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.

Suppressive effect of microRNA-126 on oral squamous cell carcinoma in vitro.

MicroRNA-126 (miR-126), an endothelial-specific miRNA located within intron 7 of epidermal growth factor­like domain 7 (EGFL7), has been demonstrated to act as a tumor suppressor in various types of human cancer. However, its role in oral squamous cell carcinoma (OSCC) remains unclear. In the present study, we revealed that the expression of miR-126 was significantly decreased in OSCC tissues, when compared with that in their matched adjacent tissues, and its expression level was also reduced in Tca8113, OSCC-15 and CAL27 cell lines compared with normal tissues. The protein expression of EGFL7 was upregulated in OSCC tissues compared with their matched adjacent tissues as well as normal tissues, and Tca8113, OSCC-15 and CAL27 cells additionally demonstrated a positive expression of EGFL7. The overexpression of miR-126 significantly reduced the protein expression of EGFL7 in OSCC-15 cells, while transfection with the miR-126 inhibitor upregulated the EGFL7 protein level in OSCC-15 cells. Furthermore, transfection with an miR-126 mimic into OSCC-15 cells markedly suppressed cell proliferation, cell cycle progression, cell invasion and colony formation, while inducing cell apoptosis, which contrasted with the effects of transfection with an miR-126 inhibitor. The overexpression of miR-126 suppressed the secretion of two key regulators of angiogenesis, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), which was also reversed by miR-126 inhibitor transfection. In conclusion, the present study demonstrated that miR-126 acts as a tumor suppressor in OSCC cells, partially at least via the downregulation of EGFL7. Thus, miR-126 may serve as a promising candidate for the treatment of OSCC.

Multifunctional polyurethane-urea nanoparticles to target and arrest inflamed vascular environment: a potential tool for cancer therapy and diagnosis.

Activation of inflammatory pathways in endothelial cells contributes to tumour growth and progression in multiple human cancers. Cellular adhesion molecules are involved in leukocyte recruitment to the vascular inflammatory environment where it plays a critical role in angiogenesis, suppression of apoptosis, proliferation, invasion and metastasis. We describe here the development of streptavidin-coated polyurethane-urea nanoparticles as multifunctional nanocarriers for fluorescence imaging or targeting of the tumour environment to identify and arrest the vascular network irrigating the tumour tissue. The design of these multifunctional nanoparticles involves incorporating streptavidin to the nanoparticle polymeric matrix. The obtained nanoparticles are spherical and exhibit an average diameter of 70-74 nm. Streptavidin-coated nanoparticles spontaneously bind biotinylated antibodies against VCAM-1 and ICAM-1 which in vitro and in vivo specifically attached to inflamed endothelial cells. Indeed the incorporation of CBO-P11 (a specific inhibitor of the vascular endothelial growth factor proangiogenic and proinflammatory pathway) to these nanoparticles allows a targeted pharmacological effect thereby decreasing the proliferation only in inflamed endothelial cells. The multiple functionalisation strategy described here could be exploited for tumour diagnostics or targeted drug delivery to tumour vasculature with a good safety profile and an attractive array of possibilities for biomedical applications.

"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.

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.

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.

Probing the interactions of phosphosulfomannans with angiogenic growth factors by surface plasmon resonance.

The binding interactions of the phosphosulfomannan anticancer agent PI-88 (1) with the angiogenic growth factors FGF-1, FGF-2, and VEGF were studied by surface plasmon resonance (SPR) on a BIAcore 3000 biosensor. Compared with heparin, PI-88 has at least 11-fold higher affinity for FGF-1 and at least 3-fold higher affinity for VEGF, but at least 13-fold lower affinity for FGF-2. To define the structural features of PI-88 that are important for growth factor binding, several analogues, such as dephosphorylated PI-88 and a sulfated pentasaccharide, were prepared. The binding interactions of these analogues with FGF-1, FGF-2, and VEGF were similarly studied by SPR, and structure-activity relationships were determined.

Dimethylarginine dimethylaminohydrolase activity modulates ADMA levels, VEGF expression, and cell phenotype.

Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthase and is metabolised by dimethylarginine dimethylaminohydrolase (DDAH). Elevated levels of circulating ADMA correlate with various cardiovascular pathologies less is known about the cellular effects of altered DDAH activity. We modified DDAH activity in cells and measured the changes in ADMA levels, morphological phenotypes on Matrigel, and expression of vascular endothelial growth factor (VEGF). DDAH over-expressing ECV304 cells secreted less ADMA and when grown on Matrigel had enhanced tube formation compared to untransfected cells. VEGF mRNA levels were 2.1-fold higher in both ECV304 and murine endothelial cells (sEnd.1) over-expressing DDAH. In addition the DDAH inhibitor, S-2-amino-4(3-methylguanidino)butanoic acid (4124W 1mM), markedly reduced human umbilical vein endothelial cell tube formation in vitro. We have found that upregulating DDAH activity lowers ADMA levels, increases the levels of VEGF mRNA in endothelial cells, and enhances tube formation in an in vitro model, whilst blockade of DDAH reduces tube formation.

Structure and inhibitory effects on angiogenesis and tumor development of a new vascular endothelial growth inhibitor.

Blocking angiogenesis is an attractive strategy to inhibit tumor growth, invasion, and metastasis. We describe here the structure and the biological action of a new cyclic peptide derived from vascular endothelial growth factor (VEGF). This 17-amino acid molecule designated cyclopeptidic vascular endothelial growth inhibitor (cyclo-VEGI, CBO-P11) encompasses residues 79-93 of VEGF which are involved in the interaction with VEGF receptor-2. In aqueous solution, cyclo-VEGI presents a propensity to adopt a helix conformation that was largely unexpected because only beta-sheet structures or random coil conformations have been observed for macrocyclic peptides. Cyclo-VEGI inhibits binding of iodinated VEGF165 to endothelial cells, endothelial cells proliferation, migration, and signaling induced by VEGF165. This peptide also exhibits anti-angiogenic activity in vivo on the differentiated chicken chorioallantoic membrane. Furthermore, cyclo-VEGI significantly blocks the growth of established intracranial glioma in nude and syngeneic mice and improves survival without side effects. Taken together, these results suggest that cyclo-VEGI is an attractive candidate for the development of novel angiogenesis inhibitor molecules useful for the treatment of cancer and other angiogenesis-related diseases.

Redesigning an antibody fragment for faster association with its antigen.

Traditional approaches for increasing the affinity of protein-protein complexes focus on constructing highly complementary binding surfaces. Recent theoretical simulations and experimental results suggest that electrostatic steering forces can also be manipulated to increase association rates while leaving dissociation rates unchanged, thus increasing affinity. Here we demonstrate that electrostatic attraction can be enhanced between an antibody fragment and its cognate antigen through application of a few simple rules to identify potential on-rate amplification sites that lie at the periphery of the antigen-antibody interface.

Expression of VEGF and its receptors Flt-1 and Flk-1/KDR is altered in lambs with increased pulmonary blood flow and pulmonary hypertension.

Utilizing in utero aortopulmonary vascular graft placement, we developed a lamb model of congenital heart disease and increased pulmonary blood flow. We showed previously that these lambs have increased pulmonary vessel number at 4 wk of age. To determine whether this was associated with alterations in VEGF signaling, we investigated vascular changes in expression of VEGF and its receptors, Flt-1 and KDR/Flk-1, in the lungs of shunted and age-matched control lambs during the first 8 wk of life. Western blot analysis demonstrated that VEGF, Flt-1, and KDR/Flk-1 expression was higher in shunted lambs. VEGF and Flt-1 expression was increased at 4 and 8 wk of age (P <0.05). However, KDR/Flk-1 expression was higher in shunted lambs only at 1 and 4 wk of age (P <0.05). Immunohistochemical analysis demonstrated that, in control and shunted lambs, VEGF localized to the smooth muscle layer of vessels and airways and to the pulmonary epithelium while increased VEGF expression was localized to the smooth muscle layer of thickened media in remodeled vessels in shunted lambs. VEGF receptors were localized exclusively in the endothelium of pulmonary vessels. Flt-1 was increased in the endothelium of small pulmonary arteries in shunted animals at 4 and 8 wk of age, whereas KDR/Flk-1 was increased in small pulmonary arteries at 1 and 4 wk of age. Our data suggest that increased pulmonary blood flow upregulates expression of VEGF and its receptors, and this may be important in development of the vascular remodeling in shunted lambs.

Pseudocowpox virus encodes a homolog of vascular endothelial growth factor.

We have identified a gene encoding a homolog of vascular endothelial growth factor (VEGF) in the Pseudocowpox virus (PCPV) genome. The predicted protein shows 27% amino acid identity to human VEGF-A. It also shows 41 and 61% amino acid identity to VEGFs encoded by orf virus (ORFV) strains NZ2 and NZ7, respectively. Assays of the expressed VEGF-like protein of PCPV (PCPV(VR634)VEGF) demonstrated that PCPV(VR634)VEGF is mitogenic for endothelial cells and is capable of inducing vascular permeability. PCPV(VR634)VEGF bound VEGF receptor-2 (VEGFR-2) but did not bind VEGFR-1 or VEGFR-3. These results indicate that PCPV(VR634)VEGF is a biologically active member of the VEGF family which shares with the ORFV-encoded VEGFs a receptor binding profile that differs from those of all cellular members of the VEGF family. It seems likely that the biological activities of PCPV(VR634)VEGF contribute to the proliferative and highly vascularized nature of PCPV lesions.

A set of loop-1 and -3 structures in the novel vascular endothelial growth factor (VEGF) family member, VEGF-ENZ-7, is essential for the activation of VEGFR-2 signaling.

The vascular endothelial growth factor (VEGF) family plays important roles in angiogenesis and vascular permeability. Novel members of the VEGF family encoded in the Orf virus genome, VEGF-E, function as potent angiogenic factors by specifically binding and activating VEGFR-2 (KDR). VEGF-E is about 45% homologous to VEGF-A at amino acid levels, however, the amino acid residues in VEGF-A crucial for the VEGFR-2-binding are not conserved in VEGF-E. To understand the molecular basis of the biological activity of VEGF-E, we have functionally mapped residues important for interaction of VEGF-E with VEGFR-2 by exchanging the domains between VEGF-E(NZ-7) and PlGF, which binds only to VEGFR-1 (Flt-1). Exchange on the amino- and carboxyl-terminal regions had no suppressive effect on biological activity. However, exchange on either the loop-1 or -3 region of VEGF-E(NZ-7) significantly reduced activities. On the other hand, introduction of the loop-1 and -3 of VEGF-E(NZ-7) to placenta growth factor rescued the biological activities. The chimera between VEGF-A and VEGF-E(NZ-7) gave essentially the same results. These findings strongly suggest that a common rule exists for VEGFR-2 ligands (VEGF-E(NZ-7) and VEGF-A) that they build up the binding structure for VEGFR-2 through the appropriate interaction between loop-1 and -3 regions.

Immunodetection and molecular forms of plasma vascular endothelial growth factor-C.

Vascular endothelial growth factor (VEGF)-C is a member of the VEGF family. VEGF-C is involved in developmental lymphangiogenesis and may be important in pathological lymphangiogenesis, lymphatic invasion and metastasis in carcinoma. We describe the development of an indirect enzyme-linked immunosorbent (ELISA) assay for the quantification of VEGF-C in plasma. Capture of VEGF-C was achieved using goat anti-human VEGF-C antibody, followed by detection with rabbit anti-human VEGF-C antibody. The sensitivity of the assay was amplified using the biotin-avidin and enhanced chemiluminescence (ECL) systems. The assay was highly sensitive and reproducible with a detection range of 0.4-100 U/ml and the intra- and inter-assay variations were less than 8%. Substitutional tests demonstrated that the assay was specific for VEGF-C with no cross-reaction with VEGF-A or VEGF-D. Practical application of the assay was evaluated in 41 colorectal cancer patients and 31 controls. Median plasma levels of VEGF-C were 35.0 U/ml (range: 17.4-75.9 U/ml) in colorectal cancer patients in contrast to 11.5 U/ml (range: 5.4-21.5 U/ml) in controls (p<0.001). Moreover, VEGF-C levels tended to be elevated in patients with advanced disease compared to early disease, but this was not statistically significant owing to a relatively small number of patients in each group. Immunoprecipitation and immunoblotting confirmed detection of VEGF-C in plasma and revealed that two forms of VEGF-C were present in the plasma corresponding to approximately 40 and approximately 80 kDa. The measurement of plasma VEGF-C offers opportunities to explore clinical applications in the management of malignancy, in particular in the prediction of lymphatic spread and in other lymphangiogenesis-related diseases.

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.

Quantitative real-time reverse transcription polymerase chain reaction: normalization to rRNA or single housekeeping genes is inappropriate for human tissue biopsies.

Careful normalization is essential when using quantitative reverse transcription polymerase chain reaction assays to compare mRNA levels between biopsies from different individuals or cells undergoing different treatment. Generally this involves the use of internal controls, such as mRNA specified by a housekeeping gene, ribosomal RNA (rRNA), or accurately quantitated total RNA. The aim of this study was to compare these methods and determine which one can provide the most accurate and biologically relevant quantitative results. Our results show significant variation in the expression levels of 10 commonly used housekeeping genes and 18S rRNA, both between individuals and between biopsies taken from the same patient. Furthermore, in 23 breast cancers samples mRNA and protein levels of a regulated gene, vascular endothelial growth factor (VEGF), correlated only when normalized to total RNA, as did microvessel density. Finally, mRNA levels of VEGF and the most popular housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were significantly correlated in the colon. Our results suggest that the use of internal standards comprising single housekeeping genes or rRNA is inappropriate for studies involving tissue biopsies.

Generation of a novel proteolysis resistant vascular endothelial growth factor165 variant by a site-directed mutation at the plasmin sensitive cleavage site.

Vascular endothelial growth factor (VEGF) is a potent angiogenic mediator in tissue repair. In non-healing human wounds plasmin cleaves and inactivates VEGF165. In the present study, we generated recombinant VEGF165 mutants resistant to plasmin proteolysis. Substitution of Arg110 with Ala110 or Gln110, and Ala111 with Pro111 yielded plasmin-resistant and biologically active VEGF165 mutants. In addition, substitution of Ala111 with Pro111 resulted in a substantial degree of stabilization when incubated in wound fluid obtained from non-healing wounds. These results suggest that the plasmin cleavage site Arg110/Ala111 and the carboxyl-terminal domain play an important role in the mitogenic activity of VEGF165.

Vascular endothelial growth factors encoded by Orf virus show surprising sequence variation but have a conserved, functionally relevant structure.

The first report of a vascular endothelial growth factor (VEGF)-like gene in Orf virus included the surprising observation that the genes from two isolates (NZ2 and NZ7) shared only 41.1% amino acid sequence identity. We have examined this sequence disparity by determining the VEGF gene sequence of 21 isolates of Orf virus derived from diverse sources. Most isolates carried NZ2-like VEGF genes but their predicted amino acid sequences varied by up to 30.8% with an average amino acid identity between pairs of NZ2-like sequences of 86.1%. This high rate of sequence variation is more similar to interspecies than intraspecies variability. In contrast, only three isolates carried an NZ7-like VEGF gene and these varied from the NZ7 sequence by no more than a single nucleotide. The VEGF family are ligands for a set of tyrosine kinase receptors. The viral VEGFs are unique among the family in that they recognize VEGF receptor 2 (VEGFR-2) but not VEGFR-1 or VEGFR-3. Comparisons of the viral VEGFs with other family members revealed some correlations between conserved residues and the ability to recognize specific VEGF receptors. Despite the sequence variations, structural predictions for the viral VEGFs were very similar to each other and to the structure determined by X-ray crystallography for human VEGF-A. Structural modelling also revealed that a groove seen in the VEGF-A homodimer and believed to play a role in its binding to VEGFR-1 is blocked in the viral VEGFs. This may contribute to the inability of the viral VEGFs to bind VEGFR-1.

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.