Recent Publications on Angiogenesis (May 2000)
series of reviews that can be downloaded from: Oncologist; 2000, Vol. 5,
translational research: the role of VEGF in tumor angiogenesis.
receptor signaling in tumor angiogenesis.
endothelial growth factor in human colon cancer: biology and therapeutic
VEGF-flk-1 activity and consequences of VEGF-flk-1 targeting In vivo using
intravital microscopy: clinical applications.
strategies and agents in clinical trials.
role of vascular endothelial growth factor (VEGF) in AIDS-related Kaposi's
hippel-lindau syndrome: target for anti-vascular endothelial growth factor
(VEGF) receptor therapy.
value of vascular endothelial growth factor in breast cancer.
vascular endothelial growth factor blockade: ascites and pleural effusion
strategy for the development of angiogenesis inhibitors.
be published in: Genes Dev.; June 1, 2000; Vol. 14, pp
signaling is essential for vascular morphogenesis in mice
a novel Notch ligand expressed in arterial endothelium
June 1, 2000; Vol. 95, pp 3387-3395
Up-regulation of vascular endothelial growth factor receptor Flt-1 after endothelial denudation: role of transcription factor Egr-1
Vidal F, Aragones J, Alfranca A, de Landazuri MO
Vascular endothelial growth factor (VEGF) is highly expressed in vascular remodeling processes and accelerates reendothelialization after mechanical denudation. Two VEGF tyrosine kinase receptors have been reported-fms-like-tyrosine kinase-1 (Flt-1) and kinase domain region (KDR). Little is known about the regulation of the expression of these receptors after vascular injury. Herein, we have analyzed the expression of Flt-1 after mechanical denudation of primary cultures of endothelial cells, which has been considered a useful in vitro model to study endothelium responses to vascular injury. After denudation, the Flt-1 protein and mRNA levels are clearly up-regulated, and transient transfection experiments showed a strong induction of the flt-1 promoter-dependent transcription. Analysis of the flt-1 promoter sequence revealed the presence of a putative binding site for the early growth response factor-1 (Egr-1) at positions -24 to -16. Electrophoretic mobility shift and supershift assays showed that Egr-1 was able to bind to this DNA sequence, and cotransfection of the flt-1 promoter reporter plasmid with an Egr-1 expression vector resulted in enhancement of its transcriptional activity. Furthermore, the mutation of the Egr-1 binding site markedly reduced the denudation-induced flt-1 promoter activity. These data demonstrate that Flt-1 is up-regulated after endothelial denudation and that Egr-1 plays a relevant role in this process.
June 1, 2000; Vol 95, pp 3403-3411
Endostatin-induced tyrosine kinase signaling through the Shb adaptor protein regulates endothelial cell apoptosis.
Dixelius J, Larsson H, Sasaki T, Holmqvist K, Lu L, Engstrom A, Timpl R, Welsh M, Claesson-Welsh L
Endostatin, which corresponds to the C-terminal fragment of collagen XVIII, is a potent inhibitor of angiogenesis. Fibroblast growth factor-2 (FGF-2)-induced angiogenesis in the chicken chorioallantoic membrane was inhibited by endostatin, but not by an endostatin mutant R158/270A, lacking heparin-binding ability. Endostatin was internalized by endothelial cells, but not by mouse fibroblasts. Treatment of murine brain endothelial (IBE) cells with endostatin reduced the proportion of cells in S phase, whereas growth-arrested IBE cells in collagen gels treated with endostatin displayed enhanced tubular morphogenesis. IBE cells overexpressing Shb, an adaptor protein implicated in angiostatin-induced apoptosis, displayed elevated apoptosis and decreased tubular morphogenesis in collagen gels in response to endostatin when added together with FGF-2. Induction of apoptosis was dependent on the heparin-binding ability of endostatin and the expression of Shb with a functional Src homology 2 (SH2)-domain. Endostatin treatment for 10 minutes or 24 hours induced tyrosine phosphorylation of Shb and formation of multiprotein complexes. An Shb SH2 domain fusion protein precipitated a 125-kd phosphotyrosyl protein in endostatin-treated cells. The 125-kd component either contained intrinsic tyrosine kinase activity or occurred in complex with a tyrosine kinase. In conclusion, our data show that endostatin induces tyrosine kinase activity and enhanced apoptosis in FGF-treated endothelial cells.
May 15, 2000; Vol. 95, pp 3106-3112
In vitro differentiation of endothelial cells from AC133-positive progenitor cells
Gehling U, Ergün S, Schumacher U, Wagener C, Pantel K, Otte M, Schuch G, Schafhausen P, Mende T, Kilic N, Kluge K, Schäfer B, Hossfeld DK, Fiedler W
Recent findings support the hypothesis that the CD34+-cell population in bone marrow and peripheral blood contains hematopoietic and endothelial progenitor and stem cells. In this study, we report that human AC133+ cells from granulocyte colony-stimulating factor-mobilized peripheral blood have the capacity to differentiate into endothelial cells (ECs). When cultured in the presence of vascular endothelial growth factor (VEGF) and the novel cytokine stem cell growth factor (SCGF), AC133+ progenitors generate both adherent and proliferating nonadherent cells. Phenotypic analysis of the cells within the adherent population reveals that the majority display endothelial features, including the expression of KDR, Tie-2, Ulex europaeus agglutinin-1, and von Willebrand factor. Electron microscopic studies of these cells show structures compatible with Weibel-Palade bodies that are found exclusively in vascular endothelium. AC133-derived nonadherent cells give rise to both hematopoietic and endothelial colonies in semisolid medium. On transfer to fresh liquid culture with VEGF and SCGF, nonadherent cells again produce an adherent and a nonadherent population. In mice with severe combined immunodeficiency, AC133-derived cells form new blood vessels in vivo when injected subcutaneously together with A549 lung cancer cells. These data indicate that the AC133+-cell population consists of progenitor and stem cells not only with hematopoietic potential but also with the capacity to differentiate into ECs. Whether these hematopoietic and endothelial progenitors develop from a common precursor, the hemangioblast will be studied at the single-cell level.
Biophys. Acta; June 1, 2000; Vol. 1466 pp 71-78
Targeting of endothelial KDR receptors with 3G2 immunoliposomes in vitro.
Benzinger P, Martiny-Baron G, Reusch P, Siemeister G, Kley JT, Marme D, Unger C, Massing U
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.
Biol. Chem.; May 22, 2000; Vol. , pp
Hypoxia inducible factor-alpha binding and ubiquitylation by the von Hippel-Lindau tumor suppressor protein.
Cockman ME, Masson N, Mole DR, Jaakkola P, Chang GW, Clifford SC, Maher ER, Pugh CW, Ratcliffe PJ, Maxwell PH
The von Hippel-Lindau tumor suppressor protein (pVHL) has emerged as a key factor in cellular responses to oxygen availability, being required for the oxygen dependent proteolysis of a subunits of hypoxia inducible factor-1 (HIF). Mutations in VHL cause a hereditary cancer syndrome associated with dysregulated angiogenesis, and upregulation of hypoxia inducible genes. Here we investigate the mechanisms underlying these processes and show that extracts from VHL deficient renal carcinoma cells have a defect in HIF-alpha ubiquitylation activity which is complemented by exogenous pVHL. This defect was specific for HIF-alpha among a range of substrates tested. Furthermore, HIF-alpha subunits were the only pVHL associated proteasomal substrates identified by comparison of metabolically labelled anti-pVHL immunoprecipitates from proteosomally inhibited cells and normal cells. Analysis of pVHL/HIF-alpha interactions defined short sequences of conserved residues within the internal transactivation domains of HIF-alpha molecules sufficient for recognition by pVHL. In contrast, whilst full length pVHL and the p19 variant interact with HIF-alpha, the association was abrogated by further N-terminal and C-terminal truncations. The interaction was also disrupted by tumor associated mutations in the beta-domain of pVHL and loss of interaction was associated with defective HIF-alpha ubiquitylation and regulation, defining a mechanism by which these mutations generate a constitutively hypoxic pattern of gene expression promoting angiogenesis. The findings indicate that pVHL regulates HIF-alpha proteolysis by acting as the recognition component of a ubiquitin ligase complex, and support a model in which its beta-domain interacts with short recognition sequences in HIF-alpha subunits.
May 23, 2000; Vol. 101, pp 2345-2348
Abnormal aortic valve development in mice lacking endothelial nitric oxide synthase.
Lee TC, Zhao YD, Courtman DW, Stewart DJ
Background-Endothelium-derived nitric oxide (NO) is produced by an oxidative reaction catalyzed by endothelial NO synthase (eNOS). NO plays a crucial role in controlling cell growth and apoptosis, as well as having well-characterized vasodilator and antithrombotic actions. More recently, endothelium-derived NO was shown to be involved in postdevelopmental vascular remodeling and angiogenesis, as well as in the formation of limb vasculature during embryogenesis. Therefore, we investigated the role of endothelium-derived NO during cardiovascular development using mice deficient in eNOS. Methods and Results-We examined the hearts of 12 mature eNOS-deficient and 26 mature wild-type mice. Five of the mature eNOS-deficient mice had a bicuspid aortic valve; none of the 26 wild-type animals exhibited identifiable valvular or cardiac abnormalities. Immunohistochemical analysis revealed prominent eNOS expression localized to the endothelium lining the valve cusps of the aorta in mature wild-type mice; expression was localized to the myocardium and endothelial cell monolayer lining the valve leaflets in the developing embryo. Conclusions-These results show a strong association between eNOS deficiency and the presence of a bicuspid aortic valve; they provide the first molecular insight into one of the most common types of congenital cardiac abnormality.
Biol. Chem.; May 17, 2000; Vol. , pp
Selectively desulfated heparin inhibits FGF-induced mitogenicity and angiogenesis.
Lundin L, Larsson H, Kreuger J, Kanda S, Lindahl U, Salmivirta M, Claesson-Welsh L
Fibroblast growth factors (FGFs) are known to induce formation of new blood vessels, angiogenesis. We show that FGF-induced angiogenesis can be modulated using selectively desulfated heparin. Chinese hamster ovary cells (CHO677) deficient in heparan sulfate biosynthesis were employed to assess the function of heparin/heparan sulfate in FGF receptor-1 (FGFR-1) signal transduction and biological responses. In the presence of FGF-2, FGFR-1 kinase and subsequent MAP kinase Erk2 activities were augmented in a dose dependant manner, whereas high concentrations of heparin resulted in decreased activity. The length of the heparin oligomer, minimally an 8/10-mer, was critical for the ability to enhance FGFR-1 kinase activity. The N- and 2-O-sulfate groups of heparin were essential for binding to FGF-2, whereas stimulation of FGFR-1 and Erk2 kinases by FGF-2 also required the presence of 6-O-sulfate groups. Sulfation at 2-O- and 6-O-positions was moreover a prerequisite for binding of heparin to a lysine-rich peptide corresponding to amino acids 160-177 in the extracellular domain of FGFR-1. Selectively 6-O-desulfated heparin, which binds to FGF-2 but fails to bind the receptor, decreased FGF-2-induced proliferation of CHO677 cells, presumably by displacing intact heparin. Furthermore, FGF-2-induced angiogenesis in chick embryos was inhibited by 6-O desulfated heparin. Thus, formation of a ternary complex of FGF-2, heparin and FGFR-1 appears critical for the activation of FGFR-1 kinase and downstream signal transduction. Preventing complex-formation by modified heparin preparations may allow regulation of FGF-2 functions, such as induction of angiogenesis.
J.; June 1, 2000; Vol. 348, pp 273-280
Tyrosine phosphorylation of the vascular endothelial-growth-factor receptor-2 (VEGFR-2) is modulated by Rho proteins.
Gingras D, Lamy S, Beliveau R
The effects of Rho-specific modifying toxins on the tyrosine phosphorylation of endothelial cell proteins were investigated. Incubation of the cells with the Rho-activating toxin cytotoxic necrotizing factor 1 (CNF1) induced a marked increase in the tyrosine phosphorylation of a number of signalling intermediates of the vascular endothelial growth factor (VEGF)-mediated cascade, including focal adhesion kinase, paxillin, phospholipase Cgamma1 and a Shc-associated protein of 195 kDa. Both CNF1- and VEGF-dependent tyrosine phosphorylation of these proteins were significantly reduced by prior incubation with C3 transferase, a known inhibitor of RhoA function, suggesting a Rho-dependent mechanism. The stimulation of endothelial cells with CNF1 resulted in a marked increase in the tyrosine phosphorylation of the VEGF receptor (VEGFR)-2, which was correlated with a stimulation of its kinase activity and with its association with downstream tyrosine phosphorylated proteins. The stimulatory effect of CNF1 was specific for VEGFR-2 since the phosphotyrosine content of VEGFR-1 was not affected by the toxin. Transient overexpression of a dominant-active RhoA mutant also induced an increase in the tyrosine phosphorylation of the VEGFR-2, whereas overexpression of a dominant-inactive form of the protein was without effect. Taken together, these results indicate that Rho proteins may play an important role in angiogenesis by modulating the tyrosine phosphorylation levels of VEGFR-2.
Thromb. Vasc. Biol.; May, 2000; Vol. 20, pp 1250-1256
Fibroblast growth factor-2 selectively stimulates angiogenesis of small vessels in arterial tree.
Parsons-Wingerter P, Elliott KE, Clark JI, Farr AG
There is a critical need for quantifiable models of angiogenesis in vivo, and in general, differential effects of angiogenic regulators on vascular morphology have not been measured. Because the potent angiogenic stimulators fibroblast growth factor (FGF)-2 (basic FGF) and vascular endothelial growth factor (VEGF) are reported to stimulate angiogenesis through distinct signaling pathways, we hypothesized that FGF-2 stimulates vascular morphology differently than does VEGF and that stimulation of angiogenesis by FGF-2 is directly correlated to FGF receptor density. FGF-2 was applied at embryonic day 7 (E7), E8, or E9 to the quail chorioallantoic membrane (CAM); subsequent response of the arterial tree was measured by the fractal dimension (D(f)), a mathematical descriptor of complex spatial patterns, and by several generational branching parameters that included vessel length density (L(v)). After application of FGF-2 at E7, arterial density increased according to D(f) as a direct function of increasing FGF-2 concentration, and FGF-2 stimulated the growth of small vessels, but not of large vessels, according to L(v) and other branching parameters. For untreated control specimens at E7, L(v) of small vessels and D(f) were 11.1+/-1.6 cm(-1) and 1.38+/-0.01, respectively; at E8, after treatment with 5 mug FGF-2/CAM for 24 hours, L(v) of small vessels and D(f) increased respectively to 22.8+/-0.7 cm(-1) and 1.49+/-0.02 compared with 16.3+/-0.9 cm(-1) and 1.43+/-0.02 for PBS-treated control specimens. Application of FGF-2 at E8 and E9 did not significantly increase arterial density. By immunohistochemistry, the expression of 4 high-affinity tyrosine kinase FGF receptors was significantly expressed at E7, when CAM vasculature responded strongly to FGF-2 stimulation, but FGF receptor expression decreased throughout the CAM by E8, when vascular response to FGF-2 was negligible. In conclusion, the "fingerprint" vascular pattern elicited by FGF-2 was distinct from vascular patterns induced by other angiogenic regulators that included VEGF(165), transforming growth factor-beta1, and angiostatin.
Res.; May 12, 2000; Vol. 86, pp 952-959
Angiopoietin-1 Induces Endothelial Cell Sprouting Through the Activation of Focal Adhesion Kinase and Plasmin Secretion.
Kim I, Kim HG, Moon SO, Chae SW, So JN, Koh KN, Ahn BC, Koh GY
Angiopoietin-1 (Ang1) is a strong inducer of endothelial cell sprouting, which is a first step in both angiogenesis and neovascularization. We examined the mechanisms underlying Ang1-induced cell sprouting using porcine pulmonary artery endothelial cells. Ang1 induced the nondirectional and directional migration of endothelial cells mediated through the Tie2 but not the Tie1 receptor. Ang1 induced tyrosine phosphorylation of p125(FAK), and this phosphorylation was dependent on phosphatidylinositol (PI) 3'-kinase activity. Ang1 induced the secretion of plasmin and matrix metalloproteinase-2 (MMP-2), which is inhibited by PI 3'-kinase inhibitors. Ang1 also induced the secretion of small amounts of proMMP-3 and proMMP-9 but not proMMP-1. Ang1 suppressed the secretion of tissue inhibitor of metalloproteinase-2 (TIMP-2), but not of TIMP-1. Addition of alpha(2)-antiplasmin, a combination of TIMP-1 and TIMP-2, or PI 3'-kinase inhibitors inhibited Ang1-induced sprouting activity. Therefore, Ang1-induced sprouting activity in endothelial cells may be accomplished by cytoskeletal changes and secretion of proteinases and may be largely mediated through intracellular PI 3'-kinase activation.
Res.; May 1, 2000; Vol. 60, pp 2520-2526
Anti-angiogenic cues from vascular basement membrane collagen.
Colorado PC, Torre A, Kamphaus G, Maeshima Y, Hopfer H, Takahashi K, Volk R, Zamborsky ED, Herman S, Sarkar PK, Ericksen MB, Dhanabal M, Simons M, Post M, Kufe DW, Weichselbaum RR, Sukhatme VP, Kalluri R
Vascular basement membrane is an important structural component of blood vessels and has been shown to interact with and modulate vascular endothelial behavior during angiogenesis. During the inductive phase of tumor angiogenesis, this membrane undergoes many degradative and structural changes and reorganizes to a native state around newly formed capillaries in the resolution phase. Such matrix changes are potentially associated with molecular modifications that include expression of matrix gene products coupled with conformational changes, which expose cryptic protein modules for interaction with the vascular endothelium. We speculate that these interactions provide important endogenous angiogenic and anti-angiogenic cues. In this report, we identify an important antiangiogenic vascular basement membrane-associated protein, the 26-kDa NC1 domain of the alpha1 chain of type IV collagen, termed arresten. Arresten was isolated from human placenta and produced as a recombinant molecule in Escherichia coli and 293 embryonic kidney cells. We demonstrate that arresten functions as an anti-angiogenic molecule by inhibiting endothelial cell proliferation, migration, tube formation, and Matrigel neovascularization. Arresten inhibits the growth of two human xenograft tumors in nude mice and the development of tumor metastases. Additionally, we show that the anti-angiogenic activity of arresten is potentially mediated via mechanisms involving cell surface proteoglycans and the alpha1beta1 integrin on endothelial cells. Collectively, our results suggest that arresten is a potent inhibitor of angiogenesis with a potential for therapeutic use.
Exp. Med.; May 15, 2000; Vol. 191, pp 1789-1798
is downregulated by anoxia and suppresses
Tenan M, Fulci G, Albertoni M, Diserens AC, Hamou MF, El Atifi-Borel M, Feige JJ, Pepper MS, Van Meir EG
Angiogenesis, the sprouting of new capillaries from preexisting blood vessels, results from a disruption of the balance between stimulatory and inhibitory factors. Here, we show that anoxia reduces expression of thrombospondin-1 (TSP-1), a natural inhibitor of angiogenesis, in glioblastoma cells. This suggests that reduced oxygen tension can promote angiogenesis not only by stimulating the production of inducers, such as vascular endothelial growth factor, but also by reducing the production of inhibitors. This downregulation may significantly contribute to glioblastoma development, since we show that an increase in TSP-1 expression is sufficient to strongly suppress glioblastoma cell tumorigenicity in vivo.
April 2000; Vol. 19, pp 2138-2146
Roles of two VEGF receptors, Flt-1 and KDR, in the signal transduction of VEGF effects in human vascular endothelial cells.
Kanno S, Oda N, Abe M, Terai Y, Ito M, Shitara K, Tabayashi K, Shibuya M, Sato Y
Vascular endothelial growth factor (VEGF) is a principal regulator of vasculogenesis and angiogenesis. VEGF expresses its effects by binding to two VEGF receptors, Flt-1 and KDR. However, properties of Flt-1 and KDR in the signal transduction of VEGF-mediated effects in endothelial cells (ECs) were not entirely clarified. We investigated this issue by using two newly developed blocking monoclonal antibodies (mAbs) against Flt-1 and KDR. VEGF elicits DNA synthesis and cell migration of human umbilical vein endothelial cells (HUVECs). The pattern of inhibition of these effects by two mAbs indicates that DNA synthesis is preferentially mediated by KDR. In contrast, the regulation of cell migration by VEGF appears to be more complicated. Flt-1 regulates cell migration through modulating actin reorganization, which is essential for cell motility. A distinct signal is generated by KDR, which influences cell migration by regulating cell adhesion via the assembly of vinculin in focal adhesion plaque and tyrosine-phosphorylation of focal adhesion kinase (FAK) and paxillin.
Gene Ther.; April, 2000; Vol. 7; pp 589-596
A novel strategy for the tumor angiogenesis-targeted gene therapy: generation of angiostatin from endogenous plasminogen by protease gene transfer.
Matsuda KM, Madoiwa S, Hasumi Y, Kanazawa T, Saga Y, Kume A, Mano H, Ozawa K, Matsuda M
When NIH 3T3 fibroblasts were transduced with a retroviral vector containing a cDNA for porcine pancreatic elastase 1 and cultured in the presence of affinity-purified human plasminogen, the exogenously added plasminogen was digested to generate the kringle 1-3 segment known as angiostatin, a potent angiogenesis inhibitor. This was evidenced by immunoblot analysis of the plasminogen digests using a monoclonal antibody specifically reacting with the kringle 1-3 segment, and by efficient inhibition of proliferation of human umbilical vein endothelial cells by the plasminogen digests isolated from the culture medium of 3T3 fibroblasts. However, when Lewis lung carcinoma cells were transduced with the same vector and injected subcutaneously into mice in their back or via the tail vein, their growth at the injection sites or in the lungs was markedly suppressed compared with the growth of similarly treated nontransduced Lewis lung carcinoma cells. Nevertheless, the transduced cells were able to grow as avidly as the control cells in vitro. Assuming that the elastase 1 secreted from the transduced cells is likely to be exempt from rapid inhibition by its physiological inhibitor, alpha1-protease inhibitor, as shown in the inflammatory tissues, the elastase 1 secreted from the tumor cells may effectively digest the plasminogen that is abundantly present in the extravascular spaces and generate the kringle 1-3 segment in the vicinity of implanted tumor cell clusters. Although the selection of more profitable virus vectors and cells to be transduced awaits further studies, such a protease gene transfer strategy may provide us with a new approach to anti-angiogenesis gene therapy for malignant tumors and their metastasis in vivo.