doi: 10.1161/hq0102.101843
© 2002 American Heart Association, Inc.
Vascular Biology |
Involvement of Endothelial Nitric Oxide in Sphingosine-1-Phosphate–Induced Angiogenesis
From the Division of Cardiovascular and Respiratory Medicine, Department of Internal Medicine (Y.R., K.-i.H., S.K., M.O., T.T., N.I., M.Y.), and the Division of Diabetes, Digestive and Kidney Diseases, Department of Clinical Molecular Medicine (W.O.), Kobe University Graduate School of Medicine, Kobe, Japan.
Correspondence to Yoshiyuki Rikitake, MD, PhD, Division of Cardiovascular and Respiratory Medicine, Department of Internal Medicine, Kobe University Graduate School of Medicine, 7-5-1, Kusunoki-cho, Chuo-ku, Kobe, 650-0017, Japan. E-mail rikitake{at}med.kobe-u.ac.jp
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Abstract |
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Nitric oxide (NO) has been implicated as a critical signaling molecule of angiogenesis. Recently, sphingosine-1-phosphate (S1P) has emerged as a mediator of angiogenesis, and S1P-induced NO synthesis in endothelial cells (ECs) has been reported. To analyze the signaling pathways involved in S1P-induced angiogenesis and clarify the role of NO in this process, we performed in vivo and in vitro angiogenesis assays. S1P activated the phosphatidylinositol-3-kinase (PI3K)/Akt/endothelial NO synthase (eNOS) pathway in ECs, since S1P-stimulated eNOS phosphorylation and NO production were blocked by inhibition of activities of PI3K and Akt. S1P increased capillary ingrowth into subcutaneously implanted Matrigel plugs in mice, and the effect of S1P was significantly reduced in mice that received NG-nitro- L-arginine methyl ester (L-NAME), an inhibitor of NOS. S1P stimulated EC migration and tube formation on Matrigel, which processes were significantly decreased by inhibition of activities of PI3K, Akt, or eNOS, whereas treatment with LY294002, a PI3K inhibitor, but not L-NAME, inhibited EC viability and proliferation. Thus, our results demonstrate the crucial role of NO in S1P-induced angiogenesis in vivo and in vitro and suggest the divergent roles of NO in the S1P-induced angiogenic response.
Key Words: angiogenesis • growth substances • signal transduction
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Introduction |
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Angiogenesis, the formation of new blood vessels from preexisting ones, plays an important role not only in normal physiological events such as embryonic development and wound healing but also in pathological conditions such as tumor growth, diabetic retinopathy, and rheumatoid arthritis.1 Angiogenesis involves complex steps, including extracellular matrix degradation, migration and proliferation of endothelial cells (ECs), and morphological differentiation of ECs into capillary-like tubes. All of these processes are tightly regulated by the action of angiogenic growth substances such as vascular endothelial growth factor (VEGF) and angiopoietin.
Nitric oxide (NO) produced by endothelial NO synthase (eNOS) has a crucial role in the regulation of vascular tone, vascular remodeling, and angiogenesis.2–4 Recent growing evidence has established the involvement of NO in VEGF-induced angiogenesis. NOS inhibitors block VEGF-induced EC migration, proliferation, and tube formation in vitro as well as VEGF-induced angiogenesis in vivo.5,6 VEGF is known to induce Akt/protein kinase B–dependent phosphorylation of eNOS, resulting in activation of eNOS and the subsequent increase in NO production.7
Recently, sphingosine-1-phosphate (S1P), a bioactive lipid released by activated platelets, has emerged as an important mediator of angiogenesis. S1P induces migration, proliferation, and cytoskeletal changes of ECs by way of the endothelial differentiation gene (Edg), a family of G protein–coupled receptors.8–11 Activation of Edg receptors triggers several signaling pathways by pertussis toxin (PTX)–sensitive Gi protein. Although the signaling pathways activated by S1P have been extensively studied in various cell types, the precise signaling mechanism by which S1P elicits angiogenesis is only recently beginning to emerge. Recent studies have revealed that S1P increases NO synthesis in ECs.12,13 However, the role of endothelial NO in S1P-induced angiogenesis remains unknown.
To address the role of eNOS in S1P-induced angiogenesis, we examined whether S1P regulates eNOS activity through the phosphatidylinositol-3-kinase (PI3K)/Akt pathway. We show in this report that S1P stimulates eNOS phosphorylation and NO production by way of a pathway dependent on PI3K and Akt. Importantly, we demonstrate that NO plays a pivotal role in S1P-induced angiogenesis in vivo. In addition, inhibition of PI3K could reduce viability, proliferation, migration, and tube formation of ECs, whereas treatment with an NOS inhibitor could inhibit migration and morphogenesis but not affect cell viability and proliferation. Taken together, these results demonstrate a critical role of NO in S1P-induced angiogenesis in vivo and in vitro and suggest the divergent roles of NO in S1P-induced angiogenic response among proliferation, migration, and differentiation.
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Methods |
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Cell Culture and Transfection
Bovine aortic ECs (BAECs) and human umbilical vein ECs (HUVECs) were purchased from Cell Systems/Clonetics. BAECs were cultured as described previously.14 HUVECs were maintained in RPMI-1640 medium supplemented with 20% fetal bovine serum (FBS), endothelial cell growth supplement, and heparin. In some experiments, cells were infected with recombinant adenoviral vectors encoding dominant-negative forms of PI3K or Akt (ie, Ad.
p8515 or Ad.Akt-AA,15 respectively) or vector alone (Ad.null) at a multiplicity of infection of 30 for 48 hours.
Immunoblot Analysis
After transfection or pretreatment with 10 ng/mL pertussis toxin (PTX, Calbiochem) for 24 hours, 100 nmol/L wortmannin (Sigma), 10 µmol/L LY294002 (Calbiochem), or 5 µmol/L PP2 (Calbiochem) for 1 hour in serum-depleted medium, cells were stimulated with S1P (BioMol). Cell lysates were resolved by SDS–polyacrylamide gel electrophoresis and then subjected to immunoblot analysis with anti–phospho-Akt (Ser473), anti–phospho-eNOS (Ser1177), or anti–phospho-extracellular signal–regulated kinase (ERK)1/2 (Thr183/Tyr185) antibodies (New England Biolabs). Bands were visualized by enhanced chemiluminescence (Amersham-Pharmacia). The membranes were reprobed with anti-Akt (New England Biolabs), anti-eNOS, or anti-ERK1 (Transduction Laboratories) antibodies.
NO Production
Nitrite production was measured by an ozone-chemiluminescence method. After transfection as described above and serum starvation for 24 hours, the media were replaced by phenol red–free Dulbecco’s modified Eagle’s medium. Cells were stimulated with S1P for 1 hour, and the culture media were assayed for nitrite production in the chemiluminescent NO analyzer. Sodium nitrite was used as a standard.
In Vivo Matrigel Plug Assay
A Matrigel plug assay was performed as previously described.16 In brief, 24 C57BL/6 male mice were divided into 2 groups: those that were left untreated and those that were treated with 1 mg/mL NG-nitro- L-arginine methyl ester (L-NAME, Sigma) in their drinking water (n=12 each). The treatment started 7 days before Matrigel implantation and continued until the end of the observation period. Growth factor–reduced Matrigel (0.5 mL, Becton Dickinson) was mixed with either 5 µmol/L S1P and 40 U/mL heparin or heparin alone and injected subcutaneously into the mice. After 7 days, the mice were humanely killed, and the plugs were recovered, fixed in 3.7% formaldehyde, embedded in paraffin, made into slides, stained with Masson’s trichrome or von Willebrand factor (vWF), and photographed. Capillaries were defined either as tubular structures containing red blood cells after being stained with Masson’s trichrome or as tubular structures that staining positively with vWF in the corresponding section. Angiogenesis was quantified by counting the number of capillaries penetrating the Matrigel plug, as averaged by section in 5 different regions throughout the Matrigel plug. For immunostaining, sections were incubated with mouse monoclonal anti-vWF antibody (1:20 dilution, Dako) overnight at 4°C. Biotinylated secondary antibody (1:200 dilution, Dako) and streptavidin–horseradish peroxidase (Dako) with 3,3'-diaminobenzidine tetrahydrochloride were used to visualize antibody binding. Endogenous peroxidase activity was blocked by 3% H2O2. Sections were then counterstained with hematoxylin.
Tube Formation Assay
A Matrigel tube formation assay was performed as previously described.11 Twenty-four-well culture plates were coated with 300 µL of growth factor–reduced Matrigel per well and then allowed to polymerize for 30 minutes at 37°C. HUVECs cultured for 24 hours in RPMI-1640 medium with 1% FBS were seeded on coated plates at a density of 2x105 cells per well in RPMI-1640 medium supplemented with 1% FBS and the agents such as 2 µmol/L S1P and various inhibitors and then incubated for 24 hours at 37°C. Pictures were taken at x40 magnification with a digital output camera (Olympus DP11) attached to an inverted phase-contrast microscope (Olympus IX70); total tube length was measured by using the NIH Image program (National Institutes of Health, Bethesda, Md).
Cell Viability
HUVECs (1x105 cells per well) were plated onto gelatin-coated 6-well plates in 1 mL of culture medium. The next day, the cells were switched to RPMI-1640 medium containing 1% FBS, pretreated with or without LY294002 or L-NAME, and then stimulated with S1P. After 24 hours, cell viability was determined by trypan blue exclusion. Counts were performed on triplicate wells.
Cell Proliferation
A cell proliferation assay was performed as previously described.14 In brief, HUVECs (5x103 cells per well) were plated onto collagen-coated 96-well plates in culture medium. The next day, the cells were pretreated with or without LY294002 or L-NAME and then stimulated with S1P. The cells were cultured in RPMI-1640 medium containing 5% FBS to avoid cell detachment. After 48 hours, cell proliferation was determined by crystal violet staining. Counts were performed on 8 wells, and experiments were performed in duplicate.
Cell Migration
Migration of HUVECs was estimated in a modified Boyden chamber as previously described.14 In brief, polyvinylpyrrolidone (PPV)-free polycarbonate filters with 8-µm pores were coated with 0.1% gelatin overnight and washed with phosphate-buffered saline to remove the excess coating. After 25 µL of RPMI-1640 medium containing 1% FBS with or without S1P was placed in the lower chamber, the filters were positioned above the wells of the lower chamber, and then 106 cells/mL suspended in 50 µL RPMI-1640 medium containing 1% FBS were added to the upper chamber. Inhibitors were given to the cells for 30 minutes before seeding. The apparatus was incubated at 37°C for 4 hours. After incubation, the filter was removed, and the upper surface of the filter was scraped off. The filter was fixed with methanol and stained with Diff-Quik. The number of cells was counted in 4 randomly chosen fields under x400 magnification. Experiments were performed in quadruplicate.
A wounding migration assay was performed as follows. BAECs, plated on gelatin-coated 60-mm culture dishes and serum starved for 24 hours, were wounded with a razor blade. After being wounded, the cultures were washed with serum-free medium and further incubated in Dulbecco’s modified Eagle’s medium in the presence of various substances. BAECs were allowed to migrate for 48 hours and were rinsed with serum-free medium, followed by fixation with methanol and staining with Diff-Quik. Migration was quantified by counting the number of cells that moved beyond the initial line.
Statistics
Data are expressed as mean±SE. Statistical significance was analyzed by unpaired Student’s t test or 1-way ANOVA, followed by the Student-Newman-Keuls test. P<0.05 was considered statistically significant.
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Results |
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S1P Stimulates Phosphorylation of Akt and eNOS
To determine whether S1P can activate Akt and eNOS in ECs, BAECs were stimulated with S1P at various concentrations. S1P induced a concentration-dependent increase in phosphorylation of Akt and eNOS within 5 minutes, with maximal response at 1 µmol/L (online Figure IA; please see http://atvb.ahajournals.org). Phosphorylation of Akt and eNOS was detected as early as 2 minutes after stimulation with 1 µmol/L S1P, peaked at 5 to 10 minutes, and then gradually decreased (online Figure IB; please see http://atvb.ahajournals.org).
PI3K/Akt-Mediated eNOS Phosphorylation and NO Production by S1P
S1P initiates intracellular signaling by the specific G protein–coupled receptor Edg.8–10 We next characterized the S1P-induced phosphorylation of Akt and eNOS. As shown in Figure 1A, PTX and an inhibitor of the Src family of tyrosine kinases, PP2, inhibited S1P-induced phosphorylation of Akt and eNOS, suggesting the involvement of Gi and the Src family of tyrosine kinases. Two selective PI3K inhibitors, wortmannin (30 nmol/L to 1 µmol/L) and LY294002 (1 to 50 µmol/L), inhibited S1P-induced phosphorylation of Akt and eNOS in a concentration-dependent manner (Figure 1A and data not shown). To directly establish that S1P-induced eNOS phosphorylation proceeds through Akt, BAECs were transfected with a dominant-negative form of Akt (Akt-AA). S1P failed to increase phosphorylation of eNOS in Ad.Akt-AA–infected cells, whereas eNOS phosphorylation was not decreased in Ad.null-infected cells compared with control cells, indicating Akt-dependent eNOS phosphorylation in response to S1P (Figure 1B). Similarly, S1P could increase phosphorylation of Akt and eNOS in HUVECs, and this increase was effectively blocked by pharmacological inhibition with LY294002 or infection with a recombinant adenoviral vector encoding a dominant-negative form of PI3K (Ad.p85) (Figure 1C). S1P-mediated eNOS phosphorylation was abolished in Ad.Akt-AA–infected HUVECs. In contrast to the effects on phosphorylation of Akt and eNOS, LY294002 did not block ERK phosphorylation induced by S1P, and S1P-induced ERK phosphorylation was not inhibited by infection with Ad.p85 or Ad.Akt-AA, suggesting specific inhibition of the activities of PI3K and Akt by these treatments. Thus, we confirmed that S1P induces eNOS phosphorylation through the PI3K/Akt pathway.
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P<0.01 compared with S1P(-).
We next evaluated the role of the PI3K/Akt pathway in NO production. S1P (1 µmol/L) increased NO production by 1.8-fold compared with control cells, and this increase was completely diminished in Ad.p85- or Ad.Akt-AA–infected cells, but not in Ad.null-infected cells (1.1±0.2-, 1.1±0.2-, and 1.5±0.1-fold increase, respectively; n=9, Figure 1D). Treatment of cells with 2 µmol/L L-NAME completely blocked NO production in response to S1P (data not shown). These data demonstrate the involvement of the PI3K/Akt pathway in S1P-induced NO production.
Role of NO in S1P-Induced In Vivo Angiogenesis
To analyze the role of NO in S1P-induced angiogenesis in vivo, we performed a Matrigel plug assay, an established in vivo angiogenesis model.16 Matrigel with or without S1P was injected subcutaneously into the abdomen of mice that had either received or not received L-NAME. In our application of the assay, we used growth factor–reduced Matrigel, which, unlike conventional Matrigel, does not stimulate angiogenesis on its own. Matrigel plugs without S1P were pale, indicating no or little blood vessel formation. In contrast, plugs containing S1P appeared yellowish (results not shown). Capillaries were defined either as tubular structures containing red blood cells after being stained with Masson’s trichrome or tubular structures staining positively with vWF. In mice not treated with L-NAME, Matrigel containing S1P produced far more neovessels within the gels compared with those with Matrigel alone (Figure 2). In contrast, there was marked reduction in the number of infiltrating capillaries containing red blood cells within Matrigel plugs taken from L-NAME–treated mice (0.5±0.3, n=12) compared with untreated mice (6.0±1.0, n=12, P<0.01; Figure 2 and online Figure II; please see http://atvb.ahajournals.org). These results demonstrate the significant role of NO in S1P-induced angiogenesis in vivo.
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Role of the PI3K/Akt/eNOS Pathway in S1P-Stimulated Tube Formation
To address the role of S1P activation of the PI3K/Akt/eNOS pathway in angiogenesis in vitro, we examined the effects of various inhibitors on S1P-induced tube formation in Matrigel. Morphogenesis of HUVECs into capillary-like tubes on Matrigel-coated plates was stimulated by S1P (2.3-fold), whereas incubation with PTX, LY294002, or L-NAME led to incomplete or degraded tube formation (1.1-, 1.6, and 1.3-fold; PTX, LY294002, and L-NAME, respectively; P<0.01, Figures 3A and 3B). Similarly, S1P-induced tube formation was significantly blocked when cells were infected with Ad.p85 or Ad.Akt-AA but not when infected with Ad.null (1.3-, 1.2-, and 2.1-fold; Ad.p85, Ad.Akt-AA, and Ad.null, respectively). These results suggest involvement of the Gi-mediated PI3K/Akt/eNOS signaling pathway in the S1P-induced morphogenic differentiation of ECs.
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Roles of PI3K and NO in S1P-Stimulated EC Viability, Proliferation, and Migration
To further analyze the mechanism by which NO regulates S1P-induced angiogenesis in vivo and in vitro, we finally assessed whether PI3K and NO are involved in S1P-stimulated proliferation and migration. As shown in Figure 4A, cell viability was slightly decreased under low-serum conditions, whereas S1P prevented cell death. Treatment of cells with LY294002 but not L-NAME significantly blocked cell survival. Similarly, S1P stimulated EC proliferation, and the effect of S1P was inhibited by LY294002 but not by L-NAME (Figure 4B). However, migration of HUVECs was reduced by treatment with LY294002 as well as with L-NAME (Figure 4C). Identical results were observed in BAECs in a wounding migration assay (Figure 4D). These results suggest that PI3K activity regulates S1P-induced cell survival, proliferation, and migration, whereas NO is involved in migration but not survival and proliferation under our experimental conditions.
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Discussion |
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The data of the present study demonstrate that S1P can induce eNOS phosphorylation and NO production by way of the PI3K/Akt pathway in cultured ECs and that NO plays a critical role in S1P-induced angiogenesis in vitro and in vivo. Platelets contain many angiogenic factors, and the angiogenic activity of those released by platelets plays an important role in initiating angiogenesis in injured tissue, especially in wound healing and tumor angiogenesis. It seems likely that S1P, which is known to be abundantly stored in platelets and released on their activation,17 may contribute to platelet-induced angiogenesis in wound repair, because it was demonstrated that S1P is the major EC chemoattractant released into serum by platelets during blood clotting.18 Therefore, it is suggested that the angiogenic activity of S1P may account for the important role of platelet interaction with ECs in angiogenesis at sites of injury. Interestingly, previous studies have demonstrated the crucial role of NO in wound healing, as wound healing is accelerated by systemic administration of L-arginine, a substrate of NOS, but delayed by NOS inhibitors.19,20 Lee et al21 reported impaired wound healing and angiogenesis in eNOS-deficient mice. Together with those findings, our data demonstrating NO-dependent angiogenesis by S1P may account for the critical role of NO in wound repair.
NO is now considered to be required for EC proliferation, migration, and organization and has been shown to be one of the key components of the angiogenic cascade. A body of recent evidence suggests a stimulatory role for NO in angiogenesis. For example, NO donors promote EC proliferation and migration in vitro; in contrast, NOS inhibitors suppress the response.22–24 NOS inhibitors block VEGF-induced EC migration, proliferation, and capillary-like network formation in vitro.5,6 We found that L-NAME could block EC migration and tube formation on Matrigel, whereas an NOS inhibitor failed to inhibit EC proliferation. Previous studies determining the role of NO in EC proliferation stimulated by growth-promoting substances have provided conflicting results. It has been reported that NO mediates EC proliferation induced by VEGF but not by basic fibroblast growth factor, endothelial cell growth supplement, or serum. It is possible that the apparent discrepancies may be related not only to differences in the various models of angiogenesis (ie, species of cell types, microvascular or macrovascular ECs, angiogenic factors, or methods to quantify mitogenic activity) but also to differences in the actions of NO, depending on the preexisting conditions.
We have also shown that L-NAME did not affect EC viability. However, Kwon et al12 have recently demonstrated that S1P-mediated NO production protects HUVECs from serum-deprived apoptosis. In their experiments, apoptosis was induced by complete serum depletion. On the other hand, in our experiments, serum was not completely depleted, but the cells were cultured in 1% FBS–containing medium. It is likely that the failure of L-NAME to antagonize S1P-induced cell survival may be due to the difference in the presence of serum.
Our results suggest that the contribution of NO was different among S1P-induced EC proliferation, migration, and tube formation in vitro. Cell-cell interaction and cell-matrix interaction are thought to be relatively important steps in migration and tube formation compared with proliferation. One possible mechanism by which L-NAME could block S1P-stimulated migration and formation of a vascular network may involve regulation of cell-cell and/or cell-matrix interaction by NO.
Previous studies have revealed the essential role of NO in angiogenesis in vivo.4,21,23 With the in vivo rabbit cornea assay, angiogenesis induced by vasoactive molecules, including substance P, was shown to have been blocked by NOS inhibition.23 Murohara et al4 reported that angiogenesis in response to tissue ischemia or VEGF administration was significantly impaired in eNOS-deficient mice. An angiogenesis-promoting role for EC-derived NO was also suggested in the report by Lee et al,21 who showed impaired wound healing and angiogenesis in eNOS-deficient mice. We observed that there was a marked reduction in the number of infiltrating capillaries containing red blood cells within Matrigel plugs taken from L-NAME–treated mice compared with untreated mice. Thus, our results are consistent with previous studies that have demonstrated that eNOS-derived NO acts as a downstream signal for growth factor–induced angiogenesis.
The precise molecular mechanisms responsible for reduced angiogenesis with NOS inhibition in our model remain to be determined. Previous studies have shown that the downstream molecules mediating angiogenic signaling of NO in VEGF-stimulated proliferation of ECs are cGMP-dependent protein kinase,6 Raf-1,25 and mitogen-activated protein kinase.26 Interestingly, it was reported that NO upregulates VEGF mRNA expression in some cell types.27 These mechanisms might be involved in S1P-mediated, NO-dependent angiogenesis. A further mechanism through which NO could modulate angiogenesis is by modifying adhesion molecule expression on ECs. NO has been shown to maintain the functional expression of 
Vß3 integrin, a mediator for EC migration, survival, and angiogenesis.28,29 Murohara et al24 have shown that L-NAME inhibits surface expression of Vß3 integrin, and Lee et al30 reported recently that an NO donor upregulates Vß3 integrin expression on HUVECs. Another important mechanism through which NO may contribute to angiogenesis is by increasing EC survival. NO can prevent apoptosis in several cell types, including ECs. It has been recognized that many growth-promoting substances such as VEGF and S1P can act as a survival factor for ECs and that NO production by VEGF and S1P protects ECs from apoptosis. Kwon et al12 have recently demonstrated that S1P-mediated NO production protects HUVECs from serum-deprived apoptosis. However, NO-dependent EC survival is unlikely a critical determinant of angiogenesis in our experiments because L-NAME did not reduce EC viability in culture media containing 1% FBS with or without S1P.
The results showing that the inhibitory effect of L-NAME on neovascularization in Matrigel plugs was more significant compared with that on EC migration suggest that NO may be involved in other angiogenic properties of ECs besides migration. It is possible that systemic administration of L-NAME may inhibit recruitment of endothelial progenitor cells, which have been recently recognized as an important participant in neovascularization of adult tissue.31 Moreover, it is also possible that treatment of mice with L-NAME may affect proteolytic degradation of the extracellular matrix by inhibition of activities of matrix metalloproteinases and may modulate angiogenic activity through an increase in systemic blood pressure. However, it is possible that the greater inhibition of in vivo angiogenesis by L-NAME compared with the inhibition of EC migration may be due to the inhibition of not only migration but also vascular network formation (Figure 4). Studies on proliferation and migration of HUVECs and BAECs cultured on plastic dishes likely reflect events of the early stages of angiogenesis involving EC activation, whereas EC vascular network formation on Matrigel in vitro is more representative of the later stages of the angiogenic response. In our experiments, L-NAME treatment showed inhibitory effects of both migration (early stage) and vascular network formation (later stage). To determine the molecular mechanism by which NO produced by S1P regulates angiogenesis, further investigations are required.
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Acknowledgments |
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This work was supported by grants-in-aid for scientific research from the Ministry of Education, Science, Sports and Culture, Japan (12835007). We are grateful to Kiyoko Matsui for her assistance with cell culture.
Received July 10, 2001; accepted October 26, 2001.
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