Abstract
Although drug-eluting stents (DESs) can decrease the risk of restenosis, this benefit is tempered by a possible increased risk of in-stent thrombosis. We assessed the effects of rapamycin on human umbilical vein endothelial cells (HUVECs) to identify the alterations in gene expression associated with thrombosis. Expression of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1) was assessed in HUVECs treated with rapamycin (final concentrations: 1, 10, 100, and 1000 ng/mL) for 24 and 48 hours. Incubation of HUVECs with rapamycin strongly reduced the expression of t-PA in a concentration-dependant manner (P < .05 to < .01). However, the expression of PAI-1 was induced by rapamycin (P < .05 to < .01). The increase in PAI-1 induction was up to 3.3-fold. In conclusion, rapamycin inhibited t-PA and induced PAI-1 expression in HUVECs. This effect may contribute to in-stent thrombosis associated with DESs.
Keywords
In-stent thrombosis remains a severe complication owing to the associated high mortality. 1 The introduction of drug-eluting stents (DESs) has been associated with late (>12 months) in-stent thrombosis. 1 Several factors are associated with the risk of in-stent thrombosis, including the procedures (eg, stent position, under expansion, number of implanted stents, stent length, and persistent slow coronary blood flow), patient and lesion characteristics, stent design (cell cycle inhibitors and/or polymers), and cessation of antiplatelet drugs. 2 Inhibitors released from DESs primarily aim at preventing vascular smooth muscle cell proliferation and migration in order to reduce restenosis. 3 However, they also delay reendothelialization and induce procoagulant expression, which can increase the risk of in-stent thrombosis. 4
Rapamycin (sirolimus) used on DESs is a bacterial macrolide that forms a complex with FK-binding protein which binds to the mammalian target of rapamycin (mTOR). 5 In the literature, there is scant evidence of a prothrombotic effect of systemic administration of rapamycin. 6,7 However, it is plausible to consider that rapamycin has prothrombotic potential. 8,9 We aimed to demonstrate the role of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1) in in-stent thrombosis induced by rapamycin.
Materials and Methods
Materials
The rapamycin for experiment was provided by LC Laboratories (Woburn, Massachusetts). It was dissolved in dimethyl sulfoxide (DMSO), purchased from Sigma (San Francisco, California), and diluted in phosphate-buffered saline (PBS) at the final concentration of 1, 10, 100, and 1000 ng/μL. Human umbilical vein endothelial cells (HUVECs) were obtained from ScienCell Research Laboratories (Carlsbad, California) and cultured in endothelial cell medium (ScienCell). Thrombin (Sigma) was dissolved in PBS at a concentration of 2 U/μL. Total ribonucleic acid (RNA) from HUVECs was isolated using the SV total RNA isolation system obtained from Promega Company (Madison, Wisconsin), and the total RNA was reverse transcribed to complementary deoxyribonucleic acid (cDNA) using the GoScript Reverse Transcriptase, which was also obtained from Promega Company. We performed real-time polymerase chain reaction (PCR) on BioRad iQ5 (Hercules, California) using SYBR Premix Ex Taq, which was purchased from Takara Belmont Corporation (Osaka, Japan). Protein concentration was determined with bicinchoninic acid (BCA) protein assay reagent, which was purchased from Pierce Thermo Fisher Scientific (Rockford, Illionis). Polyvinylidene fluoride (PVDF) membrane was obtained from Millipore (Billerica, Massachusetts). The t-PA primary antibody was purchased from Abcam (Cambridge, UK). Both PAI-1 and the β-actin primary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, California). The horseradish peroxidase–labeled secondary antibodies were obtained from Agrisera (Vännäs, Sweden).
Cell Culture
Human umbilical vein endothelial cells were cultured in endothelial cell medium under 37°C and 5% CO2 according to the manufacturer’s instructions. Endothelial cell medium consisted of 500 mL of basal medium, 25 mL of fetal bovine serum, 5 mL of endothelial cell growth supplement, and 5 mL of penicillin/streptomycin solution. We prepared poly-
Cell Treatment
At the concentration of 2 U/mL, HUVECs were stimulated with thrombin. After stimulation for 4 hours, rapamycin was added to the culture dishes at final concentrations of 1, 10, 100, and 1000 ng/mL. Human umbilical vein endothelial cells treated with rapamycin at multiple concentrations were incubated for 24 and 48 hours.
Real-Time Quantitative Reverse Transcriptase-Polymerase Chain Reaction
We prepared purified and intact total RNA from HUVECs (n = 6, for each dose) using the SV total RNA isolation system according to the manufacturer’s recommendations. The total RNA was quantified by absorbance at 260 nm and was reverse transcribed to cDNA using the GoScript Reverse Transcriptase. Heat the tube of RNA to 70°C for 5 minutes and cool it immediately on ice. Then the RT reagents were transferred to an ice bath (total volume: 20 μL). After incubation at 42°C for 1 hour, reverse transcriptase (RT) products were obtained. Quantitative PCR was performed using SYBR Premix Ex Taq on the BioRad iQ5. We performed initial denaturation at 95°C for 10 seconds. Then, we used the shuttle PCR method (95°C/5 seconds, 61°C/30 seconds; 40 cycles). Finally, melting curve was made from 55°C to 95°C by increasing the temperature 0.2°C/s. We determined the amount of t-PA and PAI-1 RNA expression using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as control. Fold changes in messenger RNA (mRNA) expression were calculated using the 2–ΔΔCt method. The sequences of the specific primers were referred to GenBank and designed as follows: PAI-1: sense, 5′-CAGACCAAGAGCCTCTCCAC-3′, antisense, 5′-ATCACTTGGCCCATGAAAAG-3; t-PA: sense, 5′-TTGGCCCAGAAGCCCTACA-3′, antisense, 5′-GGCTGACCCATTCCCAAAGTAG-3′; GAPDH: sense, 5′-TGGTCTCCTCTGACTTCAAC-3′, antisense, 5′-GTGAGGGTCTCTCTCTTCCT-3′.
Western Blot
Protein expression was determined by Western blot analysis (n = 6, for each dose). Human umbilical vein endothelial cells were lysed in 50 μL radioimmunoprecipitation assay (RIPA) lysis buffer with protease inhibitors and phosphatase inhibitors. Cell debris was removed by centrifugation. Protein concentration was determined with BCA protein assay reagent. Each lane was loaded with protein (20 µg). Protein was separated by 8% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane by wet transfer (200 mA, 2 hours). Membranes were soaked in a blocking solution containing Tris-HCl buffered salt solution (TBS) with 3% bovine serum albumin (BSA) and 0.1% Tween 20 for 1 hour at room temperature. The primary antibody of t-PA was used at 1:1000 dilution and antibody against PAI-1 was used at 1:500 dilution for 12 hours (4°C). Blots were controlled with β-actin expression (1:2000 dilution) and were incubated with horseradish peroxidase–labeled secondary antibodies for 1 hour at room temperature. Immunoreactive bands were detected with Enhanced Chemiluminescence (ECL).
Statistical Analysis
Data were expressed as mean ± standard deviation (SD). We used 1-way analysis of variance (ANOVA) to analyze the differences in variables. P < .05 (2-tailed) was considered significant. All statistical analyses were performed with SPSS 18.0.
Results
Rapamycin Inhibition of t-PA Expression in HUVECs
Stimulation of HUVECs with thrombin (2 U/mL) reduced t-PA mRNA and protein expression (Figures 1 and 2 ). Rapamycin (1-1000 ng/mL) resulted in a concentration-dependent decrease in t-PA mRNA expression compared with stimulation using thrombin alone (Figure 1A; P < .05 to < .01 for rapamycin + thrombin vs thrombin alone). Changes in t-PA mRNA expression remained low for 48 hours after adding rapamycin (Figure 1B; P < .05 to < .01 for rapamycin + thrombin vs thrombin alone). There was also a significant decrease in the protein expression of t-PA (Figure 2; P < .05 to < .01 for rapamycin + thrombin vs thrombin alone).
Effect of t-PA mRNA expression after treating rapamycin for 24 and 48 hours. A, Results of 24-hour t-PA mRNA expression. B, Results of 48-hour t-PA mRNA expression. Every bar represented the mean ± SD (n = 6). Rapamycin resulted in a concentration-dependent decrease in t-PA mRNA expression compared with stimulation using thrombin alone (*P < .05, **P < .01 for rapamycin+ thrombin vs thrombin alone). t-PA indicates tissue plasminogen activator; mRNA, messenger RNA; SD, standard deviation.
Effect of t-PA protein expression after treating rapamycin for 24 hours. A, Results of t-PA 24-hour protein expression. B, Quantitative results of t-PA protein expression. Every bar represents the mean ± SD (n = 6). There was a significant decrease in t-PA protein expression after treating with rapamycin compared with stimulation using thrombin alone (*P < .05, **P < .01 for rapamycin + thrombin vs thrombin alone). t-PA indicates tissue plasminogen activator; SD, standard deviation.
Rapamycin Induces PAI-1 Expression in HUVECs
Stimulation of HUVECs with thrombin (2 U/mL) induced PAI-1 mRNA and protein expression (Figures 3 and 4 ). Rapamycin (10-1000 ng/mL) resulted in a concentration-dependent enhancement of mRNA expression of PAI-1 compared with stimulation using thrombin alone (Figure 3A; P < .05 to < .01 for rapamycin + thrombin vs thrombin alone). The increase in PAI-1 induction was 1.9-, 3.1-, and 3.3-fold for rapamycin 10, 100, and 1000 ng/mL, respectively (Figure 3A; P < .05 for 10 ng/mL vs thrombin alone and P < .01 for 100 and 1000 ng/mL vs thrombin alone). However, there was no significant change in PAI-1 mRNA expression after rapamycin treatment for 48 hours compared with thrombin alone (Figure 3B). There was also a significant increase in PAI-1 protein expression after adding rapamycin at 1000 ng/mL (Figure 4; P < .05 for rapamycin + thrombin vs thrombin alone).
Effect of PAI-1 mRNA expression after treating rapamycin for 24 and 48 hours. A, Results of PAI-1 24-hour mRNA expression. Every bar represented the mean ± SD (n = 6). Rapamycin resulted in a concentration-dependent enhancement of PAI-1 mRNA expression compared with stimulation using thrombin alone (*P < .05, **P < .01 for rapamycin + thrombin vs thrombin alone). B, Results of PAI-1 48-hour mRNA expression. There was no significant change in PAI-1 mRNA expression after treating rapamycin for 48 hours compared with thrombin alone. PAI-1 indicates plasminogen activator inhibitor 1; mRNA, messenger RNA; SD, standard deviation.
Effect of PAI-1 protein expression after treating rapamycin for 24 hours. A, Results of PAI-1 protein expression for 24 hours. B, Quantitative results of PAI-1 protein expression. Bar represented the mean ± SD (n = 6). There was a significant increase in PAI-1 protein expression of HUVECs treated with 1000 ng/mL rapamycin compared with stimulation using thrombin alone (*P < .05 for rapamycin + thrombin vs thrombin alone). HUVECs indicates human umbilical vein endothelial cell; PAI-1, plasminogen activator inhibitor 1; SD, standard deviation.
Discussion
In the era of bare metal stents (BMSs), in-stent thrombosis was a serious complication owing to its high mortality. 1 With the introduction of P2Y12-receptor antagonists, the incidence of in-stent thrombosis decreased substantially. 10 Subsequently, DESs came into use and were superior to BMSs by decreasing the restenosis rates as well as major adverse cardiac events. 11–13 Despite reduced restenosis rates, the frequency of in-stent thrombosis has not decreased compared with BMSs, especially very late in-stent thrombosis. 14–20 The mechanisms responsible for DES-induced in-stent thrombosis are not completely defined.
Endothelial dysfunction and partial reendothelialization have been documented after DESs deployment. 21–23 Although rapamycin-eluting stents were designed so that nearly 80% of the drug is eluted by 30 days, 11,12 rapamycin easily penetrates cell walls owing to its lipophilic properties, leading to retention in arterial tissue. 24,25
In this study, we used real-time quantitative RT-PCR and Western blot analysis to assess the effects of rapamycin on endothelial gene expression, with a focus on identifying transcripts that might be associated with thrombosis. We found that HUVECs treated with rapamycin demonstrated concentration-dependent increases in PAI-1 and concentration-dependent decreases in t-PA expression. The concentrations of rapamycin occurring in vivo compared well with those used in our study. The maximal systemic concentration of rapamycin after deployment of 2 stents was reported to be nearly 1 ng/mL (1.15 × 10−9 mol/L ≈ 1.0 ng/mL). 26 However, local concentration was significantly higher (80-200 ng/mL). Because of the lipophilic properties of rapamycin, leading to its accumulation in the vessel wall, 25–27 we used 1000 ng/mL as our maximal concentration.
Some researchers have studied the potential mechanisms responsible for rapamycin-induced in-stent thrombosis. On a subcellular level, rapamycin binds to FK-binding protein 12 and subsequently inhibits the mTOR. 28 The mTOR is a downstream target of the phosphatidylinositol 3 kinase (PI3K) pathway. The mTOR is an inhibitor of tissue factor (TF) in endothelial cells and monocytes. 3,29,30 Consequently, rapamycin inhibited the PI3K/Akt/mTOR pathway so as to increase TF expression and activity in response to tumor necrosis factor α (TNF-α), histamine, thrombin, or vascular endothelial growth factor (VEGF). 31–33 Furthermore, Jin et al 34 found that rapamycin downregulated endothelial NO synthase (eNOS).
Our study has limitations. It was an in vitro investigation of HUVECs and might not represent the complex biology of the in vivo situation. Our group intends to further investigate the clinical relevance of the present findings.
In summary, rapamycin increased PAI-1 and decreased t-PA expression in vitro. This effect may contribute to in-stent thrombosis associated with DESs. This concept suggests that alternative inhibitors of proliferation may be preferable to rapamycin. However, only event-based trials can prove this point.
Footnotes
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This article is supported by the National Nature Science Foundation of China (#30971238).