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-Induced Adhesion Molecule Upregulation in Cultured Human Endothelial Cells: Role of Nuclear Factor-
B1
* Division of Life Sciences and Silver Biotechnology Research Center, Hallym University, Chuncheon, Republic of Korea, and
Department of Food and Nutrition, Cheju University, Republic of Korea
2To whom correspondence should be addressed. E-mail: yhkang{at}hallym.ac.kr.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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B (NF-B) in TNF-
-activated human umbilical vein endothelial cells (HUVEC). The effects of flavones, luteolin and apigenin, on adhesion of THP-1 monocytes to the TNF--activated HUVEC, protein expression and mRNA levels of vascular cell adhesion molecule-1 (VCAM-1), intracellular cell adhesion molecule-1 (ICAM-1) and E-selectin, and nuclear appearance and DNA binding activity of NF-B were determined. Flavanols, flavonols, and flavanones were used for comparison. TNF- significantly induced HUVEC protein expression of VCAM-1, ICAM-1, and E-selectin with increasing mRNA levels. Luteolin and apigenin inhibited the TNF--induced upregulation of THP-1 adhesion and VCAM-1 expression; these inhibitory effects were dose-dependent. The flavones at doses of 
25 µmol/L almost completely abolished the increased CAM protein and mRNA regardless of their anti-oxidative activity. With the exception of the flavonol quercetin, flavonoids had no such effect; quercetin substantially attenuated the CAM induction. The flavones inhibited nuclear translocation and DNA binding activity of the NF-B-containing binding site in the promoter region of the CAM genes in TNF--activated HUVEC. The inhibition of endothelial CAM induction by flavones is mediated by their interference with the NF-B-dependent transcription pathway. Thus, the flavones may hamper initial atherosclerotic events involving endothelial CAM induction.
KEY WORDS: • flavones • tumor necrosis factor- • cell adhesion molecules • nuclear factor-B • atherosclerosis
Epidemiological studies have demonstrated that an increased intake of polyphenolic phytochemicals such as flavonoids, proanthocyanidins, and phenolic acids found in a large number of fruits and vegetables may contribute to the low incidence of cardiovascular diseases in some populations (1,2). Dietary intakes of flavonols, flavones, and isoflavones by Japanese women are inversely correlated with the plasma LDL cholesterol concentration (3). Flavonoids and related polyphenolics have a great potential to delay LDL oxidation with their radical scavenging capacity (4). Wine flavonoids have been shown to protect against atherosclerosis by inhibition of the accumulation of oxidized LDL in atherosclerotic lesions, paraoxonase elevation, and removal of atherogenic lesions of apolipoprotein E deficient mice (5). This observation demonstrates that flavonoids may confer protection against early events in atherogenic lesion formation.
Flavonoids are anti-inflammatory agents that inhibit expression of cell adhesion molecules (CAM)3 and matrix proteases (6–11). Induction of CAM is a common feature in inflammatory environments and occurs with the early development of atherosclerosis. CAMs such as vascular cell molecule-1 (VCAM-1), intracellular adhesion molecule-1 (ICAM-1), and endothelial-leukocyte adhesion molecule-1 (E-selectin) have been observed in atherosclerotic lesions and at sites predisposed to lesion formation in the rabbit and mouse as well as in human coronary atherosclerotic plaques (12–14).
The positive regulatory domains required for cytokine induction of CAM have been defined in their promoters. DNA binding studies demonstrate a requirement for nuclear factor-B (NF-B), a pivotal transcription factor in chronic inflammatory diseases (15,16). Certain hydroxyflavones and flavanols block cytokine-induced ICAM-1, VCAM-1, and E-selectin expression on human endothelial cells through an action on NF-B transcriptional activation (9). Apigenin exhibited a reversible effect on CAM expression and inhibited CAM upregulation at the transcriptional level (9).
The present study assessed the anti-inflammatory activity of various flavonoids with respect to CAM expression and further investigated possible molecular mechanisms of the flavonoid-mediated downregulation of CAM expression in human umbilical vein endothelial cells (HUVEC). To determine the capacity of flavonoids to inhibit TNF--induced CAM expression, 4 different subgroups of flavonoids were used: flavanols, flavonols, flavanones, and flavones.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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was obtained from Roche Molecular Biochemicals. Antibodies against human VCAM-1, human ICAM-1, human ß-actin, and human NF-B were purchased from Santa Cruz Biotechnologies. Human E-selectin antibody was obtained from R&D Systems. Horseradish peroxidase–conjugated goat anti-rabbit IgG and rabbit anti-goat IgG were obtained from Jackson ImmunoResearch Laboratories. Fluorescein isothiocyanate–conjugated goat anti-mouse IgG was provided by Sigma Chemical. Reverse transcriptase, Taq DNA polymerase, T4 polynucelotide kinase, and a reagent kit for NF-B electrophoretic mobility shift assay (EMSA) were purchased from Promega.
All flavonoids were solubilized by dimethyl sulfoxide (DMSO) for culturing with cells (17); the final culture concentration of DMSO was 
5 g/L.
Cell culture. HUVEC were isolated from human umbilical cords using collagenase, as described elsewhere (18,19). Cells were incubated in 25 mmol/L HEPES-buffered M199 containing 10% FBS, 2 mmol/L glutamine, 100,000 U/L penicillin, 100 mg/L streptomycin, and growth supplements (0.9 g/L bovine brain extract, 0.75 g/L human epidermal growth factor, and 0.075 g/L hydrocortisone) at 37°C humidified atmosphere of 5% CO2 in air. Endothelial cells were identified by their cobblestone morphology and uptake of fluorescently labeled acetylated LDL (1.1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate; Molecular Probes; ref. 20).
HUVEC were plated at 90–95% confluence in all experiments. In a recent study we found that treatment of HUVEC with TNF- markedly increased VCAM-1 in a time-dependent manner and that the expression was markedly induced 4 h after activation by TNF- with peak expression at 6 h, followed by decreased expression (19). The maximal nontoxic concentration of all flavonoids used for 24-h culture experiments has been previously shown to be 50 µmol/L (21). Accordingly, in this study cells were pretreated for 30 min with 50 µmol/L of each flavonoid and exposed to 10 µg/L TNF- for 6 h.
We used a special protocol to test whether 10 µg/L TNF- affected HUVEC viability during a 6-h incubation in the presence of the tested flavonoids. TNF- had no measurable effect on cell viability as determined by MTT assay (ref. 22, data not shown).
Cell adhesion assay.
HUVEC were cultured at a density of 6.0 x 104 cells on a 4-well glass chamber slide containing 25 mmol/L HEPES-buffered M199 with 10% FBS. THP-1 cells were grown in RPMI-1640 medium containing 10% FBS in a culture flask. HUVEC were pretreated with 1–50 µmol/L of each tested flavonoid for 30 min prior to the 6-h exposure to 10 µg/L TNF-. THP-1 cells were labeled for 30 min with 5 µmol/L calcein-AM (Molecular Probes, Inc.). The labeled THP-1 (5.0 x 105) were seeded onto confluent HUVEC treated with flavonoids and/or TNF- and incubated for 24 h. Cocultured cells were washed and the images were obtained at 485 nm excitation and 538 nm emission using a SPOT II digital camera–attached fluorescence microscope with Spot II data acquisition software (Diagnostic Instrument). For the adhesion quantification, the calcein-AM fluorescent intensity was measured at 485 nm excitation and 538 nm emission by a Fluoroskan ELISA plate reader (Labsystems Oy).
Western blot analysis. Western blot analysis was performed using whole cell extracts prepared from HUVEC, as previously described (19). Cell extracts were fractionated by electrophoresis on 8% SDS-PAGE gel and transferred onto a nitrocellulose membrane. Nonspecific binding was blocked by soaking the membrane in Tris-buffered saline-Tween 20 (TBS-T) buffer [0.5 mol/L Tris-HCl (pH 7.5), 1.5 mol/L NaCl, and 1 g/L Tween 20] containing 50 g/L nonfat milk. The membrane was incubated with polyclonal rabbit anti-human VCAM-1 (1:1000), polyclonal rabbit anti-human ICAM-1 (1:1000), or polyclonal goat anti-human E-selectin (1:500). Subsequently, the membrane was incubated with a goat anti-rabbit IgG antibody (1:7500) or rabbit anti-goat IgG antibody (1:500) conjugated to horseradish peroxidase. The protein levels were determined using Supersignal West Pico chemiluminescence detection reagents (Pierce Biotech) and Konica X-ray film (Konica). Incubation with polyclonal rabbit anti-human ß-actin antibody (1:1000) was also performed as a control.
RT-PCR analysis. Total RNA was isolated from HUVEC using a commercially available Trizol reagent kit (Gibco BRL) after culture protocols. The RNA (5 µg) was reversibly transcribed with 10,000 U of reverse transcriptase and 0.5 g/L oligo-(dT)15 primer (Bioneer). The expressions of the mRNA transcripts of VCAM-1 (forward primer, 5'-ATGCCTGGGAAGATGGTCGTGA-3'; reverse primer, 5'-TGGAGCTGGTAGACCCTCGCTG-3'); ICAM-1 (forward primer, 5'-GGTGACGCTGAATGGGGTTCC-3'; reverse primer, 5'-GTCCTCATGGTGGGGCTATGACTC-3'); E-selectin (forward primer, 5'-ATCATCCTGCAACT TCACC-3'; reverse primer, 5'-ACACCTCACCAAACCCTTC-3'); and ß-actin (forward primer, 5'-GACTACCTCATGAAGATC-3'; reverse primer, 5'-GATCCACATCTGCTGGAA-3') were evaluated by RT-PCR as previously described with slight modification (19). PCR was performed in 50 µL of 10 mmol/L Tris-HCl (pH 8.3), 25 mmol/L MgCl2, 10 mmol/L dNTP, 100 U of Taq DNA polymerase, and 0.1 µmol/L of each primer and was terminated by heating at 70°C for 15 min. After thermocycling and electrophoresis of the PCR products (5 µL) on 1% agarose-formaldehyde gel, the bands were visualized using a UV transilluminator (Amersham Pharmacia Biotech) and gel photographs were obtained. The absence of contaminants was routinely checked by RT-PCR assay of negative control samples without the addition of a primer.
NF-B protein localization.
HUVEC grown on a 4-well glass chamber slide were incubated for 1 h with 10% normal goat serum to block any nonspecific binding. Cells were then fixed with 4% ice-cold formaldehyde for 30 min. After fixed cells were washed with Tris-buffered saline (TBS), rabbit polyclonal anti-human NF-B (1:50) was added and cells were incubated overnight at 4°C. Cells were washed with TBS and incubated with a fluorescein isothiocyanate–conjugated goat anti-rabbit IgG (1:200). Images were obtained by fluorescence microscopy using an Olympus BX50 fluorescent microscope with differential interference contrast and reflected light fluorescence.
Nuclear extract preparation.
Nuclear protein extracts were prepared using a detergent lysis procedure from HUVEC to assay the DNA binding activity of NF-B (19,23). Cells were lysed in a buffer of 20 mmol/L HEPES, pH 7.9, 1 mmol/L EDTA, 10 mmol/L NaCl, 1 mmol/L dithiothreitol, 1 g/L Nonidet P40, 0.4 mmol/L phenylmethylsulfonyl fluoride, 0.01 µg/L leupeptin, and 200 U of aprotinin and incubated on ice for 10 min. Proteins were extracted from nucleus pellets by incubation with a high-salt buffer containing 420 mmol/L NaCl, 1 mmol/L EDTA, 20 mmol/L HEPES (pH 7.9), 25% glycerol, 1 mmol/L dithiothreitol, 0.4 mmol/L phenylmethylsulfonyl fluoride, 0.01 µg/L leupeptin, and 200 U of aprotinin with vigorous shaking. The nuclear debris was pelleted by a brief centrifugation (400 x g for 3 min) and the supernatant was stored at –70°C.
For the determination of NF-B localization, Western blot analysis was carried out with nuclear protein extracts using anti-human NF-B primary antibody (1:1000). The Western blot analytical procedures were described above.
EMSA.
Double-stranded oligonucleotide containing the consensus sequence of the binding site for NF-B (5'-AGTTGAGGGGACTTTCCCAGGC-3') was used as the electrophoretic mobility shift assay probes (19,23,24). The NF-B probe was labeled with 30 µCi of [
-32P]ATP (Amersham Pharmacia Biotech) and T4 polynucelotide kinase. The DNA binding reaction was performed in a 25-µL reaction mixture containing 250 mmol/L NaCl, 5 mmol/L MgCl2, 2.5 mmol/L EDTA, 2.5 mmol/L dithiothreitol, 50 mmol/L Tris-HCl (pH 7.5), 25% glycerol, 0.25 g/L poly(dl-dC) · poly(dI-dC), 32P-labeled specific oligonucleotide probe, and 4 µg nuclear extract. The oligonucleotide-DNA complex was separated from free oligonucleotide probe by electrophoresis on a 5% polyacrylamide gel in 0.5X TBE buffer (90 mmol/L Tris-base, 90 mmol/L boric acid, 1 mmol/L EDTA, pH 8.0) for 3 h. Gels were dried and exposed on Konica X-films (Konica).
Data analysis. The results are presented as means ± SEM for each treatment group in each experiment. Data were tested by one-way ANOVA followed by Tukey’s test for multiple comparisons. Differences were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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-activated endothelial cells.
The observed inhibition of CAM expression by the flavonoids suggested that flavonoid treatment might inhibit mononuclear leukocyte recruitment on the TNF--induced vascular endothelium. In vitro adhesion assay of monocytes to HUVEC using a calcein-AM staining technique supported this hypothesis. A small number of monocytes adhered to unstimulated HUVEC free of TNF- (Fig. 1). There was heavy staining on the HUVEC exposed to TNF- alone, indicative of a marked increase in THP-1 adherence to the activated HUVEC. However, the treatment of TNF--exposed cells with 50 µmol/L luteolin or apigenin almost completely inhibited monocyte adherence. Quercetin- and TNF--exposed cells revealed a substantial inhibition of TNF--induced THP-1 adhesion. In contrast, the addition of catechin flavanols did not have such effects on HUVEC (Fig. 1). Flavanones and flavanone glycosides at 50 µmol/L did not inhibit the adhesion of monocytes to TNF-activated endothelial cells (Fig. 2A and B).
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-induced CAM protein expression.
Western blot analysis was used to address whether flavonoids block the TNF--activated expression of VCAM-1, ICAM-1, and E-selectin (Fig. 3). As expected, there was relatively weak expression of all three CAM in untreated quiescent cells. Expression of these CAM proteins was markedly increased by 20- to 25-fold in TNF--stimulated cells compared to the quiescent cells. TNF--exposed cells treated with 50 µmol/L luteolin and apigenin demonstrated full inhibition of expression of VCAM-1, ICAM-1, and E-selectin (Fig. 3A). Quercetin- and TNF--exposed cells exhibited a substantial but yet not full inhibition of expression of these CAM proteins, while the flavanol catechins did not inhibit their expression. When HUVEC were treated with flavanones, naringin and hesperidin, the expression of VCAM-1 was slightly decreased (Fig. 3A). Accordingly, the Western blot data (Fig. 3) supported the in vitro adhesion results (Figs. 1and 2). We also attempted to quantify the VCAM-1 expression using 50 µmol/L of naringenin and hesperetin, flavanones without the rutinose moiety of naringin and hesperidin, respectively (Fig. 3B). The presence of the rutinose moiety of flavanones had a minimal effect on VCAM-1 expression.
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decreased in a dose-dependent manner with inhibitory doses being >10 µmol/L (Fig. 4). On the other hand, only with 25 µmol/L quercetin was TNF--induced VCAM-1 expression substantially inhibited. In addition, VCAM-1 induction by TNF- was significantly blocked by treatment with10 µmol/L luteolin or apigenin. Thus, to achieve the full inhibitory effect of flavones and quercetin in the VCAM-1 expression model doses of 10–25 µmol/L were required. Consistently, the TNF--induced THP-1 adhesion was markedly attenuated in TNF--exposed cells treated with 25 µmol/L luteolin or apigenin (Fig. 5).
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-induced CAM transcription.
There were weak signals for the basal mRNA expression of these CAM proteins in quiescent cells (Fig. 6). In contrast, their CAM mRNA was greatly increased in TNF--stimulated HUVEC. However, RT-PCR data showed that the mRNA levels of all three CAM in flavone-treated cells were low but detectable. In addition, quercetin reduced TNF--induced mRNA accumulation of VCAM-1, ICAM-1, and E-selectin (Fig. 6). This was consistent with a substantial attenuation of expression of CAM proteins by quercetin and flavones shown in Figure 3. These results imply that these flavonoids inhibit CAM expression via direct effects at the level of gene transcription.
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B.
We tested whether quercetin, luteolin, and apigenin inhibit TNF--induced stimulation of CAM expression by interfering with the transactivation of transcription factors. Following exposure to 10 µg/L TNF-, nuclear p65 increased (Fig. 7A). When TNF--exposed cells were treated with 50 µmol/L quercetin, luteolin, or apigenin, a marked decrease in nuclear p65 protein occurred. In contrast, NF-B p65 protein in the cytosolic extract modestly increased following treatment with these flavonoids. In addition, intracellular localization of NF-B p65 in HUVEC was evaluated by fluorescent microscopy using specific NF-B p65 antibody (Fig. 7B). Cytoplasmic immunofluorescence staining was observed in untreated HUVEC, while heavy nuclear staining in cells exposed to TNF- alone occurred, indicative of nuclear localization of activated NF-B p65 at the single cell level. However, flavonoid- and TNF--treated cells demonstrated the diminished staining level of nuclear p65.
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B in nuclear extracts of HUVEC treated with TNF- in the absence and presence of quercetin, luteolin, or apigenin. A significant increase in NF-B binding activity was detected in nuclear extracts of TNF--exposed cells (Fig. 8), demonstrating that the protein-NF-B DNA complex was activated by TNF-. The specificity of the NF-B DNA-binding activity was evaluated by addition of excess unlabeled oligonucleotide (Fig. 8, lane 3). The activated NF-B binding was attenuated by treatment with quercetin, luteolin, or apigenin. This suggests that these flavonoids can inhibit the activation of endothelial CAM specifically by preventing the formation of NF-B-dependent DNA-protein complexes.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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-induced monocyte adhesion; the flavones were the most potent flavonoids, and the flavanols and flavanones did not prevent monocyte adherence on TNF--activated endothelial cells. 2) The flavones, luteolin and apigenin, at the nontoxic dose of 10 µmol/L, almost completely blocked the expression of VCAM-1, ICAM-1, and E-selectin proteins by a direct modulation at the level of gene transcription. 3) The flavonol quercetin substantially attenuated the expression of these CAM proteins at 25 µmol/L, while flavanol catechins and the flavanones of hesperetin and naringenin and their glycosides did not demonstrate such inhibitory activity even at 50 µmol/L. 4) The flavones appeared to inhibit CAM gene expression by blunting the activation of NF-B stimulated by TNF-. These observations demonstrate that the potential to prevent the early events in atherosclerosis differs among individual flavonoid subclasses. The results also suggest that the ability of flavones to block the TNF--induced activation of CAM expression may be mediated through the transcriptional mechanisms (Fig. 9).
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Flavonoids may inhibit early events in the atherosclerotic process by modulating monocyte adhesion and transmigration. Although definite mechanisms underlying the flavonoid protection against early atherogenic process are not fully understood, they may involve downregulation of inflammatory chemokines and cytokines, matrix proteases, and CAM (6–11,31). In this report, flavone-type flavonoids at 10–25 µmol/L almost completely inhibited and quercetin at >25 µmol/L markedly attenuated THP-1 monocyte adhesion to TNF--activated endothelial cells, at least in part by an inhibition of CAM expression. The flavanones, naringenin and hesperetin, and their glycosides, hesperidin and naringin, did not have inhibitory activity, implying that the presence of the rutinose moiety of flavanones does not facilitate blocking monocyte adhesion on the activated endothelium.
The ability of these flavonoids to block TNF--induced CAM expression could be due to their antioxidant characteristics. It has been demonstrated that oxidative stress upregulates VCAM-1 and E-selectin expression via redox-sensitive transcriptional activation and is inhibited by the antioxidants pyrrolidine dithiocarbamate and N-acetylcysteine (32,33). In addition, classical antioxidant vitamin E has been shown to inhibit expression of CAM and adhesion of monocytes to endothelial cells (34,35). (–)Epigallocatechin gallate was previously shown to have potent antioxidant activity (21). Nevertheless, this flavanol did not prevent pro-inflammatory agent–induced monocyte adhesion and CAM expression (Figs. 1 and 3). Western blot analysis showed that 5 mmol/L N-acetylcysteine did not downregulate TNF--induced VCAM-1 expression (unpublished data). Thus, it is unlikely that the antioxidant activity of flavonoids contributes to their blockade of endothelial CAM induction by TNF- and to the atheroprotective actions of flavones. It should be pointed out that coculture of HUVEC with flavones and TNF- was not cause toxic (data not shown), as determined by MTT assay. Luteolin was recently reported to reduce LPS-induced lethal toxicity possibly by inhibiting TNF- and ICAM-1 expression in vivo (36).
How CAM genes are selectively modulated in response to pro-inflammatory cytokines and which signaling pathways are involved in the selective regulation of these genes remain unknown. Clearly, the activation of endothelial expression of VCAM-1, ICAM-1, and E-selectin by TNF- was blocked possibly by a novel mechanism(s) responsive to quercetin and flavones. The inhibitory mechanism(s) of these flavonoids was inferred from the possibility that flavonoids may interrupt a signaling cascade involving CAM transcription activation of NF-B, which plays an important role in the inducible regulation of cellular inflammatory genes and which is activated in atherosclerotic tissues (16,37,38). Phenolic gallate has previously been shown to inhibit cytokine-induced nuclear translocation of NF-B and expression of leukocyte adhesion molecules in HUVEC (39). In addition, it has been shown that luteolin abolishes an LPS-induced increase in inhibitory protein IB- phosphorylation, NF-B-mediated gene expression, and pro-inflammatory cytokine production in murine macrophages (40). The anti-inflammatory activity of resveratrol, a naturally occurring phytoalexin found in grapes and wine, could be mediated by its interference with NF-B-dependent transcription (41). In this study, quercetin, luteolin, and apigenin attenuated or blocked nuclear translocation of p65 and DNA binding activity of NF-B stimulated by TNF-, which in turn attenuated CAM expression at the transcriptional level. However, the mechanisms underlying this inhibition are still unknown. Quercetin has been reported to inhibit ICAM-1 expression induced by phorbol 12-myristate 13-acetate or TNF- without NF-B activation in the human endothelial cell line ECV304 (42). In addition, a flavonoid 2-(3-amino-phenyl)-8-methoxy-chromene-4-one selectively blocked TNF--induced VCAM-1 expression in human aortic endothelial cells by an NF-B-independent mechanism (10).
The mechanism by which quercetin and flavones block endothelial CAM expression through inhibiting activation of NF-B is not fully understood. The access of flavones and quercetin to putative binding proteins in HUVEC may modulate TNF--mediated activation of NF-B signaling cascades by interrupting activation of mitogen-activated protein kinases by TNF-. Luteolin has previously been reported to interfere with LPS-triggered Akt phosphorylation and NF-B activation (40). Alternatively, these flavonoids may regulate the nuclear activity of NF-B through other signaling pathways, i.e., protein tyrosine kinase and/or protein kinase C-mediated pathways (33,40), and independent of the NF-B activation signaling pathway (42). Quercetin has been shown to inhibit agonist-induced ICAM-1 expression by interfering with the c-Jun NH2-terminal kinase pathway and activator protein-1 activation (40). It has been recently demonstrated that red wine polyphenols inhibit platelet-derived growth factor–induced vascular smooth muscle cell migration through the inhibition of signaling cascades involving phosphatidylinositol-3 kinase and p38 mitogen-activated protein kinase (43).
In summary, our studies have demonstrated that the effects of flavonoids on inducible CAM expression in cultured cells differ among individual flavonoid subclasses. Quercetin and flavones blocked monocyte adhesion on the TNF--activated endothelium and the activation of CAM expression. The selective inhibition of CAM expression by quercetin and flavones was at least in part mediated by the regulation of translocation and transactivation of NF-B. This observation might have implications for the prevention and attenuation of inflammatory diseases (Fig. 9). These inhibitory mechanisms of the flavones appear to be independent of an antioxidant sensitive transcriptional regulatory mechanism and may argue for transcriptional mechanisms as the major target of the anti-atherogenic action of flavones.
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FOOTNOTES |
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3 Abbreviations used: CAM, cell adhesion molecule; DMSO, dimethyl sulfoxide; EMSA, electrophoretic mobility shift assay; E-selectin, endothelial-leukocyte adhesion molecule-1; FBS, fetal bovine serum; HUVEC, human umbilical vein endothelial cell; ICAM-1, intracellular adhesion molecule-1; MTT, 3-(4,5-dimethylthiazol-yl)-diphenyl tetrazolium bromide; NF-B, nuclear factor-kappaB; TBS-T, Tris buffered saline-Tween 20; VCAM-1, vascular cell adhesion molecule-1.
Manuscript received 25 December 2003. Initial review completed 6 January 2004. Revision accepted 4 February 2004.
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