Blood vessel maturation in a 3-dimensional spheroidal coculture model: direct contact with smooth muscle cells regulates endothelial cell quiescence and abrogates VEGF responsiveness.

Paracrine interactions between endothelial cells (EC) and mural cells act as critical regulators of vessel wall assembly, vessel maturation and define a plasticity window for vascular remodeling. The present study was aimed at studying blood vessel maturation processes in a novel 3-dimensional spheroidal coculture system of EC and smooth muscle cells (SMC). Coculture spheroids differentiate spontaneously in a calcium-dependent manner to organize into a core of SMC and a surface layer of EC, thus mimicking the physiological assembly of blood vessels with surface lining EC and underlying mural cells. Coculture of EC with SMC induces a mature, quiescent EC phenotype as evidenced by 1) a significant increase in the number of junctional complexes of the EC surface layer, 2) a down-regulation of PDGF-B expression by cocultured EC, and 3) an increased resistance of EC to undergo apoptosis. Furthermore, EC cocultured with SMC become refractory to stimulation with VEGF (lack of CD34 expression on VEGF stimulation; inability to form capillary-like sprouts in a VEGF-dependent manner in a 3-dimensional in gel angiogenesis assay). In contrast, costimulation with VEGF and Ang-2 induced sprouting angiogenesis originating from coculture spheroids consistent with a model of Ang-2-mediated vessel destabilization resulting in VEGF responsiveness. Ang-2 on its own was able to stimulate endothelial cells in the absence of Ang-1 producing SMC, inducing lateral sheet migration as well as in gel sprouting angiogenesis. Taken together, the data establish the spheroidal EC/SMC system as a powerful cell culture model to study paracrine interactions in the vessel wall and provide functional evidence for smooth muscle cell-mediated quiescence effects on endothelial cells.

Identification of a soluble form of the angiopoietin receptor TIE-2 released from endothelial cells and present in human blood.

The transmembrane tyrosine kinase TIE-2, the receptor for the angiopoietins-1 and -2, has been shown to be involved in angiogenic processes. Investigating the regulation of TIE-2 expression on endothelial cells, we found that stimulators such as PMA induce a decrease of TIE-2 protein from the cell surface without affecting TIE-2 mRNA. In conditioned media of PMA stimulated endothelial cells, a soluble form of this receptor comprising parts of the extracellular domain can be detected. Using a sandwich ELISA, we were able to detect and quantify TIE-2 receptors in cell lysates (representing the whole transmembrane receptor) and in cell culture supernatants (representing a soluble form of this receptor, sTIE-2). Several factors influencing the shedding process e.g. basic FGF could be identified. Finally, the soluble form of TIE-2 could also be detected in human biological fluids such as sera and plasma from healthy controls.

Soluble VEGFR-1 secreted by endothelial cells and monocytes is present in human serum and plasma from healthy donors.

It was shown before that the soluble form of VEGFR-1 (sVEGFR-1) is present in serum of pregnant women. The aim of the present study was to investigate the presence of this endogenous vascular endothelial growth factor-A (VEGF-A) antagonist in human serum in more detail. sVEGFR-1 was detected in human serum and plasma from normal healthy male and female donors by ELISA. sVEGFR-1 levels ranged from non-detectable up to 440 pg/ml, with no significant difference between male and female donors. In addition, vein endothelial cells (ECs) from an intact vascular bed, the umbilical cord, were shown to secrete sVEGFR-1. Furthermore, human peripheral blood monocytes, a non-EC type expressing VEGFR-1, were shown to contribute to the sVEGFR-1 detectable in human serum and plasma for the first time. EC- and monocyte-derived sVEGFR-1 proved capable of inhibiting the VEGF-induced proliferation and migration of ECs in vitro. Finally, secretion of sVEGFR-1 was increased by the angiogenic factor basic fibroblast growth factor (bFGF) in human ECs and was also enhanced in lipopolysaccharide-activated human monocytes. In human umbilical vein endothelial cells, both the membrane-bound and the sVEGFR-1 seem to be equally regulated on the mRNA as well as the protein level. The presence of an sVEGFR-1 in human serum and plasma of normal male and female donors strongly suggests that it plays an important role as a naturally occurring VEGF antagonist in the regulation and availability of VEGF-mediated biological activities in vivo.

Donor stromal cells from human blood engraft in NOD/SCID mice.

Little is known about the presence, frequency, and in vivo proliferative potential of stromal cells within blood-derived hematopoietic transplants. In this study, nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice were injected with human CD34(+) peripheral blood cells (PBCs) or cord blood cells (CBCs, either enriched for CD34 or density-gradient separated mononuclear cells). Flow cytometric analysis 5 to 11 weeks after transplantation revealed the presence of a human lymphomyeloid hematopoiesis within the murine bone marrow. Immunohistochemical staining of bone marrow cell suspensions using human-specific antibodies showed human cells staining positive for human fibroblast markers, human von Willebrand factor (vWF) and human KDR (vascular endothelial growth factor receptor-2) in mice transplanted with CD34(+) PBCs or CBCs, with mean frequencies between 0.6% and 2.4%. In stromal layers of bone marrow cultures established from the mice, immunohistochemical staining using human-specific antibodies revealed flattened reticular cells or spindle-shaped cells staining positive with human-specific antifibroblast antibodies (mean frequency, 2.2%). Cell populations of more rounded cells stained positive with human-specific antibodies recognizing CD34 (1.5%), vWF (2.2%), and KDR (1.6%). Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis and subsequent complementary DNA sequencing detected transcripts of human KDR (endothelial specific) and human proline hydroxylase-alpha (fibroblast specific) within the bone marrow and spleen of transplanted mice. Analysis of nontransplanted control mice yielded negative results in immunocytochemistry and RT-PCR. Cells expressing endothelial and fibroblast markers were also detected in the grafts before transplantation, and their numbers increased up to 3 log in vivo after transplantation. These results indicate that stromal progenitor cells are present in human cytokine-mobilized peripheral blood or cord blood that engraft in NOD/SCID mice. (Blood. 2000;96:3971-3978)

Effects of PTK787/ZK 222584, a specific inhibitor of vascular endothelial growth factor receptor tyrosine kinases, on primary tumor, metastasis, vessel density, and blood flow in a murine renal cell carcinoma model.

Antiangiogenic therapy is a promising new strategy to inhibit tumor growth and formation of metastases. Vascular endothelial growth factor (VEGF) and its receptors, VEGF-receptor 1 (VEGF-R1; FLT-1) and VEGF-R2 (KDR), have been shown to play a major role in tumor angiogenesis. PTK787/ZK 222584, a specific inhibitor of both VEGF-receptor tyrosine kinases, was investigated for its antitumoral and antiangiogenic activity in a murine renal cell carcinoma model. After intrarenal application of the renal carcinoma cells, mice develop a primary tumor and metastases to the lung and to the abdominal lymph nodes. Daily oral therapy with PTK787/ZK 222584 at a dose of 50 mg/kg resulted in a significant decrease of 61 and 67% in primary tumors after 14 and 21 days, respectively. The occurrence of lung metastases was significantly inhibited at both time points (98% reduction and 78% reduction, respectively). After 14 days, no lymph node metastases developed in the PTK787/ZK 222584-treated group, whereas after 21 days of treatment, the lymph node metastases were reduced by 87%. Vessel density in tumor tissues, detected by immunohistochemistry with an anti-CD31 antibody, was significantly decreased by PTK787/ZK 222584. Using color Doppler imaging ultrasound, significant changes in blood flow in the tumor feeding renal artery were found under treatment with PTK787/ZK 222584. Blood flow changes correlated with changes in vessel density but not with tumor volume. The compound was well tolerated in all in vivo experiments and had no significant effects on body weight or general well-being of the animals. This was in contrast to the animals treated with the antiangiogenic agent TNP-470. s.c. therapy with 30 mg/kg TNP-470 every other day had to be discontinued after 13 days because of animal weight loss (>20%) and ataxia. These results demonstrate that PTK787/ZK 222584 is a potent inhibitor of tumor growth, metastases formation, and tumor vascularization in murine renal cell carcinoma. Furthermore, we have been able to demonstrate that color Doppler imaging ultrasound can be used to measure blood flow to a tumor and that flow correlates with vessel density. Thus, this may be a valuable noninvasive method for monitoring the effects of antiangiogenic agents such as PTK787/ZK 222584 on tumor vasculature.

New anilinophthalazines as potent and orally well absorbed inhibitors of the VEGF receptor tyrosine kinases useful as antagonists of tumor-driven angiogenesis.

The sprouting of new blood vessels, or angiogenesis, is necessary for any solid tumor to grow large enough to cause life-threatening disease. Vascular endothelial growth factor (VEGF) is one of the key promoters of tumor induced angiogenesis. VEGF receptors, the tyrosine kinases Flt-1 and KDR, are expressed on vascular endothelial cells and initiate angiogenesis upon activation by VEGF. 1-Anilino-(4-pyridylmethyl)-phthalazines, such as CGP 79787D (or PTK787 / ZK222584), reversibly inhibit Flt-1 and KDR with IC(50) values < 0.1 microM. CGP 79787D also blocks the VEGF-induced receptor autophosphorylation in CHO cells ectopically expressing the KDR receptor (ED(50) = 34 nM). Modification of the 1-anilino moiety afforded derivatives with higher selectivity for the VEGF receptor tyrosine kinases Flt-1 and KDR compared to the related receptor tyrosine kinases PDGF-R and c-Kit. Since these 1-anilino-(4-pyridylmethyl)phthalazines are orally well absorbed, these compounds qualify for further profiling and as candidates for clinical evaluation.

Targeting of endothelial KDR receptors with 3G2 immunoliposomes in vitro.

Immunoliposomes (IL) containing anti-angiogenic drugs directed selectively to the easily accessible kinase insert domain containing receptor (KDR) vascular endothelial growth factor (VEGF), which is predominantly expressed on tumour vessels are a promising tool to inhibit tumour angiogenesis. To explore this strategy, we have prepared fluorescent-labelled IL presenting antibodies against the KDR receptor (3G2) on their surface. 3G2-IL were composed of egg phosphatidylcholine and cholesterol (6:4), containing 2 mol% of the new thiol reactive linker lipid O-(3-cholesteryloxycarbonyl)propionyl-O'-m-maleimido-benzoyl tetraethylene glycol. Specific binding of 3G2-IL to immobilised recombinant KDR was used to show the maintenance of sufficient immunoreactivity of 3G2 antibodies upon the coupling procedure. 3G2-IL bound to Chinese hamster ovarian (CHO) cells stably transfected to overexpress KDR to a five times higher amount as compared to mock-transfected CHO cells. Subsequently, specific binding of 3G2-IL to KDR could also be demonstrated on KDR expressing cells, human umbilical vein endothelial cells and human microvascular endothelial cells, whereas only low binding of 3G2-IL to NIH-3T3 mouse fibroblast cells, which do not express KDR, was found. The binding of 3G2-IL to KDR receptors could not be blocked by VEGF, suggesting that the binding site for VEGF is not identical with the epitope recognised by 3G2. We could demonstrate that 3G2-IL is able to bind in vitro even in the presence of high levels of VEGF.

PTK787/ZK 222584, a novel and potent inhibitor of vascular endothelial growth factor receptor tyrosine kinases, impairs vascular endothelial growth factor-induced responses and tumor growth after oral administration.

PTK787/ZK 222584 (1-[4-chloroanilino]-4-[4-pyridylmethyl] phthalazine succinate) is a potent inhibitor of vascular endothelial growth factor (VEGF) receptor tyrosine kinases, active in the submicromolar range. It also inhibits other class III kinases, such as the platelet-derived growth factor (PDGF) receptor beta tyrosine kinase, c-Kit, and c-Fms, but at higher concentrations. It is not active against kinases from other receptor families, such as epidermal growth factor receptor, fibroblast growth factor receptor-1, c-Met, and Tie-2, or intracellular kinases such as c-Src, c-Abl, and protein kinase C-alpha. PTK787/ZK 222584 inhibits VEGF-induced autophosphorylation of kinase insert domain-containing receptor (KDR), endothelial cell proliferation, migration, and survival in the nanomolar range in cell-based assays. In concentrations up to 1 microM, PTK787/ZK 222584 does not have any cytotoxic or antiproliferative effect on cells that do not express VEGF receptors. After oral dosing (50 mg/kg) to mice, plasma concentrations of PTK787/ZK 222584 remain above 1 microM for more than 8 h. PTK787/ZK 222584 induces dose-dependent inhibition of VEGF and PDGF-induced angiogenesis in a growth factor implant model, as well as a tumor cell-driven angiogenesis model after once-daily oral dosing (25-100 mg/kg). In the same dose range, it also inhibits the growth of several human carcinomas, grown s.c. in nude mice, as well as a murine renal carcinoma and its metastases in a syngeneic, orthotopic model. Histological examination of tumors revealed inhibition of microvessel formation in the interior of the tumor. PTK787/ZK 222584 is very well tolerated and does not impair wound healing. It also does not have any significant effects on circulating blood cells or bone marrow leukocytes as a single agent or impair hematopoetic recovery after concomitant cytotoxic anti-cancer agent challenge. This novel compound has therapeutic potential for the treatment of solid tumors and other diseases where angiogenesis plays an important role.

Active interaction of human A375 melanoma cells with the lymphatics in vivo.

We have used the avian chorioallantoic membrane (CAM) to study the interaction of tumor cells with the lymphatics in vivo. The vascular endothelial growth factor-C (VEGF-C) has been shown to be lymphangiogenic. We have therefore grown VEGF-C-expressing human A375 melanoma cells on the CAM. These tumors induced numerous lymphatics at the invasive front, and compressed or destroyed VEGF receptor (R)-3-positive lymphatics were observed within the solid tumors. The lymphatics in the CAM and in the A375 melanomas could also be demonstrated with an antibody against Prox 1, a highly specific marker of lymphatic endothelial cells. Proliferation studies revealed a BrdU labeling index of 11.6% of the lymphatic endothelial cells in the tumors and at their margins. A great number of melanoma cells invaded the lymphatics. Such interactions were not observed with VEGF-C-negative Malme 3 M melanoma cells. Lymphangiogenesis was inhibited to some extent when A375 melanoma cells were transfected with cDNA encoding soluble VEGFR-3 (sflt4), and the BrdU labeling index of the lymphatics in these tumors was 3.9%. Invasion of lymphatics and growth of blood vascular capillaries were not inhibited by the transfection. Therefore, tumor-induced lymphangiogenesis seems to be dependent to some extent on VEGF-C/flt4 interactions, but invasion of lymphatics seems to be a distinct mechanism.

Two independent mechanisms essential for tumor angiogenesis: inhibition of human melanoma xenograft growth by interfering with either the vascular endothelial growth factor receptor pathway or the Tie-2 pathway.

Protein ligands and receptor tyrosine kinases that specifically regulate endothelial cell function are mainly involved in physiological as well as in disease-related angiogenesis. These ligand/receptor systems include the vascular endothelial growth factor (VEGF) and the angiopoietin (Ang) families, and their receptors, the VEGF receptor family and the tyrosine kinase with immunoglobulin-like and epidermal growth factor homology domains (Tie) family. In the present study, the contribution of these endothelium-specific ligand/receptor systems to tumor angiogenesis was evaluated. A375v human melanoma cells, which express at least the angiogenic growth factors VEGF, VEGF-C, and Ang-1, were stably transfected to overexpress the extracellular ligand-binding domains of the endothelium-specific receptor tyrosine kinases fms-like tyrosine kinase-1 (Flt-1), Flt-4, Tie-1, and Tie-2, respectively. In vitro proliferation and colony formation assays confirmed that expression of the extracellular receptor domains inhibited neither tumor cell mitogenesis nor the ability to produce anchorage-independent growth. Nude mouse xenografts revealed that interference with either the VEGF receptor pathway or the Tie-2 pathway resulted in a significant inhibition of tumor growth and tumor angiogenesis. In contrast, interference with the Flt-4 pathway or the Tie-1 pathway was without significant effect. Our results show that both the VEGF receptor pathway and the Tie-2 pathway are essential for A375v melanoma xenograft growth. The inhibition of the VEGF receptor pathway cannot be compensated by the Tie-2 pathway, nor vice versa. These findings suggest that the VEGF receptor pathway and the Tie-2 pathway have to be considered as two independent mediators essential for the process of in vivo angiogenesis.

The alpha-helical domain near the amino terminus is essential for dimerization of vascular endothelial growth factor.

Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen and a key mediator of aberrant endothelial cell proliferation and vascular permeability in a variety of human pathological situations such as tumor angiogenesis, diabetic retinopathy, or psoriasis. By amino-terminal deletion analysis and by site-directed mutagenesis we have identified a new domain within the amino-terminal alpha-helix that is essential for dimerization of VEGF. VEGF121 variants containing amino acids 8 to 121 or 14 to 121, respectively, either expressed in Escherichia coli and refolded in vitro, or expressed in Chinese hamster ovary cells, were in a dimeric conformation and showed full binding activity to VEGF receptors and stimulation of endothelial cell proliferation as compared with wild-type VEGF. In contrast, a VEGF121 variant covering amino acids 18 to 121, as well as a variant in which the hydrophobic amino acids Val14, Val15, Phe17, and Met18 within the amphipathic alpha-helix near the amino terminus were replaced by serine, failed to form biological active VEGF dimers. From these data we conclude that a domain between amino acids His12 and Asp19 within the amino-terminal alpha-helix is essential for formation of VEGF dimers, and we propose hydrophobic interactions between VEGF monomers to stabilize or favor dimerization.

An antagonistic vascular endothelial growth factor (VEGF) variant inhibits VEGF-stimulated receptor autophosphorylation and proliferation of human endothelial cells.

Vascular endothelial growth factor (VEGF) is a potent mitogen with a unique specificity for endothelial cells and a key mediator of aberrant endothelial cell proliferation and vascular permeability in a variety of human pathological situations, such as tumor angiogenesis, diabetic retinopathy, rheumatoid arthritis, or psoriasis. VEGF is a symmetric homodimeric molecule with two receptor binding interfaces lying on each pole of the molecule. Herein we report on the construction and recombinant expression of an asymmetric heterodimeric VEGF variant with an intact receptor binding interface at one pole and a mutant receptor binding interface at the second pole of the dimer. This VEGF variant binds to VEGF receptors but fails to induce receptor activation. In competition experiments, the heterodimeric VEGF variant antagonizes VEGF-stimulated receptor autophosphorylation and proliferation of endothelial cells. A 15-fold excess of the heterodimer was sufficient to inhibit VEGF-stimulated endothelial cell proliferation by 50%, and a 100-fold excess resulted in an almost complete inhibition. By using a rational approach that is based on the structure of VEGF, we have shown the feasibility to construct a VEGF variant that acts as an VEGF antagonist.

Novel antibodies directed against the extracellular domain of the human VEGF-receptor type II.

Vascular endothelial cell growth factor (VEGF) plays a pivotal role in the regulation of angiogenesis by binding to its cognate receptor molecule type II (VEGFr-II, KDR). VEGFr-II is an endothelial cell-specific transmembrane tyrosine kinase important for vascular endothelial cell development and differentiation during embryogenesis, angiogenic processes under physiological conditions, and various diseases. An increasing number of reports indicate that VEGF/VEGFr-II also play a fundamental role for tumor angiogenesis. We present the generation and in vitro characterization of the monoclonal antibodies 2-7-9 and 2-10-1. Both antibodies are highly specific for VEGFr-II as demonstrated by Western blotting and immunoprecipitation. MAbs 2-10-1 and 2-7-9 bind to a disulphide bridge-stabilized epitope within domains 6 and 7 of the human VEGFr-II with an affinity of 8 and 80 nM, respectively. Furthermore, the antibodies are suitable for immunohistochemistry and ELISA techniques. Because both antibodies recognize their epitope on living cells, they also have the potential for drug targeting and diagnostic purposes.

Vascular endothelial growth factor up-regulates its receptor fms-like tyrosine kinase 1 (FLT-1) and a soluble variant of FLT-1 in human vascular endothelial cells.

The growth of solid tumors and the formation of metastases are dependent on neoangiogenesis. One of the most important factors in inducing the formation of new blood vessels is the vascular endothelial growth factor (VEGF), which acts specifically on endothelial cells. VEGF is expressed and secreted by almost all solid tumors. The molecular mechanisms leading to enhanced production of this angiogenic mitogen are manyfold and have been elucidated to some degree. Two VEGF receptors, fms-like tyrosine kinase 1 (FLT-1) and KDR, have been identified almost specifically on human endothelial cells. They are expressed preferentially in the proliferating endothelium of vessels lining and/or penetrating solid tumors, whereas they are almost undetectable by convenient methods in vessels of healthy tissue. However, the underlying mechanisms are not understood. We could show that media conditioned by various cancer cell lines grown under hypoxic conditions were able to up-regulate expression of FLT-1 mRNA and protein but not of KDR mRNA. Furthermore, up-regulation of a shorter mRNA species was observed that most probably codes for the soluble variant of FLT-1. These effects were completely inhibited by VEGF-neutralizing extracellular VEGF receptor domains. The effect could be mimicked by adding recombinant VEGF instead of conditioned cancer cell medium to the endothelial cell cultures. Both mutant VEGF, which activates only KDR, and placenta growth factor, which activates only FLT-1, were able to enhance FLT-1 expression. VEGF-stimulated FLT-1 mRNA expression was inhibited by actinomycin D. These data suggest that VEGF itself is the main factor secreted by tumor cells that is able to enhance the expression of its receptor FLT-1 and of a soluble variant of FLT-1 in endothelial cells.

Mapping of the sites for ligand binding and receptor dimerization at the extracellular domain of the vascular endothelial growth factor receptor FLT-1.

The vascular endothelial growth factor (VEGF) receptor FLT-1 has been shown to be involved in vasculogenesis and angiogenesis. The receptor is characterized by seven Ig-like loops within the extracellular domain. Upon VEGF binding FLT-1 becomes phosphorylated, which has been thought to be preceded by receptor dimerization. To further investigate high affinity binding of VEGF to FLT-1 and ligand-induced receptor dimerization, we expressed in Sf9 cells the entire extracellular domain comprising all seven Ig-like loops: sFLT-1(7) and several truncated mutants consisting of loop one, one and two, one to three, one to four, and one to five. The corresponding proteins, named sFLT-1(1), (2), (3), (4), and (5) were purified. Only mutants sFLT-1(3) to (7) were able to bind 125I-VEGF with high affinity. No binding of VEGF was observed with sFLT-1(1) and sFLT-1(2), indicating that the first three Ig-like loops are involved in high affinity binding of VEGF. The binding of VEGF to sFLT-1(3) could be competed with placenta growth factor (PlGF), a VEGF-related ligand, suggesting that high affinity binding of VEGF and PlGF is mediated by the same or closely related contact sites on sFLT-1. Deglycosylation of the sFLT-1(3), (4), (5), and (7) did not abolish VEGF binding. Furthermore, unglycosylated sFLT-1(3), expressed in Escherichia coli, was able to bind VEGF with similar affinity as sFLT-1(3) or sFLT-1(7), both expressed in Sf9 cells. This indicates that receptor glycosylation is not essential for high affinity binding. Dimerization of the extracellular domains of FLT-1 upon addition of VEGF was detected with all mutants containing the Ig-like loop four. Although sFLT-1(3) was able to bind VEGF, dimerization of this mutant was inefficient, indicating that sites on Ig-like loop four are essential to stabilize receptor dimers.

Chemiluminescence immunoassay for vascular endothelial growth factor (vascular permeability factor) in tumor-tissue homogenates.

We developed a two-site chemiluminescence immunoassay for human vascular endothelial growth factor (VEGF). The assay recognized both VEGF121 and VEGF165 isoforms, but had no detectable cross-reactivity with platelet-derived growth factor or placenta growth factor. The range of detection was between 30 ng/L and 30 micrograms/L VEGF. Inter- and intraassay variations were 8.2-8.3% and 7.2-7.6%, respectively. VEGF concentrations were measured in the cytosolic extracts of 45 ovarian and 142 primary breast tumors. The amount of VEGF in the ovarian tumors (median = 0.46 ng/mg total protein, range 0-15.8 ng/mg) was significantly (P = 0.03) higher compared with the breast tumors (median = 0.24 ng/mg total protein, range 0-12.3 ng/mg). In 32 and 7 extracts of normal breast tissues adjacent and distant to the tumors, respectively, VEGF concentrations were significantly much lower (P < 0.0001). The detection of substantial amounts of VEGF in two invasive tumors (compared with normal tissues) suggests that the assay should be a useful tool for investigating the prognostic value of VEGF in breast and ovarian carcinomas and for selecting patients for future anti-VEGF therapy.

VEGF121 induces proliferation of vascular endothelial cells and expression of flk-1 without affecting lymphatic vessels of chorioallantoic membrane.

We have studied the effect of VEGF(121) homodimer and VEGF(121/165) heterodimer on the chorioallantoic membrane (CAM) of 13-day-old chick embryos. The factors were applied in doses of 2-4 micrograms and the effects were evaluated macroscopically after 2 and 3 days. Histological studies were performed on semi- and ultrathin sections. Proliferation was studied according to the BrdU-anti-BrdU method on whole mounts and sections. The labeling density was quantified in whole mounts. The fractal dimension, D, of the vascular tree was assessed as a value for vascular bifurcation density. Both forms of VEGF induce brush-like vessel formation in the precapillary region. New capillaries are found in the stroma of the CAM, which normally does not contain capillaries. Our results show that VEGF(121) is a specific endothelial cell mitogen. A fourfold increase of BrdU-labeled endothelial cells is found after VEGF(121) application. The fractal dimension of the vascular tree increases from 1.26 in the controls to 1.44 (VEGF(121)) and 1.41 (VEGF(121/165)). The endothelial cells of the newly formed capillaries possess many mitochondria and micropinocytotic vesicles, but no fenestrations. These capillaries are obviously formed by intussusceptive microvascular growth. Signs of sprouting are almost absent. An effect on the lymphatic vessels of the CAM is not detectable. Compared to VEGF(165) and VEGF(121/165), VEGF(121) diffuses over a slightly greater distance. Using in situ hybridization, VEGF receptor-2 (flk-1/Quek1) and the homologous flt-4 (Quek2) receptor were studied in the CAM of normal quail embryos and after VEGF(121) application on the CAM of 11-day-old quail embryos. During normal development, flk-1 expression becomes restricted to vascular endothelial cells of large vessels in the stroma of the CAM. VEGF(121) application induces expression of flk-1 in capillaries that normally do not express the receptor. In the normal development of the CAM, flt-4 becomes restricted to endothelial cells of vessels that appear to be lymphatic vessels. Application of VEGF(121) does not alter flt-4 expression.

Expression of biologically active isoforms of the tumor angiogenesis factor VEGF in Escherichia coli.

Vascular endothelial growth factor (VEGF) was identified as an endothelial cell specific mitogen that induces angiogenesis and vascular permeability in vivo. VEGF is a homodimeric protein which contains three intramolecular and two intermolecular disulfide bridges. Here, we report on an efficient procedure for recombinant production of VEGF isoforms VEGF121 and VEGF165 in Escherichia coli. The proteins were solubilized from inclusion bodies, refolded, and purified by chromatographic methods. The final protein products were almost completely in the dimeric conformation, bound to VEGF receptor FLT1 with a Kd of 30 pM, stimulated proliferation of human umbilical vein endothelial cells half-maximally at a concentration of 30 pM, and induced in vivo neovascularization and vascular permeability on the chicken chorioallantoic membrane.