|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
Prepublished online as a Blood First Edition Paper on October 24, 2002; DOI 10.1182/blood-2002-02-0582.
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Wake Forest University School of Medicine,
Section of Cardiology, Winston-Salem, NC.
Angiostatin, an inhibitor of angiogenesis, contains 3 to 4 kringle
domains that are derived from proteolytic cleavage of plasminogen. The
antiangiogenic effects of angiostatin occur, in part, from its
inhibition of endothelial cell surface adenosine
triphosphate synthase, integrin functions, and pericellular
proteolysis. Angiostatin has structural similarities to hepatocyte
growth factor (HGF; "scatter factor"), a promoter of
angiogenesis, that induces proliferation and migration of both
endothelial and smooth muscle cells via its cell surface receptor,
c-met. We hypothesized that angiostatin might block HGF-induced
signaling in endothelial and smooth muscle cells. Angiostatin inhibited
HGF-induced phosphorylation of c-met, Akt, and ERK1/2. Angiostatin
also significantly inhibited proliferation of human umbilical vein
endothelial cells (HUVECs) induced by HGF. In contrast,
angiostatin did not inhibit vascular endothelial growth factor
(VEGF)-or basic fibroblast growth factor (bFGF)-induced signaling events or HUVEC proliferation. Angiostatin bound to immobilized truncated c-met produced by A431 cells and could be immunoprecipitated as a complex with soluble c-met. HGF inhibited the
binding of 125I-angiostatin to HUVECs. Soluble c-met,
produced by several tumor cell lines, could inhibit the antiangiogenic
effect of angiostatin. The disruption of HGF/c-met signaling is a novel
mechanism for the antiangiogenic effect of angiostatin.
(Blood. 2003;101:1857-1863) Tumor growth and metastasis often depend on the
elaboration of new capillary blood vessels, a process called
angiogenesis.1 Angiogenesis inhibitors primarily target
the proliferation and migration of endothelial cells, providing a
complimentary approach, along with direct tumor cytotoxic chemotherapy,
for treating cancer.2
Angiostatin is an approximately 38-kDa protein derived from an internal
fragment of plasminogen3 that suppresses the growth of
experimental tumors in animal models.4 Angiostatin was
originally purified from the urine of mice with Lewis lung
carcinoma3 and is generated endogenously by a variety of
human tumors as well.5,6 The conversion of plasminogen to
angiostatin appears to require reduction of disulfide bonds followed by
proteolysis with one or more proteinases.7,8
Angiostatin has been observed to inhibit endothelial3 and
smooth muscle cell9 proliferation by disrupting the G2/M
transition in the cell-cycle progression.10 Angiostatin
has also been shown to induce apoptosis of human umbilical vein
endothelial cells (HUVECs).11 Angiostatin may have
an even more potent inhibitory effect on circulating, bone
marrow-derived endothelial progenitor cells than it has on mature
endothelial cells.12 However, the molecular mechanisms of
the antiangiogenesis effect of angiostatin have not been completely
elucidated. Angiostatin binds to the endothelial cell surface
F1-F0 adenosine triphosphate (ATP) synthase, where it
blocks both the ATP synthase and ATPase activities of this enzyme
complex.13,14 The antiangiogenic effects of angiostatin may also be mediated via blockade of Angiostatin has similarities with hepatocyte growth factor (HGF;
"scatter factor"), an angiogenic growth factor that acts as a
mitogen and motogen for endothelial and smooth muscle cells. Although
they have opposing effects on angiogenesis, both HGF and angiostatin
possess kringle motifs18,19 and share significant homology
at the amino acid level.19 We have previously shown that
angiostatin inhibits HGF-induced smooth muscle cell proliferation and
migration, suggesting that angiostatin might competitively inhibit the
binding of HGF to its receptor, c-met proto-oncogene.9 In
this paper, we examine this hypothesis and demonstrate that angiostatin
inhibits signaling events induced by HGF but not by vascular
endothelial growth factor (VEGF) or basic fibroblast growth
factor (bFGF).
Angiostatin (K1-3 without N-linked glycosylation) expressed in
Pichia pastoris, was a gift from Entremed (Rockville, MD). HGF and the alkaline phosphatase-conjugated anti-goat and
anti-rabbit IgG were purchased from Sigma (St Louis, MO).
Peroxidase-conjugated anti-rabbit IgG and peroxidase-conjugated
anti-mouse IgG were from Amersham Pharmacia Biotech (Piscataway, NJ).
Antihuman plasminogen and protease inhibitor cocktail were from
Calbiochem (San Diego, CA). Recombinant human insulin growth factor-1
(IGF-1) was from Bachem Bio Science (King of Prussia, PA). Monoclonal
antibodies to human c-met, specific for the extracellular Centricon-10 concentrators were purchased from Amicon (Beverly, MA).
Iodobeads and the Super Signal chemiluminescence kit were purchased
from Pierce Chemical (Rockford, IL). Alkaline phosphatase activity was
detected using the soluble substrate p-nitrophenylphosphate with
diethanolamine buffer from BIORAD (Hercules, CA).
Cell culture
Treatment of cells
Soluble c-met from tumor cell lines Tumor cell lines were seeded in 100-mm tissue culture dishes and grown in complete medium to near 100% confluency. Cells were washed with PBS and serum-free medium was added to the plate. After 16 hours, the medium was harvested (7.0 mL) and protease inhibitor cocktail was added and centrifuged at 10 000g at 4°C for 1 hour to remove any cell debris. The supernatant was filtered through a 0.22-µm filter and concentrated by using centriprep 10 (cutoff 10 000 kDa) at 3000g until the volume was reduced to 0.5 mL. The concentrated medium (35 µL) was mixed with 35 µL of 2 × sample buffer (reducing or nonreducing) then boiled for 3 minutes and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 7.5% gels).Cell proliferation assay HUVECs (5000 cells per well) were cultured in a 96-well plate in EBM-2 containing 1.0% serum in the presence or absence of HGF (10 ng/mL), bFGF (10 ng/mL), or VEGF (10 ng/mL). Angiostatin, at a final concentration of 3 to 5 µM was added to replicate wells containing growth factors. After 72 hours, 20 µL per well of CellTiter 96 AQueous One cell proliferation assay reagent (Promega, Madison, WI) was added and the plate was incubated for 1 hour at 37°C, 5% CO2. The reaction product (formazan) was then monitored at 490 nm, and the background absorbance of wells containing culture medium only was subtracted. All assays were performed at n = 6.Immunoprecipitation Concentrated A431 medium was precleared by adding 50 µL of protein L beads for 2 hours at 4°C; it was then centrifuged and supernatant was collected. Angiostatin (2.5 µg/mL) was added for 2 hours at room temperature with shaking. Anti-c-met clone DO24 (5 µL) or nonimmune IgG was added and incubated overnight at 4°C with shaking. After incubation with primary antibody, 50 µL of protein L beads was added and again incubated for 2 hours at 4°C with shaking. Beads were washed 3 times with ice-cold radioimmunoprecipitation assay (RIPA) buffer and bound protein was extracted by adding 50 µL of 2 × SDS sample buffer and boiling for 5 minutes. Beads were again centrifuged and 50 µL of extracted proteins was loaded onto a polyacrylamide gel (8%). The gel was transferred onto a nitrocellulose membrane and immunoblotting was performed using antiplasminogen to detect angiostatin.Cell-binding assay Purified angiostatin (10 µg; Calbiochem) was iodinated using Iodobeads (Pierce Chemical) according to the manufacturer's recommendations. Cell-binding assay was performed essentially as described.20 HUVECs (n = 10 000 in 100 µL) were seeded in a 96-well plate in complete medium. After 24 hours medium was removed and cells were serum starved for 24 hours in medium containing 0.1% serum. Cells were washed 3 times with ice-cold RPMI medium containing 20 mM HEPES (4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid), pH 7.4, 0.1% bovine serum albumin (BSA; binding medium) and incubated at 4°C for 30 minutes. Different concentrations of 125I-angiostatin with or without a 100-fold excess of angiostatin were added at a final volume of 100 µL/well and incubated for 1 hour at 4°C. To test the ability of HGF to inhibit 125I-angiostatin binding, 125I-angiostatin (5 nM) was incubated with the HUVECs as described in the absence or presence of increasing concentrations of unlabeled HGF (75 nM to 1.0 µM), or with 0.5 µM cold angiostatin. After incubation, cells were washed 5 times with ice-cold binding buffer and extracted with 1% Triton X-100 in PBS, then counted in a -counter. Replicates of 6 were performed. Binding studies were graphed and analyzed using
GraphPad Prism Version 3.0 (GraphPad Software, San Diego, CA).
Enzyme-linked immunosorbent assay (ELISA) Concentrated A431 serum-free medium containing soluble c-met (total protein concentration, approximately 4 mg/mL) or Voller buffer (15 mM Na2CO3, 35 mM NaHCO3, 0.2% NaN3, pH 9.6) was added in triplicate wells to a 96-well EIA/RIA plate (100 µL) and allowed to adsorb overnight at 4°C. The plates were washed 3 times with PBS between each step. To block the plates, PBS containing 1% nonfat dry milk was added for one hour at room temperature. For a sandwich ELISA, 96-well enzyme immunoassay/radioimmunoassay (EIA/RIA) plates were incubated overnight with 100 µL of a 1:100 dilution of a monoclonal antibody to the extracellular -chain of c-met in Voller buffer. The plates were
blocked and concentrated soluble c-met receptor was added. Angiostatin
at concentrations of 0 to 5 µM in PBS/1% milk was incubated on the
plate for one hour at room temperature. Antihuman plasminogen at 1:250
dilution was added for one hour at room temperature. Alkaline
phosphatase-conjugated secondary antibodies (1:250 of anti-rabbit
IgG) were incubated for one hour at room temperature. Alkaline
phosphatase activity was detected in 1 M diethanolamine (pH 9.8), 0.5 mM MgCl2, and 12 mM p-nitrophenyl phosphate. Plates were
read at OD405 (optical density at 405 nm) in
an ELISA plate reader. The Vmax (maximum velocity) was
determined using SOFTMAX Pro software (version 2.1.1; Molecular Devices, Sunnyvale, CA).
Western blotting of cell lysates and conditioned medium Cell lysates were prepared by adding 300 µL ice-cold RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA [ethylenediaminetetraacetic acid], 1% nonidet P-40 [NP-40] and 0.25% Na-deoxycholate) containing protease and phosphatase inhibitors cocktail. Cells were incubated for 30 minutes on ice containing RIPA buffer. The lysates were cleared by centrifugation and protein concentration of the supernatant was determined by using BioRad Dc Protein assay reagent. Electrophoresis was performed in reducing and nonreducing Laemmli buffer using SDS-polyacrylamide gel. After electrophoresis, proteins were transferred onto nitrocellulose membrane, then blocked in 5% dried milk or 3% BSA in TBST (10 mM Tris, pH 7.5, 100 mM NaCl, 0.1% Tween20) for 1 hour at room temperature. The membranes were washed twice in TBST for 10 minutes, then probed overnight at 4°C with primary antibody (0.5 to 1 µg/mL) in 5% nonfat milk or 3% BSA in TBST. The membranes were washed twice in TBST and incubated in secondary antibody labeled with horseradish peroxidase for 1 hour at room temperature, then washed twice in TBST for 10 minutes and developed for enhanced chemiluminescence.In some cases, immunoblots were stripped by incubating the blot at 50°C for 30 minutes in a buffer containing 62.5 mM Tris-HCl, pH 6.7, containing 2% SDS and 100 mM 2-mercaptoethanol. The blot was then washed twice with TBST for 5 minutes and blocked with 5% milk for 30 minutes at room temperature followed by a washing twice with TBST for 5 minutes, then re-probing with a primary antibody as described.
c-met was detected in immunoblots from HUVECs and HASMCs, as well
as from A431 cells, which are known to overexpress c-met (Figure
1; Galvani et al21). HGF
induced the phosphorylation of c-met, a process inhibited by
angiostatin (Figure 2A). In contrast, angiostatin did not inhibit the phosphorylation of KDR (flk-1; Figure
2B). By itself, angiostatin at 5 µM did not induce phosphorylation of
c-met, KDR, Akt, or ERK1/2 (data not shown). Angiostatin also blocked
the phosphorylation of the survival signal, Akt, in response to HGF.
This effect was demonstrated in endothelial (Figure
3) and smooth muscle (Figure
4) cells and appeared to be
competitive, since higher levels of HGF were able to reverse inhibition
by angiostatin (Figure 5). Angiostatin
failed to inhibit phospho-Akt generation in response to VEGF, bFGF
(Figure 6A-B), or IGF-1 (data not shown).
Plasminogen did not inhibit HGF-induced AKT phosphorylation in HUVECs
(Figure 7). Angiostatin inhibited ERK1
and ERK2 phosphorylation by HGF (Figure
8A) but not by VEGF or bFGF (Figure
8B).
Angiostatin significantly inhibited HGF-induced proliferation of HUVECs
(Figure 9). Although there was a trend
toward inhibition of bFGF-induced proliferation, this effect was not
statistically significant. Angiostatin did not significantly inhibit
VEGF-induced proliferation (not shown).
To test the ability of angiostatin, in the absence of HGF, to bind to
truncated c-met, we performed an ELISA with medium containing truncated
c-met or Voller buffer to control for nonspecific binding. Angiostatin
(0-5 µM) binding was detected with a polyclonal antibody to
plasminogen. Angiostatin bound to truncated c-met with a dissociation constant (Kd) of approximately 0.75 µM (Figure 10A). Similar results were
obtained with sandwich ELISA, in which soluble c-met was captured with
an antibody to the
We examined the production of soluble c-met by 5 tumor cell lines. A431
and A549 cells produced soluble c-met, but MCF-7, S180, and BT474 cells
did not (Figure 12).
In this paper, we have shown that angiostatin inhibits endothelial and smooth muscle cell responses to HGF, while not affecting VEGF- or bFGF-induced signaling events. Angiostatin inhibited autophosphorylation of the HGF receptor (c-met), as well as downstream events including the phosphorylation of Akt and ERK1/2. The inhibition appeared to be competitive, since high concentrations of HGF could overcome the inhibition by angiostatin. HGF competed with 125I-angiostatin for binding to HUVECs. Finally, angiostatin inhibited endothelial cell proliferation that was induced by HGF, but not proliferation that was induced by VEGF or bFGF. Plasminogen did not inhibit HGF-induced AKT phosphorylation, indicating that this property is unique to angiostatin. Our finding of a specific effect of angiostatin on signal transduction by HGF is consistent with previous reports. Claesson-Welsh et al11 found that angiostatin did not block the binding of 125I-VEGF to its receptor on bovine adrenal cortex capillary endothelial cells. Furthermore, angiostatin had no effect on the bFGF-induced tyrosine phosphorylation of the adapter protein Shb, on bFGF-induced p42 MAPK (ERK1) phosphorylation, or on bFGF-induced tyrosine phosphorylation of an unidentified 140-kDa protein.11 Our results are also similar to those obtained by Kuba et al who studied NK4, a derivative of HGF composed of the N-terminal hairpin and 4 kringle domains of HGF.22 NK4 has 47% amino acid homology with angiostatin.22 NK4 inhibited the HGF-induced phosphorylation of c-met but not the phosphorylation of KDR induced by VEGF. Furthermore, NK4 inhibited HGF-induced ERK1/2 activation, but did not inhibit the phosphorylation of ERK1/2 in response to bFGF or VEGF.22 The antiangiogenic effects of NK4 could not be ascribed solely to
antagonism of HGF activities, however, because NK4 also blocked
proliferation and migration induced by bFGF and VEGF, as well as
HGF.22 Similarly, angiostatin inhibited migration of cells
toward bFGF and VEGF,11 an effect that appears to be largely due to interference with integrin function. Tarui et al reported that bovine aortic endothelial cells adhered to angiostatin in
an integrin-dependent manner, with the predominant receptor being
The inhibition of Akt phosphorylation by angiostatin is not solely a
marker for the inhibition of HGF binding to c-met; instead, a
reduction in phospho-Akt could directly contribute to the disruption of
angiogenesis. Akt is a serine/threonine kinase that is rapidly activated as a downstream effector of phosphatidylinositol 3 (PI3) kinase in response to a variety of cytokines and growth
factors, including HGF.25 Akt plays multiple roles in
cellular homeostasis including the regulation of glucose
metabolism,26 the activation of eNOS,27 and
the suppression of apoptosis.28 The antiapoptotic effect
of Akt may occur through the phosphorylation of the apoptosis-inducing proteins BAD,29,30 caspase-9,31 and
FKHRL1.32 The activation of Akt is also essential for the
induction of a survival pathway involving ligation of
At first thought, the finding that angiostatin inhibits HGF but not
VEGF or bFGF signaling might appear to be an inadequate explanation for
the inhibition of angiogenesis by angiostatin. Several factors need to
be considered in this analysis, however. First, it has been
demonstrated that amplification and signaling cross-talk between
angiogenic growth factors are common. For example, HGF in cell
culture35 and when injected in vivo36 induces the expression of VEGF. The induction of VEGF expression by HGF depends
on the activation of Akt, which up-regulates HIF1 We found that angiostatin binds to soluble c-met derived from A431 epidermoid carcinoma cells. Soluble c-met was also detected in the medium of A549 lung carcinoma cells, but not in the medium of MCF-7, S180, or BT474 breast carcinoma cells. Soluble c-met is also produced by GTL-16 gastric carcinoma cells40 and by LoVo colon carcinoma cells41 We have also previously demonstrated that soluble c-met is released from cultured HUVECs and HASMCs, and can bind to the ligand HGF.41 These findings suggest that the ability of angiostatin to block the HGF/c-met interaction as well as its other antiangiogenic actions could be abrogated in the presence of high plasma levels of soluble c-met. Therefore, an important clinical implication of this work is that tumors that produce high levels of soluble c-met may be relatively resistant to the antiangiogenic effects of angiostatin, while others that do not produce soluble c-met may be more sensitive. Finally, it is also possible that angiostatin exerts effects directly at the surface of tumor cells that express c-met.
Submitted February 22, 2002; accepted October 15, 2002.
Prepublished online as Blood First Edition Paper, October 24, 2002; DOI 10.1182/blood-2002-02-0582.
Supported by the National Institutes of Health (NIH) grant CA 81233 (D.C.S.). The DNA Core Synthesis Laboratory of the Comprehensive Cancer Center and Wake Forest University School of Medicine was supported in part by National Cancer Institute grant CA 12197.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: David C. Sane, Section of Cardiology, Wake Forest University School of Medicine, Medical Center Blvd, Winston-Salem, NC 27157-1045; e-mail: dsane{at}wfubmc.edu.
1. Folkman J, Browder T, Palmblad J. Angiogenesis research: guidelines for translation to clinical application. Thromb Haemost. 2001;86:23-33[Medline] [Order article via Infotrieve]. 2. Folkman J. Tumor angiogenesis. In: Bast RC Jr,Kufe DW,Pollock RE,Weichselbaum RR,Holland JF,Frei E III, eds. Cancer Medicine 5th ed. Hamilton, Ontario, Canada: BC Decker; 2000:132-152. 3. O'Reilly MS, Holmgren L, Shing Y, et al. Angiostatin: a novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma. Cell. 1994;79:315-328[CrossRef][Medline] [Order article via Infotrieve]. 4. O'Reilly MS, Holmgren L, Chen C, Folkman J. Angiostatin induces and sustains dormancy of human primary tumors in mice. Nat Med. 1996;2:689-692[CrossRef][Medline] [Order article via Infotrieve].
5.
Kisker O, Onizuka S, Banyard J, et al.
Generation of multiple angiogenesis inhibitors by human pancreatic cancer.
Cancer Res.
2001;61:7298-7304
6.
Gately S, Twardowski P, Stack MS, et al.
Human prostate carcinoma cells express enzymatic activity that converts human plasminogen to the angiogenesis inhibitor, angiostatin.
Cancer Res.
1996;56:4887-4890
7.
Stathakis P, Fitzgerald M, Metthias LJ, Chesterman CN, Hogg PJ.
Generation of angiostatin by reduction and proteolysis of plasmin.
J Biol Chem.
1997;272:20641-20645
8.
Gately S, Twardowski P, Stack MS, et al.
The mechanism of cancer-mediated conversion of plasminogen to the angiogenesis inhibitor angiostatin.
Proc Natl Acad Sci U S A.
1997;94:10868-10872
9.
Walter JJ, Sane DC.
Angiostatin binds to smooth muscle cell in the coronary artery and inhibits smooth muscle cell proliferation and migration in vitro.
Arterioscler Thromb Vasc Biol.
1999;19:2041-2048
10.
Griscelli F, Li H, Bennaceur-Griscelli A, et al.
Angiostatin gene transfer: inhibition of tumor growth in vivo by blockage of endothelial cell proliferation associated with a mitosis arrest.
Proc Natl Acad Sci U S A.
1998;95:6367-6372
11.
Claesson-Welsh L, Welsh M, Ito N, et al.
Angiostatin induces endothelial cell apoptosis and activation of focal adhesion kinase independently of the integrin-binding motif RGD.
Proc Natl Acad Sci U S A.
1998;95:5579-5583
12.
Ito H, Rovira II, Bloom ML, et al.
Endothelial progenitor cells as putative targets for angiostatin.
Cancer Res.
1999;59:5875-5877
13.
Moser TL, Kenan DJ, Ashley TA, et al.
Endothelial cell surface F1-F0 ATP synthase is active in ATP synthesis and is inhibited by angiostatin.
Proc Natl Acad Sci U S A.
2001;98:6656-6661
14.
Moser TL, Stack MS, Asplin I, et al.
Angiostatin binds ATP synthase on the surface of human endothelial cells.
Proc Natl Acad Sci U S A.
1999;96:2811-2816
15.
Tarui T, Miles LA, Takada Y.
Specific interaction of angiostatin with integrin 16. Stack MS, Gately S, Bafetti LM, Enghild JJ, Soff GA. Angiostatin inhibits endothelial and melanoma cellular invasion by blocking matrix-enhanced plasminogen activation. Biochem J. 1999;340:77-84[Medline] [Order article via Infotrieve].
17.
Troyanovsky B, Levchenko T, Månsson G, Matvijenko O, Holmgren L.
Angiomotin: an angiostatin binding protein that regulates endothelial cell migration and tube formation.
J Cell Biol.
2001;152:1247-1254
18.
Cao Y, Ji RW, Davidson D, Schaller J, Martinas M, Folkman J.
Kringle domains of human angiostatin.
J Biol Chem.
1996;271:29461-29467 19. Nakamura T, Nishizawa T, Hagiya M, et al. Molecular cloning and expression of human hepatocyte growth factor. Nature. 1989;342:440-443[CrossRef][Medline] [Order article via Infotrieve]. 20. Naldini L, Weidner KM, Vigna E, et al. Scatter factor and hepatocyte growth factor are indistinguishable ligands for the MET receptor. EMBO J. 1991;10:2867-2878[Medline] [Order article via Infotrieve]. 21. Galvani AP, Cristiani C, Carpinelli P, Landonio A, Bertolero F. Suramin modulates cellular levels of hepatocyte growth factor receptor by inducing shedding of a soluble form. Biochem Pharmacol. 1995;50:959-966[CrossRef][Medline] [Order article via Infotrieve].
22.
Kuba K, Matsumoto K, Date K, Shimura H, Tanaka M, Nakamura T.
HGF/NK4, a four-kringle antagonist of hepatocyte growth factor, is an angiogenesis inhibitor that suppresses tumor growth and metastasis in mice.
Cancer Res.
2000;60:6737-6743 23. Brooks PC, Montgomery AM, Rosenfeld M, et al. Integrin antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels. Cell. 1994;79:1157-1164[CrossRef][Medline] [Order article via Infotrieve].
24.
Gutheil JC, Campbell TN, Pierce PR, et al.
Targeted antiangiogenic therapy for cancer using Vitaxin: a humanized monoclonal antibody to the integrin alphavbeta3.
Clin Cancer Res.
2000;6:3056-3061
25.
Nakagami H, Morishita R, Yamamoto K, et al.
Mitogenic and antiapoptotic actions of hepatocyte growth factor through ERK, STAT3, and Akt in endothelial cells.
Hypertension.
2001;37:581-586
26.
Kohn AD, Summers SA, Bimbaum MJ, et al.
Expression of a constitutively active Akt Ser/Thr kinase in 3T3-L1 adipocytes stimulated glucose uptake and glucose transporter 4 translocation.
J Biol Chem.
1996;271:31372-31378 27. Dimmeler S, Fleming I, Fisslthaler B, Hermann C, Busse R, Zeiher AM. Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation. Nature. 1999;399:601-605[CrossRef][Medline] [Order article via Infotrieve]. 28. Coffer PJ, Jin J, Woodgett JR. Protein kinase B (c-Akt): a multifunctional mediator of phosphatidylinositol 3-kinase activation. Biochemical J. 1998;335:1-13[Medline] [Order article via Infotrieve]. 29. Datta SR, Dudek H, Tao X, et al. Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell. 1997;91:231-241[CrossRef][Medline] [Order article via Infotrieve].
30.
Del Peso L, Gonzalez-Garcia M, Page C, Herrara R, Nunez G.
Interleukin-3-induced phosphorylation of BAD through the protein kinase Akt.
Science.
1997;278:687-689
31.
Cardone MH, Roy N, Stennicke HR, et al.
Regulation of cell death protease caspase-9 by phosphorylation.
Science.
1998;282:1318-1321 32. Brunet A, Bonni A, Zigmond MJ, et al. Akt promotes cell survival by phosphorylating and inhibiting a forkhead transcription factor. Cell. 1999;96:857-868[CrossRef][Medline] [Order article via Infotrieve].
33.
Matter ML, Ruoslahti E.
A signaling pathway from the
34.
Mirza AM, Kohn AD, Roth RA, McMahon M.
Oncogenic transformation of cells by a conditionally active form of the protein kinase Akt/PKB.
Cell Growth Differ.
2000;11:279-292 35. Wojta J, Kaun C, Breuss JM, et al. Hepatocyte growth factor increases expression of vascular endothelial growth factor and plasminogen activator inhibitor-1 in human keratinocytes and the vascular endothelial growth factor receptor flk-1 in human endothelial cells. Lab Invest. 1999;79:427-438[Medline] [Order article via Infotrieve].
36.
Van Belle E, Witzenbichler B, Chen D, et al.
Potentiated angiogenic effect of scatter factor/hepatocyte growth factor via induction of vascular endothelial growth factor: the case for paracrine amplification of angiogenesis.
Circulation.
1998;97:381-390
37.
Sodhi A, Montaner S, Miyazaki H, Gutkind JS.
MAPK and Akt act cooperatively but independently on hypoxia inducible factor-1
38.
Jiang BH, Zheng JZ, Masahiro A, Vogt PK.
Phosphatidylinositol 3-kinase signaling mediates angiogenesis and expression of vascular endothelial growth factor in endothelial cells.
Proc Natl Acad Sci U S A.
2000;97:1749-1753
39.
Xin X, Yang S, Ingle G, et al.
Hepatocyte growth factor enhances vascular endothelial growth factor-induced angiogenesis in vitro and in vivo.
Am J Pathol.
2001;158:1111-1120
40.
Prat M, Crepalki T, Gandino L, Giordano S, Longati P, Comoglio P.
C-terminal truncated forms of met, the hepatocyte growth factor receptor.
Mol Cell Biol.
1991;11:5954-5962
41.
Wajih N, Walter J, Sane DC.
Vascular origin of a soluble truncated form of the hepatocyte growth factor receptor (c-met).
Circ Res.
2002;90:46-52
© 2003 by The American Society of Hematology.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
|
 |
|
  T.-Y. Lee, S. Muschal, E. A. Pravda, J. Folkman, A. Abdollahi, and K. Javaherian Angiostatin regulates the expression of antiangiogenic and proapoptotic pathways via targeted inhibition of mitochondrial proteins Blood, August 27, 2009; 114(9): 1987 - 1998. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Bratt, O. Birot, I. Sinha, N. Veitonmaki, K. Aase, M. Ernkvist, and L. Holmgren Angiomotin Regulates Endothelial Cell-Cell Junctions and Cell Motility J. Biol. Chem., October 14, 2005; 280(41): 34859 - 34869. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Leksa, S. Godar, H. B. Schiller, E. Fuertbauer, A. Muhammad, K. Slezakova, V. Horejsi, P. Steinlein, U. H. Weidle, B. R. Binder, et al. TGF-{beta}-induced apoptosis in endothelial cells mediated by M6P/IGFII-R and mini-plasminogen J. Cell Sci., October 1, 2005; 118(19): 4577 - 4586. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. S. Aguzzi, C. Giampietri, F. De Marchis, F. Padula, R. Gaeta, G. Ragone, M. C. Capogrossi, and A. Facchiano RGDS peptide induces caspase 8 and caspase 9 activation in human endothelial cells Blood, June 1, 2004; 103(11): 4180 - 4187. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-H. Ahn, J.-S. Kim, H.-K. Yu, H.-J. Lee, and Y. Yoon A Truncated Kringle Domain of Human Apolipoprotein(a) Inhibits the Activation of Extracellular Signal-regulated Kinase 1 and 2 through a Tyrosine Phosphatase-dependent Pathway J. Biol. Chem., May 21, 2004; 279(21): 21808 - 21814. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Ikai, R. K. Riemer, X. Ma, O. Reinhartz, F. L. Hanley, and V. M. Reddy Pulmonary expression of the hepatocyte growth factor receptor c-Met shifts from medial to intimal layer after cavopulmonary anastomosis J. Thorac. Cardiovasc. Surg., May 1, 2004; 127(5): 1442 - 1449. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Levchenko, K. Aase, B. Troyanovsky, A. Bratt, and L. Holmgren Loss of responsiveness to chemotactic factors by deletion of the C-terminal protein interaction site of angiomotin J. Cell Sci., September 15, 2003; 116(18): 3803 - 3810. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|||||||
 |
 |
 |
 |
 |
 |
 |
 |
||
| Copyright © 2003 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
v
