雷帕霉素抑制内皮细胞增殖和迁移：PI3K/Akt/mTOR/p70S6K信号通路的作用
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摘要
研究背景
冠状动脉介入治疗发展到今天,药物洗脱支架已经取得令人瞩目的效果。相对于金属裸支架治疗而言,药物洗脱支架在减少血管成形术后再狭窄方面有着不可比拟的优势。但随着其大规模的临床应用,药物洗脱支架术后支架内血栓又引起了人们的关注。与裸支架相比,药物洗脱支架降低了术后再狭窄率却没有改变甚至增加了支架内血栓的发生。尤其是晚期支架内血栓形成更加导致人们对药物洗脱支架应用的疑虑。
随着研究的不断深入,人们发现,支架内血栓形成与支架表面的内皮化程度密切相关,即内皮化越完全,支架内血栓发生的机率越低。实验中发现,祼支架术后28天内可完全内皮化,而药物洗脱支架在6个月时还显示内膜愈合不完全,表面有纤维蛋白沉积及炎症细胞附着,易发生支架内血栓。而多数研究认为,引起药物支架表面内皮化不完全的主要原因是支架表面的涂层药物。
雷帕霉素(西罗莫司)是一种大环内酯类免疫抑制剂。由于该药有免疫抑制、抗炎和抗细胞增殖作用,所以人们将其涂层于金属支架表面,通过支架植入后药物在病变部位的局部释放,抑制血管平滑肌细胞(Vascular smooth muscle cells, VSMCs)的迁移和增殖,最终减少术后再狭窄率。雷帕霉素主要是与胞浆蛋白--FK506结合蛋白12(FKBP-12)结合形成FKBP12/雷帕霉素复合物,再与胞内特定的细胞周期调节蛋白--哺乳动物雷帕霉素靶分子(Mammalian target of rapamycin, mTOR)结合,并抑制其活性,从而干预多个G1期向S期过渡的周期调控蛋白,最终诱导细胞周期停滞于G1期的晚期。鉴于细胞普遍表达细胞周期调控蛋白,可以想象,雷帕霉素不但影响VSMCs的增殖和迁移,同样影响内皮细胞的增殖和迁移及内皮祖细胞在局部的定位及附着,而这两种细胞对内皮修复是至关重要。这也可能是药物洗脱支架术后,支架表面内皮化延迟的原因。
研究目的
1.动物在体实验观察植入祼金属支架和药物洗脱支架28天后,局部血管段内皮愈合和血小板黏附情况。
2.体外细胞实验观察雷帕霉素对内皮细胞增殖和迁移的影响;对内皮祖细胞凋亡的影响。同时在体实验观察雷帕霉素对循环中VEGF表达的影响。
3.研究PI3K/Akt及mTOR/p70S6K信号通路是否参与雷帕霉素对内皮细胞增殖和迁移的抑制作用,并明确PI3K/Akt与mTOR/p70S6K信号蛋白在其中的作用和两者的相互关系。
实验方法
1.六只18-22 kg雄性杂种犬在左胸廓内动脉远端及近端分别植入Cypher雷帕霉素药物洗脱支架和Bx sonic裸金属支架。术前及术后给予双联抗血小板治疗;术后1个月,处死杂种犬,解剖离取支架植入处血管段并固定;纵切后电镜扫描观察支架表面血管覆盖情况及是否有血小板黏附或微血栓形成。
2.原代培养大鼠心肌微血管内皮细胞及内皮祖细胞,加入不同浓度的雷帕霉素(0.1、1、10和100 ng/ml)或等体积的10%甲醇孵育24、48或72小时。MTT法检测内皮细胞及内皮祖细胞增殖;划痕法及Tranwell小室法观察内皮细胞迁移;流式细胞术检查内皮细胞及内皮祖细胞凋亡。
3. SD雄性大鼠(100-150 g)给予雷帕霉素(0.05、0.10、0.15和0.20 mg/kg/d)或者10%甲醇(0.1 ml/kg/d)连续腹腔注射5天,尾静脉采血,ELISA法检测大鼠血循环中VEGF水平。
4.雷帕霉素(10 ng/ml)孵育微血管内皮细胞24小时后,Western blot法观察FKBP-12、mTOR及p70 S6激酶磷酸化水平,并与用同等浓度甲醇孵育的对照组进行比较。
5.原代培养的微血管内皮细胞分组为:对照组;VEGF (20 ng/ml)组;VEGF (20 ng/ml)+Wortmannin (100 nM)组及VEGF (20 ng/ml)+LY294002 (20μM)组。各组细胞培养24小时后,观察内皮细胞的增殖和迁移。同时Western blot法检测mTOR和p70 S激酶的表达水平和磷酸化水平。
6.原代培养的微血管内皮细胞分组为:对照组;VEGF (20 ng/ml)组;VEGF (20 ng/ml)+Wortmannin (100 nM);VEGF (20 ng/ml)+LY294002 (20μM)组;雷帕霉素(10 ng/ml)组。各组内皮细胞培养4小时后,裂解细胞,Western blot法观察Akt和p-Akt表达水平。
实验结果
1.犬胸廓内动脉金属裸支架及药物支架植入30天后的电镜扫描发现:与金属裸支架相比,药物支架表面内皮化不完全;部分药物支架暴露于管腔;局部表面有血小板黏附形成。
2.孵育24小时后,10 ng/ml及100 ng/ml的雷帕霉素明显抑制内皮细胞增殖(71.5%±6.4% vs. Control, P < 0.01;57.0%±9.0% vs. Control, P < 0.001);48小时后,1 ng/ml的雷帕霉素也明显抑制内皮细胞增殖(55.3%±13.4% vs. Control,P < 0.01);0.1 ng/ml组于72小时后明显抑制内皮细胞增殖(26.3%±11.9 % vs. Control,P < 0.01)。孵育24小时后,雷帕霉素明显抑制内皮细胞的迁移,1 ng/ml组是对照组的75.5%±7.0%(P < 0.05);10 ng/ml组是对照组的65.2%±4.3%(P < 0.001);而100 ng/ml组是对照组的39.5%±8.0%(P < 0.001)。和对照组相比,0.1、1、10和100 ng/ml的雷帕霉素组中内皮祖细胞的增殖分别为98.7%±8.9%(P > 0.05)、84.9%±6.7%(P < 0.001)、77.2%±6.1%(P < 0.001)及69.0%±4.1% (P < 0.001)。雷帕霉素也诱导内皮祖细胞的凋亡,正常内皮祖细胞组的凋亡率为4.2‰±0.5‰,而1、10和100 ng/ml的雷帕霉素组中,内皮祖细胞的凋亡率分别为:10.8‰±0.8‰、14.4‰±1.0‰及18.6‰±1.2‰(P < 0.001)。
3. SD大鼠腹腔给药5天后与对照组相比,高浓度的雷帕霉素(0.15和0.2 mg/kg/d)明显抑制循环中VEGF的表达(P < 0.05及P < 0.01),但较低浓度的雷帕霉素(0.05和0.10 mg/kg/d)没有抑制作用。
4. Western blot法分析见雷帕霉素(10 ng/ml)孵育24小时后抑制内皮细胞中mTOR的磷酸化水平,但FKBP-12磷酸化水平未有改变,而且内皮细胞中p70 S6激酶的磷酸化也被雷帕霉素抑制。
5. VEGF作用4小时后,内皮细胞中mTOR和p70 S6K蛋白磷酸化水平明显增加,但PI3K/Akt的特异性抑制剂Wortmannin或LY294002可完全阻断VEGF的这一作用。
6. VEGF明显促进内皮细胞迁移(188.0%±5.5% vs.对照组, P < 0.001);而VEGF+LY294002或者VEGF+Wortmannin组与对照组比较,内皮细胞的迁移无差别(89.3%±11.3%及83.7%±6.8% vs.对照组, P > 0.05)。同样观察到VEGF明显促进内皮细胞增殖(175.7%±8.9% vs.对照组, P < 0.001);而VEGF+LY294002或者VEGF+Wortmannin组与对照组比较则无变化(82.8%±10.9%及99.0%±5.9% vs.对照组, P > 0.05)。
7. Wortmannin和LY294002都可抑制内皮细胞中Akt蛋白的磷酸化。但雷帕霉素孵育4小时后,Akt的磷酸化水平增高。
结论
1.雷帕霉素洗脱支架植入术后28天,支架表面内皮化不完全。其原因可能为雷帕霉素抑制内皮细胞的增殖和迁移、诱导内皮祖细胞的凋亡、并且减少局部血管中VEGF的表达,从而延缓药物洗脱支架后再内皮化进程。
2.雷帕霉素通过抑制mTOR/p70 S6K蛋白的激活进而抑制内皮细胞的增殖和迁移;PI3K /Akt信号蛋白是激活mTOR/p70 S6K蛋白所必需的,并且是上游信号蛋白。
3. PI3K /Akt信号蛋白和mTOR/p70 S6K信号蛋白并非是单纯的线性关系,在雷帕霉素抑制mTOR/p70 S6K蛋白的过程中,PI3K和mTOR之间存在反馈调节,Akt可能是反馈调节的中间分子。
Background
Nowadays, 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.
Objectives
1. 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.
Methods
1. 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.
Results
1. 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.
Conclusions
1. 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.
冠状动脉介入治疗发展到今天,药物洗脱支架已经取得令人瞩目的效果。相对于金属裸支架治疗而言,药物洗脱支架在减少血管成形术后再狭窄方面有着不可比拟的优势。但随着其大规模的临床应用,药物洗脱支架术后支架内血栓又引起了人们的关注。与裸支架相比,药物洗脱支架降低了术后再狭窄率却没有改变甚至增加了支架内血栓的发生。尤其是晚期支架内血栓形成更加导致人们对药物洗脱支架应用的疑虑。
随着研究的不断深入,人们发现,支架内血栓形成与支架表面的内皮化程度密切相关,即内皮化越完全,支架内血栓发生的机率越低。实验中发现,祼支架术后28天内可完全内皮化,而药物洗脱支架在6个月时还显示内膜愈合不完全,表面有纤维蛋白沉积及炎症细胞附着,易发生支架内血栓。而多数研究认为,引起药物支架表面内皮化不完全的主要原因是支架表面的涂层药物。
雷帕霉素(西罗莫司)是一种大环内酯类免疫抑制剂。由于该药有免疫抑制、抗炎和抗细胞增殖作用,所以人们将其涂层于金属支架表面,通过支架植入后药物在病变部位的局部释放,抑制血管平滑肌细胞(Vascular smooth muscle cells, VSMCs)的迁移和增殖,最终减少术后再狭窄率。雷帕霉素主要是与胞浆蛋白--FK506结合蛋白12(FKBP-12)结合形成FKBP12/雷帕霉素复合物,再与胞内特定的细胞周期调节蛋白--哺乳动物雷帕霉素靶分子(Mammalian target of rapamycin, mTOR)结合,并抑制其活性,从而干预多个G1期向S期过渡的周期调控蛋白,最终诱导细胞周期停滞于G1期的晚期。鉴于细胞普遍表达细胞周期调控蛋白,可以想象,雷帕霉素不但影响VSMCs的增殖和迁移,同样影响内皮细胞的增殖和迁移及内皮祖细胞在局部的定位及附着,而这两种细胞对内皮修复是至关重要。这也可能是药物洗脱支架术后,支架表面内皮化延迟的原因。
研究目的
1.动物在体实验观察植入祼金属支架和药物洗脱支架28天后,局部血管段内皮愈合和血小板黏附情况。
2.体外细胞实验观察雷帕霉素对内皮细胞增殖和迁移的影响;对内皮祖细胞凋亡的影响。同时在体实验观察雷帕霉素对循环中VEGF表达的影响。
3.研究PI3K/Akt及mTOR/p70S6K信号通路是否参与雷帕霉素对内皮细胞增殖和迁移的抑制作用,并明确PI3K/Akt与mTOR/p70S6K信号蛋白在其中的作用和两者的相互关系。
实验方法
1.六只18-22 kg雄性杂种犬在左胸廓内动脉远端及近端分别植入Cypher雷帕霉素药物洗脱支架和Bx sonic裸金属支架。术前及术后给予双联抗血小板治疗;术后1个月,处死杂种犬,解剖离取支架植入处血管段并固定;纵切后电镜扫描观察支架表面血管覆盖情况及是否有血小板黏附或微血栓形成。
2.原代培养大鼠心肌微血管内皮细胞及内皮祖细胞,加入不同浓度的雷帕霉素(0.1、1、10和100 ng/ml)或等体积的10%甲醇孵育24、48或72小时。MTT法检测内皮细胞及内皮祖细胞增殖;划痕法及Tranwell小室法观察内皮细胞迁移;流式细胞术检查内皮细胞及内皮祖细胞凋亡。
3. SD雄性大鼠(100-150 g)给予雷帕霉素(0.05、0.10、0.15和0.20 mg/kg/d)或者10%甲醇(0.1 ml/kg/d)连续腹腔注射5天,尾静脉采血,ELISA法检测大鼠血循环中VEGF水平。
4.雷帕霉素(10 ng/ml)孵育微血管内皮细胞24小时后,Western blot法观察FKBP-12、mTOR及p70 S6激酶磷酸化水平,并与用同等浓度甲醇孵育的对照组进行比较。
5.原代培养的微血管内皮细胞分组为:对照组;VEGF (20 ng/ml)组;VEGF (20 ng/ml)+Wortmannin (100 nM)组及VEGF (20 ng/ml)+LY294002 (20μM)组。各组细胞培养24小时后,观察内皮细胞的增殖和迁移。同时Western blot法检测mTOR和p70 S激酶的表达水平和磷酸化水平。
6.原代培养的微血管内皮细胞分组为:对照组;VEGF (20 ng/ml)组;VEGF (20 ng/ml)+Wortmannin (100 nM);VEGF (20 ng/ml)+LY294002 (20μM)组;雷帕霉素(10 ng/ml)组。各组内皮细胞培养4小时后,裂解细胞,Western blot法观察Akt和p-Akt表达水平。
实验结果
1.犬胸廓内动脉金属裸支架及药物支架植入30天后的电镜扫描发现:与金属裸支架相比,药物支架表面内皮化不完全;部分药物支架暴露于管腔;局部表面有血小板黏附形成。
2.孵育24小时后,10 ng/ml及100 ng/ml的雷帕霉素明显抑制内皮细胞增殖(71.5%±6.4% vs. Control, P < 0.01;57.0%±9.0% vs. Control, P < 0.001);48小时后,1 ng/ml的雷帕霉素也明显抑制内皮细胞增殖(55.3%±13.4% vs. Control,P < 0.01);0.1 ng/ml组于72小时后明显抑制内皮细胞增殖(26.3%±11.9 % vs. Control,P < 0.01)。孵育24小时后,雷帕霉素明显抑制内皮细胞的迁移,1 ng/ml组是对照组的75.5%±7.0%(P < 0.05);10 ng/ml组是对照组的65.2%±4.3%(P < 0.001);而100 ng/ml组是对照组的39.5%±8.0%(P < 0.001)。和对照组相比,0.1、1、10和100 ng/ml的雷帕霉素组中内皮祖细胞的增殖分别为98.7%±8.9%(P > 0.05)、84.9%±6.7%(P < 0.001)、77.2%±6.1%(P < 0.001)及69.0%±4.1% (P < 0.001)。雷帕霉素也诱导内皮祖细胞的凋亡,正常内皮祖细胞组的凋亡率为4.2‰±0.5‰,而1、10和100 ng/ml的雷帕霉素组中,内皮祖细胞的凋亡率分别为:10.8‰±0.8‰、14.4‰±1.0‰及18.6‰±1.2‰(P < 0.001)。
3. SD大鼠腹腔给药5天后与对照组相比,高浓度的雷帕霉素(0.15和0.2 mg/kg/d)明显抑制循环中VEGF的表达(P < 0.05及P < 0.01),但较低浓度的雷帕霉素(0.05和0.10 mg/kg/d)没有抑制作用。
4. Western blot法分析见雷帕霉素(10 ng/ml)孵育24小时后抑制内皮细胞中mTOR的磷酸化水平,但FKBP-12磷酸化水平未有改变,而且内皮细胞中p70 S6激酶的磷酸化也被雷帕霉素抑制。
5. VEGF作用4小时后,内皮细胞中mTOR和p70 S6K蛋白磷酸化水平明显增加,但PI3K/Akt的特异性抑制剂Wortmannin或LY294002可完全阻断VEGF的这一作用。
6. VEGF明显促进内皮细胞迁移(188.0%±5.5% vs.对照组, P < 0.001);而VEGF+LY294002或者VEGF+Wortmannin组与对照组比较,内皮细胞的迁移无差别(89.3%±11.3%及83.7%±6.8% vs.对照组, P > 0.05)。同样观察到VEGF明显促进内皮细胞增殖(175.7%±8.9% vs.对照组, P < 0.001);而VEGF+LY294002或者VEGF+Wortmannin组与对照组比较则无变化(82.8%±10.9%及99.0%±5.9% vs.对照组, P > 0.05)。
7. Wortmannin和LY294002都可抑制内皮细胞中Akt蛋白的磷酸化。但雷帕霉素孵育4小时后,Akt的磷酸化水平增高。
结论
1.雷帕霉素洗脱支架植入术后28天,支架表面内皮化不完全。其原因可能为雷帕霉素抑制内皮细胞的增殖和迁移、诱导内皮祖细胞的凋亡、并且减少局部血管中VEGF的表达,从而延缓药物洗脱支架后再内皮化进程。
2.雷帕霉素通过抑制mTOR/p70 S6K蛋白的激活进而抑制内皮细胞的增殖和迁移;PI3K /Akt信号蛋白是激活mTOR/p70 S6K蛋白所必需的,并且是上游信号蛋白。
3. PI3K /Akt信号蛋白和mTOR/p70 S6K信号蛋白并非是单纯的线性关系,在雷帕霉素抑制mTOR/p70 S6K蛋白的过程中,PI3K和mTOR之间存在反馈调节,Akt可能是反馈调节的中间分子。
Background
Nowadays, 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.
Objectives
1. 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.
Methods
1. 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.
Results
1. 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.
Conclusions
1. 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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