Key points are not available for this paper at this time.
Epidemiological studies suggest that tea catechins may reduce the risk of cardiovascular disease, but the mechanisms of benefit have not been determined. The objective of the present study was to investigate the effects of epigallocatechin-3-gallate (EGCG), the major constituent of green tea, on vasorelaxation and on eNOS expression and activity in endothelial cells. EGCG (1-50 μm) induced dose-dependent vasodilation in rat aortic rings. Vasodilation was abolished by pretreatment with NG-nitro l-arginine methyl ester. In bovine aortic endothelial cells, EGCG increased endothelial nitric oxide (eNOS) activity dose-dependently after 15 min. Treatment with EGCG induced a sustained activation of Akt, ERK1/2, and eNOS Ser1179 phosphorylation. Inhibition of extracellular signal-regulated kinase (ERK)1/2 had no influence on eNOS activity or Ser1179 phosphorylation. Simultaneous treatment of cells with selective inhibitors for cAMP-dependent protein kinase (PKA) and Akt completely prevented the increase in eNOS activity by EGCG after 15 min, indicating that both kinases act in concert. Specific phosphatidylinositol-3-OH-kinase inhibitors yielded identical results. Akt inhibition prevented eNOS Ser1179 phosphorylation, whereas inhibition of PKA did not influence Akt and eNOS Ser1179 phosphorylation. Pretreatment of endothelial cells with EGCG for 4 h markedly enhanced the increase in eNOS activity stimulated by Ca-ionomycin, suggesting that Akt accounts for prolonged eNOS activation. Treatment of cells for 72 h with EGCG did not change eNOS protein levels. Our results indicate that EGCG-induced endothelium-dependent vasodilation is primarily based on rapid activation of eNOS by a phosphatidylinositol 3-kinase-, PKA-, and Akt-dependent increase in eNOS activity, independently of an altered eNOS protein content. Epidemiological studies suggest that tea catechins may reduce the risk of cardiovascular disease, but the mechanisms of benefit have not been determined. The objective of the present study was to investigate the effects of epigallocatechin-3-gallate (EGCG), the major constituent of green tea, on vasorelaxation and on eNOS expression and activity in endothelial cells. EGCG (1-50 μm) induced dose-dependent vasodilation in rat aortic rings. Vasodilation was abolished by pretreatment with NG-nitro l-arginine methyl ester. In bovine aortic endothelial cells, EGCG increased endothelial nitric oxide (eNOS) activity dose-dependently after 15 min. Treatment with EGCG induced a sustained activation of Akt, ERK1/2, and eNOS Ser1179 phosphorylation. Inhibition of extracellular signal-regulated kinase (ERK)1/2 had no influence on eNOS activity or Ser1179 phosphorylation. Simultaneous treatment of cells with selective inhibitors for cAMP-dependent protein kinase (PKA) and Akt completely prevented the increase in eNOS activity by EGCG after 15 min, indicating that both kinases act in concert. Specific phosphatidylinositol-3-OH-kinase inhibitors yielded identical results. Akt inhibition prevented eNOS Ser1179 phosphorylation, whereas inhibition of PKA did not influence Akt and eNOS Ser1179 phosphorylation. Pretreatment of endothelial cells with EGCG for 4 h markedly enhanced the increase in eNOS activity stimulated by Ca-ionomycin, suggesting that Akt accounts for prolonged eNOS activation. Treatment of cells for 72 h with EGCG did not change eNOS protein levels. Our results indicate that EGCG-induced endothelium-dependent vasodilation is primarily based on rapid activation of eNOS by a phosphatidylinositol 3-kinase-, PKA-, and Akt-dependent increase in eNOS activity, independently of an altered eNOS protein content. Epidemiological evidence suggests that chronic diseases such as coronary heart disease and stroke have a lower incidence in countries with a high intake of tea. Numerous studies have demonstrated that tea drinking lowers the risk of heart disease and reduces cardiovascular mortality (1Nakachi K. Matsuyama S. Miyake S. Suganuma M. Imai K. Biofactors. 2000; 13: 49-54Crossref PubMed Scopus (378) Google Scholar, 2Arts I.C. Hollman P.C. Feskens E.J. Bueno de Mesquita H.B. Kromhout D. Am. J. Clin. Nutr. 2001; 74: 227-232Crossref PubMed Scopus (314) Google Scholar, 3Mukamal K.J. Maclure M. Muller J.E. Sherwood J.B. Mittleman M.A. Circulation. 2002; 105: 2476-2481Crossref PubMed Scopus (185) Google Scholar); others, however, do not support these findings (4Rimm E.B. Katan M.B. Ascherio A. Stampfer M.J. Willett W.C. Ann. Intern. Med. 1996; 125: 384-389Crossref PubMed Scopus (502) Google Scholar, 5Woodward M. Tunstall-Pedoe H. J. Epidemiol. Community Health. 1999; 53: 481-487Crossref PubMed Scopus (165) Google Scholar). In addition, tea consumption has been inversely associated with the development and progression of atherosclerosis (6Geleijnse J.M. Launer L.J. Hofman A. Pols H.A. Witteman J.C. Arch. Intern. Med. 1999; 159: 2170-2174Crossref PubMed Scopus (216) Google Scholar). Tea is second only to water in worldwide usage and contains important phytochemicals, the flavonoids. Tea flavonoids include catechins, quercetin, kaempferol, and numerous other polyphenols (7Kris-Etherton P.M. Keen C.L. Curr. Opin. Lipidol. 2002; 13: 41-49Crossref PubMed Scopus (360) Google Scholar). Catechins are the major components of tea; in green tea, they represent > 80% of flavonoids, and in black tea, they represent only 20-30% (8Balentine D.A. Wiseman S.A. Bouwens L.C. Crit. Rev. Food Sci. Nutr. 1997; 37: 693-704Crossref PubMed Scopus (955) Google Scholar). The suggested mechanisms involved in the prevention of coronary heart disease by green and black tea beverages involve potent antioxidative, antithrombogenic, and antiinflammatory properties of flavonoids, primarily of the major and most active catechin derivative, epigallocatechin-3-gallate (EGCG) 1The abbreviations used are: EGCGepigallocatechin-3-gallateeNOSendothelial nitric oxide synthasePKAcAMP-dependent protein kinasePKIPKA inhibitorBAECbovine aortic endothelial cellsPI3Kphosphatidylinositol-3-OH-kinasel-NAMENG-nitro l-arginine methyl esterERKextracellular signal-regulated kinase. (9Vinson J.A. Dabbagh Y.A. FEBS Lett. 1998; 433: 44-46Crossref PubMed Scopus (107) Google Scholar). In addition, there is experimental and clinical evidence that tea extracts also improve endothelial function (10Huang Y. Chan N.W.K. Lau C.W. Yao X.Q. Chan F.L. Chen Z.Y. Biochim. Biophys. Acta. 1999; 1427: 322-328Crossref PubMed Scopus (91) Google Scholar, 11Duffy S.J. Keaney Jr., J.F. Holbrook M. Gokce N. Swerdloff P.L. Frei B. Vita J.A. Circulation. 2001; 104: 151-156Crossref PubMed Scopus (493) Google Scholar). Short- and long-term tea consumption has been shown to reverse endothelial dysfunction in patients with documented coronary heart disease (11Duffy S.J. Keaney Jr., J.F. Holbrook M. Gokce N. Swerdloff P.L. Frei B. Vita J.A. Circulation. 2001; 104: 151-156Crossref PubMed Scopus (493) Google Scholar). Moreover, purified epicatechins from tea evoke endothelium-dependent vasorelaxation in precontracted rat aortic rings by means of nitric oxide (NO) release from the endothelium (10Huang Y. Chan N.W.K. Lau C.W. Yao X.Q. Chan F.L. Chen Z.Y. Biochim. Biophys. Acta. 1999; 1427: 322-328Crossref PubMed Scopus (91) Google Scholar). Although tea catechins may improve endothelial function, the underlying molecular mechanisms are unknown. epigallocatechin-3-gallate endothelial nitric oxide synthase cAMP-dependent protein kinase PKA inhibitor bovine aortic endothelial cells phosphatidylinositol-3-OH-kinase NG-nitro l-arginine methyl ester extracellular signal-regulated kinase. Endothelial-dependent NO is produced by endothelial nitric oxide synthase (eNOS). This key enzyme in cardiovascular homeostasis is regulated posttranslationally by means of rapid modulation of eNOS activity by increase in intracellular Ca2+ and/or phosphorylation and on the transcriptional level by changes in eNOS gene expression. In the present study, we show the effects of a natural plant-derived catechin, EGCG, on eNOS expression and activity in endothelial cells, and we elucidate the molecular mechanisms involved. Cell Culture—Bovine aortic endothelial cells (BAEC) were purchased from Clonetics and maintained in microvascular endothelial cell growth medium (EGM-MV), supplemented with 5% fetal bovine serum, 10 pg/ml epidermal growth factor, 1 μg/ml hydrocortisone, 12 μg/ml bovine brain extract, and 0.1% gentamicin. For experiments, the cells were seeded onto 6-cm dishes and treated with EGCG (dissolved in water), LY294002, wortmannin, Ca-ionomycin from Sigma (Deisenhofen, Germany), the cell-permeable myristoylated 14-22 amide peptide PKI, H89, and PD98059 from Calbiochem, and SH-5 from Alexis (San Diego, CA) at confluency. Cells were used between passages 4 and 10 for all experiments. For Western blots with phospho-specific antibodies and the cAMP-dependent protein kinase (PKA) assays, cells were starved in medium with 1% fetal bovine serum for 20 h. Western Blot Analysis—After treatment, cells were washed twice with phosphate-buffered saline and lysed in extraction buffer containing 50 mm Tris/HCl (pH 7.4), 154 mm KCl, 5 mm glucose, 0.5 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 2 mm dithiothreitol, and 1% Triton X-100. For Western blots with phospho-specific antibodies, cells were lysed in buffer containing 20 mm Hepes (pH 7.9), 100 mm NaCl, 1 mm Na3VO4,4 mm sodium pyrophosphate, 10 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 10 mm NaF, 0.1 mm okadaic acid, and 1% Triton X-100. Total protein (50 μg per lane) was subjected to SDS-PAGE, and membranes were probed with anti-phospho-eNOS (Ser1177), anti-phospho-Akt (Ser473), and anti-phospho-ERK1/2 (Thr202/Tyr204) from Cell Signaling Technology (Frankfurt, Germany), anti-Akt antibody from Santa Cruz Biotechnology (Santa Cruz, CA), anti-eNOS from BD Transduction Laboratories (Heidelberg, Germany), secondary anti-mouse from Santa Cruz Biotechnology, and anti-rabbit from Dianova (Hamburg, Germany). Bands were visualized by using either 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium (Sigma) or the ECL detection system (Amersham, Freiburg, Germany). Measurement of eNOS Activity in Intact Cells—eNOS activity was assessed by formation of l-3Hcitrulline from l-3Harginine after separation of the amino acids by cation-exchange chromatography. Endothelial cells were washed twice with phosphate-buffered saline and incubated for 30 min in Hepes buffer, pH 7.4, containing 145 mm NaCl, 5 mm KCl, 1 mm MgSO4, 10 mm Hepes-Na, 10 mm glucose, and 1 mm CaCl2. Stimulation was initiated by the addition of agonists, 10 μm cold l-arginine, and 3 μCi/ml l-3Harginine. Where indicated, protein kinase inhibitors were present 30 min before and during agonist stimulation. After 15 min, the reaction was terminated with ice-cold stop solution containing 5 mml-arginine and 4 mm EDTA. Cells were denatured with 96% ethanol and, after evaporation, the soluble cellular components were extracted with 20 mm Hepes-Na, pH 5.5. l-3Hcitrulline was separated from l-3Harginine by Dowex chromatography, and l-3Hcitrulline formation was quantified by liquid scintillation counting. In preincubation experiments of the cells with EGCG for 4 h, no further EGCG was added after replacing the medium with Hepes buffer. PKA Assay—Measurement of PKA activity was performed with a commercial PKA assay kit (PepTag assay kit, Promega, Madison, WI). Cells were serum-starved in medium with 1% fetal bovine serum for 20 h and stimulated for 15 min with 100 μm EGCG, or EGCG and 20 μm PKI, to demonstrate the specificity of the reaction. After stimulation, cells were lysed in extraction buffer containing 20 mm Tris (pH 7.4), 25 μg/ml aprotinin, 1 mm sodium pyrophosphate, and 1 mm EGTA. We used 5 μg of cell homogenate in the assay. The reactions were performed according to the protocol of the manufacturer. After phosphorylation, the PKA-specific fluorescent peptide substrate kemptide alters the net charge from +1 to -1, and the phosphorylated and non-phosphorylated forms can be separated on agarose gels. Vasorelaxation Studies—Thoracic aortas from male Wistar rats were rapidly excised, cleaned of connective tissue, and cut into rings 2-3 mm in length for organ-chamber experiments. The rings were then mounted on platinum hooks in 10-ml jacketed organ baths containing modified Krebs-Henseleit solution (144 mm NaCl, 5.9 mm KCl, 1.6 mm CaCl2, 1.2 mm MgSO4, 1.2 mm KH2PO4, 25 mm NaHCO3, and 11.1 mm d-glucose) and 1 μm diclofenac. Tension was gradually adjusted to 2 g over 1 h. The solution in the bath was maintained at 37 °C with a gas mixture of 5% CO2 and 95% O2. After equilibration and submaximal precontraction with 0.05 μm phenylephrine, relaxation to increasing concentrations (0.1 to 50 μm) of EGCG was performed to obtain cumulative concentration-response curves. Selected studies were conducted in rings treated with 1 mml-NAME before phenylephrine exposure and in rings in which the endothelium was removed by gently rubbing with dental floss. Removal of the endothelium was confirmed by the inhibition of acetylcholine-dependent vasodilation after contraction with phenylephrine. Statistical Analysis—All values are expressed as mean ± S.E. compared with controls. Vasorelaxation is expressed as a percentage of precontraction with phenylephrine. Statistical analysis was performed by use of ANOVA, Mann-Whitney, or Student's t test where appropriate. A level of p < 0.05 was considered significant in all statistical tests. To examine whether EGCG could induce vasorelaxation, we exposed phenylephrine-precontracted rat aortic rings to cumulative doses of EGCG dose-dependent vasorelaxation, at 1 μm The of a is shown that a contraction after EGCG by sustained The of experiments is in EGCG produced which at 1 μm and statistical at 10 Pretreatment with 1 mml-NAME abolished the vasodilation induced by μm of EGCG, whereas at 50 μm EGCG, had no Removal of the endothelium yielded identical results not indicating that of rat aortic rings induced by EGCG is to of nitric To test we incubated with increasing concentrations of EGCG, and eNOS activity in cells. EGCG a dose-dependent increase in eNOS activity after 15 min 100 μm EGCG, we a in eNOS To investigate whether EGCG can also influence eNOS protein we incubated with increasing concentrations of EGCG for 72 h. was no change in eNOS protein of the which that EGCG has no on the level of eNOS protein has been shown J. H. Y. 2002; Google that EGCG can at and that can to eNOS activation and vasorelaxation in aortic rings Chen K. Keaney Jr., J.F. J. 2002; PubMed Scopus Google Scholar). To a of to the effects of EGCG, we performed the vasorelaxation studies and eNOS activity in the of with had no influence on the indicating that the effects of EGCG are not to the of not eNOS is posttranslationally by phosphorylation. To for kinases involved in the of eNOS activity by EGCG, we assessed Akt and phosphorylation. EGCG 100 μm) induced phosphorylation of Akt and (Thr202/Tyr204) after min the activation of was and to after h, phosphorylation of Akt for at h. Akt can eNOS S. B. 1999; PubMed Scopus Google we the phosphorylation for Ser1179 of bovine eNOS Ser1179 phosphorylation a as did Akt activation at 15 and for at h The that a increase in eNOS enzyme activity by EGCG is after 15 min, whereas there was only a activation of Akt and eNOS Ser1179 phosphorylation at suggests that kinase may be involved in the rapid EGCG-induced eNOS activation. has been shown that PKA can eNOS by phosphorylation M.B. Chen H. M.A. J. 2002; PubMed Scopus Google Scholar, N. K. J. H. J. 2002; PubMed Scopus Google Scholar). Pretreatment of cells with a PKA the increase in eNOS activity after 15 min results were with PKA not To further demonstrate the of PKA in the rapid activation of eNOS by EGCG, we PKA activity in after treatment of cells with EGCG at 100 μm to an activation of PKA 15 min, which was abolished by the PKA inhibitor of kinase inhibitors on eNOS activity and phosphorylation in cells were or not with the PKA inhibitor μm) for 30 min and stimulated with 100 μm EGCG for 15 min. eNOS activity in cells was by of l-3Hcitrulline from l-3Harginine. p < of PKA activity in Cells were or not with μm) for 30 min and stimulated with 100 μm EGCG for 15 min. and indicate the phosphorylated non-phosphorylated forms of the PKA-specific fluorescent peptide substrate kemptide after separation on agarose gels. A from experiments is were or not with the inhibitor μm) or the inhibitor PD98059 μm) for 30 min. After with 100 μm EGCG for 15 min, eNOS activity in cells was p < cells were stimulated for with 100 μm EGCG, and Western blots were probed with phospho-specific antibodies ERK1/2, Akt, and eNOS antibody as for are from shown the of both kinases in rapid and sustained eNOS we whether are involved. Akt can be by means of phosphatidylinositol-3-OH-kinase S. B. 1999; PubMed Scopus Google Scholar); a for eNOS phosphorylation in was N. K. J. H. J. 2002; PubMed Scopus Google Scholar). To investigate whether is involved in rapid eNOS were with μm) before with EGCG μm) for 15 min. shown in inhibition of completely prevented EGCG-induced eNOS activation. inhibitor of yielded identical results not the of for eNOS activation by To a of to the increase in eNOS activation by the tea catechin, cells were with PD98059 an inhibitor of PD98059 had no influence on EGCG-induced increase in eNOS activity after 15 min EGCG-induced eNOS Ser1179 phosphorylation after 1 h was abolished by LY294002, whereas PD98059 was which that the activation not to either rapid 15 or sustained 1 eNOS activation. To examine whether the activation of PKA of Akt we Akt and eNOS phosphorylation after inhibition of Pretreatment of with had no on EGCG-induced Akt and eNOS Ser1179 phosphorylation after suggesting that both kinases act in SH-5 an inhibitor of Akt H. J. J. Am. 125: PubMed Scopus Google abolished both Akt and eNOS phosphorylation that Akt is for the sustained eNOS Ser1179 phosphorylation by In with the treatment of cells with and SH-5 completely prevented the increase in eNOS activity after 15 min, which that both kinases act in concert. SH-5 on only to inhibition To whether prolonged eNOS phosphorylation to a active we incubated with 100 μm EGCG for 4 h and and stimulated eNOS activity in cells. EGCG was present before and during the assay Although eNOS activity in the cells was compared with cells, the with Ca-ionomycin yielded an increase in eNOS activity in the cells The present study that endothelium-dependent vasorelaxation induced by the catechin EGCG in to a dose-dependent activation of eNOS in endothelial cells. The increase in eNOS activity is a suggesting of eNOS as an underlying of endothelial function by flavonoids may be an important by which tea reduces cardiovascular risk (11Duffy S.J. Keaney Jr., J.F. Holbrook M. Gokce N. Swerdloff P.L. Frei B. Vita J.A. Circulation. 2001; 104: 151-156Crossref PubMed Scopus (493) Google Scholar). The endothelium a in and is that nitric oxide activity is associated with the progression of evidence has demonstrated that and long-term tea consumption endothelial dysfunction in patients with documented coronary heart disease, a for the of tea in these Our studies and experiments clinical evidence by suggesting that purified epicatechins from tea evoke endothelium-dependent vasorelaxation in precontracted rat by means of NO release from the endothelium (10Huang Y. Chan N.W.K. Lau C.W. Yao X.Q. Chan F.L. Chen Z.Y. Biochim. Biophys. Acta. 1999; 1427: 322-328Crossref PubMed Scopus (91) Google Scholar). experimental on the effects of catechins are not have no on A. A. J. J. Nutr. 1998; PubMed Scopus Google as as the of inhibition of endothelium-dependent vasodilation Y. H. H. Sci. 2002; PubMed Scopus Google Scholar). in experimental may these A experimental study Clin. PubMed Scopus Google has demonstrated that epicatechins on properties may the effects in studies by sustained in rat aortic rings after EGCG In with EGCG-induced endothelium-dependent vasodilation in the rat which could be to 25 μm of EGCG by pretreatment with we a dose-dependent increase in eNOS activity in after 15 min. EGCG can J. H. Y. 2002; Google Scholar, Clin. PubMed Scopus Google and can to eNOS activation and vasorelaxation in aortic rings Chen K. Keaney Jr., J.F. J. 2002; PubMed Scopus Google Scholar). experiments in the of an of to the results. To examine mechanisms to eNOS activation in after treatment with EGCG, we the of protein kinases by Our study that ERK1/2, Akt, and PKA are involved in the cellular after EGCG stimulation. A study has shown that can induce eNOS activation M.J. J. PubMed Scopus Google Scholar). experiments indicate that is not involved in either rapid or sustained EGCG-induced eNOS activation in Treatment of cells with protein kinase inhibitors a of PKA and Akt in eNOS activation. of Akt and eNOS in and in long-term activation of Inhibition of Akt prevented phosphorylation of eNOS at in with the of Akt in eNOS phosphorylation S. B. 1999; PubMed Scopus Google Scholar). eNOS Ser1179 phosphorylation to enzyme as by increased Ca-ionomycin after treatment of cells with EGCG for 4 h. Inhibition of PKA the in eNOS activity after 15 min. The findings the of PKA in the rapid activation of eNOS enzyme activity by means of inhibitors of PKA and at rapid eNOS activation by EGCG after 15 activation of PKA 15 min after treatment of with EGCG, as shown in the PKA and inhibitors of completely prevented of eNOS activity after 15 min, whereas there was only a phosphorylation of eNOS Ser1179 at A N. K. J. H. J. 2002; PubMed Scopus Google has demonstrated that PKA eNOS Ser1179 phosphorylation in to and, in study, was shown that PKA also and eNOS at M.B. Chen H. M.A. J. 2002; PubMed Scopus Google Scholar). Inhibition of PKA did not Akt and eNOS Ser1179 phosphorylation, which that PKA did not the phosphorylation of in also suggests that these kinases do not on and that there is no between these Simultaneous of both kinases abolished the in eNOS activity after 15 min, which a of PKA and prevention of the rapid increase in eNOS activity and Akt-dependent eNOS phosphorylation by inhibition of suggests that and is for activation of both The phosphorylation and activation of eNOS by means of an in endothelial cells has only been N. K. J. H. J. 2002; PubMed Scopus Google Scholar). no into the of PKA activation by could be of PKA activation by could involve the of PKA with protein kinase A after phosphorylation. For after phosphorylation of the of PKA by the kinase changes by with protein kinase A protein H.B. K. J. Cell Sci. 2001; Google Scholar). of the of PKA by or other kinases could cellular and the PKA in to the eNOS in and of the of PKA with in the intracellular for eNOS protein has been demonstrated B. J. 1999; PubMed Scopus Google Scholar). at present there is no as to EGCG could PKA by means of a studies are to The results of study show that EGCG eNOS by and of and Akt-dependent phosphorylation. Treatment of with increasing concentrations of EGCG for 72 h did not change eNOS protein expression. In the present study, we the experimental evidence that the tea EGCG rapidly the key enzyme of homeostasis eNOS and endothelium-dependent to be whether these in may be to the in catechin concentrations after of green and black tea in the of of μm Chen M.J. D. Epidemiol. 1998; Google Scholar, K. A. K. M. J. Nutr. Sci. 2001; PubMed Scopus Google Scholar). Although the concentrations of EGCG in cell were we a vasodilation in rat aortic rings at 1 The effects of catechins in may be to the of endothelial function and atherosclerosis in In we that the plant-derived catechin EGCG to endothelium-dependent vasorelaxation and as a natural of eNOS in endothelial cells by increasing protein phosphorylation. The of endothelial function may in the effects of flavonoids on cardiovascular Our may in the development of natural to improve endothelial function and cardiovascular We are to and N. for
Lorenz et al. (Sun,) studied this question.