1 Department of Urology and
2 Department of Molecular Microbiology and Immunology, Division of Endothelial Cell Biology, Nagasaki University Graduate School of Medicine, 1-7-1, Sakamoto, Nagasaki 852-8501, Japan
*Author for correspondence (e-mail: shigeruk{at}net.nagasaki-u.ac.jp)
Accepted September 20, 2001
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Angiopoietin 2, Tie2, Fes, Fyn, PI 3-kinase, IBE cells, Chemotaxis, Tube formation
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The Angiopoietins (Ang) family is composed of four members, Ang1 to 4 (Yancopoulos et al., 2000). Ang1 and 4 stimulate their specific receptor tyrosine kinase, Tie2, whereas Ang2 and 3 inhibit Ang1-induced tyrosine phosphorylation of Tie2 (Yancopoulos et al., 2000). The expression of Tie2 is exclusively observed in vascular endothelial cells during development. Targeted disruption of Tie2 as well as Ang1 demonstrated similar defects of maturation of myocardium and endocardium as well as vascular complexity (Dumont et al., 1994; Suri et al., 1996). In addition, Ang1 was clearly involved in angiogeneis during embryonic development (Suri et al., 1998; Takakura et al., 2000). Using tetracycline-responsive transgenic mice, loss of Tie2 induced rapid endothelial cell apoptosis in vivo (Jones et al., 2001), suggesting that Tie2 signals are involved in the maintenance of vasculature. Ang2 was cloned by Ang1-based homology screening and it was found that Ang2 inhibited Ang1-mediated autophosphorylation of Tie2 in human umbilical vein endothelial cells (HUVECs) (Maisonpierre et al., 1997). In addition, overexpression of Ang2 in transgenic mice demonstrated similar vascular anomalies to mice lacking Ang1, indicating that Ang2 is a natural antagonist for Ang1 during embryonic development (Maisonpierre et al., 1997).
Angiogenesis in mature tissues is prerequisite for many physiological and pathological processes, such as ovulation, wound healing, malignant tumor growth, retinopathies and rheumatoid arthritis (Colville-Nash and Willoughby, 1997; Folkman, 1995). Angiogenesis in mature tissue is composed of a series of cellular responses. Pericytes leave from basement membranes of pre-existing capillaries, followed by activation of endothelial cells by angiogenic factors, such as fibroblast growth factors (FGFs), VEGFs, hepatocyte growth factor and Ang. Activated endothelial cells produce proteases, which digest the basement membranes of blood vessels. Endothelial cells migrate into interstitial tissue, proliferate, and form lumen-containing, tube-like structures. Finally, basement membranes of newly formed vessels are surrounded by pericytes (Darland and DAmore, 1999; Hanahan, 1997; Yancopoulos et al., 2000).
Compared with the developmental angiogenesis, the roles of Ang/Tie2 system in pathological angiogenesis were less understood. However, recent works concerning the examination of Ang/Tie2 system in adult tissues have shown new insights into the important roles of Ang2 in angiogenesis. Expressions of Ang2 and Tie2, but not Ang1, were increased in malignant endothelial cells, such as angiosarcoma and aquired-immunodeficiency syndrome-associated Kaposis sarcoma (Brown et al., 2000), as well as rodent tumor tissues (Holash et al., 1999). Ectopic expression of Ang2 in tumour cells induced angiogenesis in association with increased tumor growth in transplanted mice (Tanaka et al., 1999; Ahmad et al., 2001; Etoh et al., 2001). In the previous report, activation of Tie2 by Ang1 was antagonized by Ang2 in endothelial cells. However, when Tie2 was ectopically expressed in fibroblasts or BaF3 cells, Ang2 could induce autophosphorylation of Tie2 (Maisonpierre et al., 1997; Sato et al., 1998). Furthermore, high concentrations of Ang2 (Kim et al., 2000) or Ang2-treatment in a particular condition (Teichert-Kuliszewska et al., 2001) was found to stimulate autophosphorylation of Tie2 in HUVECs, which was associated with increased survival and tube formation in fibrin matrix, respectively. These data strongly support the idea that Ang2 has stimulatory action on angiogenesis in mature tissues.
Signal transduction pathways via Tie2 have been extensively examined previously. Tie2 directly associates with the p85 regulatory subunit of phosphoinositide 3-kinase (PI 3-kinase) at an autophsohorylation site, Y1101 (Y1100) (Jones et al., 1999; Kontos et al., 1998), which in turn activate PI 3 kinase, leading to cell motility and survival (Jones et al., 1999; Papapertropoulos et al., 2000). Tie2 also binds to Dok-R, which is structurally homologous to p62dok and insulin-receptor substrate 3 (Jones and Dumont, 1998). Dok-R recruits adaptor protein Nck and Ras-GTPase activating protein (Ras-GAP). Upstream molecules of Ras/MAPK pathways, Grb2 and Shp2 were also identified as binding partners of Tie2 (Huang et al., 1995). More recently, it was demonstrated that Grb14 bound to Y814 and Y1106, Shp2 associated with Y814 and Y1111, and Grb2 as well as Grb7 could bind to Y1100 (Jones et al., 1999). Signal transducers and activators of transcription 3 (STAT3) and STAT5 were activated by Tie2, in association with the increased expression of p21 cell cycle inhibitor (Korpelainen et al., 1999). However, neither MAPK activation nor induction of proliferation were activated by Tie2 (Huang et al., 1995; Jones et al., 1999; Witzenbichler et al., 1998). In addition, no candidate signaling molecule involved in tube formation of endothelial cells was identified downstream of Tie2.
We have previously established a murine brain capillary endothelial cell line from ts-A58-H-2Kb transgenic mice, denoted IBE (Immortomouse brain endothelial) cells. IBE cells can proliferate, migrate and form lumen-containing tube-like structures in response to FGF-2 treatment (Kanda et al., 1996; Rahmanian et al., 1997). Using this culture model, we have previously shown that expression of dominant-negative c-Src in IBE cells perturbed chemotaxis towards FGF-2 with the lack of MAPK activation within focal contacts (Shono et al., 2001a). Expression of dominant-negative c-Fes caused impaired chemotaxis independently on c-Src and MAPK (Kanda et al., 2000; Shono et al., 2001a). Dominant-negative c-Fyn inhibited FGF-2-mediated tube formation without major effect on proliferation or motility (S. Tsuda et al., unpublished).
In the present study, we examined the signal transduction pathways leading to Ang2-mediated cellular responses of parental IBE cells as well as examination of downstream signaling pathways of Tie2 by using stable IBE cell lines expressing dominant-negative c-Src, c-Fyn and c-Fes. We found that: (1) 2000 ng/ml Ang2 added to IBE cells (which express Tie2) induced autophosphorylation, suggesting that Ang2 could activate Tie2 in IBE cells; (2) Ang2 stimulated motility and tube formation; (3) PI 3-kinase was activated and was dependent on c-Fes kinase activity; (4) PI 3-kinase was involved in chemotaxis towards Ang2, as has been demonstrated in the case of Ang1 (Witzenbichler et al., 1998); and (4) expression of kinase inactive c-Fyn attenuated Ang2-mediated tube formation, suggesting that c-Fyn is involved in both FGF-2- and Ang2-mediated tube formations of IBE cells.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell culture
IBE cells were routinely cultured in Hams F-12 medium supplemented with 20% heat-inactivated fetal bovine serum (FBS), 75 µg/ml endothelial cell growth supplement (Sigma), 5 µg/ml bovine pancreas insulin (Sigma) and 10 ng/ml human recombinant epidermal growth factor (Roche Diagnostics Inc. Tokyo, Japan), as described previously (Kanda et al., 1996). All experiments were performed at 33°C, because cells at 37 or 39°C did not proliferate, migrate or form tube-like structures following FGF-2-treatment (Kanda et al., 1996). Establishment of IBE cell lines stably expressing kinase-inactive c-Fyn (S. Tsuda et al., unpublished), wild-type or kinase inactive c-Fes (Kanda et al., 2000) and kinase-inactive c-Src (Shono et al., 2001a) are described elsewhere.
Cell proliferation assay
The cell proliferation assay was performed as described previously (Kanda et al., 2000). Briefly, cells suspended in Hams F-12 medium containing 0.25% (w/v) bovine serum albumin (BSA) were inoculated into wells of fibronectin-coated 24-well-culture plates and cultured in the presence or absence of recombinant human FGF-2 (Roche Diagnostics) and/or human recombinant Ang2 (Genzyme-Techne, Minneapolis, MN) for 3 days. Cell numbers were counted with a hemacytometer. The number of cells counted in untreated samples was set as 100%.
Chemotaxis assay
The chemotaxis assay was performed as described previously (Kanda et al., 2000). In brief, cells were suspended in Hams F-12 medium containing 0.25% BSA and seeded onto the upper surface of Transwell membrane filters (Corning Coster Japan, Tokyo), of which were pre-coated with fibronectin. In the lower wells, known concentrations of FGF-2 and/or Ang2 were added to the same medium. Four hours later, cells were fixed and stained, and cells attached onto the lower surface of membranes were counted microscopically. The number of cells counted in untreated samples was set as 100%.
Tube formation assay
The tube formation assay was performed as described previously (Kanda et al., 1996). Briefly, cells were suspended in Hams F-12 medium containing 0.25% in the presence or absence of FGF-2 and/or Ang2 at indicated concentrations and seeded onto the first layer of collagen gels. After 4 hours, medium was aspirated and cells were covered with the second layer of collagen gels. After gelation, Hams F-12 medium containing 0.25% BSA was added onto the second layer of collagen gels and cultured for 16 hours, and then photographed under a phase-contrast microscope.
Immunoprecipitation and immunoblotting
Methods for immunoprecipitation and immunoblotting were described elsewhere (Kanda et al., 2000) with some modifications. Cells were incubated with serum- and growth supplement-free medium for 16 hours. Medium was changed to the fresh serum- and growth supplement-free medium, then cells were further incubated for 2 hours. After 20 minutes treatment of cells with 0.1 mM orthovanadate for 20 minutes, cells were either stimulated or left unstimulated with Ang2 (1 µg/ml) for 15 minutes. Cells were rinsed with TBS containing vanadate and lysed in NP-40 lysis buffer (50 mM Hepes, pH 7.2, supplemented with 0.15 M NaCl, 10% glycerol, 10 mM pyrophosphate, 50 mM NaF, 1% NP-40, 100 U/ml aprotinin, 1 mM PMSF, 0.1 mM orthovanadate, 10 µM leupeptin and 10 µM pepstatin A). After centrifugation to remove insoluble materials, cell lysates were immunoprecipitated with the indicated antibodies, followed by adsorption to Protein A-agarose beads. For the specification of anti-Tie2 antibody, 1 µg of antibody was incubated with 50 µg of immunized peptide (Santa Cruz) at 4°C overnight, then used for the immunoprecipitation. After washing the beads, proteins were eluted by heating in SDS-sample buffer and then separated by SDS-polyacrylamide gel electrophoresis. After the transfer of proteins onto polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA), blots were probed with the indicated antibodies. Antibody incubation was followed by incubation with horseradish peroxidase-conjugated anti-rabbit IgG or anti-mouse IgG, and detection was via the enhanced chemiluminescence reaction (ECL, Amersham Life Science, UK). Between two probings, stripping of the membrane was performed as described before (Kanda et al., 2000).
In vitro kinase assay for receptor tyrosine kinase
The in vitro kinase assay for receptor tyrosine kinase was described previously (Kanda et al., 1996). IBE cells (15 cm dishes) were serum-starved overnight and either stimulated or left unstimulated with 2 µg/ml Ang2 for 15 minutes in the presence of 100 µM orthovanadate. Cells were washed, lysed in Triton X-100 lysis buffer, and centrifuged. Cleared lysates (90%) were then incubated with anti-Tie2 antibody and Tie2 was also immunoprecipitated from the remaining 10% of lysate to examine the amount of Tie2 by immunoblotting. Incubated proteins with antibodies were absorbed with Protein A agarose beads. Beads were washed thoroughly and the autophosphorylation assay was carried out in 20 mM Hepes, pH 7.4, supplemented with 10 mM MgCl2, 2 mM MnCl2, 0.05% Triton X-100, 1 mM DTT and 0.037 MBq/sample of [-32P] ATP at 4°C for 10 minutes. The reaction was stopped by addition of 2x sample buffer, boiled, and eluted proteins were then separated by SDS-PAGE. Gels were incubated with destain for 30 minutes, fixed with 2.5% glutaraldehyde, rinsed with water, treated with 1 M KOH for 30 minutes at 55°C to remove phosphorylated serine residues, and then incubated with destain for 30 minutes at room temperature. Gels were dried and analyzed for radioactivity of particular proteins using a Bio Imager BAS 5000 (Fuji, Tokyo), followed by exposure on X-ray films.
In vitro kinase assay for Src family kinases
Serum-straved cells were either stimulated or not stimulated with Ang2 (1 µg/ml) for 15 minutes, then rinsed and lysed in NP-40 lysis buffer (Kanda et al., 2000); 10% of the lysate was mixed with SDS-sample buffer, then stored until required for immunoblotting to investigate the levels of proteins. Particular proteins were immunoprecipitated with the corresponding antibodies followed by incubation with Protein A-agarose beads with or without anti-mouse IgG rabbit IgG as a bridging antibody. Beads were washed four times with NP-40 lysis buffer, twice with TBS and twice with kinase buffer (25 mM Tris-HCl, pH 7.4, containing 10 mM MnCl2 and 2 mM MgCl2). Kinase buffer supplemented with 0.37 MBq/sample of [-32P] ATP and 0.5 mg/sample of acid-denatured rabbit muscle enolase (Roche Diagnostics) was added to the beads and incubated for 10 minutes at room temperature. The reaction was stopped by addition of 2x sample buffer, boiled, and eluted proteins were then separated by SDS-PAGE. Gels were treated to remove phosphorylated serine with 1 M KOH as described above. Gels were dried and analyzed for radioactivity of particular proteins using a Bio Imager BAS 5000 (Fuji, Tokyo), followed by exposure on X-ray films.
In vitro PI 3-kinase assay
Details of the in vitro kinase assay for PI 3-K are described elsewhere (Kanda et al., 1997). In brief, serum-starved IBE cells were either stimulated or not stimulated with Ang2 (1 µg/ml) for 15 minutes, then rinsed and lysed in Nonidet P-40 (NP-40) lysis buffer. In indicated experiments, Ang2 (1 µg) was preincubated with extracellular domain of either Tie1/Fc chimera or Tie2/Fc chimera (25 µg) at room temperature for 10 minutes, then added to the cells. Clarified cell lysates were immunoprecipitated with indicated antibodies, followed by the absorption with Protain A-agarose beads. In the examination using anti-phosphotyrosine immunoprecipitates, cells were treated with 0.1 mM orthovanadate for 20 minutes, then either stimulated or left unstimulated with Ang2. Beads were extensively washed, suspended in kinase buffer containing phosphatidylinositol (Sigma) and 0.37 MBq/sample of [-32P] ATP, and were incubated for 10 minutes. The reaction was stopped and reaction products were extracted with chloroform, and separated by thin layer chromatography on silica Gel-60 plates (Merck). Phosphorylated products were detected and incorporation of [
-32P] ATP was measured by Image Analyzer BAS 5000 (Fuji) and exposed on X-ray films (Amersham-Pharmacia BioTech).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Tie2-mediated activation of PI 3-kinase in the previous reports have suggested the direct interaction of p85 subunit with phosphorylated Y1100 (Y1101) of Tie2 (Jones et al., 1999; Kontos et al., 1998). However, these reports demonstrated this interaction by the use of a yeast two-hybrid assay, peptide competition assay, pull down of particular proteins from unstimulated endothelial cell lysate with GST fusion proteins or coprecipitation of p85 with Tie2 in 293T cells cotransfected with Tie2 and p85. To date, the increase of PI 3-kinase activity has been demonstrated in anti-p85 immunoprecipitates (Kim et al., 2000), but not in anti-Tie2 immunoprecipitates from Ang-treated endothelial cells. It is therefore possible that Ang-treated endothelial cells may use yet unidentified pathways to activate PI 3-kinase. In the present study, no increase in association of PI 3-kinase activity with Tie2 by Ang2-treatment was detected. However, kinase-inactive c-Fes exhibited the dominant-negative effect on Ang2-mediated activation of PI 3-kinase (Fig. 5D). This distinct mechanism of activation of PI 3-kinase via Tie2 may be due to the difference of cell types used between the studies, because c-Fes is exclusively expressed in endothelial and hematopoietic cells. Downstream of interleukin 4 receptor, c-Fes was not fully, but partially, involved in PI 3-kinase activation (Izuhara et al., 1996). In addition, kinase-activity-dependent association of c-Fes with two SH2 domain-containing fragments of p85 regulatory subunit was observed in yeast two-hybrid system (T.N. and S.K, unpublished). c-Fes has a typical YXXM motif surrounding Y633, which is recognized by p85 regulatory subunit of PI 3-kinase (Smithgall et al., 1998). It is therefore possible that activated c-Fes might associate with the p85 subunit of PI 3-kinase in endothelial cells. However, expression of kinase-inactive c-Fes had a dominant-negative effect on FGF-2-mediated motility without any effects on PI 3-kinase activity (Kanda et al., 2000). In IBE cells, only activated Ras seemed to be involved in PI 3-kinase activation mediated by FGF-2 and binding of the p85 regulatory subunit to tyrosine phosphorylated proteins was not involved downstream of FGF-2-treated cells (Kanda et al., 1997) (Y.M. et al., unpublished). Taken together, these data show that Ang2 could activate PI 3-kinase through c-Fes, which in turn regulates the binding of PI 3-kinase to phosphotyrosine-containing proteins in endothelial cells in a manner different from FGF-2-treatment.
Recent studies of signal transduction pathways downstream of Tie2 and the effects of Ang1 as well as Ang2 on cellular responses were examined mainly by the use of HUVECs. Ang1 could not stimulate tube formation of HUVECs in 3D collagen gels (Papapetropoulos et al., 1999). However, Ang2 mediated tube formation of HUVECs in fibrin gels in association with Tie2 activation (Teichert-Kuliszewska et al., 2001). These studies clearly demonstrate that tube formation by HUVECs depends on the type of extracellular matrix protein. Modulation of particular integrin functions may be involved in this process. Figrin is one of the ligands for vß3 integrin and inhibition of this integrin perturbed tube formation in fibrin gels (Bayless et al., 2000; Dallabrida et al., 2000). It is therefore possible that signals via Tie2 may modify the
vß3 integrin function. Interestingly, angiopoietin family molecules have a fibrinogen-like domain (Davis et al., 1996; Gale and Yancopoulos, 1999). It is also possible that angiopoietin may have an adhesive property, which can bridge cell-cell association involved in tube formation, especially in fibrin gels. By contrast, IBE cells can efficiently form lumen-containing tube-like structures in type I collagen gels; type I collagen is also a ligand of
vß3 integrin (Kanda et al., 1996). However, the inhibition of
vß3 integrin by cyclic peptide did not block tube formation in collagen gels (Kanda et al., 1999), but attenuated FGF-2-mediated motility on fibronectin (Shono et al., 2001b). The different cellular responses might be caused by the different kinds of integrin required for tube-like structure formation.
c-Src was activated by Ang2-treatment in IBE cells. However, no dominant-negative effect on Ang2-mediated cellular responses was observed. Conversely, another member of the Src kinase family, c-Fyn, was involved in Ang2-mediated tube formation. In addition, c-Fyn was shown to play a part in FGF-2-mediated tube formation (S. Tsuda et al., unpublished). However, the morphology of tube-like structure was different in FGF-2-treated and Ang2-treated cells. FGF-2-mediated activation of c-Fyn was observed only transiently and in the later phase of stimulation. The dominant-negative effect of kinase-inactive c-Fyn on FGF-2-induced tube formation demonstrated cell aggregates, but no cord-like, continuous morphology. By contrast, inhibition of Ang2-mediated tube formation resulted in the defect of aggregate formation. This difference in the dominant-negative effect of c-Fyn on tube formation in FGF-2- and Ang2-treatment seems to suggest that c-Fyn might regulate tube formation by more than one mechanism.
Many molecules are commonly expressed in endothelial cells and hematopoietic cells, because their origin is the same during development. However, hematopoietic cells do not form tube-like structures. Identification of the endothelial cell-specific signaling molecule downstream of c-Fyn and its inhibition would thus help in the development of new anti-angiogenic therapies.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ahmad, S. A., Liu, W., Jung, Y. D., Fan, F., Wilson, M., Reinmuth, N., Shaheen, R. M., Bucana, C. D. and Ellis, L. M. (2001). The effects of angiopoietin-1 and -2 on tumor growth and angiogenesis in human colon cancer. Cancer Res. 61, 1255-1259.
Bayless, K. L., Salazar, R. and Davis, G. E. (2000). RGD-dependent vacuolation and lumen formation observed during endothelial cell morphogenesis in three-dimensional fibrin matrices involves the vß3 and
5ß1 integrins. Am. J. Pathol. 156, 1673-1683.
Brown, L. F., DeZube, B. J., Tognazzi, K., Dvorak, H. F. and Yancopoulos, G. D. (2000). Expression of Tie1, Tie2, and angiopoietins 1, 2, and 4 in Kaposis sarcoma and cutaneous angiosarcoma. Am. J. Pathol. 156, 2179-2183.
Colville-Nash, P. R. and Willoughby, D. A. (1997). Growth factors in angiogenesis: Current interest and therapeutic potential. Mol. Med. Today 3, 14-23.[Medline]
Dallabrida, S. M., De Sousa, M. A. and Farell, D. A. (2000). Expression of antisense to integrin subunit ß3 inhibits microvascular endothelial cell capillary tube formation in fibrin. J. Biol. Chem. 275, 32281-32288.
Darland, D. C. and DAmore, P. A. (1999). Blood vessel maturation: vascular development comes of age. J. Clin. Invest. 103, 157-158.
Davis, S., Aldrich, T. A., Jones, P. F., Acheson, A., Compton, D. L., Jain, V., Ryan, T. E., Bruno, J., Radzeijewski, C., Maisonpierre, P. C. and Yancopoulos, G. D. (1996). Isolation of angiopoetin-1, a ligand for the TIE2 reeptor, by secretion-trap expression cloning. Cell 87, 1161-1169.[Medline]
Dumont, D. J., Gradwohl, G., Fong, G.-H., Puri, M. C., Gertsenstein, M., Auerbach, A. and Breiman, M. L. (1994). Dominant-negative and targeted null mutations in the endothelial receptor tyrosine kinase, tek, reveal a critical role in vasculogenesis of the embryo. Genes Dev. 8, 1897-1909.[Abstract]
Etoh, T., Inoue, H., Tanaka, S., Barnard, G. F., Kitano, S. and Mori, M. (2001). Angopoietin-2 is related to tumor angiogenesis in gastric carcinoma: Possible in vivoregulation via induction of proteases. Cancer Res. 61, 2145-2153.
Folkman, J. (1995). Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat. Med. 1, 27-31.[Medline]
Folkman, J. and DAmore, P. A. (1996). Blood vessel formation: what is its molecular basis? Cell 87, 1153-1155.[Medline]
Gale, N. W. and Yancopoulos, G. D. (1999). Growth factors acting via endothelial cell-specific receptor tyrosine kinases: VEGFs, angiopoietins, and ephrins in vascular development. Genes Dev. 13, 1055-1066.
Hanahan, D. (1997). Signaling vascular morphogenesis and maintenance. Science 277, 48-50.
Holash, J., Maisonpierre, P. C., Compton, D., Boland, P., Alexander, C. R., Zagzag, D., Yancopoulos, G. D. and Wiegand, S. J. (1999). Vessel cooption, regression, and growth in tumors mediated by angiopoietins and VEGF. Science 284, 1994-1998.
Huang, L., Turck, C. W., Rao, P. and Peters, K. G. (1995). GRB2 and SH-PTP2: potentially important endothelial signaling molecules downstream of the TEK/TIE2 receptor tyrosine kinase. Oncogene 11, 2097-2103.[Medline]
Izuhara, K., Feldman, R. A., Greer, P. and Harada, N. (1996). Interleukin-4 induces association of the c-fes proto-oncogene product with phosphatidylinositol-3 kinase. Blood 88, 3910-3918.
Jones, N. and Dumont, D. J. (1998). The Tek/Tie2 receptor signals through a novel Dok-related docking protein, Dok-R. Oncogene 17, 1097-1108.[Medline]
Jones, N., Master, Z., Jones, J., Bouchard, D., Gunji, Y., Sasaki, H., Daly, R., Alitalo, K. and Dumont, D. J. (1999). Identification of Tek/Tie2 binding partners. Binding to a multifunctional docking site mediates cell survival and migration. J. Biol. Chem. 274, 30896-30905.
Jones, N., Voskas, D., Master, Z., Sarao, R., Jones, J. and Dumont, D. J. (2001). Rescue of the early vascular defects in Tek/Tie2 null mice reveals an essential survival function. EMBO Rep. 2, 438-445.
Kanda, S., Landgren, E., Ljungström, M. and Claesson-Welsh, L. (1996). Fibroblast growth factor receptor 1-induced differentiation of endothelial cell line established from tsA58 large T transgenic mice. Cell Growth Differ. 7, 383-395.[Abstract]
Kanda, S., Hodgkin, M. N., Woodfield, R. J., Wakelam, M. J. O., Thomas, G. and Claesson-Welsh, L. (1997). Phosphatidylinositol 3'-kinase-independent p70 S6 kinase activation by fibroblast growth factor receptor-1 is important for proliferation but not differentiation of endothelial cells. J. Biol. Chem. 272, 23347-23353.
Kanda, S., Shono, T., Tomasini-Johansson, B., Klint, P. and Saito, Y. (1999). Role of thrombospondin-1-derived peptide, 4N1K, in FGF-2-induced angiogenesis. Exp. Cell Res. 252, 262-272.[Medline]
Kanda, S., Lerner, E. C., Tsuda, S., Shono, T., Kanetake, H. and Smithgall, T. E. (2000). The nonreceptor protein-tyrosine kinase c-Fes is involved in fibroblast growth factor-2-induced chemotaxis of murine brain capillary endothelial cells. J. Biol. Chem. 275, 10105-10111.
Kim, I., Kim, J.-H., Moon, S.-O., Kwak, H. J., Kim, N.-G. and Koh, G. Y. (2000). Angiopoietin-2 at high concentration can enhance endothelial cell survival through the phosphatidylinositol 3'-kinase/Akt signal transduction pathway. Oncogene 19, 4549-4552.[Medline]
Klippel, A., Escobedo, J. A., Fantl, W. J. and Williams, L. T. (1992). The C-terminal SH2 domain of p85 accounts for the high affinity and specificity of the binding of phosphatidylinositol 3-kinase to phosphorylated platelet-derived growth factor beta receptor. Mol. Cell Biol. 12, 1451-1459.[Abstract]
Kontos, C. D., Stauffer, T. P., Yang, W.-P., York, J. D., Huang, L., Blanar, M. A., Meyer, T. and Peters, K. G. (1998). Tyrosine 1101 of Tie2 is the major site of association of p85 and is required for activation of phosphatidylinositol 3-kinase and Akt. Mol. Cell Biol. 18, 4131-4140.
Korpelainen, E. I., Karkkainen, M., Gunji, Y., Vikkula, M. and Alitalo, K. (1999). Endothelial receptor tyrosine kinases activate the STAT signaling pathway: mutant Tie-2 causing venous malformations signals a distinct STAT activation response. Oncogene 18, 1-8.[Medline]
Lauren, J., Gunji, Y. and Alitalo, K. (1998). Is angiopoietin-2 necessary for the initiation of tumor angiogenesis? Am. J. Pathol. 153, 1333-1339.
Maisonpierre, P. C., Suri, C., Jones, P. F., Patan, S., Bartunkova, S., Wiegand, S. J., Radziejewski, C., Compton, D., McClain, J., Aldrich, T. H. et al. (1997). Angiopoietin-2, a natural antagonist for Tie2 that disrupts in vivo angiogenesis. Science 277, 55-60.
Papapertropoulos, A., Fulton, D., Mahboubi, K., Kalb, R. G., OCorner, D. S., Li, F., Altieri, D. C. and Sessa, W. C. (2000). Angiopoietin-1 inhibits endothelial cell apoptosis via the Akt/survivin pathway. J. Biol. Chem. 275, 9102-9105.
Papapetropoulos, A., Garcia-Cardena, G., Dengler, T. J., Maisonpierre, P. C., Yancopoulos, G. D. and Sessa, W. C. (1999). Direct actions of angiopoietin-1 on human endothelium: evidence for network stabilization, cell survival, and interaction with other angiogenic growth factors. Lab. Invest. 79, 213-223.[Medline]
Partanen, J., Armstrong, E., Makela, T. P., Korhonen, J., Sandgerg, M., Renkonen, R., Knuutila, S., Huebner, K. and Alitalo, K. (1992). A novel endothelial cell surface receptor tyrosine kinase with extracellular epidermal growth factor homology domains. Mol. Cell Biol. 12, 1698-1707.[Abstract]
Rahmanian, M., Pertoft, H., Kanda, S., Christofferson, R., Claesson-Welsh, L. and Heldin, P. (1997). Hyaluronan oligosaccharides induce tube formation of a brain endothelial cell line in vitro. Exp. Cell Res. 237, 223-230.[Medline]
Sato, A., Iwama, A., Takakura, N., Nishio, H., Yancopoulos, G. D. and Suda, T. (1998). Characterization of TEK receptor tyrosine kinase and its ligands, Angiopoietins, in human hematopoietic progenitor cells. Int. Immunol. 10, 1217-1227.[Abstract]
Savage, C. R. and Cohen, S. (1972). Epidermal growth factor and a new derivative. Rapid isolation procedures and biological and chemical characterization. J. Biol. Chem. 247, 7609-7611.
Shono, T., Kanetake, H. and Kanda, S. (2001a). Activation of mitogen-activated protein kinase within focal adhesions is involved in chemotaxis towards FGF-2 by murine brain capillary endothelial cells. Exp. Cell Res. 264, 275-283.[Medline]
Shono, T., Mochizuki, Y., Kanetake, H. and Kanda, S. (2001b). Inhibition of FGF-2-mediated chemotaxis of murine brain capillary endothelial cells by cyclic RGDfV peptide through blocking the redistribution of c-Src into focal adhesions. Exp. Cell Res. (in press).
Smithgall, T. E., Rogers, J. A., Peters, K. L., Li, J., Briggs, S. D., Lionberger, J. M., Cheng, H., Shibata, A., Scholtz, B., Schreiner, S. and Dunham, N. (1998). The c-Fes family of protein-tyrosine kinases. Crit. Rev. Oncogen. 9, 43-62.[Medline]
Suri, C., Jones, P. F., Patan, S., Bartunkova, S., Maisonpierre, P. C., Davis, S., Sato, T. N. and Yancopoulos, G. D. (1996). Requisite role of angiopoietin-1, a ligand for the TIE2 receptor, during embryonic angiogenesis. Cell 87, 1171-1180.[Medline]
Suri, C., McClain, J., Thurston, G., McDonald, D. M., Zhou, H., Oldmixon, E. H., Sato, T. N. and Yancopoulos, G. D. (1998). Increased vascularization in mice overexpressing angiopoietin-1. Science 282, 468-471.
Takakura, N., Watanabe, T., Suenobu, S., Yamada, Y., Noda, T., Ito, Y., Satake, M. and Suda, T. (2000). A role for hematopoietic stem cells in promoting angiogenesis. Cell 102, 199-209.[Medline]
Tanaka, S., Mori, M., Sakamoto, Y., Makuuchi, M., Sugimachi, K. and Wands, J. R. (1999). Biologic significance of angiopoietin-2 expression in human hepatocellular carcinoma. J. Clin. Invest. 103, 341-345.
Teichert-Kuliszewska, K., Maisonpierre, P. C., Jones, N., Cambell, A. I. M., Master, Z., Bendeck, M. P., Alitalo, K., Dumont, D. J., Yancopoulos, G. D. and Stewart, D. J. (2001). Biological action of angiopoietin-2 in a fibrin matrix model of angiogenesis is associated with activation of Tie2. Cardiovasc. Res. 49, 659-670.[Medline]
van Weering, D. H., de Rooij, J., Marte, B., Downward, J., Bos, J. L. and Burgering, B. M. (1998). Protein kinase B activation and lamellipodium formation are independent phosphoinositide 3-kinase-mediated events differentially regulated by endogenous Ras. Mol. Cell. Biol. 18, 1802-1811.
Witzenbichler, B., Maisonpierre, P. C., Jones, P., Yancopoulos, G. D. and Isner, J. M. (1998). Chemotactic properties of angiopoietin-1 and -2, ligands for the endothelial-specific receptor tyrosine kinase Tie2. J. Biol. Chem. 273, 18514-18521.
Yancopoulos, G. D., Davis, S., Gale, N. W., Rud, J., Wiegand, S. J. and Holash, J. (2000). Vascular-specific growth factors and blood vessel formation. Nature 407, 242-248.[Medline]