Rapamycin promoted thrombosis and platelet adhesion to endothelial cells by inducing membrane remodeling
- Ping Jiang†1Email author,
- Yong Lan†2,
- Jun Luo1,
- Ya-Li Ren3,
- Dong-Ge Liu4,
- Jian-Xin Pang4,
- Jin Liu5,
- Jian Li1,
- Chen Wang6 and
- Jian-Ping Cai1Email author
© Jiang et al.; licensee BioMed Central Ltd. 2014
Received: 22 March 2013
Accepted: 6 February 2014
Published: 24 February 2014
Recently, evidence indicated that the rapamycin-eluting stent which was used worldwide may contribute to an increased risk for thrombosis. On the contrary, other researchers found it was safe. Thus, it is necessary to clarify the effect of rapamycin on thrombosis and the corresponding mechanisms.
The effects of rapamycin in vivo were evaluated by modified deep vein thrombosis animal model. The platelets were from healthy volunteers and the platelet-endothelium (purchased from ATCC) adhesion in cultured endothelial cells was assessed. Membrane rufflings in endothelial cells were examined by confocal and electron microscope. Thrombus formation increased in rats that were injected with rapamycin. Electron microscope analysis exhibited microvilli on the rapamycin-treated endothelium in rats. Rapamycin enhanced membrane ruffling in human umbilical vein endothelial cells (HUVECs) and adhesion of platelets to HUVECs. The platelet-HUVECs adhesion was attenuated when cells were treated with cytochalacin B. Inhibition of autophagy by 3-methyladenine led to suppression of membrane ruffles in HUVECs and augmentation of platelet-endothelial adhesion.
In conclusion, we found that endothelial membrane remodeling induced by rapamycin is crucial for the adhesion of platelets to endothelial cells and thereby for thrombosis in vivo, and that the endothelial membrane remodeling is autophagy dependent.
KeywordsThrombosis Membrane remodeling Endothelial cell Platelet
Venous thrombosis, including deep vein thrombosis (DVT) and pulmonary embolism, is a major source of morbidity and mortality worldwide 1]. Although it is accepted that the combination of so-called virchow triad, namely 1) vascular abnormalities and endothelial dysfunction, 2) hypercoagulability and 3) stasis, may play a pivotal role in the pathogenesis of venous thrombosis, the underlying mechanisms are not fully elucidated.
The pathogenesis of thrombosis involves a variety of factors among which platelet adhesion to endothelial cells is one element of importance 2, 3]. The data that adhesion can occur in the mice who lack fibrinogen and VWF suggests that some pivotal mechanisms, for example, the platelet-endothelial interaction, may be involved in this process and the concomitant thrombosis 4]. We have known that the rougher the membrane surfaces are, the more the platelets adhere and the poorer the hemocompatibility is, and vice versa 5]. The above data suggested that the dynamic regulation of endothelial membrane shape may affect the process of platelet adhesion. In fact, the morphology of endothelial membrane changed with environment frequently. For example, when the transmural pressure elevated or cells were exposed to E2, projections, such as membrane ruffles, pseudopodia and microvilli, will appear on the outer surface of endothelium and stretch into the vessel lumen 6, 7].
Kadandale et al. 8] identified that autophagy plays a pivotal role in blood cell cortical remodeling, with involvement in the extension of cell protrusions, such as lamellipodia and filopodia. Rapamycin is a kind of autophagy agonist, which was reported to be associated with regulation of endothelial cytoskeleton 9]. Every year, rapamycin-eluting stents are implanted in millions of patients with coronary artery disease who undergo percutaneous coronary intervention. However, evidence indicated that rapamycin-eluting stents may be associated with an increased risk for stent thrombosis when compared with bare-metal stents 4, 10]. For example, Camici et al. 10] reported that rapamycin promoted arterial thrombosis in vivo. In endothelial cells, rapamycin can enhance the activity of tissue factor (TF) which is a key trigger of coagulation cascade. In addition, the effects that rapamycin inhibits tissue plasminogen activator (t-PA) and induces plasminogen activator inhibitor 1 (PAI-1) in human umbilical vein endothelial cells (HUVECs) may contribute to thrombosis associated with rapamycin-eluting stents 11]. On the contrary, Daemen et al. and others 12, 13] found that rapamycin-eluting stents were safe and effective compared with bare-metal ones. Thus, it is necessary to elucidate the effect of rapamycin on thrombosis.
In this study, we found that rapamycin (500 ng/kg) promoted formation of microvilli-like structure in endothelium and thrombotic occlusion in the modified DVT rat model. HUVECs treated with rapamycin demonstrated that both dorsal ruffling and platelets-endothelial adhesion were promoted. Suppressing dorsal ruffling by cytochalasin B led to inhibited platelet-endothelial adhesion. Further analysis suggested that rapamycin-mediated autophagy activation may contribute to the formation of the dorsal ruffling in endothelial cells.
Rapamycin promoted thrombus formation in vivo
Rapamycin enhanced platelet adhesion to endothelium
Rapamycin induced membrane remodeling in endothelium
As shown in Figure 3B and3C, rapamycin at the concentration of 10 to 1000 nM can lead to dramatically enhanced dorsal ruffle formation. And the formation of prominent dorsal ruffles peaks at the 30th minute after rapamycin stimulation.
The membrane remodeling is necessary for platelet-endothelium adhesion
The membrane remodeling was induced by autophagy
Anti-LC3-II antibody and anti-mouse IgG-FITC antibody were obtained from Medical & Biological Laboratoris CO., LTD (#025, #107). Rapamycin, 3-methyladenine, and Cytochalasin B were purchased from Sigma (#R878, #M9281, C6762). Rhodamine labeled Goat anti-Rabbit IgG antibody was from Kirkegaard & Perry Laboratories, Inc. (#110144).
Cell culture and preparation of washed human platelet suspensions
HUVECs were purchased from ATCC and cultured in Endothelial cell medium (containing 10% FBS, 1% ECGS, and 1% penicillin/streptomycin solution) under a 5% CO2/95% air atmosphere in a humidified incubator at 37°C. Cells were used between passages 3 and 10.
Venous blood was drawn from healthy volunteers. Platelet-rich plasma (PRP) was prepared by centrifugation at 500 g for 15 minutes at room temperature, and then platelets were pelleted by centrifugation of the PRP for 6 minutes at 2000 g and washed in HEPES buffer warmed to 37°C (0.137 M NaCl, 2.68 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 5 mM HEPES, and 0.1% glucose, pH 6.8).
The work was approved by the Ethics Committee of the National Center for Clinical Laboratories, and adhered to the tenets of the Declaration of Helsinki. Written informed consents were obtained from the donors.
Platelet adhesion assay
The platelet adhesion assay was performed as previously described 17] by some modification. In brief, HUVECs were seeded on a gelatin coated coverslips at a concentration of 5 × 105/ml and grown to confluence. Washed platelets (50 × 106/μl) were added to the coverslips and incubated for 20 minutes under vibrating at 37°C. Non-adhered platelets were removed by washing with PBS. Cells were fixed in 3.7% paraformaldehyde for 15 minutes at room temperature and then stained with crystal violet for 10 minutes at 37°C. Cells on the coverslips were mounted and imaged by microscopy. The number of cells with platelets adhered was evaluated in triplicate per × 40 magnification field.
Male SD rats (180 ± 30 g) were maintained under clean conventional conditions (24 ± 2°C, 12 h light/dark cycle) and were allowed free access to water and food. The DVT model was made as previously described 18]. In brief, the inferior vena cava (IVC) was exposed via a midline incision in the abdomen followed by retraction of intestines. The IVC was carefully dissected away from the aorta for a distance of 2–3 mm immediately inferior to the renal arteries. Upon injecting rapamycin (500 ng/kg) or DMSO (control) into iliac vein, the IVC was tied with a silk suture for 40 minutes. This procedure thus created approximately a 100% cross-sectional surface stenosis and a reduction of flow upstream of the suture. Then the IVC was removed and fixed in 3.7% paraformaldehyde for subsequent Hematoxylin and eosin staining and transmission electron microscopy (TEM) analysis.
All animal procedures were performed in accordance with the National Institutes of Health Animal Care and Use Guidelines. All animal protocols were approved by the Animal Ethics Committee at the Beijing Institute of Geriatrics.
Cells cultured on gelatin-coated coverslips were washed in PBS and fixed in 3.7% formaldehyde for 15 minutes at room temperature. After blocked with 10% goat serum for 15 minutes, the coverslips were incubated with primary antibody for 60 minutes and fluorescently labeled secondary antibodies for 45 minutes at 37°C successively, and then washed extensively and mounted. Cells were viewed by use of a Bio-Rad MRC 600 laser scanning confocal microscope.
Cells or tissues were fixed in ice-cold 1.0% glutaraldehyde in 0.1 mol/L PBS and preserved at 4°C for further processing. When processing resumed, the cells or tissues were postfixed in 1% osmium tetroxide in the same buffer, dehydrated in graded alcohols, embedded in Epon 812, sectioned with ultramicrotome (Leica, Germany), and then stained with uranyl acetate and lead citrate. The sections were examined with a transmission electron microscope (JEOL-1230, Japan).
All data were analyzed with SPSS statistics software (Version 13.0, Chicago, IL, USA). Results were represented as mean ± standard deviation. Statistical analysis was performed using the one-way analysis of variance (ANOVA) or independent t-tests. A P-value less than 0.05 was considered statistically significant.
By intravital microscopic examination, Iba et al. found that in the venous occlusion rat model adhesion of leukocytes to endothelium was the first event after clamping followed by minute leukocyte-platelet clusters. These leukocyte-platelet aggregates move from venule to vein and finally formed a venous thrombus 14]. Thus it is important to identify what kind of factors affect the adhesion between endothelium and leukocytes. One of the possibilities, we speculated, is the remodeling of cell membrane. Emerging evidence showed that the disturbance of cellular membrane plays a pivotal role in thrombosis. For example, neutrophils interact with platelets through membrane tethers, which procedure, the authors believe, will be important in the process of inflammation and thrombosis 19]. Data indicated that platelet filopodia formation mediated by Cdc42 was required for platelet aggregation 20]. And the development of membrane tethers was essential for platelets aggregation and concomitant thrombosis 21, 22]. Another sample is that tumor vessels often exhibit ‘endothelial abnormalization', characterized by a pseudostratified, hyperactive endothelium with filopodialike protrusions. In parallel, tumor vessels often show signs of thrombotic occlusion 23]. Perhaps the rough surface of endothelium is one of the reason for thrombosis.
Theoretically, the development of membrane ruffle/tethers will increase the area for platelets to contact with endothelium so that platelets have more opportunity to interact with endothelial cells. In our study, platelets from the rapamycin-treated thrombolic rat showed more filopodia (data not shown). In addition, membrane ruffles in HUVECs and microvilli-like structures in the venous endothelium increased dramatically under the condition of rapamycin incubation. It has been found that endothelial microvilli are necessary for lymphocyte-endothelium interaction 24], thus we speculate that the enhanced membrane ruffles, microvilli (or filopodia) in both endothelium and platelets, will strengthen the interaction between them and lead to thrombosis.
Hypoxia and nutrient shortage, which are apt to occur under the condition of stasis (eg. bed rest >3 days, air travel >8 hours), can induce marked upregulation of autophagy in hemocytes and endothelial cells 25, 26]. The activated autophagy affected the rates of thrombosis 27, 28] of which the detailed mechanisms are not clear. Recently, it was identified that autophagy was essential for cytoskeleton remodeling 8, 9]. Researchers found that hemocyte-targeted RNAi depletion of autophagy-related genes (Atg4, Atg6, Atg7, Atg8a and Atg9) abolished the ability of hemocytes to spread and extend F-actin protrusions. Mammalian macrophages disrupted for autophagy remained predominantly circular in shape. In addition, live cell imaging suggests that autophagy might contribute to the cell protrusion attachment or extension 8]. More and more evidence suggested that rapamycin promote thrombosis (the mechanisms include rapamycin reducing t-PA expression and inducing PAI-1/TF expression 10, 11, 27]). These conclusions were confirmed in our experiment. In addition, we believe that membrane remodeling induced by rapamycin is one of the responsible mechanisms.
In conclusion, we found that rapamycin stimulation induced membrane remodeling in endothelial cells. And the platelet-endothelial adhesion was enhanced in parallel. Further exploration suggested that autophagy induced by rapamycin promoted membrane remodeling, platelet-endothelial adhesion, and the concomitant thrombosis.
Inferior vena cava
Deep vein thrombosis
Human umbilical vein endothelial cells
This work was supported by National Natural Science Foundation of China (31371160); China Postdoctoral Science Foundation special funded project (201104043); and Scientific research foundation of the Education Ministry for returned Chinese scholars (jws1433).
- Goldhaber SZ, Bounameaux H: Pulmonary embolism and deep vein thrombosis. Lancet. 2012, 379: 1835-1846. 10.1016/S0140-6736(11)61904-1.View ArticlePubMed
- Nishimura S, Manabe I, Nagasaki M, Kakuta S, Iwakura Y, Takayama N, Ooehara J, Otsu M, Kamiya A, Petrich BG, Urano T, Kadono T, Sato S, Aiba A, Yamashita H, Sugiura S, Kadowaki T, Nakauchi H, Eto K, Nagai R: In vivo imaging visualizes discoid platelet aggregations without endothelium disruption and implicates contribution of inflammatory cytokine and integrin signaling. Blood. 2012, 119: e45-e56. 10.1182/blood-2011-09-381400.PubMed CentralView ArticlePubMed
- Barr J, Chauhan AK, Schaeffer GV, Hansen JK, Motto DG: Red blood cells mediate the onset of thrombosis in the ferric chloride murine model. Blood. 2013, 121: 3733-3741. 10.1182/blood-2012-11-468983.PubMed CentralView ArticlePubMed
- Wang Y, Andrews M, Yang Y, Lang S, Jin JW, Cameron-Vendrig A, Zhu G, Reheman A, Ni H: Platelets in thrombosis and hemostasis: old topic with new mechanisms. Cardiovasc Hematol Disord Drug. 2012, 12: 126-132. 10.2174/1871529X11202020126.View Article
- Tsunoda N, Kokubo K, Sakai K, Fukuda M, Miyazaki M, Hiyoshi T: Surface roughness of cellulose hollow fiber dialysis membranes and platelet adhesion. ASAIO J. 1999, 45: 418-423. 10.1097/00002480-199909000-00010.View ArticlePubMed
- Herman IM, Brant AM, Warty VS, Bonaccorso J, Klein EC, Kormos RL, Borovetz HS: Hemodynamics and the vascular endothelial cytoskeleton. J Cell Biol. 1987, 105: 291-302. 10.1083/jcb.105.1.291.View ArticlePubMed
- Kublickiene K, Fu XD, Svedas E, Landgren BM, Genazzani AR, Simoncini T: Effects in postmenopausal women of estradiol and medroxyprogesterone alone and combined on resistance artery function and endothelial morphology and movement. J Clin Endocrinol Metab. 2008, 93: 1874-1883. 10.1210/jc.2007-2651.View ArticlePubMed
- Kadandale P, Stender JD, Glass CK, Kiger AA: Conserved role for autophagy in Rho1-mediated cortical remodeling and blood cell recruitment. Proc Natl Acad Sci USA. 2010, 107: 10502-10507. 10.1073/pnas.0914168107.PubMed CentralView ArticlePubMed
- Barilli A, Visigalli R, Sala R, Gazzola GC, Parolari A, Tremoli E, Bonomini S, Simon A, Closs EI, Dall'Asta V, Bussolati O: In human endothelial cells rapamycin causes mTORC2 inhibition and impairs cell viability and function. Cardiovasc Res. 2008, 78: 563-571. 10.1093/cvr/cvn024.View ArticlePubMed
- Camici GG, Steffel J, Amanovic I, Breitenstein A, Baldinger J, Keller S, Lüscher TF, Tanner FC: Rapamycin promotes arterial thrombosis in vivo: implications for everolimus and zotarolimus eluting stents. Eur Heart J. 2010, 31: 236-242. 10.1093/eurheartj/ehp259.View ArticlePubMed
- Ma Q, Zhou Y, Nie X: Rapamycin affects tissue plasminogen activator and plasminogen activator inhibitor 1 expression: a potential prothrombotic mechanism of drug-eluting stents. Angiology. 2012, 63: 330-335. 10.1177/0003319711418219.View ArticlePubMed
- Daemen J, Kukreja NN, van Twisk PH, Onuma Y, de Jaegere PP, van Domburg R, Serruys PW: Four-year clinical follow-up of the rapamycin-eluting stent evaluated at Rotterdam Cardiology Hospital registry. Am J Cardiol. 2008, 101: 1105-1111. 10.1016/j.amjcard.2007.11.074.View ArticlePubMed
- Qian J, Xu B, Lansky AJ, Yang YJ, Qiao SB, Wu YJ, Chen J, Hu FH, Yang WX, Mintz GS, Leon MB, Gao RL: First report of a novel abluminal groove filled biodegradable polymer rapamycin-eluting stent in de novo coronary artery disease: results of the first in man FIREHAWK trial. Chin Med J (Engl). 2012, 125: 970-976.
- Iba T, Aihara K, Kawasaki S, Yanagawa Y, Niwa K, Ohsaka A: Formation of the venous thrombus after venous occlusion in the experimental mouse model of metabolic syndrome. Thromb Res. 2012, 129: e246-e250. 10.1016/j.thromres.2012.03.001.View ArticlePubMed
- Stroka KM, Aranda-Espinoza H: Effects of morphology vs. cell-cell interactions on endothelial cell stiffness. Cell Mol Bioeng. 2011, 4: 9-27. 10.1007/s12195-010-0142-y.PubMed CentralView ArticlePubMed
- Mizushima N, Yoshimori T, Levine B: Methods in mammalian autophagy research. Cell. 2010, 140: 313-326. 10.1016/j.cell.2010.01.028.PubMed CentralView ArticlePubMed
- Egan K, Crowley D, Smyth P, O'Toole S, Spillane C, Martin C, Gallagher M, Canney A, Norris L, Conlon N, McEvoy L, Ffrench B, Stordal B, Keegan H, Finn S, McEneaney V, Laios A, Ducrée J, Dunne E, Smith L, Berndt M, Sheils O, Kenny D, O'Leary J: Platelet adhesion and degranulation induce pro-survival and pro-angiogenic signalling in ovarian cancer cells. PLoS One. 2011, 6: e26125-10.1371/journal.pone.0026125.PubMed CentralView ArticlePubMed
- Henke PK, Wakefield TW, Kadell AM, Linn MJ, Varma MR, Sarkar M, Hawley A, Fowlkes JB, Strieter RM: Interleukin-8 administration enhances venous thrombosis resolution in a rat model. J Surg Res. 2011, 99: 84-91.View Article
- Schmidtke DW, Diamond SL: Direct observation of membrane tethers formed during neutrophil attachment to platelets or P-selectin under physiological flow. J Cell Biol. 2000, 149: 719-730. 10.1083/jcb.149.3.719.PubMed CentralView ArticlePubMed
- Akbar H, Shang X, Perveen R, Berryman M, Funk K, Johnson JF, Tandon NN, Zheng Y: Gene targeting implicates Cdc42 GTPase in GPVI and non-GPVI mediated platelet filopodia formation, secretion and aggregation. PLoS One. 2011, 6: e22117-10.1371/journal.pone.0022117.PubMed CentralView ArticlePubMed
- Maxwell MJ, Westein E, Nesbitt WS, Giuliano S, Dopheide SM, Jackson SP: Identification of a 2-stage platelet aggregation process mediating shear-dependent thrombus formation. Blood. 2007, 109: 566-576. 10.1182/blood-2006-07-028282.View ArticlePubMed
- Nesbitt WS, Westein E, Tovar-Lopez FJ, Tolouei E, Mitchell A, Fu J, Carberry J, Fouras A, Jackson SP: A shear gradient-dependent platelet aggregation mechanism drives thrombus formation. Nat Med. 2009, 15: 665-673. 10.1038/nm.1955.View ArticlePubMed
- Carmeliet P, De Smet F, Loges S, Mazzone M: Branching morphogenesis and antiangiogenesis candidates: tip cells lead the way. Nat Rev Clin Oncol. 2009, 6: 315-326. 10.1038/nrclinonc.2009.64.View ArticlePubMed
- Guezguez B, Vigneron P, Lamerant N, Kieda C, Jaffredo T, Dunon D: Dual role of melanoma cell adhesion molecule (MCAM)/CD146 in lymphocyte endothelium interaction: MCAM/CD146 promotes rolling via microvilli induction in lymphocyte and is an endothelial adhesion receptor. J Immunol. 2007, 179: 6673-6685.View ArticlePubMed
- Sadoshima J: The role of autophagy during ischemia/reperfusion. Autophagy. 2008, 4: 402-403.View ArticlePubMed
- Codogno P, Mehrpour M, Proikas-Cezanne T: Canonical and non-canonical autophagy: variations on a common theme of self-eating?. Nat Rev Mol Cell Biol. 2011, 13: 7-12.View ArticlePubMed
- Hayashi S, Yamamoto A, You F, Yamashita K, Ikegame Y, Tawada M, Yoshimori T, Shimizu S, Nakashima S: The stent-eluting drugs sirolimus and paclitaxel suppress healing of the endothelium by induction of autophagy. Am J Pathol. 2009, 175: 2226-2234. 10.2353/ajpath.2009.090152.PubMed CentralView ArticlePubMed
- Zhaorigetu S, Yang Z, Toma I, McCaffrey TA, Hu CA: Apolipoprotein L6, induced in atherosclerotic lesions, promotes apoptosis and blocks Beclin 1-dependent autophagy in atherosclerotic cells. J Biol Chem. 2011, 286: 27389-27398. 10.1074/jbc.M110.210245.PubMed CentralView ArticlePubMed
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