Arteriolar Responses to ECM Fibronectin

我们的总目标是建立一个机械力的能力之间的机械联系， 完整组织以改变ECM蛋白、纤连蛋白（FN）的构象，以及随后的信号传导事件， 导致小动脉直径的变化。我们发表的工作（REF）确定FN信号传导有助于 肌肉收缩产生的扩张，从而确定了调节小阻力的新机制 小动脉我们将使用我们构建的FN模拟肽来探索微动脉反应， 麻醉WT和基因敲除动物中完整组织的共聚焦活体显微镜检查， 在隔离的细胞中进行研究。特异性目的1将决定HSPG-和整合素-连接的作用， 维持血管张力和小动脉扩张。假设第一部分：骨骼肌的主动收缩 完整的成年动物的肌肉瞬时暴露周围ECM FN中的基质内分泌III-1位点。 随后，HSPG在细胞表面与III-1H的连接通过α 1整合素触发局部血管舒张， 依赖机制第二部分：在静息条件下，细胞表面HSPGs连接的基础水平 有助于维持静息血管张力。特异性目的2将决定eNOS，nNOS， 小窝蛋白和内皮细胞Ca ~（2+）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短暂暴露了基质蛋白III-1位点 在周围的ECM FN中。随后，细胞表面上的HSPG与III-1H的连接引发局部 通过小窝蛋白和NO依赖性机制的血管舒张。第二部分：在静息条件下， HSPGs在细胞表面的连接有助于通过NO依赖性的血管张力维持静息状态 机制等具体目标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的ECM FN构象的变化 收缩，并确定组织应变如何响应机械力暴露FNIII-1 结缔组织中的基质埋藏部位。假设：组织应变响应骨骼肌收缩 改变ECM FN原纤维的构象并暴露FN III-1中的基质裂解位点。这个项目是一个关键的 进一步了解组织中的机械力如何影响FN构象，从而影响血管 在正常和病理条件下的综合反应，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蛋白组成的变化是 记录在案。该提案将汇集一个独特的跨学科方法，结合FN的专业知识， 基质生物学和微血管功能，以使用一种新的范式来解决血管中的一个关键问题， 生物学，即机械信号转换为血管反应的机制。

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.