CD40 Ligand–Dependent Tyrosine Nitration of Prostacyclin Synthase In Vivo
http://www.100md.com
《循环学杂志》
the Vascular Research Laboratory (B.D.), Graduate School of Medicine, University of Tennessee, Knoxville, Tenn, and Medicine Endocrinology (M.-H.Z.), University of Oklahoma, Oklahoma City, Okla.
Abstract
Background— Cells in human atherosclerotic lesions express the immune mediator CD40 and its ligand, CD40L, but the mechanisms and the mediators by which CD40L contributes to atherosclerosis are poorly defined. Here, we show how CD40L increases vascular inflammation and thrombosis via tyrosine nitration and inhibition of prostacyclin synthase (PGIS), an enzyme with antithrombotic, antiproliferative, and dilatory functions in the normal vasculature.
Methods and Results— Exposure of cultured human aortic endothelial cells to clinically relevant concentrations of CD40L (20 to 80 ng/mL) dose-dependently increased the production of superoxide (O2·–), decreased nitric oxide (NO) bioactivity, and increased PGIS nitration. Furthermore, inhibition of CD40 expression by small interfering RNA blocked the effects of CD40L on O2·–, NO bioactivity, and PGIS nitration, which indicates a specific effect of CD40L. In addition, either depletion of mitochondria (0 cells, ie, mitochondria-depleted cells, to prevent mitochondrial O2·–) or adenoviral overexpression of superoxide dismutase, as well as inhibition of NO synthase, abolished the CD40L-enhanced PGIS nitration, which implies that the mitochondria might be the source of O2·– and thus peroxynitrite (ONOO–). Furthermore, SQ29548, a thromboxane A2/prostaglandin H2 receptor antagonist, significantly reduced CD40L-enhanced expression of intercellular adhesion molecule-1. Finally, administration of CD40L resulted in PGIS inhibition and nitration in the aortas of C57BL6 mice but less in mice overexpressing human superoxide dismutase, which suggests that ONOO– might be required for CD40L-enhanced PGIS nitration in vivo.
Conclusions— We conclude that CD40L might contribute to the initiation and progression of atherosclerosis by increasing O2·–- and ONOO–-dependent PGIS nitration and thromboxane A2/prostaglandin H2 receptor stimulation.
Key Words: endothelium-derived factors free radicals inflammation nitric oxide vasoconstriction
Introduction
Interaction of the multipotent immunomodulator CD40 ligand (CD40L or CD154) with its receptor CD40 has emerged as an important contributor to the inflammatory process in the vessel wall.1–3 CD40 and CD40L are expressed on endothelial cells, vascular smooth muscle cells, mononuclear cells, and platelets, and CD40-CD40L interaction has been shown to exhibit proinflammatory and proatherogenic effects in vitro and in vivo.4,5 In addition to the cell-associated form, CD40L also exists in a soluble, biologically active form (sCD40L), which has similar proinflammatory effects on vascular cells. Interestingly, sCD40L is associated with acute coronary syndromes,6,7 as well as hypercholesterolemia,8 and elevated sCD40L levels predict an increased cardiovascular risk in healthy subjects.9 Therefore, CD40L has been suggested as a potential therapeutic target to modulate vascular inflammation and possibly influence cardiovascular risks. The role of CD40L in atherosclerosis has been established recently.10–12 Mach and coworkers10 found that disruption of CD40L function in mice lacking the receptor for LDL (LDLr–/– mice), by administration of a blocking CD40L Clinical Perspective p 2192 antibody, prevented the progression of atherosclerotic disease. Lutgens et al11 targeted the CD40L gene in mice deficient for apolipoprotein E (Apo-E–/– mice), which also greatly inhibited lesion progression. However, the mechanism by which CD40L enhances vascular inflammation and atherogenesis is not fully understood.
Clinical Perspective p 2192
Under physiological conditions, the endothelium provides vasodilatory and antiaggregatory properties to the cardiovascular system and prevents growth of the underlying vascular smooth muscle cells by releasing nitric oxide (NO), endothelium-derived hyperpolarizing factor, and prostacyclin (PGI2).13,14 Each of these mediators acts by a different mechanism: NO by the stimulation of guanylyl cyclase and PGI2 by activating adenylyl cyclase, so that these mediators can act synergistically and also serve as backup systems for each other. Functional impairment of 1 or both of these enzymes may predispose to various vascular diseases, including atherosclerosis.13,14
It was interesting to study this interdependence after a selective inhibition of prostacyclin synthase (PGIS) by peroxynitrite (ONOO–) had been described.15,16 Vascular cells are capable of generating ONOO– owing to their capacity to simultaneously release O2·– and NO. Because ONOO– is generated in vivo by a rapid combination of NO and superoxide (O2·–),17,18 it appeared that not only could O2·– neutralize NO, but subsequently, through its product ONOO–, it could suppress PGI2 formation. We have previously shown that isolated PGIS is nitrated and inactivated by ONOO– at nanomolar concentrations.15,16 Using normal aortic vessel strips, we have subsequently shown that ONOO– exposure leads to nitration of PGIS, associated with impaired PGI2 production, and a defective vasorelaxation.19 The underlying mechanism for PGIS blockade has been suggested as the nitration of tyrosine 430 at the active site20 through a reaction catalyzed by the ferric iron of this heme-thiolate (P450) protein.21 In spite of the cellular antioxidant potential, ONOO– causes PGIS nitration in whole cells13,22 and in intact coronary arteries.17 We have also demonstrated that PGIS nitration and inactivation in atherosclerosis not only leads to a decreased PGI2 but also activates thromboxane receptor (TPr) via its accumulated substrate, prostaglandin H2 (PGH2).23
Previous studies demonstrated that CD40L triggered oxidant stress in lymphocytes.24 CD40 is reported to reduce NO bioactivity, likely via O2·– in cultured endothelial cells,24,25 which assists in the perpetuation of a dysfunctional endothelium. Whether CD40 enhances ONOO– remains unknown. In addition, the mechanisms by which 3-nitrotyrosine (3-NT) is formed in vivo remain unclear and controversial. Thus, the aim of the present study was to determine whether CD40L increases O2·– and ONOO–, which results in PGIS nitration and inhibition both in vitro and in vivo. In the present study, we have demonstrated that exposure of human aortic endothelial cells (HAECs) to CD40L (40 to 80 ng/mL) increased O2·–, inactivated NO to form ONOO–, caused tyrosine nitration of PGIS, and decreased PGIS activity, although it did not alter the level of PGIS expression. Furthermore, our studies have also shown that inactivation of PGIS by CD40L exposure resulted in TPr stimulation in cultured HAECs. This activation of TPr by CD40L can modulate the expression of intercellular cellular adhesion molecules (ICAM-1) in HAECs, which was mediated by O2·–, because both TPr blockade and scavenging of O2·– abolishes CD40L-enhanced expression of ICAM-1. Finally, administration of CD40L resulted in PGIS nitration and inhibition in the aorta and hearts of C57BL6 mice but had less effect in those of mice that overexpressed human superoxide dismutase (SOD-Tg), which suggests that ONOO– might be required for CD40L-enhanced PGIS nitration in vivo. We conclude that CD40L, via ONOO–, causes PGIS nitration and inhibition, as well as consequent TPr activation, to initiate atherosclerotic lesion formation and thrombosis.
Methods
Animals
Male mice overexpressing human Cu,Zn-SOD (hSOD-TG, TgHS-51) mice and their littermates, C57BL6 mice, 10 weeks of age, were obtained from the Jackson Laboratory (Bar Harbor, Maine). Mice were housed in temperature-controlled cages with a 12-hour light-dark cycle and given free access to water and normal chows. These mice were randomly divided into sham-treated (control group) and CD40L-treated groups. CD40L (1.5 mg · g–1 · d–1) was administered by tail-vein injection for 3 consecutive days, and control mice received 0.9% physiological saline injection. The mice were euthanized with inhaled isoflurane. Mice hearts and aortas were removed and immediately frozen in liquid nitrogen. The animal protocol was reviewed and approved by the institutional Animal Care and Use Committee.
Materials
Recombinant CD40L was obtained from Alex Inc and was purified before use with the EndoTrap 5/1 (Profos AG) to remove contaminated bacterial endotoxins (lipopolysaccharide). NG-nitro-L-arginine methyl ester (L-NAME), PGH2, 1S[1, 2B(5Z),3b,4]-7-[3-[2(phenylamino)carbonyl]hydrazino methyl [-7-oxabicyclo(2.2.1)hept-2-yl-5-heptenoic acid (SQ29548), indomethacin, and the ELISA kit for 6-keto-PGF1, PGE2, and cGMP were obtained from Cayman Chemicals. Embedding medium (OCT compound) was from Miles. Protein A-sepharose CL-4B was obtained from Pharmacia. Antibodies against CD40L and ICAM-1 were obtained from Santa Cruz Biotechnology, Inc (Santa Cruz, Calif). A monoclonal antibody against 3-NT was purchased from Upstate Biotechnology Inc (Waltham, Mass). Rabbit anti-PGIS antisera were kindly provided by Dr T. Klein (Altana, Inc, Konstanz, Germany). Secondary antibodies were from Pierce. Enzyme-linked chemiluminescence kits and nitrocellulose membranes (Hybond-C) were purchased from Amersham. The adenoviral constructs for Cu,Zn-SOD, uncoupling proteins (UCP)-1 and 3, and catalase were obtained from the University of Iowa Viral Vector Core facility. Other chemicals, if not otherwise indicated, were acquired from Sigma.
Cell Culture
HAECs were obtained from Clonetic, Inc (San Diego, Calif) and cultured in endothelial basal growth medium-2 with 2% fetal calf serum (FCS) until confluence was reached and then were incubated overnight in media without serum. Cells were incubated in a humidified atmosphere of 5% CO2/95% air at 37°C. To generate mitochondria-depleted HAECs (0 cells), wild-type HAECs were incubated in medium containing ethidium bromide (50 ng/mL), sodium pyruvate (1 mmol/L), and uridine (50 μg/mL) for 3 weeks, as described previously.31 The 0 status of cells was confirmed by the absence of cytochrome oxidase subunit II by both reverse transcription–polymerase chain reaction and Western blots and by the failure to grow in the absence of uridine in the media, as described previously.26
Adenoviral Infection
Confluent HAECs were infected with adenovirus expressing green fluorescence protein (GFP) as a control or adenovirus encoding Cu,Zn-SOD (SOD-1), catalase, UCP-1, or UCP-3. HAECs were infected in medium with 2% FCS overnight. The cells were then washed and incubated in fresh endothelial growth medium without FCS for an additional 18 hours before experimentation. Under these conditions, infection efficiency was typically >80% as determined by GFP expression.
RNA Interference
Small interfering RNA (siRNA) duplexes targeting human CD40L mRNA (GeneBank accession No. X60592.1) were designed according to the methods published previously.27 The sequences of siRNA (5-'GCGAAUUCCUAGACACUGUU-3', 5'-UGUCACCCUU-GGACAAGCUUU-3') were synthesized by Dharmacon Research (Lafayette, Colo). Ninety percent to 95% confluent HAECs were transfected 24 hours before CD40L treatment with 0.27 μmol/L CD40 siRNA with oligofectamine reagent (Invitrogen). Mock controls were transfected with unrelated siRNA.
Detection of Superoxide and Peroxynitrite
Detection of O2·– was performed with the SOD-inhibitable cytochrome C reduction assay in HAECs or by lucigenin chemiluminescence (5 μmol/L) in isolated aortas, as described previously.28,29 Formation of ONOO– was detected with luminol chemiluminescence, as described previously.30
Determination of cGMP
After incubation in the presence of A23187 for 2 hours in PBS, cells were quickly scraped into PBS and homogenized in ice-cold 5% trichloroacetic acid containing 0.5 mmol/L IMBX. cGMP levels were measured in cell homogenates with an enzyme immunoassay kit obtained from Cayman Chemicals, expressed as picomoles of cGMP per milligram of trichloroacetic acid–precipitable protein solubilized with 1 mol/L sodium hydroxide.
Assay of PGIS Activity
PGIS activity was assayed by the stable metabolite of PGI2, 6-keto-PGF1, after cells were incubated with its substrate, PGH2 (10–5 mol/L for 3 minutes), as described previously.15,16 Briefly, after incubation with PGH2, the reaction was stopped by acidification with 1 N HCl to pH 3.5. Incubation media were extracted with ethyl acetate (3 vol). After centrifugation, the organic phases were evaporated to dryness under nitrogen. Samples were then resuspended in 100 μL of PBS. The amount of 6-keto-PGF1 and PGE2 was subsequently determined with an ELISA kit (Cayman Chemicals) according to the instructions provided by the supplier.
Immunohistochemistry
Isolated mouse aortas or isolated human aortas were fixed in 4% paraformaldehyde and cryoprotected in 30% sucrose/0.1 mol/L sodium cacodylate buffer. Immunohistochemical stainings for CD40L and 3-NT were performed, as described previously.19
Immunoprecipitation and Western Blots
Immunoprecipitation and Western blots were performed as described previously.19
Detection of ICAM-1 Expression
Expression of ICAM-1 was detected by flow cytometry with a specific antibody against ICAM-1 and a fluorescent secondary antibody, as described previously.28 Briefly, HAEC monolayers were subsequently rinsed with PBS/BSA and fixed with 1% paraformaldehyde in PBS for 10 minutes at room temperature after incubation with the antibodies. The monolayers were rinsed twice with PBS/BSA and scraped with rubber policemen. Cell suspensions were centrifuged at 1500 rpm for 5 minutes, the supernatants discarded, and the pellets resuspended in 0.5 mL of PBS/BSA. Cytofluorographic analysis was performed with a Becton Dickinson FACScan. Acquisition was set at 5000 gated cells, and mean fluorescent intensities and the percentage of cells were measured in all samples with CellQuest software version 1.2 (Becton Dickinson).
Protein Hydrolysis and HPLC Detection of 3-NT
After incubation, the cells were scraped off in ice-cold 0.1 mol/L sodium acetate (pH 7.2) and sonicated twice on ice after being washed twice with ice-cold PBS (pH 7.4). The samples were hydrolyzed with dialyzed pronase E in the presence of 10 mmol/L CaCl2 for 60 to 72 hours at 55°C. 3-NT was quantified by its absorption at 365 nm, by its electrochemical detector response, and by co-elution with 3-NT standard. As expected, 3-NT in samples was eliminated completely by reduction with sodium dithionite, as described previously.28
Statistical Analysis
Results were analyzed with a 2-way ANOVA. Values are expressed as mean±SEM for n assays. A probability value of <0.05 is considered statistically significant.
Results
Exposure to CD40L Produces Higher Amounts of Oxidants in HAECs
We first determined whether CD40L upregulated O2·– release in HAECs. Confluent HAECs were exposed to various concentrations of CD40L (20 to 80 ng/mL) for 18 hours, and the release of O2·– was assayed with the SOD-inhibitable cytochrome C reduction assay. As shown in Figure 1A, exposure of HAECs to CD40L (20 to 80 ng/mL) for 18 hours dose-dependently increased O2·– release when stimulated with calcium ionophore A23187. To exclude any contribution of endotoxin, a possible contaminating factor in recombinant CD40L, we measured CD40L-induced O2·– in the presence of polymyxin B (5 μg/mL), which binds and neutralizes lipid A, the active moiety of lipopolysaccharide.31 Neither polymyxin B nor inactivation of CD40L by boiling or trypsin digestion abolished the CD40L-upregulated release (data not shown), which suggests that biologically active CD40L was required for O2·– release in HAECs.
Exposure to CD40L Impairs NO Bioactivity
Compared with NO, ONOO– is a much less potent activator of guanylyl cyclase,18 and thus, the formation of ONOO– decreases cGMP. As expected, cells exposed to CD40L showed a significant decrease in cGMP levels compared with control HAECs, which is consistent with increased NO inactivation by O2·– and ONOO– formation in cells exposed to CD40L (Figure 1B). These data were in line with Figure 1A, in which an increase in O2·– was seen in CD40L-exposed HAECs.
Exposure to CD40L Increases Formation of ONOO–
Increased releases of both O2·– and NO combine at a diffusion-controlled rate to form ONOO–, a potent oxidant.17,18 Luminol reacts with ONOO– but not with its precursor, NO, or O2·– alone.30 Thus, luminol chemiluminescence was used to measure the formation of ONOO– in cells exposed to CD40L. As shown in Figure 1C, CD40L dose-dependently increased ONOO–, which was attenuated by the addition of both SOD (500 IU/mL) and the NO synthase (NOS) inhibitor L-NAME (1 mmol/L; n=5; P<0.05).
Inhibition of CD40 With siRNA Blocks CD40L-Enhanced ONOO–
To investigate whether CD40L increased ONOO– by binding its ligand, CD40, CD40L (40 ng/mL) was added to the HAECs, which had been transfected with the siRNA specific for CD40. Transfection of the CD40 siRNA resulted in 86±7% reduction of CD40 protein expression in HAECs, as detected with Western blots using the antibody specific for CD40. Interestingly, transfection of the CD40-specific siRNA significantly reduced CD40L-upregulated ONOO– in HAECs (Figure 1C), which indicates that CD40 was required for CD40L-enhanced ONOO– in HAECs exposed to CD40L.
Exposure to CD40L Inhibits PGIS Activity
Our previous studies14–16 had demonstrated that ONOO– selectively inhibited and nitrated PGIS. Further studies were conducted to determine whether the increased production of ONOO– caused by CD40L exposure altered PGIS activity in HAECs. As shown in Figure 2A, CD40L dose-dependently attenuated PGIS activity, as measured by the conversion of PGH2 into 6-keto-PGF1, a stable degradation product of PGI2. Interestingly, transfection of the CD40-specific siRNA significantly reduced CD40L-upregulated PGIS inhibition (Figure 2A). The levels of thromboxane (TX) B2, a stable metabolite of TXA2, remained undetectable in CD40L-treated HAECs. Furthermore, CD40L exposure significantly increased the formation of PGE2, a major metabolite of cyclooxygenase in endothelial cells (Figure 2B). In parallel, inhibition of CD40 with CD40-specific SiRNA attenuated the CD40L-triggered increase in PGE2 (Figure 2B). Collectively, these data suggest that CD40L inhibited PGIS, whereas the synthesis of other prostaglandins was upregulated.
CD40L-Enhanced PGIS Nitration Is ONOO– Dependent
3-NT is regarded as the "footprint" of ONOO– formation,18 although other pathways of protein tyrosine nitration have also been suggested in vivo.32,33 Our previous studies20,21 demonstrated that ONOO– selectively nitrates tyrosine 450, a residue situated in the active site of PGIS. To assay whether CD40L inhibits PGIS by tyrosine nitration of the enzyme, PGIS proteins were first immunoprecipitated with a polyclonal antibody against PGIS and were further stained for a monoclonal antibody against 3-NT in Western blots. As shown in Figure 2B, CD40L significantly increased 3-NT staining in PGIS without altering PGIS expression, which indicates that CD40L selectively caused PGIS nitration. Furthermore, inhibition of CD40 expression with siRNA significantly attenuated CD40L-enhanced PGIS nitration, which suggests that CD40 was required for CD40L-enhanced PGIS nitration (Figure 2C). In addition, a SOD mimic, MnTMPyP (10 μmol/L), significantly inhibited PGIS nitration enhanced by CD40L, which suggests that CD40L might increase PGIS nitration via reactive nitrogen species, likely ONOO–.
The formation of ONOO– requires the simultaneous release of O2·– and NO. To establish whether ONOO– was involved in PGIS nitration in HAECs exposed to CD40L, HAECs were incubated with CD40L (40 ng/mL) with or without SOD (500 IU/mL, to scavenge O2·–) or the nonselective NOS inhibitor L-NAME (1 mmol/L, to inhibit NOS). These 2 inhibitors were added 1 hour before CD40L addition and were kept during CD40L exposure. As shown in Figure 2D, CD40L-enhanced PGIS nitration was attenuated by scavenging O2·– with polyethylene glycol (PEG)-SOD (500 IU/mL) or inhibition of NOS with L-NAME (1 mmol/L), which suggests that reactive nitrogen species, likely ONOO–, were involved in CD40L-enhanced PGIS nitration. Furthermore, neutralization of CD40L with the antibody against CD40L (10 μg/mL, Calbiochem) blocked CD40L-enhanced PGIS nitration, which indicates that CD40L was required for increased ONOO– in HAECs.
Identification of Mitochondria as the Source of Oxidants in HAECs Exposed to CD40L
Increased O2·– is an important feature of atherosclerosis, and potential sources of this O2·– could be NAD(P)H oxidases, xanthine oxidase, mitochondria, or NOS (see review in Zou et al14). We next investigated the source(s) of oxidants in the CD40L-exposed HAECs. Overexpression of the dominant negative mutant p47phox (p47phox-DN), which blocks assembly of the active form of NAD(P)H oxidase and then inhibits its activation,26 did not alter either O2·– (Figure 3A) or PGIS nitration (Figure 3B) enhanced by CD40L. In contrast, overexpression of UCP-1 and UCP-3, which inhibit the mitochondria-derived O2·–,26 significantly attenuated both CD40L-enhanced O2·– (Figure 3A) and PGIS nitration (Figure 3B), which suggests that mitochondria are likely to be the sources of oxidants in HAECs when exposed to CD40L.
To further establish whether mitochondria are the target of CD40L, we created HAECs that lacked functional mitochondria (so-called 0 cells).26 We next investigated whether CD40L increased intracellular reactive oxygen species and PGIS nitration in 0-HAECs. CD40L (40 ng/mL), which significantly increased PGIS nitration in the wild type of human aortic endothelial cells, did not increase PGIS nitration in 0 bovine aortic endothelial cells (Figure 3C). These observations further confirmed that CD40L requires functional mitochondria to increase O2·– and ONOO– in endothelial cells. Because 0-HAECs also failed to produce O2·– in response to CD40L (data not shown), the results strongly support the notion that CD40L-upregulated PGIS nitration is likely via mitochondria-derived reactive oxygen species.
CD40L-Enhanced Expression of ICAM-1 Is O2·– Dependent
To further explore the mechanisms of CD40L-induced endothelial dysfunction, we determined whether CD40L altered the expression of ICAM-1 in HAECs. As shown in Figure 4A, CD40L exposure dramatically increased the expression of ICAM-1 in HAECs exposed to CD40L. Interestingly, overexpression of SOD-1 or SOD-1 plus catalase significantly attenuated CD40L-enhanced ICAM-1 expression. In contrast, catalase alone had no effect. These data suggest that ONOO–, but not hydrogen peroxide, contributed to the overexpression of ICAM-1 in HAECs exposed to CD40L.
CD40L-Induced PGIS Inhibition Is Associated With ICAM-1 Overexpression Mediated by TPr Stimulation
To further explore whether PGIS nitration is associated with ICAM-1 expression, we incubated CD40L-exposed HAECs with either indomethacin (10 μmol/L), a cyclooxygenase inhibitor, or SQ29548 (10 μmol/L), a selective TPr antagonist.28 As shown in Figure 4B, either indomethacin or SQ29548 effectively attenuated the effects of CD40L on ICAM-1 expression. Because either indomethacin (which inhibits cyclooxygenase to prevent the formation of PGH2) or SQ29548 (TPr antagonist) effectively reduced the CD40L-enhanced ICAM-1 expression while PGIS was inhibited, these results indicate that CD40L, likely via the consequent activation of TPr by its substrate, PGH2, caused the overexpression of ICAM-1.
ONOO–-Dependent Tyrosine Nitration of PGIS Is Operated In Vivo
In an effort to determine whether CD40L causes ONOO– and PGIS nitration in vivo, recombinant CD40L (1.5 mg/kg) was administered into hSOD-TG and littermate C56BL6 (wild type) by tail-vein injection. Three days after being given CD40L, mice were euthanized; PGIS activity and PGIS nitration were monitored in both CD40L-infused and vehicle-treated mice (sham). hSOD-TG mice, which are extensively back-crossed to the C57BL6 background, possess 5 copies of hSOD expressed ubiquitously and display tissue SOD activity 2.5-fold greater than the wild type. The presence of hSOD activity was confirmed with nondenaturing gels of red cell lysates reacted with nitro blue tetrazolium (Zou et al, unpublished data). We have also measured SOD activity by SOD of the auto-oxidation of pyrogallol. Aortic homogenates from mice expressing hSOD show a 2.5-fold increase in activity compared with control C57BL6 (164±26 versus 33±7 U/mg protein, P<0.001).
We first measured the release of O2·– in isolated mouse aorta with lucigenin chemiluminescence. Measurements on intact arteries with lucigenin at 5 μmol/L have been corroborated with electron spin resonance and were not complicated by its redox cycling.29 As shown in Figure 5A, O2·– was significantly elevated in aortas isolated from a CD40L-exposed mouse compared with aortas in sham-treated mice (n=9; P<0.05, sham versus CD40L-treated mice). In hSOD-TG mice, basal aortic O2·– release was significantly lower than in C57BL6 mice (Figure 5A; n=7; P<0.05, C57BL6 versus hSOD-TG). Importantly, there was a significant decrease of O2·– release in CD40L-treated hSOD-TG mice compared with CD40L-treated C57BL6 mice (n=9; P<0.01, CD40L-treated C57BL6 versus CD40L-treated hSOD-TG mice; Figure 5A). However, there was no significant difference in aortic O2·– release between CD40L-treated hSOD-TG and nontreated hSOD-TG mice (Figure 5A) These results indicate that CD40L infusion significantly increased O2·–.
It was interesting to investigate whether CD40L infusion increased 3-NT, PGIS nitration and inhibition in vivo. As shown in Figure 3B, CD40L significantly increased 3-NT staining in the mouse aorta of C57BL6 mice but not in hSOD-TG (n=6; P<0.05, sham versus CD40L and CD40L versus sham-treated mice). 3-NT immunostaining was intensely present in the endothelium of CD40L-treated C57BL6 mice but was significantly less in CD40L-treated hSOD-TG mice, which suggests that CD40L infusion increased 3-NT staining in vivo. Because hSOD-TG mice had decreased contents of 3-NT in aortas, these results suggest that CD40L increased reactive nitrogen species, likely ONOO–. In parallel, CD40L decreased PGIS activity in wild types but had less effect in hSOD-Tg mice (Figure 6A) without altering PGIS expression (Figure 6B). The increased ONOO– was further confirmed by the increased PGIS nitration. As shown in Figure 6B, there was a slight decrease in PGIS expression in hSOD-Tg mice. Western blotting with antibodies against 3-NT showed a marked increase in 3-NT in PGIS in mice treated with CD40L (n=5; P<0.05, sham versus CD40L-infused mice; P<0.05, sham wild-type versus sham hSOD-TG mice and wild-type CD40L-infused mice versus hSOD-TG CD40L-infused mice), which indicates increased PGIS nitration by CD40L treatment.
Discussion
In the present study using cultured HAECs, we have demonstrated for the first time that exposure of HAECs to clinically relevant concentrations of CD40L (20 to 80 ng/mL) for 18 hours significantly increases the production of O2·– and ONOO– and consequently decreases the bioactivity of NO, as indicated by decreased levels of cGMP. Further evidence that NO is inactivated by reacting with O2·– to form the reaction product ONOO– is provided by the increased detection of PGIS nitration and inhibition in cells exposed to CD40L. Convincing evidence for ONOO– formation in cells exposed to CD40L comes from the identification of tyrosine nitration of PGIS. 3-NT is regarded as the "footprint" of ONOO– formation in vivo,18 although other pathways of tyrosine nitration have been suggested.32,33 Here, we for the first time present evidence that PGIS might be nitrated by ONOO– both in vitro and in vivo. Our previous studies have demonstrated that this enzyme undergoes tyrosine nitration by low concentrations of ONOO–.15,16 In the present study, we found that increased staining with an antibody against 3-NT was found in the immunoprecipitates obtained with antibodies against PGIS in CD40L-exposed cells, although the levels of PGIS expression were not changed. Inhibition of both endothelial NOS and scavenging O2·– with SOD attenuated CD40L-enhanced PGIS nitration, which indicated that reactive nitrogen species, likely ONOO–, are involved. In addition, inhibition of mitochondria-derived O2·– also abolished CD40L-enhanced PGIS nitration, which suggests that mitochondria might be the source of O2·– and ONOO–.
We have further demonstrated that administration of CD40L caused PGIS nitration and inhibition in C57BL6 mice in vivo. Tyrosine nitration of PGIS is most likely mediated by ONOO– formed endogenously, generated from NO, and O2·– caused by CD40L exposure. Although peroxidases such as myeloperoxidase have been reported to cause protein nitration in vivo,32,33 a role of peroxidase-catalyzed PGIS nitration is less likely, because hSOD-TG mice had significantly attenuated CD40L-enhanced PGIS nitration and inhibition. Peroxidase-catalyzed PGIS nitration is less likely because overexpression of SOD, which provides hydrogen peroxide (H2O2) to facilitate the peroxidase-catalyzed 3-NT formation, should enhance instead of decrease PGIS nitration. Although we cannot exclude the possibility of peroxidase-catalyzed PGIS nitration, the present data strongly suggest that ONOO– is likely to be responsible for the increased PGIS nitration caused by CD40L in vivo.
The selectivity of ONOO– for PGIS is explained by the exceptional sensitivity of PGIS, which catalyzes its own nitration by ONOO–.21 In addition, our studies have also shown that inactivation of PGIS results in a consequent activation of TPr through its cumulative substrate, PGH2.19 This consequent TPr stimulation triggers the overexpression of ICAM-1 in HAECs (Figure 4). Because TXA2 promotes and PGI2 prevents the initiation and progression of atherogenesis, the present study results unveil a novel mechanism by which CD40L causes atherosclerosis, ie, CD40L generates uncontrolled O2·–, which results in increased destruction of NO and a concomitant formation of a highly cytotoxic oxidant, ONOO–, which triggers nitration and inhibition of PGIS that results in consequent TPr stimulation. Thus, the present study for the first time provides evidence that CD40L via ONOO– causes vascular endothelial dysfunction by a combination of 2 mechanisms: decreased production of the vasorelaxant PGI2 and NO and accumulation of the vasoconstrictor PGH2, which stimulates the TXA2 receptor. The present data suggest that endogenous nitration of PGIS may contribute to the functional defects of the endothelium in pathological situations, not only by a lack of the vasorelaxant NO and PGI2, but more directly by causing accumulation of the prothrombotic and proconstrictive prostanoid PGH2 (Figure 3). The results of the present study provide a potential explanation for the paradoxical effects of endothelium-dependent vasorelaxants such as acetylcholine, which trigger vasoconstriction in human atherosclerotic arteries.34,35 Even individuals with significant atherosclerotic risk factors but without clinically manifest atherosclerosis have a decreased vasodilator response in parallel with higher production of vasoconstricting prostaglandins.34–36 Such abnormal responses are normalized by inhibition of cyclooxygenase.37 Aspirin has no effect under normal conditions but improves/restores acetylcholine-mediated vasodilation in patients with atherosclerosis.32,37
In summary, we have demonstrated that exposure of HAECs to clinically relevant concentrations of CD40L (20 to 80 ng/mL) increased O2·– and ONOO–, causing PGIS nitration and TPr stimulation in vitro and in vivo. These concentrations of CD40L can be found in patients. In addition, we fully envision that local concentrations of CD40L in the active areas of thrombotic or inflammatory vasculatures might be higher than those in circulating blood. Our findings might have important implications in atherosclerosis, because tyrosine nitration and inhibition of PGIS not only eliminates the vasodilatory, growth-inhibiting, antithrombotic and antiadhesive effects of prostacyclin (PGI2) but also increases release of PGH2 or TXA2, 2 potent vasoconstrictor, prothrombotic, growth- and adhesion-promoting agents. Both PGI2 and TXA2 contribute to the initiation and progression of vascular injury and thrombosis because of the downregulation of protective actions of both NO and PGI2 and because the nonmetabolized PGH2 or shunted TXA2 tips the balance toward platelet aggregation, leukocyte adherence, atheroma accumulation, and thrombus formation, which leads to the further release of CD40L from platelets and vascular cells. Thus, this biological positive-feedback loop will not only amplify proatherogenic CD40L and TXA2 but also decrease antiatherogenic factors, NO and PGI2, which can initiate and propagate clinical events characterized by vasoconstriction, adherence of platelets and monocytes, atherosclerosis, and/or micro-occlusive vascular disease, and ultimately, vascular thrombosis and tissue damage. This may not only explain why many atherothrombotic diseases in patients and animals have decreased levels of PGI2 but also why increases have been noted in its precursors, PGH2 and TXA2, both of which activate the TXA2 receptor.
Acknowledgments
We would like to acknowledge Drs Michael Brownlee and David R. Pimental for providing constructs for UCP-1 and p47phox-dominant negative adenoviruses. We thank Dr T. Klein for kindly providing an antibody against human PGIS. This work was supported by NIH grants HL079584 and HL07439, a career development grant from the American Heart Association, a grant-in-aid from Juvenile Diabetes Research Foundation, and the Graduate School of Medicine, the University of Tennessee.
References
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Abstract
Background— Cells in human atherosclerotic lesions express the immune mediator CD40 and its ligand, CD40L, but the mechanisms and the mediators by which CD40L contributes to atherosclerosis are poorly defined. Here, we show how CD40L increases vascular inflammation and thrombosis via tyrosine nitration and inhibition of prostacyclin synthase (PGIS), an enzyme with antithrombotic, antiproliferative, and dilatory functions in the normal vasculature.
Methods and Results— Exposure of cultured human aortic endothelial cells to clinically relevant concentrations of CD40L (20 to 80 ng/mL) dose-dependently increased the production of superoxide (O2·–), decreased nitric oxide (NO) bioactivity, and increased PGIS nitration. Furthermore, inhibition of CD40 expression by small interfering RNA blocked the effects of CD40L on O2·–, NO bioactivity, and PGIS nitration, which indicates a specific effect of CD40L. In addition, either depletion of mitochondria (0 cells, ie, mitochondria-depleted cells, to prevent mitochondrial O2·–) or adenoviral overexpression of superoxide dismutase, as well as inhibition of NO synthase, abolished the CD40L-enhanced PGIS nitration, which implies that the mitochondria might be the source of O2·– and thus peroxynitrite (ONOO–). Furthermore, SQ29548, a thromboxane A2/prostaglandin H2 receptor antagonist, significantly reduced CD40L-enhanced expression of intercellular adhesion molecule-1. Finally, administration of CD40L resulted in PGIS inhibition and nitration in the aortas of C57BL6 mice but less in mice overexpressing human superoxide dismutase, which suggests that ONOO– might be required for CD40L-enhanced PGIS nitration in vivo.
Conclusions— We conclude that CD40L might contribute to the initiation and progression of atherosclerosis by increasing O2·–- and ONOO–-dependent PGIS nitration and thromboxane A2/prostaglandin H2 receptor stimulation.
Key Words: endothelium-derived factors free radicals inflammation nitric oxide vasoconstriction
Introduction
Interaction of the multipotent immunomodulator CD40 ligand (CD40L or CD154) with its receptor CD40 has emerged as an important contributor to the inflammatory process in the vessel wall.1–3 CD40 and CD40L are expressed on endothelial cells, vascular smooth muscle cells, mononuclear cells, and platelets, and CD40-CD40L interaction has been shown to exhibit proinflammatory and proatherogenic effects in vitro and in vivo.4,5 In addition to the cell-associated form, CD40L also exists in a soluble, biologically active form (sCD40L), which has similar proinflammatory effects on vascular cells. Interestingly, sCD40L is associated with acute coronary syndromes,6,7 as well as hypercholesterolemia,8 and elevated sCD40L levels predict an increased cardiovascular risk in healthy subjects.9 Therefore, CD40L has been suggested as a potential therapeutic target to modulate vascular inflammation and possibly influence cardiovascular risks. The role of CD40L in atherosclerosis has been established recently.10–12 Mach and coworkers10 found that disruption of CD40L function in mice lacking the receptor for LDL (LDLr–/– mice), by administration of a blocking CD40L Clinical Perspective p 2192 antibody, prevented the progression of atherosclerotic disease. Lutgens et al11 targeted the CD40L gene in mice deficient for apolipoprotein E (Apo-E–/– mice), which also greatly inhibited lesion progression. However, the mechanism by which CD40L enhances vascular inflammation and atherogenesis is not fully understood.
Clinical Perspective p 2192
Under physiological conditions, the endothelium provides vasodilatory and antiaggregatory properties to the cardiovascular system and prevents growth of the underlying vascular smooth muscle cells by releasing nitric oxide (NO), endothelium-derived hyperpolarizing factor, and prostacyclin (PGI2).13,14 Each of these mediators acts by a different mechanism: NO by the stimulation of guanylyl cyclase and PGI2 by activating adenylyl cyclase, so that these mediators can act synergistically and also serve as backup systems for each other. Functional impairment of 1 or both of these enzymes may predispose to various vascular diseases, including atherosclerosis.13,14
It was interesting to study this interdependence after a selective inhibition of prostacyclin synthase (PGIS) by peroxynitrite (ONOO–) had been described.15,16 Vascular cells are capable of generating ONOO– owing to their capacity to simultaneously release O2·– and NO. Because ONOO– is generated in vivo by a rapid combination of NO and superoxide (O2·–),17,18 it appeared that not only could O2·– neutralize NO, but subsequently, through its product ONOO–, it could suppress PGI2 formation. We have previously shown that isolated PGIS is nitrated and inactivated by ONOO– at nanomolar concentrations.15,16 Using normal aortic vessel strips, we have subsequently shown that ONOO– exposure leads to nitration of PGIS, associated with impaired PGI2 production, and a defective vasorelaxation.19 The underlying mechanism for PGIS blockade has been suggested as the nitration of tyrosine 430 at the active site20 through a reaction catalyzed by the ferric iron of this heme-thiolate (P450) protein.21 In spite of the cellular antioxidant potential, ONOO– causes PGIS nitration in whole cells13,22 and in intact coronary arteries.17 We have also demonstrated that PGIS nitration and inactivation in atherosclerosis not only leads to a decreased PGI2 but also activates thromboxane receptor (TPr) via its accumulated substrate, prostaglandin H2 (PGH2).23
Previous studies demonstrated that CD40L triggered oxidant stress in lymphocytes.24 CD40 is reported to reduce NO bioactivity, likely via O2·– in cultured endothelial cells,24,25 which assists in the perpetuation of a dysfunctional endothelium. Whether CD40 enhances ONOO– remains unknown. In addition, the mechanisms by which 3-nitrotyrosine (3-NT) is formed in vivo remain unclear and controversial. Thus, the aim of the present study was to determine whether CD40L increases O2·– and ONOO–, which results in PGIS nitration and inhibition both in vitro and in vivo. In the present study, we have demonstrated that exposure of human aortic endothelial cells (HAECs) to CD40L (40 to 80 ng/mL) increased O2·–, inactivated NO to form ONOO–, caused tyrosine nitration of PGIS, and decreased PGIS activity, although it did not alter the level of PGIS expression. Furthermore, our studies have also shown that inactivation of PGIS by CD40L exposure resulted in TPr stimulation in cultured HAECs. This activation of TPr by CD40L can modulate the expression of intercellular cellular adhesion molecules (ICAM-1) in HAECs, which was mediated by O2·–, because both TPr blockade and scavenging of O2·– abolishes CD40L-enhanced expression of ICAM-1. Finally, administration of CD40L resulted in PGIS nitration and inhibition in the aorta and hearts of C57BL6 mice but had less effect in those of mice that overexpressed human superoxide dismutase (SOD-Tg), which suggests that ONOO– might be required for CD40L-enhanced PGIS nitration in vivo. We conclude that CD40L, via ONOO–, causes PGIS nitration and inhibition, as well as consequent TPr activation, to initiate atherosclerotic lesion formation and thrombosis.
Methods
Animals
Male mice overexpressing human Cu,Zn-SOD (hSOD-TG, TgHS-51) mice and their littermates, C57BL6 mice, 10 weeks of age, were obtained from the Jackson Laboratory (Bar Harbor, Maine). Mice were housed in temperature-controlled cages with a 12-hour light-dark cycle and given free access to water and normal chows. These mice were randomly divided into sham-treated (control group) and CD40L-treated groups. CD40L (1.5 mg · g–1 · d–1) was administered by tail-vein injection for 3 consecutive days, and control mice received 0.9% physiological saline injection. The mice were euthanized with inhaled isoflurane. Mice hearts and aortas were removed and immediately frozen in liquid nitrogen. The animal protocol was reviewed and approved by the institutional Animal Care and Use Committee.
Materials
Recombinant CD40L was obtained from Alex Inc and was purified before use with the EndoTrap 5/1 (Profos AG) to remove contaminated bacterial endotoxins (lipopolysaccharide). NG-nitro-L-arginine methyl ester (L-NAME), PGH2, 1S[1, 2B(5Z),3b,4]-7-[3-[2(phenylamino)carbonyl]hydrazino methyl [-7-oxabicyclo(2.2.1)hept-2-yl-5-heptenoic acid (SQ29548), indomethacin, and the ELISA kit for 6-keto-PGF1, PGE2, and cGMP were obtained from Cayman Chemicals. Embedding medium (OCT compound) was from Miles. Protein A-sepharose CL-4B was obtained from Pharmacia. Antibodies against CD40L and ICAM-1 were obtained from Santa Cruz Biotechnology, Inc (Santa Cruz, Calif). A monoclonal antibody against 3-NT was purchased from Upstate Biotechnology Inc (Waltham, Mass). Rabbit anti-PGIS antisera were kindly provided by Dr T. Klein (Altana, Inc, Konstanz, Germany). Secondary antibodies were from Pierce. Enzyme-linked chemiluminescence kits and nitrocellulose membranes (Hybond-C) were purchased from Amersham. The adenoviral constructs for Cu,Zn-SOD, uncoupling proteins (UCP)-1 and 3, and catalase were obtained from the University of Iowa Viral Vector Core facility. Other chemicals, if not otherwise indicated, were acquired from Sigma.
Cell Culture
HAECs were obtained from Clonetic, Inc (San Diego, Calif) and cultured in endothelial basal growth medium-2 with 2% fetal calf serum (FCS) until confluence was reached and then were incubated overnight in media without serum. Cells were incubated in a humidified atmosphere of 5% CO2/95% air at 37°C. To generate mitochondria-depleted HAECs (0 cells), wild-type HAECs were incubated in medium containing ethidium bromide (50 ng/mL), sodium pyruvate (1 mmol/L), and uridine (50 μg/mL) for 3 weeks, as described previously.31 The 0 status of cells was confirmed by the absence of cytochrome oxidase subunit II by both reverse transcription–polymerase chain reaction and Western blots and by the failure to grow in the absence of uridine in the media, as described previously.26
Adenoviral Infection
Confluent HAECs were infected with adenovirus expressing green fluorescence protein (GFP) as a control or adenovirus encoding Cu,Zn-SOD (SOD-1), catalase, UCP-1, or UCP-3. HAECs were infected in medium with 2% FCS overnight. The cells were then washed and incubated in fresh endothelial growth medium without FCS for an additional 18 hours before experimentation. Under these conditions, infection efficiency was typically >80% as determined by GFP expression.
RNA Interference
Small interfering RNA (siRNA) duplexes targeting human CD40L mRNA (GeneBank accession No. X60592.1) were designed according to the methods published previously.27 The sequences of siRNA (5-'GCGAAUUCCUAGACACUGUU-3', 5'-UGUCACCCUU-GGACAAGCUUU-3') were synthesized by Dharmacon Research (Lafayette, Colo). Ninety percent to 95% confluent HAECs were transfected 24 hours before CD40L treatment with 0.27 μmol/L CD40 siRNA with oligofectamine reagent (Invitrogen). Mock controls were transfected with unrelated siRNA.
Detection of Superoxide and Peroxynitrite
Detection of O2·– was performed with the SOD-inhibitable cytochrome C reduction assay in HAECs or by lucigenin chemiluminescence (5 μmol/L) in isolated aortas, as described previously.28,29 Formation of ONOO– was detected with luminol chemiluminescence, as described previously.30
Determination of cGMP
After incubation in the presence of A23187 for 2 hours in PBS, cells were quickly scraped into PBS and homogenized in ice-cold 5% trichloroacetic acid containing 0.5 mmol/L IMBX. cGMP levels were measured in cell homogenates with an enzyme immunoassay kit obtained from Cayman Chemicals, expressed as picomoles of cGMP per milligram of trichloroacetic acid–precipitable protein solubilized with 1 mol/L sodium hydroxide.
Assay of PGIS Activity
PGIS activity was assayed by the stable metabolite of PGI2, 6-keto-PGF1, after cells were incubated with its substrate, PGH2 (10–5 mol/L for 3 minutes), as described previously.15,16 Briefly, after incubation with PGH2, the reaction was stopped by acidification with 1 N HCl to pH 3.5. Incubation media were extracted with ethyl acetate (3 vol). After centrifugation, the organic phases were evaporated to dryness under nitrogen. Samples were then resuspended in 100 μL of PBS. The amount of 6-keto-PGF1 and PGE2 was subsequently determined with an ELISA kit (Cayman Chemicals) according to the instructions provided by the supplier.
Immunohistochemistry
Isolated mouse aortas or isolated human aortas were fixed in 4% paraformaldehyde and cryoprotected in 30% sucrose/0.1 mol/L sodium cacodylate buffer. Immunohistochemical stainings for CD40L and 3-NT were performed, as described previously.19
Immunoprecipitation and Western Blots
Immunoprecipitation and Western blots were performed as described previously.19
Detection of ICAM-1 Expression
Expression of ICAM-1 was detected by flow cytometry with a specific antibody against ICAM-1 and a fluorescent secondary antibody, as described previously.28 Briefly, HAEC monolayers were subsequently rinsed with PBS/BSA and fixed with 1% paraformaldehyde in PBS for 10 minutes at room temperature after incubation with the antibodies. The monolayers were rinsed twice with PBS/BSA and scraped with rubber policemen. Cell suspensions were centrifuged at 1500 rpm for 5 minutes, the supernatants discarded, and the pellets resuspended in 0.5 mL of PBS/BSA. Cytofluorographic analysis was performed with a Becton Dickinson FACScan. Acquisition was set at 5000 gated cells, and mean fluorescent intensities and the percentage of cells were measured in all samples with CellQuest software version 1.2 (Becton Dickinson).
Protein Hydrolysis and HPLC Detection of 3-NT
After incubation, the cells were scraped off in ice-cold 0.1 mol/L sodium acetate (pH 7.2) and sonicated twice on ice after being washed twice with ice-cold PBS (pH 7.4). The samples were hydrolyzed with dialyzed pronase E in the presence of 10 mmol/L CaCl2 for 60 to 72 hours at 55°C. 3-NT was quantified by its absorption at 365 nm, by its electrochemical detector response, and by co-elution with 3-NT standard. As expected, 3-NT in samples was eliminated completely by reduction with sodium dithionite, as described previously.28
Statistical Analysis
Results were analyzed with a 2-way ANOVA. Values are expressed as mean±SEM for n assays. A probability value of <0.05 is considered statistically significant.
Results
Exposure to CD40L Produces Higher Amounts of Oxidants in HAECs
We first determined whether CD40L upregulated O2·– release in HAECs. Confluent HAECs were exposed to various concentrations of CD40L (20 to 80 ng/mL) for 18 hours, and the release of O2·– was assayed with the SOD-inhibitable cytochrome C reduction assay. As shown in Figure 1A, exposure of HAECs to CD40L (20 to 80 ng/mL) for 18 hours dose-dependently increased O2·– release when stimulated with calcium ionophore A23187. To exclude any contribution of endotoxin, a possible contaminating factor in recombinant CD40L, we measured CD40L-induced O2·– in the presence of polymyxin B (5 μg/mL), which binds and neutralizes lipid A, the active moiety of lipopolysaccharide.31 Neither polymyxin B nor inactivation of CD40L by boiling or trypsin digestion abolished the CD40L-upregulated release (data not shown), which suggests that biologically active CD40L was required for O2·– release in HAECs.
Exposure to CD40L Impairs NO Bioactivity
Compared with NO, ONOO– is a much less potent activator of guanylyl cyclase,18 and thus, the formation of ONOO– decreases cGMP. As expected, cells exposed to CD40L showed a significant decrease in cGMP levels compared with control HAECs, which is consistent with increased NO inactivation by O2·– and ONOO– formation in cells exposed to CD40L (Figure 1B). These data were in line with Figure 1A, in which an increase in O2·– was seen in CD40L-exposed HAECs.
Exposure to CD40L Increases Formation of ONOO–
Increased releases of both O2·– and NO combine at a diffusion-controlled rate to form ONOO–, a potent oxidant.17,18 Luminol reacts with ONOO– but not with its precursor, NO, or O2·– alone.30 Thus, luminol chemiluminescence was used to measure the formation of ONOO– in cells exposed to CD40L. As shown in Figure 1C, CD40L dose-dependently increased ONOO–, which was attenuated by the addition of both SOD (500 IU/mL) and the NO synthase (NOS) inhibitor L-NAME (1 mmol/L; n=5; P<0.05).
Inhibition of CD40 With siRNA Blocks CD40L-Enhanced ONOO–
To investigate whether CD40L increased ONOO– by binding its ligand, CD40, CD40L (40 ng/mL) was added to the HAECs, which had been transfected with the siRNA specific for CD40. Transfection of the CD40 siRNA resulted in 86±7% reduction of CD40 protein expression in HAECs, as detected with Western blots using the antibody specific for CD40. Interestingly, transfection of the CD40-specific siRNA significantly reduced CD40L-upregulated ONOO– in HAECs (Figure 1C), which indicates that CD40 was required for CD40L-enhanced ONOO– in HAECs exposed to CD40L.
Exposure to CD40L Inhibits PGIS Activity
Our previous studies14–16 had demonstrated that ONOO– selectively inhibited and nitrated PGIS. Further studies were conducted to determine whether the increased production of ONOO– caused by CD40L exposure altered PGIS activity in HAECs. As shown in Figure 2A, CD40L dose-dependently attenuated PGIS activity, as measured by the conversion of PGH2 into 6-keto-PGF1, a stable degradation product of PGI2. Interestingly, transfection of the CD40-specific siRNA significantly reduced CD40L-upregulated PGIS inhibition (Figure 2A). The levels of thromboxane (TX) B2, a stable metabolite of TXA2, remained undetectable in CD40L-treated HAECs. Furthermore, CD40L exposure significantly increased the formation of PGE2, a major metabolite of cyclooxygenase in endothelial cells (Figure 2B). In parallel, inhibition of CD40 with CD40-specific SiRNA attenuated the CD40L-triggered increase in PGE2 (Figure 2B). Collectively, these data suggest that CD40L inhibited PGIS, whereas the synthesis of other prostaglandins was upregulated.
CD40L-Enhanced PGIS Nitration Is ONOO– Dependent
3-NT is regarded as the "footprint" of ONOO– formation,18 although other pathways of protein tyrosine nitration have also been suggested in vivo.32,33 Our previous studies20,21 demonstrated that ONOO– selectively nitrates tyrosine 450, a residue situated in the active site of PGIS. To assay whether CD40L inhibits PGIS by tyrosine nitration of the enzyme, PGIS proteins were first immunoprecipitated with a polyclonal antibody against PGIS and were further stained for a monoclonal antibody against 3-NT in Western blots. As shown in Figure 2B, CD40L significantly increased 3-NT staining in PGIS without altering PGIS expression, which indicates that CD40L selectively caused PGIS nitration. Furthermore, inhibition of CD40 expression with siRNA significantly attenuated CD40L-enhanced PGIS nitration, which suggests that CD40 was required for CD40L-enhanced PGIS nitration (Figure 2C). In addition, a SOD mimic, MnTMPyP (10 μmol/L), significantly inhibited PGIS nitration enhanced by CD40L, which suggests that CD40L might increase PGIS nitration via reactive nitrogen species, likely ONOO–.
The formation of ONOO– requires the simultaneous release of O2·– and NO. To establish whether ONOO– was involved in PGIS nitration in HAECs exposed to CD40L, HAECs were incubated with CD40L (40 ng/mL) with or without SOD (500 IU/mL, to scavenge O2·–) or the nonselective NOS inhibitor L-NAME (1 mmol/L, to inhibit NOS). These 2 inhibitors were added 1 hour before CD40L addition and were kept during CD40L exposure. As shown in Figure 2D, CD40L-enhanced PGIS nitration was attenuated by scavenging O2·– with polyethylene glycol (PEG)-SOD (500 IU/mL) or inhibition of NOS with L-NAME (1 mmol/L), which suggests that reactive nitrogen species, likely ONOO–, were involved in CD40L-enhanced PGIS nitration. Furthermore, neutralization of CD40L with the antibody against CD40L (10 μg/mL, Calbiochem) blocked CD40L-enhanced PGIS nitration, which indicates that CD40L was required for increased ONOO– in HAECs.
Identification of Mitochondria as the Source of Oxidants in HAECs Exposed to CD40L
Increased O2·– is an important feature of atherosclerosis, and potential sources of this O2·– could be NAD(P)H oxidases, xanthine oxidase, mitochondria, or NOS (see review in Zou et al14). We next investigated the source(s) of oxidants in the CD40L-exposed HAECs. Overexpression of the dominant negative mutant p47phox (p47phox-DN), which blocks assembly of the active form of NAD(P)H oxidase and then inhibits its activation,26 did not alter either O2·– (Figure 3A) or PGIS nitration (Figure 3B) enhanced by CD40L. In contrast, overexpression of UCP-1 and UCP-3, which inhibit the mitochondria-derived O2·–,26 significantly attenuated both CD40L-enhanced O2·– (Figure 3A) and PGIS nitration (Figure 3B), which suggests that mitochondria are likely to be the sources of oxidants in HAECs when exposed to CD40L.
To further establish whether mitochondria are the target of CD40L, we created HAECs that lacked functional mitochondria (so-called 0 cells).26 We next investigated whether CD40L increased intracellular reactive oxygen species and PGIS nitration in 0-HAECs. CD40L (40 ng/mL), which significantly increased PGIS nitration in the wild type of human aortic endothelial cells, did not increase PGIS nitration in 0 bovine aortic endothelial cells (Figure 3C). These observations further confirmed that CD40L requires functional mitochondria to increase O2·– and ONOO– in endothelial cells. Because 0-HAECs also failed to produce O2·– in response to CD40L (data not shown), the results strongly support the notion that CD40L-upregulated PGIS nitration is likely via mitochondria-derived reactive oxygen species.
CD40L-Enhanced Expression of ICAM-1 Is O2·– Dependent
To further explore the mechanisms of CD40L-induced endothelial dysfunction, we determined whether CD40L altered the expression of ICAM-1 in HAECs. As shown in Figure 4A, CD40L exposure dramatically increased the expression of ICAM-1 in HAECs exposed to CD40L. Interestingly, overexpression of SOD-1 or SOD-1 plus catalase significantly attenuated CD40L-enhanced ICAM-1 expression. In contrast, catalase alone had no effect. These data suggest that ONOO–, but not hydrogen peroxide, contributed to the overexpression of ICAM-1 in HAECs exposed to CD40L.
CD40L-Induced PGIS Inhibition Is Associated With ICAM-1 Overexpression Mediated by TPr Stimulation
To further explore whether PGIS nitration is associated with ICAM-1 expression, we incubated CD40L-exposed HAECs with either indomethacin (10 μmol/L), a cyclooxygenase inhibitor, or SQ29548 (10 μmol/L), a selective TPr antagonist.28 As shown in Figure 4B, either indomethacin or SQ29548 effectively attenuated the effects of CD40L on ICAM-1 expression. Because either indomethacin (which inhibits cyclooxygenase to prevent the formation of PGH2) or SQ29548 (TPr antagonist) effectively reduced the CD40L-enhanced ICAM-1 expression while PGIS was inhibited, these results indicate that CD40L, likely via the consequent activation of TPr by its substrate, PGH2, caused the overexpression of ICAM-1.
ONOO–-Dependent Tyrosine Nitration of PGIS Is Operated In Vivo
In an effort to determine whether CD40L causes ONOO– and PGIS nitration in vivo, recombinant CD40L (1.5 mg/kg) was administered into hSOD-TG and littermate C56BL6 (wild type) by tail-vein injection. Three days after being given CD40L, mice were euthanized; PGIS activity and PGIS nitration were monitored in both CD40L-infused and vehicle-treated mice (sham). hSOD-TG mice, which are extensively back-crossed to the C57BL6 background, possess 5 copies of hSOD expressed ubiquitously and display tissue SOD activity 2.5-fold greater than the wild type. The presence of hSOD activity was confirmed with nondenaturing gels of red cell lysates reacted with nitro blue tetrazolium (Zou et al, unpublished data). We have also measured SOD activity by SOD of the auto-oxidation of pyrogallol. Aortic homogenates from mice expressing hSOD show a 2.5-fold increase in activity compared with control C57BL6 (164±26 versus 33±7 U/mg protein, P<0.001).
We first measured the release of O2·– in isolated mouse aorta with lucigenin chemiluminescence. Measurements on intact arteries with lucigenin at 5 μmol/L have been corroborated with electron spin resonance and were not complicated by its redox cycling.29 As shown in Figure 5A, O2·– was significantly elevated in aortas isolated from a CD40L-exposed mouse compared with aortas in sham-treated mice (n=9; P<0.05, sham versus CD40L-treated mice). In hSOD-TG mice, basal aortic O2·– release was significantly lower than in C57BL6 mice (Figure 5A; n=7; P<0.05, C57BL6 versus hSOD-TG). Importantly, there was a significant decrease of O2·– release in CD40L-treated hSOD-TG mice compared with CD40L-treated C57BL6 mice (n=9; P<0.01, CD40L-treated C57BL6 versus CD40L-treated hSOD-TG mice; Figure 5A). However, there was no significant difference in aortic O2·– release between CD40L-treated hSOD-TG and nontreated hSOD-TG mice (Figure 5A) These results indicate that CD40L infusion significantly increased O2·–.
It was interesting to investigate whether CD40L infusion increased 3-NT, PGIS nitration and inhibition in vivo. As shown in Figure 3B, CD40L significantly increased 3-NT staining in the mouse aorta of C57BL6 mice but not in hSOD-TG (n=6; P<0.05, sham versus CD40L and CD40L versus sham-treated mice). 3-NT immunostaining was intensely present in the endothelium of CD40L-treated C57BL6 mice but was significantly less in CD40L-treated hSOD-TG mice, which suggests that CD40L infusion increased 3-NT staining in vivo. Because hSOD-TG mice had decreased contents of 3-NT in aortas, these results suggest that CD40L increased reactive nitrogen species, likely ONOO–. In parallel, CD40L decreased PGIS activity in wild types but had less effect in hSOD-Tg mice (Figure 6A) without altering PGIS expression (Figure 6B). The increased ONOO– was further confirmed by the increased PGIS nitration. As shown in Figure 6B, there was a slight decrease in PGIS expression in hSOD-Tg mice. Western blotting with antibodies against 3-NT showed a marked increase in 3-NT in PGIS in mice treated with CD40L (n=5; P<0.05, sham versus CD40L-infused mice; P<0.05, sham wild-type versus sham hSOD-TG mice and wild-type CD40L-infused mice versus hSOD-TG CD40L-infused mice), which indicates increased PGIS nitration by CD40L treatment.
Discussion
In the present study using cultured HAECs, we have demonstrated for the first time that exposure of HAECs to clinically relevant concentrations of CD40L (20 to 80 ng/mL) for 18 hours significantly increases the production of O2·– and ONOO– and consequently decreases the bioactivity of NO, as indicated by decreased levels of cGMP. Further evidence that NO is inactivated by reacting with O2·– to form the reaction product ONOO– is provided by the increased detection of PGIS nitration and inhibition in cells exposed to CD40L. Convincing evidence for ONOO– formation in cells exposed to CD40L comes from the identification of tyrosine nitration of PGIS. 3-NT is regarded as the "footprint" of ONOO– formation in vivo,18 although other pathways of tyrosine nitration have been suggested.32,33 Here, we for the first time present evidence that PGIS might be nitrated by ONOO– both in vitro and in vivo. Our previous studies have demonstrated that this enzyme undergoes tyrosine nitration by low concentrations of ONOO–.15,16 In the present study, we found that increased staining with an antibody against 3-NT was found in the immunoprecipitates obtained with antibodies against PGIS in CD40L-exposed cells, although the levels of PGIS expression were not changed. Inhibition of both endothelial NOS and scavenging O2·– with SOD attenuated CD40L-enhanced PGIS nitration, which indicated that reactive nitrogen species, likely ONOO–, are involved. In addition, inhibition of mitochondria-derived O2·– also abolished CD40L-enhanced PGIS nitration, which suggests that mitochondria might be the source of O2·– and ONOO–.
We have further demonstrated that administration of CD40L caused PGIS nitration and inhibition in C57BL6 mice in vivo. Tyrosine nitration of PGIS is most likely mediated by ONOO– formed endogenously, generated from NO, and O2·– caused by CD40L exposure. Although peroxidases such as myeloperoxidase have been reported to cause protein nitration in vivo,32,33 a role of peroxidase-catalyzed PGIS nitration is less likely, because hSOD-TG mice had significantly attenuated CD40L-enhanced PGIS nitration and inhibition. Peroxidase-catalyzed PGIS nitration is less likely because overexpression of SOD, which provides hydrogen peroxide (H2O2) to facilitate the peroxidase-catalyzed 3-NT formation, should enhance instead of decrease PGIS nitration. Although we cannot exclude the possibility of peroxidase-catalyzed PGIS nitration, the present data strongly suggest that ONOO– is likely to be responsible for the increased PGIS nitration caused by CD40L in vivo.
The selectivity of ONOO– for PGIS is explained by the exceptional sensitivity of PGIS, which catalyzes its own nitration by ONOO–.21 In addition, our studies have also shown that inactivation of PGIS results in a consequent activation of TPr through its cumulative substrate, PGH2.19 This consequent TPr stimulation triggers the overexpression of ICAM-1 in HAECs (Figure 4). Because TXA2 promotes and PGI2 prevents the initiation and progression of atherogenesis, the present study results unveil a novel mechanism by which CD40L causes atherosclerosis, ie, CD40L generates uncontrolled O2·–, which results in increased destruction of NO and a concomitant formation of a highly cytotoxic oxidant, ONOO–, which triggers nitration and inhibition of PGIS that results in consequent TPr stimulation. Thus, the present study for the first time provides evidence that CD40L via ONOO– causes vascular endothelial dysfunction by a combination of 2 mechanisms: decreased production of the vasorelaxant PGI2 and NO and accumulation of the vasoconstrictor PGH2, which stimulates the TXA2 receptor. The present data suggest that endogenous nitration of PGIS may contribute to the functional defects of the endothelium in pathological situations, not only by a lack of the vasorelaxant NO and PGI2, but more directly by causing accumulation of the prothrombotic and proconstrictive prostanoid PGH2 (Figure 3). The results of the present study provide a potential explanation for the paradoxical effects of endothelium-dependent vasorelaxants such as acetylcholine, which trigger vasoconstriction in human atherosclerotic arteries.34,35 Even individuals with significant atherosclerotic risk factors but without clinically manifest atherosclerosis have a decreased vasodilator response in parallel with higher production of vasoconstricting prostaglandins.34–36 Such abnormal responses are normalized by inhibition of cyclooxygenase.37 Aspirin has no effect under normal conditions but improves/restores acetylcholine-mediated vasodilation in patients with atherosclerosis.32,37
In summary, we have demonstrated that exposure of HAECs to clinically relevant concentrations of CD40L (20 to 80 ng/mL) increased O2·– and ONOO–, causing PGIS nitration and TPr stimulation in vitro and in vivo. These concentrations of CD40L can be found in patients. In addition, we fully envision that local concentrations of CD40L in the active areas of thrombotic or inflammatory vasculatures might be higher than those in circulating blood. Our findings might have important implications in atherosclerosis, because tyrosine nitration and inhibition of PGIS not only eliminates the vasodilatory, growth-inhibiting, antithrombotic and antiadhesive effects of prostacyclin (PGI2) but also increases release of PGH2 or TXA2, 2 potent vasoconstrictor, prothrombotic, growth- and adhesion-promoting agents. Both PGI2 and TXA2 contribute to the initiation and progression of vascular injury and thrombosis because of the downregulation of protective actions of both NO and PGI2 and because the nonmetabolized PGH2 or shunted TXA2 tips the balance toward platelet aggregation, leukocyte adherence, atheroma accumulation, and thrombus formation, which leads to the further release of CD40L from platelets and vascular cells. Thus, this biological positive-feedback loop will not only amplify proatherogenic CD40L and TXA2 but also decrease antiatherogenic factors, NO and PGI2, which can initiate and propagate clinical events characterized by vasoconstriction, adherence of platelets and monocytes, atherosclerosis, and/or micro-occlusive vascular disease, and ultimately, vascular thrombosis and tissue damage. This may not only explain why many atherothrombotic diseases in patients and animals have decreased levels of PGI2 but also why increases have been noted in its precursors, PGH2 and TXA2, both of which activate the TXA2 receptor.
Acknowledgments
We would like to acknowledge Drs Michael Brownlee and David R. Pimental for providing constructs for UCP-1 and p47phox-dominant negative adenoviruses. We thank Dr T. Klein for kindly providing an antibody against human PGIS. This work was supported by NIH grants HL079584 and HL07439, a career development grant from the American Heart Association, a grant-in-aid from Juvenile Diabetes Research Foundation, and the Graduate School of Medicine, the University of Tennessee.
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