"Click" conjugation of peptide on the surface of polymeric nanoparticles for targeting tumor angiogenesis.

PURPOSE: Angiogenesis plays a critical role in tumor growth. This phenomena is regulated by numerous mediators such as vascular endothelial growth factor (VEGF). CBO-P11, a cyclo-peptide, has proven to specifically bind to receptors of VEGF and may be used as targeting ligand for tumor angiogenesis. We herein report the design of novel nanoparticles conjugated to CBO-P11 in order to specifically target tumor site. METHODS: The conjugation of CBO-P11 on the surface of poly(vinylidene fluoride) (PVDF) nanoparticles was investigated using the copper(I)-catalyzed Huisgen 1,3-dipolar cycloaddition known as "click" reaction. CBO-P11 was modified with a near-infrared cyanine dye bearing an alkyne function, allowing both "click" coupling on azido-modified nanoparticles and fluorescence labelling. Each step of this nanodevice construction was judiciously performed in aqueous solution and successfully characterized. The cytotoxicity of nanoparticles was evaluated in human brain endothelial cell line and their affinity for VEGF receptors was determined via fluorescence-based uptake assays on porcine aortic endothelial cell line. RESULTS: Nanoparticles were found to be spherical, dense, monodisperse and stable. No cytotoxicity was observed after four days of incubation demonstrating the biocompatibility of nanoparticles. Fluorescence highlighted the specific interaction of these functionalized nanoparticles for VEGF receptors, suggesting that the targeting peptide bioactivity was retained. CONCLUSIONS: These results demonstrate the potential of these functionalized nanoparticles for targeting tumor angiogenesis and their possible use as multifunctional platform for cancer treatment if coupled with therapeutic agents.

Structure and interdomain interactions of a hybrid domain: a disulphide-rich module of the fibrillin/LTBP superfamily of matrix proteins.

The fibrillins and latent transforming growth factor-beta binding proteins (LTBPs) form a superfamily of structurally-related proteins consisting of calcium-binding epidermal growth factor-like (cbEGF) domains interspersed with 8-cysteine-containing transforming growth factor beta-binding protein-like (TB) and hybrid (hyb) domains. Fibrillins are the major components of the extracellular 10-12 nm diameter microfibrils, which mediate a variety of cell-matrix interactions. Here we present the crystal structure of a fibrillin-1 cbEGF9-hyb2-cbEGF10 fragment, solved to 1.8 A resolution. The hybrid domain fold is similar, but not identical, to the TB domain fold seen in previous fibrillin-1 and LTBP-1 fragments. Pairwise interactions with neighboring cbEGF domains demonstrate extensive interfaces, with the hyb2-cbEGF10 interface dependent on Ca(2+) binding. These observations provide accurate constraints for models of fibrillin organization within the 10-12 nm microfibrils and provide further molecular insights into how Ca(2+) binding influences the intermolecular interactions and biomechanical properties of fibrillin-1.

Mouse ten-m/Odz is a new family of dimeric type II transmembrane proteins expressed in many tissues.

The Drosophila gene ten-m/odz is the only pair rule gene identified to date which is not a transcription factor. In an attempt to analyze the structure and the function of ten-m/odz in mouse, we isolated four murine ten-m cDNAs which code for proteins of 2,700-2, 800 amino acids. All four proteins (Ten-m1-4) lack signal peptides at the NH2 terminus, but contain a short hydrophobic domain characteristic of transmembrane proteins, 300-400 amino acids after the NH2 terminus. About 200 amino acids COOH-terminal to this hydrophobic region are eight consecutive EGF-like domains. Cell transfection, biochemical, and electronmicroscopic studies suggest that Ten-m1 is a dimeric type II transmembrane protein. Expression of fusion proteins composed of the NH2-terminal and hydrophobic domain of ten-m1 attached to the alkaline phosphatase reporter gene resulted in membrane-associated staining of the alkaline phosphatase. Electronmicroscopic and electrophoretic analysis of a secreted form of the extracellular domain of Ten-m1 showed that Ten-m1 is a disulfide-linked dimer and that the dimerization is mediated by EGF-like modules 2 and 5 which contain an odd number of cysteines. Northern blot and immunohistochemical analyses revealed widespread expression of mouse ten-m genes, with most prominent expression in brain. All four ten-m genes can be expressed in variously spliced mRNA isoforms. The extracellular domain of Ten-m1 fused to an alkaline phosphatase reporter bound to specific regions in many tissues which were partially overlapping with the Ten-m1 immunostaining. Far Western assays and electronmicroscopy demonstrated that Ten-m1 can bind to itself.

Expression and purification of recombinant human EGFL7 protein.

The secreted epidermal growth factor-like protein 7 (EGFL7) plays an important role in angiogenesis, especially in the recruitment of endothelial and smooth muscle cells to the site of the nascent vessel and their ordered assembly into functional vasculature. However, progress in the understanding of the underlying mechanisms is to date greatly hindered by the lack of recombinant EGFL7 protein in a stable, soluble, native state, thus preventing e.g. the characterization of the proposed functional receptor as well as investigation of additional biological effects of EGFL7. So far all attempts to produce sufficient amounts of recombinant EGFL7 protein by various groups have failed. In this study we describe a procedure for the expression and purification of human EGFL7 from Sf9 cells and for the first time provide means to isolate biologically functional EGFL7 protein in sufficient quantities for its further biological characterization. We believe that the availability of EGFL7 will greatly accelerate our understanding of the precise role of EGFL7 and the underlying molecular mechanisms of EGFL7 action in the fundamentally important process of angiogenesis.

Molecular mechanisms of blood vessel formation.

The formation of new blood vessels, angiogenesis, is a tightly regulated process. Extracellular angiogenic inducers stimulate the migration and proliferation of endothelial cells, while negative regulators counteract this effect. Changes in the relative balance of inducers and inhibitors activate the 'angiogenic switch', before stabilizer molecules activate the maturation of nascent blood vessels.

Endothelial growth factor receptor-targeted and reactive oxygen species-responsive lung cancer therapy by docetaxel and resveratrol encapsulated lipid-polymer hybrid nanoparticles.

Special targeted therapy like endothelial growth factor receptor (EGFR) targeted therapy is available for the treatment of advanced non-small cell lung cancer (NSCLC). Biodegradable core-shell lipid-polymer hybrid nanoparticles (LPNs) can combine the beneficial properties of lipid and polymeric NPs for controlled drug delivery. In the present study, epidermal growth factor (EGF) conjugated LPNs were fabricated to co-deliver docetaxel (DTX) and resveratrol (RSV). In vitro and in vivo studies demonstrated that EGF DTX/RSV LPNs have significant synergistic effects, best tumor inhibition ability and the lowest systemic toxicity. The results indicate that EGF DTX/RSV LPNs may be a promising strategy for treatment of NSCLC.

Isoforms of vascular endothelial growth factor act in a coordinate fashion To recruit and expand tumor vasculature.

Vascular endothelial growth factor (VEGF) is an essential regulator of vascularization. It is expressed as several splice variants; the major forms contain 120 amino acids, 164 amino acids, and 188 amino acids. We utilized transformed cells nullizygous for VEGF to specifically express each of these isoforms in isolation, in order to determine the role of each in tumorigenic neo-vascularization. We found that only the intermediate isoform, VEGF164, could fully rescue tumor growth; VEGF120 partially rescued tumor growth, and VEGF188 failed completely to rescue tumor expansion. Surprisingly, the vascular density of VEGF188 isoform-expressing tumors is significantly greater than that of wild-type VEGF cells and the other isoform-specific tumors. The failure of the hypervascular VEGF188-expressing tumors to grow may be due to inadequate perfusion of the massive number of microvessels in these tumors; three-dimensional imaging of the tumorigenic vasculature indicated little or no recruitment of the peripheral vasculature. This demonstrates that the VEGF isoforms perform unique functions which together enable tumorigenic vascularization.

Regulation of Notch1 and Dll4 by vascular endothelial growth factor in arterial endothelial cells: implications for modulating arteriogenesis and angiogenesis.

Notch and its ligands play critical roles in cell fate determination. Expression of Notch and ligand in vascular endothelium and defects in vascular phenotypes of targeted mutants in the Notch pathway have suggested a critical role for Notch signaling in vasculogenesis and angiogenesis. However, the angiogenic signaling that controls Notch and ligand gene expression is unknown. We show here that vascular endothelial growth factor (VEGF) but not basic fibroblast growth factor can induce gene expression of Notch1 and its ligand, Delta-like 4 (Dll4), in human arterial endothelial cells. The VEGF-induced specific signaling is mediated through VEGF receptors 1 and 2 and is transmitted via the phosphatidylinositol 3-kinase/Akt pathway but is independent of mitogen-activated protein kinase and Src tyrosine kinase. Constitutive activation of Notch signaling stabilizes network formation of endothelial cells on Matrigel and enhances formation of vessel-like structures in a three-dimensional angiogenesis model, whereas blocking Notch signaling can partially inhibit network formation. This study provides the first evidence for regulation of Notch/Delta gene expression by an angiogenic growth factor and insight into the critical role of Notch signaling in arteriogenesis and angiogenesis.

Vascular endothelial growth factor and its receptors.

Vascular endothelial growth factor (VEGF) is a prime regulator of endothelial cell proliferation, angiogenesis, vasculogenesis and vascular permeability. Its activity is mediated by the high affinity tyrosine kinase receptors, KDR/Fik-1 and Fit-1. In this article, recently discovered structural, molecular and biological properties of VEGF are described. Among the topics discussed are VEGF and VEGF receptor structure and bioactivity, the regulation of VEGF expression, the role of VEGF and its receptors in vascular development, and the involvement of VEGF and its receptors in normal and pathological (ocular and tumor) angiogenesis.

Novel vascular endothelial growth factor binding domains of fibronectin enhance vascular endothelial growth factor biological activity.

Interactions between integrins and growth factor receptors play a critical role in the development and healing of the vasculature. This study mapped two binding domains on fibronectin (FN) that modulate the activity of the angiogenic factor, vascular endothelial growth factor (VEGF). Using solid-phase assays and surface plasmon resonance analysis, we identified two novel VEGF binding domains within the N- and C-terminus of the FN molecule. Native FN bound to VEGF enhanced endothelial cell migration and mitogen-activated protein (MAP) kinase activity, but FN that is devoid of the VEGF binding domains failed to do so. Coprecipitation studies confirmed a direct physical association between VEGF receptor-2 (Flk-1) and the FN integrin, alpha5beta1, which required intact FN because FN fragments lacking the VEGF binding domains failed to support receptor association. Thrombin-activated platelets released intact VEGF/FN complexes, which stimulated endothelial cell migration and could be inhibited by soluble high affinity VEGF receptor 1 and antibodies to alpha5beta1 integrin. This study demonstrates that FN is potentially a physiological cofactor for VEGF and provides insights into mechanisms by which growth factor receptors and integrins cooperate to influence cellular behavior.

Solution structure of the heparin-binding domain of vascular endothelial growth factor.

BACKGROUND: Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen and is a potent angiogenic and vascular permeabilizing factor. VEGF is also an important mediator of pathological angiogenesis associated with cancer, rheumatoid arthritis and proliferative retinopathy. The binding of VEGF to its two known receptors, KDR and Flt-1, is modulated by cell-surface-associated heparin-like glycosaminoglycans and exogenous heparin or heparan sulfate. Heparin binding to VEGF165, the most abundantly expressed isoform of VEGF, has been localized to the carboxy-terminal 55 residues; plasmin cleavage of VEGF165 yields a homodimeric 110-residue amino-terminal receptor-binding domain (VEGF110) and two 55-residue carboxy-terminal heparin-binding fragments. The endothelial cell mitogenic potency of VEGF110 is decreased significantly relative to VEGF165, indicating that the heparin-binding domains are critical for stimulating endothelial cell proliferation. RESULTS: The solution structure of the 55-residue heparin-binding domain of VEGF165 has been solved using data from two-dimensional homonuclear and three-dimensional heteronuclear NMR spectroscopy. The structure has two subdomains, each containing two disulfide bridges and a short two-stranded antiparallel beta sheet; the carboxy-terminal subdomain also contains a short alpha helix. Hydrophobic interactions are limited to sidechains packing against the disulfide bridges. CONCLUSIONS: The heparin-binding domain of VEGF has no significant sequence or structural similarity to any known proteins and thus represents a novel heparin-binding domain. Most of the positively charged amino acid sidechains are localized on one side of the carboxy-terminal subdomain or on an adjacent disordered loop in the amino-terminal subdomain. The observed distribution of surface charges suggests that these residues constitute a heparin interaction site.

The crystal structure of vascular endothelial growth factor (VEGF) refined to 1.93 A resolution: multiple copy flexibility and receptor binding.

BACKGROUND: Vascular endothelial growth factor (VEGF) is an endothelial cell-specific angiogenic and vasculogenic mitogen. VEGF also plays a role in pathogenic vascularization which is associated with a number of clinical disorders, including cancer and rheumatoid arthritis. The development of VEGF antagonists, which prevent the interaction of VEGF with its receptor, may be important for the treatment of such disorders. VEGF is a homodimeric member of the cystine knot growth factor superfamily, showing greatest similarity to platelet-derived growth factor (PDGF). VEGF binds to two different tyrosine kinase receptors, kinase domain receptor (KDR) and Fms-like tyrosine kinase 1 (Flt-1), and a number of VEGF homologs are known with distinct patterns of specificity for these same receptors. The structure of VEGF will help define the location of the receptor-binding site, and shed light on the differences in specificity and cross-reactivity among the VEGF homologs. RESULTS: We have determined the crystal structure of the receptor-binding domain of VEGF at 1.93 A resolution in a triclinic space group containing eight monomers in the asymmetric unit. Superposition of the eight copies of VEGF shows that the beta-sheet core regions of the monomers are very similar, with slightly greater differences in most loop regions. For one loop, the different copies represent different snapshots of a concerted motion. Mutagenesis mapping shows that this loop is part of the receptor-binding site of VEGF. CONCLUSIONS: A comparison of the eight independent copies of VEGF in the asymmetric unit indicates the conformational space sampled by the protein in solution; the root mean square differences observed are similar to those seen in ensembles of the highest precision NMR structures. Mapping the receptor-binding determinants on a multiple sequence alignment of VEGF homologs, suggests the differences in specificity towards KDR and Flt-1 may derive from both sequence variation and changes in the flexibility of binding loops. The structure can also be used to predict possible receptor-binding determinants for related cystine knot growth factors, such as PDGF.

VEGF and the Fab fragment of a humanized neutralizing antibody: crystal structure of the complex at 2.4 A resolution and mutational analysis of the interface.

BACKGROUND: Vascular endothelial growth factor (VEGF) is a highly specific angiogenic growth factor; anti-angiogenic treatment through inhibition of receptor activation by VEGF might have important therapeutic applications in diseases such as diabetic retinopathy and cancer. A neutralizing anti-VEGF antibody shown to suppress tumor growth in an in vivo murine model has been used as the basis for production of a humanized version. RESULTS: We present the crystal structure of the complex between VEGF and the Fab fragment of this humanized antibody, as well as a comprehensive alanine-scanning analysis of the contact residues on both sides of the interface. Although the VEGF residues critical for antibody binding are distinct from those important for high-affinity receptor binding, they occupy a common region on VEGF, demonstrating that the neutralizing effect of antibody binding results from steric blocking of VEGF-receptor interactions. Of the residues buried in the VEGF-Fab interface, only a small number are critical for high-affinity binding; the essential VEGF residues interact with those of the Fab fragment, generating a remarkable functional complementarity at the interface. CONCLUSIONS: Our findings suggest that the character of antigen-antibody interfaces is similar to that of other protein-protein interfaces, such as ligand-receptor interactions; in the case of VEGF, the principal difference is that the residues essential for binding to the Fab fragment are concentrated in one continuous segment of polypeptide chain, whereas those essential for binding to the receptor are distributed over four different segments and span across the dimer interface.

Translational control of gene expression: role of IRESs and consequences for cell transformation and angiogenesis.

Translational control of gene expression has, over the last 10 years, become appreciated as an important process in its regulation in eukaryotes. Among a series of control mechanisms exerted at the translational level, the use of alternative codons provides a very subtle means of increasing gene diversity by expressing several proteins from a single mRNA. The internal ribosome entry sites (IRESs) act as specific translational enhancers that allow translation initiation to occur independently of the classic cap-dependent mechanism, in response to specific stimuli and under the control of different trans-acting factors. It is striking to observe that the two processes mostly concern genes coding for control proteins such as growth factors, protooncogenes, angiogenesis factors, and apoptosis regulators. Here, we focus on the translational regulation of four mRNAs, with both IRESs and alternative initiation codons, which are the messengers of retroviral murine leukemia virus, fibroblast growth factor 2, vascular endothelial growth factor, and protooncogene c-myc. Four of them are involved in cell transformation and/or angiogenesis, with important consequences for such translation regulations in these pathophysiological processes.

Pathogenesis of ascites tumor growth: vascular permeability factor, vascular hyperpermeability, and ascites fluid accumulation.

Previous studies have shown that accumulation of tumor ascites fluid results in large part from increased permeability of peritoneal lining vessels (Nagy et al., Cancer Res., 49: 5449-5458, 1989; Nagy et al., Cancer Res., 53: 2631-2643, 1993). However, the specific microvessels rendered hyperpermeable have not been identified nor has the basis of peritoneal vascular hyperpermeability been established. To address these questions, TA3/St and MOT carcinomas, well-characterized transplantable murine tumors that grow in both solid and ascites form, were studied as model systems. Ascites tumor cells of either type were injected i.p. into syngeneic A/Jax and C3Heb/FeJ mice, and ascites fluid and plasma were collected at intervals thereafter up to 8 and 28 days, respectively. Beginning several days after tumor cell injection, small blood vessels located in tissues lining the peritoneal cavity (mesentery, peritoneal wall, and diaphragm) became hyperpermeable to several macromolecular tracers (125I-human serum albumin, FITC-dextran, colloidal carbon, and Monastral Blue B). Increased microvascular permeability correlated with the appearance in ascites fluid of vascular permeability factor (VPF), a tumor cell-secreted mediator that potently enhances vascular permeability to circulating macromolecules. VPF was measured in peritoneal fluid by both a functional bioassay and a sensitive immunofluorometric assay. The VPF concentration, total peritoneal VPF, ascites fluid volume, tumor cell number, and hyperpermeability of peritoneal lining microvessels were found to increase in parallel over time. The close correlation of peritoneal fluid VPF concentration with the development of hyperpermeable peritoneal microvessels in these two well-defined ascites tumors suggests that VPF secretion by tumor cells is responsible, in whole or in part, for initiating and maintaining the ascites pattern of tumor growth.

Sequencing of the human vascular endothelial growth factor (VEGF) 3' untranslated region (UTR): conservation of five hypoxia-inducible RNA-protein binding sites.

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor whose mRNA expression is induced by hypoxia. This induction is due in large part to an increase in the stability of its mRNA. The RNA sequences and cognate proteins responsible for this increased stability with hypoxia are not well understood. In order to identify regions of functional importance in the 3'UTR of VEGF mRNA, we have sequenced the human VEGF 3'UTR and compared it to the rat sequence. Overall sequence homology was 82% with complete conservation of all four potential polyadenylation signals and both nonameric instability elements. Five hypoxia-inducible RNA protein-binding (HI-RPB) sites were identified by RNA electromobility shift assay (EMSA) in the human and rat genes. EMSA and competition studies suggest that these sites bind a similar or related protein complex. On average, the five sites were 95% conserved at the nucleotide level between the rat and corresponding human sequence. This conservation taken together with several previously described, independent correlations between the presence of these RNA-protein complexes and an increase in VEGF mRNA stability suggest an important functional role for these sites in mediating hypoxia-inducible VEGF mRNA stability.

Structural requirements for dimerization, glycosylation, secretion, and biological function of VPF/VEGF.

Vascular permeability factor (VPF) also known as vascular endothelial growth factor (VEGF), is a dimeric protein that affects endothelial cell (EC) and vascular functions including enhancement of microvascular permeability and stimulation of EC growth. To investigate the structural features of VPF/VEGF necessary for efficient dimerization, secretion, and biological activities, we employed site-directed mutagenesis with a Cos-1 cell expression system. Several cysteine residues essential for VPF dimerization were identified by mutation analysis of the Cys-25, Cys-56, and Cys-67 residues. Mutant VPF isoforms lacking either of these cysteines were secreted as monomers and were completely inactive in both vascular permeability and endothelial cell mitotic assays. VPF Cys-145 mutant protein was efficiently secreted as a glycosylated, dimeric polypeptide, but had a reduction in biological activities. The site of N-linked glycosylation was directly identified as Asn-74, which, when mutated produced an inefficiently secreted dimeric protein without post-translational glycosylation, yet maintained full vascular permeability activity. Finally, we found that one VPF mutant isoform Cys-101 was not secreted and this mutant functioned as a dominant-negative suppressor of wild-type VPF secretion as demonstrated by co-expression assays in Cos-1 cells.

Structural analysis of receptor tyrosine kinases.

Receptor tyrosine kinases (RTKs) are single-pass transmembrane receptors that possess intrinsic cytoplasmic enzymatic activity, catalyzing the transfer of the gamma-phosphate of ATP to tyrosine residues in protein substrates. RTKs are essential components of signal transduction pathways that affect cell proliferation, differentiation, migration and metabolism. Included in this large protein family are the insulin receptor and the receptors for growth factors such as epidermal growth factor, fibroblast growth factor and vascular endothelial growth factor. Receptor activation occurs through ligand binding, which facilitates receptor dimerization and autophosphorylation of specific tyrosine residues in the cytoplasmic portion. The phosphotyrosine residues either enhance receptor catalytic activity or provide docking sites for downstream signaling proteins. Over the past several years, structural studies employing X-ray crystallography have advanced our understanding of the molecular mechanisms by which RTKs recognize their ligands and are activated by dimerization and tyrosine autophosphorylation. This review will highlight the key results that have emerged from these structural studies.

Crystallization and X-ray diffraction study of recombinant platelet-derived endothelial cell growth factor.

Crystals of recombinant platelet-derived endothelial cell growth factor (PD-ECGF) were obtained by the hanging drop vapour diffusion technique. The crystals belong to the space group P2(1)2(1)2(1) with unit cell dimensions a = 63.7 A, b = 70.4 A, c = 219.6, alpha = beta = gamma = 90 degrees, and probably contain a single dimer in the asymmetric unit. Diffraction to a minimum Bragg spacing of 3.5 A has been obtained using a synchrotron X-ray source.

Solution structure of the VEGF-binding domain of Flt-1: comparison of its free and bound states.

The extracellular portion of the VEGF and PlGF receptor, Flt-1 (or VEGFR-1), consists of seven immunoglobulin-like domains. The second domain from the N terminus (Flt-1D2) is necessary and sufficient for high affinity VEGF binding. The 1.7 A resolution crystal structure of Flt-1D2 bound to VEGF revealed that this domain is a member of the I-set of the immunoglobulin superfamily, but has several unusual features including a region near the N terminus that bulges away from the domain rather than pairing with the neighboring beta-strand. Some of the residues in this region make contact with VEGF, raising the possibility that this bulge could be a consequence of VEGF binding and might not be present in the absence of ligand. Here we report the three-dimensional structure of Flt-1D2 in its uncomplexed form determined by NMR spectroscopy. A semi-automated method for NOE assignment that takes advantage of the previously solved crystal structure was used to facilitate rapid analysis of the 3D NOESY spectra. The solution structure is very similar to the previously reported VEGF-bound crystal structure; the N-terminal bulge is present, albeit in a different conformation. We also report the 2.7 A crystal structure of Flt-1D2 in complex with VEGF solved in a different crystal form that reveals yet another conformation for the N-terminal bulge region. (1)H-(15)N heteronuclear NOEs indicate this region is flexible in solution; the crystal structures show that this region is able to adopt more than one conformation even when bound to VEGF. Thus, VEGF-binding is not accompanied by significant structural change in Flt-1D2, and the unusual structural features of Flt-1D2 are an intrinsic property of this domain.