miRNA-regulation at focal adhesions establishes vascular mechanohomeostasis

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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.