The Role of the Cytoskeleton in Vascular Aging

 描述（由申请人提供）：近端主动脉通常起关键的“减震器”作用，以保护下游小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，人类近端主动脉僵硬度随年龄增长而增加，是随后不良心血管结局（包括肾衰竭、高血压和血管性痴呆）的早期和独立生物标志物，也可能是其促成因素。我们在已发表的研究中表明，血管平滑肌细胞（VSMC）调节高达总主动脉僵硬度的一半，并且衰老诱导的VSMC细胞骨架调节的丧失导致主动脉执行这种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粘着斑和细胞外基质的联系是VSMC细胞骨架的一个特别动态和重要的部分。该计划的主要目标是确定血管肌动蛋白细胞骨架衰老相关功能障碍的分子机制及其与粘着斑（FA）复合物的联系，并进一步开发一种利用细胞渗透性诱饵肽逆转这种功能障碍的纳米颗粒靶向方法。在R21阶段，我们将使用小分子抑制剂和诱饵肽，以及生物力学，磁镊，去卷积显微镜，邻近连接分析（PLA），免疫沉淀和其他生化和细胞测定，在体外测试，细胞骨架和主动脉组织硬度的变化之间的因果关系。基于初步的数据，我们将特别关注抑制皮质肌动蛋白的伸长和分支机制，肌动蛋白粘着斑连接和粘着斑蛋白-蛋白质相互作用。在R33阶段，我们将扩展细胞渗透肽方法，以选择在体外老年小鼠组织中有效的肽，并与BU工程学院的Porter博士一起实施他的纳米技术方法的应用，用于组织特异性靶向释放诱饵肽。成功的纳米颗粒包装的肽将用于年轻和老年小鼠体内急性测定肽降低脉搏波速度（PWV）和血压的作用，从而证明细胞骨架功能和主动脉僵硬度之间的因果关系。此外，一项为期6个月的慢性试验将测试纳米颗粒包装肽逆转血压、PWV、MRI监测的脑血管损伤和肾损伤变化的能力，为更长时间的慢性研究提供动力。因此，我们提出了一个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及其与脂肪酸的联系的改变。这种方法如果成功，有可能预防或逆转许多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.