Arteriolar Responses to ECM Fibronectin

描述(申请人提供)：我们的总体目标是在完整组织中的机械力改变ECM蛋白、纤维连接蛋白(FN)构象的能力和导致小动脉直径改变的后续信号事件之间建立机械联系。我们发表的工作(参考文献)证实，FN信号有助于肌肉收缩产生的扩张，从而确定了一种新的调节小阻力小动脉的机制。我们将使用我们构建的FN模拟多肽，通过对麻醉的WT和基因敲除动物的完整组织进行体内共聚焦显微镜观察，并辅之以分离细胞的研究，来探索微动脉反应。具体目标1将确定HSPG和整合素结扎在维持血管张力和小动脉扩张中的作用。假设第一部分：完整的成年动物骨骼肌的主动收缩瞬间暴露了周围ECM FN中的基质解密III-1位点。随后用III-1H连接细胞表面的HSPG，通过21整合素依赖的机制触发局部血管扩张。第二部分：在静息条件下，基础水平的HSPG在细胞表面的结扎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钙离子在维持静息张力和小动脉扩张中的作用。假设：第一部分：完整的成年动物骨骼肌的主动收缩瞬间暴露了周围ECM FN中的基质III-1位点。随后用III-1H连接细胞表面的HSPG，通过小窝蛋白和NO依赖的机制触发局部血管扩张。第二部分：在静息条件下，基础水平的HSPG在细胞表面的结扎有助于维持静息血管张力，这是通过非依赖性机制实现的。具体目标3将确定Src信号在FN依赖反应中的作用。假设：连接细胞外基质FN上的FNIII-1H位点通过一种依赖于Src激酶的机制产生NO。具体目标4将直观地显示骨骼肌收缩时细胞外基质FN构象的变化，并确定组织应变如何暴露结缔组织中的FNIII-1结合位点。假设：骨骼肌收缩引起的组织应变改变了细胞外基质FN纤维的构象，并暴露了FNIII-1中的一个密码子位点。这个项目是了解组织中的机械力如何在正常和病理条件下影响FN构象从而影响血管反应的关键一步，例如运动的综合反应，或与衰老相关的外周血管功能的变化，其中ECM蛋白质组成的变化被记录在案。这一建议将结合FN基质生物学和微血管功能方面的专业知识，将独特的跨学科方法结合在一起，使用一种新的范式来解决血管生物学中的一个关键问题，即机械信号转换为血管反应的机制。 与公共健康相关：这个项目探索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。这对人类健康可能非常重要的一个例子是在老龄化人口中，周围血管功能经常恶化，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质信号的变化有关。

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.