Leveraging the mitochondrial regulator MIRO1 to prevent neointimal hyperplasia

项目摘要 血管闭塞性疾病仍然是一个严重的心血管健康问题， 每年在美国进行经皮冠状动脉成形术。新生内膜增生导致的呼吸暂停发生在 10-30%的血管成形术，仍然是心血管医学的关键问题。没有新的办法， 改进标准治疗，插入mTOR支架或紫杉醇洗脱支架，已经在 十几年解决这一关键问题需要新的策略来抑制血管平滑肌细胞的增殖。 肌肉细胞（VSMC），新生内膜增生的主要驱动力。 我们的长期目标是确定线粒体功能如何调节细胞内信号传导事件， 血管疾病中的血管平滑肌细胞和内皮细胞。拟议研究的总体目标是 确定如何高度保守的GTdR MIRO 1，它存在于外线粒体膜， 调节VSMC增殖和新生内膜形成，并测试新的机制疗法。MIRO 1已知 控制线粒体的细胞内运输。新生数据表明MIRO 1可能有额外的 功能超出线粒体运输。MIRO 1与MERS-ER联系站点（MERCS）联系 促进线粒体Ca 2+进入并维持线粒体嵴的组织，这对于 电子传递链（ETC）活性。然而，MIRO 1结合的功能性后果并不完全清楚。 这一点已经被理解，并且对人类疾病，特别是血管疾病的影响仍然存在。 未知我们以前报道过，线粒体Ca 2+摄取和能量产生是细胞增殖所必需的。 G1/S过渡期及以后的VSMC周期进展。我们的初步研究表明，血管平滑肌细胞的增殖 MIRO 1缺失的细胞从G1/S转换开始消失。因此，我们推测MIRO 1控制VSMC 通过两个迄今未被认识的过程扩散。因此，我们的中心假设是MIRO 1是必需的， 对于VSMC增殖，以及因此对于新生内膜增生，通过控制细胞内-ER Ca 2 + 交通和ETC活动。这得到了我们强有力的试验数据的支持，这些数据表明MIRO 1缺失 阻断新生内膜增生、VSMC增殖和ATP产生。我们的新工具和检测方法使我们处于一个 完美的位置来测试我们的假设，包括缺乏MIRO 1或表达MIRO 1突变体的VSMC系， 基于RNA适体的创新工具，用于在血管损伤后体内靶向VSMC。 我们的具体目标是1。为了确定在一定程度上MIRO 1调节Ca 2+转运， MERCS控制细胞周期进程，2.以确定对嵴的控制程度 MIRO 1的组织调节ETC的活动和增殖，以及3.测试特定于VSMC的 递送siMIRO 1的适体有效地减少新生内膜增生。的预期成果 本研究旨在了解MIRO 1促进VSMC增殖的机制， 具有siMIRO 1活性的RNA适体在预防血管损伤后再狭窄中的潜力。

PROJECT SUMMARY Vascular occlusive disease remains a critical cardiovascular health issue and results in about 500,000 percutaneous-coronary angioplasties annually in the US. Restenosis due to neointimal hyperplasia occurs after 10-30% of angioplasties and remains a critical problem in cardiovascular medicine. No new approaches to improve the standard treatment, the insertion of mTOR inhibitor-or taxol-eluting stents, have been developed in a decade. Addressing this critical problem will require novel strategies to inhibit proliferation of vascular smooth muscle cells (VSMCs), the main driver of neointimal hyperplasia. Our long-term goal is to determine how mitochondrial function modulates cytosolic signaling events in VSMCs and endothelial cells in vascular disease. The overall objective of the proposed research is to determine how the highly conserved GTPase MIRO1, which resides in the outer mitochondrial membrane, regulates VSMC proliferation and neointima formation and test new mechanistic therapies. MIRO1 is known to control intracellular trafficking of mitochondria. Nascent data suggest that MIRO1 may have additional functions beyond mitochondrial trafficking. MIRO1 associates with the mitochondria-ER contact sites (MERCS) that facilitate mitochondrial Ca2+ entry and maintains the organization of mitochondrial cristae that is pivotal for electron transport chain (ETC) activity. Yet, the functional consequences of MIRO1 binding are not fully understood, and the implications for human disease, in particular in vascular disease, have remained unknown. We previously reported that mitochondrial Ca2+ uptake and energy production are required for cell- cycle progression in VSMCs in G1/S transition and beyond. Our pilot studies reveal that proliferation in VSMCs with MIRO1 deletion is abolished starting at G1/S transition. Thus, we speculate that MIRO1 controls VSMC proliferation via two hitherto unrecognized processes. Thus, our central hypothesis is that MIRO1 is required for VSMC proliferation, and thus for neointimal hyperplasia, by controlling the mitochondrial-ER Ca2+ transit and ETC activity. This is supported by our strong pilot data demonstrating that MIRO1 deletion blocks neointimal hyperplasia, VSMC proliferation and ATP production. Our novel tools and assays put us in a perfect position to test our hypothesis, including VSMC lines that lack MIRO1 or express MIRO1 mutants, and innovative RNA-aptamer-based tools for targeting of VSMCs in vivo after vascular injury. Our specific aims are 1. to determine the extent to which MIRO1 regulation of Ca2+ transport at MERCS controls cell-cycle progression, 2. to establish the extent to which the control of cristae organization by MIRO1 regulates ETC activity and proliferation, and 3. to test whether VSMC-specific aptamers that deliver siMIRO1 effectively reduce neointima hyperplasia. The anticipated outcomes of the proposed study are knowledge of the mechanisms by which MIRO1 promotes VSMC proliferation, and of the potential of RNA-based aptamers with siMIRO1 activity in preventing restenosis after vascular injury.