低剪切力通过“自噬流抑制与PKC激活”正反馈机制诱导血管内皮eNOS脱偶联的生物力学研究

Endothelial dysfunction is one of the initiative factors triggering atherosclerosis. Reduction of endothelial shear stress (SS) plays an important role in inducing endothelial dysfunction. Activation of endothelial nitric oxide synthase (eNOS) Thr495 by protein kinase C (PKC) is critical to regulate eNOS uncoupling. Inhibition of PKC was found to induce autophagy. We previously reported low SS promoted eNOS Thr495 phosphorylation, and rapamycin, an autophagy inducer, alleviated low SS induced O2.- production. Whereas, it is unclear whether autophagy is involved in the regulatory circuit of low SS induced eNOS uncoupling. In the pilot study, we found low SS induced eNOS uncoupling and impaired autophagic flux. And PKC was activated by low SS. Therefore, we hypothesized autophagic flux impairment and PKC activation developed a feed-forward mechanism in low SS induced eNOS uncoupling. In the parallel flow study and mouse model, we proposed to observe the effect of autophagic flux induction or blockade on PKC/eNOS Thr495 signal during low SS exposure. Next, using pharmacological inducers and inhibitors, as well as RNA interfering to downregulate PKC, we proposed to explore the role of PKC in low SS induced autophagic flux impairment and eNOS uncoupling. Our data will help provide evidences to develop new strategies for preventing atherosclerosis.

血管内皮损伤是动脉粥样硬化的始动因素，血管局部剪切力降低导致内皮功能受损。蛋白激酶C（PKC）上调eNOS Thr495是调节eNOS脱偶联的重要机制，而抑制PKC又可以诱导自噬。我们以往报道了低剪切力（LSS）诱导血管内皮eNOS Thr495磷酸化，并发现自噬诱导剂雷帕霉素减少LSS诱导的O2.-生成，但自噬与eNOS脱偶联的关系仍不清楚。预试验发现LSS抑制血管内皮自噬流可能导致eNOS脱偶联，且LSS激活PKC。申请者在预实验基础上，提出“自噬流抑制与PKC激活”正反馈调节LSS诱导eNOS脱偶联的假说。申请者拟运用体外流体实验及小鼠模型，首先观察自噬流诱导和阻断对LSS作用下PKC/eNOS Thr495活性的影响；再利用RNA干扰和药物抑制的方式下调PKC活性，观察PKC对LSS作用下内皮自噬流和eNOS脱偶联的影响，从而验证假说，为动脉粥样硬化的防治提供新的理论和实验依据。

血管内皮损伤是动脉粥样硬化的始动因素，血管局部剪切力降低导致内皮功能受损。蛋白激酶C（PKC）上调eNOSThr495是调节eNOS脱偶联的重要机制，而抑制PKC又可以诱导自噬。我们以往报道了低剪切力（LSS）诱导血管内皮eNOSThr495磷酸化，并发现自噬诱导剂雷帕霉素减少LSS诱导的O2.-生成，但自噬与eNOS脱偶联的关系仍不清楚。我们运用体外流体实验及小鼠模型，首先观察自噬流诱导和阻断对LSS作用下eNOS 脱偶联调控的因果关系，发现：1、氧化还原调节不参与LSS诱导的eNOS脱偶联；2、LSS抑制自噬流而非正向调控自噬流；3、LSS诱导的eNOS脱偶联是自噬流抑制导致的；4、eNOS 磷酸化而非eNOS乙酰化受自噬调控，并参与LSS诱导的eNOS脱偶联。接着，我们观察PKC对LSS作用下内皮自噬流和eNOS脱偶联的影响，发现：1、与生理性剪切力不同，LSS在自噬流晚期，通过抑制自噬体与溶酶体融合，扰乱内皮自噬流；2、LSS通过抑制溶酶体酸化从而影响自噬体与溶酶体融合，而非通过调节自噬相关基因和溶酶体生物合成；3、PKC激活介导了LSS导致的溶酶体酸化，自噬流受损，eNOSThr495磷酸化和内皮损伤；4、PKC激活和自噬流抑制形成正反馈环路诱导eNOSThr495磷酸化并损伤内皮；5、PKC及eNOS的自噬性降解和UPS介导的降解均不参与LSS诱导的内皮损伤。我们的结果为动脉粥样硬化的防治提供新的理论和实验依据。

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