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Dissertation
Links between mechanics and metabolism in endothelial cells and in vivo
University of Iowa
Doctor of Philosophy (PhD), University of Iowa
Autumn 2024
DOI: 10.25820/etd.007591
Abstract
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.
Details
- Title: Subtitle
- Links between mechanics and metabolism in endothelial cells and in vivo
- Creators
- Nicholas M Wells
- Contributors
- Kris DeMali (Advisor)Sheila Baker (Committee Member)Brandon Davies (Committee Member)Tina Tootle (Committee Member)Lori Wallrath (Committee Member)Todd Washington (Committee Member)
- Resource Type
- Dissertation
- Degree Awarded
- Doctor of Philosophy (PhD), University of Iowa
- Degree in
- Biochemistry and Molecular Biology
- Date degree season
- Autumn 2024
- DOI
- 10.25820/etd.007591
- Publisher
- University of Iowa
- Number of pages
- xvii, 165 pages
- Copyright
- Copyright 2024 Nicholas M Wells
- Language
- English
- Date submitted
- 11/18/2024
- Description illustrations
- illustrations, tables, graphs
- Description bibliographic
- Includes bibliographical references (pages 140-165).
- Public Abstract (ETD)
- All cells experience different types and levels of force during their lives. How they handle these forces is crucial for their function. Cells mainly sense these forces through receptors on their surface. When these receptors are activated by contact with other cells or external forces, they trigger processes inside the cell that rearrange and strengthen the cell's internal structure, called the cytoskeleton. Cytoskeletal rearrangement helps the cell to withstand these forces. However, this process requires a lot of energy, and we do not fully understand how cells manage this increased energy demand. One family of surface receptors called cadherins are vital for connecting cells to each other at the cell-cell interface, a subcellular location known as the adherens junction. Cadherins are linked to the cytoskeleton through other proteins within the cell. They also act as sensors and responders to force. When force is applied to cadherins, they change shape, attracting other proteins that help strengthen their connection to the actin cytoskeleton. This response, termed reinforcement, allows cells to adapt to forces quickly, but this process is energetically demanding. We know little about how cells meet this energy demand. In my research, I found that endothelial cells (cells lining blood vessels) respond to force through a complex involving a specific cadherin called vascular endothelial cadherin or VE-Cadherin. I showed that fluid flow, a physiologically relevant force, activates a key energy regulating protein, AMPK, in human endothelial cells. Disrupting VE-Cadherin blocks this activation, linking VE-Cadherin to energy regulation. I further demonstrate that activation of this pathway is essential for maintaining cell junctions, glucose uptake, and activating a molecule called eNOS, all processes that are important for endothelial cell health. Additionally, I found that this energy management is crucial for maintaining the cell structure, aligning cells, and activating eNOS. If we block glycolysis (the process of breaking down glucose to produce energy) or disrupt AMPK, the cells can’t reinforce their structure or function vii properly. I also showed the importance of this mechanism in a mouse model with a mutation that disrupts the link between cadherins and glucose metabolism in epithelial cells. These mice showed body weight, muscle mass, and heart function changes. We are still investigating how these changes happen. Overall, this research shows that cells can adjust their energy use based on physical cues and that their physical responses depend on these energy changes.
- Academic Unit
- Biochemistry and Molecular Biology
- Record Identifier
- 9984774457202771
Metrics
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