Kracht-gemedieerde angiogenese: Relatie tussen mechanische mechanismen en vasculaire letselgroei

Mechanical forces play a fundamental role in regulating biological functions in both health and disease. Cells sense their microenvironment and translate external biophysical stimuli into biochemical signals to adapt their function. This is particularly relevant to sprouting angiogenesis - a primary mechanism for vessel formation during development, tissue regeneration, and pathological vascular lesion formation. However, elucidating the mechanical regulation of angiogenesis remains a challenge due to the complexities of measuring cellular forces in this dynamic, multicellular and three-dimensional (3D) process. Chapter 2 of this thesis presents a protocol for mimicking and imaging dynamic early angiogenic sprouting into biomimetic matrices compatible with 3D traction force microscopy (TFM), and for visualizing matrix degradation. It also provides protocols for immunofluorescence and downstream processing of hydrogels to retrieve cells for single cell RNA sequencing in the same system. Given that reliably acquiring sufficiently large 3D TFM datasets in multicellular systems is challenging, this protocol emphasizes best practices for higher throughput data acquisition and for accurately imaging, analyzing and interpreting forces. This assay provides a defined and reproducible system to study cellular forces and matrix degradation during angiogenesis in response to perturbations of cell-intrinsic signaling, mixed cell populations, as well as extracellular matrix cues. Force-driven cellular interactions are known to play a critical role in cancer cell invasion, but have remained largely unexplored in the context of vascular abnormalities, partly due to a lack of suitable genetic and cellular models. One such vascular abnormality, cerebral cavernous malformation (CCM) is characterized by leaky, tumor-like vessels in the brain, where CCM mutant cells recruit wild-type cells from the surrounding endothelium to form mosaic lesions and promote lesion growth; however the mechanisms underlying this recruitment remain poorly understood. In Chapter 3, we used the platform optimized in Chapter 2 to reveal that hyper-angiogenic CCM2-silenced endothelial cells enhance angiogenic invasion of neighboring wild-type cells through force and extracellular matrix-guided mechanisms. We show that mechanically hyperactive CCM2-silenced tips guide wild-type cells by exerting and transmitting pulling forces and by leaving degraded paths in the matrix as cues promoting invasion in a ROCKs-dependent manner. This transmission of forces is associated with a reinforcement of β1-integrin adhesions and actin cytoskeleton in the wild-type followers. During this process, wild-type cells are reprogrammed into stalk cells through activation of matrisome and DNA replication programs, eventually leading to cell proliferation. These observations unveil a novel vascular lesion growth mechanism where CCM2 mutants hijack the function of wild-type cells to fuel CCM lesion growth. By integrating biophysical computational methodologies to quantify cellular forces with advanced molecular techniques, we provide new insights in the etiology of vascular malformations, and open up avenues to study the role of cell mechanics in tissue heterogeneity and disease progression.