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Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:1501-1508

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:1501-1508.)
© 1996 American Heart Association, Inc.

Articles

Selective Inhibition of Tumor Necrosis Factor–Induced Vascular Cell Adhesion Molecule-1 Gene Expression by a Novel Flavonoid

Lack of Effect on Transcription Factor NF-{kappa}B

Joachim Wolle; Russell R. Hill; Erika Ferguson; Larry J. Devall; B.K. Trivedi; Roger S. Newton; Uday Saxena

the Departments of Atherosclerosis Therapeutics and Chemistry (B.K.T.), Parke-Davis Pharmaceutical Research Division, Warner Lambert Co, Ann Arbor, Mich.

Correspondence to Dr Uday Saxena, Atherogenics, Inc, 3065 Northwoods Cir, Norcross, GA 30071.


*    Abstract
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 *Abstract
down arrowIntroduction
 down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
In the present studies, we examined the effect of flavonoids on the endothelial cell expression of adhesion molecules, an early step in inflammation and atherogenesis. Addition of tumor necrosis factor-{alpha} (TNF) to human aortic endothelial cells (HAECs) led to the induction of vascular cell adhesion molecule-1 (VCAM-1) expression and enhancement in expression of intercellular adhesion molecule-1 (ICAM-1). A flavonoid, 2-(3-amino-phenyl)-8-methoxy-chromene-4-one (PD 098063), markedly inhibited TNF-induced VCAM-1 cell-surface expression in a concentration-dependent fashion with half-maximal inhibition at 19 µmol/L but had no effect on ICAM-1 expression. Another structurally distinct flavonoid, 2-phenyl-chromene-4-one, similarly selectively decreased VCAM-1 expression. The inhibition in cell-surface expression of VCAM-1 by PD 098063 correlated with decreases in steady-state mRNA levels, but there was no effect on ICAM-1 mRNA levels. The decrease in VCAM-1 mRNA levels was not due to changes in mRNA stability but rather resulted from a reduction in the rate of transcription of the gene. However, electrophoretic mobility shift assays using nuclear extracts from TNF-induced HAECs treated with PD 098063 failed to show a decrease in the activation of NF-{kappa}B, indicating that inhibition of activation of this transcription factor may not be its mode of action. Similarly, PD 098063 did not affect chloramphenicol acetyltransferase reporter gene activity in TNF-inducible minimal VCAM-1 promoter constructs containing two NF-{kappa}B sites, suggesting that the compound does not affect the transactivation driven by these sites. We conclude that this compound selectively blocks agonist-induced VCAM-1 protein and gene expression in HAECs by NF-{kappa}B–independent mechanism


Key Words: endothelium • vascular cell adhesion molecule-1 • atherosclerosis • NF-{kappa}B • flavonoids


*    Introduction
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 up arrowAbstract
 *Introduction
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 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Adhesion of monocytes to vascular endothelium and their subsequent recruitment into the artery wall are key features in the pathogenesis of atherosclerosis and inflammation.1 2 The adhesion of circulating leukocytes to the endothelial cell surface is mediated by the expression of adhesion molecules on the endothelial surface and their interaction with counter-receptors on leukocytes.3 4 VCAM-1, an adhesion molecule expressed on the endothelial cell surface, may be partly responsible for the recruitment of monocytes during atherogenesis.5 ICAM-1 and E-selectin are other adhesion molecules that participate in leukocyte adhesion.6 Endothelial cell expression of VCAM-1 in diet-induced hypercholesterolemic rabbits precedes monocyte infiltration, suggesting a causal role in monocyte recruitment.7 Immunocytochemistry studies show that VCAM-1 expression is localized to endothelium directly overlying monocyte/macrophage–containing lesions in early atherosclerosis in rabbits and in the apo E knockout mouse model of atherosclerosis.5 8 9 VCAM-1 expression has also been demonstrated in human coronary atherosclerotic plaques, which is consistent with a potential role for this adhesion molecule in the disease.10

TNF and other cytokines induce the expression of all three adhesion molecules in endothelial cells.11 The cell-surface expression of these proteins correlates with an increase in mRNA levels, indicating transcriptional activation of these genes by TNF. In addition, studies have shown that TNF induction of these genes may involve the activation of NF-{kappa}B transcriptional regulatory proteins.11 12 NF-{kappa}B is composed of a heteromer of two proteins, p50 and p65, that are localized in the cytoplasm of unstimulated cells in an inactive form.13 On stimulation of cells, eg, by TNF, the heteromer translocates to the nucleus and transactivates gene expression.13 14 VCAM-1 promoter consists of two NF-{kappa}B binding sites, whereas E-selectin has three sites and ICAM-1 one.11 Intracellular generation of reactive oxygen species in response to TNF and other stimuli has been proposed to serve as a signaling event in the activation of NF-{kappa}B and the upregulation of VCAM-1 in human endothelial cells.15 16 17 Different classes of antioxidants, such as PDTC and N-acetylcysteine, have been shown to block TNF-stimulated NF-{kappa}B activation, transcription, and cell-surface expression of VCAM-1 in endothelial cells.16 18 19

Flavonoids are a group of antioxidant phenol derivatives present in plants and vegetables.20 21 The flavonoid family of compounds contain flavones, flavonols, and flavonones as major members. They scavenge superoxide radical and singlet anions at physiological pH, which in part contributes to their antioxidant property.22 In vitro studies have also shown that flavonoids present in red wine inhibit copper-catalyzed oxidation of human LDL, a putative triggering molecule in atherogenesis.23 24 Recent epidemiological studies suggest a beneficial role of flavonoids in the prevention of coronary heart disease.25 26 27 These studies concluded that flavonoid consumption reduces the risk of death from coronary heart disease. It is largely believed that the protective effects of flavonoids are related to their ability to prevent the oxidation of LDL. Flavonoids have been shown to decrease accumulation of aortic cholesterol and lesion coverage in cholesterol-fed rabbits in the absence of plasma lipid lowering, suggesting effects on LDL oxidation and/or direct modulation of other events in the artery wall.28

To gain insights into potential molecular mechanisms of antiatherosclerotic properties of flavonoids, we examined their effects on the expression of adhesion molecules in endothelial cells, an early step in the pathophysiology of atherosclerosis. TNF-activated expression of VCAM-1 and ICAM-1 in HAECs was used as a model. We focused on the effects of flavone members because they represent a major component of the flavonoid family. Our results demonstrate that PD 098063 reduces TNF-induced VCAM-1 expression without affecting activation of NF-{kappa}B or decreasing VCAM-1 transactivation through NF-{kappa}B sites in minimal VCAM-1 promoter constructs.


*    Methods
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 up arrowAbstract
 up arrowIntroduction
 *Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Cell Culture
HAECs (Cell System) were cultured in a 50:50 mixture of CS 3.0 medium (Cell System) and MCDB-107 (Sigma Chemical Co) containing 10% fetal calf serum. The cells were seeded at 100 000 cells/well in 24-well cluster plates (Costar) and used for experiments at confluence. HAECs were used between the 4th and 10th passages. BAECs were cultured in DMEM (GIBCO BRL, Life Technologies) supplemented with 10% FCS. One day before transfection, the cells were seeded at 150 000 per well in six-well cluster plates.

Measurement of Cell-Surface ICAM-1 and VCAM-1
HAECs were treated with the compounds at indicated concentrations overnight at 37°C in the presence or absence of TNF (250 U/mL) as described.29 Cell-surface expression of VCAM-1 or ICAM-1 was determined with immunoassays exactly as described before.29 Data shown are averages of assays performed in triplicate±SEM. Cell viability was determined by lactate dehydrogenase activity and was found to be unchanged under all the conditions used here.

Northern Blot Analysis and Transcription Run-on Assay
Total RNA from HAECs or BAECs treated with TNF (250 U/mL) for 6 hours in the presence or absence of PD 098063 (50 µmol/L) was isolated. Northern blot analysis was performed exactly as described.29 For the detection of bovine VCAM-1 mRNA, the full-length human VCAM-1 cDNA was used. VCAM-1 mRNA stability was examined in untreated HAECs and cells treated with PD 098063 as described.30 31 Transcription run-on assays using HAECs were performed as described.32

Electrophoretic Mobility Shift Assay
Endothelial cells were pretreated with PD 098063 or PDTC for 1 hour and then exposed to TNF for an additional hour. Nuclear extracts were prepared and the shift assay was performed exactly as described by Marui et al.16 Two complementary oligonucleotides (VCAM-1 wt) containing the two NF-{kappa}B sites (underlined) of the human VCAM-1 promoter (49-mers, Oligos Etc Inc) wild-type sense sequence: 5'-G CTG CCC TGG GTT TCC CCT TGA AGG GAT TTC CCT CCG CCT CTG CAA CAA-3' were annealed in 50 mmol/L Tris, pH 8.0, 100 mmol/L NaCl, and 10 mmol/L MgCl. Mutated complementary oligonucleotides (VCAM-1 Mut), which differed from the wild-type sequence at eight nucleotides in the NF-{kappa}B binding region shown in bold: 5'-G CTG CCC TGA GTC ACG CCT TGA AGA GAC ATC ACT CCG CCT CTG CAA CAA-3', were annealed similarly. Double-stranded oligonucleotides were labeled on their 5' ends with 32P by use of T4 polynucleotide kinase (Promega) as described by the manufacturer. The DNA-binding reaction was performed at 30°C for 15 minutes in a total reaction volume of 20 to 25 µL, which contained 3 µg nuclear extract, 225 µg/mL BSA, 3x106 cpm 32P-labeled probe, 0.1 mg/mL polyinosinic:polycytidylic acid, and 15 µL of binding buffer (in mmol/L: HEPES 12 [pH 7.4], Tris 4, KCl 60, EDTA 1, DTT 1, PMSF 1, and 12% glycerol).

Transient Transfection Assay
BAECs were used to transiently transfect the reporter plasmids 288VCAMCAT and 933VCAMCAT,30 33 a generous gift of Dr Douglas C. Dean, Washington University School of Medicine, St Louis, Mo. BAECs rather than HAECs were used because of higher transfection efficiency. Transfections were performed with 5 µg of reporter plasmid and 0.8 µg of TKLUC,33 which contains the thymidine kinase promoter fused to the firefly luciferase gene, as internal control. Cells were transfected for 18 hours with calcium phosphate precipitation according to the manufacturer's instructions (Promega Corp). After one washing with PBS, the cells were incubated with fresh medium for 24 hours. The next day, cells were treated for 8 hours at 37°C with or without TNF (250 U/mL) in the presence or absence of added drugs. Cell extracts were then prepared by addition of lysis buffer and assayed for luciferase activity and CAT protein. Luciferase activity was measured with the luciferase assay system (catalog No. E4030, Promega) and an Auto Lumat LB 953 luminometer (EG & G), and CAT protein was determined with an ELISA kit supplied by Boehringer Mannheim (catalog No. 1363 727) exactly as suggested by the suppliers.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowReferences
 
Time Course of Cell-Surface VCAM-1 and ICAM-1 Induction by TNF
The temporal induction of ICAM-1 and VCAM-1 by TNF on the endothelial cell surface was examined. In unstimulated HAECs, surface expression of ICAM-1 was low and VCAM-1 was hardly detectable. However, addition of TNF increased the expression of both adhesion molecules in a time-dependent fashion (Fig 1Down). ICAM-1 expression reached a maximum 8-fold increase at 10 hours, remained constant for up to 24 hours, and then declined (Fig 1Down). Maximal VCAM-1 expression (21-fold) was found at 6 hours and progressively decreased between 18 and 48 hours (Fig 1Down).



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  		Figure 1. Induction of adhesion molecule surface expression by TNF. HAECs were incubated with TNF (250 U/mL) for various periods of time, then cell-surface expression of ICAM-1 or VCAM-1 was quantified by use of immunoassays, as described in "Methods." Results for ICAM-1 are shown as OD414 and for VCAM-1 as 125I-streptavidin bound to the cell surface. Data shown are the average of triplicate wells±SEM.

Effects of Flavonoids on VCAM-1 and ICAM-1 With TNF as a Stimulus
Fig 2ADown shows the effect of increasing concentrations of the flavonoid PD 098063 on HAEC-surface expression of VCAM-1 induced by TNF. PD 098063 inhibited VCAM-1 expression in a concentration-dependent fashion, with half-maximal inhibition at about 19 µmol/L (Fig 2ADown). In parallel experiments, this flavonoid showed no decrease in ICAM-1 expression (Fig 2ADown). Similarly, this compound did not significantly inhibit the cell-surface expression of E-selectin (21% decrease at 50 µmol/L), further demonstrating its selective effect on VCAM-1.




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  		Figure 2. Effect of PD 098063 on adhesion molecule expression. A, Effect on TNF-induced VCAM-1 and ICAM-1 expression. Endothelial cells were incubated overnight with TNF (250 U/mL) together with increasing concentrations of PD 098063. Cell-surface expression of VCAM-1 and ICAM-1 determined by immunoassays is shown. Experiments were performed in triplicate wells, and data shown are mean±SEM. B, Structures of flavonoids.

We next examined whether other structurally distinct flavonoids (Fig 2BUp) also share the ability to selectively inhibit agonist-stimulated VCAM-1 expression. 2-Phenyl-chromene-4-one (flavone, compound 2 in Fig 2BUp) selectively decreased VCAM-1 cell-surface expression with half-maximal inhibition at 65 µmol/L. Hydroxylation in the 5-position (compound 3, Fig 2BUp) seemed to be detrimental to the inhibitory activity (IC50 for VCAM-1>=100 µmol/L). However, the corresponding 5,7-disubstituted analog (compound 4, Fig 2BUp) was a more potent VCAM-1 inhibitor (IC50 for VCAM-1=6.29 µmol/L). Regioisomeric hydroxylation in the phenyl ring provided analogs (compounds 5 and 6, Fig 2BUp) with decreased potency (IC50 for VCAM-1=36.86 and 77.43 µmol/L). All these compounds had no effect on ICAM-1 expression up to 100 µmol/L. Overall, it appears that hydroxylation and positioning of the hydroxyl group, which may modulate the redox potential of the compound, is important for the inhibitory activity of the compounds. Similar importance for flavonoid hydroxyl groups in the inhibition of adhesion molecule gene expression was described previously by Gerritsen et al.34 Their studies described the inhibitory effects of another flavonoid, apigenin, on both ICAM-1 and VCAM-1 as well as on E-selectin and a variety of other cytokine genes. Several other flavonoids were also shown to similarly decrease adhesion molecule expression. In our HAEC system, apigenin decreased the TNF-induced cell-surface expression of both VCAM-1 and ICAM-1 with an IC50 of 20 and 32 µmol/L, respectively. The ability of apigenin to decrease ICAM-1 and E-selectin expression suggests that it has distinct effects on adhesion molecule expression relative to PD 098063. Because of the availability and potency of PD 098063, we used this compound in all other experiments.

Specificity and Time Course of Inhibition of Cell-Surface VCAM-1 Expression by PD 098063
To assess whether the inhibitory effect of PD 098063 was related to a decrease in total cellular protein synthesis, tritiated leucine incorporation in HAECs treated with the compound (18 hours, 50 µmol/L) in the presence of TNF was measured. Leucine incorporation was similar in untreated and treated cells, 7.20 and 7.46 cpm/µg cell protein, respectively.

To examine whether inhibition of VCAM-1 expression by PD 098063 was restricted to TNF, we tested the effect of this compound using a noncytokine, LPS, as a stimulus. As shown in Fig 3ADown, PD 098063 decreased LPS-induced VCAM-1 expression by 73%, comparable to its effect on TNF-stimulated VCAM-1 expression (83% decrease), suggesting that antagonism of TNF interaction with its receptor is not its mode of action.





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  		Figure 3. Mechanism studies with PD 098063. A, Effect on LPS- or TNF-stimulated adhesion molecule surface expression. Endothelial cells were stimulated with LPS (1 µg/mL) or TNF (250 U/mL) in the presence of PD 098063 (100 µmol/L), and cell-surface expression of VCAM-1 was determined after overnight incubation. B, Reversibility of PD 098063 inhibitory effect. HAECs were cotreated with TNF and PD 098063 for 8 hours, washed, and incubated in fresh medium without TNF or the compound for another 18 hours. The cells were then stimulated with TNF for 8 hours, and cell-surface VCAM-1 expression was determined. Data shown are the average of duplicate wells. Results are shown as percentage of VCAM-1 expression in fresh HAECs. C, Time course of PD 098063 effect. TNF (250 U/mL) was added to endothelial cells, followed by addition of PD 098063 (100 µmol/L) at indicated times, and cell-surface VCAM-1 expression was determined after an overnight incubation. Data shown are the mean of triplicate wells±SEM.

We also investigated whether the inhibitory effect of PD 098063 was reversible using TNF-stimulated HAECs that were either untreated or treated with the compound for 8 hours, followed by washing of the cells with medium. Fresh medium without the compound and the cytokine was added, and cells were incubated for 18 hours. After restimulation of HAECs with TNF for 8 hours, cell-surface VCAM-1 expression was determined. As shown in Fig 3BUp, the cell-surface expression of VCAM-1 in PD 098063–treated cells was similar to that in untreated cells and comparable to that seen in fresh HAECs stimulated with TNF (control cells), suggesting that its inhibitory effect is completely reversible. In a parallel experiment, cotreatment of HAECs for 8 hours with TNF and PD 098063 at 25 and 50 µmol/L decreased VCAM-1 cell-surface expression by 52% and 65%, respectively.

To gain insights into the mechanism(s) by which PD 098063 inhibits VCAM-1 expression, the compound was added to the cells at various time points after TNF addition, and cell-surface adhesion molecule expression was determined. The compound was able to show maximal inhibitory effect when added within the first 120 minutes of TNF addition (>88% inhibition), and its inhibitory effect decreased after 4 hours of TNF pretreatment (<40% inhibition) (Fig 3CUp). These results show that the maximal inhibitory effect of PD 098063 is obtained when it is added within 2 hours of TNF addition. Thus, the compound may be affecting early events in TNF-induced VCAM-1 expression.

Inhibition of TNF-Induced VCAM-1 and ICAM-1 Gene Transcription
To determine whether the inhibitory effects of PD 098063 on cell-surface expression of adhesion molecules were due to changes in steady-state mRNA levels, Northern blot analysis of total RNA from HAECs treated with TNF and the compound was performed. Untreated cells had small amounts of ICAM-1 mRNA, which increased substantially with TNF treatment. VCAM-1 mRNA was not detectable in control cells but was markedly induced by TNF. Treatment with PD 098063 resulted in >90% reduction in the mRNA level for VCAM-1 (Fig 4ADown). In contrast, this treatment did not effect the mRNA levels of either ICAM-1 or the constitutively expressed gene G3PDH. Densitometric scanning of ICAM-1/G3PDH mRNA levels yielded similar ratios for untreated and PD 098063–treated cells: 0.39 and 0.36, respectively. These effects of PD 098063 on VCAM-1 and ICAM-1 mRNA levels are consistent with its effects on the cell-surface expression of these two proteins.





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  		Figure 4. Effects of PD 098063 on adhesion molecule mRNA. A, Northern blot analysis. Total RNA (25 µg) from endothelial cells treated for 6 hours with medium alone (-TNF) or with TNF, 250 U/mL (+TNF) or TNF and 50 µmol/L PD 098063 (PD 098063) was electrophoresed on 1% agarose-formaldehyde gel. The RNA was transferred to a nitrocellulose membrane and hybridized to either 32P-labeled VCAM-1–specific (top) or ICAM-1–specific or G3PDH-specific cDNA (bottom) and visualized by autoradiography. B, Effect of PD 098063 on VCAM-1 mRNA stability. HAECs were treated with 250 U/mL TNF for 4 hours, then actinomycin D (10 µg/mL) was added together with or without PD 098063 (50 µmol/L). RNA was isolated at various time points, and Northern blot analysis for VCAM-1 and G3PDH mRNA was performed and visualized by autoradiography. C, Transcription run-on assay of VCAM-1 gene transcription in HAECs. Endothelial cells were either induced or not with TNF (250 U/mL) in the presence or absence of PD 098063 (50 µmol/L). Nuclei were isolated, and labeled RNA ({approx}2.0x106 cpm/condition) was used to probe alkali-denatured target DNAs [7 µg/slot, VCAM-1 cDNA in PGEM 11zf(+), pGEM 11zf(+), and rat G3PDH cDNA]. At top is a graphic representation of the VCAM-1 hybridization normalized to the G3PDH signal.

To examine whether PD 098063 affects the stability of VCAM-1 mRNA, HAECs were treated with TNF for 4 hours, then actinomycin D (10 µg/mL) was added to block transcription, in the presence or absence of PD 098063 (50 µmol/L). RNA was isolated at various times after addition of actinomycin D and subjected to Northern blot analysis. As shown in Fig 4BUp, there was no difference in mRNA levels between treated and untreated cells at various time points, suggesting that VCAM-1 mRNA stability is not affected by PD 098063. VCAM-1 mRNA levels were also quantified by densitometry and were found to be similar in untreated and treated cells (data not shown). The addition of actinomycin D had no effect on VCAM-1 mRNA in untreated cells (data not shown).

Since no variations in VCAM-1 mRNA stability were found in PD 098063–treated HAECs, we examined whether there were changes in promoter activity of the VCAM-1 gene. Nuclei were isolated from HAECs treated or not treated with TNF in the presence or absence of the compound, and ongoing VCAM-1 transcription was measured. As shown in Fig 4CUp, stimulation of endothelial cells with TNF increased the transcriptional rate of VCAM-1 gene expression compared with untreated cells. Treatment of HAECs with PD 098063 decreased the TNF-induced augmentation of VCAM-1 promoter activity. Only minimal hybridization was observed between endothelial nuclear RNA and the control plasmid pGEM 11zf(+) alone (Fig 4CUp). From these data we conclude that reduction in VCAM-1 expression caused by PD 098063 treatment results from a reduction in the rate of transcription of the gene.

Effect of PD 098063 on NF-{kappa}B Activation
PD 098063 may regulate TNF-induced adhesion molecule gene expression by blocking cytokine-induced activation of NF-{kappa}B transcription factors. To explore this possibility, nuclear extracts from treated and untreated cells were assayed for binding activity to a double-stranded oligonucleotide containing the two VCAM-1 NF-{kappa}B–specific binding sites (VCAM-1 wt) by electrophoretic mobility shift assays. NF-{kappa}B–specific binding activity was assessed by (1) demonstrable competition of binding activity by excess cold VCAM-1 wt, (2) lack of competition by a probe mutated in NF-{kappa}B binding sequences (VCAM-1 Mut), and (3) absence of binding to VCAM-1 Mut with nuclear proteins from TNF-treated endothelial cells (data not shown). As shown in Fig 5Down, incubation of endothelial cells with TNF induced two major bands representing NF-{kappa}B binding activity. Pretreatment of the cells with PD 098063 did not alter the binding pattern relative to TNF-treated cells (Fig 5Down). In contrast, pretreatment with PDTC, a compound known to block NF-{kappa}B activation, did result in >90% inhibition in binding activity.16 These data show that TNF activates NF-{kappa}B binding activity and that this activation is not affected by PD 098063.



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  		Figure 5. Effect of PD 098063 and PDTC on NF-{kappa}B activation. Endothelial cells were pretreated for 1 hour with either medium alone or PD 098063 (50 µmol/L) or PDTC (100 µmol/L) followed by 1 hour of incubation with or without TNF (250 U/mL) in the continued presence of the compounds. Nuclear proteins were prepared and incubated for 15 minutes at 30°C with 32P-labeled double-stranded oligonucleotide containing the two NF-{kappa}B sites present in human VCAM-1 promoter. The incubation mixture was electrophoresed on a 4% native polyacrylamide gel and exposed to an autoradiographic film.

Effect of PD 098063 on TNF-Inducible Minimal VCAM-1 Promoter–Driven Reporter Gene Expression
To test whether PD 098063 blocks transactivation of the VCAM-1 gene, we studied its effects using a minimal human VCAM-1 promoter fragment containing the two adjacent NF-{kappa}B sites (coordinates -288 to +22) fused to the CAT reporter gene. Treatment with TNF resulted in activation of this construct and an increase in CAT protein (Fig 6ADown). As reported previously,35 addition of PDTC significantly decreased VCAM-1 gene promoter activity (Fig 6ADown). In contrast, addition of PD 098063 had no inhibitory effect, demonstrating that the flavonoid may not affect transactivation driven by the NF-{kappa}B sites in VCAM-1 promoter. Similarly, PD 098063 showed no effect on the TNF-induced reporter gene activity of another VCAM-1 promoter construct (coordinates -933 to +22) that contained an AP-1 site in addition to NF-{kappa}B sites (Fig 6BDown). The compound had no effect on the constitutively active reporter gene RSVCAT (data not shown).




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  		Figure 6. Effect of PD 098063 on VCAM-1 gene promoter activity. BAECs were transfected with (A) 288VCAMCAT plasmid or (B) 933VCAMCAT plasmid as described in "Methods." The cells were then incubated with or without TNF in the presence or absence of PD 098063 (50 µmol/L) or PDTC (10 µmol/L). Next, cell lysates were prepared, and CAT protein was determined by ELISA and luciferase activity by chemiluminescence assay. Data are shown as CAT protein produced, normalized to luciferase activity. A representative experiment from at least two separate experiments is shown. C, Northern blot analysis of bovine VCAM-1 and G3PDH mRNA. BAECs were treated with TNF (250 U/mL, 6 hours) in the presence or absence of PD 098063 (50 µmol/L), and Northern blot analysis was performed.

To exclude the possibility that PD 098063 is ineffective in the BAECs used in the above experiments, we directly examined its effect on TNF-induced bovine VCAM-1 mRNA levels. As shown in Fig 6CUp, treatment of BAECs with the compound (6 hours, 50 µmol/L) markedly decreased VCAM-1 mRNA levels compared with untreated cells.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
The purpose of our studies was to examine whether PD 098063, a flavonoid, affects endothelial cell functions. To this end, we examined its effects on the endothelial cell expression of VCAM-1 and ICAM-1, both endothelial surface molecules expressed in aortic vasculature during atherogenesis. Our results showed that it selectively decreased TNF-induced expression of VCAM-1 without affecting the expression of the other TNF-inducible adhesion molecule, ICAM-1. The inhibitory effects of PD 098063 on VCAM-1 were concentration- and time-dependent and correlated with effects on mRNA levels. The compound did not affect the stability of VCAM-1 mRNA, but it did decrease the transcription rate of this gene. To further delineate the mechanism by which PD 098063 may be acting, we studied its effect on the activation of the transcription factor NF-{kappa}B. Using electrophoretic mobility shift assays, we found that PD 098063 had no apparent effects on the activation of NF-{kappa}B. It was conceivable that the compound might affect the transactivation function of NF-{kappa}B factors. However, using minimal human VCAM-1 promoter CAT reporter gene constructs containing the two NF-{kappa}B sites and the AP-1 site, we were unable to show any inhibitory effects of the compound. Collectively, these results suggest that the compound blocks VCAM-1 expression by mechanism(s) independent of NF-{kappa}B activation. Thus, the compound appears to affect regulatory step(s) downstream from NF-{kappa}B activation. It is of interest to note that another antioxidant, vitamin E, has been shown to decrease E-selectin gene expression without any apparent effects on NF-{kappa}B–like factors.12 Additionally, we have previously shown that another compound, an alkoxybenzo[b]thiophene-2-carboxamide, had also blocked TNF-induced adhesion molecule gene expression without any effect on NF-{kappa}B activation.29 Further insights into the mechanism of action of such compounds may provide the basis for design of unique molecules that regulate adhesion molecule gene expression.

The mechanism of VCAM-1 inhibition by PD 098063 differs from that of the thiol antioxidant PDTC. The thiol antioxidant inhibits VCAM-1 expression because of inhibition of NF-{kappa}B activation and VCAM-1 transcription. PDTC has several additional properties besides being an antioxidant. It is also a metal chelator, which could potentially inhibit oxygen radical formation through chelation of intracellular metal ions. Another possibility is that the thiol compound may affect the glutathione-dependent redox reactions, since PDTC is an effective thiol delivery compound that increases intracellular levels of glutathione. Finally, PDTC has also been shown to directly inhibit the binding of NF-{kappa}B–like proteins to the ICAM-1-{kappa}B site.36 Thus, PDTC may affect adhesion molecule expression by a variety of mechanisms that may not be shared by the flavonoids used in our study.

Another observation of interest is the lack of effect of the PD 098063 on ICAM-1 expression. Even though both VCAM-1 and ICAM-1 are induced by cytokines, there are several differences in their expression. Although some expression of ICAM-1 protein and mRNA is found in unstimulated cells, VCAM-1 expression is absent in unstimulated cells but is rapidly induced by cytokines. The ICAM-1 gene has only one NF-{kappa}B site, unlike the two in VCAM-1. The kinetics of expression of the two proteins are also different. Thus, distinct pathways may regulate the expression of these two proteins and PD 098063 may affect only the signaling pathways involved in VCAM-1 regulation. The selectivity of PD 098063 for VCAM-1 inhibition may be desirable, since ICAM-1 is involved in adhesion of various leukocytes, whereas VCAM-1 participates primarily in monocyte and T-lymphocyte adhesion, cell types specifically found in atherosclerotic lesions. In a recent study, Gerritsen et al34 showed that a trihydroxyflavone, apigenin, inhibited the expression of both VCAM-1 and ICAM-1. Apigenin was unable to decrease NF-{kappa}B activation in human umbilical vein endothelial cells as assessed by electrophoretic mobility shift assays. However, it did inhibit TNF-induced ß-galactosidase reporter gene activity driven by the human cytomegalovirus enhancer containing four NF-{kappa}B elements in stably transfected human SW480 colon adenocarcinoma cells. The effects of PD 098063 on adhesion molecule expression as well as the NF-{kappa}B–driven reporter gene activity are clearly distinct from the effects of apigenin.

A potential consequence of the VCAM-1 inhibitory effect of flavonoids may relate to their antiatherosclerotic effects. Recently published epidemiological studies suggest a protective role for flavonoids in the prevention of coronary heart disease. This protection was attributed to the ability of flavonoids to inhibit the oxidation of LDL. We speculate that a novel mechanism by which flavonoids may prevent the pathogenesis of atherosclerosis may be related to their ability to block adhesion molecule expression. Our in vitro results provide the basis for further in vivo analysis of flavonoid therapy in pathologies involving adhesion molecule expression. In this regard, it is of interest to note that Gerritsen et al recently demonstrated potent anti-inflammatory activity of apigenin in animal models of inflammation.34


*    Selected Abbreviations and Acronyms
 
BAEC = bovine aortic endothelial cell
CAT = chloramphenicol acetyltransferase
G3PDH = glyceraldehyde-3-phosphate dehydrogenase
HAEC = human aortic endothelial cell
ICAM-1 = intercellular adhesion molecule-1
LPS = lipopolysaccharide
NF-{kappa}B = nuclear factor-{kappa}B
PD 098063 = 2-(3-amino-phenyl)-8-methoxy-chromene-4-one
PDTC = pyrrolidine dithiocarbamate
TNF = tumor necrosis factor-{alpha}
VCAM-1 = vascular cell adhesion molecule-1


*    Acknowledgments
 
The authors wish to thank Drs Michael Iademarco (Washington University, St Louis, Mo) and Andrew Neish (Brigham and Women's Hospital, Boston, Mass) for advice on the transfection assays and Bruce J. Auerbach for critical reading of the manuscript.

Received October 20, 1995; revision received April 8, 1996;
*    References
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 up arrowDiscussion
 *References
 

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