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Journal of Cardiovascular Pharmacology:
doi: 10.1097/01.fjc.0000211754.54691.f3
Original Articles

Statins Blunt Thrombin-induced Down-regulation of Endothelial Nitric Oxide Synthase Expression in Human Endothelial Cells

Eto, Masato MD* †; Rathgeb, Lisa; Cosentino, Francesco MD* † ‡; Kozai, Toshiyuki MD* †; Lüscher, Thomas F. MD, FRCP* †

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Author Information

*Cardiology, Cardiovascular Centre, University Hospital

Cardiovascular Research, Institute of Physiology, University of Zürich, Zürich, Switzerland

Cardiology, The 2nd Faculty of Medicine, “La Sapienza” University, Rome, Italy

Supported by the Swiss National Research Foundation (Grant Nr.32-67202.01), the Swiss Heart Foundation and the Roche Research Foundation. Dr Eto was supported by an educational grant from Merck. Dr Kozai was supported by an educational grant from Bayer Pharmaceuticals.

Reprints: Thomas F. Lüscher, MD, FRCP, Cardiovascular Center, University Hospital, Rämistrasse 100, CH-8091 Zürich, Switzerland (e-mail: cardiotfl@gmd.ch)

Received for publication February 3, 2004; accepted December 24, 2004

First two authors contributed equally to this study.

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Abstract

Thrombin plays a pivotal role in the pathophysiology of acute coronary syndromes by mediating thrombus formation and endothelium-dependent vasomotor dysfunction. In human endothelial cells, prolonged incubation with thrombin down-regulates endothelial nitric oxide synthase (eNOS) expression via activation of Rho. Statins are effective in patients with acute coronary syndromes. These beneficial effects are attributed to their pleiotropic effects and also to an improved lipid profile. We hypothesized that statins may prevent the down-regulation of eNOS induced by thrombin in human endothelial cells. Human umbilical vein endothelial cells were used. Expression and activity of eNOS protein were evaluated by Western blotting and L-citrulline assay, respectively. Rho A membrane translocation was evaluated by Wesern blotting after fractionation. Stimulation of human umbilical vein endothelial cells with thrombin (4 U/mL, 24 h) significantly decreased eNOS expression. The addition of simvastatin significantly prevented thrombin-induced down-regulation of eNOS expression in a concentration-dependent manner (100 nmol/L to 10 μmol/L). Cerivastatin (10 μmol/L) also reversed the down-regulation of eNOS by thrombin. Both simvastatin and cerivastatin-blocked thrombin-induced decrease in NOS activity. Stimulation with thrombin (4 U/mL, 10 min) significantly increased the membrane translocation of Rho A. Simvastatin (10 μmol/L) and cerivastatin (10 μmol/L) significantly decreased thrombin-induced membrane translocation of Rho A. Therefore, statins blunt thrombin-induced down-regulation of eNOS expression in human endothelial cells. This finding provides a novel mechanism of the pleiotropic effects of statins, which may be beneficial for patients with acute coronary syndromes.

Acute coronary syndromes occur owing to plaque disruption, superficial erosion, and endothelial dysfunction, followed by thrombus formation.1,2 Thrombin plays a pivotal role in thrombus formation, because it cleaves fibrinogen to fibrin, induces platelet aggregation, and up-regulates tissue factor expression.3

In addition to thrombus formation, impaired vasomotor function also contributes to the pathophysiology of acute coronary syndromes, as it further impairs coronary blood flow.4,5 In endothelial cells, thrombin increases the expression and release of the potent vasoconstrictor peptide endothelin-1,6 which is up-regulated in patients with acute coronary syndromes.7 Although the immediate vascular effect of thrombin is nitric oxide (NO)-mediated endothelium-dependent vasorelaxation,8 we recently reported that prolonged incubation with thrombin down-regulates endothelial NO synthase (eNOS) expression in human endothelial cells via activation of the small G protein Rho.9 This effect may further impair coronary blood flow.

Statins are highly effective in reducing cardiovascular events, even in patients with acute coronary syndromes.10,11 It is likely that these beneficial effects are due to their pleiotropic effects and also to an improved lipid profile.12 Statins inhibit cholesterol synthesis by inhibiting the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA)-mevalonate-cholesterol pathway.13 Activation of this pathway generates not only cholesterol but also several intermediates, such as geranyl pyrophospate, that are essential for the activation of Rho.13

In the present study, we hypothesized that statins may prevent the down-regulation of eNOS induced by thrombin in human endothelial cells via inhibition of the Rho pathway.

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METHODS

Materials

All materials for cell culture were from Gibco BRL (Basel, Switzerland). Human thrombin was purchased from Sigma (Buchs, Switzerland). Rabbit polyclonal antibody against Rho A (Sc-119) was purchased from Santa Cruz Biotechnology (Basel, Switzerland). Mouse monoclonal antibody against human eNOS (N30020) was from Transduction Laboratories (Basel, Switzerland).

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Cell Culture

Endothelial cells were isolated from human umbilical veins (HUVEC) as described.9 Briefly, fresh veins were harvested in Medium199. The veins cleaned of connective tissue and adventitia were incubated with 75 U/mL collagenase for 15 minutes in phosphate-buffered saline (PBS). Cell pellets were then collected by centrifugation at 1000 rpm for 10 minutes and seeded in culture dishes coated with fibronectin and cultured in Medium199 supplemented with 20 mmol/L L-glutamine, 10 mmol/L N-2-hydroxyethylpiperazine-N′-2-ehanesulfonic acid buffer, 100 U/mL penicillin, 100 μg/mL streptomycin, 50 μg/mL endothelial cell growth supplement, 25 μg/mL heparin, and 20% fetal calf serum in a humidified atmosphere (37°C, 95% air/5% CO2). The day after, cells were washed with the medium to eliminate blood cells. Endothelial cells were characterized by typical cobblestone and nonoverlapping appearance and immunostaining using specific antibody against von Willebrand factor. Cells of third to sixth passage were used.

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eNOS Protein Expression

Confluent endothelial cells were rendered quiescent for 24 hours by changing the medium to Medium199 with 0.5% fetal calf serum. The cells were stimulated with thrombin (4 U/mL) for 24 hours and then washed twice with PBS, and harvested in the extraction buffer (120 mmol/L sodium chloride, 50 mmol/L tri (hydroxymethyl) aminomethane (Tris), 20 mmol/L sodium fluoride, 1 mmol/L benzamidine, 1 mmol/L dithiothreitol, 1 mmol/L ethylenediaminetetraacetic acid (EDTA), 6 mmol/L ethyleneglycoltetraacetic acid, 15 mmol/L sodium pyrophosphate, 0.8 μg/mL leupeptin, 30 mmol/L p-nitrophenyl phosphate, 0.1 mmol/L phenylmethysulfonyl fluoride, and 1% Nonidet P-40) for immunoblotting. The samples (30 μg) were treated with 5×Laemmli sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (0.35 mol/L Tris-hydrochloric acid (HCl), pH 6.8, 15% SDS, 56.5% glycerol, 0.0075% bromophenol blue), followed by heating at 95°C for 3 minutes and then subjected to 8% SDS-PAGE gel for electrophoresis. Protein concentration was measured by protein assay kit from Bio Rad Laboratories (Glattbrugg, Switzerland). The proteins were then transferred onto membranes (Millipore, Volketswil, Switzerland) with a semidry transfer unit. The membranes were then blocked using 5% skimmed milk and incubated with the antibody against human eNOS (1:600). The immunoreactive bands were detected by an enhanced chemiluminescence system (Amersham, Zürich, Switzerland).

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NOS Activity

The cells were harvested with PBS containing 1 mmol/L EDTA and disrupted by sonication. The enzyme reaction (per 100 μg protein) was performed at 37°C in 100 μL of the assay buffer (25 mmol/L Tris-HCl (pH 7.4), 0.6 mmol/L calcium chloride, 0.1 μmol/L calmodulin, 2 mmol/L nicotinamide adenine dinucleotide phosphate (reduced form), 3 μmol/L tetrahydrobiopterin, 1 μmol/L flavin adenosine dinucleotide, and 1 μmol/L flavin adenosine mononucleotide) containing 0.02 μCi/μL [3H]L-arginine (Amersham Pharmacia Biotech) in the presence or absence of 1 mmol/L NG-nitro-L-arginine methyl ester (L-NAME). After 20 minutes of incubation, the reaction was stopped by the addition of 400 μL of stop buffer (50 mmol/L N-2-hydroxyethylpiperazine-N′-2-ehanesulfonic acid [pH 5.5] and 5 mmol/L EDTA). The reaction mixture was applied to Dowex AG50WX-8 column, and [3H]L-citrulline of the elute was counted by the scintillation counter. NOS activity was expressed as L-NAME–inhibitable citrulline generation.

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Rho A Membrane Translocation

The confluent and quiescent endothelial cells as described above were stimulated with thrombin (4 U/mL) for 10 minutes. The cells were then washed twice with cold PBS and then harvested in PBS buffer containing 2 mmol/L EDTA, 2 mmol/L phenylmethylsulfonyl fluoride, and 0.8 μg/mL leupeptin. The cells were then disrupted by brief sonication on ice. The samples were then centrifuged at 500g for 10 minutes at 4°C to remove the nucleus. The membrane and cytosol were then separated by centrifugation at 100,000g for 1 hour at 4°C (Beckman Instruments, Inc, Nyon, Switzerland). The cell membrane was washed once with the buffer described above and then resuspended in buffer containing 100 mmol/L Tris-HCl, 300 mmol/L sodium chloride, 1% Triton X-100, 0.1% SDS, 2 mmol/L EDTA, 2 mmol/L phenylmethylsulfonyl fluoride, and 0.8 μg/mL leupeptin. Samples were loaded into 12% SDS-PAGE gel and electrophoresed. Immunoblotting was then performed as described above, except that the antibody against Rho A (1:1000) was used.

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Statistics

Data were shown as means±SEM. Statistical analyses were performed with analysis of variance followed by Fisher protected least significant difference test among more than 3 groups. A P value smaller than 0.05 was considered to indicate a significant difference.

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RESULTS

Stimulation with thrombin (4 U/mL, 24 h) significantly decreased eNOS protein expression in HUVECs (Fig. 1). Treatment with simvastatin (10 μmol/L) prevented thrombin-induced down-regulation of eNOS protein level (Fig. 1). In thrombin-stimulated cells, simvastatin increased eNOS protein levels by 112% (Fig. 1). This preventive effect of simvastatin was concentration dependent (100 nmol/L to 10 μmol/L, Fig. 2). Cerivastatin (10 μmol/L) also restored the down-regulation of eNOS by thrombin (Fig. 1). In thrombin-stimulated cells, cerivastatin increased eNOS protein levels by 106% (Fig. 1). Both statins significantly increased the basal eNOS protein expression level (Fig. 1). Consistent with the protein expression levels, prolonged treatment with thrombin (4 U/mL, 24 h) significantly decreased NOS activity (Fig. 3). Both simvastatin and cerivastatin prevented thrombin-induced decrease in NOS activity (Fig. 3). In thrombin-stimulated cells, simvastatin and cerivastatin increased NOS activity by 48% and 42%, respectively (Fig. 3).

Figure 1
Figure 1
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Figure 2
Figure 2
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Figure 3
Figure 3
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We previously reported that thrombin induces a rapid and transient activation of Rho A (maximal effect is obtained at 10 min) and that the down-regulation of eNOS expression induced by thrombin is mediated by Rho A/Rho-kinase pathway.9 Thus, to investigate the signaling mechanism underlying the inhibitory effect of statins on eNOS down-regualtion by thrombin, we examined the effect of statins on Rho A activation. In HUVECs, stimulation with thrombin (4 U/mL, 10 min) significantly increased the membrane translocation of Rho A (Fig. 4). Treatment with simvastatin (10 μmol/L) and with cerivstatin (10 μmol/L) significantly decreased thrombin-induced membrane translocation of Rho A (Fig. 4).

Figure 4
Figure 4
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DISCUSSION

In the present study, we have shown that in human endothelial cells, statins blunt the down-regulation of eNOS induced by thrombin. This restoration of eNOS expression by statins was associated with an inhibition of membrane translocation of Rho, which is the crucial step for Rho activation. Indeed, Rho negatively regulates eNOS expression via a reduction in mRNA stability.9,14 Moreover, we recently reported that prolonged incubation of thrombin induces the down-regulation of eNOS expression via activation of Rho.9 Thus, the same intracellular signaling pathway must mediate the preventive effect of statins on thrombin-induced eNOS down-regulation observed in this study.

In acute coronary syndromes, the coagulation cascade is activated and as a result, local production of thrombin is increased.15 Thrombin not only cleaves fibrinogen to fibrin and aggregates platelets but also has direct effects on vascular cells through its protease-activated receptors.16 In fact, in patients with acute coronary syndromes, vasomotor function in the coronary arteries is impaired17; this may be, at least in part, attributed to the inhibitory action of thrombin on eNOS expression. In addition to its vasodilator action, NO derived from eNOS simultaneously inhibits platelet aggregation.18 In this context, eNOS down-regulation by thrombin and the subsequent reduction in the luminal release of bioavailable NO may further reduce blood flow and facilitate thrombus formation. This sequence of events may in turn contribute to the clinical course of acute coronary syndromes. Therefore, this mechanism may represent a potential therapeutic target for pharmacologic intervention. Recent clinical trials have shown that statins are also effective in patients with acute coronary syndromes.10,11 As this beneficial effect of statins cannot be explained by their lipid-lowering effect alone,10 the inhibitory action of statins on thrombin-induced down-regulation of eNOS may contribute to the pathogenesis of acute coronary syndromes and to the reduction of events thereafter. In this study we have shown a concentration-dependent effect of simvastatin that was seen from 100 nmol/L onwards. This level is close to the peak plasma concentration in humans after the administration of relatively high doses of simvastatin,19 suggesting that the effects of simvastatin on eNOS expression in human endothelial cells observed in this study are clinically relevant. As cerivastatin acted like simvastatin, a class effect involving the specific inhibition of the HMG-CoA pathway is likely to be responsible.

In conclusion, the inhibition of the HMG-CoA pathway and in turn inactivation of the Rho pathway blunt thrombin-induced down-regulation of eNOS expression in human endothelial cells. This finding provides a novel mechanism of the pleiotropic effects of statins, which may be important in patients with acute coronary syndromes.

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REFERENCES

1. Libby P. Current concepts of the pathogenesis of the acute coronary syndromes. Circulation. 2001;104:365–372.

2. Rosenberg RD, Aird WC. Vascular-bed-specific hemostasis and hypercoagulable states. N Engl J Med. 1999;340:1555–1564.

3. Eto M, Kozai T, Cosentino F, et al. Statin prevents tissue factor expression in human endothelial cells: role of Rho/Rho-kinase and Akt pathways. Circulation. 2002;105:1756–1759.

4. Fuster V, Badimon L, Badimon JJ, et al. Mechanisms of disease: the pathogenesis of coronary artery disease and the acute coronary syndromes (part two). N Engl J Med. 1992;326:310–318.

5. Bogarty P, Hackett D, Davies G, et al. Vasoreactivity of the culprit lesion in unstable angina. Circulation. 1994;90:5–11.

6. Boulanger C, Luscher TF. Release of endothelin from the porcine aorta. Inhibition by endothelium-derived nitric oxide. J Clin Invest. 1990;85:587–590.

7. Zeiher AM, Ihling C, Pistorius K, et al. Increased tissue endothelin immunoreactivity in atherosclerotic lesions associated with acute coronary syndromes. Lancet. 1994;344:1405–1406.

8. Luscher TF, Diederich D, Siebenmann R, et al. Difference between endothelium-dependent relaxations in arterial and in venous coronary bypass grafts. N Engl J Med. 1988;319:462–467.

9. Eto M, Barandier C, Rathgeb L, et al. Thrombin suppresses endothelial nitric oxide synthase and upregulates endothelin-converting enzyme-1 expression by distinct pathways: role of Rho/ROCK and mitogen-activated protein kinase. Circ Res. 2001;89:583–590.

10. Schwartz GG, Olsson AG, Ezekowitz MD, et al. Effects of atorvastatin on early recurrent ischemic events in acute coronary syndromes: the MIRACL study: a randomized controlled trial. JAMA. 2001;285:1711–1718.

11. Aronow HD, Topol EJ, Roe MT, et al. Effect of lipid-lowering therapy on early mortality after acute coronary syndromes: an observational study. Lancet. 2001;357:1063–1068.

12. Vaughan CJ, Murphy MB, Buckley BM. Statins do more than just lower cholesterol. Lancet. 1996;348:1079–1082.

13. Laufs U, Liao JK. Targeting Rho in cardiovascular disease. Circ Res. 2000;87:526–528.

14. Laufs U, Liao JK. Post-transcriptional regulation of endothelial nitric oxide synthase mRNA stability by Rho GTPase. J Biol Chem. 1998;273:24266–24271.

15. Weitz JI, Buller HR. Direct thrombin inhibitors in acute coronary syndromes: present and future. Circulation. 2002;105:1004–1011.

16. Coughlin SR. Thrombin signalling and protease-activated receptors. Nature. 2000;407:258–264.

17. Tomai F, Crea F, Gaspardone A, et al. Unstable angina and elevated c-reactive protein levels predict enhanced vasoreactivity of the culprit lesion. Circulation. 2001;104:1471–1476.

18. Loscalzo J. Nitric oxide insufficiency, platelet activation and arterial thrombosis. Circ Res. 2001;88:756–762.

19. Davidson MH, Stein EA, Dujovne CA, et al. The efficacy and six-week tolerability of simvastatin of 80 and 160 mg/day. Am J Cardiol. 1997;79:38–42.

Cited By:

This article has been cited 2 time(s).

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American Journal of Physiology-Heart and Circulatory Physiology, 292(6): H3158-H3163.
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Keywords:

thrombin; endothelial nitric oxide synthase; stains; Rho

© 2006 Lippincott Williams & Wilkins, Inc.

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