Functional Nanocomplexes with Vascular Endothelial Growth Factor A/C Isoforms Improve Collateral Circulation and Cardiac Function.

Protein-based therapies are potential treatments for cancer, immunological, and cardiovascular diseases. However, effective delivery systems are needed because of their instability, immunogenicity, and so on. Crosslinked negatively charged heparin polysaccharide nanoparticle (HepNP) is proposed for protein delivery. HepNP can efficiently condense vascular endothelial growth factor (VEGF) because of the unique electronegative sulfonic acid and carboxyl domain of heparin. HepNP is then assembled with VEGF-C (Hep@VEGF-C) or VEGF-A (Hep@VEGF-A) protein for the therapy of myocardial infarction (MI) via intravenous (iv) injection. Hep@VEGF-A-mediated improvement of cardiac function by promoting angiogenesis is limited because of elevated vascular permeability, while Hep@VEGF-C effectively promotes lymphangiogenesis and reduces edema. On this basis, a graded delivery of VEGF-C (0.5-1 h post-MI) and VEGF-A (5 d post-MI) using HepNP is developed. At the dose ratio of 3:1 (Hep@VEGF-C vs Hep@VEGF-A), Hep@VEGF functional complexes substantially reduce the scar formation (≈-39%; p < 0.05) and improve cardiac function (≈+74%; p < 0.05). Such a HepNP delivery system provides a simple and effective therapeutic strategy for cardiovascular diseases by delivering functional proteins. Because of the unique binding ability of heparin with cytokines and growth factors, HepNP also has considerable application prospects in protein therapy for other serious diseases.

Evolutionary Differences in the Vegf/Vegfr Code Reveal Organotypic Roles for the Endothelial Cell Receptor Kdr in Developmental Lymphangiogenesis.

Lymphatic vascular development establishes embryonic and adult tissue fluid balance and is integral in disease. In diverse vertebrate organs, lymphatic vessels display organotypic function and develop in an organ-specific manner. In all settings, developmental lymphangiogenesis is considered driven by vascular endothelial growth factor (VEGF) receptor-3 (VEGFR3), whereas a role for VEGFR2 remains to be fully explored. Here, we define the zebrafish Vegf/Vegfr code in receptor binding studies. We find that while Vegfd directs craniofacial lymphangiogenesis, it binds Kdr (a VEGFR2 homolog) but surprisingly, unlike in mammals, does not bind Flt4 (VEGFR3). Epistatic analyses and characterization of a kdr mutant confirm receptor-binding analyses, demonstrating that Kdr is indispensible for rostral craniofacial lymphangiogenesis, but not caudal trunk lymphangiogenesis, in which Flt4 is central. We further demonstrate an unexpected yet essential role for Kdr in inducing lymphatic endothelial cell fate. This work reveals evolutionary divergence in the Vegf/Vegfr code that uncovers spatially restricted mechanisms of developmental lymphangiogenesis.

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.

Lymphangiogenesis and NOS Localization in Healing Process after Tooth Extraction in Akita Mouse.

Type I diabetes, an autoimmune disease, induces insulin deficiency, which then disrupts vascular endothelial cell function, affecting blood and lymphatic vessels. Nitric oxide (NO) is an immune-induced destructive mediator in type I diabetes, and inhibition of its production promotes arteriosclerosis. In this study, lymphangiogenesis and expression of NO synthase (NOS) during the healing process after tooth extraction were investigated immunohistochemically in control (C57BL) and Akita mice as a diabetes model. Between 1, 4, and 10 days after extraction, expression of NOS, vascular endothelial growth factor-C (VEGF-C), VEGF receptor-3 (VEGFR-3), and von Willebrand factor was strongest during the granulation tissue phase. This suggests that severe inflammation triggers regulation of NOS and these other angiogenic and lymphangiogenic factors. During the callus phase, a few days after extraction, induced osteoblasts were positive for VEGF-C and VEGFR-3 in both the control and Akita mice, suggesting that bone formation is active in this period. Bone formation in the Akita group exceeded that in the controls. Bone tissue formation was disrupted under hyperglycemic conditions, however, suggesting that such activity would be insufficient to produce new bone.

Pathway-related molecules of VEGFC/D-VEGFR3/NRP2 axis in tumor lymphangiogenesis and lymphatic metastasis.

Precondition for tumor lymphatic metastasis is that tumor cells induce formation of original and newborn lymphatic vessels and invade surrounding lymphatic vessels in tumor stroma, while some pathway-related molecules play an important role in mechanisms associated with proliferation and migration of lymphatic endothelial cells (LECs) and tumor cells. In lymphangiogenesis and lymphatic metastasis, the pathway-related molecules of VEGFC/D-VEGFR3/NRP2 axis, such as Furin-like enzyme, CNTN1, Prox1, LYVE-1, Podoplanin, SOX18, SDF1 and CXCR4, are direct constitutors as a portion of VEGFC/D-VEGFR3/NRP2 axis, and their biological activities rely on this ligand-receptor system. These axis-related signal molecules could gradually produce waterfall-like cascading effects, mediate differentiation and maturation of LECs, remodel original and neonatal lymphatic vessels, as well as ultimately promote tumor cell chemotaxis, migration, invasion and metastasis to lymphoid tracts. This review summarizes the structure and function features of pathway-related molecules of VEGFC/D-VEGFR3/NRP2 axis, the expression changes of these molecules in different anatomic organs or histopathologic types or development stages of various tumors, the characteristics of transduction, implementation, integration of signal networks, the interactive effects on biological behaviors between tumor cells and lymphatic endothelial cells, and their molecular mechanisms and significances in tumor lymphangiogenesis and lymphatic metastasis.

Aligned nanofibrillar collagen scaffolds - Guiding lymphangiogenesis for treatment of acquired lymphedema.

Secondary lymphedema is a common disorder associated with acquired functional impairment of the lymphatic system. The goal of this study was to evaluate the therapeutic efficacy of aligned nanofibrillar collagen scaffolds (BioBridge) positioned across the area of lymphatic obstruction in guiding lymphatic regeneration. In a porcine model of acquired lymphedema, animals were treated with BioBridge scaffolds, alone or in conjunction with autologous lymph node transfer as a source of endogenous lymphatic growth factor. They were compared with a surgical control group and a second control group in which the implanted BioBridge was supplemented with exogenous vascular endothelial growth factor-C (VEGF-C). Three months after implantation, immunofluorescence staining of lymphatic vessels demonstrated a significant increase in lymphatic collectors within close proximity to the scaffolds. To quantify the functional impact of scaffold implantation, bioimpedance was used as an early indicator of extracellular fluid accumulation. In comparison to the levels prior to implantation, the bioimpedance ratio was significantly improved only in the experimental BioBridge recipients with or without lymph node transfer, suggesting restoration of functional lymphatic drainage. These results further correlated with quantifiable lymphatic collectors, as visualized by contrast-enhanced computed tomography. They demonstrate the therapeutic potential of BioBridge scaffolds in secondary lymphedema.

Splicing and proteolytic processing in VEGF signaling: now it is the coreceptor's turn.

Alternative splicing and proteolytic processing of VEGFs generate proteins with distinct physiological roles. In this issue of Structure, Parker et al. show that proteolysis of an isoform of the VEGF-C coreceptor Nrp2 produces a soluble receptor that inhibits VEGF-C/Nrp2 interactions.

Structural basis for VEGF-C binding to neuropilin-2 and sequestration by a soluble splice form.

Vascular endothelial growth factor C (VEGF-C) is a potent lymphangiogenic cytokine that signals via the coordinated action of two cell surface receptors, Neuropilin-2 (Nrp2) and VEGFR-3. Diseases associated with both loss and gain of VEGF-C function, lymphedema and cancer, respectively, motivate studies of VEGF-C/Nrp2 binding and inhibition. Here, we demonstrate that VEGF-C binding to Nrp2 is regulated by C-terminal proteolytic maturation. The structure of the VEGF-C C terminus in complex with the ligand binding domains of Nrp2 demonstrates that a cryptic Nrp2 binding motif is released upon proteolysis, allowing specific engagement with the b1 domain of Nrp2. Based on the identified structural requirements for Nrp2 binding to VEGF-C, we hypothesized that the endogenous secreted splice form of Nrp2, s9Nrp2, may function as a selective inhibitor of VEGF-C. We find that s9Nrp2 forms a stable dimer that potently inhibits VEGF-C/Nrp2 binding and cellular signaling. These data provide critical insight into VEGF-C/Nrp2 binding and inhibition.

Dextran-based fluorescent nanoprobes for sentinel lymph node mapping.

Biopsy of sentinel lymph node (SLN) has become a common practice to predict whether tumor metastasis has occurred, so proper SLN positioning tracers are highly required. Due to many drawbacks of SLN tracers currently used, developing ideal, biosafe SLN imaging agents is always an urgent issue. The current study designed a novel fluorescent nanoprobe for accurate SLN mapping. Dextran-based nanogel (DNG) was prepared through a highly efficient self-assembly assisted approach and serves as a multi-functional platform for conjugating wide spectra emitting fluorescent agents. The newly fabricated fluorescent DNG (FDNG) could be designed with optimum size and stable fluorescent intensity for specific SLN imaging. Furthermore, a long-term dynamic course in vivo (from 1 min to 72 h) revealed the satisfactory specificity, sensitivity, and stability for SLN mapping. Most importantly, both in vitro and in vivo evaluations indicated that FDNG had fine biosafety and biocompatibility with lymphatic endothelial cells. All these results supported that FDNG could be used as highly efficient molecular imaging probes for specific, sensitive, stable, non-invasive, and safe SLN mapping, which provides efficient and accurate location for SLN biopsy and thus predicts tumor metastasis as well as directs therapies. Besides, our recent studies further demonstrated that DNG could also serve as a specific and controllable drug carrier, indicating a potential application for specific therapies of various lymph-associated diseases.

Microplate-based screening for small molecule inhibitors of neuropilin-2/vascular endothelial growth factor-C interactions.

Vascular endothelial growth factor-C (VEGF-C) is a secreted growth factor essential for lymphangiogenesis. VEGF-C functions in both physiological and pathological lymphangiogenesis, particularly in tumor metastasis, making it an attractive therapeutic target. Members of two families of cell surface receptors transduce VEGF-C signals: neuropilin-2 (Nrp2) and VEGF-receptor (VEGFR)-2/3. Nrp2 is a promising target for inhibition because it is highly expressed in lymphatic vessels. Here we describe a microplate-based assay for discovery of VEGF-C/Nrp2 inhibitors. We optimize this assay for use in screening an inhibitor library and identify three novel Nrp2/VEGF-C binding inhibitors from the National Institutes of Health (NIH) Clinical Collection small molecule library.

Structural and mechanistic insights into VEGF receptor 3 ligand binding and activation.

Vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are key drivers of blood and lymph vessel formation in development, but also in several pathological processes. VEGF-C signaling through VEGFR-3 promotes lymphangiogenesis, which is a clinically relevant target for treating lymphatic insufficiency and for blocking tumor angiogenesis and metastasis. The extracellular domain of VEGFRs consists of seven Ig homology domains; domains 1-3 (D1-3) are responsible for ligand binding, and the membrane-proximal domains 4-7 (D4-7) are involved in structural rearrangements essential for receptor dimerization and activation. Here we analyzed the crystal structures of VEGF-C in complex with VEGFR-3 domains D1-2 and of the VEGFR-3 D4-5 homodimer. The structures revealed a conserved ligand-binding interface in D2 and a unique mechanism for VEGFR dimerization and activation, with homotypic interactions in D5. Mutation of the conserved residues mediating the D5 interaction (Thr446 and Lys516) and the D7 interaction (Arg737) compromised VEGF-C induced VEGFR-3 activation. A thermodynamic analysis of VEGFR-3 deletion mutants showed that D3, D4-5, and D6-7 all contribute to ligand binding. A structural model of the VEGF-C/VEGFR-3 D1-7 complex derived from small-angle X-ray scattering data is consistent with the homotypic interactions in D5 and D7. Taken together, our data show that ligand-dependent homotypic interactions in D5 and D7 are essential for VEGFR activation, opening promising possibilities for the design of VEGFR-specific drugs.

Characterization of the interaction between endostatin short peptide and VEGF receptor 3.

Corneal angiogenesis and lymphangiogenesis are induced by vascular endothelial growth factors (VEGFs) signaling through its receptors VEGFR-1, -2, and -3. Endostatin is a peptide antagonist of these receptors that causes inhibition of bFGF-induced corneal angiogenesis and lymphangiogenesis. Here we show that binding of VEGF-C and endostatin to recombinant VEGFR-3 is competitive. Alignments of the primary amino acid sequences of VEGF-C and the C-terminal endostatin peptide (mEP: LEQKAASCHNSYIVLCIENSFMTSFSK) identified two conserved cysteine residues separated by seven amino acids. Peptides of VEGF-C and mEP containing these conserved residues bound to VEGFR-3. However, substitution of alanine for either of the cysteines in the mEP peptide perturbed the secondary structure, and this mutated peptide was unable to bind to VEGFR-3. Analysis by surface plasmon resonance demonstrated that the binding of the mEP peptide for recombinant VEGFR-3 had a Ka of 1.41 x 107 M⁻¹ s⁻¹, Kd of 0.6718 s⁻¹, and a KD of 4.78 x 10⁻8 M. Characterization of the mechanism of endostatin binding to VEGFR-3 may lead to the development of novel therapies for lymphangiogenesis-related disorders, such as transplant rejection, lymphedema, and cancer metastasis.

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.

Phage-derived fully human monoclonal antibody fragments to human vascular endothelial growth factor-C block its interaction with VEGF receptor-2 and 3.

Vascular endothelial growth factor C (VEGF-C) is a key mediator of lymphangiogenesis, acting via its receptors VEGF-R2 and VEGF-R3. High expression of VEGF-C in tumors correlates with increased lymphatic vessel density, lymphatic vessel invasion, sentinel lymph node metastasis and poor prognosis. Recently, we found that in a chemically induced skin carcinoma model, increased VEGF-C drainage from the tumor enhanced lymphangiogenesis in the sentinel lymph node and facilitated metastatic spread of cancer cells via the lymphatics. Hence, interference with the VEGF-C/VEGF-R3 axis holds promise to block metastatic spread, as recently shown by use of a neutralizing anti-VEGF-R3 antibody and a soluble VEGF-R3 (VEGF-C/D trap). By antibody phage-display, we have developed a human monoclonal antibody fragment (single-chain Fragment variable, scFv) that binds with high specificity and affinity to the fully processed mature form of human VEGF-C. The scFv binds to an epitope on VEGF-C that is important for receptor binding, since binding of the scFv to VEGF-C dose-dependently inhibits the binding of VEGF-C to VEGF-R2 and VEGF-R3 as shown by BIAcore and ELISA analyses. Interestingly, the variable heavy domain (V(H)) of the anti-VEGF-C scFv, which contains a mutation typical for camelid heavy chain-only antibodies, is sufficient for binding VEGF-C. This reduced the size of the potentially VEGF-C-blocking antibody fragment to only 14.6 kDa. Anti-VEGF-C V(H)-based immunoproteins hold promise to block the lymphangiogenic activity of VEGF-C, which would present a significant advance in inhibiting lymphatic-based metastatic spread of certain cancer types.

Structural determinants of growth factor binding and specificity by VEGF receptor 2.

Vascular endothelial growth factors (VEGFs) regulate blood and lymph vessel formation through activation of three receptor tyrosine kinases, VEGFR-1, -2, and -3. The extracellular domain of VEGF receptors consists of seven immunoglobulin homology domains, which, upon ligand binding, promote receptor dimerization. Dimerization initiates transmembrane signaling, which activates the intracellular tyrosine kinase domain of the receptor. VEGF-C stimulates lymphangiogenesis and contributes to pathological angiogenesis via VEGFR-3. However, proteolytically processed VEGF-C also stimulates VEGFR-2, the predominant transducer of signals required for physiological and pathological angiogenesis. Here we present the crystal structure of VEGF-C bound to the VEGFR-2 high-affinity-binding site, which consists of immunoglobulin homology domains D2 and D3. This structure reveals a symmetrical 22 complex, in which left-handed twisted receptor domains wrap around the 2-fold axis of VEGF-C. In the VEGFs, receptor specificity is determined by an N-terminal alpha helix and three peptide loops. Our structure shows that two of these loops in VEGF-C bind to VEGFR-2 subdomains D2 and D3, while one interacts primarily with D3. Additionally, the N-terminal helix of VEGF-C interacts with D2, and the groove separating the two VEGF-C monomers binds to the D2/D3 linker. VEGF-C, unlike VEGF-A, does not bind VEGFR-1. We therefore created VEGFR-1/VEGFR-2 chimeric proteins to further study receptor specificity. This biochemical analysis, together with our structural data, defined VEGFR-2 residues critical for the binding of VEGF-A and VEGF-C. Our results provide significant insights into the structural features that determine the high affinity and specificity of VEGF/VEGFR interactions.

The comparison of VEGFR-1-binding domain of VEGF-A with modelled VEGF-C sheds light on receptor specificity.

Receptor specificity determines the role of vascular endothelial growth factors (VEGFs), which either induce angiogenesis via VEGFR-1 and VEGFR-2 receptors or lymphangiogenesis via the VEGFR-3 receptor. Among the VEGFs, VEGF-A and VEGF-B predominantly induce angiogenesis while VEGF-C and VEGF-D induce lymphangiogenesis. The answer for the question of why VEGF-C and VEGF-D are not able to bind VEGFR-1 and behave as angiogenic growth factors may hide behind the details of the tertiary structures of these proteins. In the present study, the tertiary structure of human VEGF-C protein was modelled and the model was compared with the known human VEGF-A tertiary structure. In overall, the modelled structure highly resembled the structure of VEGF-A. The respective key residues that are involved in cysteine-knot motif formation in VEGF-A are similarly located and identically oriented in VEGF-C, indicating the presence of a VEGF-A-like homodimer. However, a VEGF-C homodimer created via monomer docking did not superimpose well with the VEGF-A homodimer. Rigid docking models of VEGF-C with the VEGFR-1 receptor revealed that in the VEGF-C-VEGFR-1 complex, the receptor-protein-interacting residues were not correctly oriented to induce angiogenesis via VEGFR-1. Mapping the electrostatic surface potentials to the protein surfaces revealed noteworthy number of dissimilarities between VEGF-A and VEGF-C, indicating that overall both proteins differ in their folding properties and stability.

Enhanced capillary formation stimulated by a chimeric vascular endothelial growth factor/vascular endothelial growth factor-C silk domain fusion protein.

Vascular endothelial growth factor (VEGF)-C and VEGF-D require proteolytic cleavage of the carboxy terminal silk-homology domain for activation. To study the functions of the VEGF-C propeptides, we engineered a chimeric growth factor protein, VEGF-CAC, composed of the amino- and carboxy-terminal propeptides of VEGF-C fused to the receptor-activating core domain of VEGF. Like VEGF-C, VEGF-CAC underwent proteolytic cleavage, and like VEGF, it bound to and activated VEGF receptor-1 and VEGF receptor-2, but not the VEGF-C receptor VEGF receptor-3. VEGF-CAC also bound to neuropilins in a heparin-dependent manner. Strikingly, when VEGF-CAC was expressed via an adenovirus vector in the ear skin of immunodeficient mice, it proved to be a more potent inducer of capillary angiogenesis than VEGF. The VEGF-CAC-induced vessels differed greatly from those induced by VEGF, as they formed a very dense and fine network of pericyte and basement membrane-covered capillaries that were functional, as shown by lectin perfusion experiments. VEGF-CAC could prove useful in proangiogenic therapies in patients experiencing tissue ischemia.

High lymphatic vessel density correlates with overexpression of VEGF-C in gastric cancer.

Lymph node metastasis is one of the most important prognostic factors in malignant tumors. In this study, we investigated vascular endothelial growth factor (VEGF)-C expression in human gastric cancer using immunohistochemical techniques and determined the number of microvessels in peritumoral tissue. VEGF-C expression was positive in 22 of 79 cases (27.8%), and correlated with the presence of lymphatic invasion and lymph node metastasis. We confirmed by reverse transcription-polymerase chain reaction (RT-PCR) that VEGF-C mRNA expression is observed more commonly in cancer tissues than normal tissues. For 59 gastric tumors, we examined lymphatic vessel density (LVD) using the specific lymphatic vessel endothelial hyaluronan receptor (LYVE) -1 antibody. VEGF-C expression was observed in 10 of 25 cases (40%) that exhibited a high LVD. Furthermore, high LVD exhibited a significant correlation with VEGF-C expression. Our findings suggest that VEGF-C plays a pivotal role for lymphangiogenesis and tumor growth in gastric cancer.

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.