miRNA-regulation at focal adhesions establishes vascular mechanohomeostasis

项目摘要 内皮细胞是组织力学特性的特殊哨兵。内皮细胞感知 细胞外基质的血管壁通过整合素为基础的粘连（局灶性粘连），和同型 邻近组织细胞之间的粘附，以调节基质组成、转换和硬化的变化。 此外，内皮细胞通过动态过程感知和调节血流动力学 包括肌动球蛋白和其他细胞骨架应力的变化，局部粘连和细胞粘附的重塑， 粘连和细胞骨架重组。内皮细胞如何缓冲相关力的变化 与血流和细胞外基质的关系尚不清楚。在这里，我们将研究内皮细胞如何调节 局部粘连时的基质和血流力，以及这些机制如何维持血管力学 体内平衡 细胞内稳态通过一种弹性机制依赖于microRNAs（miRNAs）的活性 并快速调节基因表达。我们最近发现了一个miRNA网络， 基体刚度这些miRNAs优先靶向细胞骨架、粘附和基质（CAM）基因，用于细胞凋亡后的修复。 在体外和体内的蛋白质水平的转录调控。我们的结论是， CAM基因的调节对于介导细胞-基质相互作用至关重要，并允许内皮细胞维持 在刚度变化的条件下的机械内稳态。 本申请中提供的初步数据显示了新的和令人兴奋的观察， 并且CAM mRNA独特地定位于内皮细胞中的粘着斑处。我们假设CAM mRNA定位于粘着斑并在粘着斑处翻译， 调节粘连灶处的CAM蛋白水平以缓冲基质和/或血流动力学的影响 改变并维持机械稳态。我们将通过设置2D单元来测试这个新假设 培养模型和斑马鱼血管系统，以表征基质和/或血液动力学特性， 内皮细胞在粘着斑处缺乏miRNA调控。

PROJECT SUMMARY Endothelial cells are exceptional sentinels of tissues' mechanical properties. Endothelial cells sense the extracellular matrix of the vascular wall via integrin-based adhesions (focal adhesions), and homotypic adhesion between neighboring tissue cells to regulate changes in matrix composition, turnover and stiffening. Furthermore, endothelial cells sense and adjust to blood flow hemodynamics through dynamic processes involving changes in actomyosin and other cytoskeletal stresses, remodeling of focal adhesions and cell adhesions, and cytoskeleton reorganization. How endothelial cells can buffer changes in forces associated with both blood flow and extracellular matrix remains unclear. Here, we will study how endothelial cells regulate matrix and blood flow forces at focal adhesion, and how these mechanism(s) sustain vascular mechanical homeostasis. Cell homeostasis hinges on the activity of microRNAs (miRNAs) through a mechanism that is resilient and regulates gene expression rapidly. We recently discovered a miRNA network activated by changes in matrix stiffness. These miRNAs preferentially target cytoskeletal, adhesion and matrix (CAM) genes for post- transcriptional regulation of protein levels both in vitro and in vivo. We concluded that miRNA-dependent regulation of CAM genes is critical for mediating cell-matrix interactions and allows endothelial cells to maintain mechanical homeostasis under conditions of changing stiffness. Preliminary data presented in this application shows the novel and exciting observation that miRNAs and CAM mRNAs are uniquely localized at focal adhesions in endothelial cells. We hypothesize that CAM mRNAs are localized to and translated at focal adhesions, and that miRNAs post-transcriptionally regulate CAM protein levels at focal adhesions to buffer the effect of matrix and/or hemodynamic changes and maintain mechanical homeostasis. We will test this new hypothesis by setting up 2D cell culture models and the zebrafish vascular system to characterize matrix and/or hemodynamic properties of endothelial cells that lack of miRNA regulation at focal adhesions.