Dynamic contrast-enhanced (DCE) MRI assessment of microvascular characteristics in the murine orthotopic pancreatic cancer model.

OBJECT: The aim of this study was to assess the dynamic contrast enhanced magnetic resonance imaging (DCE-MRI)-derived pharmacokinetic parameters between two contrast agents in a murine orthotopic pancreatic cancer model and to evaluate the tumor heterogeneity and the potential association between kinetic parameters and angiogenic markers such as the microvessel density (MVD) and vascular endothelial growth factor (VEGF) expression by immunohistochemistry. MATERIALS AND METHODS: Human pancreatic adenocarcinoma cell line MIAPaCa-2 was injected into the pancreas of BALB/C nu/nu mice. DCE-MRI was performed using Gd-DTPA and Gd-EOB-DTPA. Quantitative and semi-quantitative vascular parameters (K(trans), Kep, Ve and AUC) were calculated by using a dedicated postprocessing software program. Values were compared with tumor rim, tumor core and the entire tumor. The MVD and VEGF expressions between tumor rim and tumor core were also compared. RESULTS: There were no significant differences in K(trans), Kep, Ve, and AUC values of the three groups when using Gd-DTPA. However there were significant differences in K(trans), Kep, and AUC values of the three groups when using Gd-EOB-DTPA (P=0.014, 0.022, 0.007, respectively), in addition, the K(trans) and Kep values of tumor core were significantly lower than those of the entire tumor (adjusted P=0.014 and 0.027, respectively), the AUC values of core were significantly lower than those of the entire tumor and rim (adjusted P=0.039 and 0.009, respectively). Immunohistology results revealed that MVD and VEGF expression in the tumor rim was significantly higher than that in the core. There was positive correlation between AUC and MVD, VEGF. CONCLUSION: The murine orthotopic pancreatic cancer model provides an ideal animal model to study human pancreatic cancer. It can more sensitively semi-quantitatively and quantitatively analyze tumor angiogenesis through selecting the albumin-binding contrast agent.

Secretion of VEGF-165 has unique characteristics, including shedding from the plasma membrane.

Vascular endothelial growth factor (VEGF) is a critical regulator of endothelial cell differentiation and vasculogenesis during both development and tumor vascularization. VEGF-165 is a major form that is secreted from the cells via a poorly characterized pathway. Here we use green fluorescent protein- and epitope-tagged VEGF-165 and find that its early trafficking between the endoplasmic reticulum and the Golgi requires the small GTP-binding proteins Sar1 and Arf1 and that its glycosylation in the Golgi compartment is necessary for efficient post-Golgi transport and secretion from the cells. The relative temperature insensitivity of VEGF secretion and its Sar1 and Arf1 inhibitory profiles distinguish it from other cargoes using the "constitutive" secretory pathway. Prominent features of VEGF secretion are the retention of the protein on the outer surface of the plasma membrane and the stimulation of its secretion by Ca(2+) and protein kinase C. Of importance, shedding of VEGF-165 from the cell surface together with other membrane components appears to be a unique feature by which some VEGF is delivered to the surroundings to exert its known biological actions. Understanding VEGF trafficking can reveal additional means by which tumor vascularization can be inhibited by pharmacological interventions.

Coiled-coil-mediated grafting of bioactive vascular endothelial growth factor.

Chimeric growth factors may represent a powerful alternative to their natural counterparts for the functionalization of tissue-engineered scaffolds and applications in regenerative medicine. Their rational design should provide a simple, readily scalable production strategy while improving retention at the site of action. In that endeavor, we here report the synthesis of a chimeric protein corresponding to human vascular endothelial growth factor 165 being N-terminally fused to an E5 peptide tag (E5-VEGF). E5-VEGF was successfully expressed as a homodimer in mammalian cells. Following affinity purification, in vitro surface plasmon resonance biosensing and cell survival assays confirmed diffusible E5-VEGF ability to bind to its receptor ectodomains, while observed morphological phenotypes confirmed its anti-apoptotic features. Additional surface plasmon resonance assays highlighted that E5-VEGF could be specifically captured with high stability when interacting with covalently immobilized K5 peptide (a synthetic peptide designed to bind to the E5 moiety of chimeric hVEGF). This immobilization strategy was applied to glass substrates and chimeric hVEGF was shown to be maintained in a functionally active state following capture. Altogether, our data demonstrated that stable hVEGF capture can be performed via coiled-coil interactions without impacting hVEGF bioactivity, thus opening up the way to future applications in the field of tissue engineering and regenerative medicine.

Small molecule inhibitors of the neuropilin-1 vascular endothelial growth factor A (VEGF-A) interaction.

We report the molecular design and synthesis of EG00229, 2, the first small molecule ligand for the VEGF-A receptor neuropilin 1 (NRP1) and the structural characterization of NRP1-ligand complexes by NMR spectroscopy and X-ray crystallography. Mutagenesis studies localized VEGF-A binding in the NRP1 b1 domain and a peptide fragment of VEGF-A was shown to bind at the same site by NMR, providing the basis for small molecule design. Compound 2 demonstrated inhibition of VEGF-A binding to NRP1 and attenuated VEGFR2 phosphorylation in endothelial cells. Inhibition of migration of endothelial cells was also observed. The viability of A549 lung carcinoma cells was reduced by 2, and it increased the potency of the cytotoxic agents paclitaxel and 5-fluorouracil when given in combination. These studies provide the basis for design of specific small molecule inhibitors of ligand binding to NRP1.

Anti-angiogenesis effect of generation 4 polyamidoamine/vascular endothelial growth factor antisense oligodeoxynucleotide on breast cancer in vitro.

OBJECTIVE: To study the effects of the generation 4 polyamidoamine/vascular endothelial growth factor antisense oligodeoxynucleotide (G4PAMAM/VEGFASODN) compound on the expressions of vascular endothelial growth factor (VEGF) and its mRNA of breast cancer cells and on the inhibition of vascular endothelial cells. METHODS: We examined the morphology of G4PAMAM/VEGFASODN compound and its pH stability, in vitro transfection efficiency and toxicity, and the expressions of VEGF and its mRNA. Methyl thiazolyl tetrazolium assay was used to detect the inhibitory function of the compound on vascular endothelial cells. RESULTS: The compound was about 10 nm in diameter and was homogeneously netlike. From pH 5 to 10, it showed quite a buffered ability. The 48-h transfection rate in the charge ratio of 1:40 was 98.76%, significantly higher than that of the liposome group (P<0.05). None of the transfection products showed obvious toxicity on the cells. The expressions of both VEGF protein and its mRNA after G4PAMAM/VEGFASODN transfection decreased markedly. CONCLUSION: With a low toxicity, high safety, and high transfection rate, G4PAMAM/VEGFASODN could be a promising gene vector. Specifically, it inhibits VEGF gene expression efficiently, laying a basis for further in vivo animal studies.

Effects of sterilization on an extracellular matrix scaffold: part I. Composition and matrix architecture.

The impact of peracetic acid (PAA), lyophilization, and ethylene oxide (EO) sterilization on the composition and three dimensional matrix structure of small intestinal submucosa (SIS), a biologic scaffold used to stimulate the repair of damaged tissues and organs, was examined. Fibronectin and glycosaminoglycans are retained in SIS following oxidation by peracetic acid and alkylation using ethylene oxide gas. Significant amounts of FGF-2 are also retained, but VEGF is susceptible to the effects of PAA and is dramatically reduced following processing. Further, matrix oxidation, lyophilization, and sterilization with EO can be performed without irreversibly collapsing the three dimensional structure of the native SIS. These structural features and growth promoting extracellular matrix constituents are likely to be important variables underlying cellular attachment, infiltration and eventual incorporation of SIS into healing host tissues.

Structure of a VEGF-VEGF receptor complex determined by electron microscopy.

Receptor tyrosine kinases are activated upon ligand-induced dimerization. Here we show that the monomeric extracellular domain of vascular endothelial growth factor (VEGF) receptor-2 (VEGFR-2) has a flexible structure. Binding of VEGF to membrane-distal immunoglobulin-like domains causes receptor dimerization and promotes further interaction between receptor monomers through the membrane-proximal immunoglobulin-like domain 7. By this mechanism, ligand-induced dimerization of VEGFR-2 can be communicated across the membrane, activating the intracellular tyrosine kinase domains.

Localized angiogenesis induced by human vascular endothelial growth factor-activated PLGA sponge.

The objective of this study was to assess the in vitro release kinetics and the in vivo angiogenic effect of human vascular endothelial growth factor (VEGF)-activated poly(DL-lactide-co-glycolide) (PLGA) sponge. The highly porous sponges (each 3 x 4 x 4 mm(3)) were activated by soaking in a VEGF solution (2.5 or 5.0 microg) and then freeze-drying. In vitro release in PBS was investigated by a competitive enzyme immunoassay for up to 3 weeks. The burst-type initial release within the first 3 days followed a more controlled one lasting for >2 weeks. The angiogenic potential of the VEGF sponge was evaluated by subcutaneous implantation into the epigastric groin fascia of Wistar rats. Histomorphometry and SEM confirmed the formation of new capillaries infiltrating the sponge pores starting from the first week and the drastic anostomosis at weeks 2 and 3. However, the rats implanted with control sponges or receiving VEGF injection exhibited much lower or no angiogenic response, respectively. TEM revealed the neo-vessels had a single endothelial layer surrounded by the matrix inoculated with the rat circulation. The results indicate that VEGF-activated PLGA sponge can be considered as a tool to establish neovascularized subcutaneous transplantation sites for tissue-engineering applications.