Showing papers on "Vascular endothelial growth factor A published in 1991"
TL;DR: Alternative splicing of VEGF RNA can produce four polypeptides with strikingly different secretion patterns, which suggests multiple physiological roles for this family of proteins.
Abstract: Vascular endothelial growth factor (VEGF) was recently identified as a secreted, direct-acting mitogen specific for vascular endothelial cells and capable of stimulating angiogenesis in vivo. Molecular cloning revealed multiple forms of VEGF, apparently arising from alternative splicing of its RNA transcript. We have examined various human cDNA libraries by the polymerase chain reaction technique and discovered a fourth molecular form, VEGF206. This form contains a 41-amino acid insertion relative to the most abundant form, VEGF165, and includes the highly basic 24-amino acid insertion found in VEGF189. Southern blot analysis revealed that a single gene encoded these various forms, and nucleic acid sequence analysis of a portion of the VEGF gene revealed an intron/exon structure compatible with alternative splicing of RNA as a mechanism for their generation. Transient transfection of human embryonic kidney 293 cells showed that, like VEGF189, VEGF206 was predominately cell-associated and only very poorly secreted despite the presence of the signal peptide identical to that found in VEGF121 and VEGF165, both of which are efficiently exported from the cell. Vascular permeability activity was detected in the medium of 293 cells transfected with all four forms of VEGF; however, endothelial cell mitogenic activity was apparent only with VEGF121 and VEGF165. Thus, alternative splicing of VEGF RNA can produce four polypeptides with strikingly different secretion patterns, which suggests multiple physiological roles for this family of proteins.
1,469 citations
TL;DR: It is demonstrated that PlGF, derived from transfected COS-1 cells, is actually N-glycosylated and secreted into the medium and, like VPF, proves to be a dimeric protein.
Abstract: A human cDNA coding for a protein related to the vascular permeability factor (VPF) was isolated from a term placenta cDNA library; we therefore named its product placenta growth factor (PlGF). PlGF is a 149-amino-acid-long protein and is highly homologous (53% identity) to the platelet-derived growth factor-like region of human VPF. Computer analyses reveal a putative signal peptide and two probable N-glycosylation sites in the PlGF protein, one of which is also conserved in human VPF. By using N-glycosidase F, tunicamycin, and specific antibodies produced in both chicken and rabbit, we demonstrate that PlGF, derived from transfected COS-1 cells, is actually N-glycosylated and secreted into the medium. In addition, PlGF, like VPF, proves to be a dimeric protein. Finally, a conditioned medium from COS-1 cells containing PlGF is capable of stimulating specifically the growth of CPA, a line of endothelial cells, in vitro.
1,055 citations
TL;DR: Ligand autoradiography on rat tissue sections demonstrates that VEGF binding sites are associated with vascular endothelial cells of both fenestrated and non‐fenestrated capillaries and with the endothelium of large vessels, while no displaceable binding is evident on non‐endothelial cells.
Abstract: Vascular endothelial growth factor (VEGF) was identified as a heparin-binding polypeptide mitogen with a target cell specificity restricted to vascular endothelial cells. Molecular cloning reveals the existence of four species of VEGF having 121, 165, 189, and 206 amino acids. These have strikingly different secretion patterns, which suggests multiple physiological roles for this family of polypeptides. The two shorter forms are efficiently secreted, while the longer ones are mostly cell-associated. Alternative splicing of mRNA, rather that transcription from different genes, is the mechanism for their generation. In situ hybridization reveals that the VEGF mRNA is widely distributed in most tissues and organs and expressed at particularly high levels in areas of active vascular proliferation, like the ovarian corpus luteum. Ligand autoradiography on rat tissue sections demonstrates that VEGF binding sites are associated with vascular endothelial cells of both fenestrated and non-fenestrated capillaries and with the endothelium of large vessels, while no displaceable binding is evident on non-endothelial cell types. These findings support the hypothesis that VEGF plays a highly specific role in the maintenance and in the induction of growth of vascular endothelial cells.
721 citations
TL;DR: Results demonstrate the regulation of PA and PAI-1 production by VEGF in microvascular endothelial cells and are in accord with the hypothesis that extracellular proteolysis, appropriately balanced by protease inhibitors, is required for normal capillary morphogenesis.
625 citations
TL;DR: Its site of synthesis and spectrum of targets imply that this growth factor may play an important role as a paracrine mediator of the proliferation of melanocytes and endothelial cells, as well as cells of epithelial origin.
Abstract: A heparin-binding mitogen was isolated from conditioned medium of human embryonic lung fibroblasts. It exhibited broad target-cell specificity whose pattern was distinct from that of any known growth factor. It rapidly stimulated tyrosine phosphorylation of a 145-kDa protein in responsive cells, suggesting that its signaling pathways involved activation of a tyrosine kinase. Purification identified a major polypeptide with an apparent molecular mass of 87 kDa under reducing conditions. Partial amino acid sequence analysis and cDNA cloning revealed that it was a variant of hepatocyte growth factor, a mitogen thought to be specific for hepatic cells and structurally related to plasminogen. Recombinant expression of the cDNA in COS-1 cells established that it encoded the purified growth factor. Its site of synthesis and spectrum of targets imply that this growth factor may play an important role as a paracrine mediator of the proliferation of melanocytes and endothelial cells, as well as cells of epithelial origin.
530 citations
TL;DR: These studies indicate that VPF is synthesized by tumor cells in vivo and accumulates in nearby blood vessels, its target of action.
Abstract: Vascular permeability factor (VPF) is a highly conserved 34-42-kD protein secreted by many tumor cells. Among the most potent vascular permeability-enhancing factors known, VPF is also a selective vascular endothelial cell mitogen, and therefore has been called vascular endothelial cell growth factor (VEGF). Our goal was to define the cellular sites of VPF (VEGF) synthesis and accumulation in tumors in vivo. Immunohistochemical studies were performed on solid and ascites guinea pig line 1 and line 10 bile duct carcinomas using antibodies directed against peptides synthesized to represent the NH2-terminal and internal sequences of VPF. These antibodies stained tumor cells and, uniformly and most intensely, the endothelium of immediately adjacent blood vessels, both preexisting and those newly induced by tumor angiogenesis. A similar pattern of VPF staining was observed in autochthonous human lymphoma. In situ hybridization demonstrated VPF mRNA in nearly all line 10 tumor cells but not in tumor blood vessels, indicating that immunohistochemical labeling of tumor vessels with antibodies to VPF peptides reflects uptake of VPF, not endogenous synthesis. VPF protein staining was evident in adjacent preexisting venules and small veins as early as 5 h after tumor transplant and plateaued at maximally intense levels in newly induced tumor vessels by approximately 5 d. VPF-stained vessels were also hyperpermeable to macromolecules as judged by their capacity to accumulate circulating colloidal carbon. In contrast, vessels more than approximately 0.5 mm distant from tumors were not hyperpermeable and did not exhibit immunohistochemical staining for VPF. Vessel staining disappeared within 24-48 h of tumor rejection. These studies indicate that VPF is synthesized by tumor cells in vivo and accumulates in nearby blood vessels, its target of action. Because leaky tumor vessels initiate a cascade of events, which include plasma extravasation and which lead ultimately to angiogenesis and tumor stroma formation, VPF may have a pivotal role in promoting tumor growth. Also, VPF immunostaining provides a new marker for tumor blood vessels that may be exploitable for tumor imaging or therapy.
525 citations
Journal Article•
TL;DR: Together these data indicate that VPF acts directly on ECs and that rapid cellular events in its in vivo/in vitro actions are likely to involve phospholipase C activation, [Ca2+]i increase, and von Willebrand factor release.
Abstract: Vascular permeability factor (VPF), a tumor-secreted heparin-binding protein (Mr approximately 38,000), is responsible for increased vessel permeability and fluid accumulation associated with tumor growth. Vascular permeability factor also promotes the growth of human umbilical vein endothelial cells (EC) and bovine pulmonary ECs in vitro. It is shown for the first time that guinea pig VPF (half-maximal and maximal dose approximately 0.4 and 22 pmol/l (picomolar), respectively), as well as human VPF, are potent stimuli for human ECs resulting in [Ca2+]i increases (maximal three- to fourfold) and inositol triphosphate (IP3) formation. Unlike the maximal responses to thrombin and histamine, the [Ca2+]i response to a maximal VPF dose was preceded by a characteristic 10- to 15-second delay. Guinea pig VPF also selectively increased [Ca2+]i in cultured aortic and pulmonary artery ECs, but not aortic smooth muscle cells, human fibroblasts, or neutrophils. Affinity-purified rabbit antibody (raised to a synthetic peptide representing VPF N-terminal amino acids 1 to 24) adsorbed all vessel permeability-increasing activity, EC growth-promoting activity, and specifically all activity responsible for increasing EC [Ca2+]i. Similar to other mediators that increase [Ca2+]i in cultured ECs, VPF also induced a 200% increase in von Willebrand factor release. Together these data indicate that VPF acts directly on ECs and that rapid cellular events in its in vivo/in vitro actions are likely to involve phospholipase C activation, [Ca2+]i increase, and von Willebrand factor release.
348 citations
TL;DR: Vascular permeability factor, also known as vascular endothelial growth factor (VEGF), is a potent polypeptide regulator of blood vessel function and regulates the expression of tissue factor and the glucose transporter.
Abstract: Vascular permeability factor (VPF), also known as vascular endothelial growth factor (VEGF), is a potent polypeptide regulator of blood vessel function. VPF promotes an array of responses in endothelium, including hyperpermeability, endothelial cell growth, angiogenesis, and enhanced glucose transport. VPF regulates the expression of tissue factor and the glucose transporter. All of the endothelial cell responses to VPF are evidently mediated by high affinity cell surface receptors. Thus, endothelial cells have a unique and specific spectrum of responses to VPF. Since each of the responses of endothelial cells to VPF are also elicited by agonists, such as bFGF, TNF, histamine and others, it remains a major challenge to determine how post-receptor signalling pathways maintain both specificity and redundancy in cellular responses to various agonists.
331 citations
TL;DR: Results show that basic fibroblast growth factor, besides stimulating uPA production by vascular endothelial cells, also increases the production of receptors, which modulates their capacity to focalize this enzyme on the cell surface, which may be important in the degradative processes that occur during angiogenesis.
Abstract: Basic fibroblast growth factor, a potent angiogenesis inducer, stimulates urokinase (uPA) production by vascular endothelial cells. In both basic fibroblast growth factor-stimulated and -nonstimulated bovine capillary endothelial and human umbilical vein endothelial cells single-chain uPA binding is mediated by a membrane protein with a Mr of 42,000. Exposure of bovine capillary or endothelial human umbilical vein endothelial cells to pmolar concentrations of basic fibroblast growth factor results in a dose-dependent, protein synthesis-dependent increase in the number of membrane receptors for uPA (19,500-187,000) and in a parallel decrease in their affinity (KD = 0.144-0.790 nM). With both cells, single-chain uPA binding is competed by synthetic peptides whose sequence corresponds to the receptor-binding sequence in the NH2-terminal domain of uPA. Exposure of bovine capillary endothelial cells to transforming growth factor beta 1, which inhibits uPA production and upregulates type 1 plasminogen activator inhibitor, the major endothelial cell plasminogen activator inhibitor, has no effect on uPA receptor levels. These results show that basic fibroblast growth factor, besides stimulating uPA production by vascular endothelial cells, also increases the production of receptors, which modulates their capacity to focalize this enzyme on the cell surface. This effect may be important in the degradative processes that occur during angiogenesis.
182 citations
TL;DR: It is suggested that the 164 amino-acid species is the predominant molecular form of the growth factor secreted by such cells and may play a paracrine role in the maintenance of the integrity of the endothelial lining or in the abnormal proliferation of the vasa vasorum which takes place in atherosclerosis.
Abstract: We examined whether cultured bovine aortic smooth muscle (ASM) cells express VEGF. RNA blot analysis of total cellular RNA derived from ASM cells demonstrates the expression of the VEGF gene. ASM cells release in the medium a VEGF-like endothelial cell mitogen which binds to heparin-sepharose and has an apparent molecular weight of 40-45 kDa as assessed by an HPLC gel filtration column. Consistent with VEGF, this mitogen does not stimulate the proliferation of ASM cells. Immunoblot analysis of the bioactive material with an antibody specific for VEGF demonstrates the presence of a major immunoreactive band with an apparent molecular mass of 23 kDa and a minor band with a molecular mass of approximately 18 kDa, in reducing conditions. The major band has very similar apparent molecular weight as the 165 amino-acid species of human recombinant VEGF of folliculo-stellate cells derived VEGF. These data demonstrate the expression and synthesis of VEGF by cultured ASM cells and suggest that the 164 amino-acid species is the predominant molecular form of the growth factor secreted by such cells. VEGF released by ASM cells may play a paracrine role in the maintenance of the integrity of the endothelial lining or in the abnormal proliferation of the vasa vasorum which takes place in atherosclerosis.
142 citations
TL;DR: Results indicate that VAS/VEGF might be a potent inducer of neovascularization resulting from a direct interaction with endothelial cells and the angiogenic activity seems to be independent of the plasminogen activator or inhibitor system.
Abstract: Vasculotropin/vascular endothelial cell growth factor (VAS/VEGF) is a newly purified growth factor with a unique specificity for vascular endothelial cells. We have investigated the interactions of VAS/VEGF with human umbilical vein endothelial cells (HUVE cells). 125I-VAS/VEGF was found to HUVE cells in a saturable manner with a half-maximum binding at 2.8 ng/ml. Scatchard analysis did show two classes of high-affinity binding sites. The first class displayed a dissociation constant of 9 pM with 500 sites/cell. The dissociation constant and the number of binding sites of the second binding class were variable for different HUVE cell cultures (KD = 179 +/- 101 pM, 5,850 +/- 2,950 sites/cell). Half-maximal inhibition of 125I-VAS/VEGF occurred with a threefold excess of unlabeled ligand. Basic fibroblast growth factor (bFGF) and heparin did not compete with 125I-VAS/VEGF binding. In contrast, suramin and protamin sulfate completely displaced 125I-VAS/VEGF binding from HUVE cells. VAS/VEGF was shown to be internalized in HUVE cells. Maximum internalization (55% of total cell-associated radioactivity) was observed after 30 min. 125I-VAS/VEGF was completely degraded 2-3 hr after binding. At 3 hr, the trichloroacetic acid (TCA)-soluble radioactivity accumulated in the medium was 60% of the total radioactivity released by HUVE cells. No degradation fragment of 125I-VAS/VEGF was observed. Chloroquine completely inhibited degradation. VAS/VEGF was able to induce angiogenesis in vitro in HUVE cells. However, it did not significantly modulate urokinase-type plasminogen activator (u-PA), tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor (PAI-1), and tissue factor (TF). Prostacyclin production was only stimulated at very high VAS/VEGF concentrations. Taken together, these results indicate that VAS/VEGF might be a potent inducer of neovascularization resulting from a direct interaction with endothelial cells. The angiogenic activity seems to be independent of the plasminogen activator or inhibitor system.
TL;DR: Findings are not only of relevance to the unambiguous identification and characterization of cultured endothelial cells, but may explain the phenomenon of preferential organ tumour metastasis and provide novel opportunities for antitumour therapy.
TL;DR: VEGF 2083-fold is purified to apparent homogeneity from protein-free culture medium conditioned by A-431 human epidermoid carcinoma cells and induced the tyrosine phosphorylation of a 190-kDa polypeptide which was similar in mass to the largest binding site detected by affinity cross-linking.
Abstract: A distinct family of endothelial cell mitogens that are homologous to platelet-derived growth factor has recently been identified. Unlike other known endothelial cell mitogens, these vascular endothelial cell growth factors (VEGFs) are secreted and appear to act specifically on endothelial cells. We have purified VEGF 2083-fold to apparent homogeneity from protein-free culture medium conditioned by A-431 human epidermoid carcinoma cells. This A-431-derived VEGF was characterized as a homodimer composed of 22-kDa subunits with an N-terminal sequence that was similar to VEGFs produced by human HL-60 leukemic and U-937 histiocytic lymphoma cells. A-431 VEGF was used to identify specific and saturable binding sites for VEGF on human umbilical vein endothelial cells (HUVEC). By affinity cross-linking, VEGF-binding site complexes of 230, 170, and 125 kDa were detected on HUVEC. VEGF specifically induced the tyrosine phosphorylation of a 190-kDa polypeptide, which was similar in mass to the largest binding site detected by affinity cross-linking.
Journal Article•
TL;DR: The results suggest that in vivo TGF-beta may inhibit the adhesive phenotype of endothelial cells and also may limit the immunologic response occurring at the endothelial cell barrier.
Abstract: Recombinant human transforming growth factor-beta (TGF-beta) was found to inhibit the adhesive phenotype of human umbilical vein endothelial cells for human PBL, purified T lymphocytes, and PHA-activated lymphoblasts. TGF-beta inhibited lymphocyte attachment to resting human umbilical vein endothelial cells and also to endothelial monolayers stimulated with the pro-inflammatory cytokines TNF-alpha and IL-1 beta. Our investigations also show that the ability of endothelial cells to respond to TGF-beta by altering their adhesiveness is lost with prolonged culture of the cells. However, this loss is selective as TGF-beta inhibits cell proliferation in both early and late passage endothelial cells. These results suggest that in vivo TGF-beta may inhibit the adhesive phenotype of endothelial cells and also may limit the immunologic response occurring at the endothelial cell barrier.
TL;DR: It is shown that various fetal or adult endothelial cell strains originating from either capillary or large vessels possess specific high affinity and saturable binding sites for guinea pig tumor-derived [125I]VPF.
Journal Article•
TL;DR: The results demonstrate that, like macrophages, microvessel endothelial cells exposed to low levels of cytokines are capable of lysing tumor cells.
Abstract: The purpose of these studies was to determine whether vascular endothelial cells can be treated with various cytokines to become cytotoxic against tumorigenic target cells under defined conditions in vitro . Microvascular endothelial cells were isolated from immunocompetent mice by perfusion of lungs grown in culture, cloned, and then characterized. The cloned microvascular endothelial cells were activated by incubation with a combination of recombinant tumor necrosis factor α and recombinant-γ interferon (10 units/ml each) for 24 h. Activated endothelial but not control endothelial cells (incubated in medium alone, recombinant tumor necrosis factor α alone, or recombinant γ interferon alone) produced significant lysis of mouse reticulum cell sarcoma and two different mouse melanomas. Moreover, at the concentrations used here, recombinant tumor necrosis factor α and recombinant γ interferon did not produce direct target cell lysis. The activated endothelial cell-mediated tumor cell lysis depended on the continuous presence of the cytokines and was not due solely to initial target cell binding. The results demonstrate that, like macrophages, microvessel endothelial cells exposed to low levels of cytokines are capable of lysing tumor cells.
TL;DR: In vivo VAS was recognized as an inducer of angiogenesis and vascular permeability, and in vitro, despite a moderate action on proliferation, VAS strongly stimulates the cell migration and seems to act through a paracrine pathway.
Patent•
18 Sep 1991TL;DR: Vascular endothelial cell growth factor II (VEGFII) as discussed by the authors is derived from the culture media used to maintain mammalian glioma cells and is a heterodimer, which stimulates mitogenesis of mammalian vascular endothelial cells, and is useful for the promotion of vascular development and repair.
Abstract: Vascular endothelial cell growth factor II is purified from the culture media used to maintain mammalian glioma cells. The protein is a heterodimer, stimulates mitogenesis of mammalian vascular endothelial cells and is useful for the promotion of vascular development and repair. This unique growth factor is also useful in the promotion of tissue repair.
TL;DR: Several potential levels of interaction of the growth factors TGF-beta, bFGF, PDGF and IGF-I in cultured vascular endothelial cells are suggested.
Journal Article•
TL;DR: Hemparin and ECGF synergistically elicit a coordinate and selective inhibition of matrix gene expression in human smooth muscle cells, and suppression of the collagen gene expression was related to decreased collagen production by cells incubated with heparinand ECGF.
TL;DR: Lp(a), a plasminogen-like lipoprotein, by competing at the endothelial surface for plAsminogen binding down-regulates endothelial cell plasmine generation and may thus promote localized thrombogenesis that over a period of time contributes to progressive atherosclerosis.
Abstract: Endothelial cells play a critical role in thromboregulation by controlling the assembly of fibrinolytic constituents on the membrane. The assembly system illustrated in FIGURE 6 is characterized by the binding of circulating glu-plasminogen to a membrane receptor (Pathway 1). A membrane-associated protease (possibly plasmin) converts the inactive zymogen into a catalytically more efficient zymogen lys-plasminogen (Pathway 2). T-PA binds to a specific receptor, retains its catalytic activity, and is protected from its natural inhibitor PAI-1. The membrane provides a favorable environment for plasmin generation (Pathway 3) at the vessel surface and contributes to the maintenance of a physiological nonthrombogenic state. The immobilization and surface activation of plasminogen provides an important mechanism for localizing proteolytic activity at the surface of other cells such as macrophages and tumor cells. Lp(a), a plasminogen-like lipoprotein, by competing at the endothelial surface for plasminogen binding down-regulates endothelial cell plasmin generation and may thus promote localized thrombogenesis that over a period of time contributes to progressive atherosclerosis.
TL;DR: Therapy aimed at reducing serum LDL-cholesterol levels and increasing HDL-ch cholesterol levels will have multifactorial beneficial effects on cardiovascular disease.
Abstract: The major risk factors for cardiovascular disease include hypertension, hyperlipidemia, hyperinsulinemia and smoking. They promote the development of atherosclerosis and thromboembolic complications, i.e. strokes and heart attacks. There are diverse interactions between these factors, the outstanding common denominator being alterations in serum lipoproteins, mainly elevated low-density lipoprotein (LDL)-cholesterol and reduced high-density lipoprotein (HDL)-cholesterol concentrations, which results in disturbed cholesterol homeostasis (transport and metabolism) and eventually in perturbations of cellular integrity/function. In addition to their roles as cholesterol-transporting molecules, LDL and HDL directly influence inter- and intracellular communication via stimulation of pivotal signal transduction processes. The 'hormonal' effects of LDL on platelets, endothelial cells and vascular smooth muscle cells are potentially harmful, whereas the effects of HDL may be protective in as much as HDL can antagonize LDL-mediated stimulation of cells. Therapy aimed at reducing serum LDL-cholesterol levels and increasing HDL-cholesterol levels will have multifactorial beneficial effects on cardiovascular disease.
TL;DR: The authors' observations suggest that aortic endothelial cell hypoxia leads to the production of transforming growth factor beta, a known monocyte chemoattractant, which may contribute to the initial cellular events in the formation of an atherosclerotic lesion.
Journal Article•
TL;DR: It is proposed that the inductive capacity of interleukin-1 results, at least in part, from its capacity to activate cytoplasmic inhibitors of selective ribonucleases and hypothesize that this may be a mechanism that has been conserved throughout evolution.
Abstract: Vascular endothelial cells do not constitutively produce significant quantities of hematopoietic growth factors, interleukins, or adhesion molecules, but they can be induced to do so by a variety of stimuli including the inductive cytokines, interleukin-1 (IL-1), and tumor necrosis factor-alpha, as well as phorbol esters, bacterial proteins, endotoxin, certain viruses, and modified low-density lipoproteins. Recently, some groups have begun to investigate the molecular mechanism by which expression of these genes is regulated. Because induced expression of the gene products is always prefaced by an increase in mRNA the focus of attention has been largely upon the mechanisms involved in the process of transcript accumulation. Certain inducing agents drive transcription of these genes, others may induce both transcription and transcript stability. In the case of the inducing factor IL-1, it was recently demonstrated that accumulation of G-CSF, GM-CSF, and interleukin-1 beta mRNAs induced in vascular endothelial cells occurs as a result of transcript stabilization. Based on preliminary studies in a cell-free system, it is proposed that the inductive capacity of interleukin-1 results, at least in part, from its capacity to activate cytoplasmic inhibitors of selective ribonucleases and hypothesize that this may be a mechanism that has been conserved throughout evolution. Because other inducing agents also incite IL-1 gene expression, transcript stabilization may be a common ingredient of most inductive stimuli.
TL;DR: The present invention provides a homogeneous PD- ECGF in substantially greater yields than available in the past, the primary structure of PD-ECGF, antibodies against PD-ecGF, clones of its cDNA, and variants thereof, and the invention also provides a therapeutic preparation of PD -ECGF.
TL;DR: The nature of the SMC growth promoting activity that is secreted by EC, the regulation of growth factor production, and the possible role of this activity in the growth control of SMC in physiological and pathological processes are focused on.
TL;DR: Glucocorticoids appear to selectively induce endothelin release from vascular smooth muscle cells and this may be relevant to glucocortioid-induced hypertension.