Arteriolar Responses to ECM Fibronectin

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、纤维连接蛋白（FN）构象的能力和随后导致小动脉直径变化的信号事件之间的机制联系。我们发表的研究（REF）证实，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。我们将使用我们构建的fn模拟肽，在麻醉WT和敲除动物的完整组织的共聚焦活体显微镜下探索小动脉反应，并辅以分离细胞的研究。特异性目的1将确定HSPG-和整合体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：完整成年动物骨骼肌的主动收缩暂时暴露了周围ECM FN中的III-1基质。随后，III-1H将HSPGs连接到细胞表面，通过21整合素依赖机制触发局部血管舒张。第二部分：在静息条件下，细胞表面的HSPGs结扎的基础水平有助于维持静息血管张力。特异性Aim 2将确定eNOS、nNOS、小血管蛋白和内皮细胞Ca2+在维持静息张力和小动脉扩张中的作用。假设：第一部分：完整成年动物骨骼肌的主动收缩暂时暴露周围ECM FN的III-1基质。随后用III-1H在细胞表面结扎HSPGs，通过依赖于小窝蛋白和no的机制触发局部血管舒张。第二部分：在静息条件下，细胞表面HSPGs的基础结扎通过no依赖机制有助于维持静息血管张力。特异性目标3将确定Src信号在fn依赖性反应中的作用。假设：连接ECM FN上的FNIII-1H位点通过Src激酶依赖机制产生NO。特异性Aim 4将可视化骨骼肌收缩时ECM FN构象的变化，并确定组织应变对机械力的反应如何暴露结缔组织中的FNIII-1基质部位。假设：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中的基质位点。该项目是了解组织中的机械力如何影响FN构象和血管反应的关键一步，在正常和病理条件下，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下来。本提案将结合FN基质生物学和微血管功能方面的专业知识，采用一种独特的跨学科方法，利用一种新的范式解决血管生物学中的一个关键问题，即机械信号转导成血管反应的机制。

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.