Arteriolar Responses to ECM Fibronectin

动脉对 ECM 纤连蛋白的反应

• 批准号: 8021938
• 负责人: INGRID H SARELIUS
• 金额: $ 38.41万
• 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/8021938
• 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

DESCRIPTION (provided by applicant): 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 21 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 expertise 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. PUBLIC HEALTH RELEVANCE: This project explores how the connective tissue that surrounds arterioles can generate signals that modify responses in these vessels. These vessels are the ones that primarily regulate peripheral blood flow, hence it is of considerable significance to understand how changes in the connective tissue proteins can alter how the blood flow is regulated. One example of where this may be very significant for human health is in aging populations, where peripheral blood vessel function often deteriorates, but the mechanisms are unknown; our work raises the possibility that some of this change in vascular function may relate to changes in signals from connective tissue proteins.

描述（由申请人提供）：我们的总体目标是建立完整组织中机械力改变ECM蛋白质、纤连蛋白（FN）构象的能力与导致小动脉直径变化的后续信号传导事件之间的机械联系。我们发表的工作（REF）确定FN信号有助于肌肉收缩产生的扩张，从而确定了一种调节小阻力小动脉的新机制。我们将使用FN-模拟肽，我们已经构建了探索小动脉反应，使用共聚焦活体显微镜的完整组织在麻醉WT和敲除动物，在分离的细胞中的研究补充。特异性目的1将确定HSPG和整合素连接在维持血管张力和小动脉扩张中的作用。假设第一部分：完整的成年动物的骨骼肌的主动收缩瞬时暴露周围ECM FN中的matricryptic III-1位点。随后HSPG在细胞表面与III-1H的连接通过21整合素依赖性机制触发局部血管舒张.第二部分：在静息状态下，细胞表面HSPGs的基础水平的连接有助于维持静息血管张力。具体目标2将确定eNOS、nNOS、小窝蛋白和内皮细胞Ca 2+在静息张力维持和小动脉扩张中的作用。假设：第一部分：完整的成年动物的骨骼肌的主动收缩瞬时暴露周围ECM FN中的matricryptic III-1位点。随后，HSPG与III-1H在细胞表面上的连接通过小窝蛋白和NO依赖性机制触发局部血管舒张。第二部分：在静息条件下，细胞表面HSPG的基础水平的连接有助于通过NO依赖性机制维持静息血管张力。具体目标3将确定Src信号传导在FN依赖性应答中的作用。假设：ECM FN上FNIII-1H位点的连接通过Src激酶依赖性机制产生NO。具体目标4将可视化响应于骨骼肌收缩的ECM FN构象的变化，并确定响应于机械力的组织应变如何暴露结缔组织中的FNIII-1基质融合位点。假设：响应骨骼肌收缩的组织应变改变了ECM FN原纤维的构象，并暴露了FNIII-1中的matricryptic位点。该项目是了解组织中的机械力如何影响FN构象以及正常和病理条件下的血管反应的关键一步，例如对运动的综合反应，或与衰老相关的外周血管功能的变化，其中ECM蛋白组成的变化被记录。该提案将汇集一个独特的跨学科方法，结合FN基质生物学和微血管功能的专业知识，使用一种新的范式解决血管生物学中的一个关键问题，即机械信号转导机制转化为血管反应。 公共卫生关系：这个项目探讨了围绕小动脉的结缔组织如何产生信号来改变这些血管的反应。这些血管是主要调节外周血流量的血管，因此了解结缔组织蛋白的变化如何改变血流的调节方式具有相当重要的意义。这可能对人类健康非常重要的一个例子是在老龄化人群中，外周血管功能经常恶化，但机制尚不清楚;我们的工作提出了这样一种可能性，即血管功能的一些变化可能与结缔组织蛋白信号的变化有关。