The Role of the Cytoskeleton in Vascular Aging

 描述(申请人提供)：近端的主动脉通常起着关键的“减震器”的作用，保护小的下游血管免受心脏产生的高压力脉冲的影响。最近的流行病学研究表明，人的近端主动脉僵硬随着年龄的增长而增加，是包括肾功能衰竭、高血压和血管性痴呆在内的后续心血管不良后果的早期和独立的生物标志物，并可能是其可能的贡献者。我们在已发表的研究中表明，血管平滑肌细胞(VSMC)调节多达一半的总主动脉僵硬，而衰老导致VSMC细胞骨架调节的丧失导致主动脉执行这一减震功能的能力受损。我们实验室的一个主要进展是证明了皮质非肌肉肌动蛋白细胞骨架及其与局灶性粘连和细胞外基质的联系是VSMC细胞骨架中特别动态和重要的部分。该计划的主要目标是确定与衰老相关的血管肌动蛋白细胞骨架功能障碍的分子机制及其与焦点黏附(FA)复合体的联系，并进一步开发一种利用细胞诱骗多肽来逆转这一功能障碍的纳米颗粒靶向方法。在R21期，我们将使用小分子抑制剂和诱饵多肽，结合生物力学、磁钳、去卷积显微镜、邻近连接分析(PLA)、免疫沉淀等生化和细胞分析方法，在体外测试细胞骨架变化与主动脉组织硬度之间的因果关系。在初步数据的基础上，我们将特别关注抑制皮质肌动蛋白的伸长和分支机制，肌动蛋白-焦点黏附连接和焦点黏附蛋白-蛋白质相互作用。在R33阶段，我们将扩展细胞预示肽方法，以在体外选择对衰老小鼠组织有效的多肽，并与波士顿大学工程学院的Porter博士一起应用他的纳米技术方法来组织特异性靶向释放诱饵多肽。成功的纳米颗粒包装的多肽将在体内急性用于年轻和老年小鼠，以确定多肽降低脉搏波速度(PWV)和血压的效果，从而证明细胞骨架功能和主动脉僵硬之间的因果关系。此外，一项为期6个月的慢性试验将测试纳米颗粒包装的多肽逆转血压、PWV、MRI监测的脑血管损伤和肾脏损伤的能力，为更长时间的慢性研究提供动力。因此，我们提出了一种高度创新的研究策略来定义和攻击衰老诱导的血管肌动蛋白细胞骨架的变化及其与FA的联系。这种方法如果成功，有可能预防或逆转一系列与衰老相关的心血管疾病。

 DESCRIPTION (provided by applicant): The proximal aorta normally functions as a critical "shock absorber" to protect small downstream vessels from the high pulses of pressure generated by the heart. Recent epidemiological studies have made clear that human proximal aortic stiffness increases with age and is an early and independent biomarker of, and probable contributor to, subsequent adverse cardiovascular outcomes including kidney failure, hypertension and vascular dementia. We have shown in published studies that the vascular smooth muscle cell (VSMC) regulates up to half of total aortic stiffness and that aging-induced loss of regulation of the VSMC cytoskeleton leads to impairment of the ability of the aorta to perform this shock absorption function. A major advance from our lab has been the demonstration that the cortical nonmuscle actin cytoskeleton and its linkage to focal adhesions and the extracellular matrix is a particularly dynamic and important part of the VSMC cytoskeleton. The broad goal of this program is to define molecular mechanisms of aging-associated malfunction of the vascular actin cytoskeleton and its connection with focal adhesion (FA) complexes and to furthermore develop a nanoparticle-targeted approach utilizing cell permeant decoy peptides to reverse this malfunction. In the R21 phase, we will use small molecule inhibitors and decoy peptides, together with biomechanics, magnetic tweezers, deconvolution microscopy, proximity ligation analysis (PLA), immunoprecipitation and other biochemical and cellular assays to test, in vitro, the cause-and-effect relationship between changes in the cytoskeleton and aortic tissue stiffness. Based on preliminary data, we will focus specifically on inhibition of cortical actin elongation and branching mechanisms, actin-focal adhesion connections and focal adhesion protein-protein interactions. In the R33 phase we will extend the cell permeant peptide approach to select peptides that are effective in aged mouse tissues in vitro, and implement, with Dr. Porter in the BU Engineering College an application of his nanotechnology approach for tissue-specific targeted release of the decoy peptides. The successful nanoparticle-packaged peptides will be used in young and old mice acutely in vivo to determine the effect of the peptides to decrease pulse wave velocity (PWV) and blood pressure and hence demonstrate a cause-and-effect relationship between cytoskeletal function and aortic stiffness. Additionally, a 6 month chronic trial will test the ability of nanoparticle packaged peptides to reverse changes in blood pressure, PWV, MRI-monitored brain vascular damage and kidney damage to provide the impetus for longer chronic studies. Hence, we propose a highly innovative research strategy to define and attack aging-induced alterations in the vascular actin cytoskeleton and its linkage to FAs. This approach, if successful, has the potential to prevent or reverse a host of aging-associated cardiovascular disorders.