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This article has been retracted as it was submitted for publication without the knowledge and approval of the co-authors Maxine Emmett and Rowan Pritchard Jones.
Volume 2012 (2012), Article ID 546927, 7 pages
The Roles of Angiogenesis in Malignant Melanoma: Trends in Basic Science Research over the Last 100 Years
1Department of Molecular and Clinical Cancer Medicine, Mersey Academic Plastic Surgery Group, Liverpool Cancer Research UK Centre, The Duncan Building, Daulby Street, Liverpool L69 3GA, UK
2Department of Plastic Surgery, Whiston Hospital, Warrington Road, Prescott, Merseyside L35 5DR, UK
Received 30 March 2012; Accepted 28 April 2012
Academic Editors: N. Fujimoto, P. Karakitsos, and L. Saragoni
Copyright © 2012 D. Dewing et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Blood vessels arose during evolution carrying oxygen and nutrients to distant organs via complex networks of blood vessels penetrating organs and tissues. Mammalian cells require oxygen and nutrients for survival, of which oxygen has a diffusion limit of 100 to 200 μm between cell and blood vessel. For growth beyond this margin, cells must recruit new blood vessels, first by vasculogenesis, where embryonic vessels form from endothelial precursors, then angiogenesis which is the sprouting of interstitial tissue columns into the lumen of preexisting blood vessels. Angiogenesis occurs in many inflammatory diseases and in many malignant disease states, including over 90% of solid tumours. Malignant melanoma (MM) is the most lethal skin cancer, highly angiogenic, highly metastatic, and refractory to all treatments. Raised serum levels of vascular endothelial growth factor (VEGF) strongly correlate MM disease progression and poor prognosis. Melanoma cells secrete several proangiogenic cytokines including VEGF-A, fibroblast growth factor (FGF-2), platelet growth factor (PGF-1), interleukin-8 (IL-8), and transforming growth factor (TGF-1) that modulate the angiogenic switch, changing expression levels during tumour transition from radial to invasive vertical and then metastatic growth. We highlight modern and historical lines of research and development that are driving this exciting area of research currently.
Angiogenesis was first associated with malignancy 100 years ago  being new vessel growth from a pre-existing blood supply. It is physiologically tightly regulated during wound healing, embryogenesis, and female reproduction. It also occurs in several pathological conditions including malignant melanoma. In 1919, Krogh described a tissue cylinder surrounding an axial blood vessel allowing oxygen and glucose diffusion for metabolism . In 1945, vascularised tumour cell transplants used in vivo murine models survived to promote tumour survival and growth . A diffusible “angiogenic” substance was later proposed in 1968 using a hamster cheek pouch model demonstrating choriocarcinoma vasoproliferation . In 1971, Folkman postulated that tumour growth and metastasis driven by angiogenesis might be blocked by inhibiting angiogenesis (6). Later it was recognised, ischaemic stress occurs as the tumour growth exceeds the distances of Krogh’s cylinder, resulting in ischaemic necrosis, or ischaemic/hypoxic induced activation of angiogenesis  with diffusion limits of oxygen for cell survival measured at 100–200 microns . Beyond this margin, angiogenesis facilitates cell growth and survival, demonstrated experimentally with cultured tumor cells in avascular rabbit cornea attracting new capillaries and vascularizing the expanding tumor . In 1976, Gullino showed precancerous cells acquiring angiogenic capacity in a sequence leading to cancer , leading to a concept of “angiogenic switch” . This is postulated to be crucial to angiogenesis with the switch “off” when pro-angiogenic molecules are balanced by anti-angiogenic molecules, and “on” when this balance is reversed [10, 11]. “Switch” triggers include low pO2, low pH  or hyper/hypoglycaemia or hyperthermia , mechanical stress, immune/inflammatory response, and genetic mutations [14, 15].
2. Vascular Endothelial Growth Factor (VEGF)
Central to angiogenesis is VEGF, first isolated in 1989 . VEGF promotes endothelial cell proliferation, survival, migration, vasodilatation, and vasculogenesis by recruiting bone marrow-derived haematopoetic progenitor cells [17, 18]. VEGF is a heparin-binding family of glycoproteins including VEGF-A, VEGF-B, VEGF-C, and VEGF-D. VEGF-A occurs in at least four isoforms of 121, 165, 189, and 201 amino acids length, because of alternative gene splicing. VEGF-A commonly referred to as VEGF is overexpressed in almost all solid tumours and correlates with vascularity, grade, and prognosis . It is also expressed by dendritic and macrophage immune cells infiltrating into tumour stroma . VEGF ligands bind with variable affinity to tyrosine kinase receptors expressed on blood endothelial cell surfaces with vascular endothelial growth factor receptors (VEGFR) VEGFR-1 and VEGFR-2 involved in angiogenesis by their binding of VEGF-A isoforms. VEGFR-3 is expressed on lymphatic endothelial cells and is involved in lymphangiogenesis, binding VEGF-C and VEGF-D.
3. The Role of VEGF in Melanoma Angiogenesis
Neovascularisation’s importance in human cutaneous melanomas was demonstrated to indicate angiogenic activity  and VEGF’s role in melanoma angiogenesis was first demonstrated with the successful transplantation of human melanoma fragments into a hamster cheek pouch . Tumor blood flow in melanomas thicker than 0.9 mm was detected using Doppler ultrasound , and endogenous VEGF expression and secretion in melanoma tumour cells were later established . Murine studies have examined several aspects of VEGF expression and its role in tumour growth. Transfection and overexpression of VEGF isoforms in cell lines normally producing baseline VEGF levels have been an invaluable tool for identifying differences in tumorigenicity between isoforms. VEGF121 and VEGF165 promote aggressive tumour growth in mouse xenografts, contrasting VEGF189 (high heparin affinity/lower bioavailability) where overexpression demonstrates poor tumour growth .
In vivo murine studies have also shown that aggressive melanoma cell lines express higher levels of VEGF compared to nonaggressive cell lines . Nonaggressive cell lines such as Mel-2 transfected to overexpress VEGF, demonstrated conversion to an aggressive phenotype producing large vascularised nonnecrotic tumours in mouse models. These effects could be reversed with antisense VEGF transfection resulting in small poorly vascular tumours . These findings demonstrate VEGF’s role in aggressive tumour behaviour.
VEGF-A isoform behaviour may vary with environment. Nonmetastatic skin melanoma (SKMEL) cells transfected to overexpress murine VEGF164, an equivalent to human VEGF165, were subcutaneously implanted into mice, and demonstrated neovascularisation . Brain metastatic cells from the human melanoma cell line Mel57 were transfected to overexpress VEGF165, and coopted pre-existing intra- and peritumoural vessels without inducing neovascularisation . Are these clues to MM resistance to treatments, with tumour behaviour varying according to environment?
Surprisingly VEGF is difficult to detect in skin  and is localised in dermal endothelium but not epithelial keratinocytes  or benign naevi. Dysplastic melanocytes produce FGF-2 and VEGF. MM by comparison to normal melanocytes, greatly overexpresses bFGF thereby stimulating endothelial cell growth and further production of VEGF . Significantly an increase in the secretion and stromal deposition of VEGF is demonstrable during the switch from radial to vertical growth of MM  evidencing a role for VEGF in the “switch” mechanism. Clinical investigations with VEGF give conflicting conclusions.
Immunohistochemical studies demonstrated upregulation of VEGF165 and VEGF121 and increased microvascular density in primary melanomas, strongly correlating disease progression [30, 32, 33]. Conversely a similar analysis showed only tumour thickness as an independent variable associated with disease-free survival (Breslow classification) and overall survival—predicted by depth of tumor infiltration (Clark classification). Another study found increased vascularity actually correlated with survival .
Bridging this gap in understanding may be the discovery of anti-angiogenic VEGF isoforms which until recently could not be isolated from their pro-angiogenic sister isoforms. These differ from pro-angiogenic isoforms due to gene splicing in the 8th exon of the VEGF gene, resulting in same length final protein product, but with a different terminal base sequence encoded in the 8th exon due to splicing at a novel distal splicing site, conferring anti-angiogenic properties, denoted VEGFxxxb .
In vitro and in vivo evidence show VEGFxxxb isoforms competitively bind VEGFR-2 with equal affinity to pro-angiogenic isoforms, preventing phosphorylation and down-stream intracellular signaling of angiogenic processes . In vivo experiments with VEGF165b transfected melanoma cells in nude mice demonstrated reduced tumour growth and vessel density . Studies with melanoma samples demonstrated VEGFxxxb to be constitutively expressed in normal surrounding epidermis, but reduced in primary melanoma samples (both horizontal and vertical growth phases) which developed metastasis irrespective of primary tumour thickness. Total VEGF expression, however, demonstrated staining in metastatic and Nonmetastatic melanomas and normal epidermis. Diminished VEGFxxxb expression may predict metastatic spread in patients with primary melanoma being crucial in pushing the angiogenic switch, due to imbalanced VEGF gene splicing to pro-angiogenic isoforms .
Overall, these experimental endeavors have translated into modest clinical gains, the benefits of which are still being evaluated and developed. VEGF serum levels do show prognostic utility, confirming clinically angiogenesis’ importance in MM  and a role is being developed for therapy response monitoring in clinical trials.
4. Angiopoietins and the TIE Receptor Axis
Angiopoietins bind the receptor tyrosine kinase (RTK) family of Tie-1 and Tie-2 receptors and are required for embryonic and adult angiogenesis, interacting with VEGF in the regulation of angiogenesis and tumour growth through RTK receptor activation . Experimentally Ang-1 agonism of Tie-2 promotes vascular maturation through pericyte mediation maintaining endothelial cell (EC) quiescence, cell survival in pericytes treated in vivo with TNF-α (whilst pericytes treated with Ang-2 undergo apoptosis), and pericyte migration . In human umbilical vascular endothelial cells (HUVECs), VEGF induces Tie-2 and may therefore promote angiogenesis by inhibition of EC vascular stabilization . Raised serum levels of Ang-2 are also associated with advanced disease in melanoma offering a potential biomarker for disease monitoring.
5. Interleukin-8 (IL-8)
IL-8 chemokines are synthesized by macrophages and endothelial cells primarily inducting chemotaxis in target cells. Benign melanocytic lesions and normal epidermis express minimal levels; however, in MM patients IL-8 serum levels are significantly higher and correlate with advanced disease stage and overall patient survival . IL-8 expression in melanoma cells is upregulated in response to TGF-1, and is pro-angiogenic in mouse xenograft models. Tumour production of IL-8 not only drives melanoma cell growth but also promotes tumour cell migration, whilst endothelial IL-8 induces endothelial cell migration .
6. Platelet-Derived Growth Factor (PDGF) and PDGFR Axis
Pericyte and EC crosstalk is influenced by platelet derived growth factor (PDGF) which has 5 isoforms, and its TK receptor platelet derived growth factor receptor (PDGFR) PDGFRβ. Activation by PDGF leads to receptor dimerisation, autophosphorylation, and signaling transduction through the PI3K pathway important in EC migration . During angiogenesis this axis promotes recruitment of pericytes to the unstable neoangiogenic vasculature. Pericytes are relatively undifferentiated cells normally lining the outer surface of endothelial cells contributing to stability and angiogenesis by producing VEGF. Overexpressing PDGF-BB or PDGF-DD isoforms in a Nonmetastatic B12 mouse melanoma cell line demonstrated paracrine effects on tumour growth with pericyte recruitment and coverage of tumour vessel. The observed reduction in apoptosis and increased tumour growth is suggested to be due to increased pericyte expression of VEGF . Endothelial progenitor cells (EPCs) subjected to hypoxic conditioning demonstrate higher levels of PDGF-BB isoform compared to cells in normoxic conditions exerting positive effects on HUVEC cells including increased proliferation and migration, not observed in HUVECs exposed to recombinant PDGF-BB alone . These models examining the effects of hypoxic conditioning on PDGF overexpression on endothelial cell behaviour suggest an important role for the PDGF/PDGFR signaling axis in future therapeutic approaches and an important pathway through which hypoxia stimulates angiogenic behaviour.
7. Placental Growth Factor (PGF)
PGF isoforms result from gene splicing and exhibit variable heparin-binding. PGF-1 and -2, are expressed by melanoma cells binding neuropilin receptors NP-1 and -2 expressed on ECs. NPs are VEGF coreceptors involved in axon guidance and cell survival and migration . PGF may also influence MM development by binding VEGFR-1 receptors on haematopoietic bone marrow precursors inducing mobilization and recruitment of inflammatory mediators that upregulate VEGF production around the tumour, and by binding VEGFR-1 expressing smooth muscle cells and pericytes, thereby affecting blood vessel maturation and stability . Tumour vessels commonly lack functional pericytes, which are normally protective against changes in oxygen or hormonal balance by physically stabilising the vasculature. The effect of this is to allow hypoxic stimulation of tumour angiogenesis to go unchecked by the loss of vasoactive control . PGF also binds VEGF-A forming heterodimers, directly enhancing melanoma angiogenesis by VEGFR-2 activation of ECs .
8. Extracellular Matrix (ECM)
The ECM is a complex environment for biochemical and cellular regulation of gene expression, differentiation, adhesion, and migration supporting cells and related structures . EC migration into tumour stroma is essential for metastasis, and ECs and tumour cells increase metallo-matrix proteinase (MMP) expression to facilitate this process . The gelatinases MMP-2 and MMP-9 degrade basement membrane and were the first MMP’s linked with angiogenesis  correlating with invasive and metastatic phenotypes . MM expresses several MMPs including tissue inhibitor metalloproteinases (TIMPs) . Raised serum levels of MT1-MMP and MMP-9 are strongly associated with rapid disease progression. Raised serum MMP-9 expressed exclusively in the horizontal phase of melanoma growth is strongly associated with bone metastases. By contrast, nondysplastic melanocytes express basal levels of MMP-1 and -9 . Angiogenic mitogens, such as bFGF and VEGF, play a key role in stimulating capillary endothelial cells to produce MMPs . Studies also demonstrated MMPs involvement in the angiogenic switch. A tumour model reliably replicating this switch demonstrated MMP-2′s important role in developing an angiogenic phenotype . Another demonstrated MMP-9 influencing the angiogenic switch in a pancreatic tumor model . These findings suggest MMP/VEGF interaction is critical in initiating angiogenesis and promoting tumour invasiveness.
9. Basement Membrane
Basement membrane (BM) is specialised ECM separating ECs from underlying mesenchyme . BM comprises collagen type IV, laminin and fibronectin, and heparan sulphate proteoglycans (HSPG) . Heparanase secreted from melanoma cells cleaves heparan sulphate (HS), degrading this barrier that is normally protective of the basement membrane’s underlying type IV collagen structure from proteolysis. This exposes a ligand-integrin binding site for angiogenesis not found in quiescent vessels . Heparanase also liberates and activates heparin-binding growth factors bFGF and VEGF from the ECM, which may act in a paracrine manner with the tumour . Several melanoma cell lines degrade ECM through heparanase enzyme degradation  and pathological specimens of melanoma tissue demonstrate increased levels of heparanase mRNA at all stages of tumour development, with highest expression found in vertical growth phase samples . Transfection of the heparanase gene into nonaggressive MM cell lines demonstrates significantly increased enzyme activity and invasiveness . Heparan sulphate break-down products also suppress T-lymphocyte immune surveillance in the environment around the tumour . It is also hypothesized that invasive melanoma cells may arise by a macrophage-transformed melanocyte fusion, allowing the tumour to acquire a “macrophage phenotype,” improving tumour migratory potential and allowing it to bypass immune surveillance mechanisms [67–69].
10. Hypoxia-Driven Pathological Angiogenesis
Hypoxia strongly stimulates angiogenesis physiologically or pathologically, and oxygen tension is a key regulator of VEGF gene expression in vivo and in vitro , with VEGF mRNA expression induced by low oxygen concentrations in normal or transformed cultured cells . In melanoma, hypoxia independently upregulates VEGF  and tissue factor (TF) production . TF is a factor VII receptor and a pathway of hypoxic stimulation for VEGF triggering the coagulation cascade. In melanoma, this potentiates tumour cell binding, with cytoplasmic tail tumour cells required for VEGF synthesis . The second pathway for hypoxic stimulation is through hypoxia inducible factor (HIF) α “master switch” for transcriptional regulation of cellular responses to hypoxia, controlling diverse target genes including glycolytic metabolism, erythropoiesis, and vascular remodeling. HIF is a 28-base sequence found in the 5′ promoter of the VEGF gene, and mediates hypoxia-induced transcription of VEGF . It is regulated at protein level by the Von Hippel-Lindau protein (VHLP) which controls HIF degradation in normoxic conditions . VHL disease unsurprisingly is strongly associated with angiogenic neoplasms .
11. Hypoxia and Melanoma
Human epidermis lacks vasculature and is markedly hypoxic. The O2 tension (pO2) in dermis has a pO2 of 10%, whilst in some skin appendages the pO2 ranges from mildly (5%) to severely hypoxic (0.5%) . Human skin exhibits extensive binding of hypoxia-sensitive agents (such as nitroimidazole EF5) in the basal epidermal compartment with increased expression levels of the oxygen sensor HIF-1α  and it is this already hypoxic basal epidermis that may provide a permissive environment for pushing resident dysplastic melanocytes into a pro-angiogenic state with hypoxia pushing the angiogenic switch. Physiologically mild hypoxia of the skin can promote melanocyte transformation [80, 81] and correlate with increased expression of HIF-1α, HIF-2α, and VEGF, found commonly in melanoma samples, and poor prognosis and survival . Hypoxia directly stimulates tumour blood vessel formation in response to HIF induced VEGF production. In MM, this results in vessels structurally and functionally abnormal with shunts contributing disordered tumor blood flow  exacerbating hypoxic/acidic regions in the growing tumour . Mismatched vascularity and oxygenation further influences the balance of angiogenic stimulators and inhibitors in favor of angiogenesis, aiding malignant/metastatic cell selection .
Angiogenesis is a hallmark of cancer , and VEGF is a key player . Evidence shows its involvement at key elements of tumour growth; including tumour hypoxia triggering HIF-induced VEGF production, endogenous tumour production of VEGF, tumour-endothelium paracrine interaction and upregulation of VEGF, liberation of VEGF from the ECM by tumour heparanase production, or recruitment of VEGF producing macrophages. Whether these processes are sequentially linked or not, the common denominator is VEGF. Anti-angiogenic isoforms are gradually emerging in the research sphere  and may provide invaluable prognostic information , and in the future therapeutic possibilities. We hypothesise that melanoma cells express at low levels (unpublished data) VEGF receptors in pro- or anti-angiogenic isoforms, the balance of which is governed by splicing, in turn influenced by factors that modulate the angiogenic switch, and upregulating VEGF receptor expression as a tumour survival response. This process of “survival” growth may facilitate further tumour growth by causing localised intratumoral hypoxia which is a powerful stimulus to HIF-1α production and therefore more neovascularisation. Upregulating VEGF receptors at the tumour surface or tumour-endothelial cell interface may occur as a prelude to tumour ECM interaction with MMPs. Whether the angiogenic switch is pushed by a preponderance of proangiogenic VEGF or by VEGF splicing from anti- to pro-angiogenic forms is unclear; however, this critical event appears directly related to the tumour change from radial to invasive growth and may represent the culmination of a sequence of processes. This relationship has not yet been linked in a unified model that gradually upregulates pro-angiogenic VEGF to this critical level.
Melanoma cells also produce heparanase disrupting the basement membrane facilitating tumour cell invasion. Macrophages possibly add fuel to the fire bringing greater quantities of VEGF to the tumour site setting up new VEGF gradients favoring tumour growth, which in turn may upregulate MMPs and therefore metastatic behaviour. Heparin-treated tumour cells demonstrate reduced cell adhesion and migration , and given that heparin-binding sites are spliced during synthesis of VEGF isoforms, investigating how heparin may affect the efficacy of VEGF isoforms on tumour growth in vitro would be intriguing.
A tumour thus enriched by numerous sources of pro-angiogenic growth factor, and at the same time appearing to enjoy immune privilege, is able to liberate VEGF from the ECM which acts as a standing reservoir of VEGF and facilitate invasion and metastasis.
Future research might focus on tumour VEGF isoform characterization, distribution and site-specific variability, and the influence on disease progression. Examining the possible role of splice variants in hypoxic states such as pregnancy in melanoma (indeed murine models show increased lymphangiogenesis and metastasis in pregnancy ) could yield valuable insights.
- E. Goldmann, “The growth of malignant disease in man and the lower animals, with special reference to the vascular system,” The Lancet, vol. 170, no. 4392, pp. 1236–1240, 1907.
- A. Krogh, “The number and distribution of capillaries in muscles with calculations of the oxygen pressure head necessary for supplying the tissue,” Journal of Physiology, vol. 52, no. 6, pp. 409–415, 1919.
- G. H. C. H. W. Algire, “Vascular reactions of normal and malignant tissues in vivo. Vascular reactions of mice to wounds and to normal and neoplastic transplants,” Journal of the National Cancer Institute, vol. 6, pp. 73–85, 1945.
- R. L. Ehrmann and M. Knoth, “Choriocarcinoma. Transfilter stimulation of vasoproliferation in the hamster cheek pouch. Studied by light and electron microscopy,” Journal of the National Cancer Institute, vol. 41, no. 6, pp. 1329–1341, 1968.
- S. Brem, H. Brem, and J. Folkman, “Prolonged tumor dormancy by prevention of neovascularization in the vitreous,” Cancer Research, vol. 36, no. 8, pp. 2807–2812, 1976.
- P. T. Schumacker and R. W. Samsel, “Analysis of oxygen delivery and uptake relationships in the Krogh tissue model,” Journal of Applied Physiology, vol. 67, no. 3, pp. 1234–1244, 1989.
- M. A. Gimbrone Jr., S. B. Leapman, R. S. Cotran, and J. Folkman, “Tumor dormancy in vivo by prevention of neovascularization,” Journal of Experimental Medicine, vol. 136, no. 2, pp. 261–276, 1972.
- P. M. Gullino, “Angiogenesis and oncogenesis,” Journal of the National Cancer Institute, vol. 61, no. 3, pp. 639–643, 1978.
- J. Kandel, E. Bossy-Wetzel, F. Radvanyi, M. Klagsbrun, J. Folkman, and D. Hanahan, “Neovascularization is associated with a switch to the export of bFGF in the multistep development of fibrosarcoma,” Cell, vol. 66, no. 6, pp. 1095–1104, 1991.
- D. Hanahan and R. A. Weinberg, “The hallmarks of cancer,” Cell, vol. 100, no. 1, pp. 57–70, 2000.
- N. Bouck, V. Stellmach, and S. C. Hsu, “How tumors become angiogenic,” Advances in Cancer Research, vol. 69, pp. 135–174, 1996.
- J. L. Wike-Hooley, J. Haveman, and H. S. Reinhold, “The relevance of tumour pH to the treatment of malignant disease,” Radiotherapy and Oncology, vol. 2, no. 4, pp. 343–366, 1984.
- J. van der Zee, “Heating the patient: a promising approach?” Annals of Oncology, vol. 13, no. 8, pp. 1173–1184, 2002.
- R. S. Kerbel, “Tumor angiogenesis: past, present and the near future,” Carcinogenesis, vol. 21, no. 3, pp. 505–515, 2000.
- P. Carmeliet, “Controlling the cellular brakes,” Nature, vol. 401, no. 6754, pp. 657–658, 1999.
- N. Ferrara and W. J. Henzel, “Pituitary follicular cells secrete a novel heparin-binding growth factor specific for vascular endothelial cells,” Biochemical and Biophysical Research Communications, vol. 161, no. 2, pp. 851–858, 1989.
- S. Rafii, D. Lyden, R. Benezra, K. Hattori, and B. Heissig, “Vascular and haematopoietic stem cells: novel targets for anti-angiogenesis therapy?” Nature Reviews Cancer, vol. 2, no. 11, pp. 826–835, 2002.
- D. Lyden, K. Hattori, S. Dias et al., “Impaired recruitment of bone-marrow-derived endothelial and hematopoietic precursor cells blocks tumor angiogenesis and growth,” Nature Medicine, vol. 7, no. 11, pp. 1194–1201, 2001.
- H. F. Dvorak, “Vascular permeability factor/vascular endothelial growth factor: a critical cytokine in tumor angiogenesis and a potential target for diagnosis and therapy,” Journal of Clinical Oncology, vol. 20, no. 21, pp. 4368–4380, 2002.
- D. Fukumura, R. Xavier, T. Sugiura et al., “Tumor induction of VEGF promoter activity in stromal cells,” Cell, vol. 94, no. 6, pp. 715–725, 1998.
- M. C. Mihm Jr., W. H. Clark Jr., and L. From, “The clinical diagnosis, classification and histogenetic concepts of the early stages of cutaneous malignant melanomas,” The New England Journal of Medicine, vol. 284, no. 19, pp. 1078–1082, 1971.
- B. A. Warren and P. Shubik, “The growth of the blood supply to melanoma transplants in the hamster cheek pouch,” Laboratory Investigation, vol. 15, no. 2, pp. 464–478, 1966.
- A. Srivastava, L. E. Hughes, J. P. Woodcock, and E. J. Shedden, “The significance of blood flow in cutaneous malignant melanoma demonstrated by Doppler flowmetry,” European Journal of Surgical Oncology, vol. 12, no. 1, pp. 13–18, 1986.
- H. Gitay-Goren, R. Halaban, and G. Neufeld, “Human melanoma cells but not normal melanocytes express vascular endothelial growth factor receptors,” Biochemical and Biophysical Research Communications, vol. 190, no. 3, pp. 702–709, 1993.
- J. L. Yu, J. W. Rak, G. Klement, and R. S. Kerbel, “Vascular endothelial growth factor isoform expression as a determinant of blood vessel patterning in human melanoma xenografts,” Cancer Research, vol. 62, no. 6, pp. 1838–1846, 2002.
- A. J. G. Potgens, N. H. Lubsen, M. C. Van Altena, J. G. G. Schoenmakers, D. J. Ruiter, and R. M. W. De Waal, “Vascular permeability factor expression influences tumor angiogenesis in human melanoma lines xenografted to nude mice,” American Journal of Pathology, vol. 146, no. 1, pp. 197–209, 1995.
- K. P. Claffey, L. F. Brown, L. F. Del Aguila et al., “Expression of vascular permeability factor/vascular endothelial growth factor by melanoma cells increases tumor growth, angiogenesis, and experimental metastasis,” Cancer Research, vol. 56, no. 1, pp. 172–181, 1996.
- B. Küsters, R. M. W. De Waal, P. Wesseling et al., “Differential effects of vascular endothelial growth factor A isoforms in a mouse brain metastasis model of human melanoma,” Cancer Research, vol. 63, no. 17, pp. 5408–5413, 2003.
- T. Pufe, F. Paulsen, W. Peterson, R. Mentlein, and M. Tsokos, “The angiogenic peptide vascular endothelial growth factor (VEGF) is expressed in chronic sacral pressure ulcers,” Journal of Pathology, vol. 200, no. 1, pp. 130–136, 2003.
- P. Salven, P. Heikkilä, and H. Joensuu, “Enhanced expression of vascular endothelial growth factor in metastatic melanoma,” British Journal of Cancer, vol. 76, no. 7, pp. 930–934, 1997.
- S. M. Plum, J. W. Holaday, A. Ruiz, J. W. Madsen, W. E. Fogler, and A. H. Fortier, “Administration of a liposomal FGF-2 peptide vaccine leads to abrogation of FGF-2-mediated angiogenesis and tumor development,” Vaccine, vol. 19, no. 9-10, pp. 1294–1303, 2000.
- H. Erhard, F. J. R. Rietveld, M. C. Van Altena, E. B. Bröcker, D. J. Ruiter, and R. M. W. De Waal, “Transition of horizontal to vertical growth phase melanoma is accompanied by induction of vascular endothelial growth factor expression and angiogenesis,” Melanoma Research, vol. 7, supplement 2, pp. S19–S26, 1997.
- D. H. Gorski, A. D. Leal, and J. S. Goydos, “Differential expression of vascular endothelial growth factor-A isoforms at different stages of melanoma progression,” Journal of the American College of Surgeons, vol. 197, no. 3, pp. 408–418, 2003.
- S. Ilmonen, A. L. Kariniemi, T. Vlaykova, T. Muhonen, S. Pyrhönen, and S. Asko-Seljavaara, “Prognostic value of tumour vascularity in primary melanoma,” Melanoma Research, vol. 9, no. 3, pp. 273–278, 1999.
- D. O. Bates, T. G. Cui, J. M. Doughty et al., “VEGF165b, an inhibitory splice variant of vascular endothelial growth factor, is down-regulated in renal cell carcinoma,” Cancer Research, vol. 62, no. 14, pp. 4123–4131, 2002.
- J. Woolard, W. Y. Wang, H. S. Bevan et al., “VEGF165b, an inhibitory vascular endothelial growth factor splice variant: mechanism of action, in vivo effect on angiogenesis and endogenous protein expression,” Cancer Research, vol. 64, no. 21, pp. 7822–7835, 2004.
- E. S. Rennel, M. A. Hamdollah-Zadeh, E. R. Wheatley et al., “Recombinant human VEGF165b protein is an effective anti-cancer agent in mice,” European Journal of Cancer, vol. 44, no. 13, pp. 1883–1894, 2008.
- R. O. Pritchard-Jones, D. B. A. Dunn, Y. Qiu et al., “Expression of VEGFxxxb, the inhibitory isoforms of VEGF, in malignant melanoma,” British Journal of Cancer, vol. 97, no. 2, pp. 223–230, 2007.
- S. Ugurel, G. Rappl, W. Tilgen, and U. Reinhold, “Increased serum concentration of angiogenic factors in malignant melanoma patients correlates with tumor progression and survival,” Journal of Clinical Oncology, vol. 19, no. 2, pp. 577–583, 2001.
- T. M. Hansen, H. Singh, T. A. Tahir, and N. P. J. Brindle, “Effects of angiopoietins-1 and -2 on the receptor tyrosine kinase Tie2 are differentially regulated at the endothelial cell surface,” Cellular Signalling, vol. 22, no. 3, pp. 527–532, 2010.
- J. Cai, O. Kehoe, G. M. Smith, P. Hykin, and M. E. Boulton, “The angiopoietin/Tie-2 system regulates pericyte survival and recruitment in diabetic retinopathy,” Investigative Ophthalmology and Visual Science, vol. 49, no. 5, pp. 2163–2171, 2008.
- C. M. Findley, M. J. Cudmore, A. Ahmed, and C. D. Kontos, “VEGF induces Tie2 shedding via a phosphoinositide 3-kinase/Akt-dependent pathway to modulate Tie2 signaling,” Arteriosclerosis, Thrombosis, and Vascular Biology, vol. 27, no. 12, pp. 2619–2626, 2007.
- M. Bar-Eli, “Role of interleukin-8 in tumor growth and metastasis of human melanoma,” Pathobiology, vol. 67, no. 1, pp. 12–18, 1999.
- G. Liu, F. Zhang, J. Lee, and Z. Dong, “Selective induction of interleukin-8 expression in metastatic melanoma cells by transforming growth factor-β1,” Cytokine, vol. 31, no. 3, pp. 241–249, 2005.
- J. C. Yu, W. Li, L. M. Wang, A. Uren, J. H. Pierce, and M. A. Heidaran, “Differential requirement of a motif within the carboxyl-terminal domain of α-platelet-derived growth factor (αPDGF) receptor for PDGF focus forming activity chemotaxis, or growth,” Journal of Biological Chemistry, vol. 270, no. 13, pp. 7033–7036, 1995.
- M. Furuhashi, T. Sjöblom, A. Abramsson et al., “Platelet-derived growth factor production by B16 melanoma cells leads to increased pericyte abundance in tumors and an associated increase in tumor growth rate,” Cancer Research, vol. 64, no. 8, pp. 2725–2733, 2004.
- M. Wyler von Ballmoos, Z. Yang, J. Völzmann, I. Baumgartner, C. Kalka, and S. Di Santo, “Endothelial progenitor cells induce a phenotype shift in differentiated endothelial cells towards PDGF/PDGFRβ axis-mediated angiogenesis,” PloS ONE, vol. 5, no. 11, Article ID e14107, 2010.
- T. Odorisio, F. Cianfarani, C. M. Failla, and G. Zambruno, “The placenta growth factor in skin angiogenesis,” Journal of Dermatological Science, vol. 41, pp. 11–19, 2006.
- S. Donnini, et al., “Expression and localization of placenta growth factor and PlGF receptors in human meningiomas,” Journal of Pathology, vol. 189, no. 1, pp. 66–71, 1999.
- L. E. Benjamin, D. Golijanin, A. Itin, D. Pode, and E. Keshet, “Selective ablation of immature blood vessels in established human tumors follows vascular endothelial growth factor withdrawal,” Journal of Clinical Investigation, vol. 103, no. 2, pp. 159–165, 1999.
- A. Luttun, M. Autiero, M. Tjwa, and P. Carmeliet, “Genetic dissection of tumor angiogenesis: are PlGF and VEGFR-1 novel anti-cancer targets?” Biochimica et Biophysica Acta, vol. 1654, no. 1, pp. 79–94, 2004.
- D. Dogic, B. Eckes, and M. Aumailley, “Extracellular matrix, integrins and focal adhesions,” Current Topics in Pathology, vol. 93, pp. 75–85, 1999.
- J. E. Rundhaug, “Matrix metalloproteinases and angiogenesis,” Journal of Cellular and Molecular Medicine, vol. 9, no. 2, pp. 267–285, 2005.
- M. Egeblad and Z. Werb, “New functions for the matrix metalloproteinases in cancer progression,” Nature Reviews Cancer, vol. 2, no. 3, pp. 161–174, 2002.
- U. B. Hofmann, J. R. Westphal, G. N. P. Van Muijen, and D. J. Ruiter, “Matrix metalloproteinases in human melanoma,” Journal of Investigative Dermatology, vol. 115, no. 3, pp. 337–344, 2000.
- J. Nikkola, P. Vihinen, M. S. Vuoristo, P. Kellokumpu-Lehtinen, V. M. Kähäri, and S. Pyrhönen, “High serum levels of matrix metalloproteinase-9 and matrix metalloproteinase-1 are associated with rapid progression in patients with metastatic melanoma,” Clinical Cancer Research, vol. 11, no. 14, pp. 5158–5166, 2005.
- W. J. Lamoreaux, M. E. C. Fitzgerald, A. Reiner, K. A. Hasty, and S. T. Charles, “Vascular endothelial growth factor increases release of gelatinase A and decreases release of tissue inhibitor of metalloproteinases by microvascular endothelial cells in vitro,” Microvascular Research, vol. 55, no. 1, pp. 29–42, 1998.
- J. Fang, Y. Shing, D. Wiederschain et al., “Matrix metalloproteinase-2 is required for the switch to the angiogenic phenotype in a tumor model,” Proceedings of the National Academy of Sciences of the United States of America, vol. 97, no. 8, pp. 3884–3889, 2000.
- G. Bergers, R. Brekken, G. McMahon et al., “Matrix metalloproteinase-9 triggers the angiogenic switch during carcinogenesis,” Nature Cell Biology, vol. 2, no. 10, pp. 737–744, 2000.
- R. Timpl and J. C. Brown, “The laminins,” Matrix Biology, vol. 14, no. 4, pp. 275–281, 1994.
- R. V. Iozzo and A. D. Murdoch, “Proteoglycans of the extracellular environment: clues from the gene and protein side offer novel perspectives in molecular diversity and function,” FASEB Journal, vol. 10, no. 5, pp. 598–614, 1996.
- A. Srivastava, R. Ralhan, and K. Jatinder, “Angiogenesis in cutaneous melanoma: pathogenesis and clinical implications,” Microscopy Research and Technique, vol. 60, no. 2, pp. 208–224, 2003.
- M. Roy and D. Marchetti, “Cell surface heparan sulfate released by Heparanase promotes melanoma cell migration and angiogenesis,” Journal of Cellular Biochemistry, vol. 106, no. 2, pp. 200–209, 2009.
- M. Nakajima, T. Irimura, and G. L. Nicolson, “Heparanases and tumor metastasis,” Journal of Cellular Biochemistry, vol. 36, no. 2, pp. 157–167, 1988.
- D. Marchetti and G. L. Nicolson, “Human heparanase: a molecular determinant of brain metastasis,” Advances in Enzyme Regulation, vol. 41, pp. 343–359, 2001.
- K. Gohji, Y. Katsuoka, M. Okamoto et al., “Human heparanase: roles in invasion and metastasis of cancer,” Acta Urologica Japonica, vol. 46, no. 10, pp. 757–762, 2000.
- J. M. Pawelek and A. K. Chakraborty, “Chapter 10 the cancer cell-leukocyte fusion theory of metastasis,” Advances in Cancer Research, vol. 101, pp. 397–444, 2008.
- J. M. Pawelek and A. K. Chakraborty, “Fusion of tumour cells with bone marrow-derived cells: a unifying explanation for metastasis,” Nature Reviews Cancer, vol. 8, no. 5, pp. 377–386, 2008.
- J. Pawelek, A. Chakraborty, R. Lazova et al., “Co-opting macrophage traits in cancer progression: a consequence of tumor cell fusion?” Contributions to Microbiology, vol. 13, pp. 138–155, 2006.
- N. Ferrara, “Molecular and biological properties of vascular endothelial growth factor,” Journal of Molecular Medicine, vol. 77, no. 7, pp. 527–543, 1999.
- A. Minchenko, T. Bauer, S. Salceda, and J. Caro, “Hypoxic stimulation of vascular endothelial growth factor expression in vitro and in vivo,” Laboratory Investigation, vol. 71, no. 3, pp. 374–379, 1994.
- Y. G. Shellman, J. T. Chapman, M. Fujita, D. A. Norris, and I. H. Maxwell, “Expression of activated N-ras in a primary melanoma cell line counteracts growth inhibition by transforming growth factor-β,” Journal of Investigative Dermatology, vol. 114, no. 6, pp. 1200–1204, 2000.
- A. Amirkhosravi, T. Meyer, G. Warnes et al., “Pentoxifylline inhibits hypoxia-induced upregulation of tumor cell tissue factor and vascular endothelial growth factor,” Thrombosis and Haemostasis, vol. 80, no. 4, pp. 598–602, 1998.
- K. Abe, M. Shoji, J. Chen et al., “Regulation of vascular endothelial growth factor production and angiogenesis by the cytoplasmic tail of tissue factor,” Proceedings of the National Academy of Sciences of the United States of America, vol. 96, no. 15, pp. 8663–8668, 1999.
- Y. Liu, S. R. Cox, T. Morita, and S. Kourembanas, “Hypoxia regulates vascular endothelial growth factor gene expression in endothelial cells: identification of a enhancer,” Circulation Research, vol. 77, no. 3, pp. 638–643, 1995.
- K. Kondo and W. G. Kaelin Jr., “The von Hippel-Lindau tumor suppressor gene,” Experimental Cell Research, vol. 264, no. 1, pp. 117–125, 2001.
- T. Shuin, K. Kondo, S. Ashida et al., “Germline and somatic mutations in von hippel-lindau disease gene and its significance in the development of kidney cancer,” Contributions to Nephrology, vol. 128, pp. 1–10, 1999.
- S. M. Evans, A. E. Schrlau, A. A. Chalian, P. Zhang, and C. J. Koch, “Oxygen levels in normal and previously irradiated human skin as assessed by EF5 binding,” Journal of Investigative Dermatology, vol. 126, no. 12, pp. 2596–2606, 2006.
- K. Nys, H. Maes, A. M. Dudek, and P. Agostinis, “Uncovering the role of hypoxia inducible factor-1α in skin carcinogenesis,” Biochimica et Biophysica Acta, vol. 1816, no. 1, pp. 1–12, 2011.
- G. Monsel, N. Ortonne, M. Bagot, A. Bensussan, and N. Dumaz, “C-Kit mutants require hypoxia-inducible factor 1α to transform melanocytes,” Oncogene, vol. 29, no. 2, pp. 227–236, 2010.
- B. Bedogni, S. M. Welford, D. S. Cassarino, B. J. Nickoloff, A. J. Giaccia, and M. B. Powell, “The hypoxic microenvironment of the skin contributes to Akt-mediated melanocyte transformation,” Cancer Cell, vol. 8, no. 6, pp. 443–454, 2005.
- A. Giatromanolaki, E. Sivridis, C. Kouskoukis, K. C. Gatter, A. L. Harris, and M. I. Koukourakis, “Hypoxia-inducible factors 1α and 2α are related to vascular endothelial growth factor expression and a poorer prognosis in nodular malignant melanomas of the skin,” Melanoma Research, vol. 13, no. 5, pp. 493–501, 2003.
- J. W. Baish and R. K. Jain, “Fractals and cancer,” Cancer Research, vol. 60, no. 14, pp. 3683–3688, 2000.
- G. Helmlinger, F. Yuan, M. Dellian, and R. K. Jain, “Interstitial pH and pO2 gradients in solid tumors in vivo: high-resolution measurements reveal a lack of correlation,” Nature Medicine, vol. 3, no. 2, pp. 177–182, 1997.
- T. G. Graeber, C. Osmanian, T. Jacks et al., “Hypoxia-mediated selection of cells with diminished apoptotic potential in solid tumours,” Nature, vol. 379, no. 6560, pp. 88–91, 1996.
- G. Chalkiadaki, D. Nikitovic, A. Berdiaki, P. Katonis, N. K. Karamanos, and G. N. Tzanakakis, “Heparin plays a key regulatory role via a p53/FAK-dependent signaling in melanoma cell adhesion and migration,” IUBMB Life, vol. 63, no. 2, pp. 109–119, 2011.
- K. Khosrotehrani, S. N. Huu, A. Prignon et al., “Pregnancy promotes melanoma metastasis through enhanced lymphangiogenesis,” American Journal of Pathology, vol. 178, no. 4, pp. 1870–1880, 2011.