Mitochondrial CaMKII drives smooth muscle migration and neointimal hyperplasia

项目摘要/摘要 血管闭塞疾病仍然是一个严重的心血管健康问题，经皮治疗约有500,000人 仅在美国，每年就进行了50,000例冠状动脉和外周球囊血管成形术。血流动力学 在10%到30%的患者中，有10%到30%的患者通过新生内膜增生发生相关的再狭窄。血管平滑肌 血管损伤后血管内膜细胞(VSMC)迁移与新生内膜增生密切相关。VSMC迁移 是一个依赖于钙离子的过程；迁移细胞必须维持胞浆内的钙梯度并创造局部的钙离子 靠近前沿的脉冲可能完成细胞骨架的动态周转。线粒体是一种 VSMC细胞内钙的主要缓冲作用，并参与局部的钙反应。在其他单元格类型中，单元格 迁移依赖于线粒体在前沿的定位。因此，我们建议 线粒体提供对钙离子瞬变的局部控制，有助于VSMC迁移。以前的工作 来自本课题组的研究证实，多功能钙/钙调蛋白依赖性蛋白激酶II(CaMKII)是一种 VSMC细胞迁移和增殖的关键调节因子。在最近的研究中，我们发现CaMKII是 存在并活跃在线粒体基质中，在那里它被认为促进线粒体基质钙离子的内流。 在我们实验室建立的一种新的转基因模型中，MitoCaMKII抑制VSMC的初步研究 阻断体内新生内膜的增殖。此外，mitoCaMKII抑制抑制线粒体钙摄取， 线粒体运动与VSMC迁移。这些数据将线粒体CaMKII(MitoCaMKII)定位为 线粒体功能和与新生内膜增生相关的关键VSMC表型的守门人。在这 在这个应用中，我们将直接测试我们的工作假设，即mitoCaMKII抑制阻止线粒体 基质钙摄取，从而影响线粒体的流动性和VSMC的迁移，并最终影响新生内膜 增生症。我们将在两个目标上检验我们的工作假说：1.确定VSMC中是否存在mitoCaMKII 通过调节线粒体钙摄取来控制新生内膜的形成和2.解剖 MitoCaMKII调控VSMC迁移的途径和机制研究将包括在体内 利用抑制或过度表达mitoCaMKII的转基因新模型分析新生内膜形成 包括内质网/线粒体钙转换、钙波和线粒体钙离子转换在内的局部钙离子异常的体外成像 闪烁以及细胞骨架和焦点黏附翻转的分析。此外，连接的新路径 线粒体基质钙摄取对VSMC迁移的影响将被检测。预计成功的 拟议研究的完成将提供对线粒体功能如何变化的机械性洞察 导致VSMC迁移和新生内膜形成。这样的知识可能会导致一流的线粒体- 血管疾病的靶向治疗，特别是新生内膜增生。

Project Summary / Abstract Vascular occlusive disease remains a critical cardiovascular health issue with about 500,000 percutaneous coronary and 50,000 peripheral balloon angioplasties performed annually in the US alone. Hemodynamically relevant restenoses through neointimal hyperplasia occur in 10 to 30% of patients. Vascular smooth muscle cell (VSMC) migration significantly contributes to neointimal hyperplasia after vascular injury. VSMC migration is a Ca2+-dependent process; migrating cells must maintain cytosolic Ca2+ gradients and create local Ca2+ pulses near the leading edge likely to accomplish dynamic cytoskeletal turnover. Mitochondria are one of the major buffers of intracellular Ca2+ in VSMC and participate in localized Ca2+ responses. In other cell types, cell migration is dependent upon mitochondrial localization to the leading edge. Thus, we propose that mitochondria provide localized control of Ca2+ transients, serving to facilitate VSMC migration. Previous work from our group established that the multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a key regulator of VSMC cell migration and proliferation. In more recent studies, we discovered that CaMKII is present and active in the mitochondrial matrix, where it is believed to promote mitochondrial matrix Ca2+ influx. In preliminary studies, mitoCaMKII inhibition in VSMC in a novel transgenic model developed in our laboratory blocks neointimal hyperplasia in vivo. Moreover, mitoCaMKII inhibition blocks mitochondrial Ca2+ uptake, mitochondrial mobility and VSMC migration. These data position mitochondrial CaMKII (mitoCaMKII) as a gatekeeper of mitochondrial function and of key VSMC phenotypes relevant for neointimal hyperplasia. In this this application, we will directly test our working hypothesis that mitoCaMKII inhibition blocks mitochondrial matrix Ca2+ uptake, thereby affecting mitochondrial mobility and VSMC migration and ultimately neointimal hyperplasia. We will test our working hypothesis in two aims: 1. Determine whether mitoCaMKII in VSMC controls neointimal formation through regulation of mitochondrial Ca2+ uptake and 2. Dissect the pathways and mechanisms by which mitoCaMKII controls VSMC migration. Studies will include in vivo analysis of neointimal formation using novel transgenic models of mitoCaMKII inhibition or overexpression and in vitro imaging of localized Ca2+ dysregulation, including ER/mitochondrial Ca2+ transition, Ca2+ waves and flickers as well as analysis of cytoskeleton and focal adhesion turnover. Moreover, novel pathways that link mitochondrial matrix Ca2+ uptake to VSMC migration will be tested. It is anticipated that the successful completion of the proposed studies will provide mechanistic insight into how changes in mitochondrial function lead to VSMC migration and neointimal formation. Such knowledge could lead to first-in-class, mitochondria- targeted therapies for vascular disease, in particular neointimal hyperplasia.