a-simple-bioassay-for-evaluation-vascular-endothelial-growth

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

Vascular endothelial growth factor-D (VEGF-D) promotes angiogenesis, lymphangiogenesis and metastatic spread via the lymphatics, however, the mode of VEGF-D action (e.g. paracrine vs. autocrine) was unknown. We analyzed VEGF-D action in human tumors and a mouse model of metastasis. VEGF-D was localized in tumor cells and endothelium in human non-small cell lung carcinoma and breast ductal carcinoma in situ. Tumor vessels positive for VEGF-D were also positive for its receptors, VEGF receptor-2 (VEGFR-2) and/or VEGFR-3 but negative for VEGF-D mRNA, indicating that VEGF-D is secreted by tumor cells and subsequently associates with endothelium via receptor-mediated uptake. The mature form of VEGF-D was detected in tumors demonstrating that VEGF-D is proteolytically processed and bioactive. In a mouse model of metastasis, VEGF-D synthesized in tumor cells became localized on the endothelium and thereby promoted metastatic spread. These data indicate that VEGF-D promotes tumor angiogenesis, lymphangiogenesis and metastatic spread by a paracrine mechanism.

The angiogenic and lymphangiogenic factor vascular endothelial growth factor-D exhibits a paracrine mode of action in cancer.

Facteur de croissance endoth&eacute;lial vasculaire-D (VEGF-D) encourage l&#39;angiogen&egrave;se, la lymphangiogen&egrave;se et m&eacute;tastases via les vaisseaux lymphatiques, cependant, le mode d&#39;action de VEGF-D (p. ex. paracrine et autocrine) &eacute;tait inconnu. Nous avons analys&eacute; l&#39;action du VEGF-D dans les tumeurs humaines et un mod&egrave;le de souris de m&eacute;tastases. VEGF-D a &eacute;t&eacute; localis&eacute;e dans les cellules tumorales et l&#39;endoth&eacute;lium dans le carcinome du poumon humain non &agrave; petites cellules et le carcinome canalaire in situ du sein. Navires de tumeur positives pour le VEGF-D ont &eacute;t&eacute; aussi positifs pour ses r&eacute;cepteurs, les r&eacute;cepteurs VEGF-2 (VEGFR-2) et/ou VEGFR-3, mais n&eacute;gatif de VEGF-D ARNm, ce qui indique que les VEGF-D est s&eacute;cr&eacute;t&eacute;e par la tumeur des cellules et par la suite, associ&eacute; &agrave; l&#39;endoth&eacute;lium par absorption m&eacute;di&eacute;e par les r&eacute;cepteurs. La forme mature de VEGF-D a &eacute;t&eacute; d&eacute;tect&eacute;e dans les tumeurs d&eacute;montrant que le VEGF-D est clivage prot&eacute;olytique transform&eacute;s et bioactifs. Dans un mod&egrave;le murin de la m&eacute;tastase, synth&eacute;tis&eacute; dans les cellules tumorales de VEGF-D est devenu localis&eacute; sur l&#39;endoth&eacute;lium et ainsi favoris&eacute; la diss&eacute;mination m&eacute;tastatique. Ces donn&eacute;es indiquent que le VEGF-D favorise angiogen&egrave;se tumorale, lymphangiogen&egrave;se et m&eacute;tastases par un m&eacute;canisme paracrine.

L&#39;angiog&eacute;nique et lymphangiog&eacute;nique facteur expositions de D-facteur de croissance endoth&eacute;liales vasculaires un mode paracrine d&#39;action dans le cancer.

Vascular endothelial growth factor-D (VEGF-D) promuove l&#39;angiogenesi, linfangiogenesi e diffusione metastatica attraverso i vasi linfatici, tuttavia, la modalit&agrave; di azione del VEGF-D (ad esempio paracrina vs autocrino) era sconosciuta. Abbiamo analizzato l&#39;azione del VEGF-D in tumori umani e un modello del mouse di metastasi. VEGF-D &egrave; stata localizzata nelle cellule tumorali e dell&#39;endotelio nel carcinoma polmonare non a piccole cellule e carcinoma mammario duttale in situ. Vasi tumorali positivi per VEGF-D sono stati anche positivi per i suoi recettori, VEGF receptor-2 (VEGFR-2) e/o VEGFR-3, ma negativo per mRNA-VEGF-D, che indica che il VEGF-D viene secreta dal tumore cellule e successivamente associa endotelio tramite captazione mediata dal recettore. La forma adulta di VEGF-D &egrave; stata rilevata nei tumori dimostrando che il VEGF-D &egrave; proteoliticamente trasformati e bioattivi. In un modello murino di metastasi, VEGF-D sintetizzata nelle cellule tumorali &egrave; diventato localizzata sull&#39;endotelio e quindi promosso la diffusione metastatica. Questi dati indicano che il VEGF-D promuove l&#39;angiogenesi tumorale, linfangiogenesi e diffusione metastatica da un meccanismo paracrina.

L&#39;angiogenesi e lymphangiogenic Fattore vascolare endothelial growth factor-D esibisce una modalit&agrave; paracrina di azione nel cancro.

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.

&#x8840;&#x7BA1;&#x5185;&#x76AE;&#x751F;&#x957F;&#x56E0;&#x5B50; (VEGF) C&#x3001; D &#x901A;&#x8FC7;&#x6FC0;&#x6D3B;&#x5185;&#x76AE;&#x7EC6;&#x80DE;&#x8868;&#x9762;&#x7684;&#x53D7;&#x4F53;&#x916A;&#x6C28;&#x9178;&#x6FC0;&#x9176; VEGF &#x53D7;&#x4F53; (&#x751F;&#x957F;&#x56E0;&#x5B50;&#x53D7;&#x4F53;&#xFF09; 2 &#x548C;&#x751F;&#x957F;&#x56E0;&#x5B50;&#x53D7;&#x4F53;-3 &#x523A;&#x6FC0;&#x6DCB;&#x5DF4;&#x7BA1;&#x751F;&#x6210;&#x4E0E;&#x80BF;&#x7624;&#x7EC4;&#x7EC7;&#x4E2D;&#x7684;&#x8840;&#x7BA1;&#x751F;&#x6210;&#x3002;&#x8FD9;&#x4E9B;&#x751F;&#x957F;&#x56E0;&#x5B50;&#x5206;&#x6CCC;&#x4F5C;&#x4E3A; NH2 &#x548C;&#x4E8C;&#x7518;&#x9187;&#x7684;&#x7EC8;&#x7AEF; propeptides &#x548C;&#x4E2D;&#x592E; VEGF &#x540C;&#x6E90;&#x6027; (VHD) &#x5305;&#x542B;&#x57DF;&#x53D7;&#x4F53;&#x7ED3;&#x5408;&#x4F4D;&#x70B9;&#x7EC4;&#x6210;&#x7684;&#x5168;&#x957F;&#x975E;&#x6D3B;&#x52A8;&#x7A97;&#x4F53;&#x3002;&#x86CB;&#x767D;&#x6C34;&#x89E3;&#x5288;&#x4E2D;&#x79FB;&#x9664; propeptides &#x751F;&#x6210;&#x7EC4;&#x6210;&#x7684;&#x4E8C;&#x805A;&#x4F53;&#x7684; VEGF &#x540C;&#x6E90;&#x57DF;&#xFF0C;&#x7ED1;&#x5B9A;&#x6BD4;&#x5168;&#x957F;&#x7684;&#x5F62;&#x5F0F;&#x66F4;&#x4EB2;&#x548C;&#x4E0E;&#x53D7;&#x4F53;&#x7684;&#x6210;&#x719F;&#x5F62;&#x5F0F;&#x3002;&#x56E0;&#x6B64;&#xFF0C;&#x86CB;&#x767D;&#x6C34;&#x89E3;&#x5904;&#x7406;&#x6FC0;&#x6D3B; VEGF-C &#x548C; D&#xFF0C;&#x867D;&#x7136;&#x6D89;&#x53CA;&#x7684;&#x86CB;&#x767D;&#x9176;&#x662F;&#x672A;&#x77E5;&#x3002;&#x5728;&#x8FD9;&#x91CC;&#xFF0C;&#x6211;&#x4EEC;&#x62A5;&#x544A;&#x4E1D;&#x6C28;&#x9178;&#x86CB;&#x767D;&#x9176;&#x7EA4;&#x7EF4;&#x86CB;&#x767D;&#x6EB6;&#x9176;&#x4ECE;&#x4EBA;&#x7C7B; VEGF-d VEGF &#x540C;&#x6E90;&#x57DF;&#x5206;&#x89E3;&#x4E24;&#x4E2A; propeptides &#x548C;&#x7EA6;&#x675F;&#x529B;&#x548C;&#x5168;&#x957F;&#x6750;&#x6599;&#x4E0E;&#x4EA4;&#x8054;&#x7684;&#x751F;&#x957F;&#x56E0;&#x5B50;&#x53D7;&#x4F53;-2 &#x548C;&#x751F;&#x957F;&#x56E0;&#x5B50;&#x53D7;&#x4F53;-3 &#x4ECE;&#x800C;&#x751F;&#x6210;&#x6210;&#x719F;&#x5F62;&#x5F0F;&#x53C2;&#x5C55;&#x5927;&#x5927;&#x52A0;&#x5F3A;&#x3002;&#x7EA4;&#x7EF4;&#x86CB;&#x767D;&#x6EB6;&#x9176;&#x8FD8;&#x6FC0;&#x6D3B; VEGF C.&#x6DCB;&#x5DF4;&#x7BA1;&#x751F;&#x6210;&#x751F;&#x957F;&#x56E0;&#x5B50;&#x4FC3;&#x8FDB;&#x764C;&#x7EC6;&#x80DE;&#x901A;&#x8FC7;&#x6DCB;&#x5DF4;&#x7BA1;&#x8F6C;&#x79FB;&#x6269;&#x6563;&#xFF0C;&#x8FD9;&#x4E9B;&#x5206;&#x5B50;&#x7684;&#x86CB;&#x767D;&#x6C34;&#x89E3;&#x6D3B;&#x5316;&#x8FDC;&#x5904;&#x8F6C;&#x79FB;&#x4EE3;&#x7406;&#x8868;&#x793A;&#x7684;&#x6F5C;&#x5728;&#x76EE;&#x6807;&#x3002;&#x6FC0;&#x6D3B;&#x6DCB;&#x5DF4;&#x7BA1;&#x751F;&#x6210;&#x751F;&#x957F;&#x56E0;&#x5B50;&#x7684;&#x9176;&#x8BC6;&#x522B;&#x6709;&#x52A9;&#x6291;&#x5236;&#x5242;&#x8F6C;&#x79FB;&#x7684;&#x53D1;&#x5C55;&#x3002;

&#x7EA4;&#x7EF4;&#x86CB;&#x767D;&#x6EB6;&#x9176;&#x6FC0;&#x6D3B;&#x7684;&#x6DCB;&#x5DF4;&#x7BA1;&#x751F;&#x6210;&#x751F;&#x957F;&#x56E0;&#x5B50; VEGF-C &#x548C; VEGF D.

Vascular endothelial growth factor (VEGF) C and VEGF-D stimulate lymphangiogenesis and angiogenesis in tissues and tumors by activating the endothelial cell surface receptor tyrosine kinases VEGF receptor (VEGFR) 2 and VEGFR-3. These growth factors are secreted as full-length inactive forms consisting of NH2- and COOH-terminal propeptides and a central VEGF homology domain (VHD) containing receptor binding sites. Proteolytic cleavage removes the propeptides to generate mature forms, consisting of dimers of the VEGF homology domain, that bind receptors with much greater affinity than the full-length forms. Therefore, proteolytic processing activates VEGF-C and VEGF-D, although the proteases involved were unknown. Here, we report that the serine protease plasmin cleaved both propeptides from the VEGF homology domain of human VEGF-D and thereby generated a mature form exhibiting greatly enhanced binding and cross-linking of VEGFR-2 and VEGFR-3 in comparison to full-length material. Plasmin also activated VEGF-C. As lymphangiogenic growth factors promote the metastatic spread of cancer via the lymphatics, the proteolytic activation of these molecules represents a potential target for antimetastatic agents. Identification of an enzyme that activates the lymphangiogenic growth factors will facilitate development of inhibitors of metastasis.

Plasmin activates the lymphangiogenic growth factors VEGF-C and VEGF-D.

Facteur de croissance endoth&eacute;lial vasculaire (VEGF) C et VEGF-D stimulent la lymphangiogen&egrave;se et l&#39;angiogen&egrave;se dans les tissus et les tumeurs en activant le r&eacute;cepteur VEGF endoth&eacute;lial surface r&eacute;cepteurs tyrosine kinases (VEGFR) 2 et VEGFR-3. Ces facteurs de croissance sont s&eacute;cr&eacute;t&eacute;es comme formes inactives sur toute la longueur, comprenant des propeptides NH2 et COOH-terminale et un centre domaine d&#39;homologie VEGF (VHD) contenant des sites de liaison du r&eacute;cepteur. Le clivage prot&eacute;olytique supprime les propeptides pour g&eacute;n&eacute;rer des formes matures, compos&eacute;e de dim&egrave;res de la domaine d&#39;homologie VEGF, qui lient les r&eacute;cepteurs avec une affinit&eacute; beaucoup plus grande que les formes sur toute la longueur. Par cons&eacute;quent, prot&eacute;olyse active VEGF-C et VEGF-D, bien que les prot&eacute;ases impliqu&eacute;es &eacute;taient inconnus. Nous rapportons ici que la plasmine de prot&eacute;ase de s&eacute;rine cliv&eacute; deux propeptides du domaine d&#39;homologie VEGF de VEGF-D humaine et ainsi g&eacute;n&eacute;r&eacute; une forme mature expose grandement am&eacute;lior&eacute;e contraignant et r&eacute;ticulation des VEGFR-2, VEGFR-3 en comparaison de mat&eacute;riel sur toute la longueur. Plasmine activ&eacute;e aussi VEGF-C. Comme facteurs de croissance lymphangiog&eacute;nique promouvoir la propagation m&eacute;tastatique du cancer par les vaisseaux lymphatiques, l&#39;activation prot&eacute;olytique de ces mol&eacute;cules repr&eacute;sente une cible potentielle pour les agents antim&eacute;tastatiques. Identification d&#39;une enzyme qui active les facteurs de croissance lymphangiog&eacute;nique facilitera le d&eacute;veloppement d&#39;inhibiteurs de m&eacute;tastase.

Plasmine active les facteurs de croissance lymphangiog&eacute;nique VEGF-C et le VEGF-D.

Plasmin aktiviert die Lymph Wachstumsfaktoren VEGF-C und VEGF-D.

Fattore di crescita endoteliale vascolare (VEGF) C e VEGF-D stimolare linfangiogenesi e angiogenesi nei tessuti e tumori attivando le cellule endoteliali superficiale del recettore tirosina chinasi VEGF receptor (VEGFR) 2 e VEGFR-3. Questi fattori di crescita sono secreti come full-length forme inattive composto NH2-COOH terminale propeptides e un VEGF omologia dominio centrale (VHD) che contiene i siti di legame del recettore. Clivaggio proteolitico rimuove il propeptides per generare forme mature, costituiti da dimeri di dominio di omologia VEGF, che legano i recettori con affinit&agrave; molto maggiore rispetto al full-length forme. Pertanto, trattamento proteolitico attiva VEGF-C e VEGF-D, anche se la proteasi coinvolte erano sconosciute. Qui, segnaliamo che la plasmina di serina proteasi spaccati entrambi propeptides al dominio di omologia VEGF dell&#39;umano VEGF-D e quindi generato una forma matura espositrici notevolmente migliorata vincolanti e cross-linking del VEGFR-2 e VEGFR-3 rispetto a materiale full-length. Plasmina attivata anche VEGF-C. Come fattori di crescita lymphangiogenic promuovere la diffusione metastatica del tumore tramite i vasi linfatici, l&#39;attivazione proteolitica di queste molecole rappresenta un potenziale bersaglio per gli agenti antimetastatica. Identificazione di un enzima che attiva i fattori di crescita lymphangiogenic faciliter&agrave; lo sviluppo di inibitori di metastasi.

Plasmina attiva i fattori di crescita lymphangiogenic VEGF-C e VEGF-D.

&#x8840;&#x7BA1;&#x5185;&#x76AE;&#x5897;&#x6B96;&#x56E0;&#x5B50; &#xFF08;VEGF&#xFF09; C &#x3068; VEGF D &#x6027;&#x30EA;&#x30F3;&#x30D1;&#x7BA1;&#x65B0;&#x751F;&#x3068;&#x8840;&#x7BA1;&#x65B0;&#x751F;&#x7D44;&#x7E54;&#x3084;&#x816B;&#x760D;&#x306E;&#x5185;&#x76AE;&#x7D30;&#x80DE;&#x8868;&#x9762;&#x53D7;&#x5BB9;&#x4F53;&#x30C1;&#x30ED;&#x30B7;&#x30F3;&#x30AD;&#x30CA;&#x30FC;&#x30BC; &#x30AD;&#x30CA;&#x30FC;&#x30BC; VEGF &#x53D7;&#x5BB9;&#x4F53; (VEGFR) 2 &#x3068; VEGFR 3 &#x6D3B;&#x6027;&#x5316;&#x3092;&#x523A;&#x6FC0;&#x3057;&#x307E;&#x3059;&#x3002;&#x3053;&#x308C;&#x3089;&#x306E;&#x6210;&#x9577;&#x8981;&#x56E0; NH2 &#x672B;&#x7AEF; COOH &#x672B;&#x7AEF;&#x30D7;&#x30ED;&#x30DA;&#x30D7;&#x30C1;&#x30C9;&#x3068;&#x53D7;&#x5BB9;&#x4F53;&#x7D50;&#x5408;&#x90E8;&#x4F4D;&#x3092;&#x542B;&#x3080;&#x4E2D;&#x592E; VEGF &#x76F8;&#x540C;&#x30C9;&#x30E1;&#x30A4;&#x30F3; (VHD) &#x304B;&#x3089;&#x6210;&#x308B;&#x30D5;&#x30EB;&#x30EC;&#x30F3;&#x30B0;&#x30B9;&#x306E;&#x30A2;&#x30AF;&#x30C6;&#x30A3;&#x30D6;&#x3067;&#x306A;&#x3044;&#x30D5;&#x30A9;&#x30FC;&#x30E0;&#x3068;&#x3057;&#x3066;&#x5206;&#x6CCC;&#x3055;&#x308C;&#x307E;&#x3059;&#x3002;&#x86CB;&#x767D;&#x8CEA;&#x5206;&#x89E3;&#x958B;&#x88C2;&#x306F;&#x30D5;&#x30EB;&#x30EC;&#x30F3;&#x30B0;&#x30B9;&#x306E;&#x5F62;&#x614B;&#x3088;&#x308A;&#x3082;&#x306F;&#x308B;&#x304B;&#x306B;&#x5927;&#x304D;&#x3044;&#x89AA;&#x548C;&#x6027;&#x53D7;&#x5BB9;&#x4F53;&#x7D50;&#x5408;&#x30C0;&#x30A4;&#x30DE;&#x30FC;&#x306E; VEGF &#x306E;&#x76F8;&#x540C;&#x9818;&#x57DF;&#x304B;&#x3089;&#x6210;&#x308B;&#x3001;&#x6210;&#x719F;&#x3057;&#x305F;&#x30D5;&#x30A9;&#x30FC;&#x30E0;&#x3092;&#x751F;&#x6210;&#x3059;&#x308B;&#x30D7;&#x30ED;&#x30DA;&#x30D7;&#x30C1;&#x30C9;&#x3092;&#x524A;&#x9664;&#x3057;&#x307E;&#x3059;&#x3002;&#x3057;&#x305F;&#x304C;&#x3063;&#x3066;&#x3001;&#x30D7;&#x30ED;&#x30C6;&#x30A2;&#x30FC;&#x30BC;&#x306E;&#x95A2;&#x4E0E;&#x304C;&#x77E5;&#x3089;&#x308C;&#x3066;&#x3044;&#x306A;&#x304B;&#x3063;&#x305F;&#x3082;&#x306E;&#x306E;&#x86CB;&#x767D;&#x5206;&#x89E3;&#x51E6;&#x7406; VEGF C &#x3068; VEGF D &#x30A2;&#x30AF;&#x30C6;&#x30A3;&#x30D6;&#x306B;&#x306A;&#x308A;&#x307E;&#x3059;&#x3002;&#x3053;&#x3053;&#x3067;&#x306F;&#x3001;&#x30BB;&#x30EA;&#x30F3; &#x30D7;&#x30ED;&#x30C6;&#x30A2;&#x30FC;&#x30BC; &#x30D7;&#x30E9;&#x30B9;&#x30DF;&#x30F3;&#x4E21;&#x65B9;&#x30D7;&#x30ED;&#x30DA;&#x30D7;&#x30C1;&#x30C9;&#x4EBA;&#x9593; VEGF D &#x306E; VEGF &#x76F8;&#x540C;&#x30C9;&#x30E1;&#x30A4;&#x30F3;&#x304B;&#x3089;&#x5207;&#x65AD;&#x3057;&#x3001;&#x305D;&#x308C;&#x306B;&#x3088;&#x3063;&#x3066;&#x751F;&#x6210;&#x3055;&#x308C;&#x308B;&#x6210;&#x719F;&#x3057;&#x305F;&#x30D5;&#x30A9;&#x30FC;&#x30E0;&#x51FA;&#x5C55;&#x5927;&#x5E45;&#x306B;&#x30D0;&#x30A4;&#x30F3;&#x30C9;&#x3068; VEGFR 2 &#x3068; VEGFR 3 &#x306E;&#x30D5;&#x30EB;&#x30EC;&#x30F3;&#x30B0;&#x30B9;&#x306E;&#x6750;&#x6599;&#x3068;&#x6BD4;&#x8F03;&#x3057;&#x3066;&#x67B6;&#x6A4B;&#x5F37;&#x5316;&#x3092;&#x5831;&#x544A;&#x3057;&#x307E;&#x3059;&#x3002;&#x30D7;&#x30E9;&#x30B9;&#x30DF;&#x30F3;&#x306F;&#x307E;&#x305F; VEGF C. &#x30A2;&#x30AF;&#x30C6;&#x30A3;&#x30D6;Lymphangiogenic &#x6210;&#x9577;&#x56E0;&#x5B50;&#x3001;&#x8EE2;&#x79FB;&#x6027;&#x764C;&#x306E;&#x30EA;&#x30F3;&#x30D1;&#x7BA1;&#x3092;&#x4ECB;&#x3057;&#x3066;&#x306E;&#x666E;&#x53CA;&#x3068;&#x3057;&#x3066;&#x3001;&#x3053;&#x308C;&#x3089;&#x306E;&#x5206;&#x5B50;&#x306E;&#x86CB;&#x767D;&#x5206;&#x89E3;&#x6D3B;&#x6027;&#x5316;&#x306E;&#x6F5C;&#x5728;&#x7684;&#x306A;&#x30BF;&#x30FC;&#x30B2;&#x30C3;&#x30C8;&#x7528;&#x6297;&#x8EE2;&#x79FB;&#x5264;&#x3092;&#x8868;&#x3057;&#x307E;&#x3059;&#x3002;Lymphangiogenic &#x306E;&#x6210;&#x9577;&#x56E0;&#x5B50;&#x3092;&#x6D3B;&#x6027;&#x5316;&#x9175;&#x7D20;&#x306E;&#x540C;&#x5B9A;&#x8EE2;&#x79FB;&#x963B;&#x5BB3;&#x5264;&#x306E;&#x958B;&#x767A;&#x304C;&#x5BB9;&#x6613;&#x306B;&#x306A;&#x308A;&#x307E;&#x3059;&#x3002;

&#x30D7;&#x30E9;&#x30B9;&#x30DF;&#x30F3; lymphangiogenic &#x6210;&#x9577;&#x56E0;&#x5B50; VEGF C &#x3068; VEGF D &#x3092;&#x30A2;&#x30AF;&#x30C6;&#x30A3;&#x30D6;&#x306B;&#x3057;&#x307E;&#x3059;&#x3002;

&#xD608;&#xAD00; &#xB0B4; &#xD53C; &#xC131;&#xC7A5; &#xC778;&#xC790; (VEGF) C&#xC640; VEGF D &#xB0B4; &#xD53C; &#xC138;&#xD3EC; &#xD45C;&#xBA74; &#xC218;&#xC6A9; &#xCCB4; tyrosine kinases VEGF &#xC218;&#xC6A9; &#xCCB4; (VEGFR) 2&#xC640; VEGFR 3&#xC744; &#xD65C;&#xC131;&#xD654; &#xD558; &#xC5EC; lymphangiogenesis&#xC640; &#xC870;&#xC9C1; &#xBC0F; &#xC885;&#xC591; &#xD608;&#xAD00; &#xC2E0;&#xC0DD;&#xC744; &#xC790;&#xADF9;. &#xC774; &#xC131;&#xC7A5; &#xC778;&#xC790;&#xB294; NH2-COOH &#xD130;&#xBBF8;&#xB110; propeptides&#xC640; &#xD3EC;&#xD568; &#xD558;&#xB294; &#xC218;&#xC6A9; &#xCCB4; &#xBC14;&#xC778;&#xB529; &#xC0AC;&#xC774;&#xD2B8; &#xC911;&#xC559; VEGF homology &#xB3C4;&#xBA54;&#xC778; (VHD)&#xB85C; &#xAD6C;&#xC131; &#xB41C; &#xC804;&#xCCB4; &#xAE38;&#xC774; &#xBE44;&#xD65C;&#xC131; &#xC591;&#xC2DD;&#xC73C;&#xB85C; &#xBD84; &#xBE44; &#xD569;&#xB2C8;&#xB2E4;. &#xB2E8;&#xBC31;&#xC9C8; &#xBD84;&#xC5F4; &#xC804;&#xCCB4; &#xAE38;&#xC774; &#xD615;&#xD0DC; &#xBCF4;&#xB2E4; &#xD6E8;&#xC52C; &#xB354; &#xD070; &#xC120;&#xD638;&#xB3C4;&#xC640; &#xC218;&#xC6A9; &#xCCB4;&#xB97C; &#xBC14;&#xC778;&#xB529;&#xD558;&#xB294; VEGF homology &#xB3C4;&#xBA54;&#xC778;&#xC758; &#xC774;&#xD569;&#xCCB4;&#xB85C; &#xAD6C;&#xC131; &#xB41C; &#xC131;&#xC219;&#xD55C; &#xD615;&#xD0DC;&#xB97C; &#xC0DD;&#xC131; &#xD558;&#xB294; propeptides&#xB97C; &#xC81C;&#xAC70; &#xD569;&#xB2C8;&#xB2E4;. &#xB530;&#xB77C;&#xC11C;, &#xBE44;&#xB85D; &#xD504;&#xB85C; &#xD14C;&#xC544; &#xC81C; &#xCC38;&#xC5EC;&#xB294; &#xC54C;&#xB824;&#xC9C0;&#xC9C0; &#xC54A;&#xC558;&#xB358; C VEGF&#xC640; VEGF &#xCC28;&#xC6D0; &#xB2E8;&#xBC31;&#xC9C8; &#xCC98;&#xB9AC; &#xD65C;&#xC131;&#xD654; &#xD569;&#xB2C8;&#xB2E4;. &#xC5EC;&#xAE30;, &#xC6B0;&#xB9AC; &#xBCF4;&#xACE0; &#xB5A0;&#xB4E4;&#xACE0; protease plasmin &#xC778;&#xAC04;&#xC758; VEGF D&#xC758; VEGF homology &#xB3C4;&#xBA54;&#xC778;&#xC5D0;&#xC11C; &#xBAA8;&#xB450; propeptides &#xBD99;&#xC5B4; &#xD558; &#xACE0; &#xBC14;&#xC778;&#xB529; &#xBC0F; &#xC804;&#xCCB4; &#xAE38;&#xC774; &#xC790;&#xB8CC;&#xC5D0; &#xBE44;&#xD574; VEGFR 2&#xC640; VEGFR 3 &#xAD50;&#xAC00; &#xD06C;&#xAC8C; &#xD5A5;&#xC0C1; &#xC131;&#xC219;&#xD55C; &#xC591;&#xC2DD; &#xC804;&#xC2DC; &#xD568;&#xC73C;&#xB85C;&#xC368; &#xC0DD;&#xC131; &#xD569;&#xB2C8;&#xB2E4;. Plasmin &#xB610;&#xD55C; VEGF C. &#xD65C;&#xC131;&#xD654; Lymphangiogenic &#xC131;&#xC7A5; &#xC778;&#xC790;&#xB294; lymphatics &#xD1B5;&#xD574; &#xC554; &#xC804;&#xC774;&#xC131; &#xD655;&#xC0B0; &#xCD94;&#xC9C4;&#xC73C;&#xB85C; &#xC774;&#xB7EC;&#xD55C; &#xBD84;&#xC790;&#xC758; &#xB2E8;&#xBC31;&#xC9C8; &#xD65C;&#xC131;&#xD654; antimetastatic &#xC5D0;&#xC774;&#xC804;&#xD2B8;&#xC5D0; &#xB300; &#xD55C; &#xC7A0;&#xC7AC;&#xC801;&#xC778; &#xBAA9;&#xD45C;&#xB97C; &#xB098;&#xD0C0;&#xB0C5;&#xB2C8;&#xB2E4;. Lymphangiogenic &#xC131;&#xC7A5; &#xC778;&#xC790;&#xB97C; &#xD65C;&#xC131;&#xD654; &#xD558;&#xB294; &#xD6A8;&#xC18C;&#xC758; &#xC2DD;&#xBCC4; &#xC804;&#xC774; &#xC5B5;&#xC81C;&#xC81C;&#xC758; &#xAC1C;&#xBC1C;&#xC744; &#xCD09;&#xC9C4; &#xD55C;&#xB2E4;.

Plasmin &#xD65C;&#xC131;&#xD654; lymphangiogenic &#xC131;&#xC7A5; &#xC778;&#xC790; VEGF C&#xC640; VEGF 4.

Factor de crecimiento endotelial vascular (VEGF) C y VEGF-D estimulan la linfangiog&eacute;nesis y angiog&eacute;nesis en tumores y tejidos mediante la activaci&oacute;n de la c&eacute;lula endotelial receptor de superficie celular tirosina quinasas VEGF receptor (VEGFR) 2 y VEGFR-3. Estos factores de crecimiento son secretados como larga duraci&oacute;n formas inactivas de NH2-COOH-terminal y propeptides y un dominio de homolog&iacute;a (VHD) de VEGF central que contiene sitios de uni&oacute;n del receptor. Clivaje proteol&iacute;tico elimina la propeptides para generar formas maduras, consistiendo en d&iacute;meros del dominio de homolog&iacute;a VEGF, que se unen a receptores con mayor afinidad que las formas del cuerpo entero. Por lo tanto, el proceso proteol&iacute;tico activa VEGF-C y VEGF-D, aunque las proteasas implicadas eran desconocidas. Aqu&iacute;, Divulgamos que la plasmina proteasa de serina troceados ambos propeptides desde el dominio de homolog&iacute;a VEGF de VEGF-D humana y as&iacute; genera una forma madura que exhibe mucho mayor vinculante y cross-linking de VEGFR-2 y VEGFR-3 en comparaci&oacute;n con el material de larga duraci&oacute;n. Plasmina activa tambi&eacute;n VEGF-C. Como factores de crecimiento lymphangiogenic promover la diseminaci&oacute;n metast&aacute;sica de c&aacute;ncer a trav&eacute;s de los vasos linf&aacute;ticos, la activaci&oacute;n proteol&iacute;tica de estas mol&eacute;culas representa un blanco potencial para los agentes antimetast&aacute;ticos. Identificaci&oacute;n de una enzima que activa los factores de crecimiento lymphangiogenic facilitar&aacute; el desarrollo de inhibidores de la met&aacute;stasis.

Plasmina activa los factores de crecimiento lymphangiogenic VEGF-C y VEGF-D.

Vascular endothelial growth factor receptor 3 (Vegfr-3) is a tyrosine kinase that is expressed on the lymphatic endothelium and that signals for the growth of the lymphatic vessels (lymphangiogenesis). Vegf-d, a secreted glycoprotein, is one of two known activating ligands for Vegfr-3, the other being Vegf-c. Vegf-d stimulates lymphangiogenesis in tissues and tumors; however, its role in embryonic development was previously unknown. Here we report the generation and analysis of mutant mice deficient for Vegf-d. Vegf-d-deficient mice were healthy and fertile, had normal body mass, and displayed no pathologic changes consistent with a defect in lymphatic function. The lungs, sites of strong Vegf-d gene expression during embryogenesis in wild-type mice, were normal in Vegf-d-deficient mice with respect to tissue mass and morphology, except that the abundance of the lymphatics adjacent to bronchioles was slightly reduced. Dye uptake experiments indicated that large lymphatics under the skin were present in normal locations and were functional. Smaller dermal lymphatics were similar in number, location, and function to those in wild-type controls. The lack of a profound lymphatic phenotype in Vegf-d-deficient mice suggests that Vegf-d does not play a major role in lymphatic development or that Vegf-c or another, as-yet-unknown activating Vegfr-3 ligand can compensate for Vegf-d during development.

Vascular endothelial growth factor D is dispensable for development of the lymphatic system.

Vascular endothelial growth factor receptor 3 (Vegfr-3) est une kinase de tyrosine qui s&#39;exprime sur l&#39;endoth&eacute;lium lymphatique et que des signaux pour la croissance des vaisseaux lymphatiques (lymphangiogen&egrave;se). VEGF-d, une glycoprot&eacute;ine s&eacute;cr&eacute;t&eacute;e, est l&#39;un des deux ligands activation connus pour Vegfr-3, l&#39;autre &eacute;tant le Vegf-c. VEGF-d stimule la lymphangiogen&egrave;se dans les tissus et les tumeurs ; Cependant, son r&ocirc;le dans le d&eacute;veloppement embryonnaire &eacute;tait inconnue. Ici, nous rapportons la g&eacute;n&eacute;ration et l&#39;analyse des souris mutantes d&eacute;ficientes pour le Vegf-d. Souris d&eacute;ficientes en VEGF-d &eacute;taient en bonne sant&eacute; et fertile, avaient masse corporelle normale et n&#39;affichent aucun changement pathologique coh&eacute;rente avec un d&eacute;faut dans la fonction lymphatique. Les poumons, les sites de forte expression du g&egrave;ne Vegf-d au cours de l&#39;embryogen&egrave;se chez les souris de type sauvage, &eacute;taient normaux chez les souris d&eacute;ficientes en Vegf-d en ce qui concerne les tissus masse et morphologie, sauf que l&#39;abondance des vaisseaux lymphatiques adjacents aux bronchioles a &eacute;t&eacute; l&eacute;g&egrave;rement r&eacute;duite. Exp&eacute;riences d&#39;absorption de teinture a indiqu&eacute; que gros vaisseaux lymphatiques sous la peau &eacute;taient pr&eacute;sents dans des endroits normales &eacute;taient fonctionnels. Petits cutan&eacute;es lymphatics ressemblaient en nombre, l&#39;emplacement et fonction &agrave; ceux dans les contr&ocirc;les de type sauvage. L&#39;absence d&#39;un ph&eacute;notype lymphatique profond chez les souris d&eacute;ficientes en Vegf-d sugg&egrave;re que le Vegf-d ne joue pas un r&ocirc;le majeur dans le d&eacute;veloppement lymphatique ou que Vegf-c ou un autre, comme-encore-inconnu activant Vegfr-3 ligand peut compenser de Vegf-d au cours du d&eacute;veloppement.

Facteur de croissance endoth&eacute;lial vasculaire D est indispensable pour le d&eacute;veloppement du syst&egrave;me lymphatique.

Vascular endothelial growth factor receptor 3 (Vegfr-3) &egrave; una tirosina chinasi che segnali per la crescita dei vasi linfatici (linfangiogenesi) e che si esprime sull&#39;endotelio linfatico. VEGF-d, una glicoproteina secreta, &egrave; uno dei due ligandi attivante noti per Vegfr-3, l&#39;altro &egrave; il Vegf-c. VEGF-d stimola linfangiogenesi nei tessuti e tumori; Tuttavia, il ruolo dello sviluppo embrionale era precedentemente sconosciuto. Qui riportiamo la generazione e l&#39;analisi dei topi mutanti carenti per Vegf-d. VEGF-d-deficient mice erano sani e fertili, avevano massa corporea normale e non visualizzato nessun cambiamento patologico coerenza con un difetto nella funzione linfatica. I polmoni, i siti di forte espressione del gene Vegf-d durante l&#39;embriogenesi nei topi wild-type, erano normali nei topi Vegf-d-carenti rispetto al tessuto massa e morfologia, ad eccezione del fatto che l&#39;abbondanza dei vasi linfatici adiacenti a bronchioli &egrave; stata leggermente ridotta. Esperimenti di assorbimento di colorante indicato che grandi vasi linfatici sotto la pelle erano presenti nelle posizioni normali e sono stati funzionali. Piccoli vasi linfatici dermici erano simili in numero, posizione e funzione a quelli in controlli di tipo selvatico. La mancanza di un fenotipo linfatico profondo nei topi Vegf-d-carenti suggerisce che il Vegf-d non gioca un ruolo importante nello sviluppo linfatico o che Vegf-c o un altro, non ancora sconosciuto ligando Vegfr-3 attivazione pu&ograve; compensare Vegf-d durante lo sviluppo.

Fattore di crescita endoteliale vascolare D &egrave; superfluo per lo sviluppo del sistema linfatico.

Vascular endothelial growth factor (VEGF)-D is a secreted glycoprotein that induces angiogenesis and lymphangiogenesis. It consists of a central domain, containing binding sites for VEGF receptor-2 (VEGFR-2) and VEGFR-3, and N- and C-terminal propeptides. It is secreted from the cell as homodimers of the full-length form that can be proteolytically processed to remove the propeptides. It was recently shown, using adenoviral gene delivery, that fully processed VEGF-D induces angiogenesis in vivo, whereas full-length VEGF-D does not. To better understand these observations, we monitored the effect of VEGF-D processing on receptor binding using a full-length VEGF-D mutant that cannot be processed. This mutant binds VEGFR-2, the receptor signaling for angiogenesis, with approximately 17,000-fold lower affinity than mature VEGF-D, indicating the importance of processing for interaction with this receptor. Further, we show that members of the proprotein convertase (PC) family of proteases promote VEGF-D processing, which facilitates the VEGF-D/VEGFR-2 interaction. The PCs furin and PC5 promote cleavage of both propeptides, whereas PC7 promotes cleavage of the C-terminal propeptide only. The finding that PCs promote activation of VEGF-D and other proteins with roles in cancer such as matrix metalloproteinases, emphasizes the importance of these enzymes as potential regulators of tumor progression and metastasis.

Proprotein convertases promote processing of VEGF-D, a critical step for binding the angiogenic receptor VEGFR-2.

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.

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.

Vascular endothelial growth factor-D (VEGF-D) is a secreted protein that promotes tumor growth and metastatic spread in animal models of cancer. Expression of VEGF-D in prevalent human cancers was reported to correlate with lymph node metastasis and patient outcome-hence, this protein is a potential target for novel anticancer therapeutics designed to restrict tumor growth and spread. Here, we define the binding site in VEGF-D of a neutralizing antibody, designated VD1, which blocks the interaction of VEGF-D with its cell surface receptors vascular endothelial growth factor receptor (VEGFR)-2 and VEGFR-3 and is being used for the development of therapeutic antibodies. We show by peptide-based mapping and site-directed mutagenesis that the VD1 binding site includes the five residues (147)NEESL(151) and that immunization with a synthetic peptide containing this motif generates antibodies that neutralize VEGF-D. The tertiary structure of VEGF-D indicates that the (147)NEESL(151) epitope is located in the L2 loop of the growth factor, which is important for receptor binding. Mutation of any of these five residues influences receptor binding; for example, mutations to E148, which abolished binding to VD1, impaired the interaction with VEGFR-2 but enhanced binding to VEGFR-3. This structure/function study indicates that the VD1 binding epitope is part of the receptor binding site of VEGF-D, identifies a region of VEGF-D critical for binding of receptors and explains why VD1 does not bind other members of the VEGF family of growth factors.

The VD1 neutralizing antibody to vascular endothelial growth factor-D: binding epitope and relationship to receptor binding.

VEGF-D is a mitogen for endothelial cells that promotes tumor growth and metastatic spread in animal models, and expression of which correlates with lymph node metastasis in some human cancers. It is secreted from the cell as a full-length form with propeptides flanking a central region containing binding sites for VEGFR-2 and VEGFR-3, receptors that signal for angiogenesis and lymphangiogenesis. The propeptides can be cleaved from VEGF-D, enhancing affinity for VEGFR-2 and VEGFR-3 in vitro; however, the importance of this processing in cancer is unclear. To explore the necessity of processing for the effects of VEGF-D in cancer, we use a mutant full-length form that cannot be processed, and show that, in contrast to full-length VEGF-D that is processed, this mutant does not promote tumor growth and lymph node metastasis in a mouse tumor model. Processing of VEGF-D is required for tumor angiogenesis, lymphangiogenesis, and recruitment of tumor-associated macrophages. These observations may be explained by the requirement of processing for VEGF-D to bind neuropilin receptors and activate VEGFR-2. Our results indicate that proteolytic processing is necessary for VEGF-D to promote the growth and spread of cancer, and suggest that enzymes catalyzing this processing could be targets for antimetastatic therapeutics.

Proteolytic processing of vascular endothelial growth factor-D is essential for its capacity to promote the growth and spread of cancer.

Vascular endothelial growth factor-D (VEGF-D), a secreted angiogenic and lymphangiogenic glycoprotein, enhances tumor growth and metastasis in animal models, and its expression correlates with metastasis and poor patient outcome in some cancers - it is therefore considered a target for novel anti-cancer therapeutics. The definition of the structure of the complex of VEGF-D bound to its receptors would be beneficial for design of inhibitors of VEGF-D signaling aimed at restricting the growth and spread of cancer. In addition, there is interest in using VEGF-D protein for therapeutic angiogenesis and lymphangiogenesis in the settings of cardiovascular diseases and lymphedema, respectively. However, VEGF-D has proven difficult to express and purify in a highly bioactive form due to a tendency to exist as monomers rather than bioactive dimers. Here we describe a protocol for expression and purification of mature human VEGF-D, and a mutant thereof with reduced glycosylation, potentially suitable for preclinical therapeutic and structural studies, respectively. The degree of glycosylation in mature VEGF-D was reduced by eliminating one of the two N-glycosylation sites, and expressing the protein in Lec3.2.8.1 cells which had reduced glycosylation capacity. Mature VEGF-D and the glycosylation mutant were each enriched for the biologically active dimeric form by optimizing the separation of dimer from monomer via gel filtration, followed by conversion of remaining monomers to dimers via treatment with cysteine. The glycosylation mutant of VEGF-D intended for structural studies preserved all the cysteine residues of mature VEGF-D, in contrast to previous structural studies, exhibited comparable receptor binding to mature VEGF-D and might facilitate structural studies of the VEGF-D/VEGFR-3 complex.

Preparation of human vascular endothelial growth factor-D for structural and preclinical therapeutic studies.

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.

VEGF-D is an angiogenic and lymphangiogenic glycoprotein that can be proteolytically processed generating various forms differing in subunit composition due to the presence or absence of N- and C-terminal propeptides. These propeptides flank the central VEGF homology domain, that contains the binding sites for VEGF receptors (VEGFRs), but their biological functions were unclear. Characterization of propeptide function will be important to clarify which forms of VEGF-D are biologically active and therefore clinically relevant. Here we use VEGF-D mutants deficient in either propeptide, and in the capacity to process the remaining propeptide, to monitor the functions of these domains. We report for the first time that VEGF-D binds heparin, and that the C-terminal propeptide significantly enhances this interaction (removal of this propeptide from full-length VEGF-D completely prevents heparin binding). We also show that removal of either the N- or C-terminal propeptide is required for VEGF-D to drive formation of VEGFR-2/VEGFR-3 heterodimers which have recently been shown to positively regulate angiogenic sprouting. The mature form of VEGF-D, lacking both propeptides, can also promote formation of these receptor heterodimers. In a mouse tumor model, removal of only the C-terminal propeptide from full-length VEGF-D was sufficient to enhance angiogenesis and tumor growth. In contrast, removal of both propeptides is required for high rates of lymph node metastasis. The findings reported here show that the propeptides profoundly influence molecular interactions of VEGF-D with VEGF receptors, co-receptors, and heparin, and its effects on tumor biology.

The propeptides of VEGF-D determine heparin binding, receptor heterodimerization, and effects on tumor biology.

The lymphatic vasculature is important for skin biology as it maintains dermal fluid homeostasis. However, the molecular determinants of the form and function of the lymphatic vasculature in skin are poorly understood. Here, we explore the role of vascular endothelial growth factor-d (Vegf-d), a lymphangiogenic glycoprotein, in determining the form and function of the dermal lymphatic network, using Vegf-d-deficient mice. Initial lymphatic vessels in adult Vegf-d-deficient mice were significantly smaller than wild-type but collecting lymphatics were unaltered. The uptake/transport of dextran in initial lymphatics of Vegf-d-deficient mice was far less efficient, indicating compromised function of these vessels. The role of Vegf-d in modulating initial lymphatics was further supported by delivery of Vegf-d in skin of wild-type mice, which promoted enlargement of these vessels. Vegf-d-deficient mice were subjected to cutaneous wounding to challenge lymphatic function: the resulting wound epithelium was highly edematous and thicker, reflecting inadequate lymphatic drainage. Unexpectedly, myofibroblasts were more abundant in Vegf-d-deficient wounds leading to faster wound closure, but resorption of granulation tissue was compromised suggesting poorer-quality healing. Our findings demonstrate that Vegf-d deficiency alters the caliber of initial lymphatics in the dermis leading to reduced functional capacity.

Vascular endothelial growth factor-d modulates caliber and function of initial lymphatics in the dermis.

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.

Signaling for lymphangiogenesis via VEGFR-3 is required for the early events of metastasis.

Leakage of fluid from blood vessels, leading to oedema, is a key feature of many diseases including hyperoxic acute lung injury (HALI) which can occur when patients are ventilated with high concentrations of oxygen (hyperoxia). The molecular mechanisms driving vascular leak and oedema in HALI are poorly understood. VEGF-D is a protein that promotes blood vessel leak and oedema when overexpressed in tissues, but the role of endogenous VEGF-D in pathological oedema was unknown. To address these issues, we exposed Vegfd-deficient mice to hyperoxia. The resulting pulmonary oedema in Vegfd-deficient mice was substantially reduced compared to wild-type as was the protein content of bronchoalveolar lavage fluid, consistent with reduced vascular leak. VEGF-D and its receptor VEGFR-3 were more highly expressed in lungs of hyperoxic, versus normoxic, wild-type mice indicating that components of the VEGF-D signalling pathway are up-regulated in hyperoxia. Importantly, VEGF-D and its receptors were co-localised on blood vessels in clinical samples of human lungs exposed to hyperoxia, hence VEGF-D may act directly on blood vessels to promote fluid leak. Our studies show that VEGF-D promotes oedema in response to hyperoxia in mice and support the hypothesis that VEGF-D signalling promotes vascular leak in human HALI.

Article	Year
The angiogenic and lymphangiogenic factor vascular endothelial growth factor-D exhibits a paracrine mode of action in cancer. Growth factors (Chur, Switzerland)\| PubMed ID: 12148568	2002
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 mi... The Journal of biological chemistry\| PubMed ID: 12867434	2003
Plasmin activates the lymphangiogenic growth factors VEGF-C and VEGF-D. The Journal of experimental medicine\| PubMed ID: 12963694	2003
Vascular endothelial growth factor D is dispensable for development of the lymphatic system. Molecular and cellular biology\| PubMed ID: 15743836	2005
Proprotein convertases promote processing of VEGF-D, a critical step for binding the angiogenic receptor VEGFR-2. FASEB journal : official publication of the Federation of American Societies for Experimental Biology\| PubMed ID: 17242158	2007
Molecular pathways for lymphangiogenesis and their role in human disease. Novartis Foundation symposium\| PubMed ID: 17534064	2007
The VD1 neutralizing antibody to vascular endothelial growth factor-D: binding epitope and relationship to receptor binding. Journal of molecular biology\| PubMed ID: 21315726	2011
Proteolytic processing of vascular endothelial growth factor-D is essential for its capacity to promote the growth and spread of cancer. FASEB journal : official publication of the Federation of American Societies for Experimental Biology\| PubMed ID: 21515745	2011
Preparation of human vascular endothelial growth factor-D for structural and preclinical therapeutic studies. Protein expression and purification\| PubMed ID: 22261343	2012
VEGF-D promotes tumor metastasis by regulating prostaglandins produced by the collecting lymphatic endothelium. Cancer cell\| PubMed ID: 22340592	2012
The propeptides of VEGF-D determine heparin binding, receptor heterodimerization, and effects on tumor biology. The Journal of biological chemistry\| PubMed ID: 23404505	2013
Vascular endothelial growth factor-d modulates caliber and function of initial lymphatics in the dermis. The Journal of investigative dermatology\| PubMed ID: 23439394	2013
Signaling for lymphangiogenesis via VEGFR-3 is required for the early events of metastasis. Clinical & experimental metastasis\| PubMed ID: 23591595	2013
Vegf-d promotes pulmonary oedema in hyperoxic acute lung injury. The Journal of pathology\| PubMed ID: 26924464	2016

Sally Roufail

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