Antiangiogenesis for cancer therapy

Abstract

Angiogenesis is the development of new from pre-existing blood vessels. It occurs during wound healing and the menstrual cycle in ovaries and endometrium, but otherwise in adults endothelial cells divide rarely. In many diseases, including cancer, there is an increase in angiogenesis. The latest developments include methods to assess angiogenesis, the demonstration of its prognostic importance, the cloning of angiogenic and antiangiogenic factors, the understanding of a genetic basis to angiogenesis, and development of antiangiogenic drugs.Assessment of tumour angiogenesis and prognosisMonoclonal antibodies that detect vascular endothelium and stain tumours show that tumour blood-vessels occur mostly at the growing edge of turnouts in focal areas or “hot spots”.1Weidner N Folkman J Pozza F et al.Tumour angiogenesis: a new significant and independent prognostic indictor in early-stage breast carcinoma.J Natl Cancer Inst. 1992; 84: 1875-1887Crossref PubMed Scopus (1811) Google Scholar Counting these hot spots or grading vascular density shows a correlation between increasing vascular density with distant metastasis in breast cancer. Several staining methods have been used, including antibodies to cell adhesion molecules CD31 (ref 2) and CD34 and to factor-VIII-associated antigen. Computed image analysis and Chalkley counting have been used to obtain more quantitative information. Assessment of vascular density provides independent prognostic information in breast cancer and most other cancers. The generality of the observation, including even in leukaemias (with high vascular density in marrow) with poor prognosis, supports this as an important in-vivo mechanism. Although some studies have not associated angiogenesis with poor prognostic features, these were often small series or had technical problems in counting.Mechanisms of tumour angiogenesisA tumour is unable to grow above about 1 mm3Folkman J What is the evidence that tumours are angiogenesis dependent?.J Natl Cancer Inst. 1990; 82: 4-6Crossref PubMed Scopus (4376) Google Scholar without development of a new blood supply.3Folkman J What is the evidence that tumours are angiogenesis dependent?.J Natl Cancer Inst. 1990; 82: 4-6Crossref PubMed Scopus (4376) Google Scholar The vessels migrate into the tumour and the tumour towards the vessels (figure, a). The endothelial basement membrane is degraded and new endothelial cells with vascular loops sprout. These vessels are leaky and abnormal in size and shape (figure, b). A range of biochemical processes is involved in these steps, including production of proteases such as urokinase and collagenase by both tumour and endothelial cells. The vessels leak fibrinogen and plasminogen, and tissue factor is activated on them,4Contrino J Hair G Kreutzer DL Rickles FR In-situ detection of tissue factor in vascular endothelial-cells—correlation with the malignant phenotype of human breast disease.Nat Med. 1996; 2: 209-215Crossref PubMed Scopus (525) Google Scholar producing hypercoagulability and extravascular fibrin deposition (figure, c).The turnout endothelial cells are dividing much more rapidly than normal endothelial cells, up to 50 times as fast as in breast cancer. They also can increase in number by remodelling, and cell adhesion molecules such as E-selectin, known to be up-regulated in tumour endothelium, are involved in capillary morphogenesis. The newly proliferating and migrating endothelial cells induce specific integrins αvβ3 and αvβ5 which are essential for their viability while growing.5Brooks PC Cell-adhesion molecules in angiogenesis.Cancer Metastasis Rev. 1996; 15: 187-194Crossref PubMed Scopus (79) Google ScholarMany vascular growth factors have been isolated, including acidic and basic fibroblast-growth-factor and the family of vascular endothelial growth factors (panel 1). The first member of the latter family (VEGF) is the most potent and specific growth factor for endothelial cells.6Claffey KP Robinson GS Regulation of VEGF/VPF expression in tumor-cells — consequences for tumor-growth and metastasis.Cancer Metastasis Rev. 1996; 15: 165-176Crossref PubMed Scopus (182) Google Scholar VEGF is induced by hypoxia and hypoglycaemia and binds to two specific receptors of the tyrosine kinase family—KDR and Flt-1. Both receptors may be up-regulated on tumour endothelial cells and VEGF is up-regulated in many turnouts.Panel 1Angiogenic factorsVascular endothellal growth factorsVEGFA. VEGFB. VEGFCFibroblast growth factors (FGF)FGF-1 (acidic FGF)FGF-2 (basic FGF)FGF-3 (int-2)FGF-4 (K-FGF)Tumour necrosis factor-αTransforming growth factor-βThymldlne phosphorylase (platelet-derived endothelial cell growth-factor)Platelet-derived growth factorScatter factor (hepatocyte growth factor)Interieukln 8Other proteins and peptldesTransforming growth factor-aGranulocyte colony-stimulating factorAngiogeninPlatelet-activating factorProliferinSubstance PNon-peptidesLactateHyaluron fragment Erucamide Prostaglandin E1, and E2Apart from the stimulatory pathways, we now recognise that there are antiangiogenic factors which suppress normal angiogenesis and may need to be switched off to allow tumour angiogenesis (panel 2). Examples include thrombospondin, a secreted protein that interacts with several components of the extracellular matrix and inhibits angiogenesis. Folkman's group purified an antiangiogenic protein made within tumours in vivo and found it was a proteolytic product of plasminogen, angiostatin.7O'Reilly MS Holmgren L Shing Y et al.Angiostatin—a novel angiogenesis inhibitor that mediates the suppression of metastases by a lewis lung-carcinoma.Cell. 1994; 79: 315-328Summary Full Text PDF PubMed Scopus (3150) Google Scholar Several antiangiogenic proteins are proteolytic products of large molecules previously not known to be inhibitors of angiogenesis (eg, prolactin and fibronectin) or more potent products of antiangiogenic parent molecules (eg, platelet factor 4).Panel 2Antlanglogenlc factors and drugsAntlangiogenlc factorsAngiostatinEndostatinInterferons α, β, and γInterferon-finducible protein 10Interleukin 1Interleukin 12Platelet factor 4Proliferin related protein16 K prolactin fragmentThrombospondin 12-methoxyoestradiolTissue inhibitor of metalloproteinaseRetinoic acidTumour necrosis factor-α(high concertratrations)Antlanglogenlc drugsMetalloproteinase inhibitorsFumagillm analogues (AGM1470)Group B streptococcus toxin InterferonsPentosan polysulphateVEGF blocking-antibodiesVEGF-receptor-kinase inhibitorsThe angiogenic switchThe essential role of angiogenesis in tumour progression and metastasis (figure, d) and the balance of positive and negative regulated factors lead to the idea of an angiogenic “switch”, which is activated in tumour angiogenesis.8Hanahan D Folkman J Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis.Cell. 1996; 86: 353-364Summary Full Text Full Text PDF PubMed Scopus (6021) Google Scholar A small hypoxic tumour which is genetically unstable will be exposed to a hostile environment, and mutations producing a phenotype able to resist cell death (apoptosis) will have a survival advantage (figure, b). p53, a nuclear protein involved in cell-cycle regulation and apoptosis, is one of the genes most commonly mutated in cancer. Mutations enable cells to survive under hypoxic conditions9Graeber TG Osmanian C Jacks T et al.Hypoxia-mediated selection of cells with diminished apoptotic potential in solid tumors.Nature. 1996; 379: 88-91Crossref PubMed Scopus (2160) Google Scholar and also switch on the angiogenic phenotype by enhancing VEGF production10Mukhopadhyay D Tsiokas L Sukhatme VP Wild-type p53 and v-src exert opposing influences on human vascular endothelial growth-factor gene-expression.Cancer Res. 1995; 55: 6161-6165PubMed Google Scholar and down-regulation of thrombospondin. There are many other mechanisms involving loss of inhibitors and release of angiogenic factors bound to the stroma by proteins. The balance of proangiogenic and antiangiogenic factors is important in tumour dormancy and control of micrometastasis, where the apoptosis rate is high until angiogenesis occurs, providing growth advantage by reducing apoptosis.11Holmgren L O'Reilly MS Folkman J Dormancy of micrometastases—balanced proliferation and apoptosis in the presence of angiogenesis suppression.Nat Med. 1995; 1: 149-153Crossref PubMed Scopus (1662) Google Scholar The pathway regulating VEGF involves a transcription factor (hypoxia-induced factor 1, hif1), which activates transcription under hypoxic conditions.12Bunn HF Poyton RO Oxygen sensing and molecular adaptation to hypoxia.Physiol Rev. 1996; 76: 839-885Crossref PubMed Scopus (1041) Google Scholar VEGF mRNA can also be regulated post-transcriptionally. Mutations in the latter pathway increase VEGF production, and occur in familial renal cancer (von Hippel-Lindau syndrome)13Iliopoulos O Levy AP Jiang C Kaelin WG Goldberg MA Negative regulation of hypoxia-inducible genes by the von Hippel-Lindau protein.in: 4th ed. Proc Natl Acad Sci USA. 93. 1996: 10595-10599Google Scholar and sporadic renal cancer. Thus there is genetic evidence for a major role of the angiogenic pathway as a mechanism of carcinogenesis. The switch may occur, as for in-situ-carcinoma of the breast, and may involve different mechanisms at later stages of tumour progression.Antiangiogenic therapy and vascular targetingAntiangiogenesis involves therapy against biochemical targets on the neovasculature, aiming to inhibit proliferation and blood-vessel formation. Vascular targeting differs in that it aims to specifically destroy the existing vasculature acutely and will induce tumour necrosis. Many targets for antiangiogenesis are being developed based on the biochemical pathways described above.14Teicher BA Angiogenesis and cancer metastases—therapeutic approaches.Crit Rev Oncol Hematol. 1995; 20: 9-39Summary Full Text PDF PubMed Scopus (35) Google Scholar These include inhibitors of matrix metalloproteases15Brown PD Giavazzi R Matrix metalloproteinase inhibition—a review of antitumor-activity.Ann Oncol. 1995; 6: 967-974Crossref PubMed Scopus (230) Google Scholar and urokinase,16Min HY Doyle LV Vitt CR et al.Urokinase receptor antagonists inhibit angiogenesis and primary tumor-growth in syngeneic mice.Cancer Res. 1996; 56: 2428-2433PubMed Google Scholar inhibitory factors (platelet factor 4), heparin analogues that inhibit heparin-binding angiogenic factors, and inhibitors of VEGF function (panel 2). The VEGF pathway has become a prime target because many ways of inhibiting VEGF have shown potent antitumour effects in vivo in animal models. These include antisense VEGF RNA, antibodies to VEGF or its receptors, and dominant negative VEGF receptors produced by retroviruses to inhibit VEGF receptors.17Millauer B Longhi MP Plate KH et al.Dominant-negative inhibition of flk-1 suppresses the growth of many tumor types in-vivo.Cancer Res. 1996; 56: 1615-1620PubMed Google Scholar Low-molecular-weight inhibitors of VEGF receptor kinase are also under development.Vascular targeting is already highly successful in the treatment of limb metastasis in melanoma by regional arterial perfusion of tumour necrosis factor-α which specifically destroys tumour vasculature, but is too toxic systemically for treating other sites of disease. Approaches are under investigation using gene therapy to produce tumour necrosis factor-α selectively, regulated by promoters activated in tumour endothelium. Antibodies to VEGF receptors, linked to toxins are also under development and antibodies to the αvβ3 integrins produce endothelial apoptosis. The use of cytotoxic drugs activated by hypoxia may be synergistic with the above approaches.18Wilson WR Pruijn FB Hypoxia-activated prodrugs as antitumor agents—strategies for maximizing tumor-cell killing.Clin Exp Pharmacol Physiol. 1995; 22: 881-885Crossref PubMed Scopus (8) Google ScholarClinical use and assessment of antiangiogenic drugsAntiangiogenic drugs may not cause tumour regression, but inhibit growth of the invading edge and produce “disease stabilisation”. Thus in future clinical trials it may be difficult to assess response with conventional criteria. To assess antiangiogenic effects, non-invasive techniques measuring blood flow, such as colour doppler, and measurement of changes in tumour metabolism are being investigated (eg, by positron emission tomography). There is a reasonable correlation of angiogenesis assessed by magnetic resonance imaging with vascular density assessed pathologically in primary breast cancer. Other techniques involve measurement of angiogenic factors such as basic fibroblast-growth-factor and VEGF in urine and plasma.Animal experiments show that a combination of antiangiogenic drugs is more effective that single agents, similar to experience with chemotherapy.19Parangi S O'Reilly M Christofori G et al.Antiangiogenic therapy of transgenic mice impairs de-novo tumor-growth.in: 4th ed. Proc Natl Acad Sci USA. 93. 1996: 2002-2007Google Scholar Also, combination of antiangiogenic agents with conventional cytotoxic drugs enhances the effects of the latter. New antiangiogenic drugs will probably be used in combination with existing drugs but perhaps also used alone as long-term maintenance therapy. These studies also show antiangiogenic agents are more effective against small than large tumours, so early application in adjuvant therapy may be their future role.ConclusionAntiangiogenesis therapy is undergoing extensive study in phase I-III trials. The field of angiogenesis is an excellent example of how basic understanding of a key pathological process can generate better clinical understanding and generate new therapeutic approaches. We await the results of the ingenuity of the biotechnology industry to see how this finally develops into clinical practice. Angiogenesis is the development of new from pre-existing blood vessels. It occurs during wound healing and the menstrual cycle in ovaries and endometrium, but otherwise in adults endothelial cells divide rarely. In many diseases, including cancer, there is an increase in angiogenesis. The latest developments include methods to assess angiogenesis, the demonstration of its prognostic importance, the cloning of angiogenic and antiangiogenic factors, the understanding of a genetic basis to angiogenesis, and development of antiangiogenic drugs. Assessment of tumour angiogenesis and prognosisMonoclonal antibodies that detect vascular endothelium and stain tumours show that tumour blood-vessels occur mostly at the growing edge of turnouts in focal areas or “hot spots”.1Weidner N Folkman J Pozza F et al.Tumour angiogenesis: a new significant and independent prognostic indictor in early-stage breast carcinoma.J Natl Cancer Inst. 1992; 84: 1875-1887Crossref PubMed Scopus (1811) Google Scholar Counting these hot spots or grading vascular density shows a correlation between increasing vascular density with distant metastasis in breast cancer. Several staining methods have been used, including antibodies to cell adhesion molecules CD31 (ref 2) and CD34 and to factor-VIII-associated antigen. Computed image analysis and Chalkley counting have been used to obtain more quantitative information. Assessment of vascular density provides independent prognostic information in breast cancer and most other cancers. The generality of the observation, including even in leukaemias (with high vascular density in marrow) with poor prognosis, supports this as an important in-vivo mechanism. Although some studies have not associated angiogenesis with poor prognostic features, these were often small series or had technical problems in counting. Monoclonal antibodies that detect vascular endothelium and stain tumours show that tumour blood-vessels occur mostly at the growing edge of turnouts in focal areas or “hot spots”.1Weidner N Folkman J Pozza F et al.Tumour angiogenesis: a new significant and independent prognostic indictor in early-stage breast carcinoma.J Natl Cancer Inst. 1992; 84: 1875-1887Crossref PubMed Scopus (1811) Google Scholar Counting these hot spots or grading vascular density shows a correlation between increasing vascular density with distant metastasis in breast cancer. Several staining methods have been used, including antibodies to cell adhesion molecules CD31 (ref 2) and CD34 and to factor-VIII-associated antigen. Computed image analysis and Chalkley counting have been used to obtain more quantitative information. Assessment of vascular density provides independent prognostic information in breast cancer and most other cancers. The generality of the observation, including even in leukaemias (with high vascular density in marrow) with poor prognosis, supports this as an important in-vivo mechanism. Although some studies have not associated angiogenesis with poor prognostic features, these were often small series or had technical problems in counting. Mechanisms of tumour angiogenesisA tumour is unable to grow above about 1 mm3Folkman J What is the evidence that tumours are angiogenesis dependent?.J Natl Cancer Inst. 1990; 82: 4-6Crossref PubMed Scopus (4376) Google Scholar without development of a new blood supply.3Folkman J What is the evidence that tumours are angiogenesis dependent?.J Natl Cancer Inst. 1990; 82: 4-6Crossref PubMed Scopus (4376) Google Scholar The vessels migrate into the tumour and the tumour towards the vessels (figure, a). The endothelial basement membrane is degraded and new endothelial cells with vascular loops sprout. These vessels are leaky and abnormal in size and shape (figure, b). A range of biochemical processes is involved in these steps, including production of proteases such as urokinase and collagenase by both tumour and endothelial cells. The vessels leak fibrinogen and plasminogen, and tissue factor is activated on them,4Contrino J Hair G Kreutzer DL Rickles FR In-situ detection of tissue factor in vascular endothelial-cells—correlation with the malignant phenotype of human breast disease.Nat Med. 1996; 2: 209-215Crossref PubMed Scopus (525) Google Scholar producing hypercoagulability and extravascular fibrin deposition (figure, c).The turnout endothelial cells are dividing much more rapidly than normal endothelial cells, up to 50 times as fast as in breast cancer. They also can increase in number by remodelling, and cell adhesion molecules such as E-selectin, known to be up-regulated in tumour endothelium, are involved in capillary morphogenesis. The newly proliferating and migrating endothelial cells induce specific integrins αvβ3 and αvβ5 which are essential for their viability while growing.5Brooks PC Cell-adhesion molecules in angiogenesis.Cancer Metastasis Rev. 1996; 15: 187-194Crossref PubMed Scopus (79) Google ScholarMany vascular growth factors have been isolated, including acidic and basic fibroblast-growth-factor and the family of vascular endothelial growth factors (panel 1). The first member of the latter family (VEGF) is the most potent and specific growth factor for endothelial cells.6Claffey KP Robinson GS Regulation of VEGF/VPF expression in tumor-cells — consequences for tumor-growth and metastasis.Cancer Metastasis Rev. 1996; 15: 165-176Crossref PubMed Scopus (182) Google Scholar VEGF is induced by hypoxia and hypoglycaemia and binds to two specific receptors of the tyrosine kinase family—KDR and Flt-1. Both receptors may be up-regulated on tumour endothelial cells and VEGF is up-regulated in many turnouts.Panel 1Angiogenic factorsVascular endothellal growth factorsVEGFA. VEGFB. VEGFCFibroblast growth factors (FGF)FGF-1 (acidic FGF)FGF-2 (basic FGF)FGF-3 (int-2)FGF-4 (K-FGF)Tumour necrosis factor-αTransforming growth factor-βThymldlne phosphorylase (platelet-derived endothelial cell growth-factor)Platelet-derived growth factorScatter factor (hepatocyte growth factor)Interieukln 8Other proteins and peptldesTransforming growth factor-aGranulocyte colony-stimulating factorAngiogeninPlatelet-activating factorProliferinSubstance PNon-peptidesLactateHyaluron fragment Erucamide Prostaglandin E1, and E2Apart from the stimulatory pathways, we now recognise that there are antiangiogenic factors which suppress normal angiogenesis and may need to be switched off to allow tumour angiogenesis (panel 2). Examples include thrombospondin, a secreted protein that interacts with several components of the extracellular matrix and inhibits angiogenesis. Folkman's group purified an antiangiogenic protein made within tumours in vivo and found it was a proteolytic product of plasminogen, angiostatin.7O'Reilly MS Holmgren L Shing Y et al.Angiostatin—a novel angiogenesis inhibitor that mediates the suppression of metastases by a lewis lung-carcinoma.Cell. 1994; 79: 315-328Summary Full Text PDF PubMed Scopus (3150) Google Scholar Several antiangiogenic proteins are proteolytic products of large molecules previously not known to be inhibitors of angiogenesis (eg, prolactin and fibronectin) or more potent products of antiangiogenic parent molecules (eg, platelet factor 4).Panel 2Antlanglogenlc factors and drugsAntlangiogenlc factorsAngiostatinEndostatinInterferons α, β, and γInterferon-finducible protein 10Interleukin 1Interleukin 12Platelet factor 4Proliferin related protein16 K prolactin fragmentThrombospondin 12-methoxyoestradiolTissue inhibitor of metalloproteinaseRetinoic acidTumour necrosis factor-α(high concertratrations)Antlanglogenlc drugsMetalloproteinase inhibitorsFumagillm analogues (AGM1470)Group B streptococcus toxin InterferonsPentosan polysulphateVEGF blocking-antibodiesVEGF-receptor-kinase inhibitors A tumour is unable to grow above about 1 mm3Folkman J What is the evidence that tumours are angiogenesis dependent?.J Natl Cancer Inst. 1990; 82: 4-6Crossref PubMed Scopus (4376) Google Scholar without development of a new blood supply.3Folkman J What is the evidence that tumours are angiogenesis dependent?.J Natl Cancer Inst. 1990; 82: 4-6Crossref PubMed Scopus (4376) Google Scholar The vessels migrate into the tumour and the tumour towards the vessels (figure, a). The endothelial basement membrane is degraded and new endothelial cells with vascular loops sprout. These vessels are leaky and abnormal in size and shape (figure, b). A range of biochemical processes is involved in these steps, including production of proteases such as urokinase and collagenase by both tumour and endothelial cells. The vessels leak fibrinogen and plasminogen, and tissue factor is activated on them,4Contrino J Hair G Kreutzer DL Rickles FR In-situ detection of tissue factor in vascular endothelial-cells—correlation with the malignant phenotype of human breast disease.Nat Med. 1996; 2: 209-215Crossref PubMed Scopus (525) Google Scholar producing hypercoagulability and extravascular fibrin deposition (figure, c). The turnout endothelial cells are dividing much more rapidly than normal endothelial cells, up to 50 times as fast as in breast cancer. They also can increase in number by remodelling, and cell adhesion molecules such as E-selectin, known to be up-regulated in tumour endothelium, are involved in capillary morphogenesis. The newly proliferating and migrating endothelial cells induce specific integrins αvβ3 and αvβ5 which are essential for their viability while growing.5Brooks PC Cell-adhesion molecules in angiogenesis.Cancer Metastasis Rev. 1996; 15: 187-194Crossref PubMed Scopus (79) Google Scholar Many vascular growth factors have been isolated, including acidic and basic fibroblast-growth-factor and the family of vascular endothelial growth factors (panel 1). The first member of the latter family (VEGF) is the most potent and specific growth factor for endothelial cells.6Claffey KP Robinson GS Regulation of VEGF/VPF expression in tumor-cells — consequences for tumor-growth and metastasis.Cancer Metastasis Rev. 1996; 15: 165-176Crossref PubMed Scopus (182) Google Scholar VEGF is induced by hypoxia and hypoglycaemia and binds to two specific receptors of the tyrosine kinase family—KDR and Flt-1. Both receptors may be up-regulated on tumour endothelial cells and VEGF is up-regulated in many turnouts. Vascular endothellal growth factorsVEGFA. VEGFB. VEGFCFibroblast growth factors (FGF)FGF-1 (acidic FGF)FGF-2 (basic FGF)FGF-3 (int-2)FGF-4 (K-FGF)Tumour necrosis factor-αTransforming growth factor-βThymldlne phosphorylase (platelet-derived endothelial cell growth-factor)Platelet-derived growth factorScatter factor (hepatocyte growth factor)Interieukln 8Other proteins and peptldesTransforming growth factor-aGranulocyte colony-stimulating factorAngiogeninPlatelet-activating factorProliferinSubstance PNon-peptidesLactateHyaluron fragment Erucamide Prostaglandin E1, and E2 Vascular endothellal growth factors VEGFA. VEGFB. VEGFC Fibroblast growth factors (FGF) FGF-1 (acidic FGF) FGF-2 (basic FGF) FGF-3 (int-2) FGF-4 (K-FGF) Tumour necrosis factor-α Transforming growth factor-β Thymldlne phosphorylase (platelet-derived endothelial cell growth-factor) Platelet-derived growth factor Scatter factor (hepatocyte growth factor) Interieukln 8 Other proteins and peptldes Transforming growth factor-a Granulocyte colony-stimulating factor Angiogenin Platelet-activating factor Proliferin Substance P Non-peptides Lactate Hyaluron fragment Erucamide Prostaglandin E1, and E2 Apart from the stimulatory pathways, we now recognise that there are antiangiogenic factors which suppress normal angiogenesis and may need to be switched off to allow tumour angiogenesis (panel 2). Examples include thrombospondin, a secreted protein that interacts with several components of the extracellular matrix and inhibits angiogenesis. Folkman's group purified an antiangiogenic protein made within tumours in vivo and found it was a proteolytic product of plasminogen, angiostatin.7O'Reilly MS Holmgren L Shing Y et al.Angiostatin—a novel angiogenesis inhibitor that mediates the suppression of metastases by a lewis lung-carcinoma.Cell. 1994; 79: 315-328Summary Full Text PDF PubMed Scopus (3150) Google Scholar Several antiangiogenic proteins are proteolytic products of large molecules previously not known to be inhibitors of angiogenesis (eg, prolactin and fibronectin) or more potent products of antiangiogenic parent molecules (eg, platelet factor 4). Antlangiogenlc factorsAngiostatinEndostatinInterferons α, β, and γInterferon-finducible protein 10Interleukin 1Interleukin 12Platelet factor 4Proliferin related protein16 K prolactin fragmentThrombospondin 12-methoxyoestradiolTissue inhibitor of metalloproteinaseRetinoic acidTumour necrosis factor-α(high concertratrations)Antlanglogenlc drugsMetalloproteinase inhibitorsFumagillm analogues (AGM1470)Group B streptococcus toxin InterferonsPentosan polysulphateVEGF blocking-antibodiesVEGF-receptor-kinase inhibitors Antlangiogenlc factors Angiostatin Endostatin Interferons α, β, and γ Interferon-finducible protein 10 Interleukin 1 Interleukin 12 Platelet factor 4 Proliferin related protein 16 K prolactin fragment Thrombospondin 1 2-methoxyoestradiol Tissue inhibitor of metalloproteinase Retinoic acid Tumour necrosis factor-α(high concertratrations) Antlanglogenlc drugs Metalloproteinase inhibitors Fumagillm analogues (AGM1470) Group B streptococcus toxin Interferons Pentosan polysulphate VEGF blocking-antibodies VEGF-receptor-kinase inhibitors The angiogenic switchThe essential role of angiogenesis in tumour progression and metastasis (figure, d) and the balance of positive and negative regulated factors lead to the idea of an angiogenic “switch”, which is activated in tumour angiogenesis.8Hanahan D Folkman J Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis.Cell. 1996; 86: 353-364Summary Full Text Full Text PDF PubMed Scopus (6021) Google Scholar A small hypoxic tumour which is genetically unstable will be exposed to a hostile environment, and mutations producing a phenotype able to resist cell death (apoptosis) will have a survival advantage (figure, b). p53, a nuclear protein involved in cell-cycle regulation and apoptosis, is one of the genes most commonly mutated in cancer. Mutations enable cells to survive under hypoxic conditions9Graeber TG Osmanian C Jacks T et al.Hypoxia-mediated selection of cells with diminished apoptotic potential in solid tumors.Nature. 1996; 379: 88-91Crossref PubMed Scopus (2160) Google Scholar and also switch on the angiogenic phenotype by enhancing VEGF production10Mukhopadhyay D Tsiokas L Sukhatme VP Wild-type p53 and v-src exert opposing influences on human vascular endothelial growth-factor gene-expression.Cancer Res. 1995; 55: 6161-6165PubMed Google Scholar and down-regulation of thrombospondin. There are many other mechanisms involving loss of inhibitors and release of angiogenic factors bound to the stroma by proteins. The balance of proangiogenic and antiangiogenic factors is important in tumour dormancy and control of micrometastasis, where the apoptosis rate is high until angiogenesis occurs, providing growth advantage by reducing apoptosis.11Holmgren L O'Reilly MS Folkman J Dormancy of micrometastases—balanced proliferation and apoptosis in the presence of angiogenesis suppression.Nat Med. 1995; 1: 149-153Crossref PubMed Scopus (1662) Google Scholar The pathway regulating VEGF involves a transcription factor (hypoxia-induced factor 1, hif1), which activates transcription under hypoxic conditions.12Bunn HF Poyton RO Oxygen sensing and molecular adaptation to hypoxia.Physiol Rev. 1996; 76: 839-885Crossref PubMed Scopus (1041) Google Scholar VEGF mRNA can also be regulated post-transcriptionally. Mutations in the latter pathway increase VEGF production, and occur in familial renal cancer (von Hippel-Lindau syndrome)13Iliopoulos O Levy AP Jiang C Kaelin WG Goldberg MA Negative regulation of hypoxia-inducible genes by the von Hippel-Lindau protein.in: 4th ed. Proc Natl Acad Sci USA. 93. 1996: 10595-10599Google Scholar and sporadic renal cancer. Thus there is genetic evidence for a major role of the angiogenic pathway as a mechanism of carcinogenesis. The switch may occur, as for in-situ-carcinoma of the breast, and may involve different mechanisms at later stages of tumour progression. The essential role of angiogenesis in tumour progression and metastasis (figure, d) and the balance of positive and negative regulated factors lead to the idea of an angiogenic “switch”, which is activated in tumour angiogenesis.8Hanahan D Folkman J Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis.Cell. 1996; 86: 353-364Summary Full Text Full Text PDF PubMed Scopus (6021) Google Scholar A small hypoxic tumour which is genetically unstable will be exposed to a hostile environment, and mutations producing a phenotype able to resist cell death (apoptosis) will have a survival advantage (figure, b). p53, a nuclear protein involved in cell-cycle regulation and apoptosis, is one of the genes most commonly mutated in cancer. Mutations enable cells to survive under hypoxic conditions9Graeber TG Osmanian C Jacks T et al.Hypoxia-mediated selection of cells with diminished apoptotic potential in solid tumors.Nature. 1996; 379: 88-91Crossref PubMed Scopus (2160) Google Scholar and also switch on the angiogenic phenotype by enhancing VEGF production10Mukhopadhyay D Tsiokas L Sukhatme VP Wild-type p53 and v-src exert opposing influences on human vascular endothelial growth-factor gene-expression.Cancer Res. 1995; 55: 6161-6165PubMed Google Scholar and down-regulation of thrombospondin. There are many other mechanisms involving loss of inhibitors and release of angiogenic factors bound to the stroma by proteins. The balance of proangiogenic and antiangiogenic factors is important in tumour dormancy and control of micrometastasis, where the apoptosis rate is high until angiogenesis occurs, providing growth advantage by reducing apoptosis.11Holmgren L O'Reilly MS Folkman J Dormancy of micrometastases—balanced proliferation and apoptosis in the presence of angiogenesis suppression.Nat Med. 1995; 1: 149-153Crossref PubMed Scopus (1662) Google Scholar The pathway regulating VEGF involves a transcription factor (hypoxia-induced factor 1, hif1), which activates transcription under hypoxic conditions.12Bunn HF Poyton RO Oxygen sensing and molecular adaptation to hypoxia.Physiol Rev. 1996; 76: 839-885Crossref PubMed Scopus (1041) Google Scholar VEGF mRNA can also be regulated post-transcriptionally. Mutations in the latter pathway increase VEGF production, and occur in familial renal cancer (von Hippel-Lindau syndrome)13Iliopoulos O Levy AP Jiang C Kaelin WG Goldberg MA Negative regulation of hypoxia-inducible genes by the von Hippel-Lindau protein.in: 4th ed. Proc Natl Acad Sci USA. 93. 1996: 10595-10599Google Scholar and sporadic renal cancer. Thus there is genetic evidence for a major role of the angiogenic pathway as a mechanism of carcinogenesis. The switch may occur, as for in-situ-carcinoma of the breast, and may involve different mechanisms at later stages of tumour progression. Antiangiogenic therapy and vascular targetingAntiangiogenesis involves therapy against biochemical targets on the neovasculature, aiming to inhibit proliferation and blood-vessel formation. Vascular targeting differs in that it aims to specifically destroy the existing vasculature acutely and will induce tumour necrosis. Many targets for antiangiogenesis are being developed based on the biochemical pathways described above.14Teicher BA Angiogenesis and cancer metastases—therapeutic approaches.Crit Rev Oncol Hematol. 1995; 20: 9-39Summary Full Text PDF PubMed Scopus (35) Google Scholar These include inhibitors of matrix metalloproteases15Brown PD Giavazzi R Matrix metalloproteinase inhibition—a review of antitumor-activity.Ann Oncol. 1995; 6: 967-974Crossref PubMed Scopus (230) Google Scholar and urokinase,16Min HY Doyle LV Vitt CR et al.Urokinase receptor antagonists inhibit angiogenesis and primary tumor-growth in syngeneic mice.Cancer Res. 1996; 56: 2428-2433PubMed Google Scholar inhibitory factors (platelet factor 4), heparin analogues that inhibit heparin-binding angiogenic factors, and inhibitors of VEGF function (panel 2). The VEGF pathway has become a prime target because many ways of inhibiting VEGF have shown potent antitumour effects in vivo in animal models. These include antisense VEGF RNA, antibodies to VEGF or its receptors, and dominant negative VEGF receptors produced by retroviruses to inhibit VEGF receptors.17Millauer B Longhi MP Plate KH et al.Dominant-negative inhibition of flk-1 suppresses the growth of many tumor types in-vivo.Cancer Res. 1996; 56: 1615-1620PubMed Google Scholar Low-molecular-weight inhibitors of VEGF receptor kinase are also under development.Vascular targeting is already highly successful in the treatment of limb metastasis in melanoma by regional arterial perfusion of tumour necrosis factor-α which specifically destroys tumour vasculature, but is too toxic systemically for treating other sites of disease. Approaches are under investigation using gene therapy to produce tumour necrosis factor-α selectively, regulated by promoters activated in tumour endothelium. Antibodies to VEGF receptors, linked to toxins are also under development and antibodies to the αvβ3 integrins produce endothelial apoptosis. The use of cytotoxic drugs activated by hypoxia may be synergistic with the above approaches.18Wilson WR Pruijn FB Hypoxia-activated prodrugs as antitumor agents—strategies for maximizing tumor-cell killing.Clin Exp Pharmacol Physiol. 1995; 22: 881-885Crossref PubMed Scopus (8) Google Scholar Antiangiogenesis involves therapy against biochemical targets on the neovasculature, aiming to inhibit proliferation and blood-vessel formation. Vascular targeting differs in that it aims to specifically destroy the existing vasculature acutely and will induce tumour necrosis. Many targets for antiangiogenesis are being developed based on the biochemical pathways described above.14Teicher BA Angiogenesis and cancer metastases—therapeutic approaches.Crit Rev Oncol Hematol. 1995; 20: 9-39Summary Full Text PDF PubMed Scopus (35) Google Scholar These include inhibitors of matrix metalloproteases15Brown PD Giavazzi R Matrix metalloproteinase inhibition—a review of antitumor-activity.Ann Oncol. 1995; 6: 967-974Crossref PubMed Scopus (230) Google Scholar and urokinase,16Min HY Doyle LV Vitt CR et al.Urokinase receptor antagonists inhibit angiogenesis and primary tumor-growth in syngeneic mice.Cancer Res. 1996; 56: 2428-2433PubMed Google Scholar inhibitory factors (platelet factor 4), heparin analogues that inhibit heparin-binding angiogenic factors, and inhibitors of VEGF function (panel 2). The VEGF pathway has become a prime target because many ways of inhibiting VEGF have shown potent antitumour effects in vivo in animal models. These include antisense VEGF RNA, antibodies to VEGF or its receptors, and dominant negative VEGF receptors produced by retroviruses to inhibit VEGF receptors.17Millauer B Longhi MP Plate KH et al.Dominant-negative inhibition of flk-1 suppresses the growth of many tumor types in-vivo.Cancer Res. 1996; 56: 1615-1620PubMed Google Scholar Low-molecular-weight inhibitors of VEGF receptor kinase are also under development. Vascular targeting is already highly successful in the treatment of limb metastasis in melanoma by regional arterial perfusion of tumour necrosis factor-α which specifically destroys tumour vasculature, but is too toxic systemically for treating other sites of disease. Approaches are under investigation using gene therapy to produce tumour necrosis factor-α selectively, regulated by promoters activated in tumour endothelium. Antibodies to VEGF receptors, linked to toxins are also under development and antibodies to the αvβ3 integrins produce endothelial apoptosis. The use of cytotoxic drugs activated by hypoxia may be synergistic with the above approaches.18Wilson WR Pruijn FB Hypoxia-activated prodrugs as antitumor agents—strategies for maximizing tumor-cell killing.Clin Exp Pharmacol Physiol. 1995; 22: 881-885Crossref PubMed Scopus (8) Google Scholar Clinical use and assessment of antiangiogenic drugsAntiangiogenic drugs may not cause tumour regression, but inhibit growth of the invading edge and produce “disease stabilisation”. Thus in future clinical trials it may be difficult to assess response with conventional criteria. To assess antiangiogenic effects, non-invasive techniques measuring blood flow, such as colour doppler, and measurement of changes in tumour metabolism are being investigated (eg, by positron emission tomography). There is a reasonable correlation of angiogenesis assessed by magnetic resonance imaging with vascular density assessed pathologically in primary breast cancer. Other techniques involve measurement of angiogenic factors such as basic fibroblast-growth-factor and VEGF in urine and plasma.Animal experiments show that a combination of antiangiogenic drugs is more effective that single agents, similar to experience with chemotherapy.19Parangi S O'Reilly M Christofori G et al.Antiangiogenic therapy of transgenic mice impairs de-novo tumor-growth.in: 4th ed. Proc Natl Acad Sci USA. 93. 1996: 2002-2007Google Scholar Also, combination of antiangiogenic agents with conventional cytotoxic drugs enhances the effects of the latter. New antiangiogenic drugs will probably be used in combination with existing drugs but perhaps also used alone as long-term maintenance therapy. These studies also show antiangiogenic agents are more effective against small than large tumours, so early application in adjuvant therapy may be their future role. Antiangiogenic drugs may not cause tumour regression, but inhibit growth of the invading edge and produce “disease stabilisation”. Thus in future clinical trials it may be difficult to assess response with conventional criteria. To assess antiangiogenic effects, non-invasive techniques measuring blood flow, such as colour doppler, and measurement of changes in tumour metabolism are being investigated (eg, by positron emission tomography). There is a reasonable correlation of angiogenesis assessed by magnetic resonance imaging with vascular density assessed pathologically in primary breast cancer. Other techniques involve measurement of angiogenic factors such as basic fibroblast-growth-factor and VEGF in urine and plasma. Animal experiments show that a combination of antiangiogenic drugs is more effective that single agents, similar to experience with chemotherapy.19Parangi S O'Reilly M Christofori G et al.Antiangiogenic therapy of transgenic mice impairs de-novo tumor-growth.in: 4th ed. Proc Natl Acad Sci USA. 93. 1996: 2002-2007Google Scholar Also, combination of antiangiogenic agents with conventional cytotoxic drugs enhances the effects of the latter. New antiangiogenic drugs will probably be used in combination with existing drugs but perhaps also used alone as long-term maintenance therapy. These studies also show antiangiogenic agents are more effective against small than large tumours, so early application in adjuvant therapy may be their future role. ConclusionAntiangiogenesis therapy is undergoing extensive study in phase I-III trials. The field of angiogenesis is an excellent example of how basic understanding of a key pathological process can generate better clinical understanding and generate new therapeutic approaches. We await the results of the ingenuity of the biotechnology industry to see how this finally develops into clinical practice. Antiangiogenesis therapy is undergoing extensive study in phase I-III trials. The field of angiogenesis is an excellent example of how basic understanding of a key pathological process can generate better clinical understanding and generate new therapeutic approaches. We await the results of the ingenuity of the biotechnology industry to see how this finally develops into clinical practice.