| BackgroundNowadays, with rapid development of the percutaneous coronary intervention, drug-eluting stents have already achieved a remarkable success. Compared with bare-metal stents, the drug-eluting stents has unparalleled advantages in preventing restenosis after angioplasty. However, with the large-scale clinical application of drug-eluting stents, in-stent thrombosis has attracted more attention. Compared with the bare-metal stents, drug-eluting stent has not changed, or even increased the incidence of stent thrombosis. In particular, late stent thrombosis even led to concerns of the application of drug-eluting stent.With the continuous deepening of the study, it was found that stent thrombosis is closely related to re-endothelialization in local impaired blood vessels, that is, the more complete re-endothelialization, the lower the probability of in-stent thrombosis. Previous experiments found that, impaired vessel was completely re-endothelialized within 28 days after bare-metal stent implantation. However, in the drug-eluting stents, endothelium healing was not completed after more than 6 months, and local impaired vessel showed fibrin deposition and inflammatory cells attachment in the surface of the vessel, thus high incidence of stent thrombosis.Rapamycin (sirolimus) is a macrolide immunosuppressant. With potent property of immune suppression, anti-inflammatory and anti-cell proliferation, it was coated on the metal surface, released into local lesion after stent implantation. It was presumed to inhibit vascular smooth muscle cells (VSMCs) migration and proliferation, to impede the formation of neo-endothelium, thereby reducing the restenosis rate. Rapamycin mainly combines with the cytoplasmic protein-FK506 binding protein 12 (FKBP-12), and with binding a specific intracellular cell cycle regulatory proteins, mammalian target of rapamycin (mTOR). The FKBP12/rapamycin complex inhibits mTOR activity, thereby interferes with multiple cycle regulatory proteins involving in G1 to S phases, and ultimately induces cell cycle arrested in late G1 phase. In view of the universal expression of cellular cell cycle regulatory proteins, it is conceivable that rapamycin not only inhibits VSMCs proliferation and migration, but also acts on endothelial cells (ECs) proliferation and migration, and even impedes the endothelial progenitor cells (EPCs) positioning and attaching in local lesion. This may induces delayed re-endothelialization in drug-eluting stent.Objectives1. Dogs were chosen and DES and BMS were implantated in order to evaluate the effects of rapamycin on re-endothelialization.2. To determine the effects of rapamycin on endothelial cells proliferation and migration, and the effects on endothelial progenitor cells growth in vitro; In vivo, observe of rapamycin on VEGF expression in circulation.3. To investigate whether PI3K/Akt and mTOR/p70 S6 kinase signaling pathway are involved in rapamycin inhibiting endothelial cells proliferation and migration and to identify proteins'role in the inhibiting process; to determine the relationship between PI3K/Akt and mTOR/p70 S6K signaling protein in rapamycin inhibiting endothelial cells proliferation and migration.Methods1. Six 18-22 kg male mongrel dogs were given percutaneous coronary intervention. Bare metal stent and drug-eluting stent were implanted in the left distal and proximal internal thoracic artery respectively. Electron microscopy was used to evaluate the endothelial coverage and adheresion of platelets on the surface of the stents.2. Endothelial cells or endothelial progenitor cells were treated with Rapa (0.1, 1, 10 and 100 ng/ml) or carrier (0.1% methanol) in reduced-serum media (DMEM containing 5% FBS). After incubation for 24 h, 48 h or 72 h, cells proliferation was quantified by MTT assay. Cell migration was investigated by scratch assay and counted by using Transwell chamber; and cells apoptosis was detected by flow cytometry.3. SD rats, weighing 100 to 150 g, were administered Rapa (0.05, 0.10, 0.15 and 0.20 mg/kg/d) or 10% methanol (0.1 ml/kg/d) intraperitoneally for 5 days. VEGF levels in plasma were measured in triplicate or quadruplicate with an ELISA test.4. Pretreating endothelial cells with rapamycin (10 ng/ml) or carrier (0.1% methanol) for 24 h. Phosphorylation levels of FKBP-12, mTOR and p70 S6 kinase was investigated by Western blot analysis.5. Endothelial cells were randomly divided into following groups: Control; VEGF (20 ng/ml); VEGF (20 ng/ml) + Wortmannin (100 nM); VEGF (20 ng/ml) + LY294002 (20μM). After incubated in different groups for 24 h, endothelial cells proliferation and migration were observed respectively. Meanwhile, Phosphorylation levels of mTOR and p70 S kinase were detected by Western blot analysis.6. Endothelial cells were randomly divided into following groups: Control; VEGF (20 ng/ml); VEGF (20 ng/ml) + Wortmannin (100 nM); VEGF (20 ng/ml) + LY294002 (20μM). After incubated in different groups for 4 h, endothelial cells were lysised and the changes of Akt and p-Akt were recorded by Western blot analysis.Results1. All dogs survived stent implantation. Electron microscopy showed endothelium complete covering the BMS stent. However, poor endothelial cells junction formation was observed on the DES stent and part of stent exposed to the vessel lumen. Platelets were adhered to the surface of stent.2. ECs were incubated with rapamycin for 24 h, and cells proliferation was reduced by 10 ng/ml and 100 ng/ml Rapa to 71.5%±6.4% (P < 0.01) and 57.0%±9.0% (P < 0.001) of controls, respectively. Low concentration of Rapa (1 ng/ml) also decreased ECs proliferation after 48 h (55.3%±13.4% of control, P < 0.01). After 72 h, Rapa of 0.1 ng/ml caused clear reductions in cells proliferation (26.3%±11.9% vs. Control, P < 0.01). Rapamycin inhibited cells migration to 75.5%±7.0% at 1 ng/ml (P < 0.05), 65.2%±4.3% at 10 ng/ml (P < 0.001), and 39.5%±8.0% at 100 ng/ml (P < 0.001).3. Rapamycin dose- and time-dependently inhibited ECs proliferation, with 0.1 ng/ml showing 98.7%±8.9% (P > 0.05), 1 ng/ml showing 84.9%±6.7% (P < 0.001), 10 ng/ml showing 77.2%±6.1% (P < 0.001) and 100 ng/ml showing 69.0%±4.1% (P < 0.001) of control cells growth, respectively. Rapamycin also induced EPCs apoptosis. The normal apoptotic index in EPCs was 4.2‰±0.5‰, and all higher rapamycin levels increased this index, with 1, 10 and 100 ng/ml showing apoptotic levels of 10.8‰±0.8‰, 14.4‰±1.0‰, and 18.6‰±1.2‰respectively (all P < 0.001).4. A 5-day treatment with rapamycin decreased VEGF expression at higher doses (0.15 and 0.2 mg/kg/d; P < 0.05 and P < 0.01) but not lower doses (0.05 and 0.10 mg/kg/d).5. Western blot analysis showed that rapamycin inhibited mTOR phosphorylation but not FKBP12 phosphorylation after endothelial cells were incubated with rapamycin (10ng/ml) for 24 h. Similarly, rapamycin (10ng/ml) also inhibited the phosphorylation of p70 S6K, the downstream target of mTOR.6. The phosphorylation mTOR and p70 S6K were enhanced after endothelial cells were incubated with VEGF for 4 h. However, both Wortmannin (100 nM) and LY294002 (20μM) completely abolished mTOR and p70 S6K activation in response to VEGF. VEGF-induced endothelial cells proliferation (175.7%±8.9% vs. control, P < 0.001) and migration (188.0%±5.5% vs. control, P < 0.001) were abolished by LY294002 and Wortmannin (89.3%±11.3% and 83.7%±6.8% vs. control, P > 0.05; 82.8%±10.9% and 99.0%±5.9% vs. control, P > 0.05; respectively), consistent with the suppression of mTOR and p70 S6K.7. Endothelial cells were treated with 100 nM Wortmannin or 20μM LY294002 for 4 h and Western blot analysis showed that both LY294002 and Wortmannin inhibited Akt phosphorylation. However, rapamycin increased Akt phosphorylation after endothelial cells were incubated with rapamycin for 4 h.Conclusions1. Rapamycin impedes re-endothelialization after drug eluting stent (DES) implantation and the mechanism may involves with inhibiting proliferation and migration of ECs, inducing EPCs apoptosis, and decreasing VEGF expression in the circulation.2. Our data demonstrated that rapamycin inhibits ECs proliferation and migration through PI3K-mediated mTOR/p70 S6K activation. As the upstream proteins, PI3K/Akt signaling proteins are needed for the activation of mTOR/p70.3. The relationship of PI3K/Akt and mTOR/p70 S6 kinase is not just a line signaling pathway. Akt may centers a negative feedback between PI3K and mTOR in ECs during rapamycin inhibiting the activation of mTOR/p70 S6 kinase.
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