Links between mechanics and metabolism in endothelial cells and in vivo

All cells experience varying types and magnitudes of force throughout their lifetime. Cellular responses to these forces are often critical to their biological role and function. Forces are predominantly sensed by surface adhesion receptors. Activation of the adhesion receptors at the cell-cell or junctional interface triggers pathways that culminate in cytoskeletal rearrangement and reinforcement of the actin cytoskeleton. Cytoskeletal rearrangement allows the cell to counter externally and internally applied forces. The processes and pathways involved in cytoskeletal rearrangement are considered energetically demanding events. However, how the cell accounts for this increased energetic demand or the pathways that regulate these responses have not been extensively studied. In epithelial and endothelial cells, cadherins play a critical role in forming and maintaining the cellular interface known as the adherens junction. Cadherins are connected to the actin cytoskeleton through an intercellular protein complex. Through their role in cell-cell adhesion and their connection to the actin cytoskeleton, cadherins are force sensors and transducers. Specifically, force applied to cadherins elicits a response to recruit proteins that form additional links to the actin cytoskeleton, thereby reinforcing the actin cytoskeleton against external forces. Yet the structural changes in the actin cytoskeleton, myosin contractility, and signaling pathways all require energy. How cells respond to the increased metabolic demand of these processes is not well defined. In this thesis, I determine that endothelial cells respond to force through the vascular endothelial cadherin VE-Cadherin (VE-Cadherin) complex, which regulates the metabolic activation of these cells. First, I demonstrate that in primary human umbilical vascular endothelial cells (HUVECs) and bovine aortic endothelial cells (BAECs) fluid shear activates the master metabolic regulator AMP-Activating Protein Kinase (AMPK). Disruption of VE-Cadherin through physical perturbation or genetic ablation blocks AMPK activation, establishing a connection between cadherin mechanotransduction and metabolic regulation. The mechanism for how VE-Cadherin stimulates AMPK was found to be regulated through liver kinase beta 1 (LKB1). Activation of this pathway was required for endothelial junction stability, glucose uptake, and eNOS localization and activation. Moreover, I demonstrate that the metabolic activation stimulated by this pathway is critical for sustaining these effects, as inhibitors of glycolytic intermediates or genetic disruption of the pathway prevent actin reinforcement, endothelial alignment, and eNOS activity. Furthermore, I highlight the physiological importance of mechanic-metabolic activation in a mouse model that harbors a mutation that disrupts this link. Specifically, I show that uncoupling these systems via the vinculin point mutation Y822F leads to physiological changes to mouse weights and lean muscle mass. More revealing is the observation that these mice have abnormal cardiac function. The mechanisms by which these changes occur are under further investigation. Taken together, this work builds upon a paradigm where the ability of cells to respond to mechanical cues by adapting their metabolic state is paramount to proper cellular function and tissue homeostasis.