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 ...

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.

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.

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.

Plasmin aktiviert die Lymph Wachstumsfaktoren VEGF-C und 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 (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.

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.

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.

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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