Matrix metalloproteinase-9 triggers the angiogenic switch during carcinogenesis.

During carcinogenesis of pancreatic islets in transgenic mice, an angiogenic switch activates the quiescent vasculature. Paradoxically, vascular endothelial growth factor (VEGF) and its receptors are expressed constitutively. Nevertheless, a synthetic inhibitor (SU5416) of VEGF signalling impairs angiogenic switching and tumour growth. Two metalloproteinases, MMP-2/gelatinase-A and MMP-9/gelatinase-B, are upregulated in angiogenic lesions. MMP-9 can render normal islets angiogenic, releasing VEGF. MMP inhibitors reduce angiogenic switching, and tumour number and growth, as does genetic ablation of MMP-9. Absence of MMP-2 does not impair induction of angiogenesis, but retards tumour growth, whereas lack of urokinase has no effect. Our results show that MMP-9 is a component of the angiogenic switch.

Vascular permeability factor: a tumor-derived polypeptide that induces endothelial cell and monocyte procoagulant activity, and promotes monocyte migration.

Systemic infusion of low concentrations of tumor necrosis factor/cachectin (TNF) into mice that bear TNF-sensitive tumors leads to activation of coagulation, fibrin formation, and occlusive thrombosis exclusively within the tumor vascular bed. To identify mechanisms underlying the localization of this vascular procoagulant response, a tumor-derived polypeptide has been purified to homogeneity from supernatants of murine methylcholanthrene A-induced fibrosarcomas that induces endothelial tissue factor synthesis and expression (half-maximal response at approximately 300 pM), and augments the procoagulant response to TNF in a synergistic fashion. This tumor-derived polypeptide was identified as the murine homologue of vascular permeability factor (VPF) based on similar mobility on SDS-PAGE, an homologous NH2-terminal amino acid sequence, and recognition by a monospecific antibody to guinea pig VPF. In addition, VPF was shown to induce monocyte activation, as evidenced by expression of tissue factor. Finally, VPF was shown to induce monocyte chemotaxis across collagen membranes and endothelial cell monolayers. Taken together, these results indicate that VPF can modulate the coagulant properties of endothelium and monocytes, and can promote monocyte migration into the tumor bed. This suggests one mechanism through which tumor-derived mediators can alter properties of the vessel wall.

A three-step purification method of large quantities of human recombinant alpha endothelial cellular growth factor for clinical use.

The endothelial cellular growth factor alpha-ECGF is a candidate drug for the induction of therapeutic neoangiogenesis. Its use in extensive experimental and clinical trials is hampered by the fact that currently published purification procedures allow only small yields, and the absence of pyrogenic impurities is not demonstrated. The rh alpha-ECGF was expressed in E. coli. Isolation of rh alpha-ECGF from E. coli lysates to apparent homogenicity was achieved by a three step purification procedure involving ionic exchange, heparin-sepharose and polymyxin B chromatography. By this method, 200 mg of rh alpha-ECGF was purified from 15 g wet weight E. coli bacteria. The isolated protein of 18 kDa appeared as a single band after SDS gel electrophoresis and subsequent silver-staining. The biological activity was expressed in the chorion-allantois-membrane assay and in the 3H-thymidine proliferation in baby hamster kidney cells. Drug trials with rabbits revealed no increase in body temperature after intravenous injections with 1 mg rh-ECGF.

The N-myc oncogene in human neuroblastoma cells: down-regulation of an angiogenesis inhibitor identified as activin A.

Angiogenesis, the formation of new blood vessels, is seen during embryonic development and tumor progression, but the mechanisms have remained unclear. Recent data indicate that developmental and tumor angiogenesis can be induced by cellular oncogenes, leading to the enhanced activity of molecules stimulating angiogenesis. However, activated oncogenes might also facilitate angiogenesis by down-regulating endogenous inhibitors of angiogenesis. We report here that enhanced expression of the N-myc oncogene in human neuroblastoma cells down-regulates an inhibitor of endothelial cell proliferation, identified by amino acid sequencing as being identical with activin A, a developmentally regulated protein. Down-regulation appears to involve interaction of the N-Myc protein with the activin A promoter. In addition, activin A inhibits both endothelial cell proliferation in vitro and angiogenesis in vivo, and it induces hemorrhage in vivo. We suggest that the N-myc-induced down-regulation of activin A could contribute to developmental and tumor angiogenesis.

The shortest isoform of human vascular endothelial growth factor/vascular permeability factor (VEGF/VPF121) produced by Saccharomyces cerevisiae promotes both angiogenesis and vascular permeability.

Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) is a multifunctional cytokine that is expressed as four isoforms having 206, 189, 165, and 121 amino acids in humans. We constructed a system that produces the shortest isoform of human VEGF/VPF in Saccharomyces cerevisiae (yVEGF/VPF121). Active yVEGF/VPF121 was secreted from the yeast cells as a glycosylated dimeric protein. Various biological activities of the purified yVEGF/VPF121 were examined. It bound to cell surface receptor(s) and stimulated the growth of human umbilical vein endothelial cells in culture at a dose similar to that of native VEGF/VPF. Purified yVEGF/VPF121 also induced angiogenesis in mice, and promoted the extravasation of plasma proteins from the blood vessels. These observations demonstrated that the shortest isoform of VEGF/VPF with an amino acid sequence of 121 residues contains enough information necessary to trigger both angiogenesis and the induction of vascular permeability upon binding to its cognate receptor(s).

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

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

Purification and refolding of vascular endothelial growth factor-B.

Vascular endothelial growth factor (VEGF)-A interacts with the receptor tyrosine kinases VEGF-R1 and R2, and the importance of this interaction in endothelial cell (EC) function and blood vessel development has been well documented. Other ligands that interact differentially with these receptors and that are structurally related to VEGF-A include VEGF-B, VEGF-C, VEGF-D, and placenta growth factor (PLGF). Compared with VEGF-A, relatively little is known about the biological role of the VEGF-R1 specific ligand, VEGF-B. Two splice variant isoforms that differ at the COOH-terminus and which retain unique solubility characteristics are widely expressed throughout embryonic and postnatal development. Recent analysis of mice with a targeted deletion of the VEGF-B gene has revealed a defect in heart development and function consistent with an important role in vascularization of the myocardium (Bellomo D et al., 2000, Circ Res 86:E29-E35). To facilitate further characterization of VEGF-B, we have developed a protocol for expression and purification of refolded recombinant protein from Escherichia coli inclusion bodies (IBs). The approach developed resolves a number of significant issues associated with VEGF-B, including the ability to heterodimerize with endogenous VEGF-A when co-expressed in mammalian cells, a complex secondary structure incorporating inter- and intrachain disulfide bonds and hydrophobic characteristics that preclude the use of standard chromatographic resins. The resulting purified disulfide-linked homodimer was demonstrated to bind to VEGF-R1 and to compete with VEGF-A for binding to this receptor.

Identification of non heparin-binding endothelial cell growth factor from rat myofibroblasts.

Myofibroblasts (Mfs) from rat fat tissues produced a potent endothelial cell growth factor (Mf-ECGF). The growth factor activity found in the conditioned media from primary cultures of Mfs, was labile to heat (80 degrees C for 10 min) and proteinase (trypsin), and did not bind to heparin in the presence of 0.2 M NaCl. Mf-ECGF was partially purified 4760-fold with a recovery of 25% from serum-free conditioned media by sequential carboxymethyl (CM) ion-exchange column chromatography and gel filtration. This Mf-ECGF activity was recovered from the 40 kD region of a non-reducing SDS-PAGE, and from the pH region between 6.5 and 7 of isoelectric focusing, with recoveries of 20% and 65%, respectively. These results indicated that a major portion of ECGF activity in the conditioned media was clearly distinct from other well-known endothelial cell growth factors including fibroblast growth factors (FGFs).

Immunohistochemical localization of vascular endothelial growth factor in the human placenta.

To understand the role of vascular endothelial growth factor (VEGF) in placental development, we examined immunohistochemically 56 placentae ranging from 6--41-weeks gestation using rabbit antibody to a synthetic multiple antigen peptide (MAP), composed of N-terminal amino acid residues 1--20 of human VEGF. In the present study, syncytiotrophoblast and invading extravillous trophoblasts ubiquitously expressed VEGF throughout gestation. However, the expression of VEGF in syncytiotrophoblasts was uneven in the first trimester and most intense at the sprouting sites. In addition, some stromal cells in the villi and decidual cells were also positive in the first trimester. A morphometrical analysis of the ratio of the VEGF-positive cell area to the capillary area in the terminal villi statistically revealed a critical point of change at 16 weeks' gestation. These results provide further evidence to support the hypothesis that VEGF, locally expressed by trophoblasts, stromal cells in villi and decidual cells, may play an important role in the physiological growth and function of the vascular system in the villous stroma and basal plate during placental development and maturation.

Src kinase becomes preferentially associated with the VEGFR, KDR/Flk-1, following VEGF stimulation of vascular endothelial cells.

BACKGROUND: The cytoplasmic tyrosine kinase, Src, has been found to play a crucial role in VEGF (vascular endothelial growth factor) - dependent vascular permeability involved in angiogenesis. The two main VEGFRs present on vascular endothelial cells are KDR/Flk-1 (kinase insert domain-containing receptor/fetal liver kinase-1) and Flt-1 (Fms-like tyrosine kinase-1). However, to date, it has not been determined which VEGF receptor (VEGFR) is involved in binding to and activating Src kinase following VEGF stimulation of the receptors. RESULTS: In this report, we demonstrate that Src preferentially associates with KDR/Flk-1 rather than Flt-1 in human umbilical vein endothelial cells (HUVECs), and that VEGF stimulation resulted in an increase of Src activity associated with activated KDR/Flk-1. These findings were determined through immunoprecipitation-kinase experiments and coimmunoprecipitation studies, and were further confirmed by GST-pull-down assays and Far Western studies. However, Fyn and Yes, unlike Src, were found to associate preferentially with Flt-1. CONCLUSIONS: Thus, Src preferentially associates with KDR/Flk-1, rather than with Flt-1, upon VEGF stimulation in endothelial cells. Our findings further highlight the potential significance of upregulated KDR/Flk-1-associated Src activity in the process of angiogenesis, and help to elucidate more clearly the specific roles and mechanisms involving Src family tyrosine kinase in VEGF-stimulated signal transduction events.

EORTC Receptor and Biomarker Study Group Report: a sandwich enzyme-linked immunosorbent assay for vascular endothelial growth factor in blood and tumor tissue extracts.

A four-antibody sandwich enzyme-linked immunosorbent assay (ELISA) for vascular endothelial growth factor (VEGF) for application in blood (serum and plasma) and tumor tissue extracts was set up within the framework of the EORTC Receptor and Biomarker Study Group (RBSG). Polyclonal antibodies against VEGF165 were raised in chickens and rabbits, and used in a previously described assay format. The assay was validated and characterized for use in serum, plasma and tumor tissue extracts. The resulting VEGF ELISA was found to be specific for VEGF165 and VEGF121, the main isoforms of VEGF. The assay showed good precision and parallelism in serial dilutions of samples. The assay was not susceptible to interference by heterophilic antibodies because avian antibodies (duck anti-chicken and chicken anti-VEGF) were used in the pre-analyte stage and mammalian antibodies (rabbit anti-VEGF and goat anti-rabbit) in the post-analyte stage. In conclusion, a sensitive, robust and specific VEGF ELISA has been developed. Research into the prognostic value of VEGF employing this assay is currently underway.

[Culture of human blood vessel endothelial cells--a simple and inexpensive culture method].

The purpose of this review is to introduce a simple and inexpensive culture method for human umbilical blood vessel endothelial cells. The medium used is MCDB-104 supplemented with 10% fetal bovine serum, 70 ng/ml endothelial cell growth factor from new-born bovine brains, 10 ng/ml murine epidermal growth factor, and 100 micrograms/ml heparin. The culture dishes are coated with gelatin. Under these conditions, endothelial cells from human vessels were grown with doubling times of 18-22 hrs and reached saturation densities of 8-12 x 10(4) cells/cm2. To determine the lifespan of the endothelial cells, the cells were serially subcultivated weekly at an inoculum size of 1,000 cells/cm2. Human endothelial cells from umbilical vein and artery were grown for 21 to 37 passages with 55 to 125 population doublings. This culture method seems to be useful for studying cell proliferation and functions of human endothelial cells.

Developmental expression of vascular endothelial growth factor in the masseter muscle of rats.

Developmental changes in vascular endothelial growth factor (VEGF) in rat masseter after birth were investigated. VEGF was extracted efficiently and reproducibly from muscle homogenate with low concentrations of non-ionic detergents, such as Triton X-100, Nonidet P-40, and Tween 20. The amount of VEGF measured by enzyme-linked immunosorbent assay (ELISA) increased markedly by approximately 9-fold, from day 8 to 35 after birth. The increase in VEGF was closely correlated with the development of the capillary network, as shown by the capillary to muscle fibre ratio (C/F ratio). Immunoblotting revealed that the predominant molecular species of VEGF concentrated with heparin-sepharose beads was VEGF(188). These results suggest that VEGF plays an important part in the development and maintenance of the capillary network in the rat masseter.

Primary study of vascular endothelial growth factor immunohistochemical staining in the diagnosis of early acute myocardial ischemia.

The expression of vascular endothelial growth factor (VEGF) in the model of rat early acute myocardial ischemia was studied by Strept-Avidin-Biotin-Peroxidase Complex (SABC) immunohistochemical staining. After ligating the anterior descending branch of the left coronary artery, an initial rapid (30min) positive expression of VEGF in myocardial ischemic areas was observed, the intensity of positive expression of VEGF increased with the continuation of myocardial ischemia. After 5h infarction, the strongly positive myocytes of SABC-VEGF staining were predominantly limited to perimyocardial infarction areas. No positive expression of VEGF was found in the control group. These findings suggested that SABC-VEGF method could give a sensitive, specific, simple and objective morphologic evidence to the diagnosis of sudden cardiac death caused by acute early myocardial ischemia.

Distribution of transforming growth factor-beta isoforms TGF-beta 1, TGF-beta 2 and TGF-beta 3 and vascular endothelial growth factor in vulvar lichen sclerosus.

OBJECTIVE: To study the distribution of transforming growth factor beta (TGF-beta) isoforms TGF-beta 1, TGF-beta 2 and TGF-beta 3 and vascular endothelial growth factor (VEGF) in vulvar lichen sclerosus. STUDY DESIGN: Biopsies were obtained from 10 patients with vulvar lichen sclerosus, snap frozen and stained with polyclonal antibodies to TGF-beta 1, TGF-beta 2, TGF-beta 3 and VEGF. Control tissues used were uninvolved thigh tissue from two of the lichen sclerosus patients and normal vulvar tissue obtained from eight patients during gynecologic procedures. Two specimens of morphea were also examined. RESULTS: Weak TGF-beta 1 staining was demonstrated in the epidermis of all the lichen sclerosus, morphea, thigh and five of the eight normal vulvar specimens. Slight increase in TGF-beta 1 staining was seen in the upper and middermis in 6 of the 10 lichen sclerosus specimens and in the morphea specimens as compared to the control tissue, and this staining was localized within cells. TGF-beta 2 staining was present throughout the epidermis in all the normal thigh, normal vulva, lichen sclerosus and morphea specimens. TGF-beta 2 staining was increased within cells in the upper and middermis of the lichen sclerosus and morphea specimens. TGF-beta 3 staining occurred in the basal half of the epidermis of all the control, lichen sclerosus and morphea specimens, and only slight upper dermal staining of a few individual cells was seen in 3 of the 10 lichen sclerosus specimens. VEGF staining was similar in the normal tissues, lichen sclerosus and morphea. CONCLUSION: These results suggest that TGF-beta may.

Expression of human vascular endothelial growth factor (VEGF165) in Pichia pastoris and its biological activity.

OBJECTIVE: To express human vascular endothelial growth factor (hVEGF165) cDNA in Pichia pastroris, purify the expressed product and detect the biological activity of it. METHODS: By inserting hVEGF165 cDNA coding 165 amino acid residues into Pichia pastoris expression vector pPIC9K containing AOX1 promoter and the sequences of alpha secreting signal peptides, a recombinant expression plasmid pPIC9K/hVEGF165 was constructed and transformed to yeast host strain KM71, then multiple-copy insert transformants were screened out and cultured in flasks, and hVEGF165 was expressed under the induction of 1% methanol. RESULTS: SDS-PAGE showed that after being induced with 1% methanol for 4 d, the expressed product existed in supernatant in the form of soluble molecule and contained 60% of total protein expressed. Western blot showed good antigenicity and specificity of expressed product. After being purified by Heparin-Sepharose CL6B affinity chromatography, the purity of expressed product reached above 90%. Biological assays proved that the expressed product could stimulate the proliferation of HUVEC. CONCLUSION: hVEGF165 was successfully expressed. The study opened up a wide prospect for the application of VEGF165 in the prevention and treatment of ischemic heart disease and other tissue ischemic diseases such as secondary arterial occlusion in limbs.

[A basic study on omental transplantation. Vascular endothelial cell growth factor in human omentum].

Omentum tissue has potent angiogenic activity, so transplantation is often effective in treatment of moyamoya disease. Omentum was homogenized and fractionated to investigate the presence of an endothelial cell growth factor. The effectiveness of extracts was measured by the growth activity of bovine aortic endothelial cell incubated for 6 days with various omentum extracts. The lipid fraction had no growth promoting activity. However, the water soluble extracts were active. The activity could be increased by ammonium sulfate precipitation (60-80% saturation). Gel permeation chromatography of the ammonium sulfate fraction showed that the endothelial growth factor had an apparent molecular weight of 96,000. Heparin affinity chromatography revealed poor heparin affinity. The activity was stable at pH 3.5 and pH 9.5, but inactivated by heating at 70 degrees C for 10 minutes or 100 degrees C for 2 minutes. These properties clearly distinguish the omentum-derived growth factor (OmDGF) from the heparin binding growth factor. OmDGF is probably distinct from other vascular endothelial cell growth factors.

[Cloning, purification and biological activity of human vascular endothelial growth factor fragment in E. coli].

OBJECTIVE: To observe the effect of human vascular endothelial growth factor (VEGF) fragment (3 approximately 4 exon) in E. coli on anti-angiogenesis. METHODS: Through RT-PCR amplification, endonuclease cut and DNA sequence analysis identification, hVEGF fragment cDNA was inserted into E. coli expression vector pTrcHis2A. The prokaryotic expression plasmid pTrcHis2A/VEGF(3 approximately 4) was constructed and transformed into TOP10F. RESULTS: After 8hr isopropy-beta-D-thiogalactoside (IPTG) induction, VEGF fragment was expressed in 15% of total proteins through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The expressed protein was highly antigenic and specific. The VEGF fragment was further purified by affinity, which could inhibit HUVEC proliferation and neovascularization of the chick chorioallantoic membrane. CONCLUSION: VEGF fragment is anti-angiogenetic, which may potentially be used in oncologico-biological targeting therapy.