The Polyclonal Antibodies Induced by VBP3 Complex Peptide Targeting Angiogenesis and Tumor Suppression

Basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) play a critical role in tumor-associated angiogenesis and have become the targets of anti-tumor therapy. The BALB/c mice were immunized with VEGF/bFGF complex peptide (VBP3) constructed with different epitope peptides of human VEGF and bFGF. The results of the immunogenicity showed that the VBP3 could effectively stimulate immune response in mice and elicit the mice to produce high titer specific anti-VEGF and anti-bFGF antibodies (anti-VBP3 antibodies). The polyclonal anti-VBP3 antibodies separated from the mouse immune serum could effectively inhibit the proliferation, migration and tube formation of human umbilical vein endothelial cells (HUVECs) and block the proliferation and migration of lung cancer A549 cells. Besides, the anti-VBP3 antibodies could effectively inhibit tumor growth and tumor angiogenesis in BABL/c nude mice. The results demonstrated that the VBP3 complex peptide could elicit the body to produce the high titer anti-VEGF and anti-bFGF antibodies, which showed anti-tumor and anti-angiogenic effects in vitro and in vivo. The results revealed that the VBP3 complex peptide could be used as a potential peptide vaccine in tumor therapy.     

Large‐scale production,purification, and function of a tumor multi‐epitope vaccine: Peptibody with bFGF/VEGFA

In tumor tissue, basic fibroblast growth factor (bFGF) and vascular endothelial growth factor A (VEGFA) promote tumorigenesis by activating angiogenesis, but targeting single factor may produce drug resistance and compensatory angiogenesis. The Peptibody with bFGF/VEGFA was designed to simultaneously blockade these two factors. We were aiming to produce this Fc fusion protein in a large scale. The biological characterizations of Peptibody strains were identified as Escherichia coli and the fermentation mode was optimized in the shake flasks and 10‐L bioreactor. The fermentation was scaled up to 100 L, with wet cell weight (WCW) 126 g/L, production 1.41 g/L, and productivity 0.35 g/(L·h) of IPTG induction. The target protein was isolated by cation‐exchange, hydrophobic and Protein A chromatography, with total recovery of 60.28% and HPLC purity of 86.71%. The host cells protein, DNA, and endotoxin residues were within the threshold. In mouse model, immunization of Peptibody vaccine could significantly suppressed the tumor growth and angiogenesis, with inhibition rate of 57.73 and 39.34%. The Peptibody vaccine could elicit high‐titer anti‐bFGF and anti‐VEGFA antibodies, which inhibited the proliferation and migration of Lewis lung cancer cell cells by decreasing the Akt/MAPK signal pathways. Therefore, the Peptibody with bFGF/VEGFA might be used as a therapeutic tumor vaccine. The large‐scale process we developed could support its industrial production and pre‐clinical study in the future.     

Polyethylenimine/small interfering RNA‐mediated knockdown of vascular endothelial growth factor in vivo exerts anti‐tumor effects synergistically with Bevacizumab

Background

RNA interference is a powerful method for the knockdown of pathologically relevant genes. The in vivo delivery of siRNAs, preferably through systemic, nonviral administration, poses the major challenge in the therapeutic application of RNAi. Small interfering RNA (siRNA) complexation with polyethylenimines (PEI) may represent a promising strategy for siRNA‐based therapies and, recently, the novel branched PEI F25‐LMW has been introduced in vitro. Vascular endothelial growth factor (VEGF) is frequently overexpressed in tumors and promotes tumor growth, angiogenesis and metastasis and thus represents an attractive target gene in tumor therapy.

Methods

In subcutaneous tumor xenograft mouse models, we established the therapeutic efficacy and safety of PEI F25‐LMW/siRNA‐mediated knockdown of VEGF. In biodistribution and siRNA quantification studies, we optimized administration strategies and, employing chemically modified siRNAs, compared the anti‐tumorigenic efficacies of: (i) PEI/siRNA‐mediated VEGF targeting; (ii) treatment with the monoclonal anti‐VEGF antibody Bevacizumab (Avastin®); and (iii) a combination of both.

Results

Efficient siRNA delivery is observed upon systemic administration, with the biodistribution being dependent on the mode of injection. Toxicity studies reveal no hepatotoxicity, proinflammatory cytokine induction or other side‐effects of PEI F25‐LMW/siRNA complexes or polyethylenimine, and tumor analyses show efficient VEGF knockdown upon siRNA delivery, leading to reduced tumor cell proliferation and angiogenesis. The determination of anti‐tumor effects reveals that, in pancreas carcinoma xenografts, single treatment with PEI/siRNA complexes or Bevacizumab is already highly efficacious, whereas, in prostate carcinoma, synergistic effects of both treatments are observed.

Conclusions

PEI F25‐LMW/siRNA complexes, which can be stored frozen as opposed to many other carriers, represent an efficient, safe and promising avenue in anti‐tumor therapy, and PEI/siRNA‐mediated, therapeutic VEGF knockdown exerts anti‐tumor effects. Copyright © 2010 John Wiley & Sons, Ltd.     

Regulatory effects of Spirulina complex polysaccharides on growth of murine RSV‐M glioma cells through Toll‐like receptor 4

This study is the first to report that Spirulina complex polysaccharides (CPS) suppress glioma growth by down‐regulating angiogenesis via a Toll‐like receptor 4 signal. Murine RSV‐M glioma cells were implanted s.c. into C3H/HeN mice and TLR4 mutant C3H/HeJ mice. Treatment with either Spirulina CPS or Escherichia coli (E. coli) lipopolysaccharides (LPS) strongly suppressed RSV‐M glioma cell growth in C3H/HeN, but not C3H/HeJ, mice. Glioma cells stimulated production of interleukin (IL)‐17 in both C3H/HeN and C3H/HeJ tumor‐bearing mice. Treatment with E. coli LPS induced much greater IL‐17 production in tumor‐bearing C3H/HeN mice than in tumor‐bearing C3H/HeJ mice. In C3H/HeN mice, treatment with Spirulina CPS suppressed growth of re‐transplanted glioma; however, treatment with E. coli LPS did not, suggesting that Spirulina CPS enhance the immune response. Administration of anti‐cluster of differentiation (CD)8, anti‐CD4, anti‐CD8 antibodies, and anti‐asialo GM1 antibodies enhanced tumor growth, suggesting that T cells and natural killer cells or macrophages are involved in suppression of tumor growth by Spirulina CPS. Although anti‐interferon‐γ antibodies had no effect on glioma cell growth, anti‐IL‐17 antibodies administered four days after tumor transplantation suppressed growth similarly to treatment with Spirulina CPS. Less angiogenesis was observed in gliomas from Spirulina CPS‐treated mice than in those from saline‐ or E. coli LPS‐treated mice. These findings suggest that, in C3H/HeN mice, Spirulina CPS antagonize glioma cell growth by down‐regulating angiogenesis, and that this down‐regulation is mediated in part by regulating IL‐17 production.     

Molecular MRI assessment of vascular endothelial growth factor receptor‐2 in rat C6 gliomas

Angiogenesis is essential to tumour progression and a precise evaluation of angiogenesis is important for tumour early diagnosis and treatment. The quantitative and dynamic in vivo assessment of tumour angiogenesis can be achieved by molecular magnetic resonance imaging (mMRI). Vascular endothelial growth factor (VEGF) and VEGF receptors (VEGFRs) are the main regulatory systems in angiogenesis and have been used as hot targets for radionuclide‐based molecular imaging. However, little research has been accomplished in targeting VEGF/VEGFRs by mMRI. In our study, we aimed to assess the expression of VEGFR2 in C6 gliomas by using a specific molecular probe with mMRI. The differential uptake of the probe conjugated to anti‐VEGFR2 monoclonal antibody, shown by varied increases in T₁ signal intensity during a 2 hr period, demonstrated the heterogeneous expression of VEGFR2 in different tumour regions. Microscopic fluorescence imaging, obtained for the biotin group in the probe with streptavidin‐Cy3, along with staining for cellular VEGFR2 levels, laminin and CD45, confirmed the differential distribution of the probe which targeted VEGFR2 on endothelial cells. The angiogenesis process was also assessed using magnetic resonance angiography, which quantified tumour blood volume and provided a macroscopic view and a dynamic change of the correlation between tumour vasculature and VEGFR2 expression. Together these results suggest mMRI can be very useful in assessing and characterizing the expression of specific angiogenic markers in vivo and help evaluate angiogenesis associated with tumour progression.     

Diallyl trisulfide inhibits migration,invasion and angiogenesis of human colon cancer HT‐29 cells and umbilical vein endothelial cells,and suppresses murine xenograft tumour growth

Angiogenesis inhibitors are beneficial for the prevention and treatment of angiogenesis‐dependent diseases including cancer. We examined the cytotoxic, anti‐metastatic, anti‐cancer and anti‐angiogenic effects of diallyl trisulfide (DATS). In HT29 cells, DATS inhibited migration and invasion through the inhibition of focal adhesion kinase (FAK), extracellular signal‐regulated kinase, c‐Jun N‐terminal kinase and p38 which was associated with inhibition of matrix metalloproteinases‐2, ‐7 and ‐9 and VEGF. In human umbilical vein endothelial cells (HUVEC), DATS inhibited the migration and angiogenesis through FAK, Src and Ras. DATS also inhibited the secretion of VEGF. The capillary‐like tube structure formation and migration by HUVEC was inhibited by DATS. The chicken egg chorioallantoic membrane (CAM) assay indicated that DATS treatment inhibited ex‐vivo angiogenesis. We investigated the anti‐tumour effects of DATS against human colon cancer xenografts in BALB/c^nu/nu mice and its anti‐angiogenic activity in vivo. In this in‐vivo study, DATS also inhibited the tumour growth, tumour weight and angiogenesis (decreased the levels of haemoglobin) in HT29 cells. In conclusion, the present results suggest that the inhibition of angiogenesis may be an important mechanism in colon cancer chemotherapy by DATS.     

Preparation of extracellular domain 3 of human VEGF receptor‐2 and the monitoring of its real‐time binding to VEGF by biosensors

Vascular endothelial growth factor receptor‐2 (VEGFR‐2) plays an important role in stimulating the proliferation of endothelial cells and improving the permeability of blood vessels, which is involved in tumor angiogenesis, a process that is essential for tumor growth and metastasis. In this study, we describe a method for high yield of recombinant extracellular domain 3 (KDR3) of human VEGFR‐2 in an Escherichia coli system with further purification by cation exchange chromatography and immobilized metal affinity chromatography (IMAC). The biological activity of recombinant KDR3 was performed by sequestering VEGF in HUVEC proliferation assay. The real‐time binding of human VEGF to immobilized KDR3 was monitored by a label‐free biosensor, Optical waveguide lightmode spectroscopy (OWLS). Under the given experimental conditions, the association rate constant k_a was 4.2 × 10³ M^?1 s^?1 and the dissociation rate k_d was 5.1 × 10^?3 s^?1. The dissociation constant K_D was then calculated to be 1.2 × 10^?6 M. The obtained values will serve as baseline parameters for the design of improved versions of recombinant soluble VEGF receptors and the evaluation of developed anti‐KDR antibodies. In addition, such a scenario established by the use of OWLS will potentiate the kinetic study of ligand/receptor and antigen/antibody. The receptor discussed here, which block VEGF binding to cell membrane KDR, have potential clinical application in the treatment of cancer and other diseases where pathological angiogenesis is involved. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Immune Response of the VEGF/bFGF Complex Peptide Vaccine and Function of Immune Antibodies in Inhibiting Migration of HUVEC Cells and Proliferation of Cancer Cells

Overexpression of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) plays a key role in tumor angiogenesis and metastasis in tumors. VEGF/bFGF complex peptide (VBP3) was designed to elicit the body to produce both high titer anti-VEGF and anti-bFGF antibodies to inhibit tumor angiogenesis and tumor growth. BALB/c mice were immunized with the VEGF/bFGF complex peptide, and the immune responses were assayed. Splenocytes were separated from the immunized mice and the CD4, CD8 T cells and IFN-γ were assayed by Flow cytometry. The results showed that the VBP3 could effectively stimulate immune response in mice and resulted in the increase of CD4 and CD8 T cells. CD4+ T cells and CD8+ T cells were increased from 10.78 to 15.13 and 6.82 to 11.58 % respectively. Polyclonal antibodies purified from the VBP3 immunized mice showed good anti-proliferation function to lung cancer cells, and resulted in the decrease of phosphroylation level of Akt and Erk assayed by the Western-blot. Transwell assays showed that the migration of HUVEC cells was inhibited by the antibodies. The results revealed that the VBP3 have good immunogenicity and may be used as a vaccine for tumor therapy.     

Involvement of interleukin-8, vascular endothelial growth factor, and basic fibroblast growth factor in tumor necrosis factor alpha-dependent angiogenesis.

Tumor necrosis factor alpha (TNF-alpha) is a macrophage/monocyte-derived polypeptide which modulates the expression of various genes in vascular endothelial cells and induces angiogenesis. However, the underlying mechanism by which TNF-alpha mediates angiogenesis is not completely understood. In this study, we assessed whether TNF-alpha-induced angiogenesis is mediated through TNF-alpha itself or indirectly through other TNF-alpha-induced angiogenesis-promoting factors. Cellular mRNA levels of interleukin-8 (IL-8), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and their receptors were increased after the treatment of human microvascular endothelial cells with TNF-alpha (100 U/ml). TNF-alpha-dependent tubular morphogenesis in vascular endothelial cells was inhibited by the administration of anti-IL-8, anti-VEGF, and anti-bFGF antibodies, and coadministration of all three antibodies almost completely abrogated tubular formation. Moreover, treatment with Sp1, NF-kappaB, and c-Jun antisense oligonucleotides inhibited TNF-alpha-dependent tubular morphogenesis by microvascular endothelial cells. Administration of a NF-kappaB antisense oligonucleotide almost completely inhibited TNF-alpha-dependent IL-8 production and partially abrogated TNF-alpha-dependent VEGF production, and an Sp1 antisense sequence partially inhibited TNF-alpha-dependent production of VEGF. A c-Jun antisense oligonucleotide significantly inhibited TNF-alpha-dependent bFGF production but did not affect the production of IL-8 and VEGF. Administration of an anti-IL-8 or anti-VEGF antibody also blocked TNF-alpha-induced neovascularization in the rabbit cornea in vivo. Thus, angiogenesis by TNF-alpha appears to be modulated through various angiogenic factors, both in vitro and in vivo, and this pathway is controlled through paracrine and/or autocrine mechanisms.     

In vivo neurogenesis is inhibited by neutralizing antibodies to basic fibroblast growth factor

While extracellular growth factors govern neuronal precursor mitosis in culture, little is known about their roles in regulating neurogenesis in vivo. Previously, we reported that subcutaneously administered basic fibroblast growth factor (bFGF) promoted neuroblast proliferation in P1 rat brain, in regions in which bFGF and FGF receptors are expressed during development. To define the role of endogenous bFGF in neurogenesis, we employed a neutralizing monoclonal antibody to the factor. In culture, bFGF-induced granule precursor proliferation was progressively inhibited by increasing concentrations of antibody. In contrast, heat-inactivated or nonneutralizing anti-bFGF antibodies were ineffective. The inhibition was specific for bFGF, since EGF-induced [³H]dT incorporation was not altered. To study effects in vivo, neutralizing antibody was administered to newborn rats via the cisterna magnum. Four hours after injection, DNA synthesis in cerebellum and hippocampus was decreased by 53% and 63%, respectively, suggesting that endogenous bFGF was involved in brain development. To define effects on neurogenesis specifically, granule cell precursors were isolated after antibody treatment. [³H]dT incorporation in granule precursors was decreased by 50%, indicating that the neutralizing antibody inhibited neuroblast proliferation in vivo. In contrast, no reduction was observed using nonneutralizing or the heat-inactivated antibodies. The inhibition of precursor proliferation following immunoneutralization of bFGF in vivo suggests that the endogenous factor normally regulates brain neurogenesis. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 289–296, 1997     

Pharmacological inhibition of microsomal prostaglandin E synthase-1 suppresses epidermal growth factor receptor-mediated tumor growth and angiogenesis

Background

Blockade of Prostaglandin (PG) E₂ production via deletion of microsomal Prostaglandin E synthase-1 (mPGES-1) gene reduces tumor cell proliferation in vitro and in vivo on xenograft tumors. So far the therapeutic potential of the pharmacological inhibition of mPGES-1 has not been elucidated. PGE₂ promotes epithelial tumor progression via multiple signaling pathways including the epidermal growth factor receptor (EGFR) signaling pathway.

Methodology/Principal Findings

Here we evaluated the antitumor activity of AF3485, a compound of a novel family of human mPGES-1 inhibitors, in vitro and in vivo, in mice bearing human A431 xenografts overexpressing EGFR. Treatment of the human cell line A431 with interleukin-1beta (IL-1β) increased mPGES-1 expression, PGE₂ production and induced EGFR phosphorylation, and vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2) expression. AF3485 reduced PGE₂ production, both in quiescent and in cells stimulated by IL-1β. AF3485 abolished IL-1β-induced activation of the EGFR, decreasing VEGF and FGF-2 expression, and tumor-mediated endothelial tube formation. In vivo, in A431 xenograft, AF3485, administered sub-chronically, decreased tumor growth, an effect related to inhibition of EGFR signalling, and to tumor microvessel rarefaction. In fact, we observed a decrease of EGFR phosphorylation, and VEGF and FGF-2 expression in tumours explanted from treated mice.

Conclusion

Our work demonstrates that the pharmacological inhibition of mPGES-1 reduces squamous carcinoma growth by suppressing PGE₂ mediated-EGFR signalling and by impairing tumor associated angiogenesis. These results underscore the potential of mPGES-1 inhibitors as agents capable of controlling tumor growth.     

Regulation of retinal capillary cells by basic fibroblast growth factor, vascular endothelial growth factor, and hypoxia

Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) feature prominently in retinal neovascular diseases. Although the role of VEGF in retinal angiogenesis is well established, the importance of bFGF in this process requires further clarification. This study was undertaken to investigate the responses of retinal capillary cells (endothelial cells and pericytes) to bFGF under hypoxic conditions, as well as the potentially synergistic effects of bFGF and VEGF on the proliferation and cord formation of retinal endothelial cells. Cell proliferation was determined by cell number and by 3H-thymidine incorporation. Cord formation was assessed in three-dimensional gels of collagen type I. VEGF and bFGF increased 3H-thymidine incorporation by both cell types, an effect that was more pronounced in a hypoxic environment. Moreover, the proliferation of pericytes was stimulated to a greater extent by bFGF relative to VEGF. Endothelial migration in collagen gels, however, was induced more effectively by VEGF than by bFGF. A synergistic effect of VEGF and bFGF on cell invasion was observed in the collagen gel assay. VEGF and bFGF each augment proliferation of these cells, especially under hypoxia. We thus propose that these two cytokines have a synergistic effect at several stages of angiogenesis in the retina.     

Vascular endothelial growth factor accelerates compensatory lung growth after unilateral pneumonectomy

We hypothesize that compensatory lung growth after unilateral pneumonectomy in a murine model is, in part, angiogenesis dependent and can be altered using angiogenic agents, possibly through regulation of endothelial cell proliferation and apoptosis. Left pneumonectomy was performed in mice. Mice were then treated with proangiogenic factors [vascular endothelial growth factor (VEGF); basic fibroblast growth factor (bFGF)], VEGF receptor antibodies (MF-1, DC101), and VEGF receptor small molecule chemical inhibitors. Lung volume and mass were measured. The lungs were analyzed using immunohistochemistry by CD31 staining, terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling, type II pneumocytes staining, and proliferating cell nuclear antigen. Compensatory lung growth was complete by postoperative day 10 and was associated with diffuse apoptosis of endothelial cells and pneumocytes. This process was accelerated by VEGF, such that growth was complete by postoperative day 4 with similar associated apoptosis. bFGF had no effect on lung growth. MF-1 and DC101 had no effect. The VEGF receptor small molecule chemical inhibitors also had no effect. VEGF, but not bFGF, accelerates growth. VEGF receptor inhibitors do not block growth, suggesting that other proangiogenic factors play a role or can compensate for VEGF receptor blockade. Diffuse apoptosis, endothelial cell and pneumocyte, occurs at cessation of both normal compensatory and VEGF-accelerated growth. Angiogenesis modulators may control growth via regulation of endothelial cell proliferation and apoptosis, although the exact relationship between endothelial cells and pneumocytes has yet to be determined. The fact that bFGF did not accelerate growth in our model when it did accelerate regeneration in the liver model suggests that angiogenesis during organ regeneration is regulated in an organ-specific manner.     

Vascular endothelial growth factor and basic fibroblast growth factor in patients with squamous cell oesophageal cancer

It is suggested that vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) play an important role in tumor-induced angiogenesis. The purpose of this study was to estimate the correlation between VEGF and bFGF levels and tumor pathological status according to pTNM classification in patients with squamous cell oesophageal cancer. A group of 25 healthy controls and 32 consecutive patients with oesophageal cancer were included in this study. Serum VEGF and bFGF levels were determined by enzyme-linked immunosorbent assay (Quantikine R&D Systems). Serum VEGF and bFGF levels were significantly elevated in the patient groups (VEGF: 146.0 pg/ml, 79.0-386.3 pg/ml vs. 38.0 pg/ml, 6.5-135.1 pg/ml, p<0.005, and bFGF: 5.2 pg/ml, 1.2-10.6 pg/ml vs. 2.06 pg/ml, 0.07-4.0 pg/ml, p<0.02 Fisher test). The highest correlation between serum VEGF and bFGF levels were found in patients with advanced cancers, especially with: T4, N1, and M1 factors. The VEGF and bFGF levels were significantly higher in patients with pT4 (p<0.01). Patients with N1 lymph node invasion, compared with N0 factor, have higher levels of angiogenetic factors (p<0.04). Also in patients with advanced cancers with liver metastases the serum levels VEGF and bFGF were significantly higher (M1 vs. M0, VEGF p<0.001 and bFGF p<0.05). Consecutive monitoring of VEGF and bFGF serum levels may be a useful prognostic marker for patients with squamous cell oesophageal cancer.     

Placental growth factor silencing ameliorates liver fibrosis and angiogenesis and inhibits activation of hepatic stellate cells in a murine model of chronic liver disease

Placental growth factor (PlGF) is a member of the vascular endothelial growth factor (VEGF) family and is involved in pathological angiogenesis associated with chronic liver diseases. However, the precise mechanisms underlying PlGF signalling contributing to liver fibrosis and angiogenesis remain largely unexplored. This study aimed to assess the effect of reducing PlGF expression using small interfering RNA (siRNA) on experimental liver fibrosis and angiogenesis, and to elucidate the underlying molecular mechanisms. Fibrosis was induced in mice by carbon tetrachloride (CCl₄) for 8 weeks, and mice were treated with PlGF siRNA or non‐targeting control siRNA starting two weeks after initiating CCl₄ injections. The results showed that PlGF was highly expressed in cirrhotic human and mice livers; which mainly distributed in activated hepatic stellate cells (HSCs). PlGF silencing robustly reduced liver inflammation, fibrosis, intrahepatic macrophage recruitment, and inhibited the activation of HSCs in vivo. Moreover, PlGF siRNA‐treated fibrotic mice showed diminished hepatic microvessel density and angiogenic factors, such as hypoxia‐inducible factor‐1α (HIF‐1α), VEGF and VEGF receptor‐1. Moreover, down‐regulation of PlGF with siRNA in HSCs inhibited the activation and proliferation of HSCs. Mechanistically, overexpression of PlGF in activated HSCs was induced by hypoxia dependent on HIF‐1α, and PlGF induces HSC activation and proliferation via activation the phosphatidylinositol 3‐kinase (PI3K)/Akt signalling pathways. These findings indicate that PlGF plays an important role in liver fibrosis‐associated angiogenesis and that blockage of PlGF could be an effective strategy for chronic liver disease.     

Cheiradone: a vascular endothelial cell growth factor receptor antagonist

Background  

Angiogenesis, the growth of new blood vessels from the pre-existing vasculature is associated with physiological (for example wound healing) and pathological conditions (tumour development). Vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2) and epidermal growth factor (EGF) are the major angiogenic regulators. We have identified a natural product (cheiradone) isolated from a Euphorbia species which inhibited in vivo and in vitro VEGF- stimulated angiogenesis but had no effect on FGF-2 or EGF activity. Two primary cultures, bovine aortic and human dermal endothelial cells were used in in vitro (proliferation, wound healing, invasion in Matrigel and tube formation) and in vivo (the chick chorioallantoic membrane) models of angiogenesis in the presence of growth factors and cheiradone. In all cases, the concentration of cheiradone which caused 50% inhibition (IC₅₀) was determined. The effect of cheiradone on the binding of growth factors to their receptors was also investigated.     

Studies on the role of basic fibroblast growth factor in vivo: inability of neutralizing antibodies to block tumor growth

Affinity-purified polyclonal rabbit antibodies prepared against recombinant basic fibroblast growth factor (bFGF) neutralized the ability of bFGF to stimulate plasminogen activator (PA) production and endothelial cell migration in vitro. After iodination and intraperitoneal injection of the antibodies in mice, approximately 76% of the maximum circulating level of 125I-anti-bFGF antibodies (AF) was found as intact IgG after 24 hr. Furthermore, the circulating 125I-AF retained the ability to bind bFGF. Studies were performed to determine whether the growth of three different murine tumors (CT26, EHS, or B16/BL6) could be inhibited with affinity-purified neutralizing antibodies against bFGF. Tumors were injected subcutaneously in syngeneic mice, and neutralizing antibodies against bFGF were injected daily into the peritoneum. All studies, which varied in tumor burden, antibody dose, and study length, indicated that neutralizing antibodies against bFGF had no effect on tumor size, tumor growth, or tumor histology.     

Quantitative physiology of Pichia pastoris during glucose‐limited high‐cell density fed‐batch cultivation for recombinant protein production

Pichia pastoris has become one of the major microorganisms for the production of proteins in recent years. This development was mainly driven by the readily available genetic tools and the ease of high‐cell density cultivations using methanol (or methanol/glycerol mixtures) as inducer and carbon source. To overcome the observed limitations of methanol use such as high heat development, cell lysis, and explosion hazard, we here revisited the possibility to produce proteins with P. pastoris using glucose as sole carbon source. Using a recombinant P. pastoris strain in glucose limited fed‐batch cultivations, very high‐cell densities were reached (more than 200 g_CDW L^?1) resulting in a recombinant protein titer of about 6.5 g L^?1. To investigate the impact of recombinant protein production and high‐cell density fermentation on the metabolism of P. pastoris, we used ¹³C‐tracer‐based metabolic flux analysis in batch and fed‐batch experiments. At a controlled growth rate of 0.12 h^?1 in fed‐batch experiments an increased TCA cycle flux of 1.1 mmol g^?1 h^?1 compared to 0.7 mmol g^?1 h^?1 for the recombinant and reference strains, respectively, suggest a limited but significant flux rerouting of carbon and energy resources. This change in flux is most likely causal to protein synthesis. In summary, the results highlight the potential of glucose as carbon and energy source, enabling high biomass concentrations and protein titers. The insights into the operation of metabolism during recombinant protein production might guide strain design and fermentation development. Biotechnol. Bioeng. 2010;107: 357–368. © 2010 Wiley Periodicals, Inc.     

MFTZ‐1 reduces constitutive and inducible HIF‐1α accumulation and VEGF secretion independent of its topoisomerase II inhibition

The macrolide compound MFTZ‐1 has been identified as a novel topoisomerase II (Top2) inhibitor with potent in vitro and in vivo anti‐tumour activities. In this study, we further examined the effects of MFTZ‐1 on hypoxia‐inducible factor‐1α (HIF‐1α) accumulation, vascular endothelial growth factor (VEGF) secretion and angiogenesis. MFTZ‐1 reduced HIF‐1α accumulation driven by hypoxia or growth factors in human cancer cells. Mechanistic studies revealed that MFTZ‐1 did not affect the degradation of HIF‐1α protein or the level of HIF‐1α mRNA. By contrast, MFTZ‐1 apparently inhibited constitutive and inducible activation of both phosphatidylinositol‐3‐kinase (PI3K)‐Akt and p42/p44 mitogen‐activated protein kinase (MAPK) pathways. Further studies revealed that MFTZ‐1 abrogated the HIF‐1α‐driven increase in VEGF mRNA and protein secretion. MFTZ‐1 also lowered the basal level of VEGF secretion. The results reveal an important feature that MFTZ‐1 can reduce constitutive, HIF‐1α‐independent VEGF secretion and concurrently antagonize inducible, HIF‐1α‐dependent VEGF secretion. Moreover, MFTZ‐1 disrupted tube formation of human umbilical vein endothelial cells (HUVECs) stimulated by hypoxia with low‐concentration serum or by serum at normoxia, and inhibited HUVECs migration at normoxia. MFTZ‐1 also prevented microvessel outgrowth from rat aortic ring. These data reflect the potent anti‐angiogenesis of MFTZ‐1 under different conditions. Furthermore, using specific small interfering RNA targeting Top2α or Top2‐defective HL60/MX2 cells, we showed that MFTZ‐1 affected HIF‐1α accumulation and HUVECs tube formation irrelevant to its Top2 inhibition. Taken together, our data collectively reveal that MFTZ‐1 reduces constitutive and inducible HIF‐1α accumulation and VEGF secretion possibly via PI3K‐Akt and MAPK pathways, eliciting anti‐angiogenesis independently of its Top2 inhibition.     

Effect of local neutralization of basic fibroblast growth factor or vascular endothelial growth factor by a specific antibody on the development of the corpus luteum in the cow

Active angiogenesis and progesterone (P) synthesis occur in parallel during development of the corpus luteum (CL). Basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) are known to stimulate angiogenesis and P synthesis in vitro. The aim of the present study was to investigate the impact of bFGF or VEGF on the CL development in the cow by using a specific antibody against bFGF or VEGF. bFGF antibody, VEGF antibody, or saline as a control (n = 4 cows/treatment) were injected directly into the CL immediately after ovulation (Day 1), and the treatment was continued for 3 times/day over 7 days. Luteal biopsies were applied on Day 8 of the estrous cycle to determine the expression of genes associated with P synthesis and angiogenesis. Intraluteal injections with the bFGF antibody or the VEGF antibody markedly decreased the CL volume, plasma P concentration and StAR mRNA expression. bFGF antibody treatment decreased the mRNA expression of bFGF, FGF receptor-1, VEGF120, and angiopoietin (ANPT)-1, and increased ANPT-2/ANPT-1 ratio. However, VEGF antibody treatment decreased ANPT-2 mRNA expression and ANPT-2/ANPT-1 ratio. These results indicate that local neutralization of bFGF or VEGF changes genes regulating angiogenesis and P synthesis, and remarkably suppresses the CL size and P secretion during the development of CL in the cow, supporting the concept that bFGF and VEGF control the CL formation and function.