IRF-1 mediates the suppressive effects of mTOR inhibition on arterial endothelium

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-scent 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 Lorin 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 PKC zeta was responsible for the growth-inhibitory effect on HAEC. Activated PKC zeta increased IRF1 transcription through JAK/STAT-1 and NF-kappa B signaling. Finally, overexpression of wild type or mutant raptor incapable of binding mTOR indicated that mTOR-free raptor contributed to PKC zeta 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：用于药物洗脱支架（DES）以控制再狭窄的雷帕霉素靶蛋白（mTOR）抑制剂被发现会延迟内皮化，并通过尚未完全明确的机制增加晚期支架内血栓形成的发生率。我们发现mTOR抑制（mTORi）会上调原代人动脉内皮细胞（HAEC）中细胞生长抑制因子IRF - 1的表达。本研究旨在探讨mTOR调节的IRF - 1表达如何导致mTORi对动脉内皮增殖的抑制作用。 方法与结果：蛋白质印迹法、定量PCR和双荧光素酶报告基因检测表明，mTOR抑制剂雷帕霉素和洛林1上调IRF - 1的表达并增加其转录活性。如CCK - 8检测、膜联蛋白V结合检测、活化的半胱天冬酶3测定、BrdU掺入检测和基质胶管形成检测所示，IRF - 1反过来通过介导HAEC凋亡和细胞周期停滞，部分通过上调半胱天冬酶1和下调细胞周期蛋白D3，从而对mTORi的抑制作用产生影响。在股动脉钢丝损伤的小鼠模型中，雷帕霉素的使用抑制了内皮细胞的恢复，而IRF - 1在内皮细胞中的缺失可缓解这种影响。对HAEC进行的染色质免疫沉淀检测以及在从Irf1（-/-）小鼠分离的内皮细胞中野生型或显性失活IRF - 1的挽救性表达证实了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对HAEC的生长抑制作用负责。活化的PKC ζ通过JAK/STAT - 1和NF - κB信号通路增加IRF1转录。最后，野生型或不能结合mTOR的突变型raptor的过表达表明，无mTOR的raptor有助于mTOR抑制的HAEC中PKC ζ的激活。 结论：该研究揭示了一种由IRF - 1介导的机制，该机制导致mTORi对HAEC增殖产生抑制作用。进一步的研究可能有助于制定有效的策略，以减少冠状动脉介入治疗中使用的DES的副作用。