IRF-1 mediates the suppressive effects of mTOR inhibition on arterial endothelium.
IRF-1 介导 mTOR 抑制对动脉内皮的抑制作用。
- 作者列表："Peng K","Fan X","Li Q","Wang Y","Chen X","Xiao P","Passerini AG","Simon SI","Sun C
AIMS:Mammalian target of rapamycin (mTOR) inhibitors used in drug-eluting stents (DES) to control restenosis have been found to delay endothelialization and increase incidence of late-stent thrombosis through mechanisms not completely understood. We revealed that mTOR inhibition (mTORi) upregulated the expression of cell growth suppressor IRF-1 in primary human arterial endothelial cells (HAEC). This study aimed to examine how mTOR-regulated IRF-1 expression contributes to the suppressive effect of mTORi on arterial endothelial proliferation. METHODS AND RESULTS:Western blotting, quantitative PCR, and a dual-luciferase reporter assay indicated that mTOR inhibitors rapamycin and torin 1 upregulated IRF-1 expression and increased its transcriptional activity. IRF-1 in turn contributed to the suppressive effect of mTORi by mediating HAEC apoptosis and cell cycle arrest in part through upregulation of caspase 1 and downregulation of cyclin D3, as revealed by CCK-8 assay, Annexin V binding assay, measurement of activated caspase 3, BrdU incorporation assay, and matrigel tube formation assay. In a mouse model of femoral artery wire injury, administration of rapamycin inhibited EC recovery, an effect alleviated by EC deficiency of IRF-1. Chromatin immunoprecipitation assay with HAEC and rescue expression of wild type or dominant-negative IRF-1 in EC isolated from Irf1-/- mice confirmed transcriptional regulation of IRF-1 on the expression of CASP1 and CCND3. Furthermore, mTORi activated multiple PKC members, among which PKCζ was responsible for the growth-inhibitory effect on HAEC. Activated PKCζ increased IRF1 transcription through JAK/STAT-1 and NF-κB signaling. Finally, overexpression of wild type or mutant raptor incapable of binding mTOR indicated that mTOR-free raptor contributed to PKCζ activation in mTOR-inhibited HAEC. CONCLUSIONS:The study reveals an IRF-1-mediated mechanism that contributes to the suppressive effects of mTORi on HAEC proliferation. Further study may facilitate the development of effective strategies to reduce the side effects of DES used in coronary interventions.
目的: 哺乳动物雷帕霉素靶蛋白 (mTOR) 抑制剂用于药物洗脱支架 (DES) 为了控制再狭窄，已经发现通过尚未完全了解的机制延缓内皮化和增加晚期支架血栓形成的发生率。我们发现 mTOR 抑制 (mTORi) 可上调原代人动脉内皮细胞 (HAEC) 中细胞生长抑制 IRF-1 的表达。本研究旨在研究 mTOR 调节的 IRF-1 表达如何促进 mTORi 对动脉内皮增殖的抑制作用。 方法和结果: Western blotting 、定量 PCR 和双荧光素酶报告基因检测表明，mTOR 抑制剂雷帕霉素和 torin 1 上调 IRF-1 的表达，并增加其转录活性。IRF-1 反过来通过介导 HAEC 凋亡和细胞周期阻滞促进 mTORi 的抑制作用，部分是通过上调 caspase 1 和下调细胞周期蛋白 D3，如 CCK-8 试验所揭示的, annexin V 结合测定、活化 caspase 3 测定、 BrdU 掺入测定和 matrigel 管形成测定。在股动脉丝损伤的小鼠模型中，给予雷帕霉素抑制 EC 恢复，这种作用通过 EC 缺乏 IRF-1 缓解。用 HAEC 染色质免疫沉淀试验和拯救 Irf1-/-小鼠分离的 EC 中野生型或显性阴性 IRF-1 的表达证实了 IRF-1 对 CASP1 和 ccnd3 表达的转录调控。此外，mTORi 激活了多个 PKC 成员，其中 PKC ζ 负责对 HAEC 的生长抑制作用。激活的 pkc ζ 通过 JAK/STAT-1 和 NF-κ b 信号增加 IRF1 转录。最后，不能结合 mTOR 的野生型或突变型 raptor 的过表达表明，无 mTOR 的 raptor 有助于 mTOR 抑制的 HAEC 中的 pkc ζ 激活。 结论: 本研究揭示了 mTORi 抑制 HAEC 增殖的 IRF-1-mediated 机制。进一步的研究可能有助于开发有效的策略来减少冠状动脉介入治疗中使用的 DES 的副作用。
METHODS:Aims : We sought to investigate the thrombogenicity of different DES and BMS in an in vitro system of stent perfusion. Material and Methods: The experimental model consisted of a peristaltic pump connected to 4 parallel silicone tubes in which different stents were deployed. Blood was drawn from healthy volunteers and the amount of stent surfaced -induced thrombus deposition was determined using 125 I -fibrinogen. Results: Compared to Resolute, Biomatrix and Vision, Xience was associated with the lowest amount of stent surface -induced thrombus formation, with a significant difference compared to Vision (125 I -fibrinogen median value deposition [IQ range]: 50 ng [25 -98] versus 560 ng [320 - 1,520], respectively, p<0.05), but not to other DES. In the second set of experiments Fluoropolymer -coated BMS not eluting drug was associated with a significant 3 -fold reduction in 125 I -fibrinogen deposition (245 ng [80 -300]) compared to Vision (625 ng [320 -760], p<0.05), but a 7 -fold increase compared to Xience (35 ng [20 -60], p<0.05). Finally Xience was associated with a significantly greater absorption of albumin compared to BMS. Conclusions: In an in vitro system of stent perfusion, Xience was associated with the lowest amount of stent surface -induced thrombus formation compared with Resolute, Biomatrix and Vision, with a noted synergistic effect between the fluoropolymer and the drug.
METHODS::Fibronectin-splice variant containing extra domain A (Fn-EDA) is associated with smooth muscle cells (SMCs) following vascular injury. The role of SMC-derived Fn-EDA in SMC phenotypic switching or its implication in neointimal hyperplasia remains unclear. Herein, using human coronary artery sections with a bare metal stent, we demonstrate the expression of Fn-EDA in the vicinity of SMC-rich neointima and peri-strut areas. In mice, Fn-EDA colocalizes with SMCs in the neointima of injured carotid arteries and promotes neointima formation in the comorbid condition of hyperlipidemia by potentiating SMC proliferation and migration. No sex-based differences were observed. Mechanistic studies suggested that Fn-EDA mediates integrin- and TLR4-dependent proliferation and migration through activation of FAK/Src and Akt1/mTOR signaling, respectively. Specific deletion of Fn-EDA in SMCs, but not in endothelial cells, reduced intimal hyperplasia and suppressed the SMC synthetic phenotype concomitant with decreased Akt1/mTOR signaling. Targeting Fn-EDA in human aortic SMCs suppressed the synthetic phenotype and downregulated Akt1/mTOR signaling. These results reveal that SMC-derived Fn-EDA potentiates phenotypic switching in human and mouse aortic SMCs and neointimal hyperplasia in the mouse. We suggest that targeting Fn-EDA could be explored as a potential therapeutic strategy to reduce neointimal hyperplasia.
METHODS:OBJECTIVE:The goal of this study was to determine the impact of late-acquired stent malapposition (LASM) on long-term clinical outcomes in patients treated with coronary stent implantation. Approach and Results: We investigated major adverse cardiac event during 10 years after 6-month intravascular ultrasound examination using our previous studies database. A total of 732 patients treated with bare-metal stent (54 LASM versus 678 non-LASM) and 529 patients treated with first-generation drug-eluting stent (82 LASM versus 447 non-LASM), who did not have clinical event or censoring at the time of follow-up intravascular ultrasound, were included for the present analysis. major adverse cardiac event was defined as the composite of cardiac death, target vessel-related myocardial infarction, target lesion revascularization and stent thrombosis. Multivariable adjustment and inverse probability weight were performed to consider baseline differences. After multivariable adjustment, LASM was related to a greater risk of major adverse cardiac event (hazard ratio, 1.666 [95% CI, 1.041-2.665]; P=0.0333) and very-late stent thrombosis (hazard ratio, 3.529 [95% CI, 1.153-10.798]; P=0.0271) than non-LASM in patients treated with first-generation drug-eluting stent, but not in those treated with bare-metal stent. Results were consistent after inverse probability weight. Among patients with LASM of first-generation drug-eluting stent, no late stent thrombosis occurred in patients who continued to receive dual antiplatelet therapy. CONCLUSIONS:The relationship between LASM and major adverse cardiac event might depend on the type of implanted stents during the long-term follow-up, highlighting the clinical significance of polymers and drugs in drug-eluting stent system.