miRNA-regulation at focal adhesions establishes vascular mechanohomeostasis

项目总结 内皮细胞是组织机械特性的特殊哨兵。内皮细胞感觉到 血管壁细胞外基质通过基于整合素的粘连(灶性粘连)和同型 邻近组织细胞之间的黏附，以调节基质组成、周转和僵硬的变化。 此外，内皮细胞通过动态过程感知和调节血流动力学。 涉及肌动蛋白和其他细胞骨架应激的变化，局灶性粘连和细胞的重塑 粘连和细胞骨架重组。内皮细胞如何缓冲相关作用力的变化 血流和细胞外基质尚不清楚。在这里，我们将研究内皮细胞如何调节 局部粘连处的基质和血流作用力，以及这些机制如何维持血管机械(S) 动态平衡。 细胞动态平衡取决于microRNAs(MiRNAs)的活性，它通过一种有弹性的机制 并快速调节基因表达。我们最近发现了一个由基因变化激活的miRNA网络 矩阵刚度。这些miRNAs优先靶向细胞骨架、黏附和基质(CAM)基因。 体外和体内蛋白质水平的转录调控。我们得出结论，依赖miRNA的 CAM基因的调节对于调节细胞-基质相互作用至关重要，并允许内皮细胞维持 变刚度条件下的机械动态平衡。 本申请中提供的初步数据显示了miRNAs的新颖和令人兴奋的观察 而CAM mRNAs仅定位于内皮细胞的局部粘连部位。我们假设CAM MRNAs定位于焦点粘连并在其上翻译，而miRNAs在转录后 调节局部粘连的CAM蛋白水平以缓冲基质和/或血流动力学的影响 改变和维持机械动态平衡。我们将通过建立2D单元来检验这一新假设 培养模型和斑马鱼血管系统以表征基质和/或血流动力学特性 在局部粘连处缺乏miRNA调控的内皮细胞。

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.