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Departments of 1Pathology and 2Medicine, Vanderbilt University Medical Center, Nashville, Tennessee 37232-2650
Submitted 15 July 2003 ; accepted in final form 3 October 2003
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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phosphorylated endothelial nitric oxide synthase; Tie-2 receptor; neovascularization; signal transduction
The PI3K-Akt pathway is also known to increase nitric oxide (NO) production by direct phosphorylation of endothelial NO synthase (eNOS) (11, 15, 31). NO is highly regulated by various stimuli and growth factors, such as basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) (1, 3), and has been considered a downstream mediator of VEGF and other angiogenic factors. Previous work has established the involvement of PI3K-Akt-NO signaling in VEGF, bFGF, transforming growth factor (TGF)-
-, ephrin-B4-, and sphingosine-1-phosphate-induced angiogenesis (1, 33, 35, 37, 42). In coronary arteries, Ang-1 has been shown to protect endothelial cells against oxidized low-density lipoprotein-induced injury via a PI3K-Akt-dependent mechanism (25). More recently, Babaei and colleagues (2) demonstrated, using human umbilical vein endothelial cells, that Ang-1-induced angiogenesis is NO dependent. To date, little is known about the functional significance and molecular mechanisms of NO in Ang-1-induced angiogenesis in porcine coronary artery endothelial cells (PCAEC).
In vitro and in vivo evidence suggests that heat shock protein (HSP) 90 plays an important role in the regulation of eNOS and NO production. In bovine aortic endothelial cells, exposure to VEGF increases HSP90 binding to eNOS, eNOS activity, and NO production (16). Our previous data in PCAEC revealed that hypoxic stimulation of eNOS activity and NO production involved an increase in eNOS-associated HSP90 (8). Moreover, an HSP90-eNOS interaction has been considered a key target to modulate NO-dependent angiogenesis (5). However, the role of HSP90-eNOS interaction in Ang-1-induced angiogenesis has not been examined.
The present study uses PCAEC to explore the possible molecular mechanisms of Ang-1-stimulated NO production and the role of NO on Ang-1-induced angiogenesis. Our results demonstrate that Ang-1 stimulates eNOS phosphorylation and increases NO production via stimulation of HSP90 binding to eNOS and activation of the PI3K-Akt pathway. We also demonstrate that PI3K-Akt and HSP90-eNOS interactions play a pivotal role in Ang-1-induced PCAEC migration, tube formation, and sprouting.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Coronary arteries were isolated from normal pig hearts as previously described (23). Briefly, the coronary arteries were cleaned of connective tissues, and endothelial cells were carefully removed from the luminal surface of the coronary arteries with a sterile cotton bud. Cells attached to the cotton were agitated in growth medium and cultured at 37°C in a humidified 95% air-5% CO2 incubator. Single colonies of cells were subcultured in 10% fetal bovine serum (FBS) in endothelial basal growth medium (EGM, Clonetics). Each of the endothelial lines had a typical cobblestone morphology, showed uptake of acetylated low-density lipoprotein, and exhibited factor VIII-related antigen. Primary cultures of PCAEC between passages 5 and 10 were used in all experiments.
Experimental Protocols
PCAEC were pretreated for 30 min with one of the following pharmacological inhibitors: 1 µg/ml geldanamycin (GA; HSP90 inhibitor; Sigma Chemical, St. Louis, MO), 500 nM wortmannin (Wort; PI3K inhibitor; Sigma Chemical), and 2.5 mM N
-nitrio-L-arginine methyl ester [L-NAME; NO synthase (NOS) inhibitor; Sigma Chemical]. All time course and pharmacological interventions were carried out in 0.4% FBS in MEM. All pharmacological interventions were dissolved and diluted in 0.4% FBS in MEM. After 30 min of pretreatment with each of the interventions, 250 ng/ml Ang-1 (recombinant human Ang-1, R&D System) was added, and the cells were incubated for periods between 5 and 120 min. All pharmacological interventions at the concentrations used in these experiments had no obvious toxicity over the times studied.
Western Blot Analysis
PCAECs were lysed in 300 µl of lysis buffer (50 mM Tris, pH 7.4, 1% Triton X-100, 0.25% Na deoxycholate, 150 mM NaCl, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM NaF, 10 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 mM orthovanadate). Twenty-five or 50 µg of proteins were subjected to SDS-PAGE on 12% polyacryl-amide gels and transferred to nitrocellulose membranes. Immunoblotting was performed with anti-phospho-eNOS (1:1,000; Cell Signaling Technology), mouse anti-eNOS (1:2,500; BD Transduction Laboratories), mouse anti-HSP90 (1:1,000; BD Transduction Laboratories), or rabbit anti-phospho-Akt antibody (1:1,000; Santa Cruz Biotech) for 1 h at room temperature. The membrane was then washed and incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (1:4,000 for eNOS, 1:2,000 for HSP90; BD Transduction Laboratories) or horseradish peroxidase-conjugated anti-rabbit IgG (1:2,000 dilution for p-Akt and p-eNOS; Promega). Total level of Akt was detected by using anti-Akt (1:1,000; Cell Signaling Technology). The membranes were developed by using the Western blot chemiluminescence detection reagent (Perkin-Elmer, Life Science Products), and densitometric analysis was carried out by using image acquisition and analysis software (LabWorks, UVP, Upland, CA).
Immunoprecipitation of eNOS and Blotting with HSP90
Endothelial cells were washed and incubated in lysis buffer containing 20 mM Tris·HCl (pH, 7.4), 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, 1 mM phenylmethylsulfonylfluoride, 1 mM NaF, 10 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 mM orthovanadate for 30 min on ice and briefly sonicated. Immunoprecipitation was carried out by incubating the cell lysates (200 µg) with eNOS monoclonal antibody (2 µg/mg of total cell protein) for 16 h at 4°C, followed by a 2-h incubation with a 1:1 protein A-to-protein G ratio sepharose slurry. After centrifugation, the immunoprecipitates were washed in lysis buffer, resuspended in loading buffer, boiled for 5 min, subjected to SDS-PAGE on 12% polyacrylamide gels, and transferred to a nitrocellulose membrane. The primary antibody used for immunoblotting was anti-HSP90 (1:1,000, BD Transduction Laboratories). The membrane was then washed and incubated with secondary antibody. The membrane was then developed by using the Western blot chemiluminescence detection reagent (Perkin-Elmer, Life Science Products). The density of the band was measured by using image acquisition and analysis Software (LabWorks, UVP).
Measurement of NO Release
NO release was measured by using a specific chemiluminescence method and a NO analyzer (NOA 280i, Sievers, Boulder, CO) as previously described (7). Briefly, the sample was injected into a reflux chamber that contained saturated vanadium chloride in 1 N HCl. The mixture was refluxed at 55°C while being continuously flushed with nitrogen gas, which in turn was transferred to the NO analyzer, where NO reacted with ozone to produce light. Light was measured by a photomultiplier, converted into voltage, and recorded on a standard flatbed recorder. A standard curve of NO was determined by using known concentrations of sodium nitrite, and the sample NO concentrations (NOx) were calculated by using the standard curve. Each assay was carried out in duplicate.
Cell Migration Assay
Migration assays were performed as previously described (45). Briefly, polycarbonate filter wells (Transwell, Costar, 8-µm pores, 6.5-mm diameter) were coated with 10 µg/ml rat tail type I collagen (BD Biosciences). PCAEC were plated (1 x 104 cells in a volume of 200 µl) in the upper chamber of the filter well with or without each pharmacological intervention. The bottom chamber was filled with 600 µl of 0.4% MEM containing 250 ng/ml Ang-1 with or without each intervention. The cells were then allowed to migrate for 4 h at 37°C. Cells were fixed in 10% formalin (Fisher) for 20 min, rinsed in PBS, stained with 0.2% Crystal violet dye (Fisher) for 30 min, and mounted in Cytoseal-60 (Richard-Allan Scientific, Kalamazoo, MI). Cells adhering to the top of the transwell filter were removed with a cotton swab. Only those cells that migrated completely to the lower surface of the filter insert were counted. Ten randomly selected fields were counted on each filter by using a x25 objective. Experiments were performed in duplicate for each intervention.
Capillary-like Tubule Formation
PCAEC were trypsinized, counted, and placed into flat-bottom 24-well tissue culture plates (2.5 x 104 cells/well) in serum-free EGM. Cells were incubated with 250 ng/ml Ang-1 for 3 h. In the pharmacological intervention experiments, cells were incubated with each pharmacological intervention for 30 min before adding Ang-1. After cells were seeded, 300 µl of extracellular matrix gel (Sigma Chemical) was placed onto the surface of the cells, and the plate was incubated at 37°C for 1 h to allow the gel to set. After solidification, the gel was covered with EGM medium or EGM medium containing 250 ng/ml Ang-1 with or without each intervention for a further 24 h. The gels were photographed by using a phase-contrast microscope (44). Capillary tubule formation was quantified by counting the number and cumulative length of tubular structures in eight fields by using image acquisition and analysis software (LabWorks, UVP). Each assay was conducted in duplicate.
Endothelial Cell Spheroid Model of Angiogenesis
Generation of PCAEC spheroids. We utilized the recently described cell spheroid model of angiogenesis (19, 26). This model has been reported to involve the aggregation of endothelial cells into three-dimensional spheroids when embedded in collagen gels with subsequent formation of sprouts or capillary-like projections and complex networks (19). A confluent monolayer of PCAEC was trypsinized, and the cells were centrifuged at 800 rpm for 5 min. The cell pellet was suspended the cell in 0.2% methylcellulose-10% FBS EGM medium, and 1 x 103 of the suspended cells were seeded into nonadhesive 96-well plates and cultured at 37°C. After 24 h, spheroid formation and morphology were assessed by using phase-contrast microscopy. The spheroids were harvested by centrifugation at 800 rpm for 3 min and carefully vacuum aspirated to leave the spheroids in 10% of the methocel medium.
Spheroid-based in vitro angiogenesis assay. One-half milliliter of the spheroid suspension was mixed with 250 ng/ml of Ang-1 with or without each of the pharmacological interventions in 0.2% methocell-20% FBS in EGM medium. A 0.5-ml aliquot of collagen gel solution was then added to the suspension, mixed thoroughly, and added to each well of a 24-well cell culture plate. The gel was solidified at 37°C for 30 min, and 0.5 ml of 20% FBS in EGM containing 250 ng/ml Ang-1 with or without each pharmacological intervention was added to the top of the gel. After 24 h, the length of capillary sprouts from each spheroid was measured with image acquisition and analysis software (LabWorks, UVP). At least 10 spheroids per experimental group were analyzed. Experiments were performed in duplicate for each intervention.
Morphological analysis. Collagen gel-embedded PCAEC spheroids were fixed for 24 h with 4% paraformaldehyde followed by dehydration through graded ethanol at 4°C for 24 h. The gel was then vacuum infiltrated in 1:1 paraffin-to-xylene solution at 60°C for 10 h. The infiltration was completed by using fresh low melting paraplast X-tra at 60°C for 24 h before embedding. The blocks were cooled at 4°C, and serial 10-µm sections were cut and stained with hematoxylin.
Statistical Analysis
The results are expressed as means ± SD. Statistical analysis was performed by using one-way ANOVA followed by Duncan's multiple-comparison test. A P value of <0.05 was taken as significant.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Exposure to Ang-1 (250 ng/ml) for various periods of time (5, 15, 30, 60, and 120 min) resulted in a time-dependent increase in eNOS phosphorylation at Ser1177. Phosphorylated eNOS was apparent at 5 min, was significantly increased by 15 min, peaked at 60 min, and then started to decline (Fig. 1, A and B). Exposure to Ang-1 had little effect on total eNOS protein expression over the 120 min of exposure (Fig. 1A). The time frame of Ang-1-stimulated NOx-release NO production showed a similar pattern as eNOS phophorylation (Fig. 1C).
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Ang-1 Increases HSP90 Binding to eNOS and Upregulates Phosphorylated eNOS
To examine the time course of HSP90 binding to eNOS after treatment with Ang-1, we immunoprecipitated with an eNOS antibody followed by immunoblotting with a HSP90 antibody. Ang-1 led to a gradual increase in HSP90 binding to eNOS that was apparent within 5 min, compared with untreated controls, was significantly elevated by 15 min, peaked at 30 min, and then gradually decreased (Fig. 2). Ang-1 had little effect on total intracellular HSP90 levels (Fig. 2). We next examined whether pharmacological blockade of HSP90 suppressed Ang-1-induced eNOS phosphorylation. Cells were pretreated with GA (1 µg/ml) for 30 min and were exposed to Ang-1 for 60 min. GA completely abolished Ang-1-induced eNOS phosphorylation to a level similar to controls (Fig. 3, A and B). Pretreatment with GA had no effect on eNOS protein expression. Pretreatment with GA also significantly suppressed Ang-1-stimulated NOx release (Fig. 3C).
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Ang-1 Activates PI3K-Akt Pathway and Stimulates eNOS Phosphorylation
Exposure to Ang-1 for periods up to 120 min led to a dramatic increase in phosphorylation of Akt at Ser473, which was observed at 5 min, peaked at 30 min, and then declined toward baseline (Fig. 4). Pretreatment with the PI3K inhibitor Wort (500 nM) completely suppressed Ang-1-stimulated eNOS phosphorylation (Fig. 3, A and B), and this was accompanied by significant inhibition of Ang-1-stimulated NO production (Fig. 3C).
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HSP90, PI3K/Akt, NO- and Ang-1-Stimulated Endothelial Cell Migration and Capillary Tubule Formation
To investigate the effects of HSP90, PI3K-Akt, and NO on Ang-1-induced angiogenesis in vitro, we first examined endothelial cell migration by using a modification of the Boyden chamber assay. Stimulation of PCAEC with Ang-1 for 4 h caused a 2.7-fold increase in endothelial cell migration compared with untreated controls (Fig. 5). Cells treated with Ang-1 + Wort (500 nM), Ang-1 + GA (1 µg/ml), or Ang-1 + L-NAME (2.5 mM) resulted in significant suppression of Ang-1-stimulated endothelial cell migration (Fig. 5).
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Next, we determined the effect of HSP90, PI3K-Akt, and NO on Ang-1-induced capillary tubule structure formation in three-dimensional collagen gels. Exposure to Ang-1 for 24 h resulted in a 2.7- to 3-fold increase in both the number and length of capillary tubule formation in the collagen gel. Pretreatment with either Wort (500 nM) or GA (1 µg/ml) completely abolished Ang-1-induced tubule formation in three-dimensional collagen gel. Pretreatment with L-NAME (2.5 mM) also led to a significant decreasein Ang-1-stimulated capillary tubule formation (Fig. 6).
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Ang-1 Stimulates Sprouting of Capillary-like Structures
To further test the angiogenic activity of Ang-1, we examined spheroid formation and sprouting by using the multicellular three-dimensional spheroid technique (Fig. 7, top). Untreated collagen gel-embedded PCAEC spheroids had a low level of spontaneous sprouting (Fig. 7A). Twenty-four-hour exposure to Ang-1 led to an approximately threefold increase in length of sprouts from the spheroids compared with controls (Fig. 7B). Exposure to Ang-1 for 48-72 h resulted in an organized network of sprouts and branches from the collagen-embedded spheroids (Fig. 7, C and D). Morphological analysis of cross sections through Ang-1-stimulated spheroid-embedded gels revealed a single layer of endothelial cells lining the lumens of capillary-like structures (Fig. 7, bottom).
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Role of the HSP90, PI3K/Akt-NO Pathway on Ang-1-Stimulated Endothelial Cell Spheroid Sprouts
Treatment with PI3K inhibitor Wort (500 nM) or HSP90-specific inhibitor GA (1 µg/ml) completely suppressed Ang-1-stimulated PCAEC spheroid sprouting. Treatment with L-NAME (2.5 mM) caused a significant decrease in the length of the Ang-1-stimulated capillary sprouts from PCAEC spheroids (Fig. 8).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Phosphorylation of eNOS at Ser1177 is widely considered to be an important mechanism for increased NO production after stimulation with angiogenic factors such as VEGF, shear stress, and, more recently, Ang-1 (2, 12, 15, 31). Phosphorylation of eNOS correlates directly with electron flow through the eNOS enzyme, increases in eNOS activity, and NO production by endothelial cells (30). Our study demonstrates that Ang-1 stimulates both phosphorylation of eNOS at Ser1177 and NO release from PCAEC and that these changes can be suppressed by pretreatment with a specific PI3K inhibitor. These data demonstrate and confirm the notion that that eNOS phosphorylation at Ser1177 mediates Ang-1-induced NO release.
Ang-1 is a unique growth factor that induces Tie-2 receptor phosphorylation and interacts with other signal transduction molecules. Recent studies show that Ang-1 induces diverse angiogenic activities and that activation of PI3K-Akt pathway is necessary for Ang-1-induced endothelial cell migration, cell survival, and tubule formation (2, 14, 24, 29, 34). Consistent with these findings, our data reveal that Ang-1 regulates endothelial cell migration and capillary formation via a PI3K-Akt-dependent mechanism in PCAEC. Furthermore, this is the first demonstration that Ang-1 stimulates sprouting of capillary-like structures with patent endothelial-lined lumens from PCAEC spheroids with formation of complex three-dimensional networks and branches. These data indicate that Ang-1 plays an essential role in the angiogenesis and formation of mature, intact coronary capillaries.
The molecular mechanisms by which the PI3K-Akt pathway initiates angiogenesis remain largely unknown. Because the PI3K-Akt pathway is only transiently increased by Ang-1, it is likely that the downstream mediators of this pathway play a critical role in angiogenesis. So far, relatively little is known about the downstream target molecules of Akt in angiogenesis, although several candidate molecules have been identified, including Bad, procaspase-9, survivin, and eNOS (2, 6, 9, 12, 15, 24, 31, 34). It is possible that combinations of these and other unidentified Akt target molecules cooperatively and synergistically modulate vascular angiogenesis. Further studies are needed to dissect the PI3K-Akt pathway and elucidate its downstream mediators.
NO is known to mediate many physiological and pathological functions and is considered to be an important mediator of angiogenesis. For instance, stimulation of human umbilical vein endothelial cells with bFGF or VEGF increased NO production and induced formation of capillary-like structures in three-dimensional fibrin gels; these effects were inhibited by pretreatment with the NOS inhibitor L-NAME (1, 35, 48). Furthermore, L-NAME has been demonstrated to inhibit VEGF-induced angiogenesis in the rabbit cornea model of angiogenesis (47), and the recent study of Rikitake et al. (37) demonstrated that sphingosine-1-phosphate induced angiogenesis via a NO-dependent mechanism. These data highlight the importance of NO in modulating angiogenesis. Our data are in line with those findings and further demonstrate that Ang-1-induced NO production is a key event in the angiogenesis in PCAEC.
In the last few years, the PI3K-Akt signal transduction pathway has emerged as one of the main signaling routes that coordinate complex events leading to changes in cell metabolism, cell growth, cell movement, and cell survival. Akt also has been reported to regulate initiation of angiogenesis through its stimulatory effects on endothelial cell migration and actin reorganization, proliferation, and survival (12, 17, 22, 27, 32, 38, 40, 41, 46). Recent studies (11, 15, 31) have shown that stimulation with VEGF leads to transient activation of PI3K and phosphorylation of Akt, thereby regulating eNOS gene expression and NO production. The present studies are in agreement with those showing that the PI3K-Akt pathway regulates Ang-1-stimulated eNOS phosphorylation and NO production in PCAEC.
Although many studies have defined the angiogenic properties of Ang-1, the role that PI3-Akt-NO signaling axis plays on Ang-1-induced angiogenesis in PCAEC has been little studied. However, recently Babaei and colleagues (2) demonstrated that Ang-1-induced NO production and capillary formation could be inhibited in human umbilical vein endothelial cells by inhibitors of NOS and PI3K and that Ang-1-induced neovascularization was reduced in eNOS knockout mice. Our study in PCAEC reveals that inhibition of the PI3K-Akt-eNOS-NO signaling pathway by Wort significantly suppresses Ang-1-induced endothelial cell migration and tubule structure formation and completely blocks Ang-1-induced sprouting of complex capillary-like structures from PCAEC spheroids. These findings confirm that the PI3K-Akt-eNOS-NO pathway plays an important role in capillary formation and branching.
HSP90 has long been known to affect the activity and function of other proteins by acting as a molecular chaperone. Recent in vitro evidence shows that overexpression of HSP90 enhanced eNOS phosphorylation and NO production; an increase in HSP90 binding to eNOS was central to these effects (4, 16, 18). We have recently reported a similar mechanism in response to hypoxia, which showed an increase in eNOS activity, NO release, and eNOS-associated HSP90 (8). To investigate the HSP90 and eNOS interaction, a well-recognized and specific inhibitor of HSP90, the ansamycin antibiotic GA, was used in our present study. GA binds to the ATP binding site of the chaperone HSP90 to block the assembly of HSP90 heterocomplexes and inhibit its folding, as well as ATPase activity, leading to cell-cycle disruption (36, 39). Recently, GA also has been reported to prevent HSP90 and Akt binding to eNOS, and block phosphorylation of eNOS at Ser1179 and inhibition of NO production (4, 16, 18). In the present study, we further demonstrate that exposure to Ang-1 leads to a time-dependent increase in HSP90 binding to eNOS, and treatment with GA significantly suppresses Ang-1-induced eNOS phosphorylation and NO production. To our knowledge, this is the first demonstration that an interaction of HSP90 with eNOS modulates Ang-1-induced upregulation of eNOS and increases NO production.
Recently, Brouet and colleagues (5) demonstrated that HSP90 plays a key role in NO-dependent angiogenesis. They found that inhibition of HSP90 function or overexpression of HSP90 regulated NO production and NO-induced angiogenesis. Inhibition of HSP90 function has also been reported to reduce VEGF gene expression and suppress hypoxia-induced angiogenesis (21). Consistent with those studies, our data demonstrate that pharmacological inhibition of HSP90 significantly suppressed Ang-1-induced endothelial cell migration and tubule structure formation and completely blocked Ang-1-induced sprouting of capillary-like structures from spheroids. Thus it would seem that an HSP90-eNOS interaction plays a key role in Ang-1-induced angiogenesis in PCAEC.
In summary, this study demonstrates that Ang-1 stimulates HSP90 binding to eNOS, activates the PI3K-Akt pathway, leads to eNOS phosphorylation at Ser1177, and increases NO production by PCAEC. Furthermore, we demonstrated that disruption of the HSP90-eNOS interaction and of the PI3K-Akt pathway blocks Ang-1-stimulated endothelial cell migration and formation of complex capillary networks. We conclude that both a HSP90-eNOS interaction and activation of the PI3K-Akt pathway play a critical role in the regulation of Ang-1-induced eNOS phosphorylation, NO production, and angiogenesis in PCAEC. Unraveling the complex molecular mechanisms that contribute to angiogenesis should provide the foundation for future therapies for the prevention and reversal of coronary artery disease.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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1-stimulated capillary organization in vitro. Am J Pathol 150: 1835-1844, 1997.[Abstract]
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). Total intracellular HSP90 levels showed little change over the 120 min of the study (
) [n = 3; data are means ± SD; *P < 0.05 compared with controls (time 0)].
