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Ziv-aflibercept (ziv-AFL) is a complex fusion protein which is widely used in hospitals for the treatment of colorectal metastatic cancer. Charge variants are critical attributes for assessing post-transitional modifications (PMTs) that have to be controlled during the development and manufacture of these proteins and until their administration to patients. Cation exchange (CEX) chromatography is a charge-sensitive analytical method that is well suited for analysing charge variants in proteins. The aim of this paper is to analyse the charge variants of ziv-AFL in the medicine (Zaltrap®) when fresh and when degraded. Two CEX chromatographic methods were compared for this purpose. The former was an adaptation of the method used in the first published study in which charge variants were analysed via pH gradient elution using volatile, low ionic strength buffers with direct coupling to high-resolution Orbitrap mass spectrometry. The second method was developed and optimized during our research using the salt-mediated pH gradient mode and classical non-volatile, high ionic strength buffers which were incompatible with direct coupling with mass detection. Fresh and controlled degraded samples of ziv-AFL were used to evaluate the capacity of both CEX chromatographic strategies for detecting charge variants in ziv-AFL. In the controlled degradation study the samples of the medicine were subjected to three stress factors: temperature of 60 °C for three hours, freeze/thaw process -two cycles-, and exposure to light for twelve hours. The CEX chromatographic method with non-volatile salts in the mobile phase enabled better detection of charge variants degraded ziv-AFL samples than the method using volatile salts with lower ionic strength. In addition, the complexity of the mass spectra data generated made it impossible to identify the multicharge variant species of ziv-AFL. Although charge variants were not separated in ziv-AFL fresh sample, our results indicate that the method with non-volatile salts in the mobile phase could be used to characterize and track changes in the charge variant UV chromatographic profile of ziv-AFL in fresh and degraded samples, even though it cannot be coupled to a mass detector and there is therefore no information about mass. The increase of basic protein degraded compounds were the most important degradation pattern detected in ziv-AFL (Zaltrap®)., Competing Interests: Declaration of Competing Interest All authors declare that they have no conflict of interest related to the contents of this article that might influence the results of the investigation., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The increased expression of vascular endothelial growth factor (VEGF) and its receptors is associated with angiogenesis in a growing tumor, presenting potential targets for tumor-selective imaging by way of targeted tracers. Though fluorescent tracers are used for targeted in vivo imaging, the lack of photostability and biocompatibility of many current fluorophores hinder their use in several applications involving long-term, continuous imaging. To address these problems, fluorescent nanodiamonds (FNDs), which exhibit infinite photostability and excellent biocompatibility, were explored as fluorophores in tracers for targeting VEGF receptors in growing tumors. To explore FND utility for imaging tumor VEGF receptors, we used click-chemistry to conjugate multiple copies of an engineered single-chain version of VEGF site-specifically derivatized with trans-cyclooctene (scVEGF-TCO) to 140 nm FND. The resulting targeting conjugates, FND-scVEGF, were then tested for functional activity of the scVEGF moieties through biochemical and tissue culture experiments and for selective tumor uptake in Balb/c mice with induced 4T1 carcinoma. We found that FND-scVEGF conjugates retain high affinity to VEGF receptors in cell culture experiments and observed preferential accumulation of FND-scVEGF in tumors relative to untargeted FND. Microspectroscopy provided unambiguous determination of FND within tissue by way of the unique spectral shape of nitrogen-vacancy induced fluorescence. These results validate and invite the use of targeted FND for diagnostic imaging and encourage further optimization of FND for fluorescence brightness.

Angiogenesis and its involved proteins, particularly Vascular Endothelial Growth Factor family (VEGFs) and VEGF receptors (VEGFRs), have been considered as a target of therapeutic interest for numerous inflammatory and vascular diseases. Acting on this biological process through interaction with VEGFs or VEGFRs has received considerable attention. Indeed, VEGFs and VEGFRs are currently targeted by drugs such as monoclonal antibodies. The feasibility of a therapeutic strategy based on blocking the VEGF/VEGFR interaction by using ligands "other-than-biologics" is also explored. To help to the discovery of new molecules, screening assays have been developed, particularly to evaluate the VEGFA/VEGFR1 interaction. Despite the therapeutic importance of VEGFB and PlGF (Placental Growth Factor), no assays have been developed to evaluate molecules against their interactions with VEGFR1. Here, we present new versatile colorimetric immunoassays to screen and evaluate the specific interaction of discovered molecules with different growth factors (VEGFA, VEGFB, PlGF) and receptors (VEGFR1, VEGFR2). These tests, based on competitive immunoassay format, will provide essential information on specificity and selectivity of molecules for their targets and will help to work on the pharmaco-modulation of molecules for targeting one specific interaction., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Although vascular endothelial growth factor receptor (VEGF-R)-targeted antiangiogenic agents are important treatment for a number of human malignancies, there is accumulating evidence that the therapies may promote disease progression, such as invasion and metastasis. How tumors become to promote their evasiveness remains fully uncertain. One of possible mechanisms for the adaptation may be a direct effect of VEGF-R inhibitors on tumor cells expressing VEGF-R. To elucidate a direct effect of VEGF-R-targeting drug (sunitinib), we established a human colorectal cancer cell model adapted to sunitinib. The sunitinib-conditioned cells showed a significant increase in cellular motility and migration activities, compared to the vehicle-treated control cells. Consistent with the phenotype, the sunitinib-conditioned cells decreased the expression levels of E-cadherin (an epithelial marker), while significantly increased the levels of Slug and Zeb1 (mesenchymal markers). Expression profiles of VEGF-R in the sunitinib-conditioned cells showed that only neuropilin-1 (NRP1) expression was significantly increased among all VEGF-R tested. Blockade of NRP1 using its antagonist clearly repressed the migration activation in sunitinib-conditioned cells, but not in the control cells. These results suggest that inhibition of VEGF-R on colorectal cancer cells can drive the epithelial-mesenchymal transition, leading to activation of cell motility in an NRP1-dependent manner. J. Med. Invest. 64: 250-254, August, 2017.

Purpose: To measure the hydrodynamic radii of intravitreal anti-VEGF drugs ranibizumab, aflibercept and bevacizumab with μs time-resolved phosphorescence anisotropy., Methods: Ruthenium-based dye Ru(bpy)2(mcbpy - O - Su - ester)(PF6)2, whose lifetime of several hundred nanoseconds is comparable to the rotational correlation time of these drugs in buffer, was used as a label. The hydrodynamic radii were calculated from the rotational correlation times of the Ru(bpy)2(mcbpy - O - Su - ester)(PF6)2-labelled drugs obtained with time-resolved phosphorescence anisotropy measurements in buffer/glycerol solutions of varying viscosity., Results: The measured radii of 2.76±0.04 nm for ranibizumab, 3.70±0.03 nm for aflibercept and 4.58±0.01 nm for bevacizumab agree with calculations based on molecular weight and other experimental measurements., Conclusions: Time-resolved phosphorescence anisotropy is a relatively simple and straightforward method that allows experimental measurement of the hydrodynamic radius of individual proteins, and is superior to theoretical calculations which cannot give the required accuracy for a particular protein.

Background: Extracellular matrix (ECM) scaffolds, obtained through detergent-based decellularization of native kidneys, represent the most promising platform for investigations aiming at manufacturing kidneys for transplant purposes. We previously showed that decellularization of the human kidney yields renal ECM scaffolds (hrECMs) that maintain their basic molecular components, are cytocompatible, stimulate angiogenesis, and show an intact innate vasculature. However, evidence that the decellularization preserves glomerular morphometric characteristics, physiological parameters (pressures and resistances of the vasculature bed), and biological properties of the renal ECM, including retention of important growth factors (GFs), is still missing., Methods: To address these issues, we studied the morphometry and resilience of hrECMs' native vasculature with resin casting at electronic microscopy and pulse-wave measurements, respectively. Moreover, we determined the fate of 40 critical GFs post decellularization with a glass chip-based multiplex enzyme-linked immunosorbent assay array and in vitro immunofluorescence., Results: Our method preserves the 3-dimensional conformation of the native glomerulus. Resin casting and pulse-wave measurements, showed that hrECMs preserves the microvascular morphology and morphometry, and physiological function. Moreover, GFs including vascular endothelial growth factor and its receptors are retained within the matrices., Conclusions: Our results indicate that discarded human kidneys are a suitable source of renal scaffolds because they maintain a well-preserved structure and function of the vasculature, as well as GFs that are fundamental to achieve a satisfying recellularization of the scaffold in vivo due to their angiogenic properties.

Background/aim: Prognosis for patients with head and neck squamous cell carcinoma (HNSCC) is poor in most cases and has not improved despite advances in therapy. Novel therapeutic approaches are mandatory in order to improve the situation. Everolimus, an inhibitor of mammalian target of rapamycin, as well as the multi-tyrosine kinase inhibitors sorafenib and sunitinib, has demonstrated a substantial therapeutic effect in various types of human cancer with moderate side-effects. Expression of vascular endothelial growth factor receptor (VEGFR) 1 and 2, and of the tumor-suppressor protein phosphatase and tensin homolog deleted on chromosome 10 (PTEN) were evaluated in chemonaïve human papillomavirus (HPV)-positive and -negative squamous cell carcinoma (SCC) and after exposure to everolimus, sorafenib or sunitinib., Materials and Methods: p16-positive CERV196 and p16-negative HNSCC 11A and 14C cells were incubated with different drug concentrations for 48-192 h. Expression of VEGFR1 and -2 as well as PTEN were determined by enzyme-linked immunosorbent assay and was compared to a chemonaïve control., Results: VEGFR1 and -2, as well as PTEN, were expressed in all three cell lines. Sunitinib, sorafenib and everolimus significantly reduced the expression of VEGFR1 and -2, especially in p16-positive CERV196 cells. Sunitinib appeared to be more effective in reducing VEGFR1 and -2 expression than sorafenib and everolimus. PTEN levels were remarkably lower in HPV-positive CERV196 cells. PTEN expression increased significantly under sunitinib and sorafenib in HNSCC 11A and CERV196 cells. Everolimus, on the other hand, led to a significant decrease of PTEN expression in these cell lines., Conclusion: The tested drugs displayed a remarkable anti-angiogenic effect by inhibition of VEGFR1 and -2 expression. Sunitinib and sorafenib were able to increase PTEN expression, which might induce apoptosis of cancer cells. HPV-positive CERV196 cells were characterized by an increased susceptibility to these small-molecule drugs. Further studies are imperative to scrutinize HPV status-dependent differences in drug response and possible implications for future treatment options., (Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Purpose: The intracytoplasmic sperm injection (ICSI) outcome is depended mainly on oocyte quality. Cytokines and their receptors play a critical role in oocyte maturation, fertilization, and embryo implantation. The purpose of the study was to study the levels of vascular endothelial growth factors (VEGFA, VEGFR1, VEGFA) in follicular fluids (FF) women participating in ICSI-in vitro fertilization (IVF) cycles in relation to cycle's outcome., Material and Methods: One hundred and fifty three samples of 70 women participating in ICSI cycles were classified in three infertility groups: male factor, female factor, and low responders. For controlled ovarian stimulation in male and female factor group, the long agonist protocol with leuprolide and recombinant follicle stimulating hormone (FSH) was employed, while the antagonist cetrorelix was used in low responders. Cytokines levels were evaluated with enzyme-linked immunosorbent assay (ELISA)., Results: In a total of 153 samples, the overall pregnancy rate was 51.6%, the higher one observed in female factor group (59% vs. 37.5% and 28.6% in male a factor and low responders group, p = 0.013. VEGFR2 differed statistically significantly between the two groups, being higher in the pregnancy group [median (IQR): 5,630 (4,870 - 6,651) vs. 4938 (4,068 - 6,020) in the non-pregnancy group, p = 0.003]. There were significant correlations between VEGF receptors, differentiated depending on infertility groups., Conclusions: The VEGFA/VEGFR2 system is important in human reproduction and the association pattern between VEGFA receptors may serve as a marker for ICSI outcome. Examination for spermatozoa functional defects may increase pregnancy rate in male factor group.

Object: Vascular endothelial growth factor (VEGF) is the major proangiogenic factor in many solid tumors. Vascular endothelial growth factor receptor (VEGFR) is expressed in abundance in pediatric patients with medulloblastoma and is associated with tumor metastasis, poor prognosis, and proliferation. Gadolinium enhancement on MRI has been suggested to have prognostic significance for some tumors. The association of VEGF/VEGFR and Gd enhancement in medulloblastoma has never been closely examined. The authors therefore sought to evaluate whether Gd-enhancing medulloblastomas have higher levels of VEGFR and CD31. Outcomes and survival in patients with enhancing and nonenhancing tumors were also compared., Methods: A retrospective analysis of patients with enhancing, nonenhancing, and partially enhancing medulloblastomas was performed. Primary end points included risk stratification, extent of resection, and perioperative complications. A cohort of 3 enhancing and 3 nonenhancing tumors was selected for VEGFR and CD31 analysis as well as microvessel density measurements., Results: Fifty-eight patients were analyzed, and 20.7% of the medulloblastomas in these patients were nonenhancing. Enhancing medulloblastomas exhibited strong VEGFR1/2 and CD31 expression relative to nonenhancing tumors. There was no significant difference in perioperative complications or patient survival between the 2 groups., Conclusions: These results suggest that in patients with medulloblastoma the presence of enhancement on MRI may correlate with increased vascularity and angiogenesis, but does not correlate with worse patient prognosis in the short or long term.

Objective: To review the current status and progress on nuclear medical molecular imaging of angiogenesis., Data Sources: A literature search was performed in Medline and PubMed published in English up to May 31, 2012. The search terms were molecular imaging, nuclear medicine and angiogenesis., Study Selection: Articles studying molecular imaging of angiogenesis using radionuclide were selected and reviewed., Results: Molecular imaging has been used for studying angiogenesis by targeting integrin αVβ3, VEGF/VEGFR, and matrix metalloproteinases (MMPs) with radionuclide-labeled tracers. The technology has been shown to be able to assess the angiogenesis status and/or predict the efficacy of anti-angiogenic therapy. Future directions of the research on the molecular imaging of angiogenesis include development of new tracers with better tumor targeting efficacy, desirable pharmacokinetics, and easy translation to clinical applications., Conclusion: Advances in molecular imaging of angiogenesis using radioculcide will make the technology a valuable tool for personalized anti-angiogenesis treatment.

Introduction: Vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are key players in vasculogenesis and are also involved in pathologic conditions with bone destruction. Vasculogenesis is critical for disease progression, and bone resorption is a hallmark of apical periodontitis. However, the localization of VEGFs and VEGFRs and their gene signaling pathways in human apical periodontitis have not been thoroughly investigated. The aim of this study was to localize VEGFs and VEGFRs and analyze their gene expression as well as signaling pathways in human periapical lesions., Methods: Tissue was collected after endodontic surgery from patients diagnosed with chronic apical periodontitis. Periodontal ligament samples from extracted healthy wisdom teeth was also collected and used as control tissue. In lesion cryosections, VEGFs/VEGFRs were identified by immunohistochemistry/double immunofluorescence by using specific antibodies. A human VEGF signaling polymerase chain reaction array system was used for gene expression analysis comparing lesions with periodontal ligament samples., Results: The histologic evaluation revealed heterogeneous morphology of the periapical lesions with various degrees of inflammatory infiltrates. In the lesions, all investigated factors and receptors were identified in blood vessels and various immune cells. No lymphatic vessels were detected. Gene expression analysis revealed up-regulation of VEGF-A and VEGFR-3, although not significant. Phosphatidylinositol-3-kinases, protein kinase C, mitogen-activated protein kinases, and phospholipases, all known to be involved in VEGF-mediated angiogenic activity, were significantly up-regulated., Conclusions: The cellular and vascular expressions of VEGFs and VEGFRs in chronic apical periodontitis, along with significant alterations of genes mediating VEGF-induced angiogenic responses, suggest ongoing vascular remodeling in established chronic periapical lesions., (Copyright © 2013 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Bevacizumab is the first antiangiogenic therapy proven to slow metastatic disease progression in patients with cancer. Although it has changed clinical practice, some patients do not respond or gradually develop resistance, resulting in rather modest gains in terms of overall survival. A major challenge is to develop robust biomarkers that can guide selection of patients for whom bevacizumab therapy is most beneficial. Here, we discuss recent progress in finding such markers, including the first results from randomized phase III clinical trials evaluating the efficacy of bevacizumab in combination with comprehensive biomarker analyses. In particular, these studies suggest that circulating levels of short vascular endothelial growth factor A (VEGF-A) isoforms, expression of neuropilin-1 and VEGF receptor 1 in tumors or plasma, and genetic variants in VEGFA or its receptors are strong biomarker candidates. The current challenge is to expand this first set of markers and to validate it and implement it into clinical practice. A first prospective biomarker study known as MERiDiAN, which will treat patients stratified for circulating levels of short VEGF-A isoforms with bevacizumab and paclitaxel, is planned and will hopefully provide us with new directions on how to treat patients more efficiently.

We investigated whether the angiogenic profile, which is based on the local expression and systemic levels of angiogenic growth factors (VEGF, Ang-1, Ang-2, and the corresponding receptors), differs between rheumatoid arthritis (RA) and osteoarthritis (OA) patients. We determined the expression of VEGF, Ang-1, and Ang-2 together with its receptors (VEGFR-1/-2 and Tie2) in synovium tissue (ST) and muscular tissue (MT) from patients with RA and OA using quantitative PCR. Tissue samples were obtained from 15 RA and 19 OA patients during total knee arthroplasty. Control MT samples (n = 10) were obtained during spinal surgery. Results are correlated to VEGF and angiopoietin serum levels via ELISA measurements. The VEGF expressions in ST and serum levels were significantly higher in RA patients than in OA patients (P < 0.05). Furthermore, the VEGFR-1 and VEGFR-2 expression in ST from RA patients were significantly higher than in OA patients (P < 0.001 and P < 0.05). The relative concentration of angiopoietins (Ang-1/Ang-2 ratio) was significantly increased in RA (P < 0.01). Serum levels for Ang-2 showed no significant differences. Statistical analysis showed a significant higher level of Tie2 in RA patients (P < 0.001). Analysis of local levels of VEGF, VEGFR-1, VEGFR-2, Ang-1, Ang-2, and Tie2 in the muscular tissue showed no significant difference between RA and OA patients. These results underline the importance of pro-angiogenic growth factor levels for RA corroborating the assumption that VEGF and angiopoietins play an important role in the pathogenesis of RA.

The vascular endothelial growth factor (VEGF) has beneficial effects on ovine oocytes during in vitro maturation and their subsequent early embryonic development, but the biochemical pathway underlying this effect has not been elucidated. Therefore, the focus of the present study was to investigate the activation of the mitogen-activated protein kinase (MAPK) pathway in response to the addition of VEGF to the maturation medium, and to study the subcellular localization of VEGF and its receptors during ovine oocyte maturation. We concluded that: (1) VEGF mainly localized in the cytoplasm, whereas its receptors, fms-tyrosine kinase-1 and kinase domain region (KDR), were localized on the plasma membrane of oocytes; (2) the addition of 5 ng/mL VEGF increased the percentage of oocytes with extruded first polar bodies (50.9 ± 2.2% vs. 34.6 ± 2.9%; treatment vs. control, respectively; P < 0.01) and the rate of oocytes competent to undergo nuclear maturation (70.6 ± 0.9% vs. 62.9 ± 1.9%, P < 0.01); and (3) as the expression of VEGF, fms-tyrosine kinase-1, and KDR increased after supplementation with 5 ng/mL, expression of VEGF, mitogen-activated protein kinase kinase (MEK), and MAPK mRNA, as well as MAPK phosphorylation, were stimulated in a time-dependent manner. We inferred that, in a paracrine manner, exogenous VEGF bound to KDR, its main receptor, and then activated the MAPK signaling pathway, which promoted maturation of ovine oocytes. However, the VEGF system also had an autocrine regulatory loop that contributed to creating an environment optimal for oocyte maturation., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Aim: We studied expression of molecules of the vascular endothelial growth factor (VEGF) pathway and its relation to vascularization, cell proliferation and patient outcome in recurring non-anaplastic meningioma. We studied 29 tumor specimens of 8 patients with recurring meningiomas and of 8 age- and gender-matched control patients with non-recurring meningiomas (including meningothelial, transitional, fibroblastic and atypical subtypes) using immunohistochemistry and in-situ hybridization., Results: VEGF protein, VEGF-mRNA, VEGF receptor (VEGFR)-1 mRNA, VEGFR-2 mRNA and hypoxia-inducible factor (HIF)-1-α protein were expressed in 27/29 (93%), 20/27 (74%), 9/27 (33.3%), 12/27 (44.4%) and 5/29 (17.2%) specimens, respectively. VEGFR- 2 mRNA expression was found in 6/8 tumors extracted at first operation in patients with recurring tumors and in none of the control cases (p = 0.007). Microvessel density (MVD) and Ki-67 index values were generally higher in meningiomas with expression of angiogenic factors. The association of high Ki-67 index values with VEGF-mRNA expression was significant (p = 0.04). Time to recurrence was shorter in patients with high MVD than in patients with low MVD (p = 0.027)., Conclusions: High MVD correlates with unfavorable prognosis in our series of recurring meningioma. VEGF and its receptors are frequently expressed in meningiomas and seem important for tumor growth and recurrence. Thus, anti-VEGF therapy in aggressive meningioma seems rational from a pathobiological point of view.

Objective: Vascular endothelial growth factor (VEGF) signaling plays a key role in the pathogenesis of vascular remodeling, including graft arteriosclerosis. Graft arteriosclerosis is the major cause of late organ failure in cardiac transplantation. We used molecular near-infrared fluorescent imaging with an engineered Cy5.5-labeled single-chain VEGF tracer (scVEGF/Cy) to detect VEGF receptors and vascular remodeling in human coronary artery grafts by molecular imaging., Methods and Results: VEGF receptor specificity of probe uptake was shown by flow cytometry in endothelial cells. In severe combined immunodeficiency mice, transplantation of human coronary artery segments into the aorta followed by adoptive transfer of allogeneic human peripheral blood mononuclear cells led to significant neointima formation in the grafts over a period of 4 weeks. Near-infrared fluorescent imaging of transplant recipients at 4 weeks demonstrated focal uptake of scVEGF/Cy in remodeling artery grafts. Uptake specificity was demonstrated using an inactive homolog of scVEGF/Cy. scVEGF/Cy uptake predominantly localized in the neointima of remodeling coronary arteries and correlated with VEGF receptor-1 but not VEGF receptor-2 expression. There was a significant correlation between scVEGF/Cy uptake and transplanted artery neointima area., Conclusions: Molecular imaging of VEGF receptors may provide a noninvasive tool for detection of graft arteriosclerosis in solid organ transplantation.

Introduction: Apical periodontitis is a common inflammatory disease caused by persistent root canal infection and is characterized by bone resorption. Vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) have been described in many pathologic and inflammatory conditions, but their involvement in the development of apical periodontitis has not been thoroughly investigated. The aim of this study was to quantify gene expression and localize VEGF-A, VEGF-C, and VEGF-D and VEGFR-2 and VEGFR-3 in a rat model of apical periodontitis., Methods: Molar pulps were unilaterally exposed to the oral cavity for 10 or 21 days. Jaw sections were used for localization of VEGFs and VEGFRs with immunohistochemistry and identification of cells with double immunofluorescence. Gene expression analysis for VEGF-A, VEGF-C, and VEGFR-3 of periapical tissues was performed with quantitative real-time polymerase chain reaction., Results: All investigated factors and receptors were expressed immunohistochemically in blood vessels at the periodontal ligament of control teeth and were up-regulated during lesion development. In apical lesions, macrophages and neutrophils expressed all studied factors and receptors, with macrophages being an important source of VEGF-C and VEGF-D. Osteoclasts expressed VEGFR-2 and VEGFR-3, and the latter was also identified in fibroblast-like cells in the lesions. VEGF-A and VEGFR-3 gene expression was up-regulated at days 10 and 21 (P < .05)., Conclusions: The current findings indicate that the VEGF family and receptors are involved in vascular remodeling and immune functions during disease development. The presence of VEGFR-2 and VEGFR-3 on osteoclasts indicates that bone resorbing activity is influenced by VEGFs., (Copyright © 2012 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Objective: To study the expression of vascular endothelial growth factors (VEGFs), placental growth factor (PLGF) and their receptors (VEGFR-1, -2, -3) and their regulators (IL-6, CD147) in normal placenta and gestational trophoblastic disease (GTD) in order to evaluate their potential role in the biology of GTD., Study Design: Paraffin sections of 10 normal, first-trimester placentas, 10 partial moles, 10 complete moles, 5 choriocarcinomas and 5 placental site trophoblastic tumors (PSTTs) were studied immunohistochemically for expression of VEGFR-1, VEGFR-2, VEGFR-3, IL-6, PLGF and CD147. Immunolocalization of VEGF, Angiopoietin-1 and Angiopoietin-2 was performed on 5 choriocarcinomas and 5 PSTTs. The levels of VEGF and VEGFR-2 were determined in supernatants and lysates of normal trophoblast, JEG-3 and JAR choriocarcinoma cells with electrochemiluminescence assays., Results: The normal placenta had significantly stronger expression of VEGFR-2 than did those of partial and complete mole (p = 0.001, p = 0.003). VEGF, Angiopoietin-1 and Angiopoietin-2 expression in PSTT were significantly higher than those in choriocarcinoma (p = 0.002, p= 0.01, p = 0.038). Choriocarcinoma showed stronger intensity of staining for VEGFR-3 than did normal placenta, partial and complete mole (p = 0.036, p = 0.038, p = 0.05). Choriocarcinoma had significantly stronger staining of CD147 than did partial and complete mole (p<0.01, p<0.01). PSTT exhibited significantly stronger staining for IL-6 than did choriocarcinoma (p = 0.03)., Conclusion: PSTTs exhibited strong staining for VEGF, and choriocarcinoma showed strong staining for VEGFR-3. Agents that inhibit the activity of VEGF and VEGF receptors may prove to be useful in the therapy of gestational trophoblastic neoplasia.

Aim: Neonates undergoing surgery may receive phototherapy (PT) for the treatment of hyperbilirubinemia. Although the effects of PT on neonatal structures are well documented, the effect of PT on wound healing has not been previously evaluated. An experimental study was performed to evaluate the effect of PT on growth factor levels responsible for wound healing in neonatal rat skin., Materials and Methods: Eighteen Wistar newborn rats (7 ± 2 g) were included in the study. Rats were randomized into 3 groups: control (CG), PT, and sham (SG) (n = 6). Both groups had 1-cm median dorsal skin incision. In CG, 1 × 1 cm of dorsal skin was sampled including the incised skin. The PT group received 5 banks of blue light (wave density, 30-40 μw/cm(2) per nanometer; exposure distance, 45 cm). Phototherapy was started 24 hours after birth and exposed during light period (mean duration, 21 hours to 15 minutes ± 2 hour to 1.5 minutes). Sham group consisted of animals that received a bank of white light with same exposure distance and a total duration of 26 hours to 18 minutes ± 3 hours to 9.1 minutes. After exposure, 1 × 1 cm dorsal skin samples were obtained from both PT and SG groups, including the median incision. The effect of PT was evaluated with the expressions of vascular endothelial growth factor (VEGF), its receptor (VEGF receptor), and transforming growth factor β (TGF-β) in endothelial vessels and fibroblasts of neonatal skin samples., Results: There was no significant difference between groups in VEGF receptor and transforming growth factor β expressions. The VEGF levels in endothelial vessels were significantly decreased in PT and SG when compared with CG (P < .05)., Conclusion: Vascular endothelial growth factor is a mediator of angiogenesis and may decrease in neonatal rat skin after light exposure. It can be suggested that decreased levels of VEGF after PT application may alter angiogenesis and also may adversely affect the healing features of neonatal skin., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The exact nature of juvenile nasopharyngeal angiofibroma (JNA) is still in dispute. In recent years, the main controversy of its nature has focused on hemangioma and vascular malformation. In this study, the immunolocalization of vascular endothelial growth factor (VEGF), VEGF receptor-1/fms-like tyrosine kinase-1 (VEGFR-1/Flt-1), VEGF receptor-2/fetal liver kinase-1 (VEGFR-2/Flk-1), proliferating cell nuclear antigen (PCNA), and CD34 was investigated in 28 cases of JNA and 20 cases of orbital cavernous hemangiomas (OCH). The immunostaining levels of VEGF, Flt-1, and Flk-1 were higher and more frequent in vascular endothelial cells of JNA than those of OCH (p<0.05). The average microvessel density (MVD) marked by CD34 in JNA was (49.3 ± 9.1)/HPF (high power field), which was higher than OCH (29.1 ± 6.7)/HPF (p<0.05). Immunoreactivity of PCNA was localized in both endothelial and stromal cell components of JNA, but was predominantly seen in the stromal cells. However, no PCNA immunoreactivity was identified in any of the stromal and endothelial cells in cases of OCH. The immunostaining levels of CD34, VEGF, Flt-1, Flk-1, and PCNA in JNA were higher than those in OCH. These data support the view that JNA has biological characteristics of an angiogenic histogenetic tumor. In the future, anti-angiogenic therapy may represent a novel treatment strategy for JNA., (Copyright © 2010 Elsevier GmbH. All rights reserved.)

Background/aims: Angiogenesis and lymphangiogenesis are essential for tumor growth and metastasis. Vascular endothelial growth factors and their receptors (VEGFs/VEGFRs) are important to modulate vasculogenesis. Disregulation of the VEGFs/VEGFRs are closely related to tumor progression and prognosis., Methodology: We established an open-label, randomized trial to assess the effect of somatostatin on the patients with primary gastric cancer and total gastrectomy. All the 60 patients were randomly divided into somatostatin pretreatment and placebo groups and we used ELISA, real-time PCR and immunohistochemistry analysis to identify the level of VEGFs., Results: The results showed that the patients pretreated with somatostatin had a significant decrease in serum VEGF level, but not in the tissue mRNA level, and the decrease in serum VEGF was partially dependent on the synthesis and degradation of the protein but not the transcription of mRNA. We also found the tissue Flt-4 (VEGFR-3) protein decreased in somatostatin pretreatment group., Conclusions: We concluded that somatostatin exerts its anti-vasculogenesis effect by downregulating the serum VEGFs and Flt-4 level. Somatostatin can be used as an important adjuvant to improve the survival of gastric cancer patients.

The present study was conducted to demonstrate of the immunohistochemical localization of vascular endothelial growth factor (VEGF) and its receptors (flt1/fms, flk1/KDR and flt4) as well as vascular endothelial growth inhibitor (VEGI) and to determine the correlation of VEGF and its receptors and VEGI with serum sex steroids (estrogen and progesterone) in the bovine uterus during the sexual cycle. The stage of the estrous cycle in 30 Holstein cattle was assessed based on the gross and histological appearance of the ovaries and uterus and on blood steroid hormone levels. Tissue samples obtained from the uterus were fixed in 10% formaldehyde for routine histological processing. During both follicular and luteal phases, positive cytoplasmic and membrane staining was achieved for VEGF and its receptors (flt1/fms, flk1/KDR and flt4) as well as VEGI in the luminal and glandular epithelial cells, the connective tissue and smooth muscle cells, and the vascular endothelial cells and smooth muscle cells in the uterus. The intensity, proportional and total scores determined for VEGF and its receptors (flt1/fms and flt4) as well as VEGI were greater in the luminal and glandular epithelial cells compared to the connective tissue and smooth muscle cells (P < 0.05). Furthermore, the number and intensity of the flk1/KDR positive cells were greater among the connective tissue cells compared to the luminal and glandular epithelial cells (P < 0.05). As a result, it was determined that the expression of VEGF and its receptors as well as VEGI in the bovine uterus during the follicular and luteal phases varied with different cell types. This suggests that depending on the stage of the sexual cycle, these factors may mediate the establishment of an appropriate environment for the nutritional supply and implantation of the embryo primarily due to the stimulation of angiogenesis but also through the increase in the secretory activity of the epithelial cells in the uterus. Furthermore, this indicates that ovarian steroid hormones play a significant role in regulating the expression of VEGF and its receptors as well as VEGI., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Copper has five radioisotopes ((60)Cu, (61)Cu, (62)Cu, (64)Cu, and (67)Cu) that can be used in copper radiopharmaceuticals. These radioisotopes decay by mixed emissions of β+, β-, and γ with a wide range of half-lives from 9.74 min ((62)Cu) to 2.58 d ((67)Cu), which enable the design and synthesis of a variety of radiopharmaceuticals for different biomedical applications in diagnostic and therapeutic nuclear medicine. However, due to the availability and production cost, the research efforts in copper radiopharmaceuticals are mainly focused on the use of (64)Cu (t(1/2) = 12.7 h; 17.4% β+, 43% EC, 39% β-), a radioisotope with low positron energy (E β+max = 0.656 MeV) that is ideal for positron emission tomography (PET) imaging quantification and β- emissions along with Auger electron for radiotherapy. Driven by the ever-increasing availability of preclinical and clinical PET scanners, a considerable interest has been seen in the development of novel copper radiopharmaceuticals in the past decade for a variety of diseases as represented by PET imaging of cancer. To avoid unnecessary literature redundancy, this review focuses on the unrepresented research aspects of copper chemistry (e.g. electrochemistry) and their uses in the evaluation of novel nuclear imaging probe design and recent advances in the field towards the practical use of copper radiopharmaceuticals.

Vascular endothelial growth factor (VEGF) has been implicated in the regulation of vesicular transport of amniotic fluid via caveolae across the amnion. This study tested the hypothesis that VEGF regulates caveolar function by stimulating caveolin-1 expression and phosphorylation in ovine amniotic epithelial cells (oAECs). Using primary cultures of oAECs, caveolin-1 was identified by immunofluorescent staining. Caveolin-1 messenger RNA (mRNA) abundance was determined by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and protein by Western blotting. The effects of VEGF( 165) on caveolin-1 expression and phosphorylation were determined. Caveolin-1 immunoreactivity was detected in oAECs. In response to 10 ng/mL VEGF( 165), caveolin-1 mRNA levels increased whereas the protein levels were unaffected. Furthermore, VEGF stimulated caveolin-1 phosphorylation, an effect abrogated by the inhibition of c-Src protein kinase. These data suggest that VEGF upregulates caveolin-1 activity through c-Src signaling pathways. Our observations support the hypothesis that VEGF regulates amniotic fluid transport across the amnion by stimulating caveolin-1 activity to mediate caveolar function in amnion cells.

Background: Matrix metalloproteinases (MMPs) play an important role in degradation of the extracellular matrix of injured tissue. MMP-9 expression increases in fibrillating atrial tissue; however, the mechanism for this increase has not been clarified., Methods and Results: Changes in the expression of vascular endothelial growth factor (VEGF), VEGF receptors, and hypoxia-induced transcription factor-1alpha (HIF-1alpha) in fibrillating atrial tissue were investigated. Atrial tissue samples were obtained from 13 patients with atrial fibrillation (AF) and 25 patients without a history of AF (regular sinus rhythm, RSR) undergoing cardiac operations. Western blot, real-time polymerase chain reaction, and immunofluorescence analyses of the expression of VEGF, VEGF receptors, and HIF-1alpha were performed. The VEGF mRNA and protein levels increased significantly in the AF group compared with the RSR group (P<0.05), and the expression of HIF-1alpha protein was also significantly higher in the AF group. VEGF receptor-1 mRNA, a high-affinity receptor for VEGF, but not VEGF receptor-2 mRNA, was upregulated in the atria of the AF group (P<0.05). Immunofluorescence staining revealed excess production and co-localization of HIF-1alpha, VEGF and MMP-9 in the endothelium of the atrial arteries in the AF group., Conclusions: It is possible that upregulation of HIF-1/VEGF is involved in the enhancement of MMP-9 expression under hypoxic conditions.

Aim: To describe the case history and new histopathological findings of a young woman suffering from moyamoya disease., Methods: The patient underwent brain computed tomography, magnetic resonance imaging and brain angiography. Vessel samples of a. temporalis superficialis were processed by standard histopathological and immunohistochemical methods by analysis of VEGF, VEGFR and nestin expression., Results: Brain angiography revealed both internal carotid artery stenoses and stenoses of the anterior cerebral arteries. Stenotic parts of vessels were accompanied by coiled and elongated vessels with a picture of "smoke puffs carried away by breeze" after contrast medium application. Histological examination showed: obstruction of lumen, fibrocellular intimal thickening, tortuosity and disruption of internal elastic lamina. Imunohistochemistry confirmed a defect of the internal elastic membrane of the muscular arteria and progressive intimal thickening accompanied by abnormal smooth muscle cells and, VEGF/VEGFR expression in intima. Nestin positivity in endothelium of arteria indicated that endothelial cells are activated., Conclusion: We found that the endothelium of affected vessels is nestin positive. This, together with the finding of VEGF/VEGFR expression, might suggest an active angiogenetic process We present a new conception of pathogenesis but further studies with higher number of patients are necessary to elucidate the role of these growth factors in the moyamoya disease.

Tyrosine kinase inhibitors against the receptors of vascular endothelial growth factor (VEGFR), epidermal growth factor (EGFR) and the platelet derived growth factor (PDGFR) are increasingly used in the treatment of progressive cancers. However, the expression of these receptors especially in lung metastases has not been examined. Tissue specimen from 35 lung metastases of 33 patients with renal cell carcinoma (n=8), sarcoma (n=10), colorectal carcinoma (n=6), otolaryngologic carcinoma (OLC, n=4), testicular and endometrial cancer (n=1 each), malignant melanoma (n=1), adrenal cancer (n=2), malignant fibrous histiocytoma and malignant peripheral nerve sheath tumor (n=1 each) have been immunohistochemically tested for the expression of PDGFR alpha/beta, VEGFR and EGFR. None of the patients had been pretreated with angiogenic inhibitors prior to metastasectomy. PDGFRalpha was expressed in all metastases; 31% stained negative for PDGFRbeta, 86% negative for VEGFR and 45% negative for EGFR. Primary tumors revealed positive staining for PDGFRalpha in 88%, for PDGFRbeta in 59%, for VEGFR in 0% and for EGFR in 18%. Our investigation of a pilot character represents a 'biomarker-based' analysis of pulmonary metastases of different primary tumors; we conclude that an immediate 'tumor profiling' at initial diagnosis should be considered in order to guide tumor therapy individually.

Angiogenesis is a multistep process regulated by pro- and antiangiogenic factors. In order to grow and metastasize, tumors need a constant supply of oxygen and nutrients. For growth beyond 1-2 mm in size, tumors are dependent on angiogenesis. Inhibition of angiogenesis is a new cancer treatment strategy that is now widely investigated clinically. Researchers have begun to search for objective measures that indicate pharmacologic responses to antiangiogenic drugs. Therefore, there is a great interest in techniques to visualize angiogenesis in growing tumors noninvasively. Several markers have been described that are preferentially expressed on newly formed blood vessels in tumors (alpha(v)beta(3) integrin, vascular endothelial growth factor, and its receptor, prostate-specific membrane antigen) and in the extracellular matrix surrounding newly formed blood vessels (extra domain B of fibronectin, Tenascin-C, matrix metalloproteinases, and Robo-4). Several ligands targeting these markers have been tested as a radiotracer for imaging angiogenesis in tumors. The potential of some of these tracers, such as radiolabeled cyclic RGD peptides and radiolabeled anti-PSMA antibodies, has already been tested in cancer patients, while for markers such as Robo-4, the ligand has not yet been identified. In this review, an overview on the currently used nuclear imaging probes for noninvasive visualization of tumor angiogenesis is given.

Our goal was to discover genes differentially expressed in the perichondrium (PC) of the mandibular condylar cartilage (MCC) that might enhance regenerative medicine or orthopaedic therapies directed at the tissues of the temporomandibular joint. We used targeted gene arrays (osteogenesis, stem cell) to identify genes preferentially expressed in the PC and the cartilaginous (C) portions of the MCC in 2-day-old mice. Genes with higher expression in the PC sample related to growth factor ligand-receptor interactions [FGF-13 (6.4x), FGF-18 (4x), NCAM (2x); PGDF receptors, transforming growth factor (TGF)-beta and IGF-1], the Notch isoforms (especially Notch 3 and 4) and their ligands or structural proteins/proteoglycans [collagen XIV (21x), collagen XVIII (4x), decorin (2.5x)]. Genes with higher expression in the C sample consisted mostly of known cartilage-specific genes [aggrecan (11x), procollagens X (33x), XI (14x), IX (4.5x), Sox 9 (4.4x) and Indian hedgehog (6.7x)]. However, the functional or structural roles of several genes that were expressed at higher levels in the PC sample are unclear [myogenic factor (Myf) 9 (9x), tooth-related genes such as tuftelin (2.5x) and dentin sialophosphoprotein (1.6x), VEGF-B (2x) and its receptors (3-4x) and sclerostin (1.7x)]. FGF, Notch and TGF-beta signalling may be important regulators of MCC proliferation and differentiation; the relatively high expression of genes such as Myf6 and VEGF-B and its receptors suggests a degree of unsuspected plasticity in PC cells.

This is the first report of the Southwest oncology group phase II trial of single agent bevacizumab in patients with relapsed, aggressive non-Hodgkin lymphoma (NHL). Fifty-two patients in first or second relapse with diffuse large B-cell or mantle cell lymphoma were enrolled. Patients were treated with bevacizumab at 10 mg/kg every 2 weeks. Therapy was well tolerated with no unexpected toxicities observed. Six-month progression-free survival (PFS) was 16% with a response rate of 2% and median duration of response or stable disease of 5.2 months (range 3.5-72.7). Vascular endothelial growth factor A (VEGF) and VEGF receptor expression was observed in 70% and 65% of specimens, respectively. In an exploratory subgroup analysis, baseline urine VEGF and plasma vascular cell adhesion molecule-1 (VCAM) levels correlated with survival. Prolonged PFS in several patients as well as biomarker studies suggest the VEGF pathway plays an important role in aggressive NHL. Clinical trials combining active chemotherapy regimens with VEGF targeted agents are currently in progress.

Introduction: Protein expressions of angiogenic markers provide prognostic information on patients with non-small cell lung cancer (NSCLC). Both expression and its prognostic impact may be associated with patient selection. Data addressing the prognostic relevance of angiogenic marker expression in NSCLC patients treated with postoperative radiotherapy (PORT) is warranted., Methods: In 55 patients with stage I-IIIA NSCLC administered PORT between 1990 and 2005, we have reviewed the clinicopathological variables and investigated the expression of angiogenic markers in tumor and stroma in tissue micro arrays., Results: The median follow-up was 114 months and the major end point disease-specific survival (DSS). Univariate analysis showed that high expression of vascular endothelial growth factor A (VEGF-A) (p = 0.004), VEGF receptor-1 (VEGFR-1, p = 0.028), VEGFR-2 (p = 0.021), VEGFR-3 (p = 0.001) and platelet derived growth factor (PDGF) in tumors correlated significantly with a poor survival. Inversely, high basic fibroblast growth factor (bFGF) expression in stroma was associated with significantly improved DSS (p = 0.017). In multivariate analyses, tumor PDGF expression appeared independently associated with a shorter DSS (hazard ratio 5.42, p = 0.002) and stromal bFGF expression an increased DSS (hazard ratio 0.077, p < 0.001)., Conclusions: Tumor PDGF expression was an independent negative prognostic factor and stromal bFGF expression an independent positive prognostic factor for survival in NSCLC receiving PORT.

Background and Purpose: Vascular endothelial growth factor (VEGF) and VEGF receptors (VEGFRs) play important roles during neurovascular repair after stroke. In this study, we imaged VEGFR expression with positron emission tomography (PET) to noninvasively analyze poststroke angiogenesis., Methods: Female Sprague-Dawley rats after distal middle cerebral artery occlusion surgery were subjected to weekly MRI, (18)F-FDG PET, and (64)Cu-DOTA-VEGF(121) PET scans. Several control experiments were performed to confirm the VEGFR specificity of (64)Cu-DOTA-VEGF(121) uptake in the stroke border zone. VEGFR, BrdU, lectin staining, and (125)I-VEGF(165) autoradiography on stroke brain tissue slices were performed to validate the in vivo findings., Results: T2-weighed MRI correlated with the "cold spot" on (18)F-FDG PET for rats undergoing distal middle cerebral artery occlusion surgery. The (64)Cu-DOTA-VEGF(121) uptake in the stroke border zone peaked at approximately 10 days after surgery, indicating neovascularization as confirmed by histology (VEGFR-2, BrdU, and lectin staining). VEGFR specificity of (64)Cu-DOTA-VEGF(121) uptake was confirmed by significantly lower uptake of (64)Cu-DOTA-VEGF(mutant) in vivo and intense (125)I-VEGF(165) uptake ex vivo in the stroke border zone. No appreciable uptake of (64)Cu-DOTA-VEGF(121) was observed in the brain of sham-operated rats., Conclusions: For the first time to our knowledge, we successfully evaluated the VEGFR expression kinetics noninvasively in a rat stroke model. In vivo imaging of VEGFR expression could become a significant clinical tool to plan and monitor therapies aimed at improving poststroke angiogenesis.

Infantile hemangioma is a benign endothelial tumor composed of disorganized blood vessels. It exhibits a unique life cycle of rapid postnatal growth followed by slow regression to a fibrofatty residuum. Here, we have reported the isolation of multipotential stem cells from hemangioma tissue that give rise to hemangioma-like lesions in immunodeficient mice. Cells were isolated based on expression of the stem cell marker CD133 and expanded from single cells as clonal populations. The CD133-selected cells generated human blood vessels 7 days after implantation in immunodeficient mice. Cell retrieval experiments showed the cells could again form vessels when transplanted into secondary recipients. The human vessels expressed GLUT-1 and merosin, immunodiagnostic markers for infantile hemangioma. Two months after implantation, the number of blood vessels diminished and human adipocytes became evident. Lentiviral expression of GFP was used to confirm that the hemangioma-derived cells formed the blood vessels and adipocytes in the immunodeficient mice. Thus, when transplanted into immunodeficient mice, hemangioma-derived cells recapitulated the unique evolution of infantile hemangioma--the formation of blood vessels followed by involution to fatty tissue. In summary, this study identifies a stem cell as the cellular origin of infantile hemangioma and describes for what we believe is the first time an animal model for this common tumor of infancy.

Objective: To study the expression regularity of vascular endothelial growth factor (VEGF) during the process of fracture healing, and the type of VEGF receptor expressed in the vascular endothelial cells of the fracture site., Methods: The fracture model was made in the middle part of left radius in 35 rabbits. The specimens from the fracture site were harvested at 8, 24, 72 hours and 1, 3, 5, 8 weeks, and then fixed, decalcified, and sectioned frozenly to detect the expression of VEGF and its receptor at the fracture site by in situ hybridization and immunochemical assays., Results: VEGF mRNA and VEGF expression was detected in many kinds of cells at the fracture site during 8 hours to 8 weeks after fracture. Flt1 receptor of VEGF was found in the vascular endothelial cells at the fracture site during 8 hours to 8 weeks after fracture, and strong expression of flk1 receptor was detected from 3 days to 3 weeks after fracture., Conclusions: The expression of VEGF and flt1 receptor appears during the whole course of fracture healing, especially from 1 to 3 weeks. Flk1 receptor is highly expressed in a definite period after fracture. VEGF is proved to be involved in the vascular reconstruction and fracture healing.

Unlabelled: Myocardial infarction (MI) leads to left ventricular (LV) remodeling, which leads to the activation of growth factors such as vascular endothelial growth factor (VEGF). However, the kinetics of a growth factor's receptor expression, such as VEGF, in the living subject has not yet been described. We have developed a PET tracer (64Cu-DOTA-VEGF121 [DOTA is 1,4,7,10-tetraazadodecane-N,N',N'',N'''-tetraacetic acid]) to image VEGF receptor (VEGFR) expression after MI in the living subject., Methods: In Sprague-Dawley rats, MI was induced by ligation of the left coronary artery and confirmed by ultrasound (n = 8). To image and study the kinetics of VEGFRs, 64Cu-DOTA-VEGF121 PET scans were performed before MI induction (baseline) and on days 3, 10, 17, and 24 after MI. Sham-operated animals served as controls (n = 3)., Results: Myocardial origin of the 64Cu-DOTA-VEGF121 signal was confirmed by CT coregistration and autoradiography. VEGFR specificity of the 64Cu-DOTA-VEGF121 probe was confirmed by in vivo use of a 64Cu-DOTA-VEGFmutant. Baseline myocardial uptake of 64Cu-DOTA-VEGF121 was minimal (0.30 +/- 0.07 %ID/g [percentage injected dose per gram of tissue]); it increased significantly after MI (day 3, 0.97 +/- 0.05 %ID/g; P < 0.05 vs. baseline) and remained elevated for 2 wk (up to day 17 after MI), after which time it returned to baseline levels., Conclusion: We demonstrate the feasibility of imaging VEGFRs in the myocardium. In summary, we imaged and described the kinetics of 64Cu-DOTA-VEGF121 uptake in a rat model of MI. Studies such as the one presented here will likely play a major role when studying pathophysiology and assessing therapies in different animal models of disease and, potentially, in patients.

Juvenile nasopharyngeal angiofibroma (JNA) is a rare, invasive, and locally destructive tumor of the nasopharynx. The Wnt pathway, angiogenetic and hormonal factors are involved in the pathophysiology of JNA; it can result in an extracolonic manifestation of familial adenomatous polyposis (FAP) or in a sporadic tumor. All patients who underwent resection of JNA between 1991 and 2006 at the University of Modena and Reggio Emilia were studied to identify immunohistochemical markers of associated FAP syndrome. Paraffin-embedded JNA samples were analyzed immunohistochemically for the expression of adenomatous polyposis coli (APC), beta-catenin, E-cadherin, androgen receptor, and vascular endothelial growth factors receptor (VEGFR2). In one out of the 4 (25%) young patients affected by JNA the diagnosis of FAP syndrome linked to APC mutation was made. All of the sporadic and familial JNA tumors showed nuclear staining of beta-catenin, whereas altered APC expression was seen only in FAP-associated JNA. All cases were stained with VEGFR2. A combined clinical, immunohistochemical, and biomolecular screening may be useful for the identification of FAP among patients with a diagnosis of JNA. The Wnt pathway can be involved in the JNA pathogenesis either by somatic mutations of beta-catenin or by germline APC mutations. As the VEGFR has an important impact on the pathogenesis of JNA, we suggest that a targeted therapy with monoclonal antibodies against VEGFR might lead to a specific chemoprevention and treatment of these tumors and their recurrences.

Bone marrow-derived endothelial progenitor cells (EPC) play an important role in neovascularisation and tumor growth. However, the clinical relevance of EPCs on blood vessel formation in non-small cell lung cancer (NSCLC) is unclear. EPC numbers in circulation are very low and therefore their detection is technically challenging. In the present study, 10 NSCLC patients and 5 healthy controls were included. Patients underwent blood analyses before and after surgery. EPCs were isolated from whole blood by magnetic cell sorting to CD34 (MACS). Afterwards, FACS analyses using antibodies against CD133, CD34, VEGFR2 and CD45 and and immunocytological staining to CD133 on cytospins (MCA) were performed. Cryostat sections of tumor samples were stained for CD133, CD31 and cytokeratin A7. Serum levels of the vascular endothelial growth factor (VEGF) were quantified by sandwich ELISA. Compared to the control group NSCLC patients showed significantly elevated EPC counts and VEGF levels in peripheral blood before and after surgery. From a methodological point of view, the tested procedure (MCA) was validated as compared to the standard FACS analyses (CD34+/VEGFR2+). MCA proved to have a very high sensitivity and even allowed the identification of singular positive EPCs.

Recent experimental and clinical studies have placed new emphasis on the role of angiogenesis in chronic inflammatory disease. Vascular endothelial growth factor (VEGF) and its receptors are the best characterized system in the regulation of rheumatoid arthritis (RA) by angiogenesis. Furthermore, in addition to its angiogenic role, VEGF can act as a direct proinflammatory mediator during the pathogenesis of RA, and protect rheumatoid synoviocytes from apoptosis, which contributes to synovial hyperplasia. Therefore, the developments of synovial inflammation, hyperplasia, and angiogenesis in the joints of RA patients seem to be regulated by a common cue, namely, VEGF. Agents that target VEGF, such as anti-VEGF antibody and aptamer, have yielded promising clinical data in patients with cancer or macular degeneration, and in RA patients, pharmacologic modulations targeting VEGF or its receptor may offer new therapeutic approaches. In this review, the authors integrate current knowledge of VEGF signaling and information on VEGF antagonists gleaned experimentally and place emphasis on the use of synthetic anti-VEGF hexapeptide to prevent VEGF interacting with its receptor.

Immunohistochemical analysis of human colon cancers growing in the cecal walls of nude mice revealed that epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor 2 (VEGFR2) were expressed by different tumor cells and tumor-associated endothelial cells, whereas platelet-derived growth factor receptor (PDGFR)beta was expressed by tumor-associated endothelial cells and pericytes. We hypothesized that treatment of nude mice with AEE788 (an inhibitor of EGFR and VEGFR phosphorylation) and STI571 (an inhibitor of PDGFRbeta phosphorylation) combined with irinotecan would overcome the intratumoral heterogeneity of these growth factors and efficiently inhibit colon cancer growth and metastasis. We implanted HT29 and KM12SM cells into the cecal walls of nude mice. Two weeks later, the mice were treated with oral vehicle solution; oral AEE788, oral STI571, or intraperitoneal injection of irinotecan as single agents; or the various combinations of these agents. We then assessed the mice for tumor growth and metastasis. Immunohistochemical analyses revealed that oral AEE788 suppressed proliferation and increased apoptosis of tumor cells and tumor-associated endothelial cells. Oral STI571 increased apoptosis of tumor-associated endothelial cells and pericytes. The combination of AEE788, STI571, and irinotecan produced the greatest inhibition of primary tumor growth and metastasis. Collectively, these data demonstrate that only targeting multiple tyrosine kinase receptors on colon cancer cells and tumor-associated stromal cells can overcome the effects of biologic heterogeneity for resistance to treatment and has the potential to improve therapeutic outcome for patients with this disease.

Background: Angiosarcomas are malignant mesenchymal neoplasms, including sarcomas of presumptive vascular endothelial origin and sarcomas of probable lymphatic origin. It is, however, often difficult to determine whether they are from blood vascular or lymphatic endothelium. The majority of angiosarcomas are thought to originate from vascular endothelia and spread via bloodstream to lung, but lymphatic metastases can occur., Methods: We investigated immunohistochemical expression of vascular endothelial growth factors (VEGF-A, VEGF-C) and their receptors (VEGFR-1, VEGFR-2, VEGFR-3) in a series of 34 angiosarcomas., Results: VEGF-A was expressed by 32/34 (94%), VEGF-C by 4/34 (12%), VEGFR-1 by 32/34 (94%), VEGFR-2 by 22/34 (65%), and VEGFR-3 by 27/34 (79%). Patients who expressed low or no VEGFR-2 showed a significantly unfavorable prognosis by log-rank test (P = 0.010) and multivariate analysis (hazard ratio, 5.16; 95% CI, 1.40-19.04; P = 0.014). VEGFR-1 and VEGFR-3 were not significantly associated with patients' prognosis., Conclusions: VEGF-A and VEGFR-1 were detected in diverse subtypes of angiosarcomas. In cooperation, VEGF-A and VEGF-C are likely to be involved in the development of angiosarcoma associated with lymphedema. VEGF-C expression may cause susceptibility to lymphatic metastasis through tumor lymphangiogenesis. Angiosarcoma of the scalp, which is traditionally considered as a true hemangiosarcoma, may include some cases of lymphatic origin., ((c) 2007 Wiley-Liss, Inc.)

Vascular endothelial growth factor (VEGF) has been shown to be the major mediator of physiologic and pathologic angiogenesis. VEGF was initially thought to be an endothelial cell specific ligand, but recently, VEGF has been shown to mediate tumor cell function via activation of receptors on tumor cells themselves. Here, we review the expression patterns and binding profiles of the VEGF receptors and their ligands on gastrointestinal tumor cells. Furthermore, we describe the current knowledge in regards to the function of these receptors on tumor cells. Elucidating the function of VEGF receptors on tumor cells should help us to better understand the potential mechanisms of action of anti-VEGF therapies.

Purpose: The vascular endothelial growth factors (VEGF-A, -C, -D) and the VEGF receptors (VEGFR-1, -2, and -3) are important molecular markers in angiogenesis and lymphangiogenesis. This study elucidates the prognostic significance of these molecular markers in tumor cells as well as in the tumor stroma of resected non-small cell lung cancer tumors., Experimental Design: Tumor tissue samples from 335 resected patients with stage I to IIIA disease were obtained and tissue microarrays were constructed from duplicate cores of tumor cells and surrounding stromal tissue from each resected specimen. Immunohistochemistry was used to evaluate the expression of each molecular marker. Microvessel density was assessed by CD34 immunohistochemical staining., Results: In univariate analyses, high tumor cell expression of VEGF-A (P = 0.0005), VEGFR-1 (P = 0.013), VEGFR-2 (P = 0.006), and VEGFR-3 (P = 0.0003) were negative prognostic indicators for disease-specific survival (DSS). In tumor stroma, however, high expression of VEGF-A (P = 0.017), VEGF-C (P = 0.003), VEGF-D (P = 0.009), VEGFR-1 (P = 0.01), and VEGFR-2 (P = 0.019) correlated with good prognosis. There was no significant correlation between microvessel density and DSS. In multivariate analyses, high expression in tumor cells of VEGFR-3 (P = 0.007) was an independent negative prognostic factor for DSS, whereas in stromal cells, high VEGF-C (P = 0.004) expression had an independent positive survival impact., Conclusion: These are the first tissue microarray data in non-small cell lung cancers showing a positive prognostic impact by highly expressed angiogenic markers in tumor stroma, with VEGF-C as a major independent prognostic indicator.

Angiogenesis plays a central role in a variety of important biological processes such as reproduction, tissue development, and wound healing, as well as being critical to tumor formation in cancer. The development of chromosomal substitution (consomic) rat strains has permitted the chromosomal localization of genetic factors critical to angiogenesis, but many questions remain as to the mechanisms involved. Here we utilize a novel cell capture assay to assess changes in the functional expression of vascular endothelial growth factor (VEGF) receptors on the surface of vascular endothelial cells isolated from rat strains that are normal or impaired in angiogenesis. We show that functional VEGF receptor expression is increased under hypoxic conditions in rat strains that exhibit normal angiogenesis but not in a strain impaired in angiogenesis. This result implicates the dysregulation of VEGF receptor expression levels on the endothelial cell surface as a key factor in impaired angiogenesis.

Aims: Both aerobic and resistance exercise training promote skeletal muscle angiogenesis. Acute aerobic exercise increases several pro-angiogenic pathways, the best characterized being increases in vascular endothelial growth factor (VEGF). We hypothesized that acute resistance exercise also increases skeletal muscle angiogenic growth factor [VEGF and angiopoietin (Ang)] expression., Methods: Seven young, sedentary individuals had vastus lateralis muscle biopsies and blood drawn prior to and at 0, 2 and 4 h post-resistance exercise for the measurement of VEGF; VEGF receptor [KDR, Flt-1 and neuropilin 1 (Nrp1)]; Ang1 and Ang2; and the angiopoietin receptor--Tie2 expression. Resistance exercise consisted of progressive knee extensor (KE) exercise to determine one repetition maximum (1-RM) followed by three sets of 10 repetitions (3 x 10) of KE exercise at 60-80% of 1-RM., Results: Resistance exercise significantly increased skeletal muscle VEGF mRNA and protein and plasma VEGF protein at 2 and 4 h. Resistance exercise increased KDR mRNA and Tie2 mRNA at 4 h and Nrp1 mRNA at 2 and 4 h. Skeletal muscle Flt-1, Ang1, Ang2 and Ang2/Ang1 ratio mRNA were not altered by resistance exercise., Conclusions: These findings suggest that acute resistance exercise increases skeletal muscle VEGF, VEGF receptor and angiopoietin receptor expression. The increases in muscle angiogenic growth factor expression in response to acute resistance exercise are similar in timing and magnitude with responses to acute aerobic exercise and are consistent with resistance exercise promoting muscle angiogenesis.

The status of angiogenic switching was examined in alveolar capillaries of primary lung adenocarcinoma (ADC) from 10 patients and primary squamous cell carcinoma (SCC) from 11 patients, using immunostaining for CD31, thrombomodulin, von Willebrand factor (vWF), collagen types IV and VII, and alpha-smooth muscle actin (alpha-SMA). We applied the TdT-mediated dUTP nick-end labeling assay and the reverse transcription-polymerase chain reaction for vascular endothelial growth factor (VEGF) and its receptors (VEGFRs). In bronchioloalveolar and papillary subtypes of ADC, the neoplastic cells, replacing the normal alveolar epithelial cells, had spread over alveolar walls and adhered firmly to alveolar interstitium as shown by the development of type IV collagen. Neoplastic cells of SCC were characterized by local proliferation in alveolar sacs without firm attachment to alveolar walls. Tumor lesions of SCC had often developed necrotic foci of various size. In ADC and SCC, alveolar capillary endothelial cells newly obtained reactivity to vWF. Such segments of endothelial cells lost surface thrombomodulin expression. CD31 was consistently expressed in normal and ADC tissues, but each endothelial cell marker was often attenuated or even lost in SCC, suggesting degeneration or necrosis of the alveolar capillaries. The capillary pericytes and interstitial fibroblasts were often hypertrophic and developed alpha-SMA in the cytoplasm in ADC, but they became atrophic in SCC. In ADC, apoptosis occurred in cells of alveolar capillaries more frequently in the peripheral zone than in the deeper zone of the tumor, whereas the frequency was not consistent in SCC. In microdissected alveolar wall tissues, mRNA expression patterns of VEGF isoforms and VEGFRs were similar in both ADC and SCC. In ADC, de novo angiogenic switching took place in cytoplasm as a unit of cells segments in alveolar capillary endothelium. Suppression of angiogenic switching in SCC implies that factors other than VEGF-VEGFR interaction, such as physical contact and compression of tumor cells, might play a critical role in alveolar capillaries.

Polyclonal antisera for vascular endothelial growth factor (VEGF) and its two main receptor molecules, VEGF-I (Flt) and VEGF-II (KDR), were used in a conventional immunocytochemical staining method to localize these angiogenic ligand molecules in the endometrium and placenta of the mare during the oestrous cycle and pregnancy. The anti-VEGF and anti-Flt sera both labelled the lumenal and glandular epithelia of the endometrium throughout the oestrous cycle and both the invasive trophoblast cells of the endometrial cups and the non-invasive trophoblast of the allantochorion in pregnancy. The anti-KDR serum likewise stained the maternal and foetal epithelial layers during the oestrous cycle and pregnancy and it also labelled fibroblast-like cells in the endometrial and allantoic stromas and the endothelium of foetal and maternal capillaries. The results demonstrated that constant supplies of the principal vasculogenic and angiogenic factor, VEGF, and its two major receptors, Flt and KDR, are available on both the maternal and foetal sides of the placental barrier throughout gestation in the mare. They are presumed to facilitate the continuing development of the extensive foetal and maternal capillary networks that are such prominent features within the microplacentomes of the diffuse, epitheliochorial equine placenta.

Little is known about the mechanisms regulating the growth of early preantral follicles, especially in nonrodent mammalian species. To test the hypothesis that vascular endothelial growth factor (VEGF) promotes the primary to secondary follicle transition, pieces of bovine fetal ovarian cortex were cultured with VEGF (0, 1, 10, or 100 ng/ml) for 0 or 10 days, followed by morphometric analysis. On day 0, cortical pieces contained mostly primordial follicles, but after 10 days in vitro most primordial follicles had activated, differentiating into primary follicles. VEGF had no effect on the numbers of primordial or primary follicles, compared with untreated controls, but all doses increased the number of secondary follicles. In the second experiment, a range of lower doses of VEGF (0.1-10 ng/ml) increased the number of secondary follicles in a dose-dependent manner. Analysis of VEGF transcripts by RT-PCR showed expression of mRNA for three isoforms of VEGF (VEGF121, 165, and 189) in fetal bovine ovarian cortex, with VEGF121 and 165 expressed predominantly and levels of mRNA for VEGF121 and 189 increasing after day 211 of gestation, when the first secondary follicles appear. Expression of mRNA for both VEGF receptors (flt-1 and flk-1) was also detected, but did not change with the development of fetal ovaries. Immunohistochemistry revealed positive staining for VEGF in blood vessels and in follicle cells of secondary follicles, consistent with Western blot analyses showing increases in VEGF protein as ovarian development progressed. Taken together, the results provide support for a role for VEGF in the primary to secondary follicle transition.

Purpose: Gastrointestinal neuroendocrine tumors (NET) are rare heterogeneous tumors that hypersecrete neuropeptides. The scarcity of good gastrointestinal NET models has limited the ability to study potential therapeutic agents. We describe and characterize the establishment of a human midgut carcinoid tumor cell line carcinoid tumor 2 (CNDT2)., Experimental Design: Tumor cells (CNDT2) were isolated from a liver metastasis from a patient with a primary ileal carcinoid. After 9 weeks in culture, the cells were plated in soft agar, and cells from a single colony were put back in culture (CNDT2.1). Those CNDT2.1 cells were injected s.c. into nude mice. Cells were isolated from a single resultant tumor (CNDT2.5), cultured, and characterized by electron microscopy, reverse transcription-PCR, serotonin enzyme immunoassay, Western blotting, and immunohistochemical analysis for NET markers and potential therapeutic targets., Results: CNDT2 cells grew in monolayers in vitro, formed colonies in soft agar, and formed tumors in mice. Electron microscopy revealed round, pleomorphic, electron-dense neurosecretory granules characteristic of NETs. Tumor xenografts exhibited the appearance of NETs with small "salt-and-pepper" nuclei on H&E staining and chromogranin A, synaptophysin, and CD56 on immunohistochemical staining. CNDT2.5 cells produced serotonin and expressed insulin-like growth factor receptor-I, platelet-derived growth factor receptor-beta, vascular endothelial growth factor receptor-1, cMET, epidermal growth factor receptor, neuropilin-1, and somatostatin receptors 1 to 5. Cytogenetic analysis revealed the presence of deletions at 2p and 6q and numerous translocations., Conclusion: The establishment of this human midgut carcinoid tumor cell line may serve as a useful model system for studying cell biology and novel targeted agents in preclinical models.