The FASEB Journal express article 10.1096/fj.01-0556fje. Published online November 29, 2001.
Angiopoietin-1 negatively regulates expression and activity of tissue factor in endothelial cells Injune Kim,* Jong-Lark Oh,* Young Shin Ryu,* June-No So,† William C. Sessa,‡ Kenneth Walsh,§ Gou Young Koh* *National Creative Research Initiatives Center for Endothelial Cells and Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, 790-784, Republic of Korea; †Department of Biotechnology, Woosuk University, Chonju, 560-180, Republic of Korea; ‡ Departments of Pharmacology, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, Connecticutt 06536-0812; §Division of Cardiovascular Research, St. Elizabeth's Medical Center of Boston, Massachusetts 02135, USA. Corresponding author: Gou Young Koh, National Creative Research Initiatives Center for Endothelial Cells, Department of Life Science, Pohang University of Science and Technology, San 31, Hyoja-Dong, Pohang, 790-784, Republic of Korea, E-mail:
[email protected] ABSTRACT Normally, tissue factor (TF) is not expressed on the surface of endothelial cells, but its expression can be induced by vascular endothelial growth factor (VEGF) and tumor necrosis factor (TNF)-α. However, the signaling pathway(s) affecting this induction is unknown. Using human umbilical vein endothelial cells, we found that inhibitors of guanine-cytosine-rich DNA binding protein and nuclear factor (NF)-κB suppressed VEGF- and TNF-α-induced expression and activity of TF. However, unexpectedly, phosphatidylinositol (PI) 3'-kinase inhibitor enhanced the VEGF- and TNF-α-induced expression and activity of TF. Angiopoietin-1 (Ang1), a strong activator of intracellular PI 3'-kinase/Akt, inhibited the induction of TF by VEGF and TNF-α, whereas Ang1 itself did not produce any significant effect on TF. Selective activation (or inactivation) of PI 3'-kinase/Akt by using adenoviral transfer reduced (or enhanced) TNF-αinduced expression of TF mRNA and protein, regardless of Ang1 treatment. From these results, we conclude that Ang1 inhibits the up-regulation of TF expression, possibly through activation of PI 3'-kinase/Akt in endothelial cells. Ang1 may be useful as an inhibitor of VEGF- and TNFα-induced coagulation, inflammation, and cancer progression. Key words: vascular endothelial growth factor • TNF-α • Akt • phosphatidylinositol 3'-kinase
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mong more than 20 known growth factors, vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang1) are the only ones that are endothelial cell-specific (1–3). These two factors have strong effects on angiogenesis and vasculogenesis (1–3). VEGF acts as a potent angiogenic factor, whose specific activities include endothelial cell survival, proliferation, migration, and tube formation (1–4). The biological effects of VEGF on endothelial cells are exerted through its binding to Flt-1 and Flk-1/KDR, two high-affinity tyrosine kinase receptors (1, 4). In the mouse embryo, targeted gene inactivation of one VEGF allele is lethal at day 10.5 or 11.5 as a result of impaired vascular formation (5, 6). In the adult mouse, VEGF is also a primary contributor to physiologic and pathologic angiogenesis through
its strong biological effects on endothelial cells (1, 2, 4, 7). In fact, the VEGF gene and protein are strongly expressed in actively angiogenic areas (7). Moreover, many reports have shown that transgenic overexpression or gene transfer of VEGF increases angiogenesis and vascularization in adult tissues (8–11). Ang1 has been identified as a ligand of the endothelial cell-specific Tie2 receptor (12, 13). In vivo analysis by targeted gene inactivation suggests that Ang1 recruits and sustains periendothelial support cells. It is interesting that transgenic overexpression or gene transfer of Ang1 increases vascularization (11, 14, 15). In vitro experiments have shown that Ang1 has specific effects on endothelial cells: It potently induces chemotactic response (16), network formation (17), sprouting (18, 19), and survival from apoptosis (20), but it does not induce cell proliferation. Thus, VEGF and Ang1 are both involved in vascular development during embryogenesis and certain pathologic conditions. VEGF also acts as a proinflammatory cytokine by inducing adhesion molecules that bind leukocytes to endothelial cells and by increasing endothelial permeability through the Flk1/KDR receptor (21, 22). In fact, the first activity attributed to VEGF was the permeabilization of microvessels, primarily postcapillary venules and small veins (23). Although VEGF overexpressed in mouse skin increases vascularity, the vascularized skin shows signs of inflammation, including vascular leakage due to the permeability activity of VEGF, edema, and increased adhesion of leukocytes (8, 9). Ang1 overexpressed in mouse skin results in a prominent enlargement of vessels without signs of inflammation (9, 13). Moreover, when cooverexpressed in mouse skin, VEGF and Ang1 show an additive effect on angiogenesis but result in leakage-resistant vessels with little inflammation. (9). It appears that Ang1 counteracts at least some component of VEGF activity in endothelial cells during certain pathologic conditions. Furthermore, a recent in vitro experiment demonstrated that Ang1 decreases Eselectin expression and decreases basal and VEGF-induced endothelial permeability, possibly through the regulation of junctional complexes, platelet endothelial cell adhesion molecule-1, and vascular endothelial cadherin (24). However, the exact molecular mechanism by which Ang1 inhibits VEGF-induced vascular permeability and inflammation is unknown. TF is a 47-kDa transmembrane glycoprotein and a member of the cytokine-receptor superfamily. TF acts as a high-affinity receptor and cofactor for plasma factor VII/VIIa, and it is the primary cellular initiator of blood coagulation (25). TF is involved in thrombosis and inflammation associated with sepsis, atherosclerosis, and cancer, and it can participate in other cellular processes, including metastasis, tumor-associated angiogenesis, and embryogenesis (26–31). TF is not normally expressed on the surface of endothelial cells. However, several stimuli, including tumor necrosis factor (TNF)-α, interleukin-1, bacterial lipopolysaccharide, thrombin, and VEGF, induce TF expression (26, 32–35). Thus, VEGF, like TNF-α, can act as a procoagulant cytokine by increasing TF expression in endothelial cells (33–35). Nuclear factor (NF)-κB and AP-1 are major transcriptional factors for TNF-α-induced TF expression in endothelial cells (30, 36). Although the intracellular signaling pathway responsible for VEGF-induced TF expression in endothelial cells has not yet been identified, reporter gene studies have implicated that transcription factors, including EGR-1, Sp1, and nuclear factor of activated T cells, are involved (33, 34). Thus, VEGF has multiple cytokine functions in endothelial cells, and these functions are carried out through multiple intracellular signaling pathways. The other endothelial cell-specific ligand, Ang1, has varied affects on VEGF’s activity in endothelial cells. Therefore, this study seeks to determine the role of Ang1 in VEGF- and TNF-α-induced expression of TF in primary cultured
endothelial cells and to reveal the pathway through which Ang1 carries out this role. Our results indicate that Ang1 counteracts VEGF- and TNF-α-induced expression and activity of TF, possibly through activation of the PI 3'-kinase/Akt pathway. These results suggest that Ang1 may be used as an inhibitor of VEGF- and TNF-α-induced coagulation, inflammation, and tumorassociated angiogenesis and metastasis. MATERIALS AND METHODS Materials and cell culture Recombinant human VEGF165 and TNF-α were purchased from R&D Systems. Ang1*, rTie2Fc, and rTie1-Fc were obtained from Regeneron Pharmaceuticals (Tarrytown, NY). Ang1* is a recombinant version of Ang1 that is easier to produce and purify (37). Ang1* contains a modified NH2-terminus and mutated Cys245. The biological activity of recombinant Ang1 and Ang1* is similar, confirmed by their high-affinity binding to and stimulation of the Tie2 receptor in vitro. PI 3'-kinase inhibitors wortmannin and LY294002 were purchased from RBI. MEK 1/2 inhibitor PD98059 was obtained from New England Biolabs (Beverly, MA). PLC inhibitor U73122 was purchased from Biomol Research Laboratory (Plymouth Meeting, PA). Guaninecytosine- (GC)-rich DNA binding protein inhibitor mithramycin A, NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC), and protein kinase C (PKC) inhibitor chelerythrine chloride were purchased from Sigma (St. Louis, MO). Media and sera were obtained from Life Technology (Rockville, MD). Most other biochemical reagents were purchased from Sigma, unless otherwise specified. All experiments in this study were performed in human umbilical vein endothelial cells (HUVECs). These cells were prepared from human umbilical cords by collagenase digestion and maintained as previously described (19). HUVECs were maintained in M-199 medium supplemented with 20% (vol/vol) fetal bovine serum at 37°C in 5% CO2. The primary cultured cells used in this study were between passage 2 and 3. Enzyme-linked immunoassay and activity of TF Confluent monolayers of HUVECs in 30-mm dishes or 48-well dishes were incubated in phenol red-free M-199 medium supplemented with 1% serum for 24 h, washed, and then stimulated with VEGF, TNF-α, and Ang1 in the same medium for the indicated times. The protein levels of TF in the cell lysates were measured by using an IMUBIND TF ELISA kit according to manufacturer’s instructions (American Diagnostica, Greenwich, CT). The TF activity on the cell surface of HUVECs was assayed by measuring the enzymatic activity of the TF/factor VIIa complex. The cells were washed twice and incubated with Tris buffer (0.05 mol/l Tris and 0.1 mol/l NaCl; pH 7.5) containing bovine serum albumin (5 mg/ml) and 4 mmol/l CaCl2, which was followed by the addition of human factor VII. After incubation at 37°C for 30 min, the chromogenic substrate for TF/factor VIIa complex, SpectrozymefXa (American Diagnostica) was added. The reaction was incubated at 22°C for 30 min. Glacial acetic acid was added to stop the reaction. The absorbance was read at 405 nm and compared with those values obtained from a standard curve generated using known amounts of lapidated TF. To confirm the specificity of the process for TF, HUVECs were preincubated for 30 min with anti-human TF monoclonal antibody (10 µg/ml, catalog no. 4509, American Diagnostica).
Measurement of procoagulant activity of TF by one-stage clotting assay One-stage clotting assays were performed according to the method described by Nawroth et al. (38). After they were washed, cells were removed nonenzymatically by scraping in barbital buffer (pH 7.6), collected by centrifugation for 5 min at 1000 rpm, and resuspended in 100 µl of sample buffer (0.01 M HEPES, 0.015 M CaCl2, 0.01% Tween 20, and 0.0005% polybrene; pH 7.3). After adding 100 µl of citrated bovine plasma to the samples, they were incubated at 37°C. The time to the first defined fibrin strand was measured. Procoagulant activity was calculated by comparing the measured clotting time with a standard curve made with known amounts of TF. Flow cytometry analysis HUVECs were stimulated with VEGF, TNF-α, and Ang1 for the indicated times. Then, cells were washed twice with cold phosphate-buffered saline (PBS), removed by careful trypsinization, and washed again with Ca2+/Mg2+-free PBS before incubating with 20% FBS for 30 min. Following two washes, cells were incubated with a FITC-conjugated antibody against human TF (American Diagnostica) for 1 h at 4°C. Cells were then fixed with 2% paraformaldehyde and analyzed by flow cytometry in a FACS cytofluorometer (Becton Dickinson, Franklin Lakes, NJ). The results were gated for mean fluorescence intensity above the fluorescence produced by control FITC-conjugated anti-IgG1 (Becton Dickinson) antibody alone. RNase protection assay (RPA) The partial cDNA of human TF (nucleotides 638–858, GenBank accession J02931) was amplified by polymerase chain reaction and subcloned into pBluescript II KS+ (Stratagene, La Jolla, CA). After linearizing with EcoRI, 32P-labeled antisense RNA probes were synthesized by in vitro transcription, using T7 polymerase (Maxiscript kit, Ambion, Austin, TX) and gel purified. RPA was performed on total RNAs by using the Ambion RPA kit. An antisense RNA probe of human cyclophilin (nucleotides 135–239, GenBank accession X52856) was used as an internal control for RNA quantification. Akt phosphorylation assay Following treatment with VEGF, TNF-α, and Ang1, HUVECs were washed two times with PBS, dissolved in sample buffer, boiled, separated by SDS-PAGE, and transferred to nitrocellulose membrane. The membranes were treated and analyzed according to the manufacturer's protocol (New England BioLabs) for detection of Akt Ser473 phosphorylation levels. Adenoviral gene transfer Adenoviral vectors encoding a constitutively active mutant of PI 3'-kinase (Ade-myr-p110) and a mutant regulatory subunit of PI 3'-kinase that lacks the binding site for the catalytic subunit (Ade-∆p85) were obtained from Dr. Ogawa at Kobe University School of Medicine, Japan (39). Adenoviral vectors encoding β-galactosidase (Ade-β-gal), hemagglutinin (HA)-tagged inactive phosphorylation mutant Akt (Ade-Akt-AA), and carboxyl-terminal HA-tagged constitutively active Akt (Ade-myr-Akt) were generated as described previously (40). HUVECs were infected at a 100 multiplicity of infection with Ade-β-gal, Ade-∆p85, Ade-myr-p110, Ade-Akt-AA, or Ade-myr-Akt for 12 h. The virus was removed, and cells were left to recover for 12 h in
complete medium. These conditions resulted in uniform expression of the transgenes in nearly 100% of the cells (determined by infection with β-gal followed by staining for β-gal activity) and equal expression of Akt proteins, based on Western blotting (data not shown). Densitometric analyses and statistics All signals were visualized and analyzed by densitometric scanning (LAS-1000 for Western blotting, using an electrochemical luminescence system and BAS-2000 for RPA, Fuji Film, Tokyo). Data are expressed as mean ± SD. Statistical significance was tested using one-way ANOVA followed by the Student-Newman-Keuls test. Statistical significance was set at P
