- Email: [email protected]
Effects of quercetin on the release of endothelin, prostacyclin and tissue plasminogen activator from human endothelial cells in culture
Journal of Ethnopharmacology 67 (1999) 279 – 285 www.elsevier.com/locate/jethpharm
Effects of quercetin on the release of endothelin, prostacyclin and tissue plasminogen activator from human endothelial cells in culture Xueying Zhao a, Zhenlun Gu a, Anoja S. Attele b, Chun-Su Yuan b,c,* a Department of Pharmacology, Suzhou Medical College, Suzhou 215007, China Department of Anesthesia and Critical Care, The Uni6ersity of Chicago, 5841 S. Maryland A6enue, MC 4028, Chicago, IL 60637, USA c Committee on Clinical Pharmacology, The Uni6ersity of Chicago, 5841 S. Maryland A6enue, MC 2115, Chicago, IL 60637, USA b
Received 22 December 1998; received in revised form 29 March 1999; accepted 11 April 1999
Abstract Quercetin and related flavonoids are naturally occurring polyphenolic compounds with multiple pharmacological activities. Using cultured human umbilical vein endothelial cells, we investigated the effects of quercetin on endothelin (ET-1) and tissue plasminogen activator (t-PA) release induced by thrombin. We observed that when endothelial cells pretreated with 5 or 50 mM of quercetin were incubated for 4 and 24 h with thrombin, ET-1 concentration-dependently decreased (n=6, PB 0.01, at 4 h IC50 =1.54 mM, at 24 h IC50 = 2.78 mM). Under the same experimental conditions, quercetin significantly increased t-PA (n= 6, PB 0.01, at 4 h EC50 = 0.71 mM and at 24 hrs EC50 = 0.74 mM). In the same preparation, we evaluated prostacyclin (PGI2) release, induced by thrombin activated platelets, as determined by a 6-Keto-PGF1a radioimmunoassay. Following the treatment of cultured endothelial cells with activated platelets, the concentration of 6-Keto-PGF1a was significantly increased (PB 0.01). Quercetin (1, 5, and 20 mM) inhibited PGI2, in a concentration-dependent manner (n= 6, P B0.05). Our data indicate that quercetin modulates the release of ET-1, t-PA, and PGI2 from vascular endothelial cells. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Quercetin; Endothelial cell; Human umbilical vein; Endothelin; Thrombin; Prostacyclin; 6-Keto-PGF1a
1. Introduction Flavonoids are a large group of naturally occurring polyphenolic compounds found in many * Corresponding author. Tel.: +1-773-7021916; fax: +1773-8340601. E-mail address: [email protected] (C.-S. Yuan)
green plants and plant products that make up the human diet (Formica and Regelson, 1995). The largest portion of flavonoid intake comes from cocoa, coffee, tea, and wine (Pierpoint, 1986). Quercetin, a flavonol, has been shown to possess a wide spectrum of pharmacological effects.Quercetin has important palliative effects on the cardiovascular system. It decreases serum
0378-8741/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 9 9 ) 0 0 0 5 5 - 0
280
X. Zhao et al. / Journal of Ethnopharmacology 67 (1999) 279–285
LDL cholesterol concentration (Frankel et al., 1993) by acting as a free radical scavenger and an inhibitor of lipid peroxidation (Afanas’ev et al., 1989). Duarte et al. (1993) reported that quercetin inhibited the contractions induced by noradrenaline, high KCl, and Ca2 + in rat aortic strips. Here, the vasodilatory effect of quercetin was mainly related to the inhibition of protein kinase C. In another study, quercetin caused vasodilation by both endothelium-dependent and endothelium-independent mechanisms (Chen and Pace-Asciak, 1996). Some cardiovascular effects of quercetin implicate the involvement of endproducts of arachidonate metabolism such as prostaglandin eicosaniods (Kalkbrenner et al., 1992). Quercetin also blocked platelet aggregation by inhibiting cyclic phosphodiesterase (Lanza et al., 1987). The endothelium regulates vascular tone by releasing factors involved in relaxation and contraction, in coagulation and thrombus formation, and in growth inhibition and stimulation (Lusher and Tanner, 1993). Endothelial cell dysfunction is believed to be the initial step in the genesis of thrombosis and arteriosclerosis (Furchgott and Vanhoutte, 1989). Despite many previous studies on quercetin, its effects on smooth muscle relaxing and contracting factors, and fibrinolytic mediators released from vascular endothelial cells have not been reported. In this study, we investigated the effect of quercetin on thrombin-induced endothelin (ET-1) and tissue plasminogen activator (t-PA) release, as well as its effect on activated platelet-induced 6-KetoPGF1a release, in cultured endothelial cells from human umbilical cord veins.
2. Materials and methods
2.1. Preparation of endothelial cells and cell culture
buffer (0.14 M NaCl, 0.004 M KCl, 0.001 M phosphate buffer, 0.011 M glucose, at pH 7.4), and held at 4°C until processing. The vein was perfused with 100 ml of cord buffer to wash out the blood and allowed to drain. The cord buffer containing 0.2% collagenase was then infused into the umbilical vein. The umbilical cord was placed in a water bath containing cord buffer and incubated at 37°C. After incubation, the collagenase solution containing the endothelial cells was flushed from the cord vein by perfusion with cord buffer. The effluent was collected, the cells were sedimented and washed. A sterile technique was utilized in all manipulations of the cord. Endothelial cells were cultured in DMEM medium with 20% fetal calf serum, penicillin (100 unit/ml), and streptomycin (100 mg/ml). The dishes were incubated and the cells were harvested with 0.01% EDTA-0.1% collagenase.
2.2. Incubation procedures The confluent monolayers of endothelial cells were used after 7–8 days. The primary culture medium was discarded and dishes were washed twice with PBS. Next, fresh, conditioned, cultured medium without serum was added. The dishes with 2 unit/ml of thrombin were incubated at 37°C for 4 and 24 h. After being sedimented at 1500×g for 15 min, the cell suspension was used to assay the concentrations of ET-1 and t-PA. ET-1 was measured by radioimmunoassay (Fyhrquist et al., 1990) using synthetic ET-1 and ET-1 antiserum generated in rabbits. Measurement of t-PA was done by the method of MacGregor and Prowse (1983). The addition of PBS alone was used as the control. For the test group, quercetin, at different concentrations was added 10 min before the application of thrombin.
2.3. Preparation and acti6ation of platelets Endothelial cells were obtained from human umbilical cord veins by an adaptation following Ristimaki et al. (1990). In brief, the cord was severed from the placenta soon after birth, placed in a sterile container filled with cord
Venous blood with 2% EDTA Na2 as the anticoagulant (9:1, volume:volume) was drawn from healthy adults, and the blood was centrifuged at 250× g for 9 min. The platelet-rich
X. Zhao et al. / Journal of Ethnopharmacology 67 (1999) 279–285
plasma was drawn, and centrifuged at 1200× g for 9 min, to sediment into platelet concentrate. The platelet pellet was gently resuspended in modified Tyrode buffer and adjusted to 2 × 108/ ml, after being washed twice with platelet-washing liquid. The platelets were incubated together with 2 unit/ml of thrombin for 5 min at 37°C to obtain thrombin activated platelets.
2.4. 6 -Keto-PGF1a determination 6-Keto-PGF1a was measured according to a previously described radioimmunoassay (Maclouf, 1982). Briefly, 50, 100, 200, and 300 ml aliquots of unextracted cultured supernatant were incubated in a total volume of 700 ml containing specific antibody, 125I histamine-6-Keto-PGF1a (activities: 12 000 cpm), gamma globulins (5 g/l) in Tris –HCI buffer (0.05 M, pH 7.4), and EDTA (1 M). After 18 h of incubation at 4°C, 25% (wt./vol) polyethylene glycol was added, and the mixture was centrifuged. The supernatant was then removed, and the radioactivity of the precipitate was measured in a gamma counter. The concentration of 6-Keto-PGF1a released during adhesion and the subsequent washes was measured. The sensitivity of the technique permitted the detection of 5 pg of 6-Keto-PGF1a concentration.
281
3. Results
3.1. Effects of quercetin on ET-1 release from endothelial cells When endothelial cells were incubated in the presence of thrombin for 4 h, the concentrations of ET-1 in the conditioned medium increased from 399 8 pg/ml (mean 9 SD) to 136920 pg/ml (n= 6, PB 0.01). Under the same experimental conditions, at 24 h, the concentration of ET-1 in the conditioned medium increased from 354939 to 5459 28 pg/ml (n= 6, PB 0.01). When endothelial cells were pretreated with 5 or 50 mM of quercetin, the concentration of ET-1 in the conditioned medium was significantly decreased (n= 6, PB 0.01). The decrease in ET-1 was concentration-dependent and was observed when quercetin was in contact with endothelial cells for 4 and 24 h (Fig. 1). At 4 h IC50 = 1.54 mM and at 24 h IC50 = 2.78 mM. These results show that quercetin inhibits ET-1 induced by thrombin.
2.5. Drugs Quercetin, collagenase, penicillin, streptomycin, thrombin, EDTA, and DMEM medium were purchased from Sigma Co. (St. Louis, MO). Quercetin was initially dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution, and further dilutions were made in PBS. The final DMSO concentration (B0.25%) did not significantly affect the results.
2.6. Statistical analysis Results were analyzed using Student’s t-test and Mann–Whitney U test to determine if changes between the two conditions were significantly different. Probability levels less than 0.05 were considered significant differences.
Fig. 1. Effect of quercetin on the concentration of ET-1 in the conditioned medium of HUVECs pretreated with thrombin. *P B0.01 compared with saline control group; **PB0.01 compared with thrombin control group. Brackets indicate the mean 9SEM, for all groups n = 6.
282
X. Zhao et al. / Journal of Ethnopharmacology 67 (1999) 279–285
which was determined by radioimmunoassay (Maclouf, 1982). Thrombin activated platelets and endothelial cells were incubated together for 10, 30 and 120 min. As shown in Fig. 3, the concentrations of 6-Keto-PGF1a in the conditioned medium increased from 2.390.2 (mean9 SD), 5.890.8 and 10.5 9 1.9 pg/ml to 8.09 1.1 (n=6, PB0.001), 9.1 9 1.2 (n= 6, PB 0.01) and 21.2 9 1.0 pg/ml (n= 6, PB 0.001), respectively.
3.4. Effects of quercetin on 6 -Keto-PGF1a release from endothelial cells induced by thrombin acti6ated platelets
Fig. 2. Effect of quercetin on the concentration of t-PA in the conditioned medium of HUVECs pretreated with thrombin. *PB0.01 compared with saline control group; **PB 0.01 compared with thrombin control group. Brackets indicate the mean9 SEM, for all groups n= 6.
3.2. Effects of quercetin on t-PA release from endothelial cells
Endothelial cells were incubated together with quercetin 1, 5 or 20 mM for 10 min and further incubated after the addition of activated platelets for 10, 30 or 120 min. Compared with the control group, the concentration of 6-Keto-PGF1a in the conditioned medium, after quercetin pretreatment, decreased significantly (n= 6, PB 0.05) (Fig. 4). Quercetin (20 mM), at 10, 30, and 120 min, had a 4593.1% (mean9SD) maximum inhibition of induced 6-Keto-PGF1a release. At 10 min IC50 = 2.1 mM, at 30 min IC50 = 2.9 mM, and at 120 min IC50 = 0.58 mM. The decrease in 6Keto-PGF1a release was concentration-dependent.
After endothelial cells were treated with thrombin for 4 h, t-PA in the conditioned medium decreased from 4.290.4 pg/ml (mean 9 SD) to 3.1 90.3 pg/ml (n= 6, P B0.01). Under the same experimental conditions, at 24 h, t-PA decreased from 4.39 0.4 pg/ml to 3.490.3 pg/ml (n = 6, P B 0.01). When endothelial cells pretreated with 0.5, 5, or 50 mM of quercetin were incubated for 4 and 24 h, the concentration of t-PA in the conditioned medium was significantly increased (n = 6, P B 0.01) (Fig. 2). At 4 h EC50 =0.71 mM and at 24 h EC50 =0.74 mM. These results show that quercetin stimulates t-PA release, which was inhibited by thrombin.
3.3. Effects of acti6ated platelets on 6 -Keto-PGF1a release from endothelial cells PGI2 is unstable and is rapidly degraded to the stable metabolite 6-Keto-PGF1a, the level of
Fig. 3. Effect of thrombin activated platelets on the release of 6-Keto-PGF1a from the endothelial cells. Test group = Platelet activated group. *P B0.01; **PB 0.001. Brackets indicate the mean 9SEM, for both groups n = 6.
X. Zhao et al. / Journal of Ethnopharmacology 67 (1999) 279–285
Fig. 4. Effect of quercetin on the concentration of 6-KetoPGF1a in the conditioned medium of HUVECs pretreated with thrombin activated platelets. *PB0.05 compared with saline control group; **PB 0.01 compared with saline control group. Brackets indicate the mean 9 SEM, for both groups n= 6.
These results show that quercetin inhibits the release of 6-Keto-PGF1a, which was induced by thrombin activated platelets, from endothelial cells.
4. Discussion An important effect of quercetin on the cardiovascular system is its ability to modulate vasoreactivity of the vascular endothelium (Duarte et al., 1993; Chen and Pace-Asciak, 1996). The results of this study demonstrate that quercetin inhibits ET-1 release from human vascular endothelial cells. Quercetin, at concentrations of 5 and 50 mM was able to reverse the increase in ET-1 induced by thrombin. Thrombin stimulates ET-1 release by increasing the activity of phospholipase C (Yanagisawa et al., 1989). This, in turn, modulates intracellular calcium and protein kinase C which enhances expression of prepro ET-1 mRNA (Yanagisawa et al., 1989). Recent studies have demonstrated that flavonoids may be potent inhibitors of several kinases involved in signal transduction, mainly protein kinase C (PKC) and tyrosine kinases. Moreover, flavones
283
such as quercetin, and flavonols are the most active inhibitors of PKC (Ferriola et al., 1989; Agullo et al., 1997). Nitric oxide (NO) is a feedback inhibitor of ET-1 release (Boulanger and Lusher, 1990) and an increase in NO would also lead to decreased ET-1 in the media of treated cells. In the rat aorta, quercetin, at concentrations between 1×10 − 5 and 6×10 − 5 M, caused vasodilation, which depended on NO release (Chen and Pace-Asciak, 1996). However, at higher concentrations, the vasodilation was independent of NO release. The inhibitory action on ET-1 release by quercetin in our experimental model may be related to one or both of these mechanisms. Our results show that when endothelial cells pretreated with quercetin were incubated with thrombin activated platelets, PGI2 in the medium, concentration-dependently decreased as measured by a 6-Keto-PGF1a assay. Thrombin activated platelets released arachidonic acid (Skeaf and Holub, 1985) and stimulated PGI2 release (Mikkola et al., 1993). Moreover, thrombin stimulated arachidonate metabolism in endothelial cells (Alhenc-Gelas et al., 1982). Arachidonic acid is primarily metabolized via two separate enzymatic pathways, the cyclooxygenase (CO) and lipooygenase pathways (Moncada et al., 1976), both of which are present in vascular endothelial cells (Alhenc-Gelas et al., 1982). PGI2 is the major CO metabolite in blood vessels (Setty et al., 1985). Flavonoids including quercetin have been shown to competitively inhibit CO (Kalkbrenner et al., 1992; Mirzoeva and Calder, 1996). In addition, flavonoids which are potent free radical scavengers inhibited the peroxidation of polyunsaturated fatty acids required for the activation of CO (Halliwell, 1990). It is possible that in our preparation, quercetin concentration and time-dependently decreased PGI2 in the medium by inhibiting CO. The capacity to release PGI2 enables endothelial cells to regulate the state of dilation of arteries and inhibit platelet aggregation (Vane and Botting, 1995). Under physiological conditions, the contribution of PGI2 to endothelium-dependent relaxation may be negligible (Lusher and Tanner, 1993). However, it may be important in atherogenic states where endothelial function is already compromised. A reduction in
284
X. Zhao et al. / Journal of Ethnopharmacology 67 (1999) 279–285
PGI2 would be a disadvantage because it would permit vasoconstrictors such as ET-1 and angiotensin II to determine the capacity of the artery to maintain its lumen in the progression of atherogenic states. Vascular endothelium is strategically located at the interface between blood and smooth muscle. Therefore, it also plays a central role in maintaining blood fluidity. Tissue plasminogen activator (t-PA), produced by vascular endothelial cells, converts plasminogen to plasmin which degrades fibrin (Hoylaerts et al., 1982). Endothelial cells also produce plasminogen activator inhibitor (PAI-1), the main physiological inhibitor of t-PA, and it is believed that the balance between t-PA and PAI-1 is important for the regulation of fibrinolysis (Ranby, 1982). The results of our study show that quercetin reversed the significant reduction of t-PA in endothelial cells induced by thrombin at 4 and 24 h. Previous studies have shown that thrombin, at physiological concentrations, increased both PAI-1 and t-PA production (Yamamoto et al., 1994; Kimura et al., 1997). However, the net effect of thrombin was to increase the activity of PAI-1 and, therefore, decrease fibrinolytic activity of human endothelial cells (Gelehrter and Sznycer-Laszuk, 1986). Flavonoids are potent inhibitors of PAI-1 production in cultured HUVECs (Kimura et al., 1997). Flavonoids such as baicalein may inhibit PAI-1 by a direct inhibition of PKC activity or a reduction in [Ca2 + ]I elevation (Kimura et al., 1997). In our experimental model, quercetin may inhibit PAI-1 by a similar mechanism and increase the fibrinolytic activity of the vascular endothelium. In summary, this study presents data on the effects of quercetin on three mediators of endothelial cell function. Our data showed that quercetin suppressed ET-1 and PGI2 and increased t-PA from stimulated cultured human endothelial cells. Suppression of ET-1 production may partly explain the vasodilating action of quercetin. By stimulating t-PA release, quercetin may increase the fibrinolytic activity in endothelial cells. Further studies are necessary to reveal the mechanisms of action behind the observed effects.
Acknowledgements This work was support in part by the Tang Family Foundation. The authors wish to thank Ms. Tasha Lowell for her technical assistance.
References Afanas’ev, I.B., Dorozhko, A.I., Brodskii, A.V., Korstyuk, V.A., Potapovitch, A.I., 1989. Chelating and free radical scavenging mechanism of inhibitory action of rutin and quercetin in lipid peroxidation. Biochemical Pharmacology 38, 1763 – 1768. Agullo, G., Gamet-Payrastre, L., Manenti, S., Viala, C., Remesy, C., Chap, H., Payrastre, B., 1997. Relationship between flavonoid structure and inhibition of phosphatidylinositol 3-kinase: a comparison with tyrosine kinase and protein kinase C inhibition. Biochemical Pharmacology 53, 1649 – 1657. Alhenc-Gelas, F., Tsai, S.J., Callahan, K.S., Campbell, W.B., 1982. Stimulation of prostaglandin formation by vasoactive mediators in cultured human endothelial cells. Prostaglandins 24, 723 – 742. Boulanger, C., Lusher, T.F., 1990. Release of endothelin from the porcine aorta: inhibition by endothelium-derived nitric oxide. Journal of Clinical Investigation 85, 587 – 590. Chen, C.K., Pace-Asciak, C.R., 1996. Vasorelaxing activity of resveratrol and quercetin in isolated rat aorta. General Pharmacology 27, 363 – 366. Duarte, J., Perez-Vizcaino, F., Zarzuelo, A., Jimenez, J., Tamargo, J., 1993. Vasodilator effects of quercetin in isolated rat vascular smooth muscle. European Journal of Pharmacology 239, 1 – 7. Ferriola, P.C., Cody, V., Middleton, E. Jr., 1989. Protein kinase C inhibition by plant flavonoids. Kinetic mechanisms and structure – activity relationships. Biochemical Pharmacology 38, 1617 – 1624. Formica, J.V., Regelson, W., 1995. Review of the biology of quercetin and related bioflavonoids. Food and Chemical Toxicology. 33, 1061 – 1080. Frankel, E.N., Kanner, J., German, J.B., Parks, E., Kinsella, J.E., 1993. Inhibition of oxidation of human low-density lipoprotein by phenolic substances in red wine. Lancet 341, 454 – 457. Furchgott, R.F., Vanhoutte, P.M., 1989. Endothelium-derived relaxing and contracting factors. FASEB Journal 3, 2007 – 2018. Fyhrquist, F., Saijonmaa, O., Metsarinne, K., Tikkanen, I., Rosenlof, K., Timmanen, T., 1990. Raised plasma endothelin-1 concentration following cold pressor test. Biochemical and Biophysical Research Communications 169, 217 – 221. Gelehrter, T.D., Sznycer-Laszuk, R., 1986. Thrombin Induction of Plasminogen Activator – Inhibitor in Cultured Hu-
X. Zhao et al. / Journal of Ethnopharmacology 67 (1999) 279–285 man Endothelial Cells. Journal of Clinical Investigation 77, 165 – 169. Halliwell, B., 1990. How to characterize a biological antioxidant. Free Radical Research Communications 9, 1–32. Hoylaerts, M., Rijken, D.C., Lijnen, H.R., Collen, D., 1982. Kinetics of the activation of plasminogen by human tissue plasminogen activator: Role of fibrin. Journal of Biological Chemistry 257, 2912 –2919. Kalkbrenner, F., Wurin, G., von Bruchhausen, F., 1992. In vitro inhibition and stimulation of purified prostaglandin endoperoxide synthase of flavonoids: structure–activity relationship. Pharmacology 44, 1–12. Kimura, Y., Yokoi, K., Matsushita, N., Okuda, H., 1997. Effects of flavonoids isolated from Scutellariae radix on the production of tissue-type plasminogen activator and plasminogen activator inhibitor-1 induced by thrombin and thrombin receptor agonist peptide in cultured human umbilical vein endothelial cells. Journal of Pharmacy and Pharmacology 49, 816–822. Lanza, F., Beretz, A., Stierle, A., Corre, G., Cazenave, J.P., 1987. Cyclic nucleotide phosphodiesterase inhibitors prevent aggregation of human platelets by raising cyclic AMP and reducing cytoplasmic free calcium mobilization. Thrombosis Research 45, 477–484. Lusher, T.F., Tanner, F.C., 1993. Endothelial Regulation of Vascular Tone and Growth. American Journal of Hypertension 6, 283S – 293S. MacGregor, I.R., Prowse, C.V., 1983. Tissue plasminogen activator in human plasma measured by radioimmunoassay. Thrombosis Research 31, 461–474. Maclouf, J., 1982. A radioimmunoassay for 6-Keto-PGF1a. Methods in Enzymology 86, 273–286. Mikkola, T., Ristimaki, A., Viinikka, L., Ylikorkala, O., 1993. Human serum, plasma, and platelets stimulate prostacyclin and endothelin-1 synthesis in human vascular endothelial cells. Life Sciences 53, 283–289. Mirzoeva, O.K., Calder, P.C., 1996. The effect of propolis and its components on eicosanoid production during the infl-
.
285
ammatory response. Prostaglandins, Leukotrienes and Essential Fatty Acids 55, 441 – 449. Moncada, S., Gryglewski, R.J., Bunting, S., Vane, J.R., 1976. An enzyme isolated from arteries transforms prostaglandin endoperoxides to an unstable substance that inhibits platelet aggregation. Nature 263, 663 – 665. Pierpoint, W.S. Jr., 1986. Flavonoids in the human diet. In: Cody, V., Middleton, E. Jr., Harborne, J.B. (Eds.), Progress in Clinical and Biological Research. Alan R. Liss, New York. Ranby, M., 1982. Studies in the kinetics of plasminogen activation by tissue plasminogen activator. Biochimica et Biophysica Acta 704, 461 – 469. Ristimaki, A., Ylilorkala, O., Viinikka, L., 1990. Effect of growth factors on human vascular endothelial cell prostacyclin production. Arteriosclerosis 10, 653 – 657. Setty, B.N.Y., Stuart, M.J., Walenga, R.W., 1985. Formation of 11-hydroxyeicosatetraenoic acid and 15-hydroxyeicosatetraenoic acid in human umbilical arteries is catalyzed by clooxygenase. Biochimica et Biophysica Acta 833, 484 – 494. Skeaf, C.M., Holub, B.J., 1985. Altered phospholipid composition of plasma membranes from thrombin-stimulated human platelets. Biochimica et Biophysica Acta 834, 164 – 171. Vane, J.R., Botting, R.M., 1995. Pharmacodynamic profile of prostacyclin. American Journal of Cardiology 75, 3A – 10A. Yamamoto, C., Kaji, T., Sakamoto, M., Kozuka, H., Koizumi, F., 1994. Calcium regulation of tissue plasminogen activator and plasminogen activator inhibitor-1 release from cultured human vascular endothelial cells. Thrombosis Research 74, 163 – 168. Yanagisawa, M., Inoue, M.A., Takuwa, Y., Kobayashi, M., Masaki, T., 1989. The human preproendothelin-1 gene: possible regulation by endothelial phosphoinositide turnover signalling. Journal of Cardiovascular Pharmacology 13, S13 – S18.
Effects of quercetin on the release of endothelin, prostacyclin and tissue plasminogen activator from human endothelial cells in culture Xueying Zhao a, Zhenlun Gu a, Anoja S. Attele b, Chun-Su Yuan b,c,* a Department of Pharmacology, Suzhou Medical College, Suzhou 215007, China Department of Anesthesia and Critical Care, The Uni6ersity of Chicago, 5841 S. Maryland A6enue, MC 4028, Chicago, IL 60637, USA c Committee on Clinical Pharmacology, The Uni6ersity of Chicago, 5841 S. Maryland A6enue, MC 2115, Chicago, IL 60637, USA b
Received 22 December 1998; received in revised form 29 March 1999; accepted 11 April 1999
Abstract Quercetin and related flavonoids are naturally occurring polyphenolic compounds with multiple pharmacological activities. Using cultured human umbilical vein endothelial cells, we investigated the effects of quercetin on endothelin (ET-1) and tissue plasminogen activator (t-PA) release induced by thrombin. We observed that when endothelial cells pretreated with 5 or 50 mM of quercetin were incubated for 4 and 24 h with thrombin, ET-1 concentration-dependently decreased (n=6, PB 0.01, at 4 h IC50 =1.54 mM, at 24 h IC50 = 2.78 mM). Under the same experimental conditions, quercetin significantly increased t-PA (n= 6, PB 0.01, at 4 h EC50 = 0.71 mM and at 24 hrs EC50 = 0.74 mM). In the same preparation, we evaluated prostacyclin (PGI2) release, induced by thrombin activated platelets, as determined by a 6-Keto-PGF1a radioimmunoassay. Following the treatment of cultured endothelial cells with activated platelets, the concentration of 6-Keto-PGF1a was significantly increased (PB 0.01). Quercetin (1, 5, and 20 mM) inhibited PGI2, in a concentration-dependent manner (n= 6, P B0.05). Our data indicate that quercetin modulates the release of ET-1, t-PA, and PGI2 from vascular endothelial cells. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Quercetin; Endothelial cell; Human umbilical vein; Endothelin; Thrombin; Prostacyclin; 6-Keto-PGF1a
1. Introduction Flavonoids are a large group of naturally occurring polyphenolic compounds found in many * Corresponding author. Tel.: +1-773-7021916; fax: +1773-8340601. E-mail address: [email protected] (C.-S. Yuan)
green plants and plant products that make up the human diet (Formica and Regelson, 1995). The largest portion of flavonoid intake comes from cocoa, coffee, tea, and wine (Pierpoint, 1986). Quercetin, a flavonol, has been shown to possess a wide spectrum of pharmacological effects.Quercetin has important palliative effects on the cardiovascular system. It decreases serum
0378-8741/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 9 9 ) 0 0 0 5 5 - 0
280
X. Zhao et al. / Journal of Ethnopharmacology 67 (1999) 279–285
LDL cholesterol concentration (Frankel et al., 1993) by acting as a free radical scavenger and an inhibitor of lipid peroxidation (Afanas’ev et al., 1989). Duarte et al. (1993) reported that quercetin inhibited the contractions induced by noradrenaline, high KCl, and Ca2 + in rat aortic strips. Here, the vasodilatory effect of quercetin was mainly related to the inhibition of protein kinase C. In another study, quercetin caused vasodilation by both endothelium-dependent and endothelium-independent mechanisms (Chen and Pace-Asciak, 1996). Some cardiovascular effects of quercetin implicate the involvement of endproducts of arachidonate metabolism such as prostaglandin eicosaniods (Kalkbrenner et al., 1992). Quercetin also blocked platelet aggregation by inhibiting cyclic phosphodiesterase (Lanza et al., 1987). The endothelium regulates vascular tone by releasing factors involved in relaxation and contraction, in coagulation and thrombus formation, and in growth inhibition and stimulation (Lusher and Tanner, 1993). Endothelial cell dysfunction is believed to be the initial step in the genesis of thrombosis and arteriosclerosis (Furchgott and Vanhoutte, 1989). Despite many previous studies on quercetin, its effects on smooth muscle relaxing and contracting factors, and fibrinolytic mediators released from vascular endothelial cells have not been reported. In this study, we investigated the effect of quercetin on thrombin-induced endothelin (ET-1) and tissue plasminogen activator (t-PA) release, as well as its effect on activated platelet-induced 6-KetoPGF1a release, in cultured endothelial cells from human umbilical cord veins.
2. Materials and methods
2.1. Preparation of endothelial cells and cell culture
buffer (0.14 M NaCl, 0.004 M KCl, 0.001 M phosphate buffer, 0.011 M glucose, at pH 7.4), and held at 4°C until processing. The vein was perfused with 100 ml of cord buffer to wash out the blood and allowed to drain. The cord buffer containing 0.2% collagenase was then infused into the umbilical vein. The umbilical cord was placed in a water bath containing cord buffer and incubated at 37°C. After incubation, the collagenase solution containing the endothelial cells was flushed from the cord vein by perfusion with cord buffer. The effluent was collected, the cells were sedimented and washed. A sterile technique was utilized in all manipulations of the cord. Endothelial cells were cultured in DMEM medium with 20% fetal calf serum, penicillin (100 unit/ml), and streptomycin (100 mg/ml). The dishes were incubated and the cells were harvested with 0.01% EDTA-0.1% collagenase.
2.2. Incubation procedures The confluent monolayers of endothelial cells were used after 7–8 days. The primary culture medium was discarded and dishes were washed twice with PBS. Next, fresh, conditioned, cultured medium without serum was added. The dishes with 2 unit/ml of thrombin were incubated at 37°C for 4 and 24 h. After being sedimented at 1500×g for 15 min, the cell suspension was used to assay the concentrations of ET-1 and t-PA. ET-1 was measured by radioimmunoassay (Fyhrquist et al., 1990) using synthetic ET-1 and ET-1 antiserum generated in rabbits. Measurement of t-PA was done by the method of MacGregor and Prowse (1983). The addition of PBS alone was used as the control. For the test group, quercetin, at different concentrations was added 10 min before the application of thrombin.
2.3. Preparation and acti6ation of platelets Endothelial cells were obtained from human umbilical cord veins by an adaptation following Ristimaki et al. (1990). In brief, the cord was severed from the placenta soon after birth, placed in a sterile container filled with cord
Venous blood with 2% EDTA Na2 as the anticoagulant (9:1, volume:volume) was drawn from healthy adults, and the blood was centrifuged at 250× g for 9 min. The platelet-rich
X. Zhao et al. / Journal of Ethnopharmacology 67 (1999) 279–285
plasma was drawn, and centrifuged at 1200× g for 9 min, to sediment into platelet concentrate. The platelet pellet was gently resuspended in modified Tyrode buffer and adjusted to 2 × 108/ ml, after being washed twice with platelet-washing liquid. The platelets were incubated together with 2 unit/ml of thrombin for 5 min at 37°C to obtain thrombin activated platelets.
2.4. 6 -Keto-PGF1a determination 6-Keto-PGF1a was measured according to a previously described radioimmunoassay (Maclouf, 1982). Briefly, 50, 100, 200, and 300 ml aliquots of unextracted cultured supernatant were incubated in a total volume of 700 ml containing specific antibody, 125I histamine-6-Keto-PGF1a (activities: 12 000 cpm), gamma globulins (5 g/l) in Tris –HCI buffer (0.05 M, pH 7.4), and EDTA (1 M). After 18 h of incubation at 4°C, 25% (wt./vol) polyethylene glycol was added, and the mixture was centrifuged. The supernatant was then removed, and the radioactivity of the precipitate was measured in a gamma counter. The concentration of 6-Keto-PGF1a released during adhesion and the subsequent washes was measured. The sensitivity of the technique permitted the detection of 5 pg of 6-Keto-PGF1a concentration.
281
3. Results
3.1. Effects of quercetin on ET-1 release from endothelial cells When endothelial cells were incubated in the presence of thrombin for 4 h, the concentrations of ET-1 in the conditioned medium increased from 399 8 pg/ml (mean 9 SD) to 136920 pg/ml (n= 6, PB 0.01). Under the same experimental conditions, at 24 h, the concentration of ET-1 in the conditioned medium increased from 354939 to 5459 28 pg/ml (n= 6, PB 0.01). When endothelial cells were pretreated with 5 or 50 mM of quercetin, the concentration of ET-1 in the conditioned medium was significantly decreased (n= 6, PB 0.01). The decrease in ET-1 was concentration-dependent and was observed when quercetin was in contact with endothelial cells for 4 and 24 h (Fig. 1). At 4 h IC50 = 1.54 mM and at 24 h IC50 = 2.78 mM. These results show that quercetin inhibits ET-1 induced by thrombin.
2.5. Drugs Quercetin, collagenase, penicillin, streptomycin, thrombin, EDTA, and DMEM medium were purchased from Sigma Co. (St. Louis, MO). Quercetin was initially dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution, and further dilutions were made in PBS. The final DMSO concentration (B0.25%) did not significantly affect the results.
2.6. Statistical analysis Results were analyzed using Student’s t-test and Mann–Whitney U test to determine if changes between the two conditions were significantly different. Probability levels less than 0.05 were considered significant differences.
Fig. 1. Effect of quercetin on the concentration of ET-1 in the conditioned medium of HUVECs pretreated with thrombin. *P B0.01 compared with saline control group; **PB0.01 compared with thrombin control group. Brackets indicate the mean 9SEM, for all groups n = 6.
282
X. Zhao et al. / Journal of Ethnopharmacology 67 (1999) 279–285
which was determined by radioimmunoassay (Maclouf, 1982). Thrombin activated platelets and endothelial cells were incubated together for 10, 30 and 120 min. As shown in Fig. 3, the concentrations of 6-Keto-PGF1a in the conditioned medium increased from 2.390.2 (mean9 SD), 5.890.8 and 10.5 9 1.9 pg/ml to 8.09 1.1 (n=6, PB0.001), 9.1 9 1.2 (n= 6, PB 0.01) and 21.2 9 1.0 pg/ml (n= 6, PB 0.001), respectively.
3.4. Effects of quercetin on 6 -Keto-PGF1a release from endothelial cells induced by thrombin acti6ated platelets
Fig. 2. Effect of quercetin on the concentration of t-PA in the conditioned medium of HUVECs pretreated with thrombin. *PB0.01 compared with saline control group; **PB 0.01 compared with thrombin control group. Brackets indicate the mean9 SEM, for all groups n= 6.
3.2. Effects of quercetin on t-PA release from endothelial cells
Endothelial cells were incubated together with quercetin 1, 5 or 20 mM for 10 min and further incubated after the addition of activated platelets for 10, 30 or 120 min. Compared with the control group, the concentration of 6-Keto-PGF1a in the conditioned medium, after quercetin pretreatment, decreased significantly (n= 6, PB 0.05) (Fig. 4). Quercetin (20 mM), at 10, 30, and 120 min, had a 4593.1% (mean9SD) maximum inhibition of induced 6-Keto-PGF1a release. At 10 min IC50 = 2.1 mM, at 30 min IC50 = 2.9 mM, and at 120 min IC50 = 0.58 mM. The decrease in 6Keto-PGF1a release was concentration-dependent.
After endothelial cells were treated with thrombin for 4 h, t-PA in the conditioned medium decreased from 4.290.4 pg/ml (mean 9 SD) to 3.1 90.3 pg/ml (n= 6, P B0.01). Under the same experimental conditions, at 24 h, t-PA decreased from 4.39 0.4 pg/ml to 3.490.3 pg/ml (n = 6, P B 0.01). When endothelial cells pretreated with 0.5, 5, or 50 mM of quercetin were incubated for 4 and 24 h, the concentration of t-PA in the conditioned medium was significantly increased (n = 6, P B 0.01) (Fig. 2). At 4 h EC50 =0.71 mM and at 24 h EC50 =0.74 mM. These results show that quercetin stimulates t-PA release, which was inhibited by thrombin.
3.3. Effects of acti6ated platelets on 6 -Keto-PGF1a release from endothelial cells PGI2 is unstable and is rapidly degraded to the stable metabolite 6-Keto-PGF1a, the level of
Fig. 3. Effect of thrombin activated platelets on the release of 6-Keto-PGF1a from the endothelial cells. Test group = Platelet activated group. *P B0.01; **PB 0.001. Brackets indicate the mean 9SEM, for both groups n = 6.
X. Zhao et al. / Journal of Ethnopharmacology 67 (1999) 279–285
Fig. 4. Effect of quercetin on the concentration of 6-KetoPGF1a in the conditioned medium of HUVECs pretreated with thrombin activated platelets. *PB0.05 compared with saline control group; **PB 0.01 compared with saline control group. Brackets indicate the mean 9 SEM, for both groups n= 6.
These results show that quercetin inhibits the release of 6-Keto-PGF1a, which was induced by thrombin activated platelets, from endothelial cells.
4. Discussion An important effect of quercetin on the cardiovascular system is its ability to modulate vasoreactivity of the vascular endothelium (Duarte et al., 1993; Chen and Pace-Asciak, 1996). The results of this study demonstrate that quercetin inhibits ET-1 release from human vascular endothelial cells. Quercetin, at concentrations of 5 and 50 mM was able to reverse the increase in ET-1 induced by thrombin. Thrombin stimulates ET-1 release by increasing the activity of phospholipase C (Yanagisawa et al., 1989). This, in turn, modulates intracellular calcium and protein kinase C which enhances expression of prepro ET-1 mRNA (Yanagisawa et al., 1989). Recent studies have demonstrated that flavonoids may be potent inhibitors of several kinases involved in signal transduction, mainly protein kinase C (PKC) and tyrosine kinases. Moreover, flavones
283
such as quercetin, and flavonols are the most active inhibitors of PKC (Ferriola et al., 1989; Agullo et al., 1997). Nitric oxide (NO) is a feedback inhibitor of ET-1 release (Boulanger and Lusher, 1990) and an increase in NO would also lead to decreased ET-1 in the media of treated cells. In the rat aorta, quercetin, at concentrations between 1×10 − 5 and 6×10 − 5 M, caused vasodilation, which depended on NO release (Chen and Pace-Asciak, 1996). However, at higher concentrations, the vasodilation was independent of NO release. The inhibitory action on ET-1 release by quercetin in our experimental model may be related to one or both of these mechanisms. Our results show that when endothelial cells pretreated with quercetin were incubated with thrombin activated platelets, PGI2 in the medium, concentration-dependently decreased as measured by a 6-Keto-PGF1a assay. Thrombin activated platelets released arachidonic acid (Skeaf and Holub, 1985) and stimulated PGI2 release (Mikkola et al., 1993). Moreover, thrombin stimulated arachidonate metabolism in endothelial cells (Alhenc-Gelas et al., 1982). Arachidonic acid is primarily metabolized via two separate enzymatic pathways, the cyclooxygenase (CO) and lipooygenase pathways (Moncada et al., 1976), both of which are present in vascular endothelial cells (Alhenc-Gelas et al., 1982). PGI2 is the major CO metabolite in blood vessels (Setty et al., 1985). Flavonoids including quercetin have been shown to competitively inhibit CO (Kalkbrenner et al., 1992; Mirzoeva and Calder, 1996). In addition, flavonoids which are potent free radical scavengers inhibited the peroxidation of polyunsaturated fatty acids required for the activation of CO (Halliwell, 1990). It is possible that in our preparation, quercetin concentration and time-dependently decreased PGI2 in the medium by inhibiting CO. The capacity to release PGI2 enables endothelial cells to regulate the state of dilation of arteries and inhibit platelet aggregation (Vane and Botting, 1995). Under physiological conditions, the contribution of PGI2 to endothelium-dependent relaxation may be negligible (Lusher and Tanner, 1993). However, it may be important in atherogenic states where endothelial function is already compromised. A reduction in
284
X. Zhao et al. / Journal of Ethnopharmacology 67 (1999) 279–285
PGI2 would be a disadvantage because it would permit vasoconstrictors such as ET-1 and angiotensin II to determine the capacity of the artery to maintain its lumen in the progression of atherogenic states. Vascular endothelium is strategically located at the interface between blood and smooth muscle. Therefore, it also plays a central role in maintaining blood fluidity. Tissue plasminogen activator (t-PA), produced by vascular endothelial cells, converts plasminogen to plasmin which degrades fibrin (Hoylaerts et al., 1982). Endothelial cells also produce plasminogen activator inhibitor (PAI-1), the main physiological inhibitor of t-PA, and it is believed that the balance between t-PA and PAI-1 is important for the regulation of fibrinolysis (Ranby, 1982). The results of our study show that quercetin reversed the significant reduction of t-PA in endothelial cells induced by thrombin at 4 and 24 h. Previous studies have shown that thrombin, at physiological concentrations, increased both PAI-1 and t-PA production (Yamamoto et al., 1994; Kimura et al., 1997). However, the net effect of thrombin was to increase the activity of PAI-1 and, therefore, decrease fibrinolytic activity of human endothelial cells (Gelehrter and Sznycer-Laszuk, 1986). Flavonoids are potent inhibitors of PAI-1 production in cultured HUVECs (Kimura et al., 1997). Flavonoids such as baicalein may inhibit PAI-1 by a direct inhibition of PKC activity or a reduction in [Ca2 + ]I elevation (Kimura et al., 1997). In our experimental model, quercetin may inhibit PAI-1 by a similar mechanism and increase the fibrinolytic activity of the vascular endothelium. In summary, this study presents data on the effects of quercetin on three mediators of endothelial cell function. Our data showed that quercetin suppressed ET-1 and PGI2 and increased t-PA from stimulated cultured human endothelial cells. Suppression of ET-1 production may partly explain the vasodilating action of quercetin. By stimulating t-PA release, quercetin may increase the fibrinolytic activity in endothelial cells. Further studies are necessary to reveal the mechanisms of action behind the observed effects.
Acknowledgements This work was support in part by the Tang Family Foundation. The authors wish to thank Ms. Tasha Lowell for her technical assistance.
References Afanas’ev, I.B., Dorozhko, A.I., Brodskii, A.V., Korstyuk, V.A., Potapovitch, A.I., 1989. Chelating and free radical scavenging mechanism of inhibitory action of rutin and quercetin in lipid peroxidation. Biochemical Pharmacology 38, 1763 – 1768. Agullo, G., Gamet-Payrastre, L., Manenti, S., Viala, C., Remesy, C., Chap, H., Payrastre, B., 1997. Relationship between flavonoid structure and inhibition of phosphatidylinositol 3-kinase: a comparison with tyrosine kinase and protein kinase C inhibition. Biochemical Pharmacology 53, 1649 – 1657. Alhenc-Gelas, F., Tsai, S.J., Callahan, K.S., Campbell, W.B., 1982. Stimulation of prostaglandin formation by vasoactive mediators in cultured human endothelial cells. Prostaglandins 24, 723 – 742. Boulanger, C., Lusher, T.F., 1990. Release of endothelin from the porcine aorta: inhibition by endothelium-derived nitric oxide. Journal of Clinical Investigation 85, 587 – 590. Chen, C.K., Pace-Asciak, C.R., 1996. Vasorelaxing activity of resveratrol and quercetin in isolated rat aorta. General Pharmacology 27, 363 – 366. Duarte, J., Perez-Vizcaino, F., Zarzuelo, A., Jimenez, J., Tamargo, J., 1993. Vasodilator effects of quercetin in isolated rat vascular smooth muscle. European Journal of Pharmacology 239, 1 – 7. Ferriola, P.C., Cody, V., Middleton, E. Jr., 1989. Protein kinase C inhibition by plant flavonoids. Kinetic mechanisms and structure – activity relationships. Biochemical Pharmacology 38, 1617 – 1624. Formica, J.V., Regelson, W., 1995. Review of the biology of quercetin and related bioflavonoids. Food and Chemical Toxicology. 33, 1061 – 1080. Frankel, E.N., Kanner, J., German, J.B., Parks, E., Kinsella, J.E., 1993. Inhibition of oxidation of human low-density lipoprotein by phenolic substances in red wine. Lancet 341, 454 – 457. Furchgott, R.F., Vanhoutte, P.M., 1989. Endothelium-derived relaxing and contracting factors. FASEB Journal 3, 2007 – 2018. Fyhrquist, F., Saijonmaa, O., Metsarinne, K., Tikkanen, I., Rosenlof, K., Timmanen, T., 1990. Raised plasma endothelin-1 concentration following cold pressor test. Biochemical and Biophysical Research Communications 169, 217 – 221. Gelehrter, T.D., Sznycer-Laszuk, R., 1986. Thrombin Induction of Plasminogen Activator – Inhibitor in Cultured Hu-
X. Zhao et al. / Journal of Ethnopharmacology 67 (1999) 279–285 man Endothelial Cells. Journal of Clinical Investigation 77, 165 – 169. Halliwell, B., 1990. How to characterize a biological antioxidant. Free Radical Research Communications 9, 1–32. Hoylaerts, M., Rijken, D.C., Lijnen, H.R., Collen, D., 1982. Kinetics of the activation of plasminogen by human tissue plasminogen activator: Role of fibrin. Journal of Biological Chemistry 257, 2912 –2919. Kalkbrenner, F., Wurin, G., von Bruchhausen, F., 1992. In vitro inhibition and stimulation of purified prostaglandin endoperoxide synthase of flavonoids: structure–activity relationship. Pharmacology 44, 1–12. Kimura, Y., Yokoi, K., Matsushita, N., Okuda, H., 1997. Effects of flavonoids isolated from Scutellariae radix on the production of tissue-type plasminogen activator and plasminogen activator inhibitor-1 induced by thrombin and thrombin receptor agonist peptide in cultured human umbilical vein endothelial cells. Journal of Pharmacy and Pharmacology 49, 816–822. Lanza, F., Beretz, A., Stierle, A., Corre, G., Cazenave, J.P., 1987. Cyclic nucleotide phosphodiesterase inhibitors prevent aggregation of human platelets by raising cyclic AMP and reducing cytoplasmic free calcium mobilization. Thrombosis Research 45, 477–484. Lusher, T.F., Tanner, F.C., 1993. Endothelial Regulation of Vascular Tone and Growth. American Journal of Hypertension 6, 283S – 293S. MacGregor, I.R., Prowse, C.V., 1983. Tissue plasminogen activator in human plasma measured by radioimmunoassay. Thrombosis Research 31, 461–474. Maclouf, J., 1982. A radioimmunoassay for 6-Keto-PGF1a. Methods in Enzymology 86, 273–286. Mikkola, T., Ristimaki, A., Viinikka, L., Ylikorkala, O., 1993. Human serum, plasma, and platelets stimulate prostacyclin and endothelin-1 synthesis in human vascular endothelial cells. Life Sciences 53, 283–289. Mirzoeva, O.K., Calder, P.C., 1996. The effect of propolis and its components on eicosanoid production during the infl-
.
285
ammatory response. Prostaglandins, Leukotrienes and Essential Fatty Acids 55, 441 – 449. Moncada, S., Gryglewski, R.J., Bunting, S., Vane, J.R., 1976. An enzyme isolated from arteries transforms prostaglandin endoperoxides to an unstable substance that inhibits platelet aggregation. Nature 263, 663 – 665. Pierpoint, W.S. Jr., 1986. Flavonoids in the human diet. In: Cody, V., Middleton, E. Jr., Harborne, J.B. (Eds.), Progress in Clinical and Biological Research. Alan R. Liss, New York. Ranby, M., 1982. Studies in the kinetics of plasminogen activation by tissue plasminogen activator. Biochimica et Biophysica Acta 704, 461 – 469. Ristimaki, A., Ylilorkala, O., Viinikka, L., 1990. Effect of growth factors on human vascular endothelial cell prostacyclin production. Arteriosclerosis 10, 653 – 657. Setty, B.N.Y., Stuart, M.J., Walenga, R.W., 1985. Formation of 11-hydroxyeicosatetraenoic acid and 15-hydroxyeicosatetraenoic acid in human umbilical arteries is catalyzed by clooxygenase. Biochimica et Biophysica Acta 833, 484 – 494. Skeaf, C.M., Holub, B.J., 1985. Altered phospholipid composition of plasma membranes from thrombin-stimulated human platelets. Biochimica et Biophysica Acta 834, 164 – 171. Vane, J.R., Botting, R.M., 1995. Pharmacodynamic profile of prostacyclin. American Journal of Cardiology 75, 3A – 10A. Yamamoto, C., Kaji, T., Sakamoto, M., Kozuka, H., Koizumi, F., 1994. Calcium regulation of tissue plasminogen activator and plasminogen activator inhibitor-1 release from cultured human vascular endothelial cells. Thrombosis Research 74, 163 – 168. Yanagisawa, M., Inoue, M.A., Takuwa, Y., Kobayashi, M., Masaki, T., 1989. The human preproendothelin-1 gene: possible regulation by endothelial phosphoinositide turnover signalling. Journal of Cardiovascular Pharmacology 13, S13 – S18.