KCa3.1通道调控巨噬细胞极化参与易损斑块发生的新机制：线粒体Kv1.3通道的关键作用

Our previous work suggested that plasma membrane KCa3.1 channel is a novel regulator in determining macrophage phenotype and atherosclerotic plaque vulnerability. The underlying signaling mechanism, however, has not been sufficiently clarified yet. In the preliminary experiments, we proved that the KCa3.1 channel mediated cytoplasmic calcium signaling need to be further accumulated into mitochondria through the regulation of mitochondrial Kv1.3 (mKv1.3) channel in the process of pro-inflammatory M1 macrophage polarization. In this project, we assumed that mKv1.3 channel, acting as a downstream of effector of KCa3.1 channel, might plays critical of in the process of M1 polarization by inducing mitochondrial calcium overloading through its over-expression and activation. The present study was designed to investigate the potential role of macrophage KCa3.1-mKv1.3 cascade pathway in the pathogenesis of vulnerable plaque in vitro and in vivo, using whole cell/mitochondria path clamp, molecular fluorescent probe combined with laser scanning confocal microscope and gene silencing or knockout techniques. The results of our study would highlight the importance of mK1.3 signaling pathway in the setting of mitochondrial calcium overloading, abnormal energy metabolism and imbalanced macrophage polarization, which may provide an alternative strategy for the prevention and treatment of plaque vulnerability.

本课题组前期研究揭示细胞膜KCa3.1通道是一种新的巨噬细胞极化调控因子并参与易损斑块发生，但潜在机制不明。在预实验中，我们发现KCa3.1通道介导的钙信号有赖于线粒体Kv1.3通道（mKv1.3）调控以进一步内流线粒体，从而诱导促炎性M1型巨噬细胞极化。因此，我们提出如下假说：mKv1.3通道作为KCa3.1通道下游信号分子，其异常表达和活化后触发的线粒体钙超载是KCa3.1通道介导的M1型巨噬细胞极化的关键环节。本课题拟通过离体和在体实验，采用全细胞/线粒体膜片钳、分子荧光探针结合激光扫描共聚焦以及特异性基因沉默或敲除等实验方法，探讨巨噬细胞KCa3.1-mKv1.3通道级联信号在易损斑块形成中的作用及机制，重点阐明mKv1.3通道介导的信号通路在诱发线粒体钙超载、细胞能量代谢障碍和巨噬细胞极化失衡等一系列病理过程中所起的关键作用，为探索易损斑块特异性干预靶点提供新思路。

冠状动脉粥样硬化易损斑块是动脉粥样硬化的一种重要类型，其所继发的斑块破裂和血栓形成是导致临床急性冠脉综合征和心源性猝死的首要病因。因此，探索动脉粥样易损斑块特异性干预手段具有非常重要的临床意义。组织病理学证实巨噬细胞的浸润和活化是易损斑块发生的核心环节。巨噬细胞极化失衡——促炎性 M1型巨噬细胞过度活化促发的炎症级联反应在易损斑块发生发展中发挥重要作用。然而，目前尚无干预M1型巨噬细胞极化的特异性手段。本课题在前期研究基上以KCa3.1-mKv1.3通道级联信号为主要研究切入点，探讨mKv1.3通道调控线粒体钙稳态而作为巨噬细胞极化和易损斑块干预靶点的可行性。首先，我们通过体外构建M0/M1巨噬细胞极化模型。采用全细胞/线粒体膜片钳技术、免疫荧光和亚细胞组分Western Blot等多种实验手段证实在M0/M1型巨噬细胞表型转化过程中伴随有mKv1.3通道表达显著增高，而细胞膜Kv1.3通道表达无明显变化。通过间接阻断mKv1.3通道可显著抑制M1型巨噬细胞极化与功能。随后，我们在ApoE-/-小鼠采用肾动脉联合颈内外动脉缩窄方法构建易损斑块模型。结果证实巨噬细胞选择性敲除Kv1.3通道可有效改善斑块稳定性，从而减少斑块破裂发生。进一步研究显示mKv1.3通道干预通过调控线粒体膜电位ΔΨm防止其过度去极化而抑制M1型巨噬细胞极化过程。由此提示，通过阻断mKv1.3通道可有效抑制M1型巨噬细胞极化，减少易损斑块的发生。然而，目前尚未特异性阻断线粒体mKv1.3通道的手段，而Kv1.3通道表达的缺乏组织和细胞特异性，完全阻断Kv1.3通道存在诱发内皮功能失调及系统性免疫抑制等风险。因此，仍需进一步研究深入阐述mKv1.3通道表达和调控机制，以前为巨噬细胞极化和易损斑块干预提供更为特异的靶点。