Arteriolar Responses to ECM Fibronectin

动脉对 ECM 纤连蛋白的反应

• 批准号: 8385529
• 负责人: INGRID H SARELIUS
• 金额: $ 36.77万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-01-01 至 2014-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8385529
• 关键词: Address; Affect; Aging; Animals; Biology; Blood Vessels; Blood flow; Calcium; Caliber; Caveolins; Cell surface; Cells; Cellular Mechanotransduction; Complement; Confocal Microscopy; Connective Tissue; Data; Endothelial Cells; Event; Exercise; Extracellular Matrix; Fibronectins; Fluo 4; Fluorescence Microscopy; Fluorescence Resonance Energy Transfer; Generations; Health; Heparan Sulfate Proteoglycan; Heparin; Human; In Situ; Integrin Binding; Integrins; Label; Ligation; Link; Maintenance; Measurement; Mechanics; Modeling; Molecular; Molecular Conformation; Monitor; Mus; Muscle Contraction; Peptide Signal Sequences; Peptides; Peripheral; Population; Proteins; Protocols documentation; Publishing; Pulsatile Flow; Regulation; Resistance; Rest; Role; Signal Pathway; Signal Transduction; Site; Skeletal Muscle; Smooth Muscle Myocytes; System; Testing; Tissues; Vasodilation; Work; arteriole; interdisciplinary approach; intravital microscopy; knockout animal; mature animal; mimetics; novel; peripheral blood; peripheral blood vessel; response; shear stress; src-Family Kinases

Our general aim is to establish a mechanistic connection between the ability of mechanical forces in intact tissue to alter conformation of an ECM protein, fibronectin (FN), and subsequent signaling events that result in changes in arteriolar diameter. Our published work (REF) established that FN signaling contributes to the dilation produced by muscle contraction, thus identifying a new mechanism regulating small resistance arterioles. We will use FN-mimetic peptides that we have constructed to explore arteriolar responses using confocal intravital microscopy of intact tissues in anesthetized WT and knockout animals, complemented by studies in isolated cells. Specific Aim 1 will determine the roles of HSPG- and integrin-ligation in maintenance of vascular tone and in arteriolar dilation. Hypothesis Part I: Active contraction of skeletal muscle of intact, adult animals transiently exposes the matricryptic III-1 site in the surrounding ECM FN. Subsequent ligation of HSPGs on cell surfaces with III-1H triggers local vasodilation by a ¿1 integrin- dependent mechanism. Part II: Under resting conditions, a basal level of ligation of HSPGs on cell surfaces contributes to maintenance of resting vascular tone. Specific Aim 2 will determine the role of eNOS, nNOS, caveolin and endothelial cell Ca2+ in maintenance of resting tone and in arteriolar dilation. Hypothesis: Part I: Active contraction of skeletal muscle of intact, adult animals transiently exposes the matricryptic III-1 site in the surrounding ECM FN. Subsequent ligation of HSPGs on cell surfaces with III-1H triggers local vasodilation by a caveolin- and NO-dependent mechanism. Part II: Under resting conditions, a basal level of ligation of HSPGs on cell surfaces contributes to maintenance of resting vascular tone via NO-dependent mechanisms. Specific Aim 3 will identify the role of Src signaling in FN-dependent responses. Hypothesis: Ligation of the FNIII-1H site on ECM FN generates NO via a Src kinase-dependent mechanism. Specific Aim 4 will visualize changes in ECM FN conformation in response to skeletal muscle contraction and determine how tissue strain in response to mechanical force exposes the FNIII-1 matricryptic site in connective tissue. Hypothesis: Tissue strain in response to skeletal muscle contraction alters the conformation of ECM FN fibrils and exposes a matricryptic site in FNIII-1. This project is a critical step towards understanding how mechanical forces in the tissue affect FN conformation and hence vascular responses, under normal and pathological conditions, for example the integrated response to exercise, or the changes in peripheral vascular function associated with aging, where changes in ECM protein composition are documented. This proposal will bring together a unique interdisciplinary approach combining expertises in FN matrix biology and microvascular function to use a novel paradigm addressing a key question in vascular biology, that of mechanisms for transduction of mechanical signals into vascular responses.

我们的总体目的是建立机械力之间的机械连接 完整的组织以改变ECM蛋白，纤连蛋白（FN）的构象以及随后的信号事件 导致小动脉直径的变化。我们已发表的工作（参考）确定FN信令有助于 肌肉收缩产生的扩散，从而确定了调节小电阻的新机制 小动脉。我们将使用我们已经构建的FN模拟宠物来探索动脉反应 麻醉WT和基因敲除动物中完整组织的共聚焦浸润室显微镜检查，由 在分离细胞中的研究。特定的目标1将确定HSPG和整联蛋白结扎在 维持血管张力和动脉词典。假设第一部分：骨骼的主动收缩 完整的成年动物的肌肉会瞬时暴露于周围ECM FN中的基质III-1位点。 随后使用III-1H的HSPG在细胞表面上结扎，通过1个整合素触发局部血管舒张 依赖机制。第二部分：在静止条件下，HSPG在细胞表面的基本结合水平 有助于维持静止的血管张力。特定的目标2将确定eNOS，nnos的作用 可爱素和内皮细胞Ca2+在维持静息音和小动脉词典中。假设： 第一部分：成年动物完整的骨骼肌的主动收缩瞬时暴露了基质III-1位点 在周围的ECM FN中。随后使用III-1H触发本地HSPG在细胞表面上的连接 可爱素和无依赖性机制的血管舒张。第二部分：在休息条件下，基础水平 HSPG在细胞表面上的连接有助于通过无依赖性维持静止的血管张力 机制。具体目标3将确定SRC信号在FN依赖性响应中的作用。 假设：FNIII-1H位点在ECM FN上的连接可通过SRC激酶依赖性机制产生NO。 特定的目标4将对骨骼肌肉的ECM FN构象的变化可视化 收缩并确定如何响应机械力的组织应变暴露于FNIII-1 结缔组织中的基质位点。假设：响应骨骼肌收缩的组织应变 改变ECM FN原纤维的构象，并在FNIII-1中暴露了基质位点。这个项目是关键 迈向了解组织中的机械力如何影响FN构象，因此是血管 在正常和病理条件下的反应，例如对运动的综合响应或 与衰老相关的外周血管功能的变化，其中ECM蛋白质组成的变化是 记录。该建议将汇集一种独特的跨学科方法，结合FN的专业知识 基质生物学和微血管功能，使用新型范式解决血管中的关键问题 生物学，将机械信号转化为血管反应的机制。