Aspirin Attenuates Cytomegalovirus Infectivity and Gene Expression Mediated by Cyclooxygenase-2 in Coronary Artery Smooth Muscle Cells

From the Cardiology Branch, National Heart, Lung, and Blood Institute (NHLBI), National Institutes of Health (NIH), Bethesda, Md (E.S.); Washington Hospital Center, Washington, DC (S.E.E.); the Pathology Section, NHLBI, NIH, (Z.-X.Y., V.J.F.); and Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill (E.-S.H.).

Correspondence to Edith Speir, Cardiology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892-1650. E-mail speire{at}gwgate.nhlbi.nih.gov

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abstract—Human cytomegalovirus (CMV) infection of smooth muscle cells generates reactive oxygen species (ROS) and thereby activates nuclear factor B (NFB), which causes expression of viral and cellular genes involved in immune and inflammatory responses. These changes could account for the mounting evidence suggesting that CMV may contribute causally to restenosis and atherosclerosis. We found that CMV induces ROS, at least partly, through a cyclooxygenase-2 (COX-2)–dependent pathway. Moreover, the viral immediate-early (IE) gene products, IE72 and IE84, have the capacity to transactivate the COX-2 promoter. Aspirin and indomethacin, both cyclooxygenase inhibitors as well as direct ROS scavengers, reduce CMV-induced ROS, probably through both of these activities. Sodium salicylate also has antiviral effects as the result of its potent antioxidant properties. Furthermore, by reducing ROS, aspirin and sodium salicylate inhibit CMV-induced NFB activation, the ability of IE72 to transactivate its promoter, CMV IE gene expression after infection of SMCs, and CMV replication in SMCs. This is the first time aspirin has been shown to have antiviral effects. Thus, it is possible that aspirin has previously unrecognized therapeutic effects in various clinical situations, such as in viral infections (when used as an antipyretic agent) and in atherosclerosis (when used as an antiplatelet agent).

Key Words: antioxidant • atherosclerosis • cyclooxygenase • herpesvirus • salicylate

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
After primary infection, cytomegalovirus (CMV), like other herpesviruses, establishes lifelong persistence in the host. Although CMV is thought to cause clinically important infection only in newborns and in immunocompromised patients, accumulating evidence suggests that this virus may play a role in the development of restenosis after coronary angioplasty^{1 2 3} and in the genesis of atherosclerosis.⁴

We recently demonstrated that CMV infection of smooth muscle cells (SMCs) generates intracellular reactive oxygen species (ROS) within minutes of infection and that the resulting ROS contribute to nuclear factor B (NFB) activation.⁵ NFB stimulates the expression of many cellular genes and their products, including cytokines and adhesion molecules, which are involved in immune and inflammatory responses.⁶ Because the CMV major immediate-early promoter (MIEP) has 4 NFB-binding sites, activation of NFB is also critical for MIEP activation and eventual expression of all viral gene products, including the immediate-early (IE) major gene product IE72. In turn, IE72 transactivates its own promoter through the multiple NFB sites.⁷ Furthermore, angioplasty-induced injury to the vessel wall and reperfusion after balloon angioplasty produce ROS⁸ and cytokines. The resulting activation of NFB can in turn stimulate the MIEP present in latently infected cells and thereby contribute to reactivation of latent CMV.

Because of the critical role in both viral and cellular gene expression, CMV-induced ROS generation might constitute an excellent target for any therapeutic attempt to inhibit those cellular changes that are mediated by CMV infection and that might contribute to either restenosis or atherosclerosis. Indeed, we previously demonstrated that after CMV infection of SMCs, antioxidants inhibit CMV IE gene expression and viral replication.⁵ Further insights to refine such a strategy require identification of the cellular pathways responsible for generating ROS after CMV infection.

Recent studies have shown that CMV infection of human cells^{9 10 11} leads to stimulation of arachidonic acid (AA) release. Although the components downstream of AA release that are responsible for the CMV-stimulated ROS generation in SMCs have not been defined, one such component may involve cyclooxygenase (COX), a major enzyme system by which AA metabolism leads to the generation of ROS. If so, this may have therapeutic implications. Thus, aspirin is commonly used in patients with atherosclerosis. If COX is involved in CMV-induced generation of ROS and if CMV plays a causal role in restenosis and in atherosclerosis, then aspirin, a potent inhibitor of COX, might exert therapeutic effects in such patients through its antiviral properties, in addition to its antiplatelet actions.

Two isoforms of COX, encoded by different genes, catalyze the reactions whereby eicosanoids and ROS are formed from AA.¹² COX-1 is constitutively expressed and appears to mediate housekeeping functions. COX-2 is an IE gene product that is induced by various stimuli, many of which appear to exert their effects through the generation of ROS. Furthermore, the COX-2 promoter contains 2 NFB sites, which are important for cytokine-induced COX-2 transcription.¹³ In this investigation we determined whether COX-2 mediates the CMV-induced generation of ROS and whether inhibition of the generation of ROS might serve as a potential target to inhibit CMV gene expression and replication in SMCs.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cells, Viruses, Plasmids, Antibodies, and Drugs
Human coronary artery SMCs at passages 4 to 6 were grown as described previously in their optimal medium (Clonetics). Human kidney 293 cells were purchased from American Type Culture Collection (ATCC) and grown according to ATCC instructions. Human CMV, Towne strain, was passaged in human fibroblasts (HEL299, ATCC) and titrated as described before.⁵ Because the virus was harvested in the supernatant of infected fibroblasts in cell culture, we obtained virus-free supernatant by ultracentrifugation for use as negative controls in all experiments.¹⁴ The experiments were repeated with virus purified by ultracentrifugation as follows: Supernatant media of CMV-infected HEL299 cells (containing virus released by the cells) was centrifuged at 3000 rpm to remove cell debris. The supernatant (28 mL) was then carefully layered on a 0.5 mol/L sucrose sterilized in MEM cushion and centrifuged in an SW-28 rotor at 26 000 rpm for 2 hours at 4°C. The supernatant was then discarded, and the purified virus pellet was suspended in 0.6 mL sterile MEM. Titers typically are 3 to 5x10⁸ plaque-forming units per milliliter.

The following constructs have been described previously: the IE72 and IE84 expression plasmids¹⁵ ; the CMV-reporter plasmid, MIEP–chloramphenicol acetyltransferase (CAT)⁵ ; the COX-1 and COX-2 expression vectors¹⁶ ; the reporter plasmid COX-2–CAT (p102), which contains the nucleotides -582 to +101 of the COX-2 promoter upstream to a CAT reporter gene, or the deleted COX-2 promoter-CAT plasmid (p105, -92 to +101), which lacks all inducible transcription factor binding sites.¹⁶

The polyclonal anti–COX-2 antibody was raised by T. Hla.¹⁷ Aspirin (acetylsalicylic acid) and sodium salicylate were purchased from Sigma, and NS-398 was obtained from Biomol.

Assessment of Intracellular Redox State
Intracellular ROS generation after CMV infection was measured as described in detail elsewhere. Briefly, cells were grown in 4-well chamber glass slides (Nunc), infected with CMV for 1 hour, and treated with drugs for 1 hour after removal of the virus. Cells were incubated for 5 minutes with 5 µmol/L 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA, Molecular Probes), a nonpolar dye that diffuses into cells. The dye is then deacetylated, and the polar derivative becomes fluorescent only when oxidized by H₂O₂ or hydroxyl radicals. Fluorescence was monitored and recorded by laser-scanning confocal microscopy (Leica TCS4D, Leica Lasertechnik) as described.⁵

Cyclooxygenase Inhibitors
To determine whether COX-1 and COX-2 contribute to the CMV-mediated induction of ROS, the following separate experiments were performed: infected SMCs were treated with (1) aspirin and indomethacin, 2 nonselective COX-1 and COX-2 inhibitors, (2) NS-398 and dexamethasone, a selective and a nonselective COX-2 inhibitor, respectively, and (3) sodium salicylate, which has minimal COX-inhibitory effects but is a ROS scavenger more potent than aspirin.

Transfections and CAT Assays
Human coronary SMCs were grown in 10-cm dishes and transfected with 1 µg of the COX-2–CAT alone or cotransfected with 2 µg of the IE72 or IE84 plasmid or with 0.1 µg of the major IE CMV-promoter MIEP-CAT plasmid without or with 1 µg of the IE72 or the IE84 expression vector. Transfection was performed by lipofection, with N-[1-(2,3-dioleolyloxy)propyl]-N, N, N-trimethylammonium methyl sulfate (DOTAP) reagent (Boehringer). Human 293 cells were grown in 10-cm dishes, transfected with COX-1 or COX-2 expression vectors (2 µg per dish) with lipofectamine according to the manufacturer's instructions (Gibco/BRL), and harvested with a 10 mmol/L EDTA solution 24 hours later. They were then grown in 4-well chamber slides coated with human fibronectin (5 µg per cm²). After 24 hours they were infected with 5 multiplicities of infection (MOI) of CMV for 1 hour and, after removal of free virus, incubated with NS-398 for 1 hour. The drug was then removed, and the cells were prepared for detection of ROS by confocal microscopy as described above.

Immunoblots
SMCs were grown in 175-cm² flasks to 90% confluence, treated with drugs, infected with CMV, lysed, subjected to gel electrophoresis, and blotted.⁵ Steady-state protein levels of viral IE72 or human cellular COX-2 were assessed by immunoblotting with monoclonal anti-IE72 antibodies (6E1, Vancouver Biotech) as described elsewhere in detail.⁵ Polyclonal anti–COX-2 antibodies at a 1:1000 dilution and a chemiluminescence kit (Immun-star, Bio-Rad) were used for (protein) signal detection.

Viral Titer Assay and Cytopathic Effects
Human CMV (Towne strain) was passaged in our laboratory in HEL299 fibroblasts as described. Coronary SMCs were seeded in 48-well plates (15 000 per cm²) and, for immunocytochemistry of IE72 (72-kD IE CMV region-1 product), in 8-well glass chamber slides (Nunc) for 48 to 72 hours and then infected with CMV at 5 MOI. One hour after adsorption, free virus was removed and duplicate wells were treated with either vehicle, 2 mmol/L aspirin, or 2 mmol/L sodium salicylate. Infected SMCs and their growth media were sonicated 96 hours after infection and diluted 10-fold; viral titer was determined by plating aliquots of the sonicate on indicator fibroblasts. Cytopathic effects and plaque formation were assessed 3 to 10 days later by counting the number of foci of infected cells exhibiting cytomegalic changes. Additional plates of SMCs were infected or mock-infected, treated with aspirin or sodium salicylate as described above, and harvested for cell counting 24, 48, and 96 hours later to ensure that changes in viral titer were not due to changes in cell number after infection.

Cyclooxygenase Activity
Human coronary SMCs (HCSMCs) were plated in 24-well dishes for 48 hours and then infected with human CMV (5 MOI) for 1 hour. After removal of the virus, cells were treated for 1 hour with serum-free media containing 1, 5, or 10 µmol/L NS-398. The medium was then replaced with 1 mL of serum-free medium. The media supernatant was removed at 3, 6, and 12 hours and centrifuged at 3000 rpm for 10 minutes to remove cell debris; supernatants were flash-frozen in ethanol/dry ice and stored at -70°C until assay. Aliquots (50 µL) of collected samples were assayed for spontaneously released prostaglandin (PG) E₂ by Enzyme Immuno Assay as described by the manufacturer (Amersham) (Figure 5).

View larger version (14K): [in this window] [in a new window]   Figure 5. HCMV infection stimulates cultured SMC-cyclooxygenase activity, as measured by PGE2 production. Viral activation of SMCs induces marked increases in PGE2 generation throughout an 8-hour time course, in contrast to control SMC rates. The specific COX-2 inhibitor NS-398 attenuated PGE2 release in a concentration-related fashion. Data represent mean+SD. A total of 10 experiments were performed with similar results.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Effects of Aspirin and Indomethacin on CMV-Induced ROS Generation
We have previously shown that CMV infection of HCSMCs generates intracellular ROS (Figure 1A, a and b), as assessed by quantified confocal microscopic study of the fluorescence produced by the oxidation of DCFH-DA. Aspirin or indomethacin, 2 nonselective COX-1 and COX-2 inhibitors, diminishes CMV-induced ROS generation in a concentration-dependent manner (Figure 1A and Table). That this effect is not entirely due to inhibition of these enzymes is indicated by the finding that sodium salicylate also diminishes ROS generation (Figure 1A and Table). This compound has only minimal COX inhibitory activity¹⁸ but, like aspirin and indomethacin, is a potent ROS scavenger.¹⁹

View larger version (101K): [in this window] [in a new window]   Figure 1. A, Effect of aspirin, sodium salicylate, or indomethacin on endogenous ROS generation in CMV-infected SMCs: uninfected (a) and infected controls (b); aspirin (0.1 or 2 mmol/L) (c and d); sodium salicylate (0.1 or 2 mmol/L) (e and f); and indomethacin (2 or 20 µmol/L) (g and h). All cells (except in panel Aa) were infected with CMV at 5 MOI. Cells were infected with CMV, treated with drugs, and then exposed to DCFH-DA (5 µmol/L). The resulting fluorescence, indicative of ROS generation, was then assessed by confocal microscopy. The study was performed 3 times with similar results. Bar=200 µm. B, CMV-induced ROS generation is dependent on COX-2. Human 293 cells were transfected with expression plasmids containing empty vector DNA (a) or cDNA for COX-1 (b), cDNA for COX-2 (c), and cDNA for COX-2 plus treatment with 5 µmol/L NS-398 for 1 hour after infection (d). Twenty-four hours after transfection they were infected with CMV (5 MOI) for 1 hour and assessed for ROS, as in panel A. The study was performed 3 times with similar results. Bar=200 µm. C, CMV-induced ROS generation is dependent on COX-2: quantitative analysis. Relative fluorescence (RF) units from the experiments depicted in panel B were quantified, and 3 fields were analyzed for each bar depicted in the graph, as described in the Table. Cells transfected with plasmids encoding COX-1 or COX-2 but not infected with CMV exhibited little fluorescence.

Role of COX-2 in CMV-Dependent ROS Generation
Effect of NS-398 or Dexamethasone
That COX-2 is involved, at least in part, in CMV-induced ROS generation, is indicated by the finding that the selective COX-2 inhibitors NS-398 and dexamethasone also decrease CMV-induced ROS generation (Table). NS-398 irreversibly inactivates COX-2, whereas dexamethasone decreases COX-2 mRNA accumulation.^{20 21}

CMV Induction of ROS Is Dependent on Expression of COX-2
To more directly examine the role of COX-2 in CMV-induced ROS activity, we transfected 293 cells, which have minimal COX-1 or COX-2 expression,¹⁷ with expression vectors containing either the gene encoding COX-2 or COX-1, or the empty vector.¹⁶ Using immunoperoxidase staining with a specific anti-COX-2 antibody, we found that transfection efficiency was 15% to 20% (not shown). ROS generation induced by CMV is strikingly enhanced in the cells transfected with COX-2 when compared with that in the cells transfected with COX-1, with the empty vector, or with the COX-2 vector but without CMV infection (Figure 1B and 1C). These results indicate that CMV-induced ROS generation is dependent, at least in part, on the presence of COX-2. Confirmation of this conclusion is demonstrated by the decrease in CMV-induced ROS generation produced by NS-398 in the COX-2-transfected cells (Figure 1B, d).

Effects of IE72 and IE84 on Expression of COX-2–CAT in SMCs
We also found that the CMV IE gene products IE72 and IE84 have the potential to increase the transcription of the COX-2 gene (Figure 2A). We cotransfected the IE72 or IE84 expression plasmids into SMCs with either the COX-2 promoter–CAT reporter plasmid, which contains nucleotides -582 to +101 of the COX-2 promoter upstream to a CAT reporter gene, or with a deleted COX-2 promoter-CAT plasmid (-92 to +101), which lacks all inducible transcription factor–binding sites. IE72 and IE84 each transactivates the COX-2 promoter and exerts synergistic effects when cotransfected. The COX-2 promoter region that we used contains 2 NFB and 3 SP-1–binding elements, among others. IE72 is known to regulate NFB sites,⁷ and both IE72 and IE84 can activate SP-1–binding sites.¹⁵ The deleted reporter construct was unresponsive to IE72 and IE84.

View larger version (16K): [in this window] [in a new window]   Figure 2. A, IE72 and IE84 are capable of transactivating the COX-2 promoter. SMCs were transfected with either the COX-2–CAT reporter plasmid (nucleotides -582 to +101) encoding the binding elements 2 NFB, 3 SP-1, and one each, AP-2, C/EBP, Ets-1, and CRE or with the COX-2 mutant CAT reporter (all binding sites removed) and cotransfected with expression plasmids encoding either IE72 or IE84, or with both. Columns indicate the average of 3 independent experiments; error bars represent standard deviation of the means. B, Effect of aspirin (ASA) on activation of the CMV MIEP by H2O2 and by IE72. HCSMCs were transfected by lipofection with 0.1 µg of the CMV reporter plasmid MIEP-CAT or cotransfected with 1 µg of the IE72 expression plasmid. At 35 hours after transfection, SMCs were treated with ASA or vehicle for 1 hour. The drugs were removed, and for the H2O2 experiment, fresh medium containing 10 µmol/L H2O2 was added. Cells were harvested 12 hours later and processed for CAT activity. SMCs cotransfected with the reporter and IE72 (bars 8 to 10) were treated with aspirin for 12 hours at 24 to 36 hours after transfection and then assayed for CAT activity. Shown are means of 3 separate experiments. *Statistical significance of P<0.03.

Effects of Aspirin on Activation of MIEP-CAT by H₂O₂ and IE72 in SMCs
IE72 transactivates, in an ROS-dependent manner, its own promoter, the MIEP, through NFB sites.⁵ This step is critical for expression of downstream CMV genes.¹⁵ We found that aspirin inhibits, in a concentration-dependent manner, the MIEP transcriptional activity of IE72 (Figure 2B). This effect may be partly due to a decrease in ROS secondary to COX-2 inhibition; however, aspirin also inhibits the capacity of H₂O₂ to transactivate the MIEP (Figure 2B), indicating that another mechanism may be operative through a direct ROS scavenger effect.

Electrophoretic Mobility Shift Assay
We have previously shown by gel shift assay that CMV infection of SMCs activates NFB, an effect that is inhibited by antioxidants such as N-acetylcysteine.⁵ Here we determined that 1 to 2 mmol/L aspirin inhibits CMV-induced NFB activation in a concentration-related fashion (not shown).

Immunoblotting
The inhibitory effects of aspirin, sodium salicylate, and indomethacin on IE72 expression were confirmed by Western blot analysis (Figure 3). Both active CMV infection and reactivation of latent CMV leading to release of progeny follow a cascade of IE, early, and late events: activation of the MIEP, which leads to expression within 2 to 4 hours of the major IE protein IE72, a potent transcription factor for both viral and cellular promoters. IE72 then transactivates the MIEP through NFB binding sites,⁷ which leads to expression of IE84. The latter protein is a transcription factor more powerful than the former, which activates the early viral promoter and multiple cellular genes to create a milieu favorable to progression of infection and packaging of viral progeny. Because IE72 is a critical component of the infectious cycle for both acute and latent CMV, interventions aimed at inhibition of IE72 are reasonable and advantageous. We demonstrate that aspirin, indomethacin, or sodium salicylate inhibits IE72 expression by >50%. (Figure 3).

View larger version (48K): [in this window] [in a new window]   Figure 3. A, Effect of aspirin (ASA), sodium salicylate (NaSal), or indomethacin (Indo) on steady-state IE72 levels. Human coronary SMCs were first infected with human CMV (HCMV, 5 MOI) and then treated for 16 hours with ASA (2 mmol/L), NaSal (2 mmol/L), or Indo (20 µmol/L) and harvested; 80 µg of protein per lane was subjected to SDS-electrophoresis and immunoblotted with anti-IE72 antibody 6E1.1 The experiment was repeated once. B, Induction of COX-2 protein at 3 to 12 hours after CMV infection of SMCs shown by immunoblotting. Samples were processed as above. The experiment was repeated once.

We further show that steady-state levels of the COX-2 protein accumulated at 3 to 12 hours after infection, whereas COX-2 levels in uninfected cells were undetectable (Figure 3A). This confirms availability of the enzyme for PGE₂ synthesis and release (Figure 5).

Effect of Aspirin on Viral Titer
To determine whether the inhibition of ROS, NFB, and IE72 translates into impairment of viral replication, we treated infected SMCs with aspirin immediately after infection and again after renewal of the medium at 48 hours after infection. We found that aspirin or sodium salicylate at 0.5 and 2 mmol/L doses decreased viral titer by 50% to 70%. This effect was not due to infection-related reduction in cell number, as shown by counting cells in another set of experimental wells (Figure 4).

View larger version (13K): [in this window] [in a new window]   Figure 4. Effect of aspirin (ASA) or sodium salicylate (NaSal) on viral titer. HCMV-infected coronary SMCs (5 MOI) were kept in drug-supplemented maintenance medium for 96 hours. Aliquots of sonicated SMCs at 1:10 dilutions in serum-free medium were plated on indicator cells (HEL299, ATCC), and cytopathic foci of cytomegalic cells (by morphology) were recorded 96 hours later.

PGE₂ Production After Human CMV Infection
At 3 to 6 hours after infection of SMCs with human CMV, large amounts of PGE₂ appeared in the medium. In contrast, uninfected cells released only small amounts of PGE₂. Release peaked at 3 to 6 hours and then declined at 12 hours after infection (Figure 5).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The results of this investigation demonstrate that cyclooxygenases, and in particular COX-2, an IE mediator of inflammation,¹² are an important component of the pathway by which CMV infection of SMCs generate ROS and that ROS induction is prevented by both aspirin and indomethacin. ROS in turn activate NFB, which regulates COX-2¹³ and other inflammatory genes, notably intracellular adhesion molecule (ICAM)-1.²² We first found that aspirin and indomethacin inhibited ROS generation in a dose-dependent manner, as assessed by fluorescence and confocal microscopy (Figure 1A and Table). Because these agents inhibit both COX-1 and COX-2, we also determined the effects of the selective COX-2 inhibitors NS-398 and dexamethasone. Each of these agents markedly decreased the CMV-induced generation of ROS (Table). Thus, although we cannot exclude a role of COX-1, these results indicate that COX-2 is involved in CMV-induced ROS generation and in the synthesis of PGE₂ (Figure 5).

We more directly tested the conclusion that CMV induction of ROS is dependent on COX-2 by transfecting 293 cells (which, unlike SMCs, have minimal COX-1 or COX-2 expression),¹⁷ with expression vectors containing either the gene encoding COX-2 or COX-1.¹⁶ ROS were generated after CMV infection only in the cells transfected with the COX-2 expression plasmid, an effect abolished by treatment with the specific COX-2 inhibitor NS-398 (Figure 1B and 1C). We further demonstrate (1) accumulation of COX-2 protein in infected but not in uninfected coronary SMCs (Figure 3A), (2) COX enzyme activity in SMCs by measuring the release of PGE₂, and (3) COX-2 activity by concentration-related inhibition of PGE₂ release after treatment of the cells with the specific COX-2 inhibitor NS-398 (Figure 5). We speculate that CMV-induced COX-2 activation is, at least in part, caused by NFB activation.¹³

We have previously speculated that the generation of ROS can be viewed as a protective mechanism of the cell, which can contribute to the induction of apoptosis²³ and thereby prevent the infecting virus from replicating and infecting neighboring cells and to the activation of NFB, which mediates expression of cellular genes involved in the immune and inflammatory responses,⁶ such as ICAM-1.²² We also extracted RNA from infected SMCs and demonstrated induction of ICAM-1 mRNA (by Northern blotting) at 3 to 12 hours after CMV infection, whereas uninfected cells had no detectable message (not shown). CMV appears to have adapted to the cellular defense mechanism involving the release of AA and activation of COX-2, a known mediator of inflammation,²⁴ with subsequent generation of ROS and the resulting activation of NFB, which regulates transcription of COX-2, the CMV promoter, and the ICAM-1 promoter. During latent infection, stimuli to host cells that activate NFB also lead to activation of the viral promoter and to direct expression of viral IE genes. It is tempting to speculate that the virus has evolved to benefit from the cellular mechanism of ROS generation to activate its own genetic programs and to ensure frequent albeit abortive reactivation from latency.

We also found that the IE gene products IE72 and IE84 of CMV can transactivate the COX-2 promoter (Figure 2A). By increasing the expression and therefore the activity of COX-2, the virus facilitates the increase in ROS that follows infection and thereby further ensures that the intracellular environment is favorable to viral gene expression.

The IE protein IE72, a nuclear phosphoprotein, is expressed within a few hours after infection and then transactivates its own promoter, the MIEP, through multiple NFB sites.⁶ Because IE72 synthesis is critical for the ensuing cascade of CMV gene expression²⁵ in both acute infections and in reactivation from latency, interventions aimed at inhibiting this major IE CMV protein product would seem most beneficial. Because ROS are important for transactivation of the MIEP by IE72,⁵ we next determined whether aspirin interferes with this step of viral gene expression. By cotransfecting an IE72 expression plasmid and an MIEP-CAT reporter gene construct into human coronary SMCs, we found that aspirin inhibits, in a concentration-dependent manner, the transcriptional activity of IE72 (Figure 2B).

We previously showed that CMV infection of SMCs activates NFB. This effect appeared to be dependent on ROS, as NFB binding to its DNA was inhibited by antioxidants.⁵ In this investigation we found that NFB/DNA binding in SMCs is inhibited by aspirin in a concentration-related fashion (not shown).

On the basis of these findings, we predicted that aspirin would attenuate IE72 expression after CMV infection and thereby would also inhibit viral replication. We found by Western blotting analysis of infected SMCs that aspirin, sodium salicylate, or indomethacin inhibited IE72 expression by >50% (Figure 3). Moreover, both aspirin and salicylate modulated viral replication (Figure 4).

The effects of aspirin are caused, at least in part, by its capacity to inhibit COX-2. However, aspirin is a complex drug with multiple effects. In particular, it is known to be a potent ROS scavenger,¹⁹ and, therefore, part of the effects of aspirin we have observed in this investigation may be due to this activity. That the inhibition of the CMV-induced generation of ROS is not entirely due to inhibition of COX-2 is indicated by the findings that sodium salicylate also diminishes ROS generation. Also attributable to the direct ROS scavenger activity of aspirin is our finding that this drug inhibits the capacity of H₂O₂ to transactivate the MIEP (Figure 2B).

Our data suggest that aspirin, salicylate, and indomethacin have anti-CMV effects by directly scavenging ROS. We and others²⁶ also found that aspirin and salicylate inhibit NFB, which is critical for the activation of gene expression not only of CMV but also of cytokines, adhesion molecules, and other mediators of the inflammatory response in injured arteries. ROS are known to activate NFB, and antioxidants inhibit this activation. Recent reports have shown that treatment of patients with probucol, a potent antioxidant, substantially reduced luminal narrowing after balloon coronary angioplasty, presumably at least in part by inhibiting NFB.²⁷

This is the first time that aspirin has been shown to have antiviral effects. Although relatively high concentrations of aspirin were needed to achieve these effects, such concentrations are attained in the plasma of patients treated for chronic inflammatory diseases such as arthritis.¹⁸ These findings raise the possibility that aspirin has a previously unrecognized therapeutic effect in various clinical situations, such as when it is administered to patients with viral infections as an antipyretic agent, to patients undergoing angioplasty as an antiplatelet agent, and to patients with atherosclerosis to prevent thrombotic complications of the disease.

	Acknowledgments

 
We thank Dr Kazuyo Takeda for contributing her expertise on the confocal microscopy and Linda Raley for her assistance in the preparation of the manuscript.

Received November 25, 1997; accepted June 18, 1998.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Speir E, Modali R, Huang ES, Leon MB, Shawl F, Finkel T, Epstein SE. Potential role of human cytomegalovirus and p53 interaction in coronary restenosis. Science. 1994;265:391–394.[Abstract/Free Full Text]

2. Speir E, Huang ES, Modali R, Leon MB, Shawl F, Finkel T, Epstein SE. Interaction of human cytomegalovirus with p53: possible role in coronary restenosis. Scand J Infect Dis Suppl. 1995;99:78–81.[Medline] [Order article via Infotrieve]

3. Zhou Y F, Leon MB, Waclawiw MA, Popma JJ, Yu ZX, Finkel T, Epstein SE. Association between prior cytomegalovirus infection and the risk of restenosis after coronary atherectomy. N Engl J Med. 1996;335:624–630.[Abstract/Free Full Text]

4. Melnick JL, Adam E, DeBakey ME. Possible role of cytomegalovirus in atherogenesis. JAMA. 1990;263:2204–2207.[Abstract/Free Full Text]

5. Speir E, Shibutani T, Yu ZX, Ferrans VJ, Epstein SE. Role of reactive oxygen intermediates in cytomegalovirus gene expression and in the response of human smooth muscle cells to viral infection. Circ Res. 1996;79:1143–1152.[Abstract/Free Full Text]

6. Grilli M, Chiu JJ, Lenardo MJ. NF-kappa B and Rel: participants in a multiform transcriptional regulatory system. Int Rev Cytol. 1993;143:1–62.[Medline] [Order article via Infotrieve]

7. Sambucetti LC, Cherrington JM, Wilkinson GW, Mocarski ES. NF-kappa B activation of the cytomegalovirus enhancer is mediated by a viral transactivator and by T cell stimulation. EMBO J. 1989;8:4251–4258.[Medline] [Order article via Infotrieve]

8. Grech ED, Dodd NJ, Jackson MJ, Morrison WL, Faragher EB, Ramsdale DR. Evidence for free radical generation after primary percutaneous transluminal coronary angioplasty recanalization in acute myocardial infarction. Am J Cardiol. 1996;77:122–127.[Medline] [Order article via Infotrieve]

9. Tanaka J, Iida H, Sato H, Hatano M. Inhibitors of prostaglandin synthesis inhibit growth of human cytomegalovirus and reactivation of latent virus in a productively and latently infected human cell line. Virology. 1988;163:205–208.[Medline] [Order article via Infotrieve]

10. AbuBakar S, Boldogh I, Albrecht T. Human cytomegalovirus stimulates arachidonic acid metabolism through pathways that are affected by inhibitors of phospholipase A2 and protein kinase C. Biochem Biophys Res Commun. 1990;166:953–959.[Medline] [Order article via Infotrieve]

11. Shibutani T, Yu ZX, Ferrans VJ, Moss J, Epstein SE. Pertussis toxin sensitive G-proteins as mediators of the signal transduction pathways activated by cytomegalovirus infection of smooth muscle cells. J Clin Invest. 1997;100:2054–2061.[Medline] [Order article via Infotrieve]

12. Smith WL, DeWitt DL. Biochemistry of prostaglandin endoperoxide H synthase-1 and synthase-2 and their differential susceptibility to nonsteroidal anti-inflammatory drugs. Semin Nephrol. 1995;15:170–194.

13. Crofford LJ, Tan B, McCarthy CJ, Hla T. Involvement of nuclear factor B in the regulation of cyclooxygenase-2 expression by interleukin-1 in rheumatoid synoviocytes. Arthritis Rheum. 1997;40:226–236.[Medline] [Order article via Infotrieve]

14. Alcami J, Barzu T, Michelson S. Induction of an endothelial cell growth factor by human cytomegalovirus infection of fibroblasts. J Gen Virol. 1991;72:2765–2770.[Abstract/Free Full Text]

15. Yurochko AD, Kowalik TF, Huong SM, Huang ES. Human cytomegalovirus upregulates NF-kappa B activity by transactivating the NF-kappa B p105/p50 and p65 promoters. J Virol. 1995;69:5391–5400.[Abstract]

16. Appleby SB, Ristimaki A, Neilson K, Narko K, Hla T. Structure of the human cyclo-oxygenase-2 gene. Biochem J. 1994;302:723–727.

17. Ristimaki A, Narko K, Hla T. Down-regulation of cytokine-induced cyclooxygenase-2 transcript isoforms by dexamethasone: evidence for post-transcriptional regulation. Biochem J. 1996;318:325–331.

18. Weissmann G. Aspirin. Sci Am. 1991;264:84–90.[Medline] [Order article via Infotrieve]

19. Sagone AL Jr, Husney RM. Oxidation of salicylates by stimulated granulocytes: evidence that these drugs act as free radical scavengers in biological systems. J Immunol. 1987;138:2177–2183.[Abstract]

20. Copeland RA, Williams JM, Giannaras J, Nurnberg S, Covington M, Pinto D, Pick S, Trzaskos JM. Mechanism of selective inhibition of the inducible isoform of prostaglandin G/H synthase. Proc Natl Acad Sci U S A.. 1994;91:11202–11206.[Abstract/Free Full Text]

21. Evett GE, Xie W, Chipman JG, Robertson DL, Simmons DL. Prostaglandin G/H synthase isoenzyme 2 expression in fibroblasts: regulation by dexamethasone, mitogens, and oncogenes. Arch Biochem Biophys. 1993;306:169–177.[Medline] [Order article via Infotrieve]

22. Shin WS, Hong YH, Peng HB, DeCaterina R, Libby P, Liao JK. Nitric oxide attenuates vascular smooth muscle cell activation by interferon-. J Biol Chem. 1996;271:11317–11324.[Abstract/Free Full Text]

23. Jacobson MD. Reactive oxygen species and programmed cell death. Trends Biochem Sci. 1996;21:83–86.[Medline] [Order article via Infotrieve]

24. Morham SG, Langenbach R, Loftin CD, Tiano HF, Vouloumanos N, Jennette JC, Mahler JF, Kluckman KD, Ledford A, Lee CA, Smithies O. Prostaglandin synthase 2 gene disruption causes severe renal pathology in the mouse. Cell. 1995;83:473–482.[Medline] [Order article via Infotrieve]

25. Wathen MW, Stinski MF. Temporal patterns of human cytomegalovirus transcription: mapping the viral RNAs synthesized at immediate early, early, and late times after infection. J Virol. 1982;41:462–477.[Abstract/Free Full Text]

26. Kopp E, Gosh S. Inhibition of NFB by sodium salicylate and aspirin. Science. 1994;265:956–958.[Abstract/Free Full Text]

27. Tardif JC, Cote G, Lesperance J, Bourassa M, Lambert J, Doucet S, Bilodeau L, Nattel S, DeGuise P. Probucol and multivitamins in the prevention of restenosis after coronary angioplasty. N Engl J Med. 1997;337:365–372.[Abstract/Free Full Text]

This article has been cited by other articles:

				S. E. Epstein, J. Zhu, A. H. Najafi, and M. S. Burnett Insights Into the Role of Infection in Atherogenesis and in Plaque Rupture Circulation, June 23, 2009; 119(24): 3133 - 3141. [Full Text] [PDF]

				J. Schroer and T. Shenk Inhibition of cyclooxygenase activity blocks cell-to-cell spread of human cytomegalovirus PNAS, December 9, 2008; 105(49): 19468 - 19473. [Abstract] [Full Text] [PDF]

				M. M. Brinkmann, M. Pietrek, O. Dittrich-Breiholz, M. Kracht, and T. F. Schulz Modulation of Host Gene Expression by the K15 Protein of Kaposi's Sarcoma-Associated Herpesvirus J. Virol., January 1, 2007; 81(1): 42 - 58. [Abstract] [Full Text] [PDF]

				X. Yang, V. Murthy, K. Schultz, J. B. Tatro, K. A. Fitzgerald, and D. Beasley Toll-like receptor 3 signaling evokes a proinflammatory and proliferative phenotype in human vascular smooth muscle cells Am J Physiol Heart Circ Physiol, November 1, 2006; 291(5): H2334 - H2343. [Abstract] [Full Text] [PDF]

				E. Fosslien Cardiovascular Complications of Non-Steroidal Anti-Inflammatory Drugs Ann. Clin. Lab. Sci., October 1, 2005; 35(4): 347 - 385. [Abstract] [Full Text] [PDF]

				S.-P. Gravel and M. J. Servant Roles of an I{kappa}B Kinase-related Pathway in Human Cytomegalovirus-infected Vascular Smooth Muscle Cells: A MOLECULAR LINK IN PATHOGEN-INDUCED PROATHEROSCLEROTIC CONDITIONS J. Biol. Chem., March 4, 2005; 280(9): 7477 - 7486. [Abstract] [Full Text] [PDF]

				C. A. Rue, M. A. Jarvis, A. J. Knoche, H. L. Meyers, V. R. DeFilippis, S. G. Hansen, M. Wagner, K. Fruh, D. G. Anders, S. W. Wong, et al. A Cyclooxygenase-2 Homologue Encoded by Rhesus Cytomegalovirus Is a Determinant for Endothelial Cell Tropism J. Virol., November 15, 2004; 78(22): 12529 - 12536. [Abstract] [Full Text] [PDF]

				L. Hertel and E. S. Mocarski Global Analysis of Host Cell Gene Expression Late during Cytomegalovirus Infection Reveals Extensive Dysregulation of Cell Cycle Gene Expression and Induction of Pseudomitosis Independent of US28 Function J. Virol., November 1, 2004; 78(21): 11988 - 12011. [Abstract] [Full Text] [PDF]

				M. K. Froberg CMV Escapes! Ann. Clin. Lab. Sci., April 1, 2004; 34(2): 123 - 130. [Abstract] [Full Text] [PDF]

				L. Potena, G. Frascaroli, F. Grigioni, T. Lazzarotto, G. Magnani, L. Tomasi, F. Coccolo, L. Gabrielli, C. Magelli, M. P. Landini, et al. Hydroxymethyl-Glutaryl Coenzyme A Reductase Inhibition Limits Cytomegalovirus Infection in Human Endothelial Cells Circulation, February 3, 2004; 109(4): 532 - 536. [Abstract] [Full Text] [PDF]

				P. L. Nerheim, J. L. Meier, M. A. Vasef, W.-G. Li, L. Hu, J. B. Rice, D. Gavrila, W. E. Richenbacher, and N. L. Weintraub Enhanced Cytomegalovirus Infection in Atherosclerotic Human Blood Vessels Am. J. Pathol., February 1, 2004; 164(2): 589 - 600. [Abstract] [Full Text] [PDF]

				T. L. Symensma, D. Martinez-Guzman, Q. Jia, E. Bortz, T.-T. Wu, N. Rudra-Ganguly, S. Cole, H. Herschman, and R. Sun COX-2 Induction during Murine Gammaherpesvirus 68 Infection Leads to Enhancement of Viral Gene Expression J. Virol., December 1, 2003; 77(23): 12753 - 12763. [Abstract] [Full Text] [PDF]

				D. Rott, J. Zhu, M. S. Burnett, Y. i F. u Zhou, A. Zalles-Ganley, J. Ogunmakinwa, and S. E. Epstein Effects of MF-tricyclic, a selective cyclooxygenase-2 inhibitor, on atherosclerosis progression and susceptibility to cytomegalovirus replication in apolipoprotein-E knockout mice J. Am. Coll. Cardiol., May 21, 2003; 41(10): 1812 - 1819. [Abstract] [Full Text] [PDF]

				H. Yoneda, K. Miura, H. Matsushima, K. Sugi, T. Murakami, K. Ouchi, K. Yamashita, H. Itoh, T. Nakazawa, M. Suzuki, et al. Aspirin inhibits Chlamydia pneumoniae-induced NF-{kappa}B activation, cyclo-oxygenase-2 expression and prostaglandin E2 synthesis and attenuates chlamydial growth J. Med. Microbiol., May 1, 2003; 52(5): 409 - 415. [Abstract] [Full Text] [PDF]

				M.-E. Janelle, A. Gravel, J. Gosselin, M. J. Tremblay, and L. Flamand Activation of Monocyte Cyclooxygenase-2 Gene Expression by Human Herpesvirus 6. ROLE FOR CYCLIC AMP-RESPONSIVE ELEMENT-BINDING PROTEIN AND ACTIVATOR PROTEIN-1 J. Biol. Chem., August 16, 2002; 277(34): 30665 - 30674. [Abstract] [Full Text] [PDF]

				C.-J. Chen, S.-L. Raung, M.-D. Kuo, and Y.-M. Wang Suppression of Japanese encephalitis virus infection by non-steroidal anti-inflammatory drugs J. Gen. Virol., August 1, 2002; 83(8): 1897 - 1905. [Abstract] [Full Text] [PDF]

				E. S. Mocarski Jr. Virus self-improvement through inflammation: No pain, no gain PNAS, March 19, 2002; 99(6): 3362 - 3364. [Full Text] [PDF]

				H. Zhu, J.-P. Cong, D. Yu, W. A. Bresnahan, and T. E. Shenk From the Cover: Inhibition of cyclooxygenase 2 blocks human cytomegalovirus replication PNAS, March 19, 2002; 99(6): 3932 - 3937. [Abstract] [Full Text] [PDF]

				T. M. Lincoln, N. Dey, and H. Sellak Signal Transduction in Smooth Muscle: Invited Review: cGMP-dependent protein kinase signaling mechanisms in smooth muscle: from the regulation of tone to gene expression J Appl Physiol, September 1, 2001; 91(3): 1421 - 1430. [Abstract] [Full Text] [PDF]

				C.-L. Liao, Y.-L. Lin, B.-C. Wu, C.-H. Tsao, M.-C. Wang, C.-I Liu, Y.-L. Huang, J.-H. Chen, J.-P. Wang, and L.-K. Chen Salicylates Inhibit Flavivirus Replication Independently of Blocking Nuclear Factor Kappa B Activation J. Virol., September 1, 2001; 75(17): 7828 - 7839. [Abstract] [Full Text] [PDF]

				J. Cinatl Jr, S. Margraf, J.-U. Vogel, M. Scholz, J. Cinatl, and H. W. Doerr Human Cytomegalovirus Circumvents NF-{kappa}B Dependence in Retinal Pigment Epithelial Cells J. Immunol., August 15, 2001; 167(4): 1900 - 1908. [Abstract] [Full Text] [PDF]

				E. Guetta, E. M Scarpati, and P. E DiCorleto Effect of cytomegalovirus immediate early gene products on endothelial cell gene activity Cardiovasc Res, June 1, 2001; 50(3): 538 - 546. [Abstract] [Full Text] [PDF]

				D. Wu, M. G. Hayek, and S. N. Meydani Vitamin E and Macrophage Cyclooxygenase Regulation in the Aged J. Nutr., February 1, 2001; 131(2): 382S - 388. [Abstract] [Full Text]

				E. Speir, Z.-X. Yu, K. Takeda, V. J. Ferrans, and R. O. Cannon III Antioxidant Effect of Estrogen on Cytomegalovirus-Induced Gene Expression in Coronary Artery Smooth Muscle Cells Circulation, December 12, 2000; 102(24): 2990 - 2996. [Abstract] [Full Text] [PDF]

				E. Speir, Z.-X. Yu, K. Takeda, V. J. Ferrans, and R. O. Cannon III Competition for p300 Regulates Transcription by Estrogen Receptors and Nuclear Factor-{kappa}B in Human Coronary Smooth Muscle Cells Circ. Res., November 24, 2000; 87(11): 1006 - 1011. [Abstract] [Full Text] [PDF]

				J. Cinatl Jr., R. Blaheta, M. Bittoova, M. Scholz, S. Margraf, J.-U. Vogel, J. Cinatl, and H. W. Doerr Decreased Neutrophil Adhesion to Human Cytomegalovirus-Infected Retinal Pigment Epithelial Cells Is Mediated by Virus-Induced Up-Regulation of Fas Ligand Independent of Neutrophil Apoptosis J. Immunol., October 15, 2000; 165(8): 4405 - 4413. [Abstract] [Full Text] [PDF]

				S A Morre, W Stooker, W K Lagrand, A J C van den Brule, and H W M Niessen Microorganisms in the aetiology of atherosclerosis J. Clin. Pathol., September 1, 2000; 53(9): 647 - 654. [Abstract] [Full Text] [PDF]

				M. Mietus-Snyder, M. S. Gowri, and R. E. Pitas Class A Scavenger Receptor Up-regulation in Smooth Muscle Cells by Oxidized Low Density Lipoprotein. ENHANCEMENT BY CALCIUM FLUX AND CONCURRENT CYCLOOXYGENASE-2 UP-REGULATION J. Biol. Chem., June 2, 2000; 275(23): 17661 - 17670. [Abstract] [Full Text] [PDF]

				E.W.N. Lam, H.M. Hammad, R. Zwacka, C.J. Darby, K.R. Baumgardner, B.L. Davidson, T.D. Oberley, J.F. Engelhardt, and L.W. Oberley Immunolocalization and Adenoviral Vector-mediated Manganese Superoxide Dismutase Gene Transfer to Experimental Oral Tumors Journal of Dental Research, June 1, 2000; 79(6): 1410 - 1417. [Abstract] [PDF]

				Z. Yan, K. Subbaramaiah, T. Camilli, F. Zhang, T. Tanabe, T. A. McCaffrey, A. J. Dannenberg, and B. B. Weksler Benzo[a]pyrene Induces the Transcription of Cyclooxygenase-2 in Vascular Smooth Muscle Cells. EVIDENCE FOR THE INVOLVEMENT OF EXTRACELLULAR SIGNAL-REGULATED KINASE AND NF-kappa B J. Biol. Chem., February 18, 2000; 275(7): 4949 - 4955. [Abstract] [Full Text] [PDF]

				E. A. Fortunato, M. L. Dell'Aquila, and D. H. Spector Specific chromosome 1 breaks induced by human cytomegalovirus PNAS, January 18, 2000; 97(2): 853 - 858. [Abstract] [Full Text] [PDF]

				S. E. Epstein, Y. F. Zhou, and J. Zhu Infection and Atherosclerosis : Emerging Mechanistic Paradigms Circulation, July 27, 1999; 100 (4): e20 - e28. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Speir, E. Articles by Epstein, S. E. Search for Related Content PubMed PubMed Citation Articles by Speir, E. Articles by Epstein, S. E. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB ACETYLSALICYLIC ACID DEXAMETHASONE INDOMETHACIN