Mechanisms of Heterocellular Signaling at the Myoendothelial Junction

肌内皮连接处的异细胞信号传导机制

• 批准号: 8208057
• 负责人: Brant E Isakson
• 金额: $ 37.5万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-01-01 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8208057
• 关键词: Animals; Blood Vessels; Blood flow; Calcium; Calcium Signaling; Cells; Coculture Techniques; Communication; Confocal Microscopy; Connexins; Coupled; Coupling; Cultured Cells; Dyes; Electron Microscopy; Elements; Endothelial Cells; Endothelium; Engineering; Gap Junctions; Goals; In Vitro; Inositol; Link; Location; Measures; Mediating; Membrane; Methods; Modeling; Modification; Molecular; Movement; Pattern; Permeability; Play; Process; Research; Resistance; Role; Ryanodine Receptors; Second Messenger Systems; Side; Signal Transduction; Signaling Protein; Small Interfering RNA; Smooth Muscle Myocytes; Structure; System; Testing; Vascular Smooth Muscle; Vasomotor; base; cell type; hypertension control; immunocytochemistry; in vivo; insight; interest; light microscopy; novel; operation; receptor; research study; response; second messenger; solute; tripolyphosphate

DESCRIPTION (provided by applicant): Vascular smooth muscle cells (VSMC) and endothelial cells (EC) in the resistance vessels are functionally linked, and the point of contact between the two cells, the myoendothelial junction (MEJ), plays a key role in many elements of vascular function. However, the location and size of the MEJ have made it extremely difficult to study in vivo. We have developed a model of the MEJ by co-culturing VSMC and EC that show connexin- dependent dye transfer and a coupled pool of Ca2+. Moreover, the mode of intercellular calcium signaling depends on which of the two cell types is stimulated, i.e. intercellular second messenger signaling is polarized, and this appears to mimic signaling patterns seen in vivo. We propose to determine the structural and molecular basis for the intercellular coupling that occurs at the MEJ, which we believe has important implications for the operation of the MEJ in vivo. We will determine which connexins and second messengers are involved in intercellular signaling, and will test whether polarization of calcium communication is determined by selective permeability of the gap junctions at the MEJ or differential expression of second messenger receptors at the MEJ in the two cell types. We propose 3 specific experimental aims: Specific Aim 1 - Use light and electron microscopy-based immunocytochemistry to assess and compare the in vivo and in vitro placement of: a.) connexins, b.) ryanodine receptors, and c.) inositol 1,4,5 triphosphate-receptors at the MEJ; Specific Aim 2 - Measure the effect of modification of the gap junctional connexin composition on: a.) Ca2+ and b.) inositol 1,4,5 triphosphate -mediated intercellular signaling; and lastly, Specific Aim 3 - Assess the effects of cell specific deletion of a) ryanodine receptors and b) inositol 1,4,5 triphosphate-receptors on polarized calcium signaling. Our experiments should enhance understanding of the coordination of VSMC and EC and will provide insights into basic questions of vasomotor control and a variety of pathophysiological responses. The methods by which vascular cells communicate are key for understanding vascular processes such as hypertension and control of blood flow. Our model and the experiments proposed offer the first opportunity to investigate the capabilities of the myoendothelial junction and to understand its role in the vessel wall. We propose to study the ways in which vascular cells utilize this structure to maintain vascular function.

描述（由申请人提供）：电阻容器中的血管平滑肌细胞（VSMC）和内皮细胞（EC）在功能上连接，并且两个细胞之间的接触点，肌膜内皮结（MEJ），在许多血管功能的许多元素中都起着关键作用。但是，MEJ的位置和大小使在体内学习非常困难。我们通过共同培养VSMC和EC开发了MEJ模型，该模型显示了依赖性染料的染料转移和Ca2+的耦合池。此外，细胞间钙信号传导的模式取决于刺激两种细胞类型中的哪种，即细胞间的第二信号信号传导是偏振的，这似乎模仿了体内看到的信号传导模式。我们建议确定MEJ发生的细胞间耦合的结构和分子基础，我们认为这对MEJ在体内的操作具有重要意义。我们将确定哪些连接蛋白和第二个使者参与细胞间信号传导，并将测试钙通信的极化是通过MEJ上的间隙连接的选择性渗透性确定的，或者是两种细胞类型中MEJ的第二个Messenger受体的差异表达。我们提出了3个特定实验目的：具体目标1-使用基于光和电子显微镜的免疫细胞化学评估和比较体内和体外放置：具体目标2-测量间隙连接连接蛋白组成的修饰的影响：最后，具体目标3-评估a）ryanodine受体的细胞特异性缺失和b）肌醇1,4,5三磷酸蛋白受体受体对偏振钙信号传导的影响。我们的实验应增强对VSMC和EC协调的理解，并将为血管舒适控制和各种病理生理反应的基本问题提供见解。血管细胞传达的方法是理解血管过程的关键，例如高血压和控制血流。我们的模型和拟议的实验提供了第一个机会来研究肌膜内皮交界处的能力并了解其在血管壁中的作用。我们建议研究血管细胞利用这种结构维持血管功能的方式。