Ontrafelen van endotheelcel mechanica in gezondheid en ziekte aan de hand van in vitro modellen

The vascular system is a highly dynamic network that carries blood to and from tissues through large arteries and veins, and facilitates nutrient and waste exchange via its extensive microvascular (MV) network. Endothelial cells (ECs) form the inner lining of these vessels, simultaneously acting as a restrictive barrier and the primary location for molecule and cellular exchange. Maintaining appropriate cell morphology, gene expression and activity is critical for endothelial function. However, this optimal phenotype varies across vessel types and vascular beds, reflecting the remarkable heterogeneity and adaptability of ECs. Upon stimulation by proangiogenic factors, ECs undergo phenotypic changes that initiate angiogenesis - the formation of new blood vessels from the pre-existing vasculature. This process in vital in vascular maintenance and repair, with ECs playing a central role. Dysregulated angiogenesis can lead to severe pathologies, including Cerebral Cavernous Malformations (CCMs). EC mechanics are fundamental to both vascular homeostasis and angiogenesis, encompassing intercellular junction stability and interactions with the extracellular matrix (ECM) such as adhesion, matrix degradation and traction force generation during migration and invasion. Quantifying these cellular forces provides crucial insight into endothelial migration and vascular remodeling under physiological and pathological conditions. In this context, robust invitro models compatible with Traction Force Microscopy (TFM) - particularly those that are high-throughput and user friendly - are invaluable tools for elucidating endothelial mechanical behavior. Two-dimensional (2D) single-cell assays, enable detailed investigation of the fundamental mechanical behavior of individual ECs without interference from neighboring cells. Extension of this model by incorporation of paracrine signaling and ECM remodeling further allows the investigation of indirect cell-cell communication mechanisms. However, the key advantage of studying individual cells also represents the main limitation of this approach, as EC behavior within the endothelium is intrinsically influenced by interactions with adjacent cells. To address this limitation, cell patterning techniques can be applied to organize ECs in defined multicellular configurations while maintaining high throughput and compatibility with TFM. Various methods for selective collagen functionalization are available and can be chosen according to the experimental objectives and technical constraints. While 2D models are valuable tools to study the quiescent endothelium, they inherently fail to reproduce three-dimensional (3D) angiogenic invasion - an essential process in both physiological and pathological vascular remodeling. To capture this EC behavior, a 3D TFM-compatible invasion assay was developed and optimized. The utility of this model is demonstrated in two studies: (1) investigation of mechanosensitive ion channels in CCMs and (2) assessment of organ-specific mechanical heterogeneity among ECs. CCMs are mulberry-like vascular lesions that predominantly develop in low-flow regions in the central nervous system. Because of this low-flow aspect, mechanosensitive ion channels - typically sensitive to shear stress modulations - have emerged as molecules of interest in CCM research. Within the broader context of this project, upregulation of PIEZO1, TRPV2, and TRPV4 was identified in CCM lesions and CCM2-mutant primary ECs. This upregulation was shown to play a critical role in CCM pathogenesis: PIEZO1 and TRPV4 contribute to increased endothelial permeability, while PIEZO1 and TRPV2 drive excessive ECM degradation; in addition, TRPV2 modulated 2D cellular force generation. The optimized 3D invasion assay was further employed to investigate early angiogenic invasion dynamics of CCM2-mutant cells. This study revealed TRPV2 as a key regulator of angiogenic invasion and wildtype cell recruitment, whereas PIEZO1 was found to be essential in 3D force generation. Collectively, the identification of these three upregulated channels as contributors to CCM pathology highlights their potential as therapeutic targets - an especially significant finding given the current lack of pharmacological treatment for CCM. Endothelial heterogeneity manifests across organs and vessels, resulting in distinct genetic profiles and functional profiles among EC populations. However, mechanical heterogeneity across these populations remains poorly understood. To address this, ECs derived from the umbilical vein, as well as from the microvasculature of the brain, lungs, and skin, together with MV lymphatic cells from the skin, were compared using the 3D invasion assay. This study elucidated key invasion mechanisms, including the importance of tightly regulated ECM degradation during the initial sprouting phase and filopodia formation throughout the sprouting process. Furthermore, traction force quantification revealed clear organ-specific mechanical heterogeneity, with MV cells from the skin exhibiting higher force generation than those from other vascular origins. While the high-throughput in vitro assays used here have certain limitations - most notably the lack of flow - this work illustrates their strong utility for investigating EC mechanics in both health and disease.