This article has Open Peer Review reports available.
Sirolimus increases tissue factor expression but not activity in cultured human vascular smooth muscle cells
- Shengsi Zhu†1, 2,
- Hema Viswambharan†1,
- Thusitha Gajanayake1,
- Xiu-Fen Ming1 and
- Zhihong Yang1Email author
© Zhu et al; licensee BioMed Central Ltd. 2005
Received: 07 January 2005
Accepted: 15 July 2005
Published: 15 July 2005
Sirolimus-eluting stents (CYPHER stents) demonstrated remarkable efficacy in reducing restenosis rates in patients with coronary artery disease. There is a concern of sub-acute and late stent thrombosis. Tissue factor (TF) is critical in thrombosis. This study investigated the effect of sirolimus on TF expression and activity in cultured human vascular smooth muscle cells (SMCs).
SMCs were cultured from human saphenous veins and aortas. Quiescent cells were stimulated with sirolimus (0.1 – 20 ng/ml) over 24 hours. Cellular TF expression and activity released into culture medium were measured. The effect of sirolimus on activation of mammalian target of rapamycin (mTOR) was measured by phosphorylation of the substrate p70s6k at T389, and activation of RhoA was measured by pull-down assay.
Sirolimus increased TF protein level in cultured human SMCs in a concentration and time-dependent manner (about 2-fold, p < 0.01) reaching maximal effect at 5 ng/ml. The stimulation of TF expression by sirolimus was associated with inhibition of basal activity of mTOR. No effects of sirolimus on RhoA or p38mapk activation that are positive regulators of TF in vascular wall cells were observed. The stimulation of TF expression by sirolimus (20 ng/ml) was prevented by the HMG-CoA reductase inhibitor fluvastatin (1 μmol/L). However, no increase in TF activity released from SMC into culture medium was observed after sirolimus treatment.
Although sirolimus stimulates TF protein expression in human SMC associated with inhibition of mTOR, it does not enhance TF activity released from the cells, suggesting a relatively safe profile of CYPHER stents. The inhibition of TF expression by fluvastatin favors clinical use of statins in patients undergoing coronary stenting.
Since the first human study with sirolimus (rapamycin)-eluting stents (Cordis CYPHER™ stent) by Sousa , considerable promise of sirolimus-eluting stents for reducing restenosis rates and clinical parameters was subsequently demonstrated by several randomized clinical trials [2–7]. The mechanism of inhibition of restenosis by sirolimus has been suggested to be attributed to the blockade of smooth muscle cell (SMC) cycle progression from G1 to S phase via inhibition of the protein kinase, mammalian target of rapamycin (mTOR).
Despite the promising results on restenosis rates, there is concern that drug-eluting stents may be associated with increased thrombosis rates. Although stent thrombosis associated with sirolimus-eluting stents has been reported in several clinical trials, it remains a rare event and is not higher in patients receiving bare metal stents [2–4, 9, 10]. Pooled analysis of clinical trials does not reveal a higher incidence of stent thrombosis, suggesting a relative safe profile of drug-eluting stents at least under the condition of anti-platelet regiment . However, individual case reports generated some suspicion that drug-eluting stents may be prone to thrombosis . In a report, four cases of late coronary thrombosis related to drug-eluting stents were presented, all of them occurred shortly after anti-platelet therapy was interrupted , and in two patients who received both a bare metal stent and a sirolimus-eluting stent, only the sirolimus-eluting stents were closed due to thrombosis, while the bare metal stents remained open in the same patients . Based on the controversial reports and concerns, we analyzed whether sirolimus per se exerts some adverse effects related to thrombosis in vascular cells namely smooth muscle cells.
Tissue factor (TF) plays an important role in thrombosis and acute coronary syndromes . It is the principle initiator of extrinsic coagulation pathway activating thrombin and generating fibrin leading to thrombus formation. Recent study suggests that aberrant TF expression in the vascular wall cells plays a crucial role in triggering intravascular thrombosis . Under non-stimulated conditions, vascular wall cells i.e. endothelial cells and SMCs express negligible or low level of TF that can be up-regulated by cytokines and thrombin [15–17]. Several intracellular signal transduction mechanisms have been demonstrated to be involved in the regulation of TF expression. The small G-protein RhoA and the protein kinase p38mapk are positive regulators, whereas phosphatidylinositol 3-kinase (PI3-K) negatively regulates TF expression in vascular wall cells .
The HMG CoA reductase inhibitors or statins reduce cardiovascular events in patients with coronary heart disease . The non-cholesterol lowering effects i.e. pleiotropic effects of statins seem to play important roles . Experimental studies demonstrate that statins increase eNOS expression in endothelial cells, inhibit TF expression in SMC via inhibition of Rho/ROCK pathway [16, 20]. Hence, the present study is aimed to investigate whether sirolimus could promote TF expression in human SMC, and whether this is associated with an increased TF activity. The effects of statin such as fluvastatin on TF expression in SMC were also investigated.
Sirolimus was purchased from Calbiochem (Lucerne, Switzerland); fluvastatin was kindly provided by Novartis (Basel, Switzerland); tumor necrosis factor-α (TNF-α) was purchased from R & D, France); monoclonal mouse anti-TF antibody and tissue factor activity kit were purchased from American Diagnostica Inc (Socochim, Lausanne, Switzerland); anti-tubulin and all the other chemicals for immunoblotting were purchased from Sigma (Buchs, Switzerland); anti-phospho p70s6k (T389) was from Cell Signaling Technology. Alkaline phosphatase (AP)-conjugated anti-mouse IgG and BCIP/NBT substrate for AP were from Interchim (Chemie Brunschwig AG, Basel, Switzerland).
SMC and endothelial cell culture
SMC were isolated and cultured from human saphenous veins  and human aortic SMC were kindly provided by Dr. Therese Resink (University of Basel, Switzerland). Endothelial cells from human umbilical veins were isolated as previously described .
Cells were rendered quiescent for 24 hours in DMEM containing 0.2% BSA before they were treated with sirolimus (20 ng/ml, 24 hours), a concentration which fully inhibits mTOR/p70s6k pathway as previously shown . To study the effect of fluvastatin on sirolimus-induced TF expression, the cells were pre-incubated with fluvastatin (1 μmol/L) for 60 minutes. Cell lysates were prepared as described . 30 μg extracts were used for immunoblotting of TF expression . Tubulin expression was used to ensure equal protein loading. Quantification was performed using NIH Image-J software. TF expression was expressed as percentage changes of the basal level.
TF activity in cell conditioned medium
2 × 10-5 cells/ml were seeded onto each dish for overnight attachment. Cells were then rendered quiescent in phenol-red free DMEM medium containing 0.2% BSA for 24 hours and then treated with sirolimus (20 ng/ml; 24 hours) as described above except that conditioned medium was collected and TF activity was measured as instructed by the manufacturer. Briefly, the same amount of conditioned medium (25 μl) was incubated in the presence of Factor VIIa and Factor X for 15 minutes in a 96-well plate, after which the substrate was added and further incubated for another 60 minutes before the reaction was stopped with glacial acetic acid and color reaction was measured with a microplate reader at 405 nm. TF activity is expressed in picomolar obtained from the standard curve.
The activation of RhoA was assessed by pull-down assay in the cells stimulated with sirolimus (20 ng/ml) over one hour as described . Briefly, SMCs were washed with ice-cold Tris-buffered saline and lysed in RIPA buffer (50 mmol/L Tris, pH 7.2, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 500 mmol/L NaCl, 10 mmol/L MgCl2, 10 μg/ml each of leupeptin and aprotinin, and 1 mmol/L PMSF). 200 μg of cell lysates were incubated with 10 μg of GST-TRBD beads at 4°C for 60 min. The beads were washed four times with buffer B (Tris-buffer containing 1% Triton X-100, 150 mmol/L NaCl, 10 mmol/L MgCl2, 10 μg/ml each of leupeptin and aprotinin, and 0.1 mmol/L PMSF). Bound RhoA proteins were then detected by immunoblotting using a monoclonal antibody against RhoA (Santa Cruz Biotechnology). The total amount of RhoA in cell lysates was used as a control for the cross-comparison of RhoA activity (level of GTP-bound RhoA).
mTOR activation was examined by immunoblotting measuring p70s6k phosphorylation at T389 in quiescent cells with or without sirolimus treatment (20 ng/ml, 1 hour). 40 μg cell extracts were subjected to 8% SDS-PAGE and phosphorylated p70s6k was detected using anti-phospho-p70s6k (T389) antibody. Activation of p70s6k was calculated as ratio of phospho-p70s6k against tubulin.
All data were expressed as mean ± SEM and one way analysis of variance (ANOVA) with Bonferroni's post-test was used for statistical analysis. A two-tailed value of p ≤ 0.05 was considered statistically significant.
Results and discussion
It is well described that sirolimus is a natural immunosuppressant which interferes with cellular functions via blockade of the protein kinase, mTOR  which further activates its downstream effector p70s6k by phosphorylating T389 residue . In our present study, we showed a significant basal activity of mTOR in SMC as measured by p70s6k phosphorylation at T389 (Fig. 2D). The activity of mTOR was abolished by sirolimus (20 ng/ml, 1 hour treatment, Fig. 2D). Whether this data suggest an inhibitory effect of mTOR on TF expression needs further investigation. Further results demonstrate that fluvastatin does not reverse the inhibition of mTOR i.e. phosphorylation of p70s6k at T389 by sirolimus, nor it had any effect on basal mTOR activity in the cells (Fig. 2D), suggesting that statin inhibits TF expression not through regulation of mTOR.
Taken together, our results demonstrate that although sirolimus stimulates TF expression in human SMC, it does not enhance TF activity released from the cells. The results support the safe profile of CYPHER stents observed by clinical trials. The inhibition of TF expression by fluvastatin favors clinical use of statins in patients undergoing coronary stenting.
This study was supported by Swiss National Science Foundation (3100A0-105917/1) and Swiss University Conference (SUK) program. X-F. Ming was supported by Swiss Heart Foundation and Roche Research Foundation. S. Zhu and T. Gajanayake are recipients of Swiss Federal Scholarship.
- Sousa JE, Costa MA, Abizaid AC, Rensing BJ, Abizaid AS, Tanajura LF, Kozuma K, Van LG, Sousa AG, Falotico R, Jaeger J, Popma JJ, Serruys PW: Sustained suppression of neointimal proliferation by sirolimus-eluting stents: one-year angiographic and intravascular ultrasound follow-up. Circulation. 2001, 104: 2007-2011.View ArticlePubMedGoogle Scholar
- Morice MC, Serruys PW, Sousa JE, Fajadet J, Ban HE, Perin M, Colombo A, Schuler G, Barragan P, Guagliumi G, Molnar F, Falotico R: A randomized comparison of a sirolimus-eluting stent with a standard stent for coronary revascularization. N Engl J Med. 2002, 346: 1773-1780. 10.1056/NEJMoa012843.View ArticlePubMedGoogle Scholar
- Moses JW, Leon MB, Popma JJ, Fitzgerald PJ, Holmes DR, O'Shaughnessy C, Caputo RP, Kereiakes DJ, Williams DO, Teirstein PS, Jaeger JL, Kuntz RE: Sirolimus-eluting stents versus standard stents in patients with stenosis in a native coronary artery. N Engl J Med. 2003, 349: 1315-1323. 10.1056/NEJMoa035071.View ArticlePubMedGoogle Scholar
- Schofer J, Schluter M, Gershlick AH, Wijns W, Garcia E, Schampaert E, Breithardt G: Sirolimus-eluting stents for treatment of patients with long atherosclerotic lesions in small coronary arteries: double-blind, randomised controlled trial (E-SIRIUS). Lancet. 2003, 362: 1093-1099. 10.1016/S0140-6736(03)14462-5.View ArticlePubMedGoogle Scholar
- Lemos PA, Serruys PW, van Domburg RT, Saia F, Arampatzis CA, Hoye A, Degertekin M, Tanabe K, Daemen J, Liu TK, McFadden E, Sianos G, Hofma SH, Smits PC, van der Giessen WJ, de Feyter PJ: Unrestricted utilization of sirolimus-eluting stents compared with conventional bare stent implantation in the "real world": the Rapamycin-Eluting Stent Evaluated At Rotterdam Cardiology Hospital (RESEARCH) registry. Circulation . 2004, 109: 190-195. 10.1161/01.CIR.0000109138.84579.FA.View ArticlePubMedGoogle Scholar
- Popma JJ, Leon MB, Moses JW, Holmes DR, Cox N, Fitzpatrick M, Douglas J, Lambert C, Mooney M, Yakubov S, Kuntz RE: Quantitative assessment of angiographic restenosis after sirolimus-eluting stent implantation in native coronary arteries. Circulation. 2004, 110: 3773-3780. 10.1161/01.CIR.0000150331.14687.4B.View ArticlePubMedGoogle Scholar
- Fajadet J, Morice MC, Bode C, Barragan P, Serruys PW, Wijns W, Constantini CR, Guermonprez JL, Eltchaninoff H, Blanchard D, Bartorelli A, Laarman GJ, Perin M, Sousa JE, Schuler G, Molnar F, Guagliumi G, Colombo A, Ban Hayashi E, Wulfert E: Maintenance of long-term clinical benefit with sirolimus-eluting coronary stents: three-year results of the RAVEL trial. Circulation. 2005, 111: 1040-1044. 10.1161/01.CIR.0000156334.24955.B2.View ArticlePubMedGoogle Scholar
- Poon M, Badimon JJ, Fuster V: Overcoming restenosis with sirolimus: from alphabet soup to clinical reality. Lancet. 2002, 359: 619-622. 10.1016/S0140-6736(02)07751-6.View ArticlePubMedGoogle Scholar
- Babapulle MN, Joseph L, Belisle P, Brophy JM, Eisenberg MJ: A hierarchical Bayesian meta-analysis of randomised clinical trials of drug-eluting stents. Lancet. 2004, 364: 583-591. 10.1016/S0140-6736(04)16850-5.View ArticlePubMedGoogle Scholar
- Ong AT, Hoye A, Aoki J, van Mieghem CA, Rodriguez Granillo GA, Sonnenschein K, Regar E, McFadden EP, Sianos G, van der Giessen WJ, de Jaegere PP, de Feyter P, van Domburg RT, Serruys PW: Thirty-day incidence and six-month clinical outcome of thrombotic stent occlusion after bare-metal, sirolimus, or paclitaxel stent implantation. J Am Coll Cardiol. 2005, 45: 947-953. 10.1016/j.jacc.2004.09.079.View ArticlePubMedGoogle Scholar
- Moreno R, Fernandez C, Hernandez R, Alfonso F, Angiolillo DJ, Sabate M, Escaned J, Banuelos C, Fernandez-Ortiz A, Macaya C: Drug-eluting stent thrombosis: results from a pooled analysis including 10 randomized studies. J Am Coll Cardiol. 2005, 45: 954-959. 10.1016/j.jacc.2004.11.065.View ArticlePubMedGoogle Scholar
- McFadden EP, Stabile E, Regar E, Cheneau E, Ong AT, Kinnaird T, Suddath WO, Weissman NJ, Torguson R, Kent KM, Pichard AD, Satler LF, Waksman R, Serruys PW: Late thrombosis in drug-eluting coronary stents after discontinuation of antiplatelet therapy. Lancet. 2004, 364: 1519-1521. 10.1016/S0140-6736(04)17275-9.View ArticlePubMedGoogle Scholar
- Moons AH, Levi M, Peters RJ: Tissue factor and coronary artery disease. Cardiovasc Res. 2002, 53: 313-325. 10.1016/S0008-6363(01)00452-7.View ArticlePubMedGoogle Scholar
- Day SM, Reeve JL, Pedersen B, Farris DM, Myers DD, Im M, Wakefield TW, Mackman N, Fay WP: Macrovascular thrombosis is driven by tissue factor derived primarily from the blood vessel wall. Blood. 2005, 105: 192-198. 10.1182/blood-2004-06-2225.View ArticlePubMedGoogle Scholar
- Archipoff G, Beretz A, Freyssinet JM, Klein-Soyer C, Brisson C, Cazenave JP: Heterogeneous regulation of constitutive thrombomodulin or inducible tissue-factor activities on the surface of human saphenous-vein endothelial cells in culture following stimulation by interleukin-1, tumour necrosis factor, thrombin or phorbol ester. Biochem J. 1991, 273: 679-684.View ArticlePubMedPubMed CentralGoogle Scholar
- Eto M, Kozai T, Cosentino F, Joch H, Luscher TF: Statin prevents tissue factor expression in human endothelial cells: role of Rho/Rho-kinase and Akt pathways. Circulation. 2002, 105: 1756-1759. 10.1161/01.CIR.0000015465.73933.3B.View ArticlePubMedGoogle Scholar
- Viswambharan H, Ming XF, Zhu S, Hubsch A, Lerch P, Vergeres G, Rusconi S, Yang Z: Reconstituted high-density lipoprotein inhibits thrombin-induced endothelial tissue factor expression through inhibition of RhoA and stimulation of phosphatidylinositol 3-kinase but not Akt/endothelial nitric oxide synthase. Circ Res. 2004, 94: 918-925. 10.1161/01.RES.0000124302.20396.B7.View ArticlePubMedGoogle Scholar
- Grundy SM, Cleeman JI, Merz CN, Brewer HB, Clark LT, Hunninghake DB, Pasternak RC, Smith SC, Stone NJ, Coordinating Committee of the National Cholesterol Education Program: Implications of recent clinical trials for the National Cholesterol Education Program Adult Treatment Panel III Guidelines. J Am Coll Cardiol. 2004, 44: 720-732. 10.1016/j.jacc.2004.07.001.View ArticlePubMedGoogle Scholar
- Farmer JA: Pleiotropic effects of statins. Curr Atheroscler Rep. 2000, 2: 208-217.View ArticlePubMedGoogle Scholar
- Yang Z, Kozai T, van der LB, Viswambharan H, Lachat M, Turina MI, Malinski T, Luscher TF: HMG-CoA reductase inhibition improves endothelial cell function and inhibits smooth muscle cell proliferation in human saphenous veins. J Am Coll Cardiol. 2000, 36: 1691-1697. 10.1016/S0735-1097(00)00924-4.View ArticlePubMedGoogle Scholar
- Ming XF, Burgering BM, Wennstrom S, Claesson-Welsh L, Heldin CH, Bos JL, Kozma SC, Thomas G: Activation of p70/p85 S6 kinase by a pathway independent of p21ras. Nature. 1994, 371: 426-429. 10.1038/371426a0.View ArticlePubMedGoogle Scholar
- Kerner A, Gruberg L, Kapeliovich M, Grenadier E: Late stent thrombosis after implantation of a sirolimus-eluting stent. Catheter Cardiovasc Interv. 2003, 60: 505-508. 10.1002/ccd.10712.View ArticlePubMedGoogle Scholar
- Virmani R, Guagliumi G, Farb A, Musumeci G, Grieco N, Motta T, Mihalcsik L, Tespili M, Valsecchi O, Kolodgie FD: Localized hypersensitivity and late coronary thrombosis secondary to a sirolimus-eluting stent: should we be cautious?. Circulation. 2004, 109: 701-705. 10.1161/01.CIR.0000116202.41966.D4.View ArticlePubMedGoogle Scholar
- Eisenberg MJ: Drug-eluting stents: some bare facts. Lancet. 2004, 364: 1466-1467. 10.1016/S0140-6736(04)17287-5.View ArticlePubMedGoogle Scholar
- Lemos PA, Lee CH, Degertekin M, Saia F, Tanabe K, Arampatzis CA, Hoye A, van DM, Sianos G, Smits PC, de FP, van der Giessen WJ, van Domburg RT, Serruys PW: Early outcome after sirolimus-eluting stent implantation in patients with acute coronary syndromes: insights from the Rapamycin-Eluting Stent Evaluated At Rotterdam Cardiology Hospital (RESEARCH) registry. J Am Coll Cardiol. 2003, 41: 2093-2099. 10.1016/S0735-1097(03)00429-7.View ArticlePubMedGoogle Scholar
- Colombo A, Moses JW, Morice MC, Ludwig J, Holmes DR, Spanos V, Louvard Y, Desmedt B, Di MC, Leon MB: Randomized study to evaluate sirolimus-eluting stents implanted at coronary bifurcation lesions. Circulation. 2004, 109: 1244-1249. 10.1161/01.CIR.0000118474.71662.E3.View ArticlePubMedGoogle Scholar
- Murphy BE: Drug-eluting stents: some first-generation problems. Am Heart Hosp J. 2004, 2: 80-84.View ArticlePubMedGoogle Scholar
- Jeanmart H, Malo O, Carrier M, Nickner C, Desjardins N, Perrault LP: Comparative study of cyclosporine and tacrolimus vs newer immunosuppressants mycophenolate mofetil and rapamycin on coronary endothelial function. J Heart Lung Transplant. 2002, 21: 990-998. 10.1016/S1053-2498(02)00429-1.View ArticlePubMedGoogle Scholar
- Babinska A, Markell MS, Salifu MO, Akoad M, Ehrlich YH, Kornecki E: Enhancement of human platelet aggregation and secretion induced by rapamycin. Nephrol Dial Transplant. 1998, 13: 3153-3159. 10.1093/ndt/13.12.3153.View ArticlePubMedGoogle Scholar
- Walpoth BH, Pavlicek M, Celik B, Nicolaus B, Schaffner T, Althaus U, Hess OM, Carrel T, Morris RE: Prevention of neointimal proliferation by immunosuppression in synthetic vascular grafts. Eur J Cardiothorac Surg. 2001, 19: 487-492. 10.1016/S1010-7940(01)00582-6.View ArticlePubMedGoogle Scholar
- Guagliumi G, Farb A, Musumeci G, Valsecchi O, Tespili M, Motta T, Virmani R: Images in cardiovascular medicine. Sirolimus-eluting stent implanted in human coronary artery for 16 months: pathological findings. Circulation. 2003, 107: 1340-1341. 10.1161/01.CIR.0000062700.42060.6F.View ArticlePubMedGoogle Scholar
- Fukuda D, Sata M, Tanaka K, Nagai R: Potent inhibitory effect of sirolimus on circulating vascular progenitor cells. Circulation. 2005, 111: 926-931. 10.1161/01.CIR.0000155612.47040.17.View ArticlePubMedGoogle Scholar
- Iakovou I, Sangiorgi GM, Stankovic G, Corvaja N, Vitrella G, Ferraro M, Colombo A: Results and follow-up after implantation of four or more sirolimus-eluting stents in the same patient. Catheter Cardiovasc Interv. 2005, 64: 436-439. 10.1002/ccd.20305.View ArticlePubMedGoogle Scholar
- Guba M, Yezhelyev M, Eichhorn ME, Schmid G, Ischenko I, Papyan A, Graeb C, Seeliger H, Geissler EK, Jauch KW, Bruns CJ: Rapamycin induces tumor-specific thrombosis via tissue factor in the presence of VEGF. Blood. 2005, 105: 4463-4469. 10.1182/blood-2004-09-3540.View ArticlePubMedGoogle Scholar
- Wolfrum S, Jensen KS, Liao JK: Endothelium-dependent effects of statins. Arterioscler Thromb Vasc Biol. 2003, 23: 729-736. 10.1161/01.ATV.0000063385.12476.A7.View ArticlePubMedGoogle Scholar
- Hay N, Sonenberg N: Upstream and downstream of mTOR. Genes Dev. 2004, 18: 1926-1945. 10.1101/gad.1212704.View ArticlePubMedGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2261/5/22/prepub
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.