A recombinant mutant vascular endothelial growth factor-C that has lost vascular endothelial growth factor receptor-2 binding, activation, and vascular permeability activities

The vascular endothelial growth factor (VEGF) and the VEGF-C promote growth of blood vessels and lymphatic vessels, respectively. VEGF activates the endothelial VEGF receptors (VEGFR) 1 and 2, and VEGF-C activates VEGFR-3 and VEGFR-2. Both VEGF and VEGF-C are also potent vascular permeability factors. Here we have analyzed the receptor binding and activating properties of several cysteine mutants of VEGF-C including those (Cys156 and Cys165), which in other platelet-derived growth factor/VEGF family members mediate interchain disulfide bonding. Surprisingly, we found that the recombinant mature VEGF-C in which Cys156 was replaced by a Ser residue is a selective agonist of VEGFR-3. This mutant, designated DeltaNDeltaC156S, binds and activates VEGFR-3 but neither binds VEGFR-2 nor activates its autophosphorylation or downstream signaling to the ERK/MAPK pathway. Unlike VEGF-C, DeltaNDeltaC156S neither induces vascular permeability in vivo nor stimulates migration of bovine capillary endothelial cells in culture. These data point out the critical role of VEGFR-2-mediated signal transduction for the vascular permeability activity of VEGF-C and strongly suggest that the redundant biological effects of VEGF and VEGF-C depend on binding and activation of VEGFR-2. The DeltaNDeltaC156S mutant may provide a valuable tool for the analysis of VEGF-C effects mediated selectively via VEGFR-3. The ability of DeltaNDeltaC156S to form homodimers also emphasizes differences in the structural requirements for VEGF and VEGF-C dimerization.     

Human neutrophils secrete vascular endothelial growth factor

Vascular endothelial growth factor (VEGF) is a multifunctional cytokine that plays a pivotal role in mediating neovascularization as well as other endothelial cell alterations during inflammation. In this study, we demonstrate that human neutrophils are a source of VEGF. We observed that isolated blood neutrophils released VEGF in response to different stimuli and we demonstrated by immunohistochemistry that neutrophils infiltrating inflamed tissues contain VEGF. These results indicate that neutrophil-derived VEGF may be instrumental in regulating vascular responses during acute and chronic inflammation.     

Expression of vascular endothelial growth factor and placenta growth factor in human placenta

Normal development and function of the placenta requires invasion of the maternal decidua by trophoblasts, followed by abundant and organized vascular growth. Little is known of the significance and function of the vascular endothelial growth factor (VEGF) family, which includes VEGF, VEGF-B, and VEGF-C, and of placenta growth factor (PIGF) in these processes. In this study we have analyzed the expression of VEGF and PIGF mRNAs and their protein products in placental tissue obtained from noncomplicated pregnancies. Expression of VEGF and PIGF mRNA was observed by in situ hybridization in the chorionic mesenchyme and villous trophoblasts, respectively. Immunostaining localized the VEGF and PIGF proteins in the vascular endothelium, which was defined by staining for von Willebrand factor and for the Tie receptor tyrosine kinase, an early endothelial cell marker. VEGF-B and VEGF-C mRNAs were strongly expressed in human placenta as evidenced by Northern blot analysis. These data imply that VEGF and PIGF are produced by different cells but that both target the endothelial cells of normal human term placenta.     

Transforming growth factor beta 1 down-regulates vascular endothelial growth factor receptor 2/flk-1 expression in vascular endothelial cells

Although the importance of the vascular endothelial growth factor (VEGF)/VEGF tyrosine kinase receptor (VEGFR) system in angiogenesis is well established, very little is known about the regulation of VEGFR expression in vascular endothelial cells. We have cloned partial cDNAs encoding bovine VEGFR-1 (flt) and -2 (flk-1) and used them to study VEGFR expression by bovine microvascular- and large vessel-derived endothelial cells. Both cell lines express flk-1, but not flt. Transforming growth factor beta 1 (TGF-beta 1) reduced the high affinity 125I-VEGF binding capacity of both cell types in a dose-dependent manner, with a 2.0-2.7-fold decrease at 1-10 ng/ml. Cross-linking experiments revealed a decrease in 125I-VEGF binding to a cell surface monomeric protein corresponding to Flk-1 on the basis of its affinity for VEGF, molecular mass (185-190 kDa), and apparent internalization after VEGF binding. Immunoprecipitation and Western blot experiments demonstrated a decrease in Flk-1 protein expression, and TGF-beta 1 reduced flk-1 mRNA levels in a dose-dependent manner. These results imply that TGF-beta 1 is a major regulator of the VEGF/Flk-1 signal transduction pathway in endothelial cells.     

Identification of vascular endothelial growth factor receptor-1 tyrosine phosphorylation sites and binding of SH2 domain-containing molecules

Receptor tyrosine phosphorylation is crucial for signal transduction by creating high affinity binding sites for Src homology 2 domain-containing molecules. By expressing the intracellular domain of Flt-1/vascular endothelial growth factor receptor-1 in the baculosystem, we identified two major tyrosine phosphorylation sites at Tyr-1213 and Tyr-1242 and two minor tyrosine phosphorylation sites at Tyr-1327 and Tyr-1333 in this receptor. This pattern of phosphorylation of Flt-1 was also detected in vascular endothelial growth factor-stimulated cells expressing intact Flt-1. In vitro protein binding studies using synthetic peptides and immunoblotting showed that phospholipase C-gamma binds to both Y(p)1213 and Y(p)1333, whereas Grb2 and SH2-containing tyrosine protein phosphatase (SHP-2) bind to Y(p)1213, and Nck and Crk bind to Y(p)1333 in a phosphotyrosine-dependent manner. In addition, unidentified proteins with molecular masses around 74 and 27 kDa bound to Y(p)1213 and another of 75 kDa bound to Y(p)1333 in a phosphotyrosine-dependent manner. SHP-2, phospholipase C-gamma, and Grb2 could also be shown to bind to the intact Flt-1 intracellular domain. These results indicate that a spectrum of already known as well as novel phosphotyrosine-binding molecules are involved in signal transduction by Flt-1.     

青蒿琥酯对急性单核细胞白血病SHI-1细胞株血管内皮生长因子及其受体表达的影响

目的 研究青蒿琥酯对急性单核细胞白血病SHI-1细胞株血管内皮生长因子(VEGF)及其受体( VEGFR)的影响。方法酶联免疫吸附法检测非细胞毒性浓度(5、10、20 ng/ml)青蒿琥酯作用SHI-1细胞后培养上清液VEGF浓度,流式细胞术检测有或无青蒿琥酯作用时,SHI-1细胞表面VEGFR-1及VEGFR-2阳性表达率。结果培养24、48 h后,无青蒿琥酯作用的SHI-1细胞培养上清液VEGF质量浓度分别为( 980.3±2.2)、(982.4±2.3) pg/ml,VEGFR-1表达率分别为(5.40±3.11)％和(4.45±2.85)％,VEGFR-2表达率分别为(13.90.± 2.26)％和（13.95±1.96）％。5、10、20 ng/ml青蒿琥酯作用24h后,SHI-1细胞培养上清液VEGF质量浓度分别为(234.6±1.8)、(114.9±1.6)、(108.8±1.5) pg/ml,作用48 h后分别为(62.3±1.7)、(60.9±1.6)、(32.7±1.7) pg/ml,与培养相同时间无青蒿琥酯组相比,VEGF浓度明显下降（均P＜ 0.05）,且相同浓度青蒿琥酯作用24 h与48 h间差异亦有统计学意义(均P＜ 0.05)。5、10、20 ng/ml青蒿琥酯作用24 h,VEGFR-1阳性率分别为(4.30±2.21)％、(4.20±1.37)％和(3.90±1.86)％,作用48 h后分别为(3.80±2.87)％、(3.60±1.73)％和(3.00±1.82)％,相同作用时间不同浓度青蒿琥酯组间及相同浓度作用不同时间组间VEGFR-1阳性率差异均无统计学意义(均P＞ 0.05);作用24h后,SHI-1细胞VEGFR-2阳性率分别为(4.40±1.15)％、(3.10±0.68)％和(1.10±0.72)％,作用48 h后分别为(3.00±1.68)％、(2.20±0.93)％和(0.60±0.92)％,3个不同浓度青蒿琥酯作用相同时间后VEGFR-2表达率降低（均P＜ 0.05）,相同浓度作用24与48 h间差异均无统计学意义（均P＞ 0.05）。结论SHI-1细胞株高分泌VEGF,青蒿琥酯可下调VEGF分泌及VEGFR-2的表达,而对VEGFR-1表达的调节作用不显著。     

Expression of the angiogenic factors, basic fibroblast growth factor and vascular endothelial growth factor, in the ovary

In adult tissues, vascular growth (angiogenesis) occurs normally during tissue repair, such as in the healing of wounds and fractures. Inappropriate vascular growth is associated with various pathological conditions. These conditions include tumor growth, retinopathies, hemangiomas, fibroses, and rheumatoid arthritis in the case of rampant vascular growth and nonhealing wounds and fractures in the case of inadequate vascular growth. The female reproductive organs exhibit dramatic, periodic growth and regression, accompanied by equally dramatic changes in their rates of blood flow. Thus, it is not surprising that they are some of the few adult tissues in which angiogenesis occurs as a normal process. Ovarian follicles and corpora lutea contain and produce angiogenic factors. These angiogenic factors bind heparin and seem to belong to the fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) families of proteins. Based on our studies of the pattern of expression of FGF and its major receptors in bovine, ovine, and porcine corpora lutea, we have suggested that FGF may influence not only luteal cell proliferation but also cell death, thereby regulating cell turnover in the luteal vascular and nonvascular compartments. In addition, we recently have shown that luteal expression of VEGF is greatest during the early luteal phase, coincident with luteal vascularization. Moreover, VEGF is present exclusively in luteal connective tissue and perivascular (arteriolar smooth muscle and capillary pericyte) cells. In fact, the first thecal-derived cells to invade the granulosa-derived regions immediately after ovulation seem to be VEGF-containing pericytes. We have therefore hypothesized that ovarian pericytes play a key role in vascularization of developing follicles and corpora lutea. Further understanding of the specific physiological roles of these factors in follicular and luteal growth, development, and function will ultimately lead to improved methods of regulating fertility.     

Role of vascular endothelial growth factor on erythropoietin-related endothelial cell proliferation

The vascular actions of recombinant human erythropoietin (rhEPO) are of particular relevance for fully understanding rhEPO effects. This study examines the mechanisms of action of rhEPO on endothelial cells from bovine aorta (BAEC). First, the studies demonstrated that rhEPO acts on BAEC proliferation as a comitogenic growth factor in the presence of fetal calf serum (FCS). The main experimental findings disclosed that an interaction between rhEPO and vascular endothelial growth factor (VEGF) is instrumental for the growth-promoting action of rhEPO, as shown by the blockade (92.8+/-2.2% inhibition, P < 0.01) of the rhEPO-induced BAEC proliferation by a specific anti-VEGF antibody and by the capability of VEGF for substituting FCS in the induction of rhEPO-related BAEC proliferation (increase in BAEC number in the absence of FCS: 20 U/ml rhEPO alone, 0.3+/-2.8%; 5 x 10(-11) M VEGF alone, 52.9+/-3.1%; 20 U/ml rhEPO + 5 X 10(-11) M VEGF, 117.8+/-6.9%, P < 0.01 between the two agents combined with respect to each agent alone). The existence of a positive interaction between rhEPO and VEGF was further demonstrated by observing an increased cytosolic Ca2+ ([Ca2+]i) mobilization response to VEGF (10(-11)M) in BAEC pretreated or not with 20 U/ml rhEPO (delta[Ca2+]i = 704+/-111 versus 246+/-36 nM, respectively, P < 0.01). To further examine the mechanism of the potentiation of VEGF effect by rhEPO, we analyzed the mRNA expression of the VEGF receptors KDR/flk-1 and flt-1. The results disclosed that BAEC pretreatment with rhEPO upregulated the expression of both KDR/flk-1 and flt-1, therefore providing a structural basis for the aforementioned positive interactions between VEGF and rhEPO. Furthermore, inhibition by genistein suggests that tyrosine phosphorylation was involved in the VEGF receptor upregulation. The mechanisms identified in the present study disclose an interaction at the level of mRNA expression and functional effects between a hormone with predominantly hemopoietic effects, namely, erythropoietin, and an angiogenic factor, namely, VEGF. This relationship between rhEPO and VEGF might be of particular importance in neovascularization processes and in patients receiving rhEPO as a treatment.     

Enhancement of gene expression under hypoxic conditions using fragments of the human vascular endothelial growth factor and the erythropoietin genes

PURPOSE: Selective gene expression in response to tumor hypoxia may provide new avenues, not only for radiotherapy and chemotherapy, but also for gene therapy. In this study, we have assessed the extent of hypoxia responsiveness of various DNA constructs by the luciferase assay to help design vectors suitable for cancer therapy. MATERIALS AND METHODS: Reporter plasmids were constructed with fragments of the human vascular endothelial growth factor (VEGF) and the erythropoietin (Epo) genes encompassing the putative hypoxia-responsive elements (HRE) and the pGL3 promoter vector. Test plasmids and the control pRL-CMV plasmid were cotransfected into tumor cells by the calcium phosphate method. After 6 h hypoxic treatment, the reporter assay was performed. RESULTS: The construct pGL3/VEGF containing the 385 bp fragment of the 5' flanking region in human VEGF gene showed significant increases in luciferase activity in response to hypoxia. The hypoxic/aerobic ratios were about 3-4, and 8-12 for murine and human tumor cells, respectively. Despite the very high degree of conservation among the HREs of mammalian VEGF genes, murine cells showed lower responsiveness than human cells. We next tested the construct pGL3/Epo containing the 150 bp fragment of the 3' flanking region in the Epo gene. Luciferase activity of pGL3/Epo was increased with hypoxia only in human cell lines. The insertion of 5 copies of the 35-bp fragments derived from the VEGF HREs and 32 bp of the E1b minimal promoter resulted in maximal enhancement of hypoxia responsiveness. CONCLUSIONS: The constructs with VEGF or Epo fragments containing HRE may be useful for inducing specific gene expression in hypoxic cells. Especially, the application of multiple copies of the HREs and an E1b minimal promoter appears to have the advantage of great improvement in hypoxia responsiveness.     

Characterization of vascular permeability factor/vascular endothelial growth factor receptors on mononuclear phagocytes

Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) is a polypeptide mediator, elaborated by certain tumors and other cell types, that exerts multiple effects on endothelium via interaction with a class of high-affinity binding sites. In this report, the interaction of VPF/VEGF with human mononuclear phagocytes (MPs) is characterized. Radioligand binding studies at 4 degrees C showed the presence of a single class of binding sites, kd approximately 300 to 500 pmol/L (approximately 20 times lower affinity than the high-affinity binding site on endothelial cells [ECs]), the occupancy of which correlated with VPF/VEGF-induced MP migration and expression of tissue factor. These binding results were paralleled by functional experiments which indicated that the same VPF/VEGF preparations were about an order of magnitude less effective in stimulating MP chemotaxis than in inducing EC proliferation. When MPs with surface-bound 125I-VPF/VEGF were warmed to 37 degrees C, endocytosis and degradation occurred. Occupancy of VPF/VEGF binding site resulted in subsequent activation of intracellular signal transduction mechanisms, as shown by an increase in MP intracellular calcium concentration. Cross-linking studies with 125I-VPF/VEGF showed a new high-molecular weight band (corresponding to putative 125I-VPF/VEGF-receptor complex), the appearance of which was blocked by excess unlabeled VPF/VEGF. Consistent with these results, immunoprecipitation of 32PO4-labeled MPs exposed to VPF/VEGF showed a single band of similar mobility, not seen in untreated controls. These results demonstrate that the interaction of VPF/VEGF with MPs, though of lower affinity than that observed with ECs, also results from interaction of the polypeptide with a specific cell-surface protein and leads to activation of intracellular transduction mechanisms.     

Expression and regulation of vascular endothelial growth factor in osteoblasts

Bone formation is linked closely to angiogenesis. Because prostaglandin E2 (PGE2) is a potent stimulator of bone formation, its effects were evaluated on vascular endothelial growth factor, a secreted endothelial cell-specific mitogen, and a potent angiogenic protein. Prostaglandin E2 increased vascular endothelial growth factor protein in conditioned media of osteoblastic RCT-3 cells within 3 hours. Prostaglandin E2 also increased the steady-state levels of vascular endothelial growth factor mRNA in a dose-dependent manner. The increased expression of vascular endothelial growth factor mRNA produced by PGE2 was rapid (maximal at 1 hour) and was enhanced by the protein synthesis inhibitor cycloheximide (5 micrograms/ml). The increase in vascular endothelial growth factor mRNA by PGE2 was inhibited strongly by pretreatment for 3 hours with dexamethasone (10(-7) M). Stimulation of vascular endothelial growth factor by PGE2 and its suppression by dexamethasone implicate the involvement of vascular endothelial growth factor in bone metabolism.     

Characterization of the extracellular domain in vascular endothelial growth factor receptor-1 (Flt-1 tyrosine kinase)

Flt-1 tyrosine kinase, vascular endothelial growth factor (VEGF) receptor-1, binds VEGF and a new VEGF-related ligand, placenta growth factor, but KDR/Flk-1 (VEGF receptor-2) binds only VEGF. To characterize the functional regions in the Flt-1 extracellular domain such as the ligand binding region and the dimer formation of the receptor, we constructed a series of mutants of the Flt-1 extracellular domain as soluble forms in a baculovirus system. We found that a region carrying the N-terminal 1st to 3rd immunoglobulin (Ig)-like domains of Flt-1 binds both ligands with high affinity. However, for dimer formation of soluble Flt-1, a region further downstream in the Flt-1 extracellular domain was required. Mutant Flt-1 receptors expressed in COS cells confirmed the requirement of the 4th to 7th Ig region for the activation of Flt-1 tyrosine kinase. Soluble Flt-1 carrying the N-terminal 1st to 3rd Ig region suppressed VEGF-dependent endothelial proliferation in vitro to the same level as the larger forms of soluble Flt-1, suggesting that the binding of one soluble Flt-1 molecule to one subunit of the VEGF homodimer may be sufficient to block the VEGF activity.     

Expression of two angiogenic factors, vascular endothelial growth factor and platelet-derived endothelial cell growth factor in human pancreatic cancer, and its relationship to angiogenesis

Twenty-three cases of ductal carcinoma in situ (DCIS), ten of which had an associated invasive component, were studied for loss of heterozygosity (LOH) of microsatellite markers on chromosome 9p and the results compared with a panel of 20 invasive breast carcinomas. In addition to the gene encoding p16, chromosome 9p is also thought to contain other putative tumour-suppressor genes. If the three panels of breast tumours showed LOH of markers in this region this would suggest that such putative genes were important in breast carcinogenesis. By studying both preinvasive and invasive breast tumours, it should also be possible to gain further information about the relationship between lesions of a different stage and to determine whether DCIS is indeed a precursor of invasive ductal carcinoma. Levels of LOH were low in the invasive-only set of tumours. Surprisingly, considerably higher levels of loss were observed in the tumours with an in situ component. Also, much heterogeneity was observed between different DCIS ducts or invasive tumour and DCIS from the same case.     

Retinoids downregulate vascular endothelial growth factor/vascular permeability factor production by normal human keratinocytes

PURPOSE: We describe the clinical presentation, angiographic findings, and clinical outcome in a group of patients with pseudoaneurysms treated by a new endovascular technique using Guglielmi electrolytically detachable platinum coils (GDCs). METHODS: We retrospectively reviewed the angiographic and clinical findings in a series of 11 patients with pseudoaneurysms occurring in a variety of locations: seven in the cavernous carotid artery, one in the petrous carotid artery, two in the anterior cerebral artery, and one in the cervical vertebral artery. RESULTS: All aneurysms were cured with GDC embolization. The only complication was a branch occlusion, which resolved with heparinization and produced no clinical sequelae. CONCLUSION: Pseudoaneurysms can be safely and effectively treated by embolization with GDCs. Consideration needs to be given to the anatomic location of the pseudoaneurysm and the acuity of onset. Treatment efficacy may by improved if there are bony confines around the aneurysm or if therapy takes place in the subacute period, when the wall of the pseudoaneurysm has matured and stabilized.     

Levels of vascular endothelial growth factor, hepatocyte growth factor/scatter factor and basic fibroblast growth factor in human gliomas and their relation to angiogenesis

Angiogenesis is a possible target in the treatment of human gliomas. To evaluate the role of 3 growth factors, vascular endothelial growth factor (VEGF), hepatocyte growth factor/scatter factor (HGF/SF) and basic fibroblast growth factor (bFGF), in the angiogenic cascade, we determined their levels in extracts of 71 gliomas by enzyme-linked immunosorbent assay (ELISA). The levels of bFGF were only marginally different between gliomas of World Health Organization (WHO) grade II (low grade) and grades III and IV (high grade). In contrast, the mean concentrations of VEGF were 11-fold higher in high-grade tumors and those of HGF/SF 7-fold, respectively. Both were highly significantly correlated with microvessel density (p < 0.001) as determined by immunostaining for factor VIII-related antigen. In addition, VEGF and HGF/SF appeared to be independent predictive parameters for glioma microvessel density as determined by multiple regression analysis. We measured the capacity of all 3 factors to induce endothelial tube formation in a collagen gel. In this assay, bFGF was found to be an essential cofactor with which VEGF as well as HGF/SF were able to synergize independently. According to the concentrations of angiogenic factors, extracts from high-grade tumors were significantly more potent in the tube formation assay than the low-grade extracts (p = 0.02). Adding neutralizing antibodies to bFGF, VEGF and HGF/SF together with the extracts, tube formation was inhibited by up to 98%, 62% and 54%, respectively. Our findings suggest that bFGF is an essential cofactor for angiogenesis in gliomas, but in itself is insufficient as it is present already in the sparsely vascularized low-grade tumors. Upon induction of angiogenesis in high-grade tumors, bFGF may synergize with rising levels of not only VEGF but possibly also with HGF/SF, which appears here to be an independent angiogenic factor.     

Increased expression of vascular permeability factor (vascular endothelial growth factor) in bullous pemphigoid, dermatitis herpetiformis, and erythema multiforme

Vascular permeability factor (VPF), also known as vascular endothelial growth factor (VEGF), plays an important role in the increased vascular permeability and angiogenesis associated with many malignant tumors. In addition, VPF/VEGF is strongly expressed by epidermal keratinocytes in wound healing and psoriasis, disorders that are also characterized by increased microvascular permeability and angiogenesis. In this study, we investigated the expression of VPF/VEGF in three bullous diseases with subepidermal blister formation that are characterized by hyperpermeable dermal microvessels and pronounced papillary dermal edema. The expression of VPF/VEGF mRNA was strongly up-regulated in the lesional epidermis of bullous pemphigoid (n = 3), erythema multiforme (n = 3), and dermatitis herpetiformis (n = 4) as detected by in situ hybridization. Epidermal labeling was particularly intense over blisters, but strong expression was also noted in areas of the epidermis adjacent to dermal inflammatory infiltrates at a distance from blisters. Moreover, the VPF/VEGF receptors, flt-1 and KDR, were up-regulated in endothelial cells in superficial dermal microvessels. High levels of VPF/VEGF (138-238 pM) were detected in blister fluids obtained from five patients with bullous pemphigoid. Addition of blister fluid to human dermal microvascular endothelial cells exerted a dose-dependent mitogenic effect that was suppressed after depletion of VPF/VEGF by immunoadsorption. These findings strongly suggest that VPF/VEGF plays an important role in the induction of increased microvascular permeability in bullous diseases, leading to papillary edema and fibrin deposition and contributing to the bulla formation characteristic of these disorders.