Angiogenic Actions of Angiopoietin-1 Require Endothelium-Derived Nitric Oxide
2003; Elsevier BV; Volume: 162; Issue: 6 Linguagem: Inglês
10.1016/s0002-9440(10)64326-x
ISSN1525-2191
AutoresSaeid Babaei, Krystyna Teichert-Kuliszewska, Qiuwang Zhang, Nina Jones, Daniel Dumont, Duncan J. Stewart,
Tópico(s)Coronary Interventions and Diagnostics
ResumoAngiopoietin1 (Ang1) is a novel angiogenic factor with important actions on endothelial cell (EC) differentiation and vascular maturation. Ang1 has been shown to prevent EC apoptosis through activation of PI3-kinase/Akt, a pathway that is also known to activate endothelium nitric oxide synthase (eNOS). Therefore, we hypothesized that the angiogenic effects of Ang1 would also be dependent on the PI3-kinase/Akt pathway, possibly mediated by increased eNOS activity and NO release. Treatment of human umbilical vein endothelial cells with recombinant Ang1* (300 ng/ml) for 15 minutes resulted in PI3-kinase-dependent Akt phosphorylation, comparable to that observed with vascular endothelial growth factor (VEGF) (50 ng/ml), and increased NO production in a PI3-kinase/Akt-dependent manner. Capillary-like tube formation induced by Ang1* in fibrin matrix at 24 hours (differentiation index, DI: 13.74 ± 0.76 versus control 1.71 ± 0.31) was abolished in the presence of the selective PI3-kinase inhibitor, LY294002 (50 μmol/L) (DI: 0.31 ± 0.31, P < 0.01) or the NOS inhibitor, L-NAME (3 mmol/L) (DI: 4.10 ± 0.59, P < 0.01). In subcutaneous Matrigel implants in vivo, addition of recombinant Ang1* or wild-type Ang1 from conditioned media of COS-1 cells transfected with a pFLAG Ang1 expression vector, induced significant neovascularization to a degree similar to VEGF. Finally, angiogenesis in vivo in response to both Ang1 and VEGF was significantly reduced in eNOS-deficient compared with wild-type mice. In summary, our results demonstrate for the first time that endothelial-derived NO is required for Ang1-induced angiogenesis, and that the PI3-kinase signaling mediates the activation of eNOS and NO release in response to Ang1. Angiopoietin1 (Ang1) is a novel angiogenic factor with important actions on endothelial cell (EC) differentiation and vascular maturation. Ang1 has been shown to prevent EC apoptosis through activation of PI3-kinase/Akt, a pathway that is also known to activate endothelium nitric oxide synthase (eNOS). Therefore, we hypothesized that the angiogenic effects of Ang1 would also be dependent on the PI3-kinase/Akt pathway, possibly mediated by increased eNOS activity and NO release. Treatment of human umbilical vein endothelial cells with recombinant Ang1* (300 ng/ml) for 15 minutes resulted in PI3-kinase-dependent Akt phosphorylation, comparable to that observed with vascular endothelial growth factor (VEGF) (50 ng/ml), and increased NO production in a PI3-kinase/Akt-dependent manner. Capillary-like tube formation induced by Ang1* in fibrin matrix at 24 hours (differentiation index, DI: 13.74 ± 0.76 versus control 1.71 ± 0.31) was abolished in the presence of the selective PI3-kinase inhibitor, LY294002 (50 μmol/L) (DI: 0.31 ± 0.31, P < 0.01) or the NOS inhibitor, L-NAME (3 mmol/L) (DI: 4.10 ± 0.59, P < 0.01). In subcutaneous Matrigel implants in vivo, addition of recombinant Ang1* or wild-type Ang1 from conditioned media of COS-1 cells transfected with a pFLAG Ang1 expression vector, induced significant neovascularization to a degree similar to VEGF. Finally, angiogenesis in vivo in response to both Ang1 and VEGF was significantly reduced in eNOS-deficient compared with wild-type mice. In summary, our results demonstrate for the first time that endothelial-derived NO is required for Ang1-induced angiogenesis, and that the PI3-kinase signaling mediates the activation of eNOS and NO release in response to Ang1. Angiopoietin-1 (Ang1) has recently been identified as a ligand of the endothelial selective receptor tyrosine kinase (RTK), Tie2.1Davis S Aldrich TH Jones PF Acheson A Compton DL Jain V Ryan TE Bruno J Radziejewski C Maisonpierre PC Yancopoulos GD Isolation of angiopoietin-1, a ligand for the TIE2 receptor, by secretion-trap expression cloning.Cell. 1996; 87: 1161-1169Abstract Full Text Full Text PDF PubMed Scopus (1698) Google Scholar Tie2 signaling has been shown to be required for later stages of embryonic blood vessel development,2Puri MC Rossant J Alitalo K Bernstein A Partanen J The receptor tyrosine kinase TIE is required for integrity and survival of vascular endothelial cells.EMBO J. 1995; 14: 5884-5891Crossref PubMed Scopus (422) Google Scholar, 3Dumont DJ Gradwohl G Fong GH Puri MC Gertsenstein M Auerbach A Breitman ML Dominant-negative and targeted null mutations in the endothelial receptor tyrosine kinase, tek, reveal a critical role in vasculogenesis of the embryo.Genes Dev. 1994; 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20: 5919-5928Crossref PubMed Scopus (83) Google Scholar mediates EC migration.15Jones N Master Z Jones J Bouchard D Gunji Y Sasaki H Daly R Alitalo K Dumont DJ Identification of Tek/Tie2 binding partners: binding to a multifunctional docking site mediates cell survival and migration.J Biol Chem. 1999; 274: 30896-30905Crossref PubMed Scopus (185) Google Scholar In addition, the p85 subunit of phosphatidylinositol (PI) 3-kinase can interact specifically with the phosphorylated Tie2 through its SH2 domain. 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We now report that Ang1 induced the activation of Akt and increased NO release in cultured ECs by a PI3-kinase-dependent mechanism. Moreover, Ang1-induced capillary-like tube formation in three-dimensional fibrin matrices in vitro and neovascularization of Matrigel implants in response to Ang1 in vivo were dependent on endothelium-derived NO. Human Ang1* was kindly provided by Regeneron Pharmaceuticals, Inc. (Tarrytown, NY). Ang1* is a genetically engineered variant of naturally occurring Ang1 that retains similar properties in all assays. In Ang1*, the nonconserved cysteine at residue 245 has been mutated to the corresponding serine residue of Ang2, and the first 77 amino acids of human Ang1 have been replaced with the first 73 residues of Ang2.1Davis S Aldrich TH Jones PF Acheson A Compton DL Jain V Ryan TE Bruno J Radziejewski C Maisonpierre PC Yancopoulos GD Isolation of angiopoietin-1, a ligand for the TIE2 receptor, by secretion-trap expression cloning.Cell. 1996; 87: 1161-1169Abstract Full Text Full Text PDF PubMed Scopus (1698) Google Scholar The recombinant Ang1* protein was prepared in buffer containing 0.05 mol/L Tris-HCl pH 7.5, 150 mmol/L NaCl and 0.05% CHAPS. Native human Ang1 and VEGF165 were obtained from R&D Systems (Minneapolis, MN). Other sources of materials are indicated as mentioned. Human umbilical vein endothelial cells (HUVEC) and monkey kidney (COS-1) cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA). HUVECs were maintained in culture in Ham's 12 medium (Invitrogen/Gibco, Burlington, ON) supplemented with 15% fetal bovine serum (FBS), penicillin (500 U/ml), streptomycin (50 μg/ml) and heparin (100 μg/ml), (all from Invitrogen/Gibco), and EC growth factor (ECGF 20 μg/ml; Roche Diagnostics, Mannheim, Germany) and equilibrated with 95% air and 5% CO2 at 37°C. Cells between passages 13 and 18 were used in these experiments. COS-1 cells were grown in Dulbrecco's modified Eagle's medium (DMEM) supplemented with 10% FBS and antibiotics as indicated above. Plasmid (pFLAG-Ang1 kindly provided by Dr. Injune Kim, University of South Korea), encoding the human Ang1, fused with a c-Myc tag at the C terminus, was expressed in COS-1 cell line. Transient transfection was performed using Superfect reagent (Qiagen GmbH, Hilden, Germany) according to the manufacturer's instructions. Twenty hours after transfection, cells were incubated in serum-free DMEM for another 24 hours. The conditioned medium (CM) was harvested and concentrated 100× using Amicon Centricon 10-kd cutoff columns (Millipore Corp., Bedford, MA). Male C57 (WT) and eNOS KO mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Mice were housed in filter-topped cages, maintained with a day/night cycle of 12 hours under pathogen-free conditions, fed a standard diet of rodent chow, and given water ad libitum until they reached 6 to 8 weeks of age. All animal use was approved by and adhered closely to the guidelines set out by the Animal Care and Use Committee, St. Michael's Hospital. Endotoxin- and plasminogen-free human fibrinogen (10 mg/ml, Calbiochem-Novabiochem Corp., La Jolla, CA) was prepared as previously described.27Babaei S Teichert-Kuliszewska K Monge JC Mohamed F Bendeck MP Stewart DJ Role of nitric oxide in the angiogenic response in vitro to basic fibroblast growth factor.Circ Res. 1998; 82: 1007-1015Crossref PubMed Scopus (180) Google Scholar After polymerization, gels were soaked in cultured medium containing 15% FBS for 2 hours at 37°C to inactivate the thrombin. EC were plated on the surface of the three-dimensional matrix and culture for 24 hours in the presence or absence of study agents as described above. HUVECs were cultured on fibrin-matrix, pretreated with NG-nitro-l-arginine methyl ester (L-NAME, 3 mmol/L; 1 hour) or with LY294002 (50 μmol/L; 2 hours) before exposure to recombinant Ang1* (300 ng/ml). After 24 hours, total length of capillary-like structures >30 μm was derived using an Olympus BX50 inverted microscope (100×) for each of six randomly preselected fields. At the same time, the total area of residual EC monolayer was determined for the same fields, and differentiation index (DI) was calculated as the ratio of total tube length over cell area for each field. Images were taken using a digitized Sony CCD-IRIS/RGB camera (Cohu Inc., Japan) and analyzed by a computer-assisted morphometric analysis system (C-Imaging, Compix Inc., Cranberry Township, PA) by observers blinded to the experimental conditions. CM was collected from pFLAG (mock)-transfected or pFLAG-Ang1 transfected COS-1 cells. Ten microliters of 100× concentrated CM was subjected to SDS-PAGE, and proteins were transferred onto nitrocellulose membrane and probed with an anti c-Myc monoclonal antibody (1:5000 dilution according to the supplier's instruction; Invitrogen, Carlsbad, CA). For Tie2 phosphorylation studies, HUVECs were maintained overnight in F12 medium with 1% FBS, and stimulated for 10 minutes in serum-free medium with Ang1* protein (300 ng/ml), mock-CM or concentrated Ang1-CM (200 μl CM/ml). Cells were solubilized with radio immuno-precipitation assay (RIPA) lysis buffer (1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 20 mmol/L Tris, pH 7.6, 50 mmol/L sodium fluoride, 150 mmol/L sodium chloride, 1 mmol/L EDTA, 5 mmol/L benzamidine, 1 mmol/L sodium orthovanadate, 10 μg/ml aprotinin, 1 mmol/L phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, and 1 μg/ml pepstatin). The lysates were immunoprecipitated with anti-Tie2 antibodies (C-20, Santa Cruz Biotechnology, Santa Cruz, CA). Immunocomplexes were recovered on Protein G-Sepharose and separated by SDS-PAGE. Proteins were transferred onto nitrocellulose membrane, blocked with 5% bovine serum albumen, 1X TBS, 0.1% Tween-20 for 2 hours, and probed with anti-phosphotyrosine antibodies 4G10 (1:2500, Upstate Biotechnology, Inc., Lake Placid, NY), stripped and re-probed with anti-Tie2 (1:1000). Specific bands were visualized using the enhanced chemiluminescence (ECL) system (Amersham Pharmacia Biotech). Densitometry was performed by scanning the immunoblots (imaging densitometer; BioRad, Hercules, CA) and the intensity of each band was analyzed using the Molecular Analyst software (BioRad). HUVEC were grown until confluence, serum-starved for 12 hours in F12 medium containing 1% FBS, and then the medium was changed to serum-free F12 containing the inhibitors of PI3-kinase, wortmannin (100 nmol/L) or LY294002 (50 μmol/L) (both from Sigma, St. Louis, MO), or the selective Akt inhibitor, 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate (20 to 75 μmol/L; Calbiochem-Novabiochem). After a 2-hour pretreatment with inhibitors, the cells were stimulated with Ang1* (100 to 600 ng/ml, and in some cases native Ang1 for comparison) for 15 to 30 minutes before lysis with 200 μl of SDS sample buffer (10 mmol/L Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 0.4% dithiothreitol, 1 mmol/L orthovanadate). Proteins (50 μg per lane) were separated on SDS-PAGE and transferred to nitrocellulose membrane. Membranes were blotted using antibodies specific to phospho-Akt (Ser473, 1:1000; New England Biolabs, Beverly, MA), or phospho-eNOS (Ser1177, 1:1000; Cell Signaling Technology, New England Biolabs) and developed by ECL as described above. The blots were then stripped and reprobed with Akt (1:1000, New England Biolabs), eNOS (1:2500, Transduction Laboratories, Lexington, KY), or Tie2 antibodies (1:1000, Santa Cruz) and the bands were scanned and analyzed by densitometry as described above. The measurement of NO production in ECs were performed using the amino-700 NO sensor (Innovative Instruments, Inc., Tampa, FL) which is 100 times more sensitive than the Griess reagent (detection limit below 1 nmol/L). Cells were treated as described above and 50 μl of cell culture media was injected into an acid/iodide solution to reduce NO2 stoichiometrically to NO, detected by sensor. The exchange of electrons between NO and the electrode surface resulted in an electrical current, which was monitored and recorded. The analyzer was calibrated on the day of the experiment with nitrite (NO2) standards, and the results were normalized to the cell number in the plate. NO production was confirmed using the Griess reaction as previously described.27Babaei S Teichert-Kuliszewska K Monge JC Mohamed F Bendeck MP Stewart DJ Role of nitric oxide in the angiogenic response in vitro to basic fibroblast growth factor.Circ Res. 1998; 82: 1007-1015Crossref PubMed Scopus (180) Google Scholar Matrigel (0.5 ml, Collaborative Biomedical Products) containing Ang1* (300 ng/ml), VEGF (100 or 300 ng/ml, as indicated), or PBS was injected subcutaneously at either side of the abdominal midline of WT or eNOS KO mice (n = 12/group). For the CM experiments, 100 μl of concentrated CM (100×) were mixed with 400 μl of Matrigel and injected subcutaneously into the animals. On day 10, Matrigel plugs were harvested and subjected to histological analysis or determination of blood content (see below). For histology, the Matrigel plugs were fixed in 10% buffered formalin for 24 hours and paraffin blocks were cut to 5-μm sections and stained with hematoxylin and eosin (H&E). Adjacent sections from each group of animals were also collected for immunohistochemistry using vWF polyclonal rabbit anti-human antibody (1:250 dilution, Dako Corp., Santa Barbara, CA) for 1 hour at 37°C and subsequently treated with goat anti-rabbit antibody (1:300 dilution; Vector Laboratories, Burlingame, CA) for 1 hour at room temperature and then treated with streptavidin-biotin-peroxidase complex (Vectastain ABC kit; Vector Laboratories) for 30 minutes at room temperature. Diaminobenzadine was used as the peroxidase substrate and hematoxylin as the nuclear counterstain. Negative controls were prepared by substituting preimmune rabbit serum for the primary antibody. The sections were stained for vWF and the images were captured under 20× objective with the use of a Cool SNAP camera (Photometrics, Tucson, AZ) connected to an inverted microscope. On average, 6 to 8 fields were taken from each section. Results were expressed as mean number of vWF-positive vessels per mm2. Fluorescein isothiocyanate-dextran (FITC-dextran; molecular weight, 200-kd) was used as an index of functionality of the Matrigel neovessel.28Klement G Baruchel S Rak J Man S Clark K Hicklin DJ Bohlen P Kerbel RS Continuous low-dose therapy with vinblastine and VEGF receptor-2 antibody induces sustained tumor regression without overt toxicity.J Clin Invest. 2000; 105: R15-R24Crossref PubMed Scopus (1084) Google Scholar FITC-dextran was prepared fresh (25 mg/ml in PBS) and 0.02 ml was injected into the lateral tail vein of each mouse 10 days after the Matrigel injection, and then the mice were sacrificed by cervical dislocation after 20 minutes, blood samples were collected by cardiac puncture into heparinized tubes, centrifuged immediately, and plasma was separated and protected from light at 4°C. The matrix plugs were then promptly removed and placed into tubes containing 1 ml of dispase (1:10 dilution in PBS), and incubated overnight, in the dark, in a 37°C shaker. The following day, the plugs were homogenized using a polytron and centrifuged at 2000 × g for 15 minutes, and the supernatant was saved and kept in the dark until fluorescence analysis. Fluorescence readings were obtained on FL600 fluorescence plate reader using a standard curve created by serial dilution of FITC-dextran used for injection. Angiogenic response was calculated as a ratio of Matrigel plug/plasma fluorescence and presented as fluorescent unit (FU). One-way analysis of variance was used to determine the significance of differences between groups, where appropriate, with post hoc Student's t-test for unpaired observations and Bonferroni correction for multiple comparisons. Summary data are presented as means ± SEM or as percentage of control of the indicated numbers of observations. Statistical significance is defined as P < 0.05. The addition of Ang1* to HUVEC cultured on 3-D fibrin matrices resulted in the formation of extensive capillary-like networks at 24 hours (Figure 1A, panel c), as has previously been reported.9Teichert-Kuliszewska K Maisonpierre PC Jones N Campbell AI Master Z Bendeck MP Alitalo K Dumont DJ Yancopoulos GD Stewart DJ Biological action of angiopoietin-2 in a fibrin matrix model of angiogenesis is associated with activation of Tie2.Cardiovasc Res. 2001; 49: 659-670Crossref PubMed Scopus (245) Google Scholar Addition of the nonselective inhibitor of NOS, L-NAME, nearly completely inhibited this effect of Ang1* in cultured ECs (Figure 1A, panel d). Figure 1B shows summary data for six separate experiments. The addition of L-NAME markedly reduced differentiation index (DI) in Ang1*-treated wells to values not significantly different from control. Stimulation of HUVECs with Ang1* (300 ng/ml) or Ang1-CM for 10 minutes induced a marked increase in Tie2 phosphorylation (Figure 2A, left). This was associated with Akt phosphorylation at Ser473 (Figure 2B), which was nearly completely blocked by two specific inhibitors of PI3-kinase, wortmannin and LY294002. A 70-kd single band was observed in Ang1-CM but not in mock-CM subjected to Western blot analysis using anti-c-Myc antibody, consistent with the expression of the Ang1 c-Myc fusion protein (Figure 2A, right). Densitometric analysis of the Western blot bands showed that Ang1*-stimulated eNOS phosphorylation in a dose-dependent manner (100 ng/ml: 2.96 ± 0.18, 300 ng/ml: 3.27 ± 0.24* and 600 ng/ml: 3.74 ± 0.37**; *P < 0.05, **P < 0.01 versus PBS: 1.63 ± 0.27) with a maximal response similar to that of native Ang1 (600 ng/ml: 4.39 ± 0.60, P < 0.01). Based on these experiments the 300 ng/ml concentration was used for the in vivo studies. Next we tested the upstream pathways that were involved in eNOS phosphorylation by using selective inhibitors of Akt and PI3-kinase (Figure 3). Addition of Akt inhibitor (20 to 75 μmol/L) dose-dependently inhibited eNOS phosphorylation in response to Ang1* (Figure 3A); however, at 75 μmol/L total eNOS expression was also reduced, possibly due to cytotoxicity. Therefore, in further experiments a concentration of 50 μmol/L was used. Inhibition of Akt (50 μmol/L) reduced eNOS phosphorylation in response to Ang1* to a similar degree as the inhibition of PI3-kinase (Figure 3B). Capillary-like tube formation in the fibrin matrix model in response to Ang1* was completely inhibited by the PI3-kinase inhibitor, LY 294002 (50 μmol/L) (Figure 4C). To determine whether the effect of Ang1* was associated with an increase in the release of NO, HUVECs were stimulated with Ang1* for up to 24 hours and the medium was collected for measurement of NO. As shown in Figure 5, treatment of HUVECs with Ang1* resulted in an increase in NO production, which was apparent after only 30 minutes of exposure and was sustained for up to 24 hours. Increase in NO release after 24 hours of incubation with Ang1* was confirmed using the Griess reagent (data not shown). The ability of Ang1* to stimulate NO release was abrogated by pretreatment with the PI3-kinase inhibitor LY294002 (Figure 5), which itself had no effect on basal NO levels (data not shown).Figure 5Ang1* activates NO production and release. HUVEC CM was collected after incubation with Ang1* (300 ng/ml) for the periods indicated, and assayed for NO production, as described in the Methods section. Results are expressed as the mean ± SEM of three independent experiments (five experiments for the 24-hour time point). *P < 0.05 versus PBS, ¶P < 0.05 versus Ang1*.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To assess the role of Ang1 on angiogenesis in vivo, we studied neovascularization of Matrigel implants supplemented with Ang1* (300 ng/ml) or VEGF (100 ng/ml). Both of the angiogenic factors induced neovascularization of the Matrigel plugs within 10 days (Figure 6,b and c), while only minimal angiogenesis was observed in the control plugs (Figure 6a). To determine the effect of wild-type Ang1, we added 100 μl of either concentrated CM from pFLAG-Ang1 transfected cells (Ang1-CM) or CM from mock-vector transfected cells (mock-CM) to the Matrigel (PBS served as a negative control). In WT mice, Ang1-CM resulted in greater neovascularization of the Matrigel implants compared to plugs containing mock-CM or PBS alone (Figure 7A). Moreover, the response to Ang1-CM was not different from that observed with the purified recombinant Ang1* or VEGF protein (both 300 ng/ml). Similar results were seen when FITC-dextran perfusion was used as an index of Matrigel blood content (Figure 7B); however, in this case VEGF produced substantially greater increases in fluorescent signals than Ang1* or Ang1-CM, possibly due to differential effects on vascular permeability which may have resulted in greater extravasations of labeled dextran in the VEGF-treated inserts. To test whether the combination of these two factors would have additive effects, we measured the blood volume in the Matrigel plugs using FITC-dextran conten
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