Harvard-MIT Division of Health Sciences and Technology, Cambridge, MA 02139, USA.
Circulation. 2010 May 25;121(20):2192-9. doi: 10.1161/CIRCULATIONAHA.109.877282. Epub 2010 May 10.
Local modulation of vascular mammalian target of rapamycin (mTOR) signaling reduces smooth muscle cell (SMC) proliferation after endovascular interventions but may be associated with endothelial cell (EC) toxicity. The trilaminate vascular architecture juxtaposes ECs and SMCs to enable complex paracrine coregulation but shields SMCs from flow. We hypothesized that flow differentially affects mTOR signaling in ECs and SMCs and that SMCs regulate mTOR in ECs.
SMCs and/or ECs were exposed to coronary artery flow in a perfusion bioreactor. We demonstrated by flow cytometry, immunofluorescence, and immunoblotting that EC expression of phospho-S6 ribosomal protein (p-S6RP), a downstream target of mTOR, was doubled by flow. Conversely, S6RP in SMCs was growth factor but not flow responsive, and SMCs eliminated the flow sensitivity of ECs. Temsirolimus, a sirolimus analog, eliminated the effect of growth factor on SMCs and of flow on ECs, reducing p-S6RP below basal levels and inhibiting endothelial recovery. EC p-S6RP expression in stented porcine arteries confirmed our in vitro findings: Phosphorylation was greatest in ECs farthest from intact SMCs in metal stented arteries and altogether absent after sirolimus stent elution.
The mTOR pathway is activated in ECs in response to luminal flow. SMCs inhibit this flow-induced stimulation of endothelial mTOR pathway. Thus, we now define a novel external stimulus regulating phosphorylation of S6RP and another level of EC-SMC crosstalk. These interactions may explain the impact of local antiproliferative delivery that targets SMC proliferation and suggest that future stents integrate design influences on flow and drug effects on their molecular targets.
局部调节血管哺乳动物雷帕霉素靶蛋白（mTOR）信号可减少血管内介入后的平滑肌细胞（SMC）增殖，但可能与内皮细胞（EC）毒性有关。三层血管结构并列排列 EC 和 SMC，使其能够进行复杂的旁分泌共调控，但同时也使 SMC 免受血流影响。我们假设血流对 EC 和 SMC 中的 mTOR 信号有差异影响，并且 SMC 调节 EC 中的 mTOR。
在灌注生物反应器中使 SMC 和/或 EC 暴露于冠状动脉血流中。我们通过流式细胞术、免疫荧光和免疫印迹证明，EC 中磷酸化 S6 核糖体蛋白（p-S6RP）的表达（mTOR 的下游靶点）被血流增加了一倍。相反，SMC 中的 S6RP 对生长因子而不是血流有反应，并且 SMC 消除了 EC 对血流的敏感性。替西罗莫司，一种西罗莫司类似物，消除了生长因子对 SMC 的作用和对 EC 的血流作用，将 p-S6RP 降低到基础水平以下，并抑制内皮细胞的恢复。支架猪动脉中的 EC p-S6RP 表达证实了我们的体外发现：在金属支架动脉中，离完整 SMC 最远的 EC 中磷酸化程度最高，在洗脱西罗莫司支架后完全消失。
mTOR 途径在 EC 中被腔内血流激活。SMC 抑制这种由血流诱导的内皮 mTOR 途径的刺激。因此，我们现在定义了一种新的外部刺激，调节 S6RP 的磷酸化，以及另一个 EC-SMC 串扰水平。这些相互作用可能解释了针对 SMC 增殖的局部抗增殖药物输送的影响，并表明未来的支架将整合对血流的设计影响和对其分子靶点的药物作用。
