PDGF-D Prodomain Differentially Inhibits the Biological Activities of PDGF-D and PDGF-B.

As a member of PDGF/VEGF (Platelet-derived growth factor/ Vascular endothelial growth factor) growth factors, PDGF-D regulates blood vessel development, wound healing, innate immunity, and organogenesis. Unlike PDGF-A and PDGF-B, PDGF-D has an additional CUB (Complement C1r/C1s, Uegf, Bmp1) domain at the N-terminus of its growth factor domain, and thus it is secreted in a latent, inactive complex, which needs to be proteolytically activated for its biological activities. However, how the CUB domain contributes to the latency and activation of the growth factor remains elusive. In this study, we modeled the dimeric structure of PDGF-D pro-complex and studied the inhibitory functions of PDGF-D prodomain on PDGF-B and PDGF-D signaling. In our model, the growth factor domain of PDGF-D forms a VEGF-D-like dimer through their ß1 and ß3 interactions. The hinge and CUB domains of PDGF-D bind at the opposite sides of the growth factor domain and exclude the PDGFR-ß (PDGF Receptor ß) D2 and D3 domains from recognizing the growth factor. In addition, we verified that PDGF-D prodomain could inhibit both PDGF-B and PDGF-D mediated PDGFR-ß transphosphorylation in a dose-dependent manner. However, PDGF-D prodomain could only inhibit the proliferation of NIH 3T3 cells stimulated by PDGF-D but not by PDGF-B, indicating its differential inhibitory activities toward PDGF-B and PDGF-D signaling.

Differential Receptor Binding and Regulatory Mechanisms for the Lymphangiogenic Growth Factors Vascular Endothelial Growth Factor (VEGF)-C and -D.

VEGF-C and VEGF-D are secreted glycoproteins that induce angiogenesis and lymphangiogenesis in cancer, thereby promoting tumor growth and spread. They exhibit structural homology and activate VEGFR-2 and VEGFR-3, receptors on endothelial cells that signal for growth of blood vessels and lymphatics. VEGF-C and VEGF-D were thought to exhibit similar bioactivities, yet recent studies indicated distinct signaling mechanisms (e.g. tumor-derived VEGF-C promoted expression of the prostaglandin biosynthetic enzyme COX-2 in lymphatics, a response thought to facilitate metastasis via the lymphatic vasculature, whereas VEGF-D did not). Here we explore the basis of the distinct bioactivities of VEGF-D using a neutralizing antibody, peptide mapping, and mutagenesis to demonstrate that the N-terminal α-helix of mature VEGF-D (Phe93-Arg108) is critical for binding VEGFR-2 and VEGFR-3. Importantly, the N-terminal part of this α-helix, from Phe93 to Thr98, is required for binding VEGFR-3 but not VEGFR-2. Surprisingly, the corresponding part of the α-helix in mature VEGF-C did not influence binding to either VEGFR-2 or VEGFR-3, indicating distinct determinants of receptor binding by these growth factors. A variant of mature VEGF-D harboring a mutation in the N-terminal α-helix, D103A, exhibited enhanced potency for activating VEGFR-3, was able to promote increased COX-2 mRNA levels in lymphatic endothelial cells, and had enhanced capacity to induce lymphatic sprouting in vivo This mutant may be useful for developing protein-based therapeutics to drive lymphangiogenesis in clinical settings, such as lymphedema. Our studies shed light on the VEGF-D structure/function relationship and provide a basis for understanding functional differences compared with VEGF-C.

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

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.

Vascular endothelial growth factor-D: signaling mechanisms, biology, and clinical relevance.

Vascular endothelial growth factor-D (VEGF-D) is a secreted glycoprotein that promotes growth of blood vessels (angiogenesis) and lymphatic vessels (lymphangiogenesis), and can induce remodeling of large lymphatics. VEGF-D enhances solid tumor growth and metastatic spread in animal models of cancer, and in some human cancers VEGF-D correlates with metastatic spread, poor patient outcome, and, potentially, with resistance to anti-angiogenic drugs. Hence, VEGF-D signaling is a potential target for novel anti-cancer therapeutics designed to enhance anti-angiogenic approaches and to restrict metastasis. In the cardiovascular system, delivery of VEGF-D in animal models enhanced angiogenesis and tissue perfusion, findings which have led to a range of clinical trials testing this protein for therapeutic angiogenesis in cardiovascular diseases. Despite these experimental and clinical developments, our knowledge of the signaling mechanisms driven by VEGF-D is still evolving--here we explore the biology of VEGF-D, its signaling mechanisms, and the clinical relevance of this growth factor.

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

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.

Structural determinants of vascular endothelial growth factor-D receptor binding and specificity.

Vascular endothelial growth factors (VEGFs) and their tyrosine kinase receptors (VEGFR-1-3) are central mediators of angiogenesis and lymphangiogenesis. VEGFR-3 ligands VEGF-C and VEGF-D are produced as precursor proteins with long N- and C-terminal propeptides and show enhanced VEGFR-2 and VEGFR-3 binding on proteolytic removal of the propeptides. Two different proteolytic cleavage sites have been reported in the VEGF-D N-terminus. We report here the crystal structure of the human VEGF-D Cys117Ala mutant at 2.9 Å resolution. Comparison of the VEGF-D and VEGF-C structures shows similar extended N-terminal helices, conserved overall folds, and VEGFR-2 interacting residues. Consistent with this, the affinity and the thermodynamic parameters for VEGFR-2 binding are very similar. In comparison with VEGF-C structures, however, the VEGF-D N-terminal helix was extended by 2 more turns because of a better resolution. Both receptor binding and functional assays of N-terminally truncated VEGF-D polypeptides indicated that the residues between the reported proteolytic cleavage sites are important for VEGF-D binding and activation of VEGFR-3, but not of VEGFR-2. Thus, we define here a VEGFR-2-specific form of VEGF-D that is angiogenic but not lymphangiogenic. These results provide important new insights into VEGF-D structure and function.

Novel vascular endothelial growth factor D variants with increased biological activity.

Members of the vascular endothelial growth factor (VEGF) family play a pivotal role in angiogenesis and lymphangiogenesis. They are potential therapeutics to induce blood vessel formation in myocardium and skeletal muscle, when normal blood flow is compromised. Most members of the VEGF/platelet derived growth factor protein superfamily exist as covalently bound antiparallel dimers. However, the mature form of VEGF-D (VEGF-D(DeltaNDeltaC)) is predominantly a non-covalent dimer even though the cysteine residues (Cys-44 and Cys-53) forming the intersubunit disulfide bridges in the other members of the VEGF family are also conserved in VEGF-D. Moreover, VEGF-D bears an additional cysteine residue (Cys-25) at the subunit interface. Guided by our model of VEGF-D(DeltaNDeltaC), the cysteines at the subunit interface were mutated to study the effect of these residues on the structural and functional properties of VEGF-D(DeltaNDeltaC). The conserved cysteines Cys-44 and Cys-53 were found to be essential for the function of VEGF-D(DeltaNDeltaC). More importantly, the substitution of the Cys-25 at the dimer interface by various amino acids improved the activity of the recombinant VEGF-D(DeltaNDeltaC) and increased the dimer to monomer ratio. Specifically, substitutions to hydrophobic amino acids Ile, Leu, and Val, equivalent to those found in other VEGFs, most favorably affected the activity of the recombinant VEGF-D(DeltaNDeltaC). The increased activity of these mutants was mainly due to stabilization of the protein. This study enables us to better understand the structural determinants controlling the biological activity of VEGF-D. The novel variants of VEGF-D(DeltaNDeltaC) described here are potential agents for therapeutic applications, where induction of vascular formation is required.

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 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.

Heterodimerization with vascular endothelial growth factor receptor-2 (VEGFR-2) is necessary for VEGFR-3 activity.

VEGFR-3 is essential for vascular development and maintenance of lymphatic vessel's integrity. Little is known about its cooperative effect with other receptors of the same family. Contrary to VEGFR-2, stimulation of VEGFR-3 by VEGF-C and -D failed to enhance its phosphorylation either in HEK293T or in PAE cells. These ligands were unable to induce angiogenesis of PAEC expressing VEGFR-3 alone. In the presence of VEGFR-2, VEGF-C and -D induced heterodimerization of VEGFR-3 with VEGFR-2. This heterodimerization was associated with enhanced VEGFR-3 phosphorylation and subsequent cellular responses as evidenced by the formation of capillary-like structures in PAE cells and proliferation of primary human endothelial cells expressing both receptors. Taken together, these results show for the first time that VEGFR-3 needs to be associated to VEGFR-2 to induce ligand-dependent cellular responses.