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(Circulation. 1995;91:1914-1917.)
© 1995 American Heart Association, Inc.

Articles

Aspirin Inhibits Nuclear Factor–B Mobilization and Monocyte Adhesion in Stimulated Human Endothelial Cells

Christian Weber, MD; Wolfgang Erl, BS; Angelika Pietsch, BS; Peter C. Weber, MD

From the Institut für Prophylaxe der Kreislaufkrankheiten, Ludwig-Maximilians-Universität, München, Germany.

Correspondence to C. Weber, MD, Institut für Prophylaxe der Kreislaufkrankheiten, Pettenkoferstrasse 9, D-80336 München, FRG.

	Abstract

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Background The induction of vascular cell adhesion molecule–1 (VCAM-1) and E-selectin by tumor necrosis factor– (TNF) is mediated by mobilization of the transcription factor nuclear factor–B (NF-B). Since salicylates have been reported to inhibit NF-B activation by preventing the degradation of its inhibitor IB, we studied a potential inhibition of this pathway by acetylsalicylate (aspirin) in human umbilical vein endothelial cells (HUVECs).

Methods and Results Gel-shift analyses demonstrated dose-dependent inhibition of TNF-induced NF-B mobilization by aspirin at concentrations ranging from 1 to 10 mmol/L. Induction of VCAM-1 and E-selectin surface expression by TNF was dose-dependently reduced by aspirin over the same range, while induction of intercellular adhesion molecule–1 (ICAM-1) was hardly affected. Aspirin appeared to prevent VCAM-1 transcription, since it dose-dependently inhibited induction of VCAM-1 mRNA by TNF. As a functional consequence, adhesion of U937 monocytes to TNF-stimulated HUVECs was markedly reduced by aspirin due to suppression of VCAM-1 and E-selectin upregulation. These effects of aspirin were not related to the inhibition of cyclooxygenase activity, since indomethacin was ineffective.

Conclusions Our data suggest that aspirin inhibits NF-B mobilization, induction of VCAM-1 and E-selectin, and subsequent monocyte adhesion in endothelial cells stimulated by TNF, thereby providing an additional mechanism for therapeutic effects of aspirin.

Key Words: aspirin • nuclear factor--B • endothelium

	Introduction

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Monocyte recruitment after adhesion to endothelial cells crucially contributes to inflammation and atherogenesis.^{1 2} This process may involve induction of E-selectin and vascular cell adhesion molecule–1 (VCAM-1) by the inflammatory mediator tumor necrosis factor– (TNF),³ which is secreted by activated monocytes/macrophages, lymphocytes, and smooth muscle cells.⁴ VCAM-1 expression is found in endothelium covering foam cell lesions and in areas of neovascularization or inflammatory infiltration of coronary atherosclerotic plaques.^{5 6} In human umbilical vein endothelial cells (HUVECs), induction of VCAM-1 and E-selectin by TNF is mediated by mobilization of nuclear factor–B (NF-B)–like enhancer elements.⁷

The inducible transcription factor of the rel family NF-B is retained as an inactive cytoplasmic complex by IB protein. Various activators cause NF-B release and nuclear translocation by phosphorylation or oxidative or proteolytic degradation of IB.⁸ Recently, the inhibitors of cyclooxygenase and prostaglandin H synthase aspirin and sodium salicylate⁹ have been shown to inhibit activation of NF-B by preventing alteration of IB.¹⁰ In endothelial cells, cyclooxygenase products such as prostacyclin are major arachidonic acid metabolites involved in hemostasis and perfusion.^{11 12} Salicylates, in addition, exert eicosanoid-independent effects on cellular signaling.¹³ Aspirin is a widely used drug in inflammatory disorders (high dose¹⁴ ) and in thromboembolic disease (low dose¹⁵ ). We studied whether aspirin may have additional effects on NF-B–dependent induction of adhesion molecules in endothelial cells and subsequent monocyte adhesion. We demonstrated that aspirin may suppress TNF-stimulated monocyte adhesion to endothelial cells by inhibiting NF-B mobilization and VCAM-1 induction.

	Methods

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Cell Culture
HUVECs were obtained from human umbilical cord veins by chymotrypsin digestion and grown in endothelial cell growth medium (EGM, PromoCell) using T-75 flasks coated with collagen.⁷ HUVECs passage 2 were detached by 0.01% trypsin/EDTA antagonized by addition of fetal calf serum (FCS), grown to confluence and treated with TNF (8.7x10⁶ U/mg) in the presence of acetylsalicylate (aspirin) or indomethacin. Our results were not affected by pH changes. Under all conditions, cell viability was >95% as judged by ethidium bromide/acridin orange fluorescence. U937 cells were cultured in RPMI 1640 (Roswell Park Memorial Institute medium) suspension with 2 mmol/L L-glutamine and 10% FCS.

Gel-Shift Analysis
Nuclear extracts were prepared by ultrasound disruption of cell membranes followed by high salt extraction with Dignam's buffer C and diluted with buffer D, as described.¹⁶ Ten micrograms of nuclear protein was mixed with a double-stranded oligonucleotide corresponding to an NF-B binding motif located -57 bp from the initiation site in the VCAM-1 promoter (5'-TGAAGGGATTTCCC-3'¹⁷ ) and labeled with [³²P]dATP using oligonucleotide kinase. Unlabeled VCAM-1–B, a mouse IgH enhancer B motif, and an AP-1 motif from the collagenase enhancer were used as competitor DNA.⁷ After binding for 15 minutes, samples were separated by electrophoresis on nondenaturating 4% polyacrylamide gel and exposed to x-ray films.

Immunofluorescence
Cells (10⁵) were treated for 30 minutes with saturating amounts of mouse anti–VCAM-1 monoclonal antibody (mAb) 1G11, anti–ICAM-1 mAb 84H10 (Dianova), anti–E-selectin mAb, or IgG_1a isotype control in PBS containing 0.5% BSA on ice. For staining, cells were reacted with goat anti-mouse fluorescein isothiocyanate IgG_1a (Camon), fixed in 2% paraformaldehyde, and analyzed with 10 000 cells/sample by a fluorescence-activated cell sorter (Becton-Dickinson).⁷ After correction for unspecific binding, specific mean fluorescence intensity (sMFI) was expressed in channels.

Reverse Transcription Polymerase Chain Reaction
RNA was isolated from 10⁶ cells by phenol/chloroform/isoamylalcohol extraction. cDNA was produced by murine leukemia virus reverse transcriptase (Gibco-BRL). cDNA was amplified using Taq polymerase in a thermocycler 480 (Perkin-Elmer). Primers synthesized from reported sequences⁷ yielded products of 441 bp (VCAM-1) or 548 bp (ß-actin) in length. Polymerase chain reaction (PCR) products (20 µL) were subjected to high-performance liquid chromatography (HPLC) and UV detection at 280 nm (Abimed-Gilson).¹⁸ Peaks appeared at retention times predicted by weight standards; areas were integrated and normalized to ß-actin levels. The intra-assay (3.4%) and interassay variations (7.8%) of reverse transcription PCR (RTPCR) and HPLC quantification determined for a standard RNA (n=6) were minimal and in a similar range as reported.¹⁹

Adhesion Assay
U937 cells were labeled with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acethoxymethyl ester (BCECF/AM, 10 µg/mL, Boehringer-Mannheim) for 30 minutes. Dye loading was stopped by adding RPMI 1640 with 2% FCS, and cells were suspended in medium 199 (10 mmol/L HEPES, Gibco-BRL). Confluent HUVEC monolayers in 24-well plates were coincubated with 10⁶ loaded U937 cells/well for 30 minutes (37°C). U937 suspensions were removed, and inverted plates were centrifuged. Cells were lysed in 0.1% Triton X-100/0.1 mol/L Tris. Fluorescence was measured by spectrofluorometry. Adherent cells were calculated by comparison with standard curves of BCECF activity/cell as % adhesion of added cells.⁷ All other reagents were from Sigma Chemical Co.

Statistics
Based on our results of optimally effective aspirin concentrations and previous adhesion experiments with blocking mAbs, we decided to analyze differences of adhesion data for TNF-stimulated HUVECs treated with aspirin (10 mmol/L) or a mAb combination in comparison with TNF-stimulated, otherwise untreated HUVECs by multiple application of two-sided, paired Student's t test with Bonferroni correction.

	Results

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Effect of Aspirin on NF-B Activation
Salicylates have been found to inhibit NF-B activation induced by lipopolysaccharide or phorbol myristate acetate (PMA) in T cells, not affecting other transcription factors.¹⁰ We asked whether aspirin is also effective in TNF-stimulated HUVECs. Gel-shift analyses demonstrated induction of NF-B–like DNA binding activity in response to TNF (100 U/mL, 1 hour) and reduction of this NF-B mobilization in the presence of aspirin (Fig 1) over the narrow dose range reported.¹⁰ Aspirin did not modulate unstimulated NF-B activity. Competition studies with prototypic NF-B motifs (50 ng) revealed specific binding, while an irrelevant AP-1 motif (100 ng) had no effect (Fig 1A). Interestingly, the nonsalicylate cyclooxygenase inhibitor indomethacin was ineffective at concentrations known to block cyclooxygenase (10 µmol/L). Our data indicate that aspirin is able to inhibit TNF-stimulated NF-B mobilization in HUVECs and suggest that it may suppress induction of NF-B–activated genes such as VCAM-1.

View larger version (62K): [in this window] [in a new window]   Figure 1. Effects of aspirin on nuclear factor–B (NF-B) mobilization and expression of vascular cell adhesion molecule–1 (VCAM-1) mRNA. Nuclear extracts of human umbilical vein endothelial cells (HUVECs) stimulated with tumor necrosis factor (TNF; 100 U/mL, 1 hour) in the presence of aspirin at indicated concentrations (mmol/L) were admixed with 32P-oligonucleotide derived from the -57 bp VCAM-1 B motif and separated by polyacrylamide gel electrophoresis. Arrow indicates bound NF-B protein. Unlabeled VCAM-1 B, a mouse IgH B motif (50 ng each), and an AP-1 motif (100 ng) were used as competitor DNA. Shown is one representative of three experiments (A). HUVECs were stimulated with or without TNF (100 U/mL, 6 hours) in the presence of aspirin at indicated concentrations (mmol/L). Total RNA was extracted and reverse transcribed. cDNA was amplified by polymerase chain reaction (PCR) using specific primers for ß-actin or VCAM-1. PCR products were subjected to high-performance liquid chromatography and UV detection at 280 nm. Peak areas for VCAM-1 were integrated and expressed as % of ß-actin levels. Data represent mean±SD of three separate experiments (B).

Effect of Aspirin on Adhesion Molecule Induction
To test this hypothesis, we studied the induction of endothelial cell adhesion molecules by TNF. The presence of aspirin resulted in a marked suppression of TNF-induced (100 U/mL, 6 hours) VCAM-1 or E-selectin surface expression (Fig 2). In accordance with NF-B inhibition, the effect was dose dependent, first evident at 2 mmol/L and maximal at 10 mmol/L, with an IC₅₀ of approximately 4 mmol/L. A decrease in sMFI of 160 channels for VCAM-1 or E-selectin reflects a threefold reduction in surface density at 10 mmol/L. Pretreatment with aspirin 12 hours before TNF stimulation more effectively reduced VCAM-1 induction (Fig 2). In contrast, aspirin hardly affected constitutive or TNF-induced expression of ICAM-1 protein, showing specific inhibition of NF-B–dependent VCAM-1 and E-selectin induction. While indomethacin had no effect, 10 mmol/L aspirin also reduced E-selectin induction by PMA (25 nmol/L, 6 hours) from 78±11 to 9±6 channels, excluding interference with TNF-specific signal transduction and indicating an effect at the level of NF-B release. To confirm that aspirin acts by inhibiting NF-B–activated VCAM-1 transcription, we studied mRNA expression by RTPCR. In untreated HUVECs, VCAM-1 mRNA was hardly detectable. TNF induced a marked increase in specific VCAM-1 PCR products. Induction of VCAM-1 transcripts was also dose-dependently reduced in the presence of aspirin (Fig 1B), with an IC₅₀ of 5 mmol/L. HPLC analyses revealed a 75% suppression of TNF-induced VCAM-1 expression by 10 mmol/L aspirin, supporting an effect on NF-B–dependent transcription.

View larger version (43K): [in this window] [in a new window]   Figure 2. Effects of aspirin on adhesion molecule induction and monocyte adhesion. Human umbilical vein endothelial cells (HUVECs) were stimulated with tumor necrosis factor (TNF; 100 U/mL, 6 hours) in the presence of aspirin at indicated concentrations (mmol/L) or after pretreatment with 1 mmol/L (light bar); stained for vascular cell adhesion molecule–1 (VCAM-1), E-selectin, intercellular adhesion molecule–1 (ICAM-1), or isotype control; and analyzed by flow cytometry. Data expressed as specific mean fluorescence intensity (sMFI) in channels represent mean±SD of three separate experiments (A). HUVECs were pretreated with or without blocking anti–VCAM-1 monoclonal antibody (mAb), anti–E-selectin mAb, or a combination of both (15 minutes) and stimulated with TNF (50 U/mL, 6 hours) in the presence of aspirin at indicated concentrations (mmol/L) or indomethacin (indo, 10 µmol/L). Adhesion of fluorescence-labeled U937 cells was determined by comparison with standard curves. Data are expressed as percent adhesion of cells added and represent mean±SD of three separate experiments. Statistical analyses were performed by two-sided paired Student's t test in comparison with TNF-stimulated, otherwise untreated HUVECs. P values are given (B).

Effect of Aspirin on Monocytic U937 Cell Adhesion
To show the functional relevance of a modulated VCAM-1 and E-selectin induction, we studied adhesion of human premonocytic U937 cells to TNF-treated HUVECs. Stimulation with TNF (50 U/mL, 6 hours) markedly enhanced U937 cell adhesion (Fig 2), which is mediated by both E-selectin and VCAM-1, as shown by marked inhibition of adhesion only with a combination of blocking anti–VCAM-1 and anti–E-selectin mAbs (Fig 2). While aspirin had no effect on basal adhesion (not shown), the presence of aspirin caused a dose-dependent inhibition of TNF-induced adhesion, consistent with its suppressive effect on both VCAM-1 and E-selectin induction. Indomethacin had no such effect. Our data suggest that inhibition of TNF-stimulated NF-B mobilization may result in reduction of VCAM-1 and E-selectin induction and subsequent monocyte adhesion.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
TNF stimulates leukocyte adhesion to endothelial cells by inducing adhesion molecules with distinct functions. In the sequence of adhesion, the selectin E-selectin mediates leukocyte rolling, while VCAM-1 contributes to firm arrest.²⁰ Monocyte adhesion to HUVECs involves both E-selectin and VCAM-1.³ Consistently, we showed that adhesion of monocytic U937 cells was only partially reduced by preincubation of TNF-treated HUVECs with blocking of E-selectin or VCAM-1 mAbs but was almost prevented with a combination of both. We observed the same degree of inhibition in the presence of aspirin at high concentrations and furthermore demonstrated that this suppression was due to a reduction of TNF-induced VCAM-1 and E-selectin expression. In HUVECs, TNF-induced gene transcription of VCAM-1 is mediated by mobilization of NF-B–like enhancer elements.⁷ Importantly, we demonstrated that suppression of TNF-stimulated NF-B mobilization by aspirin was associated with a reduction of VCAM-1 mRNA and protein expression.

It has been well documented that radical scavengers are potent inhibitors of NF-B dissociation after radical generation in response to LPS, TNF, or PMA in intact cells.²¹ Genes activated by NF-B include the inflammatory cytokines interleukin-1 (IL-1), IL-6, and TNF. Consistently, induction of TNF is preventable by radical scavengers.¹⁶ Generation of radicals has also been implicated as an antioxidant-sensitive mechanism regulating NF-B mobilization and VCAM-1 induction in TNF-stimulated HUVECs.⁷ Possible mechanisms for NF-B release from inhibitory IB include the oxidative or proteolytic degradation of IB by an oxidosensitive protease.⁸ Besides other eicosanoid-independent actions at high concentrations,¹³ salicylates have been described to act as free radical scavengers in biological systems by selectively inactivating hydroxyl anions,²² which are required for NF-B mobilization.²¹ We showed that only at such high concentrations is aspirin equally potent as dithiocarbamate antioxidants⁷ in suppressing NF-B activation, NF-B–dependent VCAM-1 induction, and monocyte adhesion in TNF-stimulated HUVECs. Hence, aspirin may act by scavenging radicals or interfering with radical generation, leading to reduced NF-B release via proteolytic IB degradation by oxidosensitive proteases.⁸ The inhibition of NF-B by salicylates reported in T cells also may be attributable to this property,¹⁰ since it was only obvious at high concentrations. By contrast, indomethacin fails to produce any effect, excluding an involvement of cyclooxygenase blockade. Rather high plasma concentrations of salicylates are maintained for treatment of chronic inflammatory states. In addition, local conditions of inflamed areas may favor uptake and concentration of salicylates.^{9 14} Thus, concentrations sufficient to inhibit NF-B may be achieved during treatment of inflammatory conditions.

Monocyte adhesion to endothelial cells is crucial for inflammation,^{1 2} involving induction of E-selectin and VCAM-1.³ Expression of VCAM-1 and E-selectin is detectable in sections of advanced coronary lesions with monocyte infiltration.²³ Aspirin may occur at sites of inflammation or atherosclerosis in concentrations that inhibit NF-B. In addition to its effects on cyclooxygenase activity and eicosanoid metabolism, suppression of stimulated NF-B mobilization in endothelial cells, VCAM-1, and E-selectin induction and subsequent monocyte adhesion contribute to anti-inflammatory effects of aspirin.

	Acknowledgments

 
This work was supported by grants from the Deutsche Forschungsgemeinschaft (We 681) and August-Lenz Stiftung. This work partially fulfills requirements for the doctoral theses of W.E. and A.P.

Received December 12, 1994; revision received January 19, 1995; accepted January 19, 1995.

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				A. Kagawa, H. Azuma, M. Akaike, Y. Kanagawa, and T. Matsumoto Aspirin Reduces Apolipoprotein(a) (Apo(a)) Production in Human Hepatocytes by Suppression of Apo(a) Gene Transcription J. Biol. Chem., November 26, 1999; 274(48): 34111 - 34115. [Abstract] [Full Text] [PDF]

				N. Li, N. H. Wallen, and P. Hjemdahl Evidence for Prothrombotic Effects of Exercise and Limited Protection by Aspirin Circulation, September 28, 1999; 100(13): 1374 - 1379. [Abstract] [Full Text] [PDF]

				K. S. C. Weber, P. J. Nelson, H.-J. Grone, and C. Weber Expression of CCR2 by Endothelial Cells : Implications for MCP-1 Mediated Wound Injury Repair and In Vivo Inflammatory Activation of Endothelium Arterioscler. Thromb. Vasc. Biol., September 1, 1999; 19(9): 2085 - 2093. [Abstract] [Full Text] [PDF]

				K. S.C. Weber, G. Draude, W. Erl, R. de Martin, and C. Weber Monocyte Arrest and Transmigration on Inflamed Endothelium in Shear Flow Is Inhibited by Adenovirus-Mediated Gene Transfer of Ikappa B-alpha Blood, June 1, 1999; 93(11): 3685 - 3693. [Abstract] [Full Text] [PDF]

				W. Erl, G. K. Hansson, R. de Martin, G. Draude, K. S. C. Weber, and C. Weber Nuclear Factor-{kappa}B Regulates Induction of Apoptosis and Inhibitor of Apoptosis Protein-1 Expression in Vascular Smooth Muscle Cells Circ. Res., April 2, 1999; 84(6): 668 - 677. [Abstract] [Full Text] [PDF]

				P. SESTINI, R. M. REFINI, M. G. PIERONI, A. VAGHI, M. ROBUSCHI, and S. BIANCO Different Effects of Inhaled Aspirinlike Drugs on Allergen-Induced Early and Late Asthmatic Responses Am. J. Respir. Crit. Care Med., April 1, 1999; 159(4): 1228 - 1233. [Abstract] [Full Text] [PDF]

				A. F. de Arriba, F. Cavalcanti, A. Miralles, Y. Bayón, A. Alonso, M. Merlos, J. García-Rafanell, and J. Forn Inhibition of Cyclooxygenase-2 Expression by 4-Trifluoromethyl Derivatives of Salicylate, Triflusal, and Its Deacetylated Metabolite, 2-Hydroxy-4-trifluoromethylbenzoic Acid Mol. Pharmacol., April 1, 1999; 55(4): 753 - 760. [Abstract] [Full Text]

				G. Draude, P. von Hundelshausen, M. Frankenberger, H. W. L. Ziegler-Heitbrock, and C. Weber Distinct scavenger receptor expression and function in the human CD14+/CD16+ monocyte subset Am J Physiol Heart Circ Physiol, April 1, 1999; 276(4): H1144 - H1149. [Abstract] [Full Text] [PDF]

				B. JILMA, A. BLANN, T. PERNERSTORFER, P. STOHLAWETZ, H.-G. EICHLER, B. VONDROVEC, J. AMIRAL, V. RICHTER, and O. F. WAGNER Regulation of Adhesion Molecules during Human Endotoxemia . No Acute Effects of Aspirin Am. J. Respir. Crit. Care Med., March 1, 1999; 159(3): 857 - 863. [Abstract] [Full Text]

J.-I. Lee and G. J. Burckart Nuclear Factor Kappa B: Important Transcription Factor and Therapeutic Target J. Clin. Pharmacol., November 1, 1998; 38(11): 981 - 993. [Abstract] [PDF]