Leveraging the mitochondrial regulator MIRO1 to prevent neointimal hyperplasia

项目总结 血管闭塞性疾病仍然是一个严重的心血管健康问题，导致约500,000 美国每年的经皮冠状动脉血管成形术。术后发生新生内膜增生所致的再狭窄 占血管成形术的10-30%，仍然是心血管医学中的一个关键问题。没有新的方法来 为了改进标准治疗，已经开发了插入mTOR抑制剂或紫杉醇洗脱支架的方法 十年。解决这一关键问题将需要新的策略来抑制血管平滑的增殖 肌细胞(VSMCs)，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胞浆信号事件 血管疾病中的VSMCs和内皮细胞。拟议研究的总体目标是 确定高度保守的位于线粒体膜外膜的GTP酶Miro1是如何， 调节VSMC的增殖和新生内膜的形成，并测试新的机制治疗。已知Miro1 控制线粒体在细胞内的运输。新的数据表明，Miro1可能有额外的 其功能超越了线粒体交易。Miro1与线粒体-内质网接触位点(MERS)相关 促进线粒体Ca~(2+)进入，并维持线粒体嵴的组织，这对 电子传递链(ETC)活性。然而，Miro1结合的功能后果并不完全 对人类疾病，特别是对血管疾病的影响仍然存在 未知。我们之前曾报道，线粒体钙摄取和能量产生是细胞- 血管平滑肌细胞在G1/S过渡期及以后的周期进展。我们的初步研究表明，VSMC的增殖 对于Miro1，从G1/S转换开始取消缺失。因此，我们推测，Miro1控制着VSMC 通过两个迄今未被认识到的过程进行扩散。因此，我们的中心假设是需要Miro1 通过控制线粒体-内质网钙离子，促进VSMC增殖，从而促进新生内膜增生 过境和ETC活动。这一点得到了我们强大的试点数据的支持，该数据表明Miro1的删除 阻断新生内膜增生、VSMC增殖和三磷酸腺苷的产生。我们的新奇工具和化验方法让我们陷入了 完美的位置来测试我们的假设，包括缺乏Miro1或表达Miro1突变的VSMC系，以及 创新的基于RNA适体的工具，用于血管损伤后体内VSMCs的靶向。 我们的具体目标是：1.确定miro1对钙离子转运的调节程度。 Mercs控制细胞周期进程，2.建立对眉骨的控制程度 通过Miro1的组织来调节ETC的活性和增殖，以及3.检测VSMC特异性 提供siMIRO1的适配子有效地减少新生内膜的增生。的预期结果 拟议的研究是关于Miro1促进VSMC增殖的机制的知识，以及 具有siMIRO1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.