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(Circulation Research. 2003;92:732.)
© 2003 American Heart Association, Inc.

Molecular Medicine

Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand (TRAIL) Sequentially Upregulates Nitric Oxide and Prostanoid Production in Primary Human Endothelial Cells

Giorgio Zauli^*, Assunta Pandolfi^*, Arianna Gonelli, Roberta Di Pietro, Simone Guarnieri, Giovanni Ciabattoni, Rosalba Rana, Marco Vitale, Paola Secchiero

From the Department of Normal Human Morphology (G.Z., M.V.), University of Trieste, Trieste, Italy; the Department of Biomorphology (A.P., R.D.P., R.R.), "G. D’Annunzio" University of Chieti, Chieti Scalo, Chieti, Italy; the Department of Morphology and Embryology (A.G., P.S.), Human Anatomy Section, University of Ferrara, Ferrara, Italy; and the Department of Drug Sciences (G.C.), Laboratory of Cell Physiology (S.G.), "G. D’Annunzio" University of Chieti, Chieti, Italy.

Correspondence to Giorgio Zauli, MD, PhD, Department of Normal Human Morphology, University of Trieste, Via Manzoni 16, 34138 Trieste. E-mail zauli{at}units.it

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Endothelial cells express tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) receptors, but the function of TRAIL in endothelial cells is not completely understood. We explored the role of TRAIL in regulation of key intracellular signal pathways in endothelial cells. The addition of TRAIL to primary human endothelial cells increased phosphorylation of endothelial nitric oxide synthase (eNOS), NOS activity, and NO synthesis. Moreover, TRAIL induced cell migration and cytoskeleton reorganization in an NO-dependent manner. TRAIL did not activate the NF-B or COX-2 pathways in endothelial cells. Instead, TRAIL increased prostanoid production (PGE₂=PGI₂>TXA₂), which was preferentially inhibited by the COX-1 inhibitor SC-560. Because NO and prostanoids play a crucial role in the state of blood vessel vasodilatation and angiogenesis, our data suggest that TRAIL might play an important role in endothelial cell function.

Key Words: tumor necrosis factor–related apoptosis-inducing ligand • nitric oxide • prostanoids

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Tumor necrosis factor (TNF)–related apoptosis-inducing ligand (TRAIL)/Apo-2L is a member of the TNF family of cytokines, which are structurally related proteins playing important roles in regulating cell death, immune response, and inflammation.¹ TRAIL is a type II membrane protein, which can be proteolytically cleaved to a soluble form,² as previously shown also for TNF- and CD95 (Apo-1/Fas)L. The unique feature of TRAIL, compared with other members of the TNF family, is its ability to induce apoptosis in a variety of malignant cells both in vitro and in vivo, displaying minimal toxicity on normal cells and tissues.R3-127474 ^3,4

TRAIL interacts with 4 high affinity transmembrane receptors belonging to the apoptosis-inducing TNF-receptor (R) family. TRAIL-R1 (DR4) and TRAIL-R2 (DR5) transduce apoptotic signals on binding of TRAIL, whereas TRAIL-R3 (DcR1) and TRAIL-R4 (DcR2) are homologous to DR4 and DR5 in their cysteine-rich extracellular domain, but they lack the intracellular death domain and apoptosis inducing capability. It has been proposed that TRAIL-R3 and TRAIL-R4 function as decoy receptors protecting normal cells, including endothelial cells, from apoptosis.R5-127474 ^5,6 It has been shown that endothelial cells express TRAIL receptors,⁶ and TRAIL protein is expressed in the medial smooth cell layer of the aorta and pulmonary arteries.⁷ Whereas cleavage of Fas ligand from the cell surface requires the action of zinc-dependent metalloproteases, generation of soluble TRAIL involves the action of cysteine proteases.² Notably, the vessel wall is a rich source of cysteine proteases.⁸

Using human umbilical vein endothelial cells (HUVECs) as a model system, the aim of this study was to investigate the ability of TRAIL to modulate intracellular pathways that play a key role in endothelial cell biology. In particular, we have analyzed whether TRAIL was able to modulate the production of nitric oxide (NO), which regulates vascular tone, promotes endothelial cell survival and migration, and inhibits platelet adhesion and aggregation, leukocyte adherence, and vascular smooth muscle cell proliferation, therefore providing antithrombotic and antiinflammatory activity.R9-127474 R10-127474 R11-127474 ^9–12 Moreover, we have investigated the expression and/or activity of cyclooxygenases (COX) in response to TRAIL, because also these enzymes, by catalyzing the rate-limiting step in the biosynthesis of prostanoids,¹³ such as prostaglandin (PGE)₂, prostacyclin (PGI)₂, and thromboxane (TXA)₂, have a profound influence on blood pressure, regional blood flow, vascular remodeling, and angiogenesis.

	Materials and Methods

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Reagents and HUVEC Cultures
Recombinant Histidine6-tagged TRAIL was produced in bacteria as described.¹⁴ Primary HUVECs, obtained as described previously,¹⁵ were used between the 3rd and 6th passage in vitro. Cells were grown on gelatin-coated tissue culture plates in M199 endothelial growth medium (BioWhittaker) supplemented with 20% FBS, 10 µg/mL heparin, and 50 µg/mL ECGF (Sigma). In some experiments, subconfluent HUVECs were starved in M199 containing 1% FBS for 18 hours before exposure to cytokines. The optimal concentration for both TRAIL (100 ng/mL) and TNF- (50 ng/mL) was determined in preliminary experiments in which NO and/or prostanoid production was measured after exposure to serial dilutions (0.1 to 5000 ng/mL) of the cytokines. For apoptosis evaluation, cells were analyzed after propidium iodide staining.¹⁴

Analysis of TRAIL Receptor Expression
For RT-PCR analysis of TRAIL receptors, RNA was purified from HUVECs, using the SV total RNA isolation system (Promega). Synthesis of first strand cDNA and amplification were performed using the Access RT-PCR system (Promega) and specific primer sets, following the manufacturer’s protocol.

For flow cytometric analysis of surface TRAIL receptors, the following antibodies were used: polyclonal goat anti-human TRAIL-R1, TRAIL-R2, TRAIL-R3, and TRAIL-R4 (all from R&D System); PE-conjugated rabbit anti-goat Abs (Immunotech; Marseille, France). Flow cytometric analysis was performed by FACScan (Becton Dickinson).

Western Blot Analysis
Subconfluent HUVECs were harvested in lysis buffer containing 1%Triton X-100, Pefablock (1 mmol/L), aprotinin (10 µg/mL), pepstatin (1 µg/mL), leupeptin (10 µg/mL), NaF (10 mmol/L), and Na₃VO₄ (1 mmol/L). Equal amounts of protein (50 µg) for each sample were migrated in acrylamide gels, blotted onto nitrocellulose filters, and probed with the following antibodies: anti-phospho-eNOS (P-Ser1177, Cell Signaling Technology), anti-eNOS/NOS Type III (BD Transduction Laboratories), anti-tubulin antibody (Sigma), anti-poly (ADP-ribose) polymerase (PARP), anti-caspase 3, anti-IkB, and IkB antibodies (all from Santa Cruz Biotechnology); and anti-COX-1, -COX-2 (Cayman).

Measurement of Intracellular NO, Ca²⁺, NOS Activity, and cGMP Formation
For confocal measurement of intracellular NO production and Ca²⁺ flux, HUVECs were plated on glass coverslips, grown to subconfluence and assays were performed as previously described.R16-127474 ^16,17

NOS activity was assayed by measuring the ability of cell lysates to convert L-³H-arginine (185x10³ Bq, 5 µCi Amersham) into L-³H-citrulline, as previously described.R16-127474 ^16,17

For NO-dependent cGMP measurement, HUVECs were seeded in standard 96-well plates, incubated overnight at standard conditions and subsequently treated, as indicated, for 30 minutes at 37°C in culture medium containing 0.6 mmol/L IBMX. After cell lysis, cGMP levels were measured using an enzyme-immunoassay kit (Amersham) according to the manufacturer’s instruction.

When indicated, pharmacological inhibitors or the vehicle (0.25% DMSO) were added to the cells 45 minutes before TRAIL.

Cell Migration Assay and Labeling of Actin Cytoskeleton
Cell migration was assayed using a modified Boyden chamber assay as described previously.¹⁸

For labeling of actin cytoskeleton, HUVECs were seeded on gelatin-coated coverslips and maintained in serum-free media overnight. Fresh media was administered to the cells 2 hours before stimulation with either TRAIL or VEGF for 20 minutes. Cell membranes were permeabilized with 0.1% Triton X-100/PBS and labeling of filamentous actin was attained by staining with Texas Red-X phalloidin (Molecular Probes).¹⁸ Images were captured using a digital fluorescence microscope.

Measurement of Prostanoids
PGE₂, 6-keto-PGF₁, (a PGI₂ metabolite), and TXB₂ (a TXA₂ metabolite) were detected in the supernatants of cell cultures using previously validated radioimmunoassays.¹⁹

Statistical Analysis
Data were analyzed using the two-tailed, two-sample t test (Minitab, statistical analysis software, State College). Values of P<0.05 were considered significant.

An expanded Materials and Methods section can be found in the online data supplement available at http://www.circresaha.org.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
TRAIL Stimulates eNOS Activation via Ser¹¹⁷⁷ Phosphorylation
HUVECs represent a valuable model in which to assess the biological activity of TRAIL, because they express all TRAIL transmembrane receptors (TRAIL-R1, TRAIL-R2, TRAIL-R3, and TRAIL-R4) both at the mRNA and surface protein level (Figures 1A and 1B). TRAIL did not induce apoptosis (constantly <5% over background levels) in HUVECs, even when used at high concentrations (up to 5 µg/mL; data not shown), and it was unable to activate the caspase pathway and/or to induce PARP cleavage (Figure 1C). These findings are similar to those reported by other authors.R5-127474 ^5,6

View larger version (26K): [in this window] [in a new window]   Figure 1. Lack of TRAIL-induced cytotoxicity in HUVECs. A, RNA extracted from HUVECs was used as template for the amplification reaction with primer sets specific for each TRAIL receptor or ß-actin (lanes 1 to 4). Products of each RT-PCR reaction (lane 1, TRAIL-R1; lane 2, TRAIL-R2; lane 3, TRAIL-R3; lane 4, TRAIL-R4) were visualized by ethidium bromide staining. Control reactions performed by amplifying the same RNA samples before RT (-RT) are also shown. Lane M, 100-bp ladder; lane b, blank (samples without RNA). B, Surface TRAIL receptor expression was evaluated by flow cytometry. Shadowed histograms represent cells stained with antibodies specific for the indicated TRAIL receptors, whereas unshadowed histogram represents the background fluorescence obtained from the staining of the same cultures with isotype-matched control Abs. C, HUVECs were either left untreated (Unt.) or treated with TRAIL for 24 hours before Western blot analysis for PARP cleavage and caspase 3 activation. Positive control (Cont.+), lysates from HUVECs that were induced to apoptosis by trophic withdrawal. Intact (118 kDa) and cleaved (85 kDa) PARP and processed caspase 3 (17 kDa) are shown. Data are representative of 4 to 6 independent experiments.

In order to investigate whether TRAIL was able to modulate the NOS/NO pathway in endothelial cells, quiescent HUVECs were exposed to recombinant TRAIL and analyzed for NOS activation and NO production. Of the different isoforms of NOS, HUVECs constitutively express endothelial NOS (type III NOS or eNOS) (Figure 2A). Of note, whereas vascular endothelial cells are capable of generating large amounts of NO through the inducible NOS (type II or iNOS) pathway after stimulation with inflammatory cytokines,⁹ HUVECs are a notable exception, because they are unable to express iNOS even when exposed to a mixture of inflammatory cytokines, such as TNF- and IL-1.²⁰ When quiescent HUVECs were exposed to recombinant TRAIL, a significant increase in phosphorylation of eNOS at Ser¹¹⁷⁷ was noticed in Western blots, performed with a mAb directed against the phosphorylation site of eNOS (Figure 2A). The increase of eNOS phosphorylation by TRAIL started to be statistically significant (P<0.05) at 5 minutes, peaked at 15 to 30 minutes, and declined thereafter (Figures 2A and 2B). Similar/overlapping results were obtained in experiments performed in the presence of polymyxin B sulfate, used to exclude the possibility that LPS potentially contaminating the TRAIL preparations could confound the results. Moreover, neither His-control peptide nor LPS (used up to 10 µg/mL) affected eNOS phosphorylation and NO production activity, demonstrating that stimulation of NO was specifically due to TRAIL (data not shown).

View larger version (36K): [in this window] [in a new window]   Figure 2. TRAIL stimulates eNOS activation via Ser1177 phosphorylation. A, Quiescent HUVECs were stimulated with TRAIL for 0 to 90 minutes. Cell lysates were analyzed for eNOS activation by Western blot analysis of total and Ser1177-phosphorylated eNOS (P-eNOS). B, Protein bands were quantified by densitometry and the level of P-eNOS was calculated for each time point after normalization to eNOS in the same sample. Unstimulated basal expression was set as unity. In A and B, a representative of 3 separate experiments is shown. C, NOS activity was evaluated in HUVECs either left untreated or stimulated for 30 minutes with the indicated concentrations of TRAIL. NOS activity induced by stimulation with 10 nmol/L of Insulin is also shown for comparison. Data are expressed as mean±SD of 4 separate experiments performed in duplicate (*P<0.05, TRAIL and insulin vs untreated).

In parallel experiments, the NOS activity was assessed in HUVEC lysates (Figure 2C). As expected on the basis of the Western blot data illustrated above, TRAIL treatment induced a dose-dependent increase in NOS activity. It should be noticed that the peak of NOS stimulation by TRAIL (100 to 1000 ng/mL) was similar to that induced by insulin (10 nmol/L) (Figure 2C), and comparable to that reported by other authors,R21-127474 R22-127474 R23-127474 ^21–24 in response of potent NOS agonists, such as vascular endothelial growth factor (VEGF), angiotensin II, and fluid shear stress (FSS). The increase of NOS activity induced by TRAIL started to be statistically significant (P<0.05) over background levels of untreated controls at concentrations as low as 10 ng/mL. This concentration is in the range of those reported in the plasma of several groups of patients.²⁵ It should also be noticed that the local concentrations of TRAIL in the intima microenvironment are expected to be significantly higher than the plasmatic concentrations, taking into account that the major source of TRAIL in the vessel wall is represented by medial smooth cell layer.⁷

TRAIL Induces NO Production Independently of Intracellular Ca²⁺ Flux
We next investigated whether eNOS-expressing cells were able to generate bioactive NO. For this purpose, we first measured the formation of cGMP, a good proxy for NO, because soluble guanylate cyclase is activated by nanomolar concentrations of the gas.¹⁷ Exposure to TRAIL resulted in a significant (P<0.01) increase in cGMP over controls (68±2 and 28±3 fmol/10⁶ cells, respectively; n=4), which was inhibited by the presence of L-NAME (34±2 and 30±2 pmol/mg min^-1, respectively; n=4), a competitive inhibitor of NOS. In parallel, NO production was also measured by loading HUVECs with the NO-specific fluorescence dye DAF-2 DA, a cell-permeable compound that is converted to DAF-2 by intracellular esterases. In the presence of NO, DAF-2 forms a triazole derivative that emits light at 515 nm on excitation at 489 nm, in proportion to the amount of NO present. When these cultures were excited with light at 489 nm, TRAIL caused an asynchronous increase in fluorescence at 515 nm, indicative of a rise in intracellular NO in approximately 40% of the cells examined, which was completely abrogated by pretreatment of the cells with L-NAME (Figures 3A and 2B), but not by pretreatment of the cells with the calcium chelator BAPTA (Figure 3B). Moreover, calcium-imaging studies performed after exposure to TRAIL clearly indicated that TRAIL failed to induce intracellular Ca²⁺ flux in HUVECs (Figure 3C).

View larger version (43K): [in this window] [in a new window]   Figure 3. TRAIL induces NO production independently of intracellular Ca2+ flux. A, DAF-loaded HUVECs were acquired at different time intervals during TRAIL stimulation in the absence or presence of L-NAME (0.5 mmol/L). Fluorescence emissions from DAF-2DA were captured and the relative intensity values depicted in pseudocolor (blue is the minimum and red the maximum DAF intensity, respectively). Insets in the upper left corner of each image show a magnification of cell portions containing DAF-positive spots. B, Traces deriving from fluorescence temporal analysis during TRAIL stimulation of DAF-2DA–loaded cells cultured in the indicated conditions (BAPTA, 10 µmol/L). Stimulation with 5 µmol/L of LPA (a phospholipid growth factor that induce rise of NO production via mobilization of intracellular Ca2+) is shown for comparison. C, Trace deriving from fluorescence temporal analysis during TRAIL and ionomycin (Iono.) stimulation of FLUO4/AM-loaded HUVECs. In B and C, Traces deriving from total cellular area were calculated as F/F0, where F is the fluorescence emission of a single DAF-2DA- or FLUO4/AM-loaded cell, respectively, at time intervals ranging from 2 to x seconds, and F0 is the fluorescence emission of the same cell at time 0. Results are representative of 3 to 6 experiments. D, NOS activity was evaluated in HUVECs either left untreated (vehicle) or stimulated for 30 minutes with TRAIL, in the absence or presence of the indicated pharmacological inhibitors. Data are expressed as mean±SD of 4 separate experiments performed in duplicate (*P<0.05, TRAIL vs untreated).

In order to further investigate the intracellular pathway(s) involved in eNOS activation by TRAIL, HUVECs were pretreated with LY294002, a selective pharmacological inhibitor of the PI 3 kinase/Akt pathway. In agreement with other authors’ studies reporting eNOS Ser¹¹⁷⁷ phosphorylation by Akt,R21-127474 R22-127474 R23-127474 ^21–24 LY294002 completely abrogated the TRAIL-induced NO production (data not shown) and the NOS activity, at the same extent of L-NAME (Figure 3D). Taken together, these data indicate that eNOS is activated by TRAIL through a PI 3 kinase/Akt pathway and is independent of the induction of intracellular Ca²⁺ fluxes.

The apparent time-course discrepancy between NO production investigated by DAF-2 dye staining plus confocal microscopy analysis and eNOS phosphorylation, as evaluated by Western blot, can be readily explained by taking into account that Figures 3A and 3B show cells that are immediately activated by TRAIL, whereas data shown in Figure 2 are derived from the bulk HUVEC population, in which the number of responsive cells progressively increases with time.

NO-Dependent Cell Migration and Actin Reorganization Upon Exposure to TRAIL
NO has been proposed to modulate cell migration and to be essential for podokinesis.⁹ We thus investigated whether the activation of NO induced by TRAIL was sufficient to drive endothelial cell migration by measuring the transfilter migration of cells seeded on a membrane separating the lower and upper part of a 6.5-mm Transwell. TRAIL dose-dependently (P<0.05, starting at 1 to 10 ng/mL) promoted endothelial cell migration (Figure 4A). Accordingly, with the notion that cell migration is tightly associated with formation of stress fibers,²² we found that TRAIL, similarly to VEGF, induced profound cytoskeletal reorganization characterized by the formation of transcytoplasmic stress fibers (Figure 4B).

View larger version (52K): [in this window] [in a new window]   Figure 4. TRAIL induces migration and the formation of actin stress fibers in HUVECs. A, HUVECs were plated in the upper compartment of a 6.5-mm Transwell in the absence or presence of L-NAME. TRAIL was added in the lower wells, and the cells that migrated through the filter were counted after 4 hours. Data are expressed as the number of migrated cells in 5 high-power fields and are the average±SD of results from 3 experiments each performed in duplicate (*P<0.05, TRAIL vs untreated). B, HUVECs plated on gelatin-coated slides were left untreated or treated as indicated for 20 minutes. Filamentous actin was detected using Texas Red X-phalloidin. Images were captured under x40 magnification. Representative fields are shown.

The TRAIL-induced increase in cell migration and the actin reorganization into stress fibers were mediated by NOS activity, as indicated by the inhibition after preincubation with L-NAME (Figures 4A and 4B).

TRAIL Induces Prostanoid Production by HUVECs
A complex interplay between the NOS and COX pathways has been demonstrated.R26-127474 ^26,27 Therefore, also taking into consideration the key role of prostanoids in vascular biology,¹³ in the next group of experiments we have analyzed the release of prostanoids in HUVEC supernatants collected after treatment with either TRAIL or TNF-, used as positive control.²⁸ As expected, at 24 hours, TNF- induced a several fold increase in all prostanoids examined (PGE₂>PGI₂>TXA₂; Table). Of note, TRAIL also induced a significant (P<0.01) increase in prostanoid production (PGE₂=PGI₂>TXA₂) over basal levels detected in unstimulated cells, although lower that that observed in cultures treated with TNF- (Table). In time-course experiments, the peak induction of prostanoids by TRAIL was reached after 4 to 6 hours of treatment, showing a plateau thereafter. On the other hand, TNF- induced maximal prostanoid production after 24 hours (data not shown). In parallel experiments, primary HUVECs were treated with TRAIL or TNF- for 24 hours, washed, and prostanoid production was measured after incubation of equal numbers of viable cells (10⁶) with 10 µmol/L of exogenous AA. This type of examination ensures a constant substrate concentration. The results obtained under these conditions were similar to those obtained with measurement of spontaneous prostanoid release (Table). It is also noteworthy that TRAIL induced an approximately 2-fold induction of both PGI₂, which represent the key prostanoid controlling vascular tone, and PGE₂, which plays a major role in inflammation and vascular permeability.¹³ On the other hand, TNF- was significantly more efficient in inducing the production of PGE₂.

View this table: [in this window] [in a new window]   Table 1. In Vitro Prostanoid Production in HUVEC Cultures, Either Left Untreated or Treated With TRAIL or TNF- for 24 Hours

TRAIL Does Not Affect COX Isoenzyme Expression and Does Not Induce IkB Degradation in HUVECs
In the following experiments, we examined whether TRAIL was also able to modulate the expression of COX-1 and COX-2 isoenzymes, the rate limiting enzymes involved in the production of different classes of prostanoids.¹³ In Western blot analysis, unstimulated HUVECs showed a strong constitutive expression of COX-1 and a less intense, but detectable, expression of COX-2 (Figure 5). As expected,²⁸ TNF- strongly upregulated COX-2 protein from 6 hours onwards without affecting the constitutive COX-1 expression; on the other hand TRAIL did not affect either COX-1 or COX-2 expression (Figure 5).

View larger version (36K): [in this window] [in a new window]   Figure 5. TRAIL does not affect COX isoenzyme expression in HUVECs. HUVECs cells were exposed to either TNF- or TRAIL and incubated for the indicated times (0 to 24 hours). Cell lysates were analyzed for COX-1 and COX-2 expression by Western blotting using specific antibodies to each. One of 4 experiments with similar results is shown.

In parallel experiments, we investigated the ability of TRAIL and TNF- to modulate NF-B transcription factor, taking advantage of the fact that NF-B activation is normally prevented by the inhibitory family of IkB proteins.²⁹ Consistent with the requirement of NF-B for the transcriptional upregulation of COX-2 expression,R27-127474 ^27,28 TNF- induced IkB degradation, followed by NF-B–driven increase in protein due to resynthesis of the inhibitor (Figure 6). IkB degradation occurred over much the same time course as IkB. On the other hand, TRAIL did not induce any degradation of IkB and IkB, nor was there any effect on the synthesis of IkB or IkB (Figure 6).

View larger version (23K): [in this window] [in a new window]   Figure 6. TRAIL does not affect IkB degradation in HUVECs. HUVECs were exposed to either TNF- or TRAIL and incubated for the indicated time intervals (0 to 60 minutes). Cell lysates were analyzed for degradation of IkB and IkB by Western blotting using specific antibodies to each. One of 4 experiments with similar results is shown.

TRAIL-Mediated Prostanoid Production Is Preferentially Inhibited by the Selective COX-1 Inhibitor SC-560
We next investigated the inhibition of prostanoid production induced by treatment with either TRAIL or TNF- by preincubating HUVECs with serial doses of the selective COX-1 and COX-2 pharmacological inhibitors. The nonselective COX-inhibitor indomethacin suppressed basal, TRAIL-, and TNF-–mediated PGE₂ production (Figures 7A and 7B). On the other hand, in TRAIL-treated cultures, SC-560 (a COX-1 inhibitor) dose-dependently suppressed the production of PGE₂ to a significantly (P<0.05) higher extent than equimolar doses of NS-398 (a COX-2 inhibitor) (Figure 7A). In sharp contrast, in TNF-–treated cultures, NS-398 was significantly (P<0.01) more potent than SC-560 in suppressing PGE₂ production (Figure 7B). These data clearly indicate that COX-1 and COX-2 play predominant roles in the production of PGE₂ by TRAIL and TNF-, respectively. To evaluate the possible interplay of the NOS and COX signaling pathways, the effect of the NOS inhibitor L-NAME was also tested on prostanoid production. L-NAME showed minimal effects on basal PGE₂ production, whereas it induced opposite effects on TRAIL- (Figure 7A) and TNF-– (Figure 7B) treated cultures. In fact, it showed a partial but significant (P<0.01) reduction in the TRAIL-mediated increase of PGE₂ production, while it strongly enhanced (<0.01) TNF-–mediated increase of PGE₂ production.

View larger version (32K): [in this window] [in a new window]   Figure 7. Effects of specific pharmacological inhibitors on TRAIL- or TNF-–induced prostanoid production. Spontaneous release of PGE2 was measured in the culture supernatants of HUVECs treated as indicated. Serum-free culture supernatants were harvested 24 hours after treatment of cells with TRAIL (A) or TNF- (B) in the presence or absence of the indicated inhibitors (L-NAME, 0.5 mmol/L; Indomethacin, 25 µmol/L; SC-560 and NS-398, 0.1 to 100 µmol/L). Data are expressed as mean±SD of 4 experiments (*P<0.05, nil vs inhibitors).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The identification of TRAIL/Apo-2L several years ago generated a great deal of interest when it was discovered that it was able to induce apoptosis both in vitro and in vivo in a variety of tumor cell lines, but not in normal cells.R3-127474 ^3,4 Moreover, it was documented that TRAIL mRNA was constitutively expressed in a wide variety of cells and tissues.¹ These were unusual characteristics for a death-inducing molecule of the TNF family, as the expression of TNF and CD95L is tightly regulated because they can manifest toxic effects on normal tissues.¹

We have here demonstrated for the first time that the addition of TRAIL to HUVECs induces the production of NO through an Akt/eNOS pathway and increases the production of prostanoids via COX-1. Due to the complexity of the TRAIL receptor system comprising 4 transmembrane receptors that are all expressed by HUVECs to a comparable extent, it is possible that specific TRAIL receptors are differentially involved in the activation of specific intracellular pathways. In particular, TRAIL induced a time- and dose-dependent increase in NO production paralleled by a significant increase in eNOS phosphorylation, NOS activity, and cGMP production. Several studies have shown that Ca²⁺/calmodulin and phosphorylation have coordinate roles in regulating the activity of eNOS and are necessary for complete activation of eNOS in response to VEGF and angiotensin II.R21-127474 R22-127474 R23-127474 R24-127474 ^21–24,30 In the present study, we have observed that TRAIL was unable to increase the intracellular Ca²⁺ levels but it rather stimulated NO production in a way that was completely blocked by pharmacological inhibitors of the PI 3 kinase/Akt pathway.

The ability of TRAIL to upregulate eNOS activity is particularly noteworthy taking into account that eNOS is an important regulator of cardiovascular homeostasis, and it is the major source of NO production in vascular endothelial cells. NO was first identified as an endothelium-derived relaxing factor (EDRF),⁹ originally discovered by Furchgott and Zawadzki.³¹ Its critical role in hemodynamic homeostasis has been unequivocally demonstrated by the presence of hypertension in eNOS knockout mice.³² In addition, NO released from the endothelium modulates other processes, including platelet aggregation, platelet and leukocyte adhesion to the endothelium, vascular smooth muscle cell proliferation, and angiogenesis.R9-127474 R10-127474 R11-127474 R12-127474 ^9–12,33 In particular, angiogenesis, the process of new blood vessel formation from preexisting ones, is composed of several discrete steps including dissolution of matrix, endothelial cell migration, proliferation, and organization into a network structure, followed by lumen formation. NO had been implicated in all the above-mentioned processes in a manner consistent with a proangiogenic phenotype. In this respect, we have demonstrated here that TRAIL actively promoted actin reorganization and migration of HUVECs similarly to VEGF, a well-known angiogenic factor. Moreover, both TRAIL-mediated actin reorganization and migration were completely inhibited by L-NAME.

A substantial difference between TRAIL and other cytokines active on endothelial cells, such as VEGF, angiotensin II, and bradykinin, which are able to simultaneously induce NO production and NF-B activation,R23-127474 R29-127474 ^23,29,34 is represented by the fact that TRAIL does not activate NF-B in HUVECs. These findings are particularly remarkable, because it has been clearly established that NO exerts its antiinflammatory and antiarteriosclerosis actions mainly by inhibiting NF-B activation.R35-127474 ^35,36 In this respect, it is generally accepted that the vascular endothelium becomes dysfunctional in the early stages of vascular diseases, which are characterized by NF-B–dependent activation of inflammatory markers. This eventually leads to inflammation, leukocyte adhesion, and arteriosclerosis. In this respect, it is noteworthy that previous studies have demonstrated that eNOS-derived NO acts as an endogenous inhibitor of LPS- or TNF-–induced NF-B activity and COX-2 transcription.R33-127474 ^33,37 As expected due to the central role of NF-B in COX-2 upregulation and to the inhibitory role of NO on COX-2 expression and activity,R27-127474 ^27,33 TRAIL did not affect COX-2 expression. However, TRAIL induced a moderate but significant upregulation of prostanoid production/release.

Although we have not addressed the molecular mechanism by which TRAIL stimulates COX activity in HUVECs, it has been previously demonstrated that NO, besides inhibiting COX-2, is able to activate COX-1,²⁷ which under normal conditions regulates prostaglandin production and thereby affects basal vascular tone and normal cell activity.²⁹ Two lines of evidences suggest that TRAIL promotes COX-1 in a NO-dependent manner. First, the addition of the pharmacological inhibitor of the NOS pathway, L-NAME, increased the production of prostanoids in TNF-–treated cultures, whereas it reduced PGE₂ released by TRAIL-treated cultures. This is consistent with the high susceptibility of COX-2 to NO inhibition,²⁷ which is due to the NO-mediated inhibition of NF-B and to the direct nitrosylation of COX-2 protein. Second, the COX-1 selective SC-560 inhibitor was significantly more potent that the COX-2 inhibitor NS-398 in abrogating the TRAIL-mediated increase in prostanoid release. The ability of TRAIL to activate COX-1 in HUVECs is reminiscent of the effect of the angiogenic cytokine VEGF,³⁸ which stimulated the constitutive COX-1 but not the inducible isoform COX-2.

Although experiments performed in more intact paradigms besides cultured endothelial cells, such as animal models, would strengthen our results obtained in cultured HUVECs, our data indicate that TRAIL likely plays an important vasoprotective function on endothelial cells due to its ability to increase the biosynthesis and release of both PGE₂ and PGI₂ without affecting NF-B activity. In fact, PGE₂ and PGI₂, together with NO, regulate the vascular tone and permeability, calm down activated platelets and leukocytes, prevent the occurrence of parietal thrombotic events, promote thrombolysis, maintain tissue perfusion and protect vascular wall against acute damage and chronic remodeling, and promote angiogenesis.

	Acknowledgments

 
This study was supported by grants from the Italian Association for Cancer Research (AIRC) and from the Ministero dell’Universita’ e della Ricerca Scientifica e Tecnologica (MIUR).

	Footnotes

 
*Both authors contributed equally to this work.

Received August 15, 2002; revision received March 11, 2003; accepted March 11, 2003.

	References

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