.57 In prostate tumours Lind et al. found that the intensity of immunoreaction of Ang2 was significantly increased with Gleason score (GS) with GS 8-10 showing the most intense immunoreaction.50 Ang2 levels correlated with the occurrence of metastases at the time of diagnosis and high Ang2 expression was associated with lower survival time.50 Previous studies have found that VEGF is upregulated in prostate cancer and this upregulation is also related to increased Gleason Score and outcome.58e61 These data suggest that the dual upregulation of Ang2 and VEGF in high grade prostate cancer enable an angiogenic drive. Low level Ang1 and high level of Ang2 expression, therefore, may be markers for a poor prognosis with increased vascular involvement, advanced stage disease, lymph node invasion and decreased survival times. In addition, some of these studies have suggested a temporalspatial relationship between the expression of angiopoietins and VEGF in (Fig. 2). Current knowledge is limited and little is known about the mechanism of regulation of the

F. Bach et al. / EJSO 33 (2007) 7e15

2. Vessel co-option by tumour cells

1. Low level paracrine Ang1 binds to Tie2 for stable vessel

Ang1

Temporal-Spatial Relationship

PERICYTE EC

3. Autocrine Ang2 release leads to vessel regression

Y

Tie2

EC

4. Necrotic central area develops. Hypoxia causes VEGF up-regulation

5. Ang2 and VEGF leads to robust angiogenesis

VEGF Ang2

PERICYTE

Y

EC

13

EC VEGF

Tie2

VEGF Ang2

Ang2

Figure 2. Temporal-spatial relationship between the angiopoietins and VEGF (EC e endothelial cell).

angiopoietin genes. Promoter analyses have not been undertaken for the angiopoietins, therefore much of the understanding in this area is derived from in vitro observations of increased or decreased expression following certain conditions.62 It is thought that many tumours start off by co-opting vessels and thus start off as small well vascularised tumours.63 Conceivably as part of a host defence mechanism, there is regression of these initially co-opted vessels initiated by Ang2 expression, leading to a secondarily avascular tumour and massive tumour cell loss.63 The remaining tumour is rescued by robust angiogenesis at the tumour margin due to the combined expression of VEGF, following hypoxia, and Ang2.10 The angiopoietins and VEGF dosage must be exquisitely regulated in spatial, temporal and quantitative manner to avoid vascular disaster.63

with a higher rate of proliferation which can be highly metastatic. A lower ratio of Ang2:Ang1 gives a slower rate of angiogenesis and a less aggressive tumour. Where a higher relative Ang1 level is seen, smaller tumours with less tumour proliferation and fewer vessels are seen. Zagzag et al.54 considered that because Ang2 is expressed in early vascular channels of astrocytomas, it may prove crucial for initiating and perpetuating brain tumour angiogenesis. As such, it may be one of the earliest and most consistent markers for tumour-associated vascular activation, therefore making it an excellent pathological marker of vascular changes and a potential candidate for vascular targeting. Further studies on the temporal-spatial distribution of the angiopoietins and VEGF will elucidate further on the mechanisms involved and may enable development of appropriate therapies.

Summary Angiogenesis is the formation of new blood vessels from existing ones in the presence of stimulatory signals. It is usually absent from normal body tissue as the vasculature is quiescent under the stabilising influences of Ang1/Tie2. In tumours, however, the ‘angiogenic switch’ appears to be activated.4 The properties of the angiogenic growth factors must be further understood in order to determine potential mechanisms for antineoplastic therapy. Studies suggest that the temporal-spatial imbalance between the angiopoietins is crucial to the angiogenic switch. Where a higher relative Ang2 level is seen, the phosphorylation status of Tie2 changes and angiogenesis is initiated. The presence of a higher level of Ang2 compared to Ang1 leads to increased angiogenesis and a more aggressive, larger, heavier tumour

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