Coordination of Pathophysiologic endothelial cell signaling by ROS

描述（由申请人提供）：异常细胞内钙（[Ca 2 +]i）稳态与心血管疾病（CVD）有关，导致内皮功能障碍。Ca 2+作为第二信使，已知可调节多种细胞功能。尽管现有证据表明Ca 2+信号传导和内皮功能障碍之间存在联系，但血管疾病发展的潜在机制仍不完全清楚。除了Ca 2+，活性氧也与内皮细胞（EC）功能障碍有关。越来越多的人认识到，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（mROS）之间存在联系。线粒体通过单向转运体和转运体螯合Ca 2+来形成胞浆Ca 2+信号。然而，在EC功能障碍的背景下，线粒体Ca 2+超载和mROS产生之间的因果关系知之甚少。最近鉴定的线粒体Ca 2+单向转运体复合物分子MCU和MICU 1显示出调节线粒体Ca 2+摄取，然而这些分子在内皮功能中的病理生理作用尚未研究。我们的新发现表明，MICU 1通过调节MCU门控基础线粒体Ca 2+积累。此外，MICU 1的沉默促进组成型线粒体Ca 2+过载，随后升高mROS并使细胞对凋亡刺激物敏感。因此，我们的中心假设是MICU 1门控MCU孔活性，限制基础线粒体Ca 2+积累和ROS过量产生，以保持血管完整性。该假设是根据我们最近在Cell上发表的文章制定的。将使用分子和细胞生物学、生物化学和先进成像技术、原代内皮细胞、转基因动物和来自患有冠状动脉疾病的人类受试者的样本的组合来检验该假设。我们的提案将通过三个具体目标来解决这些问题：1）表征MICU 1在EC线粒体Ca 2+稳态中的功能作用; 2）研究MICU 1在EC信号传导和功能中的作用; 3）研究MICU 1在病理生理条件下在EC生物学中的作用。重要的是，我们的研究将揭示MICU 1在血管内皮细胞线粒体Ca 2+稳态和mROS产生中的作用。这些研究的预期结果将通过阐明MICU 1在限制线粒体Ca 2+负荷和氧化信号传导中发挥核心作用来显著转移EC信号传导的焦点。这些发现预计将通过推进EC Ca 2+信号传导和氧化应激领域产生直接影响，这些信息很有可能为CVD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.