Coordination of Pathophysiologic endothelial cell signaling by ROS

描述(由申请人提供)：异常的细胞内钙([Ca+]i)稳态与心血管疾病(CVD)有关，后者导致内皮功能障碍。Ca~(2+)作为第二信使，调节多种细胞功能。尽管已有证据表明钙信号与内皮功能障碍之间存在联系，但血管疾病发生的潜在机制仍不完全清楚。除了Ca~(2+)外，内皮细胞(EC)功能障碍也与活性氧有关。越来越多的人认识到，在心血管疾病中，线粒体Ca~(2+)水平升高与随后产生的线粒体活性氧物种(MRO)之间存在联系。线粒体通过单转运体和转运体隔离钙离子，从而形成胞浆钙信号。然而，在EC功能障碍的背景下，线粒体钙超载和MROS产生之间的因果联系还知之甚少。新近发现的线粒体钙单转运蛋白复合体分子MCU和MICU1对线粒体钙摄取有调节作用，但这些分子在内皮功能中的病理生理作用尚未见报道。我们的新发现表明，MICU1通过调节MCU来抑制线粒体基础钙的积累。此外，MICU1的沉默促进了结构性线粒体钙超载，从而提高了MRO，并使细胞对凋亡刺激敏感。因此，我们的中心假设是，MICU1门MCU孔活动限制基础线粒体钙离子积累和ROS过量生产，以保持血管完整性。这一假说是基于我们最近在《细胞》杂志上发表的。这一假设将结合分子和细胞生物学、生化和先进的成像技术、原代内皮细胞、转基因动物和冠状动脉疾病患者的样本进行验证。我们的建议将通过三个具体的目标来解决这些问题：1)表征MICU1在EC线粒体钙稳态中的功能；2)研究MICU1在EC信号和功能中的作用；3)研究MICU1在病理生理条件下的EC生物学中的作用。重要的是，我们的研究将揭示MICU1在线粒体钙稳态和血管内皮细胞MRO产生中的作用。这些研究的预期结果将通过阐明MICU1在限制线粒体钙负荷和氧化信号方面发挥核心作用，从而显著转移EC信号转导的焦点。这些发现有望通过推进EC、Ca~(2+)信号和氧化应激领域产生立竿见影的影响，极有可能为心血管疾病的治疗干预提供新的靶点。

DESCRIPTION (provided by applicant): Aberrant intracellular calcium ([Ca2+]i) homeostasis has been implicated in cardiovascular diseases (CVD) which contributes to endothelial dysfunction. Ca2+ acts as a second messenger and is known to regulate several cell functions. Despite the available evidence of a link between Ca2+ signaling and endothelial dysfunction, mechanisms underlying the development of vascular disease remain incompletely understood. Besides Ca2+, reactive oxygen species have also been implicated in endothelial cell (EC) dysfunction. A growing recognition exists of a link between elevated levels of mitochondrial Ca2+ and subsequent generation of mitochondrial reactive oxygen species (mROS) in cardiovascular diseases. Mitochondria shape cytosolic Ca2+ signals by sequestering Ca2+ through uniporter and transporters. Nevertheless, the causal link between mitochondrial Ca2+ overload and mROS production is poorly understood in the context of EC dysfunction. Recently identified mitochondrial Ca2+ uniporter complex molecules MCU and MICU1 are shown to regulate the mitochondrial Ca2+ uptake, however the pathophysiological role of these molecules in endothelial function has not been studied. Our new findings show that MICU1 gates the basal mitochondrial Ca2+ accumulation by regulating MCU. Further, silencing of MICU1 facilitates constitutive mitochondrial Ca2+ overload which subsequently elevates mROS and sensitizes cells to apoptotic stimuli. Accordingly, our central hypothesis is that MICU1 gates MCU pore activity limiting basal mitochondrial Ca2+ accumulation and ROS overproduction to preserve vascular integrity. The hypothesis was formulated based on our recent publication in Cell. The hypothesis will be tested using combination of molecular and cell biology, biochemical and advanced imaging technology, primary endothelial cells, genetically-modified animals and samples from human subject with coronary artery disease. Our proposal will address these issues via three specific aims: 1) Characterize the functional role of MICU1 in EC mitochondrial Ca2+ homeostasis 2) Study the role of MICU1 in EC signaling and function, and 3) Investigate the role of MICU1 in EC biology under pathophysiological conditions. Importantly, our investigations will uncover the role of MICU1 in mitochondrial Ca2+ homeostasis and mROS production in vascular endothelium. The expected outcomes from these studies will significantly shift the focus of EC signaling by elucidating that MICU1 plays a central role in limiting mitochondrial Ca2+ load and oxidative signaling. Such findings are expected to have an immediate impact through advancing the fields of EC Ca2+ signaling and oxidative stress with a strong likelihood that the information will provide new targets for therapeutic interventions in CVD.