Published online before print January 2, 2009, doi: 10.1161/CIRCRESAHA.108.191114
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© 2009 American Heart Association, Inc.
Molecular Medicine |
Induction of Prostacyclin by Steady Laminar Shear Stress Suppresses Tumor Necrosis Factor-
Biosynthesis via Heme Oxygenase-1 in Human Endothelial Cells
From the Department of Medicine and Aging (L.D.F., M.D., A.D.F., T.S., P.P.), "G. d’Annunzio" University, Chieti, Italy; CeSI (L.D.F., M.D., A.D.F., T.S., A. Pandolfi, P.P.), Chieti, Italy; Mario Negri Sud (L.T., A. Piccoli, V.E.), Santa Maria Imbaro, Chieti, Italy; and Departments of Pharmacology and Toxicology (R.A.D.) and Physiology and Biochemistry (F.S.), Queen’s University, Kingston, Ontario, Canada.
Correspondence to Paola Patrignani, PhD, Sezione di Farmacologia, Dipartimento di Medicina e Scienze dell’Invecchiamento, Università "G. d’Annunzio", Via dei Vestini, 31, 66100 Chieti, Italy. E-mail ppatrignani{at}unich.it
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Abstract |
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Cyclooxygenase (COX)-2 is among the endothelial genes upregulated by uniform laminar shear stress (LSS), characteristically associated with atherosclerotic lesion-protected areas. We have addressed whether the induction of COX-2–dependent prostanoids in endothelial cells by LSS plays a role in restraining endothelial tumor necrosis factor (TNF)-
generation, a proatherogenic cytokine, through the induction of heme oxygenase-1 (HO)-1, an antioxidant enzyme. In human umbilical vein endothelial cells (HUVECs) exposed to steady LSS of 10 dyn/cm2 for 6 hours, COX-2 protein was significantly induced, whereas COX-1 and the downstream synthases were not significantly modulated. This was associated with significant (P<0.05) increase of 6-keto-prostaglandin (PG)F1 (the hydrolysis product of prostacyclin), PGE2, and PGD2. In contrast, TNF- released in the medium in 6 hours (3633±882 pg) or detected in cells lysates (1091±270 pg) was significantly (P<0.05) reduced versus static condition (9100±2158 and 2208±300 pg, respectively). Coincident induction of HO-1 was detected. The finding that LSS-dependent reduction of TNF- generation and HO-1 induction were abrogated by the selective inhibitor of COX-2 NS-398, the nonselective COX inhibitor aspirin, or the specific prostacyclin receptor (IP) antagonist RO3244794 illuminates the central role played by LSS-induced COX-2–dependent prostacyclin in restraining endothelial inflammation. Carbacyclin, an agonist of IP, induced HO-1. Similarly to inhibition of prostacyclin biosynthesis or activity, the novel imidazole-based HO-1 inhibitor QC15 reversed TNF- reduction by LSS. These findings suggest that inhibition of COX-2–dependent prostacyclin might contribute to acceleration of atherogenesis in patients taking traditional nonsteroidal antiinflammatory drugs (NSAIDs) and NSAIDs selective for COX-2 through downregulation of HO-1, which halts TNF- generation in human endothelial cells.
Key Words: cyclooxygenase • endothelial cells • prostaglandins • prostanoids • shear stress • tumor necrosis factor-
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Prostanoids, including prostaglandin (PG)E2, PGF2
, PGD2, prostacyclin (PGI2), and thromboxane (TX)A2, are lipid autacoids, immediately released outside the cell after intracellular biosynthesis, that modulate a wide variety of physiological and pathological processes.1 They are generated by 3 sequential enzymatic steps involving: (1) phospholipase A2 enzymes, (2) cyclooxygenase (COX) enzymes (ie, COX-1 and COX-2),2 and (3) tissue-specific synthases (3 different enzymes for PGE2: cytosolic PGE2 synthase [cPGES], microsomal PGE2 synthase-1 [mPGES-1] and -2 [mPGES-2]; PGF synthase [PGFS]; 2 enzymes for PGD2: lipocalin-type PGD2 synthase [L-PGDS] and hematopoietic-type PGD2 synthase [H-PGDS]; PGI2 synthase [PGIS], thromboxane synthase [TXS]).3–9 Two isoforms of COX (COX-1 and COX-2) have been cloned and characterized.2 COX-1 is considered a "housekeeping gene" by virtue of constitutive low levels of expression in most cell types. Differently, the gene for COX-2 is a primary response gene with many regulatory sites; thus, COX-2 expression can be rapidly induced by bacterial endotoxin (lipopolysaccharide), cytokines, such as interleukin (IL)-1β, and tumor necrosis factor (TNF)-, growth factors, and tumor promoter phorbol myristate acetate.10 However, COX-2 is constitutively expressed in some cells in lung,11 brain,12 kidney,13 pancreatic β-cells,14 and gastric carcinomas.15,16 In endothelial cells, COX-2 is among the vasoprotective genes upregulated by steady laminar shear stress (LSS),17 which characterizes "atherosclerotic lesion-protected areas."18 In addition to COX-2, endothelial cells constitutively express also COX-1.19 Thus, the contribution of COX isozymes to endothelial prostanoid generation, in physiological conditions, is still controversial.20 PGI2 is considered a major prostanoid generated in the macrocirculation (both in endothelial cells and vascular smooth muscle cells), where it acts as a general restraint on endogenous stimuli to platelet activation, vascular proliferation and contraction, and cell adhesion.21 PGI2 acts mostly through I prostanoid receptor (IP), a rhodopsin-like class A, 7-transmembrane-spanning G protein–coupled receptor (GPCR), which activates membrane-bound adenylyl cyclase and the subsequent formation of the second messenger cAMP.1 cAMP triggers a host of cellular responses including inhibition of platelet aggregation, promotion of vascular smooth muscle cell relaxation, and induction of thrombomodulin, an important inhibitor of blood coagulation.22,23 Recently, it has been reported an antioxidant role for PGI2 through the induction of heme oxygenase (HO)-1.24
HO-1 (32-kDa stress-inducible protein) catalyzes the degradation of heme to liberate free iron, carbon monoxide (CO) and biliverdin in mammalian cells.25 HO-1 induction represents a cytoprotective defense mechanism against oxidative insults through the contribution of different actions: (1) the removal of free heme because this tetrapyrrole is a prooxidant that catalyzes the decomposition of organic peroxides leading to the generation of alkyl peroxyl radicals26; (2) the antioxidant activities of biliverdin and its metabolite, bilirubin; and (3) the antiinflammatory action of CO.25 There is a considerable body of evidence that suggests that HO-1 plays an important protective role in the pathogenesis of cardiovascular disease. For example, induction of HO-1 in vascular cells suppresses oxidized low-density lipoprotein–induced monocyte transmigration and inhibits atherosclerotic lesion formation in low density lipoprotein receptor knockout mice.27,28 Interestingly, the levels of bilirubin in the normal human population correlate inversely with the incidence of atherosclerotic events,29 and it has been shown that bilirubin attenuates vascular endothelial activation and dysfunction in vitro.30 Finally, it has been recently shown that in human monocytes, HO-1 activity is involved in attenuation of TNF- production.31
TNF- is a proinflammatory cytokine that contributes to atherogenesis. High levels of TNF- have been found in endothelial cells of human atheroma.32 Recently, it has been shown that human umbilical vein endothelial cells (HUVECs) stimulated with lipopolysaccharide or IL-1 can generate TNF-.33 TNF- is produced as a membrane-bound precursor protein with a relative molecular mass of 26 kDa. This precursor protein is processed by a membrane-bound metalloproteinase, TNF-–converting enzyme, to generate secreted 17-kDa mature TNF-.34 Secreted TNF- may be involved in autocrine activation of endothelial cells, and TNF- retained in cell membrane may serve as a juxtacrine system to activate target cells on the endothelial surface. Recently, it has been shown that intravascular administration of anti–TNF- antibody ameliorates endothelial function in patients with rheumatoid arthritis (RA), but does not concurrently affect systemic inflammatory changes, suggesting that its increased generation within the vessel wall may be involved in endothelial dysfunction in RA.35
We have addressed whether the induction of COX-2–dependent prostanoids in endothelial cells by uniform LSS (characteristically associated with lesion-protected areas) plays a role in restraining endothelial TNF- generation through the induction of HO-1. To accomplish this objective we have applied a strategy to examine simultaneously the release of prostanoids and the expression of COX-isozymes and downstream synthases in cultured HUVECs activated by uniform LSS. In the same experimental conditions, we assessed TNF- levels both secreted and retained in endothelial cells and HO-1 expression. COX-2 was the only protein of the biosynthetic machinery of prostanoids, which was significantly upregulated by LSS. The finding that LSS-dependent reduction of TNF- generation and HO-1 induction were completely countered by the selective inhibitor of COX-2 NS-398, the nonselective COX inhibitor aspirin, or the specific IP antagonist RO324479436 illuminates the central role played by LSS-induced COX-2–dependent PGI2 in restraining endothelial inflammation. Finally, we showed that HO-1 is a key mechanism in the vasoprotective phenotype induced by PGI2. In fact, (1) carbacyclin, an agonist of IP, induced HO-1, whereas an IP blocker reduced HO-1 expression; and (2) similarly to inhibition of PGI2 biosynthesis or activity, the novel imidazole-based HO-1 inhibitor QC1537 reversed TNF- reduction by LSS. These findings suggest that inhibition of COX-2–dependent PGI2 might contribute to acceleration of atherogenesis in patients taking traditional NSAIDs and NSAIDs selective for COX-2 through downregulation of HO-1 which halts TNF- generation in human endothelial cells.
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Materials and Methods |
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Endothelial Cell Cultures
HUVECs were isolated from normal-term umbilical cords, as previously described,38 used at passage level 2 or 3, and grown39 as described in the online data supplement, available at http://circres.ahajournals.org.
Exposure of HUVECs to Steady LSS
HUVECs (0.8 to 1x106 cells per glass slide) were shear stressed, using a parallel plate flow chamber connected to a constant pressure drop flow loop,40 maintained at 37°C and gassed continuously with a humidified mixture of 5% CO2 in air. Endothelial monolayers were continuously perfused in a closed circuit at an estimated shear stress of 10 dyn/cm2 (flow rate of 2.53 mL/min; shear rate of 1400 sec–1) with 7 mL of perfusion DMEM-medium199 (50% vol/vol), supplemented with 5% FCS, 1% glutamine, and antibiotics for 6 hours. Matched control cells were cultured under static condition in parallel.
Pharmacological Treatments
The nonselective COX inhibitor aspirin (Sigma-Aldrich, St Louis, Mo), the COX-2 inhibitor NS-398 (Sigma-Aldrich), IP receptor antagonist RO3244794 (R-3-(4-fluoro-phenyl)-2-[5-(4-fluoro-phenyl)-benzofuran-2-ylmethoxycarbonylamino]-propionic acid)36 (kindly provided by Roche, Palo Alto, Calif) were dissolved in DMSO and the HO-1 inhibitor QC15 (2-[2-(4-chlorophenyl)ethyl]-2-[(1H-imidazol-1-yl)methyl]-1,3-dioxolane hydrochloride)37 (kindly provided by Drs Walter A. Szarek and Kanji Nakatsu, Queen’s University, Kingston, Ontario, Canada) in distilled water. Seven microliters of vehicles (DMSO or distilled water) or stock solutions of the different compounds were added to 7 mL of perfusion medium to give final concentrations of 25 µmol/L for aspirin, 1 µmol/L for NS-398, 10 µmol/L for RO3244794, and 50 µmol/L for QC15, respectively. Aspirin 25 µmol/L was shown to completely inhibit the activity of platelet COX-141 and COX-2 induced in HUVECs by IL-1β (Figure I, A, in the online data supplement). NS-398 1 µmol/L was previously shown to completely suppress the activity of monocyte COX-2 without affecting platelet COX-1.42 NS-398 1 µmol/L completely suppressed COX-2 activity induced in HUVECs by IL-1β (supplemental Figure I, B).
Finally, HUVECs, in static condition, were treated with an agonist of IP, carbacyclin (Sigma-Aldrich), in the absence and in the presence of RO3244794. Carbacyclin and RO3244794 were dissolved in DMSO, and 2 µL of vehicle or stock solution of the compounds were added to 2 mL of culture medium to give final concentrations of 1 and 10 µmol/L, respectively.
Biochemical Analyses
6-keto-PGF1, PGE2, PGF2, and TXB2 levels were measured in cell culture media by previously described and validated radioimmunoassay techniques.43,44 PGD2 levels were measured by enzyme immunoassay (Cayman Chemical, Ann Arbor, Minn). TNF- levels released in the medium and in cell lysates were determined by ELISA (Pierce, Rockford, Ill), according to the protocol of the manufacturer.
Western Blot Analysis
A detailed description of the technical procedure and the dilution of antibodies used to detect COX-1, COX-2, PGIS, TXS, PGFS, HO-1, mPGES-2, mPGES-1, cPGES, L-PGDS, and H-PGDS in HUVEC lysates is reported in the online data supplement. Quantification of optical density (OD) of different specific bands was calculated using laser densitometry (Bio-Rad Laboratories, Hercules, Calif) and normalized to the OD of β-actin.
Statistical Analysis
All values were reported as means±SEM. Statistical analysis was performed with Student’s t test or 1-way ANOVA and Newman–Keuls multiple comparisons test. Values of P<0.05 were considered statistically significant. Analysis and graphing were performed in GraphPad Prism (version 4.00 for Windows, GraphPad).
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Results |
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Exposure of HUVECs to Steady LSS Induced COX-2 but Not COX-1 and Downstream Synthases
Western blot analysis showed that static HUVECs (control), cultured for 6 hours, expressed COX-1, PGIS, TXS, PGFS, L-PGDS, cPGES, and mPGES-2 but not mPGES-1 and H-PGDS. A low expression of COX-2 was detected (Figure 1A and 1B). In HUVECs subjected to steady LSS (10 dyn/cm2, characteristically associated with lesion-protected areas) for 6 hours, COX-2 was significantly (P<0.05) induced versus static condition (by
7-fold), whereas COX-1 and the downstream synthases were not significantly induced (Figure 1A and 1B). cPGES was significantly (P<0.01) reduced by 45%.
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Exposure of HUVECs to Steady LSS Induced the Synthesis of PGI2, PGE2, and PGD2
In static HUVECs cultured for 6 hours, 6-keto-PGF1 (the hydrolysis product of PGI2), PGE2, PGD2, and PGF2 concentrations were significantly higher than those detected in cell-free medium (DMEM–medium 199) containing 5% FCS (Table). High levels of TXB2 (the stable hydrolysis product of TXA2) were detected in cell-free medium containing 5% FCS (it was a contaminant of FCS) thus preventing to assess the possible release of endogenous TXB2 by HUVECs (Table). The rank order of prostanoid released into the medium was PGF2>PGE2=6-keto-PGF1>PGD2 (Table). Exposure of HUVECs to steady LSS induced (P<0.05) the synthesis of PGI2, PGE2 and PGD2 (Figure 2), whereas PGF2 generation was reduced (P<0.05) versus static condition (supplemental Figure II, A). LSS did not elicit any significant change in TXB2 levels versus those detected in cell-free medium and static condition (supplemental Figure II, B).
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Differential Contribution of COX-2 and COX-1 to LSS-Induced Prostanoid Generation
As shown in Figure 3A through 3C, NS-398 1 µmol/L, at a concentration that selectively inhibited COX-2 activity, significantly (P<0.05) reduced 6-keto-PGF1 (by 33%) without affecting PGE2 and PGD2 generation. In contrast, aspirin 25 µmol/L, at a concentration that inhibits both COX-1 and COX-2, significantly (P<0.05) reduced 6-keto-PGF1 (by 56%), PGE2 (by 27%) and PGD2 (by 31%). These results suggest that, in LSS-stimulated HUVECs, both COX-2 and COX-1 contributed to PGI2 generation, whereas only COX-1 contributed to PGE2 and PGD2.
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P<0.01 vs NS-398.
LSS-Induced COX-2–Dependent PGI2 Restrains Endothelial Inflammation
Exposure of HUVECs to steady LSS for 6 hours caused a significant reduction (versus static condition) of TNF- released in the medium (3633±882 and 9100±2158 pg, respectively; P<0.05) or in cells lysates (1091±270 and 2208±300 pg, respectively; P<0.05) (Figure 4A). In the presence of NS-398 or aspirin, reduction of TNF- by LSS was completely abrogated (P<0.05) (Figure 4B). The comparable effect obtained by the selective COX-2 inhibitor NS-398 and the nonselective COX inhibitor aspirin supports the role of LSS-induced COX-2 in constraining TNF- biosynthesis. The contribution of PGI2 over other prostanoids induced by steady LSS to halt endothelial inflammation was unraveled by using the specific IP antagonist, RO3244794. The compound caused a complete recovery of the reduction of TNF- induced by LSS (Figure 4B).
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HO-1 Induction Mediates the Vasoprotective Phenotype Induced by PGI2
LSS caused the induction of HO-1 (Figure 5A and 5B). As shown in Figure 5A and 5B, NS-398, aspirin, and RO3244794 significantly reduced HO-1 induction by exposure of HUVECs to steady LSS. These results suggest the participation of COX-2–dependent PGI2 in the induction of HO-1 by steady LSS. This is further sustained by the finding that carbacyclin, an agonist of IP, induced HO-1 in HUVECs cultured in static condition (Figure 5C). This effect was abrogated by the IP antagonist RO3244794, suggesting a specific action of carbacyclin on IP receptor (Figure 5C).
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As shown in Figure 6, similarly to inhibition of PGI2 biosynthesis by NS-398 and aspirin or IP antagonism by RO3244794, inhibition of HO-1 activity by the novel imidazole-based HO-1 inhibitor QC1537 abrogated TNF- reduction by LSS. These findings suggest that (1) upregulation of HO-1 represents a key mechanism associated with the induction of a protective phenotype by uniform LSS and (2) COX-2–dependent PGI2 contributes to this effect.
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Discussion |
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COX-2 is among the endothelial genes upregulated by uniform LSS, characteristically associated with atherosclerotic lesion-protected areas.17 In fact, COX-2 is the source of PGI2 biosynthesis in humans which exhibits properties of relevance to atheroprotection, inhibiting platelet activation, vascular smooth muscle contraction and proliferation, leukocyte–endothelial cell interactions, and cholesteryl ester hydrolase.21 PGI2 analogs retard atherogenesis,45 and atherosclerotic lesions exhibit a decreased capacity to produce PGI2 ex vivo.46 Egan et al24 showed that PGI2 serves an antioxidant function before and at initiation of atherogenesis through increased expression of the antioxidant HO-1. However, questions are under discussion on the possible contribution of endothelial COX-1 to PGI2 biosynthesis and of endothelial COX-2 to the generation of other prostanoids, in particular PGE2.20 The role of PGE2 in cardiovascular homeostasis is more complex than PGI2 because it transduces by four E prostanoid receptors (EPs), which mediate contrasting biologies. Two of them, EP2 and EP4, are linked to Gs-mediated activation of adenylyl cyclase, whereas the 2 others, EP1 and EP3, are linked to Gq and/or Gi.47 Recent results suggest a cardioprotective role of PGE2 via EP2 and EP4.48,49 On the other hand, because of its importance in inflammation, it has been suggested that PGE2 may play a role in enhanced plaque burden and plaque destabilization in humans.50
We performed the present study to distinguish between the vasoprotective function of COX-2 and COX-1 in HUVECs exposed to a physiological fluid mechanical stimulus in vitro. Furthermore, we addressed whether other prostanoids, apart from PGI2, induced by LSS in endothelial cells may contribute to vasoprotection. Because of the recognized role of TNF- in endothelial dysfunction and atherogenesis,32,35 we evaluated the effects of steady LSS on its biosynthesis in HUVECs and the interplay with COX-isozyme activity. We revealed the central role of COX-2–dependent PGI2, induced by uniform LSS, to restrain TNF- generation in the endothelium through a mechanism that involves the action of HO-1. This is a novel protective action of endothelial PGI2 which may work in physiological conditions.
COX-2, almost undetectable in endothelial cells cultured in static condition, was significantly induced by LSS. It has previously shown that both transcriptional activation and posttranscriptional mRNA stabilization contribute to the expression of COX-2 in response to shear stress.17,51
In contrast, COX-1 and downstream synthases involved in the synthesis of TXA2, PGI2, PGF2, and PGD2, ie, TXS, PGIS, PGFS, and L-PGDS, respectively, constitutively expressed in static HUVECs, were not significantly modulated by LSS. We assessed the expression of 3 different enzymes participating in PGE2 biosynthesis7,47: (1) the cytokine-induced mPGES-1, functional coupled with COX-2; (2) the constitutive cPGES, which seems to act functionally coupled with COX-1; and (3) mPGES-2, which was reported to use substrate coming from both COX-1 and COX-2 activities. As previously reported, HUVECs do not express mPGES-152 whereas the other PGESs were constitutively expressed. LSS did not significantly affect mPGES-2, whereas cPGES was significantly reduced. These data suggest the major contribution of mPGES-2 in PGE2 generation in HUVECs exposed to LSS. In addition to PGE2, PGI2 and PGD2 were induced by LSS. By comparing the impact of a COX-2 selective inhibitor (NS-398) and a nonselective COX inhibitor (aspirin), we found that in HUVECs exposed to LSS, both COX-2 and COX-1 contributed to PGI2 generation, whereas only COX-1 contributed to PGE2 and PGD2. In fact, in LSS-stimulated HUVECs, NS-398 significantly affected only PGI2, whereas aspirin reduced all the prostanoids. The more profound inhibition of PGI2 by aspirin than NS-398, unraveled the contribution of both COX-isozymes to PGI2 generation. However, a major contribution of COX-2 versus COX-1 to PGI2 generation by HUVECs in response to LSS was found. This is explained by the fact that PGIS is preferentially coupled with COX-2.7 The finding that aspirin 25 µmol/L, a concentration that suppresses completely both COX-141 and COX-2 activities (supplemental Figure I, A), inhibited LSS-induced PGI2 generation by only 56% may suggest that a rapid generation of PGI2 (presumably COX-1–dependent) occurred before the drug was able to block COX activity. This is coherent with previous results showing that flow induces a burst in PGI2 production followed by a sustained stimulation of production.53 However, this immediate but short-lasting release is not playing a functional effect because of the chemical instability of PGI2. In fact, this prostanoid is rapidly hydrolyzed to the biologically inactive 6-keto-PGF1 in neutral solutions in vitro showing an half-life of few minutes. Thus, the protective phenotype induced by PGI2 in endothelial cells requires its continuous generation which is sustained by LSS-induced COX-2. The COX-2–derived contribution to PGI2 and the COX-1–derived contribution to PGD2, found in the present study, are in accord with what has been found in humans.21,54
PGF2 was the major prostanoid generated in static conditions but it was significantly reduced by LSS. This is probably attributable to the sustained upregulation of a human prostaglandin transporter (hPGT), in cultured HUVECs exposed to a physiological fluid mechanical stimulus in vitro55 (supplemental Figure III). Basal generation of PGF2 may occur through constitutively expressed PGFS.8 However, COX-dependent excess of PGH2, which was not transformed into prostanoids, could be transformed nonenzymatically to PGF2.56 The finding that only a low expression of PGF2 receptor (FP) is detectable in HUVECs57 suggests a marginal role of PGF2 in endothelial function. To address whether PGF2 and TXA2 are of functional relevance to HO-1 induction (perhaps restraining its expression) acting via the TX receptors (TP), we studied the effect of the TP antagonist SQ 29,548. As shown in supplemental Figure IV, SQ 29,548 did not affect HO-1 induction in HUVECs exposed to steady LSS. However, endothelial-derived PGF2 could influence vascular smooth muscle physiology. It can cause cellular hypertrophy58 in addition to being a vasoconstrictor,59 thus contributing to the pathogenesis of hypertension and atherosclerosis.
We provided the prime evidence that steady LSS restrained endothelial TNF- generation. This did not involve inhibition of "shedding" of TNF- from the cell surface by TNF-–converting enzyme activity. In fact, LSS reduced both secreted mature form of TNF- and TNF- retained in endothelial cells. Understanding what level, including transcription, mRNA stability and translation, of TNF- biosynthesis was interfered by LSS signaling was not an objective of the present study but it deserves to be explored in further investigations. The use of a specific inhibitor of HO-1 activity showed that HO-1 induction played a role in LSS-dependent reduction of TNF- biosynthesis. Similarly, inhibition of PGI2 biosynthesis or activity reverted LSS-dependent reduction of TNF- and this effect was accompanied with downregulation of HO-1. These results together with the finding that the PGI2 analog carbacyclin induced HO-1 in HUVECs, strongly suggest the contribution of LSS-induced PGI2 in HO-1 induction. The expression of phase II genes, including HO-1, is regulated by the transcription factor Nrf2 (nuclear factor E2-related factor 2), which in response to oxidative stress, escapes from Keap1-mediated proteasomal degradation, resulting in prolonged protein half-life and its nuclear accumulation.60 Nrf2 interacts with the antioxidant response element in the promoters of phase II enzyme genes, such as HO-1, leading to their transcriptional activation. Glycogen synthase kinase (GSK)-3β may modulate the expression of HO-1 through the phosphorylation of Nrf2 which will translate into its destabilization and proteasomal degradation, thus excluding this transcription factor from the nucleus.61 PGI2 may induce the phosphorylation of GSK-3β (causing its inactivation) through protein kinase A62; this might lead to stabilize Nrf2 and consequently to HO-1 expression. This hypothesis will be addressed in a specific study. The finding that NS-398 reversed LSS-induced TNF- without affecting PGE2 and PGD2 biosynthesis leads to exclude the contribution of the two prostanoids. This is coherent with the finding that IP receptor is the predominant Gs-linked PG receptor expressed in HUVECs.57
In conclusion, we provide evidence that COX-2–dependent PGI2 (induced by steady LSS) upregulates HO-1 which halts TNF- generation in human endothelial cells. This vasoprotective effect is abrogated by COX inhibitors. Our findings suggest that inhibition of COX-2–dependent PGI2 may contribute to acceleration of atherogenesis in patients taking traditional NSAIDs and NSAIDs selective for COX-2.
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Acknowledgments |
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We thank Drs Walter A. Szarek and Kanji Nakatsu (Heme Oxygenase Inhibitor Team, Queen’s University) for providing the HO-1 inhibitor QC15 and fruitful suggestions. We thank Pamela Di Tomo and Sara Di Silvestre for excellent technical assistance.
Sources of Funding
Supported by a grant from the European Community’s Sixth Framework Program (Eicosanox, LSMH-CT-2004-005033) (to P.P.).
Disclosures
None.
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Footnotes |
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*Both authors contributed equally to this work.
Original received November 13, 2008; revision received December 17, 2008; accepted December 23, 2008.
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References |
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