A Simple Bioassay for the Evaluation of Vascular Endothelial Growth Factors

The analysis of receptor tyrosine kinases and their interacting ligands involved in vascular biology is often challenging due to the constitutive ...

Peter MacCallum Cancer Centre

Infections of humans and ungulates by parapoxviruses result in skin lesions characterized by extensive vascular changes that have been linked to viral-encoded homologues of vascular endothelial growth factor (VEGF). VEGF acts via a family of receptors (VEGFRs) to mediate endothelial cell proliferation, vascular permeability, and angiogenesis. The VEGF genes from independent parapoxvirus isolates show an extraordinary degree of inter-strain sequence variation. We conducted functional comparisons of five representatives of the divergent viral VEGFs. These revealed that despite the sequence divergence, all were equally active mitogens, stimulating proliferation of human endothelial cells in vitro and vascularization of sheep skin in vivo with potencies equivalent to VEGF. This was achieved even though the viral VEGFs bound VEGFR-2 less avidly than did VEGF. Surprisingly the viral VEGFs varied in their ability to cross-link VEGFR-2, induce vascular permeability and bind neuropilin-1. Correlations between these three activities were detected. In addition it was possible to correlate these functional variations with certain sequence and structural motifs specific to the viral VEGFs. In contrast to the conserved ability to bind human VEGFR-2, the viral growth factors did not bind either VEGFR-1 or VEGFR-3. We propose that the extensive sequence divergence seen in the viral VEGFs was generated primarily by selection against VEGFR-1 binding.

Viral vascular endothelial growth factors vary extensively in amino acid sequence, receptor-binding specificities, and the ability to induce vascular permeability yet are uniformly active mitogens.

The lymphatic network functions to return fluid, cells and macromolecules to the circulation. Recent characterization of growth factors that control the growth and development of the lymphatics, and markers which specify lymphatic endothelial cells have enhanced our understanding of this system. Members of the VEGF family of factors are key regulators of these vessels with VEGF-C/VEGF-D and VEGFR-3 being the best validated signalling pathways in lymphangiogenesis. The study of these molecules in various pathologies has shown that they are important in the processes of cancer metastasis and in the formation of lymphoedema. Knowledge of these molecular pathways allows for the generation of modulators of these pathways which could form the basis of novel therapeutic approaches.

Molecular pathways for lymphangiogenesis and their role in human disease.

Les fonctions de r&eacute;seau lymphatique de regagner la circulation de fluide, de cellules et de macromol&eacute;cules. R&eacute;cente caract&eacute;risation des facteurs de croissance qui contr&ocirc;lent la croissance et le d&eacute;veloppement des vaisseaux lymphatiques et les marqueurs qui sp&eacute;cifient des cellules endoth&eacute;liales lymphatiques ont am&eacute;lior&eacute; notre compr&eacute;hension de ce syst&egrave;me. Membres de la famille VEGF de facteurs sont des r&eacute;gulateurs cl&eacute;s de ces navires au VEGF-C/VEGF-D et VEGFR-3 &eacute;tant la meilleure valid&eacute;s des voies de signalisation dans la lymphangiogen&egrave;se. L&#39;&eacute;tude de ces mol&eacute;cules dans diverses pathologies a montr&eacute; qu&#39;ils sont importants dans les processus de m&eacute;tastases canc&eacute;reuses et la formation de lymphoed&egrave;me. Connaissance de ces voies mol&eacute;culaires permet la g&eacute;n&eacute;ration de modulateurs de ces voies qui pourraient constituer la base de nouvelles approches th&eacute;rapeutiques.

Voies mol&eacute;culaires de la lymphangiogen&egrave;se et leur r&ocirc;le dans la maladie humaine.

Le funzioni di rete linfatica a tornare la circolazione fluido, celle e macromolecole. Recente caratterizzazione dei fattori di crescita che controllano la crescita e lo sviluppo dei vasi linfatici e marcatori che specificano le cellule endoteliali linfatiche hanno rafforzato la nostra comprensione di questo sistema. Membri della famiglia di fattori VEGF sono regolatori chiave di queste navi con VEGF-C/VEGF-D e VEGFR-3 &egrave; il migliore convalidato segnalazione percorsi nella linfangiogenesi. Lo studio di queste molecole in varie patologie ha dimostrato che essi sono importanti nei processi di metastasi del tumore e la formazione del linfedema. Conoscenza di questi percorsi molecolari permette la generazione di modulatori di questi percorsi che potrebbero costituire le basi di nuovi approcci terapeutici.

Molecular pathways for linfangiogenesi e il loro ruolo nella malattia umana.

Lymphatic metastasis is facilitated by lymphangiogenic growth factors VEGF-C and VEGF-D that are secreted by some primary tumors. We identified regulation of PGDH, the key enzyme in prostaglandin catabolism, in endothelial cells of collecting lymphatics, as a key molecular change during VEGF-D-driven tumor spread. The VEGF-D-dependent regulation of the prostaglandin pathway was supported by the finding that collecting lymphatic vessel dilation and subsequent metastasis were affected by nonsteroidal anti-inflammatory drugs (NSAIDs), known inhibitors of prostaglandin synthesis. Our data suggest a control point for cancer metastasis within the collecting lymphatic endothelium, which links VEGF-D/VEGFR-2/VEGFR-3 and the prostaglandin pathways. Collecting lymphatics therefore play an active and important role in metastasis and may provide a therapeutic target to restrict tumor spread.

VEGF-D promotes tumor metastasis by regulating prostaglandins produced by the collecting lymphatic endothelium.

M&eacute;tastase lymphatique est facilit&eacute;e par des facteurs de croissance lymphangiog&eacute;nique VEGF-C et VEGF-D qui sont s&eacute;cr&eacute;t&eacute;es par certaines tumeurs primaires. Nous avons identifi&eacute; le r&egrave;glement de PGDH, l&#39;enzyme cl&eacute; du catabolisme de prostaglandine, dans les cellules endoth&eacute;liales de la collecte des vaisseaux lymphatiques, comme un changement mol&eacute;culaire cl&eacute; au cours de la propagation de la tumeur de VEGF-D-conduit. La r&eacute;gulation de VEGF-D-d&eacute;pendant de la voie de la prostaglandine a &eacute;t&eacute; &eacute;tay&eacute;e par le fait que collecte dilatation de vaisseau lymphatique et m&eacute;tastase ult&eacute;rieur ont &eacute;t&eacute; affect&eacute;es par les anti-inflammatoires non st&eacute;ro&iuml;diens (AINS), connu des inhibiteurs de la synth&egrave;se des prostaglandines. Nos donn&eacute;es sugg&egrave;rent un point de contr&ocirc;le pour les m&eacute;tastases du cancer dans l&#39;endoth&eacute;lium lymphatique gestion collective, qui relie le VEGF-D/VEGFR-2/VEGFR-3 et les voies de la prostaglandine. Collecteurs de vaisseaux lymphatiques donc jouent un r&ocirc;le actif et important dans les m&eacute;tastases et peuvent fournir une cible th&eacute;rapeutique visant &agrave; limiter la propagation de la tumeur.

VEGF-D favorise les m&eacute;tastases tumorales en r&eacute;glementant les prostaglandines produites par l&#39;endoth&eacute;lium lymphatique collecte.

Metastasi linfatiche sono facilitata da fattori di crescita lymphangiogenic VEGF-C e VEGF-D che sono secreti da alcuni tumori primari. Abbiamo identificato regolamento di PGDH, l&#39;enzima chiave nel catabolismo delle prostaglandine, nelle cellule endoteliali di raccolta di vasi linfatici, come un cambiamento molecolare chiave durante la diffusione del tumore VEGF-D-driven. Il regolamento di VEGF-D-dipendente del percorso prostaglandina &egrave; stato sostenuto dalla constatazione che raccogliendo la dilatazione del vaso linfatico e metastasi successive sono stati influenzati da farmaci anti-infiammatori non steroidei (FANS), noti inibitori della sintesi della prostaglandina. I nostri dati suggeriscono un punto di controllo per metastasi del cancro all&#39;interno della raccolta endotelio linfatico, che collega i percorsi della prostaglandina e VEGF-D/VEGFR-2/VEGFR-3. Raccolta di vasi linfatici pertanto svolgono un ruolo attivo e importante in metastasi e possono fornire un obiettivo terapeutico per limitare la diffusione del tumore.

VEGF-D promuove metastasi tumorali regolando le prostaglandine prodotte dall&#39;endotelio linfatico raccolta.

Tumor metastasis to lymph nodes is a key indicator of patient survival, and is enhanced by the neo-lymphatics induced by tumor-secreted VEGF-C or VEGF-D, acting via VEGFR-3 signalling. These targets constitute important avenues for anti-metastatic treatment. Despite this new understanding, clinical observations linking metastasis with tumor depth or location suggest that lymphangiogenic growth factors are not the sole determinants of metastasis. Here we explored the influence of tumor proximity to lymphatics capable of responding to growth factors on nodal metastasis in a murine VEGF-D over-expression tumor model. We found that primary tumor location profoundly influenced VEGF-D-mediated lymph node metastasis: 89 % of tumors associated with the flank skin metastasised, in contrast with only 19 % of tumors located more deeply on the body wall (p < 0.01). Lymphatics in metastatic tumors arose from small lymphatics, and displayed distinct molecular and morphological profiles compared with those found in normal lymphatics. Smaller lymphatic subtypes were more abundant in skin (2.5-fold, p < 0.01) than in body wall, providing a richer source of lymphatics for VEGF-D(+) skin tumors, a phenomenon also confirmed in human samples. This study shows that the proximity of a VEGF-D(+) primary tumor to small lymphatics is an important determinant of metastasis. These observations may explain why tumor location relative to the lymphatic network is prognostically important for some human cancers.

Tumor location and nature of lymphatic vessels are key determinants of cancer metastasis.

Metastasis to regional lymph nodes is an important and early event in many tumors. Vascular endothelial growth factor-C (VEGF-C), VEGF-D and their receptor VEGFR-3, play a role in tumor spread via the lymphatics, although the timing of their involvement is not understood. In contrast, VEGFR-2, activated by VEGF-A, VEGF-C and VEGF-D, is a mediator of angiogenesis and drives primary tumor growth. We demonstrate the critical role for VEGFR-3, but not VEGFR-2, in the early events of metastasis. In a tumor model exhibiting both VEGF-D-dependent angiogenesis and lymphangiogenesis, an antibody to VEGFR-2 (DC101) was capable of inhibiting angiogenesis (79 % reduction in PECAM + blood vessels) and growth (93 % reduction in tumor volume). However, unlike an anti-VEGFR-3 Mab (mF4-31C1), DC101 was not capable of eliminating either tumor lymphangiogenesis or lymphogenous metastasis (60 % reduction of lymph node metastasis by DC101 vs 95 % by mF4-31C1). Early excision of the primary tumors demonstrated that VEGF-D-mediated tumor spread precedes angiogenesis-induced growth. Small but highly metastatic primary human breast cancers had significantly higher lymphatic vessel density (23.1 vessels/mm(2)) than size-matched (11.7) or larger non-metastatic tumors (12.4) thus supporting the importance of lymphatic vessels, as opposed to angiogenesis-mediated primary tumor growth, for nodal metastasis. These results suggest that lymphangiogenesis via VEGF-D is more critical than angiogenesis for nodal metastasis.

Carol Caesar

Tumour Angiogenesis Program

Peter MacCallum Cancer Centre

A Simple Bioassay for the Evaluation of Vascular Endothelial Growth Factors

Carol Caesar

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Peter MacCallum Cancer Centre

A Simple Bioassay for the Evaluation of Vascular Endothelial Growth Factors

Tumour Angiogenesis Program