Sally Roufail

Biosynthesis of Vascular Endothelial Growth Factor-D Involves Proteolytic Processing Which Generates Non-covalent Homodimers

Vascular endothelial growth factor-D (VEGF-D) binds and activates the endothelial cell tyrosine kinase receptors VEGF receptor-2 (VEGFR-2) and VEGF receptor-3 (VEGFR-3), is mitogenic for endothelial cells, and shares structural homology and receptor specificity with VEGF-C. The primary translation product of VEGF-D has long N- and C-terminal polypeptide extensions in addition to a central VEGF homology domain (VHD). The VHD of VEGF-D is sufficient to bind and activate VEGFR-2 and VEGFR-3. Here we report that VEGF-D is proteolytically processed to release the VHD. Studies in 293EBNA cells demonstrated that VEGF-D undergoes N- and C-terminal cleavage events to produce numerous secreted polypeptides including a fully processed form of M r ∼21,000 consisting only of the VHD, which is predominantly a non-covalent dimer. Biosensor analysis demonstrated that the VHD has ∼290- and ∼40-fold greater affinity for VEGFR-2 and VEGFR-3, respectively, compared with unprocessed VEGF-D. In situ hybridization demonstrated that embryonic lung is a major site of expression of the VEGF-D gene. Processed forms of VEGF-D were detected in embryonic lung indicating that VEGF-D is proteolytically processed in vivo.

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

ABSTRACT 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.

VEGF-D Promotes Tumor Metastasis by Regulating Prostaglandins Produced by the Collecting Lymphatic Endothelium

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.

Plasmin activates the lymphangiogenic growth factors VEGF-C and 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.

Localization of vascular endothelial growth factor-D in malignant melanoma suggests a role in tumour angiogenesis

Expression of angiogenic and lymphangiogenic factors by tumours may influence the route of metastatic spread. Vascular endothelial growth factor (VEGF) is a regulator of tumour angiogenesis, but studies of the inhibition of solid tumour growth by neutralizing anti‐VEGF antibodies indicated that other angiogenic factors may be involved. VEGF‐D may be an alternative regulator because like VEGF it is angiogenic and it activates VEGF receptor‐2 (VEGFR‐2), an endothelial cell receptor which is a key signalling molecule in tumour angiogenesis. This study reports the generation of monoclonal antibodies to the receptor‐binding domain of VEGF‐D and the use of these antibodies to localize VEGF‐D in malignant melanoma. VEGF‐D was detected in tumour cells and in vessels adjacent to immunopositive tumour cells, but not in vessels distant from the tumours. These findings are consistent with a model in which VEGF‐D, secreted by tumour cells, activates endothelial cell receptors and thereby contributes to the regulation of tumour angiogenesis and possibly lymphangiogenesis. In addition, VEGF‐D was detected in the vascular smooth muscle, but not the endothelium, of vessels in adult colon. The endothelium of these vessels was negative for VEGFR‐2 and VEGFR‐3. As VEGF receptors can be up‐regulated on endothelium in response to vessel damage and ischaemia, these findings of a specific localization of VEGF‐D in smooth muscle of the blood vessels suggest that VEGF‐D produced by vascular smooth muscle could play a role in vascular repair by stimulating the proliferation of endothelial cells. Copyright

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

Vascular endothelial growth factor (VEGF)‐D is a secreted glycoprotein that induces angio‐genesis 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 propep‐tides. 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 ~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.—McColl, B. K., Paavonen, K., Karnezis, T., Harris, N. C., Davydova, N., Rothacker, J., Nice, E. C., Harder, K. W., Roufail, S., Hibbs, M. L., Rogers, P. A. W., Alitalo, K., Stacker, S. A., Achen, M. G. Proprotein convertases promote processing of VEGF‐D, a critical step for binding the angiogenic receptor VEGFR‐2. FASEB J. 21, 1088–1098 (2007)

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

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.

The Angiogenic and Lymphangiogenic Factor Vascular Endothelial Growth Factor-D Exhibits a Paracrine Mode of Action in Cancer

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.

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

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.—Harris, N. C., Paavonen, K., Davydova, N., Roufail, S., Sato, T., Zhang, Y. ‐F., Karnezis, T., Stacker, S. A., Achen, M. G. Proteolytic processing of vascular endothelial growth factor‐D is essential for its capacity to promote the growth and spread of cancer. FASEB J. 25, 2615–2625 (2011). www.fasebj.org

Vascular Endothelial Growth Factor-d Modulates Caliber and Function of Initial Lymphatics in the Dermis

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.