Arteriolar Responses to ECM Fibronectin

动脉对 ECM 纤连蛋白的反应

• 批准号: 8586347
• 负责人: INGRID H SARELIUS
• 金额: $ 37.85万
• 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 2015-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8586347
• 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.

我们的总体目标是在机械力的能力之间建立一种机械联系 完整的组织来改变ECM蛋白的构象，纤维连接蛋白(FN)，以及随后的信号事件 导致小动脉直径的改变。我们发表的工作(参考文献)确立了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的新机制 小动脉。我们将使用我们构建的FN模拟多肽来探索微动脉反应 共聚焦活体显微镜观察麻醉WT和基因敲除动物的完整组织，辅以 在分离的细胞中进行研究。特异性目标1将确定HSPG-和整合素-连接在 维持血管张力和微动脉扩张。假设第一部分：骨骼的主动收缩 完整的成年动物的肌肉瞬间暴露在周围的ECM FN中的基质解密III-1部位。 随后用III-1H连接细胞表面的HSPG，通过1整合素-1触发局部血管扩张- 依赖机制。第二部分：在静息条件下，细胞表面HSPG连接的基础水平 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出基质解密的III-1部位 在周围的ECM FN。细胞表面HSPG与III-1H的后续连接可局部触发 血管扩张通过小窝蛋白和一氧化氮依赖的机制。第二部分：在静息条件下，基础水平 细胞表面HSPG的结扎通过NO依赖促进静息血管张力的维持 机制。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。 特定目标4将可视化响应骨骼肌的ECM FN构象的变化 收缩并确定组织应变对机械力的响应如何暴露FNIII-1 结缔组织中的基质部位。假设：骨骼肌收缩引起的组织应变 改变ECM FN纤维的构象，并暴露FNIII-1中的基质解密位点。这个项目是一个关键的 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 有记录在案。这项建议将结合FN的专业知识，形成一种独特的跨学科方法。 基质生物学和微血管功能使用一种新的范式解决血管中的一个关键问题 生物学，将机械信号转换为血管反应的机制。

项目成果

Extracellular matrix fibronectin mediates an endothelial cell response to shear stress via the heparin-binding, matricryptic RWRPK sequence of FNIII1H.

细胞外基质纤连蛋白通过 FNIII1H 的肝素结合、基质隐性 RWRPK 序列介导内皮细胞对剪切应力的反应。

• DOI: 10.1152/ajpheart.00126.2016
• 发表时间: 2016
• 期刊: American journal of physiology. Heart and circulatory physiology
• 作者: Okech,William;Abberton,KerenM;Kuebel,JuliaM;Hocking,DeniseC;Sarelius,IngridH
• 通讯作者: Sarelius,IngridH

Extracellular matrix fibronectin initiates endothelium-dependent arteriolar dilatation via the heparin-binding, matricryptic RWRPK sequence of the first type III repeat of fibrillar fibronectin.

细胞外基质纤连蛋白通过纤维纤连蛋白第一个 III 型重复的肝素结合、基质隐性 RWRPK 序列启动内皮依赖性小动脉扩张。

• DOI: 10.1113/jp271478
• 发表时间: 2016
• 期刊: The Journal of physiology
• 作者: Sarelius,IngridH;Titus,PatriciaA;Maimon,Nir;Okech,William;Wilke-Mounts,SusanJ;Brennan,JamesR;Hocking,DeniseC
• 通讯作者: Hocking,DeniseC