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(Circulation Research. 2000;86:24.)
© 2000 American Heart Association, Inc.

Molecular Medicine

Angiopoietin-1 Regulates Endothelial Cell Survival Through the Phosphatidylinositol 3'-Kinase/Akt Signal Transduction Pathway

Injune Kim, Hwan Gyu Kim, June-No So, Joo Heon Kim, Hee Jin Kwak, Gou Young Koh

From the National Creative Research Initiatives Center for Cardiac Regeneration and Institute of Cardiovascular Research (I.K., H.G.K., H.J.K., G.Y.K.), Department of Pathology (J.H.K.), Chonbuk National University School of Medicine, and Department of Biotechnology (J.-N.S.), Woosuk University, Chonju, Korea.

Correspondence to Gou Young Koh, MD, PhD, National Creative Research Initiatives Center for Cardiac Regeneration, Chonbuk National University School of Medicine, San 2-20, Keum-Am-Dong, Chonju, 560-180, Republic of Korea. E-mail gykoh{at}moak.chonbuk.ac.kr

	Abstract

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Abstract—Angiopoietin-1 (Ang1) is a strong apoptosis survival factor for endothelial cells. In this study, the receptor/second messenger signal transduction pathway for the antiapoptotic effect of Ang1 on human umbilical vein endothelial cells was examined. Pretreatment with soluble Tie2 receptor, but not Tie1 receptor, blocked the Ang1-induced antiapoptotic effect. Ang1 induced phosphorylation of Tie2 and the p85 subunit of phosphatidylinositol 3'-kinase (PI 3'-kinase) and increased PI 3'-kinase activity in a dose-dependent manner. The PI 3'-kinase–specific inhibitors wortmannin and LY294002 blocked the Ang1-induced antiapoptotic effect. Ang1 induced phosphorylation of the serine-threonine kinase Akt at Ser473 in a PI 3'-kinase–dependent manner. Expression of a dominant-negative form of Akt reversed the Ang1-induced antiapoptotic effect. Ang1 mRNA and protein were present in vascular smooth muscle cells but not in endothelial cells. Cultured vascular smooth muscle cells, but not human umbilical vein endothelial cells, secreted Ang1. These findings indicate that the Tie2 receptor, PI 3'-kinase, and Akt are crucial elements in the signal transduction pathway leading to endothelial cell survival induced by the paracrine activity of Ang1.

Key Words: angiopoietin-1 • endothelial cell • apoptosis

	Introduction

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Vascular endothelium, a monolayer of cells lining the intima of the blood vessels, is involved in vascular permeability, vascular tonus, coagulation, vascular remodeling, and other functions.¹ Endothelial cells directly contact the plasma and cellular components of the blood and are the targets of many cytokines and growth factors.^{2 3}

Apoptosis, a process for eliminating unwanted cells, is involved in the regulation of cell number under physiological and certain pathological conditions.⁴ Apoptosis in vascular endothelial cells is prevented by several growth factors and cytokines, such as fibroblast growth factor,⁵ vascular endothelial growth factor (VEGF),⁶ and endothelin-1.⁷ These molecules not only suppress apoptosis but also stimulate cell proliferation, thereby maintaining or increasing cell number.

Angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2) have recently been identified as ligands of the endothelial cell–specific Tie2 receptor.^{8 9} In vivo analyses by targeted gene inactivation and transgenic overexpression reveal that Ang1 recruits and sustains periendothelial support cells.¹⁰ Ang2 disrupts blood vessel formation in the developing embryo by antagonizing the effects of Ang1 on Tie2.⁹ Interestingly, transgenic overexpression¹¹ or gene transfer¹² of Ang1 increases vascularization in vivo. In vitro experiments have shown that Ang1 has specific effects on endothelial cells; it has little effect on proliferation, but it potently induces sprouting,^{13 14} chemotactic response,¹⁵ and network formation.¹⁶ Also, Ang1 is a strong apoptosis survival factor in endothelial cells under serum deprivation.^{16 17 18} Because Ang1 does not have proliferative activity in endothelial cells,^{8 14} we conclude that the antiapoptotic effect of Ang1 is the result of an enhancement of cell survival, not proliferation. However, it is not known how Ang1 affects cell survival. The phosphatidylinositol 3'-kinase (PI 3'-kinase) and Akt pathways are common features in the signal transduction of the antiapoptotic effects of growth factors.¹⁹ Recently, Kontos et al²⁰ demonstrated that Tie2 activates PI 3'-kinase and Akt. They suggested that this receptor/intracellular signaling system might account for endothelial cell survival. However, their findings did not include Ang1 stimulation.

In this study, we examined the receptor/second messenger signal transduction pathway for the antiapoptotic effect of Ang1 on human umbilical vein endothelial cells (HUVECs). We found that the Tie2 receptor and the PI 3'-kinase/Akt signal transduction pathway are crucial elements in the processes leading to endothelial cell survival induced by Ang1.

	Materials and Methods

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Materials, Cell Culture, Transfection, and Induction of Apoptosis
We obtained Ang1*, soluble Tie1 receptor-Fc (rTie1-Fc), and Tie2 receptor-Fc (rTie2-Fc) fusion proteins from Regeneron Pharmaceuticals, Inc. Ang1* is a recombinant version of Ang1 with modified NH₂-terminus and mutated Cys²⁴⁵.⁹ Ang1* is easier to produce and purify. Mutation of the Cys²⁴⁵ in Ang1, which is not shared between Ang1 and Ang2, does not alter its agonistic properties. Biological activity of recombinant Ang1 and Ang1* is similar, confirmed by their high-affinity binding to and stimulation of the Tie2 receptor in vitro.⁹ Recombinant human VEGF₁₆₅ was purchased from R&D systems. Media and sera were purchased from Life Technology, Inc. All other reagents were purchased from Sigma, unless otherwise specified. HUVECs and human umbilical vascular smooth muscle cells (HUVSMCs) were prepared and grown as described previously.¹⁴ Murine endothelial cell line MS1 (CRL-2279, American Type Culture Collection) was grown in the same manner as HUVECs. Mammalian expression vector containing the Lys179Met dominant-negative mutant of Akt (Akt1) cDNA (Upstate Biotechnology, Inc) or its control empty vector (Upstate Biotechnology, Inc) was transfected into MS1 cells using LipofectAMINE Plus (Gibco-BRL) and incubated for 24 hours in M-199 medium with 10% FBS. To induce apoptosis, a serum deprivation method was used as described previously.¹⁷

Determination of Apoptosis
We used 3 methods to determine apoptosis. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assays were performed on the floating and adherent cells according to the manufacturer’s protocol (Oncor). Nuclear staining with Sytox Green (Boehringer Mannheim) was performed. DNA laddering was examined using 1.5% agarose gels containing ethidium bromide. Genomic DNAs were extracted from both floating and adherent endothelial cells using NP-40 lysis.²¹ Quantification of apoptosis was performed as described previously.¹⁷

Phosphorylation Assays of Tie2 Receptor, p85 Subunit of PI 3'-Kinase, and Akt
HUVECs were seeded to 6-well plates or 10-cm dishes at a density of 5x10⁴ cells/cm² and were grown in M-199 with 10% FBS for 24 hours. After 24 hours of serum deprivation, the medium was changed to serum-free M-199 containing wortmannin where indicated. Two hours later, Ang1* was added to the cells at the indicated amounts, and the cells were incubated for the indicated times. A phosphorylation assay of the Tie2 or p85 subunit of PI 3'-kinase was performed with anti-Tie2 antibody (Santa Cruz Biotechnology) or anti-p85 subunit of PI 3'-kinase (Upstate Biotechnology, Inc) according to the method described by Maisonpierre et al⁹ and Hu et al.²² The Akt (Ser473) phosphorylation assay was performed according to the manufacturer’s protocol (New England BioLabs). PI 3'-kinase activity was measured according to the method described by Hu et al.²²

Reverse Transcriptase–Polymerase Chain Reaction (RT-PCR) Analysis
RT-PCR was performed with specific primers for either Ang1 (sense, 5'-GGCAGTACAATGACAGTTTC-3'; antisense, 5'-CTTTGTTGCTTTCATAATCGC-3') or ß-actin (sense, 5'-ATCTGGCACCACACCTTCTACAATGAGCTGCG-3'; antisense, 5'-CGTCATACTCCTGCTTGCTGATCCACATCTGC-3') in total RNA (100 ng) from HUVSMCs or HUVECs as described previously.¹⁴

Detection of Ang1 in Tissue Sections and Culture Medium
Polyclonal anti-Ang1 antibody was produced by immunization of rabbits using standard methods with a recombinant NH₂-terminal portion of Ang1 protein (amino acids, 53 to 246) produced in Escherichia coli. Immunohistochemistry was performed in human umbilical cords, normal human uterine cervix, and pig hearts as described previously.²³ Serum-free defined medium (100 mL) that was on confluent HUVSMCs or HUVECs for 24 hours was incubated with 5 µg of rTie2-Fc for 1 hour at room temperature. Ang1/rTie2-Fc complexes were recovered on protein A–Sepharose beads, and Ang1 was determined by Western blotting with anti-Ang1 antibody.

An expanded Materials and Methods section is available online at http://www.circresaha.org.

	Results

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Ang1* Blocks Apoptosis in Endothelial Cells Through Tie2 Receptor Binding
Serum deprivation caused apoptosis in HUVECs, evidenced by more floating and less adherent cells seen with phase-contrast microscopy (Figure 1A), fragmented DNA detected with TUNEL assay (Figure 1B), condensed or fragmented nuclei in cells stained with Sytox Green (Figure 1C), and increased DNA laddering (Figure 1D). Approximately 30% of total cells undergo apoptosis during serum deprivation at 24 hours after serum deprivation (Figures 1 and 2). The proportion of floating apoptotic cells to total apoptotic cells was 45% to 50%. Similarly to our previous report,¹⁷ Ang1* at 200 ng/mL inhibited 55% to 60% of the apoptotic events (Figure 2). A 5-fold molar excess (2 µg/mL) of rTie2-Fc, but not of rTie1-Fc, completely blocked the antiapoptotic effect of Ang1* (Figure 2). These results indicate that Ang1 exerts its antiapoptotic effect in endothelial cells through Tie2 receptor binding, but not through Tie1.

View larger version (68K): [in this window] [in a new window]   Figure 1. Effect of Ang1* on serum deprivation–induced apoptosis in HUVECs. Ang1* (200 ng/mL) or control buffer was added to cells after changing to serum-free medium. Apoptosis was assayed 24 hours after serum deprivation. A, Phase-contrast microscopy. Note that there are fewer adherent cells and more floating cells in the serum-deprived (SD) culture than in the control with 10% serum. Cells exposed to Ang1* are more adherent than cells in the SD culture. Magnification, x200. B, Light microscopy of TUNEL assay. C, Fluorescence microscopy of Sytox Green staining. Arrowheads or arrows indicate brown or yellow apoptotic cells with fragmented or condensed DNA. Magnification, x400. D, Agarose gel electrophoresis. DNA from SD cells has notable DNA laddering, whereas DNA from control cells (CT) shows no DNA laddering. DNA from cells exposed to Ang1* (SA) shows reduced DNA laddering compared with that of SD cells. MM indicates -HindIII molecular weight marker.

View larger version (41K): [in this window] [in a new window]   Figure 2. Effect of rTie1-Fc or rTie2-Fc on the antiapoptotic effect of Ang1*. Ang1* (200 ng/mL) and a 5-fold molar excess (2 µg/mL) of rTie1-Fc or rTie2-Fc were added to cells in serum-free medium and incubated for 24 hours. Apoptotic cells are quantified as described in Materials and Methods. Data are mean±SD of 8 experiments. Statistical comparisons were performed using 1-way ANOVA followed by the Student-Newman-Keuls test. *P<0.01 vs 10% serum control; #P<0.01 vs serum-deprived control.

Ang1* Induces Phosphorylation of Tie2 and p85 Subunit of PI 3'-Kinase and Increases PI 3'-Kinase Activity in HUVECs
The protein level of the Tie2 receptor in HUVECs decreased slightly at 24 hours after serum deprivation (Figure 3A). Under these conditions, Ang1* induced the phosphorylation of Tie2 (Figure 3B) and the p85 subunit of PI 3'-kinase (Figure 3C) in a dose-dependent manner. Ang1* increased PI 3'-kinase activity in a dose-dependent manner (Figure 3D). Thus, the Ang1*-induced antiapoptotic effect may be mediated through activation of the Tie2 receptor and PI 3'-kinase.

View larger version (45K): [in this window] [in a new window]   Figure 3. Effect of Ang1* on phosphorylation of Tie2 and p85 subunit of PI 3'-kinase, and PI 3'-kinase activity in HUVECs. A, Western blot analysis of the Tie2 protein in 50 µg of total protein from cell lysates at 0, 12, and 24 hours after serum deprivation. B and C, Phosphorylation assays of Tie2 and p85 subunit of PI 3'-kinase, respectively. Cells were incubated for 24 hours in serum-free medium before Ang1* addition. Indicated amounts of Ang1* were added to cells and incubated for 10 minutes. Cell lysates were immunoprecipitated with corresponding antibodies and separated by SDS-PAGE. After Western blotting with antiphosphotyrosine antibody (top), the membranes were stripped and reincubated with corresponding antibodies (bottom) for detection of total Tie2 or p85 protein. D, Autoradiogram of thin-layer chromatography for PI 3'-kinase activity. Cells exposed to Ang1* for 10 minutes generated phosphatidylinositol-3-monophosphate (PIP) in a dose-dependent manner. Results from 3 experiments were similar. E, Densitometric analysis shows the relative ratios of phosphorylated Tie2/total Tie2, phosphorylated p85/total p85, and PIP/origin. The ratio for the control is arbitrarily presented as 1. Data are mean±SE of the ratios from 3 experiments.

Ang1*-Induced Antiapoptotic Effect in HUVECs Is Mediated by PI 3'-Kinase
Two specific inhibitors of PI 3'-kinase, wortmannin and LY294002, almost completely blocked the Ang1*-induced antiapoptotic effect (Figure 4). Both reagents slightly enhanced the degree of apoptosis observed in the absence of Ang1*, possibly because of inhibition of basal PI 3'-kinase activity present in serum-deprived cells. In addition, wortmannin also completely blocked the VEGF₁₆₅-induced antiapoptotic effect (Figure 4). These results suggest that Ang1, like VEGF₁₆₅, exerts its antiapoptotic effect in endothelial cells through a PI 3'-kinase–mediated pathway.

View larger version (47K): [in this window] [in a new window]   Figure 4. Effect of PI 3'-kinase inhibitors on the antiapoptotic effect of Ang1* in HUVECs. Cells were pretreated with control buffer (Cont), wortmannin (WT, 30 nmol/L) or LY294002 (LY, 100 nmol/L) before addition of control buffer, Ang1* (200 ng/mL), or VEGF (20 ng/mL) and incubated in serum-free medium for 24 hours. Data are mean±SD of 5 experiments. Statistical comparisons were performed using 1-way ANOVA followed by the Student-Newman-Keuls test. *P<0.01 vs control.

Akt Activation Is Involved in the Antiapoptotic Effect of Ang1*
To examine whether Akt activation is involved in the antiapoptotic effect of Ang1*, Akt phosphorylation at Ser473 was examined in whole-cell lysates of HUVECs by means of a phosphospecific antibody. In initial time course experiments, Ang1* caused maximal activation of Akt in 20 to 30 minutes through the phosphorylation of Ser473. The response gradually decreased after prolonged incubation (data not shown). Ang1* increased Akt phosphorylation at Ser473 in a dose-dependent manner (Figure 5A). Densitometric analysis of the signals revealed that Akt phosphorylation was 8.8-fold higher in HUVECs treated with 200 ng/mL of Ang1* (Figure 5B). The PI 3'-kinase inhibitor wortmannin completely abolished Akt activation in response to Ang1* (Figure 5A and 5B). It is known that the expression of Lys179Met Akt mutant causes a loss of Akt kinase activity with a dominant-negative effect on endogenous Akt.²⁴ We chose the MS1 cell line because it expresses the Tie2 receptor assessed by Western blot analysis, and we were able to achieve a transfection efficiency of 60%, assessed by immunofluorescent detection of the tagged protein of Lys179Met Akt mutant, c-Myc, with anti–c-Myc antibody (Invitrogen) (data not shown). MS1 endothelial cells transfected with Lys179Met Akt mutant showed an Ang1*-induced antiapoptotic effect, but the degree of antiapoptotic effect was significantly less than that in the cells transfected with control vector (Figure 6). Thus, Ang1*-induced endothelial cell survival is mediated by PI 3'-kinase through Akt phosphorylation at Ser473.

View larger version (46K): [in this window] [in a new window]   Figure 5. Effect of Ang1* on Akt phosphorylation at Ser473 in HUVECs. Cells were incubated for 24 hours in serum-free medium before Ang1* addition. Indicated amounts of Ang1* were added to cells and incubated for 25 minutes in serum-free medium. In lane 200W, wortmannin (30 nmol/L) was added to the cells 2 hours before exposure to Ang1* (200 ng/mL). A, After Western blot analysis with anti–phospho-Akt (Ser473) antibody (top), the membrane was stripped and reincubated with anti-Akt antibody (bottom) for detection of total Akt protein. B, Densitometric analysis of 3 blots, showing the relative ratio of phosphorylated Akt (Ser473)/total Akt. The ratio for the control is arbitrarily presented as 1. Data are mean±SE of ratios from 3 experiments.

View larger version (27K): [in this window] [in a new window]   Figure 6. Effect of Akt inactivation on Ang1*-induced antiapoptotic effect in endothelial cells. A, MS1 cells transfected with Lys179Met dominant-negative mutant of Akt cDNA (DNAkt) or control vector (CV) were incubated with 10% serum or under serum deprivation (SD), and apoptosis was measured at 24 hours in the absence or presence (SD+A) of Ang1* (200 ng/mL). B, Comparison of Ang1*-induced antiapoptotic effect in CV- and DNAkt-transfected cells. Data are drawn from panel A and presented as a percentage inhibition of apoptosis by Ang1*. Data are mean±SD of 5 experiments. Statistical comparisons were performed using the Student t test. *P<0.01 vs serum; #P<0.05 vs SD; P<0.05 vs CV.

Ang1 May Exert Its Antiapoptotic Effect in Endothelial Cells by Paracrine Action
Ang1 mRNA is mainly present in periendothelial cells, including vascular smooth muscle,^{8 10} whereas Tie2 is mainly present in endothelial cells.²⁵ RT-PCR revealed the correctly sized PCR product for Ang1 (1581 bp) in HUVSMCs, but not in HUVECs (Figure 7A, top). As an internal control, an equal amount of each cDNA was assayed by PCR using human ß-actin primers (Figure 7A, bottom). The identity of the PCR products was further confirmed by sequencing (data not shown). Ang1 protein is detected in culture medium from HUVSMCs but not from HUVECs (Figure 7B). Immunohistochemical analysis indicated that Ang1 protein is mainly located in perivascular or muscular, but not endothelial, areas of blood vessels (Figure 8). These results indicate that Ang1 probably exerts its antiapoptotic effect on endothelial cells by paracrine action.

View larger version (40K): [in this window] [in a new window]   Figure 7. Expression and secretion of Ang1. A, RT-PCR analysis of Ang1 mRNA in HUVSMCs and HUVECs. Total cellular RNA (100 ng) from HUVSMCs or HUVECs was reverse-transcribed and PCR-amplified. PCR products for Ang1 (top) or ß-actin (bottom) were separated on 0.8% agarose gels; bands were visualized by ethidium bromide staining. MM, DNA molecular marker (1-kb DNA ladder). B, Detection of Ang1 in the culture medium. Culture medium (100 mL) from HUVSMCs or HUVECs was incubated with 5 µg of rTie2-Fc for 1 hour, and then 10 mg of protein A–Sepharose beads was added. Protein samples were separated by SDS-PAGE and Western blotted with anti-Ang1 antibody. PC, positive control of recombinant Ang1 (10 ng).14 Molecular weight markers shown were used to estimate molecular masses. Results from 3 experiments were similar.

View larger version (135K): [in this window] [in a new window]   Figure 8. Immunohistochemistry of Ang1 in blood vessels. A, Human umbilical cord. Bar=125 µm. B, Human uterine cervix. Bar=50 µm. C, Porcine coronary artery. Bar=25 µm. Arrowheads indicate reddish-brown immunopositive signals.

	Discussion

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Although the significance of Ang1 and its endothelial cell–specific receptor, Tie2, in vasculogenesis is well established, the signal transduction cascades initiated by the binding of Ang1 to the Tie2 receptor have not been characterized. Jones and Dumont²⁶ isolated a novel Tie2 receptor–interacting protein, Dok-R, that is a novel docking protein. Dok-R is the first downstream substrate of the activated Tie2 receptor and is constitutively bound to Crk. This result suggests that Tie2 may have multiple signaling pathways. A recent study indicates that Tie2 activates PI 3'-kinase through an association with the p85 regulatory unit.²⁰ Although these findings have been observed without Ang1 stimulation, this result suggests that Ang1 can activate PI 3'-kinase through Tie2 binding. Our results demonstrate that Ang1 induced phosphorylation of the Tie2 receptor and p85 subunit of PI 3'-kinase and increased the activity of PI 3'-kinase in HUVECs. The Ang1-induced antiapoptotic effect was reversed by soluble Tie2 receptor or PI 3'-kinase inhibitors. Therefore, the signaling pathway between Tie2 and PI 3'-kinase is an essential step for an Ang1-induced antiapoptotic effect in endothelial cells.

The serine-threonine protein kinase Akt²⁷ is a downstream effector of PI 3'-kinase. Activation of PI 3'-kinase increases the intracellular amount of phosphatidylinositol-4,5-biphosphate and phosphatidylinositol-3,4,5-triphosphate, which positively regulate Akt. Thus, Akt is activated by phospholipid binding and phosphorylation within the activation loop at Thr308 and within the COOH terminus at Ser473.²⁸ The PI 3'-kinase and Akt pathways are common features in the signal transduction of the antiapoptotic effects of growth factors.¹⁹ In this study, we demonstrate that Ang1 induces Akt phosphorylation at Ser473, and this induction is PI 3'-kinase dependent. In addition, our findings indicated that inactivation of Akt reversed the Ang1-induced antiapoptotic effect in endothelial cell lines. Thus, Ang1-induced PI 3'-kinase activation and Akt phosphorylation could be crucial steps in the antiapoptotic effect of Ang1 on endothelial cells. Interestingly, VEGF is also a strong survival factor in endothelial cells under serum deprivation, and it also induces PI 3'-kinase activation and Akt phosphorylation.²⁹ Thus, Ang1 and VEGF have a common intracellular second messenger signaling pathway for preventing apoptosis in endothelial cells under serum deprivation. Recently, Akt has been shown to promote cell survival or nitric oxide production through its ability to phosphorylate Bad³⁰ and procaspase-9³¹ or endothelial nitric oxide synthase.^{32 33} Thus, the downstream pathways and processes following from Ang1-induced Akt phosphorylation in endothelial cells will be examined in future studies.

Although Ang1 is mainly synthesized in periendothelial cells, including vascular smooth muscle cells,^{8 10} its receptor, Tie2, is mainly located in the endothelial cells of normal adult vessels in which vasculogenesis or angiogenesis is not occurring.²⁵ Thus, we reach 2 conclusions. First, Ang1 may serve a cell survival function in nonproliferating endothelial cells. Second, Ang1 may be a paracrine factor. Our RT-PCR and immunohistochemical analyses indicate that Ang1 mRNA and protein are present in vascular smooth muscle cells but not in the endothelial cells where it is active. In addition, Ang1 is detected in culture medium from HUVSMCs but not from HUVECs. These facts suggest that Ang1 has a paracrine activity. The constitutive expression of Ang1 in vascular smooth muscle cells suggests that it may be involved in endothelial cell survival. This survival effect may help to maintain endothelial tissue integrity.

In summary, we found a receptor/signal transduction pathway by which Ang1 promotes the survival of endothelial cells. Our results indicate that the Tie2 receptor and the PI 3'-kinase/Akt signal transduction pathway are crucial elements in the processes leading to endothelial cell survival induced by the paracrine activity of Ang1.

	Acknowledgments

 
This work was supported by the Creative Research Initiatives of the Korean Ministry of Science and Technology. We thank Peter C. Maisonpierre and George D. Yancopoulos (Regeneron Phramaceuticals, Inc, Tarrytown, NY) for providing critical angiopoietin and Tie reagents. We thank Jennifer Macke for help in preparing the manuscript.

Received September 14, 1999; accepted October 15, 1999.

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