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Quercetin 3,7,3',4'-tetrasulphated isolated from Flaveria bidentis inhibits tissue factor expression in human monocyte.

ABSTRACT

Sulphated esters of the flavonoids sulphated quercetin 3,7,3',4'-tetrasulphated (QTS) and quercetin 3-acetyl-7,3,4'-trisulphate (ATS), isolated from Flaveria bidentis, have demonstrated anticoagulant and antiplatelet properties. In this study, we examined if both compounds affected the expression of the procoagulant tissue factor (TF) induced by lipopolysaccharide (LPS) on human monocyte. Monocytes were pretreated with different concentrations of each flavonoid (0.1-500 [micro]M), followed by a 4 h incubation with LPS in order to induce TF expression. Results of the TF expression showed different behaviors for the two flavonoids studied. A slight inhibitory effect on the TF expression was detected at a QTS concentration of 0.1 [micro]M, but from 1 [micro]M onwards a significant inhibitory effect that remained up to 500 [micro]M could be observed. In contrast, ATS induced a poor inhibitory effect on TF expression at all concentrations tested. These results suggest that QTS has another antithrombotic property, to be added to its already renowned ability as an anticoagulant and antiplatelet compound.

[c] 2012 Elsevier GmbH. All rights reserved.

ARTICLE INFO

Keywords: Flaveria bidentis Sulphated flavonoids Tissue factor Monocyte

Introduction

Tissue factor (TF), a 47-kDa membrane-bound glycoprotein, is the predominant activator of blood coagulation that interacts with coagulation factor VII/VIIa and initiates blood coagulation (Manly et al. 2011). It is normally absent from the surface of endothelial cells, but when the vasculature is injured or cells such as monocytes, macrophages or fibroblasts come into contact with different stimuli, the TF is induced to express itself on the cell surface ([empty set] sterud 2010). The catalytic complex TF-VIIa activates factor X to factor Xa, leads to prothrombinase activity on the TF-bearing cell surface and ultimately drives the thrombin to burst, which cleaves fibrinogen to fibrin, thus initiating the formation of the fibrin meshwork critical for secondary coagulation and clot stabilization. TF expression on the cell surface is associated with thromboembolic complications in inflammation, sepsis, and atherosclerotic disease (Mackman 2009).

Flavonoids are polyphenolic compounds widespread in the plant kingdom and are important dietary components. Some of these have remarkable biological activities, such as inhibitory effects on enzymes, modulatory effects on some cell types, and protection against allergies, as well as antiviral, anti-malarial, anti-oxidant, anti-inflammatory, anti-tumor and antithrombotic properties (Suer et al. 2010). Furthermore, epidemiologic studies suggest a protective role of dietary flavonoids against coronary heart disease and strokes. When the association between flavonoid intake and the long term effects on mortality was studied, it was suggested that flavonoid intake is inversely correlated with mortality due to coronary heart disease (Nijveldt et al. 2001). Sulphated esters of flavonoids represent an interesting group of sulphured compounds, especially the fully sulphated quercetin 3,7,3,4-tetrasulphated (QTS) and quercetin 3-acetyl-7,3,4'-trisulphate (ATS) isolated from Flaveria bidentis, which have demonstrated antithrombotic properties. In fact, we were able to demonstrate that sulphated flavonoids, mainly QTS, had an anticoagulant effect by acting as heparin cofactor ll agonist, as well as showing an antiplatelet effect, because they induced a deleterious effect on the production of Tx [A.sub.2] and a marked interference on the Tx [A.sub.2] receptor (Guglielmone et al. 2002, 2005).

The antithrombotic properties of these sulphated flavonoids and the assumption that FT inhibition could be a new and attractive target for the treatment of thrombosis, encouraged us to examine whether these compounds (ATS and QTS) affect the expression of TF induced by LPS on human monocyte.

Materials and methods

Plant material

Flaveria bidentis was collected in Cordoba city (Cordoba, Argentina) when the plant was in bloom. The plant material was identified by Prof. Dr. Luis Ariza Espinar and voucher specimen number 2813 was deposited at CORD. ATS and QTS were isolated, purified and identified as previously reported, and it is important to point out that according to their chemical structures both flavonoids are completely soluble in distilled water (Guglielmone et al. 2002).

Monocyte preparation

Human peripheral blood mononuclear cells (PBMC) from healthy donors (aged 25-40 years) were isolated by centrifugation over Hystopaque gradients (Sigma Chemical Co., CA). Monocyte enriched populations (MEPs) were obtained by depletion of CD4 and CD8 positive cells from total PMBC using CD4 or CD8 Microbeads (Miltenyi Biotech GmbH, Germany), according to the manufacture's instructions. MEPs were resuspended in RPMI 1640 containing 10% heat-inactivated newborn calf serum and adjusted to a final concentration of 5 x [10.sup.6] cells/ml. The conditions to achieve LPS-induction of TF were experimentally determined, and 1.25 x [10.sup.6] cells/ml incubated with 100 ng/mI LPS for 4h at 37 [degrees]C-5% [CO.sub.2] were chosen for the optimal condition assay. After the incubation, MEPs were sedimented by centrifugation at 400 x g and lysed by repeated freeze-thaw cycles (three times).

Determination of TF

TF antigen levels were determined in the cell lysate after MEPs disruption, and TF extraction was achieved according to the manufacturer's instructions using Tris buffered saline (50 mM Tris, 100mM NaC1) pH 7.4, containing 0.1% Triton X-100 for 18h of incubation at 2 [degrees]C, followed by centrifugation of the lysed cells to remove cell debris. TF-containing MEPs-lysates were stored at--8 [degrees]C until being assayed using an ELISA kit (Imubind Tissue Factor, American Diagnostic Inc., CT, USA), and the values were expressed in percentages taking into account the total protein.

Experimental design

To test the effect of QTS and ATS on the expression of TF, the MEPs were pretreated with different concentrations of each flavonoid (0.1-500 [micro]M), followed by a 4 h incubation with LPS. As control 0% and 100%, MEPs without/with stimulation with LPS were used with vehicle instead of QTS or ATS. The toxic effect of the flavonoids on the MEPs was checked by a cell survival test using a tetrazolium-based colorimetric assay (Mossman 1983). A value greater than 10% of disrupted cells was considered as a significant indication of cytotoxicity.

Statistical analysis

Values are expressed as means [+ or -] S.D. Statistical significance was determined using Student's t-test, with p < 0.05 being considered significant.

Results and discussion

Results on the TF expression in MEPs showed different behaviors for the two flavonoids studied, with it being evident in Fig. 1 that treatment of MEPs with LPS increased the TF activity about 20-fold compared to the unstimulated control cells. In fact, a nonsignificant inhibitory effect on the TF expression was detected at a QTS concentration of 0.1 [micro]M, but from 1 [micro]M onwards a significant inhibitory effect (p < 0.05) was observed which remained up to 500 [micro]M. In contrast, ATS did not induce an inhibitory effect on TF expression when stimulated with LPS at any of the concentrations tested. The only structural difference among these two compounds is that ATS has one acetyl group at position 3 (C-ring), whereas QTS possesses a sulphate group at the same position, which is more polar than ATS. The effect of ATS and QTS on cell viability was also investigated since some flavonoids had been previously shown to be cytotoxic and had induced apoptosis (Kanadaswami et al. 2005). Under our experimental conditions, it was found that only concentrations greater than 500 [micro]M in both flavonoids showed a cytotoxic effect (data not shown).

[FIGURE 1 OMITTED]

Flavonoids have received much attention in the literature over recent years, with a variety of potential beneficial effects having been elucidated. However, the study of flavonoids is complex because of the heterogeneity of the different molecular structures and the scarcity of data on bioavailability. In the present study, we demonstrated that of the sulphated flavonoids isolated from Flave-ria bidentis, QTS but not ATS was capable of inhibiting the induction of TF on monocytes after LPS stimulation. The origin of this difference found in inhibitory activity was unclear, but may have been due to the presence of an acetyl group on ATS which was absent in QTS. Several publications have reported the effects of the different flavonoids on the expression of TF in cell cultures, with these being linked to the chemical structure of these compounds (Lale and Herbert, 1996; Kaur et al. 2007; Jiang et al. 2009; Holzer et al. 2007). In these reports, the studied compounds had chemical structures quite different from each other in the substituent chemical group, thus making it difficult to carry out a comparison of the effectiveness that these polyphenols exerted on the expression of FT on a chemical basis.

Another possible explanation for the flavonoids effects could be related to the pathway of thromboxane [A.sub.2] (Tx [A.sub.2]) and its specific receptor (TP). TPs have been characterized in human peripheral blood monocytes ([alpha] isoform), with their intervention in the regulation of TF regulation of LPS-induced cells having been delineated ex vivo (Eilertsen and [empty set]sterud 2002). In fact, an increased synthesis of Tx [A.sub.2] and procoagulant activity have been detected in arachidonic acid challenged monocytes from diabetic patients compared to controls, which reinforces the finding of the positive role played by Tx [A.sub.2] in TF induction by LPS (Konieczkowski and Skrinnska 2001). In the present study, the decrease in TF expression on MEPs could have been due to the deleterious effect that QTS had on the production of Tx[A.sub.2] and its marked interference on TPs as reported previously (Guglielmone et al. 2005).

Finally, the endothelial cells and monocytes stimulated with LPS leads to the activation of various ROS-sensitive intracellular signaling pathways and transcription factors (NF-[kappa]B, AP-1, and Egr-1) that mediate the expression of FT mRNA (Di Santo et al. 2003). These authors demonstrated that monocyte IF mRNA was significantly reduced in the presence of the flavonoid quercetin in a dose-dependent fashion, originating from a reduction in the nuclear binding activity of the transacting factor c-Rel/p65, a member of the NF-[kappa]B family, thereby inducing a marked decrease in the expression of TF.

In summary, the experimental evidence obtained in the present study indicates that QTS possesses the ability to suppress the TF expression which adds another antithrombotic property to its already renowned ability as an anticoagulant and antiplatelet compound. Further investigation is currently in progress to attempt to clarify the details of the molecular mechanisms of action of QTS on LPS-induced IF expression in monocytes.

Disclosure

The authors state that they have no conflict of interests.

Acknowledgements

This work was supported by grants from FONCyT and Secre-taria de Ciencia y Tecnologia de la Universidad Nacional de Cordoba (SECyT).

We thank biochemistry Cecilia Ramello for MEPs obtention. We thank Dr. Paul Hobson, native speaker, for revision of the manuscript. AMA, JLC, ACD and CGP are established researchers at CON ICET.

References

Buer, C.S., Imin, N., Djordjevic, M.A., 2010. Flavonoids: new roles for old molecules. Journal of Integrative Plant Biology 52, 98-111.

Di Santo, A., Mezzeti, A., Napoleone, E., Di Tommaso, R., Donati, M.B., De Gaetano, G., 2003. Resveratrol and quercetin down-regulate tissue factor expression by human stimulated vascular cell. Journal of Thrombosis and Haemostasis 1, 1089-1095.

Eilertsen, K.E., [empty set]sterud, B., 2002. The central role of thromboxane and platelet activating factor receptors in ex vivo regulation of endotoxin-induced monocyte tissue factor activity in human whole blood. Journal of Endotoxin Research 8, 285-293.

Guglielmone, H.A., Agnese, A.M., Nunez-Montoya, S.C., Cabrera, J.L., 2002. Anticoagulant effect and action mechanism of sulphated flavonoids from Flaveria bidentis. Thrombosis Research 105, 183-188.

Guglielmone, H.A., Agnese, A.M., Nunez-Montoya, S.C., Cabrera, J.L., 2005. Inhibitory effects of sulphated flavonoids isolated from Flaveria bidentis on platelet aggregation. Thrombosis Research 115, 495-502.

Holzer, G., Esterbauer, H., Kronke, G., Exner, M., Kopp, C.W., Leitinge r, N.. Wagner, 0., Gmeiner, B.M.K., Kapiotis, S., 2007. The dietary soy flavonoid genistein abrogates tissue factor induction in endothelial cells induced by the atherogenic oxidized phospholipid oxPAPC. Thrombosis Research 120,71-79.

Jiang, W., Kou, J., Zhang, Z., Yu, B., 2009. The effects of twelve representative flavonoids on tissue factor expression in human monocytes: structure-activity relationships. Thrombosis Research 124,714-720.

Kanadaswami, C., Lee, Lee, P., Hwang, J., Ke, F., Huang, Y., Lee, M., 2005. The antitumor activities of flavonoids. In Vivo 19, 895-909.

Kaur, G., Roberti, M., Raul, F., Pendurthi, U.R., 2007. Suppression of human monocyte tissue factor induction by red wine phenolics and synthetic derivatives of resveratrol. Thrombosis Research 119,247-256.

Konieczkowski, M., Skrinnska, V.A., 2001. Increased synthesis of thromboxane A2 and expression of procoagulant activity by monocytes in response to arachidonic acid in diabetes mllitus. Prostaglandins Leukotrienes and Essential Fatty Acids 65,33-138.

Lale, A., Herbert, J.M., 1996. Ability of different flavonoids to inhibit the procoagulant activity of adherent human monocytes. Journal of Natural Products 59,273-276. Mackman, N., 2009. The many faces of tissue factor. Journal of Thrombosis and Haemostasis (Suppl. 1), 136-139.

Manly, D.A., Boles, J., Mackman, N., 2011. Role of tissue factor in venous thrombosis. Annual Review of Physiology 73,515-525.

Mossman, T., 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxic assays. Journal of Immunological Methods 65, 55-63.

Nijveldt, R.J., van Nood, E., van Hoorn, D.E.C., Boelens, P.G., van Norren, K., van Leeuwen, P.A.M., 2001. Flavonoids: a review of probable mechanisms of action and potential applications. American Journal of Clinical Nutrition 74,418-425.

[empty set]sterud, B., 2010. Tissue factor expression in blood cells. Thrombosis Research 125 (Suppl. 1), S31-534.

* Corresponding author at: Departamento de Bioquimica Clinica, CIBICl-CONICET, Facultad de Ciencias Quimicas, Universidad Nacional de Cordoba, Medina Allende y Haya de la Torre, AP4-Ciudad Universitaria, Cordoba, Argentina.

Tel.: +54 351 4334187; fax: +54 351 4334187.

E-mail address: hgugli@bioclin.fcq.unc.edu.ar (H.A. Guglielmone).

0944-7113/$ - see front matter 0 2012 Elsevier GmbH. All rights reserved.

http://dx.doi.org/10.1016/j.phymed.2012.06.013

H.A. Guglielmone (a), * S.C. Nunez-Montoya (b), A.M. Agnese (b), C.G. Pellizas (a), J.L. Cabrera (b), A.C. Donadio (a)

(a) Departamento de Bioquimica Clinica, CIBCI-CONICET Facultad de Ciencias Quimicas, Universidad Nacional de Cordoba, Cordoba, Argentina

(b) Farmacognosia, Departamento de Farmacia, IMBIV-CONICET, Facultad de Ciencias Quimicas, Universidad Nacional de Cordoba, COrdoba, Argentina
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Title Annotation:Short communication
Author:Guglielmone, H.A.; Nunez-Montoya, S.C.; Agnese, A.M.; Pellizas, C.G.; Cabrera, J.L.; Donadio, A.C.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:3ARGE
Date:Sep 15, 2012
Words:2361
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