Crosstalk between angiogenesis and lymphangiogenesis in tumor ...

Leukemia (2004) 18, 1054–1058 & 2004 Nature Publishing Group All rights reserved 0887-6924/04 $30.00 www.nature.com/leu

REVIEW Crosstalk between angiogenesis and lymphangiogenesis in tumor progression C Scavelli1, A Vacca1, G Di Pietro1, F Dammacco1 and D Ribatti2 Deparment of Biomedical Sciences and Human Oncology, University of Bari Medical School, Bari, Italy; and 2Department of Human Anatomy and Histology, University of Bari Medical School, Bari, Italy 1

SPOTLIGHT

Extensive studies have identified reliable markers of lymphatic endothelial cells, and mechanisms and molecules that regulate development and growth of the lymphatic vessels. Vascular endothelial growth factors (VEGF)-C and VEGF-D, and their cognate receptor tyrosine kinase, VEGF receptor-3 (VEGFR-3), are critical regulators of lymphangiogenesis. By binding to its endothelial cell surface receptors VEGFR-1 and VEGFR-2, VEGF-A mediates vascular leakage, endothelial proliferation and migration. Angiopoietin-2 (Ang-2) is expressed at sites of blood vessel remodeling and invasion, and factors that induce angiogenesis in vivo, such as VEGF-A, upregulate Ang-2 in endothelial cells. In this review, we summarize the literature concerning the crosstalk between angiogenesis and lymphangiogenesis in tumor progression, that is, involvement of VEGFC, VEGF-D and VEGFR-3 in angiogenesis, and the role played by VEGF-A and Ang-2 in lymphangiogenesis, respectively. Leukemia (2004) 18, 1054–1058. doi:10.1038/sj.leu.2403355 Published online 1 April 2004 Keywords: angiogenesis; Ang-2; lymphangiogenesis; tumor progression; VEGF-A; VEGF-C; VEGF-D; VEGFR-3

transgenic mice of a mutant form of VEGF-C (VEGF-C156S) that binds to VEGFR-3 but not VEGFR-2.5 When expressed in the dermis, VEGF-C156S induces lymphatic, but not blood vessel, hyperplasia. VEGF-C is four to five times less potent than VEGF-A in the vascular permeability assay.6 Its mRNA levels are increased by the serum and its main component growth factors, PDGF and epidermal growth factor (EGF), as well as by transforming growth factor-b and the tumor promoter phorbol myristate 12, 13-acetate (PMA).3 Conversely, hypoxia, Ras oncoprotein and mutant p53 tumor suppressor gene do not have an influence on VEGF-C mRNA levels. Interleukin-1b (IL-1b) and tumor necrosis factor-a stimulate VEGF-C expression in human lung fibroblasts and human umbilical vein endothelial cells (HUVECs).7 Furthermore, the anti-inflammatory dexamethasone inhibits IL1b-induced VEGF-C mRNA expression. VEGF-C may mediate inflammatory reactions.8

Involvement of VEGF-C in angiogenesis Involvement of VEGF-C in lymphangiogenesis Recent advances in understanding the development of the lymphatic system and the growth of lymphatic vessels derive from the discovery of a number of mediators of lymphangiogenesis, such as vascular endothelial growth factor (VEGF)-C/VEGFD/VEGF receptor-3 (VEGFR-3). VEGF-C is a member of the VEGF growth factor family. It is closely related to VEGF-D and characterized by the presence of unique amino- and carboxy-terminal extensions flanking the common VEGF-homology domain.1 It is synthesized as a precursor protein, which undergoes proteolysis reminiscent of platelet-derived growth factor (PDGF)-A and PDGF-B chain processing, suggesting an evolutionary relationship.2 Proteolytic processing may provide a regulatory mechanism for fine tuning the biological functions of VEGF-C. Unprocessed VEGF-C binds to VEGFR-3, and the stepwise processing of VEGF-C generates several VEGF-C forms with increased activity towards VEGFR-3. VEGFR-3, the tyrosine kinase receptor for VEGF-C and VEGFD, is expressed on lymphatic endothelium. VEGF-C/VEGFR-3 signaling has been suggested to play a role in maintenance of the lymphatic endothelium and/or in lymphangiogenesis. VEGF-C induces lymphangiogenesis in the ears of mice and lymphatic vessel enlargement in the skin.3,4 Lymphangiogenesis induced by VEGF-C is preferentially driven by the activation of VEGFR-3. This suggestion is supported by the overexpression in Correspondence: Professor D Ribatti, Department of Human Anatomy and Histology, Piazza Giulio Cesare, 11, Policlinico, Bari I-70124, Italy; Fax: þ 39 080 5478310; E-mail: [email protected] Received 11 February 2004; accepted 1 March 2004; Published online 1 April 2004

VEGF-C may cooperate with VEGF-A in regulating embryonic vascular development. Fully processed VEGF-C alone activates VEGFR-2. Since VEGFR-2 is present in many types of endothelia and is broadly expressed, VEGF-C biosynthesis as a precursor may prevent unnecessary angiogenic effects elicited via VEGFR2 and allows VEGF-C to signal preferentially via VEGFR-3, which is restricted to the venous endothelia during early development, and to the lymphatic endothelium later. In certain circumstances, proteolytic processing seems to release mature VEGF-C, which can signal via both VEGFR-3 and VEGFR-2. The activation of both VEGFR-3 and VEGFR-2, either as homo- or as heterodimers, may also be necessary to elicit a complete biological response to VEGF-C.7 VEGF-C may play several functions in the organization and of vascular tree. A role in early vasculogenesis is suggested by the finding that VEGFR-2-deficient mice die at an earlier stage in embryo than mice deficient in VEGF-A.9 Other VEGFR-2 ligands, including VEGF-C, may compensate for the loss of VEGF-A. VEGF-C’s potent effect on blood vessels occur because its fully processed form also binds to the VEGFR-2 of blood vessels and stimulates angiogenesis under certain experimental conditions.10 VEGFR-3 signaling by one of its ligands is essential in embryonic angiogenesis, as VEGFR-3 knockout mice fail to develop a proper vasculature and die at embryonic day 9.5.9 In the later stages of embryogenesis, and in the adult, VEGFR-3 expression is lost from vascular endothelial cells, but retained in lymphatic endothelial cells. In adults, arteries and veins are generally negative for VEGFR-3, while fenestrated capillaries of several adult organs, including bone marrow, splenic and

Angiogenesis and lymphangiogenesis in tumor progression C Scavelli et al

Involvement of VEGF-D in lymphangiogenesis VEGF-D was first isolated as a partial cDNA from a differential display screening of murine genes expressed in fibroblasts from normal mice, but not expressed in those fibroblasts from mice carrying a targeted inactivation of the c-fos gene.20 It was first called c-fos-induced growth factor, but was later renamed VEGF-D to indicate that it was a novel VEGF homologue. Like VEGF-C, VEGF-D is synthesized as a prepropeptide of B53 kDa, which is rapidly secreted from the cell. Both mature VEGF-D and a form consisting of the VHD and the N-terminal propeptide were detected in embryonic mouse lung, indicating that proteolytic processing of VEGF-D occurs in vivo. This processing is required to produce a growth factor that binds VEGFR-2 and VEGFR-3 with high affinity. VEGF-D promotes lymphangiogenesis21 and metastatic spread via the lymphatics.22

Involvement of VEGF-D in angiogenesis The fully processed form of VEGF-D binds VEGFR-2 and VEGFR-3 with B290- and B40-fold greater affinity, respectively, than unprocessed VEGF-D, and processing probably regulates VEGF-D bioactivity in vivo. As the increase in affinity for VEGFR-2 is B7-fold more than that for VEGFR-3, processing

may, in effect, modulate receptor specificity in vivo, as unprocessed VEGF-D at physiological concentrations may bind VEGFR-3, but not VEGFR-2. In this scenario, processing would be an absolute requirement for VEGFR-2 but not for VEGFR-3 binding. VEGF-D displays similar angiogenic and lymphangiogenic properties to VEGF-C in a variety of animal models. VEGFD is angiogenic in the rabbit cornea assay;23 however, it induces lymphatic, but not blood vessel, hyperplasia when expressed in mouse skin under the control of the K-14 promotor.6 Byzova et al24 have demonstrated that the biologic effects of VEGF-D are tissue specific and dependent on the abundance of blood vessels and lymphatics expressing the receptors for VEGFD in a given tissue. They show that an adenovirus encoding the mature form of human VEGF-D (Ad-VEGF-D DN DC) induces predominantly blood vessel growth in the rat cremaster muscle and both angiogenesis and lymphangiogenesis when injected into the epigastric skin.24 Immunohistochemical analysis of this muscle demonstrated that the neovascularization was composed primarily of laminin and VEGFR-2-positive vessels containing red blood cells, thus indicating a predominantly angiogenic response.

Involvement of VEGF-C and VEGF-D in tumor progression The means by which tumor cells gain access to the lymphatics have long remained the subject of a controversy in the field of metastasis research. Two essentially conflicting views have been advanced. The first maintains that tumors metastasize solely by invasion of pre-existing tissue lymphatics at their margin, arguing that the high interstitial pressure exerted within tumors would destroy such vessels even if existed. The second maintains that metastasization could also occur through the formation of new lymphatics within the body of the tumor itself.25 Nonetheless, intratumoral lymphatics have been well described in solid organ cancers and the origin of leaky tumor vessels may not be clear origin. Convincing evidence for tumor lymphangiogenesis has begun to accumulate and show that it can be stimulated in a variety of experimental cancers by VEGF-C and VEGF-D. Furthermore, metastases to regional lymph nodes and the lung are increased, suggesting an indirect role for tumor lymphangiogenesis in the lymphogenous spread of cancer. Both lymphangiogenesis and the formation of lymph node metastases can be inhibited by antagonists of VEGF-C and VEGF-D. In contrast, several studies in various human cancers26–36 have revealed a significant correlation between VEGF-C levels in primary tumors and lymph node metastases without establishing a clear association between upregulation of VEGF-C, tumor lymphangiogenesis and metastases. Two studies have described a strong correlation between lymphatic vessel density (LVD) and VEGF-C expression, and between LVD and the grade of lymphatic invasion in mesothelial and gastric tumors.37,38 However, no correlation was observed between LVD and lymph node metastases. Amioka et al39 examined the relationship between VEGF-C expression and lymph node metastases in invading gastric carcinomas. VEGF-C immunoreactivity was associated with greater depth of tumor invasion, lymphatic invasion and lymph node metastases. In addition, vessel count was also significantly higher in the VEGF-C immunoreactive tumors. These results suggest that VEGF-C may be involved in the progression of human gastric carcinoma, particularly via lymphangiogenesis. The ability of VEGF-D to induce the formation of lymphatics was investigated in a mouse tumor model.40 Staining with

SPOTLIGHT

1055 hepatic sinusoids, kidney glomeruli, adenohypophysis, thyroid gland, adrenals and choroid plexus, are positive.11 Salven et al12 demonstrated that a subset of CD34 þ cells coexpress VEGFR-3 and CD 133. These cells are functionally a unique population of progenitor cells that differentiate into lymphatic or vascular endothelial cells. The number of circulating CD34 þ VEGFR-3 þ cells is very small in healthy adult blood donors. They can, however, be mobilized to the peripheral circulation in pathological conditions, such as tumor growth and wound healing. Makinen et al13 have shown that up to 50% of freshly isolated endothelial cells (HUVECs and dermal microvascular endothelial cells) are positive for VEGFR-3, and that the number of VEGFR3 þ endothelial cells decreases upon repeated subculture in vascular endothelial cell growth medium. Valtola et al14 demonstrated VEGFR-3 expression in blood capillaries of normal breast tissues and fibroadenomas, while Paavonen et al15 showed that VEGFR-3 was weakly expressed in the vascular endothelium of chronic wounds. Furthermore, VEGFR-3 is expressed in the vascular endothelial cells of tumors, such as gliomas and colon carcinomas, which are known to produce large amounts of VEGF-A, and it is thought to play a role in their angiogenesis.14,16,17 This is important as antiangiogenesis treatments could possibly be aimed at VEGFR-3 or its ligands. Binding of VEGF-C to VEGFR-3 regulates VEGFR-2 signaling synergistically with VEGF-A in a coculture system consisting of paraaortic splanchnopleural mesoderm explants and OP9 stromal cells.18 Saaristo et al19 studied the effects of VEGF-C on blood and lymphatic vessels in the skin and respiratory tract of athymic nude mice. They found a dose-dependent enlargement and tortuosity of veins expressing VEGFR-2 along with the collecting lymphatic vessels. Moreover, expression of angiopoietin 1 (Ang1) blocked the increased leakiness of the blood vessels induced by VEGF-C, whereas vessel enlargement and lymphangiogenesis were not affected, and angiogenic sprouting was not observed.

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1056 markers specific for lymphatic endothelium demonstrated that VEGF-D induced the formation of lymphatics within tumors, and that this could be blocked with an antibody specific for VEGF-D. Tumor vessels VEGF-D þ were also VEGFR-2 þ and/ or VEGFR-3 þ but VEGF-D for mRNA, indicating that VEGF-D is secreted by tumor cells and subsequently associates with endothelium via receptor-mediated uptake. These data indicate that VEGF-D promotes tumor angiogenesis, lymphangiogenesis and metastatic spread by a paracrine mechanism.

Involvement of VEGF-A in lymphangiogenesis

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Nagy et al41 have demonstrated that in addition to angiogenesis, VEGF-A also induces proliferation of lymphatic endothelium, resulting in the formation of greatly enlarged and poorly functioning lymphatic channels. Unlike angiogenesis, the lymphangiogenic response became VEGF-A independent, since the newly formed giant lymphatics persisted indefinitely after VEGF-A expression and tissue edema ceased. The new giant lymphatics generated by VEGF-A42 were structurally and functionally abnormal: greatly enlarged with incompetent valves, sluggish flow and delayed lymph clearance. They closely resembled the large lymphatics found in lymphangiomas/lymphatics malformations, perhaps implicating VEGF-A in the pathogenesis of these lesions. The formation of giant lymphatics was largely attributable to the replication of lymphatic endothelial cells without the need for basement membrane degradation, pericyte detachment or endothelial cell thinning, as was necessary for the formation of mother vessels. These findings raise the possibility that abnormal lymphangiogenesis may also be expected in other circumstances characterized by VEGF-A overexpression, such as malignant tumors. Using an adenoviral vector engineered to express VEGFA, the steps and mechanisms by which this cytokine induced the formation of new blood vessels in adult immunodeficient mice have been investigated, and it has been demonstrated that newly formed blood vessels closely resembled those found in VEGF-Aexpressing tumors.43,44 The mechanisms by which VEGF-A induces abnormal lymphangiogenesis have not been clarified. Skobe and Detmar45 reported that VEGF-A stimulates expression of VEGF-C in cultured vascular endothelium. VEGF-A may induce lymphangiogenesis by upregulating the expression of VEGF-C.3,9 However, neither VEGF-C nor VEGF-D expression was ever detected by in situ hybridization in ears injected with Ad-VEGFA.41 Another possibility is that VEGF-A acts directly to induce lymphatic endothelial cell proliferation through VEGFR-2, since this is strongly expressed on normal lymphatic endothelium.46 Otherwise, placenta growth factor (PlGF) which induces angiogenesis but not lymphangiogenesis, does not interact with VEGFR-2.47

Ang-2 seems to be required for the proper development of the lymphatic vessels and may be involved in the regulation of the lymphatic endothelial–periendothelial cell interaction. Synergism between the VEGF and Ang growth factor families seems to be required for both the formation of the blood vascular system, and development and function of the lymphatic vascular system. Stimulation of human primary lymphatic endothelial cells with VEGF-C strongly increased Ang-2 expression via VEGFR2.51 This indicates that in addition to its role in initiating angiogenesis, Ang-2 may also play a role in lymphangiogenesis. This is supported by the phenotype of Ang-2 knockout mice, characterized by hypoplasia of the lymphatic capillaries in the skin and small intestine, and by the presence of collecting lymphatic vessels not properly invested by smooth muscle cells.52

Concluding remarks Like VEGF-A, VEGF-C stimulates the migration and proliferation of endothelial cells of blood vessel origin in vitro, and promotes blood vessel angiogenesis and increased vascular permeability in the adult in vivo. Moreover, VEGF-C may cooperate with VEGF-A in regulating embryonic vascular development. The absolute and relative expression levels of VEGFR-2 and VEGFR-3 in endothelium may determine whether VEGF-C/D elicits angiogenic or lymphangiogenic effects. Strict demarcation of the effects of VEGFR-2 and VEGFR-3 activation in terms of angiogenesis and lymphangiogenesis is complicated by observations that VEGFR-3 is involved in maintenance of tumor blood vessels in a mouse model17 and in human tumors,14,16 and the detection of VEGFR-2 expression on lymphatic endothelial cells.53,54 Furthermore, the influence of other positive and negative regulators of angiogenesis and lymphangiogenesis may modulate the biological consequences of VEGFC and VEGF-D expression. Several important questions remain unresolved, regarding the mechanisms by which expression of VEGF-C and VEGF-D is increased in primary tumors, and nonlymphangiogenic functions of these molecules in promoting lymph node metastases may exist. These functions might include the activation of existing lymphatic endothelium causing an increase of size with the production of mitogenic or chemotactic factors or alterations in lymphatic endothelial–tumor cell adhesion, sufficient to increase the lymphogenous spreading.

Acknowledgements This work is supported by Ministry for Education, the University and Research (MIUR, Interuniversity Funds for Basic Research (FIRB, Rome), Associazione Italiana per la Ricerca sul Cancro (AIRC, Milan) and Fondazione Italiana per la Lotta al Neuroblastoma (Genoa, Italy).

Involvement of ANG-2 in lymphangiogenesis Ang-1 and Ang-2 are secreted factors that mediate their effects by binding to the endothelial-specific Tie-2 receptor tyrosine kinase.48 In vivo analysis has revealed that Ang-1 recruits periendothelial cells, whereas Ang-2 is presumed to destabilize blood vessels by interfering with constitutive Ang-1/Tie-2 signals in the vessel wall, leading to detachment of the perivascular cells and allowing the vessel endothelium to revert to a more plastic state of angiogenesis.49,50 Leukemia

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