Engineering models for mechanobiology of cellular migration during angiogenesis

Angiogenesis – the formation of new blood vessels from pre-existing vasculature – is driven by endothelial cell migration, which is tightly regulated by mechanical interactions with the extracellular matrix (ECM) and neighboring cells. Precise coordination of the mechanical forces is essential for proper vascular development, and dysregulation can contribute to pathological conditions. Despite extensive research on cellular migration, the unique mechanical dynamics governing endothelial movement in sprouting angiogenesis remain insufficiently understood. This thesis addresses this gap by developing and applying in vitro and ex vivo models to dissect the mechanobiology of endothelial migration and its implications in angiogenesis. Chapter 1 introduces the key mechanobiological regulators of endothelial migration in sprouting angiogenesis. It presents mechanoreciprocity as the dynamic process by which endothelial cells generate and respond to mechanical forces from their environment. It reviews the roles of essential components like the actin cytoskeleton and focal adhesions, and introduces juxtacrine Notch signaling and the intermediate filament Vimentin as regulators of cell mechanics and migration. The chapter discusses various models, from in vivo to in vitro, emphasizing techniques such as traction force microscopy (TFM) for quantitative insights. While these models provide control and precision, their limitations in capturing the full complexity of endothelial migration underscore the need for further refinement. This chapter establishes the mechanobiological foundation for the experimental frameworks explored in subsequent chapters. Chapter 2 presents a 2D in vitro framework designed to investigate the mechanics of collective migration under controlled conditions. By integrating TFM, this model quantifies cellular forces and reveals how substrate stiffness, confinement, and intercellular interactions influence endothelial migration. This approach provides valuable insights into how mechanical forces regulate cell migration during essential biological processes, including tissue morphogenesis, wound healing, and cancer metastasis. Additionally, the model enables high-resolution analysis of both single-cell and collective migration behaviors, facilitating a deeper understanding of how cells coordinate movement in response to their mechanical environment. Chapter 3 expands on this framework by examining the role of Notch signaling in endothelial collective migration. Using high-resolution force mapping, this study demonstrates that Notch inhibition alters migration speed and force distribution in cell collectives during directed collective migration. These findings suggest that Notch signaling modulates mechanical cohesion within endothelial collectives. The results provide new insights into angiogenesis regulation, highlighting the potential for targeted therapeutic interventions that manipulate Notch-related mechanotransduction pathways. Chapter 4 explores the role of the intermediate filament Vimentin in maintaining mechanical homeostasis. The study reveals that Vimentin-expressing cells dynamically adapt to mechanical changes in their microenvironment, ensuring stability and responsiveness to mechanical cues. In contrast, Vimentin-depleted cells initially fail to maintain this balance, exhibiting altered mechanical properties. However, these cells later compensate by increasing ECM synthesis and expression of ECM crosslinking enzymes to effectively modify their microenvironment and restore homeostasis. These findings highlight Vimentin’s role in mechanoreciprocity and cellular adaptation, offering potential avenues for modulating cell mechanics in disease contexts. Chapter 5 introduces a 2.5D ex vivo model to study sprouting angiogenesis in living tissue, using TFM to characterize the mechanical forces driving cellular sprouting. This model demonstrates that leader cells generate pulling forces, while follower cells exert pushing forces, and that sprouting dynamics are matrix stiffness-dependent. By enabling the manipulation of both chemical and mechanical cues, the model provides a powerful tool for investigating the role of mechanotransduction in angiogenesis. These insights have broad implications, ranging from fundamental vascular biology to applications in tissue engineering and therapeutic strategies for vascular diseases and tumor angiogenesis. Collectively, this thesis advances vascular mechanobiology by refining in vitro and ex vivo models to dissect the mechanical regulation of endothelial migration. By integrating experimental approaches and computational analyses, these studies enhance our understanding of how mechanical forces shape angiogenesis. Chapter 6 evaluates the strengths, limitations, and assumptions of the models and methods used, and explores potential mechanisms underlying the mechanobiological observations in this thesis. This knowledge is particularly crucial in tissue engineering, where promoting angiogenesis can enhance the development of functional tissue grafts, and in disease treatment, where targeting angiogenesis can help manage conditions like cancer by controlling abnormal blood vessel growth.